yöntem - Spektrotek

Transkript

yöntem - Spektrotek

GIDA & ÇEVRE UYGULAMALARI
www.spektrotek.com
Spektrotek A.Ş.
www.spektrotek.com
[email protected]
t: 0 216 688 57 78
f: 0 216 688 57 69
Katalog taleplerinizi [email protected] e-mail adresine, adres bilgilerinizi göndererek talep edebilirsiniz.
SPEKTROTEK A.Ş. Kurumsal tanıtım ve ürün kataloğunda paylaşılan tüm görsel ve yazılı bilgilerin hakları SPEKTROTEK A.Ş. ve ilgili üreticilere aittir.
Tüm hakları saklıdır. İzinsiz kopyalanamaz, alıntılanamaz, başka yerde kullanılamaz.
Katalogda kullanılan ürünler ile orjinalleri arasında farklılık görülebilir.
Tasarım, İçerik, Teknik Hazırlık ve Üretim:
www.prosigma.net • [email protected]
SPEKTROTEK A.Ş., 2010 yılında İstanbul merkezli olarak kurulmuş alanında
Dünya lideri global üreticilerin Türkiye, Kuzey Kıbrıs ve Azerbaycan bölgesinde
temsilini yapan Laboratuvar Cihaz ve Ekipmanları sağlayıcısı firmadır.
Değişik bilim dallarının eğitim ve araştırma amaçlı olarak ağırlıklı
Laboratuvarda kullandığı Analitik Cihazlar, Ekipman ve Aksesuarlar, Kimyasal
ve Sarflar SPEKTROTEK A.Ş.' nin büyük özen ve uzmanlıkla sağladığı ürün
gruplarındandır.
Hakkımızda
Gıda ve Çevre Uygulamaları
Spektrotek A.Ş.
SPEKTROTEK uzman grubu, Klasik ihtiyaçların yanı sıra Dünyada gelişen yeni
teknoloji ve uygulamaları da son kullanıcılara aktarmayı bir görev bilmektedirler.
Laboratuvar ihtiyacının iyi anlaşılıp değerlendirmesine istinaden alternatifli marka ve özellikli sistemlerin önerilmesi ile başlayan
süreç, ne olursa olsun sat değil, ‘Doğru sistemleri, Doğru fiyatla Sat’ mantığıyla yürütülmekte ve son kullanıcılar/Araştırmacılar
müşteri olarak değil partner olarak değerlendirilmektedir.
Herbiri Yüksek Lisans ve/veya Doktora seviyesinde alanında uzmanlığa sahip çalışanlarımız aynı zamanda şirket içi detaylı
eğitimler ve sürekli yenilenen bilgi akışı sayesinde Dünya ve üreticilerimizin Know/How bilgilerini laboratuvarlara taşımaktadır.
Kaliteli servis hizmeti olmadan hiçbir ürün satışına girmemek şiarı ile yetkili personelimiz üretici tesislerinde detaylı eğitimler
alarak sertifikalandırılmaktadır. Aynı zamanda TSE Hizmet Yeri Yeterlilik Belgesi ve başka pek çok kalite belgeleriyle de hizmet
kalitemiz belgelenmiştir.
Ayrıca SPEKTROTEK A.Ş., Kendi tescilli markası olan ChromXpert ile önde gelen üreticilere yüksek kalite ile OEM ürettirdiği
ürünlerle kendi markası ile de Laboratuvar ürünleri pazarında yerini almıştır.
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Vizyonumuz;
Bilim ve teknolojik yenilikleri, Dünyanın en iyi üreticileri yoluyla Türk Araştırmacılara ileterek ülkemizin kalkınması ve gelişmesine
katkıda bulunmaktır.
3
“ Yapılırken heyecan duyulmayan işler başarılamaz “
Emerson
Profesyonel ihtiyaçlarınıza, profesyonel çözümler
Analiz ihtiyaçlarınıza profesyonel çözümler...
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Spektrotek tüm resmi kurum laboratuvarları, özel
laboratuvarlar ve üniversite araştırma laboratuvarlarının tüm
ihtiyaçlarını karşılamak üzere yapılanmıştır.
4
Bünyesinde tecrübeli uygulamacıların bulunduğu Spektrotek,
Dünyanın önde gelen firmalarının Türkiye’de temsili ve
analitik cihazların satışının yanı sıra, laboratuvarların
gerçekten ihtiyacı olan satış sonrası destek, servis ve metot
oluşturulması konusunda da tecrübelerini müşteriyle
paylaşmaktadır. Firmamız sektördeki yerini alırken işini bir
profesyonel ciddiyetinde yapmak, amatör heyecanını da
kaybetmemek şiarı ile yola çıkmıştır.
Spektrotek ailesinde, konusunda uzman farklı branşlardan
mühendisler, müşterilerin taleplerini analiz etmek, uygun
sistem ya da çözümleri belirlemek ve partner olarak
gördüğümüz müşterilerimiz ile pozitif bir iletişim içinde
tecrübelerini aktarmak üzere şirket içi eğitimler almaktadırlar.
Bunun yanı sıra tüm teknik kadronun başta Almanya ve
Amerika olmak üzere üretici firmaların laboratuvarlarında
aldığı eğitimler ve akabinde sınavlardaki dereceleri ile
kazandıkları uzmanlık sertifikaları mevcuttur. Tüm teknik
kadro, konusu ile ilgili konferans, kongre ve seminerlere
katılarak güncel gereklilikleri ve yeni gelişmeleri yakalamak
üzere teşvik edilmektedir.
Spektrotek, temsilini yürüttüğü firmaların know-how
bilgilerini Türkiye’deki laboratuvarlara aktarmak, yeni
teknolojik gelişmeler ile firmaların ve laboratuvarların mevcut
problemlerine çözümler getirmek, orta vadede Türkiye’de
Analitik Cihaz sektöründe liderliği üstlenmek üzere sağlam
adımlarla gelişmesini sürdürmektedir.
Hizmetlerimiz
Analitik Cihazların Satış ve Satış Sonrası Teknik Desteği
Metot Geliştirme ve Validasyon Hizmetleri
Laboratuvar Projelendirme
Akreditasyon Danışmanlığı
Kromatografik ve Spektroskopik Tekniklere Ait Teknik Eğitimler
Laboratuvar Sarf Malzemeleri Satışı
Temel Laboratuvar Cihaz ve Ekipmanları Satışı
Referans Standart Kimyasalların Satışı
Ürünlerimiz
Sıvı Kromatografi Sistemleri (HPLC)
PreparatifLC
Ultra/UHPLC
NanoLC
MikroLC
Online SPE Numune Hazırlık Sistemleri
Protein Saflaştırma/BioChromatography
Osmometreler
Atomik Absorpsiyon Spektrofotometresi (AAS)
ICP-OES/ICP-TOFMS
UV-VIS Spektofotometre
Gaz Jeneratörleri
Azot Jeneratörleri
Kuru Hava Jeneratörleri
Hidrojen Jeneratörleri
Numune Ön Hazırlık Ekipmanları
Temel Laboratuvar Cihaz, Ekipman ve Sarfları
Vakum Pompaları
Su Banyoları
Otoklavlar
Etüv & İnkübatörler
Çalkalayıcılar
Isıtıcı Tablalar
Homojenizatörler
Fırınlar
Karıştırıcılar & Manyetik Karıştırıcılar
Ceketli Isıtıcılar
Rotary Evaporatörler
Santrifüjler
Dondurucu & Soğutucular
Vorteks
Thermal Cycler
Jel Görüntüleme Sistemi
Pipet & Otomatik Pipet ve Dispenserlar
Cam & Plastik Laboratuvar Malzemeleri vb.
Kromatografi Sarfları
Vial
SPE Ürünleri
Membran Filtre
Şırınga Ucu Filtre
HPLC /GC Kolonları
QuEChERS Kitleri
Spektroskopi Sarfları
Spektrofotometre Küvetleri
Referans Maddeler
Filtreler
FTIR aksesuarları
Biyokimya LC-MS/MS ve HPLC analiz kitleri
Referans Standart Kimyasallar
Organik Referans Standartlar
İnorganik Referans Standartlar
USP Standartları
Kalite Kontrol (QC) Numuneleri
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Kütle Spektrometre
Triple Quadrapole (Tandem MS)
QTrap Hibrid Triple Quadrapole Lineer İyon Trap
MALDI TOF/TOF
Yüksek Rezolüsyon QqTOF
5
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6
Analitik Cihaz Satışı ve
Satış Sonrası Desteği
Tamamlayıcı Cihazlar,
Sarf Malzemeler ve Yedek Parça Satışı
Spektrotek, Kromatografi ve Spektroskopide
Dünya liderliğine sahip firmaların Türkiye, KKTC
ve Azerbaycan’da temsil hakkına sahiptir.
Spektrotek, bu firmaların ürün pörtföyünde olan
LCMSMS, MALDI TOF, Nano ve Mikro HPLC,
UHPLC, Online – SPE, Atomik Absorpsiyon
Spektrofotometresi, UV-VIS Spektrometre, ICP-OES,
ICP-MS, Osmometre vb. ürünlerin satışı ile ilgili
teknik görüşmeleri yaparak laboratuvarların
ihtiyaçlarını anlamak ve bu ihtiyaçlara en uygun
çözümleri üretmek, satışın ardından üretici ve
Spektrotek Kalite Yönergeleri gereğince kurulum
testlerini yaparak sağlıklı çalışır vaziyette cihazın
teslimatını yapmak ve ilgili raporları (IQ/OQ vb.)
laboratuvar yönetimine sunmak, laboratuvar
ihtiyaçlarının karşılanması ve bilgi transferi de dahil
olmak üzere kusursuz cihaz eğitimini verirken,
garanti içinde ve sonrasında satış sonrası destekleri
sağlamak üzere satış sürecini yönetir.
Laboratuvarların ihtiyaçları sadece analitik cihazların
temini ile bitmemektedir. Gerek hızlı sonuçlar
açısından basit sistemler olsun, gerek numune
hazırlık aşamalarında olsun, her laboratuvar
pHmetre, Rotary Evaporatör, Etüv, Karıştırıcı, Isıtıcı,
Hassas ve Analitik teraziler gibi temel laboratuvar
cihazları ismini verdiğimiz tamamlayıcı cihazlara
ihtiyaç duymaktadır.
Yine aynı şekilde analitik sistemlerde kullanılan
ve analizlerde sarf edilen Kolon, SPE kartuş, Vial/
Kapak/Septum, Kuartz Küvet, Filtreler vb. pek çok
sarf malzemesi mevcuttur. Spektrotek, tüm temel
laboratuvar cihazları, sarf malzemeler ve satışını
yaptığı tüm ürünler için gerekli yedek parçaları,
dünyanın en saygın üreticilerinden temin ederek
laboratuvarların tüm ihtiyaçlarına toplam çözüm
üretmektedir.
Danışmanlık ve Metot Bilgisi Transferi
Günümüzde tüm üretim firmalarının hem kendi
ürünlerinin kalite kontrol ve Ar-Ge’si amacıyla hem
de tedarikçilerden gelen hammaddelerinin kontrolü
amacıyla kapasiteli bir laboratuvara ihtiyacı
vardır. Bunun yanı sıra pek çok hizmet sağlayan
laboratuvar çeşitli kaynaklardan gelen numuneleri
analiz ederek raporlama yapmaktadır.
Biyoeşdeğerlik, Gıda, Çevre, Ekotest ve Biyokimya
gibi birbirinden farklı alanlarda da olsa tüm
laboratuvarların tabi olduğu evrensel kalite
kuralları söz konusudur.
Türkak, DAR vb. akreditasyon verme yetkisine
sahip kuruluşlardan akredite olmak, ISO /IEC 17025
gibi test ve kalibrasyon yapan laboratuvarların ya
da ISO 15189 gibi tıbbi laboratuvarların şartlarına
kavuşmak her laboratuvarın hedeflerindendir.
Bu süreçte elbette pek çok kriter söz konusudur.
Laboratuvar boyasından tezgahta kullanılan
materyale, çalışanların eğitim sertifikalarından,
analitik cihazlardaki metodların validasyonlarına ve
SOP’lerine kadar laboratuvarların değerlendirmesi
gereken pek çok parametre bulunmaktadır.
Spektrotek olarak, boş bir binanın akredite bir
laboratuvara dönüşünceye kadar tüm ihtiyaçlarına
cevap verecek yapılanmayı hazırladık.
Sağladığımız cihazlarla, yılların tecrübesi ve
alanında uzman firmalarla işbirliği çerçevesinde
gururla sunacağınız laboratuarınızda partneriniz
olmaya hazırız.
Çözüm ortaklığı yaptığımız uluslararası firmaların
Know-How bilgileri ve teknik ekibimizin tecrübeleri
ile laboratuvarlar için uzun ve yorucu olan metot
geliştirme, validasyon süreçlerinde yine
yanınızdayız.
Pestisit/veteriner ilaç kalıntı analizlerinden doping
kontrolüne, yeni doğan taramasından tekstil
ürünlerindeki yasaklı bileşiklerin tayinine, içme suyu
ve toprak numunelerindeki yüzlerce parametrenin
tayininden yaşam bilimlerindeki proteomik /
metabolomik çalışmalarına kadar pek çok hazır
metodun hızlıca laboratuvarınıza aktarılması
işimizin en iyi bildiğimiz ve en sevdiğimiz kısmını
oluşturmaktadır.
Bununla birlikte laboratuvar yatırımlarının en verimli
şekilde yapılabilmesi için çeşitli kaynaklardan
projeler ile yardım alınması noktasındaki
danışmanlık hizmetlerimiz de geleceğiniz için
beklediğinizden de büyük adımlar atmanıza
yardımcı olacaktır.
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Anahtar Teslim Laboratuvar Projeleri
7
İçindekiler
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SPEKTROTEK GIDA VE ÇEVRE UYGULAMALAR
8
Bebek Mamalarında Polisiklik Aromatik Hidrokarbon (PAH) Analizi 12
Bebek Mamalarında Multi-Toksin Analizi
13
Gıda Ürünlerinde LC-MS/MS ile Akrilamid Tayini
14
Süt ve Süt Ürünlerinde Melamin Analizi 15
Yaş Meyve ve Sebzede Multi-Pestisit Analizi 16
Gıdalarda Polar Pestisit Tayini 17
Gıda Ürünlerinde Paraquat/Diquat Analizi
18
Jelibonda Jelatin Tür Analizi
20
Ette Tür Tayini
22
Zeytinyağında Tağşiş ve Orijin Belirleme
23
LC-MS/MS ile Gıdalarda Alerjen Analizi
24
Bebek Mamalarında B Vitamini Analizi
26
Veteriner İlaç Kalıntıları/Antibiyotik Analizi
27
Gıda ve İçeceklerde Tatlandırıcı Tayini
28
İçme Sularında Polisiklik Aromatik Hidrokarbon (PAH) Analizi
30
İçme Sularında Akrilamid Analizi 31
İçme Sularında Multi-Pestisit Analizi
32
İçindekiler
SCIEX APPLICATION NOTES
Simultaneous Analysis of 14 Mycotoxins and 163 Pesticides in Crude Extracts of Grains by LC-MS/MS 62
Detection of Underivatized Glyphosate and Similar Polar Pesticides in Food of Plant Origin by LC-MS/MS 65
Improving the LC-MS/MS Selectivity of Triazole Derivative Metabolites with AB SCIEX SelexION™ Technology 69
Fast and Sensitive Analysis of Paraquat and Diquat in Drinking Water
74
The Quantitation and Identification of Coccidiostats in Food by LC-MS/MS using the AB SCIEX 4000 Q TRAP® System 78
Quantitation and Identification of 13 Azo-dyes in Spices using LC-MS/MS
83
Increasing Selectivity and Confidence in Detection when Analyzing Phthalates by LC-MS/MS 88
Quantitative Analysis and Identification of Migrants in Food Packaging Using LC-MS/MS
93
Analysis of Perfluoroalkyl Acids Specified Using the QTRAP® 6500 LC/MS/MS System 97
LC-(DMS)-MS/MS Analysis of Emerging Food Contaminants 102
Analysis of Endocrine Disruptors, Pharmaceuticals, and Personal Care Products in River Water
106
Analysis of Selected Microcystins in Drinking and Surface Water Using a Highly Sensitive Direct Injection Technique 111
Quantitative Analysis of Explosives in Surface Water Comparing Off-Line Solid Phase Extraction and Direct Injection LC-MS/MS 114
Trap LC-MS/MS System 118
Quantitation and Identification of Organotin Compounds in Food, Water, and Textiles Using LC-MS/MS 121
www.spektrotek.com
Screening and Identification of Unknown Contaminants in Untreated TapWater Using a Hybrid Triple Quadrupole Linear Ion
9
GIDA&ÇEVREUYGULAMALARI
BEBEK MAMALARINDA POLİSİKLİK
AROMATİK HİDROKARBON (PAH) ANALİZİ
GİRİŞ
Polisiklik aromatik hidrokarbon (PAH)’lar karbon ve hidrojen içeren organik
maddelerin pirolizi veya tam olmayan yanmalar sonucu oluşan, iki veya daha
fazla aromatik halka içeren bileşiklerdir.
PAH’lar hava, su, toprakta ve dolayısıyla da gıdalarda bulunabildiğinden;
insanlar bu bileşiklere mesleki, çevresel, tıbbi ve diyetle ilgili kaynaklar
aracılığıyla maruz kalmaktadır. Özellikle, endüstriyel üretim yapılan
bölgelerdeki kirli hava bileşenlerinin bitkisel ürünler üzerindeki birikimleri
sonucunda tahıl, meyve ve sebzeler kontamine olabilmektedir. Öte yandan
kavurma, dumanlama ve ızgara uygulamaları gibi işleme prosesleri de,
gıdada PAH’ların oluşumuna neden olabilmektedir.
Gıdanın direkt alevle teması durumunda PAH’ların miktarı daha da
yükselmektedir. Bu bileşiklerin oluşması pişirme metodu ve uygulanan
sıcaklıkla yakından ilişkilidir.
PAH bileşiklerinin oluşumu açısından riskli bulunan gıdalardan biri ve en
önemlisi bebek mamalarıdır. Bebek mamalarının PAH’larla kontaminasyon
seviyesi, gerek mama üretiminde uygulanan kurutma sıcaklıklarına, gerekse mama bileşimine giren süt ve/veya meyve, sebze,
tahılların çevresel faktörler nedeniyle PAH bileşikleri ile kontamine olmasına bağlı olarak, çeşitli düzeylerde benzo(a)piren ve diğer
PAH bileşiklerini içerebilmektedir. İnsanlar üzerinde toksik ve kanserojenik etkiye sahip oldukları için gıdalardaki miktarlarının
kontrol edilmesi oldukça önemlidir.
Maksimum izin verilen miktarları ulusal ve uluslararası gıda ve sağlık örgütleri tarafından belirlenmiştir. Avrupa Birliği uyum
süreci çerçevesinde ülkemizde de PAH için belirlenen limitler “Türk Gıda Kodeksi Bulaşanlar Yönetmeliği” ile yürürlüğe girmiştir.
Bu kirleticilerin uygun analiz yöntemleri ile tayin edilmesi ve çevremizden mümkün olduğunca uzaklaştırılması için önlemler
alınması gerekir.
YÖNTEM
Analitik Koşullar
Numune Hazırlığı
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
Yönetmelikte maksimum kalıntı limitleri
belirlenen 4 adet PAH bileşiği için numuneler,
basit bir numune hazırlama yöntemi olan sıvısıvı ekstraksiyon ile hazırlanarak LC-MS/MS’e
enjeksiyonu yapılır.
: Phenomenex Kinetex Phenyl-Hexyl 100x2.1 mm 1.7 um
: 8 dakika
: Su ve Asetonitril
: APCI
: Scheduled MRM™
MRM çiftleri:
U
ntitled1(bap2):"Linear"R
egression("N
o"w
eighting):y=178x+818(r=0.9994)
3.7e4
Polarite
pozitif
pozitif
pozitif
pozitif
Q1/Q3
228/226
252/250
252/250
228/226
3.6e4
Q1/Q3
228/199
252/226
252/224
228/200
3.4e4
3.2e4
3.0e4
2.8e4
2.6e4
2.4e4
2.2e4
2.0e4
stnuoc ,aerA
Bileşik
Benzo(a)anthracene
Benzo(a)pyrene
Benzo(b)fluorenthene
Chrysene
1.8e4
1.6e4
1.4e4
1.2e4
2
1.0e4
8000.0
6000.0
4000.0
2000.0
3
4
0.0
10
20
30
40
50
60
70
80
90
100
110
C
oncentration,pg/m
l
120
130
140
150
160
170
180
190
200
1
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SONUÇ
12
Gelişen teknoloji ile birlikte hızlı, doğru, güvenilir ve düşük maliyetli
analiz tekniklerinin geliştirilmesi bir ihtiyaç haline gelmiştir. Bu
nedenle, basit ve zahmetsiz bir numune hazırlığına sahip olan
LC-MS/MS metotları tercih edilmektedir. Scheduled MRM™
algoritması kullanılarak yapılan çalışmada elde edilen sonuçlar
değerlendirildiğinde bebek mamaları için yönetmelikte verilen
limitleri rahatlıkla karşılamaktadır.
Şekil 1- Bebek mamasında Scheduled MRM™ ile
yapılan PAH analizine ait kromatogram: (1) Benzo(a)
anthracene, (2) Chrysene, (3) Benzo(b)fluorenthene,
(4) Benzo(a)pyrene
GİRİŞ
Hayvanlar ve insanlar için toksik özellik gösteren mikotoksinler, tarlada
veya gıda maddelerinin depolama ve dağıtımı aşamasında oluşabilirler.
Mikotoksinler; aflatoksinler, okratoksinler, fumonisin, zearalenone (ZON),
trichlothecenes gibi Fusarium toksinleri ve ergot alkoloidleri gibi farklı ana
gruplara ayrılırlar.
Mikotoksinlerin gıda ve yemlerde belirlenmesi için günümüzde hassas ve
güvenli birçok yöntem bulunmaktadır. Klasik mikotoksin analizleri, her
bir mikotoksin için ya da benzer kimyasal özelliklere sahip grupların (ör;
aflatoksinler) her biri için ayrı ayrı metot kullanılarak yapılmaktadır. Bu
yöntem hedef bileşiklerin geniş polarite aralığında olması ve maddelerin
fiziksel özelliklerin farklı olmasından kaynaklanmaktadır.
Tüm metotlar immuno-affinity kolon ile cleanup aşamasının ardından
fluoresans dedektörü ile HPLC analizine dayanmaktadır. Fakat numune
içerisinde mikotoksin grupları ayrı ayrı bulunabileceği gibi aynı numune
içerisinde bir arada bulunabilirler. Bu sebeple numune hazırlık aşaması uzun
sureli ve yüksek maliyetli olduğundan dolayı pratik bir uygulama sayılmamaktadır. Bunun yerine numune hazırlığı için basit
ve hızlı bir metot olan sıvı-sıvı ekstraksiyonu ardından immuno-affinity kolon kullanımına ihtiyaç kalmadan negatif ve pozitif
polariteye sahip bileşiklerin tek bir LC-MS/MS enjeksiyonu ile analizi tercih edilmektedir.
Avrupa Birliği uyum süreci çerçevesinde ülkemizde gıdalardaki maksimum mikotoksin limitleri “Türk Gıda Kodeksi Bulaşanlar
Yönetmeliği” ile yürürlüğe girmiştir.
YÖNTEM
Analitik Koşullar
Basit, hızlı ve ucuz bir metot olan sıvı-sıvı ekstraksiyonu
ardından immuno-affinity kolon kullanımına ihtiyaç kalmadan
negatif ve pozitif polariteye sahip bileşikler, tek bir LC-MS/
MS enjeksiyonu ile analiz edilmektedir.
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
BEBEK MAMALARINDA MULTİ-TOKSİN ANALİZİ
: Phenomenex Gemini 5 μm (150x4.6 mm)
: 9 dakika
: Su (Amonyum Asetat) ve Metanol (Amonyum Asetat ve Asetik Asit)
: ESI + / ESI –
: Scheduled MRM™
MRM çiftleri:
Q1/Q3
313/128
315/259
329/200
331/189
329/272
355/59
317/175
722/704
706/688
404/102
153/135
SONUÇ
Hızlı Polarite değişimi ve Scheduled MRM™ ile hassasiyette
azalma olmadan kısa sürede analiz imkanı sağlanmış
olup çalışmada elde edilen sonuçlar bebek mamaları için
yönetmelikte verilen limitleri rahatlıkla karşılamaktadır
Max. 9.7e4 cps.
XIC of +MRM (16 pa rs): Exp 1, 313.000/128.100 amu Expected RT: 5.8 ID: Aﬂatox n
AFG1
Pozi f polarite
2.0e5
1.8e5
OKRA
1.6e5
1.4e5
1.2e5
AFB1
1.0e5
5.87
8.0e4
AFM1
6.0e4
AFB2
4.0e4
2.0e4
0.0
AFG2
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
T me, m n
5.0
FB1
5.5
6.0
FB2
6.5
7.0
7.5
8.0
8.5
Max. 9.8e4 cps.
XIC of -MRM (7 pa rs): Exp 2, 152.444/108.500 amu Expected RT: 3.2 ID: patul n
ZON
Nega f polarite
5.5e5
5.0e5
4.5e5
4.0e5
3.5e5
3.0e5
2.5e5
2.0e5
1.5e5
patulin
3.23
1.0e5
5.0e4
DON
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
T me, m n
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
Şekil 2- Bebek mamasında Negatif-pozitif polarite değişimi
ve Scheduled MRM™ algoritması ile tek metotta analiz edilen
mikotoksinlere ait kromatogram
www.spektrotek.com
Q1/Q3
313/241
315/287
329/243
331/245
329/311
355/295
317/131
722/334
706/336
404/239
153/109
In te n s t y, cps
Polarite
pozitif
pozitif
pozitif
pozitif
Pozitif
Negatif
Negatif
Pozitif
Pozitif
Pozitif
Negatif
In te n s t y, cps
Bileşik
Aflatoksin B1 (AFB1)
Aflatoksin B2 (AFB2)
Aflatoksin G1 (AFG1)
Aflatoksin G2 (AFG2)
Aflatoksin M1 (AFM1)
Deoxynivalenol (DON)
Zearalenon (ZON)
Fumonisin B1 (FB1)
Fumonisin B2 (FB2)
Okratoksin A (OTA)
Patulin
13
GIDA ÜRÜNLERİNDE LC-MS/MS İLE
AKRİLAMİD TAYİNİ
GİRİŞ
Akrilamid, gıdaların doğal yapısında bulunmayan, karbonhidrat ve protein
içerikli gıdaların yüksek sıcaklıklarda (kızartma, fırın ve ızgara) pişirilmesi
sonunda ortaya çıkan bir bileşiktir. Akrilamid, gıda analizcileri için oldukça yeni
bir konu olmasına rağmen, 2002’de gıdalarda oluştuğu tespit edildiğinden bu
yana akrilamid ile ilgili çok sayıda yöntem geliştirme çalışması yapılmış ve
halen bu çalışmalar devam etmektedir. Akrilamid içeriği açısından önemli
gıda grupları patates cipsi, kızarmış patates, kızarmış ekmek, kahvaltılık
hububatlar, unlu mamuller ve kahve gibi ürünlerdir.
Akrilamid, Uluslararası Kanser Araştırmaları Ajansı tarafından “insan için
muhtemel kanserojenik madde” olarak tanımlanmıştır. Bu sebeple gıdalarda
miktarlarının kontrol edilmesi oldukça önemlidir.
Gıdalarda akrilamid tespit ve tayini için en yaygın kullanılan yöntemler GCMS ve LC-MS/MS’dir. GC-MS analiz yönteminde türevlendirme (bromlama)
işlemine ihtiyaç duyulmaktadır. Bu işlem zaman alıcı ve yüksek maliyetli
olup aynı zamanda kullanılan toksik maddeler yönü ile de sağlığa zararlıdır. Ayrıca numune hazırlığı sırasında kişiye bağlı hatalar
oluşabilmektedir. Bu sebeple türevlendirme aşamasına ihtiyaç duymayan, kolay ve daha düşük maliyetli numune hazırlama olan
sıvı-sıvı ekstraksiyonun ardından direk olarak numunenin enjeksiyonuna dayanan LC-MS/MS yöntemleri önem kazanmıştır.
Ülkemizde konu ile ilgili çeşitli bilimsel araştırmalar yürütülmekte ve bir çok gıda kontrol laboratuvarında akrilamid analizi
yapılmaktadır. Akrilamidin gıdalar içinde bulunması bütün gıdalar çiğ tüketilmedikçe önlenemez ve gıdaların pişirilmesi sonucu
doğal olarak oluşan bir madde olduğu için bu tür gıda gruplarının yasaklanması söz konusu değildir. Ancak, ilgili sağlık
risklerinin belirlenmesinden sonra, gıdalarda izin verilen maksimum akrilamid seviyeleri ile ilgili yasal limitler getirilebilecektir.
Diğer yandan, bütün dünyada gıdalarda akrilamid içeriğinin düşürülmesi veya oluşumunun önlenmesi ile ilgili çalışmalar da
yoğun bir şekilde sürdürülmektedir.
YÖNTEM
Analitik Koşullar
Numuneler homojenize edildikten sonra sıvı-sıvı
ektraksiyonu ile hazırlanarak direk olarak LC-MS/
MS’e enjeksiyonu yapılır.
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
: Phenomenex Luna C18 3 um (150x3 mm)
: 3 dakika
: Su (Formik Asit) ve Metanol (Formik Asit)
: ESI +
: Scheduled MRM™
MRM çiftleri:
Bileşik
Akrilamid 1
Akrilamid 2
Akrilamid 3
Polarite
Pozitif
Pozitif
Pozitif
Max. 3.3e5 cps.
XIC of +MRM (3 pa rs): 72.100/55.000 Da ID: Acrylam de 1 from Sample 53 (2-2) of 26022015 acrylam de.w ff (Turbo Spray)
Q1/Q3
72/55
72/44
72/27
1.26
3.2e5
3.0e5
2.8e5
2.6e5
2.4e5
2.2e5
I n t e n s t y, cp s
2.0e5
1.8e5
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
www.spektrotek.com
SONUÇ
14
Yüksek hassasiyet ve seçicilikten dolayı basit bir numune
hazırlığı olan sıvı-sıvı ekstraksiyonu ile kısa zamanda onlarca
numune rahatlıkla çalışılabilmektedir. Yapılan çalışmalar
değerlendirildiğinde 0.02 ug/L dedeksiyon limitlerinde
çalışıldığı görülmüştür. Bu metot ile su numunelerinde
de rahatlıkla analiz yapılabilmektedir. Direk enjeksiyon
ile elde edilen sonuçlar yönetmelikteki limitleri rahatlıkla
karşılamaktadır.
4.0e4
2.0e4
0.0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
T me, m n
1.8
2.0
2.2
2.4
2.6
2.8
Şekil 3 - Ciğer numunesine ait 1 ppb konsantrasyonda
akrilamid etken maddesine ait kromatogram
GİRİŞ
Melamin, yüzey kaplamaları, plastik, ticari filtre, yapıştırıcı ve mutfak
malzemelerinin üretiminde kullanılan bir maddedir. Özellikle tabakların
yapımında genişçe kullanılması, bu materyallerden yüksek ısı (~120 °C) ve
asidik pH şartlarında gıdalara geçmesi, potansiyel olarak gıda zehirlenmelerine
neden olabileceği yorumlarına neden olmuştur.
Melaminin zehirliliğine ilişkin ilk bulgular Amerika Birleşik Devletlerinde
2007 yılında kedi ve köpeklerin böbrek yetmezliğine bağlı ani ölümü üzerine
yapılan araştırmalardan sonra ortaya konmuştur. Kedi ve köpek mamalarının
hazırlanmasında kullanılmak üzere Çin’den ithal edilen buğday ve pirinç
konsantrelerinde melamin tespit edildiği ve ölümlerin buna bağlı olduğu
bildirilmiştir. Kedi ve köpek mamalarında yaşanan bu skandalın hemen
ardından, 2008 yılında Çin Halk Cumhuriyeti’ndeki süt ürünleri üreticilerinin
de ürünlerindeki protein düzeyini yüksek göstermek için hileli bir şekilde
melamini kullandığı belirlenmiştir. Çin’deki süt skandalının ardından yapılan
geniş çaplı araştırmalarda bebek mamalarının yanı sıra normal süt ve
yoğurtlarda, süt tozu ve tahıl ürünlerinde, donmuş tatlılarda, şekerlemelerde, kek ve bisküvilerde, protein tozları ve bazı işlenmiş
gıdalarda melamin bulunduğu bildirilmiştir.
Gıda ve gıda maddelerinde melamin kullanılmasının yasal olduğu bir ülke bulunmamaktadır. Ancak çevrede yaygın bir şekilde
kullanılmasının sonucu olarak gıda zincirine girebileceği rapor edilmiştir. Bu nedenle birçok ülke yem ve gıdalarda bulunmasına
izin verilen maksimum melamin düzeylerini belirlemiştir. Avrupa Birliği ülkeleri %15 ve üzeri süt ve süt ürünü içeren tüm gıda
ürünlerinin ithalatından önce analizini zorunlu kılmakta ve 2.5 mg/kg’ı aşan ürünlerin imha edilmesi gerektiği ifade etmektedirler
Melamin analiz için sıkça kullanılan GC-MS metotlarının numune hazırlığında sağlığa zararlı solventler ile clean-up aşaması
ve ardından türevlendirmeye ihtiyaç duyulmaktadır. Buna karşılık olarak geliştirilen LC-MS/MS metotlarında daha az numune
hazırlığı, daha kısa analiz süresi ve daha güvenilir sonuç elde edilmesi sebepleriyle tercih edilen analiz yöntemi olmuştur.
Ülkemizde “Türk Gıda Kodeksi Bulaşanlar Yönetmeliği” ile gıdalarda ve bebek mamalarındaki maksimum limitleri belirlenmiştir.
SÜT VE SÜT ÜRÜNLERİNDE MELAMİN ANALİZİ
Analitik Koşullar
YÖNTEM
Numuneler basit bir numune hazırlama yöntemi
olan sıvı-sıvı ekstraksiyon ile hazırlanarak LC-MS/
MS’e enjeksiyonu yapılır.
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
MRM çiftleri:
: KNAUER Eurospher II 100-3 HILIC (150 x 3 mm)
: 10 dakika
: Su (Amonyum Asetat) ve Asetonitril
: ESI +
: Scheduled MRM™
Max. 3,1e6 cps.
XIC of +MRM (4 pa rs): 126,960/84,900 Da ID: melam n1 from Sample 3
2,14
3,0e6
2,8e6
Q1/Q3
127/85
127/68
127/60
Sıvı-sıvı ekstraksiyon ile hazırlanan bebek maması numunesinin
enjeksiyonu yapılmış ve elde edilen veriler değerlendirildiğinde
yönetmelikte belirlenen limitleri rahatlıkla karşıladığı görülmüştür.
Hızlı ve kolay numune hazırlama yöntemi ile onlarca numune kısa
sürede çalışılabilmektedir.
2,6e6
2,4e6
2,2e6
2,0e6
1,8e6
1,6e6
1,4e6
1,2e6
1,0e6
8,0e5
6,0e5
4,0e5
2,0e5
0,0
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0
5,5
T me, m n
6,0
6,5
7,0
7,5
8,0
8,5
9,0
9,5
Şekil 4- Bebek mamasına ait melamin analiz kromatogramı
www.spektrotek.com
SONUÇ
Polarite
Pozitif
Pozitif
Pozitif
In te n s ty , c p s
Bileşik
Melamin 1
Melamin 2
Melamin 3
15
YAŞ MEYVE VE SEBZELERDE
MULTİ-PESTİSİT ANALİZİ
GİRİŞ
Dünya nüfusunun hızlı artışı ve sanayinin gelişmesine karşın bitkisel üretime
ayrılan toprak alanları hızla azalmaktadır. Bu sebeple gıda maddelerine
duyulan gereksinim de yoğun bir şekilde artmaktadır. Pestisit kullanımı,
tarımsal ürünleri hastalık, zararlı ve yabancı otların zararından koruyabilmek,
kaliteli üretimi güvence altına alabilmek için kullanılan bir tarımsal mücadele
şekli olup, kısa sürede etki göstermesi ve kullanımının kolay olması nedeniyle
en çok tercih edilen yöntemdir.
Bu ilaçların gereğinden fazla, zamansız ve bilgisizce kullanılması dayanıklı
ırkların meydana gelmesine, üründe kalite düşmesine, hayvanların ve
insanların akut ve kronik zehirlenmelerine neden olmaktadır. Biyolojik
aktiviteye sahip, öldürücü olan kimyasal maddeler kullanılarak yapılan
tarımsal alanlardaki hastalıklarla mücadele ile esas hedef dışında çevre
ve canlılar için potansiyel tehlikeler ortaya çıkmakta, sonuç olarak çevre
bulaşması gibi arzu edilmeyen bazı problemlere neden olunmaktadır.
Son yıllarda kullanımı hızla artan pestisit ilaç kalıntılarının analiz edilmesi bir zorunluluk haline gelmiştir. Çeşitli ülkelerde,
ürünlerde pestisit kalıntısı taramaları yaparak gıda güvenilirliği ve potansiyel risk hakkında bilgi edinmek yolunda yoğun çalışmalar
sürdürülmektedir. Özellikle gelişmiş ülkeler kendi ürünlerinde ve ithal ürünlerde bulunabilecek pestisitlerin maksimum oranlarını
belirlemiş olup ilgili yönetmeliklerle sıkı bir şekilde denetimini yapmaktadır.
Ülkemizde Türk Gıda Kodeksi “Pestisitlerin Maksimum Kalıntı Limitleri Yönetmeliği” ile pestisitlerin taze, işlenmemiş, işlenmiş
veya kompozit bitkisel ve hayvansal gıdalarda bulunmasına izin verilen maksimum kalıntı limitleri ve bu limitlerin uygulama
esasları belirlenmiştir.
YÖNTEM
Analitik Koşullar
Numuneler, AOAC Official Method 2007.01 yöntemine göre
ChromXpert QUECHERS kitleri ile hazırlandıktan sonra LCMS/MS’e enjeksiyonu yapılır.
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
: Phenomenex Synergi Fusion 2.5 μm (50x2.1 mm)
: 17 dakika
: Su (Amonyum Format) ve Metanol (Amonyum Format)
: ESI + / ESI –
: Scheduled MRM™
XIC of +MRM (853 pa rs): Exp 1, 113.932/58.100 amu Expected RT: 0.0 ID: Mep quat 1 from Sample 17
Max. 4.1e5 cps.
9.5e6
9.0e6
8.5e6
8.0e6
7.5e6
7.0e6
6.5e6
I n t e n s
6.0e6
t y ,
4.5e6
5.5e6
5.0e6
c p s
4.0e6
3.5e6
3.0e6
2.5e6
2.0e6
1.5e6
1.0e6
5.0e5
0.0
0.87
4.46
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
T me, m n
9.0
10.0
11.0
12.0
13.0
14.0
15.0
XIC of -MRM (100 pa rs): Exp 2, 184.893/116.700 amu Expected RT: 6.7 ID: 1-Napthyl Acet cac d 2 from Sample 17
QUECHERS KİTLERİ
16.0
Max. 1.8e4 cps.
8.3e6
8.0e6
7.5e6
7.0e6
6.5e6
6.0e6
I n t e n s
5.5e6
5.0e6
4.5e6
t y ,
4.0e6
c p s
3.5e6
3.0e6
2.5e6
2.0e6
1.5e6
1.0e6
www.spektrotek.com
SONUÇ
16
Bu çalışmada; Hızlı Polarite değişimi ve Scheduled MRM™ ile
hassasiyette azalma olmadan kısa sürede analiz edilmiş ve elde
edilen sonuçlar yaş meyve ve sebze için yönetmelikte verilen
limitleri rahatlıkla karşılamaktadır. Aynı zamanda QTrap™
teknolojisi kullanılarak mevcut olan 666 adet pestisite ait
kütüphane taraması ile konfirmasyon sonucu rapor güvenliği
sağlanmaktadır.
5.0e5
0.0
1.0
2.0
3.0
4.0
5.0
6.10
6.0
7.42
7.0
8.0
T me, m n
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
Şekil 5-Negatif-pozitif polarite değişimi ve Scheduled MRM™
algoritması ile tek metotta analiz edilen 1 ppb 450 adet
pestisit etken maddesine ait kromatogram.
GİRİŞ
Son yıllarda kullanımı hızla artan pestisit ilaç kalıntılarının analiz edilmesi
bir zorunluluk haline gelmiştir. Çeşitli ülkelerde, ürünlerde pestisit kalıntısı
taramaları yaparak gıda güvenilirliği ve potansiyel risk hakkında bilgi edinmek
yolunda yoğun çalışmalar sürdürülmektedir. Özellikle gelişmiş ülkeler
kendi ürünlerinde ve ithal ürünlerde bulunabilecek pestisitlerin maksimum
oranlarını belirlemiş olup ilgili yönetmeliklerle sıkı bir şekilde denetimini
yapmaktadır.
TÜİK tarafından tarım ilaçlarında 1979’da 8.396 ton dolayında olan tüketimin,
2008’de 20.000 tonu geçmiş olduğu belirlenmiştir. Ülkemizdeki kimyasal
tarım ilaçlarının kullanımı insan sağlığı kadar çevreyi de etkileyebilecek
bir biçimdedir. Bu sorunun en temel nedeni kontrolsüz ve bilinçsiz ilaç
kullanımıdır.
Dünya Sağlık Örgütü’nün (WHO) uzmanlaşmış kanser kuruluşu olan
Uluslararası Kanser Araştırmaları Kurumu GDO’lu ürünlerin %80’inde
kullanılan ot ilacı (herbisit) etken maddesi olan Glifosat (Glyphosate)’ın
insanlarda muhtemelen kanser yaptığını açıklamıştır. Raporda da belirtildiği gibi, Glifosat en yaygın kullanılan herbisit olup
değişik tarım, orman, şehir ve konut uygulamalarında yaygın olarak kullanılmaktadır. Kullanımı, genetiği değiştirilmiş Glifosat
herbisitine dayanıklı ürünlerin geliştirilmesiyle daha da artmıştır. Genellikle GDO’lu soya ve mısır üretiminde kullanılan glifosat,
havada, suda ve yiyeceklerin yanı sıra ilaca maruz kalan tarım işçilerinin kan ve idrarlarında da tespit edilmiştir.
Rutin olarak yürütülen AOAC 2007 Quechers metodu ile yapılan pestisit analiz çalışmalarından farklı bir numune hazırlığına ve
analiz metoduna sahip olan polar pestisitler gün geçtikçe önem kazanarak bu pestisitlerin analizi zorunlu hale gelmektedir.
YÖNTEM
Analitik Koşullar
Numuneler Quppe metodu doğrultusunda basit bir
numune ön hazırlığı olan olan sıvı-sıvı ekstraksiyon
ile hazırlanır ve 10 kat seyreltilerek direk olarak LCMS/MS’e enjeksiyonu yapılır.
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
GIDALARDA POLAR PESTİSİT TAYİNİ
: Thermo Hypercarb 5um (2.1 x 100 mm)
: 30 dakika
: Su (Asetik Asit) ve Metanol (Asetik Asit)
: ESI : Scheduled MRM™
SONUÇ
Bu çalışmada; Scheduled MRM™ ile farklı matrikslerde (elma, limon, tarçın, kuru incir) analiz çalışması yapılmıştır. Elde edilen
sonuçlar değerlendirildiğinde yönetmelikte verilen limitleri rahatlıkla karşıladığı görülmüştür. Aynı zamanda QTrap™ teknolojisi
kullanılarak mevcut olan 666 adet pestisite ait kütüphane taraması ile konfirmasyon sonucu rapor güvenliği sağlanmaktadır
Max. 3.7e4 cps.
1.AMPA
2.Glufosinate
3.Phosphonic acid
4.Glyphosate
5.HEPA
6.MPPA
7.Fosetyl Al
8.Maleic Hydrazide
9.N-Acetyl-AMPA
10.Chlorate
11.Ethephon
12.N-Acetyl-Glufosinate
13.Perchlorate
13
4.2e5
4.0e5
(b)
3.8e5
3.6e5
3.4e5
3.2e5
3.0e5
I n t e n s t y, c p s
(a)
4.3e5
2.8e5
2.6e5
2.4e5
2.2e5
2.0e5
1.8e5
2
1.6e5
1.4e5
5
3
1.2e5
1.0e5
6.0e4
4.0e4
1
10
8
4
1.19
9
2.0e4
0.0
6
7
8.0e4
11
5.24
2
4
6
8
12
10
12
14
T me, m n
16
18
Şekil 6- Scheduled MRM™ kullanılarak yapılan pestisit analizine ait kromatogram (a) Kuru incir (b) Elma
20
22
24
26
28
30
www.spektrotek.com
XIC of -MRM (29 pa rs): 109.800/62.900 amu Expected RT: 1.1 ID: AMPA 1 from Sample 4
1.AMPA
2.Glufosinate
3.Phosphonic acid
4.Glyphosate
5.HEPA
6.MPPA
7.Fosetyl Al
8.Maleic Hydrazide
9.N-Acetyl-AMPA
10.Chlorate
11.Ethephon
12.N-Acetyl-Glufosinate
13.Perchlorate
17
GIDA ÜRÜNLERİNDE PARAQUAT/DIQUAT
ANALİZİ
GİRİŞ
Dünya nüfusunun hızla arttığı çağımızda açlık sorununun çözümlenebilmesi
için tarımsal üretimi arttırmada ilaçlar kullanılmaktadır. Tarım ürünlerinin
üretimi sırasında, ilaçlama ile bu ürünlere kontaminasyon ile bulaşan ve
daha sonra mamül gıda maddelerine yansıyan, kimyasal ilaç kalıntılarına
“Pestisit” adı verilmektedir. Tarımsal ilaçların kullanımı; bir taraftan tarımsal
üretimi artırırken diğer taraftan bilinçsiz ve hatalı kullanım sonucu doğrudan
ya da dolaylı yollardan insan ve çevre sağlığı problemlerini de beraberinde
getirirler.
Pestisitler tavsiye edilen dozların üzerinde kullanıldıklarında, gereğinden
fazla sayıda ilaçlama yapıldığında, gerekmediği halde birden fazla ilaç
karıştırılarak kullanıldığında veya son ilaçlama ile hasat dönemi arasında
bırakılması gereken süreye riayet edilmediği durumlarda gıda maddelerinde
fazla miktarda kalıntı bırakabilirler. Yüksek dozda pestisit kalıntısı içeren
gıdalarla beslenen insanlarda ve çevredeki diğer canlılarda akut veya kronik
zehirlenmelere neden olabildikleri gibi, özellikle bazı ürünlerde aroma ve kalite değişimleri meydana getirebilirler.
Gıdalardaki pestisit analizlerinde genellikle birden fazla pestisit aktif maddesi ile karşılaşılabilmektedir. Pestisit kullanmanın
tartışılmaz faydalarına rağmen, özellikle gıdalar vasıtasıyla insan vücudunda akümüle olması ve çevre kirliliği üzerine olumsuz
etkisi bu bileşiklerin zararları konusunda insanoğlunu gün geçtikçe daha fazla endişeye sevketmektedir. Pestisit kalıntıları gıda
maddelerinde, insan, hayvan ve çevre sağlığına zarar vermeyecek düzeylerde bulunmalıdır. Gıda maddelerindeki pestisit kalıntı
miktarlarının bilinmesi insan sağlığı açısından olduğu kadar ihraç gıda ürünleri içinde oldukça büyük önem arzetmektedir. Gıda
maddelerindeki pestisit kalıntı miktarlarının daha önceden tesbit edilip tolerans sınırlarını geçmemesi gerek tüketici sağlığı
açısından ve gerekse ihraç gıda ürünlerinin geri dönmemesi açısından büyük öneme sahiptir.
Bu nedenle üretilen her bir yeni pestisit, piyasaya arzından önce farmakolojik ve toksikolojik denemelere tabii tutularak, tolerans
sınırlarının önceden belirlenmesi mutlak surette gereklidir.
Ülkemizde Türk Gıda Kodeksi “Pestisitlerin Maksimum Kalıntı Yönetmeliği” ile pestisitlerin taze, işlenmiş veya kompozit bitkisel
ve hayvansal gıdalarda bulunmasına izin verilen maksimum kalıntı limitleri belirlenmiştir.
YÖNTEM
Numuneler Quppe metodu doğrultusunda basit bir numune ön hazırlığı olan sıvı-sıvı ekstraksiyon ile hazırlanarak LC-MS/MS’e
Analitik Koşullar
Analitik Kolon : SUPELCO Ascentis HILIC, 2.7um
(2.1 x 100 mm)
Analiz Süresi : 10 dakika
Mobil Faz : Su (Ammonium Format) ve Asetonitril
İyonizasyon : ESI +
Tarama Modu : Scheduled MRM™
www.spektrotek.com
MRM çiftleri:
18
Bileşik
Paraquat 1
Paraquat 2
Diquat 1
Diquat 2
Polarite
Pozitif
Pozitif
Pozitif
Pozitif
Q1/Q3
186/155
171/155
184/127.8
184/156
Bu çalışmada; Scheduled MRM™ ile zor bir matrix olan kekik ile analiz çalışması yapılmıştır. Elde edilen sonuçlar
değerlendirildiğinde yönetmelikte verilen limitleri rahatlıkla karşıladığı görülmüştür. Aynı zamanda QTrap™ teknolojisi
kullanılarak mevcut olan 666 adet pestisite ait kütüphane taraması ile konfirmasyon sonucu rapor güvenliği sağlanmaktadır.
(a)
(b)
SONUÇ
Şekil 7- Scheduled MRM™ kullanılarak yapılan kekik numunesi ait pestisit analiz kromatogramı (a) Paraquat (b) Diquat
Şekil 8- Direk enjeksiyon ile analiz edilen su numunesine ait pestisit kromatogramı
www.spektrotek.com
Aynı zamanda su numunelerinde direk enjeksiyon ile analiz çalışması yapılmaktadır. Numune ön hazırlık işlemine
ihtiyaç duyulmamasından dolayı hem analiz maliyeti oldukça düşüktür hem de kısa sürede onlarca analiz yapma imkanı
sağlamaktadır.
19
JELİBONDA JELATİN TÜR ANALİZİ
GİRİŞ
Gıda bilimcileri; uzun yıllardır et ve et ürünlerinin türünün belirlenmesi
konusunda birçok araştırma yapmaktadırlar. Toplum sağlığı, istenmeyen et
ve sakatatların karıştırılması, dini inançları doğrultusunda bazı hayvanlara ait
etleri tüketmeyen toplumların varlığı sebeplerinden dolayı bu araştırmalar her
geçen gün artmakta ve yeni yöntemlere ihtiyaç duyulmaktadır. İçeriği yanlış
beyan edilerek toplumu kandırmaya yönelik yapılan üretim sebebiyle özellikle
dünya nüfusunun yaklaşık %23’ünü oluşturan Müslüman toplumlarda sadece
domuz eti değil aynı zamanda domuz ürünlerinin de tüketilmemesinden
dolayı gıda ürünlerinde tür tayini önem kazanmaktadır.
Piyasada bulunan jelibon ve şekerlemeler, dondurmalar, ilaç kapsülleri ve
kozmetik ürünler içerisinde kullanılan jelatinin tür tespiti için PCR, ELISA
gibi analiz yöntemleri kullanılmakta olup bu yöntemlere ait bazı kısıtlamalar
bulunmaktadır.
Jelatinin üretim aşamasında yüksek sıcaklık ve asidik koşulların
kullanılmasından dolayı hayvan DNA’sı zarar görmekte ve bu sebeple tür tespitinde kullanılacak olan PCR yöntemi zor veya
imkansız olmaktadır. Bir diğer yöntem olarak ELISA protein bazlı bir metottur. Yöntemin, proteinin sadece bir parçasının tespit
edilmesine olanak vermesi ve birden fazla protein belirleyicisinin olmamasından dolayı yanlış pozitif ve yanlış negatif sonuçlar
elde edilebilmektedir.
Bu kısıtlamaların dışında tolerans değerinin sıfır olmasından dolayı hassas cihazlar ile düşük tespit limitlerinde analiz yapılmasına
ihtiyaç duyulmaktadır. Bu sebeple LC-MS/MS sistemleri tercih edilmektedir. Aynı zamanda proteinlerin tripsin ile parçalanmasının
ardından elde edilen peptitler ile çoklu analiz imkanı sağlanır ve böylece jelatin türü belirlenmesinde kesin ve tekrarlanabilirliği
yüksek sonuçlar elde edilir.
YÖNTEM
Analitik Koşullar
Numuneler, NH4HCO3 içerisinde çözündürüldükten sonra
tripsin ile peptitlerine parçalanır ve LC-MS/MS’e enjeksiyonu
yapılır.
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
: Phenomenex Gemini 5 μm (150x4.6 mm)
: 8 dakika
: Su (Formik Asit) ve Asetonitril (Formik Asit)
: ESI +
: Scheduled MRM™
MRM çiftleri:
www.spektrotek.com
Bileşik
Domuz Jelatini 1
Domuz Jelatini 2
Domuz Jelatini 3
Domuz Jelatini 4
Sığır Jelatini 1
Sığır Jelatini 2
Sığır Jelatini 3
20
Polarite
Pozitif
Pozitif
Pozitif
Pozitif
Pozitif
Pozitif
Pozitif
Q1/Q3
1103.0/850.9
486.2/786.4
921.5/1050.6
620.8/618.3
659.3/766.5
781.4/991.6
644.8/971.5
SONUÇ
Saf halde alınan sığır ve domuz jelatinlerinin QTrap™-EPI ile kütle spektrumları elde edilerek kütüphaneye eklenmiştir.
Domuz jelatini ve sığır jelatini kullanılarak üretilen iki farklı jelibon numunesi hazırlanarak enjeksiyon yapılmıştır. Aynı zamanda
QTrap™ teknolojisi kullanılarak numunelere ait kütle spektrumları elde edilmiş ve kütüphane taraması ile konfirmasyon
sağlanmıştır. Buna göre %87 oranında domuz jelatini, %83 oranında sığır jelatini benzeşmesi sağlanmıştır.
Max. 1.6e5 cps.
XIC of +MRM (7 pa rs): 1103.000/850.900 Da ID: Pork gelat n 1 from Sample 6 (pork jel bon) of 28042015.w ff (Turbo Spray), Sm...
1.5e5
1.4e5
1.4e5
1.3e5
1.3e5
1.2e5
1.2e5
1.1e5
1.1e5
1.0e5
1.0e5
9.0e4
9.0e4
Max. 1.6e5 cps.
6.07
6.07
1.6e5
1.5e5
(a)
(a)
(b)
(b)
8.0e4
8.0e4
7.0e4
7.0e4
6.0e4
6.0e4
5.0e4
5.0e4
4.0e4
4.0e4
3.0e4
3.0e4
2.0e4
2.0e4
1.0e4
1.0e4
0.0
0.0
0.0
0.0
0.5
0.5
1.0
1.0
1.5
1.5
2.0
2.0
2.5
2.5
3.0
3.0
3.5
3.5
4.0
T me, 4.0
mn
T me, m n
4.5
4.5
5.0
5.0
5.5
5.5
6.0
6.0
6.5
6.5
7.0
7.0
7.5
7.5
%87
%87 oranında
oranında domuz
domuz jelat
jelat n
n benzeşmes
benzeşmes
%83oranında
oranındasığır
sığırjelat
jelatnn benzeşmes
benzeşmes
%83
XIC of +MRM (7 pa rs): 1103.000/850.900 Da ID: Pork gelat n 1 from Sample 6 (pork jel bon) of 28042015.w ff (Turbo Spray), Sm...
1.6e5
Şekil 9- Jelibon numunelerine ait kromatogram ve kütle spektrumları (a) Domuz jelatini içeren jelibon numunesine ait
kromatogram ve kütle spektrumları (b) Sığır jelatini içeren jelibon numunesine ait kromatogram ve kütle spektrumları
Sığır jelatini içerisine %50 ve %1 oranlarında domuz jelatini spike edilmiş ve analiz yapılmıştır. Buna göre basit bir numune
hazırlığı sonunda analiz edilen numunelerde %1 oranında domuz jelatini bulunmasına rağmen tespit edilmiş ve QTrap™
teknolojisi kullanılarak kütüphane taraması yapılarak konfirmasyon sağlanmış ve güvenilir sonuçlar elde edilmiştir.
Sığır jelat n ç n MRM ç ftler
Domuz jelat n ç n MRM ç ftler
Şekil 10- %1 oranında domuz jelatini içeren sığır jelatin numunesine ait kromatogram
www.spektrotek.com
n benzeşmes
21
ETTE TÜR TAYİNİ
GİRİŞ
Et, insanın beslenmesinde çok önemli yeri olan temel gıda maddelerinden
birisidir. Uluslararası et ticaretinin artması, yapılan hileleri de artırmıştır.
Bu bakımdan et türlerinin belirlenmesi tüketicilerin korunması ve hilelerin
önlenmesi bakımından önem arz etmektedir.
Gıda bilimcileri; uzun yıllardır et ve et ürünlerinin türünün belirlenmesi
konusunda birçok araştırma yapmaktadırlar. Toplum sağlığı, istenmeyen et
ve sakatatların karıştırılması, dini inançları doğrultusunda bazı hayvanlara ait
etleri tüketmeyen toplumların varlığı sebeplerinden dolayı bu araştırmalar
her geçen gün artmakta ve yeni yöntemlere ihtiyaç duyulmaktadır. İçeriği
yanlış beyan edilerek toplumu kandırmaya yönelik yapılan üretim sebebiyle
özellikte dünya nüfusunun yaklaşık %23’ünü oluşturan Müslüman
toplumlarda özellikle domuz etinden dolayı gıda ürünlerinde tür tayini önem
kazanmaktadır.
Türk Gıda Kodeksi Çiğ Kırmızı Et ve Hazırlanmış Kırmızı Et Karışımları
Tebliği’nin 13. maddesindeki ambalajlama, etiketleme ve işaretleme bilgisi gereğince, ürünün ait olduğu kasaplık hayvan türü,
ürün ismi ile birlikte etikette belirtilmelidir. Koyun, keçi, sığır, manda etlerinden hazırlanmış kırmızı et karışımlarının etiketinde
ürünün elde edildiği türe ait yüzde miktarlarının etikette belirtilmesi gerekmektedir. Yine aynı şekilde Türk Gıda Kodeks’i Çiğ
Kanatlı Eti ve Hazırlanmış Kanatlı Eti Karışımları Tebliği’nin 12. maddesi gereğince ürünlerin etiketinde, ürünün ait olduğu kanatlı
hayvan türü ürünün ismi ile birlikte etikette belirtilmesi gerekmektedir. Tür tayininde PCR gibi DNA bazlı analiz yöntemleri
kullanılmakta olup bu yöntemlere ait bazı kısıtlamalar bulunmaktadır. Çünkü DNA et proses edilirken zarar görebilir ya da
değişebilir. ELISA gibi protein bazlı metotlarda ise proteinin sadece küçük bir kısmı dedekte edildiği için sıkıntılar yaşanmaktadır.
Ayrıca her tür için farklı bir kit kullanılması analiz maliyeti açısından değerlendirildiğinde yüksek olmaktadır. Tolerans değerinin
sıfır olmasından dolayı hassas cihazlar ile düşük tespit limitlerinde analiz yapılmasına ihtiyaç duyulmaktadır. Bu sebeple LC-MS/
MS sistemleri tercih edilmektedir.
YÖNTEM
SONUÇ
Numuneler sıvı-sıvı ekstraksiyonu, tripsin ile parçalama
ardından SPE ekstraksiyonu ile hazırlanarak LC-MS/MS’e
Farklı et türleri çalışılarak peptitlere ait iyon çiftleri
belirlenmiştir. Aynı zamanda QTrap™ teknolojisi kullanılarak
kütle spektrumları elde edilmiş ve kütüphane ile karşılaştırma
yapılarak konfirmasyon sağlanmıştır. Sonuçlar incelendiğinde
%1 oranında bile karışım olsa bile tespit edilebilmektedir.
Analitik Koşullar
www.spektrotek.com
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
22
: HALO C18 2.7um (50 x 0.5 mm) (Micro LC)
: 11 dakika
: Su (Formik Asit) ve Asetonitril (Formik Asit)
: ESI +
: Scheduled MRM™
Şekil 11- MIDAS Akış şeması ve et numunesine ait elde edilen peptitler ve kütle spektrumları
GİRİŞ
Zeytinyağının son zamanlardaki popülaritesi duyusal özellikleriyle ve
sağlık açısından faydalarıyla ilişkilendirilebilir. Oleik asitin ana bileşenini
oluşturduğu iyi dengelenmiş yağ asitleri kompozisyonu ile vitamin ve doğal
antioksidanlar gibi iz miktardaki biyomoleküller zeytinyağının faydalarıyla
bağdaştırılmaktadır. Zeytinyağının sağlık açısından öneminin ortaya konması
ile birlikte bu ürüne olan talep de artmıştır. Ürüne olan talepten ve üretim
maliyetinin de yüksekliğinden dolayı zeytinyağı diğer yemeklik yağlara göre
ekonomik değeri daha yüksek bir yağdır.
Yenilebilir yağların kaliteleri, elde edildikleri hammadde, uygulanan tarım
yöntemi, hasat zamanı ve şekli, işleme tekniği ve depolama koşulları gibi
çeşitli faktörlerden etkilenmektedir. Bu yüzden yenilebilir yağların kalite
özelliklerinin belirlenmesi üretici ve tüketiciler için önem taşımaktadır.
Bu amaçla çok çeşitli enstrümantal ve kimyasal analiz yöntemlerinden
yararlanılmaktadır. Yenilebilir yağların kalite parametrelerinin belirlenmesinde
kullanılan geleneksel yöntemler güvenilir sonuç vermelerinin yanında, uzun
zaman almaları, uzman analiste gereksinim duymaları ve kullanılan kimyasalların analist sağlığı için tehlike arz etmesi gibi
olumsuzlukları bulunmaktadır. Zeytinyağı gibi yenilebilir yağların çeşit ve orijinlerinin belirlenmesinde en önemli bileşenler yağ
asitleri, steroller, fenoller, trigliseritler ve tokoferoller olduğu bildirilmiştir. Bu bileşenlerin yağlardaki miktar ve kompozisyonları
çeşit, çevre ve yetiştirme koşullarına bağlı olarak farklılık göstermektedir.
Bitkisel yağların katkılandırılması özellikle tüketiciler için önem arz eden bir konudur. Çoğunlukla üretim maliyeti fazla olan, satışı
pahalı, besin kalitesi yüksek yağlar daha ucuz yağlar ile tağşişe uğratılabilmektedir. Bu konuda en fazla tağşişe uğrayan yağlardan
bir tanesi sızma zeytinyağıdır. Sızma zeytinyağı pirina veya daha ucuz sınıf zeytinyağları ile tağşiş edilmektedir. Zeytinyağının
tağşişi yağ asitleri kompozisyonu ve/veya sterol kompozisyonun belirlenmesi suretiyle saptanabilmektedir.
Ancak bu yöntemlerin pahalı olmaları, tekrar edilebilirliklerinin düşük olması gibi bazı dezavantajları bulunmaktadır. Bu sebeple
numune hazırlama işleminin olmadığı ve kesin, doğru, tekraredilebilir sonuçların elde edildiği LC-MS/MS yöntemleri öne
çıkmaktadır.
ZEYTİNYAĞINDA TAĞŞİŞ VE ORİJİN BELİRLEME
YÖNTEM
Numuneler hekzan/izopropanol karışımı ile seyreltilerek direk
olarak LC-MS/MS’e enjeksiyonu yapılır.
Analitik Koşullar
: Spherisorb Silica column 5 um
(250 x 4.6 mm) (Normal-Phase Chromatography)
: 15 dakika
: Hekzan ve İzopropanol
: APCI
: MRM
SONUÇ
Piyasada bulunan farklı yağ türleri (susam, sındık, mısır,
kanola, ayçiçeği, soya vb.) zeytinyağı ile farklı oranlarda
karıştırılmış ve QTrap™ teknolojisi kullanılarak analiz
edilmiştir. Elde edilen sonuçlar MarkerView™ PCA algoritması
ile değerlendirilmiş ve tağşiş yağlar eser miktarda dahi olsa
tespit edilebilmiştir.
Şekil 12- Zeytinyağı ve tağşiş yağlara ait MasterView™ PCA
analiz sonuçları
www.spektrotek.com
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
23
LC-MS/MS İLE GIDALARDA ALERJEN ANALİZİ
GİRİŞ
Gıda alerjisi, insan bağışıklık sistemi tarafından belirli bir gıdaya karsı
başlatılan aşırı duyarlılık reaksiyonudur. İnsan sağlığı ve gıda güvenliğini
tehdit eden bir unsur olan alerjenler yetişkinlerin yaklaşık %2-4’ünde ve
çocukların %6’sında gıda alerjisine sebep olmaktadır.
Gıda güvenliğinin sağlanmasında alerjenlerle ilgili yapılan çalışmalar oldukça
önemlidir. Gıda ile ilgili hastalıkların doğru değerlendirilmesinde, bağışıklık
sistemi kaynaklı gıda alerjisi ve bağışıklık sistemine bağlı olmayan gıda
intoleransı arasındaki farkın anlaşılması kritik önem taşımaktadır.
Moleküler düzeyde gıda alerjenlerinin karakterizasyonu ve fonksiyonlarının
ayrıntılı olarak anlaşılması, gıda alerjisinin teşhisi ve tedavisi ile ilgili
yaklaşımların gelişmesine yol açabilecektir. Ayrıca, tüketicilerin ve üreticilerin
gıda alerjisi konusunda bilinçlenmesi, üreticilerin gıdalarda bulunabilecek
alerjen bileşenleri etikette açıkça belirtmesi gerekliliğine titizlikle uyması,
alerjisi olan kişilerin alerjiden sorumlu alerjen gıdayı tüketmemeye dikkat
etmesi ile gıda alerjilerinin engellenmesine yardım edebilecektir.
Allerjenlerin tayin ve tespitinde kullanılan farklı yöntemler bulunmaktadır. ELISA yöntemlerinde yanlış pozitif ve yanlış negatif
sonuçlar elde edilebilmektedir. Ayrıca her allerjen için ayrı ELISA kitine ihtiyaç duyulmaktadır. Diğer bir tarafta kullanılan PCR
yönteminde ise allerjenin varlığı değil organizmadan gelen bir madde olup olmadığı bakıldığı için yine yanlış pozitif ve yanlış
negatif sonuçlar ortaya çıkabilir. Bu sebeple çoklu alerjenik proteinlerin belirlenmesi güvenilir sonuçlar elde edilmesi anlamında
bir zorunluluktur. Bu sebeple hem daha düşük limitlerde çalışma imkanı, hem de çoklu peptid analizinden dolayı güvenilir
sonuçların elde edilmesi sebebiyle LC-MS/MS vazgeçilmez bir çözümdür.Avrupa Birliği’nin oluşturduğu ve AB uyum yasaları
çerçevesinde ülkemizde de kabul edilen yasal düzenlemeye göre, gluten/gliadin, yumurta, yerfıstığı, fındık, badem, soya, sülfit,
süt ve laktoz etikette belirtilmesi gereken alerjenler arasındadır. İzin verilen seviyeler, alerjik reaksiyonu tetikleyebilecek eşik
değerler hakkındaki bilimsel bulgulara dayanmaktadır.
Ülkemizde etiketlendirme ve alerjenler ile ilgili yasal düzenleme Türk Gıda Kodeksi’nde “Gıda Maddelerinin Genel Etiketleme
ve Beslenme Yönünden Etiketleme Kuralları Tebliği”nde yer almaktadır.Bu bileşenler son üründe farklı bir formda olsalar bile,
etikette açıkça belirtilmelidirler.
YÖNTEM
Numuneler sıvı-sıvı ekstraksiyonu, tripsin ile parçalama
ardından SPE ekstraksiyonu ile hazırlanarak LC-MS/MS’e
SONUÇ
www.spektrotek.com
Farklı alerjenler ile çalışma yapılmış ve her alerjen için
belirleyici olan MRM çiftleri ile peptit haritaları çıkarılmıştır
24
Analitik Koşullar
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
: Phenomenex Synergi Hydro-RP 4 um (150 x 2.1 mm)
: 18 dakika
: Su (Formik Asit ve Trifloroasetik Asit) ve Asetonitril (Formik Asit ve Trifloroasetik Asit)
: ESI +
: Scheduled MRM™
Şekil 13- Yerfıstığı, yumurta ve süt için belirlenen alerjenlerin peptit haritası ve süt için elde edilen kalibrasyon grafiği
Süt ve yumurta alerjenleri makarna ve ekmek numunesine spike edilerek analiz edilmiştir. Proteinlerin tripsin ile parçalanması
yöntemine dayanan numune hazırlığının ardından enjeksiyon yapılan numunede aynı zamanda QTrap™ teknolojisinden
faydalanılarak kütle spektrumları elde edilmiştir. Aynı enjeksiyonda hem MRM oranı hem de kütle spektrumları ile karşılaştırma
yapılarak güvenilir sonuçlar elde edilmektedir. Böylece false pozitif sonuçlar ortadan kaldırılmış olur.
Şekil 15- Makarna numunesinde analiz edilen yumurta ve süt peptidlerine ait kromatogram ve kütle spektrumları
www.spektrotek.com
Şekil 14- Ekmek numunesinde analiz edilen yumurta ve süt peptidlerine ait kromatogram ve kütle spektrumları
25
BEBEK MAMALARINDA B VİTAMİNİ
ANALİZLERİ
GİRİŞ
Ülkemizde bebek ve çocuklar için özel süt ve süt ürünleri, sindirime ve kolesterol
düşürmeye yardımcı yoğurt ve yoğurt bazlı içecekler gibi fonksiyonel gıdalar
tüketicilere sunulmaktadır. Fonksiyonel gıdalar, besinlerin yanı sıra sağlığa
yararlı bileşenler içeren gıdalardır. Bu bileşenler, gıdanın içinde doğal olarak
bulunabilir, işleme sırasında eklenebilir veya doğal olarak bulunan miktara
ekleme yapılarak kuvvetlendirme yapılabilir. Dayanıklılıklarının düşük olması
nedeniyle, vitamin katkılarının dikkatle izlenmesi gerekmektedir.
Vitamin B bileşenleri hücre metabolizmasında oldukça önemli rol
oynamaktadırlar. B vitamini yönünden eksik olan gıdalar ile beslenme
depresyon ve yüksek tansiyon problemlerine yol açmaktadır. Amerika FDA
tarafından bireylerin alması gereken günlük B vitamini değerleri belirlenmiştir.
Matriksin oldukça kompleks yapıda olmasından dolayı vitamin analizleri
sıkıntılıdır. Yüksek seçiciliğe sahip clean-up aşamasına sahip numune
hazırlık yöntemleri uygulanmalıdır. Çoğunlukla ELISA ve HPLC yöntemleri
kullanılmaktadır. Fakat bu yöntemlerde vitaminler tekli olarak ya da sınıf olarak analiz edilebilmektedir. Numune hazırlığının basit
ve tüm vitaminlerin tek bir analizde tespit edildiği LC-MS/MS yöntemleri yaygınlaşmaya başlamıştır. Numune hazırlığının basit ve
hızlı oluşu ve tüm grupların tek seferde hazırlanmasından dolayı analiz maliyeti düşüktür. Ayrıca yüksek seçicilik ve hassasiyete
sahip olan LC-MS/MS sistemleri ile interferans riski yoktur ve bu sayede daha doğru analiz sonuçları elde edilmektedir.
Ülkemizde B vitaminlerinin olması gereken en düşük ve en yüksek miktarları Türk Gıda Kodeksi’nin “Devam Fomülleri tebliği”
ile belirlenmiştir.
YÖNTEM
Numuneler basit bir numune hazırlığı olan sıvı-sıvı
ekstraksiyon ile hazırlanarak direk olarak LC-MS/MS’e
Analitik Koşullar
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
: Phenomenex Luna HILIC 3 µm (100 x 2 mm)
: 11 dakika
: ESI +
: Scheduled MRM™
Vitamin B5
Vitamin B7
www.spektrotek.com
Şekil 16- Bebek mamasında analiz edilen B vitaminlerine ait
kromatogram
26
SONUÇ
Basit bir numune hazırlığı olan sıvı-sıvı ekstraksiyon ile
hazırlanan numuneler analiz edilmiştir. Buna göre tüm B
vitaminleri tek enjeksiyon ile çalışılabilmektedir. Elde edilen
veriler incelendiğinde 0.1-100 µg/kg arasında lineer aralığa
sahip olan B vitaminleri hızlı bir şekilde analiz edilmektedir.
GİRİŞ
Antibiyotikler enfeksiyöz hastalıkların tedavisinde ve gıda değeri olan çiftlik
hayvanlarının büyümelerini ve verimlerini teşvik edici olarak geniş çapta
kullanılmaktadırlar. ß-laktam, tetrasiklinler, kloramfenikol, makrolidler,
spektinomisin, linkozamid, sulfonamid, nitrofuran, nitroimidazol,
trimethoprim, polimiksin, kinolon ve makrosiklik grubu ilaçlar belirtilen
amaçlar için sahada en fazla kullanılan ilaçlardır.
Ancak bu ilaçların sahada uygun olmayan şekillerde ve yasal olmayan
kullanımları sonucu et, süt, yumurta, bal ve hayvanların yenilebilir diğer
dokularında kalıntılar oluşmaktadır. Antibiyotik kalıntı varlığı insanlarda
alerjik reaksiyonlara yol açabildiği gibi tehlikeli sağlık problemlerine yol
açabilecek olan patojenik bakterilerde antibiyotik direncinin artması gibi
ciddi durumlara da sebep olur. Bunlara ek olarak kalıntılar fermente gıdaların
kalitelerinde düşüklüğe yol açabilir. Tüm bu tehlikeli ve ciddi problemlerden
dolayı da, gıda maddelerinde ilaç kalıntılarının tespiti tüketiciler için önemli
bir konudur. Etkin bir gıda güvenliğinin sağlanması için sahada bilinçsiz
antibiyotik kullanımından kaçınılması ve gıdalardaki olası antibiyotik kalıntılarının sorumlu yasal otorite tarafından sıklıkla
izlenmesi de gereklidir.
Günümüzde antibiyotik kalıntılarının farklı gıda maddelerinde tespiti için birçok gelişmiş ve kantitatif ölçüm yeteneğine sahip
analitik metotlar kullanılmaktadır. ELISA, GC, HPLC ve LC-MS/MS kullanılan metotlar arasındadır. Bu yöntemler arasında
seçiciliği ve hassasiyeti en yüksek olan LC-MS/MS yöntemleri tercih edilmektedir. Ayrıca hızlı ve kolay numune hazırlama
yöntemleri sayesinde analiz maliyetleri oldukça düşmektedir. Kısa sürede hazırlanan numuneler analize hazır olmakta ve bu
sayede kısa sürede onlarda analiz yapılabilemektedir.
Ülkemizde hayvansal gıdalarda bulunabilecek veteriner ilaçların sınıflandırılması ve maksimum kalıntı limitleri; Türk Gıda
Kodeksi’nin “Hayvansal Gıdalarda Bulunabilecek Farmakolojik Aktif maddelerin sınıflandırılması ve maksimum kalıntı limitleri
yönetmeliği” ile belirlenmiştir.
VETERİNER İLAÇ KALINTILARI / ANTİBİYOTİK ANALİZİ
YÖNTEM
Numuneler sıvı-sıvı ekstraksiyonun ardından
clean-up işlemi uygulandıktan sonra LC-MS/MS’e
Pozitif Polarite
Analitik Koşullar
: Phenomenex Gemini C18 3 μm (50 x 2.0 mm)
: 10 dakika
: ESI + / ESI : Scheduled MRM™
SONUÇ
Bu çalışmada; Hızlı Polarite değişimi ve Scheduled
MRM™ ile hassasiyette azalma olmadan kısa sürede
analiz edilmiş ve elde edilen sonuçlar yönetmelikte verilen
limitleri rahatlıkla karşılamaktadır. Aynı zamanda QTrap™
teknolojisi kullanılarak mevcut olan 244 adet veteriner ilaç/
antibiyotiklere ait kütüphane taraması ile konfirmasyon
sonucu rapor güvenliği sağlanmaktadır.
Negatif Polarite
Şekil 17-Negatif-pozitif polarite değişimi ve Scheduled
MRM™ algoritması ile tek metotta analiz edilen balda
antibiyotik analizine ait kromatogram.
www.spektrotek.com
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
27
GIDA VE İÇECEKLERDE TATLANDIRICI TAYİNİ
GİRİŞ
Tatlandırıcılar, günlük yaşamda kullandığımız şekerin yerini almak
üzere üretilen, aynı miktardaki şekerden daha tatlı olan ve daha az enerji
içeren kimyasal maddelerdir. Başlangıçta şeker hastalarının tatlandırma
gereksiniminin giderilmesi için kullanılmış olmakla birlikte, günümüzde
fazla kilolu insanlar, vücut şeklini korumaya çalışanlar ve şekerin diş sağlığı
üzerindeki olumsuz etkilerinden korunmak isteyenler tarafından da yaygın
olarak kullanılmaktadırlar. Fakat Aspartam, sakarin veya sukraloz gibi yapay
tatlandırıcılar, diyabetin ilk evresi olan şeker duyarsızlığını (glukoz intoleransı)
tetikleyerek, diyabet hastalığına sebep olmaktadır.
Koruyucular, tatlandırıcılar, renklendiriciler ve uyarıcılar gibi besleyici olmayan
gıda katkı maddeleri gıda ve içecek ürünlerinde sıklıkla kullanılırlar. Gıda
kalite kontrol sürecinde gıda katkı maddelerinin analizi, bu katkı maddelerinin
uluslararası gıda kalite kontrol kriterlerini karşıladığından emin olmak açısından önemlidir. Gıda katkı maddelerinin kullanımı
dikkatli bir şekilde incelenmektedir ve gıda üreticileri ürünlerinin belli kriterleri karşıladığını göstermelidir. Bu regülasyonlar Gıda
ve Tarım Örgütü (FAO) ve Dünya Sağlık Örgütü (WHO) gibi örgütler tarafından yürütülmektedir. Ayrıca, yapay tatlandırıcıların
kullanımı çoğu ülkede düzenlenmektedir ve Amerikan Gıda ve İlaç Dairesi (FDA) tüm tatlandırıcılar için “Günlük Kabul Edilebilir
Alım Miktarı (ADI)” belirlemiştir. Yapılan regülasyonlar, gıda katkı maddelerinin gıda güvenliğinin tehlikeye atılmadığından emin
olmaya yöneliktir.
Gıda katkı maddelerinin analizinde kullanılabilecek pek çok analitik metot bulunmaktadır. Yüksek seçicilik ve hassasiyete sahip
olan LC-MS/MS sistemleri ile numune ön hazırlık işlemine ihtiyaç duyulmadan hızlı ve güvenilir analiz yapma imkanı sağlanır.
YÖNTEM
Analitik Koşullar
Numuneler 100/1000 kat su ile seyreltildikten sonra direk
olarak LC-MS/MS’e enjeksiyonu yapılır.
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
: Phenomenex Synergi 4 μm
(150 x 2.1 mm)
: 7 dakika
: ESI : Scheduled MRM™
MRM çiftleri:
www.spektrotek.com
Bileşik
Acesulfame
Aspartame
Cyclamate
Glycyrrhizin
Neohesperidin
Saccharin
Sucralose
28
Polarite
Negatif
Negatif
Negatif
Negatif
Negatif
Negatif
Negatif
Q1/Q3
162/82
293/200
178/80
821/351
611/303
182/42
395/359
Q1/Q3
162/78
293/261
178/79
821/113
611/166
182/106
397/361
SONUÇ
Limonata ve Kola numuneleri için 100 kat seyreltme ile analiz yapılmıştır. LC-MS/MS ile elde edilen sonuçlar diğer metotlar ile
karşılaştırıldığında 5 kat daha hızlı sonuç alınmaktadır. Ayrıca hassasiyetin yüksek olmasından dolayı numune hazırlığı sadece
seyreltme ile yapılmakta ve elde edilen sonuçlar incelendiğinde LOQ seviyelerinin belirlenmiş yasal limitlerin altında olduğu
görülmüştür.
Max. 2.1e5c ps.
Aspartame
Intensity, cps
2.0e5
1.5e5
(a)
Saccharine
1.0e5
5.0e4
0.0
1.02
.0
3.04
.0
5.06
Time,m in
XICo f- MRM (14 pairs): 292.928/261.200D aI D: Aspartame 2f romS ample5 7( cola 1/1000...
Intens ity, cps
7.0
Max. 2.9e4c ps.
Aspartame
2.9e4
2.5e4
.0
XICo f- MRM (14 pairs): 292.928/261.200D aI D: Aspartame 2f romS ample6 1( lemona de 1/1000...
(b)
2.0e4
1.5e4
Acesulfame
1.0e4
5000.0
0.0
1.02
.0
3.04
.0
Time,m in
5.06
.0
7.0
Şekil 18- Scheduled MRM™ ile yapılan tatlandırıcı analiz kromatogramı (a) Limonata (b) Kola
Kola numunesi aynı zamanda QTrap™ teknolojisi kullanılarak analiz edilmiş ve elde edilen kütle spektrumları ile kütüphane
karşılaştırması yapılmıştır. Böylece tek enjeksiyonda hem kantitatif sonuçlar elde edilir, hem de kütüphane taraması ile
konfirmasyon sonucu güvenilirlik sağlanır.
2.0e5
1.5e5
1.0e5
4.7
5.0e4
1.0
1.5
2.0
-EPI (161.88) Charge (+0) FT (25...
Intensity, cps
1.00e6
5.00e5
3.5
4.0
4.5
5.0
5.5
Time, min
Max. 1.0e6 cps. -EPI (292.93) Ch arge (+0) FT (25...
Acesulfame in cola
77.9
162.1
150
200
m/z, Da
-EPI (161.88) Charge (+0) FT (25...
100
5.2e5
78.0
162.1
1.0e6
100
150
200
m/z, Da
250
2.0e5
81.0
97.0
157.2
100
173.2 217.1
1.0e6
5.0e5
261.2
200.2
1.5e6
Aspartame
in cola
275.2 293.2
150
1.9e6
6.5
Max. 5.2e5 cps.
200
250
m/z, Da
-EPI (292.93) Ch arge (+0) FT (25...
Max. 3.6e6 cps.
Acesulfame standard
4.0e5
6.0
261.2
200.2
0.0
50
250
82.0
2.0e6
0.0
50
3.0
82.0
0.00
50
3.6e6
3.0e6
2.5
300
350
400
Max. 1.9e6 cps.
Aspartame
standard
217.2
97.1 118.1
81.0
0.0
50
100
150
174.2
275.2 293.2
200
250
m/z, Da
Şekil 19- QTrap-EPI kullanılarak analiz edilen kola numunesine ait tatlandırıcı kütle spektrumları
300
350
400
www.spektrotek.com
0.5
(c)
Intensity, cps
0.0
Intensity, cps
Max. 2.2e5 cps.
2.6
cola
Intensity, cps
Inten sity, cps
TIC of -MRM (18 pairs): Exp 1, from Sample 4 (cola 1 in 100 dilution) of samples MRM -EPI..
29
İÇME SULARINDA POLİSİKLİK AROMATİK
HİDROKARBON (PAH) ANALİZİ
GİRİŞ
Günümüzde hızla gelişen sanayileşme insan yaşamını önemli ölçüde
kolaylaştırırken birçok çevre sorununu da bir arada getirmiştir. İnsan
nüfusundaki ve şehirleşme oranındaki hızlı artış çevre kirliliğine neden olan
diğer önemli etkenlerdir. Bu etkenler içerisinde yer alan Polisiklik aromatik
hidrokarbonlar (PAH), karbon ve hidrojen atomunun iki ya da daha fazla
aromatik zincirle oluşturduğu çeşitli organik bileşiklerin oluşturduğu bir
grubu temsil eder.
Pek çok PAH, çeşitli yanma prosesleri sonucu (orman yangınları, fosil
yakıtların yanması vb.) ve piroliz kaynaklarından atmosfer yoluyla çevreye
giriş yapar. Ancak düşük çözünürlüğü ve partiküler maddeye olan çekimi
nedeniyle genellikle suda kayda değer konsantrasyonlarda görülmez.
İçme suyunda PAH konsantrasyonlarının ana kaynağı, içme suyu dağıtım
şebekesinde boruları korozyondan korumak için kullanılan kömür katranı
kaplamasıdır.
PAH’lar yağ içeren bütün vücut dokularımıza girebilir, çoğunlukla karaciğer, yağ ve böbrekte depolanma eğilimindedir. PAH’lar
tümör başlatıcı, geliştirici ve ilerletici özellikleri olan bileşiklerdir. Hayvanlar ile yapılan çalışmalarda kısa ya da uzun vadede
PAH’lara maruz kaldıklarında bağışıklık sisteminde, vücut sıvılarında sorunlara, akciğer, mesane ve deri kanserlerine neden
olduğu görülmüştür.
Doğada 100’ün üzerinde PAH bileşiği tespit edilmiştir. Ancak kanserojen ve toksik etkisinin daha fazla olduğu düşünülen 16 PAH
bileşiği öncelikli kirleticiler arasında kabul edilmiştir. Su için limitler “İnsani Tüketim Amaçlı Sular Hakkında Yönetmeliği”nde
belirlenmiştir. Bu bileşiklerin tespit ve tayini için birçok analitik yöntem kullanılmaktadır. Bu yöntemlerden en yaygın olarak
kullanılan HPLC yöntemidir. Fakat bu yöntemde, oldukça uzun ve zahmetli aynı zamanda yüksek maliyetli bir numune ön
hazırlığına ihtiyaç duyulmaktadır. Ayrıca elde edilen sonuçlar değerlendirildiğinde HPLC yöntemi ile yönetmelikte verilen
limitleri karşılamakta zorluk çekildiği görülmüştür. Buna karşılık numune hazırlığına ihtiyaç duyulmayan direk olarak numunenin
enjeksiyonuna dayanan LC-MS/MS metodu; hem zaman açısından hem de analiz maliyeti açısından avantaj getirmektedir
YÖNTEM
Analitik Koşullar
Numuneler hiçbir ön işleme tabi tutulmadan direk olarak viale
alınır ve LC-MS/MS’e enjeksiyonu yapılır.
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
: Phenomenex Kinetex Phenyl-Hexyl 1.7 um (100x2.1 mm)
: 6 dakika
: Su ve Asetonitril
: APCI
: Scheduled MRM™
MRM çiftleri:
Max. 1,1e4 cps.
XIC of +MRM (13 pa rs): 253,100/250,100 amu Expected RT: 3,6 ID: benzo(a)pyrene-2 from Sample 3
Polarite
Pozitif
Pozitif
Pozitif
Pozitif
Pozitif
Q1/Q3
253/252
252/250
252/250
276/274
276/274
Q1/Q3
253/250
252/224
252/224
276/272
276/272
2,6e4
2,5e4
2,4e4
2,3e4
2,2e4
(1) Benzo(b) fluoranthene
(2) Benzo(k) fluoranthene
(3) Benzo(a) pyrene
(4) Benzo(g,h,i) fluoranthene
(5) Indeno(1,2,3-c,d) pyrene
2,1e4
2,0e4
1,9e4
1,8e4
1,7e4
1,6e4
Bileşik
Benzo(a)pyrene
Benzo(b)fluorenthene
Benzo(k)fluorenthene
Benzo(g,h,i) perylene
Indeno(1,2,3-c,d) pyrene
3
1,5e4
1,4e4
1,3e4
1,2e4
1,1e4
3,62
1,0e4
9000,0
8000,0
7000,0
4
6000,0
www.spektrotek.com
SONUÇ
30
Su numunesinin hiçbir ön işleme tabi tutulmadan direk
olarak LC-MS/MS sistemine verilmesi ile elde edilen sonuçlar
incelendiğinde yönetmelikte belirlenen limitleri rahatlıkla
karşıladığı görülmüştür.
5000,0
4000,0
3000,0
1
2000,0
2
5
1000,0
0,0
1,6
1,8
2,0
2,2
2,4
2,6
2,8
3,0
3,2
3,4
3,6
3,8
T me, m n
4,0
4,2
4,4
4,6
4,8
5,0
5,2
5,4
5,6
Şekil 20- Su numunesi için Scheduled MRM™ ile yapılan
PAH analizine ait kromatogram
5,8
6,0
GİRİŞ
Akrilamid, yıllardır ticari olarak üretilen ve çeşitli endüstri dallarında kullanılan
bir monomerdir. En geniş kullanım alanı içme suyu ve endüstriyel atık suların
arıtımında flokülant (topaklaştırıcı) olarak kullanılan suda çözünebilen
poliakrilamidlerin üretimidir. Akrilamidin insanlarda ve deney hayvanlarında
güçlü toksik etkileri bilinmektedir. Akrilamidin endüstride yaygın olarak
kullanımı ve kalıntı akrilamid monomeri içeren polimerlerin suların
arıtımında kullanılması, içme ve yüzey sularında akrilamid bulunabileceğini
düşündürmektedir.
Akrilamid, Uluslararası Kanser Araştırmaları Ajansı tarafından “İnsan İçin
Muhtemel Kanserojenik Madde” olarak tanımlanmıştır. Bu sebeple içme
sularında miktarlarının kontrol edilmesi oldukça önemlidir.
Akrilamidin tayin ve tespiti için bir çok yöntem mevcuttur. Bu yöntemler
arasından numune hazırlığına ihtiyaç duyulmadan numunenin direk olarak
cihaza enjeksiyonu ile analiz edilmesine olanak sağlayan LC-MS/MS
yöntemleri tercih edilmektedir. Numune ön hazırlığına ihtiyaç duyulmaması
sebebiyle analiz maliyeti yok denecek kadar azdır. Yüksek seçicilik ve hassasiyet ile ön planda olan LC-MS/MS sistemleri hızlı
ve kolay analiz imkanı sağlamaktadır.
Ülkemizde Su için limitler “İnsani Tüketim Amaçlı Sular Hakkında Yönetmeliği”nde belirlenmiştir.
YÖNTEM
İÇME SULARINDA AKRİLAMİD ANALİZİ
Analitik Koşullar
Analitik Kolon : Phenomenex Luna C18 3 um
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
MRM çiftleri:
(150x3 mm)
: 5 dakika
: Su (Formik Asit) ve Asetonitril
: ESI +
: MRM
XIC of +MRM (2 pa rs): 72,176/27,000 Da ID: Acrylam de 2 from Sample 8 (1 PPB) of 14102015 ACRYLAMIDE.w ff (Turbo Spray), Smoothed
Polarite
Q1/Q3
Akrilamid 1
Akrilamid 2
Akrilamid 3
Pozitif
Pozitif
Pozitif
72/55
72/44
72/27
SONUÇ
Su numunesinin hiçbir ön işleme tabi tutulmadan direk
olarak LC-MS/MS sistemine verilmesi ile elde edilen sonuçlar
incelendiğinde yönetmelikte belirlenen limitleri rahatlıkla
karşıladığı görülmüştür.
7,0e4
6,5e4
6,0e4
5,5e4
5,0e4
4,5e4
Bileşik
Max. 6103,2 cps.
7,2e4
4,0e4
3,5e4
3,0e4
2,5e4
2,0e4
1,5e4
1,0e4
3,53
5000,0
0,0
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
2,2
2,4
2,6
T me, m n
2,8
3,0
3,2
3,4
3,6
3,8
4,0
4,2
4,4
4,6
Şekil 21- Su numunesi ile yapılan Akrilamid analizine ait
kromatogram
4,8
www.spektrotek.com
Numuneler hiçbir ön işleme tabi tutulmadan direk
olarak viale alınır ve LC-MS/MS’e enjeksiyonu
yapılır.
31
İÇME SULARINDA MULTİ-PESTİSİT ANALİZİ
GİRİŞ
İnsan yaşamını kolaylaştırmak için üretilen birçok kimyasal, üretim
aşamasından tüketim aşamasına kadar, insan sağlığı ve çevre açısından
küresel bir tehdit oluşturmaktadır. Dünyadaki kimyasal sanayi üreticileri para
kazanma içgüdüsüyle ve çoğunlukla insan sağlığı ve çevreye olan etkilerini
ciddi boyutlarda araştırmadan her yıl binlerce kimyasal bileşiği üretip piyasaya
sürmektedir. İnsan yaşam kalitesini arttırmak amacıyla kullanılmakta olan
kimyasallar ve özellikle tarımsal üretimde kullanılan pestisitler; kontrolsüz,
bilinçsiz ve gereksiz yere kullanılmaları sonucunda bireyin yaşamını ve
yaşadığı çevreyi çok ciddi anlamda tehdit eder konuma gelmiştir.
Mevcut sorun, sadece ülkemizin bir sorunu olmayıp, küresel bir problem
olarak karşımıza çıkmaktadır. Tarımda kullanılan pestisitler, ayrıca
kalıntılarıyla soframıza kadar sebze ve meyve olarak gelmekte ve pek çok
hastalığa neden olabilmektedir. Tarımsal alanlara, orman veya bahçelere
uygulanan pestisitler havaya, su ve toprağa, oradan da bu ortamlarda
yaşayan diğer canlılara geçmekte ve ölmelerine sebep olmaktadır.
Dünya sağlık örgütünün (WHO) 1995 yılında yayınlanan raporuna göre, her yıl dünyada kabaca 1 milyon insan pestisit sebebiyle
zehirlenmekte, 20.000 kadarı da ölmektedir. Pestisitlerle insanların teması, ilaç üretimi, taşıma, depolama, kullanma ve ilaç
kalıntısı içeren ürünlerin tüketimi sırasında olmaktadır. Bu etkileşim sonunda insan vücuduna girmeleri ise ağız, deri ve solunum
yoluyla olmaktadır. Pestisitlerin yanı sıra, parçalanma ürünleri olan metabolitleri de insanlara zehir etkili olabilmektedir. Bu
maddelerin bir kısmı birikime uğradığı, bir kısmı da birikmediği halde sinir hücrelerinde tahribat yaptığı için tehlikeli sonuçlar
doğurabilmektedir.
Bu kimyasalların tespiti için birçok analitik yöntem mevcuttur. Numuneye hiçbir ön işlem uygulanmadan direk olarak
enjeksiyonuna dayanan LC-MS/MS metotları avantajları göz önüne alındığında birinci sıraya yerleşmiştir. Su için limitler “İnsani
Tüketim Amaçlı Sular Hakkında Yönetmeliği”nde belirlenmiştir. Yapılan çalışmalar sonunda belirlenmiş limitlerin rahatlıkla
karşılandığı görülmüştür.
YÖNTEM
Numuneler hiçbir ön işleme tabi tutulmadan direk olarak viale
alınır ve LC-MS/MS’e enjeksiyonu yapılır.
Analitik Koşullar
Analitik Kolon
Analiz Süresi
Mobil Faz
İyonizasyon
Tarama Modu
: Phenomenex Kinetex C18 2.6 um
(50x2.1 mm)
: 10 dakika
: Su (Amonyum Format) ve Metanol (Amonyum Format)
: ESI + / ESI : MRM
SONUÇ
www.spektrotek.com
Bu çalışmada; Hızlı Polarite değişimi ve Scheduled MRM™ ile hassasiyette azalma olmadan kısa sürede analiz edilmiş ve elde
edilen sonuçlar su için yönetmelikte verilen limitleri rahatlıkla karşılamaktadır. Aynı zamanda QTrap™ teknolojisi kullanılarak
mevcut olan 666 adet pestisite ait kütüphane taraması ile konfirmasyon sonucu rapor güvenliği sağlanmaktadır.
32
Pozitif Polarite
Negatif Polarite
Şekil 22-Negatif-pozitif polarite değişimi ve Scheduled MRM™ algoritması ile tek metotta analiz edilen 1ppb 30 adet pestisit
etken maddesine ait kromatogram.
Şekil 23-Negatif-pozitif polarite değişimi ve QTrap-EPI ile tek metotta analiz edilen su numunesine ait kromatogram.
www.spektrotek.com
Aynı zamanda su numunesi QTrap-EPI teknolojisi kullanılarak mevcut olan 666 adet pestisite ait kütüphane taraması ile
konfirmasyon sonucu rapor güvenliği sağlanmaktadır.
33
SPEKTRALKÜTÜPHANETABLOLARI
Spektral Kütüphane Tabloları
Antibiotics High Resolution MS/MS Spectral Library Version 1.0 for Use in
MasterView™ Software and LibraryView™ Software
Antibiotics High Resolution MS/MS Library Version 1.0
An overview of the licensed mass spectral library for antibiotics and veterinary drugs compatible with MasterView™ and
LibraryView™ software
The MasterView™ and LibraryView™ software package is a
fast way to analyze large batches of MS/MS data for accurate
and efficient MS/MS library searching, data mining, and
compound database management. We have assembled
a high resolution, accurate mass MS/MS spectral library
containing 244 antibiotic and veterinary drug entries. This
library was created using certified reference materials and
can be used in MasterView™ and LibraryView™ software
to perform MS/MS library searching or to create custom
screening and/or quantitation methods using the integrated
MS and MS/MS information.
www.spektrotek.com
Features of this high resolution MS/MS spectra library:
• Includes data on 244 antibiotics and veterinary drugs
commonly tested for in food products of animal origin.
• Includes 259 high resolution MS/MS spectral library
entries including individual spectra acquired using distinct
collision energies, as well as a single spectra using
collision energy spread representing the sum of three
collision energies.
• Contains spectra for both positive and negative ionization
for compounds that ionize in both polarities.
36
Compound
17a-Methyltestosterone
4,6-Dimethyl-2-Hydroxypyrimidine
5-Hydroxymebendazole
5-Hydroxymebendazole-D3
5-Hydroxythiabendazole
a-Nortestosteron
Acepromazine
Acetoxyprogesterone-17a
Acetyltylosin, 3-OAlbendazole
Albendazole-2-amino-sulfone-D3
Albendazole-D3
Albendazole sulfoxide
Albendazole sulfoxide-d3
Albendazolsulfon
Albendazolsulfon-D3
Albendazolsulfonamin
Aminoflubendazol
Aminomebendazol
Aminophenazon
Formula
C20H30O2
C6H8N2O
C16H15N3O3
C16H12(2H)3N3O3
C10H7N3OS
C18H26O2
C19H22N2OS
C23H32O4
C48H79NO18
C12H15N3O2S
C10H10(2H)3N3O2S
C12H12 2H3N3O2S
C12H15N3O3S
C12H15N3O3S
C12H15N3O4S
C12H12 2H3N3O4S
C10H13N3O2S
C14H10FN3O
C14H11N3O
C13H17N3O
Advantages of using this MS/MS spectral library:
• Use the integrated MS and MS/MS information to build
methods without the need to infuse standards and optimize
conditions for a given compound.
• Easily create processing methods for a TOF-MS-IDA-MS/
MS workflow for use on TripleTOF® LC/MS/MS systems.
• Quickly set-up XIC tables for quantitation and identification
in MasterView™ software.
• Set-up smaller customer libraries by simply selecting only
the compounds of interest from the list in LibraryView™
Software.
The following is a list of the compounds currently in the
library. This library is verified for use on AB SCIEX
TripleTOF® and QTRAP® LC/MS/MS systems.
Please note that this library is continuously being expanded
to include additional compounds.
Formula Weight
302.4572
124.1425
297.3137
300.3323
217.2499
274.4034
326.4613
372.5048
958.1505
265.3347
242.3153
268.3532
281.334
284.3525
297.3333
300.3518
239.2967
255.2518
237.2613
231.2978
CAS Number
58-18-4
108-79-2
60254-95-7
948-71-0
68-22-4
61-00-7
302-23-8
63409-10-9
54965-21-8
54029-12-8
75184-71-3
80983-34-2
82050-13-3
52329-60-9
58-15-1
# of Spectra
1
Formula Weight
CAS Number
302.3767
3690-04-8
Amoxicillin
C16H19N3O5S
365.4085
26787-78-0
Ampicillin
C16H19N3O4S
349.4092
69-53-4
Arprinocid
C12H9ClFN5
277.6887
55779-18-5
Avermectin B1a
C48H72O14
873.091
71751-41-2
Azaperone
C19H22FN3O
327.4021
1649-18-9
b-Boldenone
C19H26O2
286.4144
846-48-0
Bamethan
C12H19NO2
209.2884
3703-79-5
Baquiloprim
C17H20N6
308.3867
102280-35-3
Benzylpenicillin, (Penicillin G)
C16H18N2O4S
334.3945
61-33-6
Brillant green
C27H34N2O4S
482.6427
633-03-4
Brombuterol
C12H18Br2N2O
366.095
419337-02-4
Carazolol
C18H22N2O2
298.3852
57775-29-8
Carbuterol
C13H21N3O3
267.3282
34866-47-2
Carprofen
C15H12ClNO2
273.7188
53716-49-7
Cefalonium
C20H18N4O5S2
458.5159
5575-21-3
Cefazolin
C14H14N8O4S3
454.51
25953-19-9
Cefoperazon
C25H27N9O8S2
645.6742
62893-20-3
Ceftiofur
C19H17N5O7S3
523.5667
80370-57-6
Cephalexin
C16H17N3O4S
347.3933
15686-71-2
Cephapirin
C17H17N3O6S2
423.4674
21593-23-7
Chloramphenicol
C11H12Cl2N2O5
323.1321
56-75-7
Chlorbrombuterol
C12H18BrClN2O
321.6442
37153-52-9
Chlormadinone acetate
C23H29ClO4
404.9337
302-22-7
Chlorprothixene
C18H18ClNS
315.8652
113-59-7
Chlortetracyclin
C22H23ClN2O8
478.886
57-62-5
Cimaterol
C12H17N3O
219.2868
54239-37-1
Cimbuterol
C13H19N3O
233.3137
54239-39-3
Ciprofloxacin
C17H18FN3O3
331.347
85721-33-1
Clenbuterol
C12H18Cl2N2O
277.1934
21898-19-1
Clencyclohexerol
C14H20Cl2N2O2
319.2307
157877-79-7
Clenhexerol
C14H22Cl2N2O
305.2472
78982-88-4
Clenisopenterol
C13H20Cl2N2O
291.2203
157664-68-1
Clenpenterol
C13H20Cl2N2O
291.2203
37158-47-7
Clenproperol
C11H16Cl2N2O
263.1665
38339-18-3
Clenproperol-D7
C11H9 2H7Cl2N2O
270.2098
1173021-09-4
Clobendazole
C16H12ClN3O3
329.7427
Clorprenaline
C11H16ClNO
213.707
3811-25-4
Clorsulon
C8H8Cl3N3O4S2
380.6563
60200-06-8
Closantel
C22H14Cl2I2N2O2
663.0804
57808-65-8
Closantel-13C(6)
(13C6)C16H14Cl2I2N2O2
669.0343
Cloxacillin
C19H18ClN3O5S
435.8864
61-72-3
Cyclopentylalbendazole
C14H17N3O2S
291.3726
77723-30-9
Danofloxacin
C19H20FN3O3
357.3849
112398-08-0
Dapson
C12H12N2O2S
248.3041
80-08-0
Decoquinat
C24H35NO5
417.5457
18507-89-6
Desacetylcephapirin
C15H15N3O5S2
381.4301
38115-21-8
Dexamethasone
C22H29FO5
392.4677
50-02-2
# of Spectra
Formula
C16H22N4O2
1
2
1
2
1
www.spektrotek.com
Compound
Aminopropylon
37
www.spektrotek.com
38
Compound
Diclofenac
Formula
C14H11Cl2NO2
Formula Weight
296.1526
CAS Number
Dicloxacillin
C19H17Cl2N3O5S
470.3312
3116-76-5
Dienestrol
C18H18O2
266.34
84-17-3
Dienestrol-D2
C18H16O2(2H)2
268.3523
Diethylstilbestroldipropionat
C24H28O4
380.4841
130-80-3
Difloxacin
C21H19F2N3O3
399.3974
98106-17-3
Dimetridazol
C5H7N3O2
141.1296
551-92-8
Dimetridazol-D3
C5H4 2H3N3O2
144.1481
64678-69-9
Dinitrocarbanilid-D8
C13H2 2H8N4O5
310.2958
Doramectin
C50H74O14
899.1289
117704-25-3
Doxycycline (Tautomer)
C22H24N2O8
444.4412
564-25-0
Emamectin benzoate (B1a)
C49H75NO13
886.1332
155569-91-8
Enrofloxacin
C19H22FN3O3
359.4007
93106-60-6
Enrofloxacin-D5
C19(2H)5H17FN3O3
364.4316
epi-Chlortetracyclin
C22H23ClN2O8
478.886
14297-93-9
epi-Oxytetracyclin
C22H24N2O9
460.4405
35259-39-3
epi-Tetracyclin
C22H24N2O8
444.4412
23313-80-6
Eprinomectin B1a
C50H75NO14
914.1436
133305-88-1
Erythromycin A
C37H67NO13
733.9374
59319-72-1
Erythromycin A-(13C)2
(13C)2C35H67NO13
735.922
114-07-08
Erythromycin A Anhydrid
C37H65NO12
715.9222
23893-13-2
Ethidimuron
C7H12N4O3S2
264.3262
30043-49-3
Febantel
C20H22N4O6S
446.4825
58306-30-2
Fenbendazol-D3
C15H10 2H3N3O2S
302.3704
1228182-47-5
Fenbendazole
C15H13N3O2S
299.3519
43210-67-9
Fenbendazole sulfone-D3
C15H10 2H3N3O4S
334.3691
1228182-49-7
Fenoterol
C17H21NO4
303.3581
13392-18-2
Florfenicol
C12H14Cl2FNO4S
358.2157
73231-34-2
Flubendazole
C16H12FN3O3
313.2884
31430-15-6
Flubendazole-D3
C16H9 2H3FN3O3
316.3069
1173021-08-3
Flufenamic Acid
C14H10F3NO2
281.2344
530-78-9
Flumequine
C14H12FNO3
261.2528
42835-25-6
Flumethasone
C22H28F2O5
410.4582
2135-17-3
Flunixin
C14H11F3N2O2
296.2491
38677-85-9
Flunixin-D3
C14H8(2H)3F3N2O2
299.2676
1015856-60-6
Gamithromycin
C40H76N2O12
777.0493
145435-72-9
Halofuginon
C16H17BrClN3O3
414.6859
55837-20-2
Heliotrin
C16H27NO5
313.394
303-33-3
Hydroxy-Ipronidazole-D3
C7H8(2H)3N3O3
188.2012
Hydroxymethylclenbuterol
C12H18Cl2N2O2
293.1928
38339-18-3
14885-29-1
Ipronidazole
C7H11N3O2
169.1834
Ipronidazole-D3
C7H8(2H)3N3O2
172.2019
Ipronidazole-OH
C7H11N3O3
185.1827
# of Spectra
15307-86-5
35175-14-5
Isochlortetracyclin
C22H23ClN2O8
478.886
514-53-4
Isoxsuprine
C18H23NO3
301.3857
395-28-8
Ivermectin
C48H74O14
875.1068
70288-86-7
Josamycin
C42H69NO15
828.0071
56689-45-3
Ketoprofen
C16H14O3
254.2855
22071-15-4
1
2
1
2
1
2
1
Formula Weight
CAS Number
329.5693
Kristallviolett
C25H30ClN3
407.9867
548-62-9
Labetalol
C19H24N2O3
328.4114
36894-69-6
Lasalocid A
C34H53NaO8
612.7797
25999-20-6
Leuco Malachite Green
C23H26N2
330.4734
129-73-7
Leuco Malachite Green-D5
C23H21(2H)5N2
335.5043
# of Spectra
2
Leucocrystal Violet
C25H31N3
373.5419
603-48-5
Leucomycin
C35H59NO13
701.8519
1392-21-8
Levamisole
C11H12N2S
204.2944
14769-73-4
Lincomycin
C18H34N2O6S
406.542
154-21-2
Mabuterol
C13H18ClF3N2O
310.7469
56341-08-3
Mapenterol
C14H20ClF3N2O
324.7738
95656-68-1
Marbofloxacin
C17H19FN4O4
362.361
115550-35-1
Mebendazol
C16H13N3O3
295.2979
31431-39-7
Mebendazol-D3
C16H10 2H3N3O3
298.3164
Meclofenamic acid
C14H11Cl2NO2
296.1526
644-62-2
Medroxyprogesterone acetate
C24H34O4
386.5317
71-58-9
Mefenamic acid
C15H15NO2
241.2899
61-68-7
Megestrol acetate
C24H32O4
384.5158
595-33-5
Melengestrol acetate
C25H32O4
396.5268
2919-66-6
Meloxicam
C14H13N3O4S2
351.4039
71125-38-7
Meloxicam-D3
C14H10 2H3N3O4S2
354.4224
942047-63-4
Methapyrilene
C14H19N3S
261.3898
135-23-9
Methicillin
C17H20N2O6S
380.42
61-32-5
Methotrimeprazine
C19H24N2OS
328.4772
851-68-3
Meticlorpindol, (Clopidol)
C7H7Cl2NO
192.0443
2971-90-6
Metoprolol
C15H25NO3
267.3684
37350-58-6
Metronidazol
C6H9N3O3
171.1558
444-48-1
Metronidazol-D3
C6H6(2H)3N3O3
174.1743
Metronidazole-OH
C6H9N3O4
187.1551
4812-40-2
Monensin
C36H62O11
670.8813
17090-79-8
Monocrotalin
C16H23NO6
325.3616
315-22-0
Nafcillin
C21H22N2O5S
414.4807
147-52-4
Nalidixic acid
C12H12N2O3
232.239
389-08-2
Nandrolon
C18H26O2
274.4034
434-22-0
Naproxen
C14H14O3
230.2635
22204-53-1
Netobimin-Micronized
C14H20N4O7S2
420.4641
88255-01-0
Niflumic acid
C13H9F3N2O2
282.2222
4394-00-7
Nitroxinil
C7H3IN2O3
290.017
1689-89-0
Norfloxacin
C16H18FN3O3
319.3359
70458-96-7
Oleandomycin
C35H61NO12
687.8684
2751-09-9
Orciprenaline
C11H17NO3
211.2609
586-06-1
Oxacillin
C19H19N3O5S
401.4416
61-72-3
Oxfendazole
C15H13N3O3S
315.3512
53716-50-0
Oxfendazole-D3
C15H10 2H3N3O3S
318.3698
1228182-54-4
Oxfendazolsulfon
C15H13N3O4S
331.3506
54029-20-8
Oxibendazole
C12H15N3O3
249.2696
20559-55-1
Oxibendazole-D7
C12H8 2H7N3O3
256.3128
1173019-44-7
1
Formula
C13H7Cl3N2O2
2
1
www.spektrotek.com
Compound
Ketotriclabendazole
39
www.spektrotek.com
40
Compound
Oxolinic acid
Formula
C13H11NO5
Formula Weight
261.234
CAS Number
Oxyphenbutazone
C19H20N2O3
324.3797
129-20-4
Oxytetracycline
C22H24N2O9
460.4405
79-57-2
Phenylbutazone
C19H20N2O2
308.3804
129-18-0
Phenylbutazone-D10
C19H10(2H)10N2O2
318.4422
Pirbuterol
C12H20N2O3
240.3025
38677-81-5
Pirlimycin
C17H31ClN2O5S
410.9606
79548-73-5
Praziquantel
C19H24N2O2
312.4121
55268-74-1
Prednisolone
C21H28O5
360.4503
50-24-8
Progesterone
C21H30O2
314.4682
57-83-0
Promethazine
C17H20N2S
284.4241
60-87-7
Propionylpromazine
C20H24N2OS
340.4882
3568-24-9
Ractopamine
C18H23NO3
301.3857
97825-25-7
Rafoxanide
C19H11Cl2I2NO3
626.0161
22662-39-1
Ramifenazon
C14H19N3O
245.3247
3615-24-5
Retrorsin
C18H25NO6
351.3995
480-54-6
Ritodrine
C17H21NO3
287.3588
26652-09-5
Robenidine
C15H13Cl2N5
334.2079
25875-51-8
Ronidazole
C6H8N4O4
200.154
7681-76-7
Ronidazole-D3
C6H5(2H)3N4O4
203.1725
1015855-87-4
Roxithromycin
C41H76N2O15
837.0583
80214-83-1
Salbutamol
C13H21NO3
239.3147
18559-94-9
Salmeterol
C25H37NO4
415.5732
89365-50-4
Salmeterol-D3
C25H34 2H3NO4
418.5918
497063-94-2
Sarafloxacin
C20H17F2N3O3
385.3705
98105-99-8
Secinidazol
C7H11N3O3
185.1827
3366-95-8
Selamectin
C43H63NO11
769.9732
220119-17-5
Semduramycin
C45H76O16
873.0882
113378-31-7
Senecionin
C 18H25NO5
335.4002
130-01-8
Seneciophyllin
C18H23NO5
333.3843
480-81-9
Sotalol
C12H20N2O3S
272.3668
3930-20-9
Spectinomycin
C14H24N2O7
332.3535
1695-77-8
Spiramycin
C43H74N2O14
843.0652
8025-81-8
Stanozolol
C21H32N2O
328.4982
10418-03-8
Sulfabenzamide
C13H12N2O3S
276.3145
127-71-9
Sulfacetamide
C8H10N2O3S
214.2434
144-80-9
Sulfachloropyridazine
C10H9ClN4O2S
284.7245
80-32-0
Sulfadiazine
C10H10N4O2S
250.2797
68-35-9
122-11-2
Sulfadimethoxine
C12H14N4O4S
310.3321
Sulfadimethoxine-D6
C12H8 2H6N4O4S
316.3692
# of Spectra
14698-29-4
Sulfadimidin
C12H14N4O2S
278.3335
57-68-1
Sulfadoxine
C12H14N4O4S
310.3321
2447-57-6
Sulfaguanidin
C7H10N4O2S
214.2466
57-67-0
Sulfamerazine
C11H12N4O2S
264.3066
127-79-7
Sulfameter
C11H12N4O3S
280.3059
651-06-9
Sulfamethizol
C9H10N4O2S2
270.3331
144-82-1
Sulfamethoxazole
C10H11N3O3S
253.2802
723-46-6
Sulfamethoxypyridazine
C11H12N4O3S
280.3059
80-35-3
1
2
1
Formula
Formula Weight
CAS Number
C11H13N3O3S
267.3071
729-99-7
Sulfanilamide
C6H8N2O2S
172.2062
63-74-1
Sulfaphenazole
C15H14N4O2S
314.3666
526-08-9
Sulfapyridin
C11H11N3O2S
249.2919
144-83-2
Sulfaquinoxaline
C14H12N4O2S
300.3397
59-40-5
Sulfathiazole
C9H9N3O2S2
255.3184
72-14-0
Terbutaline
C12H19NO3
225.2878
23031-25-6
Testosterone
C19H28O2
288.4303
58-22-0
Tetracycline
C22H24N2O8
444.4412
60-54-8
Tetramisole-D5
C11H7 2H5N2S
209.3253
1173021-85-6
Thiabendazole
C10H7N3S
201.2505
148-79-8
Thiamphenicol
C12H15Cl2NO5S
356.2246
15318-45-3
Tiamulin
C28H47NO4S
493.75
55297-95-5
Tilmicosin
C46H80N2O13
869.1465
108050-54-0
Tinidazol
C8H13N3O4S
247.2733
19387-91-8
Tolfenamic Acid
C14H12ClNO2
261.7078
13710-19-5
Tolfenamic Acid-(13C)6
C8(13C)6H12ClNO2
267.6617
Trenbolone
C18H22O2
270.3717
10161-33-8
Triamcinolone
C21H27FO6
394.4401
124-94-7
Triclabendazole
C14H9Cl3N2OS
359.6613
68786-66-3
Triclabendazole-D3
C14H6 2H3Cl3N2OS
362.6798
Triclabendazole sulfone
C14H9Cl3N2O3S
391.66
100648-14-4
Triclabendazole sulfoxide
C14H9Cl3N2O2S
375.6606
100648-13-3
Trimethoprim
C14H18N4O3
290.3222
738-70-5
1
2
1
2
1
2
0
Tulobuterol
C12H18ClNO
227.7339
41570-61-0
Tylosin A
C46H77NO17
916.1133
8026-48-0
Tylosin B
C39H65NO14
771.9429
11032-98-7
Tylvylosin (Aivlosin)
C53H87NO19
1042.268
63409-12-1
Valnemulin
C31H52N2O5S
564.8288
101312-92-9
Xylazine
C12H16N2S
220.3372
23076-35-9
Zilpaterol
C14H19N3O2
261.324
117827-79-9
Product Name
Antibiotics High Resolution MS/MS Spectral Library Version 1.0
1
License
Part Number
permanent
5038640
one-year
5038642
© 2015 AB Sciex. For Research Use Only. Not for use in diagnostic procedures.
The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being
used under license. Publication number: 11120615-01
www.spektrotek.com
Tulathromycin Marker
# of Spectra
Compound
Sulfamoxol
41
Mycotoxin Spectral Library v1.1 for LibraryView™ Software
An overview of the licensed mass spectral library for mycotoxins compatible with
LibraryView™ Software
LibraryView™ Software is a fast way to analyze large
batches of MS/MS data for accurate and efficient MS/MS
library searching, data mining, and compound database
management. We’ve assembled an MS/MS spectral library
compatible with LibraryView™ Software containing 248
mycotoxin residue entries. This library was created using
certified reference materials and can be used in LibraryView™
Software to perform MS/MS library searching or to create
custom screening and/or quantitation methods using the
integrated MRM information.
Features of this MS/MS spectra library:
• Includes data on 245 mycotoxins and other fungal/bacterial
metabolites commonly tested for in grains, cereals, or
other food products.
• Contains 245 integrated MRM entries with up to 3
transitions per compound.
• Includes 236 full MS/MS spectral library entries including
individual spectra acquired using three distinct collision
energies (20 eV, 35 eV, 50 eV), as well as a single spectra
representing the sum of all three collision energies.
• Contains spectra for both positive and negative ionization
for compounds that ionize in both polarities.
www.spektrotek.com
Compound
15-Acetyl-deoxynivalenol
15-Monoacetoxyscirpenol
16-Ketoaspergillimide
2-Amino-14,16-dimethyloctadecan-3-ol
3-Acetyl-deoxynivalenol
3-Nitropropionic acid
3-O-Methylviridicatin
A23187
AAL-TA1 Toxin
Actinomycin D
Aflatoxin B1
Aflatoxin B2
Aflatoxin G1
Aflatoxin G2
Aflatoxin M1
Aflatoxin M2
Agroclavine
Alamethicin F30
42
Formula
C17H22O7
C17H24O6
C20H27N3O4
C20H43ON
C17H22O7
C3H5NO4
C16H13NO2
C29H37N3O6
C25H47NO10
C62H86N12O16
C17H12O6
C17H14O6
C17H12O7
C17H14O7
C17H12O7
C17H14O7
C16H18N2
C92H150N22O25
Advantages of using this MS/MS spectral library:
• Use the integrated MRM information to build methods
without the need to re-infuse standards and optimize MRM
transitions for a given compound.
• Easily create screening methods for an MRM triggered
EPI workflow for use on QTRAP® LC/MS/MS systems.
• Quickly set-up MRM quantitation methods for traditional
MRM ratio quantitation and confirmation on a QTRAP® or
Triple Quad™ LC/MS/MS system.
• Set-up smaller customer libraries by simply selecting only
the compounds of interest from the list in LibraryView™
Software.
• Use LibraryView™ Software to automatically create your
custom acquisition and quantitation methods.
The following is a list of the antibiotics currently in the library.
This library is verified for use on AB SCIEX QTRAP® LC/MS
MS systems.
Please note that this library is continuously being expanded
to include additional compounds.
Formula Weight
338.3573
324.3738
373.4523
313.5676
338.3573
119.0768
251.285
523.6296
521.6484
1255.436
312.2787
314.2945
328.278
330.2939
328.278
330.2939
238.3328
1964.336
CAS Number
88337-96-6
2623-22-5
199784-50-4
540770-33-0
50722-38-8
504-88-1
6152-51-4
52665-69-7
79367-52-5
50-76-0
1162-65-8
7220-81-7
1165-39-5
7241-98-7
6795-23-9
6885-57-0
548-42-5
27061-78-5
alpha-Zearalenol
C18H24O5
320.3855
36455-72-8
alpha-Zearalenol-4-O-glucoside
C24H34O10
482.5276
135626-94-7
Altenuene
C15H16O6
292.2883
29752-43-0
Altenusin
C15H14O6
290.2725
31186-12-6
Alternariol
C14H10O5
258.2304
641-38-3
Alternariolmethylether
C15H12O5
272.2573
26894-49-5
Altersolanol
C16H16O7
320.2987
22268-16-2
# of Spectra
4
8
0
4
8
4
8
Formula Weight
CAS Number
# of Spectra
352.3435
56258-32-3
4
Altertoxin-II
C20H14O6
350.3276
56257-59-1
Amphotericin B
C47H73NO17
924.0926
1397-89-3
Anisomycin
C14H19NO4
265.3092
22862-76-6
4
Apicidin
C34H49N5O6
623.7934
183506-66-3
8
Ascomycin
C43H69NO12
792.0201
104987-12-4
4
Aspercolorin
C25H28N4O5
464.5215
29123-52-2
8
Aspergillimide
C20H29N3O3
359.4688
195966-93-9
Asperlactone
C9H12O4
184.1917
76375-62-7
Asperloxine A
C21H19N3O5
393.3993
223130-52-7
Aspinonene
C9H16O4
188.2234
157676-96-5
Aspyrone
C9H12O4
184.1917
17398-00-4
Asterric acid
C17H16O8
348.309
577-64-0
Atpenin A5
C15H21Cl2NO5
366.2409
119509-24-9
Aureobasidin A
C60H92N8O11
1101.438
127757-30-6
Aurofusarin
C30H18O12
570.4657
13191-64-5
Austdiol
C12H12O5
236.2242
53043-28-0
Austocystin A
C19H13ClO6
372.7614
55256-58-1
Avenacein Y
C15H10O8
318.2394
102426-44-8
Bacitracin
C66H103N17O16S
1422.713
22601-59-8
Bafilomycin A1
C35H58O9
622.8399
88899-55-2
Beauvericin
C45H57N3O9
783.9626
26048-05-5
beta-Ergocryptine
C32H41N5O5
575.7086
511-09-1
beta-Ergocryptinine
C32H41N5O5
575.7086
511-10-4
beta-Zearalenol
C18H24O5
320.3855
71030-11-0
beta-Zearalenol-4-O-glucoside
C24H34O10
482.5276
135626-93-6
Brefeldin A
C16H24O4
280.3641
20350-15-6
Brevicompanine B
C22H29N3O2
367.4916
215121-47-4
Calphostin C
C44H38O14
790.7773
121263-19-2
Cephalosporin C
C16H21N3O8S
415.4223
61-24-5
Cerulenin
C12H17NO3
223.2719
17397-89-6
Chaetocin
C30H28N6O6S4
696.8471
28097-03-2
Chaetoglobosin A
C32H36N2O5
528.6487
50335-03-0
Chanoclavine
C16H20N2O
256.348
2390-99-0
Chetomin
C31H30O6N6S4
710.874
1403-36-7
Chlamydosporol
C11H14O5
226.229
135063-30-8
Chloramphenicol
C11H12Cl2N2O5
323.1321
56-75-7
5
Chromomycin A3
C57H82O26
1183.261
7059-24-7
0
Citreoviridin
C23H30O6
402.4876
25425-12-1
4
Citrinin
C13H14O5
250.2511
518-75-2
8
Citromycetin
C14H10O7
290.229
478-60-4
4
Cochliodinol
C32H30N2O4
506.6018
11051-88-0
Curvularin
C16H20O5
292.3317
10140-70-2
Cycloaspeptide A
C36H43N5O6
641.7679
109171-13-3
Cycloechinulin
C20H21N3O3
351.4054
143086-29-7
Cycloheximide
C15H23NO4
281.3519
66-81-9
Cyclopenin
C17H14N2O3
294.3101
19553-26-5
Cyclopeptine
C17H16N2O2
280.3266
50886-63-0
Cyclopiazonic acid
C20H20N2O3
336.3908
18172-33-3
Cyclosporin A
C62H111N11O12
1202.63
59865-13-3
0
4
Formula
C20H16O6
8
4
8
4
8
4
8
4
8
4
8
4
8
www.spektrotek.com
Compound
Altertoxin-I
43
www.spektrotek.com
44
Compound
Cyclosporin C
Formula
C62H111N11O13
Formula Weight
1218.63
CAS Number
# of Spectra
59787-61-0
Cyclosporin D
C63H113N11O12
1216.657
63775-96-2
Cyclosporin H
C62H111N11O12
1202.63
83602-39-5
Cytochalasin A
C29H35O5N
477.6009
14110-64-6
Cytochalasin B
C29H37O5N
479.6167
14930-96-2
Cytochalasin C
C30H37O6N
507.6271
22144-76-9
Cytochalasin D
C30H37O6N
507.6271
22144-77-0
Cytochalasin E
C28H33O7N
495.5726
36011-19-5
Cytochalasin H
C30H39NO5
493.6436
53760-19-3
Cytochalasin J
C28H37NO4
451.6063
56144-22-0
Decarestrictine
C10H16O5
216.2338
127393-89-9
4
Dechlorogriseofulvin
C17H18O6
318.3262
3680-32-8
Deepoxy-deoxynivalenol
C15H20O5
280.3207
88054-24-4
7
Deoxybrevianamide E
C21H25N3O2
351.4489
34610-68-9
8
Deoxynivalenol
C15H20O6
296.32
51481-10-8
6
Deoxynivalenol-3-glucoside
C21H30O11
458.4621
131180-21-7
8
Diacetoxyscirpenol
C19H26O7
366.411
2270-40-8
Dihydroergosine
C30H39N5O5
549.6706
7288-61-1
Dihydroergotamine
C33H37N5O5
583.6879
511-12-6
Dihydrolysergol
C16H20N2O
256.348
18051-16-6
Dinactin
C42H68O12
764.9944
20261-85-2
Elymoclavine
C16H18N2O
254.3321
548-43-6
Elymoclavine fructoside
C22H28N2O6
416.4742
12379-50-9
Emodin
C15H10O5
270.2414
518-82-1
Enniatin A
C36H63N3O9
681.9109
144446-20-8
Enniatin A1
C35H61N3O9
667.884
4530-21-6
Enniatin B
C33H57N3O9
639.8302
917-13-5
Enniatin B1
C34H59N3O9
653.8571
19914-20-6
Enniatin B2
C32H55N3O9
625.8033
632-91-7
Enniatin B3
C31H53N3O9
611.7764
864-99-3
Equisetin
C22H31NO4
373.4926
57749-43-6
Ergine
C16H17N3O
267.3309
478-94-4
Erginine
C16H17N3O
267.3309
N/A
Ergocornine
C31H39N5O5
561.6817
564-36-3
Ergocorninine
C31H39N5O5
561.6817
564-37-4
Ergocristine
C35H39N5O5
609.7258
511-08-0
Ergocristinine
C35H39N5O5
609.7258
511-07-9
Ergocryptine
C32H41N5O5
575.7086
511-09-1
Ergocryptinine
C32H41N5O5
575.7086
511-10-4
Ergometrine
C19H23N3O2
325.4109
60-79-7
Ergometrinine
C19H23N3O2
325.4109
479-00-5
Ergosine
C30H37N5O5
547.6548
561-94-4
Ergosinine
C30H37N5O5
547.6548
596-88-3
Ergotamine
C33H35N5O5
581.6721
113-15-5
Ergotaminine
C33H35N5O5
581.6721
639-81-6
4
0
4
8
4
8
4
5
4
Ergovaline
C29H35N5O5
533.6279
2873-38-3
Ergovalinine
C29H35N5O5
533.6279
3263-56-7
0
Erythromycin
C37H67NO13
733.9374
114-07-8
8
Festuclavine
C16H20N2
240.3486
569-26-6
4
Formula Weight
CAS Number
# of Spectra
804.0312
104987-11-3
Fulvic acid
C14H12O8
308.2442
479-66-3
Fumagillin
C26H34O7
458.5517
23110-15-8
Fumigaclavine A
C18H22N2O2
298.3852
6879-59-0
Fumitremorgin C
C22H25N3O3
379.4592
118974-02-0
Fumonisin B1
C34H59NO15
721.8395
116355-83-0
Fumonisin B2
C34H59NO14
705.8402
116355-84-1
Fumonisin B3
C34H59NO14
705.8402
136379-59-4
Fumonisin B4
C34H59NO13
689.8409
136379-60-7
0
Fusaproliferin
C27H40O5
444.6116
152469-17-5
4
Fusarenon-X
C17H22O8
354.3566
23255-69-8
8
Fusaric acid
C10H13NO2
179.2188
536-69-6
0
Fusarielin A
C25H38O4
402.5744
132341-17-5
Fusidic acid
C31H48O6
516.7185
6990/06/03
Geldanamycin
C29H40N2O9
560.6445
30562-34-6
Geodin
C17H12Cl2O7
399.1835
427-63-4
Gibberellic acid
C19H22O6
346.38
1977/06/05
Gliotoxin
C13H14O4N2S2
326.394
67-99-2
Griseofulvin
C17H17O6Cl
352.7711
126-07-8
HC-Toxin
C21H32N4O6
436.5084
83209-65-8
HT-2-Toxin
C22H32O8
424.491
26934-87-2
hydrolyzed Fumonisin B1
C22H47NO5
405.6187
145040-09-1
Ionomycin
C41H72O9
709.0171
56092-82-1
K252a
C27H21N3O5
467.4813
97161-97-2
K252b
C26H19N3O5
453.4545
99570-78-2
Kojic acid
C6H6O4
142.1111
501-30-4
Lincomycin
C18H34N2O6S
406.542
154-21-2
Lolitrem B
C42H55NO7
685.9015
81771-19-9
8
Lysergol
C16H18N2O
254.3321
602-85-7
4
Macrosporin
C16H12O5
284.2683
22225-67-8
8
Malformin C
C23H39N5O5S2
529.7222
59926-78-2
Marcfortine A
C28H35N3O4
477.604
75731-43-0
Meleagrin
C23H23N5O4
433.4673
71751-77-4
Methysergide
C21H27N3O2
353.4647
361-37-5
Mevastatin
C23H34O5
390.5199
73573-88-3
Mevinolin
C24H36O5
404.5468
75330-75-5
Mithramycin
C52H76O24
1085.16
18378-89-7
Mitomycin C
C15H18N4O5
334.3319
1950/07/07
Monactin
C41H66O12
750.9675
7182-54-9
4
Moniliformin
C4H2O3
98.05797
71376-34-6
0
Mycophenolic acid
C17H20O6
320.3421
24280-93-1
7
Myriocin
C21H39NO6
401.5436
35891-70-4
Neosolaniol
C19H26O8
382.4104
36519-25-2
Neoxaline
C23H25N5O4
435.4831
71812-10-7
NG012
C32H38O15
662.6442
141731-76-2
Nidulin
C20H17Cl3O5
443.7103
10089-10-8
Nigericin
C40H68O11
724.973
28643-80-3
Nivalenol
C15H20O7
312.3193
23282-20-4
8
Nonactin
C40H64O12
736.9406
6833-84-7
4
8
4
8
Formula
C44H69NO12
4
8
4
8
4
8
4
4
8
4
4
8
4
www.spektrotek.com
Compound
FK 506
45
www.spektrotek.com
46
Compound
Formula
Formula Weight
CAS Number
# of Spectra
Nornidulin
C19H15Cl3O5
429.6834
33403-37-1
4
Ochratoxin A
C20H18NO6Cl
403.8188
303-47-9
8
Ochratoxin alpha
C11H9ClO5
256.6421
19165-63-0
7
Ochratoxin B
C20H19NO6
369.374
4825-86-9
Oligomycin A
C45H74O11
791.0757
579-13-5
Oligomycin B
C45H72O12
805.0592
11050-94-5
O-Methylsterigmatocystin
C19H14O6
338.3166
17878-69-2
Ophiobolin A
C25H36O4
400.5585
4611/05/06
Ophiobolin B
C25H38O4
402.5744
5601-74-1
Oxaspirodion
C13H14O5
250.2511
774538-95-3
oxidized Elymoclavine
N/A
N/A
N/A
8
4
8
4
oxidized Luol
N/A
N/A
N/A
Paraherquamide A
C28H35N3O5
493.6033
77392-58-6
6
Paspaline
C28H39NO2
421.6235
11024-56-9
4
Paspalinine
C27H31NO4
433.5478
63722-91-8
8
Paspalitrem A
C32H39NO4
501.6663
63722-90-7
Paspalitrem B
C32H39NO5
517.6657
63764-58-9
Patulin
C7H6O4
154.1221
149-29-1
0
Paxilline
C27H33NO4
435.5636
57186-25-1
8
Penicillic acid
C8H10O4
170.1649
90-65-3
Penicillin G
C16H18O4N2S
334.3945
61-33-6
4
4
Penicillin V
C16H18N2O5S
350.3938
1987/08/01
Penigequinolone A
C27H33NO6
467.5623
180045-91-4
Penitrem A
C37H44O6NCl
634.2125
12627-35-9
Pentoxyfylline
C13H18N4O3
278.3112
6493/05/06
Pestalotin
C11H18O4
214.2614
34565-32-7
Phomopsin A
C36H45ClN6O12
789.2392
64925-80-0
6
Phomopsin B
C36H46N6O12
754.7944
64925-81-1
0
Physcion
C16H12O5
284.2683
521-61-9
8
Pseurotin A
C22H25NO8
431.4423
58523-30-1
7
Puromycin
C22H29N7O5
471.5166
53-79-2
Pyrenophorol
C16H24O6
312.3628
22248-41-5
Pyripyropene A
C31H37NO10
583.6354
147444-03-9
Radicicol
C18H17ClO6
364.7821
12772-57-5
Rapamycin
C51H79NO13
914.187
53123-88-9
Roquefortine C
C22H23N5O2
389.4576
58735-64-1
Roridin A
C29H40O9
532.631
14729-29-4
Rubellin D
C30H22O10
542.4987
121325-49-3
Rugulosin
C30H22O10
542.4987
23537-16-8
Satratoxin G
C29H36O10
544.5986
53126-63-9
Satratoxin H
C29H36O9
528.5993
53126-64-0
Secalonic acid
C32H30O14
638.5815
56283-72-8
Setosusin
C29H38O8
514.6158
182926-45-0
Stachybotrylactam
C23H31NO4
385.5036
163391-76-2
Staurosporine
C28H26N4O3
466.5401
62996-74-1
Sterigmatocystin
C18H12O6
324.2897
10048-13-2
Sulochrin
C17H16O7
332.3097
519-57-3
T2-Tetraol
C15H22O6
298.3359
34114-99-3
T2-Toxin
C24H34O9
466.5283
21259-20-1
T2-Triol
C20H30O7
382.4538
34114-98-2
8
4
4
8
4
8
0
8
4
8
4
Formula Weight
CAS Number
# of Spectra
853.9203
33069-62-4
Tentoxin
C22H30N4O4
414.5049
28540-82-1
Tenuazonic acid
C10H15O3N
197.234
610-88-8
Terphenyllin
C20H18O5
338.36
52452-60-5
Territrem B
C29H34O9
526.5835
70407-20-4
4
Tetracycline
C22H24N2O8
444.4412
64-75-5
9
Thiolutin
C8H8N2O2S2
228.2927
1987/11/06
Trichodermin
C17H24O4
292.3751
4682-50-2
Trichostatin A
C17H22N2O3
302.3735
58880-19-6
Tryprostatin A
C22H27N3O3
381.4751
171864-80-5
Ustiloxin A
C28H43N5O12S
673.7399
143557-93-1
Ustiloxin B
C26H39N5O12S
645.6862
151841-41-7
Ustiloxin D
C23H34N4O8
494.545
158243-18-6
Valinomycin
C54H90N6O18
1111.338
2001-95-8
Vancomycin
C66H75Cl2N9O24
1449.273
1404-93-9
Verrucarin A
C27H34O9
502.5614
3148/09/02
Verrucarol
C15H22O4
266.3372
2198-92-7
Verrucofortine
C24H31N3O3
409.5288
113706-21-1
Verruculogen
C27H33O7N3
511.5751
12771-72-1
Viomellein
C30H24O11
560.5139
55625-78-0
Viridicatin
C15H11NO2
237.2581
129-24-8
Wortmannin
C23H24O8
428.4387
19545-26-7
Zearalenone
C18H22O5
318.3697
17924-92-4
Zearalenone-4-glucoside
C24H32O10
480.5118
105088-14-0
Zearalenone-4-sulfate
C18H22O8S
398.432
132505-04-5
Product Name
Part Number
Mycotoxin Spectral Library Version 1.1
5023887
For Research Use Only. Not for use in diagnostic procedures.
© 2012 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or
their respective owners. AB SCIEX™ is being used under license.
Publication number: 6860212-01
8
4
Formula
C47H51NO14
8
4
8
4
8
4
www.spektrotek.com
Compound
Taxol
47
Pesticide LC/MS/MS Library Version 1.0 for Cliquid® Software
iMethods™ Test Pesticides Library Version 1.0 for Cliquid® Software
The following description outlines the 603 pesticide MRM
catalogue and 544 pesticide spectral library that is available
for use with AB SCIEX API or QTRAP® LC/MS/MS systems
and Cliquid® Software. This library and MRM catalogue,
created using certified reference materials, can be used alone
to create custom screening and/or quantitation methods or in
conjunction with the iMethod™ test for pesticide screening.
The iMethod™ test for pesticide screening is sold separately
and provides a pre-configured test for the screening of 494
pesticides from vegetable, nut and citrus plant samples
using the QuEChERS extraction and cleanup technique. This
library is verified for use on AB SCIEX 3200 QTRAP®, 4000
QTRAP® and AB SCIEX QTRAP® 5500 LC/MS/MS Systems.
www.spektrotek.com
The MRM catalogue and spectral library contain information
on the most common 603 pesticides and their metabolites
that need to be monitored in vegetables or other food
products. The MRM catalogue contains up to three transitions
per compound. Each compound in the library has individual
spectra acquired using three distinct collision energies (20
eV, 35 eV, 50 eV), as well as a single spectra representing
48
Compound
2.4-D
2.4-DB
2-Naphthyloxyacetic acid
3,4,5-Trimethacarb
3-Hydroxycarbofuran
4-CPA
5-Hydroxy-clethodim-sulfone
5-Hydroxy-imidacloprid
5-Hydroxy-thiabendazol
6-Chlor-3-phenyl-pyridazin-4-ol (Pyridate-Metabolit)
Acephate
Acequinocyl
Acetamiprid
Acetochlor
Acibenzolar-S-methyl
Formula
C8H6Cl2O3
C10H10Cl2O3
C12H10O3
C11H15NO2
C12H15NO4
C8H7ClO3
C17H26ClNO6S
C9H10ClN5O3
C10H7N3OS
C10H7ClN2O
C4H10NO3PS
C24H32O4
C10H11ClN4
C14H20ClNO2
C8H6N2OS2
the sum of all three collision energies. If compounds ionize
in both polarities, spectra for both are included, bringing the
potential total number of spectra per compound to eight.
The MRM catalogue can be used to build methods without the
need to re-infuse standards and optimize MRM transitions for
a given compound. Screening and/or quantitation methods
can be created for use with an MRM triggered EPI workflow,
for use on QTRAP® instruments, or for traditional quantitation
where the ratio of the response of two or more transitions is
used for compound confirmation. The latter MRM approach
can be performed on either an API triple quadrupole or a
QTRAP® series instrument. Users simply need to select the
compounds of interest as well as the number of transitions
to be monitored from the MRM catalogue. Once selected, the
Cliquid® Software automatically creates the acquisition and
processing methods.
The following is a list of the 544 pesticides currently in the
library. Please note that this library is continuously being
expanded to include additional compounds.
Formula Weight
219.9694
248.0007
202.063
193.1103
237.1001
186.0084
407.1169
271.0472
217.031
206.0247
183
384.2301
222.0672
269.1183
209.9922
CAS Number
94-75-7
94-82- 6
120-23-0
2686-99-9
16655-82-6
122-88 -3
not available
not available
948-71-0
40020-01-7
40020-01-7
57960-19 -7
135410-2 0-7
34256-82 -1
135158-54-2
# of Spectra
4
Acifluorfen
C14H7ClF3NO5
360.9965
50594-66 -6
Aclonifen
Acrinathrin
Alachlor
Aldicarb-sulfoxid
C12H9ClN2O3
C26H21F6NO5
C14H20ClNO2
C7H14N2O3S
264.0302
541.1324
269.1183
206.0725
74070-46 -5
101007-0 6-1
15972-60 -8
1646-87-3
4
1
Formula Weight
CAS Number
222.0674
1646-88-4
Alloxydim
C17H25NO5
323.1733
55634-91-8
Ametryn
C9H17N5S
227.1205
834-12-8
Amidosulfuron
C9H15N5O7S2
369.0413
120923-37-7
Aminocarb
C11H16N2O2
208.1212
2032-59-9
Aminopyralid
C6H4Cl2N2O2
205.965
150114-71-9
Amitraz
C19H23N3
293.1892
33089-61-1
Amitrol
C2H4N4
84.0436
61-82-5
AMPA (Aminomethyl phosphonic acid)
CH6O3PN
111.0085
1066-51-9
Anilazine
C9H5Cl3N4
273.958
101-05-3
Anilofos
C13H19ClNO3PS2
367.0232
64249-01-0
Aramite
C15H23ClO4S
334.1006
140-57-8
Atrazin
C8H14ClN5
215.0938
1912-24-9
Atrazine-2-hydroxy
C8H15N5O
197.1277
2163-68-0
Atrazine-desethyl
C6H10N5Cl
187.0625
6190-65-4
Atrazine-desethyl-2-hydroxy
C6H11ON5
169.0964
6190-65-4
Atrazine-desisopropyl
C5H8N5Cl
173.0468
1007-28-9
Avermectin B1a
C48H72O14
872.4922
65195-55-3
Azaconazole
C12H11Cl2N3O2
299.0228
60207-31-0
Azamethiophos
C9H10ClN2O5PS
323.9737
35575-96-3
Azimsulfuron
C13H16N10O5S
424.1026
120162-55-2
Azinphos-ethyl
C12H16N3O3PS2
345.0371
2642-71-9
Azinphos-methyl
C10H12N3O3PS2
317.0058
86-50-0
Azoxystrobin
C22H17N3O5
403.1168
131860-33-8
Beflubutamid
C18H17F4NO2
355.1195
113614-08-7
Benalaxyl
C20H23NO3
325.1678
71626-11-4
Benazolin
C9H6ClNO3S
242.9757
5/6/3813
Bendiocarb
C11H13NO4
223.0845
22781-23-3
Benfuracarb
C20H30N2O5S
410.1875
82560-54-1
Benfuresate
C12H16O4S
256.0769
68505-69-1
Benomyl
C14H18N4O3
290.1379
17804-35-2
Benoxacor
C11H11Cl2NO2
259.0167
98730-04-2
Bensulfuron-methyl
C16H18N4O7S
410.0896
83055-99-6
Bensulide
C14H24NO4PS3
397.0605
741-58-2
Bensultap
C17H21NO4S4
431.0353
17606-31-4
Bentazone
C10H12N2O3S
240.0569
25057-89-0
Benzoximate
C18H18ClNO5
363.0874
29104-30-1
Bifenazate
C17H20N2O3
300.1474
149877-41-8
Bifenox
C14H9Cl2NO5
340.9858
42576-02-3
Bifenthrin
C23H22ClF3O2
422.126
82657-04-3
Bioresmethrin
C22H26O3
338.1882
28434-01-7
Bitertanol
C20H23N3O2
337.179
55179-31-2
Boscalid
C18H12Cl2N2O
342.0327
188425-85-6
Brodifacoum
C31H23BrO3
522.083
56073-10-0
Bromacil
C9H13BrN2O2
260.016
314-40-9
Bromadiolone
C30H23BrO4
526.078
28772-56-7
Bromophos
C8H8BrCl2O3PS
363.8492
2104-96-3
Bromophos-ethyl
C10H12BrCl2O3PS
391.8805
4824-78-6
Bromoxynil
C7H3Br2NO
274.8581
1689-84-5
Bromuconazole
C13H12BrCl2N3O
374.9
116255-48-2
Bupirimate
C13H24N4O3S
316.1569
41483-43-6
# of Spectra
Formula
C7H14N2O4S
4
www.spektrotek.com
Compound
Aldoxycarb
49
www.spektrotek.com
50
Compound
Buprofezin
Formula
C16H23N3OS
Formula Weight
305.1562
CAS Number
# of Spectra
69327-76-0
Butafenacil
C20H18ClF3N2O6
474.0805
134605-64-4
Butocarboxim-sulfoxid
C7H14N2O3S
206.0725
34681-24-8
Butoxycarboxim
C7H14N2O4S
222.0674
34681-23-7
Butralin
C14H21N3O4
295.1532
33629-47-9
Buturon
C12H13ClN2O
236.0716
3766-60-7
Butylate
C11H23NOS
217.15
2008-41-5
Cadusafos
C10H23O2PS2
270.0877
95465-99-9
Carbaryl
C12H11NO2
201.079
63-25-2
Carbendazim
C9H9N3O2
191.0695
10605-21-7
Carbetamide
C12H16N2O3
236.1161
16118-49-3
Carbofuran
C12H15NO3
221.1052
1563-66-2
Carbosulfan
C20H32N2O3S
380.2134
55285-14-8
Carboxin
C12H13NO2S
235.0667
5234-68-4
Carfentrazone-ethyl
C15H14Cl2F3N3O3
411.0364
128639-02-1
Carpropamid
C15H18Cl3NO
333.0454
104030-54-8
Cartap
C7H15N3O2S2
237.0606
15263-52-2
Chinomethionate
C10H6N2OS2
233.9922
1/2/2439
Chlorbromuron
C9H10BrClN2O2
291.9614
13360-45-7
Chlorbufam
C11H10ClNO2
223.04
1967-16-4
Chlorethoxyfos-oxon
C6H11Cl4O4P
317.9149
54593-83-8
Chlorfenvinphos
C12H14Cl3O4P
357.9695
470-90-6
Chlorfluazuron
C20H9Cl3F5N3O3
538.963
71422-67-8
Chloridazon
C10H8ClN3O
221.0356
1698-60-8
Chlorimuron-ethyl
C15H15ClN4O6S
414.0401
90982-32-4
Chlormephos
C5H12ClO2PS2
233.9705
24934-91-6
Chlorophacinone
C23H15ClO3
374.071
3691-35-8
Chlorotoluron
C10H13ClN2O
212.0716
15545-48-9
Chloroxuron
C15H15ClN2O2
290.0822
1982-47-4
Chlorpropham
C10H12ClNO2
213.0557
101-21-3
Chlorpyrifos
C9H11Cl3NO3PS
348.9263
2921-88-2
Chlorpyrifos-methyl
C7H7Cl3NO3PS
320.895
5598-13-0
Chlorsulfuron
C12H12CIN5O4S
357.0299
64902-72-3
Chlorthiamid
C7H5Cl2NS
204.952
1918-13-4
Chlorthiophos
C11H15Cl2O3PS2
359.9
60238-56-4
Cinerin I
C20H28O3
316.2038
25402-06-6
Cinerin II
C21H28O5
360.1937
121-20-0
Cinidon-ethyl
C19H17Cl2NO4
393.0535
142891-20-1
Cinosulfuron
C15H19N5O7S
413.1005
94593-91-6
Clethodim
C17H26ClNO3S
359.1322
99129-21-2
Clethodim-imin-sulfone
C14H23NO4S
301.1348
not available
Clethodim-imin-sulfoxide
C14H23NO3S
285.1399
not available
Clethodim-sulfone
C17H26ClNO5S
391.122
not available
Clethodim-sulfoxide
C17H26ClNO4S
375.1271
not available
Clodinafop-propargyl
C17H13ClFNO4
349.0517
105512-06-9
Clofentezine
C14H8Cl2N4
302.0126
74115-24-5
Clomazone
C12H14ClNO2
239.0713
81777-89-1
Clomeprop
C16H15Cl2NO2
323.048
84496-56-0
Clopyralid
C6H3Cl2NO2
190.9541
1702-17-6
Cloquintocet-mexyl
C18H22ClNO3
335.1288
99607-70-2
Clothianidin
C6H8ClN5O2S
249.0087
210880-92-5
4
Formula Weight
CAS Number
362.0145
56-72-4
Coumatetralyl
C19H16O3
292.1099
5836-29-3
Crotoxyphos
C14H19O6P
314.0919
7700-17-6
Cyanazine
C9H13ClN6
240.089
21725-46-2
Cyanofenphos
C15H14NO2PS
303.0483
13067-93-1
Cyanophos
C9H10NO3PS
243.0119
2636-26-2
Cyazofamid
C13H13ClN4O2S
324.0448
120116-88-3
Cyclanilide
C11H9Cl2NO3
272.9959
113136--77-9
Cycloate
C11H21NOS
215.1344
1134-23-2
Cycloxydim
C17H27NO3S
325.1712
101205-02-1
Cyfluthrin
C22H18Cl2FNO3
433.0648
68359-37-5
Cyhalofop-butyl
C20H20FNO4
357.1376
122008-85-9
Cymoxanil
C7H10N4O3
198.0753
57966-95-7
Cypermethrin
C22H19Cl2NO3
415.0742
52315-07-8
Cyphenothrin
C24H25NO3
375.1834
39515-40-7
Cyproconazole
C15H18ClN3O
291.1138
113096-99-4
Cyprodinil
C14H15N3
225.1266
121552-61-2
Cyromazine
C6H10N6
166.0967
66215-27-8
Daminozide
C6H12N2O3
160.0848
1596-84-5
Deltamethrin
C22H19Br2NO3
502.9732
52918-63-5
Demeton-S-methyl
C6H15O3PS2
230.02
919-86-8
Demeton-S-methyl-sulfon
C6H15O5PS2
262.0098
17040-19-6
Desmedipham
C16H16N2O4
300.111
13684-56-5
Desmethyl-formamido-pirimicarb
C11H16N4O3
252.1222
59333-83-4
Desmethyl-pirimicarb
C10H16N4O2
224.1273
30614-22-3
Desmetryne
C8H15N5S
213.1048
1014-69-3
Dialifos
C14H17ClNO4PS2
393.0025
10311-84-9
Di-allate
C10H17Cl2NOS
269.0408
2303-16-4
Diazinon
C12H21N2O3PS
304.101
333-41-5
Dichlofenthion
C10H13Cl2O3PS
313.97
97-17-6
Dichlofluanid
C9H11Cl2FN2O2S2
331.9623
1085-98-9
Dichlorprop-P
C9H8Cl2O3
233.985
15165-67-0
Dichlorvos
C4H7Cl2O4P
219.9459
62-73-7
Diclobutrazol
C15H19Cl2N3O
327.0905
75736-33-3
Diclofop-methyl
C16H14Cl2O4
340.0269
51338-27-3
Dicloran
C6H4Cl2N2O2
205.965
99-30-9
Diclosulam
C13H10Cl2FN5O3S
404.9865
145701-21-9
Dicrotophos
C8H16NO5P
237.0766
3735-78-3
Dicyclanil
C8H10N6
190.0967
112636-83-6
Diethofencarb
C14H21NO4
267.1471
87130-20-9
Difenacoum
C31H24O3
444.1725
56073-07-5
Difenoconazole
C19H17Cl2N3O3
405
119446-68-3
Difenoxuron
C16H18N2O3
286.1317
14214-32-5
Difenzoquat
C17H17N2
249.1392
49866-87-7
Diflubenzuron
C14H9ClF2N2O2
310.0321
35367-38-5
Diflufenican
C19H11F5N2O2
394.0741
83164-33-4
Diflufenzopyr
C15H12F2N4O3
334.0877
109293-97-2
Dimefuron
C15H19ClN4O3
338.1146
34205-21-5
Dimepiperate
C15H21NOS
263.1344
61432-55-1
Dimethachlor
C13H18ClNO2
255.1026
50563-36-5
Dimethametryn
C11H21N5S
255.1518
22936-75-0
# of Spectra
Formula
C14H16ClO5PS
4
www.spektrotek.com
Compound
Coumaphos
51
www.spektrotek.com
52
Compound
Dimethenamide
Formula
C12H18ClNO2S
Formula Weight
275.0747
CAS Number
# of Spectra
87674-68-8
Dimethoate
C5H12NO3PS2
228.9996
60-51-5
Dimethomorph
C21H22ClNO4
387.1237
110488-70-5
Dimetilan
C10H16N4O3
240.1222
644-64-4
Dimoxystrobin
C19H22N2O3
326.163
149961-52-4
Diniconazole
C15H17Cl2N3O
325.0749
83657-24-3
Dinoseb
C10H12N2O5
240.0746
88-85-7
Dinoterb
C10H12N2O5
240.0746
1420-07-1
Dioxathion
C12H26O6P2S4
456.0087
78-34-2
Diphacinone
C23H16O3
340.1099
82-66-6
Diphenamid
C16H17NO
239.131
957-51-7
Diphenylamine
C12H11N
169.0891
122-39-4
Diquat
C12H12N2
184.1
2764-72-9
Dithianon
C14H4N2O2S2
295.9714
3347-22-6
Dithiopyr
C15H16F5NO2S2
401.0543
97886-45-8
Diuron
C9H10Cl2N2O
232.017
330-54-1
DNOC
C7H6N2O5
198.0277
534-52-1
Dodemorph
C18H35NO
281.2719
1593-77-7
Dodine
C15H33N3O2
287.2573
10/3/2439
Edifenphos
C14H15O2PS2
310.0251
17109-49-8
Endosulfansulfate
C9H6Cl6O4S
419.8118
1031-07-8
EPN
C14H14NO4PS
323.0381
2104-64-5
Epoxiconazole
C17H13ClFN3O
329.0731
106325-08-0
EPTC
C9H19NOS
189.1187
759-94-4
Esfenvalerate
C25H22ClNO3
419.1288
66230-04-4
Ethametsulfuron-methyl
C15H18N6O6S
410.1009
97780-06-8
Ethidimuron
C7H12N4O3S2
264.0351
30043-49-3
Ethiofencarb
C11H15NO2S
225.0823
29973-13-5
Ethiofencarb-sulfon
C11H15NO4S
257.0722
53380-23-7
Ethiofencarb-sulfoxid
C11H15NO3S
241.0773
53380-22-6
Ethion
C9H22O4P2S4
383.9876
563-12-2
Ethirimol
C11H19N3O
209.1528
23947-60-6
Ethofumesate
C13H18O5S
286.0875
26225-79-6
Ethoprophos
C8H19O2PS2
242.0564
13194-48-4
Ethoxyquin
C14H19NO
217.1467
91-53-2
Ethoxysulfuron
C15H18N4O7S
398.0896
126801-58-9
Ethylenthiourea
C3H6N2S
102.0252
96-45-7
Etofenprox
C25H28O3
376.2038
80844-07-1
Etoxazole
C2H23F2NO2
359.1697
153233-91-1
Etrimfos
C10H17N2O4PS
292.0647
38260-54-7
Famoxadone
C22H18N2O4
374.1267
131807-57-3
Famphur
C10H16NO5PS2
325.0207
52-85-7
Fenamidone
C17H17N3OS
311.1092
161326-34-7
Fenamiphos
C13H22NO3PS
303.1058
22224-92-6
Fenarimol
C17H12Cl2N2O
330.0327
60168-88-9
Fenazaquin
C20H22N2O
306.1732
120928-09-8
Fenbuconazole
C19H17ClN4
336.1142
114369-43-6
Fenfuram
C12H11NO2
201.079
24691-80-3
Fenhexamid
C14H17Cl2NO2
301.0636
126833-17-8
Fenitrothion
C9H12NO5PS
277.0174
122-14-5
Fenobucarb
C12H17NO2
207.1259
3766-81-2
4
Formula Weight
CAS Number
267.9461
93-72-1
Fenothiocarb
C13H19NO2S
253.1136
62850-32-2
Fenoxaprop-ethyl
C18H16ClNO5
361.0717
71283-80-2
Fenoxycarb
C17H19NO4
301.1314
79127-80-3
Fenpiclonil
C11H6Cl2N2
235.9908
74738-17-3
Fenpropathrin
C22H23NO3
349.1678
39515-41-8
Fenpropidin
C19H31N
273.2456
67306-00-7
Fenpropimorph
C20H33NO
303.2562
67306-03-0
Fenpyroximate
C24H27N3O4
421.2002
111812-58-9
Fenthion
C10H15O3PS2
278.02
55-38-9
Fentin
C18H15Sn
351.0196
668-34-8
Fenuron
C9H12N2O
164.095
101-42-8
Fenvalerate
C25H22ClNO3
419.1288
51630-58-1
Fipronil
C12H4Cl2F6N4OS
435.9387
120068-37-3
Fipronil-desulfinyl
C12H4Cl2F6N4
387.9717
205650-65-3
Fipronil-sulfide
C12H4Cl2F6N4S
419.9438
120067-83-6
Fipronil-sulfone
C12H4Cl2F6N4O2S
451.9336
120068-36-2
Flamprop-M-isopropyl
C19H19ClFNO3
363.1037
63782-90-1
Flamprop-M-methyl
C17H15ClFNO3
335.0724
63729-98-6
Flazasulfuron
C13H12F3N5O5S
407.0511
104040-78-0
Florasulam
C12H8F3N5O3S
359.03
145701-23-1
Fluazifop (free acid)
C15H12F3NO4
327.0718
83066-88-0
Fluazifop-butyl
C19H20F3NO4
383.1344
69806-50-4
Fluazinam
C13H4Cl2F6N4O4
463.9514
79622-59-6
Flucycloxuron
C25H20ClF2N3O3
483.1161
113036-88-7
Flucythrinate
C26H23F2NO4
451.1595
70124-77-5
Fludioxonil
C12H6F2N2O2
248.0397
131341-86-1
Flufenacet
C14H13F4N3O2S
363.0665
142459-58-3
Flufenoxuron
C21H11ClF6N2O3
488.0362
101463-69-8
Flumetsulam
C12H9F2N5O2S
325.0445
98967-40-9
Flumioxazin
C19H15FN2O4
354.1016
103361-09-7
Fluometuron
C10H11F3N2O
232.0823
2164-17-2
Fluoroglycofene-ethyl
C18H13ClF3NO7
447.0333
77501-90-7
Fluoxastrobin
C21H16ClFN4O5
458.0793
361377-29-9
Flupyrsulfuron-methyl
C15H14F3N5O7S
465.0566
144740-54-5
Fluquinconazole
C16H8Cl2FN5O
375.009
136426-54-5
Flurenol
C14H10O3
226.063
467-69-6
Fluridone
C19H14F3NO
329.1027
59756-60-4
Flurochloridone
C12H10Cl2F3NO
311.0092
61213-25-0
Fluroxypyr
C7H5Cl2FN2O3
253.9661
69377-81-7
Fluroxypyr-meptyl
C15H21Cl2FN2O3
366.0913
81406-37-3
Flurprimidole
C15H15F3N2O2
312.1086
56425-91-3
Flurtamone
C18H14F3NO2
333.0977
96525-23-4
Flusilazole
C16H15F2N3Si
315.1003
85509-19-9
Flusulfamide
C13H7Cl2F3N2O4S
413.9456
106917-52-6
Fluthiacet-methyl
C15H15ClFN3o3S2
403.0227
117337-19-6
Flutolanil
C17H16F3NO2
323.1133
66332-96-5
Flutriafol
C16H13F2N3O
301.1027
76674-21-0
Fluxofenim
C12H11ClF3NO3
309.038
88485-37-4
Fomesafen
C15H10ClF3N2O6S
437.99
72178-02-0
Fonofos
C10H15OPS2
246.0302
944-22-9
# of Spectra
Formula
C9H7Cl3O3
4
www.spektrotek.com
Compound
Fenoprop
53
www.spektrotek.com
54
Compound
Foramsulfuron
Formula
C17H20N6O7S
Formula Weight
452.1114
CAS Number
# of Spectra
173159-57-4
Formetanate
C11H15N3O2
221.1164
22259-30-9
Fosthiazate
C9H18NO3PS2
283.0466
98886-44-3
Fuberidazole
C11H8N2O
184.0637
3878-19-1
Furalaxyl
C17H19NO4
301.1314
57646-30-7
Furathiocarb
C18H26N2O5S
382.1562
65907-30-4
Glufosinate
C5H12NO4P
181.0504
77182-82-2
Halfenprox
C24H23BrF2O3
476.0799
111872-58-3
Halofenozide
C18H19ClN2O2
330.1135
112226-61-6
Halosulfuron-methyl
C13H15ClN6O7S
434.0411
100784-20-1
Haloxyfop-etotyl
C19H19ClF3NO5
433.0904
87237-48-7
Haloxyfop-P
C15H11ClF3NO4
361.0329
95977-29-0
Haloxyfop-P-methyl
C16H13ClF3NO4
375.0485
72619-32-0
Heptenophos
C9H12ClO4P
250.0162
23560-59-0
Hexaconazole
C14H17Cl2N3O
313.0749
79983-71-4
Hexaflumuron
C16H8Cl2F6N2O3
459.9816
86479-06-3
Hexazinone
C12H20N4O2
252.1586
51235-04-2
Hexythiazox
C17H21ClN2O2S
352.1012
78587-05-0
Hydramethylnon
C25H24F6N4
494.1905
67485-29-4
Imazalil
C14H14Cl2N2O
296.0483
35554-44-0
Imazamethabenz-methyl
C16H20N2O3
288.1474
81405-85-8
Imazapic
C14H17N3O3
275.127
104098-48-8
Imazapyr
C13H15N3O3
261.1113
81334-34-1
Imazaquin
C17H17N3O3
311.127
81335-37-7
Imazosulfuron
C14H13ClN6O5S
412.0357
122548-33-8
Imibenconazole
C17H13Cl3N4S
409.9926
86598-92-7
Imidacloprid
C9H10ClN5O2
255.0523
138261-41-3
Imidacloprid-Olefin
C9H8ClN5O2
253.0367
not available
Indoxacarb
C22H17ClF3N3O7
527.0707
173584-44-6
Iodosulfuron-methyl-sodium
C14H13IN5NaO6S
506.971
185119-76-0
Ioxynil
C7H3I2NO
370.8304
1689-83-4
Iprobenfos
C13H21O3PS
288.0949
26087-47-8
Iprodione
C13H13Cl2N3O3
329.0334
36734-19-7
Iprovalicarb
C18H28N2O3
320.21
140923-17-7
Isazofos
C9H17ClN3O3PS
313.0417
42509-80-8
Isofenphos
C15H24NO4PS
345.1164
25311-71-1
Isofenphos-oxon
C15H24NO5P
329.1392
31120-85-1
Isoprocarb
C11H15NO2
193.1103
2631-40-5
Isoprothiolane
C12H18O4S2
290.0646
50512-35-1
Isoproturon
C12H18N2O
206.1419
34123-59-6
Isoxaben
C18H24N2O4
332.1736
82558-50-7
Isoxadifen-ethyl
C18H17NO3
295.1208
163520-33-0
Isoxaflutole
C15H12F3NO4S
359.0439
141112-29-0
Isoxathion
C13H16NO4PS
313.0538
18854-01-8
Jasmolin I
C21H30O3
330.2195
4466-14-2
Jasmolin II
C22H30O5
374.2093
1172-63-0
Kresoxim-methyl
C18H19NO4
313.1314
143390-89-0
lambda-Cyhalothrin
C23H19ClF3NO3
449.1006
91465-08-6
Lenacil
C13H18N2O2
234.1368
8/1/2164
Linuron
C9H10Cl2N2O2
248.0119
330-55-2
Lufenuron
C17H8Cl2F8N2O3
509.9784
103055-07-8
4
Formula Weight
CAS Number
314.0589
1634-78-2
Malathion
C10H19O6PS2
330.0361
121-75-5
Maleic hydrazide
C4H4N2O2
112.0273
123-33-1
MCPA
C9H9ClO3
200.024
94-74-6
MCPA-2-Ethylhexylester
C17H25ClO3
312.1492
29450-45-1
MCPA-butotyl
C14H21ClO4
300.1128
19480-43-4
MCPB
C11H13ClO3
228.0553
94-81-5
Mecarbam
C10H20NO5PS2
329.052
2595-54-2
Mecoprop-P
C10H11ClO3
214.0397
16484-77-8
Mefenacet
C16H14N2O2S
298.0776
73250-68-7
Mefenpyr-diethyl
C16H18Cl2N2O4
372.0644
135590-91-9
Mepanipyrim
C14H13N3
223.1109
110235-47-7
Mepiquat
C7H16N
114.1283
24307-26-4
Mepronil
C17H19NO2
269.1416
55814-41-0
Mesosulfuron-methyl
C17H21N5O9S2
503.0781
208465-21-8
Mesotrione
C14H13NO7S
339.0413
104206-82-8
Metalaxyl
C15H21NO4
279.1471
70630-17-0
Metamitron
C10H10N4O
202.0855
41394-05-2
Metazachlor
C14H16ClN3O
277.0982
67129-08-2
Metconazole
C17H22ClN3O
319.1451
125116-23-6
Methabenzthiazuron
C10H11N3OS
221.0623
18691-97-9
Methacrifos
C7H13O5PS
240.0221
30864-28-9
Methamidophos
C2H8NO2PS
141.0013
10265-92-6
Methfuroxam
C14H15NO2
229.1103
28730-17-8
Methidathion
C6H11N2O4PS3
301.9619
950-37-8
Methiocarb
C11H15NO2S
225.0823
2032-65-7
Methiocarb-sulfoxid
C11H15NO3S
241.0773
10/1/2635
Methomyl
C5H10N2O2S
162.0463
16752-77-5
Methomyl-oxime
C3H7NOS
105.0248
13749-94-5
Methoxyfenozide
C22H28N2O3
368.21
161050-58-4
Metobromuron
C9H11BrN2O2
258.0004
3060-89-7
Metolachlor
C15H22ClNO2
283.1339
51218-45-2
Metolcarb
C9H11NO2
165.079
1129-41-5
Metosulam
C14H13Cl2N5O4S
417.0065
139528-85-1
Metoxuron
C10H13ClN2O2
228.0666
19937-59-8
Metrafenone
C19H21BrO5
408.0572
220899-03-6
Metribuzin
C8H14N4OS
214.0888
21087-64-9
Metsulfuron-methyl
C14H15N5O6S
381.0743
74223-64-6
Mevinphos
C7H13O6P
224.045
7786-34-7
Molinate
C9H17NOS
187.1031
2212-67-1
Monocrotophos
C7H14NO5P
223.061
6923-22-4
Monolinuron
C9H11ClN2O2
214.0509
1746-81-2
Monuron
C9H11ClN2O
198.056
150-68-5
Myclobutanil
C15H17ClN4
288.1142
88671-89-0
Naled
C4H7Br2Cl2O4P
377.7826
300-76-5
Napropamide
C17H21NO2
271.1572
15299-99-7
Neburon
C12H16Cl2N2O
274.064
555-37-3
Nicarbazin (1,3- N,N’-bis (4-nitrophenyl)urea)
C13H10N4O5
302.0651
330-95-0
Nicosulfuron
C15H18N6O6S
410.1009
111991-09-4
Nicotine
C10H14N2
162.1157
54-11-5
# of Spectra
Formula
C10H19O7PS
4
www.spektrotek.com
Compound
Malaoxon
55
www.spektrotek.com
56
Compound
Nitenpyram
Formula
C11H15ClN4O2
Formula Weight
270.0884
CAS Number
# of Spectra
120738-89-8
Norflurazon
C12H9ClF3N3O
303.0386
27314-13-2
Norflurazon-desmethyl
C11H7ClF3N3O
289.023
23576-24-1
Novaluron
C17H9ClF8N2O4
492.0123
116714-46-6
Nuarimol
C17H12ClFN2O
314.0622
63284-71-9
Ofurace
C14H16ClNO3
281.0819
58810-48-3
Omethoate
C5H12NO4PS
213.0225
1113-02-6
Orbencarb
C12H16ClNOS
257.0641
34622-58-7
Oxadiargyl
C15H14Cl2N2O3
340.0381
39807-15-3
Oxadiazon
C15H18Cl2N2O3
344.0694
19666-30-9
Oxadixyl
C14H18N2O4
278.1267
77732-09-3
Oxamyl
C7H13N3O3S
219.0678
23135-22-0
Oxamyl-oxime
C5H10N2O2S
162.0463
30558-43-1
Oxasulfuron
C17H18N4O6S
406.0947
144651-06-9
Oxycarboxin
C12H13NO4S
267.0565
5259-88-1
Oxydemeton-methyl
C6H15O4PS2
246.0149
301-12-2
Oxyfluorfen
C15H11ClF3NO4
361.0329
42874-03-3
Paclobutrazol
C15H20ClN3O
293.1295
76738-62-0
Paraoxon
C10H14NO6P
275.0559
311-45-5
Paraoxon-methyl
C8H10NO6P
247.0246
950-35-6
Parathion
C10H14NO5PS
291.033
56-38-2
Parathion-methyl
C8H10NO5PS
263.0017
298-00-0
Pebulate
C10H21NOS
203.1344
1114-71-2
Penconazole
C13H15Cl2N3
283.0643
66246-88-6
Pencycuron
C19H21ClN2O
328.1342
66063-05-6
Pendimethalin
C13H19N3O4
281.1376
40487-42-1
Permethrin
C21H20Cl2O3
390.0789
52645-53-1
Pethoxamid
C16H22ClNO2
295.1339
106700-29-2
Phenmedipham
C16H16N2O4
300.111
13684-63-4
Phenthoate
C12H17O4PS2
320.0306
3/7/2597
Phorate
C7H17O2PS3
260.0128
298-02-2
Phorat-sulfon
C7H17O4PS3
292.0027
4/7/2588
Phorat-sulfoxide
C7H17O3PS3
276.0077
3/6/2588
Phosalone
C12H15ClNO4PS2
366.9869
2310-17-0
Phosmet
C11H12NO4PS2
316.9945
732-11-6
Phosphamidon
C10H19ClNO5P
299.0689
13171-21-6
Phoxim
C12H15N2O3PS
298.0541
14816-18-3
Picolinafen
C19H12F4N2O2
376.0835
137641-05-5
Picoxystrobin
C18H16F3NO4
367.1031
117428-22-5
Piperonyl butoxide
C19H30O5
338.2093
51-03-6
Piperophos
C14H28NO3PS2
353.1248
24151-93-7
Pirimicarb
C11H18N4O2
238.143
23103-98-2
Pirimiphos-ethyl
C13H24N3O3PS
333.1276
23505-41-1
Pirimiphos-methyl
C11H20N3O3PS
305.0963
29232-93-7
Primisulfuron-methyl
C15H12F4N4O7S
468.0363
86209-51-0
Prochloraz
C15H16Cl3N3O2
375.0308
67747-09-5
Procymidone
C13H11Cl2NO2
283.0167
32809-16-8
Profenofos
C11H15BrClO3PS
371.9351
41198-08-7
Prohexadione
C10H12O5
212.0685
88805-35-0
Promecarb
C12H17NO2
207.1259
2631-37-0
Prometon
C10H19N5O
225.159
1610-18-0
4
Formula
Formula Weight
CAS Number
C10H19N5S
241.1361
7287-19-6
Propachlor
C11H14ClNO
211.0764
1918-16-7
Propamocarb
C9H20N2O2
188.1525
24579-73-5
Propanil
C9H9Cl2NO
217.0061
709-98-8
Propaquizafop
C22H22ClN3O5
443.1248
111479-05-1
Propargite
C19H26O4S
350.1552
2312-35-8
Propazin-2-hydroxy
C9H17N5O
211.1433
not available
Propazine
C9H16ClN5
229.1094
139-40-2
Propetamphos
C10H20NO4PS
281.0851
31218-83-4
Propham
C10H13NO2
179.0946
122-42-9
Propiconazole
C15H17Cl2N3O2
341.0698
60207-90-1
Propoxur
C11H15NO3
209.1052
114-26-1
Propoxycarbazone sodium
C15H18N4O7S
398.0896
181274-15-7
Propyzamide
C12H11Cl2NO
255.0218
23950-58-5
Prosulfocarb
C14H21NOS
251.1344
52888-80-9
Prosulfuron
C15H16F3N5O4S
419.0875
94125-34-5
Prothioconazole
C14H15Cl2N3OS
343.0313
178928-70-6
Prothioconazole, Desthiometabolit (JAU C14H15Cl2N3O
6476-desthio)
311.0592
not available
Prothiofos
C11H15Cl2O2PS2
343.9628
34643-46-4
Pymetrozine
C10H11N5O
217.0964
123312-89-0
Pyraclofos
C14H18ClN2O3PS
360.0464
89784-60-1
Pyraclostrobin
C19H18ClN3O4
387.0986
175013-18-0
Pyrazophos
C14H20N3O5PS
373.0861
13457-18-6
Pyrethrin I
C21H28O3
328.2038
121-21-1
Pyrethrin II
C22H28O5
372.1937
121-29-9
Pyridaben
C19H25ClN2OS
364.1376
96489-71-3
Pyridaphenthion
C14H17N2O4PS
340.0647
119-12-0
Pyridate
C19H23ClN2O2S
378.1169
55512-33-9
Pyrifenox
C14H12Cl2N2O
294.0327
88283-41-4
Pyrimethanil
C12H13N3
199.1109
53112-28-0
Pyriproxyfen
C20H19NO3
321.1365
95737-68-1
Pyroquilon
C11H11NO
173.0841
57369-32-1
Quinalphos
C12H15N2O3PS
298.0541
13593-03-8
Quinmerac
C11H8ClNO2
221.0244
90717-03-6
Quinoclamine
C10H6ClNO2
207.0087
2797-51-5
Quinoxyfen
C15H8Cl2FNO
306.9967
124495-18-7
Quizalofop-ethyl
C19H17ClN2O4
372.0877
76578-14-8
Quizalofop-P (free acid)
C17H13ClN2O4
344.0564
76578-12-6
Resmethrin
C22H26O3
338.1882
10453-86-8
Rimsulfuron
C14H17N5O7S2
431.0569
122931-48-0
Rotenone
C23H22O6
394.1416
83-79-4
Sebuthylazine
C9H16ClN5
229.1094
7286-69-3
Sebuthylazine-desethyl
C7H12ClN5
201.0781
not available
Sethoxydim
C17H29NO3S
327.1868
74051-80-2
Siduron
C14H20N2O
232.1576
1982-49-6
Silthiofam
C13H21NOSSi
267.1113
175217-20-6
Simazine
C7H12ClN5
201.0781
122-34-9
Simazine-2-hydroxy
C7H13N5O
183.112
11/3/2599
Simetryn
C8H15N5S
213.1048
1014-70-6
129630-17-7
4
www.spektrotek.com
Pyraflufen-ethyl
# of Spectra
Compound
Prometryne
57
www.spektrotek.com
58
Compound
Spinosyn A
Formula
C41H65NO10
Formula Weight
731.4608
CAS Number
# of Spectra
131929-60-7
Spinosyn D
C42H67NO10
745.4765
131929-63-0
Spiroxamine
C18H35NO2
297.2668
118134-30-8
Sulcotrione
C14H13ClO5S
328.0172
99105-77-8
Sulfentrazone
C11H10Cl2F2N4O3S
385.9819
122836-35-5
Sulfometuron-methyl
C15H16N4O5S
364.0841
74222-97-2
Sulfosulfuron
C16H18N6O7S2
470.0678
141776-32-1
Sulfotep
C8H20O5P2S2
322.0227
3689-24-5
Sulprofos
C12H19O2PS3
322.0285
35400-43-2
tau-Fluvalinate
C26H22ClF3N2O3
502.1271
102851-06-9
Tebuconazol
C16H22ClN3O
307.1451
107534-96-3
Tebufenozide
C22H28N2O2
352.2151
112410-23-8
Tebufenpyrad
C18H24ClN3O
333.1608
119168-77-3
Tebupirimfos
C13H23N2O3PS
318.1167
96182-53-5
Tebutam
C15H23NO
233.178
35256-85-0
Tebuthiuron
C9H16N4OS
228.1045
34014-18-1
Teflubenzuron
C14H6Cl2F4N2O2
379.9742
83121-18-0
Temephos
C16H20O6P2S3
465.9897
3383-96-8
TEPP
C8H20O7P2
290.0684
107-49-3
Tepraloxydim
C17H24ClNO4
341.1394
149979-41-9
Terbumeton
C10H19N5O
225.159
33693-04-8
Terbuthylazine
C9H16ClN5
229.1094
5915-41-3
Terbuthylazine-2-hydroxy
C9H17N5O
211.1433
not available
Terbuthylazine-desethyl
C7H12ClN5
201.0781
30125-63-4
Terbutryn
C10H19N5S
241.1361
886-50-0
Tetrachlorvinphos
C10H9Cl4O4P
363.8993
22248-79-9
Tetraconazole
C13H11Cl2F4N3O
371.0215
112281-77-3
Tetramethrin
C19H25NO4
331.1784
7696-12-0
Thiabendazole
C10H7N3S
201.0361
148-79-8
Thiacloprid
C10H9ClN4S
252.0236
111988-49-9
Thiamethoxam
C8H10ClN5O3S
291.0193
153719-23-4
Thidiazuron
C9H8N4OS
220.0419
51707-55-2
Thifensulfuron-methyl
C12H13N5O6S2
387.0307
79277-27-3
Thiobencarb
C12H16ClNOS
257.0641
28249-77-6
Thiodicarb
C10H18N4O4S3
354.049
59669-26-0
Thiofanox-sulfone
C9H18N2O4S
250.0987
39184-59-3
Thiofanox-sulfoxide
C9H18N2O3S
234.1038
39184-27-5
Thiophanate
C14H18N4O4S2
370.0769
23564-06-9
Thiophanate-methyl
C12H14N4O4S2
342.0456
23564-05-8
Tolclofos-methyl
C9H11Cl2O3PS
299.9544
57018-04-9
Tolylfluanid
C10H13Cl2FN2O2S2
345.978
731-27-1
Tralkoxydim
C20H27NO3
329.1991
87820-88-0
Triadimefon
C14H16ClN3O2
293.0931
43121-43-3
Triadimenol
C14H18ClN3O2
295.1088
55219-65-3
Tri-allate
C10H16Cl3NOS
303.0018
2303-17-5
Triasulfuron
C14H16ClN5O5S
401.0561
82097-50-5
Triazamate
C13H22N4O3S
314.1413
112143-82-5
Triazophos
C12H16N3O3PS
313.065
24017-47-8
Triazoxide
C10H6ClN5O
247.0261
72459-58-6
Tribenuron-methyl
C15H17N5O6S
395.09
101200-48-0
Trichlorfon
C4H8Cl3O4P
255.9226
52-68-6
4
Formula
Formula Weight
CAS Number
C7H4Cl3NO3
254.9257
55335-06-3
Tricyclazole
C9H7N3S
189.0361
41814-78-2
Tridemorph
C19H39NO
297.3032
24602-86-6
Trietazine
C9H16ClN5
229.1094
1912-26-1
Trifloxystrobin
C20H19F3N2O4
408.1297
141517-21-7
Triflumizole
C15H15ClF3N3O
345.0856
68694-11-1
Triflumuron
C15H10ClF3N2O3
358.0332
64628-44-0
Triflusulfuron-methyl
C17H19F3N6O6S
492.1039
126535-15-7
Trinexapac-ethyl
C13H16O5
252.0998
95266-40-3
Triticonazole
C17H20ClN3O
317.1295
131983-72-7
Tritosulfuron
C13H9F6N5O4S
445.0279
142469-14-5
Uniconazole
C15H18ClN3O
291.1138
83657-22-1
Vamidothion
C8H18NO4PS2
287.0415
2275-23-2
Warfarin
C19H16O4
308.1049
81-81-2
Ziram
C6H12N2S4Zn
303.9175
137-30-4
# of Spectra
4
Compound
Triclopyr
Ordering Information
Product Name
Part Number
Pesticide LC/MS/MS Library V.1.0 for Cliquid Software
®
1037032
Legal Acknowledgements/Disclaimers
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or representations as to the fitness or the continued fitness
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© 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB
SCIEX™ is being used under license.
www.spektrotek.com
The iMethod™ test described above has been developed
by AB SCIEX to provide all the sample prep and instrument
parameters required to accelerate the adoption of this method
for routine testing. The performance of this method will
need to be verified in a given lab due to potential variations
in instrument performance, maintenance, chemicals and
procedures used, technical experience, sample matrices
and environmental conditions It is the responsibility of the
end user to make adjustments to this method to account for
slight differences in equipment and/or materials from lab to
lab as well as to determine and validate the performance of
this method for a given instrument and sample type. Please
note that a working knowledge of Analyst® Software may be
required to do so.
59
GIDA&ÇEVREUYGULAMALARI
Simultaneous Analysis of 14 Mycotoxins and 163 Pesticides
in Crude Extracts of Grains by LC-MS/MS
Kristin Von Czapiewski1; Angela Voller2; Birgit Schlutt1; André Schreiber3
1
AB SCIEX, Darmstadt (Germany); 2SGS, Hamburg (Germany); 3AB SCIEX, Concord, ON
(Canada)
Overview
Multi-component methods for the detection of different compound classes,
such as mycotoxins or pesticides, have been established and are widely
used to analyze a broad range of food or feed. However, there is a continuing
demand to test for a larger number of compounds in shorter times. The
development of a combined method for different compound classes can help
to meet those new challenges. In this paper we present a fast, robust, and
reliable method, which has been validated for the detection of 14 mycotoxins
and 163 pesticides in the matrix grain. The LC-MS/MS method using the
Scheduled Multiple Reaction Monitoring (Scheduled MRM™ algorithm)
detects all mycotoxins with Limits of Quantitation (LOQ) between 1µg/kg
and 10µg/kg. The LOQ for pesticides were found to be 10µg/kg and less. All
LOQ meet the requirements of the EU.
Introduction
Pesticides and mycotoxins are known to harm the health of humans and animals. Many of these compounds are known either
as carcinogenic, cytotoxic, or ecotoxic. Therefore, different countries have set regulations on pesticides and mycotoxins. For
example, in the EU, maximum residue levels of pesticides in or on certain products are regulated by EC/396/2005 and the
amended regulation EC/839/2008 and, in Japan, by the Japanese Positive List Syoku-An No.0124001 January 14, 2005 and
amendments May 26, 2006. Mycotoxin limits are harmonized in the regulation for contaminants in foodstuffs EC 1881/2006
and the amended regulation EC 1126/2007 in the EU.1-6
Regulations on food and environmental analysis require the analysis of contaminants using confirmatory techniques, such
as GC-MS and LC-MS/MS. More than 1000 pesticides are used worldwide and, along with their metabolites and degradation
products, are present in food and the environment. Thus, there is a demand for powerful and rapid analytical methods that can
detect very low concentrations of pesticides in mycotoxins in a variety of sample matrices.
Over the last years, LC-MS/MS replaced traditional GC and LC methods for the screening of pesticides and mycotoxins because
of its ability to analyze a wider range of compounds in a single analysis and the unmatched selectivity and sensitivity of Multiple
Reaction Monitoring (MRM).
Traditionally, mycotoxins and pesticides require different sample preparation. A simplified extraction procedure was established
to analyze the two compound classes simultaneously in one sample, without additional cleanup steps by SPE or immunoaffinity
columns. This new simplified sample preparation in combination with high resolution LC, and sensitive MRM detection allows
detecting pesticides and mycotoxins faster and less labor-intensive and time-saving.
www.spektrotek.com
Experimental
62
Sample Preparation
LC
10g of grain sample was extracted using a mixture
acetonitrile/water. The extract was filtered and diluted with
water + 5 mM ammonium acetate to optimize LC peak
shape.7
A Shimadzu Prominence LC system with an Agilent
ZORBAX Eclipse XDB C18, 100x4.6 mm, 1.8µm column
at 40°C with a gradient of eluent A water/methanol
(80/20) + 5 mM ammonium acetate and eluent B water/
methanol (10/90) + 5 mM ammonium acetate was used
at a flow rate of 500 µL/min. The injection volume was set
to 100 µL.
An AB SCIEX API 4000™ LC/MS/MS system with Turbo V™ source and Electrospray Ionization (ESI) probe was used. A
number of 14 mycotoxins and 163 pesticides were detected using 2 MRM transitions per compound to allow quantitation and
identification based on the ratio of quantifier and qualifier transitions as defined by regulation 2002/657/EC. The Scheduled
MRM™ algorithm was used for best accuracy and reproducibility (Figure 1). Every sample was injected twice in positive and
negative polarity.
Results and Discussion
A method for quantitation and identification of 9 fusarium toxins: Nivalenol (NIV), Deoxynivalenol (DON), Fusarenon X (FUS
X), 3-Acetyldeoxynivalenol (3-AcDON), 15-Acetyldeoxynivalenol (15-AcDON), Diacetoxyscirpenol (DAS), HT-2 toxin, T-2 toxin,
Zearalenon (ZON), and Ochratoxin A (OTA) was developed
(Figure 1). This method was extended to also detect aflatoxins B1, B2, G1, and G2 (Figure 2). The complete method was
validated for the analysis of wheat, barley, corn, and oat samples (Table 1).7-8
(p
),
p(
_
p
x5 0
.
NIV:
DON:
FUS X:
3-AcDON:
15-AcDON:
DAS:
OTA:
HT-2:
T-2:
ZON:
1.10e5
1.00e5
9.00e4
8.00e4
7.00e4
6.00e4
371/281
295/265
413/353
337/307
337/219
384/307
404/239
447/345
484/215
317/131
5.00e4
FUS X
6.4min
4.00e4
3.00e4
3.54
.0
4.55
B1:
B2:
G1:
G2:
1.8e4
1.6e4
313/285
315/287
329/243
331/245
G1
313/241
315/259
329/311
331/189
B1
1.4e4
OTA
8.1min
1.2e4
1.0e4
T-2
8.8min
AcDON
7.2min
B2
8000.0
HT-2
8.5min
6000.0
4000.0
G2
2000.0
NIV
4.4min
1.00e4
7.5
1.9e4
ZON
9.3min
DON
5.8min
2.00e4
0.00
DAS
8.0min
311/281
295/138
413/263
337/173
337/150
384/105
404/358
447/285
484/185
317/175
.0
5.56
.0
MS/MS
6.57
.0
Time, min
7.58
.0
8.59
.0
9.51
0.0
Figure 1. Detection of fusarium toxins and Ochratoxin A by
LC-MS/MS
0.0
3.54
.0
4.55
.0
5.56
.0
6.57
.0
Time, min
7.58
.0
8.59
.0
9.51
0.0
Figure 2. Detection of aflatoxins by LC-MS/MS
Table 1. LOQ and linear range of detected mycotoxins
LOQ (µg/kg)
Linear Range (µg/kg)
EU MRL#
10
400
(1)
15-AcDON
10
150
(1)
DON
10
10000
1750*
1250** (2)
FUS X
10
2000
(1)
DAS
10
400
(1)
NIV
10
4000
(1)
OTA
1
>10
5***
HT-2
5
200
(2)
T-2
5
1000
(2)
ZON
5
80
100*** (2)
Aflatoxin B1
1
>20
2
Aflatoxins
1
>20
1=4
Footnotes to Table 1:
EC 1881/2006 and the amended EC 1126/2007
* Unprocessed durum wheat and oats
** Unprocessed cereals other than durum wheat and oats *** Unprocessed cereals
(1) Due to co-occurrences and as “generally low” considered levels no MRL was estimated
(2) Appropriateness of setting a maximum level should be considered by 1 July 2008
www.spektrotek.com
Mycotoxin
3-AcDON
63
XIC o f +MRM (331 pairs): 4 04 .1/372. 1 a...
1.0e 6
9.0e 5
Pesticides
Positive ESI
Max. 8.9e5 cps.
X IC of -MRM (36 pa irs): 421.0/97.0 a mu ...
1 .0e6
8.7
9 .0e5
8.0e 5
8 .0e5
7.0e 5
7 .0e5
6.0e 5
5 .0e5
4.0e 5
4 .0e5
3.0e 5
3 .0e5
2.0e 5
2 .0e5
1.0e 5
1 .0e5
0.00
5
10
Time , min
0.0
15
XIC o f +MRM (331 pairs): 4 84 .1/215. 2 a...
Max. 5.2e4 cps.
4.5e 4
5
Time, min
10
X IC of -MRM (36 pa irs): 337.1/307.1 amu...
8.8
5.0e 4
9.8
6 .0e5
5.0e 5
Ma x. 5.7e 5 cp s.
Pesticides
Negative ESI
In tensity, cp s
Inten sity, cps
The developed method was recently updated to also quantify and identify 163 pesticides (Figure 3). The use of the Scheduled
MRM™ algorithm allows the monitoring of such a large panel of analytes without sacrificing sensitivity and reproducibility.
The method was validated in different grain matrices. Limits of Quantitation (LOQ) of all mycotoxins were found between
1 µg/kg and 10 µg/kg. Pesticides were quantified at 10 µg/kg and less. All LOQ meet the requirements of the EU. Positive
findings in two selected grain samples are shown in Figure 4.
1 .0e5
Mycotoxins
Positive ESI
9 .0e4
15
Ma x. 5.7e 4 cp s.
Mycotoxins
Negative ESI
8 .0e4
4.0e 4
In tensity, cp s
Inten sity, cps
7 .0e4
3.5e 4
7.2
6 .0e4
3.0e 4
5 .0e4
2.5e 4
4 .0e4
2.0e 4
1.5e 4
3 .0e4
1.0e 4
2 .0e4
1 .0e4
5000.0
0 .0
5
10
Time, min
0.00
15
5
Time, min
10
15
Figure 3. Detection of 14 mycotoxins and 163 pesticides using LC/MS/MS in two injections (positive and negative polarity)
using the Scheduled MRM™ algorithm for best sensitivity and reproducibility
XIC of +MRM (331 pairs): 331.1/285.1 amu Expect...
2.0e6
1.0e6
5.0e5
0.0
1.5e5
DON
NIV
ZEA
6
10
12
14
Max. 1.0e5 cps .
1850 g/kg
500 g/kg
30 g/kg
5.0e4
0.0
2
4
1.0e5
6
8
Time, min
10
12
14
3 g/kg
3 g/kg
5.0e4
0.0
2
4
6
8
Time, min
XIC of -MRM (36 pairs): 295.1/265.0 amu Expected...
3.8e5
5.7
1.0e5
Max. 795.2 cps.
Azoxystrobin
Pyraclostrobin
1.5e5
Intensity , cps
Intens ity, cps
2.0e5
4
9.0
8
Time, min
XIC of -MRM (36 pairs): 295.1/265.0 amu Expected...
2.4e5
2
g/kg
g/kg
g/kg
g/kg
g/kg
XIC of +MRM (331 pairs): 384.2/105.2 amu Expect...
Intensity, cps
Intens ity, cps
1.5e6
T-2
1850
HT-2
500
Malathion
2000
Pirimiphos-methyl 2400
Piperonylbutoxide 680
Max. 2.0e6 cps .
3.0e5
2.0e5
1.0e5
0.0
DON
290
NIV
300
ZEA
20
3-AcDON 20
MCPA
60
Bentazone 6
2
4
10
12
14
Max. 3.7e4 cps.
g/kg
g/kg
g/kg
g/kg
g/kg
g/kg
5.7
6
8
Time, min
10
12
14
Figure 4. Detection of mycotoxins and pesticides in a durum wheat sample (left) and a barley sample (right)
Summary
www.spektrotek.com
A fast, robust, and reliable method, for the detection 14 mycotoxins and 163 pesticides in the matrix grain was developed
and validated. A generic extraction procedure followed by a dilution step was used to cover the large panel of analytes. High
resolution LC was combined with high sensitivity detection using an AB SCIEX API 4000™
LC/MS/MS system. Multiple Reaction Monitoring (MRM) was used because of its high selectivity and sensitivity. With the
Scheduled MRM™ algorithm activated for accuracy and reproducibility.
The method was validated in different grain matrices. Limits of Quantitation (LOQ) of all mycotoxins were found between 1µg/
kg and 10µg/kg. Pesticides were quantified at 10µg/kg and less. All LOQ meet the requirements of the EU.
64
References
1 D. Elbert et al.: presentation at AOAC conference (2008) in Dallas
2 A. Voller et al.: presentation at AOAC conference (2009) in Philadelphia
Publication number: 2110210-
Stephen Lock1 and Hermann Unterluggauer2
1
AB SCIEX Warrington (UK)
2
Austrian Agency for Health and Food Safety (AGES GmbH), Innsbruck (Austria)
Introduction
Glyphosate is a common broad-spectrum systemic herbicide used widely to kill
weeds especially annual broadleaf weeds and grasses known to compete with
crops. Usually Glyphosate, as it is very polar, undergoes FMOC derivatization
by reacting the native glyphosate with fluorenylmethyloxycarbonyl chloride
(FMOC-Cl) before analysis. This derivatization step complicates the
analysis and there is a growing need for a method which can detect not
only Glyphosate (and it´s major metabolite AMPA) but also Glufosinate and
similar highly polar compounds, in their underivatized states. In addition
a simplified approach to sample extraction using either QuEChERS (Quick,
Easy, Cheap, Effective, Rugged, and Safe) or a solvent extraction would be
beneficial.
Here we present initial data using a new LC-MS/MS method which combines
the use of a HILIC type chromatography on an AB SCIEX LC/MS/MS system
to detect underivatized glyphosate and other polar pesticides which have
been spiked in different food matrices. A simple solvent extraction has been
used and initial data will be presented to show how applicable this approach
is to food analysis.
Detection of Underivatized Glyphosate and Similar Polar
Pesticides in Food of Plant Origin by LC-MS/MS
Experimental
Sample Preparation
LC
For linearity and sensitivity tests calibration standards were
prepared in 50/50 methanol/water from concentrations 1
to 500 ng/mL. Matrix samples were prepared by spiking
the polar pesticides into 50% aqueous methanol extracts of
onion, wheat, rice and grapes prepared as per the QuPPe
(Quick Polar Pesticides) Method from the EU Reference
Laboratories for Residues of Pesticides.1 These extracts
were then diluted 5x with 50% aqueous methanol before
injection to reduce possible matrix effects.
The LC system used for this analysis was a ShimadzuXR
LC system consisting of two Shimadzu LC20AD pumps,
SIL 20AC autosampler, and a CTO20A column oven. The
analyses were performed at 50ºC on an Obelisc N phase
HPLC column. An injection volume of 50 µL was used
using the gradient separation as shown in Table 1 where
mobile phase A was acidified 85% acetonitrile/water
(85/15) containing ammonium acetate and mobile phase
B was acidified water containing ammonium acetate. The
gradient used is shown in Table 1.
HO
NH
O
HO
OH
P
H2N
O
O
Glyp hosate
OH
AMPA
H3C
OH
P
O
O
NH2
Glufos inate
OH
P
HO
Cl
OH
P
O
Ethephon
www.spektrotek.com
OH
65
XICo f- MRM( 11 pairs):1 68.000/63.000D a ID:G lyphosate3 ...M
Time
Flow (mL/min)
A (%)
B (%)
0
2
1.2
100
0
1.6e4
1
3
1.2
100
0
1.2e4
2
3.6
1.2
0
100
8
1.2
0
100
4
8
1.5
0
100
5
8.2
1.5
100
0
6
13.5
1.5
100
0
7
13.8
1.2
100
0
8
14
1.2
100
0
Intensity, cps
1.4e4
ax.1 .8e4 cps.
ax.2 .3e4 cps.
6.01
2.2e4
Glyphosate
2.0e4
1.8e4
Glufosinate
1.6e4
1.0e4
8000.0
1.4e4
1.2e4
1.0e4
8000.0
6000.0
6000.0
4000.0
4000.0
2000.0
0.0
XICo f- MRM( 11 pairs):1 80.000/62.900D aI D: Glufosinate1 ...M
6.82
2000.0
4.04
.5
5.05
.5
6.06
.5
7.07
.5
Time,m in
XICo f- MRM( 11 pairs):1 09.923/62.900D a ID: AMPA 2f rom. ..
8.08
.5
9.09
.5
Max.4 .2e4 cps.
4.29
4.2e4
4.0e4
0.0
3.54
.0
4.55
.0
5.56
.0
6.57
Time,m in
XICo f- MRM( 11 pairs):1 43.000/106.900 Da ID:E thephon2 f...
7.58
.0
8.59
.0
Max.5 .7e4 cps.
Ethephon
5.0e4
4.5e4
3.0e4
.0
7.43
5.5e4
AMPA
3.5e4
4.0e4
2.5e4
Intensity,c ps
3
1.8e4
Intensity, cps
Step
Intensity, cps
Table 1. Gradient conditions used for separation
2.0e4
1.5e4
3.5e4
3.0e4
2.5e4
2.0e4
1.5e4
1.0e4
1.0e4
5000.0
0.0
5000.0
1.52
.0
2.53
.0
3.54
.0
4.55
Time,m in
.0
5.56
.0
6.57
.0
7.5
0.0
4.55
.0
5.56
.0
6.57
.0
7.58
Time,m in
.0
8.59
.0
9.51
0.0
Figure 1. Injection of a 10 ng/mL standard
MS/MS
The analyses were performed on an AB SCIEX QTRAP® 5500
LC/MS/MS system using the Turbo V™ source operated in
electrospray ionization and negative polarity with an IonSpray
(IS) voltage of -4500 V. The curtain gas was set at 35 psi,
nebulizer gas (Gas 1) set at 60 psi, drying gas (Gas 2) set
at 70 psi, CAD gas set at medium, and the temperature set
at 650ºC. The MRM transitions used as well as the retention
times for the compounds are shown in Table 2. Each MRM
was monitored with a dwell time of 50 ms.
Table 2. LC-MS/MS parameters for the analyzed compounds
Retention
time (min)
Q1
(amu)
Q3
(amu)
DP (V)
CE (V)
AMPA
4.3
110
79
-60
-24
110
63
-60
-26
Ethephon
7.4
143
79
-45
-26
143
107
-45
-12
180
63
-60
-66
180
95
-60
-24
168
79
-110
-54
168
63
-110
-32
Step
Glufosinate
Glyphosate
6.0
6.8
Figure 2 and Table 3 shows typical calibration lines obtained
the target pesticides. The response over the range tested, 1
to 500 ng/mL, was linear with a 1/x weighting. All accuracy
values were well in between 80 and 120%.
The sensitivity of the different pesticides is also shown in
Table 3. All pesticides were easily identified and quantified
at the maximum residue limits (MRL) set by EU and CODEX
Alimentarius where limits for most fruit and vegetables are
0.1 mg/kg. 2, 3 The extra sensitivity also allowed dilution of
sample extract to minimize possible matrix effects.
Table 3. Linearity with a 1 /x weighting (1 to 500 ng/mL range)
and signal-to-noise (S/N*) from a 1 ng/mL standard injected
Step
AMPA
Ethephon
www.spektrotek.com
Figure 1 shows a typical chromatogram obtained from an
injection of a 10 ng/mL standard of all studied pesticides.
The monitoring of two transitions per compound also allows
compound identification using the MRM ratio.
66
Glufosinate
Glyphosate
MRM
transition
Linear fit (r value)
S/N at 1 ng/mL
110/79
0.999
131
110/63
0.999
234
143/79
0.979
58
143/107
0.984
155
180/63
1.000
88
180/95
0.999
47
168/79
0.999
52
168/63
0.999
102
* S/N values were calculated in MultiQuant™ software
Ethephon
Glufosinate
Glufosinate
1.00e5
Intensity, cps
Glyphosate
Intensity, cps
Intensity, cps
Intensity, cps
2.0
2.0
2.5
2.5
3.0
3.0
2.0e4
1.0e5 0.0
Intensity, cps
Intensity, cps
Intensity, cps
1.5
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
Time, min
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
Time, min
3.5
7.5
7.55
8.0
8.5
9.0
Max. 1.7e5 cps.
4.5
0.0
8.0e4 4.5
5.0
5.0
5.5
5.5
6.0
6.0
6.5
6.5
7.0
7.5
8.0
Time, min
7.0
7.5
8.0
Time, min
8.5
8.5
9.0
9.0
9.5
9.5
10.0
10.0
6.0e4
4.0e4
3.0e4
Figure 3. 100 μg/kg spike into rice extract diluted 5x with
acetonitrile
2.0e4
2.0e4
1.0e4
2.5
3.0
3.5
4.0
4.5
Time, min
5.0
5.5
6.0
6.5
3.5e4 3.0e4
5.5e4
4.5e4
Intensity, cps
0.0
5.0
5.5
Intensity, cps
6.35
6.0
6.5
7.0
7.5
Time, min
XIC of -MRM (11 pairs): 109.923/62.900 Da ID: AMPA 2 from ...
2.0e4
0.0
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
4.24
Time, min
8.5e4
XIC
of -MRM
1.5e4
8.0e4(11 pairs): 109.923/62.900 Da ID: AMPA 2 from ...
8.5e4
1.0e4 7.0e4
8.0e4
5000.0 6.0e4
7.0e4
AMPA
AMPA
Intensity, cps
2.0e4
6.0e4
0.0
1.0e4
5.0e4
0.0
4.0e4
1.5
AMPA
1.5
2.0
2.0
2.5
2.5
3.0
3.0
3.5
3.0e4
8.5
8.0
8.5
9.0
9.0
9.5
7.0
7.5
Time, min
8.0
8.5
9.0
9.5
10.0
Max. 7.1e4 cps.
Max. 7.1e4 cps.
Glufosinate
Glufosinate
5.77
4.5e4
3.5e4
XIC of -MRM (11 pairs): 180.000/62.900 Da ID: Glufosinate 1 ...
3.0e4
4.0e4
5.77
2.5e4
3.5e4
7.0e4
2.0e4
3.0e4
6.5e4
1.5e4
2.5e4
6.0e4
1.0e4
2.0e4
5.5e4
Max. 7.1e4 cps.
Glufosinate
8.00e4
5000.0
1.00e5
3.21
7.00e4
0.0
9.00e4
9.5
Max. 8.5e4 cps.
Ethephon
Ethephon
7.65 7.77 8.118.61
8.0
8.5
9.0
3.49 4.24 4.39 4.71 5.11 5.43
6.33 6.54 7.06
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
6.00e4
Time, min
8.00e4
XIC of -MRM (11 pairs): 143.000/106.900 Da ID: Ethephon 2 f...
5.00e4
7.00e4
7.14
4.00e4
6.00e4
1.20e5
3.00e4
5.00e4
1.10e5
2.00e4
4.00e4
1.00e5
1.00e4
3.00e4
9.00e4
0.00
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5 2.00e4
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.00e4
Time, min
Time, min
1.00e4
7.00e4
0.00
6.00e4 4.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
Time, min
Time, min
5.00e4
7.5
Max. 1.2e5 cps.
Ethephon
8.5
9.0
9.5
10.0
8.5
9.0
9.5
10.0
8.5
9.0
9.5
10.0
4.00e4
3.00e4
2.0e4
2.00e4
1.0e4
0.0
6.5
5000.0
1.5e4
5.0e4
7.65 7.77 8.118.61
3.21 3.49 4.24 4.39 4.71 5.11 5.43
6.33 6.54 7.06
0.0
1.0e4
4.5e4
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
Time, min
5000.0
Max. 8.5e4 cps. 4.0e4
XIC of3.21
-MRM 3.49
(11 pairs):
143.000/106.900
Da ID: Ethephon
1.2e5 cps.
7.65 7.77 Max.
4.24 4.39
6.332 f...
6.54 7.06
8.118.61
4.71 5.11 5.43
0.0
3.5e4
9.0
9.5
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
7.14
Time, min
3.0e4
Max. 8.5e4 cps.
XIC 1.20e5
of -MRM (11 pairs): 143.000/106.900 Da ID: Ethephon 2 f...
Max. 1.2e5 cps.
2.5e4
1.10e5
7.14
2.0e4
1.00e5
1.20e5
1.5e4
9.00e4
1.10e5
1.0e4
8.5
4.24
5.0e4
0.0
6.0e4 4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
Time, min
4.0e4
5.0e4
3.0e4
4.24
8.5e4
4.0e4
8.0e4 2.0e4
3.0e4
7.0e4 1.0e4
8.0
8.0
Intensity, cps
4.5
6.0
5.0e4
4.0e4
Max. 2.4e4 cps.
Glyphosate
4.0
5.5
Intensity, cps
Intensity, cps
Intensity, cps
6.0e4
5.0e4
2.0e4 1.5e4
5000.0
3.0e4
1.0e4
5.0
5.77
XIC of7.0e4
-MRM (11 pairs): 180.000/62.900 Da ID: Glufosinate 1 ...
6.5e4
5.5e4
6.35
2.5e4 2.0e4
XIC of -MRM (11 pairs): 168.000/63.000 Da ID: Glyphosate 3 ...
1.0e4
3.5e4
1.5e4
4.5
XIC of -MRM (11 pairs): 180.000/62.900 Da ID: Glufosinate 1 ...
6.5e4
7.0e4
6.0e4
6.35
3.0e4 2.5e4
Intensity, cps
Max. 2.4e4 cps.
Glyphosate
Glyphosate
3.5e4
2.5e4
5000.0
0.0
7.5
Max. 2.4e4 cps.
Intensity, cps
7.0
Intensity, cps
2.0
Intensity, cps
1.5
Intensity, cps
0.0
The method was then applied to spiked matrices. Figures 3
and 4 show that all the different polar pesticides, spiked at the
level of the EU MRL (0.1 mg/kg), can be detected in different
matrices.
The S/N data is also shown in Table 4 for the results of spiking
experiments in four different matrices. What can also be seen
is that even with a 5x dilution of the food extract matrix effects
can be observed as there is a slight shift in retention times
and some suppression / enhancement was also observed.
For that reason it is recommended to use isotopically labeled
standards for quantitation.
7.0
8.0
8.5
9.0
8.0
8.5
9.0
Max. 1.7e5 cps.
Max. 1.7e5 cps.
Ethephon
4.0e4
Figure 2. Calibration lines (2 MRM transitions each) for
analyzed polar pesticides from 1 to 500 ng/mL
7.5
7.5
4.0e4
2.0e4
1.2e5
5.0e4
Glyphosate
7.55
7.55
7.0
7.0
Ethephon
Ethephon
AMPA
3.0e4 2.0e4
1.0e5
1.0e4
2.0e4
9.0e4
0.0
1.0e4
8.0e4
0.0
7.0e4
1.5
6.0e4
Max. 7.4e4 cps.
Glufosinate
AMPA
AMPA
Glufosinate
Glyphosate
Glyphosate
Max.7.4e4
7.4e4cps.
cps.
Max.
Glufosinate
Glufosinate
Intensity, cps
Glyphosate
Glyphosate
Intensity, cps
Intensity, cps
Intensity, cps
Intensity, cps
Area
1.00e4
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Time, min
5.0
5.5
6.0
6.5
7.0
7.5
0.00
4.5
5.0
5.5
6.0
6.5
7.0
7.5
Time, min
8.0
Figure 4. 100 μg/kg spike into grape extract diluted 5x with
acetonitrile
Table 4. Signal-to-noise (S/N*) observed from 0.1 mg/kg spikes into different matrices indicating matrix suppression and
enhancement even after sample dilution.
AMPA
110/79
Ethephon
110/63
143/79
Glufosinate
143/107
180/63
180/95
Glyphosate
168/79
168/63
Rice
2900
5483
998
998
3058
180/95
911
3110
Onion
761
1379
281
2514
2310
180/95
249
1312
Grapes
1187
2344
133
1149
2892
180/95
534
1799
Wheat
1636
3117
174
1014
3062
180/95
588
2708
www.spektrotek.com
Area
AMPA
Ethephon
Ethephon
Area
5.83
5.83
7.4e4
7.4e4
7.0e4
7.0e4
6.5e4
9.00e4
6.5e4
9.00e4
6.0e4
6.0e4
8.00e4
8.00e4
5.5e4
5.5e4
5.0e4
7.00e4
5.0e4
7.00e4
6.59
6.59
4.5e4
6.00e4(11 pairs): 168.000/63.000 Da ID: Glyphosate 3 ...
4.5e4
XIC
of -MRM (11 pairs): 180.000/62.900 Da ID: Glufosinate 1 ...
XIC of -MRM
Max. 6.4e4 cps.
6.00e4
4.0e4
4.0e4
5.00e4
3.5e4
5.83
7.4e4
5.00e4
3.5e4
1.00e5
3.0e4
7.0e4
4.00e4
3.0e4
4.00e4
2.5e4
6.5e4
9.00e4
2.5e4
3.00e4
2.0e4
6.0e4
3.00e4
2.0e4
8.00e4
1.5e4
2.00e4
5.5e4
1.5e4
2.00e4
1.0e4
5.0e4
7.00e4
1.00e4
1.0e4
6.59
5000.0
4.5e4
1.00e4
6.00e4
5000.0
0.00
4.0e4 0.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
0.00
0.0
Time, min
5.00e4 4.0
3.5e4
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
3.5
4.0
4.5
5.0
5.5 Time,
6.0 min6.5
ID: AMPA
Max. 1.4e5 cps.
XIC of -MRM (11 pairs): 143.000/106.900 Da ID:Time,
Ethephon
XIC of -MRM (11 pairs): 109.923/62.900 DaTime,
min 2 from ...
min 2 f...
3.0e4
4.00e4
XIC
of -MRM (11 pairs): 109.923/62.900 Da ID: AMPA 2 from ...
Max. 1.4e5 cps.
4.28
7.21
2.5e4
1.7e5
3.00e4 1.4e5
4.28
7.21
2.0e4
1.6e5
1.7e5
1.4e5 1.3e5
1.6e5
1.5e4
2.00e4
1.3e5 1.2e5
1.4e5
1.0e4
1.2e5 1.1e5
1.00e4
1.4e5
5000.0
1.2e5
1.1e5 1.0e5
0.0
0.00
1.2e5
3.5
4.0
4.5
5.0
5.5
6.0
6.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
1.0e5 9.0e4
1.0e5
Time, min
Time, min
9.0e4 8.0e4
Max. 1.4e5 cps.
1.0e5
8.0e4 7.0e4
8.0e4
4.28
7.21
1.7e5
7.0e4 6.0e4
1.4e5
8.0e4
1.6e5
6.0e4
6.0e4 5.0e4
1.3e5
6.0e4
5.0e4 4.0e4
1.2e5
4.0e4
1.4e5
4.0e4 3.0e4
1.1e5
1.00e5
of -MRM
pairs):
180.000/62.900
Glufosinate
6.4e4
Max.Max.
6.4e4
cps.cps. XICXIC
of -MRM
(11(11
pairs):
180.000/62.900
DaDa
ID:ID:
Glufosinate
11
......
XIC of (11
-MRM
(11 pairs):
168.000/63.000
ID: Glyphosate
XIC of -MRM
pairs):
168.000/63.000
Da ID:Da
Glyphosate
3 ... 3 ...
AMPA
AMPA
67
Table 5. Recoveries observed from 100 μg/kg spikes into
different matrices without the use of any internal standards.
This shows the need for internal standards or matrix matched
calibration lines to counter matrix effects which lead to
recoveries varying with matrix.
AMPA
Ethephon
Glufosinate
Glyphosate
151%
159%
148%
156%
Onion
48%
243%
99%
92%
Grapes
110%
167%
145%
95%
Wheat
106%
213%
155%
155%
Data was processed using MultiQuant™ software version 2.1
with the ‘Multicomponent’ query. Query files are customizable
commands to perform custom querying of the result table.
The ‘Multicomponent’ query automatically calculates and
compares MRM ratios for compound identification and
highlights concentrations above a user specified level. An
example of the results and peak review after running the query
file is shown in Figure 5.
Summary
This study has clearly demonstrated that Glyphosate and
other polar pesticides can be detected at low levels in their
underivatized state using a highly sensitive LC-MS/MS
system, like the AB SCIEX QTRAP® 5500, and separation
using a new HILIC type LC column. The detection of these
compounds is quick even in the non-optimal acidic mobile
phase conditions and is additionally only possible due to the
ability of the Turbo V™ source to deal with highly aqueous
solvents at high flow rates in excess of 1 mL/min. This means
that FMOC derivatization or lengthy ion chromatography is no
longer needed.
All the compounds were identified and quantified using two
MRM transitions at 0.1 mg/kg after 5x dilution of QuPPe
extraction. However, matrix effects were observed so in routine
analysis it is recommended that matrix matched calibration
standards or ideally heavy labeled internal standards are used.
References
1 http://www.crl-pesticides.eu/library/docs/srm/meth_
QuPPe.pdf
2 Regulation (EC) ‘concerning the placing of plant protection
products on the market’ No 1107/2009
3 Commission Regulation (EU) ‘regards maximum residue
levels’ No 441/2012
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Figure 5. Automatic reporting of pesticides using the ‘Multicomponent’ query in MultiQuant™ software: the
query calculates MRM ratios and flags samples with MRL violations.
68
Julia Jasak1, Yves LeBlanc2, Ralf Schöning3, Uwe Thuss3, Karl Speer1, and André Schreiber2
1
Technische Universität, Food Chemistry, Dresden (Germany); 2 AB SCIEX, Concord, Ontario
(Canada) 3 Bayer CropScience AG, Residue Analysis, Monheim (Germany)
Introduction
Table 1. Structure of studied triazole derivative metabolites
Compound
Structure
1,2,4-triazole
TRZ
N
NH
N
HO
Triazole acetic
acid (TAA)
O
N
N
N
Triazole lactic
acid (TLA)
Triazole alanine
TAL
N
N
N
N
N
O
HO
OH
H2N
OH
N
O
Orificep late
DMSc ell
Curtainp late
SelexION™ Technology
The SelexION™ Technology is a planar differential mobility
device (DMS) that attaches between the curtain plate and
orifice plate of the 5500 QTRAP® system. Gas draws the ions
through the DMS cell towards the orifice while an asymmetric
waveform applied to the plates, which alternates between high
field and low field. Unlike traditional ion mobility, ions are not
separated in time as they traverse the cell. They are separated
in trajectory based on difference in their mobility between
the high field and low field portions of the applied Separation
Voltage (SV). As the ions migrate towards the walls of the
DMS cell at different rates, they will be separated. By applying
a second voltage offset (the Compensation Voltage, CoV) the
trajectory of a desired ions can be corrected along the axis of
the DMS cell and transmitted to the mass analyzer (Figure 1).
Chemical modifiers, like isopropanol, methanol, or acetonitrile,
can be and introduced into the transport gas via the curtain
gas, to alter the separation characteristics of analytes.
The planar design of SelexION™ Technology yields a stable,
easy to tune system with high resolving power over a short
distance. This gives high speeds and short residence times,
resulting in minimal diffusion losses and enabling the use of
short MS/MS cycle times. By simply turning off the separation
voltage, the cell becomes transparent with ions moving
normally along the centre line of the device. Thus it is possible
to transmit ions through the mobility cell when not using the
DMS mode.
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1,2,4-triazole (TRZ), triazole alanine (TAL),triazole acetic
acid (TAA) and triazole lactic acid (TLA) are metabolites
that commonly occur as plant or soil metabolites of
triazole fungicides and they are collectively known as the
‘triazole derivative metabolites’ (Table 1). Therefore, the
determination of levels of triazole derivative metabolites in
soils and plant materials is the key in assessing the fate of
triazole fungicides.
However, analysis of these metabolites by Liquid
Chromatography coupled to tandem Mass Spectrometry
(LC-MS/MS) is challenging because of their polar nature
and their poor fragmentation efficiency (fragmentation into
a single fragment only). In addition, when dealing with soil
and plant extracts, LC-MS/MS analysis typically suffers
from high chemical noise and many interferences. Here
we evaluated the use of differential mobility spectrometry
(DMS) using the AB SCIEX SelexION™ technology coupled
to a QTRAP® 5500 LC/MS/MS system to improve the
selectivity of LC-MS/MS detection of triazole derivative
metabolites
Improving the LC-MS/MS Selectivity of Triazole Derivative
Metabolites with AB SCIEX SelexION™ Technology
69
SelexION™ Settings
Gasf low
To MS/MS
SV
Compensation
Voltage( COV)
CoV
Figure 1. Differential Mobility Separation Process. Innovative
planar design of the DMS cell uses an asymmetric RF
waveform (SV) to separate ions based on differential mobility
between the high and low fields. The compensation voltage
(CoV) is used to correct the trajectory of the ion of interest
which traverses the cell and into the orifice while interferences
are deflected into the cell walls.
Method Details
Sample preparation
The following matrices were evaluated in the present study;
carrot leafs, carrot roots, 2 different lots of rape green material,
rape seeds, lettuce head, grape, and water. Each matrix was
extracted using the following procedure:
• Weighing 5g of material
• Homogenization in methanol/water (4/1) with an Ultra
Turrax
• Filtration with Celite
• SPE cleanup using C18 material
• Evaporation of eluate to dryness
• Reconstitution in water
• Addition of 15N-labeled internal standard
Each sample was prepared at three different concentrations:
control (0), recovery LOQ (0.01 mg/kg) and 10x LOQ
(0.1 mg/kg
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LC separation
LC was performed using a Shimadzu UFLCXR system
with an Aquasil C18 (3x150 mm; 3 μm) column using a 2
minute gradient of 100% to 90% aqueous. The mobile phase
consisted of (A) water + 0.5% acetic acid and (B) methanol +
0.5% acetic acid.
70
SV was set to 3400 V and CoV were tuned for each analyte
of interests to obtain highest selectivity (Figure 2). No
chemical modifier was introduced. The DMS cell was used
in ‘transparent’ mode (SV and CoV turned off) to mimic
conventional MS/MS operation.
MRM transitions for all compounds, retention time (RT) and
CoV values are listed in Table 2.
Table 2. MRM transitions, optimized CoV, and retention time
(RT) of each triazole derivative metabolite
Compound
MRM
CoV
RT (min)
TRZ
70/43
-17.0
1.70
TAA
128/70
-4.0
1.91
TLA
158/70
0.5
1.96
TAL
157/70
2.0
1.30
TRZ
TAA
TLA
TAL
Figure 2. Optimization of CoV of each triazole derivative
metabolite to obtain highest selectivity
MS/MS Detection
Results
An AB SCIEX QTRAP® 5500 system with Turbo V™ source
and the Electrospray Ionization (ESI) probe was used. The
source was operated at 600°C with Gas 1 and Gas 2 at 40
and 80 psi, respectively. Curtain gas was set at 20 psi.
High background and matrix interferences are the analytical
challenges associated with the LC-MS/MS analysis of triazole
derivative metabolites (Figure 3). As can be seen, each analytes
exhibits variable interferences (high background levels as well
as multiple LC peaks) that depends on the matrix analyzed.
arrotR oots
Rape Green( 1)
Rape Green( 2)
Rape Seed
LettuceH eadG
rapes
TAA
TLA
TAL
Carrot LeafsC
TRZ
Figure 3. MRM traces for recovery LOQ (0.01 mg/kg) in various matrices when acquisition was performed with DMS cell in
transparent mode
Carrot LeafsC
arrotR oots
Rape Green( 1)
Rape Green( 2)
Rape Seed
LettuceH eadG
rapes
TAA
TLA
TAL
Figure 4. MRM traces for recovery LOQ (0.01 mg/kg) in various matrices when acquisition was performed with DMS cell
optimized for each analyte
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TRZ
71
Furthermore, minimal chromatographic separation was
achieved due to the polar nature of the analytes, but still
required to minimize isotope contribution in MRM channels.
Figure 4 shows the same matrix spiked samples analyzed with
DMS optimized for each triazole derivative metabolite. Due to
the increased selectivity single LC peaks were observed for
each analyte, with the exception of TAL in some matrices.
Even in cases where LC interferences were observed the
dominant LC peaks were associated with TAL. In addition, the
noise level was significantly reduced.
In order to quantify the reduction of the noise level, all spiked
samples (at 0.01 and 0.1 mg/kg) were integrated by summing
all intensities within a 15 sec window around the retention time
of the analyte (LC peak width at peak base). This value was
divided by the sum of all intensities within a 60 sec window (4x
LC peak width). If the noise levels (either chromatographically
resolved or unresolved) around the peak of interest is low,
than this ratio approaches a value of 1. A value significantly
below 1 indicates strong matrix interferences. Figure 5 shows
the results obtained for all spiked samples when DMS was
operated in transparent mode (A) and optimized for each
analyte (B).
1
0.9
Figure 5 A shows that the noise around the LC peaks is
elevated since the ratio is still <0.7 in many cases even when
the analytes are spiked at 10x LOQ. In contrast, Figure 5 B
shows that the ratio is greater than 0.8 in all but 3 cases (TAL
in 3 matrices), at both the LOQ and 10x LOQ level when DMS
is used.
Thus, the SelexION™ Technology provided additional
selectivity that increases confidence in the detection of
triazole derivative metabolites, reduced the LC requirements,
and simplified the data review and peak integration process.
Figure 6 shows the MRM signal across multiple CoV values
over the entire LC analysis. This is performed by monitoring
the MRM transition while ramping CoV throughout the
chromatographic run. This provides a ‘map’ in CoV space
of the analyte versus interferences of the same MRM. Rape
green spiked at 10x LOQ was used to generate the CoV map
of TRZ and TAA. Figure 6 clearly shows that the analytes of
interest are clearly separated from the chemical interferences
in terms of CoV values, in addition to LC time.
(A)
TIC7 0/43
TIC1 28/70
0.8
0.7
0.6
TAA
0.5
TAL
TLA
0.4
TRZ
2.0
0.0
0.2
GF (LOQ) GF (10xLOQ) CR (LOQ) CR (10xLOQ) CL (LOQ)
CL (10xLOQ)
LH (LOQ) LH (10xLOQ)
RGM-1
(LOQ)
RGM-1
(10xLOQ)
RGM-2
(LOQ)
RGM-2
(10xLOQ)
RS (LOQ)
-4.0
-6.0
7.6
6.0
4.4
2.8
-8.0
-10.0
RS (10xLOQ)
(B)
TRZ
CoV( V)
CoV( V)
0
1
map 70/43
-2.0
0.1
0.9
9.2
4.0
0.3
0.4
2.0
-12.0
-3.6
-14.0
-16.0
-5.2
-18.0
-8.4
-10.0
-20.0
TAA
1.2
-6.8
map1 28/70
0.8
0.7
0.6
TAA
0.5
TAL
TLA
0.4
TRZ
0.3
Figure 6. Separation of interferences of TRZ (left) and TAA
(right) at 10x LOQ in rape green in the CoV space and on the
LC time scale
0.2
0.1
0
GF (LOQ) GF (10xLOQ) CR (LOQ) CR (10xLOQ) CL (LOQ)
CL (10xLOQ)
LH (LOQ) LH (10xLOQ)
RGM-1
(LOQ)
RGM-1
(10xLOQ)
RGM-2
(LOQ)
RGM-2
(10xLOQ)
RS (LOQ)
RS (10xLOQ)
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Figure 5. Complexity of noise around LC peak of interest
across for all matrices at the LOQ and 10x LOQ with DMS
operated in (A) transparent mode and (B) optimized for each
analytes
72
Finally, quantitative performance under three different LCMS/MS configurations was compared: DMS on, DMS off
(cell mounted and operated in transparent mode) and DMS
removed (cell physically removed). Linearity (linear regression
with 1/x weighting), precision, and accuracy were found to be
similar using all three configurations (Table 3).
DMS on
DMS off
DMS removed
TAA
TLA
Actual conc.
(ng/mL)
Calculated
conc.
(ng/mL)
Accuracy
Calculated
conc.
(ng/mL)
TAL
TRZ
Accuracy
Calculated
conc. (ng/mL)
Accuracy
Calculated
conc. (ng/mL)
Accuracy
0.5
0.57
114.2
0.53
106.0
0.58
115.7
0.50
100.1
1.0
1.07
107.1
0.94
94.4
0.89
89.0
1.03
102.5
2.5
2.42
96.7
2.52
100.6
2.43
97.4
2.59
103.4
5.0
4.66
93.1
4.88
97.6
4.90
98.0
4.84
96.8
10
8.54
85.4
10.2
101.5
9.95
99.5
9.65
96.5
50
51.8
103.5
50.0
100.0
50.3
100.5
50.4
100.8
0.5
0.55
110.6
0.55
109.0
0.45
89.6
0.42
84.5
1.0
1.02
101.8
0.97
96.6
1.06
105.8
1.16
115.8
2.5
2.43
97.4
2.43
97.3
2.37
94.8
2.67
106.8
5.0
4.63
92.6
4.96
99.3
5.11
102.2
4.73
94.6
10
9.62
96.2
9.72
97.2
10.1
109.5
9.79
97.9
50
50.8
101.5
50.4
100.8
49.1
98.1
50.2
100.5
0.5
0.386
77.3
0.30
59.1
0.30
59.1
0.48
96.3
1.0
1.05
105.0
0.98
97.5
0.98
97.5
1.03
102.5
2.5
2.60
104.1
2.77
110.8
2.77
110.8
2.29
91.5
5.0
5.30
106.0
5.90
120.5
5.90
118.0
5.28
105.6
10
11.0
110.4
12.1
120.5
12.1
120.5
10.5
105.2
50
48.6
97.3
47.0
94.0
47.0
94.0
49.4
98.8
Summary
References
The combination of LC-DMS-MS/MS provides a high degree
of selectivity for the analysis to triazole derivative metabolites
across several matrices extracted. Significant reduction in
noise levels was obtained when using the AB SCIEX SelexION™
Technology. Single LC peaks were obtained for TRZ, TAA, and
TLA in all matrices and for TAL in most matrices.
Overall, combining the DMS with the AB SCIEX QTRAP® 5500
system enabled the detection of triazole derivative metabolites
with high confidence at desired LOQ levels of 0.01 mg/kg and
excellent precision. This technique proved to be extremely
useful in the detection and monitoring of these species.
1 B.B. Schneider, T. R. Covey, S.L. Coy, E.V. Krylov, E.G.
Nazarov: Int. J. Mass Spectrom. 298 (2010) 45-54
2 B.B. Schneider, T. R. Covey, S.L. Coy, E.V. Krylov, E.G.
Nazarov: Anal.Chem. 82 (2010) 1867-1880
Compound
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Table 3. Precision and accuracy obtained for single injections of solvent standards using three instrument configurations (DMS
on, DMS off, and DMS removed)
73
Fast and Sensitive Analysis of Paraquat and Diquat in
Drinking Water
Houssain El Aribi
AB SCIEX Concord, Ontario (Canada)
Overview
This application note describes a fast and sensitive LC-MS/MS method
for the determination of Paraquat and Diquat in drinking water. Using the
Ultra Quat HPLC column and the AB SCIEX API 3200™ LC/MS/MS System
equipped with a Turbo V™ source, the limits of quantitation (LOQ) for this
method in drinking water are 0.1 μg/L and 5 μg/L respectively for Diquat and
Paraquat using a 10 μL injection volume without sample preparation prior
to analysis.
Introduction
Paraquat (1,1’-dimethyl-4,4’-bipyridylium dichloride, C12H14N2Cl2), and
Diquat (1,1’-ethylene-2,2’-bipyridilium dibromide, C12H12N2Br2), are nonselective and nonsystematic contact herbicides widely used in agriculture
to control broadleaf and grassy weeds. The use of these herbicides is
very important because weeds compete vigorously with crops for water, light and other nutrients. As a result, if they are
not suppressed they reduce crop yields by up to 80%. However both Parquat and Diaquat are toxic and ingestion of either
compound can have serious effects as they can alter reduction-oxidation activities in biological systems. The analysis of these
highly charged dual quaternary amines is complicated because of their ionic nature and therefore Paraquat and Diquat are
difficult to retain by standard reversed phase HPLC. For drinking water the United States Environmental Protection Agency
(EPA) has established a maximum contaminant level of 20 μg/L for Diquat.1 Paraquat is currently not regulated in drinking
water to our knowledge.
The EPA 549.2 method for the analysis of Paraquat and Diquat uses reversed phase/ion-pair extraction utilizing C8 SPE
cartridges followed by ion-pair LC with ultraviolet (UV) and/or photodiode array (PDA) detection.2 This method is timeconsuming and requires large sample volume, and suffers from stability and reproducibility problems associated with ionexchange chromatography. Recently, various mass spectrometry (MS) methods have been developed for the analysis of these
herbicides. Although these methods have lower limit of detection, an extensive cleanup is generally required.3-4
Experimental
Chemicals
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Paraquat (1,1’-dimethyl-4,4’-bipyridylium dichloride, C12H14N2Cl2), and Diquat (1,1’-ethylene-2,2’-bipyridilium dibromide,
C12H12N2Br2), are non-selective and nonsystematic contact herbicides widely used in agriculture to control broadleaf and
grassy weeds. The use of these herbicides is very important because weeds compete vigorously with crops for water, light
and other nutrients. As a result, if they are not suppressed they reduce crop yields by up to 80%. However both Parquat and
Diaquat are toxic and ingestion of either compound can have serious effects as they can alter reduction-oxidation activities in
biological systems. The analysis of these highly charged dual quaternary amines is complicated because of their ionic nature
and therefore Paraquat and Diquat are difficult to retain by standard reversed phase HPLC. For drinking water the United States
Environmental Protection Agency (EPA) has established a maximum contaminant level of 20 μg/L for Diquat.1 Paraquat is
currently not regulated in drinking water to our knowledge.
The EPA 549.2 method for the analysis of Paraquat and Diquat uses reversed phase/ion-pair extraction utilizing C8 SPE
cartridges followed by ion-pair LC with ultraviolet (UV) and/or photodiode array (PDA) detection.2 This method is timeconsuming and requires large sample volume, and suffers from stability and reproducibility problems associated with ionexchange chromatography. Recently, various mass spectrometry (MS) methods have been developed for the analysis of these
herbicides. Although these methods have lower limit of detection, an extensive cleanup is generally required.3-4
74
LC
An Agilent 1100 series equipped with degasser, quaternary pump, and autosampler was used. HPLC separation was performed
on a Restek Ultra Quat 3μm (50x2.1mm) with guard column Ultra Quat 3 μm (20x2.1 mm) and an isocratic mobile phase
of 95% water + 5% acetonitrile + 10mM of HFBA at a flow rate of 500 μL/min. The injection volume was set to 10 μL. Low
concentrations of HFBA effectively shield the positive charges of Paraquat and Diquat, increasing interaction with the Ultra Quat
stationary phase, resulting in more retention required to separate analytes from matrix components.
An AB SCIEX API 3200™ LC/MS/MS system equipped with a
Turbo V™ source operating in Electrospray Ionization (ESI)
mode and positive polarity was used. The following source
and gas parameters were applied: TEM=700°C; CUR=15 psi;
GS1=70 psi; GS2=60 psi; IS=5500 V; and CAD=7.
Compound dependent parameters, such as Declustering
Potentials (DP), Collision Energies (CE), and Collision Cell Exit
Potential (CXP) for each detected MRM transition are listed
in Table 1. Two transitions a quantifier and a qualifier were
monitored. A dwell time of 200 ms were used per transition.
At the analytical conditions used, Paraquat and Diquat
preferentially form the singly charged [M2+-H+] ions as the
MS base peaks. However, the doubly charged [M2+] ions at
m/z 92 (Diquat) and m/z 93 (Paraquat) and the radical [M+.]
cations at m/z 184 (Diquat) and m/z 186 (Paraquat) have
also been formed at much lower relative intensities. The MS/
MS spectrum of singly charged Diquat (m/z 183) is quiet
simple compared to that of the singly charged Paraquat (m/z
185) as illustrated in Figure 1.
MS/MS
Table 1. Detected MRM transitions for the analysis of Paraquat and Diquat
Analyte Name
MRM transition
Diquat [M2+-H+]
( 3 0 ): 2 0
M C A
40
35
193/178
29
183/157
32
183/168
D4-Diquat [M2+-D+]
35
fr o m
S a m p le 2
( C ID
o f 1 8 5 _ C E = 3 0 V _ Q 3
3
35
186/158
s c a n s
CXP (V)
30
185/169
D8-Paraquat [M2+-H+]
(1 8 5 . 0 0 ) C E
CE (V)
185/170
Paraquat [M2+-H+]
+ M S 2
DP (V)
32
h ig h
R e s ) o f D iq u a t a n d P a r a q u a t . w i f f ( T u r b o S p r a y )
M a x . 3 . 2 e 5
c p s .
1 8 5 .3
3 .2 e 5
3 .0 e 5
Paraquat
2 .8 e 5
[M2+ - H+]
a – quantifier MRM 185/170
b – qualifier MRM 185/169
2 .6 e 5
2 .4 e 5
2 .2 e 5
a
1 7 0 .2
2 .0 e 5
1 .8 e 5
1 .6 e 5
b
1 5 8 . 2
1 .4 e 5
1 4 4 .3
1 .2 e 5
1 .0 e 5
1 1 8 .3
8 .0 e 4
1 4 3 .2
6 .0 e 4
1 0 7 .2
1 8 3 .2
4 .0 e 4
1 4 2 . 1
2 .0 e 4
4 3 .2
2 0
+ M S 2
3 0
(1 8 3 . 0 0 ) C E
4 0
( 2 5 ): 2 0
5 5 . 2
5 7 .0
5 0
M C A
s c a n s
9 2 .3
7 7 . 1
6 5 . 1 6 9 .2
6 0
fr o m
7 0
S a m p le 3
( C ID
1 0 6 .1
8 3 .2
1 1 5 .2
1 1 7 .3
1 2 8 .3
1 3 9 .2
1 2 1 .3
1 6 8 .0
1 5 5 .0
1 3 1 .2
1 4 5 .3
8 0
9 0
1 0 0
1 1 0
1 2 0
1 3 0
1 4 0
m /z , a m u
o f 1 8 3 _ D iq u a t _ C E = 2 5 V ) o f F r a g m e n t a t i o n _ M a r c h 7 _ 0 6 . w i f f ( T u r b o S p r a y )
1 5 9 .2
1 5 3 . 2
1 5 0
1 6 0
1 8 2 .1
1 7 0
1 8 0
1 9 0
M a x . 2 . 8 e 6
2 0 0
c p s .
1 5 6 .9
2 .8 e 6
Diquat
2 .6 e 6
2 .4 e 6
a – quantifier MRM 183/157
b – qualifier MRM 183/168
2 .2 e 6
2 .0 e 6
1 .8 e 6
[M2+- H+]
a
1 .6 e 6
1 8 2 .9
1 .4 e 6
1 .2 e 6
1 .0 e 6
8 .0 e 5
6 .0 e 5
b
2 .0 e 5
4 3 .3
3 0
4 0
5 4 .8
5 0
5 6 .7
7 8 . 1
6 6 .8
6 0
7 0
7 9 .8
8 0
8 2 . 7
9 2 .7
9 0
Figure 1. MS/MS spectra of Paraquat and Diquat
9 8 .8
1 0 0
1 0 4 .2
1 6 8 .1
1 3 0 .0
1 1 7 .2 1 2 2 . 9
1 1 4 .8
1 1 0
1 2 0
m /z , a m u
1 3 9 .7
1 3 0
1 5 5 .3
1 4 2 .1
1 4 0
1 5 0
1 6 5 .1
1 6 0
1 7 0
1 8 2 .0
1 8 0
1 8 4 .2
1 9 0
2 0 0
www.spektrotek.com
4 .0 e 5
75
XIC of +MRM( 6 pairs): 183.0/157.0 amu from Sample 21( P_D_Quats_500ng_mL_500ng_mL ISTD) of P_D_Quats_ISTDs_CAL_Marc...
Max. 3.1e5 cps
5.03
100%
95%
Diquat
90%
Paraquat
MRM1 83/157
MRM1 83/168
MRM1 86/158 (ISTD)
85%
80%
75%
MRM1 85/170
MRM1 85/169
MRM1 93/178 (ISTD)
70%
65%
60%
55%
50%
45%
40%
35%
30%
25%
20%
15%
10%
5%
0%
0.51
.0
1.52
.0
2.5
3.03
.5
4.04
.5
5.05
Time, min
.5
6.06
.5
7.07
.5
8.08
.5
9.09
.5
Figure 2. Separation and detection Diquat and Paraquat in drinking water (500 μg/L) by LC-MS/MS
For the highest sensitivity and selectivity used Multiple
Reaction Monitoring (MRM) was used to quantify Paraquat
and Diquat in DI water and drinking water.
Figure 2 shows the analysis of drinking water spiked with
500 μg/L of Paraquat, D8-Paraquat, Diquat, and D4-Diquat.
Two MRM transitions were selected for each analyte.
Internal standards were used to improve the accuracy of
quantitation, to compensate for matrix effects, and to correct
for random and systematic errors in separation and detection.
Triplicate injections of 9 concentrations of analytes in DI water
and in drinking water, from 0.1 to 500 μg/L for Diquat and
from 0.5 to 500 μg/L for Paraquat were used to investigate the
performance of the developed method.
Correlation coefficients for calibration curves were >0.999,
using a linear fit and 1/x weighting factor. These results
indicate that quantification can be performed with good
linearity and sensitivity. Figure 3 and 4 show the calibration
curve for Diquat (MRM 183/157) and Paraquat (185/170) in
the case of drinking water.
Table 2 summarizes the statistical parameters for the
analysis of Diquat and Paraquat in drinking water. The limits
of quantitation (LOQ) of the analytes were calculated from
the chromatograms, at a signal-to-noise-ratio of >10.
Table 2. Summary of statistical parameters
www.spektrotek.com
Analyte Name
76
MRM transition
LOQ (µg/L)
Linear range ( µg/L)
R2
Diquat
183/157
0.1
0.1 – 5000
0.9996
Paraquat
185/170
0.5
0.5 – 5000
0.9999
10.0
Di_Para_Quats_ISTD_Mar ch 9_06.rdb (183.0 /1 57.0): "Linear" Regression ("1/ x" weighting):y =1 .01x +0 .000817( r= 0.9996)
Diquat
9.5
MRM1 83/157
R2 =0 .9996
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.00
.5
1.01
.5
2.02
.5
3.03
.5
4.04
.5
5.05
.5
Analyte Conc./ IS Conc.
6.06
.5
7.07
.5
8.08
.5
9.09
.5
10.0
Figure 3. Calibration curve for Diquat (183/157) in drinking water from 0.1 μg/L to 5000 μg/L
Di_Para_Quats_ISTD_March9 _06.rdb (185.0/ 170.0): "Linear" Regression ("1 / x" weighting):y = 1.28 x+ -0.00099( r= 0.9999)
13
13
12
12
11
11
10
Paraquat
MRM1 85/170
R2 =0 .9999
10
9
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
0
Figure 4. Calibration curve for Paraquat (185/170) in drinking water from 0.5 μg/L to 5000 μg/L
Summary
The use of the Restek Ultra Quat 3 μm HPLC column with an eluent containing Heptafluorobutyric acid allows sufficient
separation of Paraquat and Diquat. Coupled to an API 3200™ LC/MS/MS systems enough sensitivity of detection is provided
to inject water samples directly without any time-consuming sample preparation prior to analysis. The method was found to
be robust, selective and sensitive.
References
US EPA, Drinking Water Health Advisory: Pesticides, US Environmental Protection Agency, Lewis, Chelsea, MI, 1989
J. W. Munch, W. J. Bashe, US EPA 549.2, US Environmental Protection Agency, Cincinnati, OH, 1997
R. Castro, E. Moyano, M. T. Galceran J. Chromatogr. A 2001, 914, 111-121
L. Grey, B. Nguyen, P. Yang J. Chromatogr. A 2002, 958, 25-33
www.spektrotek.com
1
2
3
4
77
The Quantitation and Identification of Coccidiostats in Food
by LC-MS/MS using the AB SCIEX 4000 Q TRAP® System
Bertram Nieland1 and Stephen Lock2
1
AB SCIEX Nieuwerkerk aan den Ijssel, The Netherlands; 2 AB SCIEX Warrington, UK
Introduction
Coccidiostats are antiprotozoal agents that act upon parasites. In animal
production, particularly in intensive animal rearing coccidiostats are used to
treat infections and as such meat, chicken, egg and milk are regularly tested
for these compounds. Recently maximum levels for these compounds (due
to unavoidable carry-over of authorized coccidiostats to non-target feed)
were set by the EU in Commission Regulations [(EC) No 124/2009]1 so
methods for their detection were required. This work compares the traditional
approach to sample preparation of solid phase extraction (SPE) followed by
separation on a conventional 5µm particle column with that of the quicker
and simpler QuEChERS2-3 technique followed by separation with a newer
2.6 µm particle column and shows how liquid chromatography coupled to
tandem mass spectrometry (LC-MS/MS) can be used to detect coccidiostats
including Narasin, Diclazuril and Monensin in milk.
Experimental
1) Conventional Approach
Sample Preparation
LC
Milk (2.0 g) in a Polypropylene Tube was mixed with
acetonitrile (2 mL) and vortexed for 40 seconds. Another 2
mL of acetonitrile was added and the the tube was sealed,
shaken by hand and then continually mixed using a head over
head mixer for 15 minutes. The sample was then centrifuged
for 15 minutes (3600 g at 4°C). The supernatant was removed
and water (16 mL) and ammonia solution (1 mL, 25%) were
added and this mixture was shaken. The whole extract was
loaded onto an OASIS HLB SPE cartridge (3 cm3, 60 mg)
which previously had been conditioned with methanol (3 mL)
and water (3 mL). The cartridge was washed with ammonia (5
mL, 1.25%) dried for 2 minutes under vacuum and eluted with
methanol (5 mL). The eluent was evaporated to dryness, the
sample was reconstituted in methanol/water (1 mL, 50/50),
vortexed, and sonicated for 5 minutes before injection
Column
: Agilent Zorbax Eclipse XDB-C8, 5 µm,
150 x 4.6 mm
Flow rate
: 400 μL/min
Oven temperature: 40 ºC
Injection Volume : 40 µL
Mobile Phase A : water + 0.2% acetic acid
Mobile Phase B : methanol + 0.2% acetic acid
www.spektrotek.com
Table 1. LC gradient profile of conventional approach
78
Step
Time (min)
A (%)
B (%)
1
0.5
100
0
2
1.5
20
80
3
10
10
90
4
13
0
100
5
18
0
100
6
18.5
100
0
7
23
100
0
The sample extraction was based on a QuEChERS method
by Anastassiades et al. and Lehotay et al.2-3 Milk in a
polypropylene tube (50 mL) was roller mixed with acetonitrile.
To this mixture anhydrous magnesium sulfate and sodium
acetate were added and samples were shaken vigorously and
centrifuged. Anhydrous magnesium sulfate, PSA and C18
were added to an aliquot (2 mL) of the upper layer and these
samples were shaken by hand. This mixture was centrifuged
and the supernatant transferred into an autosampler vial for
analysis.
were additionally acquired to increase confidence in compound
identification using mass spectral library searching. In this
mode an information dependent acquisition (IDA) experiment
was used to automatically trigger the MS/MS spectra
acquisition when a chromatographic MRM signal exceeded a
threshold of 1000 cps.
The maximum residue limits for the coccidiostats vary with
analyte (Table 3). The analysis is further complicated by the
fact that Diclazuril ionizes in negative polarity so to maximize
sensitivity the method contains periods, so it switches from
positive to negative and back to positive as shown in Figure 1.
2) New Approach
Sample Preparation
LC
Table 3. Maximum Residue limits (MRL) for some
coccidiostats1
Coccidiostats
Table 2. LC gradient profile of new approach with a
Phenomenex Kinetex column using 2.6 μm core-shell
particles for increased efficiency and improved performance
1
1.0
100
0
2
2.5
20
80
3
5.0
10
90
4
7.5
0
100
5
9.2
0
100
6
9.5
100
0
7
11.5
100
0
MS/MS
1
Maduramycin
2
Monensin
2
Narasin
1
Robenidine
5
Salinomycin
2
XICo f +MRM (12p airs): Period 3, 787.596/431.200 Da ID: Narasin0 2f romS ample1 0( 2.00...M
2.6e5
positive polarity
2.4e5
Negative and positive polarities were used with polarity
switching during, the chromatographic run, to cover all target
analytes.
For best selectivity and sensitivity Multiple Reaction
Monitoring (MRM) mode was used for detection. Two MRM
transitions were detected per compound to allow quantitation
and identification by MRM ratios (Table 4). However, since
detection in MRM mode only can lead to false positive results
full scan MS/MS spectra
positive polarity
2.0e5
1.8e5
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
Robenidine
4.0e4
The AB SCIEX 4000 Q TRAP system was used with Turbo V™
source and Electrospray Ionization (ESI) probe. The source
was heated to 600ºC with 45 psi nebulizer and heater gas.
ax.2 .6e5 cps.
16.0
2.2e5
6.0e4
®
negative
polarity
2.0e4
0.0
2468
10
12
Time,m in
14
16
18
20
Figure 1. Example of an LC-MS/MS chromatogram from a
milk matrix matched calibration standard (concentration
of coccidiostats ranging from 2 to 10 μg/kg) prepared and
analyzed using the conventional approach
www.spektrotek.com
B (%)
5
Lasalocid
Narasin
A (%)
Diclazuril
Monensin A
Lasalocid
Maduramycin
Salinomycin
Time (min)
Intensity, cps
Step
MRL in milk (μg/kg)
Diclazuril
Column
: Phenomenex Kinetex C8, 2.6 µm,
100 x 4.6 mm
Flow
: 600 μL/min
Oven temperature: 40 ºC
Injection Volume : 40 µL
Mobile Phase A : water + 0.2% acetic acid
Mobile Phase B : methanol + 0.2% acetic acid
79
Table 4. Targeted coccidiostats with retention times, polarity, and detected MRM transitions using the Phenomenex Kinetex
C8 column
Coccidiostats
CAS
Structure
RT (min)
Polarity
Q1 (amu)
Q3 (amu)
N
Cl
Diclazuril
O
101831-37-2
Cl
Cl
Decoquinate
18507-89-6
H3C
CH3
O
O
OC
25999-31-9
O
HO
O
CH3
CH3
204
372
7.1
613
377
595
7.2
939
877
895
7.0
693
461
479
6.8
787
279
431
531
7.8
747
703
501
4.6
334
6.6
773
431
531
265
6.4
423
377
OH
OH
O
OO
H3C
H
CH3
CH3
H3C
CH3
O
O
O
H3C
H3C
CH3
O
H3C
Maduramycin
418
H3
CH3
H3C
Lasalocid
6.4
O
O
N
H
H3C
334
336
negative
NH
N
O
405
407
5.3
N
O
O
84878-61-5
CH3
OH
O
H3C
O
CH3
OH
O
OH
O
CH3
O
OH
CH3
CH3
O
O
H3C
CH3
H3C
H3C
Monensin A
O
O
17090-79-8
OH
OH
O
H3C
CH3
O
O
OH
O
CH3
CH3
O
CH3
CH3
CH3
CH3
CH3
CH3
positive
Narasin
O
H3C
55134-13-9
O
OH
O
CH3
O
O
H3C
O
O
H3C
CH3
OH
OH
CH3
HO
H3C
CH3
H3C
H3C
Nigericin
O
O
OH
OH
O
28643-80-3
H3C
O
CH3
O
OH
O
H3C
H3C
CH3
O
Robenidine
N
25875-51-8
CH3
NN
Cl
H3C
www.spektrotek.com
H3C
80
N
Cl
CH3
O
O
CH3
CH3
OH
O
O
O
O
OH
138
111
CH3
O
53003-10-4
H3C
Decoquinate D5
(internal standard)
H
NH2
CH3
Salinomycin
O
CH3
OH
CH3
HO
XICo f +MRM (3 pairs):P eriod1 ,3 34.055/138.100 Da ID: Robenidine1 from Sample 25 (2 ...M
positive polarity
4.5e4
Robenidine
4.6
4.0e4
3.0e4
2.5e4
Diclazuril
Intensity,c ps
3.5e4
2.0e4
1.5e4
1.0e4
5000.0
0.0
1.02
.0
3.04
.0
5.06
.0
Time,m in
Lasalocid
Decoquinate
5.0e4
ax.4 .2e4 cps.
positive polarity
negative
polarity
5.5e4
When both approaches, the conventional using SPE and the
new one using QuEChERS, were compared both showed
coefficients of variation (% CV) of less than 10% at or below
the LOD levels needed except for Robenidine whose CV was
19% using the SPE methodology (Table 5). This showed
that both methods could be applied to food samples. Both
approaches produced linear responses and r values > 0.985
(see examples in Figure 3). This included the QuEChERS
method which used spiked calibration standards whose
concentration ranged from 0.2 to 50 μg/L with the exception
of Decoquinate whose fit was quadratic over this range. The
internal standard Decoquinate D5 was later used to correct the
non linearity and additional internal standards could further
improve these results.
Salinomycin
Narasin
Monensin A
Maduramycin
Nigericin
5.9e4
To assess the sensitivity of the developed method the
coccidiostats were spiked into milk and extracted using the
QuEChERS procedure. The results showed that this technique
was capable of detecting all the coccidiostats reproducibly in
milk at concentrations below 1 μg/L.
7.08
.0
9.01
0.01
The conventional approach using a 5 µm column, as shown
in Figure 1, produced peaks with peak widths in the range of
12 to 30 seconds and a run time of 23 minutes. When this
method was switched to the Kinetex core-shell particle column
the peak widths were reduced to between 7 and 12 seconds
and the run time could be reduced to 11.5 minutes (Figure 2).
1.0
milk matrix matched calibration standard (concentration
of coccidiostats ranging from 2 to 10 μg/kg) prepared and
analyzed using the new approach
Robenidine
To further speed up the analysis the off-line SPE was replaced
by the simpler QuEChERS sample preparation technique,
which is commonly used in pesticide residue analysis. The
resulting simplification of the extraction produced dirtier
extracts but the background interferences did not co-elute
with analytes so this approach was shown to be a feasible
alternative.
� ��
Diclazuril
Figure 3. Calibration line for Robenidine (top) and Diclazuril
(bottom) 0.2 to 50 μg/L in milk using the new approach with
QuEChERS extraction and fast chromatography
Table 5. Reproducibility from the repeat analysis of a low spiked matrix matched standard
Concentration of spiked
SPE extract (μg/L)
% CV (4 replicates)
using the conventional
approach
Concentration of QuEChERS extract (μg/L)
% CV (4 replicates) using
the new approach
1
7.7
Diclazuril
1.25
2.6
Lasalocid
0.25
6.3
Maduramycin
0.5
0.7
Monensin
0.5
2.9
Narasin
0.25
4.7
Robenidine
1.25
18.8
1
Salinomycin
0.5
3.6
0.5
5.1
0.5
3.5
3.8
4.7
7.9
www.spektrotek.com
Coccidiostats
81
There are known cases, especially in food analysis, when
MRM ratios can be misleading and produce false positive
results therefore additional information for identification is
beneficial.
So in addition to collecting MRM data there is the possibility
of automatically acquiring full scan MS/MS spectra when an
MRM signal exceeds a defined threshold. These full scan MS/
MS spectra [Enhanced Product Ion (EPI) spectra] are highly
characteristic and sensitive using this unique scan function
of a Q TRAP® system. Figure 4 shows two examples of how
MRM triggered EPI spectra further aids identification of
coccidiostats in food samples.
XICo f+ MRM( 16 pairs) : Exp1 ,7 47.600 /703.600 Da I...
65 00
positive polarity
60 00
55 00
Max. 6600.0 cp s.
4500
4000
In te n s it y , cp s
45 00
40 00
35 00
30 00
25 00
20 00
3500
3000
2500
2000
1500
15 00
1000
10 00
500
500
5.0
6.07
.0
Time ,m in
+EPI (747.60) Ch arge (+ 0) CE (35)C ES (15)F T( 25 0)...M
1.02
.0
3.04
.0
8.09
.0
10 .0
11.0
ax.7 .4 e5 cps.
0
24
68
Time ,m in
-EPI (406 .93) Ch arge (+0) FT (50) :E xp 2, 5.3 26 minf r...
74 7.5
7. 4e5
7. 0e5
1.8e6
EPIs pectrumf or
Diclazuril
1.6e6
1.4e6
I n te ns ity , c ps
5.0e5
4. 0e5
3. 0e5
1.2e6
1.0e6
8.0e5
6.0e5
2. 0e5
437. 3
1. 0e5
10 02
00
465.2
3004
00
325.2
4.0e5
729. 5
333. 2
187. 1
10
Ma x. 1.9e 6c ps
406. 9
EPI spectrum for
Nigericin
6. 0e5
0. 0
negative polarity
5000
7.76
ax.5 36 7.5 cps
5.3 0
5368
5000
0
XICo f- MR M( 2p airs): Exp1 ,4 06.931 /335.7 00 Da ID...M
500
6007
m/z, Da
2.0e5
706.7
00
8009
00
10 00
0.0
10 02
00
3004
00
500
6007
m/z, Da
00
8009
00
10 00
Summary
The LC-MS/MS approaches discussed in this work have been
shown to be suitable for the detection of coccidiostats in food
at the required sanctioned levels.
When the sample preparation was simplified using a
QuEChERS procedure and a core-shell particle column was
used the additional sensitivity of this assay enabled the
detection of these residues below the MRL required but at
over twice the speed of the conventional method which
enables a reduction in cost of the analysis.
References
1. Commission Regulation (EC) No 124/2009 ‘Setting
maximum levels for the presence of coccidiostats or
histomonostats in food resulting from the unavoidable
carry-over of these substances in non-target feed’
2. M. Anastassiades et al.: ‘Fast and easy multi-residue
method employing acetonitrile extraction/partitioning and
dispersive solid-phase extraction for the determination
of pesticide residues in produce’ J. AOAC Int. 86 (2003)
412-431
3. S. J. Lehotay et al.: ‘Validation of a fast and easy
method for the determination of residues from 229
pesticides in fruits and vegetables using gas and liquid
chromatography and mass spectrometric detection’ J.
AOAC Int. 88 (2005) 595-614
www.spektrotek.com
2 μg/L matrix matched calibration standard run in positive
polarity with an EPI spectrum of Nigericin (left) and an LCMS/MS chromatogram from the same sample run in negative
polarity where a spectrum of Diclazuril has been automatically
acquired
82
Quantitation and Identification of 13 Azo-dyes in Spices
using LC-MS/MS
André Schreiber1, Kristin von Czapiewski2
AB SCIEX Concord, Ontario (Canada), AB SCIEX Darmstadt (Germany)
Overview
This application note describes a new and simple method including
extraction, HPLC separation and MS/MS detection for the analysis of 13
different azo-dyes in spices using Multiple Reaction Monitoring (MRM) on
a 3200 QTRAP® LC/MS/MS System. The developed method is available as
an iMethod™ test for Cliquid® Software which can be used for the analysis
and automatic reporting. In addition the results of a study of ion suppression
in various spice matrices comparing quantitation with solvent standards,
matrix matched standards and standard addition are presented. Standard
addition gave highest accuracies while quantifying azo-dyes in extracts of
spices.
Introduction
Methods described in literature apply GC-MS, LC-UV, LCMS and LC-MS/MS to analyze azo-dyes.1 Extensive sample
preparation is typically necessary to achieve required limits
of quantitation (10 μg/kg).
Spices are very complex, concentrated and variable matrices
and matrix effects (ion suppression or ion enhancement)
can be very strong and can depend on the origin of the spice
sample. Ideally, isotopically labeled internal standards of all
azo-dyes should be used to improve accuracy of detection
in unknown samples, but such internal standards are not
available. Three different possibilities to quantify unknown
samples (calibration with solvent standards, calibration
with matrix matched standards and standard addition)
were investigated. The results were compared regarding
their accuracy when analyzing azo-dyes in different spice
matrices.
Figure 1 illustrates theoretical calibration curves obtained
using these three different procedures.
In general, when using a calibration curve the signal intensity
of an unknown sample is compared to an external set of
standard samples.
These standards can be prepared in solvent or in matrix. The
smaller slope of the calibration curve with matrix matched
standards in comparison to solvent standards in Figure 1
indicates ion suppression effects.
100 0
Solvents tandards
Matrix matcheds tandards
Standard addition
800
600
unknown 400
concentration
200
0
-40-
30
-20-
10
01
02
03
04
05
06
07
08
09
01
00
Figure 1. Calibration curves using solvent standards, matrix
matched standards, and standard addition
www.spektrotek.com
The International Agency for Research on Cancer (IARC)
classified azo-dyes as potential carcinogenic substances.
After oral uptake azo-dyes can be reduced to amines which
are classified as partially carcinogenic substances. As a
result various azo-dyes are banned as food additives and
maximum residue levels exist in several countries.
83
When using standard addition, defined concentration(s) of
pure standards are added to aliquots of the unknown sample.
These standards, along with an aliquot which does not contain
any added standard, are analyzed. The resulting calibration
curve is extrapolated and the absolute value of the intercept
with the concentration axis determines the concentration of
the target compound in the unknown sample as shown in
Figure 1. Generally, standard addition requires more time for
analysis because one calibration curve per unknown sample
and per analyte has to be prepared. However, standard addition
can be used to solve the matrix effect problem because all
analytes are quantified in the matrix itself.
Experimental
Chemicals
Solvents, reagents and dye standards were obtained at
highest available purity from Sigma-Aldrich (dye content
80-98%). Internal standards (D5-Sudan I and D6-Sudan IV)
were obtained from WITEGA laboratories (Berlin, Germany).
Stock solutions were prepared in acetonitrile freshly due to
degradation of some azo-dyes. Solvent standards were diluted
in the starting mobile phase.
Spice Samples
Spice samples were purchased on local markets in India
(Garam Masala), Korea (Red Chili), and Egypt (Saffron) and
analyzed by LC-MS/MS. Not one of the 13 investigated azodyes was detected in the selected spice samples.
Matrix matched standards were prepared in Garam Masala
extract. In addition every matrix was spiked with known
concentrations of a mix of azo-dyes prior to analysis. These
samples were used to investigate standard addition.
Sample Preparation
The goal was to develop a generic sample preparation
procedure that is easy extendable to other emerging azo-dyes.
www.spektrotek.com
1.Weigh 1 g of homogenized sample (multiple times for
standard addition).
2.Add 20 μL of internal standard solution (1 μg/mL of D5Sudan I and D6-Sudan IV).
3.Add standard solution(s) in case of standard addition.
4.Add 10 mL of acetonitrile.
5.Shake for 10 min.
6.Add 10 mL of water.
7.Shake and centrifuge (or filtrate) before injection.
84
HPLC
The goal was to develop a flexible HPLC method to separate
a variety of emerging dyes. A gradient of 30 min was
chosen to allow sufficient separation of analytes from matrix
components. This method can be shortened easily, but matrix
effects might increase significantly. No HPLC conditions
could be identified for the separation of the two isomeric
dyes Sudan IV and Sudan Red B, although various columns
(C8 and C18), mobile phases (water, methanol, acetonitrile),
buffers (ammonium formate, ammonium acetate, formic, and
acetic acid), and pH values were investigated.
An Agilent 1100 HPLC system with binary pump (without static
mixer), well plate autosampler, and column oven was used. A
Phenomenex LUNA 5u C8,150x2 mm column and a gradient of
eluent A: water + 0.2% formic acid + 2 mM ammonium formate
and eluent B: water/acetonitrile (10/90) + 0.2% formic acid +
2 mM ammonium formate was used at a flow rate of 300 μL/
min. Details of the gradient are given in Table 1. The column
oven temperature was set to 30°C. A volume of 50 μL of each
sample was injected.
Table 1. HPLC gradient
Step
Total Time (min)
0
10
1
15
2
29
3
30
A (%)
B (%)
80
20
0
100
80
20
MS/MS
A 3200 QTRAP® LC/MS/MS System equipped with Turbo V™
Source and Electrospray Ionization (ESI) probe was used. ESI
was found to be suitable for the ionization of azo-dyes. The
ion source temperature (450°C) was optimized for the highest
sensitivity of Orange II and Para Red, the two compounds
showing lowest sensitivity in positive polarity. Two MRM
transitions were monitored per analyte to allow quantitation
and identification using ion ratios (Table 2).
Two additional MRM transitions were detected for Sudan
IVand Sudan Red B to allow differentiating between both coeluting and isomeric compounds.2
Analyte Name
CAS
Q1 (amu)
Q3-1 (amu)
Q3-2 (amu)
Q3-3 (amu)
Q3-3 (amu)
tR (min)
S/N at 10ng/mL
Dimethyl Yellow
60-11-7
226.1
120.1
105.1
-
-
14.5
980
Fast Garnet GBC
97-56-3
226.1
91.1
107.1
-
-
13.5
300
Orange II
(positive)
633-96-5
329.1
156.0
128.0
-
-
13.0
30
Orange II
(negative)
633-96-5
327.0
171.0
80.0
-
-
13.0
220
Para Red
6410-10-2
294.1
156.1
128.1
-
-
14.2
300
Rhodamine B
81-88-9
443.2
399.1
355.1
-
-
8.7
10600
Sudan I
842-07-9
249.1
93.0
156.1
-
-
15.0
500
Sudan II
3118-97-6
277.1
121.1
106.1
-
-
16.6
1090
Sudan III
85-86-9
353.1
197.1
128.1
-
-
17.4
200
Sudan IV
85-83-6
381.1
224.1
225.1
143.1
104.1
18.8
80
Sudan Orange G
2051-85-6
215.1
93.1
122.1
-
-
11.8
310
Sudan Red 7B
6368-72-5
380.2
183.1
115.1
-
-
18.9
1860
Sudan Red B
3176-79-2
381.2
224.1
225.1
156.1
134.1
18.8
140
Sudan Red G
1229-55-6
279.1
123.1
108.1
-
-
14.7
1910
D5-Sudan Red I
254.1
156.0
-
-
-
14.9
-
D6-Sudan Red IV
387.1
106.0
-
-
-
18.7
-
Table 2. MRM transitions, retention times (tR), of detected azo-dyes and signal-to-noise (S/N) of the qualifier MRM transition
at a concentration of 10 ng/mL
Standard chromatograms in positive and negative polarity
using Electrospray Ionization are given in Figures 2 and 3.
Orange II had ~10 times higher sensitivity in negative polarity.
The method developed provides enough sensitivity to
detect all 13 azo-dyes at required concentration of 10 μg/
kg in matrix. This is indicated by Signal-to-Noise ratios
(S/N) calculated using 3x standard deviation (Table 2).
The complete range of linearity was not of interest for this
study. Only the range from one order below to one order
above the level of 10 μg/kg was investigated. The following
quality control parameters were observed: r2>0.99 with
accuracy between 90-110% for each concentration and
%CV<15% at 1 μg/kg and <5% at 10 μg/kg (n=3).
XICof-MRM(2pairs):327.0/171.0amufromSample9(10ng/mL)o fCalibration_neg.wiff( TurboSpray),Smoothed
8.7
RhodamineB
4.0e4
3.5e4
3.0e4
1.5e4
1.0e4
5000.0
67
89
10
11
12
13
Para Red
2.0e4
Orange II
SudanO range G
2.5e4
14
15
Time,min
8000.00
SudanR ed 7B
SudanI V+ SudanR ed B+ D6-Sudan IV
4.5e4
SudanI I
5.0e4
8500.00
7500.00
7000.00
6500.00
6000.00
5500.00
5000.00
4500.00
4000.00
3500.00
3000.00
2500.00
2000.00
1500.00
SudanI II
5.5e4
9000.00
DimethylYellow
6.0e4
0.0
Orange II
1.00e4
9500.00
SudanR ed G
6.5e4
1.05e4
SudanI + D5-Sudan I
7.0e4
Fast Garnet GBC+ Red2 G
7.5e4
Max.1.1e4cps.
12.9
1.09e4
16
17
1000.00
500.00
18
19
20
21
22
Figure 2. Detection of 13 selected azo-dyes in positive polarity
0.00
67
89
10
11
12
13
14
Time,min
15
16
17
18
19
Figure 3. Detection of Orange II in negative polarity
20
21
22
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8.0e4
Matrix effects and how to compensate them using different
calibration procedures were investigated. A comparison of
accuracies based on calibration with solvent standards, matrix
matched standards and standard addition is summarized in
Table 3. Values ~100% indicate that no matrix effects occur
or that they were compensated completely. These results
indicate that ion suppression varies strongly depending
on the spice matrix. Using a calibration curve based on
solvent standards did not provide sufficiently accurate data
when analyzing spices. A calibration curve based on matrix
matched standards provided more accuracy and can be used
when matrices of similar composition have to be analyzed.
But standard addition provided the best accuracy and is highly
recommended if a broad range of complex matrices, such as
different spices, have to be analyzed.
85
Table 3. Accuracy of quantifying azo-dyes in 3 different spice matrices using calibration with solvent standards, matrix matched
standards (prepared in Masala extract), and standard addition
Matrix Matched Standards
(Prepared in Masala Extract)
Solvent Standards
Analyte Name
Masala
Chili
Saffron
Masala
Chili
Standard Addition
Saffron
Masala
Chili
Saffron
Dimethyl Yellow
10%
43%
20%
96%
288%
143%
96%
97%
95%
Fast Garnet GBC
36%
67%
51%
97%
195%
143%
97%
99%
94%
Orange II (positive)
25%
24%
29%
101%
101%
150%
101%
82%
95%
Rhodamine B
52%
44%
47%
101%
73%
95%
101%
89%
104%
Sudan I
47%
77%
48%
100%
176
105%
100%
91%
110%
Sudan II
35%
44%
34%
97%
126%
104%
97%
94%
108%
Sudan III
66%
80%
53%
97%
133%
81%
97%
98%
111%
The colors represented in this table reference the data from the calibration curves in Figure 1.
®
Figure 4. Cliquid® Software; easy-to-use LC-MS/MS software with preconfigured iMethod™ Tests and automatic reporting
www.spektrotek.com
Cliquid® Software and iMethod™ Tests
86
Cliquid® Software was specifically developed for LC-MS/MS analysis in routine food testing laboratories. The software provides
an easy-to-use interface with a four step wizard to perform sample analysis and automatic report generation. These four steps
include choosing a test to perform, building the sample list, customizing reporting options, and submitting the samples for
analysis. The developed method for the analysis of azo-dyes in spices is available as an iMethod™ Test.
Screenshots illustrating the wizard and example reports generated when analyzing unknown contaminated spice samples are
shown in Figure 4 and 5.
Summary
A new analytical procedure was developed
to determine 13 azo-dyes, which are of
high priority in many European and Asian
countries, by simple solvent extraction and
LC-MS/MS analysis.
Ion suppression varied strongly from
matrix to matrix. Thus, standard addition
is recommended to quantify dyes in spices
due to a lack of isotopically labeled internal
standards. The detection of two MRM
transitions per compound is needed to match
regulatory requirements.
Cliquid™ Software is easy-to-use software
focusing on the typical workflow from LC-MS/
MS analysis to automatic report generation.
The described method for the analysis of azodyes in spices is available as an iMethod™
Test.
References
1.Lutz Hartig et al.: ‘Detection of 6 Sudan
Dyes, Dimethyl Yellow and Para Red in
Spices and Sauces with HPLC/MS/MS’
poster presented at ASMS conference on
Mass Spectrometry (2005) San Antonio,
Texas, USA
2.André Schreiber et al.: ‘Accuracy of
quantitation using external and internal
calibration to analyze dyes in extracts
of spices’ poster presented at ASMS
conference on Mass Spectrometry (2006)
Seattle, Washington, USA
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Figure 5. Example reports generated automatically by Cliquid® Software
showing calibration curves, statistical information of accuracy and
reproducibility, and detected azo-dyes in unknown samples with highlighted
analytes when identified by MRM ratio
87
Increasing Selectivity and Confidence in Detection when
Analyzing Phthalates by LC-MS/MS
André Schreiber1, Fanny Fu2, Olivia Yang2, Eric Wan3, Long Gu4, and Yves LeBlanc1
1
AB SCIEX, Concord, Ontario (Canada)
2
AB SCIEX, Taipei, (Taiwan)
3
AB SCIEX, Hong Kong (Hong Kong)
4
AB SCIEX, Shanghai (China)
Overview
Recent issues with the determination of phthalates in food and beverages
like yogurt, sport drinks and fruit juices have highlighted the need for both
food manufacturers and regulatory agencies to utilize fast and accurate
analytical techniques to proactively ensure product safety.
A fast and sensitive LC-MS/MS method was developed for the analysis
of 22 phthalates utilizing a simple extraction, fast LC separation using
a Phenomenex Kinetex™ C18 column with a run time of 10 minutes, and
selective MS/MS detection using an AB SCIEX QTRAP® 5500 system
operated in Multiple Reaction Monitoring (MRM) mode. Major challenges
of method development were the presence of chemical background and
matrix interferences. To address these challenges we successfully applied
the unique MRM3 mode to enhance detection selectivity by detecting
second generation product ions and Enhanced Product Ion (EPI) scanning
to increase confidence in identification using the molecular fingerprint of
each target analyte saved into the MS/MS spectrum. In addition, the AB
SCIEX SelexION™ technology was used to separate critical isomers using
Differential Mobility Spectrometry (DMS).
Introduction
Phthalates are widely used industrial chemicals with an
estimated annual production of over 8,000,000 tons.
Phthalates are added to plastics to increases flexibility,
transparency, and longevity. By weight, they contribute 1060% of plastic products. Phthalates are used in a variety
of products, including building materials (caulk, paint,
adhesives), household products (vinyl upholstery, shower
curtains, food containers and wrappers), and cosmetics.1
The use of various phthalates is restricted in many countries
because of health concerns.2-3
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In 2011, the illegal use of bis(2-ethylhexyl) phthalate (DEHP)
and Diisononyl phthalate (DINP) in clouding agents for use
in food and beverages has been reported in Taiwan.4
88
As a result fast and reliable methods for the detection of
different phthalates in food and beverages are needed.
Chromatographic techniques coupled to mass spectrometry
are methods of choice because of their sensitivity and
selectivity.5
Here we present a new and unique LC-MS/MS method
using the AB SCIEX QTRAP® 5500 system operated in
MRM, MRM3, and EPI mode to detect 22 phthalates. In
comparison to GC-MS the developed LC-MS/MS method
has several advantages:
•Reduced sample preparation and no need for
derivatization
•Superior quantitative results with shorter run times
•Higher degree of confidence due to the presence of the
quasi-molecular ion and characteristic fragment ions
In addition, DMS was used to separate isomeric phthalates
using the AB SCIEX SelexION™ technology.
Sample Preparation
One gram sample was homogenized and extracted with 45 mL
methanol using ultra sound for 30 min. An aliquot of 5 mL was
transferred into a vial and centrifuged for 10 min (3500 rpm).
The supernatant was further diluted for LC-MS/MS analysis.
LC Separation
LC separation was achieved using an Agilent 1200 system with
a Phenomenex Kinetex C18 (100 x 4.6 mm; 2.6 μm) column
and a fast gradient of water + 10 mM ammonium acetate and
methanol at a flow rate of 500 μL/min.
MS/MS Detection
The AB SCIEX QTRAP® 5500 system was used with Turbo V™
source and Electrospray Ionization (ESI) source. Two selective
MRM transitions were monitored for each targeted analyte
(Table 1). MRM3 was used to differentiate between isomers
and to increase selectivity to reduce interferences.
DMS Separation
The AB SCIEX SelexION™ technology was used to selectively
detect isomeric phthalates. A Separation voltage (SV) of
3800 V was used with acetonitrile as chemical modifier. The
Compensation Voltage (CoV) was optimized for each target
analyte specifically.
Results
Solid Phase Extraction (SPE) is known to be a major source of
phthalate contamination resulting in over-estimation and false
positive results.5 Thus, a simple and fast procedure using
liquid extraction was developed and successfully applied to
the analysis of food and beverage samples.
Different LC conditions were evaluated during method
development. In general C18 material with a neutral buffer
of ammonium acetate was found to give good separation.
Methanol is organic modified was more efficient in separating
isomers. The Phenomenex Kinetex C18 column was finally
chosen because of its UHPLC like efficiency and resolution
at significantly lower column pressure resulting in high
robustness and long instrument up time.
Experimental
The final gradient started at 50% methanol and included a
cleanup step at 98% methanol at a flow rate of 1000 μL/min
to reduce background levels.
In addition, a trap column was used between pump and
autosampler to retain any phthalates originating from the
HPLC system.
MRM transitions were fully optimized with M+H+ as
precursor ion and two compound dependent fragment ions.
The dominating fragment ions were protonated phthalic
acid (167), phthalic anhydride (149), and different esters of
phthalic acid and phthalic anhydride (Figure 1).
Phthalates are esters of 1,2-benzenedicarboxylic acid.
O
R1
O
O
R2
Targeted analytes of this project are listed in Table 1.
All plastic material (i.e. pipette tips) was avoided when
handling samples and making dilutions. All glassware was
cleaned carefully to avoid contamination. Different organic
solvents (LC and LC-MS grade) were evaluated and distilled
water was used to minimize background interferences.
Figure 1. EPI spectrum of BBP, the molecular fingerprint
saved into the MS/MS spectrum was used for compound
identification with highest confidence
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O
89
Table 1. Targeted phthalates, compound information, and optimized MRM transitions (Q1 and Q3 ions)
Phthalate
CAS
Formula
M.W.
Q1
Q3
Dimethyl phthalate
DMP
131-11-3
C10H10O4
194.18
195
163 / 133
Diethyl phthalate
DEP
84-66-2
C12H14O4
222.24
223
149 / 177
Diallyl phthalate
DAP
131-17-9
C14H14O4
246.26
247
189 / 149
Dipropyl phthalate
DPrP
131-16-8
C14H18O4
250.29
251
149 / 191
Diisopropyl phthalate
DIPrP
605-45-8
C14H18O4
250.29
251
149 / 191
Dibutyl phthalate EU, EPA
DBP
84-74-2
C16H22O4
278.34
279
149 / 205
Diisobutyl phthalate EPA
DIBP
84-69-5
C16H22O4
278.34
279
149 / 205
Bis(2-methoxyethyl) phthalate
DMEP
117-82-8
C14H18O6
282.29
283
207 / 59
Dipentyl phthalate EPA
DPP
131-18-0
C18H26O4
306.40
307
219 / 149
Diisopentyl phthalate
DIPP
605-50-5
C18H26O4
306.40
307
219 / 149
Bis(2-ethoxyethyl) phthalate
DEEP
605-54-9
C16H22O6
310.34
311
221 / 149
Benzyl butyl phthalate EU, EPA
BBP
85-68-7
C19H20O4
312.37
313
149 / 205
Diphenyl phthalate
DPhP
84-62-8
C20H14O4
318.32
319
225 / 77
Dicyclohexyl phthalate
DCHP
84-61-7
C20H26O4
330.42
331
167 / 249
Bis(4-methyl-2-pentyl) phthalate
BMPP
146-50-9
C20H30O4
334.46
335
167 / 251
Dihexyl phthalate
DHXP
84-75-3
C20H30O4
334.46
335
149 / 233
Di-n-heptyl phthalate
DHP
3648-21-3
C22H34O4
362.51
363
149 / 233
Bis(2-n-butoxyethyl) phthalate
DBEP
117-83-9
C20H30O6
366.45
367
101 / 249
Bis(2-ethylhexyl) phthalate EU, EPA
DEHP
117-81-7
C24H38O4
390.56
391
167 / 279
Di-n-octyl phthalate EU, EPA
DNOP
117-84-0
C24H38O4
390.56
391
261 / 149
Diisononyl ortho-phthalate EU, EPA
DINP
28553-12-0
C26H42O4
418.61
419
275 / 149
Diisodecyl ortho-phthalate EU, EPA
DIDP
26761-40-0
C28H46O4
446.66
447
149 / 289
Bold
EU
EPA
llegally used in food and beverages in Taiwan in 20114
Restricted use in toys and childcare articles in Europe2
Addressed in the phthalates action plan of the U.S. Environmental Protection Agency3
Table 2. Accuracy and linearity of six high priority phthalates
Phthalate
Accuracy (%)
Regression
DBP
97-103
0.9998
BBP
91-108
0.9999
DEHP
88-108
0.9989
DNOP
85-113
0.9982
DINP
92-111
0.9998
DIDP
94-109
0.9931
DPhP
DPP
DPrP
DMEP
DIPrP
BBP
DMP
DEHP
DBP/ DIBP
DEEP
DEP DAP
DBEP
DIPP
DCHP
BMPP
DHXP
DHP
DNOP
DINP
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An example chromatogram of LC-MS/MS detection of 22
phthalates is shown in Figure 2.
90
Limits of detection (LOD), linearity and accuracy of
quantitation were determined. Example chromatograms of six
high priority phthalates (from 1 to 100 ng/mL) are shown in
Figure 3a and 3b.
For all targeted phthalates an LOD of at least 1 ng/mL was
achieved. Please note that the final LOD greatly depends
on background interferences which can greatly vary from
laboratory to laboratory.
DIDP
Figure 2. Example LC-MS/MS chromatogram showing the
separation and detection of 22 phthalates at a concentration of
10 ng/mL
1.5e5
6.2
4.9
1.4e5
Intensity, cps
4.0e4
3.5e4
3.0e4
1.1e5
8.0e4
1.0e5
7.0e4
6.0e4
3.8 4.1
5.0e4
2.5e4
1.0e4
0.0
2
4
6
Time, min
Sample Name: "20ppb-sch" Sample ID: "" File: "1000620-1.wiff"
Peak Name: "DBP 1" Mass(es): "279.000/149.000 Da"
Comment: "" Annotation: ""
20
2.6e5
2.4e5
2
5.0
4
9.0e5
8.5e5
200
2
Time, min
6
6
Time, min
Peak Name: "BBP 1" Mass(es): "313.000/149.000 Da"
6.5e5
5.0e5
4.5e5
4.0e5
3.5e5
8.0e4
3.0e5
4
6
Time, min
6000.0
2000.0
4000.0
5.3 5.8
2000.0
0.0
8
2
4
1.6e5
2.5e4
8
BBP
1.8e5
3.0e4
6
Time, min
4.7
100
2.2e5
1.4e5
1.2e5
1.0e5
6.0e4
4.0e4
5000.0
3.7 4.2 4.3
0.0
8
8000.0
3000.0
8.0e4
5.0e4
2
1.0e4
4000.0
2.4e5
6.16.2
1.0e5
0.0
5000.0
4.7
1.0e4
1.5e5
2.0e4
20
1.6e4
1.4e4
1.2e4
1.5e4
2.0e5
4.0e4
6000.0
0.0
2
4
6
Time, min
8
2.0e4
2.5e5
3.8 4.4
6.0e4
7000.0
2.0e5
5.5e5
1.0e5
4
3.5e4
Intensity, cps
Intensity, cps
6.0 6.1
1.2e5
2
4.0e4
6.0e5
1.4e5
8000.0
4.5e4
DIBP/DBP
7.0e5
1.6e5
1.8e4
1000.0
0
8
5.0e4
7.5e5
1.8e5
4
4.7
8.0e5
2.0e5
5.4
400
4.9
100
9.5e5
2.2e5
5.8
3.5 3.7 4.3
600
0.0
8
1200
800
1.0e4
6
Time, min
4.7
3.8 4.3
2.0e4
1.0e4
1400
4.7
10
2.2e4
1.1e4
1000
2.0e4
0.0
8
7.0e4
3.0e4
1.0e4
5000.0
8.0e4
2.6e4
2.4e4
9000.0
1600
9.0e4
5
4.7
1.2e4
1800
4.0e4
2.0e4
1.3e4
2000
5.0e4
3.0e4
1.5e4
1.4e4
2200
6.2
5.8
6.0e4
4.0e4
2.0e4
2400
1.2e5
9.0e4
Intensity, cps
4.1 4.3
3.8
4.7
4.7
1
2600
1.3e5
1.0e5
4.9
4.5e4
5.0
10
1.6e5
1.1e5
5.0e4
Intensity, cps
4.7
1.2e5
4.6
5.5e4
Intensity, cps
5
1.3e5
Intensity, cps
6.0e4
6.2
Intensity, cps
6.5e4
6.0
Intensity, cps
1
7.0e4
Intensity, cps
2
4
Time, min
6
2.0e4
0.0
8
2
4
Time, min
6
0.0
8
2
4
6
Time, min
8
Figure 3a. MRM chromatograms of the high priority phthalates DBP and BBP at 1, 5, 10, 20, and 100 ng/mL
3500
1.0e4
2500
8000.0
12.0
13.0
14.0
15.0
Time, min
Peak Name: "DEHP 1" Mass(es): "391.000/167.000 Da"
9.0
10.0
11.0
20
16.0
0.0
17.0
12.8
3.6e5
3.4e5
3.2e5
6.0e4
2.8e5
5.5e4
2.6e5
4.0e4
3.5e4
11.0
100
16.0
0.0
17.0
9.0
10.0
11.0
12.0
13.0
Time, min
14.0
15.0
16.0
13.0
14.0
15.0
Time, min
Peak Name: "DNOP 1" Mass(es): "391.000/261.000 Da"
12.8
1.0e4
11.6
5000.0
16.0
17.0
0.0
18.0
DEHP
2.5e4
2.0e4
14.4
5
Peak Name: "DINP 1" Mass(es): "419.200/127.000 Da"
5.0e4
7000.00
4000.00
14.3
1.8e4
14.0
1.6e4
1.4e4
12.1
3500.00
3000.00
12.9
1.2e4
2000.00
1.0e4
1500.00
2000.0
0.00
0.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Time, min
20
16.0
17.0
18.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Time, min
13.0
4.0e5
3.8e5
3.6e5
8.0e4
100
16.0
17.0
18.0
7.0e4
2.8e5
6.0e4
2.6e5
5.0e4
4.5e4
4.0e4
10.0
11.0
12.0
13.0
14.0
Time, min
15.0
16.0
17.0
2.0e5
1.8e5
2.0e4
9.0
10.0
11.0
12.0
13.0
14.0
Time, min
15.0
16.0
17.0
18.0
0.0
2400
2200
2000
1600
1400
1200
15.7
13.4
1600
1400
1200
800
600
0
8
10
12
14
16
Time, min
Peak Name: "DIDP 1" Mass(es): "447.000/149.000 Da"
20
15.7
400
200
20
13.4
1000
0
18
14.7
1800
200
8
10
12
14
16
Time, min
10
2600
400
100
2.2e4
18
20
8
10
12
14
Time, min
16
18
20
14.4
2.0e4
4000
DIDP
1.8e4
3500
1.6e4
1.4e4
2500
2000
1.2e4
1.0e4
8000.0
15.1
6.0e4
4.0e4
2800
0
1000
8.0e4
13.4 14.2
1.0e4
3000
6000.0
1.0e5
2.0e4
1.5e4
5000.0
3200
50
1500
1.2e5
2.5e4
18.0
800
13.3
1.4e5
3.0e4
17.0
18.0
5
1000
3000
2.4e5
2.2e5
1.6e5
3.5e4
18.0
Intensity, cps
Intensity, cps
5.5e4
250
100
DINP
3.0e5
6.5e4
9.0
17.0
1800
15.7
4500
3.2e5
15.6
300
13.0
3.4e5
7.5e4
0.0
16.0
600
12.1
5000.0
16.0
12.8
2000
150
14.2
12.4
12.0
4000.0
500.00
15.0
1.0e5
2400
200
12.8
6000.0
1000.00
2.5e4
1.5e4
12.3
8000.0
3.0e4
2.0e4
12.5
1.0e4
11.8
2500.00
13.3
350
Intensity, cps
Intensity, cps
4500.00
2.0e4
13.0
14.0
Time, min
DNOP
2200
400
3.5e4
2.2e4
12.5
14.1
12.6
12.0
1.2e5
2600
15.4
14.3
4.0e4
2.4e4
12.7
6500.00
6000.00
11.0
1.4e5
0.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Time, min
18.0
450
2.6e4
5500.00
17.0
4.5e4
2.8e4
5000.00
1
550
16.0
9.0
10.0
11.0
12.0
13.0
14.0
Time, min
15.0
16.0
17.0
18.0
The accuracy was typically between 85 and 115% and
quantitation was performed with linear regression and 1/x
weighting. The coefficient of regression was above 0.999 for
all analytes. Examples for accuracy and linearity are of six
high priority phthalates are listed in Table 2.
The unique scan function of MRM3 of the AB SCIEX
QTRAP® 5500 system was investigated for its potential to
differentiate isomeric species.
An example of successfully differentiating between the
isomers DIBP and DBP using the different fragmentation
pattern in MRM3 mode is shown in Figure 4. Using traditional
MRM mode both compounds had the exact same transitions
and needed to be separated on the LC time scale. Thus, MRM3
allows speeding up the LC method if throughput requires.
4000.0
13.4
15.7
500
15.2
2000.0
0
8
10
12
14
Time, min
16
18
20
0.0
12.0
13.0
14.0
15.0
Time, min
16.0
17.0
18.0
XICo f+ MRM( 44 pairs):2 79.200/205.100 Da ID:D BP 1 from Sample1 5 (Std20...M
0%
2468
10
Time,m in
XICo f+ MRM( 44 pairs):2 79.200/149.000 Da ID:D BP 2 from Sample1 5 (Std20...M
MRM2 79/205
12
16
MRM2 79/149
2468
10
Time,m in
XICo f+ MS3( 279.20),(223.10):E xp 2, 166.789t o1 67.289 Da from Sample3 (S...M
DIBP
5.80
100%
14
ax.4 .5e5 cps.
DBP
5.99
DIBP
100%
0%
ax.2 .3e5 cps.
DBP
5.99
DIBP
100%
...
7500.00
10
13.0
500
3.0e4
14.3
10.0
2.0e4
Intensity, cps
3.2e4
8500.00
8000.00
0.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Time, min
17.0
5.5e4
12.0
9.0
4.0e4
5000.0
13.0
0.0
18.0
13.2
6.0e4
1.0e4
16.0
17.0
8.0e4
12.8
Intensity, cps
3.4e4
12.0
1.6e5
3.0e4
...
1
13.0
11.0
1.8e5
1.5e4
0.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Time, min
17.0
10.0
100
2.2e5
3.5e4
4.0e4
16.0
9.0
2.4e5
2.0e4
0.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Time, min
14.4
2000.0
13.0
14.0
15.0
16.0
Time, min
13.2
12.8
4000.0
2.0e5
6.0e4
13.3 14.0
12.6
12.0
4.0e4
8.0e4
1.5e4
11.0
20
5.0e4
4.5e4
1.0e5
2.0e4
13.8
12.6
2000.0
10.0
1.2e4
6000.0
3000.0
9.0
1.4e4
8000.0
4000.0
0
17.0
1.6e4
1.0e4
14.5
12.8
5000.0
500
8.0
1.6e5
1.2e5
0.0
10.0
1.8e5
1.4e5
8000.0
1000.0
9.0
2.0e5
2.5e4
8.5e4
5000.0
8.0
2.2e5
3.0e4
12.5
12.0
1.8e4
9000.0
6000.0
13.7
13.2
10
2.0e4
1.0e4
7000.0
12.7
1000
13.3 13.9
Intensity, cps
Intensity, cps
Intensity, cps
4.5e4
9.0e4
12.6
2.2e4
1.1e4
12.0
2.4e5
5.0e4
2000
1500
1.0e4
1.4e4
1.2e4
2500
12.4
12.0
13.0
14.0
15.0
Time, min
3.0e5
9.5e4
2.0e4
1.5e4
Intensity, cps
8.0
6.5e4
9000.00
2.5e4
2.4e4
1.3e4
13.2
3000
2000.0
500
1.00e4
11.7
13.9
4000.0
1000
9500.00
13.7
6000.0
1500
Intensity, cps
1.2e4
3000
2000
Intensity, cps
1.4e4
2.6e4
12
14
16
ax.3 .0e6 cps.
MRM3 279/223/167
0%
24
68
10
Time,m in
XICo f+ MS3( 279.20),(223.10):E xp 2, 148.710t o1 49.210 Da from Sample3 (S...M
DIBP
5.82
100%
0%
24
68
12
14
16
ax.7 .9e6 cps.
MRM3 279/223/149
Time,m in
10
12
14
16
Figure 4. Differentiation of DIBP and DBP using the different
fragmentation pattern in MRM3 mode in comparison to
MRM mode
www.spektrotek.com
4000
2.8e4
1.5e4
Intensity, cps
4500
1.6e4
5
13.2
Intensity, cps
5000
1.6e4
3500
3.0e4
1.8e4
Intensity, cps
Intensity, cps
2.0e4
1.7e4
4000
3.5e4
2.2e4
13.9
12.5
11.8
5500
Intensity, cps
6000
14.4
1
4500
4.0e4
Intensity, cps
13.5
6500
7.0e4
12.8
10
4.5e4
2.4e4
7000
7.5e4
12.8
Intensity, cps
2.6e4
7500
0
5
2.8e4
...
8000
3.0e4
...
8500
12.7
1
9000
Intensity, cps
91
Another possibility to enhance selectivity of detection is the
use of Differential Mobility Spectrometry (DMS). The new AB
SCIEX SelexION™ technology uses a planar DMS cell attached
between the curtain plate and orifice plate of the mass
spectrometer. Ions are separated based on difference in their
high field and low field mobility.SV and CoV are optimized to
correct the trajectory of a desired ion. In addition, a chemical
modifier can be introduced to alter separation characteristics.
BMPP
DHXP
CoV= 3.0
CoV= -2.0
Figure 5a. Separation of the isomers BMPP and DHXP,
both phthalates can be separated in the LC and DMS space
resulting in increased selectivity
DMSo ff
BMPP
DMSo n( CoV= 3.0)
DMSo n( CoV= -2.0)
The example presented in Figure 5a and 5b highlights the
unique selectivity achieved using DMS. The isomers BMPP
and DHXP were separated using different CoV. Acetonitrile
was introduced as chemical modifier to enhance separation.
Summary
A fast and sensitive LC-MS/MS method was developed for
the detection of 22 phthalates in food and beverage samples.
All possible precautions were taken to reduce chemical
background. This included the avoidance of plastic material,
careful handling of laboratory glassware, systematic evaluation
of different LC solvents, a simple extraction procedure, and
the use of a trap column inside the LC system.
All 22 phthalates were detected with an LOD of 1 ng/mL or
lower, good accuracy, and linearity using two MRM transitions
per analyte. Characteristic EPI spectra can be used to further
increase confidence of compound identification based on
characteristic MS/MS spectra and library searching.
In addition, the unique scan function MRM3 of the QTRAP®
5500 system and the new AB SCIEX SelexION™ technology
were successfully used to separate isomeric species
enhancing the selectivity of LC-MS/MS detection.
Acknowledgement
The authors wish to thank Ching-Hsin Tung (Food and Drug
Administration, Taiwan), Dr. Sheng-Che Lin (Tainan city health
bureau, Taiwan) and Dr. Dunming Xu (CIQ Xiamen, China) for
their assistance and advice during method development.
DHXP
References
BMPP
DHXP
1.R.A. Rudel and L.A. Perovich: Atmospheric Environment 43
(2009) 170-181
2.DIRECTIVE 2005/84/EC on ‘phthalates in toys and childcare
articles
3.EPA ‘Phthalates Action Plan Summary’ 2010
4.Taipei Times: ‘FOOD SCARE WIDENS: New chemical adds
to food scare’ May 29, 2011
5.Zhuokun Li et al.: J. Chromatogr. Sci. 49 (2011) 338-343
www.spektrotek.com
Figure 5b. Selective detection of BMPP and DHXP by
compound specific CoV for each analyte, acetonitrile was
introduced as chemical modifier
92
Quantitative Analysis and Identification of Migrants in Food
Packaging Using LC-MS/MS
Cécile Busset1 and Stephen J. Lock2
1
AB SCIEX, Paris (France); 2 AB SCIEX, Warrington, Cheshire (U.K.)
Introduction
Packaging improves the quality and safety assurance of food, especially
from micro-organisms, biological and chemical contaminants. Packaging is
therefore an essential component for the food industry and the manufacturing
processes.
However, over the last couple of years there has been a growth in the number
of materials and substances used in food packaging so in order to improve
food safety a migration study for compounds is becoming more important
to prevent the use of compounds that can migrate into food.
Currently, an upper limit for the overall migration of 60 mg/kg or 10 mg/dm2
has been set by the European Union (EU).1
In the USA, the regulations for food packaging material are more complex,
because the types of raw and processed foods, and conditions of use are
separated.2
In this study three compounds: ITX, Irgacure, and TRP are investigated (Figure 1). ITX is a mixture of 2-Isopropylthioxanthone
and 4-Isopropylthioxanthone. Irgacure contains Irgacure 819 (Phenylbis (2,4,6-trimethylbenzoyl) phosphine oxide. Both are
used as photo-initiators in UV cured inks. TRP (Tri(propylene glycol) diacrylate is an ingredient of cured inks.
The data presented discusses linearity of response, robustness and the use of the Multiple Reaction Monitoring combined with
Enhanced Product Ion scanning (MRM-EPI) using an AB SCIEX 3200 QTRAP® LC/MS/MS System as a way of gaining additional
information for the presence of these migrants.
Experimental
Sample Extraction
Standards were prepared in the solvent composition at the
start of the LC run (water/acetonitrile + 0.1% formic acid
70/30). Three sorts of real samples were analyzed: a packaging
cap with only decoration (inks), a packaging cap with only
varnish and a packaging cap with decoration and varnish.
1 dm2 of each sample was extracted with acetonitrile. The
extracted sample was evaporated and reconstituted in initial
mobile phase before analysis.
2-ITX4
O
CH3
CH3
S
S
H3C
Irgacure
CH3
TRP
CH2
CH3
LC
O
H3C
H3C
O
CH3
O
H2C
P
O
CH3
O
CH3
O
O
O
H3C
O
CH3
H3C
Figure 1. Investigated migrants from food packaging
www.spektrotek.com
An Agilent 1200 system containing a binary pump flowing at
250 µL/min, autosampler, and a column oven set at 20°C were
used with a Hypersil BDS C18 column (5 µm, 100 x 2 mm).
10 µL injections of standards and extracts were separated
using a gradient (Table 1) of mobile phase A (0.1 % formic
acid in water) and B (0.1 % formic acid in acetonitrile). 5
minutes column equilibration time was used between runs.
-ITX
O
93
Step
Time (min)
0
5.0
(%) A
70
(%) B
30
1
2.0
5
95
2
7.0
5
95
3
7.1
70
30
4
12.0
70
30
Compound
ITX
Irgacure
TRP
MS/MS
All experiments were performed on an AB SCIEX 3200
QTRAP® LC/MS/MS System with Turbo V™ source at 450°C
using Electrospray Ionization (ESI) in positive polarity. The
following source conditions were used:
25 psi
5000 V
40 psi
50 psi
Medium
450 °C
+EPI (255.13) Charge (+0) CE (20) FT (250): Exp2 ,5 .572 t...M
255.2
50
2002
50
3003
m/z, Da
213.1
1.00e5
184.1
50
1001
50
50
213.1
50
3003
m/z, Da
+EPI (255.13) Charge (+0) CE (35) CES( 15)F T( 250):E xp ...M
2002
213.1
184.2
1.0e5
www.spektrotek.com
50
94
1001
152.2
50
400
450
255.2
66
35
184.1
66
61
419.2
147.2
21
23
419.2
119.2
21
57
301.2
113.2
21
13
500
ax.1 .2e5 cps.
50
400
quant.rdb (ITX 1): "Linear" Regression ("1 / x" weighting): y = 5.88e+003 x + 3.14e+003 (r = 0.9960)
450
500
ax.4 .6e4 cps.
CE =5 0V
184.1
1001
50
CE =3 5V
2002
152.2
50
213.1
255.1
Figure 3 shows calibration lines that were obtained from
standards analyzed in MRM-EPI mode with each standard
analyzed in duplicate. The ‘r’ values obtained from these
calibration lines (0.5 – 500 ng/mL for ITX, 2 – 1000 ng/mL
for Irgacure and 0.5 – 1000 ng/mL for TRP) were greater than
0.996 when a linear fit with 1/x weighting was applied.
255.2
50
3003
m/z, Da
5.0e4
ax.2 .6e5 cps.
CE =2 0V
213.1
1001
255.1
Standards at 10 ng/mL were used to build a mass spectral
library. An example of reference spectra is shown in Figure
2. Standards were used over a range 0.1 to 1000 ng/mL to
produce calibration lines.
50
400
400
2.0e6
1.0e6
0
50
100
150
200
250
300
350
400
Concentration, ng/mL
quant.rdb (Irga 1): "Linear" Regression ("1 / x" weighting): y = 226 x + 109 (r = 0.9994)
500
ax.1 .8e5 cps.
50
ITX
r = 0.9960
3.0e6
0.0
450
CE =3 5V with CES= 15V
239.2
215.6
2002
50
3003
m/z, Da
Area, counts
50
DP (V) CE (V)
Analyses were based on two different Information Dependent
Acquisition (IDA) experiments using Multiple Reaction
Monitoring (MRM) in the survey scan and dependent
Enhanced Product Ion (EPI) scanning. MRM transitions were
previously optimized (see Table 2). A dwell time of 100 ms
was used for each transition and the pause time was set to
5 ms.
2.0e5
Q3 Mass (amu)
MS/MS spectra, in comparison to dedicated and fixed
Collision Energies, and thus greatly enhancing the quality
of library searching. The scan speed of the EPI scans were
4000 amu/s and Dynamic Fill Time (DFT) was used for all EPI
scans. In both experiments peaks were identified in the MRM
survey using Dynamic Background Subtraction (DBS).
Identification of analytes in the real samples was based on
searching against the mass spectral library created from
MRM-EPI analyses of standards.
450
500
Figure 2. An example of the effect of collision energy on the
EPI spectra of a migrant standard used for generating library
data (10 ng/mL ITX standard)
Area, counts
Curtain Gas (CUR)
IonSpray Voltage (IS)
Gas1
Gas2
CAD Gas
Temperature
Q1 Mass (amu)
Experiment 1 triggered three EPI scans at collision energies
(CE) of 20; 35 and 50 V. Experiment 2 used a single dependent
scan with a CE of 35 V and Collision Energy Spread (CES) of
15 V. CES was found to give more reproducible and richer
450
500
0
100
200
300
400
500
600
700
800
900
Concentration, ppb
quant.rdb (TRP 1): "Linear" Regression ("1 / x" weighting): y = 3.27e+003 x + 274 (r = 0.9993)
1000
Irgacure
r = 0.9994
2.4e5
2.0e5
1.0e5
0.0
Area, counts
Figure 1. Investigated migrants from food packaging
Table 1. LC gradient
TRP
r = 0.9993
3.0e6
2.0e6
1.0e6
0.0
0
100
200
300
400
500
600
700
800
900
1000
Figure 3. Calibration lines obtained from ITX, Irgacure and
TRP with r values > 0.996 (no internal standard used)
XICo f+ MRM( 6p airs): 301.2/113.2D a fromS ample7 (std2...
5.4e4
4.5e4
4.0e4
3.5e4
Intensity,c ps
Compound
Transition
ITX
255.1/213.1
0.5
8.2
Irgacure
419.2/147.2
0.844
5.2
2.0e4
TRP
301.2/113.2
0.515
9.5
1.5e4
% CV (n=5)
Max. 920.0c ps.
XIC. ..
5.7
800
400
200
0
0.5n g/mL
ITX
S/N =1 8.4S
100
2n g/mL
Irgacure
50
0
46
Time,m in
XIC. ..
5.6
Irgacure
ITX
5000. 0
0.0
1.02
.0
3.04
.0
Time,m in
5.06
.0
7.0
Figure 4. 10 µL injection of migrants standards in initial
mobile phase
4.1
Table 4. Estimates for limits of detection (LOD), limits of
quantitation LOQ), and linearity for food migrants
/N =2 3.2
250
200
0.5n g/mL
TRP
150
100
50
0
46
Time,m in
2.5e4
Max. 337.5c ps.
338
300
Intensity,c ps
600
Intensity,c ps
Intensity, cps
S/N =4 0.4
Max. 145.0c ps.
145
3.0e4
1.0e4
Figure 4 shows a typical trace obtained from the analysis
of migrant standard prepared in the initial mobile phase,
all migrants were detected below 1 ng/mL as shown in
Table 4 with Figure 5 giving the sensitivity of migrants at
a concentration of 0.5 ng/mL (ITX and TRP) and 2 ng/mL
(Irgacure).
XIC. ..
TRP
5.0e4
Table 3. Reproducibility data from 5 replicate injections
Concentration (ng/mL)
Max. 5.4e4c ps.
4.3
Repeatability and %CV were assayed by 5 repeat injections of
a standard close to the limits of quantitation of each analyte
and results are summarized in Table 3 with all coefficients of
variation <10% (no internal standard was used).
46
Time,m in
Figure 5. Signal to noise (S/N) of low level migrant standards
(S/N calculated using peak-to-peak algorithm)
Compound
S/N (at ng/mL)
LOD
(ng/mL)
LOQ
(ng/mL)
Linearity
(ng/mL)
ITX
40.4 (0.5)
0.04
0.12
0.12 - 500
Irgacure
18.4 (2.0)
0.33
0.5
0.5 - 1000
TRP
23.2 (0.5)
0.2
0.6
0.6 - 1000
This MRM data was then used to quantify migrants in cap
extracts, examples of various extracts are given in Figures 6
and concentrations of migrants were summarized in Table 5.
2.5e5
Max. 2.6e5 cps.
Deco*
5.5
XIC of +MRM (6 pairs) ...
1.4e5
Irgacure
Intensity, cps
Intensity, cps
1.5e5
1.0e5
0.0
1.0
2.0
3.0 4.0 5.0
Time, min
1.5e5
6.0
7.0
Max. 1.6e5 cps.
6.0
7.0
8.0
3.0 4.0 5.0
Time, min
6.0
5.5
Max. 7.1e4 cps.
Irgacure
2.0e4
1.2e5
Irgacure
7.0
5.5
Varnish
5.2
ITX
TRP
1.0
2.0
3.0 4.0 5.0
Time, min
2.5e5
6.0
7.0
Intensity, cps
Intensity, cps
2.0e5
1.0e5
5.0e4
ITX
TRP
5.5
Deco + varnish
Irgacure
1.0
2.0
3.0 4.0 5.0
Time, min
6.0
7.0
8.0e4
6.0e4
4.0e4
2.0e4
0.0
ITX
TRP
1.0
2.0
8.0
Max. 1.3e5 cps.
1.0e5
1.5e5
0.0
2.0
Figure 6. A comparison of food packaging
samples extracted with acetonitrile and where the
acetonitrile extract of the same sample had been
evaporated to dryness and reconstituted in mobile
phase* (cap with decoration (top), cap sealed
with varnish (middle), and cap with decoration
and sealed with varnish (bottom))
4.0e4
0.0
Max. 3.1e5 cps.
Deco + varnish*
1.0
ITX
6.0e4
Intensity, cps
Intensity, cps
3.0 4.0 5.0
Time, min
3.0e5
TRP
ITX
TRP
2.0
4.0e4
7.1e4
5.0e4
1.0
6.0e4
Irgacure
1.0e5
0.0
8.0e4
0.0
5.5
Varnish*
1.0e5
2.0e4
ITX
TRP
Irgacure
1.2e5
2.0e5
5.0e4
Max. 1.4e5 cps.
5.5
Deco
3.0 4.0 5.0
Time, min
6.0
7.0
www.spektrotek.com
95
Table 5. Quantitation results from real samples (* sample was
evaporated to dryness and reconstituted in the same volume
of mobile phase A to improve HPLC peak shape)
ITX
(ng/dm²)
Irgacure (ng/
dm²)
TRP
(ng/dm²)
Deco
4.43
6320
5.39
Deco*
4.96
4347
5.77
Varnish
0.08
3940
0.67
Varnish*
0.54
2100
0.69
Deco + varnish
4.65
6750
3.97
Deco + varnish*
4.26
3687
3.99
Extract
To further identify the migrant the automatically acquired
EPI spectra was searched against a mass spectral library
previously created with spectra obtained from 10 ng/mL
standards. DBS enabled the acquisition of high quality MS/MS
spectra even for co-eluting compounds. The Purity Fit shown
in Table 6 indicated if the spectrum, in the extract, was a good
match for the library spectrum, generally a fit above 70%
indicated a positive identification of the migrant in the extract.
Summary
The LC-MS/MS method developed can be used for quantitation
of migrants in food packaging material. The sensitivity levels
of the 3200 QTRAP® system were high enough to detect
migrants at 0.01 mg/kg in extracts. A mass spectral library
containing of EPI spectra at different standardized Collision
Energy and Collision Energy Spread values can then be used
to identify the compound at the required matrix detection
levels, enabling direct injection analysis on extracts.
Acknowledgements
We acknowledge Mr Philippe Tourelle and Gilles Jarry of
the society Impress Metal Packaging (France) for supplying
extracts and samples.
References
1 European Commission – Health & Consumer Protection
Directorate general - SANCO D3/AS D(2005)
2 FDA 21 CFR 170.100 - Submission of a premarket
notification for a food contact substance (FCN) to the Food
and Drug Administration (FDA). Code of Federal Regulations
(December 2005)
Table 6. The Purity Fit (%) results taken from the spectra
obtained from contaminants in real samples when compared
with those in a library of spectra of standards (* sample was
evaporated to dryness and reconstituted in the same volume
of mobile phase A to improve HPLC peak shape)
www.spektrotek.com
Extract
96
ITX %
Irgacure %
TRP %
Deco
78
88
81
Deco*
87
28
31
Varnish
63
60
98
Varnish*
34
81
44
Deco + varnish
97
44
65
Deco + varnish*
91
57
95
SCIEX™ is being used under license. Publication number: 1830210-01
Lily Sanchez 1, Lee Yoo 1, Mike Wehner 1, and Matthew R. Noestheden 2
1
Orange County Water District, Fountain Valley, California (USA); 2AB SCIEX Concord, Ontario
(Canada)
Overview
Analysis of Perfluoroalkyl Acids Specified Under the UCMR3
Using the QTRAP® 6500 LC/MS/MS System
This application note highlights the sensitivity and precision of the QTRAP®
6500 LC/MS/MS system for the analysis of perfluoroalkyl acids (PFAAs)
in drinking water. The PFAAs analyzed are a subset of EPA Method 537
(Determination of Selected Perfluorinated Alkyl Acids in Drinking Water
by Solid Phase Extraction and Liquid Chromatography/Tandem Mass
Spectrometry [LC/MS/MS])1, comprising the PFAAs outlined in the
Unregulated Contaminant Monitoring Rule 3 Assessment Monitoring list
(UCMR3).2 Statistically validated method detection limits range from 1.4 –
35.9 ng/L.
Introduction
PFAAs are ubiquitous chemicals that are used in a variety of industrial and consumer products including carpets, cookware,
paints, shampoos, food packaging, etc.3 PFAAs have high thermal and chemical stability and are highly resistant to degradation
in aquatic environments. Typical concentrations of PFAAs found in various water sources range from pg/L to µg/L levels.
Within the scope of EPA 537 there are 14 PFAAs (Table 1). Of these 14, six are specified in the UCMR3 Assessment Monitoring
list: PFBS, PFHpA, PFHxS, PFOA, PFOS and PFNA.
This paper describes the performance of the QTRAP® 6500 system for the evaluation of the PFAAs in the UCMR3 using the
guidelines laid out in EPA 537.
Table 1. PFAAs in EPA Method 537. Those compounds in bold type face are included in the UCMR3 Assessment Monitoring list.
Abbreviation
CASRN
UCMR3 MRL(ng/L)
PFHxA
307-24-4
-
Perfluoroheptanoic acid
PFHpA
375-85-9
10
Perfluorooctanoic acid
PFOA
335-67-1
20
Perfluorononanoic acid
PFNA
375-95-1
20
Perfluorodecanoic acid
PFDA
335-76-2
-
Perfluoroundecanoic acid
PFUnA
2058-94-8
-
Perfluorododecanoic acid
PFDoA
307-55-1
-
Perfluorotridecanoic acid
PFTrDA
72629-94-8
-
Perfluorotetradecanoic acid
PFTA
376-06-7
-
Perfluorobutanesulfonic acid
PFBS
375-73-5
90
Perfluorohexanesulfonic acid
PFHxS
355-46-4
30
Perfluorooctanesulfonic acid
PFOS
1763-23-1
40
N-methyl perfluorooctane-sulfonamidoacetic acid
NMeFOSAA
-
-
N-ethyl perfluorooctane-sulfonamidoacetic acid
NEtFOSAA
-
-
www.spektrotek.com
Compound
Perfluorohexanoic acid
97
Experimental
Sample preparation and data processing were carried out
according to EPA Method 537 without deviation (EPA 537
sections 10, 11 and section 12), unless specifically noted. All
required quality control parameters (EPA 537 section 9.3) were
met or exceeded for each batch of calibrators and/or samples
analyzed. Quantitation was performed using MultiQuant™
3.0 software. All calibration curves had a 1/x concentration
weighting and were forced through the intercept as specified
in EPA 537 section 10.2.6. For carboxylic acids 13C2-PFOA
was used as the internal standard (ISTD), while all sulfonic
acids used 13C4-PFOS as the ISTD. The surrogates used were
13C2-PFHxA and 13C2-PFDA, both of which were fortified
into samples at 40 ng/L.
Table 3. ESI source parameters
Analyses were carried out using the SCIEX QTRAP® 6500
system coupled with an Agilent 1260 HPLC (degasser,
binary pump and column oven) with an Eksigent ULC 100
HTC-xt autosampler. The mobile phase consisted of 20mM
ammonium acetate with methanol. Gradient parameters are
provided in Table 2. All samples were analyzed with a 5 µL
injection (vs. 10 µL in EPA 537) onto an Atlantis T3 analytical
column (150 x 2.1 mm, 5 µm) heated to 35˚C. An Atlantis T3
column (50 x 2.1mm, 5 µm) was also used as a delay column.
Table 4. MRM transitions, retention time (RT), Declustering
Potential (DP), and Collision Energy (CE) for target PFAAs,
ISTDs (*) and surrogates (^)
Table 2. LC gradient conditions
Time (min)
Flow Rate
(µL/min)
A (%)
B (%)
www.spektrotek.com
Value
Polarity
negative
Curtain Gas
30 psi
Collision Gas
12 psi
IonSpray Voltage
-4500 V
Temperature
400˚C
GS1
30 psi
GS2
30 psi
Compound
Q1
Q3
RT
DP (V)
CE (V)
PFBS 1
298.8
79.8
6.8
-60
-68
PFBS 2
298.8
98.9
6.8
-60
-36
PFHpA 1
362.8
318.8
10.7
-5
-12
PFHpA 2
362.8
168.8
10.7
-5
-22
PFHxS 1
398.9
79.7
10.7
-70
-86
PFHxS 2
398.9
98.7
10.7
-70
-74
PFOA 1
412.8
368.9
12.1
-5
-14
PFOA 2
412.8
168.7
12.1
-5
-24
PFOS 1
498.9
79.8
13.2
-60
-122
PFOS 2
498.8
98.9
13.2
-60
-98
PFNA 1
462.9
418.9
13.3
-30
-14
462.9
218.9
13.3
-30
-24
0.0
450
60
40
PFNA 2
1.0
450
60
40
13C2-PFOA*
414.9
369.8
12.1
-20
-14
6.0
450
35
65
13C4-PFOS*
502.9
79.8
13.3
-10
-102
6.1
350
35
65
13C2-PFHxA^
314.8
269.8
8.9
-15
-12
14.0
350
10
90
13C2-PFDA^
514.9
469.9
14.3
-25
-16
15.0
350
10
90
15.1
350
60
40
16.0
450
60
40
18.0
450
60
40
The QTRAP® 6500 system was operated in negative polarity
Electrospray Ionization (ESI) using Multiple Reaction
Monitoring (MRM) and the Scheduled MRM™ algorithm. ESI
source and MRM parameters are outlined in Tables 3 and 4.
98
Parameter
EPA 537 permits deviation from the LC conditions provided in
the method. To that end, the method presented here used an
Atlantis T3 column (5 µm) and a gradient that was designed
to increase method throughput, while still providing sufficient
chromatographic resolution (Figure 1).
Figure 1. Final chromatography using a 20mM ammonium
acetate / methanol mobile phase. Targets are shown on
top with branched isomers of PFHxS and PFOS indicated.
ISTDs (13C2-PFOA and 13C4-PFOS) and surrogates
are shown on the bottom (SUR1 = 13C2-PFHxA and
SUR2 = 13C2-PFDA)
The correlation (r) value for all calibration curves were > 0.99
(Figure 3).
Figure 3. Calibration lines and regression equations for all six
PFAAs
For PFHxS and PFOS the presence of additional small peaks
points to the presence of branched isomers, which are known
contaminants in the technical PFAAs suggested for purchase
in EPA 537. When present, these isomers were summed into
a combined value for the branched and linear isomers. This
adheres to section 12.4 of EPA 537.
Initial Calibration
The Initial Calibration (EPA 537 section 10.2) was carried
out using the UCRM3 Assessment Monitoring list as a guide,
with the lowest calibration level for each target compound
corresponding to ½ of the UCMR3 reporting limit (Table 1).
Owing to the high sensitivity of the QTRAP® 6500 system
these low ng/L levels were easily obtained for all compounds,
with Signal-to-noise values (S/N) of 50 to 1700 after 1-point
Gaussian smoothing using a peak-to-peak algorithm (Figure
2). All calibration acceptance criteria specified in EPA 537
section 10.2 were met.
Initial Demonstration of Capability
To demonstrate method suitability for EPA 537 it is necessary
to perform an Initial Demonstration of Capability (IDC)
following the Initial Calibration. In addition to the ongoing QC
criteria specified in EPA 537 section 9.3, adhering to the IDC
necessitates the following:
1.Extraction of four Laboratory Fortified Blanks (LFB) to
assess Accuracy (±30%) and Precision (RSD <20%).
Fortification should correspond to a mid-level calibrator.
2.PFBS and 13C2-PFHxA (surrogate) must have peaks
Asymmetry Factors between 0.8 to 1.5.
3.Extraction of seven LFBs that must meet a Prediction
Interval of Results (PIR) of 50 to 150% to define the Method
Reporting Limits (MRL).
4.Determination of Method Detection Limits (MDL). This is
an optional part of the IDC that requires seven replicates
prepared over three days. In this study the MRL replicates
were used.
5.All targets compounds in a Laboratory Reagent Blank (LRB)
and Field Reagent Blank (FRB) after the Initial Calibration
must quantify to <1/3 of MRL.
6.Evaluate method accuracy (±30%) using a Quality Control
Sample (QCS) that is sourced from a vendor other than the
one that provided the calibration samples.
Each of these criteria are discussed below.
www.spektrotek.com
Figure 2. Signal-to-noise values (S/N) for the low calibrators.
Low calibration levels for each compound are ½ of the UCMR3
reporting limits
99
Accuracy and Precision
Method Reporting Limits
Fortification for evaluation of Accuracy and Precision was
done at 200 ng/L. This corresponded to calibration level four
of six. For the four replicates extractions analyzed the relative
standard deviations (RSD) ranged from 3.1 to 9.8%, while
the recoveries ranged from 89 to 96% (Table 5). All of these
values were within the EPA 537 specified ranges of < 20%
RSD and ±30% recoveries.
As the current method was designed to meet the UCMR3
reporting limits, the levels used to fortify the seven extractions
required for the calculation of the Method Reporting Limit
(MRL) correspond to the UCMR3 reporting limits. To be a
valid MRL the results of the seven replicate extractions must
meet a set of statistical criteria, which are outlined in detail in
section 9.2.5 of EPA 537. Briefly, the calculations are:
Table 5. Method performance
Compound
Precision
(%)
Accuracy
(%)
QCS (%)
Batch 1
Batch 2
RPD
(%)
PFBS
3.5
91
71.2
87.6
5.65
PFHpA
6.1
89
86.0
109.0
0.20
PFHxS
3.3
93
95.3
116.0
4.81
PFOA
4.7
96
96.8
101.4
3.84
PFOS
3.1
92
91.9
111.5
5.11
PFNA
9.8
91
72.8
103.6
9.21
Asymmetry Factor
To ensure acceptable chromatography of the two earliest
eluting peaks in the method, the user is required to calculate the
Asymmetry Factor (AS) for every batch of samples analyzed.
In the present method this corresponded to PFBS and 13C2PFHxA. The AS was calculated from a mid-level calibrator of
200 ng/L. Figure 4 demonstrates that the AS for PFBS (1.31)
and 13C2-PFHxA (1.37) meet the EPA 537 acceptance criteria
of: AS must fall in the range of 0.8 to 1.5. The AS values were
calculated automatically using MultiQuant™ software version
3.0.
The PIR must be within 50 and 150% to be a validated
MRL. Using the above equations on samples that had been
fortified at the UCMR3 reporting limits yielded acceptable
PIR values (Table 6). Based on these calculations and the
UCMR3 reporting limits that were used as sample fortification
guidelines, all compounds in the current method were
validated.
Table 6. MRL and MDL determination and statistical
verification
Fortification
Level (ng/L)
Lower PIR
(%)
Upper PIR
(%)
MDL
(ng/L)
PFBS
90
81
86
56
PFHpA
10
99
99
75
PFHxS
30
8.3
1.6
144
PFOA
20
75
77
98
PFOS
40
114
109
35.9
PFNA
20
1.4
3.1
7.0
Compound
Method Detection Limits
www.spektrotek.com
The Method Detection Limit (MDL) was calculated using the
following equation:
100
Figure 4. Asymmetry Factor for PFBS (left) and 13C2PFHxA (right). The example on the left demonstrates how
MultiQuant™ software 3.0 calculates AS.
Using the MRL extracts, the calculated MDLs ranged from 1.4 to
35.9 ng/L. It is conceivable that the QTRAP® 6500 could detect
lower concentrations based on the S/N for the low calibrators
(Figure 2).
In the present method, all target compounds were observed
well under 1/3 of their respective MRLs.
Quality Control Sample and Ongoing QC Results
The Quality Control Sample (QCS) was evaluated at 200 ng/L
for all compounds to verify the validity of the Initial Calibration.
All compounds met the ±30% accuracy criterium for the QCS
samples (Table 5).
Three components of the ongoing QC requirements specified
in EPA 537, the LRB, Asymmetry Factor and QCS, have
already been discussed as they are also specified components
of the IDC. In addition, the following ongoing QC criteria were
required:
1.Laboratory fortified blank (LFB) should be analyzed with
each batch. Acceptance criteria will depend on the fortified
concentration, which should change from batch-to-batch.
2.Internal standard (ISTD) responses should not deviate more
than 50% from the average ISTD response in the initial
calibration and the ISTD in all samples should be 70-140%of
the response in the latest continuing calibration check
(CCC).
3.Surrogate recovery should be ±30% of the expected value.
4.Laboratory fortified sample matrix (LFSM) and a duplicate
(LFSMD) should yield accuracies within ±30% of expected
values and the relative percent difference (RPD) between
the LFSM and LFSMD must be < 50%.
5.A field reagent blank (FRB) should not contain residue levels
> 1/3 of the calculated MRLs.
Figure 5. LRB (top) and FRB (bottom) results. Both LRB and
FRB results showed background levels that were all < 1/3 of
the calculated MRLs. The FRB matrix was finished tap water.
Table 6. LRB and FRB background levels in comparison to
the MRL
(ng/L)
1/3 MRL
PFBS
PFHpA
PFHxS
PFOA
PFOS
PFNA
30
3.3
10
6.7
13.3
6.7
LRB
-
-
0.06
-
0.2
0.2
FRB
0.3
0.3
0.4
0.8
0.3
0.2
Conclusion
The QTRAP® 6500 LC/MS/MS system is a sensitive and
robust platform for the analysis of PFAAs in drinking water.
The demonstrated MRLs easily meet the UCMR3 reporting
limits.
A Laboratory Reagent Blank (LRB) is a system blank that has
been taken through the entire extraction procedure to assess
for background contamination. Following the Initial Calibration
a LRB was assessed. Once MRLs were established, the LRB
was evaluated with regards to the background levels relative
to the calculated MRLs (Figure 5).
The first four of these criteria were all met or exceeded in all
samples discussed herein. The RPD results ranged from 0.2
to 9.2, well within the ±30% RPD permitted in EPA 537
(Table 5). The FRB matrix in this study was finished tap
water. Figure 5 demonstrates that all compounds were < 1/3
of the calculated MRLs, which meets EPA 537 criteria and
further validates the RPD results since there was negligible
background PFAA contamination in the sample matrix.
There is also criteria for CCCs (low CCC accuracy 50-150%;
mid/high CCC accuracy 70-130%; surrogate accuracy 70130%) that were met for all samples analyzed.
References
1.EPA Method 537 ‘Determination of Selected Perfluorinated
Alkyl Acids in Drinking Water by Solid Phase Extraction
and Liquid Chromatography / Tandem Mass Spectrometry
LC/MS/MS)’ version 1.1 (2009)
http://www.epa.gov/microbes/documents/Method%20
537_FI NAL_rev1.1.pdf
2.Unregulated Contaminant Monitoring Rule 3 (UCMR3)
http://water.epa.gov/lawsregs/rulesregs/sdwa/ucmr/
ucmr3/
3.M.F. Rahman et al.: ‘Behavior and Fate of Perfluoroalkyl
substances (PFAs) in Drinking Water Treatment: A
Review.’ Water Research 50 (2014) 318-340
© 2015 AB Sciex. For Research Use Only. Not for use in
diagnostic procedures.
The trademarks mentioned herein are the property of AB
Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is
being used under license.
Abbreviations
As – asymmetry factor
CASRN – chemical abstracts registration number CCC – continuing
calibration check
CE – collision energy
DP – declustering potential
EPA – environmental protection agency
ESI – electrospray ionization
FRB – field reagent blank
HRPIR – half range prediction interval of results
IDC – initial demonstration of capability
ISTD – internal standard
LFB – laboratory fortified blank
LFSM – laboratory fortified sample matrix
LFSMD – laboratory fortified sample matrix duplicate LRB – laboratory
reagent blank
MDL – method detection limit
MRL – method reporting limit
MRM – multiple reaction monitoring
PFAAs – perfluoroalkyl acids
PIR – prediction interval of results
QCS – quality control sample
RPD – relative percent difference
RSD – relative standard deviation
RT – retention time
S/N – signal-to-noise
UCMR3 – unregulated contaminant monitoring rule 3 assessment
monitoring list
www.spektrotek.com
Laboratory Reagent Blank
101
LC-(DMS)-MS/MS Analysis of Emerging Food Contaminants
Quantitation and Identification of Maleic Acid in Starch-Rich Foods
Fanny Fu1 and André Schreiber2
AB SCIEX Taipei (Taiwan), 2AB SCIEX Concord, Ontario (Canada)
1
Experimental
Sample Preparation
Simple liquid extraction of food samples was performed
using the following procedure developed by the Taiwan FDA4
Introduction
Recent findings (in May 2013) of maleic acid in foods, such
as tapioca starch, tapioca balls, rice noodles, and hotpot
ingredients, caused the recall of many starch-based food
products in Asia.1-3
Maleic acid is usually not used in manufacturing of food
products, and it is an unapproved food additive.
• Weigh 1 g of homogenized samples into polypropylene
centrifuge tubes (50 mL).
• Add 25 mL of 50% methanol.
• Shake vigorously for 30 min using a shaker.
• Add 20 mL of 0.5 N KOH.
• Vortex and let stand for two hours.
• Add 3 mL of 5 N HCl and bring to a final volume of 50 mL
with deionized water.
• Vortex and centrifuge.
• Transfer an aliquot of 100 μL of the extract into an
autosampler vial and dilute with 900 μL of water resulting
in a total dilution factor of 500.
Further dilution of the extract might be necessary if the
sample is heavily contaminated.
LC
Maleic acid and fumaric acid were analyzed using an Agilent
1260 system with a gradient on a Poroshell EC C18 column
Occasional consumption of maleic acid at low levels does
not pose any significant health risk; however, long term
consumption of high levels of the compound can cause
kidney damage.
The substance has been traced to a modified starch containing
maleic anhydride, a chemical used in the production of food
packing materials.
Reliable analytical methods are needed to detect maleic
acid in foods to identify potential trace contamination in
food production, processing, and packaging and to ensure
consumer health.
www.spektrotek.com
Maleic acid is cis-butenedioic acid (Figure 1) and is closely
related to fumaric acid (trans-butenedioic acid). The LC-MS/
MS-based method presented here can be used to confidently
identify and accurately quantify maleic acid even in presence
of fumaric acid.
102
OH
O
O
OH
O
maleic acid
OH
HO
O
fumarica cid
Figure 1. Chemical structures of maleic acid (left) and fumaric
acid (right)
maleic acid
3.0e6
Intensity, cps
2.5e6
2.0e6
1.5e6
fumaric acid
5.24
1.0e6
5.0e5
0.0
0.00
.5
1.01
.5
2.02
.5
3.03
.5
Figure 1. LC-MS/MS analysis of maleic acid and fumaric acid
4.04
Time,m in
.5
5.05
.5
6.06
.5
7.07
.5
8.0
LC-MS/MS data were processed using the MultiQuant™
software version 2.1.
4.59
3.5e6
(150 x 3.0 mm, 2.7 μm) and a mobile phase of water containing
0.1% formic acid (A) and methanol containing 0.1% formic
acid (B). The flow rate was set to 0.3 mL/min. Gradient details
are listed in Table 1. A sample volume of 10 μL was injected.
Time (min)
Flow (mL/min)
0.0
98
1.0
5.0
7.0
7.5
16.0
A (%)
B (%)
2
Compound
Q1 (amu)
Maleic acid 1
Maleic acid 2
Fumaric acid 1
115
Fumaric acid 2
0.3
5
95
98
2
MS/MS
The AB SCIEX QTRAP® 5500 was used with the Turbo V™
source and an Electrospray Ionization (ESI) probe. The mass
spectrometer was operated in Multiple Reaction Monitoring
(MRM) mode using negative polarity. Two selective MRM
transitions were monitored using the ratio of quantifier and
qualifier ion for identification (Table 2).
In addition, SelexION™ differential mobility separation was
investigated to increase selectivity, improve Signal-to-Noise
(S/N), and increase confidence in identification.
Q3 (amu)
CE (V)
71
-11
32
-28
71
-11
32
-28
An example chromatogram of the detection of maleic acid and
fumaric acid is shown in Figure 1.
First, the limit of quantitation (LOQ), linearity, and repeatability
were evaluated using injections of maleic and fumaric acid
standards ranging from 0.5 to 200 ng/mL and spiked matrix
samples.
Both compounds had LOQ values in the sub ng/mL range,
allowing a sample extract dilution to minimize possible matrix
effects. Linearity was excellent with a regression coefficient of
0.999 for quantifier and qualifier transitions. The accuracy values
ranged from 89.6 to 107.6% across the linear dynamic range
(Figure 2).
www.spektrotek.com
Table 1. LC gradient used for the separation of maleic acid and
fumaric acid
Table 2. MRM transitions and retention times of maleic acid
and fumaric acid
103
blank
(115/71)
0.5
1.0
2.0
blank
(115/32)
0.5
1.0
2.0
Figure 2. Chromatograms of the quantifier and qualifier transition of maleic acid of the blank sample and at concentration of 0.5,
1.0, and 2.0 ng/mL (top) and calibration lines from 0.5 to 200 ng/mL (bottom)
Repeatability was evaluated using 7 injections at 5 ng/mL.
The coefficient of variation (%CV) was 2.9% for the quantifier
transition (115/71) and 1.8% for the qualifier transition
(115/32).
A number of food samples were analyzed for maleic and
fumaric acids, including noodles, tapioca starch, and
processed foods. The analysis of a 20 ppb spiked blank
extract gave 91.9% recovery.
Table 3. Maleic acid findings in different food samples
www.spektrotek.com
Compound
104
Q1 (amu)
Q3 (amu)
Noodles
0.18
0.052
Tapioca starch
4790
0.057
Processed food
36.7
0.055
20 ppb spike in
blank extract
18.4
(91.9% recovery)
0.057
CE (V)
0.049
Figure 3. Results for maleic acid in different food samples, the
‘Multicomponent’ query in MultiQuant™ software was used to
identify target analytes based on their MRM ratio
In a last experiment we investigated the use of SelexION™
differential mobility separation (DMS) to increase selectivity
and confidence in identification.
SelexION™ uses a planar differential mobility device that
attaches between the curtain plate and orifice plate of the
QTRAP® 5500 system (Figure 4). An asymmetric waveform,
called Separation Voltage (SV), combined with a Compensation
Voltage (CoV) is used to separate ions based on difference in
their mobility.5-6
Chemical modifiers, like isopropanol, methanol, or acetonitrile,
can be introduced into the transport gas via the curtain gas to
alter the separation characteristics of analytes.
SV and CoV were optimized for maleic and fumaric acids to
separate these two isomers with identical MRM transitions.
Best separation and highest selectivity was achieved using an
SV of 3600 V and CoV of -8.0 V and -10.5 V, respectively
(Figure 5). The added selectivity resulted in reduced
background interferences. The presence of an MRM signal in
combination with an optimized CoV value can also be utilized
as an additional ‘identification point’ to increase confidence in
data quality.
Figure 4. SelexION™ differential mobility separation (DMS)
Summary
The method and data presented here showcase the fast,
easy, and accurate solutions for the analysis of maleic acid
and fumaric acid in starch-rich foods by LC-MS/MS and LCDMS-MS/MS. The AB SCIEX QTRAP® 5500 systems provide
excellent sensitivity and repeatability for this analysis, with
minimal sample preparation allowing maximized throughput
for the analysis of many samples in a short time period.
Maleic acid was quantified in different food samples. MRM
ratio calculations in MultiQuant™ software used for compound
identification. SelexION™ differential mobility separation was
also used successfully to further increase selectivity and to
clearly differentiate between isomeric species adding another
‘identification point’ and increased confidence to the results.
Table 3 and Figure 3 show quantitative and qualitative results.
MRM ratios were calculated using the ‘Multicomponent’ query
in MultiQuant™ software.
References
maleic acid
fumarica cid
Figure 5. Compensation voltage (CoV) ramps for maleic and
fumaric acid, best separation and highest selectivity was
achieved using CoV of -8.0 V and -10.5V, respectiviely
XIC of -MRM (2 pairs): 115.000/71.000 Da ID: Maleic acid 1 from Sample 6 (2mix-10ppb) of 20130705 MA.wiff (Turbo Spray), Smoothed
Intensity, cps
DMS off
maleic acid
1.5e5
1.0e5
5.0e4
0.0
0.0
fumaric acid
3.36
1.0
2.0
3.0
6.15
4.0
5.0
6.0
10.44
8.0
9.0
10.0
Time, min
XIC of -MRM (30 pairs): 115.000/71.000 Da ID: CoV -7.5 from Sample 1 (2mix) of 20130705-DMS.wiff (Turbo Spray), Smoothed
7.0
11.19
11.0
11.86
12.58
12.0
13.00
13.0
13.97 14.20 14.79
14.0
15.0
Max. 2.8e4 cps.
© 2013 AB SCIEX. The trademarks mentioned herein are the
property of AB Sciex Pte. Ltd. or their respective owners. AB
SCIEX™ is being used under license. Publication number:
7830213-01
4.90
2.8e4
2.5e4
Intensity, cps
Max. 2.0e5 cps.
5.01
2.0e5
1.http://www.fda.gov.tw/EN/newsContent.
aspx?ID=9918&chk =454d1df8-1f26-43a3-9a855540dc07caae
2.http://www.ava.gov.sg/NR/rdonlyres/9253E7B2-E57D4992-982C-1304E73748D6/26074/Pressrelease_
Recallofstarchba sedproductsfromTaiwan.pdf
3.http://www.fda.gov.ph/advisories/food/76474-fdaadvisory-on-maleic-acid
4.http://www.fda.gov.tw/TC/siteList.aspx?sid=3503
5.B.B. Schneider, T. R. Covey, S.L. Coy, E.V. Krylov, E.G.
Nazarov: Int. J. Mass Spectrom. 298 (2010) 45-54
6.B.B. Schneider, T. R. Covey, S.L. Coy, E.V. Krylov, E.G.
Nazarov: Anal.Chem. 82 (2010) 1867-1880
maleic acid
2.0e4
DMS on
(CoV = – 8.0 V)
1.5e4
1.0e4
5000.0
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
Time, min
XIC of -MRM (30 pairs): 115.000/71.000 Da ID: CoV -10.5 from Sample 1 (2mix) of 20130705-DMS.wiff (Turbo Spray), Smoothed
12.0
13.0
14.0
15.0
Max. 5175.0 cps.
DMS off
fumaric acid
4000
(CoV = – 10.5 V)
3000
2000
1000
0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Time, min
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Figure 6. Selective detection of maleic acid and fumaric acid
using LC-DMS-MS/MS, the added selectivity resulted in lower
background noise and interferences and increased confidence
in identification
www.spektrotek.com
6.04
5000
Intensity, cps
15.69
11.0
105
Analysis of Endocrine Disruptors, Pharmaceuticals, and
Personal Care Products in River Water
Christopher Borton1, Loren Olson2
1
AB SCIEX, Golden, CO; 2 AB SCIEX, Foster City, CA
Overview
Endocrine disrupting compounds (EDC) encompass a wide range of
pollutants, including pharmaceuticals and personal care products (PPCP),
pesticides, and steroids to name a few. EDC are thought to disrupt the
endocrine function of mammals and fish, and as a result their biological
effects are a growing concern. In order to properly assess the effects of
these compounds on our environment, it is necessary to accurately monitor
their presence. A method is presented for analyzing up to 100 EDC and PPCP
compounds using LC-MS/MS. This method is a straight forward approach
for the quantitation and identification of these compounds with excellent
sensitivity and ruggedness.
Introduction
A wide range of endocrine disrupting compounds were
determined in river water sampled near a water treatment
plant. Compound levels upstream and downstream from the
plant were quantified and compared. A combination of Solid
Phase Extraction (SPE) and LC-MS/MS analysis in Multiple
Reaction Monitoring (MRM) mode achieved low parts per
trillion detection limits across multiple compound classes with
a linear range of 3-4 orders of magnitude for all compounds.
Both positive and negative ionization modes were utilized.
APCI and ESI ionization techniques were investigated using
the DuoSpray™ ionization source. Electrospray ionization
with polarity switching on the Turbo V™ source yielded the
broadest coverage across compound classes. Two MRM
transitions were monitored for each compound to achieve
sensitive and specific quantitation as well as ion ratio
identification. A total of 160 MRM transitions were monitored
on a chromatographic time scale.
Two sets of river water samples were collected from a rural
river (River 1) and an urban city river (River 2) both upstream
and downstream of a sewage treatment plant in North America.
The upstream and downstream samples for these two areas
were then compared to determine environmental impact
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Experimental
106
An AB SCIEX API 4000™ LC/MS/MS System equipped with
a Shimadzu Prominence autosampler and binary LC pump
was used. Ionization was achieved by Electrospray Ionization
(ESI) and Atmospheric Pressure Chemical Ionization (APCI)
using the DuoSpray™ and Turbo V™ ionization sources. All
compounds were monitored using two Multiple Reaction
Monitoring (MRM) transitions per compound. Each MRM
transition had a dwell time of 5ms/sec. The most sensitive,
first MRM transition was used for quantitation while the
second MRM transition was used for qualitative identification
using ion ratio determination. See Figure 3 and 4 for examples.
The total cycle time for the method with polarity switching
was approximately 3 seconds. Instrument conditions were
as follows: CUR 20, CAD 7, GS1 75, GS2 65, IS 5000, and
TEM 600. Chromatography was performed on a Phenomenex
Ultracarb (20) C18 250 X 4.5 mm 5 μm reverse phase
column at 30°C. The total flow rate was 600 μL/min and used
a gradient starting at 95% A and held for 1 minute before
ramping to 50% over 24 minutes. At a run time of 25 minutes
the gradient was then ramped to 4% A over 10 minutes and
held for an additional 10 minutes. Re-equilibration time was
10 minutes for a total run time of 55 minutes. Eluent A was
0.01% formic acid in water and eluent B was 0.01% formic
acid in acetonitrile.
Laboratory control samples and matrix spike samples were
prepared to monitor extraction efficiency. After conditioning
with 20 mL of methanol followed by 40 mL of water, 1.0 L of
sample was loaded onto the cartridge at a flow rate of 25.0
mL/min. After loading, nitrogen was then pulled through the
cartridge for 15 minutes to allow for sample drying. Then 5.0
mL of acetonitrile was added to the SPE bed and allowed to
stand for 15 minutes. The SPE cartridges were then eluted at
gravity flow into a 12 mL amber vial. Finally, water was added
to the extract to a final volume of 10.0 mL. Samples were kept
at 4°C ± 1°C until analysis. Figure 1 shows a schematic of the
sample preparation procedure.
Quantifier
Compound
Type
Qualifier
Quantifier
Q1
Q3
Q1
Acetaminophen
Analgesic
152
110
152
65
Q3
Estradiol
Compound
Estrogen
Type
255
Q1
159
Q3
Ketoprofen
Analgesic
255
105
255
77
Ethinylestradiol Estrogen
271
133
Estrogen
331
97
Qualifier
Q1
Q3
Codeine
Analgesic
300
215
300
17α-Hy165 droxy-progesterone
Hydrocodone
Analgesic
300
199
300
171 Progesterone
Estrogen
315
109
315
97.
Androstenedione
Androgen
287
97
287
97
Equilin
Estrogen
replacement
269
211
269
157
Testosterone
Androgen
289.5 97
289
109
Diethylstilbestrol
Estrogen
replacement
269
135
269
107
Dilantin
Anti-convulsant 253
182
TCEP
Flame
retardant
285
223
285
239
Meprobamate
Anti-anxiety
219
158
219
115 Simazine
Herbicide
202
132
202
124
Sulfadiazine
Antibiotic
251
92
251
65
Herbicide
207
72
Sulfamethoxazole
Antibiotic
254
92
254
108 Chlorotoluron
Herbicide
213
72
213
140
Sulfathiazole
Antibiotic
256
156
256
92
Herbicide
216
174
216
68
Sulfamerazine
Antibiotic
265
92
265
108 Chloridazon
Herbicide
222
104
222
92
Sulfamethizole
Antibiotic
271
156
271
92
Herbicide
230
146
230
188
Sulfamethazine
Antibiotic
279
92
279
124 Diuron
Herbicide
233
72
233
46
Sulfachlorop-yridazine
Antibiotic
285
92
285
65
Herbicide
253
171
253
85
Trimethoprim
Antibiotic
291
230
291
123 Bromacil
Herbicide
261
205
261
188
Sulfadimethoxine
Antibiotic
311
156
311
92
Metazachlor
Herbicide
278
134
278
210
Ciprofloxacin
Antibiotic
332
288
Metolachlor
Herbicide
278
134
284
175
Penicillin G
Antibiotic
335
176
335
217 DEET
Insect repellant
284
252
Amoxicillin
Antibiotic
366
114
366
208 Bezafibrate
Lipid regulator 192
119
Lincomycin
Antibiotic
407
126
407
359 Diazepam
Muscle-relaxant
362
139
362
121
Doxycycline
Antibiotic
445
428
445
339 Norethisterone
Ovulation
Inhibitor
285
154
285
193
Tetracycline
Antibiotic
445
410
445
154 Theophylline
Stimulant
299
109
299
91
Oxytetracycline
Antibiotic
461
426
461
443 Theobromine
Stimulant
181
124
181
96
Chlortetracycline
Antibiotic
479
462
479
154 Caffeine
Stimulant
181
138
181
110
Isoproturon
Atrazine
Propazine
Hexazinone
Table 1. Compound list including MRM transitions (positive polarity)
Table 1 (continued). Compound list including MRM transitions (negative polarity)
Type
Q1
Q3
Q1
Q3
Compound
Oxybenzone
Type
Q1
Q3
Q1
Q3
Virginiamycin
Antibiotic
526
109
526
67
Sunscreen
229
151
229
105
Monensin
Antibiotic
694
461
694
479 Sildenafil
Virility regulator
475
100
475
283
Erythromycin
Antibiotic
735
158
735
576 Vardenafil
Virility regulator
490
72
490
114
Roxithromycin
Antibiotic
838
679
838
158 Salicylic Acid Skin care, acne
139
61
139
79
Tylosin
Antibiotic
917
174
917
772 Cotinine
177
80
177
98
Meclocycline
Sulfosalinicyclate
Antibiotic
477
460
204
56
Sulfadimethoxine
Nicotine metabolite
4-AminoantiAminopyrine
pyrine
Antibiotic
311
156
Ketorolac
metabolite
256
105
256
77
Sulfachloro-Pyridazine Antibiotic
285
156
Fenoprop
Anti-inflammatory
269
181
269
85
Norifloxacin
Antibiotic
320
276
MeclofenamHerbicide
ic acid
296
278
296
243
Enroflofacin
Antibiotic
360
316
Piroxicam
332
95
332
121
Fluoxetine
Antidepressant 310
148
Nifedipine
347
315
Carbamazepine
Anti-seizure
194
358
139
358
75
Pentoxifylline
Blood viscosity
279
reducing agent
615
361
237
181
237
279
Dihydropyridine
calcium channel
blocker
193 Indomethacin Anti-inflammatory
138 Diatrizoate
Radiocontrasting
agent
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Compound
107
Quantifier
Compound
Type
Qualifier
Q1
Q3
Q1
Acetylsalicylic acid
Analgesic
179
137
179
93
Q3
Compound
Ibuprofen
Analgesic
205
161
205
Naproxen
Analgesic
229
183
229
Warfarin
Anti-coagulant
307
161
Diclofenac
Anti-arthritic
294
250
Carbadox
Antibiotic
261
122
2,4-D
Triclosan (Irgasan)
Antibiotic
287
35
Clofibric acid
Chloramphenicol
Antibiotic
321
257
Gemfibrozil
Anti-cholesterol 249
121
Quantifier
Qualifier
Q1
Q3
Q1
Q3
Type
Estrogen
269
159 Estradiol
Estrogen
271
155 Estriol
Estrogen
287
307
250 Ethinylestradiol
Estrogen
295
294
214 Tetrabromo-bisphenol A
Flame retardant
443
103
443
239
Herbicide
219
161
219
125
Metabolite of lipid
regulator
213
127
213
85
X-ray contrast agent 790
127
189
101
189
145
321
Estrone
152 Iopromide
2,4-Dichloro- benzoic acid
Quantitative optimization in Analyst® Software was utilized
to streamline method development for this large list of
compounds. The final method contains the analytes and MRM
transitions listed in Table 1.
XICo f+ MRM (137 pairs): Exp 1, 138.901/60.800 Da from Sample8 (100 ppb STD)o fD ata...
Filter
SPE new
Max. 8417.7 cps.
ESI positive
5.0e6
Intensity, cps
1L Riverw ater
6.6e6
6.0e6
Condition
4.0e6
3.0e6
2.0e6
1.0e6
1L filtered
water
SPE ready
10 mL extract
0.0
51
01
52
02
53
03
54
Time, min
XICo f -MRM( 24 pairs): Exp2 , 178.814/136.900 Da from Sample8 (100 ppb STD) ofD ataE...M
2.2e6
5
ax. 6.7e4 cps.
ESI negative
2.0e6
1.5e6
Intensity, cps
50 Li njection
04
1.0e6
5.0e5
0.0
www.spektrotek.com
108
52
XIC of -MRM (24 pairs): Exp 2, 17...
02
53
Time,m in
Max. 6.8e4 cps.
03
54
04
5
4.0e4
2.0e4
Acetylsalicylic
acid
0.0
22
24
Time, min
2.0e4
1.5e4
20
In tensity, cps
6.0e4
26
28
Max. 2.2e4 cps.
24.6
1.0e4
24
26
Time, min
1.5e6
1.0e6
22
Clofibric
acid
28
Max. 1.9e6 cps.
33.2
30
Warfarin
5.0e5
1.0e6
Diclofenac
38
Max. 1.4e6 cps.
36.0
5.0e5
0.0
32
34
36
Time, min
1.26e5
1.00e5
Ibuprofen
38
40
Max. 1.3e5 cps.
36.7
5.00e4
5.0e5
0.0
Max. 2.3e6 cps.
34.4
1.0e6
Intensity, cps
1.9e6
20
1.5e6
1.4e6
Chloramphenicol
5000.0
0.0
2.0e6
0.0
30
32
34
36
Time, min
Intensity, cps
In tensity, cps
23.1
Intensity, cps
A calibration curve was prepared in water/acetonitrile (1/1)
at the following concentrations, 0.2, 0.4, 1.6, 3.1, 6.3, 25,
and 100 ng/mL. Linearity was achieved for all monitored
compounds. Examples of linearity are shown in Figure 4.
Samples were collected and extracted using the procedure
described above. To monitor the extraction efficiency of the
sample preparation a laboratory control sample (LCS) was
prepared. This sample consisted of tap water being free of all
target compounds. This water was then spiked with all of the
target analytes. The final concentration of all analytes in the
LCS was 20 ng/L.
Recoveries in the LCS ranged from 30 to 115% across all
compounds. Based on these results, it was determined that
the sample preparation procedure used is adequate for a full
screen of the compounds reported. For future work, once
the final sample list is determined, surrogate compounds
will be selected for each compound class to closely monitor
the sample preparation procedure. If possible, a deuterated
surrogate will be chosen for each compound class and will
only be used to monitor sample preparation efficiency and not
instrument variability. It has been shown in previous work that
an internal standard, used to monitor instrument variability,
may introduce more error in the quantitation results of this
large list of compounds.
01
Figure 2. Polarity switching is utilized to encompass a large
list of analytes – 100 ng/mL standard injection
Intensity, cps
Figure 1. Sample preparation procedure for solid phase
extraction
51
32
34
Time, min
36
38
0.00
32
34
36
38
Time, min
40
Figure 3. Overlay of two MRM transitions used for six selected
analytes. The most sensitive transition in blue for each analyte
is used for quantitation. The area ratio of the second MRM in
red is used for identification
Analyte
LLOQ (ng/L) ppt
Analyte
LLOQ (ng/L) ppt
DEET
11.6
Propazine
0.46
Ketoprofen
3.3
Progesterone
3.9
Sulfadiazine
13.0
Trimethoprim
6.4
Fluoxetine
280
Androstenedione
4.7
2,4-D
2.3
Erythromycin
14.0
Ketorolac
0.0
5.0e5
MRM ratio = 0.576
Max. 3.6e4 cps.
MRM ratio
22.2= 0.525
1.0e6
5.0e5
5.0e5
0.0
22
Ketorolac
MRM ratio = 0.576
Mepobramate
MRM ratio = 0.077
32.8
30
32
34
Time, min
XIC 0.0
of +MRM (137 pairs): Exp 1, 2...
Max. 1.3e6 cps.
18
20
22
24
26
Time, min
22.7
1.3e6
5.0e5
0.0
28
Mepobramate
MRM ratio = 0.077
18
20
22
24
Time, min
2.0e4
3.0e5
1.0e4
2.0e5
0.0
1.0e5
26
Ketorolac
MRM ratio = 0.525
Mepobramate
30.2
MRM ratio = 0.071
30
Time, min
0.0
XIC
18
20
22
24
Time, min
22.7
4.0e5
Intensity, cps
Intensity, cps
1.0e6
26
Inten sity, cps
Intensity, cps
Inten sity,
cps cps
Intensity,
30.2
24
26
28
30
32
20
22
24
26
28
30
32
Time, min
Time, min
32.8
XIC 0.0
Max. 1.4e6 cps. XIC
Max. 3.6e4 cps.
0.0
26
28
30
32
34
26
28
30
32
34
Time, min
Time,30.2
min
27.6Exp 1, 2...
1.4e6
XIC
Max. 1.3e6 cps. XIC 3.8e4
of +MRM (137 pairs):
Max. 4.0e5 cps.
3.0e4
22.7
22.7
1.0e6
1.3e6
4.0e5
20
3.0e5
2.0e5
26
28
34
0.01.0e7
0
20
40
60
80
Concentration, n g/mL
8.0e6
EDC an d PPCP calibration .rdb (Carbamazepine 2...
6.0e6
2.8e64.0e6
Carbamazepine 2
40
60
80
Area, cou nts
Sulfathiazole 1
0
20
40
60
80
Sulfathiazole 1
2.00e6
1.00e7
100
4.00e6
8.7e6
8.0e6
2.00e6
7.0e6
6.0e60.00
0
20
40
60
80
Concen tration, ng/mL
EDC and PPCP calibration .rdb (Sulfathiazole 2)…
4.0e6
100
5.0e6
Sulfathiazole 2
0.0
6.0e6
0
5.0e6
20
40
60
Concen tration, n g/mL
80
20
40
60
80
2.0e7
EDC an d PPCP calibration.rdb (2,4-D 1): "Li…
1.5e7
3.5e7
1.0e7
3.0e7
5.0e6
100
4.0e6
3.0e6
100
2,4-D 1
2.5e7
0.0
0
20
40
60
2.0e7
EDC an d PPCP calibration.rdb (2,4-D 2): "Li...
1.5e7
Sulfathiazole 2
3.0e6
8.7e6
2.0e6
8.0e6
1.0e6
7.0e6
100
2,4-D 1
EDC an d PPCP calibration.rdb (Su lfathiazole 1)…)
6.00e6
4.00e6
1.20e7
80
0.0
100 2.5e7
0
Area, counts
8.00e6
20
40
60
EDC an d PPCP calibration.rdb (2,4-D 1): "Li…
1.0e6
3.5e7
5.0e5
3.0e7
5.0e5
1.20e7
0.0
2.0e6
0.0
0
1.5e6
100
100
Carbamazepine 2
2.5e6
5.0e5
EDC an d PPCP calibration.rdb (Su lfathiazole 1)…)
1.00e7
1.0e62.8e6
Area, cou nts
20
0.0
0
20
40
60
80
EDC an d PPCP calibration .rdb (Carbamazepine 2...
1.5e6
Area, counts
Area, cou nts
100 2.0e6
Theophylline 2
0
1.0e6
100
2.5e62.0e6
0
20
40
60
80
1.0e6
EDC and PPCP calibration .rdb (Th eophyllin.e 2)…
80
3.9e6
1.0e7
3.5e6
5.0e6
3.0e6
2.5e6 0.0
100
2,4-D 2
0
20
40
60
2.0e6
EDC an d PPCP calibration.rdb (2,4-D 2): "Li...
1.5e6
3.9e6
1.0e6
3.5e6
5.0e5
3.0e6
0.0
0
20
40
60
80
2.5e6
2.0e6
80
100
2,4-D 2
100
1.5e6
1.0e6
2.0e6
Figure
5. Example calibrations for selected
analytes
1.0e6
5.0e5
0.0
32
Area, counts
Theophylline 2
0.0
Carbamazepine 1
6.0e61.6e7
Area, counts
Area, counts
Area, counts
100
1.5e6 0.0
Max. 1.4e5 cps.
30
32
28
Erythromycin
27.6
MRM
ratio = 0.917
3.0e4 Ketorolac
5.0e4
2.0e4
0.0
1.0e4
5.0e6
0
20
40
60
80
4.0e6
3.0e6
EDC and
PPCP calibration .rdb (Th eophyllin.e 2)…
2.0e6
1.8e6
1.0e6
1.8e6
5.0e5
1.5e6
1.0e7
EDC and PPCP calibration .rdb (Carbamazepine 1…
8.0e6
4.0e61.4e7
2.0e61.2e7
20
40
60
80
Concen tration, ng/mL
EDC6.00e6
and PPCP calibration .rdb (Sulfathiazole 2)…
MRM ratio = 0.917
1.0e5
3.8e4
MRM ratio = 0.965
Theophylline 1
7.0e6
1.0e6
6.0e6
0.0
0.00
8.00e6
0
Erythromycin
22.2
XIC
0.0
20
22
24
26
Time,24.8
min
1.4e5
XIC of +MRM (137 pairs): Exp 1, 2...
3.0e6
8.0e6
2.0e6
Carbamazepine 1
1.2e7
0
20
40
60
Concen tration, n g/mL
80
100
0.0
0
20
40
60
80
100
Max. 4.0e5 cps.
26
Mepobramate
MRM ratio = 0.071
1.0e5
0.0
18
20
22
24
Time, min
26
Figure 4. Measured ion ratios of three select analytes
(Erythromycin, Ketorolac, and Meprobamate) in the
standard and the upstream and downstream sample of river
2, respectively. Despite low level detection like that seen
for Ketorolac in the River 2 sample, the ion ratios of the
two MRM transitions still confirm with the standard. MRM
ratio calculation was done automatically using the Analyst®
Reporter software
www.spektrotek.com
Max. 1.4e6 cps.
5.0e4
Intensity, cps
Inten sity, cps
Intensity,
cpssity, cps
Inten
Erythromycin
Max. 2.0e5 cps.
28
30
32
1.4e7
Area, cou nts
5.0e4
XIC 0.0
20
22
24
26
24.7min
Time,
2.0e5
XIC of +MRM (137 pairs): Exp 1, 2...
1.5e5
30.2
1.4e6
1.0e5
1.0e6
5.0e4
1.0e5
1.6e7
Area, cou nts
MRM ratio = 0.965
Theophylline 1
5.0e6
4.0e6
Area, cou nts
Erythromycin
24.8
EDC and PPCP calibration .rdb (Carbamazepine 1…
6.0e6
Area, coun ts
1.0e5
1.4e5
Intensity, cps
Intensity, cps
1.5e5
24.7
7.0e6
Area, coun ts
2.0e5
8.0e6
Area, cou nts
Detection of each analyte was identified using the area ratio of
two MRM’s collected. For River 2, Erythromycin, Ketorolac,
and Meprobamate along with 20 other compounds were
detected in either the upstream and downstream samples.
Ion ratios on the samples were compared to the ion ratios
measure on the standards for compound identification. See
Figure 5. Final results of River 1 and River 2 are shown in
XIC of +MRM
Max. 2.0e5 cps. XIC of +MRM (137 pairs): Exp 1, 7...
Max. 1.4e5 cps.
Table
3.(137 pairs): Exp 1, 7...
Area, cou nts
Result of both River 1 and River 2 showed detection of several
compound classed. As expected, a significantly larger number
of compound classes were detected in the urban river (River
2). Lower limit of quantitation (LLOQ) was determined to be
the level at which a peak is detected with a signal to noise of
at least 10:1. This level was theoretically determined using the
standards and assuming linearity down to zero concentration.
Table 2 shows a selected list of compounds and their LLOQ.
All compounds had LLOQ in the sub part per billion (ppb)
range.
Table 2. Lower Limits of Quantitation (LLOQ) of selected analytes
109
Table 3. Eight EDC and PPCP compounds were detected in the samples of river 1. Despite the rural nature of this location,
low level of these widely used herbicides and pharmaceuticals are still detected. As expected a larger list of compounds were
detected in the river 2 samples because of it urban origin. In total 23 EDC and PPCP compounds were founds at low to mid part
per trillion (ppt) levels. These results show that it is possible to scan for a functionally diverse set of compounds in one analysis
and achieve high sensitivity and accurate quantitation
Analytes in River 1
Concentration
(ng/L) upstream
Concentration
(ng/L) downstream
Concentration (ng/L)
downstream
Erythromycin
3.08
53.5
Oxybenzone
ND
6.25
65.5
152
Bromacil
ND
7.40
2,4-D
ND
9.35
Diazepam
ND
0.388
DEET
1.49
7.67
Warfarin
ND
0.930
Sulfamethoxazole
13.2
13.3
Triclosan (Irgasan)
5.90
31.4
Caffeine
41.0
23.5
Codeine
17.1
77.5
Ciprofloxacin
3.81
ND
Diuron
1.38
4.35
Cotinine
2.05
ND
Trimethoprim
58.5
123
Lincomycin
1.53
3.02
Carbamazepine
870
1305
DEET
24.0
29.9
Ketorolac
2.49
3.06
Meprobramate
85.5
97.5
Atrazine
1.08
0.88
Sulfamethoxazole
95.5
74.5
Pentoxifylline
6.60
3.39
ND not detected
Caffeine
57.0
13.5
Cotinine
14.4
ND
Increases by more than 2x
Simazine
1.01
ND
Norethisterone
1.15
ND
Erythromycin
135
ND
Tylosone Tartrate
4.28
ND
2,4-D
3.24
ND
Decreases by more than 2x
Summary
LC-MS/MS analysis has been shown to be a highly feasible
approach for the monitoring of a large set of endocrine
disrupting compounds spanning multiple categories and
chemical classes. MRM mode allows for the determination
of these compounds in river water matrix with low detection
limits and high selectivity.
Additional compound identification was achieved by the
simultaneous monitoring of a second MRM transition and
calculation of the corresponding ion ratio, which was done
automatically by Analyst Reporter™ software. Electrospray
ionization with polarity switching was found to be the most
suitable approach.
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Concentration
(ng/L) upstream
Carbamazepine
Within ± 2x
110
Analytes in River 2
References
1.Brett J. Vanderford, Rebecca A. Pearson, David J. Rexing,
Shane A. Snyder: ‘Analysis of Endocrine Disruptors,
Pharmaceuticals, and Personal Care Products in Water
Using Liquid Chromatography/Tandem Mass Spectrometry’
Anal. Chem. 75 (2003) 6265-6274
2.Paul E. Stackelberg, Edward T. Furlong, Michael T. Meyer,
Steven D. Zaugg, Alden K Henderson, Dori B Reissman:
‘Persistence of pharmaceutical compounds and other
organic wastewater contaminants in a conventional
drinking-water-treatment plant’ Science of the Total
Environment 329 (2004) 99-113
3.Susan D. Richardson and Thomas Ternes: ‘Water Analysis:
Emerging Contaminants and Current Issues’ Anal. Chem.
77 (2005) 3807-3838.
4.Axel. Besa, D. Loeffler, M. Ramil, T. Ternes, M. Suter,
R. Schonenberger, H.-R. Aerni, S. Koenig: ‘Detection of
Estrogens in Aqueous and Solid Environmental Matrices
with the API 5000™ LC/MS/MS System’ Application Note
AB SCIEX (2006)
SCIEX™ is being used under license. Publication number: 1120610-01
Jens Dahlmann1 and Bernd Luckas2
1
AB SCIEX Darmstadt (Germany) and 2University of Jena, Department of Food Chemistry,
Jena (Germany)
Overview
This application note describes a direct injection
method using Liquid Chromatography (LC) coupled to
tandem Mass Spectrometry (MS/MS) to analyze several
microcystins, including MC-LR, in drinking and surface
water. Time consuming and laborious extraction steps,
i.e. SPE, are not required due to the high sensitivity and
selectivity of the MS/MS detection using the AB SCIEX API
4000™ LC/MS/MS system. A limit of quantitation (LOQ)
of 0.1 µg/L was achieved which is 10 times below the
guideline value proposed by the World Health Organization
(WHO).
Analysis of Selected Microcystins in Drinking and Surface
Water Using a Highly Sensitive Direct Injection Technique
Figure 1. Bloom (left) and microscopic image (right) of
Planktothrix rubescense
Introduction
Microcystins (MC) are naturally occurring toxins produced by
certain genera of cyanobacteria (Figure 1). Reports suggest
that microcystins are hepatotoxic and they might also be
tumor initiators.1
Experimental
Standard
Microcystin standards are available from Enzo Life Science
International (http://www.enzolifescience.com).
Traditionally MC were analyzed by HPLC with UV detection2-3
but nowadays analytical methods are shifting more towards
mass spectrometric detection.4-5
The Microcystin MC-LR (Figure 2) is typically tested as a
marker for cyanobacteria occurrence and is regulated by the
WHO in the guidelines for drinking-water quality at a value of
1 µg/L.7
O
OH
CH3
D-Glu
Adda
O
HN
H3C
CH3
O
O
CH3
CH3
H
N
O
O
NH2
HN
NH
Y
Mdha
NH
CH3
CH2
O
NH
CH3
O
N
OO
D-Ala
HN
H
N
H
X
O
H3C
CH3
D-Me-Asp
Figure 2. Structure of MC-LR where X is leucine and Y is
arginine
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MC are cyclic peptides and the general structure is cyclo
[-D-Ala-X-D-MeAsp-Y-Adda-D-Glu-Mdha-], where X and Y are
variable L-amino acids, e.g. leucine (L), arginine (R), tyrosine
(Y), tryptophan (W), and phenylalanine (F) as X, as well as
arginine (R), alanine (A), and methionine (M) as Y (Figure
2). Due to the two variable amino acids and methylation/
demethylation of the other amino acids, there is a large variety
of microcystin compounds. More than 100 microcystins have
been identified to date. In contrast to microcystins, nodularin
(NOD) is a cyclic pentapeptide produced by Nodularia
spumigena with the structure cyclo [-D-MeAsp-L-Arg-AddaD-Glu-Mdhb-], where Mdhb stands for 2-(methylamino)2-dehydrobutyric acid.
111
LC
An Agilent 1100 LC system was used with a Phenomenex LUNA
C18 3u (150x3 mm) column and a mobile phase of water and
acetonitrile + 5 mM ammonium acetate + 0.1% formic acid
(Table 1).
The injection volume was set to 25 and 100 μL, respectively.
Table 1. LC gradient
Time (min)
Flow (µL/min)
A (%)
B (%)
70
30
MRM 1
65
35
MRM 1
15
85
70
30
0
10
250
20
re-equilibration
Transition
Q1 (amu)
995.7
Q3 (amu)
135.2
213.0
CE (V)
115
Figure 4. MRM transitions and Collision Energy (CE) to detect
MC-LR
MS/MS
MRM transitions are shown in Figure 4.
An AB SCIEX API 4000™ LC/MS/MS system equipped with
Turbo V™ source and Electrospray Ionization (ESI) probe was
used for compound detection in positive polarity.
Multiple Reaction Monitoring (MRM) was used for its
superior selectivity and sensitivity. Two MRM transitions for
simultaneous quantitation and identification based on ion
ratio calculation and compound dependent parameters were
automatically optimized by direct infusion experiments and
the ‘Compound Optimization’ tool in Analyst® software.
The full scan MS/MS spectrum is shown in Figure 3. Two
product ions at m/z 135.0 amu (characteristic ADDA fragment
for and MC) m/z 213.0 amu (Glu-Mdha) were measured.
+MS2 (995.67) CE (54): 26 MCA scans from Sample 1 (TuneSampleName) of MC_LR_InitProduct_Pos.wiff (Turbo Spray)
Due to the thermal stability of MC-LR the nitrogen gas
temperature to dry the eluent in the ion source was set to
650°C which evaporated the mobile phase completely yielding
in enhanced sensitivity of the measurement.
The Turbo V™ ion source was designed and optimized
(geometry, ceramic materials, orthogonal sprayers etc.) for
highest sensitivity, reproducibility, robustness, and lowest
carry-over.
The Signal-to-Noise values (S/N) of MC-LR, MC-RR, and
MC-YR at a concentration of 0.1 µg/L were >10 (3 x standard
deviation) resulting in a Limit of Detection (LOD) of 0.04 µg/L
for MC-LR for example (Figure 4).
Max. 1.4e6 cps.
x 5.0
995.4
1.4e6
1.2e6
Intensity, cps
1.0e6
135.0
8.0e5
6.0e5
4.0e5
127.0
70.0
111.8
2.0e5
50
100
212.8
154.8
200.0 226.0
92.8
150
200
250
268.2
300
375.0
350
400
967.6
450
500
550
m/z, amu
600
650
700
750
800
850
900
950
1000
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Figure 3. MS/MS spectrum of MC-LR with highlighted product
ions used for quantitation
112
Figure 4. MRM chromatogram of MC-LR spiked drinking
water at a concentration of 0.1 μg/L (injection volume of 25
µL)
The reproducibility of the developed method was tested by
injecting spiked drinking water. The coefficients of variation
were less than 4% (n=15) at all calibration levels.
Linearity was proven for MC-LR, MC-RR, and MC-YR standard
solutions ranging from 0.1 µg/L to 100 µg/L.
Figure 5. Chromatograms of various microcystins and nodularin (NOD), at a concentration of 1 µg/L, except desmethyl-MC-RR
and MC-WR at 10 µg/L (injection volume of 25 µL)
In addition, the API 4000™ system is equipped with a Linear
Accelerator (LINAC®) collision cell. The axial field gradient
of the LINAC® collision cell accelerates product ions after
fragmentation allowing fast MS/MS experiments without
cross-talk and without loss in sensitivity, such as fast MRM
using short dwell times for each transition.
This allows multi-target quantitation. The developed method
can easily be extended to quantify other microcystins of
interest. An example chromatogram for the quantitation of 9
microcystins, including MC-LR, MC-LF, MC-LA, MC-RR, MCYR, MC-LW, MC-WR, desmethyl-MC-RR, and Nodularin is
shown in Figure 5.
Summary
The AB SCIEX API 4000™ LC/MS/MS system offers sufficient
sensitivity for the direct analysis of microcystins, including
MC-LR, in drinking water with an LOQ of 0.1 µg/L. Time
consuming and extensive sample clean-up and concentration
is not required resulting in better reproducibility and accuracy.
The methodology is designed to also allow for the inclusion
of other water soluble cyanobacterial toxins such as anatoxins
and cylindrospermopsins.
1.W. W. Carmichael, W. W.: ‘The toxins of cyanobacteria’ Sci. Amer. 270 (1994) 64-72
2.L. Lawton, C. Edwards, and G. A. Codd: ‘Extraction and High Performance Liquid Chromatographic Method for the
Determination of Microcystins in Raw and Treated Waters’ Analyst 119 (1994) 1525-1530
3.J. Dahlmann, W. R. Budakowski, and B. Luckas: ‘Liquid chromatography–electrospray ionisation-mass spectrometry based
method for the simultaneous determination of algal and cyanobacterial toxins in phytoplankton from marine waters and
lakes followed by tentative structural elucidation of microcystins’ J. Chromatogr, A 994 (2003) 45-57
4.L. Cong, B. Huang, Q. Chen, B. Lu, J. Zhang, and Y. Ren: ‘Determination of trace amount of microcystins in water samples
using liquid chromatography coupled with triple quadrupole mass spectrometry’ Analytica Chimica Acta 569 (2006) 157168
5.‘Determination of trace amount of microcystins in water samples using liquid chromatography coupled with triple
quadrupole mass spectrometry’ Analytica Chimica Acta 569 (2006) 157-168 detection of cyanobacterial toxins in precursor
ion mode by liquid chromatography tandem mass spectrometry’ J. Mass Spectrom. 42 (2007) 1238–1250
6.K. A. Loftin, M. T. Meyer, F. Rubio, L. Kemp, E. Humpries, and E. Whereat: ‘Comparison of Two Cell Lysis Procedures
for Recovery of Microcystins in Water Samples from Silver Lake in Dover, Delaware, with Microcystin Producing
Cyanobacterial Accumulations’ Open-File Report 1341
(2008) USGS (http://pubs.usgs.gov/of/2008/1341/)
7.http://www.who.int/water_sanitation_health/dwq/fulltext.pdf
www.spektrotek.com
References
113
Quantitative Analysis of Explosives in Surface Water
Comparing Off-Line Solid Phase Extraction and Direct
Injection LC-MS/MS
J.D. Berset1, N.Schiesser1, Th. Schnyder1, A. Affolter1, St. König2, A. Schreiber3
1
Water and Soil Protection Laboratory (GBL) Bern (Switzerland); 2 AB SCIEX Rotkreuz,
(Switzerland); 3 ABSCIEX Concord, Ontario (Canada)
Overview
Presented is an efficient method for measuring selected explosives
in lake water at the sub-ng/L level applying either off-line Solid Phase
Extraction (SPE) with LC-MS/MS detection and comparing it to direct
injection LC-MS/MS.
Introduction
Between 1918 and 1967 some 8200 tons of ammunition, Trinitrotoluene
(TNT) being the main explosive, was dumped to the lakes of Thun, Brienz
and Lucerne in Switzerland.1
The amount of ecologically harmful compounds was considered to be
negligible. In order for explosives to leak to the environment the casing
must have rusted.2-3 This corrosion process very much depends on environmental water conditions such as: temperature,
oxygen content and pH value. Meanwhile a sediment layer of 20-30 cm covers the ammunition at the lakes’ bottom and
represents a natural barrier preventing the compounds to enter the aqueous phase.
Nevertheless water quality of the lakes should be monitored as lake water is frequently used as a source for drinking water.
Due to the very low concentrations of explosive residues expected in the lakes a powerful analytical set-up is important for a
reliable detection and quantitation. LC-MS/MS analysis with Electrospray Ionization (ESI) is the method of choice to analyze
polar and thermally labile compounds, such as explosives and their degradation products.
Experimental
The following explosives and degradation products were
investigated:
2,4,6-Trinitrotoluene (TNT)
• 2,4-Diamino-6-nitrotoluene (2,4-DA-6-NT)
• 2,6-Diamino-4-nitrotoluene (2,6-DA-4-NT)
• 2-Amino-4,6-dinitrotoluene (2-A-4,6-DNT
• 4-Amino-2,6-dinitrotoluene (4-A-2,6-DNT
• Hexogen (RDX)
• Nitroglycerin (NG)
• Octogen (HMX)
• Pentaerythritol tetranitrate (PETN)
• Tetryl
OH
O
O
OH
O
maleic acid
OH
HO
O
fumaric acid
Figure 1. Chemical structures of maleic acid (left) and fumaric
acid (right)
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Sample Preparation
114
50 mL of water samples were extracted on Phenomenex
StrataX SPE cartridges. These extracts were analyzed by LCMS/MS and compared to direct injections of filtered water
samples.
Liquid Chromatography
• HPLC column: Xbridge Phenyl (2.1x150 mm), 3.5 μm
• Eluent A: water + 2.5 mM ammonium acetate
• Eluent B: methanol + 2.5 mM ammonium acetate
• Gradient (A/B): 55/45 to 30/70 within 13 min and reequilibration
• Flow: 200 μL/min
• Injection volume: 100 μL
• Oven temperature: 40°C
Mass Spectrometry
• API 5000™ LC/MS/MS System
• Turbo V™ source with ESI probe
• Gas and source parameters: CUR: 20 psi, GS1: 40 psi, GS2:
40 psi, TEM: 350°C, CAD: 7, IonSpray voltage (IS): 5500 V
(positive) and -4500 V (negative)
• Two periods with detection in positive and negative
polarity using Multiple Reaction Monitoring (MRM) were
programmed: 0 to 4.5 min (positive) and 4.5 to 15 min
(negative). MRM transitions of detected explosives and
MRM ratios are listed in Table 1.
Calibration
Standards were prepared in MilliQ water and blank matrix
water (matrix matched standards) over a range of 1-100
ng/L for off-line SPE and 0-1 ng/L for direct injection LCMS/MS. Serial dilutions were obtained starting with a 10
ng/mL standard. All standards were prepared in water and
kept at 4°C in the dark. Under these conditions standards
were stable for at least three months – with the exception
of TNT and Tetryl, which degrade rapidly and thus must be
prepared freshly.
Method validation data
• Recoveries (SPE): between 89% and 110% for all analytes
• Blank analysis: field blanks, travel blanks and laboratory
blanks did not contain any traces of explosives (< 10% of
lowest calibration standard)
• Linearity: 7 point equidistant calibration, statistical tests
(Mandel, sensitivity plots and residual analysis) proved
linearity of regression lines, residual analysis with normal
distribution of the calibration points around the zero line
• Limit of Quantification (LOQ) with S/N=10 and Limit of
Detection (LOD) S/N=3
• LOQ: 1 ng/L for DANT, NG and TNT, 0.03 ng/L for HMX,
RDX, PETN and ADNT
Samples from different depths were analyzed within 48 hours
after sampling. If water had to be stored for a longer period of
time it was stabilized by acidifying to pH 3.5 with acetic acid
and adding 2% of acetonitrile.
Table 1. Retention times, MRM transitions of explosives with detected MRM ratio and tolerance intervals regarding the guideline
2002/657/EC5
MW
2,4-DA-6-NT
3.7
167
168 [M+H]+
2,6-DA-4-NT
4.1
167
168 [M+H]
168/121 168/77
0.37
HMX
5.0
296
355 [M+CH3COO]-
355/46 355/147
0.4
RDX
6.5
222
281 [M+CH3COO]-
281/46 281/93
0.04
50
0.02-0.06
NG
9.6
227
286 [M+CH3COO]-
286/62 286/46
0.83
20
0.67-1.00
4-A-2,6-DNT
9.9
197
196 [M-H]-
196/46 196/136
0.06
50
0.03-0.09
2-A-4,6-DNT
10.2
197
196 [M-H]-
196/46 196/136
0.26
25
0.20-0.33
Tetryl
11.9
287
286 [M-H]-
286/240 286/206
0.83
20
0.67/1.00
TNT
12.0
227
226 [M-H]-
226/46 226/196
0.49
PETN
13.1
316
375 [M+CH3COO]-
375/62 375/46
0.44
+
MRM Transition
MRM Ratio
168/121 168/77
0.43
Tolerance
Interval
tR (min)
0.32-0.54
25
0.28-0.46
0.30-0.50
25
0.37-0.61
0.33-0.56
www.spektrotek.com
Precursor Ion
Tolerance
(%)
Compound
115
Clearly, Electrospray Ionization turned out to be the method of
choice for detecting traces of explosives in water samples.4
Tests using either APCI or APPI were generally less sensitive
(results not shown). As shown in Table 1 precursor ions of
explosives were either detected as [M+H]+ or [M-H]- for the
DANT, ADNT, Tetryl and TNT, as [M+CH3COO]- for HMX,
RDX, NG and PETN.
Selective detection was performed in MRM mode using two
characteristic transitions for each compound. The ratio of both
transitions was used to identify the presence of explosives in
lake water regarding the guideline 2002/657/EC.5
Optimization of the compound dependent parameters was
obtained by automatic Quantitative Optimization in Analyst®
Software. The ion source temperature was a crucial parameter
during source optimization. TNT, Nitroglycerine and above all
Tetryl, known as being very labile, could only be detected
using a rather low temperature of 350°C. As Nitroglycerine
and Tetryl are not expected to persist for a longer time in the
environment they were not included in the final target method.
The separation of the different isomers of the
diaminonitrotoluenes and aminodinitrotoluenes became
difficult on traditional C18 stationary phases. Figure 1 presents
a total ion chromatogram (TIC) with baseline separated
analytes on the selected phenyl type phase.
Concentrations of explosive residues in lake water were
assumed to be very low if present at all. Therefore, in a first
attempt an off-line SPE enrichment procedure of the water
samples was performed using an enrichment factor of
100. Using this procedure a typical TIC as shown in Figure
2 was obtained. Quantitation of the compounds revealed
concentrations between 0.1-0.4 ng/L. Concentrations at
different depths were very similar assuming a homogeneous
distribution of the explosives in the water body.
www.spektrotek.com
In a second step, direct injection of 100 μL of water samples
was performed. A representative chromatogram of HMX is
shown in Figure 3. The calibration curve (working range 0-1
ng/L) is presented in Figure 4. Quantitation of the sample
resulted in a concentration of 0.21 ng/L. The calculated MRM
ratio of 0.42 was well within the limits of the ratio obtained
from the calibration line (0.40). Note the excellent agreement
between the intensity (cps) of HMX in the concentrated
sample (2.0 x 104 cps; enrichment factor 100) and the directly
injected sample (200 cps).
116
Figure 1. Total ion chromatogram of a 100 ng/L standard: 0 to
4.5 min in positive polarity 4.5 to 15 min in negative polarity
Figure 2. TIC of a lake water sample taken at a depth of 212 m
showing the presence of HMX, RDX and PETN using off-line
SPE
Figure 3. Direct injection of a lake water sample taken at a
depth of 212 m showing the two transitions of HMX: 355/46
(upper trace), 355/147 (lower trace)
Figure 4. Calibration curve of HMX with a working range
of 0-1 ng/L
(r = 0.9996) used for direct injection analysis
A comparison of the concentrations of direct injection
and SPE enriched samples from different depths of
the lake for HMX, RDX and PETN is shown in Figure
5. Concentrations of direct injection do not significantly
deviate from the SPE samples. The lower concentrations
detected after SPE can be explained by a recovery
less than 100% and/or stronger ion suppression due
to increased matrix concentration after extraction.
However, uncertainty of measurement can drastically be
reduced using direct injection LC-MS/MS.
0.5
A highly sensitive LC-MS/MS method for the analysis of
sub-ng/L levels of selected explosives such as TNT and the
corresponding monoamino and diamino metabolites, HMX,
RDX, and PETN has been presented. Specificity was obtained
using Multiple Reaction Monitoring with identification based
on ion ratio calculation using two transitions for each analyte.
Sensitivity turned out to be optimal using Electrospray
Ionization (ESI) with positive or negative polarity on an API
5000™ LC/MS/MS System equipped with a Turbo V™ source.
Using direct injection analysis of water samples comparable
results were obtained as from SPE enriched samples for
the three main explosives HMX, RDX and PETN. In addition
reproducibility was found to be much better using direct
injection LC-MS/MS analysis.
Summary
Acknowledgements
The authors would like to thank Dr. M. Zeh for his help with
lake water sampling from different depths.
References
1.Van Stuijvenberg et al: Gefahrdungsabschatzung zu
militarischen Munitionsversenkungen in Schweizer Seen,
Generalsekretariat VBS, September 2005
2.J. Sjostrom et al: Environmental Risk Assessment of
Dumped Ammunition in Natural Waters of Sweden, User
Report, September 2004
3.H. Stucki: Chimia 58, 409-413, 2004
4.X. Xu et al: J Forensic Sci, 49, 6, 1-10, 2004
5.J.D. Berset et al: Chimia 61, 532, 2007
6.Chr. Borton et al: Application Note AB SCIEX #1282110-01
measurementu ncertainty
0.4
ng/L
0.3
0.2
0.1
0
11
HMXS PE
RDXS PE
02
01
depth( m)
PETNSPE
HMXD I
00
RDXD I
212
PETNDI
© 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective
owners. AB SCIEX™ is being used under license.
www.spektrotek.com
Figure 5. Comparison of concentrations between direct
injection and off-line SPE for HMX, RDX and PETN with
error bars for uncertainty of measurement
117
Screening and Identification of Unknown Contaminants in
Untreated Tap Water Using a Hybrid Triple Quadrupole
Linear Ion Trap LC-MS/MS System
M.T. Baynham1, St. Lock1, D. Evans2, and P. Cummings2
1
AB SCIEX Warrington Cheshire (UK), 2 ALcontrol Laboratories Rotherdam Yorkshire (UK)
Introduction
Protection of our drinking water resources from contaminants is a major
responsibility for both government and water producing bodies. The
response taken to a potential drinking water emergency will depend upon
both the composition and the nature of the identified contaminant(s).
Furthermore it is essential that there is a high degree of confidence in
the correct and rapid identification of the problem before remedial action
is taken. To date it has been a necessity to employ a combination of
multiple analytical techniques to meet this end.
Screening Using Accurate Mass Measurements and
MS/MS
One method of detecting contaminants is the use of accurate mass as a
way to predict the formula and identity of a contaminant. In this approach the mass spectrometer has to be accurately calibrated
because the greater the error the more potential contaminants would be a match for the detected peak, as <2ppm mass error
is ideal.
In this example two structural related but different pesticides (Prometryn and Terbutryn) produce the same molecular ion
because they have identical molecular formulae. In the environment there are hundreds of compound with the same mass
(Figure 2). Thus, a complete identification of unknown contaminants by accurate mass alone may not yield to a complete
answer as this does not provide any structural information. In the example above separation of these two pesticides by HPLC
was not clear-cut as they eluted with very similar retention times (Figure 3). However, Prometryn and Terbutryn have different
MS/MS fragmentation patterns (Figure 2). Therefore product ion spectra are essential for confident identification of unknown
contaminants.
frequency ofo ccurrence
2000
more than 1500
compoundsh avea similar
molecularw eighto f
~250amu
1500
1000
500
www.spektrotek.com
2004
118
00
600
800
1000
molecularw eight
Figure 1. Abundance of compounds over molecular weight range of 100-1000 amu
Multi Target Screening
Compound Class
Polarity
MRM
Intensity at
1 µg/mL
~LOD
(µg/mL)
Q3 Mass
Intensity at
10 µg/mL
~LOD (µg/mL)
Brodifacoum
Rat poison
Negative
521.0/79.0
5.80E+04
0.05
521.0
7.70E+05
5.00
Chlorophacinone
Rat poison
Negative
373.0/201.1
1.23E+04
0.20
373.0
3.40E+05
15.0
Difenacoum
Rat poison
Negative
443.1/135.0
1.40E+04
0.25
443.1
1.80E+06
1.25
Difethialone
Rat poison
Negative
537.0/79.0
6.00E+04
0.07
537.0
1.40E+06
5.00
Flocoumafen
Rat poison
Negative
541.1/161.0
1.30E+04
0.12
541.1
1.40E+06
2.00
Warfarin
Rat poison
Negative
307.0/161.1
1.80E+04
0.20
307.0
1.80E+05
40.0
Endothal
Rat poison
Negative
185.0/141.0
6.00E+03
2.0
185.0
-
100
DNOC
Cresol
Negative
197.0/137.1
5.00E+04
0.10
197.0
2.00E+06
1.25
Azinphos-ethyl
Organo-phosphorus
Positive
346.0/160.1
5.13E+03
1.00
346.0
6.50E+04
200
Demeton-S-methyl
Organo-phosphorus
Positive
231.0/89.0
1.00E+04
0.50
231.0
2.30E+05
20.0
Dichlorvos
Organo-phosphorus
Positive
221.0/127.0
9.33E+02
10.0
221.0
4.00E+04
200
Disulfoton
Organo-phosphorus
Positive
275.1/89.0
2.00E+03
5.00
275.1
2.00E+04
2000
Propetamphos
Organo-phosphorus
Positive
282.1/156.0
2.20E+03
2.50
282.1
5.20E+04
200
Tebupirimfos
Organo-phosphorus
Positive
319.0/153.1
1.90E+04
0.50
319.0
2.90E+05
20.0
Parathion-ethyl
Organo-phosphorus
Positive
292.1/236.0
4.73E+03
2.00
292.1
1.00E+04
500
Parathion-methyl
Organo-phosphorus
Positive
281.1/264.3
5.00E+02
10.0
264.1
2.00E+04
400
General Unknown Screening and Multi Target
Screening
There are two possible approaches of screening methods. The
first would to screen for a complete unknown. This General
Unknown Screening (GUS) would use a single ‘universal’
survey scan over a defined mass range and could either be a
Time-of-Flight (TOF), quadrupole or ion trap scan. This survey
scan can be used to trigger automatically the acquisition of
a product ion spectrum if a signal of a detected compound
is above a defined threshold. Finally, this spectrum can be
searched against a mass spectral library for identification.
Comparison of Total Ion Chromatograms (TIC) of unknown
samples to that of the control reveal compounds that are either
unique to the sample or those that are present at significantly
higher concentrations than in the control.
The other approach is often called Multi Target Screening
(MTS). In this approach a predefined list of compounds is
looked for in a Single Ion Monitoring (SIM) or Multiple Reaction
Monitoring (MRM) experiment. MRM mode is generally
preferred because of higher selectivity and sensitivity. Once
a compound is detected above a defined threshold a product
ion scan is collected and compared against a library. Dynamic
exclusion of compounds where MS/MS spectra are already
acquired allows the data collection of co-eluting compounds
(Figure 4).
MS/MS
A 4000 QTRAP® LC/MS/MS system was used for both MTS
and GUS experiments which triggered dependant Enhanced
Product Ion scanning (mass range of 50 to 750 amu at 4000
amu/s) with a Collision Energy (CE) of 35 V and Collision
Energy Spread (CES) of 20 V. The MTS survey scan used MRM
transitions which have been optimized for each targeted analyte
while the GUS screen used a Q3 scan with a mass range of 90
to 750 amu and a Declustering Potential (DP) of 60 V.
The source and gas settings for both MTS and GUS experiments
were the same (Table 2)
Table 2. Ion source and gas parameters
Parameter
Value
Curtain gas
25 psi
Gas 1
50 psi
Gas 2
60 psi
CAD
10
Temperature
650°C
IonSpray™ source voltage
-4500 V
+5500 V
www.spektrotek.com
Compound Name
General Unknown Screening
Table 1. Comparison of sensitivities between the General Unknown Screening (GUS) and Multi Target Screening
(MTS) approaches
119
0.7
1.8e8
1.6e8
1.5e8
1.4e8
1.3e8
Tapw ater
MRM2 30/146
1.6e8
1.5e8
1.4e8
1.3e8
1.2e8
1.2e8
1.1e8
1.1e8
1.0e8
1.0e8
9.0e7
9.0e7
8.0e7
8.0e7
7.0e7
7.0e7
6.0e7
6.0e7
5.0e7
5.0e7
4.0e7
4.0e7
3.0e7
3.0e7
2.0e7
2.0e7
1.0e7
1.0e7
0.0
0.10
.2
0.30
.4
0.50
Time,m in
1.16e7
174.0
1.10e7
MS/MSo f
Terbuthylazine
1.00e7
9.00e6
.6
0.70
.8
0.0
0.9
230.0
0.10
.2
0.30
.4
0.50
Time,m in
.6
0.70
.8
0.9
230.0
1.19e7
MS/MSo f
Terbuthylazine
1.10e7
174.0
1.00e7
9.00e6
8.00e6
8.00e6
7.00e6
7.00e6
6.00e6
6.00e6
5.00e6
5.00e6
4.00e6
4.00e6
103.8
3.00e6
3.00e6
146.0
2.00e6
1.00e6
Summary
0.7
1.7e8
Mineralw ater
MRM2 30/146
1.7e8
78.9
80
100
1.00e6
138.1
109.8
1201
103.8
2.00e6
132.0
95.9
40
160
180
200
m/z, amu
220
240
2602
80
300
78.9
95.9
80
146.0
132.0
109.91
100
38.1
1201
40
160
180
200
m/z, amu
220
2402
60
280
300
Figure 5. 100 ng/mL Terbuthylazine spiked into mineral
and tap water analyzed in positive polarity MRM and EPI
0.6
3.8e7
3.4e7
3.2e7
3.0e7
2.8e7
0.6
3.4e7
3.2e7
Mineralw ater
MRM2 13/141
3.6e7
Tapw ater
MRM2 13/141
3.0e7
2.8e7
2.6e7
The 4000 QTRAP® LC/MS/MS system allows Multi Target
Screening (MTS) and General Unknown Screening (GUS) of
water samples to identify emerging contaminants. The MTS
approach is the most rapid and sensitive method to screen for
and detect the presence of targeted organic contaminants in
water. More than 2000 targeted compounds can be screened
in less than 20 minutes at low and sub μg/L level using the
described procedure and multiple sample injections. The GUS
method is an alternative to identify unknown compounds as it
does not rely on any knowledge of the analytes. Here, a sample
control comparison will detect unknown contaminants. In
both approaches automatically generated Enhanced Product
Ion spectra can be searched against a comprehensive mass
spectral library and the fragmentation information can be
used for identification and identification. However, the GUS
approach is lower in sensitive and requires significantly
longer run times.
2.4e7
2.6e7
2.2e7
2.0e7
1.8e7
n
1.8e7
1.6e7
1.6e7
1.4e7
t
6.0e7
1.4e7
1.2e7
e
TIC: from Sample 1 (SAMPLE B) of Q3 5.wiff (Turbo Spray), Smoothed
2.0e7
I
2.4e7
4.0e7
2.2e7
1.2e7
8.0e7
1.0e7
.
1.0e7
8.0e6
6.0e6
.
6.0e6
.
8.0e6
4.0e6
4.0e6
0.0
0.0
0.10
.2
0.30
.4
0.50
Time, min
.6
0.70
.8
0.9
0.10
.2
.4
0.50
Time,m in
.6
.8
0.9
8.00e6
6.0e5
4.0e5
.
7.50e6
7.00e6
2.5e6
5.00e6
2.0e6
e
1.5e6
4.00e6
.
3.00e6
100
1.50e6
140
104.7
5.00e5
120.9
160
180
200
260
2803
00
320
340
360
380
400
80
100
120
140
160
180
200
260
280
300
320
340
360
380
8.0e7
400
The GUS approach shows the comparison of a blank control
sample to a sample that has been spiked with 0.1 μg/L of a
compound to be identified (Figure 7). The presence of the
compound with m/z=350 amu is detected in the sample by
comparing the two Q3 scan chromatograms. Acquisition of
an Enhanced Product Ion scan spectrum followed by library
searching allows to identification of Chlorpyrifos.
.
Mineral water typically contains high levels of sodium, which
may affect sensitivity due to adduct formation. However,
Figure 5 and 6 indicate that there is nearly no effect on S/N
to detect Terbuthylazine and MCPP in these water samples.
4.0e7
.
Figure 5 and 6 present data obtained for an injection of
100 ng/mL Terbuthylazine and MCPP in both mineral
and tap water, using the MRM to EPI MTS approach. The
LINAC® collision cell of the 4000 QTRAP® system allows
the simultaneous monitoring of up to hundreds of MRM
transitions (contaminants) in a single sample injection.
These MRM transitions triggered Enhanced Product Ion scan
spectra in a cycle time of approximately 2.5 s without loss in
sensitivity and full spectral quality.
6.0e7
.
www.spektrotek.com
80
e
2202
40
m/z, amu
Figure 6. 100 ng/mL MCPP spiked into mineral and tap water
analyzed in negative polarity MRM and EPI
120
114.9
5.0e5
t
220 240
m/z, amu
8
Time, min
322.0
9
12.0
10
11
12
13
14
15
16
Max. 8.7e5 cps.
350.0
Survey Q3 spectrum at 12min
354.0
100
133.8 150.7
120
140
179.8
160
213.8
180
4 20
440
460
4 80
500
520
540
Max. 2.7e6 cps.
EPI spectrum at 12min
200
220
TIC: from Sample 1 (BLK) of Q3 2.wiff (Turbo Spray), Smoothed
1.00e6
120
7
197.8
n
80
6
13.7 14.0
14.9
260
280
349.7
321.7
293.7
275.7
240
m/z, amu
300
320
340
360
380
400
Max. 8.7e7 cps.
I
104.7
213.0
.
1.00e6
5
198.0
.
2.50e6
2.00e6
213.0
2.00e6
4
153.0
1.0e6
3.50e6
3.00e6
3
13.3
2.0e5
5.50e6
4.50e6
4.00e6
2
11.5
9.8
12 0
140
160
18 0
200
220
2 40
260
280
3 00
320
340
3 60
380
400
m/z, amu
+EPI (350.01) Charge (+0) CE (35) CES (20) FT (10): Exp 2, 12.058 min from Sample 1 (SAMPLE B) of Q3 5.wiff (Turbo Spra y)
t
6.00e6
n
5.00e6
6.50e6
I
6.00e6
9.4
10.7
.
8.50e6
7.00e6
1
8.0e5
.
9.00e6
8.00e6
0.70
MS/MSo f
MCPP
9.50e6
e
1.00e7
MS/MSo f
MCPP
9.00e6
0.30
140.9
1.04e7
1.00e7
0.0
+Q3: Exp 1, 12.046 min from Sample 1 (SAMPLE B) of Q3 5.wiff (Turbo Spray)
t
140.8
1.10e7
8.5
0.9
2.0e7
n
2.0e6
10.2
I
2.0e6
0.3
Max. 8.7e7 cps.
Water sample
0.3
10.7
Control sample
11.4
9.8 10.2
8.4 8.5
13.3
13.7 14.0
14.6
12.6
2.0e7
0.0
1
2
3
4
5
6
7
8
Time, min
9
10
11
12
13
14
15
16
Figure 7. Comparison of a water sample to a blank control
water with resulting Q3 scan and EPI spectrum of Chlorpyrifos
detected and identified by library searching
In order to compare the relative sensitivities of both
approaches, GUS and MTS, over 70 compounds were tested
including compounds such asorganophosphorus pesticides
and rat poisons. Limits of Detection (LOD) were determined
to be the triggering threshold of both approaches. In the
GUS method the LOD was set at 500,000 cps of the parent
ion in Q3 scan (background noise was generally lower than
500,000 cps). For
the MTS approach LOD was 5000 cps in MRM which was
determined as 2-3 times the background level of the most
intense MRM trace. The chromatographic conditions of MTS
were applied for this comparison work. Examples of results
for 16 different compounds are given in Table 1 highlighting
the higher sensitivity of the MTS approach. An average of 2
orders of magnitude comparing LOD of both approaches was
found.
For Research Use Only. Not for use in diagnostic
procedures.
© 2010 AB SCIEX. The trademarks mentioned herein
are the property of AB Sciex Pte. Ltd. or their respective
owners. AB SCIEX™ is being used under license.
Yun Yun Zou and André Schreiber AB SCIEX Concord, Ontario (Canada)
Overview
Organotin compounds are chemicals composed of tin linked
to hydrocarbons, used in industrial materials and various
biocides and fungicides. As a result, organotin compounds
can enter the environment through a number of channels, and
can often be found in seawater, seafood, fruits and vegetables,
and consumer goods. Due to the toxicity of these compounds,
there is a need for analytical methods allowing accurate
quantitation and identification. Here we present an LC-MS/
MS method to measure tributyltin, fentin, cyhexatin, and
fenbutatin oxide in different matrices. Triphenyl phosphate
was used as the internal standard.
Introduction
Organotin (organostannic) compounds are chemical
compounds comprised of tin with hydrocarbon substituents.
Organotin compounds are widely used as additives in
plastic material, wood preservatives, marine biocides, and
agricultural pesticides.
Tri-substituted organotin compounds were previously widely
used as antifouling agents in paints on ships. However,
such paints were found to release organotin compounds
into the aquatic environment, where they can accumulate
in sediments and organisms or degrade to less substituted
toxic compounds. Studies have shown that trace amounts of
organotin compounds can have significant detrimental effects
on aquatic organisms. For instance, tributyltin (TBT), present
in sea water at ng/L levels, has been identified as an endocrine
disruptor promoting harmful effects on aquatic organisms.
Therefore, the use of organotin compounds in antifouling
paints is prohibited or restricted in many countries.1-3
The use of organotin compounds in consumer products, such
as textiles, footwear, wall and floor coverings, etc., has been
found
to pose a risk to human health, particularly for children.
Therefore, the use of tri-substituted and di-substituted
organotin compounds, including TBT, tributyltin (TPhT),
dibutyltin (DBT), and dioctyltin (DOT) in consumer products
is restricted.4-5
Finally, organotin compounds enter the human diet through
contaminated seafood and the use as agricultural pesticides.
International maximum residue limits (MRL) have been
established by Codex Alimentarius and the EU for many food
commodities, with some MRL as low 50 μg/kg.
Traditionally gas chromatography coupled to mass
spectrometry (GC-MS) was used for analysis of organotin
compounds. However, the analysis by GC requires time
consuming derivatization, because of poor compound
volatility, and long chromatographic run times. Liquid
chromatography with tandem mass spectrometry (LC-MS/
MS) allows simplifying sample preparation and shortening
run times due to increased selectivity and sensitivity and,
thus, is evolving as a preferred technique for the analysis of
organotin compounds.
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Spiked apple, potato, synthetic seawater, and textile samples
were prepared using a quick and easy acetonitrile extraction.
Organotin compounds were detected using an AB SCIEX 4000
QTRAP® system with Electrospray Ionization (ESI) using
Multiple Reaction Monitoring (MRM). Detection limits were
determined to be well below regulated levels, enabling extra
dilution of the sample extract to minimize possible matrix
effects.
Quantitation and Identification of Organotin Compounds in
Food, Water, and Textiles Using LC-MS/MS
121
Method Details
Sample Preparation
TBT chloride, fentin acetate, cyhexatin and fenbutatin oxide were
purchased from Sigma-Aldrich and spiked into four matrices
(apple, potato, synthetic seawater (drinking water with 35 g
salt per liter), and textile material). Triphenyl phosphate (TPP)
was used as the internal standard.
CH3
Hom ogenize and weigh 10 g
of apple and potato.
Shred and weigh 1 g
of textile m aterial.
Add internal standard
(50 L of 10 g/m L TPP).
(50 L of 10 g/m L TPP).
Transfer 1 m L of water
sam ple into autosam pler vial.
Add 10 m L acetonitrile and
shake vigorously for 1 m inute.
Add 20 m L acetonitrile and
sonicate for 5 m inutes.
(10 L of 10 ng/m L TPP).
Centrifuge at 5 rpm for 5 m in.
Centrifuge at 5 rpm for 5 m in.
(Dilute water sam ple to
reduce possible m atrix
effects.)
Transfer 100 L of the extract
into autosam pler vial and add
900 L water.
Transfer 100 L of the extract
into autosam pler vial and add
900 L water.
Inject directly.
O
Sn
Cl
Sn
OC
Sn
H3
OH
H3C
Figure 2. Sample preparation protocols for the analysis of
organotin compounds in fruit and vegetable, textiles, and
water
CH3
H3C
CH3
H3C
Sn
O
Sn
O
CH3
O
P
O
O
MS/MS Detection
33
Figure 1. Target organotin compounds: TBT chloride, fentin
acetate, cyhexatin, fenbutatin oxide, and internal standard
triphenyl phosphate (top left to bottom right)
Spiked samples were extracted using acetonitrile and diluted
10x with LC grade water prior to LC-MS/MS analysis. The
spiked synthetic seawater was directly injected for detection
of organotin compounds. Note that additional dilution is
possible depending on required limits of detection to reduce
possible matrix effects (Figure 2).
UHPLC Separation
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A Shimadzu UFLCXR system was used with a Phenomenex
Kinetex 2.6u C18 50x3mm column at 40ºC. A gradient of
water with 2% formic acid + 5 mM ammonium formate and
methanol with 2% formic acid + 5 mM ammonium formate
at a flow rate of 800 μL/min resulted in a total run time of 12
minutes.
The injection volume was set to 20 μL for apple and potato
extracts and 50 μL for textile extracts and synthetic seawater.
122
The AB SCIEX 4000 QTRAP® LC/MS/MS system with
Turbo V™ source and ESI probe was used. All the analytes
and internal standard were detected in positive polarity
using MRM for best selectivity and sensitivity. Two MRM
transitions were monitored for each compound to allow
quantitation and identification using the characteristic MRM
ratio. The Scheduled MRM™ algorithm was activated for best
data quality (Table 1).
The data was processed in MultiQuant™ software version
2.1.
Table 1. MRM transitions and retention times (RT) of
targeted organotin compounds
Q1 (amu)
Q3 (amu)
RT (min)
TBT 1
291.0
123.0
3.8
TBT 2
291.0
235.1
3.8
Fentin 1
351.0
120.0
3.0
Fentin 2
351.0
197.0
3.0
Cyhexatin 1
369.0
205.0
5.3
Cyhexatin 2
369.0
287.1
5.3
Fenbutatin oxide 1
519.1
351.0
6.2
Fenbutatin oxide 2
517.1
349.0
6.2
Chromatography conditions were important for successful
determination of organotin compounds by LC-MS/MS.
Organotin compounds are known for strong interaction with
reversed phase material resulting in peak broadening. A
strong acidic mobile phase was used to reduce this effect and
to optimize peak shape.8
TPP (internal standard)
326.9
152.1
4.4
Two chromatographic interferences were observed for TBT in
all matrices. Thus, stable retention times and good separation
was important. A core-shell column (Phenomenex Kinetex)
was used for improved UHPLC performance while operating
at reduced column pressure (Figure 3).
Organotin compound
p2
Table 2. Signal-to-noise (S/N) in different matrices
Organotin
compound
Apple
(2 μg/kg)
Potato
(2 μg/kg)
Textile
(0.1 mg/kg)
Seawater
(50 ng/L)
TBT 1
105
71
93
53
Fentin 1
355
315
209
186
Cyhexatin 1
240
197
51
133
Fenbutatin
oxide 1
339
377
66
176
2200
2000
1800
1600
1500
1000
3.96
3.96
2.93
1200
1000
1000
800
400
200
2.53
.0
3.54
.0
4.55
6.57
.0
7.58
2000
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Time, min
0
2.5291.000/235.100
3.0amu Expected3.5
4.0 chloride 2 from
4.5 Sample 10 (potato
5.0
5.5of 21010117-apple
6.0 &...
XIC of +MRM (12 pairs):
RT: 3.8 ID: tributylin
2ngml 1/10)
Time, min
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 10 (potato 2ngml 1/10) of 21010117-apple &...
3.84
5.27
1800
6.14
3.84
5.27
1800
1600
3.73
6.14
3.97
1600
3.73
1400
3.97
.0
ax.1 .6e5 cps.
Internals tandard( TPP)
1.4e5
1.2e5
Fentin
2.95
Fentin
1400
1200
Intensity,
cps cps
Intensity,
1.0e5
8.0e4
6.0e4
4.0e4
1200
1000
6.5
7.0
7.5
6.5
7.0
7.5Max. 1826.2 cps.
8.0
8.0
Max. 1826.2 cps.
Fenbutatin oxide
Fenbutatin oxide Potato extract
Potato extract
Cyhexatin
Cyhexatin
TBT
TBT
4.41
1.6e5
Intensity, cps
1400
1200
600
400
.0
5.56
.0
Time,m in
XICo f+ MRM( 12 pairs):3 26.900/77.000a mu Expected RT:4 .4 ID: triphenyl phosphate1 from Sample4( SW)o f2 0120118-saltyw ater test.wiff( Tu...M
2.95
1000
800
800
600
600
400
400
200
2.0e4
0.0
6.13
Fentin
2.93
Fentin
1600
1400
Max. 2499.1 cps.
Apple extract
Apple extract
800
600
500
0
3.83
2000
1800
Intensity,
cps cps
Intensity,
3.97
3.72
2400
2200
Twoc hromatographic interferences forT BT
ares eparated well from the target analyte
2500
Cyhexatin
5.27
Cyhexatin
5.27
Fenbutatin oxide
6.13
Fenbutatin
oxide
TBT
3.83
TBT
2400
Blanks ynthetic seawater
3000
2000
Max. 2499.1 cps.
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 4 (apple 2ngml 1/10) of 21010117-apple & p...
3.72
3500
Intensity, cps
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 4 (apple 2ngml 1/10) of 21010117-apple & p...
ax.4 000.4c ps.
3.73
4000
2.53
.0
3.54
.0
4.55
.0
Time,m in
5.56
.0
6.57
.0
7.58
200
0
.0
0
Figure 3. Blank synthetic seawater, two chromatographic
interferences for TBT are separated well from the target
analyte (top) and internal standard (bottom)
2.5
3.0
3.5
4.0
4.5
2.5
3.0
3.5
4.0
4.5
5.0
Time, min
5.0
Time, min
5.5
6.0
6.5
7.0
7.5
8.0
5.5
6.0
6.5
7.0
7.5
8.0
Figure 4. Apple (top) and potato (bottom) sample spiked at 2
μg/kg and diluted 10x after extraction
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 3 (textile 0.1mg/kg 1/10) of 20120118-textile...
Max. 7547.7 cps.
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8 ID:
tributylin chloride 2 from Sample 3 (textile 0.1mg/kg 1/10) of 20120118-textile...
3.84
7548
3.84
7000
7548
7000
6000
5000
4000
Max. 7547.7 cps.
TBT
TBT
Fentin
2.93
Fentin
6000
5000
Intensity,
cps cps
Intensity,
Apple, potato, textile, and synthetic seawater samples were
spiked at different concentrations, extracted, and analyzed
using the fast LC-MS/MS method. Example chromatograms
are shown in Figures 4 and 5.
XICo f+ MRM( 12 pairs):2 91.000/235.100 amu Expected RT:3 .8 ID:t ributylin chloride 2f romS ample4 (SW) of 20120118-salty water test.wiff( Tu...M
Textile extract
Textile extract
Cyhexatin
5.27
Cyhexatin
5.27
Fenbutatin oxide
6.13
Fenbutatin
oxide
2.93
4000
3000
3000
2000
3.74
6.13
3.98
3.74
3.98
1000
0
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
Time, min
0
2.5291.000/235.100
3.0amu Expected3.5
4.0 chloride 2 from
4.5 Sample 7 (SW
5.050ppt) of 20120118-salty
5.5
XIC of2.0
+MRM (12 pairs):
RT: 3.8 ID: tributylin
water6.0
test....
Time, min
XIC of +MRM (12 pairs): 291.000/235.100 amu Expected RT: 3.8
ID: tributylin chloride 2 from Sample 7 (SW 50ppt) of 20120118-salty water test....
3.73
1903
1800
3.73
1903
1800
1600
2000
1000
6.5
7.0
7.5
6.5
7.0
7.5Max. 1903.4 cps.
8.0
1400
1200
3.97
Synthetic seawater
Synthetic seawater
Cyhexatin
Cyhexatin
Fenbutatin oxide
5.27
6.14
Fenbutatin
oxide
5.27
1200
1000
1000
800
Fentin
2.95
Fentin
800
600
600
400
8.0
Max. 1903.4 cps.
TBT
TBT
3.97
1600
1400
Intensity,
cps cps
Intensity,
The achieved Signal-to-noise (S/N) ratios are listed in Table
1. S/N values were measured in MultiQuant™ software after
applying a 2x Gaussian smooth. S/N values were used to
estimate limits of quantitation (LOQ) for all analytes in each
matrix.
6.14
2.95
400
200
Fentin
��
��
2.5
3.0
3.5
4.0
4.5
2.5
3.0
3.5
4.0
4.5
6.0
6.5
7.0
7.5
8.0
6.0
6.5
7.0
7.5
8.0
Table 3. Estimated limits of quantitation (LOQ) in different
matrices based on S/N of 10
Organotin
compound
Apple
(μg/kg)
Potato
(μg/kg)
Textile
(μg/kg)
Seawater
(ng/L)
0.2
0.3
10
10
< 0.1
< 0.1
< 10
< 10
Cyhexatin
0.1
0.1
20
< 10
Fenbutatin
oxide
< 0.1
< 0.1
15
< 10
TBT
Fentin
TBT
Fentin
��
� ��
5.5
5.5
enbutatin oxide
Figure 6. Calibration lines of organotin compounds in apple
matrix (2 to 100 μg/kg)
enbutatin oxide
��
CyhexatinF
��
5.0
Time, min
5.0
Time, min
Figure 5. Textile material spiked with 0.1 mg/kg and diluted
10x after extraction (top) and synthetic seawater spiked at 50
ng/L and analyzed by direct injection (50 μL)
��
CyhexatinF
2000
2.0
0
2.0
Figure 7. Calibration lines of organotin compounds in
synthetic seawater (50 to 2000 ng/L)
The linear dynamic range was evaluated from 2 to 100 μg/
kg for apple and potato, from 0.1 to 1 mg/kg for textiles,
and from 50 to 2000 ng/L for seawater. Example calibration
lines of all four organotin compounds in apple and synthetic
seawater are shown in Figures 6 and 7.
Repeatability was found to be less than 15% coefficient of
variation (%CV) and accuracy between 85 and 115% for all
compounds at all concentrations (Table 4).
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��
TBT
123
Table 4. Repeatability (%CV) and accuracy of organotin compounds at the lowest point of the calibration line
Apple (2 μg/kg)
Organotin compound
%CV
Potato (2 μg/kg)
Textile (0.1 mg/kg)
Seawater (50 ng/L)
Accuracy (%)
%CV
Accuracy (%)
%CV
Accuracy (%)
%CV
Accuracy (%)
TBT
10.0
97.0
13.9
86.4
7.3
95.6
6.3
113.1
Fentin 1
9.9
101.4
12.4
96.8
4.7
95.8
7.9
112.6
Cyhexatin
5.9
108.5
2.4
88.4
3.6
93.3
4.2
115.0
Fenbutatin oxide
11.4
104.4
11.8
99.5
13.2
97.3
3.6
107.4
Compound identification was achieved using the
‘Multicomponent’ query in MultiQuant™ software. This
query automatically calculates and compares MRM ratios
for identification and highlights concentrations above a user
specified residue level. Examples of the result table and peak
review after running the query file are shown in Figures 8 and
9.
Summary
A quick, easy, and robust LC-MS/MS method for the
determination of different organotin compounds in food,
seawater, and textile materials was developed. The method
allows accurate and reproducible quantitation using
the selectivity and sensitivity provided by the AB SCIEX
4000 QTRAP® system operated in MRM mode. Detection
limits well below regulated levels allow sample extract
dilution to minimize possible matrix effects. Confident
compound identification was achieved through the automatic
calculation of MRM ratios using the ‘Multicomponent’ query
in MultiQuant™ software.
References
Figure 8. Automatic compound identification using the
‘Multicomponent’ query (example cyhexatin in potato)
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Figure 9. Automatic compound identification using the
‘Multicomponent’ query (example fentin in textile)
124
1.K. Fent: ‘Ecotoxicology of organotin compounds’ Crit. Rev.
Toxicol. 26 (1996) 1-117
2.E. Gonzalez-Toledo et al.: ‘Detection techniques in speciation
analysis of organotin compounds by liquid chromatography’
Trends Anal. Chem. 22 (2003) 26-33
3.Regulation (EC) ‘on the prohibition of organotin compounds
on ships’ No 782/2003
4.Commission Decision ‘restrictions on the marketing and
use of organostannic compounds’ 2009/425/EC
5.International Association for Research and Testing in the
Field of Textile Ecology: OEKO-TEX Standard 100, Edition
4 (2012)
6.http://www.codexalimentarius.org/standards/pesticidemrls/en/
7.Council Directive ‘maximum levels for pesticide residues’
96/32/EC
8.EU Reference Laboratory for Single Residue Methods:
‘Analysis of Organotin Compounds via QuEChERS and LCMS/MS – Brief Description’ www.crl-pesticides.eu
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125
126
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Notlar
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Notlar
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