GIDA & ÇEVRE UYGULAMALARI.

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1 GIDA & ÇEVRE UYGULAMALARI

2 Spektrotek A.Ş. t: f: Katalog taleplerinizi [email protected] adresine, adres bilgilerinizi göndererek talep edebilirsiniz. SPEKTRTEK A.Ş. Kurumsal tanıtım ve ürün kataloğunda paylaşılan tüm görsel ve yazılı bilgilerin hakları SPEKTRTEK 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: [email protected]

3 Spektrotek A.Ş. SPEKTRTEK 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 SPEKTRTEK A.Ş.' nin büyük özen ve uzmanlıkla sağladığı ürün gruplarındandır. SPEKTRTEK 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 SPEKTRTEK A.Ş., Kendi tescilli markası olan ChromXpert ile önde gelen üreticilere yüksek kalite ile EM ürettirdiği ürünlerle kendi markası ile de Laboratuvar ürünleri pazarında yerini almıştır. Vizyonumuz; Bilim ve teknolojik yenilikleri, Dünyanın en iyi üreticileri yoluyla Türk Araştırmacılara ileterek ülkemizin kalkınması ve gelişmesine katkıda bulunmaktır. Gıda ve Çevre Uygulamaları Hakkımızda 3

4 Yapılırken heyecan duyulmayan işler başarılamaz Emerson Analiz ihtiyaçlarınıza profesyonel çözümler... Spektrotek tüm resmi kurum laboratuvarları, özel laboratuvarlar ve üniversite araştırma laboratuvarlarının tüm ihtiyaçlarını karşılamak üzere yapılanmıştır. 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. 4

5 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 Kütle Spektrometre Triple Quadrapole (Tandem MS) QTrap Hibrid Triple Quadrapole Lineer İyon Trap MALDI TF/TF Yüksek Rezolüsyon QqTF Sıvı Kromatografi Sistemleri (HPLC) PreparatifLC Ultra/UHPLC NanoLC MikroLC nline SPE Numune Hazırlık Sistemleri Protein Saflaştırma/BioChromatography smometreler Atomik Absorpsiyon Spektrofotometresi (AAS) ICP-ES/ICP-TFMS UV-VIS Spektofotometre Gaz Jeneratörleri Azot Jeneratörleri Kuru Hava Jeneratörleri Hidrojen Jeneratörleri Numune Ön Hazırlık Ekipmanları Temel Laboratuvar Cihaz, Ekipman ve Sarfları Fırınlar Karıştırıcılar & Manyetik Karıştırıcılar Ceketli Isıtıcılar Rotary Evaporatörler Santrifüjler Dondurucu & Soğutucular Vorteks Thermal Cycler Jel Görüntüleme Sistemi Pipet & tomatik 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 Vakum Pompaları Su Banyoları toklavlar Etüv & İnkübatörler Çalkalayıcılar Isıtıcı Tablalar Homojenizatörler Biyokimya LC-MS/MS ve HPLC analiz kitleri Referans Standart Kimyasallar rganik Referans Standartlar İnorganik Referans Standartlar USP Standartları Kalite Kontrol (QC) Numuneleri 5

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 TF, Nano ve Mikro HPLC, UHPLC, nline SPE, Atomik Absorpsiyon Spektrofotometresi, UV-VIS Spektrometre, ICP-ES, ICP-MS, smometre 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/Q 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. 6

7 Anahtar Teslim Laboratuvar Projeleri 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, IS /IEC gibi test ve kalibrasyon yapan laboratuvarların ya da IS 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 SP 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. 7

8 İçindekiler SPEKTRTEK GIDA VE ÇEVRE UYGULAMALAR 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 rijin 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 8

9 SCIEX APPLICATIN NTES 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 rigin by LC-MS/MS 65 Improving the LC-MS/MS Selectivity of Triazole Derivative Metabolites with AB SCIEX SelexIN 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 ff-line Solid Phase Extraction and Direct Injection LC-MS/MS 114 Screening and Identification of Unknown Contaminants in Untreated TapWater Using a Hybrid Triple Quadrupole Linear Ion Trap LC-MS/MS System 118 Quantitation and Identification of rganotin Compounds in Food, Water, and Textiles Using LC-MS/MS İçindekiler 9

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11 GIDA&ÇEVREUYGULAMALARI

12 3.6e4 3.7e4 3.2e4 3.4e4 2.8e4 3.0e4 2.4e4 2.6e4 2.0e4 2.2e4 1.6e4 1.8e4 1.2e4 1.4e e Untitled1(bap2): "Linear"Regression("No"weighting):y=178x+818(r =0.9994) Concentration,pg/ml Gıda ve Çevre Uygulamaları BEBEK MAMALARINDA PLİSİKLİK ARMATİK HİDRKARBN (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 Numune Hazırlığı 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. Analitik Koşullar Analitik Kolon : Phenomenex Kinetex Phenyl-Hexyl 100x2.1 mm 1.7 um Analiz Süresi : 8 dakika Mobil Faz : Su ve Asetonitril İyonizasyon : APCI Tarama Modu : Scheduled MRM MRM çiftleri: Bileşik Polarite Q1/Q3 Q1/Q3 Benzo(a)anthracene pozitif 228/ /199 Benzo(a)pyrene pozitif 252/ /226 Benzo(b)fluorenthene pozitif 252/ /224 Chrysene pozitif 228/ / SNUÇ 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 12

13 BEBEK MAMALARINDA MULTİ-TKSİN ANALİZİ GİRİŞ Hayvanlar ve insanlar için toksik özellik gösteren mikotoksinler, tarlada veya gıda maddelerinin depolama ve dağıtımı aşamasında oluşabilirler. Mikotoksinler; aflatoksinler, okratoksinler, fumonisin, zearalenone (ZN), 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. Gıda ve Çevre Uygulamaları YÖNTEM Numune Hazırlığı 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 Koşullar Analitik Kolon : Phenomenex Gemini 5 μm (150x4.6 mm) Analiz Süresi : 9 dakika Mobil Faz : Su (Amonyum Asetat) ve Metanol (Amonyum Asetat ve Asetik Asit) İyonizasyon : ESI + / ESI Tarama Modu : Scheduled MRM MRM çiftleri: Bileşik Polarite Q1/Q3 Q1/Q3 Aflatoksin B1 (AFB1) pozitif 313/ /128 Aflatoksin B2 (AFB2) pozitif 315/ /259 Aflatoksin G1 (AFG1) pozitif 329/ /200 Aflatoksin G2 (AFG2) pozitif 331/ /189 Aflatoksin M1 (AFM1) Pozitif 329/ /272 Deoxynivalenol (DN) Negatif 355/ /59 Zearalenon (ZN) Negatif 317/ /175 Fumonisin B1 (FB1) Pozitif 722/ /704 Fumonisin B2 (FB2) Pozitif 706/ /688 kratoksin A (TA) Pozitif 404/ /102 Patulin Negatif 153/ /135 SNUÇ 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 AFG1 KRA AFB1 AFM1 AFB2 AFG2 FB1 FB2 ZN patulin DN Şekil 2- Bebek mamasında Negatif-pozitif polarite değişimi ve Scheduled MRM algoritması ile tek metotta analiz edilen mikotoksinlere ait kromatogram 13

14 Gıda ve Çevre Uygulamaları 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 GC- MS 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 Numune Hazırlığı Numuneler homojenize edildikten sonra sıvı-sıvı ektraksiyonu ile hazırlanarak direk olarak LC-MS/ MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : Phenomenex Luna C18 3 um (150x3 mm) Analiz Süresi : 3 dakika Mobil Faz : Su (Formik Asit) ve Metanol (Formik Asit) İyonizasyon : ESI + Tarama Modu : Scheduled MRM MRM çiftleri: Bileşik Polarite Q1/Q3 Akrilamid 1 Pozitif 72/55 Akrilamid 2 Pozitif 72/44 Akrilamid 3 Pozitif 72/ SNUÇ Yüksek hassasiyet ve seçicilikten dolayı basit bir numune hazırlığı olan sıvı-sıvı ekstraksiyonu ile kısa zamanda onlarca numune rahatlıkla çalışılabilmektedir. Yapılan çalışmalar değerlendirildiğinde 2 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. Şekil 3 - Ciğer numunesine ait 1 ppb konsantrasyonda akrilamid etken maddesine ait kromatogram

15 SÜT VE SÜT ÜRÜNLERİNDE MELAMİN ANALİZİ 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. Gıda ve Çevre Uygulamaları YÖNTEM Numune Hazırlığı Numuneler basit bir numune hazırlama yöntemi olan sıvı-sıvı ekstraksiyon ile hazırlanarak LC-MS/ MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : KNAUER Eurospher II HILIC (150 x 3 mm) Analiz Süresi : 10 dakika Mobil Faz : Su (Amonyum Asetat) ve Asetonitril İyonizasyon : ESI + Tarama Modu : Scheduled MRM MRM çiftleri: Bileşik Polarite Q1/Q3 Melamin 1 Pozitif 127/85 Melamin 2 Pozitif 127/68 Melamin 3 Pozitif 127/60 SNUÇ 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. Şekil 4- Bebek mamasına ait melamin analiz kromatogramı 15

16 Gıda ve Çevre Uygulamaları 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 Numune Hazırlığı Numuneler, AAC fficial Method yöntemine göre ChromXpert QUECHERS kitleri ile hazırlandıktan sonra LC- MS/MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : Phenomenex Synergi Fusion 2.5 μm (50x2.1 mm) Analiz Süresi : 17 dakika Mobil Faz : Su (Amonyum Format) ve Metanol (Amonyum Format) İyonizasyon : ESI + / ESI Tarama Modu : Scheduled MRM QUECHERS KİTLERİ SNUÇ 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. Şekil 5-Negatif-pozitif polarite değişimi ve Scheduled MRM algoritması ile tek metotta analiz edilen 1 ppb 450 adet pestisit etken maddesine ait kromatogram. 16

17 GIDALARDA PLAR PESTİSİT TAYİNİ 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 ton dolayında olan tüketimin, 2008 de 200 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 (WH) uzmanlaşmış kanser kuruluşu olan Uluslararası Kanser Araştırmaları Kurumu GD 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 GD 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 AAC 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. Gıda ve Çevre Uygulamaları YÖNTEM Numune Hazırlığı Numuneler Quppe metodu doğrultusunda basit bir numune ön hazırlığı olan olan sıvı-sıvı ekstraksiyon ile hazırlanır ve 10 kat seyreltilerek direk olarak LC- MS/MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : Thermo Hypercarb 5um (2.1 x 100 mm) Analiz Süresi : 30 dakika Mobil Faz : Su (Asetik Asit) ve Metanol (Asetik Asit) İyonizasyon : ESI - Tarama Modu : Scheduled MRM SNUÇ 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 (a) 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 Şekil 6- Scheduled MRM kullanılarak yapılan pestisit analizine ait kromatogram (a) Kuru incir (b) Elma (b) 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

18 Gıda ve Çevre Uygulamaları 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 Numune Hazırlığı Numuneler Quppe metodu doğrultusunda basit bir numune ön hazırlığı olan sıvı-sıvı ekstraksiyon ile hazırlanarak LC-MS/MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : SUPELC 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 MRM çiftleri: Bileşik Polarite Q1/Q3 Paraquat 1 Pozitif 186/155 Paraquat 2 Pozitif 171/155 Diquat 1 Pozitif 184/127.8 Diquat 2 Pozitif 184/156 18

19 SNUÇ 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) Gıda ve Çevre Uygulamaları Şekil 7- Scheduled MRM kullanılarak yapılan kekik numunesi ait pestisit analiz kromatogramı (a) Paraquat (b) Diquat 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. Şekil 8- Direk enjeksiyon ile analiz edilen su numunesine ait pestisit kromatogramı 19

20 Gıda ve Çevre Uygulamaları JELİBNDA 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 Numune Hazırlığı Numuneler, NH4HC3 içerisinde çözündürüldükten sonra tripsin ile peptitlerine parçalanır ve LC-MS/MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : Phenomenex Gemini 5 μm (150x4.6 mm) Analiz Süresi : 8 dakika Mobil Faz : Su (Formik Asit) ve Asetonitril (Formik Asit) İyonizasyon : ESI + Tarama Modu : Scheduled MRM MRM çiftleri: Bileşik Polarite Q1/Q3 Domuz Jelatini 1 Pozitif /850.9 Domuz Jelatini 2 Pozitif 486.2/786.4 Domuz Jelatini 3 Pozitif 921.5/ Domuz Jelatini 4 Pozitif 620.8/618.3 Sığır Jelatini 1 Pozitif 659.3/766.5 Sığır Jelatini 2 Pozitif 781.4/991.6 Sığır Jelatini 3 Pozitif 644.8/ SNUÇ 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. 20

21 (a) (b) Gıda ve Çevre Uygulamaları 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. Şekil 10- %1 oranında domuz jelatini içeren sığır jelatin numunesine ait kromatogram 21

22 Gıda ve Çevre Uygulamaları 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 Numune Hazırlığı Numuneler sıvı-sıvı ekstraksiyonu, tripsin ile parçalama ardından SPE ekstraksiyonu ile hazırlanarak LC-MS/MS e enjeksiyonu yapılır. SNUÇ 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 Analitik Kolon : HAL C18 2.7um (50 x 0.5 mm) (Micro LC) Analiz Süresi : 11 dakika Mobil Faz : Su (Formik Asit) ve Asetonitril (Formik Asit) İyonizasyon : ESI + Tarama Modu : Scheduled MRM Şekil 11- MIDAS Akış şeması ve et numunesine ait elde edilen peptitler ve kütle spektrumları 22

23 ZEYTİNYAĞINDA TAĞŞİŞ VE RİJİN BELİRLEME GİRİŞ Zeytinyağının son zamanlardaki popülaritesi duyusal özellikleriyle ve sağlık açısından faydalarıyla ilişkilendirilebilir. leik 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. Gıda ve Çevre Uygulamaları YÖNTEM Numune Hazırlığı Numuneler hekzan/izopropanol karışımı ile seyreltilerek direk olarak LC-MS/MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : Spherisorb Silica column 5 um (250 x 4.6 mm) (Normal-Phase Chromatography) Analiz Süresi : 15 dakika Mobil Faz : Hekzan ve İzopropanol İyonizasyon : APCI Tarama Modu : MRM SNUÇ 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 23

24 Gıda ve Çevre Uygulamaları 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 Numune Hazırlığı Numuneler sıvı-sıvı ekstraksiyonu, tripsin ile parçalama ardından SPE ekstraksiyonu ile hazırlanarak LC-MS/MS e enjeksiyonu yapılır. SNUÇ Farklı alerjenler ile çalışma yapılmış ve her alerjen için belirleyici olan MRM çiftleri ile peptit haritaları çıkarılmıştır Analitik Koşullar Analitik Kolon Analiz Süresi Mobil Faz : 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) İyonizasyon : ESI + Tarama Modu : Scheduled MRM 24 Şekil 13- Yerfıstığı, yumurta ve süt için belirlenen alerjenlerin peptit haritası ve süt için elde edilen kalibrasyon grafiği

25 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. Gıda ve Çevre Uygulamaları Şekil 14- Ekmek numunesinde analiz edilen yumurta ve süt peptidlerine ait kromatogram ve kütle spektrumları Şekil 15- Makarna numunesinde analiz edilen yumurta ve süt peptidlerine ait kromatogram ve kütle spektrumları 25

26 Gıda ve Çevre Uygulamaları 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 Numune Hazırlığı Numuneler basit bir numune hazırlığı olan sıvı-sıvı ekstraksiyon ile hazırlanarak direk olarak LC-MS/MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : Phenomenex Luna HILIC 3 µm (100 x 2 mm) Analiz Süresi : 11 dakika Mobil Faz : Su (Formik Asit) ve Metanol (Formik Asit) İyonizasyon : ESI + Tarama Modu : Scheduled MRM Vitamin B5 Vitamin B7 Şekil 16- Bebek mamasında analiz edilen B vitaminlerine ait kromatogram SNUÇ 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 µg/kg arasında lineer aralığa sahip olan B vitaminleri hızlı bir şekilde analiz edilmektedir. 26

27 VETERİNER İLAÇ KALINTILARI / ANTİBİYTİK ANALİZİ 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. Gıda ve Çevre Uygulamaları YÖNTEM Numune Hazırlığı Numuneler sıvı-sıvı ekstraksiyonun ardından clean-up işlemi uygulandıktan sonra LC-MS/MS e enjeksiyonu yapılır. Pozitif Polarite Analitik Koşullar Analitik Kolon Analiz Süresi Mobil Faz : Phenomenex Gemini C18 3 μm (50 x 2.0 mm) : 10 dakika : Su (Formik Asit) ve Metanol (Formik Asit) İyonizasyon : ESI + / ESI - Tarama Modu : Scheduled MRM Negatif Polarite SNUÇ 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. Şekil 17-Negatif-pozitif polarite değişimi ve Scheduled MRM algoritması ile tek metotta analiz edilen balda antibiyotik analizine ait kromatogram. 27

28 Gıda ve Çevre Uygulamaları 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 (FA) ve Dünya Sağlık Örgütü (WH) 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 Numune Hazırlığı Numuneler 100/1000 kat su ile seyreltildikten sonra direk olarak LC-MS/MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : Phenomenex Synergi 4 μm (150 x 2.1 mm) Analiz Süresi : 7 dakika Mobil Faz : Su (Formik Asit) ve Metanol (Formik Asit) İyonizasyon : ESI - Tarama Modu : Scheduled MRM MRM çiftleri: Bileşik Polarite Q1/Q3 Q1/Q3 Acesulfame Negatif 162/82 162/78 Aspartame Negatif 293/ /261 Cyclamate Negatif 178/80 178/79 Glycyrrhizin Negatif 821/ /113 Neohesperidin Negatif 611/ /166 Saccharin Negatif 182/42 182/106 Sucralose Negatif 395/ /361 SNUÇ 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 LQ seviyelerinin belirlenmiş yasal limitlerin altında olduğu görülmüştür. 28

29 Intensity,cps Intensity,cps XICo f- MRM (14 pairs): / D ai D: Aspartame 2f roms ample6 1( lemonade 1/ e5 1.5e5 1.0e5 Max. 2.1e5c ps. 5.0e Time,m in XICo f- MRM (14 pairs): / D ai D: Aspartame 2f roms ample5 7( cola 1/ Max. 2.9e4c ps. 2.9e4 2.5e4 2.0e4 1.5e4 1.0e4 (a) (b) Acesulfame Saccharine Aspartame Aspartame Gıda ve Çevre Uygulamaları Time,m in 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. Intensity,cps TIC of -MRM (18 pairs): Exp 1, from Sample4(cola 1 in 100 dilution)ofsamples MRM-EPI.. 2.0e5 1.5e5 1.0e5 5.0e4 Şekil 19- QTrap-EPI kullanılarak analiz edilen kola numunesine ait tatlandırıcı kütle spektrumları 2.6 Max. 2.2e5 cps (c) Time,min -EPI (161.88) Charge (+0) FT (25... Max. 1.0e6 cps. -EPI (292.93) Charge (+0) FT (25... Max. 5.2e5 cps. 1.00e6 5.00e m/z, Da 250 -EPI (161.88) Charge (+0) FT (25... Max. 3.6e6 cps. 3.6e6 3.0e6 2.0e6 1.0e cola Acesulfame in cola Acesulfame standard m/z, Da Intensity,cps 5.2e5 4.0e e m/z, Da -EPI (292.93) Charge (+0) FT (25... Max. 1.9e6 cps. 1.9e6 1.5e6 1.0e Aspartame in cola Aspartame standard 5.0e m/z, Da 29

30 Gıda ve Çevre Uygulamaları İÇME SULARINDA PLİSİKLİK ARMATİK HİDRKARBN (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 Numune Hazırlığı Numuneler hiçbir ön işleme tabi tutulmadan direk olarak viale alınır ve LC-MS/MS e enjeksiyonu yapılır. MRM çiftleri: Bileşik Polarite Q1/Q3 Q1/Q3 Benzo(a)pyrene Pozitif 253/ /250 Benzo(b)fluorenthene Pozitif 252/ /224 Benzo(k)fluorenthene Pozitif 252/ /224 Benzo(g,h,i) perylene Pozitif 276/ /272 Indeno(1,2,3-c,d) pyrene Pozitif 276/ /272 Analitik Koşullar Analitik Kolon : Phenomenex Kinetex Phenyl-Hexyl 1.7 um (100x2.1 mm) Analiz Süresi : 6 dakika Mobil Faz : Su ve Asetonitril İyonizasyon : APCI Tarama Modu : Scheduled MRM (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 SNUÇ 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. Şekil 20- Su numunesi için Scheduled MRM ile yapılan PAH analizine ait kromatogram 30

31 İÇME SULARINDA AKRİLAMİD ANALİZİ 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. Gıda ve Çevre Uygulamaları YÖNTEM Numune Hazırlığı Numuneler hiçbir ön işleme tabi tutulmadan direk olarak viale alınır ve LC-MS/MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : Phenomenex Luna C18 3 um (150x3 mm) Analiz Süresi : 5 dakika Mobil Faz : Su (Formik Asit) ve Asetonitril İyonizasyon : ESI + Tarama Modu : MRM MRM çiftleri: SNUÇ Bileşik Polarite Q1/Q3 Akrilamid 1 Pozitif 72/55 Akrilamid 2 Pozitif 72/44 Akrilamid 3 Pozitif 72/27 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. Şekil 21- Su numunesi ile yapılan Akrilamid analizine ait kromatogram 31

32 Gıda ve Çevre Uygulamaları 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 (WH) 1995 yılında yayınlanan raporuna göre, her yıl dünyada kabaca 1 milyon insan pestisit sebebiyle zehirlenmekte, 200 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 Numune Hazırlığı Numuneler hiçbir ön işleme tabi tutulmadan direk olarak viale alınır ve LC-MS/MS e enjeksiyonu yapılır. Analitik Koşullar Analitik Kolon : Phenomenex Kinetex C um (50x2.1 mm) Analiz Süresi : 10 dakika Mobil Faz : Su (Amonyum Format) ve Metanol (Amonyum Format) İyonizasyon : ESI + / ESI - Tarama Modu : MRM SNUÇ 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

33 Pozitif Polarite Gıda ve Çevre Uygulamaları 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. Aynı zamanda su numunesi QTrap-EPI teknolojisi kullanılarak mevcut olan 666 adet pestisite ait kütüphane taraması ile konfirmasyon sonucu rapor güvenliği sağlanmaktadır. Şekil 23-Negatif-pozitif polarite değişimi ve QTrap-EPI ile tek metotta analiz edilen su numunesine ait kromatogram. 33

34

35 SPEKTRALKÜTÜPHANETABLLARI

36 Spektral Kütüphane Tabloları 36 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. 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. 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 TF-MS-IDA-MS/ MS workflow for use on TripleTF 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 TripleTF and QTRAP LC/MS/MS systems. Please note that this library is continuously being expanded to include additional compounds. Compound Formula Formula Weight CAS Number # of Spectra 17a-Methyltestosterone C20H ,6-Dimethyl-2-Hydroxypyrimidine C6H8N Hydroxymebendazole C16H15N Hydroxymebendazole-D3 C16H12(2H)3N Hydroxythiabendazole C10H7N3S a-nortestosteron C18H Acepromazine C19H22N2S Acetoxyprogesterone-17a C23H Acetyltylosin, 3-- C48H79N Albendazole C12H15N32S Albendazole-2-amino-sulfone-D3 C10H10(2H)3N32S Albendazole-D3 C12H12 2H3N32S Albendazole sulfoxide C12H15N33S Albendazole sulfoxide-d3 C12H15N33S Albendazolsulfon C12H15N34S Albendazolsulfon-D3 C12H12 2H3N34S Albendazolsulfonamin C10H13N32S Aminoflubendazol C14H10FN Aminomebendazol C14H11N Aminophenazon C13H17N

37 Compound Formula Formula Weight CAS Number # of Spectra Aminopropylon C16H22N Amoxicillin C16H19N35S Ampicillin C16H19N34S Arprinocid C12H9ClFN Avermectin B1a C48H Azaperone C19H22FN b-boldenone C19H Bamethan C12H19N Baquiloprim C17H20N Benzylpenicillin, (Penicillin G) C16H18N24S Brillant green C27H34N24S Brombuterol C12H18Br2N Carazolol C18H22N Carbuterol C13H21N Carprofen C15H12ClN Cefalonium C20H18N45S Cefazolin C14H14N84S Cefoperazon C25H27N98S Ceftiofur C19H17N57S Cephalexin C16H17N34S Cephapirin C17H17N36S Chloramphenicol C11H12Cl2N Chlorbrombuterol C12H18BrClN Chlormadinone acetate C23H29Cl Chlorprothixene C18H18ClNS Chlortetracyclin C22H23ClN Cimaterol C12H17N Cimbuterol C13H19N Ciprofloxacin C17H18FN Clenbuterol C12H18Cl2N Clencyclohexerol C14H20Cl2N Clenhexerol C14H22Cl2N Clenisopenterol C13H20Cl2N Clenpenterol C13H20Cl2N Clenproperol C11H16Cl2N Clenproperol-D7 C11H9 2H7Cl2N Clobendazole C16H12ClN Clorprenaline C11H16ClN Clorsulon C8H8Cl3N34S Closantel C22H14Cl2I2N Closantel-13C(6) (13C6)C16H14Cl2I2N Cloxacillin C19H18ClN35S Cyclopentylalbendazole C14H17N32S Danofloxacin C19H20FN Dapson C12H12N22S Decoquinat C24H35N Desacetylcephapirin C15H15N35S Dexamethasone C22H29F Spektral Kütüphane Tabloları 37

38 Spektral Kütüphane Tabloları Compound Formula Formula Weight CAS Number # of Spectra Diclofenac C14H11Cl2N Dicloxacillin C19H17Cl2N35S Dienestrol C18H Dienestrol-D2 C18H162(2H) Diethylstilbestroldipropionat C24H Difloxacin C21H19F2N Dimetridazol C5H7N Dimetridazol-D3 C5H4 2H3N Dinitrocarbanilid-D8 C13H2 2H8N Doramectin C50H Doxycycline (Tautomer) C22H24N Emamectin benzoate (B1a) C49H75N Enrofloxacin C19H22FN Enrofloxacin-D5 C19(2H)5H17FN epi-chlortetracyclin C22H23ClN epi-xytetracyclin C22H24N epi-tetracyclin C22H24N Eprinomectin B1a C50H75N Erythromycin A C37H67N Erythromycin A-(13C)2 (13C)2C35H67N Erythromycin A Anhydrid C37H65N Ethidimuron C7H12N43S Febantel C20H22N46S Fenbendazol-D3 C15H10 2H3N32S Fenbendazole C15H13N32S Fenbendazole sulfone-d3 C15H10 2H3N34S Fenoterol C17H21N Florfenicol C12H14Cl2FN4S Flubendazole C16H12FN Flubendazole-D3 C16H9 2H3FN Flufenamic Acid C14H10F3N Flumequine C14H12FN Flumethasone C22H28F Flunixin C14H11F3N Flunixin-D3 C14H8(2H)3F3N Gamithromycin C40H76N Halofuginon C16H17BrClN Heliotrin C16H27N Hydroxy-Ipronidazole-D3 C7H8(2H)3N Hydroxymethylclenbuterol C12H18Cl2N Ipronidazole C7H11N Ipronidazole-D3 C7H8(2H)3N Ipronidazole-H C7H11N Isochlortetracyclin C22H23ClN Isoxsuprine C18H23N Ivermectin C48H Josamycin C42H69N Ketoprofen C16H

39 Compound Formula Formula Weight CAS Number # of Spectra Ketotriclabendazole C13H7Cl3N Kristallviolett C25H30ClN Labetalol C19H24N Lasalocid A C34H53Na Leuco Malachite Green C23H26N Leuco Malachite Green-D5 C23H21(2H)5N Leucocrystal Violet C25H31N Leucomycin C35H59N Levamisole C11H12N2S Lincomycin C18H34N26S Mabuterol C13H18ClF3N Mapenterol C14H20ClF3N Marbofloxacin C17H19FN Mebendazol C16H13N Mebendazol-D3 C16H10 2H3N Meclofenamic acid C14H11Cl2N Medroxyprogesterone acetate C24H Mefenamic acid C15H15N Megestrol acetate C24H Melengestrol acetate C25H Meloxicam C14H13N34S Meloxicam-D3 C14H10 2H3N34S Methapyrilene C14H19N3S Methicillin C17H20N26S Methotrimeprazine C19H24N2S Meticlorpindol, (Clopidol) C7H7Cl2N Metoprolol C15H25N Metronidazol C6H9N Metronidazol-D3 C6H6(2H)3N Metronidazole-H C6H9N Monensin C36H Monocrotalin C16H23N Nafcillin C21H22N25S Nalidixic acid C12H12N Nandrolon C18H Naproxen C14H Netobimin-Micronized C14H20N47S Niflumic acid C13H9F3N Nitroxinil C7H3IN Norfloxacin C16H18FN leandomycin C35H61N rciprenaline C11H17N xacillin C19H19N35S xfendazole C15H13N33S xfendazole-d3 C15H10 2H3N33S xfendazolsulfon C15H13N34S xibendazole C12H15N xibendazole-d7 C12H8 2H7N Spektral Kütüphane Tabloları 39

40 Spektral Kütüphane Tabloları Compound Formula Formula Weight CAS Number # of Spectra xolinic acid C13H11N xyphenbutazone C19H20N xytetracycline C22H24N Phenylbutazone C19H20N Phenylbutazone-D10 C19H10(2H)10N Pirbuterol C12H20N Pirlimycin C17H31ClN25S Praziquantel C19H24N Prednisolone C21H Progesterone C21H Promethazine C17H20N2S Propionylpromazine C20H24N2S Ractopamine C18H23N Rafoxanide C19H11Cl2I2N Ramifenazon C14H19N Retrorsin C18H25N Ritodrine C17H21N Robenidine C15H13Cl2N Ronidazole C6H8N Ronidazole-D3 C6H5(2H)3N Roxithromycin C41H76N Salbutamol C13H21N Salmeterol C25H37N Salmeterol-D3 C25H34 2H3N Sarafloxacin C20H17F2N Secinidazol C7H11N Selamectin C43H63N Semduramycin C45H Senecionin C 18H25N Seneciophyllin C18H23N Sotalol C12H20N23S Spectinomycin C14H24N Spiramycin C43H74N Stanozolol C21H32N Sulfabenzamide C13H12N23S Sulfacetamide C8H10N23S Sulfachloropyridazine C10H9ClN42S Sulfadiazine C10H10N42S Sulfadimethoxine C12H14N44S Sulfadimethoxine-D6 C12H8 2H6N44S Sulfadimidin C12H14N42S Sulfadoxine C12H14N44S Sulfaguanidin C7H10N42S Sulfamerazine C11H12N42S Sulfameter C11H12N43S Sulfamethizol C9H10N42S Sulfamethoxazole C10H11N33S Sulfamethoxypyridazine C11H12N43S

41 Compound Formula Formula Weight CAS Number # of Spectra Sulfamoxol C11H13N33S Sulfanilamide C6H8N22S Sulfaphenazole C15H14N42S Sulfapyridin C11H11N32S Sulfaquinoxaline C14H12N42S Sulfathiazole C9H9N32S Terbutaline C12H19N Testosterone C19H Tetracycline C22H24N Tetramisole-D5 C11H7 2H5N2S Thiabendazole C10H7N3S Thiamphenicol C12H15Cl2N5S Tiamulin C28H47N4S Tilmicosin C46H80N Tinidazol C8H13N34S Tolfenamic Acid C14H12ClN Tolfenamic Acid-(13C)6 C8(13C)6H12ClN Trenbolone C18H Triamcinolone C21H27F Triclabendazole C14H9Cl3N2S Triclabendazole-D3 C14H6 2H3Cl3N2S Triclabendazole sulfone C14H9Cl3N23S Triclabendazole sulfoxide C14H9Cl3N22S Trimethoprim C14H18N Tulathromycin Marker 0 Tulobuterol C12H18ClN Tylosin A C46H77N Tylosin B C39H65N Tylvylosin (Aivlosin) C53H87N Valnemulin C31H52N25S Xylazine C12H16N2S Zilpaterol C14H19N Product Name License Part Number Antibiotics High Resolution MS/MS Spectral Library Version 1.0 permanent one-year AB Sciex. For Research Use nly. 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: Spektral Kütüphane Tabloları 41

42 Spektral Kütüphane Tabloları 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. 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. Compound Formula Formula Weight CAS Number # of Spectra 15-Acetyl-deoxynivalenol C17H Monoacetoxyscirpenol C17H Ketoaspergillimide C20H27N Amino-14,16-dimethyloctadecan-3-ol C20H43N Acetyl-deoxynivalenol C17H Nitropropionic acid C3H5N Methylviridicatin C16H13N A23187 C29H37N AAL-TA1 Toxin C25H47N Actinomycin D C62H86N Aflatoxin B1 C17H Aflatoxin B2 C17H Aflatoxin G1 C17H Aflatoxin G2 C17H Aflatoxin M1 C17H Aflatoxin M2 C17H Agroclavine C16H18N Alamethicin F30 C92H150N alpha-zearalenol C18H alpha-zearalenol-4--glucoside C24H Altenuene C15H Altenusin C15H Alternariol C14H Alternariolmethylether C15H Altersolanol C16H

43 Compound Formula Formula Weight CAS Number # of Spectra Altertoxin-I C20H Altertoxin-II C20H Amphotericin B C47H73N Anisomycin C14H19N Apicidin C34H49N Ascomycin C43H69N Aspercolorin C25H28N Aspergillimide C20H29N Asperlactone C9H Asperloxine A C21H19N Aspinonene C9H Aspyrone C9H Asterric acid C17H Atpenin A5 C15H21Cl2N Aureobasidin A C60H92N Aurofusarin C30H Austdiol C12H Austocystin A C19H13Cl Avenacein Y C15H Bacitracin C66H103N1716S Bafilomycin A1 C35H Beauvericin C45H57N beta-ergocryptine C32H41N beta-ergocryptinine C32H41N beta-zearalenol C18H beta-zearalenol-4--glucoside C24H Brefeldin A C16H Brevicompanine B C22H29N Calphostin C C44H Cephalosporin C C16H21N38S Cerulenin C12H17N Chaetocin C30H28N66S Chaetoglobosin A C32H36N Chanoclavine C16H20N Chetomin C31H306N6S Chlamydosporol C11H Chloramphenicol C11H12Cl2N Chromomycin A3 C57H Citreoviridin C23H Citrinin C13H Citromycetin C14H Cochliodinol C32H30N Curvularin C16H Cycloaspeptide A C36H43N Cycloechinulin C20H21N Cycloheximide C15H23N Cyclopenin C17H14N Cyclopeptine C17H16N Cyclopiazonic acid C20H20N Cyclosporin A C62H111N Spektral Kütüphane Tabloları 43

44 Spektral Kütüphane Tabloları Compound Formula Formula Weight CAS Number # of Spectra Cyclosporin C C62H111N Cyclosporin D C63H113N Cyclosporin H C62H111N Cytochalasin A C29H355N Cytochalasin B C29H375N Cytochalasin C C30H376N Cytochalasin D C30H376N Cytochalasin E C28H337N Cytochalasin H C30H39N Cytochalasin J C28H37N Decarestrictine C10H Dechlorogriseofulvin C17H Deepoxy-deoxynivalenol C15H Deoxybrevianamide E C21H25N Deoxynivalenol C15H Deoxynivalenol-3-glucoside C21H Diacetoxyscirpenol C19H Dihydroergosine C30H39N Dihydroergotamine C33H37N Dihydrolysergol C16H20N Dinactin C42H Elymoclavine C16H18N Elymoclavine fructoside C22H28N Emodin C15H Enniatin A C36H63N Enniatin A1 C35H61N Enniatin B C33H57N Enniatin B1 C34H59N Enniatin B2 C32H55N Enniatin B3 C31H53N Equisetin C22H31N Ergine C16H17N Erginine C16H17N N/A Ergocornine C31H39N Ergocorninine C31H39N Ergocristine C35H39N Ergocristinine C35H39N Ergocryptine C32H41N Ergocryptinine C32H41N Ergometrine C19H23N Ergometrinine C19H23N Ergosine C30H37N Ergosinine C30H37N Ergotamine C33H35N Ergotaminine C33H35N Ergovaline C29H35N Ergovalinine C29H35N Erythromycin C37H67N Festuclavine C16H20N

45 Compound Formula Formula Weight CAS Number # of Spectra FK 506 C44H69N Fulvic acid C14H Fumagillin C26H Fumigaclavine A C18H22N Fumitremorgin C C22H25N Fumonisin B1 C34H59N Fumonisin B2 C34H59N Fumonisin B3 C34H59N Fumonisin B4 C34H59N Fusaproliferin C27H Fusarenon-X C17H Fusaric acid C10H13N Fusarielin A C25H Fusidic acid C31H /06/03 4 Geldanamycin C29H40N Geodin C17H12Cl Gibberellic acid C19H /06/05 8 Gliotoxin C13H144N2S Griseofulvin C17H176Cl HC-Toxin C21H32N HT-2-Toxin C22H hydrolyzed Fumonisin B1 C22H47N Ionomycin C41H K252a C27H21N K252b C26H19N Kojic acid C6H Lincomycin C18H34N26S Lolitrem B C42H55N Lysergol C16H18N Macrosporin C16H Malformin C C23H39N55S Marcfortine A C28H35N Meleagrin C23H23N Methysergide C21H27N Mevastatin C23H Mevinolin C24H Mithramycin C52H Mitomycin C C15H18N /07/07 Monactin C41H Moniliformin C4H Mycophenolic acid C17H Myriocin C21H39N Neosolaniol C19H Neoxaline C23H25N NG012 C32H Nidulin C20H17Cl Nigericin C40H Nivalenol C15H Nonactin C40H Spektral Kütüphane Tabloları 45

46 Spektral Kütüphane Tabloları Compound Formula Formula Weight CAS Number # of Spectra Nornidulin C19H15Cl chratoxin A C20H18N6Cl chratoxin alpha C11H9Cl chratoxin B C20H19N ligomycin A C45H ligomycin B C45H Methylsterigmatocystin C19H phiobolin A C25H /05/06 phiobolin B C25H xaspirodion C13H oxidized Elymoclavine N/A N/A N/A 4 oxidized Luol N/A N/A N/A Paraherquamide A C28H35N Paspaline C28H39N Paspalinine C27H31N Paspalitrem A C32H39N Paspalitrem B C32H39N Patulin C7H Paxilline C27H33N Penicillic acid C8H Penicillin G C16H184N2S Penicillin V C16H18N25S /08/01 Penigequinolone A C27H33N Penitrem A C37H446NCl Pentoxyfylline C13H18N /05/06 Pestalotin C11H Phomopsin A C36H45ClN Phomopsin B C36H46N Physcion C16H Pseurotin A C22H25N Puromycin C22H29N Pyrenophorol C16H Pyripyropene A C31H37N Radicicol C18H17Cl Rapamycin C51H79N Roquefortine C C22H23N Roridin A C29H Rubellin D C30H Rugulosin C30H Satratoxin G C29H Satratoxin H C29H Secalonic acid C32H Setosusin C29H Stachybotrylactam C23H31N Staurosporine C28H26N Sterigmatocystin C18H Sulochrin C17H T2-Tetraol C15H T2-Toxin C24H T2-Triol C20H

47 Compound Formula Formula Weight CAS Number # of Spectra Taxol C47H51N Tentoxin C22H30N Tenuazonic acid C10H153N Terphenyllin C20H Territrem B C29H Tetracycline C22H24N Thiolutin C8H8N22S /11/06 Trichodermin C17H Trichostatin A C17H22N Tryprostatin A C22H27N Ustiloxin A C28H43N512S Ustiloxin B C26H39N512S Ustiloxin D C23H34N Valinomycin C54H90N Vancomycin C66H75Cl2N Verrucarin A C27H /09/02 Verrucarol C15H Verrucofortine C24H31N Verruculogen C27H337N Viomellein C30H Viridicatin C15H11N Wortmannin C23H Zearalenone C18H Zearalenone-4-glucoside C24H Zearalenone-4-sulfate C18H228S Product Name Part Number Mycotoxin Spectral Library Version For Research Use nly. Not for use in diagnostic procedures 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: Spektral Kütüphane Tabloları 47

48 Spektral Kütüphane Tabloları 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. 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 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. nce 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. Compound Formula Formula Weight CAS Number # of Spectra 2.4-D C8H6Cl DB C10H10Cl Naphthyloxyacetic acid C12H ,4,5-Trimethacarb C11H15N Hydroxycarbofuran C12H15N CPA C8H7Cl Hydroxy-clethodim-sulfone C17H26ClN6S not available 5-Hydroxy-imidacloprid C9H10ClN not available 5-Hydroxy-thiabendazol C10H7N3S Chlor-3-phenyl-pyridazin-4-ol (Pyridate-Metabolit) C10H7ClN Acephate C4H10N3PS Acequinocyl C24H Acetamiprid C10H11ClN Acetochlor C14H20ClN Acibenzolar-S-methyl C8H6N2S Acifluorfen C14H7ClF3N Aclonifen C12H9ClN Acrinathrin C26H21F6N Alachlor C14H20ClN Aldicarb-sulfoxid C7H14N23S

49 Compound Formula Formula Weight CAS Number # of Spectra Aldoxycarb C7H14N24S Alloxydim C17H25N Ametryn C9H17N5S Amidosulfuron C9H15N57S Aminocarb C11H16N Aminopyralid C6H4Cl2N Amitraz C19H23N Amitrol C2H4N AMPA (Aminomethyl phosphonic acid) CH63PN Anilazine C9H5Cl3N Anilofos C13H19ClN3PS Aramite C15H23Cl4S Atrazin C8H14ClN Atrazine-2-hydroxy C8H15N Atrazine-desethyl C6H10N5Cl Atrazine-desethyl-2-hydroxy C6H11N Atrazine-desisopropyl C5H8N5Cl Avermectin B1a C48H Azaconazole C12H11Cl2N Azamethiophos C9H10ClN25PS Azimsulfuron C13H16N105S Azinphos-ethyl C12H16N33PS Azinphos-methyl C10H12N33PS Azoxystrobin C22H17N Beflubutamid C18H17F4N Benalaxyl C20H23N Benazolin C9H6ClN3S /6/3813 Bendiocarb C11H13N Benfuracarb C20H30N25S Benfuresate C12H164S Benomyl C14H18N Benoxacor C11H11Cl2N Bensulfuron-methyl C16H18N47S Bensulide C14H24N4PS Bensultap C17H21N4S Bentazone C10H12N23S Benzoximate C18H18ClN Bifenazate C17H20N Bifenox C14H9Cl2N Bifenthrin C23H22ClF Bioresmethrin C22H Bitertanol C20H23N Boscalid C18H12Cl2N Brodifacoum C31H23Br Bromacil C9H13BrN Bromadiolone C30H23Br Bromophos C8H8BrCl23PS Bromophos-ethyl C10H12BrCl23PS Bromoxynil C7H3Br2N Bromuconazole C13H12BrCl2N Bupirimate C13H24N43S Spektral Kütüphane Tabloları 49

50 Spektral Kütüphane Tabloları 50 Compound Formula Formula Weight CAS Number # of Spectra Buprofezin C16H23N3S Butafenacil C20H18ClF3N Butocarboxim-sulfoxid C7H14N23S Butoxycarboxim C7H14N24S Butralin C14H21N Buturon C12H13ClN Butylate C11H23NS Cadusafos C10H232PS Carbaryl C12H11N Carbendazim C9H9N Carbetamide C12H16N Carbofuran C12H15N Carbosulfan C20H32N23S Carboxin C12H13N2S Carfentrazone-ethyl C15H14Cl2F3N Carpropamid C15H18Cl3N Cartap C7H15N32S Chinomethionate C10H6N2S /2/2439 Chlorbromuron C9H10BrClN Chlorbufam C11H10ClN Chlorethoxyfos-oxon C6H11Cl44P Chlorfenvinphos C12H14Cl34P Chlorfluazuron C20H9Cl3F5N Chloridazon C10H8ClN Chlorimuron-ethyl C15H15ClN46S Chlormephos C5H12Cl2PS Chlorophacinone C23H15Cl Chlorotoluron C10H13ClN Chloroxuron C15H15ClN Chlorpropham C10H12ClN Chlorpyrifos C9H11Cl3N3PS Chlorpyrifos-methyl C7H7Cl3N3PS Chlorsulfuron C12H12CIN54S Chlorthiamid C7H5Cl2NS Chlorthiophos C11H15Cl23PS Cinerin I C20H Cinerin II C21H Cinidon-ethyl C19H17Cl2N Cinosulfuron C15H19N57S Clethodim C17H26ClN3S Clethodim-imin-sulfone C14H23N4S not available Clethodim-imin-sulfoxide C14H23N3S not available Clethodim-sulfone C17H26ClN5S not available Clethodim-sulfoxide C17H26ClN4S not available Clodinafop-propargyl C17H13ClFN Clofentezine C14H8Cl2N Clomazone C12H14ClN Clomeprop C16H15Cl2N Clopyralid C6H3Cl2N Cloquintocet-mexyl C18H22ClN Clothianidin C6H8ClN52S

51 Compound Formula Formula Weight CAS Number # of Spectra Coumaphos C14H16Cl5PS Coumatetralyl C19H Crotoxyphos C14H196P Cyanazine C9H13ClN Cyanofenphos C15H14N2PS Cyanophos C9H10N3PS Cyazofamid C13H13ClN42S Cyclanilide C11H9Cl2N Cycloate C11H21NS Cycloxydim C17H27N3S Cyfluthrin C22H18Cl2FN Cyhalofop-butyl C20H20FN Cymoxanil C7H10N Cypermethrin C22H19Cl2N Cyphenothrin C24H25N Cyproconazole C15H18ClN Cyprodinil C14H15N Cyromazine C6H10N Daminozide C6H12N Deltamethrin C22H19Br2N Demeton-S-methyl C6H153PS Demeton-S-methyl-sulfon C6H155PS Desmedipham C16H16N Desmethyl-formamido-pirimicarb C11H16N Desmethyl-pirimicarb C10H16N Desmetryne C8H15N5S Dialifos C14H17ClN4PS Di-allate C10H17Cl2NS Diazinon C12H21N23PS Dichlofenthion C10H13Cl23PS Dichlofluanid C9H11Cl2FN22S Dichlorprop-P C9H8Cl Dichlorvos C4H7Cl24P Diclobutrazol C15H19Cl2N Diclofop-methyl C16H14Cl Dicloran C6H4Cl2N Diclosulam C13H10Cl2FN53S Dicrotophos C8H16N5P Dicyclanil C8H10N Diethofencarb C14H21N Difenacoum C31H Difenoconazole C19H17Cl2N Difenoxuron C16H18N Difenzoquat C17H17N Diflubenzuron C14H9ClF2N Diflufenican C19H11F5N Diflufenzopyr C15H12F2N Dimefuron C15H19ClN Dimepiperate C15H21NS Dimethachlor C13H18ClN Dimethametryn C11H21N5S Spektral Kütüphane Tabloları 51

52 Spektral Kütüphane Tabloları 52 Compound Formula Formula Weight CAS Number # of Spectra Dimethenamide C12H18ClN2S Dimethoate C5H12N3PS Dimethomorph C21H22ClN Dimetilan C10H16N Dimoxystrobin C19H22N Diniconazole C15H17Cl2N Dinoseb C10H12N Dinoterb C10H12N Dioxathion C12H266P2S Diphacinone C23H Diphenamid C16H17N Diphenylamine C12H11N Diquat C12H12N Dithianon C14H4N22S Dithiopyr C15H16F5N2S Diuron C9H10Cl2N DNC C7H6N Dodemorph C18H35N Dodine C15H33N /3/2439 Edifenphos C14H152PS Endosulfansulfate C9H6Cl64S EPN C14H14N4PS Epoxiconazole C17H13ClFN EPTC C9H19NS Esfenvalerate C25H22ClN Ethametsulfuron-methyl C15H18N66S Ethidimuron C7H12N43S Ethiofencarb C11H15N2S Ethiofencarb-sulfon C11H15N4S Ethiofencarb-sulfoxid C11H15N3S Ethion C9H224P2S Ethirimol C11H19N Ethofumesate C13H185S Ethoprophos C8H192PS Ethoxyquin C14H19N Ethoxysulfuron C15H18N47S Ethylenthiourea C3H6N2S Etofenprox C25H Etoxazole C2H23F2N Etrimfos C10H17N24PS Famoxadone C22H18N Famphur C10H16N5PS Fenamidone C17H17N3S Fenamiphos C13H22N3PS Fenarimol C17H12Cl2N Fenazaquin C20H22N Fenbuconazole C19H17ClN Fenfuram C12H11N Fenhexamid C14H17Cl2N Fenitrothion C9H12N5PS Fenobucarb C12H17N

53 Compound Formula Formula Weight CAS Number # of Spectra Fenoprop C9H7Cl Fenothiocarb C13H19N2S Fenoxaprop-ethyl C18H16ClN Fenoxycarb C17H19N Fenpiclonil C11H6Cl2N Fenpropathrin C22H23N Fenpropidin C19H31N Fenpropimorph C20H33N Fenpyroximate C24H27N Fenthion C10H153PS Fentin C18H15Sn Fenuron C9H12N Fenvalerate C25H22ClN Fipronil C12H4Cl2F6N4S Fipronil-desulfinyl C12H4Cl2F6N Fipronil-sulfide C12H4Cl2F6N4S Fipronil-sulfone C12H4Cl2F6N42S Flamprop-M-isopropyl C19H19ClFN Flamprop-M-methyl C17H15ClFN Flazasulfuron C13H12F3N55S Florasulam C12H8F3N53S Fluazifop (free acid) C15H12F3N Fluazifop-butyl C19H20F3N Fluazinam C13H4Cl2F6N Flucycloxuron C25H20ClF2N Flucythrinate C26H23F2N Fludioxonil C12H6F2N Flufenacet C14H13F4N32S Flufenoxuron C21H11ClF6N Flumetsulam C12H9F2N52S Flumioxazin C19H15FN Fluometuron C10H11F3N Fluoroglycofene-ethyl C18H13ClF3N Fluoxastrobin C21H16ClFN Flupyrsulfuron-methyl C15H14F3N57S Fluquinconazole C16H8Cl2FN Flurenol C14H Fluridone C19H14F3N Flurochloridone C12H10Cl2F3N Fluroxypyr C7H5Cl2FN Fluroxypyr-meptyl C15H21Cl2FN Flurprimidole C15H15F3N Flurtamone C18H14F3N Flusilazole C16H15F2N3Si Flusulfamide C13H7Cl2F3N24S Fluthiacet-methyl C15H15ClFN3o3S Flutolanil C17H16F3N Flutriafol C16H13F2N Fluxofenim C12H11ClF3N Fomesafen C15H10ClF3N26S Fonofos C10H15PS Spektral Kütüphane Tabloları 53

54 Spektral Kütüphane Tabloları 54 Compound Formula Formula Weight CAS Number # of Spectra Foramsulfuron C17H20N67S Formetanate C11H15N Fosthiazate C9H18N3PS Fuberidazole C11H8N Furalaxyl C17H19N Furathiocarb C18H26N25S Glufosinate C5H12N4P Halfenprox C24H23BrF Halofenozide C18H19ClN Halosulfuron-methyl C13H15ClN67S Haloxyfop-etotyl C19H19ClF3N Haloxyfop-P C15H11ClF3N Haloxyfop-P-methyl C16H13ClF3N Heptenophos C9H12Cl4P Hexaconazole C14H17Cl2N Hexaflumuron C16H8Cl2F6N Hexazinone C12H20N Hexythiazox C17H21ClN22S Hydramethylnon C25H24F6N Imazalil C14H14Cl2N Imazamethabenz-methyl C16H20N Imazapic C14H17N Imazapyr C13H15N Imazaquin C17H17N Imazosulfuron C14H13ClN65S Imibenconazole C17H13Cl3N4S Imidacloprid C9H10ClN Imidacloprid-lefin C9H8ClN not available Indoxacarb C22H17ClF3N Iodosulfuron-methyl-sodium C14H13IN5Na6S Ioxynil C7H3I2N Iprobenfos C13H213PS Iprodione C13H13Cl2N Iprovalicarb C18H28N Isazofos C9H17ClN33PS Isofenphos C15H24N4PS Isofenphos-oxon C15H24N5P Isoprocarb C11H15N Isoprothiolane C12H184S Isoproturon C12H18N Isoxaben C18H24N Isoxadifen-ethyl C18H17N Isoxaflutole C15H12F3N4S Isoxathion C13H16N4PS Jasmolin I C21H Jasmolin II C22H Kresoxim-methyl C18H19N lambda-cyhalothrin C23H19ClF3N Lenacil C13H18N /1/2164 Linuron C9H10Cl2N Lufenuron C17H8Cl2F8N

55 Compound Formula Formula Weight CAS Number # of Spectra Malaoxon C10H197PS Malathion C10H196PS Maleic hydrazide C4H4N MCPA C9H9Cl MCPA-2-Ethylhexylester C17H25Cl MCPA-butotyl C14H21Cl MCPB C11H13Cl Mecarbam C10H20N5PS Mecoprop-P C10H11Cl Mefenacet C16H14N22S Mefenpyr-diethyl C16H18Cl2N Mepanipyrim C14H13N Mepiquat C7H16N Mepronil C17H19N Mesosulfuron-methyl C17H21N59S Mesotrione C14H13N7S Metalaxyl C15H21N Metamitron C10H10N Metazachlor C14H16ClN Metconazole C17H22ClN Methabenzthiazuron C10H11N3S Methacrifos C7H135PS Methamidophos C2H8N2PS Methfuroxam C14H15N Methidathion C6H11N24PS Methiocarb C11H15N2S Methiocarb-sulfoxid C11H15N3S /1/2635 Methomyl C5H10N22S Methomyl-oxime C3H7NS Methoxyfenozide C22H28N Metobromuron C9H11BrN Metolachlor C15H22ClN Metolcarb C9H11N Metosulam C14H13Cl2N54S Metoxuron C10H13ClN Metrafenone C19H21Br Metribuzin C8H14N4S Metsulfuron-methyl C14H15N56S Mevinphos C7H136P Molinate C9H17NS Monocrotophos C7H14N5P Monolinuron C9H11ClN Monuron C9H11ClN Myclobutanil C15H17ClN Naled C4H7Br2Cl24P Napropamide C17H21N Neburon C12H16Cl2N Nicarbazin (1,3- N,N -bis (4-nitrophenyl)urea) C13H10N Nicosulfuron C15H18N66S Nicotine C10H14N Spektral Kütüphane Tabloları 55

56 Spektral Kütüphane Tabloları 56 Compound Formula Formula Weight CAS Number # of Spectra Nitenpyram C11H15ClN Norflurazon C12H9ClF3N Norflurazon-desmethyl C11H7ClF3N Novaluron C17H9ClF8N Nuarimol C17H12ClFN furace C14H16ClN methoate C5H12N4PS rbencarb C12H16ClNS xadiargyl C15H14Cl2N xadiazon C15H18Cl2N xadixyl C14H18N xamyl C7H13N33S xamyl-oxime C5H10N22S xasulfuron C17H18N46S xycarboxin C12H13N4S xydemeton-methyl C6H154PS xyfluorfen C15H11ClF3N Paclobutrazol C15H20ClN Paraoxon C10H14N6P Paraoxon-methyl C8H10N6P Parathion C10H14N5PS Parathion-methyl C8H10N5PS Pebulate C10H21NS Penconazole C13H15Cl2N Pencycuron C19H21ClN Pendimethalin C13H19N Permethrin C21H20Cl Pethoxamid C16H22ClN Phenmedipham C16H16N Phenthoate C12H174PS /7/2597 Phorate C7H172PS Phorat-sulfon C7H174PS /7/2588 Phorat-sulfoxide C7H173PS /6/2588 Phosalone C12H15ClN4PS Phosmet C11H12N4PS Phosphamidon C10H19ClN5P Phoxim C12H15N23PS Picolinafen C19H12F4N Picoxystrobin C18H16F3N Piperonyl butoxide C19H Piperophos C14H28N3PS Pirimicarb C11H18N Pirimiphos-ethyl C13H24N33PS Pirimiphos-methyl C11H20N33PS Primisulfuron-methyl C15H12F4N47S Prochloraz C15H16Cl3N Procymidone C13H11Cl2N Profenofos C11H15BrCl3PS Prohexadione C10H Promecarb C12H17N Prometon C10H19N

57 Compound Formula Formula Weight CAS Number # of Spectra Prometryne C10H19N5S Propachlor C11H14ClN Propamocarb C9H20N Propanil C9H9Cl2N Propaquizafop C22H22ClN Propargite C19H264S Propazin-2-hydroxy C9H17N not available Propazine C9H16ClN Propetamphos C10H20N4PS Propham C10H13N Propiconazole C15H17Cl2N Propoxur C11H15N Propoxycarbazone sodium C15H18N47S Propyzamide C12H11Cl2N Prosulfocarb C14H21NS Prosulfuron C15H16F3N54S Prothioconazole C14H15Cl2N3S Prothioconazole, Desthiometabolit (JAU C14H15Cl2N not available 6476-desthio) Prothiofos C11H15Cl22PS Pymetrozine C10H11N Pyraclofos C14H18ClN23PS Pyraclostrobin C19H18ClN Pyraflufen-ethyl Pyrazophos C14H20N35PS Pyrethrin I C21H Pyrethrin II C22H Pyridaben C19H25ClN2S Pyridaphenthion C14H17N24PS Pyridate C19H23ClN22S Pyrifenox C14H12Cl2N Pyrimethanil C12H13N Pyriproxyfen C20H19N Pyroquilon C11H11N Quinalphos C12H15N23PS Quinmerac C11H8ClN Quinoclamine C10H6ClN Quinoxyfen C15H8Cl2FN Quizalofop-ethyl C19H17ClN Quizalofop-P (free acid) C17H13ClN Resmethrin C22H Rimsulfuron C14H17N57S Rotenone C23H Sebuthylazine C9H16ClN Sebuthylazine-desethyl C7H12ClN not available Sethoxydim C17H29N3S Siduron C14H20N Silthiofam C13H21NSSi Simazine C7H12ClN Simazine-2-hydroxy C7H13N /3/2599 Simetryn C8H15N5S Spektral Kütüphane Tabloları 57

58 Spektral Kütüphane Tabloları 58 Compound Formula Formula Weight CAS Number # of Spectra Spinosyn A C41H65N Spinosyn D C42H67N Spiroxamine C18H35N Sulcotrione C14H13Cl5S Sulfentrazone C11H10Cl2F2N43S Sulfometuron-methyl C15H16N45S Sulfosulfuron C16H18N67S Sulfotep C8H205P2S Sulprofos C12H192PS tau-fluvalinate C26H22ClF3N Tebuconazol C16H22ClN Tebufenozide C22H28N Tebufenpyrad C18H24ClN Tebupirimfos C13H23N23PS Tebutam C15H23N Tebuthiuron C9H16N4S Teflubenzuron C14H6Cl2F4N Temephos C16H206P2S TEPP C8H207P Tepraloxydim C17H24ClN Terbumeton C10H19N Terbuthylazine C9H16ClN Terbuthylazine-2-hydroxy C9H17N not available Terbuthylazine-desethyl C7H12ClN Terbutryn C10H19N5S Tetrachlorvinphos C10H9Cl44P Tetraconazole C13H11Cl2F4N Tetramethrin C19H25N Thiabendazole C10H7N3S Thiacloprid C10H9ClN4S Thiamethoxam C8H10ClN53S Thidiazuron C9H8N4S Thifensulfuron-methyl C12H13N56S Thiobencarb C12H16ClNS Thiodicarb C10H18N44S Thiofanox-sulfone C9H18N24S Thiofanox-sulfoxide C9H18N23S Thiophanate C14H18N44S Thiophanate-methyl C12H14N44S Tolclofos-methyl C9H11Cl23PS Tolylfluanid C10H13Cl2FN22S Tralkoxydim C20H27N Triadimefon C14H16ClN Triadimenol C14H18ClN Tri-allate C10H16Cl3NS Triasulfuron C14H16ClN55S Triazamate C13H22N43S Triazophos C12H16N33PS Triazoxide C10H6ClN Tribenuron-methyl C15H17N56S Trichlorfon C4H8Cl34P

59 Compound Formula Formula Weight CAS Number # of Spectra Triclopyr C7H4Cl3N Tricyclazole C9H7N3S Tridemorph C19H39N Trietazine C9H16ClN Trifloxystrobin C20H19F3N Triflumizole C15H15ClF3N Triflumuron C15H10ClF3N Triflusulfuron-methyl C17H19F3N66S Trinexapac-ethyl C13H Triticonazole C17H20ClN Tritosulfuron C13H9F6N54S Uniconazole C15H18ClN Vamidothion C8H18N4PS Warfarin C19H Ziram C6H12N2S4Zn rdering Information Product Name Part Number Pesticide LC/MS/MS Library V.1.0 for Cliquid Software Legal Acknowledgements/Disclaimers 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. The suppliers identified in this document are provided for information purposes only and is not intended to be an exhaustive representation of all manufacturers or suppliers of the referenced product. AB SCIEX makes no warranties or representations as to the fitness or the continued fitness of a specific product by any of the manufacturers/suppliers referenced herein or the supplier. AB SCIEX assumes no responsibility or contingent liability, including indirect or consequential damages, for any use to which the purchaser may put the referenced suppliers products, or for any adverse circumstances arising therefrom. For Research Use nly. Not for use in diagnostic procedures 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: Spektral Kütüphane Tabloları 59

60

61 GIDA&ÇEVREUYGULAMALARI

62 Gıda ve Çevre Uygulamaları Simultaneous Analysis of 14 Mycotoxins and 163 Pesticides in Crude Extracts of Grains by LC-MS/MS Kristin Von Czapiewski 1 ; Angela Voller 2 ; Birgit Schlutt 1 ; André Schreiber 3 1 AB SCIEX, Darmstadt (Germany); 2 SGS, Hamburg (Germany); 3 AB SCIEX, Concord, N (Canada) Introduction Experimental Sample Preparation verview 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 (LQ) between 1µg/kg and 10µg/kg. The LQ for pesticides were found to be 10µg/kg and less. All LQ meet the requirements of the EU. 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 January 14, 2005 and amendments May 26, 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. ver 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. 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 LC A Shimadzu Prominence LC system with an Agilent ZRBAX 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. 62

63 MS/MS 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 (DN), Fusarenon X (FUS X), 3-Acetyldeoxynivalenol (3-AcDN), 15-Acetyldeoxynivalenol (15-AcDN), Diacetoxyscirpenol (DAS), HT-2 toxin, T-2 toxin, Zearalenon (ZN), and chratoxin A (TA) 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) e5 1.00e5 9.00e4 8.00e4 7.00e4 6.00e4 5.00e4 4.00e4 3.00e4 2.00e4 1.00e4 0 (p ), p( _ p x5 0. NIV: 371/ /281 DN: 295/ /138 FUS X: 413/ /263 3-AcDN: 337/ / AcDN: 337/ /150 DAS: 384/ /105 TA: 404/ /358 HT-2: 447/ /285 T-2: 484/ /185 ZN: 317/ /175 NIV 4.4min DN 5.8min FUS X 6.4min AcDN 7.2min DAS 8.0min TA 8.1min T-2 8.8min HT-2 8.5min Figure 1. Detection of fusarium toxins and chratoxin A by LC-MS/MS Table 1. LQ and linear range of detected mycotoxins ZN 9.3min 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 Figure 2. Detection of aflatoxins by LC-MS/MS Mycotoxin LQ (µg/kg) Linear Range (µg/kg) EU MRL# 3-AcDN (1) 15-AcDN (1) DN * 1250** (2) FUS X (1) DAS (1) NIV (1) TA 1 >10 5*** HT (2) T (2) ZN *** (2) Aflatoxin B1 1 >20 2 Aflatoxins 1 >20 1=4 (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 e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e B1: 313/ /241 B2: 315/ /259 G1: 329/ /311 G2: 331/ / G1 G2 B2 B Gıda ve Çevre Uygulamaları 63

64 Gıda ve Çevre Uygulamaları 64 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 (LQ) of all mycotoxins were found between 1 µg/kg and 10 µg/kg. Pesticides were quantified at 10 µg/kg and less. All LQ meet the requirements of the EU. Positive findings in two selected grain samples are shown in Figure 4. XIC of +MRM (331 pairs): 404.1/ a... Max. 8.9e5 cps. XIC of -MRM (36 pa irs): 421.0/97.0 a mu... Max. 5.7e 5 cp s. 1.0e 6 9.0e 5 8.0e 5 7.0e 5 6.0e 5 5.0e 5 4.0e 5 4.0e5 3.0e 5 3.0e5 2.0e 5 2.0e5 1.0e 5 1.0e XIC of +MRM (331 pairs): 484.1/ a... Max. 5.2e4 cps. XIC of -MRM (36 pa irs): 337.1/307.1 amu... Max. 5.7e 4 cp s e5 5.0e 4 Mycotoxins Mycotoxins 9.0e4 4.5e 4 Positive ESI Negative ESI 4.0e 4 8.0e4 3.5e 4 7.0e4 3.0e 4 6.0e e 4 5.0e4 2.0e 4 4.0e4 1.5e 4 3.0e4 1.0e 4 2.0e e Pesticides Positive ESI 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 Intens ity, cps Intens ity, cps Figure 4. Detection of mycotoxins and pesticides in a durum wheat sample (left) and a barley sample (right) Summary 8.7 In tensity, cps In tensity, cps 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 Pesticides Negative ESI XIC of +MRM (331 pairs): 331.1/285.1 amu Expect... Max. 2.0e6 cps. 2.0e6 1.5e6 1.0e6 5.0e5 2.4e5 2.0e5 1.5e5 1.0e5 5.0e4 T g/kg HT g/kg Malathion 2000 g/kg Pirimiphos-methyl 2400 g/kg Piperonylbutoxide 680 g/kg XIC of -MRM (36 pairs): 295.1/265.0 amu Expected... Max. 1.0e5 cps. DN NIV ZEA g/kg 500 g/kg 30 g/kg 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 (LQ) of all mycotoxins were found between 1µg/ kg and 10µg/kg. Pesticides were quantified at 10µg/kg and less. All LQ meet the requirements of the EU. For Research Use nly. Not for use in diagnostic procedures 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: XIC of +MRM (331 pairs): 384.2/105.2 amuexpect e5 1.0e5 5.0e XIC of -MRM (36 pairs): 295.1/265.0 amu Expected e5 3.0e5 2.0e5 1.0e5 References 1 D. Elbert et al.: presentation at AAC conference (2008) in Dallas 2 A. Voller et al.: presentation at AAC conference (2009) in Philadelphia DN 290 g/kg NIV 300 g/kg ZEA 20 g/kg 3-AcDN 20 g/kg MCPA 60 g/kg Bentazone 6 g/kg Azoxystrobin Pyraclostrobin Max cps. 3 g/kg 3 g/kg 12 Max. 3.7e4 cps

65 Detection of Underivatized Glyphosate and Similar Polar Pesticides in Food of Plant rigin by LC-MS/MS Stephen Lock 1 and Hermann Unterluggauer 2 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 FMC derivatization by reacting the native glyphosate with fluorenylmethyloxycarbonyl chloride (FMC-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. Experimental Sample Preparation 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. H H NH H Glyphosate NH 2 H 3 C Glufosinate P P H H LC The LC system used for this analysis was a ShimadzuXR LC system consisting of two Shimadzu LC20AD pumps, SIL 20AC autosampler, and a CT20A column oven. The analyses were performed at 50ºC on an belisc 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. Cl N H 2 H AMPA H P P Ethephon H H Gıda ve Çevre Uygulamaları 65

66 Gıda ve Çevre Uygulamaları Table 1. Gradient conditions used for separation Step Time Flow (ml/min) A (%) B (%) 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 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 Step Retention time (min) Q1 (amu) Results and Discussion Q3 (amu) DP (V) CE (V) AMPA Ethephon Glufosinate Glyphosate 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. XICo f- MRM( 11 pairs): /63.000D a ID:G lyphosate3...m 1.8e4 1.6e4 1.4e4 1.2e4 1.0e ax.1.8e4 cps Time,m in XICo f- MRM( 11 pairs): /62.900D a ID: AMPA 2f rom... Max.4.2e4 cps. 4.2e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 500 Glyphosate AMPA Time,m in XICo f- MRM( 11 pairs):1 800/62.900D ai D: Glufosinate1...M Figure 1. Injection of a 10 ng/ml standard 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 CDEX 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 MRM transition Linear fit (r value) S/N at 1 ng/ml AMPA 110/ / Ethephon 143/ / Glufosinate 180/ / Glyphosate 168/ / * S/N values were calculated in MultiQuant software Intensity,c ps ax.2.3e4 cps e4 2.0e4 Glufosinate 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Time,m in XICo f- MRM( 11 pairs): / Da ID:E thephon2 f... Max.5.7e4 cps e4 5.0e4 Ethephon 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Time,m in 66

67 Area Area AMPA AMPA AMPA Ethephon Ethephon Area Ethephon Glufosinate Glufosinate Glufosinate Glyphosate Glyphosate Glyphosate Figure 2. Calibration lines (2 MRM transitions each) for analyzed polar pesticides from 1 to 500 ng/ml 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. XIC of -MRM XIC of (11 -MRM pairs): (11 pairs): / / Da ID: Da Glyphosate ID: Glyphosate e5 1.00e5 9.00e4 9.00e4 8.00e4 8.00e4 7.00e4 7.00e XIC of -MRM 6.00e4(11 pairs): / Da ID: Glyphosate e4 Max. Max. 6.4e4 6.4e4 cps. cps. 5.00e4 5.00e4 1.00e5 4.00e4 4.00e4 9.00e4 3.00e4Glyphosate 3.00e4 8.00e4 2.00e4 2.00e4 7.00e4 1.00e e4 6.00e e XIC of -MRM (11 pairs): / Da Time, ID: AMPA min 2 from... Max. 1.4e5 cps. XIC 4.00e4 of -MRM (11 pairs): / Da ID: AMPA 2 from... Max. 1.4e5 cps e4 1.4e e5 1.3e5 2.00e4 1.3e5 1.2e5 1.00e4 1.2e5 1.1e5 1.1e5 1.0e e5 9.0e e4 8.0e4 XIC of -MRM (11 pairs): / Da ID: AMPA 2 from... Max. 1.4e5 cps. 8.0e4 7.0e4 6.0e e5 7.0e4 5.0e4 1.3e5 6.0e4 5.0e4 4.0e4 1.2e5 AMPA 4.0e4 3.0e4 1.1e5 3.0e4 2.0e4 1.0e5 1.0e4 9.0e4 2.0e4 8.0e4 1.0e e e4 5.0e4 Glyphosate AMPA AMPA XIC of -MRM (11 pairs): / Da ID: Glyphosate 3... XIC of -MRM (11 pairs): / Da ID: Glyphosate e4 3.0e4 3.5e4 3.0e4 2.5e4 2.5e4 2.0e XIC of -MRM (11 pairs): / Da ID: Glyphosate e4 3.5e4 1.5e4 1.5e4 1.0e e4 1.0e Max. 2.4e4 cps e XIC of -MRM (11 pairs): / Da ID: AMPA 2 from... Max. 8.5e4 cps. 2.0e e XIC 1.5e4 of -MRM 8.0e4(11 pairs): / Da ID: AMPA 2 from... Max. 8.5e4 cps. 8.5e4 1.0e4 8.0e e4 7.0e4 6.0e4 5.0e4 6.0e e4 XIC of -MRM (11 pairs): / Da ID: AMPA 2 from... Max. 8.5e4 cps. 5.0e4 3.0e4 8.5e e4 8.0e4 2.0e4 3.0e4 7.0e4 2.0e4 6.0e4 1.0e4 5.0e4 4.0e4 1.0e4 Glyphosate Glyphosate AMPA AMPA 4.24 Max. 6.4e4 cps. Max. 2.4e4 cps XIC XIC of -MRM of -MRM (11 (11 pairs): pairs): 1800/ / Da Da ID: ID: Glufosinate Glufosinate Max. Max. 7.4e4 7.4e4cps. 7.4e e4 7.0e4 7.0e4 6.5e4 6.5e4 6.0e4 Glufosinate 6.0e4 5.5e4 5.5e4 5.0e4 5.0e4 4.5e4 XIC 4.5e4 of -MRM (11 pairs): 1800/ Da ID: Glufosinate 1... Max. 7.4e4 cps. 4.0e4 4.0e4 3.5e e4 3.5e4 7.0e4 3.0e4 3.0e4 6.5e4 2.5e4 2.5e4 2.0e4 Glufosinate 6.0e4 2.0e4 5.5e4 1.5e4 1.5e4 5.0e4 1.0e e e e e Time, 6.0 min XIC of -MRM (11 pairs): / Da ID: Time, Ethephon min 2 f... Max. 1.7e5 cps. 3.0e4 XIC of -MRM (11 pairs): / Da ID: Ethephon 2 f... Max. 1.7e5 cps. 2.5e4 1.7e e4 1.7e5 1.6e e4 1.6e5 1.4e5 1.0e e5 Ethephon e5 1.2e e5 XIC of -MRM (11 pairs): / Da ID: Ethephon 2 f... Max. 1.7e5 cps. 1.0e5 8.0e4 1.7e e4 1.6e5 6.0e4 6.0e4 1.4e5 4.0e4 4.0e4 1.2e5 2.0e4 Ethephon e4 4.0e4 3.0e4 2.0e4 2.0e4 Figure 1.0e μg/kg spike into rice extract diluted 5x with acetonitrile 3.0e4 2.0e4 1.0e4 Glyphosate AMPA Max. 2.4e4 cps XIC of -MRM (11 pairs): 1800/ Da ID: Glufosinate XIC of 7.0e4 -MRM (11 pairs): 1800/ Da ID: Glufosinate e4 7.0e4 6.0e4 6.5e4 5.5e4 6.0e4 5.0e4 5.5e4 4.5e4 5.0e4 4.0e4 Max. 7.1e4 cps. 4.5e4 3.5e4 XIC of -MRM (11 pairs): 1800/ Da ID: Glufosinate 1... Max. 7.1e4 cps. 4.0e4 3.0e4 3.5e4 2.5e e4 3.0e4 2.0e4 6.5e4 2.5e4 1.5e4 6.0e4 Glufosinate 2.0e4 1.0e4 5.5e4 1.5e e e4 4.5e e4 XIC of3.21 -MRM 3.49 (11 pairs): / Da ID: Ethephon f Max e5 cps. 3.5e Time, e4 min XIC1.20e5 of -MRM (11 pairs): / Da ID: Ethephon 2 f... Max. 1.2e5 cps. 2.5e4 1.10e5 2.0e Ethephon 1.20e5 1.00e5 1.5e4 1.10e5 1.0e4 9.00e4 1.00e e e4 7.00e e4 8.00e4 XIC of -MRM (11 pairs): / Da ID: Ethephon 2 f... Max. 1.2e5 cps. 5.00e4 7.00e4 4.00e e4 1.20e5 3.00e4 5.00e4 1.10e5 2.00e4 4.00e4 Ethephon 1.00e5 1.00e4 3.00e4 9.00e e e4 1.00e4 7.00e4 2.0e4 1.0e5 8.0e4 6.0e e4 5.00e4 4.00e4 3.00e4 2.00e4 1.00e4 0 Ethephon 5.77 Glufosinate Glufosinate Ethephon Max. 7.1e4 cps Figure 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 Ethephon Glufosinate Glyphosate 110/79 110/63 143/79 143/ /63 180/95 168/79 168/63 Rice / nion / Grapes / Wheat / Gıda ve Çevre Uygulamaları 67

68 Gıda ve Çevre Uygulamaları 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% nion 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 FMC 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 QuPPe.pdf 2 Regulation (EC) concerning the placing of plant protection products on the market No 1107/ Commission Regulation (EU) regards maximum residue levels No 441/2012 Figure 5. Automatic reporting of pesticides using the Multicomponent query in MultiQuant software: the query calculates MRM ratios and flags samples with MRL violations. For Research Use nly. Not for use in diagnostic procedures 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:

69 Improving the LC-MS/MS Selectivity of Triazole Derivative Metabolites with AB SCIEX SelexIN Technology Julia Jasak 1, Yves LeBlanc 2, Ralf Schöning 3, Uwe Thuss 3, Karl Speer 1, and André Schreiber 2 1 Technische Universität, Food Chemistry, Dresden (Germany); 2 AB SCIEX, Concord, ntario (Canada) 3 Bayer CropScience AG, Residue Analysis, Monheim (Germany) Introduction 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 SelexIN technology coupled to a QTRAP 5500 LC/MS/MS system to improve the selectivity of LC-MS/MS detection of triazole derivative metabolites Table 1. Structure of studied triazole derivative metabolites 1,2,4-triazole TRZ Compound Triazole acetic acid (TAA) Triazole lactic acid (TLA) Triazole alanine TAL N N Structure N N N NH N H N N H N N N N H 2 H H Curtainp late DMSc ell rificep late SelexIN Technology The SelexIN 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 SelexIN 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. Gıda ve Çevre Uygulamaları 69

70 Gıda ve Çevre Uygulamaları Gasf low SV 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 LQ (1 mg/kg) and 10x LQ (0.1 mg/kg 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. MS/MS Detection Compensation Voltage( CV) CoV To MS/MS 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. SelexIN Settings 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/ TAA 128/ TLA 158/ TAL 157/ TRZ TLA TAA Figure 2. ptimization of CoV of each triazole derivative metabolite to obtain highest selectivity Results 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. TAL 70

71 TAA TLA TAL TRZ Figure 3. MRM traces for recovery LQ (1 mg/kg) in various matrices when acquisition was performed with DMS cell in transparent mode TAA TLA TAL TRZ Carrot LeafsC arrotr oots Rape Green( 1) Rape Green( 2) Rape Seed LettuceH eadg rapes Carrot LeafsC arrotr oots Rape Green( 1) Rape Green( 2) Rape Seed LettuceH eadg rapes Figure 4. MRM traces for recovery LQ (1 mg/kg) in various matrices when acquisition was performed with DMS cell optimized for each analyte Gıda ve Çevre Uygulamaları 71

72 Gıda ve Çevre Uygulamaları 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 1 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) (A) GF (LQ) GF (10xLQ) CR(LQ) CR(10xLQ) CL (LQ) CL(10xLQ) LH(LQ) LH (10xLQ) RGM-1 (LQ) (B) GF (LQ) GF (10xLQ) CR(LQ) CR(10xLQ) CL (LQ) CL(10xLQ) LH(LQ) LH (10xLQ) RGM-1 (LQ) Figure 5. Complexity of noise around LC peak of interest across for all matrices at the LQ and 10x LQ with DMS operated in (A) transparent mode and (B) optimized for each analytes RGM-1 (10xLQ) RGM-1 (10xLQ) RGM-2 (LQ) RGM-2 (LQ) RGM-2 (10xLQ) RGM-2 (10xLQ) RS (LQ) RS (LQ) RS(10xLQ) RS(10xLQ) TAA TAL TLA TRZ TAA TAL TLA TRZ 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 LQ. 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 LQ and 10x LQ level when DMS is used. Thus, the SelexIN 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 LQ 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. CoV( V) TRZ TIC7 0/43 map 70/43 Figure 6. Separation of interferences of TRZ (left) and TAA (right) at 10x LQ in rape green in the CoV space and on the LC time scale Finally, quantitative performance under three different LC- MS/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). CoV( V) TAA TIC1 28/70 map1 28/70 72

73 Table 3. Precision and accuracy obtained for single injections of solvent standards using three instrument configurations (DMS on, DMS off, and DMS removed) Compound TAA TLA TAL TRZ Summary Actual conc. (ng/ml) Calculated conc. (ng/ml) Accuracy Calculated conc. (ng/ml) 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 SelexIN Technology. Single LC peaks were obtained for TRZ, TAA, and TLA in all matrices and for TAL in most matrices. verall, combining the DMS with the AB SCIEX QTRAP 5500 system enabled the detection of triazole derivative metabolites with high confidence at desired LQ levels of 1 mg/kg and excellent precision. This technique proved to be extremely useful in the detection and monitoring of these species. Accuracy References Calculated conc. (ng/ml) Accuracy Calculated conc. (ng/ml) 1 B.B. Schneider, T. R. Covey, S.L. Coy, E.V. Krylov, E.G. Nazarov: Int. J. Mass Spectrom. 298 (2010) B.B. Schneider, T. R. Covey, S.L. Coy, E.V. Krylov, E.G. Nazarov: Anal.Chem. 82 (2010) For Research Use nly. Not for use in diagnostic procedures 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: Accuracy DMS on DMS off DMS removed Gıda ve Çevre Uygulamaları 73

74 Gıda ve Çevre Uygulamaları 74 Fast and Sensitive Analysis of Paraquat and Diquat in Drinking Water Houssain El Aribi AB SCIEX Concord, ntario (Canada) Experimental Chemicals LC verview 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 (LQ) 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 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 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 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 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.

75 MS/MS 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. Table 1. Detected MRM transitions for the analysis of Paraquat and Diquat Figure 1. MS/MS spectra of Paraquat and Diquat Results and Discussion 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. + M S 2 ( ) C E (30 ): 2 0 M C A sca n s fro m S am ple 2 (C ID o f 1 8 5_C E = 3 0 V _ Q 3 h ig h R e s ) o f D iq u a t a n d P a ra qua t.w iff ( T u rb o S pray ) M a x. 3.2e5 c p s e e e e e e e e e e e e 5 Paraquat a quantifier MRM185/170 b qualifiermrm 185/ e e e e m / z, a m u n ta tion_ + M S 2 ( ) C E (25 ): 2 0 M C A sca n s fro m S am ple 3 (C ID o f 1 8 3_D iq u a t _ C E = 25 V ) o f F ra g m e M a r ch 7 _ 0 6.w iff ( T u rb o S p r ay ) M a x. 2.8e6 c p s e e e e e e e e e e e e e 5 Analyte Name MRM transition DP (V) CE (V) CXP (V) 185/ Paraquat [M2+-H+] 185/ D8-Paraquat [M2+-H+] 193/ / Diquat [M2+-H+] 183/ D4-Diquat [M2+-D+] 186/ Diquat a quantifiermrm183/157 b qualifiermrm 183/ b [M 2+ -H + ] 2. 0 e m / z, a m u a b a [M 2+ -H + ] Gıda ve Çevre Uygulamaları 75

76 Gıda ve Çevre Uygulamaları 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 % 95% 90% 85% 80% 75% 70% 65% 60% 55% 50% 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% Diquat MRM1 83/157 MRM1 83/168 MRM1 86/158 (ISTD) 5.03 Paraquat MRM1 85/170 MRM1 85/169 MRM1 93/178 (ISTD) 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. Table 2. Summary of statistical parameters Max. 3.1e5 cps 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 (LQ) of the analytes were calculated from the chromatograms, at a signal-to-noise-ratio of >10. Analyte Name MRM transition LQ (µg/l) Linear range ( µg/l) R 2 Diquat 183/ Paraquat 185/

77 Di_Para_Quats_ISTD_March 9_06.rdb (183.0 /1 57.0): "Linear" Regression ("1/ x" weighting):y =1.01x ( r= ) Analyte Conc./ IS Conc. Figure 3. Calibration curve for Diquat (183/157) in drinking water from 0.1 μg/l to 5000 μg/l Figure 4. Calibration curve for Paraquat (185/170) in drinking water from 0.5 μg/l to 5000 μg/l Summary Diquat MRM1 83/157 R 2 = Di_Para_Quats_ISTD_March9 _06.rdb (185.0/ 17): "Linear" Regression ("1 / x" weighting):y = 1.28 x ( r= ) Paraquat MRM1 85/170 R 2 = 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 1 US EPA, Drinking Water Health Advisory: Pesticides, US Environmental Protection Agency, Lewis, Chelsea, MI, J. W. Munch, W. J. Bashe, US EPA 549.2, US Environmental Protection Agency, Cincinnati, H, R. Castro, E. Moyano, M. T. Galceran J. Chromatogr. A 2001, 914, L. Grey, B. Nguyen, P. Yang J. Chromatogr. A 2002, 958, For Research Use nly. Not for use in diagnostic procedures 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: Gıda ve Çevre Uygulamaları 77

78 Gıda ve Çevre Uygulamaları The Quantitation and Identification of Coccidiostats in Food by LC-MS/MS using the AB SCIEX 4000 Q TRAP System Bertram Nieland 1 and Stephen Lock 2 1 AB SCIEX Nieuwerkerk aan den Ijssel, The Netherlands; 2 AB SCIEX Warrington, UK Experimental 1) Conventional Approach Sample Preparation Introduction 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 ASIS 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 Table 1. LC gradient profile of conventional approach 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. LC Column : Agilent Zorbax Eclipse XDB-C8, 5 µm, 150 x 4.6 mm Flow rate : 400 μl/min ven temperature : 40 ºC Injection Volume : 40 µl Mobile Phase A : water + 0.2% acetic acid Mobile Phase B : methanol + 0.2% acetic acid Step Time (min) A (%) B (%)

79 2) New Approach Sample Preparation 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. LC Column : Phenomenex Kinetex C8, 2.6 µm, 100 x 4.6 mm Flow : 600 μl/min ven temperature : 40 ºC Injection Volume : 40 µl Mobile Phase A : water + 0.2% acetic acid Mobile Phase B : methanol + 0.2% acetic acid 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 MS/MS Step Time (min) A (%) B (%) 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. 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 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. Results and Discussion 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. Table 3. Maximum Residue limits (MRL) for some coccidiostats1 Coccidiostats MRL in milk (μg/kg) Diclazuril 5 Lasalocid 1 Maduramycin 2 Monensin 2 Narasin 1 Robenidine 5 Salinomycin 2 XICo f +MRM (12p airs): Period 3, / Da ID: Narasin0 2f roms ample1 0( M 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 positive polarity Robenidine negative polarity Diclazuril Monensin A Lasalocid Maduramycin Salinomycin Narasin Time,m in ax.2.6e5 cps. 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 16.0 positive polarity Gıda ve Çevre Uygulamaları 79

80 Gıda ve Çevre Uygulamaları 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) Diclazuril negative Decoquinate H 3 C Lasalocid H 3 C H Maduramycin Monensin A positive Narasin Nigericin N N Robenidine Salinomycin Decoquinate D5 (internal standard) H H 3 C H 3 C H 3 C Cl H 3 C H 3 C H 3 C Cl H H 3 C H N H H 3 C H 3 C H 3 C H 3 C H 3 C H 3 C H 3 C H 3 C N Cl H 3 C H 3 C H 3 C H 3 C H 3 C CH 3 CH 3 CH 3 Cl N N C H 3 CH 3 H 3 C CH 3 H H 3 C NN CH 3 H CH 3 H 3 C H CH 3 NH 2 CH 3 H CH 3 CH 3 H NH CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 H CH 3 H CH 3 CH 3 H CH 3 CH 3 H CH 3 CH 3 H H H CH 3 CH 3 CH 3 CH 3 CH 3 H CH 3 CH 3 CH 3 CH 3 H Cl CH 3 H H H H CH 3 H CH 3 CH

81 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). Intensity,c ps XICo f +MRM (3 pairs):p eriod1, / Da ID: Robenidine1 from Sample 25 (2...M 5.9e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 500 positive polarity Robenidine negative polarity 4.6 Diclazuril Decoquinate positive polarity ax.4.2e4 cps Time,m in Lasalocid Salinomycin Monensin A Narasin Maduramycin Nigericin Figure 2. 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 new approach 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. Table 5. Reproducibility from the repeat analysis of a low spiked matrix matched standard Coccidiostats Concentration of spiked SPE extract (μg/l) % CV (4 replicates) using the conventional approach 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. 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 LD 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 > (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. Robenidine 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 Concentration of QuECh- ERS extract (μg/l) % CV (4 replicates) using the new approach Diclazuril Lasalocid Maduramycin Monensin Narasin Robenidine Salinomycin Gıda ve Çevre Uygulamaları 81

82 Gıda ve Çevre Uygulamaları 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, / Da I... Max. 660 cps positive polarity Time,m in +EPI (747.60) Charge (+0) CE (35)C ES (15)F T( 250)...M ax.7.4e5 cps. 7.4e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 EPI spectrum for Nigericin m/z, Da In te n s ity, cp s I n te ns ity, c ps XICo f- MR M( 2p airs): Exp1, / Da ID...M ax cps Time,m in -EPI (406.93) Charge (+0) FT (50):E xp 2, minf r... Ma x. 1.9e6c ps Figure 4. Example of an LC-MS/MS chromatogram from a 2 μg/l matrix matched calibration standard run in positive polarity with an EPI spectrum of Nigericin (left) and an LC- MS/MS chromatogram from the same sample run in negative polarity where a spectrum of Diclazuril has been automatically acquired e6 1.6e6 1.4e6 1.2e6 1.0e6 8.0e5 6.0e5 4.0e5 2.0e negative polarity EPIs pectrumf or Diclazuril m/z, Da 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. AAC Int. 86 (2003) 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. AAC Int. 88 (2005) For Research Use nly. Not for use in diagnostic procedures 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:

83 Quantitation and Identification of 13 Azo-dyes in Spices using LC-MS/MS André Schreiber 1, Kristin von Czapiewski 2 AB SCIEX Concord, ntario (Canada), AB SCIEX Darmstadt (Germany) verview 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 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. Methods described in literature apply GC-MS, LC-UV, LC- MS 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 unknown concentration 200 Solvents tandards Matrix matcheds tandards Standard addition Figure 1. Calibration curves using solvent standards, matrix matched standards, and standard addition Gıda ve Çevre Uygulamaları 83

84 Gıda ve Çevre Uygulamaları 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. 1. Weigh 1 g of homogenized sample (multiple times for standard addition). 2. Add 20 μl of internal standard solution (1 μg/ml of D5- Sudan 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. 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) A (%) B (%) 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 range 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 84

85 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 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 Fast Garnet GBC range II (positive) range II (negative) Para Red Rhodamine B Sudan I Sudan II Sudan III Sudan IV Sudan range G Sudan Red 7B Sudan Red B Sudan Red G D5-Sudan Red I D6-Sudan Red IV Results and Discussion Standard chromatograms in positive and negative polarity using Electrospray Ionization are given in Figures 2 and 3. range 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. nly 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 % for each concentration and %CV<15% at 1 μg/kg and <5% at 10 μg/kg (n=3). 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 500 RhodamineB 8.7 Sudan range G FastGarnetGBC+ Red2 G range II DimethylYellow SudanR ed G Time,min Figure 2. Detection of 13 selected azo-dyes in positive polarity Para Red SudanI + D 5 -Sudan I SudanI I SudanI II SudanI V+ SudanR ed B+ D 6-Sudan IV SudanRed 7B 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. XICof-MRM(2pairs):327.0/171.0amufromSample9(10ng/mL)ofCalibration_neg.wiff( TurboSpray),Smoothed e4 1.05e4 1.00e Figure 3. Detection of range II in negative polarity range II Max.1.1e4cps Time,min Gıda ve Çevre Uygulamaları 85

86 Gıda ve Çevre Uygulamaları 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 Solvent Standards Matrix Matched Standards (Prepared in Masala Extract) Standard Addition Analyte Name Masala Chili Saffron Masala Chili 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% range 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% % 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 Cliquid Software and imethod Tests 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. 86

87 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 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 For Research Use nly. Not for use in diagnostic procedures 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: Gıda ve Çevre Uygulamaları 87

88 Gıda ve Çevre Uygulamaları Increasing Selectivity and Confidence in Detection when Analyzing Phthalates by LC-MS/MS André Schreiber 1, Fanny Fu 2, livia Yang 2, Eric Wan 3, Long Gu 4, and Yves LeBlanc 1 1 AB SCIEX, Concord, ntario (Canada) 2 AB SCIEX, Taipei, (Taiwan) 3 AB SCIEX, Hong Kong (Hong Kong) 4 AB SCIEX, Shanghai (China) Introduction verview 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 10-60% 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 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 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 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 SelexIN technology was used to separate critical isomers using Differential Mobility Spectrometry (DMS). 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 SelexIN technology. 88

89 Experimental Sample Preparation ne 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 SelexIN 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 Phthalates are esters of 1,2-benzenedicarboxylic acid. 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. R 1 R 2 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. 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). Figure 1. EPI spectrum of BBP, the molecular fingerprint saved into the MS/MS spectrum was used for compound identification with highest confidence Gıda ve Çevre Uygulamaları 89

90 Gıda ve Çevre Uygulamaları Table 1. Targeted phthalates, compound information, and optimized MRM transitions (Q1 and Q3 ions) Phthalate CAS Formula M.W. Q1 Q3 Dimethyl phthalate DMP C 10 H / 133 Diethyl phthalate DEP C 12 H / 177 Diallyl phthalate DAP C 14 H / 149 Dipropyl phthalate DPrP C 14 H / 191 Diisopropyl phthalate DIPrP C 14 H / 191 Dibutyl phthalate EU, EPA DBP C 16 H / 205 Diisobutyl phthalate EPA DIBP C 16 H / 205 Bis(2-methoxyethyl) phthalate DMEP C 14 H / 59 Dipentyl phthalate EPA DPP C 18 H / 149 Diisopentyl phthalate DIPP C 18 H / 149 Bis(2-ethoxyethyl) phthalate DEEP C 16 H / 149 Benzyl butyl phthalate EU, EPA BBP C 19 H / 205 Diphenyl phthalate DPhP C 20 H / 77 Dicyclohexyl phthalate DCHP C 20 H / 249 Bis(4-methyl-2-pentyl) phthalate BMPP C 20 H / 251 Dihexyl phthalate DHXP C 20 H / 233 Di-n-heptyl phthalate DHP C 22 H / 233 Bis(2-n-butoxyethyl) phthalate DBEP C 20 H / 249 Bis(2-ethylhexyl) phthalate EU, EPA DEHP C 24 H / 279 Di-n-octyl phthalate EU, EPA DNP C 24 H / 149 Diisononyl ortho-phthalate EU, EPA DINP C 26 H / 149 Diisodecyl ortho-phthalate EU, EPA DIDP C 28 H / 289 Bold EU EPA llegally used in food and beverages in Taiwan in Restricted use in toys and childcare articles in Europe 2 Addressed in the phthalates action plan of the U.S. Environmental Protection Agency 3 Table 2. Accuracy and linearity of six high priority phthalates Phthalate Accuracy (%) Regression DBP BBP DEHP DNP DINP DIDP An example chromatogram of LC-MS/MS detection of 22 phthalates is shown in Figure 2. Limits of detection (LD), 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 LD of at least 1 ng/ml was achieved. Please note that the final LD greatly depends on background interferences which can greatly vary from laboratory to laboratory. DMEP DMP DEEP DPrP DIPrP DEP DAP DPhP BBP DBP/ DIBP DBEP DIPP Figure 2. Example LC-MS/MS chromatogram showing the separation and detection of 22 phthalates at a concentration of 10 ng/ml DPP DCHP BMPP DHXP DHP DEHP DNP DINP DIDP 90

91 Sample Name: "1ppb-sch" Sample ID: "" File: " wiff" Sample Name: "5ppb-sch" Sample ID: "" File: " wiff" Sample Name: "10ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DBP 1" Mass(es): " / Da" Peak Name: "DBP 1" Mass(es): " / Da" Peak Name: "DBP 1" Mass(es): " / Da" Comment: "" Annotation: "" Comment: "" Annotation: "" Comment: "" Annotation: "" e e5 1.6e5 6.5e e e e5 6.0e4 1.1e5 1.3e5 5.5e e5 1.2e e4 9.0e4 1.1e e e4 1.0e e4 9.0e4 7.0e4 3.5e4 8.0e4 6.0e4 3.0e e4 5.0e4 6.0e4 2.5e e4 5.0e4 2.0e4 4.0e4 3.0e4 1.5e4 3.0e4 1.0e4 2.0e4 2.0e e4 1.0e Sample Name: "20ppb-sch" Sample ID: "" File: " wiff" Sample Name: "100ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DBP 1" Mass(es): " / Da" Peak Name: "DBP 1" Mass(es): " / Da" Comment: "" Annotation: "" Comment: "" Annotation: "" e5 9.5e e e e e5 8.0e5 7.5e5 2.0e5 7.0e5 DIBP/DBP 1.8e5 6.5e5 6.0e5 1.6e5 5.5e5 1.4e5 5.0e5 1.2e e5 4.0e5 1.0e5 3.5e5 8.0e4 3.0e5 2.5e5 6.0e e5 4.0e4 1.5e e5 2.0e e Figure 3a. MRM chromatograms of the high priority phthalates DBP and BBP at 1, 5, 10, 20, and 100 ng/ml Sample Name: "1ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DEHP 1" Mass(es): " / Da" Comment: "" Annotation: "" e4 4.5e4 2.8e e4 4.0e Sample Name: "5ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DEHP 1" Mass(es): " / Da" Comment: "" Annotation: "" Sample Name: "20ppb-sch" Sample ID: "" File: " wiff" Sample Name: "100ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DEHP 1" Mass(es): " / Da" Peak Name: "DEHP 1" Mass(es): " / Da" Comment: "" Annotation: "" Comment: "" Annotation: "" e5 7.5e e e4 3.2e5 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e 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 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 Sample Name: "10ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DEHP 1" Mass(es): " / Da" Comment: "" Annotation: "" 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Sample Name: "1ppb-sch" Sample ID: "" File: " wiff" Sample Name: "5ppb-sch" Sample ID: "" File: " wiff" Sample Name: "10ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DINP 1" Mass(es): " / Da" Peak Name: "DINP 1" Mass(es): " / Da" Peak Name: "DINP 1" Mass(es): " / Da" Comment: "" Annotation: "" Comment: "" Annotation: "" Comment: "" Annotation: "" e4 5.5e e e4 5.0e Sample Name: "20ppb-sch" Sample ID: "" File: " wiff" Sample Name: "100ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DINP 1" Mass(es): " / Da" Peak Name: "DINP 1" Mass(es): " / Da" Comment: "" Annotation: "" Comment: "" Annotation: "" e4 4.0e5 9.0e e e4 3.6e5 8.0e4 3.4e5 7.5e4 3.2e5 7.0e4 3.0e5 6.5e4 2.8e5 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 500 DEHP DINP Sample Name: "1ppb-sch" Sample ID: "" File: " wiff" Peak Name: "BBP 1" Mass(es): " / Da" Comment: "" Annotation: "" Sample Name: "20ppb-sch" Sample ID: "" File: " wiff" Peak Name: "BBP 1" Mass(es): " / Da" Comment: "" Annotation: "" e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Sample Name: "5ppb-sch" Sample ID: "" File: " wiff" Peak Name: "BBP 1" Mass(es): " / Da" Comment: "" Annotation: "" e4 1.3e4 1.2e4 1.1e4 1.0e Sample Name: "100ppb-sch" Sample ID: "" File: " wiff" Peak Name: "BBP 1" Mass(es): " / Da" Comment: "" Annotation: "" e Sample Name: "1ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DNP 1" Mass(es): " / Da" Comment: "" Annotation: "" e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e e4 1.7e e4 1.6e Sample Name: "5ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DNP 1" Mass(es): " / Da" Comment: "" Annotation: "" Sample Name: "20ppb-sch" Sample ID: "" File: " wiff" Sample Name: "100ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DNP 1" Mass(es): " / Da" Peak Name: "DNP 1" Mass(es): " / Da" Comment: "" Annotation: "" Comment: "" Annotation: "" e5 5.0e e e e4 1.4e4 1.3e4 1.2e4 1.1e4 1.0e e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e Sample Name: "10ppb-sch" Sample ID: "" File: " wiff" Peak Name: "BBP 1" Mass(es): " / Da" Comment: "" Annotation: "" Figure 4. Differentiation of DIBP and DBP using the different fragmentation pattern in MRM3 mode in comparison to MRM mode e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e BBP Sample Name: "10ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DNP 1" Mass(es): " / Da" Comment: "" Annotation: "" 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Sample Name: "1ppb-sch" Sample ID: "" File: " wiff" Sample Name: "5ppb-sch" Sample ID: "" File: " wiff" Sample Name: "10ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DIDP 1" Mass(es): " / Da" Peak Name: "DIDP 1" Mass(es): " / Da" Peak Name: "DIDP 1" Mass(es): " / Da" Comment: "" Annotation: "" Comment: "" Annotation: "" Comment: "" Annotation: "" Sample Name: "20ppb-sch" Sample ID: "" File: " wiff" Sample Name: "100ppb-sch" Sample ID: "" File: " wiff" Peak Name: "DIDP 1" Mass(es): " / Da" Peak Name: "DIDP 1" Mass(es): " / Da" Comment: "" Annotation: "" Comment: "" Annotation: "" e e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e XICo f+ MRM( 44 pairs): / Da ID:D BP 1 from Sample1 5 (Std20...M 100% DNP DIDP ax.2.3e5 cps. 0% Time,m in XICo f+ MRM( 44 pairs): / Da ID:D BP 2 from Sample1 5 (Std20...M ax.4.5e5 cps. 100% 0% Time,m in XICo f+ MS3( ),(223.10):E xp 2, t o Da from Sample3 (S...M ax.3.0e6 cps. 100% % Time,m in XICo f+ MS3( ),(223.10):E xp 2, t o Da from Sample3 (S...M ax.7.9e6 cps. 100% 0% DBP 5.99 DIBP DBP 5.99 DIBP DIBP DIBP 5.82 MRM2 79/205 MRM2 79/149 MRM 3 279/223/167 MRM 3 279/223/ Time,m in Gıda ve Çevre Uygulamaları 91

92 Gıda ve Çevre Uygulamaları Another possibility to enhance selectivity of detection is the use of Differential Mobility Spectrometry (DMS). The new AB SCIEX SelexIN 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. 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 CoV= 3.0 DMSo n( CoV= 3.0) DMSo n( CoV= -2.0) BMPP BMPP DHXP CoV= -2.0 DHXP DHXP Figure 5b. Selective detection of BMPP and DHXP by compound specific CoV for each analyte, acetonitrile was introduced as chemical modifier 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 LD 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 SelexIN 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. References 1. R.A. Rudel and L.A. Perovich: Atmospheric Environment 43 (2009) DIRECTIVE 2005/84/EC on phthalates in toys and childcare articles 3. EPA Phthalates Action Plan Summary Taipei Times: FD SCARE WIDENS: New chemical adds to food scare May 29, Zhuokun Li et al.: J. Chromatogr. Sci. 49 (2011) For Research Use nly. Not for use in diagnostic procedures 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:

93 Quantitative Analysis and Identification of Migrants in Food Packaging Using LC-MS/MS Cécile Busset 1 and Stephen J. Lock 2 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. LC 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. H 3 C 2-ITX4 S Irgacure CH 3 CH 3 P H 3 C H 3 C CH 3 CH 3 CH 3 H 2 C H 3 C -ITX S TRP H 3 C CH 3 Figure 1. Investigated migrants from food packaging CH 3 CH 2 CH 3 Gıda ve Çevre Uygulamaları 93

94 Gıda ve Çevre Uygulamaları 94 Table 1. LC gradient Step Time (min) (%) A (%) B 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: Curtain Gas (CUR) IonSpray Voltage (IS) Gas1 Gas2 CAD Gas Temperature 25 psi 5000 V 40 psi 50 psi Medium 450 C 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. +EPI (255.13) Charge (+0) CE (20) FT (250): Exp2,5.572 t...m e CE =2 0V ax.2.6e5 cps m/z, Da +EPI (255.13) Charge (+0) CE (35) FT (250): Exp2,5.531 t...m ax.1.2e5 cps e5 CE =3 5V m/z, Da +EPI (255.13) Charge (+0) CE (50) FT (250): Exp2,5.562 t...m ax.4.6e4 cps. 5.0e CE =5 0V m/z, Da +EPI (255.13) Charge (+0) CE (35) CES( 15)F T( 250):E xp...m ax.1.8e5 cps CE =3 5V with CES= 15V 1.0e m/z, Da 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) Figure 1. Investigated migrants from food packaging Compound Q1 Mass (amu) Q3 Mass (amu) DP (V) CE (V) ITX Irgacure TRP 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 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. Results and Discussion 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. 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 ( ng/ml for ITX, ng/ml for Irgacure and ng/ml for TRP) were greater than when a linear fit with 1/x weighting was applied. Area,counts Area,counts Area,counts quant.rdb(itx1): "Linear"Regression("1/x" weighting):y=5.88e+003 x+3.14e+003(r=0.9960) 3.0e6 2.0e6 1.0e Concentration, ng/ml quant.rdb (Irga 1):"Linear"Regression("1 /x"weighting): y=226x+109 (r =0.9994) 2.4e5 2.0e5 1.0e Concentration,ppb quant.rdb(trp1): "Linear" Regression("1/x" weighting):y=3.27e+003x+274(r=0.9993) 3.0e6 2.0e6 ITX r= Irgacure r= TRP r= e Concentration, ng/ml Figure 3. Calibration lines obtained from ITX, Irgacure and TRP with r values > (no internal standard used)

95 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). Table 3. Reproducibility data from 5 replicate injections Compound Transition Concentration (ng/ml) % CV (n=5) ITX 255.1/ Irgacure 419.2/ TRP 301.2/ 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 Max. 92c ps Time,m in Intensity,c ps XIC Max c ps Time,m in XIC Max c ps Time,m in Figure 5. Signal to noise (S/N) of low level migrant standards (S/N calculated using peak-to-peak algorithm) 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. S/N =4 0.4 S/N =1 8.4S /N = n g/ml ITX XICof+MRM (6pairs) e5 2.0e5 1.5e5 1.0e5 Deco* Varnish* Deco +varnish* 2n g/ml Irgacure Max. 2.6e5cps. 5.0e4 TRP ITX Time,min XICof+MRM (6pairs)... Max. 1.6e5cps. 1.5e5 1.0e5 5.0e4 5.5 Irgacure ITX TRP Time,min XICof+MRM (6pairs)... Max. 3.1e5 cps. 3.0e5 2.5e5 2.0e5 1.5e5 1.0e5 5.0e4 TRP 5.5 Irgacure 5.5 Irgacure ITX Time,min Intensity,c ps Deco 0.5n g/ml TRP XICof +MRM (6 pairs) e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 Varnish Deco+varnish TRP Intensity,c ps XICo f+ MRM( 6p airs): 301.2/113.2D a froms ample7 (std e4 5.0e4 TRP 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 500 Figure µl injection of migrants standards in initial mobile phase Table 4. Estimates for limits of detection (LD), limits of quantitation LQ), and linearity for food migrants Compound Time,m in S/N (at ng/ml) LD (ng/ml) Irgacure ITX LQ (ng/ml) Max. 5.4e4c ps. Linearity (ng/ml) ITX 40.4 (0.5) Irgacure 18.4 (2.0) TRP 23.2 (0.5) Max.1.4e5 cps. 5.5 Irgacure ITX Time,min XICof +MRM (6 pairs)... Max.7.1e4 cps. 7.1e4 6.0e4 4.0e4 5.5 Irgacure 2.0e4 5.2 ITX TRP Time,min XICof+MRM (6pairs)... Max. 1.3e5 cps. 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 TRP 5.5 Irgacure ITX Time,min 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)) Gıda ve Çevre Uygulamaları 95

96 Gıda ve Çevre Uygulamaları 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) Extract ITX (ng/dm²) Irgacure (ng/ dm²) TRP (ng/dm²) Deco Deco* Varnish Varnish* Deco + varnish Deco + varnish* 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. 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) Extract ITX % Irgacure % TRP % Deco Deco* Varnish Varnish* Deco + varnish Deco + varnish* 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 1 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 - SANC D3/AS D(2005) 2 FDA 21 CFR Submission of a premarket notification for a food contact substance (FCN) to the Food and Drug Administration (FDA). Code of Federal Regulations (December 2005) For Research Use nly. Not for use in diagnostic procedures 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:

97 Analysis of Perfluoroalkyl Acids Specified Under the UCMR3 Using the QTRAP 6500 LC/MS/MS System Lily Sanchez 1, Lee Yoo 1, Mike Wehner 1, and Matthew R. Noestheden 2 1 range County Water District, Fountain Valley, California (USA); 2 AB SCIEX Concord, ntario (Canada) verview 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 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). f these 14, six are specified in the UCMR3 Assessment Monitoring list: PFBS, PFHpA, PFHxS, PFA, PFS 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. Compound Abbreviation CASRN UCMR3 MRL(ng/L) Perfluorohexanoic acid PFHxA Perfluoroheptanoic acid PFHpA Perfluorooctanoic acid PFA Perfluorononanoic acid PFNA Perfluorodecanoic acid PFDA Perfluoroundecanoic acid PFUnA Perfluorododecanoic acid PFDoA Perfluorotridecanoic acid PFTrDA Perfluorotetradecanoic acid PFTA Perfluorobutanesulfonic acid PFBS Perfluorohexanesulfonic acid PFHxS Perfluorooctanesulfonic acid PFS N-methyl perfluorooctane-sulfonamidoacetic acid NMeFSAA - - N-ethyl perfluorooctane-sulfonamidoacetic acid NEtFSAA Gıda ve Çevre Uygulamaları 97

98 Gıda ve Çevre Uygulamaları 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 For carboxylic acids 13C2-PFA was used as the internal standard (ISTD), while all sulfonic acids used 13C4-PFS as the ISTD. The surrogates used were 13C2-PFHxA and 13C2-PFDA, both of which were fortified into samples at 40 ng/l. 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 2. LC gradient conditions Time (min) Flow Rate (µl/min) A (%) B (%) 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. Table 3. ESI source parameters Parameter Polarity Curtain Gas Collision Gas IonSpray Voltage Temperature GS1 GS2 Table 4. MRM transitions, retention time (RT), Declustering Potential (DP), and Collision Energy (CE) for target PFAAs, ISTDs (*) and surrogates (^) Compound Q1 Q3 RT DP (V) CE (V) PFBS PFBS PFHpA PFHpA PFHxS PFHxS PFA PFA PFS PFS PFNA PFNA C2-PFA* C4-PFS* C2-PFHxA^ C2-PFDA^ Results and Discussion Value negative 30 psi 12 psi V 400 C 30 psi 30 psi 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). 98

99 Figure 1. Final chromatography using a 20mM ammonium acetate / methanol mobile phase. Targets are shown on top with branched isomers of PFHxS and PFS indicated. ISTDs (13C2-PFA and 13C4-PFS) and surrogates are shown on the bottom (SUR1 = 13C2-PFHxA and SUR2 = 13C2-PFDA) For PFHxS and PFS 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). wing 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. 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 The correlation (r) value for all calibration curves were > 0.99 (Figure 3). Figure 3. Calibration lines and regression equations for all six PFAAs 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 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. Gıda ve Çevre Uygulamaları 99

100 Gıda ve Çevre Uygulamaları Accuracy and Precision 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. Table 5. Method performance Compound Precision Accuracy QCS (%) RPD ( % ) (%) Batch 1 Batch 2 (%) PFBS PFHpA PFHxS PFA PFS PFNA 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 13C2- PFHxA. 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. Figure 4. Asymmetry Factor for PFBS (left) and 13C2- PFHxA (right). The example on the left demonstrates how MultiQuant software 3.0 calculates AS. Method Reporting Limits 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 of EPA 537. Briefly, the calculations are: 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 Compound Fortification Level (ng/l) Lower PIR (%) Upper PIR (%) MDL (ng/l) PFBS PFHpA PFHxS PFA PFS PFNA Method Detection Limits The Method Detection Limit (MDL) was calculated using the following equation: 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). 100

101 Laboratory Reagent Blank 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. nce MRLs were established, the LRB was evaluated with regards to the background levels relative to the calculated MRLs (Figure 5). In the present method, all target compounds were observed well under 1/3 of their respective MRLs. Quality Control Sample and ngoing 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 %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) PFBS PFHpA PFHxS PFA PFS PFNA 1/3 MRL LRB FRB 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 %; mid/high CCC accuracy %; surrogate accuracy %) that were met for all samples analyzed. 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. 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) 537_FI NAL_rev1.1.pdf 2. Unregulated Contaminant Monitoring Rule 3 (UCMR3) ucmr3/ 3. M.F. Rahman et al.: Behavior and Fate of Perfluoroalkyl substances (PFAs) in Drinking Water Treatment: A Review. Water Research 50 (2014) AB Sciex. For Research Use nly. 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: 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 Gıda ve Çevre Uygulamaları 101

102 Gıda ve Çevre Uygulamaları LC-(DMS)-MS/MS Analysis of Emerging Food Contaminants Quantitation and Identification of Maleic Acid in Starch-Rich Foods Fanny Fu 1 and André Schreiber 2 1 AB SCIEX Taipei (Taiwan), 2 AB SCIEX Concord, ntario (Canada) 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. ccasional 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. 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. Experimental Sample Preparation Simple liquid extraction of food samples was performed using the following procedure developed by the Taiwan FDA4 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 KH. 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 H maleic acid H H H Figure 1. Chemical structures of maleic acid (left) and fumaric acid (right) fumarica cid 102

103 3.5e6 3.0e6 2.5e6 2.0e6 1.5e6 1.0e6 5.0e5 Figure 1. LC-MS/MS analysis of maleic acid and fumaric acid (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. Table 1. LC gradient used for the separation of maleic acid and fumaric acid Time (min) Flow (ml/min) A (%) B (%) 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, SelexIN differential mobility separation was investigated to increase selectivity, improve Signal-to-Noise (S/N), and increase confidence in identification. maleic acid Time,m in 4.59 fumaric acid 5.24 LC-MS/MS data were processed using the MultiQuant software version 2.1. Table 2. MRM transitions and retention times of maleic acid and fumaric acid Compound Q1 (amu) Q3 (amu) CE (V) Maleic acid Maleic acid Fumaric acid Fumaric acid Results and Discussion An example chromatogram of the detection of maleic acid and fumaric acid is shown in Figure 1. First, the limit of quantitation (LQ), 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 LQ 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 for quantifier and qualifier transitions. The accuracy values ranged from 89.6 to 107.6% across the linear dynamic range (Figure 2). Gıda ve Çevre Uygulamaları 103

104 Gıda ve Çevre Uygulamaları 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 Compound Q1 (amu) Q3 (amu) CE (V) Noodles Tapioca starch Processed food ppb spike in blank extract blank (115/71) blank (115/32) 18.4 (91.9% recovery) 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 104

105 Table 3 and Figure 3 show quantitative and qualitative results. MRM ratios were calculated using the Multicomponent query in MultiQuant software. In a last experiment we investigated the use of SelexIN differential mobility separation (DMS) to increase selectivity and confidence in identification. SelexIN 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 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. 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): / Da ID: Maleic acid 1 from Sample 6 (2mix-10ppb) of MA.wiff (Turbo Spray), Smoothed 2.0e5 1.5e5 1.0e Max. 2.0e5 cps. 5.0e XIC of -MRM (30 pairs): / Da ID: CoV -7.5 from Sample 1 (2mix) of DMS.wiff (Turbo Spray), Smoothed Max. 2.8e4 cps. 2.8e4 2.5e4 2.0e4 1.5e4 1.0e XIC of -MRM (30 pairs): / Da ID: CoV from Sample 1 (2mix) of DMS.wiff (Turbo Spray), Smoothed Max cps maleic acid maleic acid fumaric acid 6.04 fumaric acid DMS off DMS on (CoV = 8.0 V) DMS off (CoV = 10.5 V) 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 Figure 4. SelexIN 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 LC- DMS-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. SelexIN 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. References 1. aspx?id=9918&chk =454d1df8-1f26-43a3-9a dc07caae C-1304E73748D6/26074/Pressrelease_ Recallofstarchba sedproductsfromtaiwan.pdf B.B. Schneider, T. R. Covey, S.L. Coy, E.V. Krylov, E.G. Nazarov: Int. J. Mass Spectrom. 298 (2010) B.B. Schneider, T. R. Covey, S.L. Coy, E.V. Krylov, E.G. Nazarov: Anal.Chem. 82 (2010) For Research Use nly. Not for use in diagnostic procedures 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: Gıda ve Çevre Uygulamaları 105

106 Gıda ve Çevre Uygulamaları 106 Analysis of Endocrine Disruptors, Pharmaceuticals, and Personal Care Products in River Water Christopher Borton 1, Loren lson 2 1 AB SCIEX, Golden, C; 2 AB SCIEX, Foster City, CA Introduction verview 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 Experimental 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 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. 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) C 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 1% formic acid in water and eluent B was 1% 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 1 ml. Samples were kept at 4 C ± 1 C until analysis. Figure 1 shows a schematic of the sample preparation procedure.

107 Table 1. Compound list including MRM transitions (positive polarity) Quantifier Qualifier Quantifier Qualifier Compound Type Q1 Q3 Q1 Q3 Compound Type Q1 Q3 Q1 Q3 Acetaminophen Analgesic Estradiol Estrogen Ketoprofen Analgesic Ethinylestradiol Estrogen Codeine Analgesic α-Hydroxy-progesterone Estrogen Hydrocodone Analgesic Progesterone Estrogen Androstenedione Androgen Equilin Estrogen replacement Testosterone Androgen Diethylstilbestrol Dilantin Anti-convulsant TCEP Estrogen replacement Flame retardant Meprobamate Anti-anxiety Simazine Herbicide Sulfadiazine Antibiotic Isoproturon Herbicide Sulfamethoxazole Antibiotic Chlorotoluron Herbicide Sulfathiazole Antibiotic Atrazine Herbicide Sulfamerazine Antibiotic Chloridazon Herbicide Sulfamethizole Antibiotic Propazine Herbicide Sulfamethazine Antibiotic Diuron Herbicide Sulfachlorop-yridazine Antibiotic Hexazinone Herbicide Trimethoprim Antibiotic Bromacil Herbicide Sulfadimethoxine Antibiotic Metazachlor Herbicide Ciprofloxacin Antibiotic Metolachlor Herbicide Penicillin G Antibiotic DEET Amoxicillin Antibiotic Bezafibrate Lipid regulator Table 1 (continued). Compound list including MRM transitions (negative polarity) Compound Type Q1 Q3 Q1 Q3 Compound Type Q1 Q3 Q1 Q3 Virginiamycin Antibiotic xybenzone Sunscreen Monensin Antibiotic Sildenafil Virility regulator Erythromycin Antibiotic Vardenafil Virility regulator Roxithromycin Antibiotic Salicylic Acid Skin care, acne Tylosin Antibiotic Cotinine Nicotine metabolite Meclocycline Sulfosalinicyclate Antibiotic Aminopyrine Insect repellant Lincomycin Antibiotic Diazepam Muscle-relaxant Doxycycline Antibiotic Norethisterone vulation Inhibitor Tetracycline Antibiotic Theophylline Stimulant xytetracycline Antibiotic Theobromine Stimulant Chlortetracycline Antibiotic Caffeine Stimulant Aminoantipyrine Sulfadimethoxine Antibiotic Ketorolac metabolite Sulfachloro-Pyridazine Antibiotic Fenoprop Anti-inflammatory Norifloxacin Antibiotic Meclofenamic acid Herbicide Enroflofacin Antibiotic Piroxicam Fluoxetine Antidepressant Nifedipine Dihydropyridine calcium channel blocker Carbamazepine Anti-seizure Indomethacin Anti-inflammatory Pentoxifylline Blood viscosity reducing agent Diatrizoate Radiocontrasting agent Gıda ve Çevre Uygulamaları 107

108 Gıda ve Çevre Uygulamaları 108 Quantifier Qualifier Quantifier Qualifier Compound Type Q1 Q3 Q1 Q3 Compound Type Q1 Q3 Q1 Q3 Acetylsalicylic acid Analgesic Estrone Estrogen 269 Ibuprofen Analgesic Estradiol Estrogen 271 Naproxen Analgesic Estriol Estrogen 287 Warfarin Anti-coagulant Ethinylestradiol Estrogen 295 Diclofenac Anti-arthritic Tetrabromo-bisphenol A Flame retardant Carbadox Antibiotic ,4-D Herbicide Metabolite of lipid Triclosan (Irgasan) Antibiotic Clofibric acid regulator Chloramphenicol Antibiotic Iopromide X-ray contrast agent Gemfibrozil Anti-cholesterol ,4-Dichloro- benzoic acid Results and Discussion 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. 1L Riverw ater Filter 1L filtered water SPE new Condition SPE ready 50 Li njection 10 ml extract Figure 1. Sample preparation procedure for solid phase extraction 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. Intensity,cps Intensity,cps XICof+ MRM (137pairs): Exp 1, / Da from Sample8 (100ppb STD)o fd ata e6 6.0e6 5.0e6 4.0e6 3.0e6 2.0e6 1.0e6 ESI positive ESI negative Max cps XICo f -MRM( 24 pairs): Exp2, / Da from Sample8 (100 ppb STD) ofd atae...m ax. 6.7e4 cps. 2.2e6 2.0e6 1.5e6 1.0e6 5.0e Time,m in Figure 2. Polarity switching is utilized to encompass a large list of analytes 100 ng/ml standard injection In tensity, cps XIC of-mrm (24 pairs): Exp 2, e4 4.0e4 2.0e Max. 6.8e4 cps XICof-MRM (24 pairs): Exp 2, Max. 2.2e4 cps. 2.0e4 1.5e4 1.0e XICof-MRM (24 pairs): Exp 2, Max. 1.9e6 cps. 1.9e6 1.5e6 1.0e6 5.0e5 Acetylsalicylic acid 24.6 Chloramphenicol Clofibric acid In tensity, cps XIC of -MRM (24 pairs): Exp 2, Max. 2.3e6 cps XIC of -MRM (24 pairs): Exp 2, Max. 1.4e6 cps. Figure 3. verlay 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 2.0e6 1.5e6 1.0e6 5.0e5 1.4e6 1.0e6 5.0e XIC of -MRM (24 pairs): Exp 2, Max. 1.3e5 cps. 1.26e5 1.00e5 5.00e4 Warfarin Diclofenac Ibuprofen

109 Table 2. Lower Limits of Quantitation (LLQ) of selected analytes 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 (LLQ) 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 LLQ. All compounds had LLQ in the sub part per billion (ppb) range. 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 Table 3. XIC of +MRM (137 pairs): Exp 1, 7... Max. 2.0e5 cps. XIC of +MRM (137 pairs): Exp 1, 7... Max. 1.4e5 cps. 2.0e e5 1.5e5 Erythromycin Erythromycin 1.0e5 1.0e5 MRM ratio = MRM ratio = e4 5.0e XIC of +MRM (137 pairs): Exp 1, 2... Max. 1.4e6 cps. XIC of +MRM (137 pairs): Exp 1, 2... Max. 3.6e4 cps e6 3.8e Ketorolac 3.0e4 Ketorolac 1.0e6 MRM ratio = e4 MRM ratio = e e XIC of +MRM (137 pairs): Exp 1, 2... Max. 1.3e6 cps. XIC of +MRM (137 pairs): Exp 1, 2... Max. 4.0e5 cps. 1.3e e5 1.0e6 Mepobramate 3.0e5 Mepobramate MRM ratio = e5 MRM ratio = e5 1.0e5 Intensity,cps Intensity,cps Analyte LLQ (ng/l) ppt Analyte LLQ (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 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 Intensity,cps Intensity,cps Area, counts Concentration,ng/mL EDC and PPCP calibration.rdb (Theophyllin.e 2) Area, counts Area, counts EDC and PPCP calibration.rdb (Theophyllin.e 1) 8.0e6 7.0e6 6.0e6 5.0e6 4.0e6 3.0e6 2.0e6 1.0e6 1.8e6 1.5e6 1.0e6 5.0e5 Theophylline 1 Theophylline Concentration,ng/mL Concentration, ng/ml EDC and PPCP calibration.rdb (Sulfathiazole 2) Area, counts EDC and PPCP calibration.rdb (Sulfathiazole 1) ) 1.20e7 1.00e7 8.00e6 6.00e6 4.00e6 2.00e6 8.7e6 8.0e6 7.0e6 6.0e6 5.0e6 4.0e6 3.0e6 2.0e6 1.0e6 Sulfathiazole 1 Sulfathiazole Concentration, ng/ml EDC and PPCP calibration.rdb (Carbamazepine 1 1.6e7 1.4e7 1.2e7 1.0e7 8.0e6 6.0e6 4.0e6 2.0e Concentration, ng/ml EDC and PPCP calibration.rdb (Carbamazepine 2... Figure 5. Example calibrations for selected analytes Area, counts Area, counts Area, counts Area, counts 2.8e6 2.5e6 2.0e6 1.5e6 1.0e6 5.0e5 Carbamazepine 1 Carbamazepine Concentration, ng/ml EDC and PPCP calibration.rdb (2,4-D 1): "Li 3.5e7 3.0e7 2.5e7 2.0e7 1.5e7 1.0e7 5.0e Concentration, ng/ml EDC and PPCP calibration.rdb (2,4-D 2): "Li e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e6 1.0e6 5.0e5 2,4-D 1 2,4-D Concentration, ng/ml Gıda ve Çevre Uygulamaları 109

110 Gıda ve Çevre Uygulamaları 110 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 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. Analytes in River 2 Concentration (ng/l) upstream Concentration (ng/l) downstream Erythromycin xybenzone ND 6.25 Carbamazepine Bromacil ND ,4-D ND 9.35 Diazepam ND DEET Warfarin ND Sulfamethoxazole Triclosan (Irgasan) Caffeine Codeine Ciprofloxacin 3.81 ND Diuron Cotinine 2.05 ND Trimethoprim Lincomycin Carbamazepine DEET Ketorolac Meprobramate Atrazine Sulfamethoxazole Pentoxifylline ND not detected Caffeine Cotinine ND Increases by more than 2x Simazine Norethisterone ND ND Within ± 2x Erythromycin 135 ND Decreases by more than 2x Tylosone Tartrate 4.28 ND 2,4-D 3.24 ND 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) 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) Susan D. Richardson and Thomas Ternes: Water Analysis: Emerging Contaminants and Current Issues Anal. Chem. 77 (2005) 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) For Research Use nly. Not for use in diagnostic procedures 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:

111 Analysis of Selected Microcystins in Drinking and Surface Water Using a Highly Sensitive Direct Injection Technique Jens Dahlmann 1 and Bernd Luckas 2 1 AB SCIEX Darmstadt (Germany) and 2 University of Jena, Department of Food Chemistry, Jena (Germany) 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 Traditionally MC were analyzed by HPLC with UV detection2-3 but nowadays analytical methods are shifting more towards mass spectrometric detection. 4-5 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 (ND) is a cyclic pentapeptide produced by Nodularia spumigena with the structure cyclo [-D-MeAsp-L-Arg-Adda- D-Glu-Mdhb-], where Mdhb stands for 2-(methylamino)- 2-dehydrobutyric acid. The Microcystin MC-LR (Figure 2) is typically tested as a marker for cyanobacteria occurrence and is regulated by the WH in the guidelines for drinking-water quality at a value of 1 µg/l.7 verview 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 (LQ) of 0.1 µg/l was achieved which is 10 times below the guideline value proposed by the World Health rganization (WH). Experimental Standard Microcystin standards are available from Enzo Life Science International ( Adda CH 3 CH 3 CH 3 HN D-Glu H 3 C NH 2 NH HN NH Figure 2. Structure of MC-LR where X is leucine and Y is arginine Y H H N CH 3 CH 3 N CH 2 H N D-Me-Asp NH HN H H 3 C Mdha CH 3 D-Ala CH 3 X Gıda ve Çevre Uygulamaları 111

112 Gıda ve Çevre Uygulamaları 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 MS/MS 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 ptimization tool in Analyst software. The full scan MS/MS spectrum is shown in Figure 3. Two product ions at m/z amu (characteristic ADDA fragment for and MC) m/z amu (Glu-Mdha) were measured. Intensity,cps Time (min) Flow (µl/min) A (%) B (%) re-equilibration MS2 (995.67) CE (54):26MCA scansfromsample1(tunesampl ename) ofmc_lr_initproduct_pos.wi ff (Turbo Spray) x e6 1.2e6 1.0e6 8.0e Max. 1.4e6cps e e e m/z, amu Figure 3. MS/MS spectrum of MC-LR with highlighted product ions used for quantitation Transition Q1 (amu) Q3 (amu) CE (V) MRM MRM Figure 4. MRM transitions and Collision Energy (CE) to detect MC-LR MRM transitions are shown in Figure 4. 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. Results and Discussion 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 (LD) of 4 µg/l for MC-LR for example (Figure 4). Figure 4. MRM chromatogram of MC-LR spiked drinking water at a concentration of 0.1 μg/l (injection volume of 25 µl) 112

113 Linearity was proven for MC-LR, MC-RR, and MC-YR standard solutions ranging from 0.1 µg/l to 100 µg/l. 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, MC- YR, MC-LW, MC-WR, desmethyl-mc-rr, and Nodularin is shown in Figure 5. 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. Figure 5. Chromatograms of various microcystins and nodularin (ND), at a concentration of 1 µg/l, except desmethyl-mc-rr and MC-WR at 10 µg/l (injection volume of 25 µl) 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 LQ 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. References 1. W. W. Carmichael, W. W.: The toxins of cyanobacteria Sci. Amer. 270 (1994) 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) 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) 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) Determination of trace amount of microcystins in water samples using liquid chromatography coupled with triple quadrupole mass spectrometry Analytica Chimica Acta 569 (2006) detection of cyanobacterial toxins in precursor ion mode by liquid chromatography tandem mass spectrometry J. Mass Spectrom. 42 (2007) 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 pen-file Report 1341 (2008) USGS ( Gıda ve Çevre Uygulamaları 113

114 Gıda ve Çevre Uygulamaları Quantitative Analysis of Explosives in Surface Water Comparing ff-line Solid Phase Extraction and Direct Injection LC-MS/MS J.D. Berset1, N.Schiesser 1, Th. Schnyder1, A. Affolter 1, St. König 2, A. Schreiber 3 1 Water and Soil Protection Laboratory (GBL) Bern (Switzerland); 2 AB SCIEX Rotkreuz, (Switzerland); 3 ABSCIEX Concord, ntario (Canada) verview 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 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) ctogen (HMX) Pentaerythritol tetranitrate (PETN) Tetryl Sample Preparation 50 ml of water samples were extracted on Phenomenex StrataX SPE cartridges. These extracts were analyzed by LC- MS/MS and compared to direct injections of filtered water samples. H H maleic acid H fumaric acid H Figure 1. Chemical structures of maleic acid (left) and fumaric acid (right) 114

115 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. Liquid Chromatography HPLC column: Xbridge Phenyl (2.1x150 mm), 3.5 μm Eluent A: water mm ammonium acetate Eluent B: methanol mm ammonium acetate Gradient (A/B): 55/45 to 30/70 within 13 min and reequilibration Flow: 200 μl/min Injection volume: 100 μl ven 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 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 ng/l for off-line SPE and 0-1 ng/l for direct injection LC- MS/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 (LQ) with S/N=10 and Limit of Detection (LD) S/N=3 LQ: 1 ng/l for DANT, NG and TNT, 3 ng/l for HMX, RDX, PETN and ADNT Table 1. Retention times, MRM transitions of explosives with detected MRM ratio and tolerance intervals regarding the guideline 2002/657/EC5 Compound tr (min) MW Precursor Ion MRM Transition MRM Ratio Tolerance (%) Tolerance Interval ,4-DA-6-NT [M+H] + 168/ / ,6-DA-4-NT [M+H] + 168/ / HMX [M+CH 3 C]- 355/46 355/ RDX [M+CH3C]- 281/46 281/ NG [M+CH3C]- 286/62 286/ A-2,6-DNT [M-H]- 196/46 196/ A-4,6-DNT [M-H]- 196/46 196/ Tetryl [M-H]- 286/ / /1.00 TNT [M-H]- 226/46 226/ PETN [M+CH3C]- 375/62 375/ Gıda ve Çevre Uygulamaları 115

116 Gıda ve Çevre Uygulamaları Results and Discussion 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+CH3C]- 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 ptimization of the compound dependent parameters was obtained by automatic Quantitative ptimization 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 ng/l. Concentrations at different depths were very similar assuming a homogeneous distribution of the explosives in the water body. 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). 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) 116

117 Figure 4. Calibration curve of HMX with a working range of 0-1 ng/l (r = ) 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. ng/l measurementu ncertainty depth( m) HMXSPE RDXS PE PETNSPE HMXDI RDXDI PETNDI Figure 5. Comparison of concentrations between direct injection and off-line SPE for HMX, RDX and PETN with error bars for uncertainty of measurement Summary 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. 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 J. Sjostrom et al: Environmental Risk Assessment of Dumped Ammunition in Natural Waters of Sweden, User Report, September H. Stucki: Chimia 58, , X. Xu et al: J Forensic Sci, 49, 6, 1-10, J.D. Berset et al: Chimia 61, 532, Chr. Borton et al: Application Note AB SCIEX # For Research Use nly. Not for use in diagnostic procedures 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: Gıda ve Çevre Uygulamaları 117

118 Gıda ve Çevre Uygulamaları Screening and Identification of Unknown Contaminants in Untreated Tap Water Using a Hybrid Triple Quadrupole Linear Ion Trap LC-MS/MS System M.T. Baynham 1, St. Lock 1, D. Evans 2, and P. Cummings 2 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 ne 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 more than 1500 compoundsh avea similar molecularw eighto f ~250amu molecularw eight Figure 1. Abundance of compounds over molecular weight range of amu 118

119 Table 1. Comparison of sensitivities between the General Unknown Screening (GUS) and Multi Target Screening (MTS) approaches Multi Target Screening General Unknown Screening Compound Name Compound Class Polarity MRM Intensity at ~LD Intensity at Q3 Mass 1 µg/ml (µg/ml) 10 µg/ml ~LD (µg/ml) Brodifacoum Rat poison Negative 521.0/ E E Chlorophacinone Rat poison Negative 373.0/ E E Difenacoum Rat poison Negative 443.1/ E E Difethialone Rat poison Negative 537.0/ E E Flocoumafen Rat poison Negative 541.1/ E E Warfarin Rat poison Negative 307.0/ E E+05 4 Endothal Rat poison Negative 185.0/ E DNC Cresol Negative 197.0/ E E Azinphos-ethyl rgano-phosphorus Positive 346.0/ E E Demeton-S-methyl rgano-phosphorus Positive 231.0/ E E+05 2 Dichlorvos rgano-phosphorus Positive 221.0/ E E Disulfoton rgano-phosphorus Positive 275.1/ E E Propetamphos rgano-phosphorus Positive 282.1/ E E Tebupirimfos rgano-phosphorus Positive 319.0/ E E+05 2 Parathion-ethyl rgano-phosphorus Positive 292.1/ E E Parathion-methyl rgano-phosphorus Positive 281.1/ E E 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 (TF), 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. nce 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 Curtain gas Gas 1 Gas 2 Parameter CAD 10 Value 25 psi 50 psi 60 psi Temperature 650 C IonSpray source voltage V V Gıda ve Çevre Uygulamaları 119

120 Gıda ve Çevre Uygulamaları 1.8e8 1.7e8 1.6e8 1.5e8 1.4e8 1.3e8 1.2e8 1.1e8 1.0e8 9.0e7 8.0e7 7.0e7 6.0e7 5.0e7 4.0e7 3.0e7 2.0e7 1.0e7 1.16e7 1.10e7 1.00e7 9.00e6 8.00e6 7.00e6 6.00e6 5.00e6 Mineralw ater MRM2 30/ Time,m in MS/MSo f Terbuthylazine Figure ng/ml Terbuthylazine spiked into mineral and tap water analyzed in positive polarity MRM and EPI Figure ng/ml MCPP spiked into mineral and tap water analyzed in negative polarity MRM and EPI Results and Discussion e8 1.6e8 1.5e8 1.4e8 1.3e8 1.2e8 1.1e8 1.0e8 9.0e7 8.0e7 7.0e7 6.0e7 5.0e7 4.0e7 3.0e7 2.0e7 1.0e Time,m in e6 4.00e e6 3.00e e6 2.00e e e m/z, amu m/z, amu 3.8e7 3.6e7 3.4e7 3.2e7 3.0e7 2.8e7 2.6e7 2.4e7 2.2e7 2.0e7 1.8e7 1.6e7 1.4e7 1.2e7 1.0e7 8.0e6 6.0e6 4.0e6 2.0e6 1.10e7 1.00e7 9.00e6 8.00e6 7.00e6 6.00e6 5.00e6 4.00e6 3.00e6 2.00e6 1.00e e7 3.2e7 Mineralw ater 3.0e7 2.8e7 MRM2 13/ e7 2.4e7 2.2e7 2.0e7 1.8e7 1.6e7 1.4e7 1.2e7 1.0e7 8.0e6 6.0e6 4.0e6 2.0e e7 1.00e7 9.50e6 MS/MSo f 9.00e6 8.50e6 MCPP 8.00e6 7.50e6 7.00e6 6.50e6 6.00e6 5.50e6 5.00e6 4.50e6 4.00e6 3.50e6 3.00e6 2.50e6 2.00e e6 1.00e e m/z, amu 1.19e7 1.10e7 1.00e7 9.00e6 8.00e6 7.00e6 6.00e6 5.00e6 Tapw ater MRM2 30/ MS/MSo f Terbuthylazine Tapw ater MRM2 13/ Time,m in m/z, amu 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. 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. 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 MS/MSo f MCPP Summary 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. I n t e... I n t e... TIC: from Sample 1 (SAMPLE B) of Q3 5.wiff (Turbo Spray), Smoothed Max. 8.7e7cps e7 Water sample e e e Q3: Exp 1, min from Sample 1 (SAMPLE B) of Q3 5.wiff (TurboSpray) Max. 8.7e5cps. 8.0e5 6.0e5 4.0e5 2.0e m/z, amu +EPI (351) Charge (+0) CE (35) CES (20) FT (10): Exp 2, min from Sample 1 (SAMPLE B) of Q3 5.wiff (TurboSpray) Max. 2.7e6cps. 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 e6 2.0e6 1.5e6 1.0e e m/z, amu TIC: from Sample 1 (BLK) of Q3 2.wiff (TurboSpray), Smoothed Max. 8.7e7cps. 8.0e7 6.0e7 4.0e7 2.0e Control sample 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 (LD) were determined to be the triggering threshold of both approaches. In the GUS method the LD 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 LD 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 LD of both approaches was found. For Research Use nly. Not for use in diagnostic procedures 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: Survey Q3 spectrum at 12min EPI spectrum at 12min 120

121 Quantitation and Identification of rganotin Compounds in Food, Water, and Textiles Using LC-MS/MS Yun Yun Zou and André Schreiber AB SCIEX Concord, ntario (Canada) verview rganotin 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. Spiked apple, potato, synthetic seawater, and textile samples were prepared using a quick and easy acetonitrile extraction. rganotin 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. Introduction rganotin (organostannic) compounds are chemical compounds comprised of tin with hydrocarbon substituents. rganotin 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 (DT) 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. Gıda ve Çevre Uygulamaları 121

122 Gıda ve Çevre Uygulamaları 122 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. H 3 C Sn CH 3 H 3 C H 3 C CH 3 Sn Sn 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 33 Cl CH 3 CH 3 Sn C H 3 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. Results and Discussion Chromatography conditions were important for successful determination of organotin compounds by LC-MS/MS. rganotin 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 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). P Sn H Hom ogenize and weigh 10 g of apple and potato. Add internal standard (50 L of 10 g/m L TPP). Add 10 m L acetonitrile and shake vigorously for 1 minute. Centrifuge at5rpm for5 min. Transfer 100 L of the extract into autosampler vial and add 900 L water. Figure 2. Sample preparation protocols for the analysis of organotin compounds in fruit and vegetable, textiles, and water MS/MS Detection Shred and weigh 1 g of textile m aterial. Add internal standard (50 L of 10 g/ml TPP). Add 20 ml acetonitrile and sonicate for 5 m inutes. Centrifuge at 5 rpm for 5 min. Transfer 100 L of the extract into autosam pler vial and add 900 L water. Transfer 1 ml of water sample into autosampler vial. Add internal standard (10 L of 10 ng/ml TPP). (Dilute water sample to reduce possible matrix effects.) Inject directly. 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 rganotin compound Q1 (amu) Q3 (amu) RT (min) TBT TBT Fentin Fentin Cyhexatin Cyhexatin Fenbutatin oxide Fenbutatin oxide TPP (internal standard) Table 2. Signal-to-noise (S/N) in different matrices rganotin compound Apple (2 μg/kg) Potato (2 μg/kg) Textile (0.1 mg/kg) Seawater (50 ng/l) TBT Fentin Cyhexatin Fenbutatin oxide

123 XICo f+ MRM( 12 pairs): / amu Expected RT:3.8 ID:t ributylin chloride 2f roms ample4 (SW) of salty water test.wiff( Tu...M ax c ps Time,m in XICo f+ MRM( 12 pairs): /77.000a mu Expected RT:4.4 ID: triphenyl phosphate1 from Sample4( SW)o f saltyw ater test.wiff( Tu...M ax.1.6e5 cps. 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e Blanks ynthetic seawater Twoc hromatographic interferences fort BT ares eparated well from the target analyte Internals tandard( TPP) Time,m in Figure 3. Blank synthetic seawater, two chromatographic interferences for TBT are separated well from the target analyte (top) and internal standard (bottom) 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. 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 (LQ) for all analytes in each matrix. TBT CyhexatinF Figure 6. Calibration lines of organotin compounds in apple matrix (2 to 100 μg/kg) TBT CyhexatinF Fentin enbutatin oxide Fentin enbutatin oxide Figure 7. Calibration lines of organotin compounds in synthetic seawater (50 to 2000 ng/l) Intensity, cps Intensity, cps XIC of +MRM (12 pairs): / amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 4 (apple 2ngml 1/10) of apple & p... Max cps. XIC of +MRM (12 pairs): / amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 4 (apple 2ngml 1/10) of apple & p... Max cps TBT Cyhexatin Apple extract TBT Cyhexatin Apple extract Fenbutatin oxide Fenbutatin 6.13 oxide Fentin Fentin XIC of +MRM (12 pairs): / amu Expected3.5 RT: 3.8 ID: tributylin 4.0 chloride 2 from 4.5 Sample 10 (potato 5.0 2ngml 1/10) 5.5of apple 6.0 & Max cps. 8.0 XIC of +MRM (12 pairs): / amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 10 (potato 2ngml 1/10) of apple &... Max cps TBT Fenbutatin oxide 6.14 Potato extract TBT Fenbutatin oxide Cyhexatin Potato extract Cyhexatin Fentin Fentin Figure 4. Apple (top) and potato (bottom) sample spiked at 2 μg/kg and diluted 10x after extraction Intensity, cps Intensity, cps XIC of +MRM (12 pairs): / amu Expected RT: 3.8 ID: tributylin chloride 2 from Sample 3 (textile 0.1mg/kg 1/10) of textile... Max cps. XIC of +MRM (12 pairs): / amu Expected RT: 3.8 ID: 3.84 tributylin chloride 2 from Sample 3 (textile 0.1mg/kg 1/10) of textile... Max cps TBT Textile extract TBT Textile extract Fentin 5000 Fentin Cyhexatin 4000 Cyhexatin Fenbutatin oxide Fenbutatin oxide XIC of 2.0 +MRM (12 pairs): / amu Expected3.5 RT: 3.8 ID: tributylin 4.0 chloride 2 from 4.5 Sample 7 (SW 5.050ppt) of salty 5.5 water 6.0 test Max cps. 8.0 XIC of +MRM (12 pairs): / amu Expected RT: ID: tributylin chloride 2 from Sample 7 (SW 50ppt) of salty water test... Max cps Synthetic seawater Synthetic seawater 1600 TBT TBT Cyhexatin Cyhexatin Fenbutatin oxide 5.27 Fentin Fenbutatin oxide 5.27 Fentin 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) Table 3. Estimated limits of quantitation (LQ) in different matrices based on S/N of 10 rganotin compound Apple (μg/kg) Potato (μg/kg) Textile (μg/kg) Seawater (ng/l) TBT Fentin < 0.1 < 0.1 < 10 < 10 Cyhexatin < 10 Fenbutatin oxide < 0.1 < < 10 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). Gıda ve Çevre Uygulamaları 123

124 Gıda ve Çevre Uygulamaları Table 4. Repeatability (%CV) and accuracy of organotin compounds at the lowest point of the calibration line Apple (2 μg/kg) Potato (2 μg/kg) Textile (0.1 mg/kg) Seawater (50 ng/l) rganotin compound %CV Accuracy (%) %CV Accuracy (%) %CV Accuracy (%) %CV Accuracy (%) TBT Fentin Cyhexatin Fenbutatin oxide 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. Figure 8. Automatic compound identification using the Multicomponent query (example cyhexatin in potato) Figure 9. Automatic compound identification using the Multicomponent query (example fentin in textile) 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 1. K. Fent: Ecotoxicology of organotin compounds Crit. Rev. Toxicol. 26 (1996) E. Gonzalez-Toledo et al.: Detection techniques in speciation analysis of organotin compounds by liquid chromatography Trends Anal. Chem. 22 (2003) Regulation (EC) on the prohibition of organotin compounds on ships No 782/ 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: EK-TEX Standard 100, Edition 4 (2012) Council Directive maximum levels for pesticide residues 96/32/EC 8. EU Reference Laboratory for Single Residue Methods: Analysis of rganotin Compounds via QuEChERS and LC- MS/MS Brief Description For Research Use nly. Not for use in diagnostic procedures 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:

125 Nitrogen Generators Zero Air Generators Hydrogen Generators HPLC Preperative HPLC BioLC/Protein Purification smometer Chromatography Columns UV- VIS Spectrophotometer Gel Documentation System DNA /Protein Analyzer Atomic Absorbption Spectrophotometer ICP ES ICP TF MS XRD UV-VIS Spectrophotometer Compact Mass Spectrometers Stirrers Heaters Shakers Hot Plates Vaccum Pumps Vaccum Filtration Sets Homogenizers Incubators Vial/Cap/Septa Syringe Filters Membrane Filters Quartz Cuvettes Quechers Sample Prep Kits SPE Cartridges Laboratory Equipments Laboratory Intruments HPLC& LCMSMS Diagnostic Kits Thermo Cyclers Thermo Mixers Centrifuges Liquid Handling (Pippets etc) Mixers and Shakers Tube Rollers and Rotators nline SPE Systems HPLC Autosamplers Gıda ve Çevre Temsilcilikler Uygulamaları Triple Quad LCMSMS QTRAP QTF MALDI TF/TF HPLC MicroLC NanoLC 125

126 Gıda ve Çevre Uygulamaları Notlar 126

127 Notlar 127

128 Spektrotek A.Ş. t: f: