The Combination of Immuno-Affinity Column IAC and High Performance Liquid Chromatography HPLC for Detection of Aflatoxins in Rumen Fluid

Year 2015, Volume: 4 Issue: 2, 439 - 443, 01.06.2015

Murat Reis Akkaya Ali İhsan Atalay Mehmet Ali Bal

Abstract

Background/Aim: This study was performed for internal validation of immuno-affinity column and HPLC methods for determining aflatoxins B1, B2, G1, G2 in ruminal fluid. Material and Method: Aflatoxin free rumen fluid was sampled from a lactating cow, filtered and kept at -35°C for 24 h. Then it was brought to room temperature for further analysis. The certified aflatoxin mix standard was added to rumen fluid in varying concentrations. The performed following steps were; methanol-water 70:0/v:v extraction, separating aflatoxins from the extract by immuno-affinity column, taking methanol and aflatoxins from immuno-affinity column and determining the amount of aflatoxins by HPLC having fluorescence detector. The data was used for limit of detection LOD , limit of quantification LOQ , linearity, recovery and repeatability of the method’s performance criteria. Results and Conclusion: The detection and measurement limits and recovery rate for aflatoxin B1 were 0.01 ppb, 0.02 ppb and 91%, respectively. The calculated variation coefficients in repeatability study were 3.15 and 4.56% for 1 and 2 ppb concentrations. It can be done accurate and reliable analysis in ruminal fluid that specified performance criteria for the determination of aflatoxins.

Keywords

Aflatoxin, HPLC, Immuno-affinity column, Rumen fluid.

Rumen Sıvısında İmmuno-Affinite Kolon IAK ve Yüksek Performanslı Sıvı Kromatografisi HPLC Kombinasyonu ile Aflatoksinlerin Tayini

Abstract

Özbilgi/Amaç: Bu çalışmada rumen sıvısında aflatoksin B1, B2, G1, G2 tayini için immuno-affinite kolon ve yüksek performanslı sıvı kromatografisi HPLC kullanılan bir yöntem geliştirilerek laboratuar içi validasyonu yapılmıştır. Materyal ve Metot: Bu amaç için laktasyondaki bir inekten alınan aflatoksin içermeyen rumen sıvısı süzülüp -35ºC’de 24 saat bekletildikten sonra oda sıcaklığına getirilerek kullanılmıştır. Rumen sıvısına değişik konsantrasyonlarda sertifikalı aflatoksin karışım standardı ilave edilerek yapılan çalışmada, metanol-su 70:30/v:v ekstraksiyonu, immuno-affinite kolon ile aflatoksinlerin ekstrakttan ayrılması, metanol ile aflatoksinlerin immuno-affinite kolondan alınması ve floresan detektörlü HPLC ile aflatoksin miktarlarının tespit edilmesi aşamaları gerçekleştirilmiştir. Örneklerin analizi ile elde edilen veriler tespit limiti LOD , ölçüm limiti LOQ , doğrusallık, geri kazanım ve tekrarlanabilirlik gibi metot performans kriterlerinin belirlenmesi için kullanılmıştır. Bulgular ve Sonuç: Araştırmada, aflatoksin B1 için belirlenen tespit limiti, ölçüm limiti ve geri kazanım oranı sırasıyla 0.01 ppb, 0.02 ppb ve % 91 olarak bulunmuştur. Tekrarlanabilirlik çalışmasında ise 1 ve 2 ppb konsantrasyonlar için varyasyon katsayıları % 3.15 ve % 4.56 olarak hesaplanmıştır. Rumen sıvısında aflatoksinlerin tayini için belirlenen bu performans kriterleri ile doğru ve güvenilir bir şekilde analiz yapılabilir.

Keywords

Aflatoksin, HPLC, İmmuno-affinite kolon, Rumen sıvısı

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