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

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

References

1. AOAC (2000). Official Method 999.07. Aflatoxin B1 and Total Aflatoxins in Peanut Butter, Pistachio Paste, Fig Paste, and Paprika Powder. Immunoaffinity Column Liquid Chromatography with Post-Column Derivatization First Action 1999. P.49.2.29.
2. AOAC (2005). Official Method 991.31. Aflatoxin in corn, raw peanuts and peanut butter: immunoaffinity column (aflatest) method. AOAC International, p. 49.2.18.
3. Auerbach H, Maas RFM, Op Den Camp HJM, Pol A, Fink-Gremmels J (1998). Biodegradation of aflatoxin B1 by bovine rumen microorganisms in vitro and its effects on rumen fermentation. Revue de Médecine Vétérinaire. 149:573.
4. Bersjo B, Matre T, Nafstad I (1992). Effect of diets with graded levels of deoxynivalenol on performance in growing pigs. Journal of Veterinary Medicine Series A. 39:752-758.
5. Bersjo B, Langseth W, Nafstad I, Hogset Jansen J, Larsen HJS (1993). The effects of naturally deoxynivalenol-contaminated oats on the clinical condition, blood parameters, performance and carcass composition of growing pigs. Veterinary Research Communications. 17:283-294.
6. Doyle MP, Beuchat LR, Montville TJ (1997). Food Microbiology: Fundementals and Frontiers. Washington, DC, ASM Press 768p.
7. Edrington TS, Harvey RB, Kubena L (1994). Effects of aflatoxin in growing lambs fed ruminally degradable or escape protein sources. Journal of Animal Science 72:1274-1281.
8. Engel VG, Hagemeister H (1978). Untersuchungen über den verbleib von aflatoxin B1 im verdauungstrakt von kühen. Milchwissenenschaft. 33:21-23.

9. Gremmels JF (2008). The role of mycotoxins in the health and performance of dairy cows. The Veterinary Journal. 176: 84-92.
10. Jouany JP (2001). The impact of mycotoxins on performance and health of dairy cattle. Science and Technology in the Feed Industry Proceedings of Alltech’s 17th Annual Symposium. 191-223
11. Kiessling KH, Pettersson H, Sandholm K, Olsen M (1984). Metabolism of aflatoxin, ochratoxin, zearalenone and three tricothecenes by intact rumen fluid, rumen protozoa and rumen bacteria. Applied and Environmental Microbiology 47:1070-1073.
12. Nibbelink, SK (1986). Aflatoxicosis in food animals: A clinical review. Iowa State Univ. Vet. 48:28-31.
13. Rotter BA, Thompson BK, Lessard M (1995). Effects of deoxynivalenol- contaminated diet on performance and blood parameters in growing swine. Canadian Journal of Animal Science. 75:297-302.
14. Sanco/10684 (2009). Method validation and quality control procedures for pesticide residues analysis in food and feed.
15. Stark AA (2001). Mechanisms of action of aflatoxin B1 at the biochemical and molecular levels. (Edited by Charles L. Wilson and Samir Droby.), Microbial Food Contamination. CRC Press. ISBN 0-8493-2229-4, p:81-94.
16. Şenyuva HZ, Gilbert J (2006). Metot geliştirme ve validasyonu için basit kullanım klavuzu. Hacettepe Üniversitesi Hastaneleri Basımevi, Ankara. ISBN 975-00884-0-9.
17. Van Genderen H (1997). Adverse effects of naturally occurring nonnutritive substances (Edited by John De Vries). Food Safety and Toxicity, CRC Press, ISBN 0-8493-9488-0, p:153-168.
18. Yiannikouris A, Jouany JP (2002). Mycotoxins in feed and their fate in animals: a review. Animal Research 51:81-99