Journal of The Korean Society of Grassland and Forage Science (한국초지조사료학회지)

The Korean Society of Grassland and Forage Science (한국초지조사료학회)
권호 표지
DOI QR코드

DOI QR Code

Optimization of Analytical Methods for Ochratoxin A and Zearalenone by UHPLC in Rice Straw Silage and Winter Forage Crops

UHPLC를 이용한 볏짚 사일리지와 동계사료작물의 오크라톡신과 제랄레논 분석법 최적화

  • Ham, Hyeonheui (Microbial Safety Division, National Institute of Agricultural Sciences) ;
  • Mun, Hye Yeon (Microbial Safety Division, National Institute of Agricultural Sciences) ;
  • Lee, Kyung Ah (Microbial Safety Division, National Institute of Agricultural Sciences) ;
  • Lee, Soohyung (Microbial Safety Division, National Institute of Agricultural Sciences) ;
  • Hong, Sung Kee (Microbial Safety Division, National Institute of Agricultural Sciences) ;
  • Lee, Theresa (Microbial Safety Division, National Institute of Agricultural Sciences) ;
  • Ryu, Jae-Gee (Microbial Safety Division, National Institute of Agricultural Sciences)
  • 함현희 (국립농업과학원 유해생물팀) ;
  • 문혜연 (국립농업과학원 유해생물팀) ;
  • 이경아 (국립농업과학원 유해생물팀) ;
  • 이수형 (국립농업과학원 유해생물팀) ;
  • 홍성기 (국립농업과학원 유해생물팀) ;
  • 이데레사 (국립농업과학원 유해생물팀) ;
  • 류재기 (국립농업과학원 유해생물팀)
  • Received : 2016.07.20
  • Accepted : 2016.08.29
  • Published : 2016.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

The objective of this study was to optimize analytical methods for ochratoxin A (OTA) and zearalenone (ZEA) in rice straw silage and winter forage crops using ultra-high performance liquid chromatography (UHPLC). Samples free of mycotoxins were spiked with $50{\mu}g/kg$, $250{\mu}g/kg$, or $500{\mu}g/kg$ of OTA and $300{\mu}g/kg$, $1500{\mu}g/kg$, or $3000{\mu}g/kg$ of ZEA. OTA and ZEA were extracted by acetonitrile and cleaned-up using an immunoaffinity column. They were then subjected to analysis with UHPLC equipped with a fluorescence detector. The correlation coefficients of calibration curves showed high linearity ($R^2{\geq_-}0.9999$ for OTA and $R^2{\geq_-}0.9995$ for ZEA). The limit of detection and quantification were $0.1{\mu}g/kg$ and $0.3{\mu}g/kg$, respectively, for OTA and $5{\mu}g/kg$ and $16.7{\mu}g/kg$, respectively, for ZEA. The recovery and relative standard deviation (RSD) of OTA were as follows: rice straw = 84.23~95.33%, 2.59~4.77%; Italian ryegrass = 79.02~95%, 0.86~5.83%; barley = 74.93~97%, 0.85~9.19%; rye = 77.99~96.67%, 0.33~6.26%. The recovery and RSD of ZEA were: rice straw = 109.6~114.22%, 0.67~7.15%; Italian ryegrass = 98.01~109.44%, 1.65~4.81%; barley = 98~113.53%, 0.25~5.85%; rye = 90.44~108.56%, 2.5~4.66%. They both satisfied the standards of European Commission criteria (EC 401-2006) for quantitative analysis. These results showed that the optimized methods could be used for mycotoxin analysis of forages.

조사료에서 오크라톡신 A (OTA) 및 제랄레논(ZEA)의 단성분 분석법을 최적화하기 위하여 곰팡이 독소에 오염되지 않은 볏짚 사일리지, 이탈리안 라이그라스, 청보리, 호밀을 대상으로 각 시료 별로 OTA는 50, 250, $5,000{\mu}g/kg$, ZEA는 300, 1,500, $3,000{\mu}g/kg$의 농도로 스파이킹하고 면역친화성 컬럼을 이용하여 독소를 정제한 후 UHPLC-FLD를 이용하여 분석하였다. 분석 결과 표준용액의 검량선은 OTA가 결정계수 0.9999, ZEA가 0.9995로 높은 직선성을 나타냈으며, 검출 및 정량한계는 OTA가 각각 $0.1{\mu}g/kg$, $0.3{\mu}g/kg$, ZEA는 각각 $5{\mu}g/kg$, $16.7{\mu}g/kg$을 나타냈다. 유효성 및 정밀성 검정결과 OTA는 볏짚이 회수율 84.23~95.33%, 상대표준편차 (RSD) 2.59~4.77%, 이탈리안 라이그라스는 회수율 79.02~95%, RSD 0.86~5.83%, 청보리는 회수율 74.93~97%, RSD 0.85~9.19%, 호밀은 회수율 77.99~96.67%, RSD 0.33~6.26%를 나타냈으며, ZEA는 볏짚이 회수율 109.6~114.22%, RSD 0.67~7.15%, 이탈리안 라이그라스는 회수율 98.01~109.44%, RSD 1.65~4.81%, 청보리는 회수율 98~113.53%, RSD 0.25~5.85%, 호밀은 회수율 90.44~108.56%, RSD 2.5~4.66%를 나타내어 유럽연합에서 제시한 기준을 만족하였다. 따라서 본 연구에서 제시한 분석법은 국내의 볏짚 등 조사료 4종에서 OTA와 ZEA의 분석에 활용할 수 있음을 시사하였다.

Keywords

References

  1. Bhat, R., Rai, R.V. and Karim, A.A. 2010. Mycotoxins in food and feed: Present status and future concerns. Food Science and Food Safety. 9:57-81. https://doi.org/10.1111/j.1541-4337.2009.00094.x
  2. Binder, E.M., Tan, L.M., Chin, L.J., Handl, J. and Richard, J. 2007. Worldwide occurrence of mycotoxins in commodities, feeds and feed ingredients. Animal Feed Science and Technology. 137: 265-282. https://doi.org/10.1016/j.anifeedsci.2007.06.005
  3. CODEX Alimentarius. 1995. General standard for contaminants and toxins in food and feed (CODEX STAN 193-1995).
  4. Commission regulation (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. 2006. Official Journal of the European Union. L70:12-34.
  5. Commission recommendation of 17 August 2006 on the presence of deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 and fumonisins in products intended for animal feeding (2006/576/EC). 2006. Official Journal of the European Union. L229:7-9.
  6. Dalcero, A., Magnoli, C., Hallak, C., Chiacchiera, S.M., Palacio, G. and Rosa, C.D.R. 2002. Detection of ochratoxin A in animal feeds and capacity to produce this mycotoxin by Aspergillus section Nigri in Argentina. Food Additives and Contaminants. 19:1065-1072. https://doi.org/10.1080/02652030210151895
  7. Danicke, S., Brussow, K., Valenta H., Ueberschar, K., Tiemann, U. and Schollenberger, M. 2005. On the effects of graded levels of Fusarium toxin contaminated wheat in diets for gilts on feed intake, growth performance and metabolism of deoxynivalenol and zearalenone. Molecular Nutrition and Food Research. 49: 932-943. https://doi.org/10.1002/mnfr.200500050
  8. Driehuis, F., Spanjer, M.C., Scholten, J.M. and Te Giffel, M.C. 2008. Occurrence of mycotoxins in maize, grass and wheat silage for dairy cattle in the Netherlands. Food Additives and Contaminants. 1:41-50. https://doi.org/10.1080/19393210802236927
  9. Dzuman, Z., Zachariasova, M., Lacina, O., Veprikova, Z., Slavikova, P. and Hajslova, J. 2014. A rugged high-throughput analytical approach for the determination and quantification of multiple mycotoxins in complex feed matrices. Talanta. 121:263-272. https://doi.org/10.1016/j.talanta.2013.12.064
  10. Fazekas, B. and Tar, A. 2001. Determination of zearalenone content in cereals and feedstuffs by immunoaffinity column coupled with liquid chromatography. Journal of AOAC International. 84:1453-1459.
  11. Fraga, M.E., Curvello, F., Gatti, M.J., Cavaglieri, L.R., Dalcero, A.M. and da Rocha Rosa, C.A. 2007. Potential aflatoxin and ochratoxin A production by Aspergillus species in poultry feed processing. Veterinary Research Communications. 31:343-353.
  12. Kao, C. and Robinson, R.J. 1972. Aspergillus flavus deterioration of grain: Its effect on amino acids and vitamins in whole wheat. Journal of Food Science. 37:261-263. https://doi.org/10.1111/j.1365-2621.1972.tb05831.x
  13. Kim, D.H., Choi, K.I., Hong, K.S., Kim, H.J., Jang, H.S., Cho, H.J. and Han, G.S. 2011. Analysis and survey for contamination of deoxynivalenol and zearalenone in feed by high performance liquid chromatography. Journal of Food Hygiene and Safety. 26:214-221.
  14. Monbaliu, S., Van Poucke, C., Detavernier, C.L., Dumoulin, F., Van De Velde, M., Schoeters, E. and De Saeger, S. 2009. Occurrence of mycotoxins in feed as analyzed by a multi-mycotoxin LC-MS/MS method. Journal of Agricultural and Food Chemistry. 58:66-71.
  15. Muscarella, M., Iammarino, M., Nardiello, D., Lo Magro, S., Palermo, C., Centonze, D. and Palermo, D. 2009. Validation of a confirmatory analytical method for the determination of aflatoxins B1, B2, G1 and G2 in foods and feed materials by HPLC with on-line photochemical derivatization and fluorescence detection. Food Additives and Contaminants. 26:1402-1410. https://doi.org/10.1080/02652030903107906
  16. Muscarella, M., Iammarino, M., Nardiello, D., Lo Magro, S., Palermo, C. and Centonze, D. 2011. Simultaneous determination of aflatoxins B1, B2, G1, and G2 in foods and feed materials. Methods in Molecular Biology. 739:203-210.
  17. Pfohl-Leszkowicz, A. and Manderville, R.A. 2007. Ochratoxin A: An overview on toxicity and carcinogenicity in animals and humans. Molecular Nutrition & Food Research. 51:61-99. https://doi.org/10.1002/mnfr.200600137
  18. Roige, M.B., Aranguren, S.M., Riccio, M.B., Pereyra, S., Soraci, A.L. and Tapia, M.O. 2009. Mycobiota and mycotoxins in fermented feed, wheat grains and corn grains in Southeastern Buenos Aires Province, Argentina. Revista Iberoamericana de Micologia. 26: 233-237. https://doi.org/10.1016/j.riam.2009.03.003
  19. Schenzel, J., Forrer, H.R., Vogelgsang, S. and Bucheli, T.D. 2012. Development, validation and application of a multi-mycotoxin method for the analysis of whole wheat plants. Mycotoxin research. 28:135-147. https://doi.org/10.1007/s12550-012-0125-z
  20. Song, T.H., Park, T.I., Han, O.K., Park, H.H., Cho, S.K., Oh, Y.J., Kang, H.J., Jang, Y.W. and Park, K.G. 2013. Effect of harvesting time and making method on feed value and fermentative quality in silage of whole crop barley. Korean Journal of Crop Science, 58:362-366. https://doi.org/10.7740/kjcs.2013.58.4.362
  21. Stroka, J., von Holst, C., Anklam, E. and Reutter, M. 2003. Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxin B1 in cattle feed: collaborative study. Journal of AOAC International. 86:1179-1186.
  22. Sung, H.G., Lee, J.K. and Seo, S. 2011. Studies on fungal contamination and mycotoxins of rice straw round bale silage. Journal of Korean Society of Grassland and Forage Science. 31:451-462. https://doi.org/10.5333/KGFS.2011.31.4.451
  23. Zollner, P., Jodlbauer, J., Kleinova, M., Kahlbacher, H., Kuhn, T., Hochsteiner, W. and Lindner, W. 2002. Concentration levels of zearalenone and its metabolites in urine, muscle tissue, and liver samples of pigs fed with mycotoxin-contaminated oats. Journal of Agricultural and Food Chemistry. 50:2494-2501. https://doi.org/10.1021/jf0113631