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Establishment of Biotin Analysis by LC-MS/MS Method in Infant Milk Formulas

LC-MS/MS를 이용한 조제유류 중 비오틴 함량 분석법 연구

  • Shin, Yong Woon (Nutrition and Functional Food Research Team, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Lee, Hwa Jung (Nutrition and Functional Food Research Team, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Ham, Hyeon Suk (Nutrition and Functional Food Research Team, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Shin, Sung Cheol (Imported Food Analysis Division, Center for Food and Drug analysis, Busan Regional Ministry of Food and Drug Safety) ;
  • Kang, Yoon Jung (Nutrition and Functional Food Research Team, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Hwang, Kyung Mi (Nutrition and Functional Food Research Team, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Kwon, Yong Kwan (Nutrition and Functional Food Research Team, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Seo, Il Won (Nutrition and Functional Food Research Team, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Oh, Jae Myoung (Nutrition and Functional Food Research Team, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Koo, Yong Eui (Nutrition and Functional Food Research Team, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety)
  • 신용운 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 영양기능연구팀) ;
  • 이화정 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 영양기능연구팀) ;
  • 함현숙 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 영양기능연구팀) ;
  • 신성철 (부산지방식품의약품안전청 수입식품분석과) ;
  • 강윤정 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 영양기능연구팀) ;
  • 황경미 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 영양기능연구팀) ;
  • 권용관 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 영양기능연구팀) ;
  • 서일원 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 영양기능연구팀) ;
  • 오재명 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 영양기능연구팀) ;
  • 구용의 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 영양기능연구팀)
  • Received : 2016.07.25
  • Accepted : 2016.09.20
  • Published : 2016.10.28

Abstract

This study was conducted to establish the standard method for the contents of biotin in milk formulas. To optimize the method, we compared several conditions for liquid extraction, purification and instrumental measurement using spiked samples and certified reference material (NIST SRM 1849a) as test materials. LC-MS/MS method for biotin was established using $C_{18}$ column and binary gradient 0.1% formic acid/acetonitrile, 0.1% formic acid/water mobile phase is applied for biotin. Product-ion traces at m/z 245.1 ${\rightarrow}$ 227.1, 166.1 are used for quantitative analysis of biotin. The linearity was over $R^2=0.999$ in range of $5{\sim}60{\mu}g/L$. For purification, chloroform was used as a solvent for eliminating lipids in milk formula. The linearity was over 0.999 in range of 5~60 ng/mL. The detection limit and quantification limit were 0.10, 0.31 ng/mL. The accuracy and precision of LC-MS/MS method using CRM were 103%, 2.5% respectively. Optimized methods were applied in sample analysis to verify the reliability. All the tested milk formulas were acceptable contents of biotin compared with component specification and standards for nutrition labeling. The standard operating procedures were prepared for biotin to provide experimental information and to strengthen the management of nutrient in milk formula.

본 연구는 "축산물의 가공기준 및 성분규격"에 기준규격은 설정되어 있지 않으나, 국제 기준과의 조화를 위해 규격 신설 검토 대상 성분인 비오틴의 분석법을 마련하고자 수행하였다. 조제유류에 미량으로 함유된 비오틴함량 분석을 위해 선택성과 정밀성이 뛰어난 LC-MS/MS를 이용한 분석법을 확립하고 시중에 유통중인 제품을 대상으로 적용성을 검토하였다. 비오틴 표준품을 이용하여 LC-MS/MS를 이용한 기기분석조건을 확립하고, 식품의 기준 및 규격 분석법을 참고로 0.01 M 인산이수소칼륨(pH 4.8) 용액을 이용하여 시료중의 비오틴을 추출하여 분석하였다. 분석법 검증은 특이성, 직선성, 검출한계 및 정량한계, 정확성, 정밀성에 대해 수행되었다. 5~60 ng/mL의 농도범위에서 $R^2=0.999$ 이상의 우수한 직선성을 확인할 수 있었으며, LOD와 LOQ는 각각 0.10, 0.31 ng/mL 이었다. CRM (NIST SRM 1849a) 및 표준물질 첨가법을 이용하여 정확성을 검토하였으며, CRM에서 103%, 조제분유에서 101~104%, 조제우유에서 99~101%의 회수율을 확인할 수 있었다. 정밀성을 검토한 결과 시료 채취량에 따른 상대표준편차가 조제분유 0.4~0.9%, 조제우유1.4~1.6%로 확인하였으며, 실험일자에 따른 재현성은 조제분유에서 1.3%, 조제우유에서 1.2%로 확인하였다. 본 연구에서 확립된 분석법을 적용하여 조제분유 39건, 조제우유 3건, 성장기용조제분유 23건 등 국내 유통중인 조제유류 65건에 대해 적용성 검토를 실시한 결과 전체시료에서 분석이 용이하였으며, 모두 표시기준에 적합함을 확인하였다.

Keywords

References

  1. Wood, H.G., and Barden, R.E.: Biotin enzymes. Annual review of biochemistry, 46(1), 385-413 (1977). https://doi.org/10.1146/annurev.bi.46.070177.002125
  2. Zempleni, J., and Mock, D.: Biotin biochemistry and human requirements. J. nutr. biochem, 10(3), 128-138 (1999). https://doi.org/10.1016/S0955-2863(98)00095-3
  3. Pacheco-Alvarez, D., Solorzano-Vargas, R.S. and Del Rio, A.L.: Biotin in metabolism and its relationship to human disease. Archives of medical research, 33(5), 439-447 (2002). https://doi.org/10.1016/S0188-4409(02)00399-5
  4. The Korean Nutrition Society: 2015 Dietary reference intakes for koreans, The Korean Nutrition Society, Seoul, Korea (2015).
  5. Standard for livestock products. Ministry of Food and Drug Safety, Cheongju, Korea (2015).
  6. Korea Food Code. Ministry of Food and Drug Safety, Cheongju, Korea (2015).
  7. Foodstuffs-Determination of d-biotin by HPLC, European standard, BS EN 15607:2009 (2009).
  8. Kim D.S., Lee Y,J, Jeong D.Y., Lee D.Y., Ahn M.K.: Determination of biotin by HPLC, J. Analytical Science & Technology, 16, 499-503 (2003).
  9. Study on instrumental analysis of biotin and choline in foods, Korea Food and Drug Administration, Korea (2003).
  10. Baiyi L., Yiping R., Baifen H., Wenqun L., Zengxuan C., and Xiaowei T.: Simultaneous determination of four water-soluble vitamins in fortified infant foods by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry, J. Chrmatographic science, 46, 225-232 (2008). https://doi.org/10.1093/chromsci/46.3.225
  11. Yanling G., Fei G., Sumangala G., Andrew C., Qinghai S., and Mingruo G.: Quantification of water-soluble vitamins in milk-based infant formulae using biosensor-based assays. Food Chemistry, 110, 769-776 (2008). https://doi.org/10.1016/j.foodchem.2008.03.007
  12. Evangelia L., Danae C., Irene V., Leondios L., John O.N., Dionyssis S.I. and Gregory P.E.: Analytical techniques for determining biotin. Journal of chromatography A, 881, 331-343 (2000). https://doi.org/10.1016/S0021-9673(00)00118-7
  13. Chikako Y., Yukiko O.: Determination of biotin following derivatization with 2-nitrophenylhydrazine by high-performance liquid chromatography with on-line UV detection and electrospray-ionization mass spectrometry. Journal of Chromatography A, 1142, 231-235 (2007). https://doi.org/10.1016/j.chroma.2006.12.058
  14. Bryant C.N., Katherine E.S., and Lane C.S.: Improved liquid chromatography methods for the separation and quantification of biotin in NIST standard reference material 3280: Multivitamin/Multielement Tablets. J. Argic. Food Chem., 54, 8710-8716 (2006). https://doi.org/10.1021/jf062000+
  15. Staggs, C.G., Sealey, W.M., McCabe, B.J., Teagure, A.M., Mock, D.M.: Determination of the biotin content of select foods using accurate and sensitive HPLC/avidin binding, Journal of Food Composition and Analysis, 17, 767-776 (2004). https://doi.org/10.1016/j.jfca.2003.09.015