Immunoaffinity-HPLC/DAD법을 이용한 과자류와 시리얼류의 비타민 B12 분석 및 검증
Immunoaffinity-HPLC/DAD Assay and Validation for Vitamin B12 in and Cereals
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초록
식품에 존재하는 주요 비타민 B12는 hydroxycobalamin, adenosylcobalamin, methylcobalamin이며cyanocobalamin은 시리얼과 같은 강화식품에 사용되는 합성형이다. 비타민 B12는 식품 중에 낮은 농도로 존재하기 때문에 추출 및 농축과정이 매우 중요하다. 본 연구에서는 과자와 시리얼 같은 탄수화물식품 중의 비타민 B12 추출을 위해 α-amylase처리와 산성조건과 cyanide 존재시 열처리, immunoaffinitycolumn(IAC)로 농축과 세척, HPLC/DAD로 분리 검출하였다. 분석법 수행특성지표들을 측정함으로 분석법을 검증하였다. 정확성과 정밀성은 AOAC 가이드라인을 충족시켰고, LOD 0.01 μg/100g과 LOQ0.03 μg/100 g을 얻었다. 국제정도관리 시험에서 z-score 1.4로 분석수행이 우수함을 확인하였고 QCchart를 이용한 분석품질관리로 모든 분석이 관리하에 있음을 확인하였다. 검증된 분석법으로 과자류12종과 시리얼 11종의 비타민 B12 함량을 분석하여 각각 0.02-0.26과 0.03-2.67 μg/100 g 범위를 얻었고 이는 신뢰도높은 데이터로 식품성분표에 활용 가능할 것이며 IAC-HPLC/DAD법이 탄수화물 식품의비타민 B12 데이터베이스 구축에 활용될 수 있을 것으로 보인다.
Abstract
Naturally occurring vitamin B12 include hydroxycobalamin, adenosylcobalamin andmethylcobalamin. Cyanocobalamin is a synthetic form observed in fortified food. Becausevitamin B12 exists at very low concentration in food, concentration and clean-up are thecrucial steps for accurate quantification. In this study, vitamin B12 assay using enzyme andheating extraction under acidic condition with cyanide and immunoaffinity-HPLC/DAD wasvalidated and applied for vitamin B12 determination(μg cyanocobalamin/100 g) in confectioneryand cereals. Accuracy and precision were good enough to meet the AOAC guidelines. Limitof detection and quantification were 0.01 and 0.03 μg/100 g, respectively. The z-score of thevitamin B12 assay in a FAPAS Proficiency test was less than 2, assuring reliability ofanalytical performance. The validated method was applied for determining vitamin B12 fromconfectionery and cereals: 0.02-0.26 and 0.03-2.67 μg/100 g, respectively. All analysis was under the control based on quality control chart of vitamin B12. This study shows that the IAC-HPLC/DAD is acceptable for the construction of reliable database on vitamin B12 forcarbohydrate products.
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