Determination of vitamin B12 in commercial pepsin preparations using immunoaffinity chromatography and LC‐MS/MS

Abstract

AbstractVitamin B12 is a critical micronutrient in a vegan diet and thus dietary sources are limited and often low in the vitamin. Analytical determination of vitamin B12 requires laborious sample preparations including the proteolytic breakdown of the (food) matrix with proteases and the enrichment of the vitamin via immunoaffinity methods. Proteases like pepsin have an animal origin and thus might contain significant amounts of vitamin B12. We hypothesize that pepsin preparations potentially transfer residual vitamin B12 into the sample and falsify results. Hence the aim of our study was to determine trace amounts of cyano‐cobalamin by LC‐MS/MS in commercial protease enzyme preparations. The use of a stable 15N‐labeled cyano‐cobalamin as internal standard enabled us to detect vitamin B12 with a LOQ as low as 0.7 µg/l. Significant amounts of the vitamin were found in pepsin whereas the vitamin B12 content of papain preparations was below the detection limit. We conclude that sample preparation with pepsin bears the risk of false positive results, especially for samples with low vitamin B12 content, and we therefore recommend papain as an alternative protease for food sample preparation.