Quantification of biotin in feed, food, tablets, and premixes using HPLC–MS/MS

Two sensitive and specific methods for quantification of biotin in feed, food, tablets, and premixes based on HPLC–MS/MS have been developed and validated. Depending on sample matrix and biotin content different extraction procedures and HPLC conditions were applied. Key steps in sample preparation were an alkaline extraction or a hydrolysis with sulphuric acid followed by enzymatic digest with papain. For many samples with low biotin content the latter combination of extraction steps was shown to be necessary for an optimal release of biotin from the matrix. The first time synthesis of deuterated biotin for use as internal standard allowed the compensation of losses during sample work-up and ion suppression during HPLC–MS/MS analysis. The new methods are faster than the commonly used microbiological assay using Lactobacillus plantarum. Additionally, they have a higher specificity as results for biotin are based on determination of a chemically defined compound, and not of a biological activity. Quantification is applicable to samples with a biotin content >100 μg/kg. Results obtained with the new methods have been compared with those of the microbiological assay, and were in good agreement.