ABSTRACT
A reversed-phase HPLC method with postcolumn protein conjugation and fluorescence detection for the quantitative determination of biotin in infant, pediatric, and adult nutritionals was developed and evaluated in a single-laboratory validation (SLV). Sample of appropriate size is mixed with 2% metaphosphoric acid to precipitate out the protein. The filtrate is injected onto a C18 HPLC column in which biotin and riboflavin are separated with an appropriate mobile phase. The biotin, after eluting from the column, binds with the streptavidin fluorescein to become a fluorescent conjugate. The conjugate is then detected by fluorescence at λex = 495 nm and λem = 518 nm. A column switch is used in the method as an option to shorten the run time from 30 to 15 min, by eluting out riboflavin at a higher flow rate. In this SLV, a total of 19 AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals matrixes representing a range of infant, pediatric, and adult formulas were evaluated for their biotin content. The analytical range was 1.66-142 µg/100 g reconstituted final product. The repeatability and intermediate precision ranged from 0.5 to 3.0% RSDr and from 1.3 to 4.5% RSDiR, respectively. Recovery from spiked matrixes varied from 95 to 111%, and accuracy of quantification using Standard Reference Material 1849a ranged from 99 to 105%. The LOQ in reconstituted product was estimated to be 0.8 µg/100 g. The method was approved by the Expert Review Panel as First Action at the 2016 AOAC INTERNATIONAL Mid-Year Meeting.
