Extension of a LC‐UV/fluorescence/mass spectrometry method for vitamin B6 in dietary supplements to additional formulations

As part of the NIH/FDA/AOAC initiative, we are conducting a single laboratory validation (SLV) for the determination of vitamin B 6 in dietary supplements by LC‐UV/FL/MS. An AOAC Expert Review Panel reviewed over 50 methods using AOAC Acceptance Criteria for SLV. The panel selected pyridoxine (PN) as the analyte of interest, as it is the form of vitamin B 6 most commonly used in supplements. The inclusion of UV, Fl and MS detection in the SLV, as well as the reversed phase chromatographic method using water/acetonitrile with formic acid as the mobile phase from our reported method was recommended. The panel recommended a set of PN‐containing study materials, which includes tablets, capsules, soft gel, gummy, and liquid multivitamins, containing nominal PN contents ranging from 50 ppm to 20 %. Although sample preparation strategies for extracting PN from the various sample matrices were also recommended, prior to carrying out the SLV significant method development and optimization was required. These steps must be demonstrated to effectively break down the sample matrix, efficiently extract pyridoxine, and dilute it to a level appropriate for detection. While the chromatography/detection steps of the method are common, the front‐end extraction procedures must be optimized for the different formulations. This research received funding support from the Office of Dietary Supplements, NIH.