DOE-Assisted Method Development of LC-MS/MS for Four Vitamin B₁₂ Forms in Commercial Supplements

Commercial nutritional supplements are fortified with various forms of vitamin B₁₂ (VB₁₂), making accurate determination of both content and form essential. An LC-MS/MS method was developed for the simultaneous quantification of adenosylcobalamin (AdCbl), cyanocobalamin (CNCbl), hydroxocobalamin (OHCbl), and methylcobalamin (MeCbl), the most common VB₁₂ forms used in supplements. A three-factor three-level full-factorial design of experiments (DOE) was applied to optimize extraction solvent (water, 20% aqueous DMSO, or 50% aqueous methanol), mobile-phase composition (water/acetonitrile with formic acid and/or ammonium formate), and column temperature (30, 40, or 50°C). Twenty-seven experimental conditions were evaluated in triplicate (81 injections). Factorial ANOVA demonstrated significant effects of extraction solvent and mobile-phase composition, as well as extraction-mobile-phase interactions, for all four VB₁₂ forms. The optimized method was further applied to 11 commercial supplements containing 400-5000 µg VB₁₂ per serving in capsule, tablet, soft-gel, and powder formats. Matrix effects (ME) were assessed by comparing calibration slopes obtained from external calibration (EC) and the standard addition method (SAM). Most products showed minimal matrix effects (within ±15%), indicating negligible signal suppression or enhancement. Only one juice-powder formulation exhibited slightly greater suppression for CNCbl (-16%), suggesting that SAM may improve quantification for this matrix. The optimized extraction and chromatographic conditions provide a robust and sensitive LC-MS/MS method for simultaneous VB₁₂ determination. Matrix-effect evaluation further supports its suitability for routine analysis of commercial supplements, product formulation studies, and label-claim verification.