Determination of active vitamin B12 (cobalamin) in dietary supplements and ingredients by reversed-phase liquid chromatography: Single-laboratory validation

•Simultaneous determination of four bioactive vitamin B12s from dietary supplements.•The method is simpler, less time-consuming, and more economical than other methods.•The LOQs are less than 0.52 µg/mL.•The method is validated and meets all AOAC SMPR 2016.017 requirements. Vitamin B12 dietary supplement can be critical to the alleviation strategies against micronutrient malnutrition and food insecurity. An HPLC-DAD method has been developed and validated, per AOAC SMPR 2016.017 (Standard Method Performance Requirements), for the quantitation of four bioactive forms of vitamin B12 (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, methylcobalamin) from dietary ingredients and supplements. The method achieves chromatographic baseline resolution of vitamin B12 forms on a modern column platform without the expensive requirement of an ultra-high pressure liquid chromatography and/or mass spectrometry. The method has a wide analytical range (0.0005%w/w–85%w/w), high precision (reproducibility relative standard deviations ranged from 1.43% to 4.67%), and high accuracy (>96% spike recovery rate for 11 out of 12 accuracy testing data points). The method detection and quantification limits are less than 0.16 and 0.52 µg/mL, respectively. To our best knowledge, it is simpler, less time-consuming, and more economical than other published methods for its intended uses.