Accurate determination of vitamin B₁₂ in infant formula using isotope dilution-ultrahigh-performance liquid...

A method for the accurate determination of vitamin B₁₂ (cobalamin) in infant formula was developed using isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). As an internal standard, a [¹³C₇]-cyanocobalamin isotope-labeled analogue was used. Because of the hygroscopic property of cobalamins, the concentration of the cyanocobalamin standard solution was determined via quantitative nuclear magnetic resonance (qNMR) spectroscopy and further validated by capillary electrophoresis with ultraviolet detection as an independent technique. The cobalamins contained in infant formula were converted to cyanocobalamin in the presence of cyanide. Incomplete conversion of cobalamins was observed, and the residual amount of hydroxocobalamin was quantified to obtain the total amount of cobalamins. The expanded measurement uncertainty ranged from 1.7% to 2.4%, accounting for the contribution of residual hydroxocobalamin. Recovery, calculated by comparing the measured CN-Cbl concentration with the gravimetric fortification level, ranged from 101 to 103%. Both intra- and inter-day RSDs were below 1%. The method was applied to commercially available infant formula, with cobalamin concentrations ranging from 31.0 to 40.6 µg/kg.