Determination of cyanocobalamin by isotope dilution LC-MS/MS

Cyanocobalamin (CNCbl) is an active form of vitamin B12, commonly employed for the preparation of multivitamin supplements and fortified food. In this study, we present a novel analytical method for its determination based on stable isotope dilution liquid chromatography electrospray tandem mass spectrometry (ID LC-MS/MS). Isotopically enriched ¹³C¹⁵NCbl was synthesized in-house and used as internal standard. The method was validated using NIST SRM 3280 multivitamin reference material and by comparison with an independent methodology based on LC-ICPMS. The proposed method provided a detection limit of 57 pg/g and could be applied for the determination of trace level of CNCbl in multivitamin supplements with a relative standard uncertainty better than 3%. The novel ID LC-MS/MS is a primary ratio method that could become a reference for CNCbl determination in multivitamins and food supplements. The method was applied for the characterization of two NRC multivitamin tablet Certified Reference Material (CRM) candidates, VITA-1 and VITB-1 whose CNCbl levels were quantified as 2.64 ± 0.09 and 1.75 ± 0.12 μg/g, respectively.