Simultaneous determination of vitamin B12 and cobalt (II) in infant milk power using liquid chromatography-inductively coupled plasma mass spectrometry

A new method was developed for simultaneous determination of cobalamin compounds [cyanocobalamin (CN-Cbl), hydroxocobalamin (OH-Cbl), 5'-deoxyadenosylcobalamin (Ado-Cbl) and methylcobalamin (Me-Cbl)] and inorganic cobalt ions [Co(II)] in infant milk powder by liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS). The samples were enzymatic hydrolyzed by pepsin (porcine source) and the analytes extracted in the hydrolysate were measured directly by LC-ICP-MS after filtration. The chromatographic separation was performed on a Phenomenex Luna 5 μm C₁₈ (250 mm × 4.6 mm) column. The mobile phase was consist of 1.6 mM ethylenediaminetetraacetic acid disodium salt (EDTA•2Na) and 0.4 mM potassium dihydrogen phosphate in 60% (v/v) methanol-water (pH = 6.0) (mobile phase A) and ultrapure water (mobile phase B). After LC separation with gradient elution, Co(II), OH-Cbl, CN-Cbl, Ado-Cbl, and Me-Cbl were measured as ⁵⁹Co by ICP-MS. The method detection limit of Co(II), OH-Cbl, CN-Cbl, Ado-Cbl, and Me-Cbl were 0.144 µg/kg, 0.197 µg/kg, 0.267 µg/kg, 0.209 µg/kg and 0.278 µg/kg, respectively. At three spiked levels (1.0, 10.0, 50.0 µg/kg), the average recoveries (%) of Co(II), OH-Cbl, CN-Cbl, Ado-Cbl, and Me-Cbl ranged from 70.4 to 79.6, 74.5 to 77.4, 91.0 to 93.1, 83.0 to 83.8, and 84.3 to 90.2, respectively. The relative standard deviations (RSD, %) of Co(II), OH-Cbl, CN-Cbl, Ado-Cbl, and Me-Cbl ranged from 0.50 to 2.57, 0.70 to 6.29, 0.80 to 2.37, 1.33 to 2.50, and 1.16 to 2.40, respectively. These results indicating that the method had a good accuracy and precision. The preparation of sample was simple and less time consuming since it avoiding solid phase extraction and follow steps.