Determination of vitamin B_(12) in infant milk powder by immunoaffinity column purification/chromatography method

A method was established for determining VB12 in infant milk powder by ultra performance liquid chromatography-tandem mass spectrometry(UPLC/MS/MS) and ultra performance liquid chromatography. The analytes were extracted with ammonium acetate(pH=4.0) and cleaned up with immunoaffinity column. The target analytes were separated by Waters BEH C18 column. The mobile phases of UPLC were 0.03 mol/L potassium dihydrogen phosphate(pH=3.5) and acetonitrile(85:15). The detection wavelength was 361 nm. The mobile phases of UPLC-MS/MS were acetonitrile and 0.1% formate in water with gradient elution. The analytes were detected by tandem quadrupole mass spectrometry after positive electrospray ionization by multiple reaction monitoring(MRM). External standard method was used to determine the results. The linear range of the calibration curve was 10~500 μg/L. The limit of detection(LOD) were 0.5 μg/kg, and the limit of quantitation(LOQ) were 1.5 μg/kg. The mean recoveries of LC method were 90.2%~98.3%. The relative standard deviation(RSD) were 1.4%~2.5%. The mean recoveries of LC-MS/MS method were 89.3%~95.7%. The relative standard deviation(RSD) were 1.9%~3.1%. The simplicity, sensitivity and good precision of the method made it be well suitable for determination of VD in infant milk powder.