Application of UPLC-MS/MS Method for Analyzing B-vitamins in Human Milk

Objective To determine ten B-vitamins in human milk by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods The pretreated human milk samples were adequately separated and quantified within 11 min by UPLC-MS/MS with an Acquity UPLC HSS T3 column (2.1x100 mm, 1.8 mu m). The mobile phase was a gradient of 2.5 mmol/L ammonium formate aqueous solution and acetonitrile at a flow rate of 0.35 mL/min. Stable isotope internal standards were used in the analysis, to correct for the method variability, including matrix and ionization effects. The homogenized human milk samples were deproteinzed using methanol, unknown contaminants were extracted with diethyl ether and hydrophobic phase was discarded. The analytes were monitored via ESI+ionization and detected in multiple reaction monitoring (MRM) with three acquisition functions. Results Calibration curves ranged from 0.5-160 ng/mL (thiamin, riboflavin, biotin, nicotinic acid, pyridoxine, pyridoxamine, pyridoxal), and 2.5-800 ng/mL (pantothenic acid, FAD and nicotinamide) (R-2 = 0.990-0.999). The relative recovery ranged from 80.1% to 120.2%; accuracy was determined to be 98.3% to 108.0%. Intra-day and inter-day variation were 3.4%-19.9% and 5.9%-18.1%, respectively. The limit of quantification (LOQ) for all vitamins was between 0.25 and 3 mu g/L. Conclusion This method was successfully applied for simultaneous analysis of ten B-vitamins in human milk.