Rapid Determination of Water-Soluble Vitamins in Human Serum by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry

Water-soluble vitamins play essential roles in normal body functions and metabolic activities. However, few methods have simultaneously measured all nine water-soluble vitamins in biological matrices. In this study, we developed a sensitive and accurate method for the simultaneous measurement of thiamine (B1), riboflavin (B2), nicotinamide (B3), pantothenic acid (B5), 4-pyridoxic acid (B6), biotin (B7), 5-methyltetrahydrofolic acid (B9), ascorbic acid (VC), and methylmalonic acid (MMA) in human serum. Samples were analyzed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with Waters HSS T₃ and BEH C₁₈ columns, each measuring 2.1 × 50 mm with a particle size of 1.7 μm. The mass spectrometer was equipped with an electrospray ionization source, and optimized multiple reaction monitoring was employed to detect all compounds in positive ionization mode. The capillary voltage was set at 0.5 kV. Nitrogen was used as the desolvation gas, with a flow rate of 1000 L/h at 500 °C. Argon was used as the collision gas. The method's performance was validated according to the Clinical and Laboratory Standards Institute guidelines assessing linearity, limit of quantitation, precision, accuracy, carryover, matrix effects, recovery, and dilution. The results confirmed the successful validation of this method for water-soluble vitamins in serum. However, the presence of common endogenous interferences and the type of blood collection tube influenced the results for certain water-soluble vitamins. The results showed that this method was satisfactory, offering significant improvements in analytical performance, including shorter analysis time, higher sensitivity, and the requirement of a smaller sample volume.