Rapid Determination\nof Water-Soluble Vitamins in Human\nSerum by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry

Water-soluble vitamins\nplay essential roles in normal\nbody functions\nand metabolic activities. However, few methods have simultaneously\nmeasured all nine water-soluble vitamins in biological matrices. In\nthis study, we developed a sensitive and accurate method for the simultaneous\nmeasurement of thiamine (B1), riboflavin (B2), nicotinamide (B3),\npantothenic acid (B5), 4-pyridoxic acid (B6), biotin (B7), 5-methyltetrahydrofolic\nacid (B9), ascorbic acid (VC), and methylmalonic acid (MMA) in human\nserum. Samples were analyzed using ultrahigh-performance liquid chromatography-tandem\nmass spectrometry (HPLC-MS/MS) with Waters HSS T<sub>3</sub> and BEH\nC<sub>18</sub> columns, each measuring 2.1 × 50 mm with a particle\nsize of 1.7 μm. The mass spectrometer was equipped with an electrospray\nionization source, and optimized multiple reaction monitoring was\nemployed to detect all compounds in positive ionization mode. The\ncapillary voltage was set at 0.5 kV. Nitrogen was used as the desolvation\ngas, with a flow rate of 1000 L/h at 500 °C. Argon was used as\nthe collision gas. The method’s performance was validated according\nto the Clinical and Laboratory Standards Institute guidelines assessing\nlinearity, limit of quantitation, precision, accuracy, carryover,\nmatrix effects, recovery, and dilution. The results confirmed the\nsuccessful validation of this method for water-soluble vitamins in\nserum. However, the presence of common endogenous interferences and\nthe type of blood collection tube influenced the results for certain\nwater-soluble vitamins. The results showed that this method was satisfactory,\noffering significant improvements in analytical performance, including\nshorter analysis time, higher sensitivity, and the requirement of\na smaller sample volume.