Simultaneous Determination of 10 Water-Soluble Vitamins in Infant Formula by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

To develop a method for simultaneous determination of 10 water-soluble vitamins (nicotinic acid, nicotinamide, vitamin B₂, pyridoxine, pyridoxal, pyridoxine, pantothenic acid, biotin, folic acid and vitamin B₁₂) in infant milk by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were dissolved in water, and adjusted to pH 1.7 with 5 mol/L HCl solution and then to pH 4.5 with 5 mol/L NaOH solution to precipitate proteins. The supernatant was added with a mixed solution of isotope internal standard, and separated on an ACQUITY UPLC HSS T3 column (2.1 mm × 50 mm, 1.8 μm) by gradient elution using a mobile phase composed of 10 mmol/L ammonium formate aqueous solution (containing 0.1% formic acid) and 10 mmol/L ammonium formate methanol solution (containing 0.1% formic acid). Detection was performed by electrospray ionization in the positive ion mode with multiple reaction monitoring (MRM). The external standard method was used for the quantification of pyridoxine, pyridoxal and pyridoxine, and the internal standard method for the other seven vitamins. Good linearity of the calibration curves for the 10 water-soluble vitamins was obtained in the range of 10–5000 ng/mL with correlation coefficients (R²) above 0.99, and the recoveries for spiked samples ranged from 73.9% to 106.0%, with relative standard deviations (RSDs) of 0.9%–8.0%. The proposed method is simple, rapid, sensitive and accurate, and can meet the requirements for the determination of the 10 water-soluble vitamins in infant formula.