Simultaneous determination of four water-soluble vitamins in fortified infant foods by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry

A novel ultra-performance liquid chromatography electrospray ionization tandem triple quadrupole mass spectrometry method for the simultaneous determination of four water-soluble vitamins, including vitamin B5 (VB5), vitamin B8 (VB8), vitamin B9 (VB9), and vitamin B12 (VB12) in fortified infant foods is developed and validated. A reverse phase UPLC separation system consisting of a Waters ACQUITY UPLC BEH C-18 column (2.1 mm x 100 mm i.d., 1.7 μm) and a binary gradient acetonitrile-water mobile phase is applied for the separation of the four water-soluble vitamins. Formic acid is spiked into the mobile phase to enhance the ionization efficiency. Tandem MS-MS analysis is performed in multi-reaction monitoring mode (MRM). Product-ion traces at m/z 220.1 → 89.9 for VB5, 245.1 → 227.1 for VB8, 442.3 → 295.2 for VB9, and 678.9 → 147.0 for VB12 are used for quantitation of the corresponding vitamins, and traces at m/z 455.5 → 308.0 are used for methotrexate (internal standard). Limits of quantitation (LOQs) are 0.016, 0.090, 0.020, and 0.019 μg/L for VB5, VB8, VB9, and VB12, respectively. Intra- and inter-day precisions for the determination of the four vitamins are better than 6.84% and 12.26% in relative standard deviations, and recoveries for the four vitamins are in the range of 86.0∼101.5%. The developed approach is applied for the determination of the trace amounts of the vitamins in fortified milk powers and fortified rice powers.