2.6 Vitamin B complex analysis using UHPLC-MS/MS

Analysis of target B vitamins (thiamin, riboflavin, total vitamin B3, total vitamin B6) was completed using AOAC Method 2015.14 (28). In brief, 1.0 g of sample plus 100 μL of internal standard mixture (containing ²H₄-nicotinamide, ²H₄-nicotinic acid, ¹³C₄-pyridoxine stock, ²H₃-pyridoxal, ²H₃-pyridoxamine, ¹³C₄-thiamine stock, and ¹³C₄,¹⁵N₂-riboflavin acid) was added to a 50 mL centrifuge tube and quickly mixed using a vortex. An enzyme cocktail (5 mL) consisting of 1 mg/mL acid phosphatase and 1 mg/mL papain in 50 mM ammonium formate buffer (pH 4.0) was added to the centrifuge tube, which was then placed in a shaking water bath overnight at 37°C. After the sample was incubated, 50 mM ammonium formate was added to dilute the sample to approximately 30 mL. The sample was filtered through a 0.45 μm PTFE syringe filter, and further diluted ~17-fold with 50 millimolar (mM) ammonium formate before being transferred to an HPLC vial for analysis.

Target B vitamins were quantified using ultrahigh-performance liquid chromatography (UHPLC; Agilent model 1260 Infinity I; Santa Clara, CA, USA) in line with a 6460 series triple quadrupole mass spectrometer (UHPLC-MS/MS; Agilent Technologies, Santa Clara, CA, USA) with an ESI probe operated in positive ionization mode. Separations were achieved using a Waters Acquity BEH C18 column (2.1 × 100 mm, 1.7 μm; Waters Co., Bedford, MA, USA). Gradient elution mode was used with 20 mM ammonium formate in water as mobile phase A and methanol as mobile phase B. The flow rate was set to 0.35 mL/min. Quantification of the B vitamin compounds was conducted using multiple-reaction monitoring (MRM) mass transitions. Optimization of collision energies, fragmentor voltages, and MRM mass transitions was done using the Mass Hunter Optimizer. MRM mass transitions were used as reported by McClure (28). Quality control measures for analysis included validating each analytical worklist by analyzing a sample of SRM 1869.

Total vitamin B3 is reported as the sum of nicotinamide and nicotinic acid. Total vitamin B6 is the sum of pyridoxal, pyridoxamine, and pyridoxine. Thiamine and riboflavin are reported as the single measured form.