Ascorbic acid, riboflavin, pyridoxine, and thiamine reference standards were obtained from the United States Pharmacopeia.
Ammonium acetate was obtained from Fisher Scientific. HPLC-grade formic acid and acetonitrile were from EMD. Deionized water
(DI H2O) was prepared on a Milli-Q purification system from Millipore.
For HPLC–UV studies, a Hewlett–Packard 1100 HPLC system consisting of an autosampler, degasser, binary pump, and variable
wavelength UV detector set at 266 nm was used. The system was interfaced with Agilent Chemstation software. The flow rate
was 1.0 mL/min for all method steps. The injection volume was 1.0 µL in each method step. The mobile phase solvents used for
steps 1–6 are given in Table I. The gradient programs used in steps 1–6 are given in Table II. For step 1, a 75 mm × 4.6 mm
analytical column was packed with Bidentate C18 stationary phase with a particle diameter of 4 µm and a pore size of 100 Å
(MicroSolv Technology Corporation). For steps 2–6, a 75 mm × 4.6 mm analytical column was packed with Diamond Hydride (low
carbon-bonded silica hydride column) stationary phase with a particle diameter of 4 µm and a pore size of 100 Å (MicroSolv
Table I: Solvents used in each of the six method steps
For the LC–MS method, the HPLC system was an Agilent 1100 Series LC system, including a degasser, binary pump, temperature-controlled
autosampler, and temperature-controlled column compartment. The mass spectrometer was an Agilent Model 6210 MSD TOF with a
dual sprayer electrospray ionization (ESI) source. The method consisted of an injection volume of 1.0 µL, a flow rate of 1.0
mL/min, a Diamond Hydride column of the same specifications as in HPLC–UV, and step 6 mobile phase and gradient conditions
(see Tables I and II).
Table II: Gradients used for each method step
1000 mg/L stock solutions of ascorbic acid, pyridoxine, riboflavin, and thiamine were prepared. The diluents used were 50:50:0.1
(v/v) acetonitrile–deionized water–formic acid for steps 1–2 and 50:50 acetonitrile–5 mM ammonium acetate for steps 3–6. These
stock solutions were diluted to final concentrations in the range 10–300 mg/L for each of the analytes. For LC–MS, a mixture
of all four analytes was prepared as well.