2.9. Vitamin B1

The mobile phase was prepared in a 75% buffer solution (0.033M KH₂PO₄) and 25% methanol. Using ortho-phosphoric acid, the pH was adjusted to 7.1 ± 0.1 and with a 0.22 μm CA, filtered under vacuum. Vitamin B₁ separation, which converted to thiochrome form, was accomplished using an Eclipse X08-C18 column (4.6 × 150 mm, 5 μm) (Agilent Technologies, USA). The temperature of the column was set at 25 °C and the flow rate was 1 mL/min. The excitation wavelength of the fluorescence detector was 366 nm, and the emission wavelength was 445 nm.