Agilent 1260 system quaternary pump

Thiamine was measured based on previous documents and national standards (China, 2016b ; Zeeb et al., 2010 ). The standard used for thiamine was thiamine hydrochloride purchased from Sigma. Thiamine was extracted with acid hydrolysis and then derivatized with potassium ferricyanide. Then the sample was made up the volume with n-butyl alcohol and filtrated through a 0.45 μm nylon syringe filter after shaking thoroughly and ready to inject into the HPLC system (Agilent, NYSE.A, Palo Alto, California, USA). The system consisted of an Agilent 1260 System Quaternary pump and a fluorescence detector. The column was an Agilent 5 TC C18 column, 250 × 4.6 mm, particle diameter 5 μm. The mobile phase consisted of methanol/sodium acetate solution (35:65, v/v), and was pumped at a flow rate of 0.8 mL/min. The column temperature was 30 °C and the injection volume was 20 μL. The optimum wavelength for the detection of thiamine was set as: λex = 375 nm, λem = 435 nm.

Vitamin A and E were measured according to previous studies (China, 2016a ; Wen et al., 2020 ). The standard used for vitamins A was retinol purchased from Sigma. The standard used for vitamins E were α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol, which were purchased from Sigma, respectively. After saponified, tocopherols and retinols were extracted with diethyl ether/petroleum ether (200:200, v/v). Then the solution was washed and condensed, following by making up the volume with methanol and filtrating through a 0.22 μm nylon syringe filter after shaking thoroughly. Then the sample was waiting to inject into the high performance liquid chromatography (HPLC) system (Agilent, NYSE.A, Palo Alto, California, USA), which consisted of an Agilent 1260 System Quaternary pump and a ultraviolet detector. The column was a Shimsen VD C30 column, 250 × 4.6 mm, particle diameter 3 μm. The mobile phase was operated in gradient elution (eluent A: water, eluent B: methanol) and the procedure was as follows: 0–13 min, 4% A and 96% B; 13–20 min, 100% B; 20–24 min, 100% B; 24–24.5 min, 4% A and 96 % B; 24.5–30 min, 4% A and 96% B. The sample was separated at 0.8 mL/min flow rate with 10 μL injection volumes and the column temperature was 20 °C. The optimum wavelength selected for the detection of vitamin A (λ = 325 nm) and E (λ = 294 nm).