The fat- and water-soluble vitamins in the various samples were analyzed following the methods described by Maiyo et al. [31 (link)] and Bhatnagar-Panwar et al. [32 (link)]. Liquid chromatography mass spectroscopy (LC-MS) coupled with a diode array detector (HPLC-30AC; Shimadzu, Tokyo, Japan) and a C18 column (100 × 3.00 mm, 2.6 µm polar; Phenomenex, Torrance, CA, USA) at 30 °C, was used and each sample and standards were analyzed for 12 min at a flow rate of 0.4 mL/min and injection volume of 10 µL. The number of water-soluble vitamins in the sample were calculated by comparing peak area of samples to peak area of the standards.
For the fat-soluble vitamins, analyses were performed using reverse-phase HPLC (Shimadzu, Tokyo, Japan) linked to a SPD-M2A detector. The injection volume of each sample was 10 µL with a total flow rate of 0.4 mL/min for 10 min. Standards for retinol and α- and γ-tocopherol were prepared at four different concentrations each and used for the calibration curve. Peaks were identified by their retention time and the absorption spectra were compared to the standardized spectra. Vitamin concentrations were calculated by comparing the peak area of samples to the peak area of the standardized spectra.
For the fat-soluble vitamins, analyses were performed using reverse-phase HPLC (Shimadzu, Tokyo, Japan) linked to a SPD-M2A detector. The injection volume of each sample was 10 µL with a total flow rate of 0.4 mL/min for 10 min. Standards for retinol and α- and γ-tocopherol were prepared at four different concentrations each and used for the calibration curve. Peaks were identified by their retention time and the absorption spectra were compared to the standardized spectra. Vitamin concentrations were calculated by comparing the peak area of samples to the peak area of the standardized spectra.
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