High performance liquid chromatography (hplc)

Post-column derivatization of aflatoxins was performed according to the AOAC official method 2005.08 (AOAC, 2005 ). Briefly, the dried sample extracts/standards were dissolved in 200 μl hexane. TFA (50 μl) was added and the vial was closed and placed in dark for 5 min. The solution containing aflatoxins was mixed with 1.95 ml mixture of double distilled water and acetonitrile (9:1) and vortexed for 1 min. The aqueous layer (lower layer) containing aflatoxins was removed and filtered through a 0.45 μm syringe filter before loading on HPLC (Sykam, Germany).

Following the methodology laid out by Dahl-Lassen and colleagues, HPLC (SYKAM, Korea) was utilized to determine the quantity of different amino acids present in glutathione samples (Dahl-Lassen et al., 2018 (link)). The volume-to-volume ratio of the mobile phase’s components was as follows: 60% acetonitrile, 20% methanol, and 20% formic acid. After dissolving 3 g of each sample in 25 mL of 6% hydrochloric acid (HCl) at a temperature of 150 °C for 3 h, the samples were then allowed to dry. Following the addition of 5 mL of sodium citrate (Na₂C₆H₅O₇) to the samples that had been dried at a pH of 2.2, the samples were filtered using a polypropylene filter. After that, 1 mL was taken out of the extracted form, and 200 μL of ortho-phthalaldehyde (OPA) or paraldehyde with a concentration of 5% was added to it. Two minutes were spent shaking the mixture well. The volume of the injection was 100 µL, and the flow rate was 0.8 mL/min. The ZORBAX Eclipse-AAA column that was utilized had an internal diameter of 3.5 µm, 4.6 × 150 mm, and a temperature of 25 °C. Fluorescence was used as the technique for determining the presence of glutathione (Ex = 445 nm, Em = 465 nm). All of the data were reported in terms of mg/100 g of dry weight (DW), and their values were presented in terms of the mean as well as the standard deviation of three separate observations.