Lc 2000 plus series hplc system

The extraction of AF was done according to Bertuzzi et al. (2011) (link) using 50 mL of chloroform for 1 h on a rotary-shaker, and extracts were purified as described previously (Shannon et al., 1983 (link)). The extracted AF samples were analyzed by thin layer chromatography (TLC) using a C₁₈ reversed-phase TLC plate (aluminum sheets measuring 20 × 20 cm with 200-μm layers, Merck, Germany) and the chromatogram was developed using a solvent system of chloroform/acetone (90:10, v/v). After development, the spot with the same Rf-value as the AFB₁ standard was scraped off, re-extracted with chloroform and evaporated to dryness under nitrogen. The extract was taken up in 100 μL acetonitrile in an amber-colored vial under refrigeration. AF was analyzed using a Jasco LC-2000 plus series HPLC system (Jasco, United States) with a fluorescence detector (RF-10Axl, Shimadzu) (λexc 247 nm; λem 480 nm) and a C18 column (Eclipse XDB-C18 150 × 4.6 mm, 5 μm; Agilent, United States, column temperature 35°C) as described previously (Hanano et al., 2015 (link)).

The same filtrate that was used for the analysis of organic acids was analyzed using a JASCO LC-2000 Plus Series HPLC system (JASCO, Tokyo, Japan). Chromatographic separation was conducted with an AccQ-Tag Amino Acids C18 Column (3.9 mm × 150 mm, 4 μm; Waters Corporation, Milford, MA, USA). Gradient elution was carried out with AccQ-Tag/water (solvent A; 10:90, v/v) and acetonitrile/water (solvent B; 60:40, v/v). The following binary mobile phase linear gradients were used: 100% A at 0 min, 98% A at 5 min, 93% A at 15 min, 90% A at 19 min, 67% A at 32–33 min, 0% A at 34–37 min, and 100% A at 38 min. Injection volumes were 10 μL, and the column temperature and flow rate were 37 °C and 1 mL/min, respectively. The detection was performed using a fluorescent detector. AccQ-Fluor Reagent Kit (WAT052880, Waters Corporation, Milford, MA, USA) was simultaneously used as derivatizing agents, according to the manufacturer’s instructions. The photodiode array (PDA) detector (MD-2018 Plus, JASCO, Tokyo, Japan) wavelength was set to 254 nm for the determination of the AccQ-Fluor Reagent-derivatized amino acids. The concentrations of individual free amino acids were determined using five-point calibration curves of the amino acid standard (WAT088122, Waters Corporation, Milford, MA, USA). The free amino acid contents of two samples prepared in the same conditions were analyzed twice.