4.5. Determination of the Absolute Configurations of Amino Acids in MAAs by the Advanced Marfey’s Method Using LC-MS

The general procedure was adapted from a previously published protocol [55]. Approximately 0.1 mg of each MAA was stirred with HCl 37% (250 μL) at 90 °C for 120 min. The hydrolysates were evaporated to dryness and then resuspended in H₂O (100 μL).

Each amino acid (200 μg) or hydrolysate (ca. 100 µg) was dissolved in 1 M NaHCO3 (200 μL) and 1% d- or l-1-fluoro-2,4-dinitrophenyl-5-l-leucinamide (FDLA) derivatization reagent in acetone (25 μL) was added. The reaction vials were incubated and stirred for 30 min at 50 °C. The reactions were then quenched with 2 N HCl (100 μL). MeOH (800 μL) was added to prepare LC-MS samples. The reaction products were analyzed by HPLC-MS, using a Luna 5µ C-8 column (150 × 4.6 mm, 5 µm; Phenomenex, Torrance, CA, USA). H₂O containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) were used as eluents applying the following gradient: 0 min: 5% B, 1 min: 5% B, 20 min: 20% B, 40 min: 60% B, 45 min: 95% B, 45.1 min: 5% B and 55 min: 5% B. The DAD detector was set to 210, 254, 280, 320, 330, 350, and 400 nm, the flow rate, injection volume, and column temperature were adjusted to 0.9 mL min^-1, 20 μL, and 40 °C, respectively. MS spectra were recorded in positive-ESI mode (capillary voltage 4.5 kV), with a drying gas temperature of 300 °C, the nebulizer gas (nitrogen) set to 25 psi, and a nebulizer flow (nitrogen) of 12.0 L min⁻¹. The scanned mass range was set between m/z 50 and 600 (Figures S8, S16, S23, S30, S38, S39 and S47).