Marfey’s analysis

Nemamide A (purified from worms from 2.5 L of culture) was hydrolyzed with 200 μL of 6 N HCl at 110 °C for 12 h. The reaction was then dried down by rotovap, and the residue was dissolved in 50 μL of water. 50 mM stock solutions of the amino acid standards (L-Asp, D-Asp, L-Asn, D-Asn) were made in water. 20 μL of 1 M NaHCO₃ and 100 μL of 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (L-FDAA, Marfey’s reagent; 1% w/v in acetone) were added to 50 μL of the sample or the amino acid standards. After heating at 37 °C for 60 min, reactions were quenched by addition of 20 μL of 1 N HCl. The sample reaction was diluted with 100 μL of acetonitrile while the reactions of the amino acid standards were diluted with 810 μL of acetonitrile. The reactions of the sample and standards were subjected to LC-MS analysis (Phenomenex Luna C_18, 4.6 × 100 mm, 5 μm) using a linear gradient of water with 0.1% formic acid and acetonitrile with 0.1% formic acid (holding at 10% acetonitrile for 5 min, then ramping to 50% acetonitrile over 30 min; flow rate, 0.7 mL/min; UV and ESI-MS detection, 340 nm and negative ion mode). Analysis with both Asn and Asp amino acids indicated the conversion of Asn to Asp during the acid hydrolysis step for both the sample and the Asn amino acid standards. Retention times for L-FDAA-L-Asp and L-FDAA-D-Asp were 22.0 and 22.7 min, respectively. The extracted ion chromatogram (m/z 384) for the sample indicated the presence of L-FDAA-L-Asp and L-FDAA-D-Asp in a 1:2.16 ratio.