Quantification of L-arginine by HPLC

To 50 µl cell culture supernatants, 4 nmol N^G-monomethyl-L-arginine (L-NMMA) were added as internal standard, then supplemented with 0.9 ml PBS (pH 6.9) and applied to an Oasis MCX ion exchange column (Waters, Eschborn, Germany). The column was washed with 1 ml each, 0.1 N HCl and methanol, and subsequently cationic amino acids (CAA) were eluted with 1 ml methanol:water: 25% NH₃ (5:4:1), vacuum dried and resuspended in 0.2 ml sodium borate buffer (0.5 mol/l, pH 9.6). L-arginine levels were determined in cell culture supernatants by High Performance Liquid Chromatography (HPLC) using precolumn derivation, exactly as described before (20 (link), 21 (link)).