Determination of N-G-monomethyl-L-arginine in human and dog serum using pre-column O-phthaldialdehyde derivatization and high performance liquid chromatography

N-G-monomethyl-L-arginine (NMA) is a selective inhibitor of the enzyme nitric oxide synthase (NOS), which converts arginine to nitric oxide and citrulline. NMA may prove useful in the treatment of autoimmune and inflammatory disorders, as well as in preventing the cardiovascular effects associated with endotoxins and cytokines. An automated high performance liquid chromatographic method for determination of NMA in human serum samples was developed using pre-column derivitization with o-phthaldialdehyde and mercaptoethanol. NMA was extracted from serum using 5-sulfosalicylic acid and methanol, then injected into the HPLC system for on-line derivitization. A two mobile phase gradient (A: 70:30 methanol-water containing 0.01M K2HPO4, pH 7.9; B: water containing 0.01M K2HPO4, pH 6.9) was employed with 70:30 (A:B) for 15 min, 10:90 through 21 min, and 70:30 through 25 min. A Beckman ODS 3 mu m 75 x 3.6 mm column was used with a Hitachi L-6200A pump set at a now rate of 1.8 ml/min and fluorescence detection at 340 nm excitation and 450 nm emission. The sensitivity of the assay was <50 ng/mL of plasma and the assay was linear over the range 0.05-500 mu g/mL. The method was sensitive, reproducible, and specific for NMA.