Quantitative determination of circulating and urinary asymmetric dimethylarginine (ADMA) in humans by gas chromatography–tandem mass spectrometry as methyl ester tri(N-pentafluoropropionyl) derivative

Asymmetric dimethylarginine (ADMA; NG,NG-dimethyl-l-arginine) is the most important endogenous inhibitor of nitric oxide synthase and a potential risk factor for cardiovascular diseases. This article describes a gas chromatographic–tandem mass spectrometric (GC–tandem MS) method for the accurate quantification of ADMA in human plasma or serum and urine using de novo synthesized [2H3]-methyl ester ADMA (d3Me-ADMA) as the internal standard. Aliquots (100 μl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 μM for plasma and serum; 20 μM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 μl aliquot of 2 M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 μl aliquot of 2 M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 °C. After cooling to room temperature, the plasma or urine sample is combined with the d3Me-ADMA sample, the mixture is evaporated to dryness, the residue treated with a solution of pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v) and the sample is incubated for 30 min at 65 °C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 μl aliquot of 0.4 M borate buffer, pH 8.5, and toluene (0.2 ml for plasma, 1 ml for urine). Reaction products are extracted by vortexing for 1 min, the toluene phase is decanted, and a 1 μl aliquot is injected into the GC–tandem MS instrument. Quantitation is performed by selected reaction monitoring (SRM) of the common product ion at m/z 378 which is produced by collision-induced dissociation of the ions at m/z 634 for endogenous ADMA and m/z 637 for d3Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39±0.06 μM (n=12) and 3.4±1.1 μmol/mmol creatinine (n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d3Me-ADMA, respectively.