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; N(G),N(G)-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 [2H(3)]-methyl ester ADMA (d(3)Me-ADMA) as the internal standard. Aliquots (100 microl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 microM for plasma and serum; 20 microM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 microl aliquot of 2M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 microl aliquot of 2M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 degrees C. After cooling to room temperature, the plasma or urine sample is combined with the d(3)Me-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 degrees C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 microl aliquot of 0.4M 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 microl 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 d(3)Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39+/-0.06 microM (n=12) and 3.4+/-1.1 micromol/mmol creatinine (n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d(3)Me-ADMA, respectively.
Tsikas
Dimitrios
D
Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. tsikas.dimitros@mh-hannover.de
Schubert
Bibiana
B
Gutzki
Frank-Mathias
FM
Sandmann
Jörg
J
Frölich
Jürgen C
JC
D000328:Adult; D001120:Arginine / Q000031:analogs & derivatives / Q000097:blood* / Q000652:urine*; D004791:Enzyme Inhibitors / Q000097:blood* / Q000652:urine*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D015203:Reproducibility of Results
D004791:Enzyme Inhibitors; C487735:dimethylarginine; D001120:Arginine; D019001:Nitric Oxide Synthase