Determination of NG, NG-dimethyl- l-arginine, an endogenous NO synthase inhibitor, by gas chromatography–mass spectrometry

Abstract

A fully validated gas chromatographic–mass spectrometric (GC–MS) method for the accurate and precise quantification of N G, N G-dimethyl- l-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography–chemical ionization mass spectrometry, fragment ions at m/ z 634 and m/ z 640 were obtained for ADMA and for N G, N G-[ 2H 6]-dimethyl- l-arginine ([ 2H 6]-ADMA) as internal standard, respectively. [ 2H 6]-ADMA was synthesized by reaction of l-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 μM. The method was validated in a concentration range of 0–1.2 μM in cell culture medium and 0–2 μM in 50 μl aliquots of human plasma. The precision was ≥97% and the accuracy was determined to be ≥94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.