Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde

An analytical method is described for the quantification of S-nitrosoglutat hione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmalei mide, the conversion of GSNO by S-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricy clic isoindole derivative, and subsequent high-performance liquid chromatog raphic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to b e identical by mass spectrometry, GSH (up to 50 mu M) did not interfere wit h the analysis of GSNO (up to 1000 nM). The limits of detection of the meth od for buffered aqueous solutions of GSNO were determined as 3 nM using flu orescence and 70 nM using UV absorbance detection, isolation of GSNO by HPL C analysis (pH 7.0) of plasma ultrafiltrate samples (200 mu l) prior to der ivatization allows specific and artifact-free quantification of GSNO in hum an and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LO&) of the combined method was determined as 100 n M of GSNO in human plasma ultrafiltrate using fluorescence detection. No en dogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 he althy humans at concentrations exceeding the LOQ of the method. After iv in fusion of GSNO (125 mu mol/kg body wt) in a rat for 20 min GSNO and GSH wer e detected in rat plasma at 60 and 130 mu M, respectively. The method shoul d be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations. (C) 1999 Acad emic Press.