Gas chromatographic-tandem mass spectrometric quantification of free 3-nitrotyrosine in human plasma at the basal state

A fully validated gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate and precise quantification of free 3-nitrotyros ine in human plasma at the basal state is described. In the plasma of 11 he althy humans a mean concentration of 2.8 nM (range 1.4-4.2 nM) for free 3-n itrotyrosine was determined by this method. This is the lowest concentratio n reported for free 3-nitrotyrosine in plasma of healthy humans. The presen ce of endogenous free 3-nitrotyrosine in human plasma was unequivocally sho wn by generating a daughter mass spectrum. Various precautions had to be ta ken to avoid artifactual formation of 3-nitrotyrosine from nitrate during s ample treatment. Endogenous plasma 3-nitrotyrosine and 3-nitro-L-[H-2(3)]ty rosine added for use as internal standard were isolated by high-performance liquid chromatographic (HPLC) analysis of 200-mu l aliquots of plasma ultr afiltrate samples (20 kDa cut-off), extracted from a single HPLC fraction b y solid-phase extraction, derivatized to their n-propyl ester-pentafluoropr opionyl amide-trimethylsilyl ether derivatives, and quantified by GC-tandem MS. Overall recovery was determined as 50 +/- 5% using 3-nitro-L-[C-14(9)] tyrosine. The limit of detection of the method was 4 amol of 3-nitrotyrosin e, while the limit of quantitation was 125 pM using 3-nitro-L-[C-14(9)]tyro sine, 3-Nitrotyrosine added to human plasma at 1 nM was quantitated with an accuracy of greater than or equal to 80% and a precision of greater than o r equal to 94%. The method should be useful to investigate the utility of p lasma free 3-nitrotyrosine as an indicator of nitric oxide ((NO)-N-.)-assoc iated oxidative stress in vivo in humans. (C) 1999 Academic Press.