A fully validated gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate and precise quantification of free 3-nitrotyrosine in human plasma at the basal state is described. In the plasma of 11 healthy humans a mean concentration of 2.8 nM (range 1.4-4.2 nM) for free 3-nitrotyrosine was determined by this method. This is the lowest concentration reported for free 3-nitrotyrosine in plasma of healthy humans. The presence of endogenous free 3-nitrotyrosine in human plasma was unequivocally shown by generating a daughter mass spectrum. Various precautions had to be taken to avoid artifactual formation of 3-nitrotyrosine from nitrate during sample treatment. Endogenous plasma 3-nitrotyrosine and 3-nitro-l-[(2)H(3)]tyrosine added for use as internal standard were isolated by high-performance liquid chromatographic (HPLC) analysis of 200-microl aliquots of plasma ultrafiltrate samples (20 kDa cut-off), extracted from a single HPLC fraction by solid-phase extraction, derivatized to their n-propyl ester-pentafluoropropionyl amide-trimethylsilyl ether derivatives, and quantified by GC-tandem MS. Overall recovery was determined as 50 +/- 5% using 3-nitro-l-[(14)C(9)]tyrosine. The limit of detection of the method was 4 amol of 3-nitrotyrosine, while the limit of quantitation was 125 pM using 3-nitro-l-[(14)C(9)]tyrosine. 3-Nitrotyrosine added to human plasma at 1 nM was quantitated with an accuracy of > or = 80% and a precision of > or = 94%. The method should be useful to investigate the utility of plasma free 3-nitrotyrosine as an indicator of nitric oxide ((.)NO)-associated oxidative stress in vivo in humans.
Copyright 1999 Academic Press.