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Gas Chromatographic–Tandem Mass Spectrometric Quantification of Free 3-Nitrotyrosine in Human Plasma at the Basal State

✍ Scribed by Edzard Schwedhelm; Dimitrios Tsikas; Frank-Mathias Gutzki; Jürgen C. Frölich

Publisher
Elsevier Science
Year
1999
Tongue
English
Weight
114 KB
Volume
276
Category
Article
ISSN
0003-2697

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✦ Synopsis

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-l 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 >80% and a precision of >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.

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