DETERMINATION OF HYDROXYL RADICALS USING SALICYLATE AS A TRAPPING AGENT BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

Objectives: To establish a sensitive method for measuring hydroxyl rad ical formation in biological systems using salicylate as probe. Method s: Salicylate hydroxylation products and related aromatic compounds we re extracted and converted to trimethylsilyl (TMS) derivatives. The de rivatives were analyzed by gas chromatography-mass spectrometry (GC-MS ). Quantitation was achieved by selected-ion-recording (SIR) with benz oic acid (ring-D5) as an internal standard. Results: All compounds wer e well separated and specifically quantitated by a GC-MS procedure. St andard curves were linear in the concentration ranges investigated (0. 1-10 nmol) for all individual compounds. Recovery from human plasma wa s in the range of 90-102%. The detection limit was between 50 fmol-l p mol per 1 mu L injection. The within-run and between-run coefficients of variation were between 4.6-9.1%. We were able to detect the baselin e levels of hydroxylation products in human fibroblasts after incubati on with salicylate. Conclusions: The GC-MS assay presented here can sp ecifically identify and quantitate salicylate hydroxylation products a nd related aromatic compounds, which can be used as an in vivo marker of oxidative stress. This sensitive method has broad applications, bot h in the area of free radical medicine and in the pharmacological stud y of aspirin and its metabolites. Copyright (C) 1997 The Canadian Soci ety of Clinical Chemists.