Determination of hydroxyl radicals using salicylate as a trapping agent by gas chromatography-mass spectrometry

Objectives: To establish a sensitive method for measuring hydroxyl radical formation in biological systems using salicylate as probe. Methods: Salicylate hydroxylation products and related aromatic compounds were extracted and converted to trimethylsilyl (TMS) derivatives. The derivatives were analyzed by gas chromatography-mass spectrometry (GC-MS). Quantitation was achieved by selected-ion-recording (SIR) with benzoic acid (ring-D5) as an internal standard. Results: All compounds were well separated and specifically quantitated by a GC-MS procedure. Standard curves were linear in the concentration ranges investigated (0.1–10 nmol) for all individual compounds. Recovery from human plasma was in the range of 90–102%. The detection limit was between 50 fmol–1 pmol per 1 μL injection. The within-run and between-run coefficients of variation were between 4.6–9.1%. We were able to detect the baseline levels of hydroxylation products in human fibroblasts after incubation with salicylate. Conclusions: The GC-MS assay presented here can specifically identify and quantitate salicylate hydroxylation products and related aromatic compounds, which can be used as an in vivo marker of oxidative stress. This sensitive method has broad applications, both in the area of free radical medicine and in the pharmacological study of aspirin and its metabolites.