Xp. Luo et Dc. Lehotay, DETERMINATION OF HYDROXYL RADICALS USING SALICYLATE AS A TRAPPING AGENT BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY, Clinical biochemistry, 30(1), 1997, pp. 41-46
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.