LC/MS analysis of hydroxylation products of salicylate as an indicator of in vivo oxidative stress.
Hydroxyl radical attack upon salicylate leads to the generation of 2,3-dihydroxybenzoic acid (2,3-DHBA) and therefore can be used to assess hydroxyl radical formation both in vitro and in vivo. Evidence is presented for a highly sensitive LC/MS assay for the quantification of 2,3-DHBA. Calibration curves showed linearity within the concentration range tested (0.5-6.5 pmol/microl rat plasma) with a coefficient of determination (r2) greater than 0.99. A detection limit of less than 0.25 pmol for 2,3-DHBA has been achieved. The intra-assay and inter-assay variability were determined to be 4.1% and 12.5%, respectively. This method was evaluated for the determination of drug-induced in vivo generation of oxidative stress by means of 1,1,1-trichloroethane (TCE) a compound that is a pseudosubstrate for cytochrome P450 and is known to induce oxygen reductase activity of this enzyme(s). TCE treated rats had a 6.4-fold increase in the mean maximal plasma 2,3-DHBA concentration as compared to the saline treated rats (p = .009). The developed LC/MS assay requires minimal sample preparation and provides a rapid and sensitive method for quantification of 2,3-DHBA as a specific indicator of hydroxyl radical generation.
Tabatabaei
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Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Abbott
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FS
D000818:Animals; D015415:Biomarkers; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods; D062385:Hydroxybenzoates / Q000032:analysis*; D017665:Hydroxyl Radical; D006900:Hydroxylation; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D018384:Oxidative Stress; D051381:Rats; D020156:Salicylic Acid / Q000737:chemistry* / Q000493:pharmacokinetics
D015415:Biomarkers; D062385:Hydroxybenzoates; D017665:Hydroxyl Radical; C009135:2,3-dihydroxybenzoic acid; D020156:Salicylic Acid