Simultaneous liquid-chromatographic quantitation of salicylic acid, salicyluric acid, and gentisic acid in plasma.

Salicylic acid, salicyluric acid, and gentisic acid in plasma are specifically quantitated (ultraviolet detection at 313 nm) with an octadecyl silane reversed-phase chromatographic column by peak-height determination or by peak-height ratios (internal standard, o-methoxybenzoic acid). Sample preparation requires addition of an equal volume of methyl cyanide (with or without the internal standard) to precipitate proteins. After centrifugation, an aliquot of the supernate is directly injected onto the column. Salicylic acid and metabolites are eluted with a mixture of methanol and dilute acetic acid. Sensitivity is 20 ig/L, with linear response to at least 2000 mg/L for salicylic acid and its metabolites. Analytical recovery of salicylic acid and metabolites from plasma is complete. Intra-assay precision (CV) is 2.94% at 8.11 mg/L for salicylic acid; 4.10% at 7.95 mg/L for salicyluric acid; and 3.10% at 7.94 mg/L for gentisic acid. Interassay precision is 2.70% at 8.87 mg/L for salicylic acid; 4.47% at 9.02 mg/L for salicyluric acid; and 4.24% at 8.75 mg/L for gentisic acid. We saw no interference in sara from patients being treated with various drugs other than aspirin. This method estimates salicylic acid and metabolites within 12 mm; the limit of detection is 3 ng. Under normal laboratory conditions microorganisms can drastically alter salicyluric acid and salicylic acid concentrations, leading to erroneous results. Treatment of samples with sodium azide overcomes this problem. We estimated gentisic acid, salicyluric acid, and salicylic acid in plasma from a human volunteer after a single 21 mg/kg oral dose of aspirin.