High-performance liquid chromatographic determination of aspirin and its metabolites in plasma and urine.

A simple quantitative method for the rapid determination of aspirin and its metabolites, salicylic acid, salicyluric acid, and gentisic acid, in plasma and urine using o-toluic and o-anisic acids, respectively, as internal standards was developed. Plasma proteins were precipitated by the addition of acetonitrile and, after centrifugation, the supernatant fluid was injected directly onto a reverse-phase column. The mobile phase consisted of an isocratic mixture of water, methanol, and glacial acetic acid (64:25:1, v/v/v) and the separated components were detected at 238 nm using a UV detector. Concentrations greater than or equal to 0.5 microgram/ml could be quantitated for aspirin or its metabolites in plasma. The peak heights and peak height ratios to the internal standard, o-toluic acid, were linear for the concentration range of 0.5-200 micrograms/ml. The aspirin metabolites in urine were isolated by extracting the acidified urine with either and then reextracting the material into an aqueous buffer solution at pH 7.0. Twenty microliters of the buffer extract was directly injected onto the column. The separated components were detected and quantitated at 305 nm. Concentrations greater than or equal to 5 micrograms/ml of salicyluric acid, salicylic acid, and gentisic acid could be determined accurately. The peak heights and peak height ratios to the internal standard, o-anisic acid, were found to be linear for the concentration range of 5-200 micrograms/ml in urine.