Direct Measurement of Salicylphenolic Glucuronide in Human Urine

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Abstract
Indirect measurement of salicylphenolic glucuronide (SPG) has suggested that the formation of this metabolite from therapeutic doses of salicylic acid (SA) is capacity-limited in humans. A direct high performance liquid chromatographic (HPLC) assay for SPG in human urine is described. SPG was prepared by a published method and purified by HPLC. On treatment with .beta.-glucuronidae, SPG yielded the expected amount of SA. Spectroscopic data, melting point, and optical rotation of the glucuronide and/or its triacetyl dimethyl ester derivative were consistent with the proposed structure. SPG was assayed using a 5-.mu.m C18 column (temperature 55.degree. C) and fluorescence detection. A nonlinear gradient mobile phase at a flow rate of 2 ml/min was used, beginning with 100% 0.1 M pH 2.1 phosphate buffer and finishing with 84% buffer, 16% acetonitrile. Total run time was 25 min. Urine (10 .mu.l) was injected directly on the column, and quantitation was performed using urine standards. Within-run precision for SPG ranged from 1.2% at 150 mg/L to 2.4% at 5 mg/L. The limit of detection was < 1 mg/L. A pilot study in two volunteers, each receiving a single 500-mg dose of sodium salicylate, was carried out to validate the usefulness of the assay.