SOLID-PHASE EXTRACTION AND SIMULTANEOUS HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OF ANTIPYRINE AND ITS MAJOR METABOLITES IN URINE

A reversed phase gradient high performance liquid chromatographic method utilizing solid phase extraction has been described for the simultaneous determination of antipyrine (AP), 4-hydroxyantipyrine (4-OHAP), norantipyrine (NorAP) and 3-hydroxymethylantipyrine (3-OHMAP) in human urine after hydrolysis with beta-glucuronidase. The C-18 sorbent cartridges were conditioned and urine samples were applied, washed with 1 X 4 mL of phosphate buffer and eluted with 3 X 100 muL of 20% v/v of acetonitrile in methylene chloride. The eluent was evaporated to dryness, reconstituted in 100 muL phosphate buffer and injected. The calibration ranges were 2.0-250 mug/mL (AP), 2.5-250 mug/mL (NorAP), 2.0-250 mug/mL, (3-OHMAP) and 5.0-500 mug/mL (4-OHAP) with regression coefficients of 0.998 or greater. Specificity was indicated by the absence of interferences in chromatogram of blank urine from normal as well as cirrhotic patients. The average recovery was 86.7% for AP, 90.5% for NorAP, 85.2% for 4-OHAP and 74.2% for 3-OHMAP. The within-assay precision as indicated by the reproducibility of the assayed spiked urine was less than 9% in all cases and the between-assay precision was less than 12%. The method was applied to studies on antipyrine metabolism in stable cirrhotic patients. Following administration of a single oral dose of about 1000 mg to nine stable cirrhotic patients and eight age-matched healthy volunteers, the cumulative account excreted in the urine up to 48 h for AP and the three metabolites was comparable to other literature reports.