Simultaneous determination of primidone and its three major metabolites in rat urine by high-performance liquid chromatography using solid-phase extraction

A new high-performance liquid chromatographic method for simultaneous determination of primidone (PRM) and of its three major metabolites, phenobarbital (PB), p-hydroxyphenobarbital (p-HO-PB) and phenylethylmalonamide (PEMA), in rat urine, was developed. After acid hydrolysis, these compounds were extracted from urine by means of a Bond Elut Certify LRC column with good clean-up. The extracts were chromatographed on a C-18 reversed-phase column using isocratic elution at 40 degrees C, with UV detection at 227 nm. The Limit of detection was 0.5 mg/ml for the four compounds. Good linearity (r(2) > 0.99) was observed within the calibration ranges studied: 37.4-299.3 mu g/ml for PRM, 26.4-211.2 mu g/ml for PB, 22.5-100.2 mu g/ml for p-HO-PB and 12.1-97.0 mu g/ml for PEMA. Repeatability was in the range 3.1-6.8%. This method constitutes a useful tool for studies on the influence of various parameters on primidone metabolism. (C) 1998 Elsevier Science B.V. All rights reserved.