Micellar electrokinetic capillary chromatography method for direct determination of glucuronides of entacapone and its (Z)-isomer in human urine

This paper describes the validation of a micellar electrokinetic capillary chromatography method for the direct determination of the 3-O-glucuronides of entacapone and its (Z)-isomer, the main urinary metabolites of entacapon e in humans. Entacapone is a novel drug which, as a potent inhibitor of cat echol-O-methyltransferase (COMT), is used as an adjunct in the standard the rapy of Parkinson's disease. The 3-O-glucuronide of another COMT inhibitor, nitecapone, was used as internal standard (I.S.). The validation experimen ts were performed by using spiked urine samples that were extracted with Se p-Pak C-18 cartridges before analysis. Determinations were carried out in a buffer of pH 7.0 containing 25 mM of phosphate, 50 mM of berate and 20 mM of sodium dodecyl sulfate, and by applying 15 kV over a 67 cm (60 cm to the detector)x75 mu m fused-silica capillary. UV detection was at 335 nm. The validity of the method was assessed by investigating the identity of the an alytes, selectivity, limit of quantitation, linearity, within-day precision , extraction recovery, between-day precision and accuracy, electroosmotic f low stability and analyte stability. The method proved to be reproducible, sufficiently selective and accurate. Extraction recoveries of the analytes were >94%. The limit of quantitation (LOQ) was 2 mu g/ml and the assay was linear in the range 2-150 mu g/ml with correlation coefficients better than 0.999 for both glucuronides. The repeatability of the method, expressed as the ratio of corrected peak area of the analytes to that of I.S., gave RSD values of <5% even at the LOQ. Between-day precision (RSD) was <7.5% for b oth glucuronides at 7.5 mu g/ml. Determination of the glucuronide concentra tions in urine samples of 34 patients treated with entacapone either orally (200 mg) or intravenously (25 mg) showed the method to be suitable for mon itoring the concentrations of the glucuronide of entacapone after both oral and intravenous administration and those of the glucuronide of its (Z)-iso mer after oral administration. The limited long term stability of the syste m requires, however, frequent recalibration in applications involving long sample series. (C) 1999 Published by Elsevier Science B.V. All rights reser ved.