Separation of the glucuronides of entacapone and its (Z)-isomer in urine by micellar electrokinetic capillary chromatography

A micellar electrokinetic capillary chromatography (MECC) method was develo ped for the separation of the 3-O-glucuronides of entacapone and its (Z)-is omer, the two main urinary metabolites of entacapone in humans. Entacapone is a novel, potent inhibitor of catechol-O-methyltransferase (COMT) intende d for use as an adjunct in the treatment of Parkinson's disease. Urine samp les spiked with synthetic 3-O-glucuronides were used to study the effects o f running buffer pH, composition and applied voltage on separation of the c losely migrating glucuronides. The 3-O-glucuronide of nitecapone, was used as internal standard. The greatest improvement in separation was achieved b y increasing the running buffer ionic concentration. Changes in pH had litt le effect on the separation, whereas increase in sodium dodecyl sulfate (SD S) concentration slightly improved resolution. Baseline separation and good selectivity relative to urine components were achieved by using a phosphat e (25 mM)-borate (50 mM)-SDS (20 mM) running buffer, pH 7.0, in a 75 mu m X 60/67 cm fused-silica capillary at 15 kV and a 335 nm cut-off filter in th e UV detector. The limits of detection (LOD) at a signal-to-noise ratio of 3 were about 0.25 mu g/ml (5.2.10(-7) M) (injection 0.5 p.s.i./8 s). The li near detection range was 2-100 mu g/ml (r(2) > 0.999). Good repeatability o f injection and relative migration times were obtained. (C) 1999 Elsevier S cience B.V. All rights reserved.