Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography-tandem mass spectrometry

A column-switching liquid chromatography-electrospray ionization-tandem mas s spectrometric (LC-ESI-MS-MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 mul) were injec ted onto a C-18-alkyl-diol silica (ADS) column and the matrix compounds wer e washed to waste with a mixture of 20 mM ammonium acetate solution at pH 4 .0-acetonitrile (97:3). The retained analyte fraction containing (E)- and ( Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (interna l standard) was backflushed to the analytical C-18 column, with a mixture o f 20 mM ammonium acetate-acetonirrile (85:15) for the final separation at p H 7.0. The eluate was directed to the mass spectrometer after splitting (1: 100). The mass spectrometer was operated in the negative ion mode and the d eprotonated molecules [M-H] were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M-H]- in MS-M S resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M-H-Glu](-) which were chosen as the product ions for single reaction monitoring. Quantitative studies show ed a wide dynamic range (0.0025-100 mug/ml) with correlation coefficients b etter than 0.995. The method was repeatable within-day (relative standard d eviation, RSD <7%) and between-day (RSD < 14%) and the recovery (78-103%) w as better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of (E) and (Z)-isomers of entacapone glucuronide in plasma of rats used in absorpt ion studies. (C) 2001 Elsevier Science B.V. All rights reserved.