Development and validation of a semi-automated method for L-dopa and dopamine in rat plasma using electrospray LC/MS/MS

A semi-automated alumina-based extraction method for the determination of L -dopa and dopamine in plasma using liquid chromatography/mass spectrometry was validated. The method exploited the use of a Tomtec Quadra 96 liquid ha nding robot to expedite aluminum oxide extraction for sample clean up. Two 96-well sample plates can be processed in less than 2 h and extracts, colle cted in a 96-well plate format, can be directly injected onto the ESI/LC/MS /MS instrumentation. Chromatographic separation of the analytes was perform ed on a reverse-phase ODS column (TosoHaas ODS-80) with a mobile phase of a cetonitrile/0.1%, formic acid (5/95 v/v) at a flow rate of 0.22 ml/min. Ana lytes were detected by a triple-quadruple mass spectrometer equipped with a n electrospray ionization source (ESI). Recoveries were evaluated for a num ber of pH modifiers and elution solvents. Under optimized conditions. the m ean recoveries of L-dopa and dopamine were 56 and 67%, respectively. Intra- run and inter-run precision, calculated as percent relative standard deviat ion of replicate quality controls, was in the range of 1.45-10.8% for both L-dopa and dopamine. Intra-run and inter-run accuracy, calculated as percen t error, was in the range - 2.5 to 6.69% for both analytes. The limit of qu antitaiton was 2.5 ng/ml for both L-dopa and dopamine when 100 mul of plasm a was extracted. The method is simple, rapid, accurate and suitable for the quantification of L-dopa and dopamine in plasma or other biological fluid samples from clinical, preclinical, or pharmacological studies. (C) 1000 El sevier Science B.V. All rights reserved.