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 handing 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, collected in a 96-well plate format, can be directly injected onto the ESI/LC/MS/MS instrumentation. Chromatographic separation of the analytes was performed on a reverse-phase ODS column (TosoHaas ODS-80) with a mobile phase of acetonitrile/0.1% formic acid (5/95 v/v) at a flow rate of 0.22 ml/min. Analytes were detected by a triple-quadruple mass spectrometer equipped with an electrospray ionization source (ESI). Recoveries were evaluated for a number of pH modifiers and elution solvents. Under optimized conditions, the mean recoveries of L-dopa and dopamine were 56 and 67%, respectively. Intra-run and inter-run precision, calculated as percent relative standard deviation 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 percent error, was in the range -2.5 to 6.69% for both analytes. The limit of quantitaiton was 2.5 ng/ml for both L-dopa and dopamine when 100 microl of plasma 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.
Li
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Department of Pharmacokinetics, Dynamics, Metabolism, Pfiezer Global Research and Development, Ann Arbor, MI 48105, USA.
Rossi
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Fountain
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D000818:Animals; D001331:Automation; D002853:Chromatography, Liquid / Q000379:methods*; D004298:Dopamine / Q000097:blood*; D006863:Hydrogen-Ion Concentration; D007980:Levodopa / Q000097:blood*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D007980:Levodopa; D004298:Dopamine