Simultaneous determination of levodopa, carbidopa and their metabolites inhuman plasma and urine samples using LC-EC

In this study levodopa (L-DOPA), carbidopa (C-DOPA) and their metabolites w ere resolved from other endogenous components present in human plasma and u rine and determined quantitatively. The developed technique involved the us e of a second pump, a switching valve, and a pre-column in the LC system in order to perform on-line sample clean-up and enrichment. This procedure is dependent on an effective removal of the many interfering matrix component s that vitiate HPLC analysis. Several unknown endogenous electroactive comp ounds, present in plasma, were eliminated by the purification step, or supp ressed by the pre-treatment or detection conditions. The analyses were sepa rated on an Octyl-bonded reversed-phase column followed by amperometric det ection using a carbon fibre microelectrode flow cell operated at +0.8 V ver sus silver/silver phosphate reference electrode. The cell was compatible wi th the mobile and the stationary phase used in the how system without any c omplex surface reaction. The peak currents obtained for the different analy tes were directly proportional to the analyse over the concentration range 0.02-4.0 mu g ml(-1). Using this method, the minimum detectable concentrati on was estimated to be 5 and s ng ml(-1) for L-DOPA and C-DOPA, respectivel y. Recovery studies performed on human plasma samples ranged from 93.83 to 89.76%, with a relative standard deviation of < 6%. The intra- and inter-as say coefficients of variation were < 7%. The accuracy of the assay, which w as defined as the percentage difference between the mean concentration foun d and the theoretical (true) concentration, was 12% or better. The electroc hemical pre-treatment regime described in this work permitted a longer appl ication of the same microelectrode. The method showed a good agreement with other available methods described in the introduction and offers the advan tages of being simple, less time and labour consuming, does not require add itional solvents for extraction, inexpensive and suitable for routine analy sis and kinetic purposes. (C) 2000 Elsevier Science B.V. All rights reserve d.