Simultaneous determination of levodopa, its main metabolites and carbidopa in plasma by liquid chromatography

An ion-pair reversed-phase liquid chromatographic method for the simultaneous determination of levodopa, 3-O-methyldopa, 3,4-dihydroxyphenylacetic acid, homovanillic acid and carbidopa in plasma designed for clinical trials performed to study the effect of peripheral catechol-O-methyltransferase inhibitors on the metabolism of levodopa is described. The high sample throughput of over 50 samples per day of the method makes it ideal for the assay of the large number of samples encountered in clinical trials. After protein precipitation with perchloric acid the analytes are completely separated within 15 min and determined down to a plasma concentration of 20 ng ml-1 using amperometric detection at 800 mV relative to an Ag/AgCl reference electrode. For all analytes the within-day precision defined as a relative standard deviation (n = 8) is lower than 7 and 3% at plasma concentrations of 20 and 40 ng ml-1, respectively. As the method is specific and highly reproducible, the most important factor affecting accuracy is the stability of the analytes during storage and analysis.