Determination of l-dopa, carbidopa, 3-O-methyldopa and entacapone in human plasma by HPLC–ED

The aim of the study was the development of analytical methods suitable for the quantification of l-dopa, carbidopa and entacapone in plasma of Parkinsonian patients treated with Stalevo®. The metabolite 3-O-methyldopa was also determined to obtain some indications on the pharmacokinetics of l-dopa.For the simultaneous analysis of l-dopa, 3-O-methyldopa and carbidopa, a RP C18 column as the stationary phase and a mixture of methanol and a pH 2.88 phosphate buffer (8:92, v/v) as the mobile phase were used. A feasible plasma pre-treatment based on protein precipitation was implemented, obtaining extraction yield higher than 94% for all the analytes. For the analysis of entacapone a RP C8 column and a mixture of methanol, acetonitrile and a pH 1.90 phosphate buffer as the mobile phase (17.5:22.5:60, v/v/v) were used. A plasma pre-treatment procedure was developed, based on solid phase extraction of entacapone using Oasis HLB cartridges. Extraction yields were good, being always higher than 96%.Both methods, based on HPLC–ED (V = +0.8 V), have been fully validated. Good linearity was obtained over the following concentration ranges: 100–4000 ng mL−1 for l-dopa, 200–10,000 ng mL−1 for 3-O-methyldopa, 25–4000 ng mL−1 for carbidopa and 20–4000 ng mL−1 for entacapone. Precision data were satisfactory, being R.S.D.% values lower than 5.64%; accuracy also resulted very good with recovery data higher than 90%. The proposed methods have been successfully applied to the analysis of patient plasma samples and seem to be suitable for therapeutic drug monitoring purposes.