ASSAY OF R-APOMORPHINE, S-APOMORPHINE, APOCODEINE, ISOAPOCODEINE AND THEIR GLUCURONIDE AND SULFATE CONJUGATES IN PLASMA AND URINE OF PATIENTS WITH PARKINSONS-DISEASE

Analytical methods are described for the selective, rapid and sensitiv e determination of R- and S-apomorphine, apocodeine and isoapocodeine and the glucuronic acid and sulfate conjugates in plasma and urine. Th e methods involve liquid-liquid extraction followed by high-performanc e liquid chromatography with electrochemical detection. The glucuronid e and sulfate conjugates are determined after enzymatic hydrolysis. Fo r the assay of R- and S-apomorphine a 10 mu m Chiralcel OD-R column is used and the voltage of the detector is set at 0.7 V. The mobile phas e is a mixture of aqueous phase (pH 4.0)-acetonitrile (65:35, v/v). At a flow-rate of 0.9 ml min(-1) the total run time is ca. 15 min. The d etection limits are 0.3 and 0.6 ng ml(-1) for R- and S-apomorphine, re spectively (signal-to-noise ratio 3). The intra- and inter-assay varia tions are <5% in the concentration range of 2.5-25 ng ml(-1) for plasm a samples, and <4% in the concentration range of 40-400 ng ml(-1) for urine samples, For the assay of apomorphine, apocodeine and isoapocode ine, a 5 mu m C-18 column was used and the voltage of the detector set at 0.825 V. Ion-pairing chromatography was used. The mobile phase is a mixture of aqueous phase (pH 3.0)-acetonitrile (75:25, v/v). At a fl ow-rate of 0.8 ml min(-1) the total run time is ca. 14 min, The detect ion limits of this assay are 1.0 ng ml(-1) for apomorphine and 2.5 ng ml(-1) for both apocodeine and isoapocodeine (signal-to-noise ratio 3) . The inter-assay variations are 5% in the concentration range of 5-40 ng ml(-1) for plasma samples and 7% in the concentration range of 50- 500 ng ml(-1) for urine samples. The glucuronic acid and sulfate conju gates of the various compounds are hydrolysed by incubation of the sam ples with beta-glucuronidase and sulfatase type H-1, respectively. Hyd rolysis was complete after 5 h of incubation. No measurable degradatio n of apomorphine, apocodeine and isoapocodeine occurred during the inc ubation. A pharmacokinetic study of apomorphine, following the intrave nous infusion of 30 mu g kg(-1) for 15 min in a patient with Parkinson 's disease, demonstrates the utility of the methods: both the pharmaco kinetic parameters of the parent drug and the appearance of apomorphin e plus metabolites in urine could be determined. (C) 1997 Elsevier Sci ence B.V.