Analytical methods are described for the selective, rapid and sensitive determination of R- and S-
apomorphine,
apocodeine and
isoapocodeine and the
glucuronic acid and
sulfate conjugates in plasma and urine. The methods involve liquid-liquid extraction followed by high-performance liquid chromatography with electrochemical detection. The
glucuronide and
sulfate conjugates are determined after enzymatic hydrolysis. For the assay of R- and S-
apomorphine a 10 microm
Chiralcel OD-R column is used and the voltage of the detector is set at 0.7 V. The mobile phase 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 detection limits are 0.3 and 0.6 ng ml(-1) for R- and S-
apomorphine, respectively (signal-to-noise ratio 3). The intra- and inter-assay variations are <5% in the concentration range of 2.5-25 ng ml(-1) for plasma samples, and <4% in the concentration range of 40-400 ng ml(-1) for urine samples. For the assay of
apomorphine,
apocodeine and
isoapocodeine, a 5 microm C18 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 flow-rate of 0.8 ml min(-1) the total run time is ca. 14 min. The detection 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 conjugates of the various compounds are hydrolysed by incubation of the samples with
beta-glucuronidase and
sulfatase type H-1, respectively. Hydrolysis was complete after 5 h of incubation. No measurable degradation of
apomorphine,
apocodeine and
isoapocodeine occurred during the incubation. A pharmacokinetic study of
apomorphine, following the
intravenous infusion of 30 microg kg(-1) for 15 min in a patient with
Parkinson's disease, demonstrates the utility of the methods: both the pharmacokinetic parameters of the parent
drug and the appearance of
apomorphine plus metabolites in urine could be determined.