Abstract
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 μm 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 μm 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 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 β-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 μg 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.
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