Assay of R-apomorphine, S-apomorphine, apocodeine, isoapocodeine and their glucuronide and sulfate conjugates in plasma and urine of patients with Parkinson's disease.
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.
van der Geest
R
R
Leiden/Amsterdam Center for Drug Research, Division of Pharmacology, Sylvius Laboratory, Leiden, The Netherlands.
Kruger
P
P
Gubbens-Stibbe
J M
JM
van Laar
T
T
Bodde
H E
HE
Danhof
M
M
D019342:Acetic Acid / Q000494:pharmacology; D000978:Antiparkinson Agents / Q000008:administration & dosage / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D001058:Apomorphine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002940:Circadian Rhythm; D005965:Glucuronates / Q000652:urine; D020723:Glucuronic Acid; D005966:Glucuronidase / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007262:Infusions, Intravenous; D016014:Linear Models; D008297:Male; D010300:Parkinson Disease / Q000097:blood* / Q000378:metabolism / Q000652:urine*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D013429:Sulfatases / Q000378:metabolism; D013431:Sulfates / Q000652:urine; D013997:Time Factors
D000978:Antiparkinson Agents; D005965:Glucuronates; D013431:Sulfates; C044752:isoapocodeine; D020723:Glucuronic Acid; D013429:Sulfatases; D005966:Glucuronidase; D001058:Apomorphine; C006613:apocodeine; D019342:Acetic Acid