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- RESULTS AND DISCUSSION
A method was developed and validated for the analysis of R(−)-apomorphine, (R−)-apocodeine and R(−)-norapomorphine in human plasma and urine with N-propylnorapomorphine as internal standard using gas chromatography/mass spectrometry (GC/MS) and single-ion monitoring after a single liquid–liquid extraction and silylation of compounds. The quantification limits were 1 ng/ml for apomorphine and apocodeine and 25 ng/ml for norapomorphine. Calibration curves were linear, within the range 1–100 ng/ml. Variation in intraday and interday precision was below 10%. This method was applied to study apomorphine bioavailability in nine patients with Parkinson's disease before and after coadministration of a catechol-O-methyl transferase inhibitor. Copyright © 2005 John Wiley & Sons, Ltd.
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- RESULTS AND DISCUSSION
Apomorphine (APO) is a powerful nonselective agonist of dopaminergic receptors that is used to treat refractory motor complications of Parkinson's disease in late-stage levodopa therapy by reducing the incidence and duration of the ‘off’ periods.1, 2 Owing to its limited oral bioavailability, APO is commonly administered by the subcutaneous or sublingual route. APO is metabolized through glucuronidation and methylation. O-methylation by catechol-O-methyl transferase (COMT) produces the less active metabolite apocodeine (APC) in extrahepatic tissues (Fig. 1). Other metabolic pathways include N-demethylation, leading to the potentially active metabolite R(−)-norapomorphine (NPO) in the liver, oxidation into O-quinone and interconversion of R(−)-APO into S(−)-APO and glucuronide and sulfate conjugation.3–5 The relative importance of these different metabolic pathways in man remains unknown. In the rat, administration of COMT inhibitors prolongs the duration of APO-induced stereotypic behaviors, suggesting that this coadministration could increase APO bioavailability.6 In order to evaluate human pharmacokinetics of APO before and after administration of drugs interacting with APO metabolism, a quantification method to assess APO, APC and NPO levels had to be developed.
Many analytical methods described in the literature demonstrated low APO detection levels,4, 7–14 but few quantified APC and none assessed NPO in plasma or urine. High-performance liquid chromatography (HPLC) with various detection methods as well as gas chromatography (GC) have been used extensively since 1979. Using coulometric detection, Nicolle et al.8 and Sam et al.9 detected low levels of APO but no metabolites. In 1997, Bolner et al. detected no metabolite using electrochemical detection.10 With the same detector, Van der Geest et al.4 quantified APO enantiomers and APC, but with a nevertheless high limit of detection (LOD). Using a GC method coupled with flame ionization detection, Baaske et al.13 analyzed APO, APC and iso-APC. Using mass spectrometric (MS) detection, Watanabe et al.14 detected APO but no APC or NPO.
This report describes a GC/MS method to analyze APO, NPO and APC in plasma and urine using rapid, single-step, liquid–liquid extraction to assess the metabolism of APO. This method was validated and used to investigate the kinetics of APO before and after administration of a COMT inhibitor, entacapone.
RESULTS AND DISCUSSION
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- RESULTS AND DISCUSSION
Our goal was to develop a convenient analytical method for processing APO, APC and NPO, as there is only limited information on APO metabolism in the literature. Analysis of APO and metabolites is made difficult by the rapid degradation of APO by oxidation at 37 °C and pH 7.4, necessitating the use of antioxidants such as disodium edetate, ascorbic acid or sodium metabisulfite to increase the stability of APO in the samples during all extraction phases. Therefore, in this study, blood was always collected in tubes containing disodium edetate and centrifuged at 4 °C within 30 min of collection. This paper is the first to describe not only APO but also APC and NPO in the same analytical run. Extraction was derived from general conditions of extraction described by Bianchi G. and Landi M.11 The nature of the organic extraction solvents, the pH value of the solution and the extraction steps were precisely studied. Initial attempts to use one organic solvent such as chloroform, diethyl ether, ethyl acetate, hexane or methanol with different pH phosphate buffers (pH 7, 8, 9, 10 and 11) were unsuccessful. None of the solvents permitted the extraction of both target compounds. It must be emphasized that hexane was never able to extract NPO. Owing to the problem of cross contamination inducing significant interferences in the chromatograms, such as phthalates leached from plastic materials like Vacutainer® tubes and serum storage bottles, we avoided the use of chlorinated solvents. Therefore, various mixtures of two solvents were tested. The best results were obtained with ethyl acetate, especially with the diethyl ether/ethyl acetate mixture. The extraction pH was determined at a value where the analytes are uncharged prior to extraction and to avoid pollutant acidic compounds and phthalates.
Derivatization was completed by silylation with BSTFA and TMCS in carefully closed tubes. As shown in Fig. 2, chromatographic separation of target substances was achieved in 8.4, 8.9, 9.1 and 9.8 min for APO, APC, NPO and IS, respectively. Characteristic fragmentations of the structures were obtained from the reports published by Green et al.15 and Neumeyer et al.16 Molecular ions of BSTFA-TMCS derivative of APO used for quantification and qualification had a mass spectrum of m/z 410 and 411, 322 and 149, respectively. APC quantification ion was m/z 352 with ions m/z 322, 310 and 264 for qualification. NPO quantification and qualification ions were m/z 396, 322 and 308, respectively. N-propylnorapomorphine was quantified using ion m/z 438 and qualified by ions m/z 410 and 350.
Figure 2. Reconstituted chromatogram of an extracted calibration standard in plasma with specific ions of R(−)-apomorphine (m/z 410), R(−)-norapomorphine (m/z 438) and R(−)-apocodeine (m/z 352) (25 ng/ml each) by GC/MS. Chromatographic separations were achieved on an HP-5 MS column (30 m × 0.25 mm i.d.; 0.25-µm film thickness. Injector temperature was 260 °C; detector temperature was 290 °C. Oven temperature programming was 60–245 °C at 70 °C/min, 245–265 °C at 20 °C/min, and 265–285 °C at 70 °C/min.
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Analytical recoveries were determined from control samples whose alkaline pH was previously checked. The values obtained in these conditions were between 95.0 and 106.2% for APO and APC, respectively, but only 22.0% for NPO. Physical losses of NPO could occur from the sample during the various steps, especially during partition or washing, or by adsorption onto glass walls of containers. This low recovery rate is a commonly encountered problem in the analyses of other basic drugs, such as tricyclic antidepressants. This classical interaction of lipophilic drugs may be prevented by silanization of tubes, and the losses during the evaporation steps may be avoided by carefully stopping the tubes. The unsubstituted nitrogen at position 6 in NPO may also have an acidic character at high pH values, giving an ionic character and thus decreasing the partitioning into the organic phase.
As shown in Table 1, calibration curves were linear from 1 to 100 ng/ml for APO and APC and for 25 to 100 ng/ml for NPO, with satisfactory correlation coefficients. Both intraday and interday reproducibility values were satisfactory, with CVs in the ranges 2.18–8.28 and 0.82–9.30%, respectively (Table 2).
Table 1. Linearity data of apomorphine (APO), apocodeine (APC) and norapomorphine (NPO) determination in plasma and urines. All concentrations are expressed in ng/ml
|Concentration range||Slope||Y-intercept||R2||Concentration range||Slope||Y-intercept||R2|
|APO||1–100||6.1 × 10−4 ± 3.6 × 10−5||−2.0 × 10−3 ± 1.8 × 10−3||0.997 ± 0.003||5–100||0.5 × 10−3 ± 1.6 × 10−3||−5.2 × 10−4 ± 1.3 × 10−3||0.999 ± 0.001|
|APC||1–100||2.3 × 10−3 ± 2.3 × 10−4||−4.4 × 10−4 ± 3.2 × 10−3||0.996 ± 0.004||5–100||0.34 × 10−3 ± 1.10 × 10−3||−6.35 × 10−4 ± 6.40 × 10−3||0.998 ± 0.001|
|NPO||25–100||5.0 × 10−3 ± 7.9 × 10−4||−7.5 × 10−4 ± 2.3 × 10−3||0.996 ± 0.010||25–100||7.06 × 10−4 ± 1.68 × 10−4||1.34 × 10−2 ± 1.96 × 10−3||0.994 ± 0.006|
Table 2. Inter- and intraday reproducibility of apomorphine (APO), apocodeine (APC) and norapomorphine (NPO) determination in plasma and urines. All concentrations are expressed in ng/ml
|Added concentration||Calculated concentration||SEM||CV||Accuracy||Calculated concentration||SEM||CV||Accuracy|
|Interday reproducibility (N = 8)|
|Intraday reproducibility (N = 8)|
The LOQ was established as the lowest point of the calibration graph, i.e. 1 ng/ml for APO and APC and 25 ng/ml for NPO. The LODs were 0.25, 0.25 and 5 ng/ml for APO, APC and NPO, respectively. Compared to previously published data for APO and its metabolites, this method is one of the most sensitive. Some HPLC methods, used with electrochemical9–11 or fluorescence7 detection, demonstrated similar sensitivity for APO, but none of them were as sensitive for APC quantification. Moreover, our method is the first-described analytical method allowing quantification of NPO.
In the pharmacokinetic study, the two-compartment model and parameters were similar to those previously described. T1/2 and tmax were 61.6 ± 16.4 and 20 ± 8 min in our study compared to 69.7 ± 25.8 and 16 ± 11 min in the study of Nicolle et al.8, respectively. No significant difference was observed in APO plasma Cmax and AUC levels after entacapone administration compared with control patients (37.7 ± 18.7 versus 25.05 ± 14.3 ng/ml and 2258 ± 543 versus 2164 ± 456 ngmin/ml). Whatever the sampling times, no measurable APC or NPO levels were detected in plasma, as previously shown in the literature.17 Urinary amounts of APO excreted during the first 6 h were similar between entacapone-treated patients and nontreated patients (622 ± 104 versus 557 ± 105 ng/ml), but APC amounts and on a lower level NPO ones, were increased (369 ± 210 versus 109 ± 77 ng/ml and 384 ± 150 versus 217 ± 104 ng/ml, respectively). These results confirm the importance of metabolism of APO giving a lot of conjugated metabolites and the low percentage of APO excreted unchanged in the urine. However, it also emphasized the great, but not studied so far, contribution of NPO to the elimination of APO2–4 and the effect of entacapone on the APO metabolism, although nonreflected in blood probably because of the significance of the conjugation reactions of APO metabolites.
In conclusion, we established a new reliable analytical method for APO, APC and NPO quantification in plasma and urine by the complementary use of a liquid–liquid extraction and a GC/MS method. A large pool of kinetic data has been generated to date. Future analysis and interpretation of these data will be the subject of a forthcoming article.