Determination of 3,4-methylenedioxymethamphetamine and its five main metabolites in rat urine by solid-phase extraction and high performance liquid chromatography with on line mass spectrometry

The consumption of psychostimulant amphetamine-like drugs has increased significantly in recent years. Some MDMA metabolites are probably involved in the neurotoxicity and neurodegeneration caused by prolonged use rather than MDMA itself. We recently developed a method to analyze MDMA and its five main metabolites in rat plasma [7]. We have now fully validated this method to the quantification of these drugs in rat urine. We extracted MDMA and its metabolites with Oasis WCX cartridges, separated them on a Nucleodur C18 analytical column and quantified them by ion-trap mass spectrometry. Linearity was excellent: 12.5–1250 ng/mL urine for HMA, HMMA, MDA and MDMA, 25–2500 ng/mL for HHMA, and 150–7500 ng/mL for HHA (r² > 0.993 for all analytes). The lower limits of quantification were 12.5 ng/mL urine for MDMA, MDA, HMA and HMMA, 25 ng/mL for HHMA and 150 ng/mL for HHA. Reproducibility was good (intra-assay precision = 1.7–6.1%; inter-assay precision = 0.6–5.7%), as was accuracy (intra-assay deviation = 0.1–4.8%; inter-assay deviation = 0.7–7.9%). Average recoveries were around 85.0%, except for HHMA (66.2%) and HHA (53.0%) (CV < 8.3%). We also checked the stability of stock solutions and the internal standards after freeze-thawing and in the autosampler. Lastly, we measured the MDMA, MDA, HHMA, HHA, HMMA and HMA in urine samples taken over 24 h from rats given subcutaneous MDMA.     

Toxicological detection of the designer drug 3,4-methylenedioxyethylamphetamine (MDE, “Eve”) and its metabolites in urine by gas chromatography — mass spectrometry and fluorescence polarization immunoassay

Studies are presented on the toxicological detection of the designer drug methylenedioxyethylapphetamine [MDE, rac-N-ethyl-(3,4-methylenedioxyphenyl)-propane-2-amine] in urine after a single oral dose of 140 mg of MDE by GC-MS and fluorescence polarization immunoassay (FPIA). After acid hydrolysis, extraction and acetylation MDE and its metabolites could be detected by mass chromatography with the selected ions m/z 72, 86, 114, 150, 162 and 164, followed by identification of the peaks underlying full mass spectra by computer library search. The following metabolites could be detected: unchanged MDE and 3,4-dihydroxyethylamphetamine (DHE) for 33,62 h, 3,4-methylenedioxyamphetamine (MDA) for 32–2036 h, and 4-hydroxy-3-methoxyethylamphetamine (HME) for 7 4-hydroxy-3-methoxyamphetamine (HMA), piperonul aceton, 3,4-Dihydroxyphenyl acetone and 4-hydroxy-3-methoxy-phenyl acetone could only be detected in trace amounts within the first few hours. The Abbott TD_x FPIA assay amphetamine/metamphetamine II gave positive results in urine for 33--62 h. Therefore, positive immunoassay results could be confirmed by the GC-MS procedure which also allowed the differentiation of MDE and its homologues 3,4-methylenedioxymethamphetamine (MDMA) and MDA as well as other amphetamine derivatives interfering with the TD_x assay. Furthermore, this GC-MS procedure allowed the simultaneous detection of most of the toxicologically relevant drugs.     

Simultaneous quantitation of ephedrines in urine by gas chromatography–nitrogen–phosphorus detection for doping control purposes

A gas chromatographic method for the simultaneous quantitation of ephedrine, pseudoephedrine, norephedrine (phenylpropanolamine), norpseudoephedrine (cathine) and methylephedrine in urine is described. The method consists of a liquid–liquid extraction with tert.-butyl methyl ether at pH 14. The extracts are analysed on a GC system equipped with an Rtx-5 Amine column and a nitrogen–phosphorus detector. Method validation shows excellent separation, linearity, specificity, accuracy, precision, intra-laboratory repeatability and reproducibility, making the method especially suitable for quantitation of ephedrines in urine samples for doping control purposes. A statistical analysis on the abuse of the different ephedrines in urine from athletes controlled in the Flemish doping control laboratory during the period 1993–2000 is included.     

Analysis of cyclophosphamide and five metabolites from human plasma using liquid chromatography–mass spectrometry and gas chromatography–nitrogen–phosphorus detection

An assay method for the quantification of cyclophosphamide (CY) and five metabolites from human plasma is presented. The procedure is adapted to the chemical properties of the compounds of interest: non-polar compounds are extracted into methylene chloride, concentrated and analyzed by GC–NPD after derivatization, and the remaining aqueous fraction is deproteinated with acetonitrile–methanol prior to separation via reversed-phase HPLC and detection using atmospheric pressure ionization (API)-MS. Standard curves are linear over the required range and reproducible over five months. Plasma concentration–time profiles of CY and metabolites from a patient receiving CY by intravenous infusion (60 mg/kg, once a day for two days) are presented.     

Quantitation of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma by high-performance liquid chromatography and fluorescence detection

A HPLC method has been developed for the analogue of Ecstasy MDE and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA) in human plasma. In the course of our investigations we found that the methylenedioxyamphetamines and HME exhibit fluorescence at 322 nm. Therefore the detection could be carried out with a fluorescence (FL) detector. Solid-phase extraction was used for sample preparation and yielded high recovery rates greater than 95%. The limit of quantitation for MDE and its metabolites in the extracts was between 1.5 and 8.9 ng/ml and the method standard deviations were less than 5%. This sensitive, rapid and reliable analytical method has been used successfully in the quantitation of the substances in plasma samples obtained from 14 volunteers in two clinical studies after p.o. administration of 100 to 140 mg MDE*HCl. The maximum plasma concentrations were 235–465 ng/ml (MDE), 67–673 ng/ml (HME) and 7–33 ng/ml (MDA), respectively. Pharmacokinetic parameters have been investigated using the plasma concentration curves.     

Quantification of ketotifen and its metabolites in human plasma by gas chromatography mass spectrometry

Gas chromatography mass spectrometry with selected ion monitoring has been used to develop a method for the quantification of ketotifen and its demethylated, 10-hydroxy and 10-hydroxy demethylated metabolites in human plasma. The minimum detectable concentrations for ketotifen and its demethylated metabolites were 50 pg ml-1 and 300 pg ml-1 for the 10-hydroxy metabolite. The methodology has been applied in studies of the kinetics of the drug in man, and plasma levels of the unchanged drugs and its metabolites in free and conjugated form are reported.     

On-line identification of 3,4-methylenedioxymethamphetamine in human urine by non-aqueous capillary electrophoresis–fluorescence spectroscopy at 77 K

The analytical profiles for 3,4-methylenedioxymethamphetamine (3,4-MDMA) and related amphetamines in urine samples are described for non-aqueous capillary electrophoresis–fluorescence spectroscopy. 3,4-MDMA was detected and identified on-line, using a cryogenic molecular fluorescence technique at 77 K. Under optimized conditions, baseline separation of the selected compounds was achieved in less than 12 min. Precision was evaluated by measuring the repeatability and intermediate precision of the migration times and corrected peak areas. The non-aqueous CE separation conditions and the spectral characteristics of 3,4-MDMA with respect to solvent and temperature effects are also discussed.     

Determination of sulfinpyrazone and two of its metabolites in human plasma and urine by gas chromatography and selective detection

A selective and sensitive gas chromatographic method for simultaneous determination of sulfinpyrazone and two of its metabolites (the para-hydroxylated metabolite and the sulfone metabolite) in biological fluids using alkali flame ionization detection (AFID), electron capture detection (ECD) and mass fragmentographic detection is described. The compounds are extracted from the samples, methylated and separated on 2% OV-17 or 8% OV-225 columns. Phenylbutazone is used as internal standard. Standard curves are linear. The coefficient of variation at 10 μg/ml of sulfinpyrazone in plasma was shown to be 1.8% (AFID), and the detection limits were 0.1 μg/ml (AIFD) and 10 ng/ml (ECD). Mass spectra of the methylated compounds are shown and serum concentration curves after oral administration of 100 mg sulfinpyrazone to two persons are determined together with the excreted amounts of drug and metabolites.     

Quantification of phenytoin and its metabolites in equine plasma and urine using high-performance liquid chromatography

A reliable and sensitive method for the extraction and quantification of phenytoin (5,5′-diphenylhydantoin), its major metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and minor metabolite, 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) in horse urine and plasma is described. The method involves the use of solid-phase extraction (SPE), liquid–liquid extraction (LLE), enzyme hydrolysis (EH) and high-performance liquid chromatography (HPLC). The minor metabolite, 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) was not present in a reliably quantifiable concentration in all samples. The new method described was successfully applied in the pharmacokinetic studies and elimination profile of phenytoin and p-HPPH following oral or intravenous administration in the horse.     

Carrier-Mediated Release of Serotonin by 3,4-Methylenedioxymethamphetamine: Implications for Serotonin-Dopamine Interactions

Abstract: In vivo microdialysis was used to determine whether the 3,4-methylenedioxymethamphetamine (MDMA)-induced release of serotonin (5-HT) in vivo involves a carrier-mediated process and to investigate further the state-dependent interaction between 5-HT and dopamine. MDMA produced a dose-dependent increase in the extracellular concentration of 5-HT in the striatum and prefrontal cortex that was attenuated by treatment with fluoxetine but not by tetrodotoxin. Suppression by fluoxetine of the MDMA-induced release of 5-HT was accompanied by a suppression of the MDMA-induced release of dopamine. Administration of MDMA to rats treated with carbidopa and l -5-hydroxytryptophan resulted in a synergistic elevation of the extracellular concentration of 5-HT that was much greater than that produced by either treatment alone. The MDMA-induced release of dopamine by MDMA also was potentiated in 5-hydroxytryptophan-treated rats. These data are consistent with the view that MDMA increases the extracellular concentration of 5-HT by facilitating carrier-mediated 5-HT release, which can be enhanced greatly under conditions in which 5-HT synthesis is stimulated. Moreover, these data are supportive of a state-dependent, stimulatory role of 5-HT in the regulation of dopamine release.     

Determination of chloral hydrate and its metabolites in blood plasma by capillary gas chromatography with electron capture detection

A sensitive, accurate, and reliable method is described for the quantitative determination of chloral hydrate (CH) and its metabolites in blood plasma of mice and rats. Metabolites of CH include trichloroacetic acid (TCA), trichloroethanol (TCE), and trichloroethanol glucuronide (TCE-Glu). This new method uses capillary gas chromatography with electron-capture detection (GC/ECD). Procedures for improving sample stability and quality assurance are also described that were not mentioned in previous literature. Rat or mouse plasma (50 microl) is acidified (or treated enzymatically for TCE-Glu determination) and extracted with peroxide free methyl t-butyl ether. Distilled diazomethane (CH(2)N(2)) is added to derivatize TCA to its methyl ester. Detection limits were estimated at 0.2 microg/ml for CH and TCE, and 0.1 microg/ml for TCA. Detector response to TCA and TCE were shown to be linear in the range of 3.125-200 microg/ml (r> or =0.9996). For CH, the response fits a second-order equation in this same range (r=0.99994)     

Determination of pethidine and its major metabolites in human urine by gas chromatography

Procedures based on gas chromatography were established to determine pethidine and its major metabolites in human urine. The chromatographic system consisted of a glass column packed with 3% (w/w) SP2250 on Chromosorb W (80–100 mesh) linked to a nitrogen—phosphorus detector. Diethyl ether was used as the extraction solvent. Pethidinic and norpethidinic acids, and their conjugated metabolites (after β-glucuronidase treatment) were determined after conversion into pethidine and norpethidine by acid-catalysed esterification. The retention times of pethidine, norpethidine and chlorpheniramine (internal standard) were 3.3, 4.5 and 7.5 min, respectively. The amount of unchanged drugs and metabolites excreted varied considerably among the subjects. The mean 24-h urinary recoveries in eight patients of pethidine, norpethidine, pethidinic acid, norpethidinic acid, and the glucuronides of pethidinic and norpethidinic acids were 6.62 ± 5.05, 4.33 ± 1.19, 18.9 ± 6.29, 9.10 ± 4.26, 15.1 ± 3.02 and 7.57 ± 2.28%, respectively. This indicates that the major metyabolic pathways of pethidine in the eight patients were hydrolysis followed by conjugation. Over 60% of the dose was accounted for in 24 h after intramuscular administration of 1 mg/kg pethidine.     

Simultaneous determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by capillary gas chromatography with electron-capture detection

A method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites (2- and 5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed. Two internal standards were used: isomannide dinitrate (IMDN) for the determination of ISDN and isomannide mononitrate (IMMN) for the determinations of 2- and 5-ISMN. After addition of the internal standards, the compounds were isolated from plasma by solid-liquid extraction. They were determined by gas chromatography using an electron-capture detector. The reproducibility and accuracy of the method were found suitable in the range of concentrations 2.5–83 ng/ml for ISDN, 2.6–208 ng/ml for 2-ISMN and 2.3–1010 ng/ml for 5-ISMN. The limit of quantitation (LOQ) was about 2.5 ng/ml for each compound. The method was applied to clinical samples.     

Effect of 3,4-Methylenedioxymethamphetamine on 3,4-Dihydroxyphenylalanine Accumulation in the Striatum and Nucleus Accumbens

The effect of the racemic mixture of 3,4-methylenedioxymethamphetamine (MDMA) on the synthesis of dopamine in the terminals of nigrostriatal and mesolimbic neurons was estimated by measuring the accumulation of 3,4-dihydroxyphenylalanine (DOPA) in the striatum and nucleus accumbens 30 min following the administration of the L-aromatic amino acid decarboxylase inhibitor, 3-hydroxybenzylhydrazine. MDMA produced an increase in DOPA accumulation in the striatum which was greater in magnitude and longer in duration than that in the nucleus accumbens. Although the concentrations of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in both the striatum and nucleus accumbens were reduced 3 h following an injection of MDMA (20 mg/kg), 5-HT and 5-HIAA concentrations were significantly reduced only in the striatum 7 days after the administration of MDMA. Pretreatment with a 5-HT2 antagonist, ketanserin, significantly attenuated the reduction in 5-HT concentration in the striatum 3 h following MDMA administration and completely blocked 5-HT depletion at 7 days post administration. Moreover, ketanserin completely blocked MDMA-induced DOPA accumulation in the striatum. The results obtained in these studies suggest that MDMA activates nigrostriatal dopaminergic pathways via 5-HT2 receptors. In addition, these data are supportive of the hypothesis that dopamine plays a role in MDMA-induced 5-HT depletion.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

Determination of theophylline and its metabolites in human urine and plasma by high-performance liquid chromatography

A method for the quantitation of theophylline (13DMX) and the three metabolites, 1-methyluric acid (1MU), 3-methylxanthine (3MX) and 1,3-dimethyluric acid (13DMU) in human plasma and urine has been developed. The method is based on a simple one-step liquid-liquid extraction with ethylacetate-2 propanol followed by isocratic, reversed-phase high-performance liquid chromatography with UV detection (detection wavelength: 273 nm). The overall mean recoveries ranged from 86 to 95% for the four compounds. The detection limit was 1 μm for 1MU, 3MX and 13DMU and 2 μM for 13DMX in urine, and 0.1 μM for 1MU, 3MX and 13DMU and 0.2 μM for 13DMX in plasma. The intra-day and inter-day coefficient of variation was <6% and <9%, respectively, and the accuracy was within ±10% in both urine and plasma.The simple but sensitive method is highly suitable for the development of theophylline as a probe drug for assessing CYP1A2 activity in man.     

Quantification of BNP7787 (dimesna) and its metabolite mesna in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A sensitive and accurate assay was developed and validated to determine BNP7787 (dimesna), a new protector against cisplatin-induced toxicities, and its metabolite mesna in plasma and urine of patients. Both analytes were measured as mesna in deproteinized plasma or in urine diluted with mobile phase using high-performance liquid chromatography with an electrochemical detector provided with a wall-jet gold electrode. The assays for BNP7787 and mesna in deproteinized plasma were linear over the range of 1.6–500 μM and 0.63–320 μM, respectively. In plasma, the mean recovery of BNP7787 over the whole concentration range was 100.6% and of mesna 94.6%. The lower limits of quantitation (LLQs) of BNP7787 and mesna in deproteinized plasma were 1.6 μM and 0.63 μM, respectively. For both compounds the within- and between-day accuracy and precision of the assay was better than 12%. The assays for BNP7787 and mesna in urine were linear over the range of 0.8–1200 μM and 0.63–250 μM, respectively. In urine, the mean recovery of BNP7787 over the whole concentration range was 94.1% and of mesna 93.1%. The LLQ of BNP7787 in urine was 0.8 μM and of mesna 1.6 μM. The within- and between-day accuracy and precision of the assay for BNP7787 and mesna was lower than 15%. The stability of mesna in urine increased with an increasing concentration of mesna, lower temperature and addition of EDTA (1 g/l) and hydrochloric acid (0.2 M). BNP7787 in urine was stable for at least 24 h at temperatures in the range of −20°C up to 37°C and independent of the concentration. The developed assays are currently applied for samples of patients with solid tumors participating in a phase I trial of BNP7787 in combination with cisplatin.     

Mazindol Attenuates the 3,4-Methylenedioxymethamphetamine-Induced Formation of Hydroxyl Radicals and Long-Term Depletion of Serotonin in the Striatum

The formation of hydroxyl radicals following the systemic administration of 3,4-methylenedioxymethamphetamine (MDMA) was studied in the striatum of the rat by quantifying the stable adducts of salicylic acid and D-phenylalanine, namely, 2,3-dihydroxybenzoic acid (2,3-DHBA) and p-tyrosine, respectively. The repeated administration of MDMA produced a sustained increase in the extracellular concentration of 2,3-DHBA and p-tyrosine, as well as dopamine. The MDMA-induced increase in the extracellular concentration of both dopamine and 2,3-DHBA was suppressed in rats treated with mazindol, a dopamine uptake inhibitor. Mazindol also attenuated the long-term depletion of serotonin (5-HT) in the striatum produced by MDMA without altering the acute hyperthermic response to MDMA. These results are supportive of the view that MDMA produces a dopamine-dependent increase in the formation of hydroxyl radicals in the striatum that may contribute to the mechanism whereby MDMA produces a long-term depletion of brain 5-HT content.