The NTCP-inhibitor Myrcludex B: Effects on Bile Acid Disposition and Tenofovir Pharmacokinetics.

Myrcludex B acts as a hepatitis B and D virus entry inhibitor blocking the sodium taurocholate cotransporting polypeptide (SLC10A1). We investigated the effects of myrcludex B on plasma bile acid disposition, tenofovir pharmacokinetics, and perpetrator characteristics on cytochrome P450 (CYP) 3A. Twelve healthy volunteers received 300 mg tenofovir disoproxil fumarate orally and 10 mg subcutaneous myrcludex B. Myrcludex B increased total plasma bile acid exposure 19.2-fold without signs of cholestasis. The rise in conjugated bile acids was up to 124-fold (taurocholic acid). Coadministration of tenofovir with myrcludex B revealed no relevant changes in tenofovir pharmacokinetics. CYP3A activity slightly but significantly decreased by 29% during combination therapy. Myrcludex B caused an asymptomatic but distinct rise in plasma bile acid concentrations and had no relevant impact on tenofovir pharmacokinetics. Changes in CYP3A activity might be due to alterations in bile acid signaling. Long-term effects of elevated bile acids will require critical evaluation.

High-resolution mass spectrometry in toxicology: current status and future perspectives.

This paper reviews high-resolution mass spectrometry (HRMS) approaches using time-of-flight or Orbitrap techniques for research and application in various toxicology fields, particularly in clinical toxicology and forensic toxicology published since 2013 and referenced in PubMed. In the introduction, an overview on applications of HRMS in various toxicology fields is given with reference to current review articles. Papers concerning HRMS in metabolism, screening, and quantification of pharmaceuticals, drugs of abuse, and toxins in human body samples are critically reviewed. Finally, a discussion on advantages as well as limitations and future perspectives of these methods is included.

A systematic comparison of four different workup procedures for systematic toxicological analysis of urine samples using gas chromatography-mass spectrometry.

Sample preparation for systematic toxicological screening analysis (STA) in urine by gas chromatography-mass spectrometry (GC-MS) generally involves cleavage of conjugates by acid hydrolysis (Hy) or enzymatic hydrolysis (Gluc) followed by liquid-liquid extraction (LLE) or solid-phase extraction (SPE), and derivatization, e.g., acetylation (Ac). LLE and derivatization can be performed simultaneously, e.g., in extractive methylation (ExMe). The work presented consisted of two separate studies. In study I, 350 urine samples from 168 inpatients from an internal medicine ward were worked up by Hy-LLE-Ac, the standard workup in the authors' laboratory, Gluc-SPE-Ac, and Gluc-ExMe. In study II, 100 urine samples from psychiatric inpatients were worked up by Hy-LLE-Ac and Hy-SPE-Ac. The samples prepared were analyzed by full-scan GC-MS, and the drugs and/or their metabolites/artifacts detected after the different workup procedures were compared. The results obtained after Hy-LLE-Ac and Gluc-SPE-Ac showed only little differences, e.g., salicylic acid not being detectable with the latter. Hy-SPE-Ac covered a similar range of analytes as Hy-LLE-Ac but was much more time-consuming. Comparison of Hy-LLE-Ac and Gluc-ExMe showed that the former was better suited for basic drugs and the latter for acidic drugs, but the overlap was considerable. In conclusion, Hy-LLE-Ac remains the method of choice for STA in clinical toxicology owing to its wide analyte spectrum and short workup time. Gluc-ExMe is an ideal complementary method when acidics need to be covered. Gluc-SPE-Ac can be used as an alternative to Hy-LLE-Ac when turnaround is not critical or when automated analysis is required.

Identification of cytochrome P450 enzymes involved in the metabolism of 3',4'-methylenedioxy-alpha-pyrrolidinopropiophenone (MDPPP), a designer drug, in human liver microsomes.

The metabolism of 3',4'-methylenedioxy-a-pyrrolidinopropiophenone (MDPPP), a novel designer drug, to its demethylenated major metabolite 3',4'-dihydroxy-pyrrolidinopropiophenone (di-HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes. CYP2C19 catalysed the demethylenation with apparent Km and Vmax values of 120.0+/-13.4 microM and 3.2+/-0.1 pmol/min/pmol CYP, respectively (mean+/-standard deviation). CYP2D6 catalysed the demethylenation with apparent Km and Vmax values of 13.5+/-1.5 microM and 1.3+/-0.1 pmol/min/pmol CYP, respectively. HLM exhibited a clear biphasic profile with an apparent Km,1 value of 7.6+/-9.0 and a Vmax,1 value of 11.1+/-3.6 pmol/min/mg protein, respectively. Percentages of intrinsic clearances of MDPPP by specific CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4'-hydroxylation or bufuralol-1'-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively. MDPPP, di-HO-PPP and the standard 4'-methyl-pyrrolidinohexanophenone (MPHP) were separated and analysed by liquid chromatography-mass spectrometry in the selected-ion monitoring (SIM) mode. The CYP2D6-specific chemical inhibitor quinidine (3 microM) significantly (p<0.001) inhibited di-HO-PPP formation by 75.8%+/-1.7% (mean+/-standard error of the mean) in incubation mixtures with HLM and 2 microM MDPPP. It can be concluded from the data obtained from kinetic and inhibition studies that polymorphically expressed CYP2D6 and CYP2C19 are almost equally responsible for MDPPP demethylenation.

In vivo metabolism of the new designer drug 1-(4-methoxyphenyl)piperazine (MeOPP) in rat and identification of the human cytochrome P450 enzymes responsible for the major metabolic step.

1. The in vivo metabolism of 1-(4-methoxyphenyl)piperazine (MeOPP), a novel designer drug, was studied in male Wistar rats. 2. MeOPP was mainly O-demethylated to 1-(4-hydroxyphenyl)piperazine (4-HO-PP) in addition to degradation of the piperazine moiety. 3. O-demethylation, the major metabolic step, was studied with cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes in pooled human liver microsomes (pHLM) and in single donor human liver microsomes with CYP2D6 poor metabolizer genotype (PM HLM). 4. CYP2D6 catalysed O-demethylation with apparent Km and Vmax values of 48.34 +/- 14.48 microM and 5.44 +/- 0.47 pmol min(-1) pmol(-1) CYP, respectively. pHLM catalysed the monitored reaction with an apparent Km = 204.80 +/- 51.81 microM and Vmax = 127.50 +/- 13.25 pmol min(-1) mg(-1) protein. 5. The CYP2D6-specific chemical inhibitor quinidine (1 and 3 microM) significantly inhibited 4-HO-PP formation by 71.9 +/- 4.8% and by 98.5% +/- 0.5%, respectively, in incubation mixtures with pHLM and 200 microM MeOPP. 6. O-demethylation was significantly lower in PM HLM compared with pHLM (70.6% +/- 7.2%). 7. These data suggest that polymorphically expressed CYP2D6 is the enzyme mainly responsible for MeOPP O-demethylation.

Concentrations and ratios of amphetamine, methamphetamine, MDA, MDMA, and MDEA enantiomers determined in plasma samples from clinical toxicology and driving under the influence of drugs cases by GC-NICI-MS.

Enantiomers of amphetamine (AM), methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) exhibit different pharmacological properties. This may be important for the interpretation of analytical results. Plasma samples were analyzed using validated negative ion chemical ionization gas chromatography-mass spectrometry procedures. The results for clinical toxicology cases, divided into screening (SCR) and intoxication (ITX) cases, and those of driving under the influence of drugs (DUID) cases were compared. The concentrations of all enantiomers, except R-(-)-MDA and R-(-)- and S-(+)-MA, in the SCR samples were lower than in ITX and DUID samples. Differences between concentrations in ITX and DUID samples were only significant for both enantiomers of AM (DUID higher). These findings suggested impairment in drugged drivers. Different enantiomer ratios (R vs. S) were found for AM between DUID and SCR samples, for MDMA between ITX and SCR samples, and for MDA between DUID and ITX and DUID and SCR samples. Higher MDMA enantiomer ratios in SCR compared to ITX samples are in accordance with a previously described increase of those ratios over time, possibly allowing differentiation of recent from nonrecent ingestion. Pharmacokinetic analysis of a MDMA poisoning yielded elimination half-lives of 6.0 h for R-(-)-MDMA and 4.1 h for S-(+)-MDMA. The enantiomer ratios rose exponentially over time.

Hormonal counterregulation and consecutive glimepiride serum concentrations during severe hypoglycaemia associated with glimepiride therapy.

OBJECTIVE: To examine the release of counterregulatory hormones and consecutive glimepiride serum concentrations during severe hypoglycaemia (SH) associated with glimepiride therapy. METHODS: In nine type-2 diabetic patients [age 81+/-9 (65-93) years; diabetes duration 9+/-4 (3-15) years; initial blood glucose 33+/-16 (10-54) mg/dl (1.8+/-0.9 mmol/l); HbA1c 7.2+/-1.1 (5.6-8.7)%; creatinine clearance 49+/-33 (15-107) ml/min] who experienced SH associated with glimepiride therapy with neuroglucopenic presentation, insulin, C-peptide, glucagon, epinephrine, norepinephrine, cortisol, adenocorticotrophic hormone (ACTH), human growth hormone (HGH) and pancreatic polypeptide (PP) were determined in blood samples taken at 4-h intervals prior to and during treatment with glucose i.v. Serum from the same samples was screened for sulphonylurea-type oral antidiabetics. Glimepiride concentrations were determined by a validated atmospheric pressure chemical ionization liquid chromatographic-mass spectrometry (APCI-LC-MS) assay. RESULTS: Once treatment had begun, normoglycaemia was maintained; most glimepiride levels were below the limit of detection (LOD <0.01 mg/l) and further sulphonylureas could be excluded. The secretion of glucagon and epinephrine as counterregulatory hormonal responses was unaffected. In addition, protracted marked increases of cortisol and norepinephrine levels were demonstrated. Protracted stimulation of insulin and C-peptide occurred in a period of up to 24 h after SH. No significant protracted responses were observed for ACTH, HGH or PP. CONCLUSION: In SH associated with glimepiride therapy, no correlation between glimepiride serum concentrations and the protracted stimulation of insulin and C-peptide was observed. The secretion of glucagon and epinephrine as counterregulatory hormonal responses was unaffected. Protracted increased release of cortisol might be a medium-term indicator of glimepiride-associated SH.

Identification of cytochrome P450 enzymes involved in the metabolism of 4'-methoxy-alpha-pyrrolidinopropiophenone (MOPPP), a designer drug, in human liver microsomes.

1. The metabolism of 4'-methoxy-alpha-pyrrolidinopropiophenone (MOPPP), a novel designer drug, to its demethylated major metabolite 4'-hydroxy-pyrrolidinopropio-phenone (HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes. 2. CYP2C19 catalysed the demethylation with apparent Km and Vmax values of 373.4 +/- 45.1 microM and 6.0 +/- 0.3 pmol min(-1) pmol(-1) CYP, respectively (mean +/- SD). Both CYP2D6 and HLM exhibited clear biphasic profiles with apparent K(m,1) values of 1.3 +/- 0.4 and 22.0 +/- 6.5 microM, respectively, and V(max,1) values of 1.1 +/- 0.1 pmol min(-1) pmol(-1) CYP and 169.1 +/- 20.5 pmol min(-1) mg(-1) protein, respectively. 3. Percentages of intrinsic clearances of MOPPP by particular CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4'-hydroxylation or bufuralol-1'-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively. 4. MOPPP, HO-PPP and the standard 3',4'-methylenedioxy-pyrrolidinopropio-phenone (MDPPP) were separated and analysed by liquid chromatography-mass spectrometry in the selected-ion monitoring (SIM) mode. 5. The CYP2D6 specific chemical inhibitor quinidine (3 microM) significantly (p<0.0001) inhibited HO-PPP formation by 91.8 +/- 0.5% (mean +/- SEM) in incubation mixtures with HLM and 2 microM MOPPP. 6. It can be concluded from the data obtained from kinetic and inhibition studies that polymorphically expressed CYP2D6 is the enzyme mainly responsible for MOPPP demethylation.

Prevalence of illicit drug use in plasmapheresis donors.

BACKGROUND AND OBJECTIVES: No data are presently available concerning the frequency of illicit drug use in plasmapheresis donors. We therefore examined source plasma units produced in the United States (US) and in Germany for evidence of illicit drug use among donors. MATERIALS AND METHODS: Seventy-five US plasma units from 10 different US states and 75 German plasma units that had been analysed principally for their protein composition were screened for drugs. Determinations were made, using automated immunoassays, of the presence of cannabis, cocaine, amphetamine, methamphetamine, methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDE) and opiates. Positive results were confirmed by gas chromatography-mass spectrometry. RESULTS: Eleven US plasma units were found to be positive for cocaine (14.6%), whereas all German samples were cocaine negative (P = 0.0007). Fifteen US plasma units (20%) and one German unit (1.3%) were confirmed as positive for cannabis (P = 0.0003). Three out of 75 US plasma units were positive for both cannabis and cocaine. In none of the 150 samples were amphetamine, methamphetamine, MDMA, MDE or opiates detected. CONCLUSIONS: Our results strongly suggest differences in cocaine and cannabis consumption between US and German plasmapheresis donors. If these results are confirmed by larger-scale studies, random drug screening (including cocaine) of donors should be implemented in order to reduce the number of drug-containing plasma units, especially in the USA.

The antispasmodic drug mebeverine leads to positive amphetamine results by fluorescence polarization immunoassay (FPIA)--studies on the toxicological analysis of urine by FPIA and GC-MS.

Mebeverine (Duspatal, MB), an antispasmodic drug, is the veratric acid ester of 4-[ethyl-[2-(4-methoxyphenyl)-1-methylethyl]amino]butan-1-ol (MB-OH), which is a N-substituted ethylamphetamine derivative. MB is metabolized via ester hydrolysis to MB alcohol (MB-OH) and veratric acid. N-Dehydroxybutylation leads to methoxyethylamphetamine (MO-EA) and, after O-demethylation, to hydroxy EA (HO-EA). N-Bisdealkylation leads to p-methoxyamphetamine (PMA). MO-EA and PMA are also known as designer drugs. Fluorescence polarization immunoassay (FPIA) and gas chromatographic-mass spectrometric studies on the toxicological analysis of MB after ingestion of a single 405-mg oral dose of MB were performed. We could show that intake of MB leads to positive FPIA results for amphetamine. The N-dehydroxybutyl metabolites of MB, MO-EA, HO-EA, and the bis-dealkyl metabolite PMA should be responsible for the positive immunoassay results. Using our systematic toxicological analysis procedure, every positive amphetamine immunoassay could be explained by detection of MO-EA, HO-EA, and/or PMA. Misinterpretation of the origin of MO-EA, HO-EA, or PMA can be avoided by detecting the specific (non-dehydroxybutylated) metabolites of MB, which are excreted for a much longer time after ingestion.

Screening procedure for detection of non-steroidal anti-inflammatory drugs and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography-mass spectrometry after extractive methylation.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used as analgesic and anti-rheumatic drugs, and they are often misused. A gas chromatographic-mass spectrometric (GC-MS) screening procedure was developed for their detection in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The compounds were separated by capillary GC and identified by computerized MS in the full-scan mode. Using mass chromatography with the ions m/z 119, 135, 139, 152, 165, 229, 244, 266, 272, and 326, the possible presence of NSAIDs and their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of acemetacin, acetaminophen (paracetamol), acetylsalicylic acid, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, flufenamic acid, flurbiprofen, ibuprofen, indometacin, kebuzone, ketoprofen, lonazolac, meclofenamic acid, mefenamic acid, mofebutazone, naproxen, niflumic acid, phenylbutazone, suxibuzone, tiaprofenic acid, tolfenamic acid, and tolmetin in urine samples. The overall recoveries of the different NSAIDs ranged between 50 and 80% with coefficients of variation of less than 15% (n = 5), and the limits of detection of the different NSAIDs were between 10 and 50 ng/mL (S/N = 3) in the full-scan mode. Extractive methylation has proved to be a versatile method for STA of various acidic drugs, poisons, and their metabolites in urine. It has also successfully been used for plasma analysis.

Validated gas chromatographic-mass spectrometric assay for determination of the antifreezes ethylene glycol and diethylene glycol in human plasma after microwave-assisted pivalylation.

A gas chromatographic-mass spectrometric assay is described for identification and quantification of the antifreezes ethylene glycol (EG) and diethylene glycol (DEG) in plasma for early diagnosis of a glycol intoxication. After addition of 1,3-propanediol as internal standard, the plasma sample was deproteinized by acetone and an aliquot of the supernatant was evaporated followed by microwave-assisted pivalylation. After gas chromatographic separation, the glycols were first identified by comparison of the full mass spectra with reference spectra and then quantified. The quantification has been validated according to the criteria established by the Journal of Chromatography B. The assay was found to be selective. The calibration curves for EG and DEG were linear from 0.1 g/l to 1.0 g/l. The limit of detection for EG and DEG was 0.01 g/l and the limit of quantification for both was 0.1 g/l. The absolute recoveries were 50 and 65% for the low quality control samples and 51 and 73% for the high quality control samples of EG and DEG, respectively. Intra- and inter-day accuracy and precision were inside the required limits. The glycols in frozen plasma samples were stable for more than 6 months. The method was successfully applied to several authentic plasma samples from patients intoxicated with glycols. It has also been suitable for analysis of EG and DEG in urine.

Screening for detection of new antidepressants, neuroleptics, hypnotics, and their metabolites in urine by GC-MS developed using rat liver microsomes.

A gas chromatography-mass spectrometry (GC-MS) procedure for the detection of new antidepressants, neuroleptics, hypnotics, and their metabolites in urine is presented. The metabolites were first identified in rat liver microsome preparations by GC-MS after isolation and derivatization. Using these GC-MS data, a GC-MS screening was developed for urine as part of the authors' modified systematic toxicologic analysis procedure. After acid hydrolysis of a 2.5-mL aliquot of urine, a further aliquot was added. The mixture was then liquid-liquid extracted at pH 8-9, acetylated, and GC separated. Using mass chromatography with the ions m/z 58, 100, 120, 182, 195, 235, 261, 276, 284. and 293, the presence of new antidepressants, neuroleptics, hypnotics, and their metabolites could be indicated. Positive peaks could be identified by library search using the reference mass spectra recorded during the microsome studies. The intake of therapeutic doses of the following drugs could be monitored in urine: dosulepin, mirtazapine, moclobemide, nefazodone, trazodone, venlafaxine, and zolpidem. Olanzapine and zotepine were detectable in human urine only under steady-state conditions, and low-dose zopiclone was detectable only in overdose. The detection limit was less than 100 ng/mL (signal-to-noise ratio = 3) for the parent drugs.

Screening procedures for simultaneous detection of several drug classes used for high throughput toxicological analyses and doping control. A review.

This paper reviews high throughput screening procedures for the simultaneous detection of several drug classes relevant to clinical and forensic toxicology or doping control in urine or blood using gas chromatography-mass spectrometry (GC-MS), liquid chromatography coupled with a diode-array detector (LC-DAD) or mass spectrometry (LC-MS). Basic information describing these systematic toxicological analysis (STA) procedures such as the analytes, the biosample, work-up, separation column, mobile phase or separation buffer, detection mode and detection limits are summarized in tables arranged according to the analytical method. Examples of typical applications are presented in 2 figures. Analysis of alternative matrices, like sweat, saliva, nails or hair, was not reviewed.

Validated electrospray liquid chromatographic-mass spectrometric assay for the determination of the mushroom toxins alpha- and beta-amanitin in urine after immunoaffinity extraction.

Specific detection of amanitins in body fluids is necessary for an early diagnosis of an intoxication with amanita mushrooms. In this paper, a liquid chromatographic-mass spectrometric assay after immunoaffinity extraction (IAE-LC-MS) is described for the determination of alpha- and beta-amanitin in urine. The method has been validated according to the criteria established by the Journal of Chromatography B. The assay was found to be selective. The calibration curves for alpha- and beta-amanitin were linear from 5 to 75 ng/ml. Intra- and inter-day accuracy and precision were inside the required limits. Amatoxins in frozen urine samples or immunoaffinity extracts were stable for more than 6 months, and the IAE columns could be used more than fifty times without remarkable loss in performance. LOD for alpha- and beta-amanitin was 2.5 ng/ml and LOQ for both was 5.0 ng/ml. The absolute recoveries of alpha- and beta-amanitin were 63% and 58% for the low quality control and 61% and 57% for the high quality control. The absolute recovery for the internal standard gamma-amanitin methyl ether at 25 ng/ml was 60%. The analysis of 5 authentic urine samples from patients intoxicated by amanita mushrooms showed a good correlation between the results measured by radioimmunoassay and the IAE-LC-MS assay. A partial validation showed that the assay was also suitable for plasma analysis.

Screening procedure for detection of antidepressants of the selective serotonin reuptake inhibitor type and their metabolites in urine as part of a modified systematic toxicological analysis procedure using gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) screening procedure was developed for detection of selective serotonin reuptake inhibitors (SSRIs) in urine as part of a systematic toxicological analysis procedure. After acid hydrolysis of one aliquot of urine, another aliquot was added. The mixture was then liquid-liquid extracted at pH 8-9, acetylated, and GC separated. Using mass chromatography with the ions m/z 58, 72, 86, 173, 176, 234, 238, and 290, the possible presence of SSRIs and/or their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. The overall recoveries of citalopram, sertraline, and paroxetine ranged between 60 and 80%, and those of fluoxetine and fluvoxamine, which were destroyed during acid hydrolysis, were between 40 and 45%. The coefficients of variation were less than 10-20%, and the limit of detection was at least 100 ng/mL (signal-to-noise ratio = 3). This method allowed the detection of therapeutic concentrations of citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline in human urine samples.

Studies on the metabolism and toxicological detection of the amphetamine-like anorectic fenproporex in human urine by gas chromatography-mass spectrometry and fluorescence polarization immunoassay.

Studies on the metabolism and the toxicological analysis of fenproporex (R,S-3-[(1-phenyl-2-propyl)-amino]-propionitrile, FP) using GC-MS and fluorescence polarization immunoassay are described. The metabolites were identified in urine samples of volunteers by GC-MS after cleavage of conjugates, extraction and acetylation. Besides unchanged FP, fourteen metabolites, including amphetamine, could be identified. Two partially overlapping metabolic pathways could be postulated: ring degradation by one- and two-fold aromatic hydroxylation followed by methylation and side chain degradation by N-dealkylation to amphetamine (AM). A minor pathway leads via beta-hydroxylation of AM to norephedrine. For GC-MS detection, the systematic toxicological analysis procedure including acid hydrolysis, extraction at pH 8-9 and acetylation was suitable (detection limits 50 ng/ml for FP and 100 ng/ml for AM). Excretion studies showed, that only AM but neither FP nor its specific metabolites were detectable 30-60 h after ingestion of 20 mg of FP. Therefore, misinterpretation can occur. The Abbott TDx FPIA amphetamine/methamphetamine II gave positive results up to 58 h. All the positive immunoassay results could be confirmed by the described GC-MS procedure.

Gas chromatographic-mass spectrometric procedures for determination of the catechol-O-methyltransferase (COMT) activity and for detection of unstable catecholic metabolites in human and rat liver preparations after COMT catalyzed in statu nascendi derivatization using S-adenosylmethionine.

A procedure is presented for determination of the catechol-O-methyltransferase (COMT) activity in liver cytosolic preparations using 3,4-dihydroxyphenethylamine as substrate and by quantifying the product 3-methoxy-4-hydroxyphenethylamine (3-MHP). For quantification of 3-MHP in liver cytosolic preparations a gas chromatographic-mass spectrometric procedure after liquid-liquid extraction and acetylation was established and validated. The intra- and inter-day accuracy and precision were better than 15% and 20%, respectively. Extraction efficiency and selectivity were also sufficient. For in statu nascendi derivatization of unstable catecholic metabolites in liver microsome preparations, cytosolic preparations with COMT activities of at least 1 nmol product/min/mg protein were used after addition of S-adenosylmethionine. Such catecholic metabolites, which are claimed to be responsible for toxic effects in vivo, e.g., neurotoxicity or carcinogenesis, must not be overlooked in in vitro metabolism studies. Using this trick, gas chromatography-mass spectrometry (GC-MS) was suitable for the determination of catecholic metabolites in human and rat liver preparations after the same sample preparation as for 3-MHP quantification. The applicability was exemplified for the antidepressant paroxetine.

Toxicokinetics and analytical toxicology of amphetamine-derived designer drugs ('Ecstasy').

The phase I and II metabolites of the designer drugs methylenedioxyamphetamine (MDA), R,S-methylenedioxymethamphetamine (MDMA), R,S-methylenedioxyethylamphetamine (MDE), R, S-benzodioxazolylbutanamine (BDB) and R, S-N-methyl-benzodioxazolylbutanamine (MBDB) were identified by gas chromatography-mass spectrometry (GC-MS) or liquid chromotography-mass spectrometry (LC-MS) in urine and liver microsomes of humans and rats. Two overlapping pathways could be postulated: (1) demethylenation followed by catechol-O-methyl-transferase (COMT) catalyzed methylation and/or glucuronidation/sulfatation; (2) N-dealkylation, deamination and only for MDA, MDMA, MDE oxidation to the corresponding benzoic acid derivatives conjugated with glycine. Demethylenation was mainly catalyzed by CYP2D1/6 or CYP3A2/4, but also by CYP independent mechanisms. In humans, MDMA and MBDB could also be demethylenated by CYP1A2. N-demethylation was mainly catalyzed by CYP1A2, N-deethylation by CYP3A2/4. Based on these studies, GC-MS procedures were developed for the toxicological analysis in urine and plasma. Finally, toxicokinetic parameters are reviewed.