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.

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.

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.

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 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.

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.

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.

On the metabolism of the amphetamine-derived antispasmodic drug mebeverine: gas chromatography-mass spectrometry studies on rat liver microsomes and on human urine.

We describe gas chromatography-mass spectrometry studies of the metabolism of the antispasmodic drug mebeverine [Duspatal, (MB)]. MB is the veratric acid (VA) ester of 4-¿ethyl-[2-(4-methoxyphenyl)-1-methylethyl]amino¿butan-1-ol (MB-OH), which is an N-substituted ethylamphetamine derivative. The metabolites were first identified in rat liver microsome incubates and then detected in urine samples of volunteers through the use of electron impact and positive chemical ionization gas chromatography-mass spectrometry. Urinary conjugates were enzymatically cleaved before analysis. The following phase I metabolites of MB could be identified: VA, O-demethyl VA (vanillic and/or isovanillic acid), O-bisdemethyl VA (protocatechuic acid), MB-OH, hydroxy MB-OH, O-demethyl MB-OH, O-demethyl-hydroxy MB-OH, N-desethyl MB-OH, N-desethyl-O-demethyl MB-OH, N-de(hydroxybutyl) MB-OH (methoxy-ethylamphetamine), N-de(hydroxybutyl)-O-demethyl MB-OH (hydroxy-ethylamphetamine), and N-bisdealkyl MB-OH (p-methoxy-amphetamine, known as the designer drug PMA). The following, partly overlapping metabolic pathways of MB could be postulated: ester hydrolysis, O-demethylation, ring hydroxylation, N-deethylation, and N-de(hydroxybutylation). The latter pathway led to ethylamphetamine derivatives and bisdealkylation led to PMA, which are substances of forensic interest. The metabolites containing alcoholic or phenolic hydroxy groups were partly excreted into urine as conjugates.

Screening procedure for detection of dihydropyridine calcium channel blocker metabolites in urine as part of a systematic toxicological analysis procedure for acidic compounds by gas chromatography-mass spectrometry after extractive methylation.

A gas chromatographic-mass spectrometric (GC-MS) screening procedure was developed for the detection of dihydropyridine calcium channel blocker ("calcium antagonist") metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. 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 139, 284, 297, 298, 310, 312, 313, 318, 324, and 332, the possible presence of calcium channel blocker 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 amlodipine, felodipine, isradipine, nifedipine, nilvadipine, nimodipine, nisoldipine, and nitrendipine in human urine samples. Because urine samples from patients treated with nicardipine were not available, the detection of nicardipine in rat urine was studied. The overall recovery ranged between 67 and 77% with a coefficient of variation of less than 10%, and the limit of detection was at least 10 ng/mL (signal-to-noise ratio = 3) in the full-scan mode.

Detection of 4-hydroxycoumarin anticoagulants 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.

A gas chromatography-mass spectrometry (GC-MS) procedure was developed for the detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. 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 291, 294, 295, 309, 313, 322, 324, 336, 343 and 354, the possible presence of 4-hydroxycoumarin anticoagulants 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. This method allowed the detection of therapeutic concentrations of phenprocoumon and warfarin in human urine samples. In absence of human urine, acenocoumarol, coumachlor, coumatetrayl, pyranocoumarin (cyclocumarol) could be detected only in rat urine.

Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) in toxicological analysis. Studies on the detection of clobenzorex and its metabolites within a systematic toxicological analysis procedure by GC-MS and by immunoassay and studies on the detection of alpha- and beta-amanitin in urine by atmospheric pressure ionization electrospray LC-MS.

GC-MS is the method of choice for toxicological analysis of toxicants volatile in GC while non-volatile and/or thermally labile toxicants need LC-MS for their determination. Studies are presented on the toxicological detection of the amphetamine-like anorectic clobenzorex in urine by GC-MS after acid hydrolysis, extraction and acetylation and by fluorescence polarization immunoassay (FPIA, TDx (meth)amphetamine II). After ingestion of 60 mg of clobenzorex, the parent compound and/or its metabolites could be detected by GC-MS for up to 84 h or by FPIA for up to 60 h. Since clobenzorex shows no cross-reactivity with the used immunoassay, the N-dealkylated metabolite amphetamine is responsible for the positive TDx results. The intake of clobenzorex instead of amphetamine can be differentiated by GC-MS detection of hydroxyclobenzorex which is detectable for at least as long as amphetamine. In addition, the described GC-MS procedure allows the simultaneous detection of most of the toxicologically relevant drugs. Furthermore, studies are described on the atmospheric pressure ionization electrospray LC-MS detection of alpha- and beta-amanitin, toxic peptides of amanita mushrooms, in urine after solid-phase extraction on RP-18 columns. Using the single ion monitoring mode with the ions m/z 919 and 920 the amanitins could be detected down to 10 ng/ml of urine which allows us to diagnose intoxications with amanita mushrooms.

Analytical development for low molecular weight xenobiotic compounds.

Specific and sensitive detection or precise quantification of xenobiotics in biosamples (e.g. blood, urine, saliva, sweat, hair) are great challenges in analytical toxicology. GC-MS is the most sensitive, specific and universal analytical method for low mass xenobiotics. Precise quantification can be performed using the selected ion mode (SIM) and stable isotopes as internal standards. Negative chemical ionization (NCI) can improve severalfold the sensitivity for the determination of compounds with electronegative sites (e.g. halogens). For screening and identification of most of the basic and neutral drugs (e.g. drugs of abuse, psychotropics, hypnotics, analgesics, cardiacs) in urine, a systematic toxicological analysis procedure (STA) was developed using GC-MS after acid hydrolysis, extraction and acetylation. for detection of acidic xenobiotics (e.g. anticoagulants, ACE inhibitors, diuretics, antirheumatics) in urine, a further GC-MS procedure was developed using extractive alkylation. For the detection of non-volatile xenobiotics (e.g. toxic peptides like alpha- and beta-amanitin or phase II metabolites) electrospray LC-MS procedures were developed. The procedures and examples show that in analytical toxicology GC-MS is the method of choice for low mass xenobiotics while LC-MS is that for non-volatiles.

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

Studies on the metabolism and on the toxicological analysis of mefenorex [R,S-N-(3-chloropropyl)-alpha-methylphenethylamine, MF] using gas chromatography-mass spectrometry (GC-MS) and fluorescence polarization immunoassay (FPIA) are described. The metabolites were identified in urine samples of volunteers by GC-MS. Besides MF, thirteen metabolites including amphetamine (AM) could be identified and three partially overlapping metabolic pathways could be postulated. 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 MF and 100 ng/ml for AM). Excretion studies showed, that only AM but neither MF nor its specific metabolites were detectable between 32 and 68 h after ingestion of 80 mg of MF. Therefore, misinterpretation can occur. The Abbott TDx FPIA amphetamine/methamphetamine II gave positive results up to 68 h. All the positive immunoassay results could be confirmed by the described GC-MS procedure.

On the metabolism and the toxicological analysis of methylenedioxyphenylalkylamine designer drugs by gas chromatography-mass spectrometry.

Designer drugs of the methylenedioxyphenylalkylamine type are increasingly abused. Studies on their metabolism in humans are necessary to develop a reliable gas chromatography--mass spectrometry (GC-MS) screening procedure. Such a method must allow their detection in urine for drug testing in clinical and forensic toxicology. Studies on racemic methylenedioxyamphetamine (MDA), methylenedioxymetamphetamine (MDMA), methylenedioxyethylamphetamine (MDE), benzodioxazolylbutanamine (BDB), and N-methylbenzodioxazolylbutanamine (MBDB) are presented. The metabolites were identified by GC-MS after enzymatic hydrolysis, isolation (pH 4.5 and 8-9), and derivatization (acetylation followed by methylation). The drugs undergo two overlapping metabolic pathways: O-dealkylation of the methylenedioxy group to dihydroxy derivatives followed by methylation of one of the hydroxy groups and successive degradation of the side chain to N-dealkyl and deaminooxo metabolites. MDA, MDMA, and MDE are subsequently metabolized to glycine conjugates of the corresponding 3,4-disubstituted benzoic acids. The hydroxy metabolites are excreted in a conjugated form. Based on these results, a GC-MS procedure was developed for simultaneous screening and identification of these designer drugs and/or their metabolites in urine after acid hydrolysis, isolation at pH 8-9, and acetylation. With use of mass chromatography with the most characteristic fragment ions m/z 58, 72, 86, 150, 162, 164, 176, and 178, the presence of the designer drugs was indicated and the peak underlying spectra could be identified by computerized comparison with reference spectra recorded during the presented studies. The procedure was suitable to detect an abuse of or an intoxication with the studied designer drugs (detection limit 5-50 ng/ml).

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 methylenedioxyethylamphetamine [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 32-36 h and 4-hydroxy-3-methoxyethylamphetamine (HME) for 7-8 days. 3,4-Dihydroxyamphetamine (DHA), 4-hydroxy-3-methoxyamphetamine (HMA), piperonyl acetone, 3,4-dihydroxyphenyl acetone and 4-hydroxy-3-methoxyphenyl 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.

Toxicological detection of ibuprofen and its metabolites in urine using gas chromatography-mass spectrometry (GC-MS).

Ibuprofen, (R,S)-2-(4-isobutylphenyl)propionic acid, is a non-opioid analgesic which is prescription-free available in pharmacies since 1989. Consequently, use and misuse of this analgesic strongly increased. For diagnosis of an abuse or an intoxication a GC-MS procedure was developed for the toxicological detection of ibuprofen in urine after extraction at pH 5 and methylation. For screening, mass chromatography with the masses m/z 220, 161 (methylated ibuprofen), 178, 119 (methylated hydroxy ibuprofen) and 264, 145 (methylated carboxy ibuprofen) was used to indicate the presence of these compounds in urine. The identity of the peaks underlying full mass spectra was confirmed by comparison with the reference spectra using computer library search. After ingestion of one single oral dose of 400 mg ibuprofen, the parent compound could be detected for 27 to 34 h, hydroxy ibuprofen for 34 to 40 h and carboxy ibuprofen for 5 to 6 d. Using this procedure ibuprofen which was taken even several days ago could be detected.

Systematic toxicological analysis of drugs and their metabolites by gas chromatography-mass spectrometry.

Gas chromatographic-mass spectrometric (GC-MS) procedures for the systematic toxicological analysis of several categories of drugs relevant to clinical toxicology, forensic toxicology and doping control are reviewed. Papers from 1981 to 1991 are taken into consideration. They describe the detection of acute or chronic intoxication and the detection of drug abuse. Screening procedures are included for the following categories: barbiturates and other sedative-hypnotics, anticonvulsants, benzodiazepines, antidepressants, phenothiazine and butyrophenone neuroleptics, central stimulants (amphetamines, cocaine), hallucinogens (LSD, phencyclidine, tetrahydrocannabinol), opioid (narcotic) and other potent analgesics, non-opioid analgesics, antihistamines (histamine H1-receptor blockers), antiparkinsonian drugs, beta-blockers (beta-adrenoceptor blockers), antiarrhythmics (class I and IV), diuretics, laxatives and their metabolites. Methods for confirmation of results obtained by screening procedures using immunoassay or chromatographic techniques are also included. GC-MS procedures for the simultaneous detection of several categories of drugs, the so-called "general unknown analysis", are reviewed. The toxicological question to be answered and the consequence for the choice of an adequate method, the sample preparation and the chromatography itself are discussed. The basic information about the biosample assayed, work-up, GC column, mass spectral detection mode, reference data and sensitivity of each procedure are summarized in tables, arranged according to the category of drug. Examples of typical GC-MS applications are presented. Fragment ions that are suitable for mass spectral screening for particular categories of drugs and for general unknown are tabulated.

Toxicological detection of selegiline and its metabolites in urine using fluorescence polarization immunoassay (FPIA) and gas chromatography-mass spectrometry (GC-MS) and differentiation by enantioselective GC-MS of the intake of selegiline from abuse of methamphetamine or amphetamine.

Selegiline (R(-)-N-methyl-N-(1-phenyl-2-propyl)-2-propinylamine), a selective MAO-B inhibitor used as an antiparkinsonian, is excreted in urine as N-desmethyl selegiline (norselegiline), R(-)-methamphetamine (R(-)-MA), R(-)-amphetamine (R(-)-AM) and their conjugated p-hydroxy derivatives. We found that the fluorescence polarization immunoassays (FPIA) TDx amphetamine/methamphetamine II (AM/MA II) and TDx amphetamine class (AM class) lead to positive results for up to 2 days after a single oral dose of 10 mg selegiline (detection limit: 0.1 mg/l, each). Every urine specimen from long term selegiline patients (10 mg/day) showed positive TDx results during the selegiline regimen. Positive TDx results were confirmed using gas chromatography-mass spectrometry (GC-MS). Selegiline metabolites, particularly MA, could be detected in urine for up to 7 days after intake of a single oral dose of 10 mg selegiline (detection limit: 0.01 mg/l for MA and AM). Norselegiline, the only specific selegiline metabolite, was only detectable for about 12 h. Moreover, norselegiline was not detected in all urine samples from long term selegiline patients (10 mg/day). Since differentiation of selegiline intake from MA/AM abuse by detecting norselegiline was not possible in most cases, an enantioselective GC-MS procedure was developed. It allowed differentiation of the enantiomers of the selegiline metabolites and thereby separation of selegiline intake (only R(-)-enantiomers) from methamphetamine and/or amphetamine abuse (racemates or S(+)-enantiomers). After derivatization with S(-)-N-trifluoroacetyl-prolyl chloride (TPC), the two enantiomers of MA and AM were each separated as diastereomers employing the routinely used achiral GC capillary.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicological detection of pholcodine and its metabolites in urine and hair using radio immunoassay, fluorescence polarisation immunoassay, enzyme immunoassay, and gas chromatography-mass spectrometry.

Pholcodine (3-O-(2'-morpholinoethyl)-morphine) is used in many countries as an antitussive without analgesic or addictive properties. It is of forensic relevance that pholcodine interferes with opiate immunoassays. In this paper a gas chromatographic-mass spectrometric (GC-MS) procedure for the precise and sensitive detection of pholcodine and its metabolites in urine and hair, after acid hydrolysis, extraction and acetylation, is presented. Furthermore, detection of pholcodine using radio immunoassay (RIA), fluorescence polarisation immunoassay (FPIA) and enzyme immunoassay (EIA) for opiates is described. Using GC-MS, unmodified pholcodine could be detected in urine samples 4-7 weeks after ingestion of a single therapeutic dose of 50mg of pholcodine, the desmorpholinohydroxy metabolite for 1-2 weeks and the other metabolites (nor-, nordesmorpholinohydroxy-, hydroxy-, oxo- and noroxo-pholcodine) only during the first few hours. Morphine could also be detected in urine samples for the first few days. It was however mainly formed artificially during acid hydrolysis and only in trace amounts by metabolism. All the immunoassays tested gave positive results in urine samples during the first week taking the cut-off values recommended by the manufacturer into consideration. If values between the cut-off and the detection limit were taken into consideration, RIA and FPIA gave positive results for 2-4 weeks and EIA up to 2 weeks. Pholcodine could also be detected by RIA and GC-MS in samples of head hair clipped 10 weeks after ingestion of 50mg and in daily shaved samples of beard hair over a period of three weeks after ingestion of three doses of 60mg. It can be concluded that the widely used immunoassays for opiates show positive results in urine and hair samples for a long time after ingestion of the non-opioid pholcodine and that these results can be confirmed by the GC-MS procedure described in this paper.