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New designer drug p-methoxymethamphetamine: Studies on its metabolism and toxicological detection in urine using gas chromatography-mass spectrometry
Abstract
Studies are described on the metabolism and the toxicological analysis of the new designer drug rac-p-methoxymethamphetamine (PMMA) in rat urine using gas chromatography-mass spectrometry (GC-MS). The identified metabolites indicated that PMMA was extensively metabolized mainly by O-demethylation to pholedrine and to a minor extent to p-methoxyamphetamine (PMA), 1-hydroxypholedrine diastereomers (one being oxilofrine), 4'-hydroxy-3'-methoxymethamphetamine and 4'-hydroxy-3'-methoxyamphetamine. The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of the main metabolites of PMMA in rat urine after a dose corresponding to that of drug users. Therefore, this procedure should be suitable for detection of PMMA intake in human urine via its metabolites. However, it must be considered that pholedrine and oxilofrine are also in therapeutic use. Differentiation of PMMA, PMA and/or pholedrine intake is discussed.
... Di-OH-MA is not neurotoxic in itself (Zhao et al., 1992;Monks et al., 2004), but it is a precursor of conjugated GSH/N-acetylcysteine compounds that have been implicated in serotonergic neurotoxicity and neurodegeneration (Molliver et al., 1986;Schmidt and Taylor, 1988;Hiramatsu et al., 1990;McCann and Ricaurte, 1991;Paris and Cunningham, 1992;Miller et al., 1995Miller et al., , 1996Chu et al., 1996;Bai et al., 1999;Esteban et al., 2001;Carvalho et al., 2004b;de la Torre and Farre, 2004;Monks et al., 2004;Jones et al., 2005;Carmo et al., 2006;Perfetti et al., 2009). Di-OH-MA, which is well known as the main intermediate metabolite of MDMA (Segura et al., 2001;, is reported to be formed in rats after administration of PMMA (Staack et al., 2003). ...
... The high toxicity of PMMA, compared with MDMA, requires additional research on PMMA's metabolite pattern and pharmacogenetics in humans. The existing knowledge of the metabolism of PMMA is limited to two microsomal studies (Staack et al., 2004b;Lai et al., 2015) and experimental studies in rats (Staack et al., 2003;Rohanova and Balikova, 2009a,b;Pálení cek et al., 2011). The aim of this study was to examine the metabolism of PMMA in human liver microsomes (HLMs) and in authentic blood samples from fatal PMMA intoxications. ...
... The high substrate recovery observed in our study indicates, however, that the formation of glucuronide/sulfate/ thiol conjugates was low, which is in accordance with a previous HLM study (Lai et al., 2015). Regarding the fatal PMMA intoxications, we did not detect di-OH-MA in the postmortem blood samples, probably because of the highly unstable nature of catechol compounds, detection of which depends on rapid analysis of fresh biologic samples (Hiramatsu et al., 1990;Helmlin et al., 1996;Maurer et al., 2000a;Staack et al., 2003;Carvalho et al., 2004a;Perfetti et al., 2009;Vevelstad et al., 2016b). A low concentration of di-OH-MA has been measured in rats administered PMMA; however, this analysis was performed in fresh urine samples (Staack et al., 2003;Rohanova and Balikova, 2009a). ...
Para-methoxymethamphetamine (PMMA) has caused numerous fatal poisonings worldwide and appears to be more toxic than other ring-substituted amphetamines. Systemic metabolism is suggested to be important for PMMA neurotoxicity, possibly through activation of minor catechol metabolites to neurotoxic conjugates. The aim of this study was to examine the metabolism of PMMA in humans, and for this purpose we used human liver microsomes (HLM) and blood samples from three cases of fatal PMMA intoxication. We also examined the impact of CYP2D6 genetics on PMMA metabolism using genotyped HLM isolated from CYP2D6 poor, population average and ultrarapid metabolizers. In HLM, PMMA was metabolized mainly to 4-hydroxymethamphetamine (OH-MA), while low concentrations of para-methoxyamphetamine (PMA), 4-hydroxyamphetamine (OH-A), dihydroxymethamphetamine (di-OH-MA) and oxilofrine were formed. The metabolite profile in the fatal PMMA intoxications were in accordance with the HLM study, with OH-MA and PMA being the major metabolites, while OH-A, oxilofrine, HM-MA and HM-A were detected in low concentrations. A significant influence of CYP2D6 genetics on PMMA metabolism in HLM was found. The catechol metabolite di-OH-MA has previously been suggested to be involved in PMMA toxicity. Our studies show that the formation of di-OH-MA from PMMA was 2-7 times lower than from an equimolar dose of the less toxic drug MDMA, and do not support the hypothesis of catechol metabolites as major determinants of fatal PMMA toxicity. Altogether, the present study revealed the metabolite pattern of PMMA in humans and demonstrated a great impact of CYP2D6 genetics on human PMMA metabolism.
... The systematic clinical study of this drug with human volunteers remains restricted due to ethical considerations. However, PMMA biotransformation routes have been studied in animal models using experimental rats (Staack et al., 2003). Staack et al. suggested two prevalent ways of biotransformation of PMMA after peroral administration: O-demethylation to form the dominant metabolite p-hydroxymethamphetamine (pholedrine, OH-MAM), and N-demethylation leading to the minor but more specific metabolite p-methoxyamphetamine (PMA). ...
... Some metabolites were excreted in urine in conjugated forms as O-glucuronides or O-sulphates (Shima et al., 2007). Considering the available published information on the metabolism of structurally related compounds, phenylalkylamines (Staack et al., 2003;Scheline, 1978;Shima et al., 2007;Rohanova et al., 2008), the potential biotransformation scheme of PMMA also considered in our study is depicted in Fig. 1. ...
... The prevailing PMMA biodegradation pathway in our study was O-demethylation with the primary product being pholedrine which was also previously confirmed by Staack et al. (2003Staack et al. ( , 2004, even though, unlike Staack et al., a different route of drug administration reducing the first pass effect was used. Pholedrine is a nonspecific metabolite and can be a significant metabolite of methamphetamine and other drugs as well (Kanamori et al., 2005). ...
p-Methoxymethamphetamine (PMMA) is an illegal psychedelic drug of abuse derived from an amphetamine structure with a risk to health and reports of several cases of intoxications and fatalities caused by its ingestion. However, its pharmacokinetics based on a controlled study is unknown and only partial information on its biotransformation in animal models is available. Our experimental design aimed to study the disposition and kinetic profile of PMMA and its metabolites in rat plasma and selected tissues after the bolus subcutaneous dose of 40mg/kg, using a GC-MS method. Prior to this, we performed a qualitative verification of its metabolites appearing in excreted urine fractions. PMMA maximum plasma concentration of 4014+/-1122ng/mL was reached 30min after dosing, whereas the appearance of metabolites was rather delayed. The disposition of PMMA was characterized by its approximate half-life of 1.0h, volume of distribution of 6.4L/kg and plasma clearance of 4.4L/h. PMMA tissue concentration exceeded plasma and the highest one was found in the lungs (c(max) 42,988+/-10,223ng/g). Penetration through the blood/brain barrier was more efficient considering PMMA and its N-desmethylated metabolite PMA (para-methoxyamphetamine) than hydroxylated metabolites. The maximum brain/plasma ratio value of PMMA (15.8) and PMA (11.8) was reached after 8h of observation. The experimental results ascertained could be useful for subsequent evaluation of the psychotropic or neurotoxic effects of PMMA and for diagnostic concern of intoxication.
... The systematic clinical study of this drug with human volunteers remains restricted due to ethical considerations. However, PMMA biotransformation routes have been studied in animal models using experimental rats (Staack et al., 2003). Staack et al. suggested two prevalent ways of biotransformation of PMMA after peroral administration: O-demethylation to form the dominant metabolite p-hydroxymethamphetamine (pholedrine, OH-MAM), and N-demethylation leading to the minor but more specific metabolite p-methoxyamphetamine (PMA). ...
... Some metabolites were excreted in urine in conjugated forms as O-glucuronides or O-sulphates (Shima et al., 2007). Considering the available published information on the metabolism of structurally related compounds, phenylalkylamines (Staack et al., 2003;Scheline, 1978;Shima et al., 2007;Rohanova et al., 2008), the potential biotransformation scheme of PMMA also considered in our study is depicted in Fig. 1. ...
... The prevailing PMMA biodegradation pathway in our study was O-demethylation with the primary product being pholedrine which was also previously confirmed by Staack et al. (2003Staack et al. ( , 2004, even though, unlike Staack et al., a different route of drug administration reducing the first pass effect was used. Pholedrine is a nonspecific metabolite and can be a significant metabolite of methamphetamine and other drugs as well (Kanamori et al., 2005). ...
para-Methoxymethamphetamine (PMMA) is an abused psychedelic compound with reports of several intoxications and deaths after ingestion. However, its pharmacokinetics based on a controlled study is unknown and only partial information on its biotransformation is available. Our experimental study was designed for the time disposition profile of PMMA and its metabolites para-methoxyamphetamine (PMA), para-hydroxymethamphetamine (OH-MAM) and para-hydroxyamphetamine (OH-AM) in blood and biological tissues in rats after the bolus subcutaneous dose 40 mg/kg using a validated GC-MS method. The experimental results ascertained could be useful for subsequent evaluation of PMMA psychotropic or neurotoxic effects and the diagnostic concern of intoxication.
... The current knowledge about the PMMA metabolism and toxicity in humans is limited. The available information about metabolism is based solely on two rat studies [12,13], one study using human liver microsomes [14] and one urine sample from a single individual [12]. As is the case with many synthetic amphetamine analogues, PMMA metabolism probably occurs mainly through O-demethylation via the polymorphic cytochrome P450 (CYP) 2D6 enzyme to the psychoactive and potentially toxic metabolite 4-hydroxymethamphetamine (OH-MA, pholedrine) followed by conjugation with glucuronide or sulphate [14,15]. ...
... The current knowledge about the PMMA metabolism and toxicity in humans is limited. The available information about metabolism is based solely on two rat studies [12,13], one study using human liver microsomes [14] and one urine sample from a single individual [12]. As is the case with many synthetic amphetamine analogues, PMMA metabolism probably occurs mainly through O-demethylation via the polymorphic cytochrome P450 (CYP) 2D6 enzyme to the psychoactive and potentially toxic metabolite 4-hydroxymethamphetamine (OH-MA, pholedrine) followed by conjugation with glucuronide or sulphate [14,15]. ...
... Similarly, CYP2D6 has been found to mediate the in vitro formation of PMMA-M1 in humans [19,20,38]. To our knowledge, in vivo metabolism studies of PMMA are not available for humans, only for rats [39]. In urine samples collected from rats administered with PMMA, PMMA-M1 and PMMA-M2 were the major and the minor metabolites detected, respectively (Table S1). ...
... Glucuronidated PMA-M1 was occasionally detected in the urine samples of the three volunteers [37] (Table S1). For glucuronidated and/or sulfated metabolites of PMMA, minor amounts were found in the rat urine [39] (Table S1). Similarly, urinary excretion of conjugated metabolites for amphetamine and methamphetamine was observed only to a small extent in humans [34][35][36]. ...
Phenethylamine-based designer drugs are prevalent within the new psychoactive substance market. Characterisation of their metabolites is important in order to identify suitable biomarkers which can be used for better monitoring their consumption. Careful design of in vitro metabolism experiments using subcellular liver fractions will assist in obtaining reliable outcomes for such purposes. The objective of this study was to stepwise investigate the in vitro human metabolism of seven phenethylamine-based designer drugs using individual families of enzymes. This included para-methoxyamphetamine, para-methoxymethamphetamine, 4-methylthioamphetamine, N-methyl-benzodioxolylbutanamine, benzodioxolylbutanamine, 5-(2-aminopropyl) benzofuran and 6-(2-aminopropyl) benzofuran. Identification and structural elucidation of the metabolites was performed using liquid chromatography-quadrupole-time-of-flight mass spectrometry. The targeted drugs were mainly metabolised by cytochrome P450 enzymes via O-dealkylation as the major pathway, followed by N-dealkylation, oxidation of unsubstituted C atoms and deamination (to a small extent). These drugs were largely free from Phase II metabolism. Only a limited number of metabolites were found which was consistent with the existing literature for other phenethylamine-based drugs. Also, the metabolism of most of the targeted drugs progressed at slow rate. The reproducibility of the identified metabolites was assessed through examining formation patterns using different incubation times, substrate and enzyme concentrations. Completion of the work has led to a set of metabolites which are representative for specific detection of these drugs in intoxicated individuals and also for meaningful evaluation of their use in communities by wastewater-based drug epidemiology.
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... This implies that if HO-MAMP were selected as the SEM, not only would one need to measure its free form but also its sulfate conjugate. Furthermore, presence of HO- MAMP in sewers could also be ascribed to an upstream population's use of the illicit designer drug para-methoxymethamphetamine (PMMA) (Staack et al., 2003). Compared to the use of HO-MAMP as the SEM, the only major disadvantage of using MAMP would be that its sewer load would always be susceptible to direct dumping events. ...
... Nevertheless, at present, clinical use of HO-AMP is primarily limited to ophthalmic preparations (e.g., Paremyd® (US FDA, 2010)) and, hence, the contribution to the sewer loads of HO-AMP arising from such licit use should be of negligible importance. The presence of HO-AMP in sewers could also be ascribed to an upstream population's use of the illicit designer drug para-methoxyamphetamine (PMA) and/or paramethoxymethamphetamine (PMMA) (Kitchen et al., 1979; Staack et al., 2003). However, these illicit designer drugs are not metabolized to AMP. ...
In sewer epidemiology, mass balances are used to back-extrapolate measurements of wastewater influent concentrations of appropriate drug residues to assess the parent illicit drug's level of use in upstream populations. This study focussed on developing and refining mass balances for the use of illicit methamphetamine, amphetamine and tetrahydrocannabinol. As a first step, a multi-criteria evaluation was used to select unchanged methamphetamine, unchanged amphetamine and 11-nor-9-carboxy-tetrahydrocannabinol as the most appropriate drug residues to track a selected population's use of illicit methamphetamine, amphetamine and tetrahydrocannabinol, respectively. For each of these selected drug residues, mass balances were developed by utilizing all disposition data available for their release from all their respective sources, incorporating route-of-administration considerations where relevant, and accounting for variations in the metabolic capacity of users of the various relevant licit and illicit sources. Further, since the selected drug residues for the use of methamphetamine and amphetamine cannot only result from their use but numerous other licit and illicit sources, comprehensive general source models were developed for their enantiomeric-specific release to sewers. The relative importance of the sources identified in the general source model was evaluated by performing national substance flow analyses for a number of countries. Results suggested that licit sources of methamphetamine are expected to be only of significance in populations where its illicit use is minor. Similarly, in populations where the use of illicitly produced amphetamine is currently of relevance, licit contributions to the sewer loads of amphetamine are likely to be of negligible importance. Lastly, the study of tetrahydrocannabinol back-extrapolation mass balances suggested that further research is required to assess the importance of fecal elimination of 11-nor-9-carboxy-tetrahydrocannabinol.
... According to Shulgin and Shulgin [2], the effective dosage for a human is within 1-3 mg. Severe cramps with pain, flashes of depersonalisation, and hallucinations have been described with doses of 2. 8 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 ...
... Kanamori et al. [5] indicated that O-desmethyl-N-acetyl metabolites of 2C-B are major metabolites excreted into rat urine. It is also known from other studies that the methoxy-substituted phenylalkylamines ( p-methoxyamphetamine, mescaline, etc.) produce the O-desmethylmetabolites by oxidation in the organism [7][8][9]. Our limited investigation so far does not claim to present the entire extent of biotransformation of DOB in humans. ...
2,5-Dimethoxy-4-bromoamphetamine (DOB) is a strongly acting hallucinogen with an estimated effective dose of 2 mg for an 80 kg man. The case of two men who ingested a new "hallucinogen LSD-like" designer drug is reported here. They experienced intense hallucinations (onset after 15 min) and vomiting. The men became unconscious and fell into deep coma lasting several days. After an unknown period of time elapsed they were admitted to a hospital in a comatose state. One subject (AX, body mass 113 kg) survived, while the second subject (BX, body mass 65 kg) experienced convulsions, metabolic acidosis and died 6 days later. Gastric, blood, and urine specimens collected on admission into the hospital were sent for toxicological examination. Ethanol concentrations in the blood samples were less than 0.2 g/kg. CEDIA urine screening indicated the presence of THCOOH in both cases, while cocaine and its metabolites were only indicated in the urine from AX. Immunoassay for amphetamines was negative in urine specimens collected from both subjects. GC-MS analysis for unknown drugs allowed for the discovery of the presence of DOB in the gastric and urine samples of both persons. DOB-targeted analysis for the acetylated analyte confirmed its presence in both subjects. Quantitative analysis yielded concentrations in serum of 13 ng/ml (AX) and 19 ng/ml (BX). This report on both nonfatal and fatal DOB overdose cases is based on clear toxicological evidence, and is the first documentation of DOB consumption in the Czech Republic.
... Pholedrine is used therapeutically in eye drops used for diagnostic purposes (Bates et al., 1995) and is a metabolite of the new psychoactive substance PMMA (Staack et al., 2003). ...
Consumption of methamphetamine has primarily been estimated in wastewater-based epidemiology by measuring the parent compound. However, this could lead to overestimation when methamphetamine is directly disposed into the sewer system. In this respect, it would be advantageous to measure a specific metabolite of methamphetamine instead. We identified 4-hydroxymethamphetamine (pholedrine) as a potential marker. Stability experiments were performed in both filtered and unfiltered wastewater. Correlations with relative loads in wastewater were used to establish its potential as a marker of direct disposal of methamphetamine, or even as a wastewater-based epidemiology biomarker of methamphetamine consumption. This study then investigated the use of pholedrine in combination with methamphetamine to better detect direct disposal events and its potential as a marker of methamphetamine consumption. Examples from both South Australia and New Zealand exemplify the use of pholedrine to identify potential instances of direct disposal of methamphetamine.
... Detection and identification of PMMA and DHM TPs was performed by a postacquisition approach [36]. A suspect database of potential TPs was built using two in silico prediction tools, the Eawag-Biocatalysis/Biodegradation Database Pathway Prediction System and the MetabolitePredict software, and a list of reported metabolites and TPs from the literature [5,15,16,40,41,51,53]. Samples were screened using specific criteria [52] and an in-house retention time prediction model [37]. ...
There is a paucity of information on biotransformation and stability of new psychoactive substances (NPS) in wastewater. Moreover, the fate of NPS and their transformation products (TPs) in wastewater treatment plants is not well understood. In this study, batch reactors seeded with activated sludge were set up to evaluate biotic, abiotic, and sorption losses of p-methoxymethylamphetamine (PMMA) and dihydromephedrone (DHM) and identify TPs formed during these processes. Detection and identification of all compounds was performed with target and suspect screening approaches using liquid chromatography quadrupole-time-of-flight mass spectrometry. Influent and effluent 24 h composite wastewater samples were collected from Athens from 2014 to 2020. High elimination rates were found for PMMA (80%) and DHM (97%) after a seven-day experiment and degradation appeared to be related to biological activity in the active bioreactor. Ten TPs were identified and the main reactions were O- and N-demethylation, oxidation, and hydroxylation. Some TPs were reported for the first time and some were confirmed by reference standards. Identification of some TPs was enhanced by the use of an in-house retention time prediction model. Mephedrone and some of its previously reported human metabolites were formed from DHM incubation. Retrospective analysis showed that PMMA was the most frequently detected compound.
... However, the phenethylamine core is shared among several compounds including cathinones and catecholamines. Several metabolism studies have been conducted in an effort to understand their metabolic profiles (Ewald et al., 2008(Ewald et al., , 2006Lai et al., 2015b;Staack et al., 2003) but more information is needed. ...
The information obtained from the chemical analysis of specific human excretion products (biomarkers) in urban wastewater can be used to estimate the exposure or consumption of the population under investigation to a defined substance. A proper biomarker can provide relevant information about lifestyle habits, health and wellbeing, but its selection is not an easy task as it should fulfil several specific requirements in order to be successfully employed. This paper aims to summarize the current knowledge related to the most relevant biomarkers used so far. In addition, some potential wastewater biomarkers that could be used for future applications were evaluated. For this purpose, representative chemical classes have been chosen and grouped in four main categories: (i) those that provide estimates of lifestyle factors and substance use, (ii) those used to estimate the exposure to toxicants present in the environment and food, (iii) those that have the potential to provide information about public health and illness and (iv) those used to estimate the population size. To facilitate the evaluation of the eligibility of a compound as a biomarker, information, when available, on stability in urine and wastewater and pharmacokinetic data (i.e. metabolism and urinary excretion profile) has been reviewed. Finally, several needs and recommendations for future research are proposed.
... The analytical procedures usually consisted of an OT with ion traps or quadrupoles in front. Using LC-QTOF analysis, Lai et al. (2015) mimicked this in vitro strategy to reinvestigate the already published in vitro and/or in vivo metabolism of para-methoxyamphetamine, para-methoxymethamphetamine (Staack et al. 2003, 2004), 4-methylthioamphetamine (Ewald et al. 2005, N-methyl-benzodioxolylbutanamine (MBDB) (Maurer 1996), benzodioxolylbutanamine (BDB) (Maurer 1996), 5-(2-aminopropyl) benzofuran (Welter et al. 2015b), and 6-(2-aminopropyl) benzofuran (Welter et al. 2015a). The aim of this study was to discover the best urine screening targets in case of overdose or for wastewater-based drug epidemiology. ...
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.
... 19,20 PMMA has several active metabolites, including paramethoxyamphetamine and pholedrine. 21 It has a narrow margin of safety, with a two-to fourfold difference between the stimulant-producing and lethal doses observed in rats. 1 Hyperthermia from use of the drug is secondary to both serotonin syndrome and monoamine oxidase inhibition, and toxicity is increased in crowded conditions because of an accelerated rate of temperature change and longer duration of hyperthermia. 20 The delayed onset of desired effects may lead to frequent and early redosing, which increases the risk of lifethreatening toxicity. ...
Background: Paramethoxymethamphetamine (PMMA) is a ring-substituted amphetamine similar in structure to 3,4-methylenedioxy-methamphetamine (MDMA or “ecstasy”), but substantially more toxic. We describe the clinical features of fatal exposures in the provinces of Alberta and British Columbia, Canada.
Methods: We conducted a retrospective case series on deaths in Alberta and BC between June 2011 and April 2012 for which forensic toxicologic analysis was positive for PMMA and the drug was implicated as the primary toxic agent. Data collected included patient demographics, exposure history, clinical features, investigations, therapy provided and postmortem toxicologic findings.
Results: A total of 27 PMMA-associated deaths (20 in Alberta, 7 in BC) were reported in the 11-month period. The median age was 24 (range 14–52) years, and 22 (81%) were male. Ten patients were pronounced dead at the scene, and 17 died in hospital. The median time from exposure to death was 17 (range 5–264) hours. The median first-recorded vital signs (and ranges) were: heart rate 160 (86–201) beats/min, blood pressure 89/43 (69/30–162/83) mm Hg, respiratory rate 40 (26–48) breaths/min, oxygen saturation 81% (68%–100%) and temperature 39.4°C (34–43.8°C). Sixteen of the 17 people who died in hospital presented with clinical features consistent with serotonin syndrome. End-organ dysfunction included hepatic (30%) and acute kidney injury (85%), rhabdomyolysis (54%), coagulopathy (61%) and cardiac ischemia (15%). Other drugs identified on toxicologic analysis were MDMA (n = 27), cocaine or its metabolite benzoylecgonine (n = 14) and methamphetamine (n = 12).
Interpretation: Exposure to PMMA was characterized by multiorgan dysfunction and serotonin syndrome, followed by cardiovascular collapse. In addition to PMMA, multiple synthetic amphetamines were present on toxicologic analysis. When evaluating patients suspected of exposure to sympathomimetic drugs of abuse, clinicians must anticipate multiple clinical effects from the increased release of dopamine, serotonin, norepinephrine and other neurotransmitters
... 19,20 PMMA has several active metabolites, including paramethoxyamphetamine and pholedrine. 21 It has a narrow margin of safety, with a two-to fourfold difference between the stimulant-producing and lethal doses observed in rats. 1 Hyperthermia from use of the drug is secondary to both serotonin syndrome and monoamine oxidase inhibition, and toxicity is increased in crowded conditions because of an accelerated rate of temperature change and longer duration of hyperthermia. 20 The delayed onset of desired effects may lead to frequent and early redosing, which increases the risk of lifethreatening toxicity. ...
Paramethoxymethamphetamine (PMMA) is a ring-substituted amphetamine similar in structure to 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy"), but substantially more toxic. We describe the clinical features of fatal exposures in the provinces of Alberta and British Columbia, Canada.
We conducted a retrospective case series on deaths in Alberta and BC between June 2011 and April 2012 for which forensic toxicologic analysis was positive for PMMA and the drug was implicated as the primary toxic agent. Data collected included patient demographics, exposure history, clinical features, investigations, therapy provided and postmortem toxicologic findings.
A total of 27 PMMA-associated deaths (20 in Alberta, 7 in BC) were reported in the 11-month period. The median age was 24 (range 14-52) years, and 22 (81%) were male. Ten patients were pronounced dead at the scene, and 17 died in hospital. The median time from exposure to death was 17 (range 5-264) hours. The median first-recorded vital signs (and ranges) were: heart rate 160 (86-201) beats/min, blood pressure 89/43 (69/30-162/83) mm Hg, respiratory rate 40 (26-48) breaths/min, oxygen saturation 81% (68%-100%) and temperature 39.4°C (34-43.8°C). Sixteen of the 17 people who died in hospital presented with clinical features consistent with serotonin syndrome. End-organ dysfunction included hepatic (30%) and acute kidney injury (85%), rhabdomyolysis (54%), coagulopathy (61%) and cardiac ischemia (15%). Other drugs identified on toxicologic analysis were MDMA (n = 27), cocaine or its metabolite benzoylecgonine (n = 14) and methamphetamine (n = 12).
Exposure to PMMA was characterized by multiorgan dysfunction and serotonin syndrome, followed by cardiovascular collapse. In addition to PMMA, multiple synthetic amphetamines were present on toxicologic analysis. When evaluating patients suspected of exposure to sympathomimetic drugs of abuse, clinicians must anticipate multiple clinical effects from the increased release of dopamine, serotonin, norepinephrine and other neurotransmitters.
... However, the phenethylamine core is shared among several compounds including cathinones and catecholamines. Several metabolism studies have been conducted in an effort to understand their metabolic profiles (Ewald et al., 2008(Ewald et al., , 2006Lai et al., 2015b;Staack et al., 2003) but more information is needed. ...
... 19,20 PMMA has several active metabolites, including paramethoxyamphetamine and pholedrine. 21 It has a narrow margin of safety, with a two-to fourfold difference between the stimulant-producing and lethal doses observed in rats. 1 Hyperthermia from use of the drug is secondary to both serotonin syndrome and monoamine oxidase inhibition, and toxicity is increased in crowded conditions because of an accelerated rate of temperature change and longer duration of hyperthermia. 20 The delayed onset of desired effects may lead to frequent and early redosing, which increases the risk of lifethreatening toxicity. ...
http://www.bcmj.org/bc-centre-disease-control/increasing-access-naloxone-bc-reduce-opioid-overdose-deaths
... PMMA appears in two isoforms with S(+)-enantiomer being the more active compound (Young et al., 1999). It is metabolized in the liver by CYP2D6 on cytochrome P450 mainly by O-demethylation to para-hydroxymethamphetamine (pholedrine) in rats (Bach et al., 1999; Staack et al., 2004; Staack and Maurer, 2005) and to a minor extent to PMA, and other metabolites (Staack et al., 2003; Rohanova and Balikova, 2009). Subcutaneous (s.c.) administration of PMMA 40 mg/kg to rats led to a peak in serum at 30 min and its metabolite PMA at 2 h. ...
Despite poisoning with the ecstasy substitute para-methoxymethamphetamine (PMMA) being typically associated with severe hyperthermia and death, behavioral and toxicological data on this drug are missing. Herein we present the behavioral profile of PMMA, its hyperthermic potency and pharmacokinetic profile in rats. The effects of PMMA 5 and 20 mg/kg on locomotion, on prepulse inhibition (PPI) of acoustic startle reaction (ASR), on body temperature under isolated and crowded conditions and on the pharmacokinetics analyzed with gas chromatography mass spectrometry (GC-MS) were evaluated. PMMA increased overall locomotion with the higher dose showing a biphasic effect. PPI was decreased dose-dependently. The hyperthermic response was present only with PMMA 20 mg/kg and was accompanied by extensive perspiration under crowded conditions. Serum levels of PMMA peaked at approximately 30 min after both treatments; on the contrary the maximum brain concentrations of PMMA at 20 mg/kg peaked approximately 1h after the administration, which was rather delayed compared to maximum after 5mg/kg dose. These data indicate that PMMA has a similar behavioral profile to stimulants and hallucinogens and that the toxicity might be increased in a crowded environment. High doses of PMMA have a gradual penetration to the brain which might lead to the delayed peak concentrations and prolonged effects of the drug.
... Previous in vivo studies in the rat have shown that PMMA is metabolized mainly by O-demethylation to para-hydroxymethamphetamine (HO-MA) ( Fig. 1) (Staack et al., 2003). The aim of the current study was to identify the human hepatic cytochrome P450 (P450) enzymes involved in the O-demethylation, to determine the kinetic constants for this metabolic reaction, and to compare the metabolite formation in pooled human liver microsomes (pHLMs) and in single donor human liver microsomes with CYP2D6 poormetabolizer genotype (PM HLMs). ...
p-Methoxymethamphetamine (PMMA) is a new designer drug, listed in many countries as a controlled substance. Several fatalities have been attributed to the abuse of this designer drug. Previous in vivo studies using Wistar rats had shown that PMMA was metabolized mainly by O-demethylation. The aim of the study presented here was to identify the human hepatic cytochrome P450 (P450) enzymes involved in the biotransformation of PMMA to p-hydroxymethamphetamine. Baculovirus-infected insect cell microsomes, pooled human liver microsomes (pHLMs), and CYP2D6 poor-metabolizer genotype human liver microsomes (PM HLMs) were used for this purpose. Only CYP2D6 catalyzed O-demethylation. The apparent K(m) and V(max) values in baculovirus-infected insect cell microsomes were 4.6 +/- 1.0 microM and 92.0 +/- 3.7 pmol/min/pmol P450, respectively, and 42.0 +/- 4.0 microM and 412.5 +/- 10.8 pmol/min/mg protein in pHLMs. Inhibition studies with 1 microM quinidine showed significant inhibition of the metabolite formation (67.2 +/- 0.6%; p < 0.0001), and comparison of the metabolite formation between pHLMs and PM HLMs revealed significantly lower metabolite formation in the incubations with PM HLMs (87.3 +/- 1.1%; p < 0.0001). According to these studies, CYP2D6 is the major P450 involved in O-demethylation of PMMA.
... A second pathway was the O-demethylation of the parent compound, followed by N-acetylation and sulfoxidation to the corresponding sulfone. Such O-dealkylation has also been observed for the related compounds 4-bromo-2,5dimethoxyphenethylamine (2C-B), 24 para-methoxyamphetamine (PMA), 42,43 or para-methoxymethamphetamine (PMMA), 37,43,44 while sulfoxidation has been described for 2C-T-7. 7 A third pathway was the N-acetylation of the parent compound, followed either by O-demethylation and sulfoxidation or by S-deethylation to the corresponding thiol with subsequent methylation of the thiol moiety. ...
Studies are described on the metabolism and the toxicological analysis of the phenethylamine-derived designer drug 2,5-dimethoxy-4-ethylthio-beta-phenethylamine (2C-T-2) in rat urine using gas chromatography/mass spectrometry (GC/MS) after enzymatic cleavage of conjugates, liquid-liquid extraction and derivatization. The structures of 14 metabolites were assigned tentatively by detailed interpretation of their mass spectra. Identification of these metabolites indicated that 2C-T-2 was metabolized by sulfoxidation followed by N-acetylation and either hydroxylation of the S-ethyl side chain or demethylation of one methoxy group, O-demethylation of the parent compound followed by N-acetylation and sulfoxidation, deamination followed by reduction to the corresponding alcohol followed by partial glucuronidation and/or sulfation or by oxidation to the corresponding acid followed either by partial glucuronidation or by degradation to the corresponding benzoic acid derivative followed by partial glucuronidation. Furthermore, 2C-T-2 was metabolized by N-acetylation of the parent compound followed either by O-demethylation and sulfoxidation or by S-dealkylation, S-methylation and sulfoxidation. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-T-2 in rat urine, which corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-T-2 in human urine.
The emergence of ever novel psychoactive substances (NPS) on the recreational drug market is an ongoing challenge for analytical toxicologists. If still unknown, their structure must first be elucidated. Once this has been achieved and reference substances have become available, it is important that laboratories working in the field of clinical and forensic toxicology either update existing methods or develop new ones in order to also cover the latest NPS. The present chapter provides an overview on analytical methods for analysis of NPS in biological matrices such as blood, urine, hair etc. and discusses pitfalls and interpretative issues. This includes analytical methods covering compounds from several drug classes as well as methods dedicated to particular structural or pharmacological classes of NPS: piperazines, 2,5-dimethoxyphenethylamine-derived drugs (2Cs and NBOMes), 2,5-dimethoxyamphetamine-derived drugs, 4-substituted amphetamines, β-keto-amphetamines (cathinones), pyrrolidinophenones, tryptamines, synthetic cannabinoid receptor agonists, designer benzodiazepines, and designer opioids. Finally, some recommendations for setting up procedures for analysis of NPS are given.
Abuse of new psychoactive substances (NPSs) has become a health and social issue of global concern. p-Methoxyamphetamine (PMA)/p-methoxymethamphetamine (PMMA) with fluoro- or chloro-derivatives of amphetamine and methamphetamine were among the most common drugs found in specimens from fatal cases in Taiwan during the January 2011 to December 2018 period. A liquid-liquid extraction sample preparation protocol with highly sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) approach was developed for the simultaneous analysis of seven phenethylamine-type drugs - PMA, PMMA, p-methoxyethylamphetamine (PMEA), 4-fluoroamphetamine (4-FA), 4-fluoromethamphetamine (4-FMA), 4-chloroamphetamine (4-CA), and 4-chloromethamphetamine (4-CMA) - in postmortem blood and urine specimens. Separation by liquid chromatography was performed by Agilent Zorbax SB-Aq column. Tandem mass spectrometry was operated in Agilent Jet Stream Technology electrospray ionization in positive-ion multiple reaction monitoring (MRM) mode. An analytical methodology was evaluated using drug-free blood and urine after fortification with 100-2000 ng/mL of the seven target analytes. Average extraction recoveries were >80%; slightly higher ion suppression was observed for PMA and 4-CA; intra-/inter-day precision (%CV) and accuracy were in the ranges of 0.52-12.3% and 85-110%, respectively. Limit of detection (LOD) and lower limit of quantitation (LLOQ) for these seven analytes were both in the 0.5-5 ng/mL range. Interference and carryover were not significant. This relatively simple methodology was found effective and reliable for routine identification and quantitation of these seven analytes in postmortem and antemortem blood and urine specimens received in 2018. Analytical data obtained from these actual cases indicated: (i) compared to findings reported during the 2007-2011 period, the use of substituted phenethylamine-type drugs decreased in 2018; (ii) ketamine and 7-aminonimetazepam (the main metabolite of nimetazepam) were the most common co-ingested substances in specimens containing PMA/PMMA, 4-FA/4-FMA, or 4-CA/4-CMA; and (iii) in drug fatalities, the concentration of PMA was significantly higher than the concentration of PMMA in both urine and blood, while the reverse was true in urine specimens from antemortem cases.
This paper reviews various pitfalls observed during developing, validation, application, and interpretation of drug testing approaches using GC-MS and low- and high-resolution LC-MS. They include sampling and storage of body samples, sample adulteration and contamination, analyte stability, sample preparation without or with cleavage of conjugates, extraction, derivatization, internal standardization, false negative and positive results by GC-MS or LC-MS screening and/or confirmation procedures including artifact formation, ion suppression or enhancement by electrospray ionization, and finally pitfalls in data interpretation. Conclusions and prospects close the Tutorial.
Background:
New psychoactive substances (NPS) are substitutes for classical drugs of abuse, and there are now compounds available from all groups of classical drugs of abuse. During 2014 the number of synthetic cathinones increased dramatically and, together with phenylethylamines, they dominate the NPS markets in the EU. In total, 31 cathinones and 9 phenylethylamines were encountered in 2014. The aim of this paper was to summarize the existing knowledge about the basic pharmacology, metabolism, and human toxicology of relevant synthetic cathinones and phenylethylamines. Compared to existing reviews, we have also compiled the existing case reports from both fatal and non-fatal intoxications.
Method:
We performed a comprehensive literature search using bibliographic databases PubMed and Web of Science, complemented with Google Scholar. The focus of the literature search was on original articles, case reports and previously published review articles published 2014 or earlier.
Results:
and Discussion: The rapid increase of NPS is a growing concern and sets new challenges for societies in drug prevention and legislation but also in clinical and forensic toxicology. In vivo and in vitro studies have demonstrated that the pharmacodynamic profile of cathinones is similar to other psychomotor stimulants. Metabolism studies show that cathinones and phenylethylamines are extensively metabolized, however, the parent compound is usually detectable in human urine. In vitro studies have shown that many cathinones and phenylethylamines are metabolized by CYP2D6 enzymes. This indicate that these drugs may have many possible drug-drug interactions, and that genetic polymorphism may influence their toxicity. However, the clinical and toxicological relevance of CYP2D6 in adverse effects of cathinones and phenylethylamines is questionable, as these compounds are metabolized by other enzymes as well. The toxidromes commonly encountered after ingestion of cathinones and phenylethylamines are mainly of sympathomimetic and hallucinogenic character with a risk of excited delirium and life-threatening cardiovascular effects.
Conclusion:
The acute and chronic toxicity of many NPS is unknown or very sparsely investigated. There is a need for evidence based treatment recommendations for acute intoxications as well as a demand for new strategies to analyze these compounds in clinical and forensic cases.
The characterisation of new psychoactive drugs which were identified in the last years in continuously increasing numbers is a challenge for different academic institutions. This paper gives an overview on new psychoactive drugs in regard of their chemistry, pharmacology, metabolism, analytics and legal aspects in Germany.
The emergence of ever-novel psychoactive substances on the recreational drug market is an ongoing challenge for analytical toxicologists. If still unknown, their structure must first be elucidated. Once this has been achieved and reference substances have become available, it is important that laboratories working in the field of clinical and forensic toxicology either update existing methods or develop new ones that cover the novel psychoactive substances (NPS). This chapter provides an overview of analytical methods for analysis of NPS in biological matrices such as blood, urine and hair and discusses pitfalls and interpretative issues. It includes analytical methods covering compounds from several drug classes as well as methods dedicated to analysis of compounds from the following drug classes: piperazines, 2,5-dimethoxyphenethylamine-derived drugs (2Cs), 2,5-dimethoxyamphetamine-derived drugs, 4-substituted amphetamines, β-keto-amphetamines (cathinones), pyrrolidinophenones, tryptamines, and synthetic cannabinoid receptor agonists. Finally, some recommendations for setting up procedures for analysis of NPS are given.
The flowing atmospheric pressure afterglow (FAPA) ion source operates in the ambient atmosphere and has been proven to be a promising tool for direct and rapid determination of numerous compounds. Here we linked a FAPA-MS system to an electrochemical flow cell for the identification of drug metabolites generated electrochemically in order to study simulated metabolic pathways. Psychostimulants and their metabolites produced by electrochemistry (EC) were detected on-line by FAPA-MS. The FAPA source has never been used before for an on-line connection with liquid flow, neither for identification of products generated in an electrochemical flow cell. The system was optimized to achieve the highest ionization efficiency by adjusting several parameters, including distances and angles between the ion source and the outlet of the EC system, the high voltage for plasma generation, flow-rates, and EC parameters. Simulated metabolites from tested compounds [methamphetamine (MAF), para-methoxy-N-methylamphetamine (PMMA), dextromethorphan (DXM), and benzydamine (BAM)] were formed in the EC cell at various pH levels. In all cases the main products were oxidized substrates and compounds after N-demethylation. Generation of such products and their thorough on-line identification confirm that the cytochrome P450 - driven metabolism of pharmaceuticals can be efficiently simulated in an electrochemical cell; this approach may serve as a step towards predictive pharmacology using a fast and robust design.
In the recent years, analytical toxicologists have been facing difficulties in detecting designer drugs due to the chemical modifications on the existing structures and the speed in which they are released into the market, requiring the development and improvement of specific and appropriate analytical methods. This work is a review of the literature which summarizes the characteristics of the drugs and the analytical validated methods using conventional and unconventional matrices currently used for correct identification and quantification of the following classes of emerging drugs of abuse: derivatives of opiates, amphetamines, tryptamines, piperazines and cannabinoids.
The aim of this review is to present the chemical aspects, pharmacology, acute toxicities, and metabolisms of α-pyrrolidinophenone derivatives, a new group of synthetic cathinones. Compared to other synthetic cathinones, α-pyrrolidinophenone derivatives have high lipophilicity due to the pyrrolidine ring substitution at the nitrogen atom, resulting in higher blood–brain barrier permeability. To date, some acute intoxication and fatal cases involving α-pyrrolidinophenone derivatives have been reported, and the symptoms induced by their high dosages are due to central nervous system and cardiovascular toxicities. Based on the previous metabolism studies, reduction of the β-ketone moiety to the corresponding alcohol metabolites and oxidation to the 2′′-oxo metabolites are the main metabolic pathways observed among α-pyrrolidinophenone derivatives. In addition to such pathways, specific metabolic pathways like hydroxylation followed by oxidation of the 4′-methyl group, O-demethylation of the 4′-methoxyl group, and demethylenation followed by O-methylation of the 3′,4′-methylenedioxy group can be observed for the corresponding ring-substituted compounds.
This chapter describes multi-analyte screening procedures for the simultaneous detection of several drug classes in blood and urine using gas chromatography–mass spectrometry (GC–MS). They are indispensable tools in forensic and clinical toxicology because the compounds that have to be analyzed are often unknown. Therefore, the first step is screening for and identification of the compounds of interest followed by quantification. Immunoassays can be used for preliminary screening to differentiate between negative and presumptively positive samples, if only a single drug or drug class has to be monitored with target screening procedures. Positive results must be confirmed by a second independent method that is at least as sensitive as the screening test and that provides the highest level of confidence in the result. High-throughput drug screening procedures in analytical toxicology mean that thousands of relevant toxicants can simultaneously be screened for systematic toxicological analysis (STA).
In order to prove the intake of a newly encountered designer drug, Methylone, a sensitive and useful method, which allows us to simultaneously detect Methylone and its metabolites in human urine, has been established by means of a combination of gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS). GC-MS accompanied with trifluoroacetyl (TFA) derivatization and LC-MS analyses were performed following acid hydrolysis and liquid-liquid extraction with a chloroform-2-propanol mixture (3:1, v/v). Methylone and its metabolites, 3,4-methylenedioxycathinone (MDC), 4-hydroxy-3-methoxymethcathinone (HMMC) and 3-hydroxy-4-methoxymethcathinone (3-OH-4-MeO-MC) could be satisfactorily separated on a semi-micro ODS column using linear gradient elution with a binary mobile phase of methanol and 10 mM ammonium formate buffer (pH 3.5).
The detection limits of the four analytes by GC-MS were over the range of 5-25 ng/ml in a scan mode, and 0.5-2.5 ng/ml in a selected ion monitoring (SIM) mode. Upon applying the LC-ESI MS technique, the linear calibration curves were obtained using the SIM mode in the range of 25-500 ng/ml for Methylone, HMMC and 3-OH-4-MeO-MC, and 50-1000 ng/ml for MDC. The detection limits were over the range of 25-100 ng/ml in the scan mode, and 2.5-25 ng/ml in the SIM mode.
Because of its high sensitivity, this analytical procedure will be applicable for proof of Methylone intake in forensic toxicology and clinical chemistry.
La connaissance des isoenzymes du cytochrome P450 (CYP), qui sont responsables du metabolisme des xenobiotiques, est indispensable pour predire la toxicocinetique et l'evaluation des risques toxicologiques. La 1-(3-trifluoro-methyl-phenyl)piperazine (TFMPP) est une nouvelle drogue synthetique. Des etudes anterieures ont montre qu'elle est principalement metabolisee en hydroxy-TFMPP (OH-TFMPP). L'objet de cette etude a ete de verifier, a partir de modeles in vivo et in vitro, si cette reaction est catalysee par le cytochrome CYP2D6. dans ce travail, nous avons compare le rapport TFMPP / OH-TFMPP dans l'urine de rats femelles Dark Agouti (fDA, modele du phenotype CYP2D6 metaboliseur lent), de rats Wistar (Wl, modele du phenotype CYP2D6 metaboliseur rapide) et de rats males Dark Agouti (mDA, modele intermediaire). Les resultats montrent clairement l'influence de cette enzyme. L'identification des CYP450 hepatiques humains a ete realisee in vitro a l'aide de microsomes hepatiques humaines et d'enzymes humaines recombinantes. Ces etudes ont montre que CYP1A2, CYP2D6 et CYP3A4 catalysent cette reaction, CYP2D6 etant l'enzyme la plus importante, responsable a 81 % de la clairance hepatique. Des etudes d'inhibition ont confirme ces resultats. La formation de metabolites etait par ailleurs, significativement plus basse dans les microsomes des metaboliseurs lents. Les concentrations plasmatiques des differentes souches des rats ont ete comparees pour verifier si les pharmacocinetiques etaient differentes. Les rats Wl avaient les concentrations les plus faibles. Elles etaient deja plus elevees chez les rats mDA et de loin plus importantes chez les rats fDA.
A high performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-QTOF-MS) method was employed in investigation of benzbromarone metabolites in rat plasma, urine, feces and bile samples. Meanwhile, the metabolic pathways of benzbromarone in rats were discussed. The identification was achieved on a reversed-phase C(18) column with mobile phase gradient method. The QTOF-MS was operated under full scan of MS or MS/MS in negative mode. The fragments were acquired by raising collision induced dissociation (CID) energy for speculating the structures of parent ions. According to the information from the chromatograms and mass spectra, 17 metabolites were obtained. Among them, the deoxidized phase I metabolites and an array of phase II metabolites-sulfate conjugates detected in the biological samples made the work more significant.
A number of analogues of phenethylamine and tryptamine, which are prepared by modification of the chemical structures, are
being developed for circulation on the black market. Often called “designer drugs,” they are abused in many countries, and
cause serious social problems in many parts of the world. Acute deaths have been reported after overdoses of designer drugs.
Various methods are required for screening and routine analysis of designer drugs in biological materials for forensic and
clinical purposes. Many sample preparation and chromatographic methods for analysis of these drugs in biological materials
and seized items have been published. This review presents various colorimetric detections, gas chromatographic (GC)–mass
spectrometric, and liquid chromatographic (LC)–mass spectrometric methods proposed for designer drug analyses. Basic information
on extractions, derivatizations, GC columns, LC columns, detection limits, and linear ranges is also summarized.
KeywordsDesigner drug–GC–MS–LC–MS(–MS)–Phenethylamines–Tryptamines–Piperazines
Designer drugs are introduced periodically to avoid detection and to provide new drugs with different pharmacological activities. During our routine analysis of amphetamine in urine samples, we observed one sample that reacted with immunoassay with high activity. There is one prominent peak in the gas chromatography- mass spectrometry (GC-MS) chromatogram. However, no amphetamine, methamphetamine, MDA, MDMA, MDEA, or ephedrine was detected with GC-MS. Careful examination of the mass spectrum indicated the presence of one fragment ion (m/z 140), which is similar to the base peak of trifluoroacetic anhydride derivative of amphetamine. The characteristic ion cluster representing the presence of one chlorine atom was observed. Investigation with liquid chromatography (LC)-MS detected an unknown compound with molecular ion of m/z 170. This compound was tentatively identified as chloroamphetamine. Pure standard material of p-chloroamphetamine (PCA) was purchased and analyzed with both GC-MS and LC-MS. Identical GC-MS spectra and LC-MS-MS fragmentation patterns were obtained. A GC-MS procedure was developed for the quantitation of PCA. The limits of detection and quantification were 10 μg/L. Precision was between 1.26% and 4.26%, and bias was between -0.91% and 4.27%. The prevalence PCA positive rate is 0.35% of the samples screened positive for amphetamine.
Since the late 1990s the illicit drug market has undergone considerable change: along with the traditional drugs of abuse that still dominate, more than 100 psychotropic substances designed to bypass controlled substances legislation have appeared and led to intoxications and fatalities. Starting from the huge class of phenylalkylamines, containing many subgroups, the spectrum of structures has grown from tryptamines, piperazines, phenylcyclohexyl derivates and pyrrolidinophenones to synthetic cannabinoids and the first synthetic cocaine. Due to the small prevalence and high number of unknown substances, the detection of new designer drugs is a challenge for clinical and forensic toxicologists. Standard screening procedures might fail because a recently discovered or yet unknown substance has not been incorporated in the library used. Nevertheless, many metabolism studies, case reports, screening methods and substance-profiling papers concentrating on single compounds have been published. This review provides an overview of the developed bioanalytical and analytical methods, the matrices used, sample-preparation procedures, concentration of analytes in case of intoxication and also gives a résumé of immunoassay experiences. Additionally, six screening methods for biological matrices with a larger spectrum of analytes are described in more detail.
The emergence of ever new drugs of abuse on the illicit drug market is an ongoing challenge for analytical toxicologists. Because most of these new drugs or drug classes are not detected by established analytical methods targeting classic drugs of abuse, analytical procedures must be adapted or new procedures must be developed to cover these new compounds. This review summarizes the analytical toxicology of the following classes of emerging drugs of abuse: piperazines, phenethylamines (2Cs and FLYs), 4-substituted amphetamines, β-keto-amphetamines, 2,5-dimethoxy-amphetamines, pyrrolidinophenones, and synthetic cannabinoids.
In several cases, fluoxetine, its metabolites, its known artifacts, and supposedly tranylcypromine were detected in urine using the authors' systematic toxicological analysis (STA) procedure based on acid hydrolysis, extraction, and acetylation. As fluoxetine and tranylcypromine are absolutely contraindicated drugs and in none of the cases was tranylcypromine prescribed, the question of whether the detected compound might have been formed by fluoxetine and/or its metabolites arose. Therefore, rat urine taken after dosing with fluoxetine was screened in the same way. In addition, aqueous solutions of fluoxetine, norfluoxetine, tranylcypromine, and a mixture of the latter two drugs were worked-up and analyzed according to the STA and without hydrolysis. In urine specimens obtained from rats dosed with fluoxetine, tranylcypromine was detected as well as in the solution of worked-up norfluoxetine including hydrolysis. Its underlying mass spectrum could be identified by detailed interpretation of the fragmentation patterns as acetylated 3-phenyl-propyl-2-ene-amine. This compound could be postulated as hydrolysis product of norfluoxetine formed by ether cleavage and water elimination. Although this spectrum shows nearly the same fragmentation patterns as that of acetylated tranylcypromine, both compounds could finally be differentiated by their retention indices and by using the positive-ion chemical ionization mode.
Meconium analysis can detect fetal exposure to drugs taken by the mother during pregnancy. Methamphetamine (MAMP) and amphetamine (AMP) have previously been observed in meconium of MAMP-exposed neonates; the presence of other metabolites has not been investigated. Detection of such analytes may lead to more sensitive identification and thus improved medical treatment of affected infants. Forty-three MAMP-positive meconium specimens were analyzed for newly identified MAMP biomarkers, p-hydroxymethamphetamine, p-hydroxyamphetamine, and norephedrine. Due to MAMP adulteration in illicit ecstasy and to simultaneously monitor 3,4-methylenedioxymethamphetamine and MAMP prenatal exposure, 3,4-methylenedioxymethamphetamine, its metabolites, and related sympathomimetic amines were assayed. MAMP, AMP, and unconjugated p-hydroxymethamphetamine were the most prevalent and abundant analytes present in meconium; however, unconjugated p-hydroxyamphetamine and norephedrine also were identified. It is possible that one of these additional analytes could be important for predicting toxicity or maternal or neonatal outcome measures in fetuses exposed to MAMP at specific gestational ages or with different metabolic capabilities. Although these new biomarkers were present in lower concentrations than MAMP and AMP in the meconium of previously confirmed specimens, additional research will determine if inclusion of these analytes can increase identification of MAMP-exposed neonates. Novel methamphetamine biomarker concentrations were characterized in meconium of infants exposed in utero to MAMP.
Methoxyphenamine (o-methoxy-N,alpha-dimethylphenethylamine, Orthoxine) used in earlier times as a bronchodilator is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). The drug and several of its metabolites are commonly analysed in doping control screening assays using gas chromatography-mass spectrometry requiring extraction from urine specimens. A complementary method employing liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry and direct injection of urine aliquots was developed, which provided a fast and sensitive alternative to confirm the presence of the prohibited compound and degradation products in sports drug testing samples. In particular, the chromatographic separation of the active drug from isomeric compounds such as the designer drug p-methoxymetamphetamine (PMMA) was of particular interest to unambiguously identify the applied substance and was accomplished using a C6-phenyl reverse-phase column with isocratic elution. The established procedure was validated for methoxyphenamine with regard to specificity, limit of detection (0.7 ng mL(-1)), intraday- and interday precision (2.5-5.8% and 10.8-16.2%, respectively) and its applicability was demonstrated with an authentic doping control sample which tested positive for the prohibited compound early in 2008.
The classical stimulants amphetamine, methamphetamine, ethylamphetamine and the amphetamine-derived designer drugs MDA, MDMA ('ecstasy'), MDEA, BDB and MBDB have been widely abused for a relatively long time. In recent years, a number of newer designer drugs have entered the illicit drug market. 4-Methylthioamphetamine (MTA), p-methoxyamphetamine (PMA) and p-methoxymethamphetamine (PMMA) are also derived from amphetamine. Other designer drugs are derived from piperazine, such as benzylpiperazine (BZP), methylenedioxybenzylpiperazine (MDBP), trifluoromethylphenylpiperazine (TFMPP), m-chlorophenylpiperazine (mCPP) and p-methoxyphenylpiperazine (MeOPP). A number of severe or even fatal intoxications involving these newer substances, especially PMA, have been reported. This paper describes a method for screening for and simultaneous quantification of the above-mentioned compounds and the metabolites p-hydroxyamphetamine and p-hydroxymethamphetamine (pholedrine) in human blood plasma. The analytes were analyzed by gas chromatography/mass spectrometry in the selected-ion monitoring mode after mixed-mode solid-phase extraction (HCX) and derivatization with heptafluorobutyric anhydride. The method was fully validated according to international guidelines. It was linear from 5 to 1000 micro g l(-1) for all analytes. Data for accuracy and precision were within required limits with the exception of those for MDBP. The limit of quantification was 5 micro g l(-1) for all analytes. The applicability of the assay was proven by analysis of authentic plasma samples and of a certified reference sample. This procedure should also be suitable for confirmation of immunoassay results positive for amphetamines and/or designer drugs of the ecstasy type.
Studies are described on the toxicological analysis of the piperazine-derived designer drug 1-(4-methoxyphenyl)piperazine (MeOPP) in rat urine using gas chromatography-mass spectrometry (GC-MS). The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of MeOPP and its metabolites 1-(4-hydroxy phenyl)piperazine and 4-hydroxyaniline in rat urine after administration of a single dose corresponding to doses commonly taken by drug users. Therefore, this procedure should also be suitable for detection of a MeOPP intake in human urine. However, the metabolites of MeOPP are not unique and can be produced from other drugs. Therefore, differentiation of use of this designer drug from use of the medicaments dropropizine, oxypertine or others, which are metabolized to the MeOPP isomer 1-(2-methoxyphenyl)piperazine, is discussed.
1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.
Studies are described on the metabolism and toxicological analysis of the piperazine-derived designer drug 1-(3,4-methylenedioxybenzyl)piperazine (MDBP) in rat urine using gas chromatography/mass spectrometry (GC/MS). The identified metabolites indicated that MDBP was metabolized by demethylenation and subsequent methylation to N-(4-hydroxy-3-methoxybenzyl)piperazine followed by partial glucuronidation or sulfation. Additionally, degradation of the piperazine moiety to N-(3,4-methylenedioxybenzyl)ethylenediamine and 3,4-methylenedioxybenzylamine and N-dealkylation to piperazine were observed. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid/liquid extraction and microwave-assisted acetylation allowed the detection of MDBP and its above-mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of MDBP by analysis of human urine.
Qualitative studies are described on the metabolism and the toxicological analysis of the nootropic fipexide (FIP) in rat urine using gas chromatography-mass spectrometry (GC-MS). FIP was extensively metabolized to 1-(3,4-methylenedioxybenzyl)piperazine (MDBP), 4-chlorophenoxyacetic acid, 1-[2-(4-chlorophenoxy)acetyl]piperazine, N-(4-hydroxy-3-methoxy-benzyl)piperazine, piperazine, N-(3,4-methylenedioxybenzyl)ethylenediamine, and N-[2-(4-chlorophenoxy)acetyl]ethylenediamine. The authors' systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis of one urine aliquot, liquid-liquid extraction and acetylation allowed the detection of FIP via its metabolites in rat urine after administration of a common FIP dose. Therefore, this qualitative procedure should also be suitable for detection of a FIP intake in human urine. Differentiation of an intake of FIP from that of other drugs which form common metabolites is discussed.
Studies are described on the metabolism and the toxicologic analysis of the nonopioid cough suppressant dropropizine [R,S-3-(4-phenyl-1-piperazinyl)1,2-propandiol, DRO] in human urine using gas chromatography-mass spectrometry (GC-MS). The metabolism studies showed that DRO was metabolized in humans mainly by hydroxylation of the aromatic ring, by N-dealkylation of the parent drug and of the hydroxyl-metabolite to the corresponding N-phenylpiperazines, and by degradation of the piperazine moiety. The authors' systematic toxicologic analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction, and microwave-assisted acetylation allowed the unambiguous detection of DRO and its above-mentioned metabolites in human urine up to about 32 hours after intake of a single common therapeutic dose. The target analytes were found to be the parent compound DRO (earlier phase of excretion) and the hydroxylated metabolite para-hydroxy-DRO (later phase of excretion). Both allowed unambiguous detection of an intake of DRO and also differentiation from other phenylpiperazine derivatives.
This paper reviews chromatographic screening procedures for 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 a mass spectrometer (LC-MS). The pros and cons of the different techniques and procedures are discussed leading to the following conclusions and perspectives. GC-MS, especially in the electron ionization full-scan mode, is still the method of choice for comprehensive screening providing best separation power, specificity and universality, although requiring derivatization. LC-DAD is also often used for screening, but its separation power and its specificity are still inferior to those of GC-MS. Finally, LC-MS has shown to be an ideal supplement, especially for the detection of more polar, thermolabile and/or low-dose drugs, especially in blood plasma. It may become the gold standard in clinical and forensic toxicology and doping control if, at a later date, the costs of the apparatus will be markedly reduced, the current disadvantages like irreproducibility of fragmentation, reduction of ionization by matrix, etc. will be overcome, and finally if one of the increasing number of quite different techniques will become the apparatus standard.
Studies are described on the metabolism and toxicological analysis of the phenethylamine-derived designer drug 2,5-dimethoxy-4-propylthio-beta-phenethylamine (2C-T-7) in rat urine using gas chromatography/mass spectrometry (GC/MS). The identified metabolites indicated that 2C-T-7 was metabolized by hydroxylation of the propyl side chain followed by N-acetylation and sulfoxidation and also by deamination followed by oxidation to the corresponding acid or by reduction to the corresponding alcohol. To a minor extent, 2C-T-7 was also metabolized by S-dealkylation followed by N-acetylation, S-methylation and sulfoxidation. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-T-7 in rat urine that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-T-7 in human urine.
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.
4-Methylthioamphetamine (4-MTA) is a scheduled designer drug that has appeared on the illicit drug market and led to several non-fatal or even fatal poisonings. Only few data are available on its metabolism. The first aim of this study was to identify the 4-MTA metabolites in human urine and then to study whether the authors' STA procedure is suitable for screening for and identification of 4-MTA and/or its metabolites in urine. After enzymatic cleavage of conjugates, solid-phase extraction (SPE) and acetylation the following metabolites could be identified by full-scan gas chromatography-mass spectrometry (GC-MS): deamino-oxo 4-MTA, deamino-hydroxy 4-MTA, ring hydroxy and beta-hydroxy 4-MTA. 4-MTA sulfoxide could be identified as possible artifact. In urine samples after enzymatic hydrolysis, acidic extraction, and methylation, 4-methylthiobenzoic acid could be identified. The authors' systematical toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction (LLE) and acetylation allowed detection of 4-MTA as target analyte plus all the above-mentioned metabolites with the exception of 4-methylthiobenzoic acid. The extraction efficiency of 4-MTA was approximately 70% and the limit of detection (LOD) was 30 ng/ml (S/N 3).
The study of the biotransformation of a new synthetic drug 2,5-dimethoxy-4-bromamphetamine (DOB) and identification of its metabolites in urine of a poisoned person is described using gas chromatography mass spectrometry (GC-MS) with various ways of derivatization. It has been confirmed that one of its metabolic pathways leads to the corresponding 2-O-desmethyl and 5-O-desmethyl metabolites when the latter is prevailing. It is important to know the metabolism of this neurotoxic and hallucinogenic substance as it is a prerequisite for developing reliable toxicological diagnostic procedures and for assessment of toxicological risks.
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.
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.
Two recent cases of death due to paramethoxyamphetamine (PMA), a methoxylated phenylethylamine derivative, are described and compared with a previous PMA death that occurred in this province in 1985. The deceased were 18 or 19 years of age and were reported to have ingested either methylenedioxymethamphetamine (MDMA, Ecstasy) or methylenedioxyamphetamine (MDA) prior to their deaths. Concentrations of PMA were measured in both peripheral and heart blood samples using gas chromatography equipped with a nitrogen-phosphorus detector. PMA results in the most recent cases were 0.6 mg/L and 1.3 mg/L in the peripheral blood samples, and corresponding heart blood samples were 0.7 mg/L and 2.3 mg/L, respectively. In the 1985 case, the femoral blood concentration was 0.6 mg/L, and the heart blood concentration was 0.8 mg/L. Significant differences between heart and peripheral blood concentrations were observed in two of the three cases, which may indicate the potential for postmortem redistribution of PMA.
para-Methoxymethamphetamine (PMMA), a structural hybrid of two central stimulants, lacks stimulant properties but behaves in a manner similar to that of MDMA [N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane]. PMMA has been established as a training drug in drug discrimination studies, and in the present investigation we sought to determine which optical isomer of PMMA is primarily responsible for its stimulus effects. Because PMMA is a conformationally flexible molecule, it was also of interest to determine what conformation is most important for its actions., Accordingly, we prepared and examined S(+)PMMA, R(−)PMMA, and conformationally restricted forms of PMMA: PMMA-AT, TIQ-1, and TIQ-2. S(+)PMMA (ED50 = 0.32 mg/kg) was found to be at least as potent as PMMA (ED50 = 0.41 mg/kg), whereas R(−)PMMA failed to result in complete stimulus generalization. An aminotetralin-like conformation, as found in PMMA-AT (ED50 = 0.29 mg/kg), seems to better account for the actions of PMMA than a tetrahydroisoquinoline-like conformation because TIQ-1 and TIQ-2 failed to result in stimulus generalization. The results of the present study further support the concept that PMMA and MDMA share considerable similarity with respect to their stimulus properties in animals except that PMMA lacks the amphetaminergic stimulant component of action associated with MDMA.
Second-order sufficient optimality conditions for nonlinear optimal control problems arereviewed. Applications of these conditions to convex-concave models of economic controlare presented. On the basis of second-order conditions, recent stability results are discussedthat ensure differentiability of optimal solutions with respect to perturbation parameters inthe system. Methods for computing the sensitivity differentials of optimal solutions areoutlined and numerical results for two economic control problems are given.
p-Hydroxyamphetamine (PHA) and p-hydroxybenzoic acid (PHBA) have been isolated by gas-liquid chromatography (GC) as urinary metabolites of p-methoxyamphetamine (PMA) in dogs and rhesus monkeys. The amounts of PMA excreted unchanged in the total 24-hr urine samples in dogs and monkeys were 26% and 3%, respectively, of the total administered doses. The amounts of the pharmacologically active metabolite PHA excreted in 24-hr urine samples were 13% in the dog and 44% in the monkey. In the dog, 72% of the PHA was present as free (unconjugated) PHA, whereas in the monkey the amount of PHA present in the free form was only 14%.
1. The qualitative and quantitative aspects of the urinary elimination of orally administered 4-methoxy[14C]amphetamine have been examined in the rat and guinea-pig and in three volunteer human subjects, to determine interspecies and interindividual variations in disposition of the drug. 2. Both rat and guinea-pig excreted 70--80% of the administered dose(6 mg/kg) in the urine within 24 h, mainly as metabolites. 3. In the guinea-pig, the drug was metabolized by O-demethylation to give 4-hydroxyamphetamine, which was excreted free (4% dose) and conjugated (73%). No other metabolite was detected. 4. The rat metabolizes the drug both by O-dealkylation and by side-chain oxidation, the products being 4-hydroxyamphetamine (5% of dose free and 60% conjugated) and 1-(4'-methoxyphenyl)propan-2-one oxime (5% dose, free and conjugated). 5. In man the drug (dose 5 mg) is metabolized by O-demethylation and by side-chain oxidation. Marked intersubject variations were observed both in the array and quantitative aspects of metabolite excretion. Two subjects excreted mainly 4-hydroxyamphetamine (free and conjugated) together with smaller amounts of 1-(4'-methoxyphenyl)propan-2-one oxime and 4-hydroxynorephedrine. The third subject, however, who was previously known to exhibit a genetically determined defect in drug oxidation, was defective in O-dealkylation of 4-methoxyamphetamine, and the main excretion products were the unchanged drug together with products of side-chain oxidation, namely, 1-(4'-methoxyphenyl)propan-2-one oxime, 1-(4'-methoxyphenyl)propan-2-one and 4-methoxybenzoic acid. 6. Inter-individual differences in oxidative O-demethylation of the drug are discussed in relation to current theories on the aetiology of schizophrenia and reported fatalities arising from abuse of the drug.
To determine whether metabolite-related analogs of N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) produce stimulus effects similar to those of the parent compound, and to determine the structural requirements associated with the MDMA stimulus, several MDMA analogs were examined in tests of stimulus generalization using rats trained to discriminate 1.5 mg/kg MDMA from saline. Although several of the analogs produced up to 50-60% MDMA-appropriate responding, none [with the exception of N-methyl-1-(4-methoxyphenyl)-2-aminopropane (PMMA)] resulted in stimulus generalization. The partial generalization, coupled with the possible reduced ability of certain of the agents to penetrate the blood-brain barrier relative to MDMA, suggests that these agents are not behaviorally inactive. PMMA, although not a metabolite of MDMA, is closely related in chemical structure to MDMA and its metabolites; PMMA produces > 80% MDMA-appropriate responding and is approximately three times more potent (ED50 = 0.2 mg/kg) than MDMA itself (ED50 = 0.76 mg/kg). PMMA is a newer scheduled substance with an as yet unknown mechanism of action; however, on the basis of the stimulus generalization observed PMMA may share some behavioral and mechanistic similarity with MDMA. These results also indicate that an intact methylenedioxy ring, such as that found in MDMA but absent in PMMA, is not a prerequisite for MDMA-like activity and further support the notion that ring-opened MDMA metabolites may produce effects that contribute to the actions of MDMA.
These studies assessed the neurotoxic potential of N-methyl-1-(4-methoxyphenyl)-2-aminopropane (para-methoxymethamphetamine; PMMA), an amphetamine analog that has surfaced in the illicit drug market. Repeated subcutaneous injections of PMMA caused lasting, dose-related reductions in regional brain concentrations of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), and in the density of [3H]paroxetine-labelled 5-HT uptake sites. Comparison of the neurotoxic potential of PMMA to that of para-methoxyamphetamine (PMA) and 3,4-methyl-enedioxymethamphetamine (MDMA) showed that equivalent doses of PMMA and PMA (80 mg/kg) produced comparable depletions of 5-HT, but that these depletions were not as pronounced as those induced by a lower dose of MDMA (20 mg/kg). Striatal DA was not affected on a long-term basis by any of the ring-substituted amphetamines evaluated in this study. These data suggest that PMMA, like PMA and MDMA, produces long-term (possibly neurotoxic) effects on brain serotonin neurons, but that PMMA is less potent than MDMA as a 5-HT neurotoxin. Further, they raise concern over the illicit use of PMMA since humans could be more sensitive than rodents to the 5-HT neurotoxic effects of PMMA and related drugs.
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.
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)
Four biotransformation pathways of 3,4-(methylenedioxy)methamphetamine (MDMA) in the rat have been identified: N-demethylation, O-dealkylation, deamination, and conjugation (O-methylation, O-glucuronidation, and/or O-sulfation). The specific MDMA metabolites that have been identified are 3-hydroxy-4-methoxymethamphetamine, 4-hydroxy-3-methoxymethamphetamine, 3,4-dihydroxymethamphetamine, 4-hydroxy-3-methoxyamphetamine, 3,4-(methylenedioxy)amphetamine (MDA), (4-hydroxy-3-methoxyphenyl)acetone, [3,4-(methylenedioxy)phenyl]acetone, and (3,4-dihydroxyphenyl)acetone. All except 3,4-dihydroxymethamphetamine were present in the urine. The hydroxylated metabolites were excreted in the urine as the O-glucuronide and/or O-sulfate conjugates, but traces of free 4-hydroxy-3-methoxymethamphetamine and 4-hydroxy-3-methoxyamphetamine were also present in unhydrolyzed urine. N-Demethyl and 3-O-methyl phenolic amine metabolites of MDMA were consistently present in brain, liver, blood, and feces. MDMA was metabolized by the 10000g rat liver supernatant to 4-hydroxy-3-methoxymethamphetamine, 3,4-dihydroxymethamphetamine, MDA, and [3,4-(methylenedioxy)phenyl]acetone. Also, the 10000g rat brain supernatant metabolized MDMA to 4-hydroxy-3-methoxymethamphetamine, 3,4-dihydroxymethamphetamine, 4-hydroxy-3-methoxyamphetamine, and MDA.
The controlled-substance analog N-monomethyl-1-(4-methoxyphenyl)-2-aminopropane (PMMA) may be viewed as being either the 4-methoxy analog of methamphetamine or the N-methyl analog of 1-(4-methoxyphenyl)-2-aminopropane (PMA). Because of its abuse potential, PMMA was examined with regard to (a) its stimulus properties in rats trained to discriminate either 1.0 mg/kg of (+)amphetamine or (+/-)DOM from saline, (b) its toxicity (isolated and aggregated) in mice relative to (+/-)PMA, and (c) its locomotor stimulant activity in mice relative to (+/-)amphetamine, (+/-)methamphetamine, and (+/-)PMA. Racemic PMMA produced neither DOM-like nor, unlike PMA, amphetamine-like stimulus effects. There was no significant difference between the 24-hr isolated (LD50 = 63 mg/kg) and aggregated (LD50 = 53 mg/kg) toxicity, and PMMA did not produce significant locomotor stimulation at doses of up to 30 mg/kg. The present results suggest that while PMMA may produce central effects it does not appear to behave as a simple amphetamine-like agent.
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).
Studies on the isolation, identification, quantification, and synthesis of the urinary metabolites of racemic 3,4-methylenedioxyethylamphetamine (MDE) in humans are presented. After oral administration of 140 mg of racemic MDE to healthy volunteers, the following phase I metabolites could be isolated and identified by GC/MS: unchanged racemic MDE (I), racemic 3,4-dihydroxyethylamphetamine (II), racemic 4-hydroxy-3-methoxyethylamphetamine (IIIa), racemic 3,4-methylenedioxyamphetamine (IV), racemic 3,4-dihydroxyamphetamine (V), racemic 4-hydroxy-3-methoxyamphetamine (VIa), methylenedioxyphenylacetone (IXa), 3,4-methylenedioxyhippuric acid (X), and hydroxymethoxyhippuric acid (XII). The probable intermediate metabolite 3,4-dihydroxyhippuric acid (XI) could not be detected. Therefore, two overlapping phase I metabolic pathways for racemic MDE in humans could be postulated. The first and predominant pathway leads, via ring degradation by O-dealkylation, to the corresponding 3,4-dihydroxy metabolites, which are subsequently methylated at the hydroxyl group at position 3 of the aromatic ring. The second pathway leads, via side chain degradation by N-dealkylation, to the corresponding primary amines (IV, V, and VI). Oxidative N-deamination forms the substituted phenylacetones, which are degraded to the corresponding benzoic acids. This is followed by conjugation with glycine to form substituted hippurates. The structures of all of these metabolites were confirmed by chemical syntheses, which are described in this paper. All of the metabolites containing hydroxy groups are partly excreted in a conjugated form, because the amounts of these metabolites were much higher in urine extracts after enzymatic cleavage of conjugates. Quantification of the urinary excretion by HPLC revealed that 19% of the MDE dose was eliminated as I, 31.6% as IIIa, and 2.8% as IV within 32 hr.
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.
Amphetamine its methylendioxy (methylendioxyamphetamine methylenedioxymethylamphetamine, methylenedioxyethylamphetamine) and methoxy derivatives (p-methoxyamphetamine and p-methoxymethylamphetamine) are widely abused in Spanish society. We present here the results of a systematic study of all cases of deaths brought to the attention of the Madrid department of the Instituto Nacional de Toxicologia from 1993 to 1995 in which some of these drugs have been found in the cadaveric blood. The cases were divided into three categories: amphetamine and derivatives, amphetamines and alcohol, amphetamines and other drugs. Data on age, sex, clinical symptoms, morphological findings, circumstances of death, when known, and concentration of amphetamine derivatives, alcohol and other drugs in blood are given for each group. The information provided here may prove to be useful for the forensic interpretation of deaths which are directly or indirectly related to abuse of amphetamine derivatives.
The phenylisopropylamine PMMA or N-methyl-1-(4-methoxyphenyl)-2-aminopropane, a structural hybrid of paramethoxyamphetamine (PMA) and methamphetamine, has been previously shown to unexpectedly lack amphetamine-like or hallucinogen-like stimulus properties in animals. For example, in tests of stimulus generalization, neither a (+)amphetamine stimulus nor a DOM stimulus generalized to PMMA. It has also been shown, however, that stimulus generalization does occur in animals trained to discriminate the designer drug MDMA ("Ecstasy" or N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane) from vehicle. In order to further characterize this unique agent, we trained a group of six Sprague-Dawley rats to discriminate 1.25 mg/kg of PMMA (ED50 = 0.44 mg/kg) from saline vehicle. The PMMA stimulus failed to generalize to the phenylisopropylamine stimulant (+)amphetamine, or to the phenylisopropylamine hallucinogen DOM. Stimulus generalization occurred to (+/-)MDMA (ED50 = 1.32 mg/kg) and S(+)MDMA (ED50 = 0.48 mg/kg). Partial generalization occurred with R(+)MDMA, PMA, 3.4-DMA, and fenfluramine. The PMMA stimulus also generalized to the alpha-ethyl homolog of PMMA (EH/PMMA, ED50 = 1.29 mg/kg). Taken together, the results of these studies suggest that PMMA is an MDMA-like agent that lacks the amphetamine-like stimulant character of MDMA. These findings support our previous suggestion that PMMA be considered the structural parent of the MDMA-like family of designer drugs.
p-hydroxymethamphetamine (OHMAP) is one of the major metabolites of the widely abused drug methamphetamine (MAP). The demethylation of OHMAP to p-hydroxyamphetamine (OHAP) has been shown in vitro but has never been reported in vivo. The disposition kinetics as well as the metabolism of OHMAP was investigated employing a sensitive HPLC method which can separate the enantiomers of OHMAP and OHAP. Both conjugated and unconjugated forms of these compounds can be quantitated. Male Sprague-Dawley rats were given an iv bolus of racemic OHMAP (20 mg kg-1) and serum and urine samples were collected at selected times. The serum concentration-time data for OHMAP enantiomers could be described by a biexponential equation. The clearance of D-OHMAP (93.5 mL min-1 kg-1) was slightly, but statistically significantly, greater than that of the L-enantiomer (83.9 mL min-1 kg-1). The steady-state volumes of distribution of L- and D-OHMAP were (mean +/- SD) 3.15 +/- 0.84 and 4.23 +/- 1.76 L kg-1, respectively. No significant concentrations or amounts of OHAP enantiomers could be detected in any serum or urine sample. Rats excreted more unchanged L-OHMAP (34%) than D-OHMAP (29%). In contrast, more conjugated D-OHMAP (57%) was recovered compared to the conjugated L-OHMAP (52%). The results suggest that there is slight stereoselectivity in the disposition of OHMAP enantiomers. The N-demethylation product (OHAP) was not produced in vivo.
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.
Investigation of a suspicious death became confused when apparently contradictory findings arose on examination of the body. Subsequent identification of amphetamine abuse including PMA resolved these issues. The reported case highlights some atypical features which may cause difficulty in this small but increasing group of deaths. Three rapid deaths attributed to PMA have been identified in Adelaide in the 12 months before June 1996.
Paramethoxyamphetamine (PMA) is a methoxylated phenethylamine derivative that has been used illicitly in Australia since late 1994. It is purportedly sold under the guise of "ecstasy", which is the colloquial name for methylenedioxymethamphetamine (MDMA). Methods for extraction, identification, and quantitation are presented. Toxicology findings in six fatalities involving the drug are discussed. Femoral blood PMA levels ranged from 0.24 to 4.9 mg/L (mean, 2.3 mg/L). Liver PMA levels ranged from 1.4 to 21 mg/kg (mean, 8.9 mg/kg). Other amphetamines were found in five of the six cases. Blood PMA levels in three nonfatal cases are also presented. PMA appeared to be more toxic than MDMA, and blood levels greater than 0.5 mg/L seemed likely to be associated with toxic effects.
This paper reviews procedures for the determination of amphetamine, methamphetamine and amphetamine-derived designer drugs or medicaments in blood and urine. Papers published from 1991 to early 1997 were taken into consideration. Gas chromatographic and liquid chromatographic procedures with different detectors (e.g., mass spectrometer or diode array) were considered as well as the seldom used thin-layer chromatography and capillary electrophoresis. Enantioselective procedures are also discussed. A chapter deals with amphetamine-derived medicaments, e.g. anoretics, antiparkinsonians or vasodilators, which are metabolized to amphetamine or methamphetamine. Differentiation of an intake of such medicaments from amphetamine or methamphetamine intake is discussed. Basic information about the biosample assayed, internal standard, work-up, GC column or LC column and mobile phase, detection mode, reference data and validation data of each procedure is summarized in Tables. Examples of typical applications are presented.
To analyze features of a series of fatalities caused by amphetamine-derivative designer drugs marketed as "Ecstasy" in South Australia, and to identify reasons for the recent marked increase in number of these deaths.
Following the death of a 26-year-old woman after alleged ingestion of Ecstasy tablets, a retrospective search of files at State Forensic Science, Adelaide and the South Australian State Coroner's Department was undertaken from February 1992 to January 1997 to identify similar cases.
Six fatalities were found, all of which have occurred since September 1995 (M:F ratio, 1:1; age range, 22 to 36 years; average age, 27.7 years). All individuals had histories of recent ingestion of illegal drugs thought to be Ecstasy (methylenedioxymethamphetamine, MDMA) at the time of purchase. Delay occurred in seeking medical attention, despite severe symptoms. Causes of death involved documented hyperthermia in 3 cases (temperatures of 41.5-46.1 degrees C), with features of hyperthermia in one other case, and intracranial hemorrhage in another. Drugs in toxic/lethal amounts identified at postmortem included paramethoxyamphetamine (PMA) in all cases, amphetamine/methamphetamine in 4 cases, and methylenedioxymethamphetamine (MDMA or Ecstasy) in only 2 cases. Interaction with a prescription medication (fluoxetine) may have occurred in 1 case.
The number of deaths due to amphetamine derivatives apparently due to substitution of PMA for MDMA (Ecstasy) have recently increased markedly in Adelaide. Potential users should be warned that PMA has been associated with a much higher rate of lethal complications than other designer drugs, and that no guarantee can be made that tablets sold as Ecstasy are not PMA.
The method for the quantitation of pholedrine in human serum involves extraction of the sample with benzene utilizing an ion-pairing reagent, bis(2-ethylhexyl)phosphoric acid, and re-extraction into diluted phosphoric acid. Analysis is carried out on a ODS reversed-phase column with heptanesulfonate as the ion-pairing reagent. The procedure allows quantitation at the lower nanogram level and is useful for pharmacokinetic investigations.
1. Amphetamine (AM) and five amphetamine derivatives, N-ethylamphetamine (NEA), N-butylamphetamine (NBA), 4-methoxyamphetamine (M-AM), 4-methoxy-N-ethylamphetamine (M-NEA) and 4-methoxy-N-butylamphetamine (M-NBA) were incubated with microsomal preparations from cells expressing human CYP2D6 to determine whether the enzyme was capable of catalyzing the direct ring oxidation of all substrates; the N-dealkylation of NEA, NBA, M-NEA and M-NBA; and the O-demethylation of M-AM, M-NEA and M-NBA. 2. None of the six compounds examined was N-dealkylated to any extent. 3. The only metabolites produced from AM, NEA and NBA were the corresponding ring 4-hydroxylated compounds, and the rates of formation were low. 4. All ring 4-methoxylated substrates were efficiently O-demethylated by CYP2D6 to their corresponding phenols. The size of the N-alkyl group influenced the rates of formation of these phenolamines. In contrast to reported findings with 2- and 3-methoxyamphetamines, none of the 4-methoxyamphetamines was ring-oxidized in the CYP2D6 enzyme system to 2- or 3-hydroxy-4-methoxyamphetamines or to dihydroxyamphetamines.
para-Methoxymethamphetamine (PMMA), a structural hybrid of two central stimulants, lacks stimulant properties but behaves in a manner similar to that of MDMA [N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane]. PMMA has been established as a training drug in drug discrimination studies, and in the present investigation we sought to determine which optical isomer of PMMA is primarily responsible for its stimulus effects. Because PMMA is a conformationally flexible molecule, it was also of interest to determine what conformation is most important for its actions. Accordingly, we prepared and examined S(+)PMMA, R(-)PMMA, and conformationally restricted forms of PMMA: PMMA-AT, TIQ-1, and TIQ-2. S(+)PMMA (ED50 = 0.32 mg/kg) was found to be at least as potent as PMMA (ED50 = 0.41 mg/kg), whereas R(-)PMMA failed to result in complete stimulus generalization. An aminotetralin-like conformation, as found in PMMA-AT (ED50 = 0.29 mg/kg), seems to better account for the actions of PMMA than a tetrahydroisoquinoline-like conformation because TIQ-1 and TIQ-2 failed to result in stimulus generalization. The results of the present study further support the concept that PMMA and MDMA share considerable similarity with respect to their stimulus properties in animals except that PMMA lacks the amphetaminergic stimulant component of action associated with MDMA.
This paper reviews systematic toxicological analysis (STA) procedures for acidic drugs and/or metabolites relevant to clinical and forensic toxicology or doping control using gas chromatography, gas chromatography-mass spectrometry, liquid chromatography, thin-layer chromatography and capillary electrophoresis. Papers from 1992 to 1998 have been taken into consideration. Screening procedures in biosamples (whole blood, plasma, serum, urine, vitreous humor, brain, liver or hair) of humans or animals (horse, or rat) are included for the following drug classes: angiotensin-converting enzyme (ACE) inhibitors and angiotensin II (AT-II) blockers, anticoagulants of the 4-hydroxy coumarin type, barbiturates, dihydropyridine calcium channel blockers (calcium antagonists), diuretics, hypoglycemic sulfonylureas and non-steroidal anti-inflammatory drugs (NSAIDs). Methods for confirmation of preliminary results obtained by screening procedures using immunoassay or chromatographic techniques are also included. Furthermore, procedures for the simultaneous detection of several drug classes are reviewed. The toxicological question to be answered and the consequences 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, separation column, mobile phase or separation buffer, detection mode and validation data of each procedure is summarized in 16 tables. They are arranged according to the drug class and the analytical method. Examples of typical applications are presented. Finally, STA procedures are reviewed and described allowing simultaneous screening for different (acidic) drug classes.
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.
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.
This study describes screening and identifying amphetamines, methamphetamines, and their derivatives in urine using immunochemical (Triage™8, FPIA) and chromatographic techniques (REMEDi™ HS). Amphetamines, methamphetamines, MDMA (3,4-methylenedioxymethamphetamine), MDA (3,4-methylenedioxyamphetamine), MDE (3,4-methylenedioxyethylamphetamine), MBDB (N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine), BDB (3,4-(methylenedioxyphenyl)-2-butanamine), PMA (4-methoxyamphetamine), DOM (2,5-dimethyloxy-4-methylamphetamine), DOB (4-bormo-2,5-dimethyloxyamphetamine), amphetaminil, pholedrine, fenfluramine, and amfepramone were subjected to a comparative study. For this, the substances were analyzed to determine their specific threshold concentration for a positive detection in the Triage test and their limit of detection and positive threshold concentration for the FPIA test and the results compared.
Furthermore, the capabilities of a more detailed analysis with the REMEDi system were studied. This HPLC system was able to produce information on the single drugs and main metabolites found in the sample with the danger of false-positive or false-negative screening results greatly minimized.
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.
Recently, tablets inscribed with the Mitsubishi 3-diamond logo, and sold as 3,4-methylenedioxymethamphetamine (MDMA), were found to contain p-methoxymethamphetamine (PMMA), a compound with MDMA-like effects. Shortly after this first submission, similarly inscribed tablets were encountered containing both PMMA and p-methoxyamphetamine (PMA). This second tablet composition has been implicated in several recent deaths in the US. Because two other positions are available for mono-methoxy substitution on the phenyl ring, it is essential that the correct identification be made for these compounds. Analytical data are supplied to enable differentiation of these ring isomers as well as the ketones that serve as their precursors.
To describe the clinical features of paramethoxyamphetamine (PMA; "death") poisoning and to compare these with those of people with self-reported "ecstasy" poisoning.
Retrospective casenote review.
22 patients who presented to the Emergency Department of the Royal Adelaide Hospital (RAH), a major metropolitan teaching hospital, between 1 January 1996 and 31 December 1998 with PMA poisoning identified through urine drug screens; and 61 patients with self-reported ecstasy poisoning between 1 September 1997 and 31 December 1998 found through the hospital databases.
Patients with PMA poisoning presented with tachycardia (64%), hyperthermia (temperature > 37.5 degrees C; 36%), coma (41%), seizures (32%), arrhythmias (23%), and QRS intervals > or = 100 ms (50%) with greater frequency and often greater severity than those with self-reported ecstasy poisoning. Two patients with PMA poisoning presented with severe hypoglycaemia (blood glucose level, < 1.5 mmol/L) accompanied by hyperkalaemia (K+ concentration, > 7.5 mmol/L).
At our hospital, PMA poisonings accounted for most of the severe reactions among people who believed they had taken ecstasy. Hypoglycaemia and hyperkalaemia may be specific to PMA poisoning. PMA toxicity should be suspected with severe or atypical reactions to "ecstasy", and confirmed by chromatographic urine drug screens.
Psychoactive phenylisopropylamines can produce one or more of several different stimulus effects in animals. These effects are typified by the hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM), the central stimulant amphetamine, and by N-methyl-1-(4-methoxyphenyl)-2-aminopropane (PMMA), an agent whose actions are not yet well understood. The optical isomers of two phenylisopropylamines known to lack DOM and amphetamine-stimulus character, that is N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminobutane (MBDB) and 1-(3,4-dimethoxyphenyl)-2-aminopropane (3,4-DMA), were examined in rats trained to discriminate 1.25 mg/kg of PMMA from vehicle. The PMMA stimulus (ED(50)=0.4 mg/kg) generalized to all four agents: S(+)-MBDB (ED(50)=0.8 mg/kg), R(-)-MBDB (ED(50)=2.0 mg/kg), S(+)-3,4-DMA (ED(50)=2.6 mg/kg) and R(-)-3,4-DMA (ED(50)=3.9 mg/kg). The results show that these agents produce stimulus effects similar to those produced by PMMA. Both isomers of MBDB have been previously demonstrated to substitute for N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) in rats trained to discriminate MDMA from vehicle, but MBDB-trained animals failed to recognize DOM or amphetamine. Similar results were obtained with the 3,4-DMA optical isomers in the present investigation using rats trained to discriminate MDMA, DOM or (+)-amphetamine from vehicle; both isomers of 3,4-DMA substituted for an MDMA stimulus, but not for a DOM or amphetamine stimulus. Taken together, the evidence suggests that PMMA, S(+)-MBDB, R(-)-MBDB, S(+)-3,4-DMA, R(-)-3,4-DMA, and S(+)-MDMA can produce common stimulus effects in rats. The present findings also better define the PMMA stimulus and the structural requirements necessary to produce this type of stimulus effect.
The past several years have seen a marked increase in the recreational use of 3,4-methylenedioxymethamphetamine (MDMA) or "Ecstasy". MDMA use is especially common among young people participating in dance parties called "raves". Paramethoxyamphetamine (PMA) exhibits both structural and pharmacological similarity to MDMA. It may, however, be a more potent central stimulant, particularly in its effects on serotonergic transmission. Several fatalities from PMA have been reported in Australia, and here we report three recent fatalities that occurred in the midwestern United States in which each of the decedents believed that they were ingesting MDMA. Symptoms observed included agitation and bruxism, progressing to severe hyperthermia, convulsions, and hemorrhage. Blood was screened for drugs of abuse by enzyme immunoassay with the presence of amphetamines indicated in each case. Confirmation and quantitation for amphetamines was performed by gas chromatography-mass spectrometry. The deceased, two males ages 19 and 24 and a female age 18, had postmortem blood PMA concentrations of 1.07, 0.60, and 1.90 mg/L, respectively. PMA is not a contaminant of MDMA, and no MDMA was found in any of these cases. The primary metabolite of PMA is produced by O-demethylation to 4-hydroxyamphetamine, a reaction catalyzed by cytochrome P450 2D6. This enzyme is noted to be genetically polymorphic. Those with the "slow metabolizer" phenotype may be likely to have higher peak blood concentrations of PMA. Whether any of the decedents described herein were of the slow metabolizer phenotype is not known. Several groups have advocated the onsite use of the Marquis Test for the purpose of pill screening in efforts to distinguish PMA from MDMA. A dark purple is consistent with MDMA, whereas PMA imparts no color change in this test. PMA is often in the form of a white pill with a Mitsubishi symbol on one side. This design has been identified in at least one of these fatalities.
This paper reviews the toxicokinetics of amphetamines. The designer drugs MDA (methylenedioxy-amphetamine, R,S-1-(3;,4;-methylenedioxyphenyl)2-propanamine), MDMA (R,S-methylenedioxymethamphetamine), and MDE (R,S-methylenedioxyethylamphetamine), as well as BDB (benzodioxolylbutanamine; R,S-1-(1;,3;-benzodioxol-5;-yl)-2-butanamine or R,S-1-(3;,4;-methylenedioxyphenyl)-2-butanamine) and MBDB (R,S-N-methyl-benzodioxolylbutanamine), were taken into consideration, as were the following N-alkylated amphetamine derivatives: amphetaminil, benzphetamine, clobenzorex, dimethylamphetamine, ethylamphetamine, famprofazone, fencamine, fenethylline, fenproporex, furfenorex, mefenorex, mesocarb, methamphetamine, prenylamine, and selegiline. English-language publications from 1995 to 2000 were reviewed. Papers describing identification of metabolites or cytochrome P450 isoenzyme-dependent metabolism and papers containing pharmacokinetic/toxicokinetic data were considered and summarized. The implications of toxicokinetics for toxicologic assessment or for interpretation in forensic cases are discussed.
Studies are described on the metabolism and on the toxicological analysis of the piperazine-like designer drug N-benzylpiperazine (BZP, scene name "A2") in rat and human urine using gas chromatography-mass spectrometry (GC-MS). The identified metabolites indicated that BZP was hydroxylated at the aromatic ring and that the piperazine moiety is metabolically degraded. Our systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of the parent compound as well as of the above mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. It has also proved to be applicable in authentic clinical or forensic cases. However, it should be considered that BZP is also a metabolite of the medicament piberaline.
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