Chiral analysis of amphetamine, methamphetamine, MDMA and MDA enantiomers in human hair samples.

A novel analytical method was developed for the simultaneous quantification of the R/S-enantiomers of amphetamine, methamphetamine, MDA and MDMA in hair samples using liquid chromatography-tandem mass spectrometry (LC-MS-MS). This method involved a straightforward derivatization step with dansyl chloride and the use of a chiral column, enabling the separation and quantification of all eight enantiomers in a single analysis. The method exhibited excellent linearity across a concentration range of 0.03-3.00 ng/mg for each enantiomer. Precision and accuracy were within acceptable limits, with bias and relative standard deviation (RSD) values consistently below 6% and 9%, respectively. Selectivity and specificity assessments confirmed the absence of any interference from contaminants or co-extracted drugs. The method demonstrated high sensitivity, with limits of detection (LOD) below 8 pg/mg and limits of quantification (LOQ) below 19 pg/mg for all analytes. Extraction recovery exceeded 79%, and matrix effects were minimal for all analytes. Processed sample stability evaluations revealed consistent results with deviations below 11% for all analytes. Application of the method to 32 authentic human hair samples provided valuable insights into amphetamine use patterns, allowing differentiation between medical amphetamine consumption and illicit use based on enantiomeric composition. Additionally, the method detected co-use of methamphetamine, MDA or MDMA in some samples, highlighting its applicability in drug monitoring and real-life case scenarios within a forensic institute. This innovative analytical approach offers a sensitive and selective method for enantiomeric differentiation of amphetamine, methamphetamine, MDA and MDMA in human hair samples, providing a valuable tool for forensic and clinical investigations.

Derivatization-free determination of chiral plasma pharmacokinetics of MDMA and its enantiomers.

3,4-Methylenedioxymethamphetamine (MDMA) is an entactogen with therapeutic potential. The two enantiomers of MDMA differ regarding their pharmacokinetics and pharmacodynamics but the chiral pharmacology of MDMA needs further study in clinical trials. Here, an achiral and an enantioselective high performance liquid chromatography-tandem mass spectrometry method for the quantification of MDMA and its psychoactive phase I metabolite 3,4-methylenedioxyamphetamine (MDA) in human plasma were developed and validated. The analytes were detected by positive electrospray ionization followed by multiple reaction monitoring. The calibration range was 0.5-500 ng/mL for the achiral analysis of both analytes, 0.5-1,000 ng/mL for chiral MDMA analysis, and 1-1,000 ng/mL for chiral MDA analysis. Accuracy, precision, selectivity, and sensitivity of both bioanalytical methods were in accordance with regulatory guidelines. Furthermore, accuracy and precision of the enantioselective method were maintained when racemic calibrations were used to measure quality control samples containing only one of the enantiomers. Likewise, enantiomeric calibrations could be used to reliably quantify enantiomers in racemic samples. The achiral and enantioselective methods were employed to assess pharmacokinetic parameters in clinical study participants treated with racemic MDMA or one of its enantiomers. The pharmacokinetic parameters assessed with both bioanalytical methods were comparable. In conclusion, the enantioselective method is useful for the simultaneous quantification of both enantiomers in subjects treated with racemic MDMA. However, as MDMA and MDA do not undergo chiral inversion, enantioselective separation is not necessary in subjects treated with only one of the enantiomers.

3,4-Methylenedioxymethamphetamine quantification via benchtop 1H qNMR spectroscopy: Method validation and its application to ecstasy tablets collected at music festivals.

We describe a method validation for the quantification of 3,4-methylenedioxymethamphetamine (MDMA) in tablets based on the United Nations Office on Drugs and Crime (UNODC) guideline for quantitative Nuclear Magnetic Resonance analysis (qNMR). qNMR experiments were carried out on a 60 MHz benchtop NMR spectrometer employing ethylene carbonate as an internal calibrant. A series of 'ecstasy' tablets seized at music events were quantified and the results discussed regarding their within-batch variation and yearly median dose. The method showed good specificity and selectivity, with linearity, precision, accuracy, and recovery well within the UNODC recommended criteria. The limit of detection and quantification are 0.33 mg/mL and 0.10 mg/mL respectively, proving the method works well on small amounts of MDMA. Overall, the lowest amount of MDMA free base detected in this study was 9.35 mg in a piperazine mix, while the highest dosed tablet contained 237.55 mg MDMA free base, with a 9.1% decrease in median amount compared to the pre-pandemic data (2019), but still higher than the data collected in a previous study (105 mg median amount of MDMA free base in 2018). The within-batch variation was insignificant for one of the seizures but showed greater variation for the other, which confirmed that the MDMA content of a single tablet may not reflect that of the whole batch. This dynamic upward change in tablet dosage highlights the importance of ongoing trend monitoring and specific prevention intervention to counteract the negative consequences associated with MDMA use. Benchtop NMR has been successfully employed in quality control, material science and more recently, drug analysis. The present study demonstrates its beneficial application in forensic science overcoming the limitations of currently available instruments and techniques employed in harm reduction and field testing.

Determination of the enantiomeric composition of amphetamine, methamphetamine and 3,4-methylendioxy-N-methylamphetamine (MDMA) in seized street drug samples from southern Germany.

Amphetamine (speed), methamphetamine (crystal meth), and 3,4-methylenedioxy-N-methylamphetamine (MDMA, ecstasy) represent the most frequently abused amphetamine-type stimulants (ATS). Differences in pharmacological potency and metabolism have been shown for the enantiomers of all three stimulants. Legal consequences in cases of drug possession may also differ according to the German law depending on the enantiomeric composition of the seized drug. Therefore, enantioselective monitoring of seized specimens is crucial for legal and forensic casework. Various kinds of samples of amphetamine (n = 143), MDMA (n = 94), and methamphetamine (n = 528) that were seized in southern Germany in 2019 and 2020 were analyzed for their chiral composition using different chromatographic methods. Whereas all samples of amphetamine and MDMA were racemic mixtures, the chiral composition of the methamphetamine specimens was diverse. Although the vast majority (n = 502) was present as (S)-methamphetamine, also specimens containing pure (R)-methamphetamine (n = 7) were found. Furthermore, few samples (n = 8) were of racemic nature or contained non-racemic mixtures of both enantiomers (n = 10). Because methamphetamine appears in varying enantiomeric compositions, any seizure should be analyzed using an enantioselective method. Amphetamine and MDMA, on the other hand, currently appear to be synthesized exclusively via racemic pathways and are not chirally purified. Nevertheless, regular monitoring of the chiral composition should be ensured.

Identification of a new M-ALPHA analog and MDMA in an illegal health product.

The widespread abuse of illicit psychoactive substances is one of the most serious public health and social problems. A suspicious airmail package was seized by Korean customs, and two psychoactive substances in the grayish-green pills in the package were detected by ultra-performance liquid chromatography. The structures of the two substances were elucidated by a combination of liquid chromatography quadrupole time-of-flight mass spectrometry, nuclear magnetic resonance spectroscopy, and comparison with reported or newly generated spectral data of the suggested structures. One of the psychoactive substances proved to be MDMA (commonly known as "Ecstasy"), and the other compound was an M-ALPHA analog bearing a hydroxyl group and an N-methylcarboxamide group. The new M-ALPHA analog was determined as 3-(benzo[d][1,3]dioxol-5-yl)-2-hydroxy-N,2-dimethyl-3-(methylamino)propanamide and named as M-ALPHA-HMCA, wherein HMCA denotes hydroxymethylcarboxamide. Although psychoactivity of this compound has not been assessed, M-ALPHA-HMCA should be considered a potential new psychoactive substance and/or a by-product of MDMA.

Quantitative Estimation of 38 Illicit Psychostimulants in Blood by GC-APCI-QTOFMS with Nitrogen Chemiluminescence Detection Based on Three External Calibrators.

A method was developed for quantitative estimation of illicit psychostimulants in blood, with an emphasis on new psychoactive substances, based on gas chromatography nitrogen chemiluminescence detection coupled with atmospheric pressure chemical ionization quadrupole time-of-flight mass spectrometry (GC-NCD-APCI-QTOFMS). Quantitative estimation relied on the NCD's N-equimolar response to nitrogen, using amphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and methylenedioxypyrovalerone as external calibrators for prim-, sec- and tert- amines, respectively. After spiking with 38 stimulants at 3 concentration levels, the donor blood samples were submitted to liquid-liquid extraction at a basic pH followed by acylation with trifluoroacetic anhydride. All but 3 psychostimulants could be analyzed with a limit of quantification (LOQ) of 0.05 mg/L. At LOQ, the coefficient of variation (CV) values for between-day accuracy was 62.3-143.3% (mean, 93.5%; median, 88.5%) and precision 6.6-22.4% (mean, 15.8%; median, 16.1%). In addition, 11 post-mortem blood samples, containing 0.08-2.4 mg/L of amphetamine (n = 5), methamphetamine (n = 4) or MDMA (n = 4), were analyzed by the GC-NCD-APCI-QTOFMS method, and the results were compared with an established electron ionization GC-MS method with appropriate calibration. The agreement between the 2 methods was 62.5-117.3%. Regarding identification, the APCI source permitted detection of the intact precursor ion, or the respective acylation product, for all of the measured compounds. The GC-NCD-APCI-QTOFMS method developed here enables instant quantitative estimation of illicit psychostimulants in blood at reasonable accuracy, without the necessity of possessing the true reference standards for each analyte.

Locomotor effects of 3,4-methylenedioxymethamphetamine (MDMA) and its deuterated form in mice: psychostimulant effects, stereotypy, and sensitization.

RATIONALE: There is a renewed interest in the use of 3,4-methylenedioxymethamphetamine (MDMA) for treating psychiatric conditions. Although MDMA has entered phase II clinical trials and shows promise as an adjunct treatment, there is an extensive literature detailing the potential neurotoxicity and adverse neurobehavioral effects associated with MDMA use. Previous research indicates that the adverse effects of MDMA may be due to its metabolism into reactive catechols that can enter the brain and serve directly as neurotoxicants. One approach to mitigate MDMA's potential for adverse effects is to reduce O-demethylation by deuterating the methylenedioxy ring of MDMA. There are no studies that have evaluated the effects of deuterating MDMA on behavioral outcomes. OBJECTIVES: The purpose of the present study was to assess the motor-stimulant effects of deuterated MDMA (d2-MDMA) and compare them to MDMA in male mice. METHODS: Two experiments were performed to quantify mouse locomotor activity and to vary the drug administration regimen (single bolus administration or cumulative administration). RESULTS: The results of Experiments 1 and 2 indicate that d2-MDMA is less effective at eliciting horizontal locomotion than MDMA; however, the differences between the compounds diminish as the number of cumulative administrations increase. Both d2-MDMA and MDMA can elicit sensitized responses, and these effects cross-sensitize to the prototypical drug of abuse methamphetamine. Thus, d2-MDMA functions as a locomotor stimulant similar to MDMA, but, depending on the dosing regimen, may be less susceptible to inducing sensitization to stereotyped movements. CONCLUSIONS: These findings indicate that d2-MDMA is behaviorally active and produces locomotor effects that are similar to MDMA, which warrant additional assessments of d2-MDMA's behavioral and physiological effects to determine the conditions under which this compound may serve as a relatively safer alternative to MDMA for clinical use.

Variability in content and dissolution profiles of MDMA tablets collected in the UK between 2001 and 2018 - A potential risk to users?

3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) tablets are widely used recreationally, and not only vary in appearance, but also in MDMA content. Recently, the prevalence of high-content tablets is of concern to public health authorities. To compare UK data with other countries, we evaluated MDMA content of 412 tablets collected from the UK, 2001-2018, and investigated within-batch content variability for a sub-set of these samples. In addition, we investigated dissolution profiles of tablets using pharmaceutical industry-standard dissolution experiments on 247 tablets. All analyses were carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our data supported other studies, in that recent samples (2016-2018) tend to have higher MDMA content compared to earlier years. In 2018, the median MDMA content exceeded 100 mg free-base for the first time. Dramatic within-batch content variability (up to 136 mg difference) was also demonstrated. Statistical evaluation of dissolution profiles at 15-minutes allowed tablets to be categorized as fast-, intermediate-, or slow-releasing, but no tablet characteristics correlated with dissolution classification. Hence, there would be no way of users knowing a priori whether a tablet is more likely to be fast or slow-releasing. Further, within-batch variation in dissolution rate was observed. Rapid assessment of MDMA content alone provides important data for harm reduction, but does not account for variability in (a) the remainder of tablets in a batch, or (b) MDMA dissolution profiles. Clinical manifestations of MDMA toxicity, especially for high-content, slow-releasing tablets, may be delayed or prolonged, and there is a significant risk of users re-dosing if absorption is delayed.

Identification of the New Psychoactive Substance Dibutylone.

ABSTRACT: Objective To establish a method to identify unknown samples based on combined use of gas chromatography-mass spectrometry （GC-MS）, high resolution mass spectrometry （HRMS） and nuclear magnetic resonance spectrum （NMR） technique. Methods The unknown samples were dissolved in methanol solution containing internal standard SKF525A and detected by GC-MS and HRMS. The mixed samples were separated and purified by silica gel column chromatography, and then dissolved in methanol-d4 solution for structural analysis of 1H nuclear magnetic resonance spectroscopy （1H NMR）. Results The characteristic fragment ions （m/z） were 86.1 （base peak）, 71.2, 121.1, and 149.0, and the accurate mass number of molecular ion peak was measured by HRMS to be 236.128 89. By combined use of data analysis and database comparison, a new psychoactive substance of the cathinone class, Dibutylone, was detected in the sample, and the sample also contained a small amount of caffeine. The sample was purified, then identified using 1H NMR, and was further confirmed to be Dibutylone. In addition, the GC-MS retention time and characteristic fragment ions of the main components of the sample were consistent with those of Dibutylone reference material. Conclusion The method established in this study can be used for the identification of Dibutylone in mixed samples.

Kinetic mechanism of time-dependent inhibition of CYP2D6 by 3,4-methylenedioxymethamphetamine (MDMA): Functional heterogeneity of the enzyme and the reversibility of its inactivation.

We investigate the mechanism of time-dependent inhibition (TDI) of human cytochrome P450 2D6 (CYP2D6) by 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), one of the most widespread recreational drugs of abuse. In an effort to unravel the kinetic mechanism of the formation of metabolic inhibitory complex (MIC) of CYP2D6 with MDMA-derived carbene we carried out a series of spectrophotometric studies paralleled with registration of the kinetics of time-dependent inhibition (TDI) in CYP2D6-incorporated proteoliposomes. The high amplitude of spectral signal in this system allowed us to characterize the spectral properties of the formed MIC in details and obtain an accurate spectral signature of MIC formation. This information was then used in the studies with CYP2D6-containing microsomes of insect cells (CYP2D6 Supersomes™). Our results demonstrate that in both systems the formation of the ferrous carbene-derived MIC is relatively slow, reversible and is not associated with the accumulation of the ferric carbene intermediate, as takes place in the case of CYP3A4 and podophylotoxin. Furthermore, the limited amplitude of MIC formation suggests that only a fraction (∼50%) of spectrally detectable CYP2D6 in both proteoliposomes and Supersomes participates in the formation of MIC and is therefore involved in the MDMA metabolism. This observation reveals yet another example of a cytochrome P450 that exhibits persistent functional heterogeneity of its population in microsomal membranes. Our study provides a solid methodological background for further mechanistic studies of MIC formation in human liver microsomes and demonstrates that the potency and physiological relevance of MDMA-dependent TDI of CYP2D6 may be overestimated.

Dark Classics in Chemical Neuroscience: 3,4-Methylenedioxymethamphetamine.

Better known as "ecstasy", 3,4-methylenedioxymethamphetamine (MDMA) is a small molecule that has played a prominent role in defining the ethos of today's teenagers and young adults, much like lysergic acid diethylamide (LSD) did in the 1960s. Though MDMA possesses structural similarities to compounds like amphetamine and mescaline, it produces subjective effects that are unlike any of the classical psychostimulants or hallucinogens and is one of the few compounds capable of reliably producing prosocial behavioral states. As a result, MDMA has captured the attention of recreational users, the media, artists, psychiatrists, and neuropharmacologists alike. Here, we detail the synthesis of MDMA as well as its pharmacology, metabolism, adverse effects, and potential use in medicine. Finally, we discuss its history and why it is perhaps the most important compound for the future of psychedelic science-having the potential to either facilitate new psychedelic research initiatives, or to usher in a second Dark Age for the field.

Acute effects of 3,4-methylenedioxymethamphetamine (MDMA) and R(-) MDMA on actigraphy-based daytime activity and sleep parameters in rhesus monkeys.

3,4-Methylenedioxymethamphetamine (MDMA) affects monoaminergic pathways that play a critical role in sleep-wake cycles. Dopaminergic mechanisms are thought to mediate the sleep-disrupting effects of stimulant drugs. However, the mechanisms underlying the effects of MDMA on sleep-wake cycles and the effects of R(-) MDMA, a stereoisomer that lacks dopaminergic activity, on sleep remain unknown. The aim of the present study was to investigate the effects of racemic MDMA and R(-) MDMA on daytime activity and sleep-like parameters evaluated with actigraphy in adult rhesus macaques (Macaca mulatta, n = 6). Actiwatch monitors were attached to the monkeys' collars and actigraphy recording was conducted during baseline conditions and after the administration of acute intramuscular injections of saline (vehicle), racemic MDMA (0.3, 1.0, or 1.7 mg/kg), or R(-) MDMA (0.3, 1.0, or 1.7 mg/kg) at 9 or 16 h (3 h before "lights off"). Morning treatments had no effects on sleep-like parameters. Racemic MDMA decreased general daytime activity during the first hour after injection and increased daytime activity at 3 hr posttreatment. Although afternoon administration of racemic MDMA increased sleep latency, it improved other sleep parameters, decreasing wake time after sleep onset (WASO) and increasing sleep efficiency for subjects with low baseline sleep efficiency. Afternoon treatment with R(-) MDMA improved sleep measures, increasing sleep efficiency and decreasing sleep latency and WASO, while having no effects on daytime activity. The stimulant and sleep-disrupting effects of racemic MDMA are likely mediated by dopaminergic and noradrenergic mechanisms, while serotonergic pathways appear to be involved in the sleep-promoting effects of MDMA. (PsycINFO Database Record

Identification and characterization of 4-chloromethamphetamine (4-CMA) in seized ecstacy - a risk to public health.

This paper reports the structure elucidation and full characterization of 4-chloromethamphetamine (4-CMA), a compound which was never reported previously outside of laboratory settings in seized drug samples, or samples actively being used at large dance festivals. Identification of 4-CMA was obtained by liquid chromatography with diode array detector (HPLC-PDA) and gas chromatography mass spectrometry (GC-MS). Further structure elucidation was performed by fragment pattern analysis of the trimethylsilyl and trifluoroacetyl derivatives with GC-MS. The region-isomeric form was confirmed by 1H nuclear magnetic resonance spectroscopy (1H NMR). HPLC-PDA was used for quantitation of 4-CMA in the seized tablet to obtain an indication of the potency. A literature review of the toxic effects of 4-CMA was performed, and mechanisms for serotonin neurotoxicity were proposed and discussed. Finally the risks for potential widespread harm to the public in events where similar substances or tablets start appearing and circulating on a larger scale in the general population is discussed.

The DARK Side of Total Synthesis: Strategies and Tactics in Psychoactive Drug Production.

Humankind has used and abused psychoactive drugs for millennia. Formally, a psychoactive drug is any agent that alters cognition and mood. The term "psychotropic drug" is neutral and describes the entire class of substrates, licit and illicit, of interest to governmental drug policy. While these drugs are prescribed for issues ranging from pain management to anxiety, they are also used recreationally. In fact, the current opioid epidemic is the deadliest drug crisis in American history. While the topic is highly politicized with racial, gender, and socioeconomic elements, there is no denying the toll drug mis- and overuse is taking on this country. Overdose, fueled by opioids, is the leading cause of death for Americans under 50 years of age, killing ca. 64,000 people in 2016. From a chemistry standpoint, the question is in what ways, if any, did organic chemists contribute to this problem? In this targeted review, we provide brief historical accounts of the main classes of psychoactive drugs and discuss several foundational total syntheses that ultimately provide the groundwork for producing these molecules in academic, industrial, and clandestine settings.

What's in a Name? Correlates of Ecstasy Users Knowing or Agreeing that Molly is Ecstasy/MDMA.

Ecstasy (MDMA) has regained popularity in powder and crystalline form, known as Molly. However, it is unknown whether all Molly users are aware that Molly is ecstasy. A total of 1045 nightclub/festival-attending adults in New York City were surveyed about ecstasy/MDMA/Molly use in 2016. Users were asked if they agreed that "Molly is (or is supposed to be) ecstasy/MDMA." Of the 43.5% reporting lifetime use, 84.6% agreed that Molly is ecstasy, 9.5% disagreed, and 5.9% reported not knowing that Molly is ecstasy. Prevalence of use of other drugs (e.g., ketamine, opioids, methamphetamine, NBOMe, 2C series) was lowest among those not knowing that Molly is ecstasy, and highest among those not agreeing that Molly is ecstasy. Those not knowing that Molly is ecstasy were less likely to have used powder or crystal MDMA and less likely to have used in the past 12 months or to report intention to use again. Those disagreeing or not knowing that Molly is ecstasy were at over six times the odds of obtaining ecstasy from an unknown dealer, and those disagreeing were at four times higher odds of having suspected or found out that their ecstasy was adulterated. Results suggest that knowing or agreeing that Molly is ecstasy/MDMA can help indicate ecstasy-related risk.

MDA, MDMA, and other "mescaline-like" substances in the US military's search for a truth drug (1940s to 1960s).

This article describes the context in which 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and other mescaline-like compounds were explored as hallucinogens for military and intelligence purposes from the 1940s to the 1960s. Germans first tested mescaline as a "truth drug" in a military context. In the 1940s, the United States military started testing hallucinogenic substances as truth drugs for interrogation and behavior manipulation. After tests carried out using mescaline and other drugs in 1950, some derivatives of mescaline were synthesized by the Army for the exploration of possible "speech-inducing" effects. After insufficient animal testing, the substances were given to patients at the New York State Psychiatric Institute (NYSPI). 3,4-Methylenedioxy-N-ethylamphetamine (MDE), a compound almost identical to MDMA, was among the compounds delivered for testing at the NYSPI. During tests with other derivatives (3,4-dimethoxyphenethylamine (DMA), 3,4-methylenedioxyphenethylamine (MDPEA), MDA) in 1952-53, an unwitting patient died in these tests, which was kept secret from the public. Research was interrupted and toxicological animal testing procedures were initiated. The secret animal studies run in 1953/1954 revealed that some of the "mescaline derivatives" tested (e.g. MDA, MDE, DMA, 3,4,5-trimethoxyamphetamine (TMA), MDMA) were considered for further testing in humans. In 1955, the military changed focus to lysergic acid diethylamide (LSD), but some interest in mescaline-like compounds remained for their ability to change mood and habit without interfering with cognition and sensory perception. Based on the known documents, it remains unclear (but probable) whether any of the mescaline derivatives tested were being used operationally.

Intermolecular interactions between DNA and methamphetamine, amphetamine, ecstasy and their major metabolites.

In this work, we carried out a theoretical investigation regarding amphetamine-type stimulants, which can cause central nervous system degeneration, interacting with human DNA. These include amphetamine, methamphetamine, 3,4-Methylenedioxymethamphetamine (also known as ecstasy), as well as their main metabolites. The studies were performed through molecular docking and molecular dynamics simulations, where molecular interactions of the receptor-ligand systems, along with their physical-chemical energies, were reported. Our results show that 3,4-Methylenedioxymethamphetamine and 3,4-Dihydroxymethamphetamine (ecstasy) present considerable reactivity with the receptor (DNA), suggesting that these molecules may cause damage due to human-DNA. These results were indicated by free Gibbs change of bind (ΔGbind) values referring to intermolecular interactions between the drugs and the minor grooves of DNA, which were predominant for all simulations. In addition, it was observed that 3,4-Dihydroxymethamphetamine (ΔGbind = -13.15 kcal/mol) presented greater spontaneity in establishing interactions with DNA in comparison to 3,4-Methylenedioxymethamphetamine (ΔGbind = -8.61 kcal/mol). Thus, according with the calculations performed our results suggest that the 3,4-Methylenedioxymethamphetamine and 3,4-Dihydroxymethamphetamine have greater probability to provide damage to human DNA fragments.