Structural and functional perspectives on interactions between synthetic cathinones and monoamine transporters.

Synthetic cathinone derivatives comprise a family of psychoactive compounds structurally related to amphetamine. Over the last decade, clandestine chemists have synthesized a consistent stream of innovative cathinone derivatives to outpace governmental regulatory restrictions. Many of these unregulated substances are produced and distributed as designer drugs. Two of the principal chemical scaffolds exploited to expand the synthetic cathinone family are methcathinone and α-pyrrolidinopentiophenone (or α-pyrrolidinovalerophenone, α-PVP). These compounds' main physiological targets are monoamine transporters, where they promote addiction by potentiating dopaminergic neurotransmission. This chapter describes techniques used to study the pharmacodynamic properties of cathinones at monoamine transporters in vitro. Biochemical techniques described include uptake inhibition and release assays in rat brain synaptosomes and in mammalian expression systems. Electrophysiological techniques include current measurements using the voltage clamp technique. We describe a Ca2+ mobilization assay wherein voltage-gated Ca2+ channels function as reporters to study the action of synthetic cathinones at monoamine transporters. We discuss results from systematic structure-activity relationship studies on simple and complex cathinones at monoamine transporters with an emphasis on identifying structural moieties that modulate potency and selectivity at these transporters. Moreover, different profiles of selectivity at monoamine transporters directly predict compounds associated with behavioral and subjective effects within animals and humans. In conclusion, clarification of the structural aspects of compounds which modulate potency and selectivity at monoamine transporters is critical to identify and predict potential addictive drugs. This knowledge may allow prompt allocation of resources toward drugs that represent the greatest threats after drugs are identified by forensic laboratories.

Impurity profiling of methamphetamine synthesised from α-phenylacetoacetonitrile (APAAN).

The rise in popularity of 'designer' precursor compounds for the synthesis of amphetamine-type stimulants poses a significant challenge to law enforcement agencies. One such precursor is α-phenylacetoacetonitrile (APAAN). APAAN emerged in Europe in 2010 and quickly became one of the most popular precursors for amphetamine synthesis in that region. Previous literature has identified four APAAN-specific impurities formed in the synthesis of amphetamine; however, there is currently no research on the use of APAAN in the synthesis of methamphetamine, which is more likely to be employed in a non-European market. In this study methamphetamine was synthesised via three common clandestine methods: the Leuckart method and two reductive amination methods. We report the identification of five new impurities and two previously identified impurities characteristic for the use of APAAN in the synthesis of methamphetamine. The newly identified impurities were characterised by MS and NMR and determined to be (E)-3-(methylamino)-2-phenylbut-2-enenitrile, 3-(methylamino)-2-phenylbutanenitrile, 3-methyl-2,4-diphenylpentanedinitrile, 2-phenylbutyronitrile and 3-hydroxy-2-phenylbutanenitrile.

Comparative Neuropharmacology and Pharmacokinetics of Methamphetamine and Its Thiophene Analog Methiopropamine in Rodents.

Methiopropamine is a novel psychoactive substance (NPS) that is associated with several cases of clinical toxicity, yet little information is available regarding its neuropharmacological properties. Here, we employed in vitro and in vivo methods to compare the pharmacokinetics and neurobiological effects of methiopropamine and its structural analog methamphetamine. Methiopropamine was rapidly distributed to the blood and brain after injection in C57BL/6 mice, with a pharmacokinetic profile similar to that of methamphetamine. Methiopropamine induced psychomotor activity, but higher doses were needed (Emax 12.5 mg/kg; i.p.) compared to methamphetamine (Emax 3.75 mg/kg; i.p.). A steep increase in locomotor activity was seen after a modest increase in the methiopropamine dose from 10 to 12.5 mg/kg, suggesting that a small increase in dosage may engender unexpectedly strong effects and heighten the risk of unintended overdose in NPS users. In vitro studies revealed that methiopropamine mediates its effects through inhibition of norepinephrine and dopamine uptake into presynaptic nerve terminals (IC50 = 0.47 and 0.74 µM, respectively), while the plasmalemmal serotonin uptake and vesicular uptake are affected only at high concentrations (IC50 > 25 µM). In summary, methiopropamine closely resembles methamphetamine with regard to its pharmacokinetics, pharmacodynamic effects and mechanism of action, with a potency that is approximately five times lower than that of methamphetamine.

[ß-Glu2]TRH Is a Functional Antagonist of Thyrotropin-Releasing Hormone (TRH) in the Rodent Brain.

Selective antagonists of thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), in order to enable a better understanding of this peptide's central functions, have not been identified. Using pGlu-Glu-Pro-NH2 ([Glu2]TRH) as a lead peptide and with modification at its central residue, our studies focused on some of its analogues synthesized as potential functional antagonists of TRH in the rodent brain. Among the peptides studied, the novel isomeric analogue [ß-Glu2]TRH was found to suppress the analeptic and antidepressant-like pharmacological activities of TRH without eliciting intrinsic effects in these paradigms. [ß-Glu2]TRH also completely reversed TRH's stimulation of acetylcholine turnover in the rat hippocampus without a cholinergic activity of its own, which was demonstrated through in vivo microdialysis experiments. Altogether, [ß-Glu2]TRH emerged as the first selective functional antagonist of TRH's prominent cholinergic actions, by which this endogenous peptide elicits a vast array of central effects.

Characteristics and antifatigue activity of graded polysaccharides from Ganoderma lucidum separated by cascade membrane technology.

In this paper, cascade membrane technology was utilized to classify polysaccharides from Ganoderma lucidum (GLPs). The properties and antifatigue activity of graded polysaccharides were identified and compared. GLPs were separated using cascade ultrafiltration membranes of 100 kDa, 10 kDa and 1 kDa in sequence. The molecular weights of polysaccharides in these GLP fractions were approximately 322.0 kDa, 18.8 kDa and 6.4 kDa, and all polysaccharides were in active ß-configurations. This showed that all graded GLPs could elongate swimming time, improve endurance and promote fatigue recovery, especially polysaccharides with molecular weights above 10 kDa. This demonstrated that GLPs could decrease the activities of SUN and CK and the levels of MDA and BLA. They also increased the level of Gly, accelerated fat transformation, and improved the activities of GPx, SOD and LDH in all treated mice. Accordingly, GLPs above 10 kDa might be potential agents with antifatigue activity.

Modulatory Effects of Caffeine and Pentoxifylline on Aromatic Antibiotics: A Role for Hetero-Complex Formation.

Antimicrobial resistance is a major healthcare threat globally. Xanthines, including caffeine and pentoxifylline, are attractive candidates for drug repurposing, given their well-established safety and pharmacological profiles. This study aimed to analyze potential interactions between xanthines and aromatic antibiotics (i.e., tetracycline and ciprofloxacin), and their impact on antibiotic antibacterial activity. UV-vis spectroscopy, statistical-thermodynamical modeling, and isothermal titration calorimetry were used to quantitatively evaluate xanthine-antibiotic interactions. The antibacterial profiles of xanthines, and xanthine-antibiotic mixtures, towards important human pathogens Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Enterobacter cloacae were examined. Caffeine and pentoxifylline directly interact with ciprofloxacin and tetracycline, with neighborhood association constant values of 15.8-45.6 M-1 and enthalpy change values up to -4 kJ·M-1. Caffeine, used in mixtures with tested antibiotics, enhanced their antibacterial activity in most pathogens tested. However, antagonistic effects of caffeine were also observed, but only with ciprofloxacin toward Gram-positive pathogens. Xanthines interact with aromatic antibiotics at the molecular and in vitro antibacterial activity level. Given considerable exposure to caffeine and pentoxifylline, these interactions might be relevant for the effectiveness of antibacterial pharmacotherapy, and may help to identify optimal treatment regimens in the era of multidrug resistance.

Assessing Palatability of a New Amphetamine Extended-Release Tablet Formulation for the Treatment of ADHD.

INTRODUCTION/OBJECTIVE: ADHD is, for many people, a lifelong disease that requires chronic medication use. Stimulant therapy is often recommended as first-line treatment for ADHD. Adherence to stimulant treatment among patients diagnosed with ADHD is poor. Major regulatory agencies have recommended measurement of palatability for new tablet formulations. A new amphetamine extended-release tablet (AMPH ER TAB) for the treatment of attention-deficit/hyperactivity disorder (ADHD) was developed. The AMPH ER TAB has a bubblegum flavor and can be chewed or swallowed whole. In 2016, the FDA developed a draft guidance document on the topic of chewable drug tablet formulation palatability. METHODS: A palatability study of the AMPH ER TAB using the 2016 FDA guidance was conducted. Subjects were asked to assess the taste, aftertaste, and mouthfeel of the tablet formulation using a short questionnaire. Scores from the questionnaire were rated and presented. RESULTS: The substudy assessed 35 subjects with a mean age of 38 (±11) years. Subjects were predominantly male, non-Hispanic, and White. Most subjects rated the oral sensation/mouth feel and taste of the tablet as positive (pleasant to very pleasant) (70.1% and 83.6%, respectively). Additionally, 86.6% of the subjects rated the strength of the taste as neutral (moderate taste) or positive (mild to no taste). Finally, 82.1% of all subjects rated the aftertaste as positive (pleasant to very pleasant) and 92.5% of subjects rated the strength of the aftertaste as neutral or positive (mild to no taste). The trends in evaluation scores for each question were similar regardless of whether the ER chewable tablet was administered under fasted or fed conditions. CONCLUSION: The positive palatability data presented here will be useful for future "real-world" assessments of adherence to treatment with the AMPH ER TAB. Enhanced adherence may bolster the argument that taste, mouthfeel, and aftertaste are critical determinants of treatment adherence.

An updated review on synthetic cathinones.

Cathinone, the main psychoactive compound found in the plant Catha edulis Forsk. (khat), is a ß-keto analogue of amphetamine, sharing not only the phenethylamine structure, but also the amphetamine-like stimulant effects. Synthetic cathinones are derivatives of the naturally occurring cathinone that largely entered the recreational drug market at the end of 2000s. The former "legal status", impressive marketing strategies and their commercial availability, either in the so-called "smartshops" or via the Internet, prompted their large spread, contributing to their increasing popularity in the following years. As their popularity increased, the risks posed for public health became clear, with several reports of intoxications and deaths involving these substances appearing both in the social media and scientific literature. The regulatory measures introduced thereafter to halt these trending drugs of abuse have proved to be of low impact, as a continuous emergence of new non-controlled derivatives keep appearing to replace those prohibited. Users resort to synthetic cathinones due to their psychostimulant properties but are often unaware of the dangers they may incur when using these substances. Therefore, studies aimed at unveiling the pharmacological and toxicological properties of these substances are imperative, as they will provide increased expertise to the clinicians that face this problem on a daily basis. The present work provides a comprehensive review on history and legal status, chemistry, pharmacokinetics, pharmacodynamics, adverse effects and lethality in humans, as well as on the current knowledge of the neurotoxic mechanisms of synthetic cathinones.

Low-normal doses of methiopropamine induce aggressive behaviour in mice.

Recreational use of illicit methiopropamine (MPA) is a public health concern because it produces neurochemical effects comparable with those induced by methamphetamine (METH). The present study investigated the effects of MPA on the expression of an aggressive behaviour. Eighty CD-1 male mice, after receiving intraperitoneal injection of saline, MPA (0.01-10 mg/kg), METH (0.01-10 mg/kg), or AMPH (0.01-10 mg/kg), once a week over a 5-week period, underwent the resident-intruder test and spontaneous locomotor activity measurement. Results showed that all psychostimulants induce aggressive behaviour even at low doses, with a dose-dependent increase and a time-dependent sensitisation. MPA potency was similar to METH and superior to AMPH. Therefore, MPA-induced aggressive behaviour may appear even at MPA dosages free of cardiovascular or other behavioural adverse effects and could become a non-intentional side effect that users experience after increasing and repeating MPA consumption.

Role of amino terminal substitutions in the pharmacological, rewarding and psychostimulant profiles of novel synthetic cathinones.

The emergence of new synthetic cathinones continues to be a matter of public health concern. In fact, they are quickly replaced by new structurally related alternatives. The main goal of the present study was to characterize the pharmacological profile, the psychostimulant and rewarding properties of novel cathinones (pentedrone, N-ethyl-pentedrone, α-PVP, N,N-diethyl-pentedrone and α-PpVP) which only differs in their amino terminal substitution. Rat synaptosomes were used for [3H]dopamine uptake experiments. HEK293 transfected cells (hDAT, hSERT, hOCT; human dopamine, serotonin and organic cation transporter) were also used for [3H]monoamine uptake and transporter binding assays. Molecular docking was used to investigate the effect of the amino substitutions on the biological activity. Hyperlocomotion and conditioned place preference paradigm were used in order to study the psychostimulant and rewarding effects in mice. All compounds tested are potent inhibitors of DAT with very low affinity for SERT, hOCT-2 and -3, and their potency for inhibiting DAT increased when the amino-substituent expanded from a methyl to either an ethyl-, a pyrrolidine- or a piperidine-ring. Regarding the in vivo results, all the compounds induced an increase in locomotor activity and possess rewarding properties. Results also showed a significant correlation between predicted binding affinities by molecular docking and affinity constants (Ki) for hDAT as well as the cLogP of their amino-substituent with their hDAT/hSERT ratios. Our study demonstrates the role of the amino-substituent in the pharmacological profile of novel synthetic cathinones as well as their potency inhibiting DA uptake and ability to induce psychostimulant and rewarding effects in mice.

Discriminative stimulus effects of 3,4-methylenedioxypyrovalerone (MDPV) and structurally related synthetic cathinones.

The 3,4-methylenedioxypyrovalerone (MDPV), and other structurally related synthetic cathinones, are popular alternatives to prototypical illicit psychostimulants, such as cocaine and methamphetamine. These drugs are often referred to as 'bath salts' and function either as cocaine-like inhibitors of monoamine uptake, or amphetamine-like substrates for dopamine, norepinephrine and serotonin transporters. These studies used male Sprague-Dawley rats trained to discriminate MDPV from saline to evaluate the substitution profiles of structurally related synthetic cathinones, cocaine, and other direct-acting dopamine and noradrenergic receptor agonists in order to characterize the relative contributions of dopamine, norepinephrine and serotonin to the discriminative stimulus effects of MDPV. As expected, each of the cathinones and cocaine dose-dependently increased MDPV-appropriate responding, with a rank-order potency that was positively correlated with their potency to inhibit dopamine and norepinephrine, but not serotonin, a relationship that is consistent with the rank order to maintain self-administration. The dopamine D2/3 receptor-preferring agonist quinpirole produced a modest increase in MDPV-appropriate responding, whereas the dopamine D1/5 receptor agonist, SKF 82958, nonselective dopamine receptor agonist, apomorphine, as well as the α-1, and α-2 adrenergic receptor agonists, phenylephrine and clonidine, respectively, failed to increase MDPV-appropriate responding at doses smaller than those that suppressed responding altogether. Although these studies do not support a role for serotonergic or adrenergic systems in mediating/modulating the discriminative stimulus effects of MDPV, convergent evidence is provided to suggest that the discriminative stimulus effects of MDPV are primarily mediated by its capacity to inhibit dopamine uptake, and the subsequent activation of dopamine D2 or D3 receptors.

Mechanisms underlying cognitive impairment induced by prenatal caffeine exposure.

OBJECTIVE: Caffeine is one of the most commonly used stimulants among pregnant women. Human and animal studies have shown that prenatal caffeine exposure affects fetal brain development and results in persistent cognitive deficits in offspring. Studies have found that caffeine consumption during pregnancy may alter many activities that are ultimately associated with cognitive dysfunction in offspring. Despite these important findings, there is a fundamental gap in understanding the mechanism underlying cognitive impairment due to prenatal caffeine exposure. Filling this knowledge gap would provide further insights into caffeine-mediated cognitive dysfunction. The objective of this review was to evaluate the findings of studies showing that prenatal caffeine exposure induces cognitive dysfunction and the potential underlying mechanisms.

Discovery of Potent and Brain-Penetrant GPR52 Agonist that Suppresses Psychostimulant Behavior.

The G protein-coupled receptor 52 (GPR52) is an orphan receptor that is selectively expressed in the striatum and regulates various brain functions through activation of cAMP-dependent pathways. GPR52 has been identified as a promising therapeutic target for central nervous system disorders including schizophrenia and substance use disorders. Here, a series of novel GPR52 agonists were designed, synthesized, and evaluated based on compound 4. Several potent and efficacious GPR52 agonists (12c, 23a, 23d, 23e, 23f, and 23h) were identified with nanomolar range potency based on a systematic structure-activity relationship exploration. Further studies of 12c indicate enhanced efficacy, excellent target selectivity, and pharmacokinetic properties including good brain permeability. In vivo proof-of-concept investigations revealed that 12c displayed antipsychotic-like activity by significantly inhibiting amphetamine-induced hyperlocomotor behavior in mice. Collectively, our findings have resulted in an efficacious, brain-penetrant GPR52 agonist as a valuable pharmacological tool for investigating the physiological and therapeutic potential of GPR52 activation.

Enantiomeric separation and quantification of R/S-amphetamine in serum using semi-automated liquid-liquid extraction and ultra-high performance supercritical fluid chromatography-tandem mass spectrometry.

The amphetamine molecule contains a chiral center and its enantiomers exhibit differences in pharmacological effects, with the S-enantiomer mediating most of the central nervous system stimulating activity. The majority of prescribed amphetamine consists of the pure S-enantiomer, but therapeutic formulations containing the R-enantiomer in various proportions are also available. Illegal amphetamine remains available mainly as a racemic mixture of the R- and S-enantiomers. To distinguish between legal and illegal consumption of amphetamine a method for enantiomeric separation and quantification of R/S-amphetamine in serum was developed and validated using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS). Sample preparation prior to UHPSFC-MS/MS analysis was performed by a semi-automated liquid-liquid extraction method. The UHPSFC-MS/MS method used a Chiralpak AD-3 column with a mobile phase consisting of CO2 and 0.1% ammonium hydroxide in 2-propanol/methanol (50/50, v/v). The injection volume was 2 µL and run time was 4 minutes. MS/MS detection was performed with positive electrospray ionization and two multiple reaction monitoring transitions (m/z 136.1 > 119.0 and m/z 136.1 > 91.0). The calibration range was 12.5-1,000 nM for each analyte. The between-assay relative standard deviations were in the range of 1.3-3.0%. Recovery was 73% and matrix effects ranged from 95 to 100% when corrected with internal standard. After development and validation, the method has been successfully implemented in our laboratory for both separation and quantification of R/S-amphetamine and has proved to be a reliable and useful tool for distinguishing intake of R- and S-amphetamine in authentic patient samples.

Methylenedioxymethamphetamine-like discriminative stimulus effects of pyrrolidinyl cathinones in rats.

BACKGROUND: Synthetic cathinone derivatives are used as alternatives both for stimulant drugs such as cocaine and methamphetamine and for club drugs such as 3,4-methylenedioxymethamphetamine (MDMA), but little is known about their MDMA-like subjective effects. METHODS: In order to determine their similarity to MDMA, the discriminative stimulus effects of 10 pyrrolidinyl cathinones (α-pyrrolidinopropiophenone, 4'-methyl-α-pyrrolidinopropiophenone (4'-MePPP), α-pyrrolidinobutiophenone, 3',4'-methylenedioxy-α-pyrrolidinobutyrophenone (MD-PBP), α-pyrrolidinovalerophenone, 3,4-methylenedioxy-pyrovalerone (MDPV), α-pyrrolidinopentiothiophenone, napthylpyrovalerone (naphyrone), α-pyrrolidinohexiophenone, and 4'-methyl-α-pyrrolidinohexiophenone (4'-MePHP)) were assessed in Sprague-Dawley rats trained to discriminate 1.5 mg/kg racemic ±-MDMA from vehicle. RESULTS: Compounds with no substitutions on the phenyl ring and the thiophene produced 44-67% MDMA-appropriate responding. In contrast, the substituted pyrrolidinyl cathinones produced a range of MDMA-appropriate responding dependent upon the length of the alpha side chain. 4'-MePPP, with a single carbon on the alpha position, produced 99.8% MDMA-appropriate responding, MD-PBP (two carbons) produced 83%, naphyrone (three carbons) produced 71%, MDPV (three carbons) produced, 66%, and 4'-MePHP (four carbons) produced 47%. CONCLUSIONS: Many cathinone compounds have discriminative stimulus effects similar to those of MDMA. However, the pyrrolidine substitution appears to reduce serotonergic effects, with a commensurate decrease in MDMA-like effects. Substitutions on the phenyl ring appear to be able to restore MDMA-like responding, but only in compounds with short alpha side chains. These findings agree with earlier findings of increasing dopaminergic effects and stronger reinforcing effects with increasing side chain. Assessment of more compounds is necessary to establish the replicability/robustness of this phenomenon. These findings may be of use in predicting which compounds will have MDMA/club drug-like effects versus psychostimulant-like effects.

Application of a chiral high-performance liquid chromatography-tandem mass spectrometry method for the determination of 13 related amphetamine-type stimulants to forensic samples: Interpretative hypotheses.

Interpretation of amphetamine-type stimulant (ATS) findings in urine samples can be challenging without chiral information. We present a sensitive enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantification of (R)-amphetamine, (S)-amphetamine, (R)-methamphetamine, (S)-methamphetamine, (1R,2R)-pseudoephedrine, (1S,2S)-pseudoephedrine, (1R,2S)-ephedrine, (1S,2R)-ephedrine, (1R,2S)-norephedrine, (1S,2R)-norephedrine, (R)-cathinone, (S)-cathinone, and (1S,2S)-norpseudoephedrine (cathine) in urine. The method was successfully applied to more than 100 authentic urine samples from forensic casework. In addition, samples from a controlled self-administration of (1S,2S)-pseudoephedrine (Rinoral, 1200 mg within 6 days) were analyzed. The results strengthen the hypothesis that (1R,2S)-norephedrine is a minor metabolite of amphetamine and methamphetamine. We suggest cathine and (1S,2R)-norephedrine as minor metabolites of amphetamine racemate in humans. Small methamphetamine concentrations detected in samples with high concentrations of amphetamine could result from a metabolic formation by methylation of amphetamine although in samples with an (R)/(S) ratio for methamphetamine < 1 an additional (previous) (S)-methamphetamine consumption seems likely. Our data suggest that even amphetamine concentrations exceeding methamphetamine concentrations in urine can be caused by the biotransformation of methamphetamine to amphetamine as long as no (R)-amphetamine is detected. However, without chiral information, such findings might be (falsely) assumed as a co-consumption of both substances. Cathinone enantiomers detected in urine samples with high amphetamine concentrations can be interpreted as metabolites of amphetamine. In addition, the results of the self-administration study revealed that both cathinone enantiomers are minor metabolites of (1S,2S)-pseudoephedrine, which is the active ingredient of various medicines used for cold. The enantioselective analysis is a powerful tool to avoid the misinterpretation of ATS findings in urine samples.

Syntheses and analytical characterizations of novel (2-aminopropyl)benzo[b]thiophene (APBT) based stimulants.

Two groups of amphetamine-like drugs with psychostimulant properties that were first developed during the course of scientific studies and later emerged as new psychoactive substances (NPS) are based on the (2-aminopropyl)indole (API) and (2-aminopropyl)benzofuran (APB) structural scaffolds. However, sulfur-based analogs with a benzo[b]thiophene structure (resulting in (2-aminopropyl)benzo[b]thiophene (APBT) derivatives) have received little attention. In the present investigation, all six racemic APBT positional isomers were synthesized in an effort to understand their structure-activity relationships relative to API- and APB-based drugs. One lesson learned from the NPS phenomenon is that one cannot exclude the appearance of such substances on the market. Therefore, an in-depth analytical characterization was performed, including various single- and tandem mass spectrometry (MS) and ionization platforms coupled to gas chromatography (GC) and liquid chromatography (LC), nuclear magnetic resonance spectroscopy (NMR), and solid phase and GC condensed phase infrared spectroscopy (GC-sIR). Various derivatizations have also been explored; it was found that all six APBT isomers could be differentiated during GC analysis after derivatization with heptafluorobutyric anhydride and ethyl chloroformate (or heptafluorobutyric anhydride and acetic anhydride) under non-routine conditions. Discriminating analytical features can also be derived from NMR, GC-EI/CI- single- and tandem mass spectrometry, LC (pentafluorophenyl stationary phase), and various infrared spectroscopy approaches (including GC-sIR). Availability of detailed analytical data obtained from these novel APBT-type stimulants may be useful to researchers and scientists in cases where forensic and clinical investigations are warranted.

Prodrugs of New Psychoactive Substances (NPS): A New Challenge.

The concept of a substance acting as a prodrug for an intended drug is not new and has been known and utilized with particular benefits within medicine for efficacy and patient safety. Prodrugs of psychoactive substances are also not particularly new but this has also extended to considerations of prodrugs of new psychoactive substances (NPS). The continuing evolution of NPS has been a constant forensic challenge. In some countries, this constant evolution has led to the introduction of various alternative methods of drug control. Whether for this reason or in the pursuit of user experimentation, prodrugs of NPS have been discussed, developed, and exploited, posing some distinct forensic challenges. This is especially the case within toxicological analysis of biological fluids and for some substances, also forensic chemical analysis, through inherent instability of the prodrug or metabolism in the body. Particular examples of NPS prodrugs include 1-propanoyl-LSD, 1-butanoyl-LSD, 1-acetyl-LSD, and 2C-B-AN. This is in addition to associated substances and medicines that may be used for an intended pharmacological effect. Various prodrugs for stimulant and hallucinogenic substances in particular have appeared in the literature and have been discussed within drug user forums and made available for purchase online. Presently, drug monitoring data from national and international systems indicate that prodrugs are not widely available or problematic. Nevertheless, it is important that there is sufficient awareness of the prodrug concept and potential impact and associated forensic implications, not just for chemical analysis but also for toxicological considerations when a substance has been used.

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.

Identification of specific markers for amphetamines synthesized from glycidic acid pre-precursors and retrospective search in German profiling database.

The pre-precursor market and the clandestine production of amphetamine-type stimulants (ATS) has become more diverse in recent years. Besides α-phenylacetoacetonitrile (APAAN) and α-phenylacetoacetamide (APAA), glycidic acid derivatives and methyl α-phenylacetoacetate (MAPA) are gaining importance. This conclusion is based on seizure data of police and customs. However, analytical data are needed to confirm and quantify the actual prevalence of new pre-precursors by elucidating the percentage of seized ATS that have been produced from them. A recent study showed that APAAN use is currently declining, which supports the view that new pre-precursors are being used. In this study, several conversion procedures using different batches of glycidic acid derivatives and a complete Leuckart reaction to produce amphetamine were carried out. The resulting organic phases were analyzed using gas chromatography - mass spectrometry to identify possible marker compounds. Three marker compounds were discovered and characterized using mass spectra and nuclear magnetic resonance spectroscopy. They were identified as phenyl-1-propanone, N-(1-phenylpropyl)formamide and 1-phenylpropan-1-amine. Their prevalence was investigated by searching the markers in an amphetamine impurity profiling database to determine to what extent they occurred in amphetamine samples from recent years. Data from the central German amphetamine profiling database of more than 250 cases were used for this purpose. The yearly occurrence of the three glycidate marker compounds was determined going back as far as 2009, revealing an increasing trend from 2016 on. This article presents experimental proof that APAAN is currently being replaced by other pre-precursors, such as glycidic acid derivatives.