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The accumulation of iron in dopaminergic neurons can cause oxidative stress and dopaminergic neuron degeneration. Iron chelation therapy may reduce dopaminergic neurodegeneration, but chelators should be targeted towards dopaminergic cells. In this work, two series of compounds based on 8-hydroxyquinoline and deferiprone, iron chelators that have amphetamine-like structures, have been designed, synthesized and characterized. Each of these compounds chelated iron ions in aqueous solution. The hydroxyquinoline-based compounds exhibited stronger iron-binding constants than those of the deferiprone derivatives. The hydroxyquinoline-based compounds also exhibited greater free radical scavenging activities compared to the deferiprone derivatives. Molecular dynamics simulations showed that the hydroxyquinoline-based compounds generally bound well within human dopamine transporter cavities. Thus, these compounds are excellent candidates for future exploration as drugs against diseases that are affected by iron-induced dopaminergic neuron damage, such as Parkinson's disease.

Magnetic surface imprinted polymer microspheres (Fe 3 O 4 @MIPs) were successfully synthesized via Pickering emulsion polymerization, utilizing N-Methylphenethylamine as a surrogate template for amphetamine-type drugs. Fe 3 O 4 @MIPs not only possessed excellent dispersibility and enough magnetic properties in aqueous solutions, but also displayed good selectivity towards six amphetamines, with an imprinting factor ranging from 1.8 to 2.6. The adsorption kinetics closely aligned with the pseudo-second-order model, and the adsorption efficiency exceeds 80 % for each amphetamine at equilibrium. Fe 3 O 4 @MIPs were then employed as the efficient adsorbents for the extraction of amphetamine drugs. Extraction parameters, including sample pH, the mass of adsorbent, and the type and volume of eluting solvent, were carefully optimized. In combination with the high performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-MS/MS), a selective magnetic solid-phase extraction (MISPE) method utilizing Fe 3 O 4 @MIPs was developed for the detection of six amphetamines in water samples. The limits of detection and limits of quantitation were determined to be 5.2∼23 ng L -1 and 17∼77 ng L -1 , respectively. Recoveries for the six target drugs from lake water and sewage samples fell within the range of 87.2∼110 %. Additionally, the MISPE-HPLC-MS/MS method exhibited excellent repeatability, with a precision below 8.5 % at two spiking levels. The prepared Fe 3 O 4 @MIPs possessed the advantages of high selectivity, straightforward preparation, facile separation and good reusability, and was highly suitable for the efficient extraction of amphetamine-type substances in complex environmental water., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hydrogen/deuterium (H/D) isotope effects are not unusual in chromatography and such phenomena have been observed in both gas- and liquid-phase separations. Despite the numerous reports on this topic, the understanding of mechanisms and the underlying noncovalent interactions at play remains rather challenging. In our recent study, we reported baseline separation of isotopologoues of some amphetamine (AMP) derivatives on achiral and polysaccharide-based chiral columns, as well as some correlations between the degree of separation of enantiomers and isotopologues on (the same) polysaccharide-based chiral column(s). Following our previous findings on isotope effects in high-performance liquid chromatography, we report herein a comparative study on the isotope effects observed with AMP and methamphetamine (MET). The impact of some pivotal factors such as the number of deuterium atoms part of AMP isotopologues, the structure of its isotopomers, the chemical structure of the achiral and chiral stationary phases used in this study, and the use of methanol- vs acetonitrile-containing mobile phases on the isotope effects was examined and discussed. Quantitative correlations between the observed isotope effects and the enantioselectivity of the chiral columns used are also shortly discussed. Furthermore, considering the chromatographic results as benchmark experimental data, we attempted to elucidate the molecular bases of the observed phenomena using quantum mechanics calculations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sample pretreatment technology is crucial for drug analysis and detection, because the effect of sample pretreatment directly determinates the final analysis results. In recent years, with the continuous innovation of microextraction and other technologies like material preparation technologies and assistant technologies for extraction, the sample pretreatment techniques in the process of drug analysis have become more and more mature and diverse. This article takes amphetamine (AM) or methamphetamine as an example to review the recent development of pretreatment methods for AM-containing biological samples from the perspectives of extraction techniques, extraction media and auxiliary technologies. Extraction techniques are summarized with the categories of contact microextraction, separate microextraction and membrane-based microextraction for better guidance of application according to their features. Prevailing and innovative extraction media including carbon-based material, silicon-based material, metal organic framework, molecularly selective materials, supramolecular solvents and ionic liquids are reviewed. Auxiliary technologies like magnetic field, electric field, microwave, ultrasound and so on which can enhance extraction efficiency and accuracy are also reviewed. In the last, prospects of the future development of pretreatment technology for the analysis of AM biological samples are provided., (© 2024 Wiley‐VCH GmbH.)

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., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Biogenic monoamines-vital transmitters orchestrating neurological, endocrinal and immunological functions 1-5 -are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release 6,7 . Harnessing proton antiport, VMATs enrich monoamines around 10,000-fold and sequester neurotoxicants to protect neurons 8-10 . VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension and drug addiction 1,8,11-16 . However, the structural mechanisms underlying these actions remain unclear. Here we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines (dopamine, noradrenaline, serotonin and histamine), the Parkinsonism-inducing MPP + , the psychostimulant amphetamine and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, whereas the other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favoured transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket that possesses polar sites for specificity and a wrist-and-fist shape for versatility. Variations in this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop therapeutics for neurodegenerative diseases and substance abuse., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Separation and quantification of amphetamine enantiomers are commonly used to distinguish between consumption of prescription amphetamine (mostly S-amphetamine) and illicit forms of the drug (racemate). In this study, electromembrane extraction with prototype conductive vials was combined with ultra-high performance supercritical fluid chromatography (UHPSFC-MS/MS) to quantify R- and S-amphetamine in urine. Amphetamine was extracted from 100 μL urine, diluted with 25 μL internal standard solution and 175 μL 130 mM formic acid, across a supported liquid membrane (SLM) consisting of 9 μL of a 1:1(w/w) mixture of 2-nitrophenyloctyl ether (NPOE) and bis(2-ethylhexyl)phosphite (DEHPi) into an acceptor phase containing 300 μL 130 mM formic acid. The extraction was facilitated by the application of 30 V for 15 min. Enantiomeric separation was achieved using UHPSFC-MS/MS with a chiral stationary phase. The calibration range was 50-10,000 ng/mL for each enantiomer. The between-assay CV was ≤5%, within-assay CV ≤ 1.5%, and bias within ±2%. Recoveries were 83%-90% (CV ≤ 6%), and internal standard corrected matrix effects were 99-105 (CV ≤ 2%). The matrix effects ranged from 96% to 98% (CV ≤ 8%) when not corrected by the internal standard. The EME method was compared with a chiral routine method that employed liquid-liquid extraction (LLE) for sample preparation. Assay results were in agreement with the routine method, and the mean deviation between methods was 3%, ranging from -21% to 31%. Finally, sample preparation greenness was assessed using the AGREEprep tool, which resulted in a greenness score of 0.54 for conductive vial EME, opposed to 0.47 for semi-automated 96-well LLE., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

This case report demonstrates the impact of different sampling sites on the quantification of narcotic substances. In 2020, officers secured a syringe containing a light-yellow paste-like substance, for which a drug pre-test indicated a positive result for amphetamine, inducing subsequent analyses of the sample by means of a gaschromatographic-mass spectrometric method (GC-MS) and liquid chromatography-(tandem) mass spectrometry (LC-MS/MS). Depending on the sample location, different results were obtained, with amphetamine not being detected in each sample. Amphetamine was particularly found at the outlet of the syringe, while amphetamine detection on the inside of the syringe at the plunger seal was only possible occasionally and, moreover, in lower concentrations. Based on this and with regard to the comparatively small amphetamine concentrations, contamination of the syringe (especially on the tip of the syringe) was assumed. Hence, the results strengthened the importance of the implication of different sampling sites, when either homogenization of the sample is not feasible or is not performed for reasons of plausibility checks concerning possible contamination of the sample., (© 2022. The Author(s).)

Reliable and feasible tools for detecting ( S )-methamphetamine [( S )-MAP] and ( S )-amphetamine [( S )-AP] are required for regulating their illicit circulation. Antibodies that react equally to these stimulants are desirable for this purpose, but have been difficult to generate because of the crucial difference between their characteristic structures: i.e ., N -methylamino (MAP) and amino (AP) groups. Furthermore, their small molecular masses ( M r < 150) have hampered the generation of high-affinity antibodies. To overcome these problems, we converted ( S )-MAP and -AP into their 2-(trimethylsilyl)ethyl carbamate forms, Teoc-( S )-MAP and -AP, respectively, as surrogate analytes. The Teoc-derivatization not only increases their molecular masses, but also masks their structural differences. We generated a novel monoclonal antibody that showed a satisfactory affinity to Teoc-( S )-MAP residues ( K d = 13 nM as the IgG form) and developed a competitive enzyme-linked immunosorbent assay (ELISA) using microplates containing immobilized Teoc-( S )-MAP residues. Almost overlapping dose-response curves were obtained for Teoc-( S )-MAP and -AP, with the limit of detection of 0.078 and 0.10 ng per assay, respectively. A fixed amount of test powder sample (1 mg) was derivatized with Teoc- O -succinimidyl for 5 min, and subjected to ELISA using Teoc-( S )-MAP as the calibration standard. Under this protocol, ( S )-MAP and -AP were converted to their Teoc derivatives with 30% and 34% yield, respectively, determined using ELISA as "Teoc-( S )-MAP equivalent," being distinguished from the derivatization products of ( R )-MAP, ( R )-AP, ephedrine, ( S )-methylenedioxymethamphetamine, tyramine, dopamine, and β-alanine. This ELISA detected as little as 10 μg of ( S )-MAP and -AP, and ( S )-MAP in urine obtained from ( S )-MAP-administered rats. Immunochromatography devices were also developed using gold nanoparticles coated with the monoclonal antibody, with which 0.10 mg of ( S )-MAP and -AP was detected by the naked eye. We conclude that the present derivatization-assisted immunoassays may be useful for the detection of ( S )-MAP and/or -AP in early stage screening of suspicious substances.

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., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The host-guest interactions of cucurbit[7]uril (CB[7]) as host and amphetamine (AMP), methamphetamine (MET) and their enantiomeric forms (S-form and R-form) as guests were computationally investigated using density functional theory calculations with the recent D4 atomic-charge dependent dispersion corrections. The analysis of energetic, structural and electronic properties with the aid of frontier molecular orbital analysis, charge decomposition analysis (CDA), extended charge decomposition analysis (ECDA) and independent gradient model (IGM) approach allowed to characterize the host-guest interactions in the studied systems. Energetic results indicate the formation of stable non-covalent complexes where R-AMP@CB[7] and S-AMP@CB[7] are more stable thermodynamically than R-MET@CB[7] and S-MET@CB[7] in gas phase while the reverse is true in water solvent. Based on structural analysis, a recognition mechanism is proposed, which suggests that the synergistic effect of van der Waals forces, ion-dipole interactions, intermolecular charge transfer interactions and intermolecular hydrogen bonding is responsible for the stabilization of the complexes. The geometries of the complexes obtained theoretically are in good agreement with the X-ray experimental structures and indicate that the phenyl ring of amphetamine and methamphetamine is deeply buried into the cavity of CB[7] through hydrophobic interactions while the ammonium group remains outside the cavity to establish hydrogen bonds with the portal oxygen atoms of CB[7].

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., Competing Interests: AP, ER, and JCK are compensated employees of Tris Pharma, Inc., the developer and manufacturer of the product that is the subject of this work. JCK owns stock from Johnson and Johnson and Takeda. TRK was an employee of Tris Pharma, Inc. at the time the study was conducted. The authors report no other conflicts of interest in this work., (© 2021 Pardo et al.)

The density functional theory (DFT) was used to examine the electronic reactivity and sensitivity of a pristine, Si, and Al-doped fullerene C 70 with AM drug. AM drug has been shown to be physically absorbed by its N-head on the pristine C 70 with an adsorption energy of about - 1.09 kcal/mol and to have no impact on the electric conductivity of that cluster. The atom substitution of Si and Al for C atoms at C 70 significantly increases C 70 fullerene reactivity, with adsorption energy predictions of approximately - 31.09 and - 45.59 kcal/mol, respectively. The energy difference of LUMO and HOMO, i.e., Eg from C 70 fullerene, significantly affects AM drug. Significant LUMO destabilization in Al-C 70 by adsorption of the drug AM boosts the electrical conductivity of Al-C 70 while generating electric signals that are related to the environmental presence of AM drug. Hence, Al-doped C 70 is demonstrated to be an effective electronic AM drug sensor. In contrast to Si-C 70 fullerene, significant AM-drug adsorption effects on Fermi and Si-C 70 work functions make Si-C 70 an Ф-type candidate for AM drug sensor applications. The time-dependent theory of the functional density shows that the AM/Al-C 70 complex is steadily situated at a maximum peak of 784.15 nm.

The linkage of molecular components into functional heterogeneous framework materials has revolutionized modern materials chemistry. Here, we use this principle to design polyoxometalate-based frameworks as high affinity adsorbents for drugs of abuse, leading to their application in solid-phase extraction analysis. The frameworks are assembled by the reaction of a Keggin-type polyanion, [SiW 12 O 40 ] 4- , with lanthanoids Dy(III), La(III), Nd(III), and Sm(III) and the multidentate linking ligand 1,10-phenanthroline-2,9-dicarboxylic acid (H 2 PDA). Their reaction leads to the formation of crystalline 1D coordination polymers. Because of the charge mismatch between the lanthanoids (+3) and the dodecasilicotungstate (-4), we observe incorporation of the PDA 2- ligands into crystalline materials, leading to four polyoxometalate-based frameworks where Keggin-type heteropolyanions are linked by cationic {Ln n (PDA) n } groups (Ln = Dy ( 1 ), La ( 2 ), Nd ( 3 ), and Sm ( 4 )). Structural analysis of the polyoxometalate-based frameworks suggested that they might be suitable for surface binding of common drugs of abuse via supramolecular interactions. To this end, they were used for the extraction and quantitative determination of four model drugs of abuse (amphetamine, methamphetamine, codeine, and morphine) by using micro-solid-phase extraction (D-μSPE) and high-performance liquid chromatography (HPLC). The method showed wide linear ranges, low limits of detection (0.1-0.3 ng mL -1 ), high precision, and satisfactory spiked recoveries. Our results demonstrate that polyoxometalate-based frameworks are suitable sorbents in D-μSPE for molecules containing amine functionalities. The modular design of these networks could in the future be used to expand and tune their substrate binding behavior.

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 CO 2 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., (© 2020 John Wiley & Sons, Ltd.)

Combating brain tumors (glioblastoma multiforme or GBM) is a formidable challenge because of the existence of blood-brain barrier (BBB), a tight cellular junction that separates the central nervous system (CNS) and systemic circulation. Such a selectively permeable barrier prevents the entry of therapeutic molecules from blood circulation to brain parenchyma. Towards enhancing the efficacy of brain tumor-selective drug delivery without perturbing the BBB integrity, nanometric drug carriers are increasingly becoming an efficient therapeutic modality in preclinical studies. Psychostimulant drugs such as amphetamine and methylated amphetamine (METH) are known to penetrate the BBB. Still, little effort has been made to exploit them in nano-drug delivery, largely due to their toxicities. Herein, for the first time, we design, synthesize, and formulate three different β-amphetaminylated cationic lipid nanoparticles. We show that the β-amphetaminylated cationic lipid nanoparticles are nontoxic and can cross the BBB presumably through active transcytosis. The BBB penetrating ability also depends on the hydrophilic-hydrophobic balance of the lipids, with hexadecyl lipid (16-BACL) nanoparticle showing maximum accumulation in the brain. The lipid nanoparticle of 16-BACL can simultaneously encapsulate paclitaxel and PDL1-siRNA. The dual drug-loaded lipid nanoparticles showed apoptosis driven cellular cytotoxicity against GL261 cells and improved the overall survivability of orthotopic glioblastoma bearing mice compared to their non-targeting counterpart. The present work describes a new class of BBB-crossing lipid nanoparticles and delineates their therapeutic promise against glioblastoma.

Halogenation of amphetamines and methcathinones has become a common method to obtain novel psychoactive substances (NPS) also called "legal highs". The para -halogenated derivatives of amphetamine and methcathinone are available over the internet and have entered the illicit drug market but studies on their potential neurotoxic effects are rare. The primary aim of this study was to explore the neurotoxicity of amphetamine, methcathinone and their para -halogenated derivatives 4-fluoroamphetamine (4-FA), 4-chloroamphetamine (PCA), 4-fluoromethcathinone (4-FMC), and 4-chloromethcathinone (4-CMC) in undifferentiated and differentiated SH-SY5Y cells. We found that 4-FA, PCA, and 4-CMC were cytotoxic (decrease in cellular ATP and plasma membrane damage) for both cell types, whereby differentiated cells were less sensitive. IC 50 values for cellular ATP depletion were in the range of 1.4 mM for 4-FA, 0.4 mM for PCA and 1.4 mM for 4-CMC. The rank of cytotoxicity observed for the para -substituents was chloride > fluoride > hydrogen for both amphetamines and cathinones. Each of 4-FA, PCA and 4-CMC decreased the mitochondrial membrane potential in both cell types, and PCA and 4-CMC impaired the function of the electron transport chain of mitochondria in SH-SY5Y cells. 4-FA, PCA, and 4-CMC increased the ROS level and PCA and 4-CMC induced apoptosis by the endogenous pathway. In conclusion, para- halogenation of amphetamine and methcathinone increases their neurotoxic properties due to the impairment of mitochondrial function and induction of apoptosis. Although the cytotoxic concentrations were higher than those needed for pharmacological activity, the current findings may be important regarding the uncontrolled recreational use of these compounds.

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., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

An amphetamine interference was observed during the development of an liquid chromatography-high-resolution mass spectrometry (LC-HRMS) multi-class confirmation method for the determination of 47 drugs and metabolites in urine. The interference passed all qualitative criteria for amphetamine leading to potential false-positive results. Upon investigation, it was found that the amphetamine interference was correlated with the presence of high levels of gabapentin. Gabapentin is routinely detected in patient urine specimens at levels in excess of 1 mg/mL as it is widely prescribed at high doses and does not undergo significant metabolism. The source of the interference was identified as a gabapentin in-source fragment isomeric with protonated amphetamine. Here we describe the characterization of this interference and how its effect was mitigated in the LC-HRMS method., (© The Author(s) 2019. Published by Oxford University Press.)

Bath salts, fumigations, cleaners and air fresheners, behind these terms substances are hidden, which count as "Legal Highs". These fancy names are used to pretend Legal Highs as harmless compounds, to circumvent legal regulations for marketing as well as to increase the sales. Besides classic illicit drugs of synthetic origin such as amphetamines, cocaine and MDMA, the trade of these compounds, also known as new psychoactive substances (NPS), is not uncommon today. In many countries, NPS are still not subject to drug control. Among them, there are stimulants such as new amphetamine derivatives or cathinones, which possess a chiral centre. Little is known about the fact that the two possible enantiomers may differ in their pharmacological effect. The aim of this study was to test a novel HPLC column for the enantioseparation of a set of 112 NPS coming from different chemical groups and collected by internet purchases during the years 2010-2018. The CSP, namely Lux® 5 μm i-Amylose-1, LC Column 250 x 4.6 mm, was run in normal phase mode under isocratic conditions, UV detection was performed at 245 nm and 230 nm, injection volume was 10 μl and flow rate was 1 ml/min. With a mobile phase consisting of n-hexane/isopropanol/diethylamine (90:10:0.1), herein, 79 NPS were resolved into their enantiomers successfully, for 37 of them baseline resolution was achieved. After increase of lipophily of the mobile phase to 99:1:0.1, another 27 compounds were baseline separated. It was found that all separated NPS are traded as racemic compounds., (© 2019 Wiley Periodicals, Inc.)

Currently, consumption of illicit drugs and pharmaceuticals has increased significantly. Many of these substances are chiral and can be available as racemates or enantiomerically pure. Determination of the enantiomeric fraction (EF) in wastewater is useful for: i) distinguishing between the consumption of prescribed and illicit drugs; ii) identification of possible local of illegal synthesis; iii) illegal discharge of sewage and estimation of illicit drugs and pharmaceuticals consumption by a community (wastewater-based epidemiology). This work describes the development of an indirect method by gas chromatography-mass spectrometry (GC-MS) for enantiomeric quantification of chiral substances namely psychoactive drugs and β-blockers based on the formation of diastereomers using (R)-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride ((R)-MTPA-Cl) as chiral derivatization reagent. The developed method presented linearity (R 2  > 0.99) for 20 compounds, 9 diastereomer pairs and paroxetine (PAR) and sertraline (SER). Recovery ranged from 80.7 to 114.5% (RSD < 9.1%) and accuracy between 84.6 and 118% (RSD < 9.9%). The limits of detection (LOD) varied from 0.03 and 26.0 ngL -1 and limits of quantification (LOQ) from 0.15 and 104 ngL -1 . Results showed the occurrence of amphetamine (AMP), illicit drugs as 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (MAMP), alprenolol (ALP), norfluoxetine (NFLX), (SER), metoprolol (MET) and propranolol (PHO) at concentrations ranging from 21.7 ngL -1 (MDMA) to 622 ngL -1 (PHO). Measured concentrations were used to estimate the drug loads of the target chiral substances in a specific population. The EF was determined providing valuable information about the consumption and origin of the target drugs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A chiral derivatizing agent (CDA) with the aldehyde function has been widely used in discriminating chiral amines because of the easy formation of imines under mild conditions. There is a preference for the use of cyclic aldehydes as a CDA since their lower conformational flexibility favors the differentiation of the diastereoisomeric derivatives. In this study, the imines obtained from the reaction between ( S )-citronellal and the chiral amines ( sec -butylamine, methylbenzylamine, and amphetamine) were analyzed by the nuclear Overhauser effect (NOE). Through NOE, it was possible to observe that the ends of the molecules were close, suggesting a quasi-folded conformation. This conformation was confirmed by theoretical calculations that indicated the London forces and the molecular orbitals as main justifications for this conformation. This conformational locking explains the good separation of 13 C NMR signals between the diastereomeric imines obtained and, consequently, a good determination of the enantiomeric excess using the open chain ( S )-citronellal as a CDA.

Sample preparation is an important step in the isolation of target compounds from complex matrices to perform their reliable and accurate analysis. Hair samples are commonly pulverized or processed as fine cut, depending on preference, before extraction by techniques such as solid-phase extraction (SPE), liquid-liquid extraction, and other methods. In this study, a method based on hybrid solid-phase extraction (hybridSPE) and gas chromatography-mass spectrometry (GC-MS) was developed and validated for the determination of methamphetamine (MA) and amphetamine (AP) in hair. The hair samples were mechanically pulverized after washing with de-ionized water and acetone. The samples were then sonicated in methanol at 50 °C for 1 h and centrifuged at 50,000× g for 3 min. The supernatants were transferred onto the hybridSPE cartridge and extracted using 1 mL of 0.05 M methanolic hydrogen chloride. The combined solutions were evaporated to dryness, derivatized using pentafluoropropionic anhydride, and analyzed by GC-MS. Excellent linearity (R 2 > 0.9998) was achieved in the ranges of 0.05-5.0 ng/mg for AP and 0.1-10.0 ng/mg for MA. The recovery was 83.4-96.8%. The intra- and inter-day accuracies were -9.4% to 5.5% and -5.1% to 3.1%, while the intra- and inter-day precisions were within 8.3% and 6.7%, respectively. The limits of detections were 0.016 ng/mg for AP and 0.031 ng/mg for MA. The validated hybridSPE method was applied to dyed hair for MA and AP extraction and compared to a methanol extraction method currently being used in our laboratory. The results showed that an additional hybridSPE step improved the recovery by 5.7% for low-concentration quality control (QC) samples and by 24.1% for high-concentration QC samples. Additionally, the hybridSPE method was compared to polymeric reversed-phase SPE methods, and the absolute recoveries for hybridSPE were 50% and 20% greater for AP (1.5 ng/mg) and MA (3.0 ng/mg), respectively. In short, the hybridSPE technique was shown to minimize the matrix effects, improving GC-MS analysis of hair. Based on the results, the proposed method proved to be effective for the selective determination of MA and AP in hair samples.

Objectives: Fluoroquinolone antibiotics are commonly used in the treatment of infections and have previously been confirmed to cross-react with previous generations of opiates immunoassays. In this work we evaluated the cross-reactivity of the three fluoroquinolones in use at our institution with a panel of 10 urine drug screens., Design and Methods: Drug preparations of levofloxacin, ciprofloxacin, and moxifloxacin that were designed for intravenous delivery were added to drug-free urine at varying concentrations. Spiked urine samples were screened for illicit and therapeutic drugs on an Abbott Architect c16000 automated chemistry analyzer. Percent cross-reactivity was calculated., Results: Levofloxacin displayed clinically relevant cross-reactivity with the Abbott MULTIGENT opiates and Thermo CEDIA® buprenorphine immunoassays but did not cross-react with the Abbott MULTIGENT oxycodone or methadone immunoassays. Moxifloxacin displayed clinically relevant cross-reactivity only with the Abbott MULTIGENT amphetamine/methamphetamine assay. Ciprofloxacin did not cross-react with any of the 10 immunoassays., Conclusions: This study demonstrates that levofloxacin cross-reacts with modern immunoassays for two related opioids (buprenorphine and morphine) and moxifloxacin cross-reacts with the amphetamine/methamphetamine assay. Urine concentrations of these fluoroquinolones that are consistent with therapeutic use produced results above commonly used-cutoffs for positivity. This underscores the necessity of confirmatory testing of presumptively positive urine drug screens., (Copyright © 2019 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

A methamphetamine-based molecularly imprinted polymer (MIP) has been prepared by bulk polymerization to recognize new psychoactive substances (NPS) of the amphetamine, cathinones and 2C families in oral fluid samples, being the first precedent of a synthetized MIP for the extraction and preconcentration 32 NPS including amphetamine type substances and synthetic cathinones from oral fluids. Pre-polymerization complex and resulting materials were appropriately characterized by infrared spectroscopy, scanning electron microscopy, and nitrogen adsorption-desorption isotherms. Appropriateness of the material for the specific recognition of the target analytes was also evaluated through computational calculations and experimentally assessed by solid phase extraction (SPE). The most appropriate SPE conditions were evaluated and recoveries of 32 different NPS were obtained, ranging from 80 to 120% with a relative standard deviation (RSD) in all cases lower than 12%. Amphetamine-related NPS were analyzed by a fast and portable methodology based on ion mobility spectrometry (IMS) and a rearguard procedure based on ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) providing limit of detection values from 10 to 80 μg L -1 and from 0.03 to 1.3 μg L -1 , respectively. Oral fluid samples, containing different interferents like caffeine, fluticasone and cetirizine, were spiked with 300 μg L -1 amphetamine and subsequently analyzed, showing recoveries ranging from 81 to 115% using both methodologies. Thus, this paper shows preliminary results to demonstrate the applicability of the developed procedure which could be used with minor modifications as screening technique in on-road drug analysis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The differentiation between single methamphetamine consumption and co-consumption with amphetamine is difficult, however possible by enantioselective analysis due to different preferred synthesis pathways of both substances. We quantified (R)-(-) and (S)-(+)-enantiomers of methamphetamine and amphetamine by a fast liquid chromatographic tandem-mass spectrometric method using a Lux® 3-μm AMP 150 × 3.0 mm analytical column after simple protein precipitation with methanol. Method validation for quantitative detection showed limits of quantification < 5 ng/mL, linearity in a range between 5 and 300 ng/mL and bias and imprecision data < 15%. Overall, 134 plasma samples of police cases from the German regions of Franconia and Northrhine-Westphalia were analyzed for the enantiomers of methamphetamine and amphetamine. In 28 cases, the intake of racemic illicit amphetamine could be demonstrated; (R)-(-) / (S)-(+)-amphetamine concentration ratios in these cases were between 1.38 and 4.50 with most of the ratios being < 2.0. These ratios were compared to a subgroup of 25 consumers with a co-consumption of (S)-(+)-methamphetamine and racemic amphetamine detected by the qualitative proof of (R)-(-)-amphetamine but also by (R)-(-) / (S)-(+)-amphetamine concentration ratios (< 1 in 11 of 25 cases). Within our collective of 106 plasma samples after methamphetamine use, 25 samples showed co-consumption with amphetamine which shows that co-consumption of both stimulants is not a rare scenario. Furthermore, we could show that if non-stereoselective methods are used and the concentration ratio of total methamphetamine/total amphetamine is determined, a reliable estimation of co-consumption is not possible.

Objective: Psychostimulants are the first-line treatment in adults with attention-deficit hyperactivity disorder (ADHD). This meta-analysis aimed to evaluate the efficacy, acceptability, and tolerability of lisdexamfetamine (LDX), mixed amphetamine salts (MASs), modafinil (MDF), and methylphenidate (MPH) in comparison with placebo., Data Sources: We systematically searched PubMed/MEDLINE and Clinicaltrials.gov in May 2016, along with CENTRAL and EU Clinical Trials Register in February 2016, for the randomized, double-blind, placebo-controlled, parallel-group clinical trials conducted on adults diagnosed with ADHD., Study Selection and Data Extraction: Substantial comorbidity, substance abuse or dependence, and nonpharmacological interventions represented grounds for exclusion. Published reports were the sole source for data extraction. Improvement in ADHD symptoms was the primary outcome. Random-effects model meta-analysis was applied to calculate the standardized mean difference (SMD) with 95% CIs., Data Synthesis: The search retrieved 701 records, of which 20 studies were eligible for analysis. High effect size (expressed as SMD) in reducing ADHD symptoms was observed for LDX (-0.89; 95% CI = -1.09, -0.70), whereas MASs (-0.64; 95% CI = -0.83, -0.45) and MPH (-0.50; 95% CI = -0.58, -0.41) reduced symptoms moderately compared with placebo. No efficacy was shown for MDF (0.08; 95% CI; -0.18, 0.34). Relevance to Patient Care and Clinical Practice: In this meta-analysis, the efficacy, tolerability, and acceptability of psychostimulants were compared with that for placebo. Five of the included trials have not been evaluated in any of the previously published meta-analyses., Conclusions: The results suggest that LDX has the largest effect size and has a promising potential for treating adults with ADHD.

Enantiomeric profiling was used in this study to investigate the consumption of amphetamine and methamphetamine in regional and urban Southeast Queensland, Australia over a period of seven years. S(+) methamphetamine was predominantly consumed in both urban and regional areas, showing a two and three fold increase in urban and regional catchments respectively between 2011 and 2017. The ratio of amphetamine to methamphetamine (AMP/METH) in wastewater reflected the expected excretion profile of methamphetamine consumption indicating the presence of amphetamine in this study was primarily the result of methamphetamine metabolism. However, the occasional occurrence of R(-) amphetamine in samples containing higher AMP/METH ratios, suggested the consumption of racemic amphetamine. The R(-) methamphetamine enantiomer was also identified in several samples, possibly indicative that the phenyl-2-propanone (P2P) synthesis process rather than the more typical reduction of ephedrines was also being used to manufacture methamphetamine. Furthermore, we identified two samples with a significantly different enantiomer ratio for the METH and AMP as well as a much lower AMP/METH concentration ratio suggesting contribution from direct disposal of methamphetamine into the sewer. This study demonstrated that enantiomeric profiling in wastewater-based epidemiology can provide valuable information for evaluating the origin of amphetamine in wastewater as either a metabolite of methamphetamine consumption or amphetamine itself., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Impurity profiling is a well described forensic tool that may be applied to gain information about the illegal drug market. However, it requires experience to assess the correlation between chemical profiles thereby separating linked from unlinked samples. One of the challenges in this context is that the chemical profiles may change over time, thus complicating an assessment if samples are stored under different conditions. In this study, the impact of different storage conditions on the stability of amphetamine impurity profiles was investigated. We examined the influence of storage time, temperature, sample purity, sample quantity and the presence of methanol on the amphetamine profile stability when stored for up to 12 months. We find that the target compounds in amphetamine impurity profiles are susceptible to all the examined storage conditions. Consequently, this unstable nature of amphetamine profiles may complicate the assessment when comparing amphetamine seizures that has been separated for longer time periods or stored under different conditions prior to seizure. Knowledge about the seizure history is rarely available to the forensic analyst. Therefore, sample stability issues should be taken into account when comparisons of chemical profiles are made., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 (ΔG bind ) 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 (ΔG bind  = -13.15 kcal/mol) presented greater spontaneity in establishing interactions with DNA in comparison to 3,4-Methylenedioxymethamphetamine (ΔG bind  = -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.

The aim of this paper is to present the first study on spatial and temporal variation in the enantiomeric profile of chiral drugs in eight European cities. Wastewater-based epidemiology (WBE) and enantioselective analysis were combined to evaluate trends in illicit drug use in the context of their consumption vs direct disposal as well as their synthetic production routes. Spatial variations in amphetamine loads were observed with higher use in Northern European cities. Enantioselective analysis showed a general enrichment of amphetamine with the R-(-)-enantiomer in wastewater indicating its abuse. High loads of racemic methamphetamine were detected in Oslo (EF = 0.49 ± 0.02). This is in contrast to other European cities where S-(+)-methamphetamine was the predominant enantiomer. This indicates different methods of methamphetamine synthesis and/or trafficking routes in Oslo, compared with the other cities tested. An enrichment of MDMA with the R-(-)-enantiomer was observed in European wastewaters indicating MDMA consumption rather than disposal of unused drug. MDA's chiral signature indicated its enrichment with the S-(+)-enantiomer, which confirms its origin from MDMA metabolism in humans. HMMA was also detected at quantifiable concentrations in wastewater and was found to be a suitable biomarker for MDMA consumption. Mephedrone was only detected in wastewater from the United Kingdom with population-normalised loads up to 47.7 mg 1000 people -1 day -1 . The enrichment of mephedrone in the R-(+)-enantiomer in wastewater suggests stereoselective metabolism in humans, hence consumption, rather than direct disposal of the drug. The investigation of drug precursors, such as ephedrine, showed that their presence was reasonably ascribed to their medical use., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

To distinguish between legal and illegal consumption of amphetamine reliable analytical methods for chiral separation of the R- and S-enantiomers of amphetamine in biological specimens are required. In this regard, supercritical fluid chromatography (SFC) has several potential advantages over liquid chromatography, including rapid separation of enantiomers due to low viscosity and high diffusivity of supercritical carbon dioxide, the main component in the SFC mobile phase. A method for enantiomeric separation and quantification of R- and S-amphetamine in urine 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 a semi-automatic solid phase extraction method. The UHPSFC-MS/MS method used a Chiralpak AD-3 column with a mobile phase consisting of CO 2 and 0.2% cyclohexylamine in 2-propanol. The injection volume was 2 μL and run-time was 6 min. 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 50-10,000 ng/mL for each enantiomer. The between-assay relative standard deviations were in the range of 3.7-7.6%. Recovery was 92-93% and matrix effects ranged from 100 to 104% corrected with internal standard. After development and validation, the method has been successfully implemented in routine use at 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A method was developed for the screening and quantification of seven amphetamine-type stimulants (ATS) and 22 cathinones, including three metabolites, in urine with Gas Chromatography-Mass Spectrometry. This method allowed the detection and quantification of ATS and cathinones group molecules using one procedure. A study of the stability of the drug mixtures for a period of 201 days in human urine samples under three different conditions has been carried. The ATS and cathinones include amphetamine, methamphetamine, MDA, MDEA, MDMA, PMA, PMMA, cathinone, methcathinone, 3'-position-substituted, ring-substituted, methylenedioxy-substituted, N-alkyl-substituted and pyrrolidinyl-substituted. Twenty drugs out of twenty-nine were validated with a quantitative method. This method can be applied to the nine remaining drugs as a screening method. The linearity of the assay was from 50 to 2000 ng/ml, with limits of detection of 0.5 to 10 ng/ml. In terms of accuracy, between-run and within-run precision were ≤20% for 20 compounds with good selectivity. No carryover was seen, and the recovery was between 80 and 120% for most drugs tested. ATS and pyrrolidinyl-substituted groups were conducted to be stable compounds under all conditions. All compounds tested were stable at -20 °C. Some cathinones were primarily degraded after 21 days at 4 °C. They were detectable but unstable after 201 days at 4 °C. Most cathinones were unstable after a day and completely lost after 28 days at RT., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This work investigates the Y326I point mutation effect on the kinetics of oxidative deamination of phenylethylamine (PEA) catalyzed by the monoamine oxidase B (MAO B) enzyme. PEA is a neuromodulator capable of affecting the plasticity of the brain and is responsible for the mood enhancing effect caused by physical exercise. Due to a similar functionality, PEA is often regarded as an endogenous amphetamine. The rate limiting step of the deamination was simulated at the multiscale level, employing the Empirical Valence Bond approach for the quantum treatment of the involved valence states, whereas the environment (solvated protein) was represented with a classical force field. A comparison of the reaction free energy profiles delivered by simulation of the reaction in the wild type MAO B and its Y326I mutant yields an increase in the barrier by 1.06 kcal mol -1 upon mutation, corresponding to a roughly 6-fold decrease in the reaction rate. This is in excellent agreement with the experimental kinetic studies. Inspection of simulation trajectories reveals possible sources of the point mutation effect, namely vanishing favorable electrostatic interactions between PEA and a Tyr326 side chain and an increased amount of water molecules at the active site due to the replacement of tyrosine by a less spacious isoleucine residue, thereby increasing the dielectric shielding of the catalytic environment provided by the enzyme.

Amphetamine (AMP) is used as an illicit drug and also for the treatment of attention deficit hyperactivity disorder (ADHD). Respective drugs most often contain the enantiomer (S)-AMP as active compound or (S)-AMP is formed from the prodrug lisdexamfetamine (Elvanse®) whereas the illicit drug is usually traded as racemate ((R/S)-AMP). A differentiation between the use of the medically prescribed drug and the abuse of illicit street amphetamine is of great importance, for example in retrospective consumption monitoring by hair analysis. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the chiral separation and quantitation of (S)- and (R)-AMP in hair was developed. For this purpose, 20 mg hair was extracted and derivatized with N-(2,4-dinitro-5-fluorophenyl)-L(S)-valinamide L(S)-(DNPV) to yield amphetamine diastereomers. Baseline separation of the resulting diastereomers was achieved on a high-pressure liquid-chromatography system (HPLC) coupled to a Sciex QTRAP® 5500 linear ion trap quadrupole mass spectrometer. The method was successfully validated. Analysis of hair samples from nine Elvanse® patients revealed only (S)-AMP in eight cases; one subject showed both enantiomers indicating a (side-) consumption of street amphetamine. The analysis of the 16 amphetamine users' samples showed only racemic amphetamine. Furthermore, it could be shown in a controlled study that (S)-AMP can be detected after administration of even very low doses of lisdexamfetamine and dexamphetamine, which can be of interest in forensic toxicology and especially in drug-facilitated crime (DFC). The method now enables the retrospective compliance-monitoring of ADHD patients and the differentiation between medically prescribed intake of (S)-amphetamine and abuse of illicit street amphetamine., (Copyright © 2017 John Wiley & Sons, Ltd.)

Identifying possible new biological activities of psychoactive substances belonging to various chemical classes may lead to a better understanding of their mode of action and side effects. We report here that amines structurally related to amphetamine, a widely used psychoactive substance, such as amphetamine, methamphetamine, phentermine, mephentermine, and chlorphenteramine, potently activate several carbonic anhydrase (CA, EC 4.2.1.1) isoforms involved in important physiological functions. Of the 11 investigated human (h) isoforms, the widespread hCA I and II, the secreted hCA VI, as well as the cytosolic hCA XIII, and membrane-bound hCA IX and XIV were poorly activated by these amines, whereas the extracellular hCA IV, the mitochondrial enzymes hCA VA/VB, the cytosolic hCA VII, and the transmembrane isoform hCA XII were potently activated. Some of these enzymes are abundant in the brain, raising the possibility that some of the cognitive effects of such psychoactive substances might be related to their activation of these enzymes.

To which extent illicit drugs are transformed during in-sewer transport, depends on a number of factors: i) substance-specific transformation rates, ii) environmental conditions, iii) point of discharge (location of drug user) and iv) sewer network properties, primarily hydraulic residence time (HRT) and the ratio of biofilm contact area to wastewater volume (A/V eq ). Assessing associated uncertainties typically requires numerous simulations. Therefore, we propose a new two-step modeling framework: 1) Quantify hydrodynamic conditions. This computationally demanding step was performed once in SWMM to derive HRT and A/V eq for each potential point of discharge (node) in three catchments of different size. 2) Estimate biomarker loss. In this step, Monte Carlo simulations were performed for defined scenarios. Depending on assumptions about drug user distribution and prevalence, a number of nodes was sampled. For each node an empirical first-order transformation model was applied with flow-path-corresponding HRT and A/V eq from step 1. Biotic and abiotic transformation rates were sampled from distributions combining variability of different biofilms. In our modeling study, median losses were >30% for amphetamine, 6-monoacetylmorphine and 6-acetylcodeine in all three catchments with high uncertainty (5%-100% loss), which would imply a systematic underestimation of consumption when neglecting in-sewer processes. Median losses for 21 other investigated biomarkers were <10% with different uncertainty ranges - "no substantial transformation" was confirmed for nine substances in a real sewer segment with a 2-h residence time. Transferability of these results must be tested for other catchments. To further reduce uncertainty, mainly additional knowledge on transformation rates, particularly in biofilm, and their distribution across a sewer network is needed to update model input objectively. Our approach allows efficient testing and, furthermore, can be expanded for many other human biomarkers. Accounting for biomarker stability during in-sewer transport will avoid biased estimates and further improve wastewater-based epidemiology., (Copyright © 2017. Published by Elsevier Ltd.)

Amphetamine selective molecularly imprinted sol-gel polymer tablets, MIP-tablets, for solid-phase microextraction of biofluid samples were prepared. An acetonitrile solution of deuterated amphetamine template and silane precursor, 3-(propylmethacrylate) trimethoxysilane, was soaked into the pores of polyethylene tablet substrates and polymerized by an acid-catalysed sol-gel process. Application of the resultant MIP-tablets to extract amphetamine from human urine samples followed by LC-MS/MS analysis was investigated. The extraction protocol was optimised with respect to pH of sample, addition of sodium chloride, extraction time, desorption solvent and desorption time. The final analysis method determined amphetamine in human urine with a limit of detection (LOD) of 1.0ng/mL and a lower limit of quantification (LLOQ) of 5ng/mL. Validation demonstrated accuracy of the method was 91.0-104.0% and inter-assay precision was 4.8-8.5% (RSD). Extraction recovery was 80%. The MIP-tablets could be re-used and the same tablet could be employed for more than twenty extractions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Analysis of student consumption of illicit drugs (ID) by school population surveys (SPS) provides information useful for prevention, but the results may be influenced by subjective factors. We explored wastewater (WW) analysis to improve the information., Methods: We used WW analysis to measure ID consumption in eight secondary schools in Italy in 2010-13 (students aged 15-19). Samples were collected from the sewage pipes of the schools during lessons for one week each year. Samples were analysed by mass spectrometry to measure ID and consumption by students was compared to that of the general population., Results: We found THCCOOH (human metabolite of THC) concentrations in 2010 indicating significant consumption of cannabis in all the schools and benzoylecgonine (human metabolite of cocaine) suggesting a limited consumption of cocaine in all but one school. Morphine was only found in traces, and amphetamine, methamphetamine, ecstasy, ketamine and mephedrone were not detectable. Repeated analysis showed cannabis stable until 2012 with increases in 2013, low cocaine and morphine levels, and none of the other ID., Discussion: WW analysis suggested that students used amounts of cannabis comparable to the general population, with low, sporadic use of cocaine and opioids, but excluded the use of significant amounts of amphetamine, methamphetamine, ecstasy, ketamine and mephedrone. WW analysis was useful to confirm SPS figures and provides complementary findings for effective prevention strategies. This is the first time WW analysis has been used to investigate consumption of a large number of ID and new psychoactive substances (NPS) in schools., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Fenethylline, also known by the trade name Captagon, is a synthetic psychoactive stimulant that has recently been linked to a substance-use disorder and 'pharmacoterrorism' in the Middle East. Although fenethylline shares a common phenethylamine core with other amphetamine-type stimulants, it also incorporates a covalently linked xanthine moiety into its parent structure. These independently active pharmacophores are liberated during metabolism, resulting in the release of a structurally diverse chemical mixture into the central nervous system. Although the psychoactive properties of fenethylline have been reported to differ from those of other synthetic stimulants, the in vivo chemical complexity it manifests upon ingestion has impeded efforts to unambiguously identify the specific species responsible for these effects. Here we develop a 'dissection through vaccination' approach, called DISSECTIV, to mitigate the psychoactive effects of fenethylline and show that its rapid-onset and distinct psychoactive properties are facilitated by functional synergy between theophylline and amphetamine. Our results demonstrate that incremental vaccination against a single chemical species within a multi-component mixture can be used to uncover emergent properties arising from polypharmacological activity. We anticipate that DISSECTIV will be used to expose unidentified active chemical species and resolve pharmacodynamic interactions within other chemically complex systems, such as those found in counterfeit or illegal drug preparations, post-metabolic tissue samples and natural product extracts.

This perspective examines amphetamine importations into Ireland. Some novel by-products were detected and linked to a change in the method of production of P2P from APAAN. These by-products remained present during subsequent Leuckart reaction conditions. Novel by-products from substituted cathinone synthesis reactions were also isolated and characterized., (Copyright © 2017 John Wiley & Sons, Ltd.)

Background and Aims: On-line drug markets flourish and consumers have high expectations of on-line quality and drug value. The aim of this study was to (i) describe on-line drug purchases and (ii) compare on-line with off-line purchased drugs regarding purity, adulteration and price., Design: Comparison of laboratory analyses of 32 663 drug consumer samples (stimulants and hallucinogens) purchased between January 2013 and January 2016, 928 of which were bought on-line., Setting: The Netherlands., Measurements: Primary outcome measures were (i) the percentage of samples purchased on-line and (ii) the chemical purity of powders (or dosage per tablet); adulteration; and the price per gram, blotter or tablet of drugs bought on-line compared with drugs bought off-line., Findings: The proportion of drug samples purchased on-line increased from 1.4% in 2013 to 4.1% in 2015. The frequency varied widely, from a maximum of 6% for controlled, traditional substances [ecstasy tablets, 3,4-methylenedioxy-methamphetamine (MDMA) powder, amphetamine powder, cocaine powder, 4-bromo-2,5-dimethoxyphenethylamine (2C-B) and lysergic acid diethylamide (LSD)] to more than a third for new psychoactive substances (NPS) [4-fluoroamphetamine (4-FA), 5/6-(2-aminopropyl)benzofuran (5/6-APB) and methoxetamine (MXE)]. There were no large differences in drug purity, yet small but statistically significant differences were found for 4-FA (on-line 59% versus off-line 52% purity for 4-FA on average, P = 0.001), MDMA powders (45 versus 61% purity for MDMA, P = 0.02), 2C-B tablets (21 versus 10 mg 2C-B/tablet dosage, P = 0.49) and ecstasy tablets (131 versus 121 mg MDMA/tablet dosage, P = 0.05). The proportion of adulterated samples purchased on-line and off-line did not differ, except for 4-FA powder, being less adulterated on-line (χ 2  = 8.3; P < 0.02). Drug prices were mainly higher on-line, ranging for various drugs from 10 to 23% higher than that of drugs purchased off-line (six of 10 substances: P < 0.05)., Conclusions: Dutch drug users increasingly purchase drugs on-line: new psychoactive substances in particular. Purity and adulteration do not vary considerably between drugs purchased on-line and off-line for most substances, while on-line prices are mostly higher than off-line prices., (© 2016 Society for the Study of Addiction.)

Amphetamine (AMPH) is a psychostimulant used worldwide by millions of patients in the clinical treatment of attention deficit hyperactivity disorder, narcolepsy or even obesity, and is also a drug of abuse. 4-Hydroxynorephedrine (4-OHNE) and 4-hydroxyamphetamine (4-OHAMPH) are two major metabolites known to persist in the brain longer than AMPH. The contribution of AMPH metabolites for its neurotoxicity is undetermined. We evaluated the toxicity of AMPH and its metabolites 4-OHNE and 4-OHAMPH, obtained by chemical synthesis, in human dopaminergic differentiated SH-SY5Y neurons. Cells were exposed to AMPH (concentration range 0-5mM) or 4-OHAMPH or 4-OHNE (concentration range 0-10mM) for 24 or 48h, and the viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) leakage assays. Results showed that for both AMPH and the metabolites a concentration-dependent toxicity was observed. The toxic concentration 50% (TC 50 ) for AMPH and 4-OHNE following 24h exposure was circa 3.5mM and 8mM, respectively. For 4-OHAMPH the TC 50 was not reached in the tested concentration range. N-acetyl cysteine, cycloheximide, l-carnitine, and methylphenidate were able to reduce cell death induced by AMPH TC 50 . Acridine orange/ethidium bromide staining showed evident signs of late apoptotic cells and necrotic cells following 24h exposure to AMPH 3.50mM. The 4-OHAMPH metabolite at 8.00mM originated few late apoptotic cells, whereas 4-OHNE at 8.00mM resulted in late apoptotic cells and necrotic cells, in a scenario similar to AMPH. In conclusion, the AMPH metabolite 4-OHNE is more toxic than 4-OHAMPH, nonetheless both are less toxic than the parent compound in vitro. The most toxic metabolite 4-OHNE has longer permanence in the brain, rendering likely its contribution for AMPH neurotoxicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

1-Phenyl-2-propanone (P2P) is an internationally monitored precursor that has become increasingly difficult for illicit amphetamine producers to source, which means that alternative routes to its preparation have become increasingly important. One such approach includes the hydrolysis of alpha-phenylacetoacetonitrile (APAAN) with sulfuric acid. Previously, we reported the identification of 4,6-dimethyl-3,5-diphenylpryid-2-one following implementation of hydrolysis conditions and it was proposed that this compound might serve as one route specific by-product in the APAAN to P2P conversion. This study continued to explore the presence of impurities formed during this conversion and expanded also into a second route of P2P synthesis starting from alpha-methylstyrene (AMS). All P2P products underwent the Leuckart procedure to probe the presence of P2P-related impurities that might have carried through to the final product. Two by-products associated with the APAAN hydrolysis route to P2P were identified as 2,3-diacetyl-2,3-diphenylsuccinonitrile (1) and 2-methyl-1-phenyl-1,3-dicarbonitrile-1H-indene (2), respectively. Two by-products associated with the AMS route to P2P and subsequent Leuckart reaction were 1,1,3-trimethyl-3-phenyl-2,3-dihydro-1H-indene (3) and 1-phenyl-N-(phenylethyl)propan-2-amine (4), respectively. The two indenes (2 and 3) identified in synthesized amphetamine originating from P2P suggested that it might be possible to differentiate between the two synthetic routes regarding the use of APAAN and AMS. Furthermore, the association of these compounds with amphetamine production appears to have been reported for the first time. The presence of compounds 1 - 4 in seized amphetamine samples and waste products could facilitate the suggestion whether APAAN or AMS were employed in the synthesis route to the P2P. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Chromatographic differentiation of the ring-substituted regioisomers of amphetamine (AMP) and methamphetamine (MA) was performed by supercritical fluid chromatography (SFC). The behaviour of the retention against the changes of column temperature and co-solvent proportion was studied. The obtained information facilitated the optimization of the each regioisomer. As a result, 2-, 3-, and 4-ring-substituted analogues of AMP and MA with methyl, methoxy, fluoro, chloro, and bromo groups were separated, generally within 6 min. In addition, we found that the separation pattern of the examined regioisomers was classified into two, which depended on the electron donating/withdrawing effect of the substituent. Our results indicate that SFC could be used in forensic drug analysis for fast, reliable identification of structurally similar drugs of abuse. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Amphetamine (AM) is a powerful psychostimulant existing in two enantiomeric forms. Stereoselective analysis of AM in biosamples can assist clinicians and forensic experts in differentiating between abuse of illicitly synthesized racemic AM and ingestion of pharmaceutical AM formulations containing either S-AM or different proportions of the S- and R-enantiomers. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying AM enantiomers in urine was newly developed. The method comprised dilution with water, followed by injection of the diluted sample onto an achiral C18 trapping column for purification and subsequent backflush elution to a chiral Lux 3 μm AMP LC column by means of a switching valve. An isocratic mobile phase of 25 % acetonitrile in 0.1 M aqueous ammonia was used for enantiomeric separation. Injection, cleanup, and backflush of the next sample were performed before the previous sample had eluted from the analytical column, thus enabling simultaneous enantioseparation of up to three samples within the analytical column. This novel chromatographic concept allowed for increased sample throughput by accelerating both the sample preparation and the LC analysis. Analyte detection was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was successfully validated through assessment of its linearity, lower limit of quantification, accuracy and precision, selectivity, matrix effect, carry-over, dilution integrity, and re-injection reproducibility. Linearity ranged from 0.05 to 25 mg/L for both enantiomers. Proof of the method included analysis of urine samples obtained from drug abusers and patients receiving an S-AM prodrug. Graphical Abstract Enantioselective determination of amphetamine in human urine using liquid chromatography with achiral-chiral column-switching and tandem mass spectrometry.

Amphetamine and methamphetamine are emerging contaminants-those for which no regulations currently require monitoring or public reporting of their presence in our water supply. In this research, a protocol for weak cation-exchange (WCX) SPE coupled with LC-MS/MS was developed for determination of emerging contaminants amphetamine and methamphetamine in a complex wastewater matrix. Gradient LC parameters were adjusted to yield baseline separation of methamphetamine from other contaminants. Methamphetamine-D5 was used as the internal standard (IS) to compensate for sample loss during SPE and for signal loss during MS (matrix effects). Recoveries were 102.1 ± 7.9% and 99.4 ± 4.0% for amphetamine and methamphetamine, respectively, using WCX sorbent. Notably, methamphetamine was determined to be present in wastewater influent at each sampling date tested. Amphetamine was present in wastewater influent on two of four sampling dates. Amphetamine concentrations ranged from undetectable to 86.4 ng/L in influent, but it was undetectable in wastewater effluent. Methamphetamine was detected in influent at concentrations ranging from 27.0-60.3 ng/L. Methamphetamine concentration was reduced but incompletely removed at this facility. Although absent in one post-UV effluent sample, concentrations of methamphetamine ranged from 10.8-14.8 ng/L., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An advantage of differential mobility spectrometry (DMS) is it provides an orthogonal mechanism to mass spectrometry (MS). The DMS-MS/MS detects analytes in the gas phase on the basis of differences in ion mobility in low and high electric fields, which makes DMS-MS/MS an alternative to chromatographic separation-MS. One drawback of DMS is its limited resolution and sensitivity, especially for detecting small molecules when using a nonpolar inert gas as the carrier gas. The present work has evaluated the effects on peak capacity of adding chemical modifiers to inert carrier gases (nitrogen, helium, argon and carbon dioxide). Use of a methanol-helium mixture gave improvements in both separation and sensitivity. Nine structurally similar amphetamine-type stimulants were determined in urine without pretreatment of the samples before analysis. After optimization of carrier gas, nature and concentration of chemical modifier, and DMS temperature, limits of detection ranging from 1.1 to 2.7 ng mL -1 , with a linear range of three orders of magnitude (5-5000 ng mL -1 ) were achieved. Precision was <15% and the accuracy of the quality control samples was 87.6-113.7%. For the quantitation of urine samples from drug abusers, data obtained using DMS-MS/MS showed reasonable agreement (within ±19.5%) with that obtained using LC-MS/MS. The analysis time for DMS-MS/MS was only 1.1 min and a paired sample t-test between the two methods gave a p-value of 0.0894, which indicates that DMS-MS/MS is a reliable method, with comparable precision and sensitivity to LC-MS/MS., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Identification and quantification of new psychoactive substances (NPS), both in biological and non-biological samples, represent a hard challenge for forensic toxicologists. NPS are increasingly emerging on illegal drug market. Many cases of co-consumption of NPS and other substances have also been reported. Hence, the development of analytical methods aiming at the detection of a broad-spectrum of compounds (NPS and "traditional" drugs) could be helpful. In this paper, a fully validated screening method in blood for the simultaneous detection of 69 substances, including 64 NPS (28 synthetic cannabinoids, 19 synthetic cathinones, 5 phenethylamines, 3 indanes, 2 piperazines, 2 tryptamines, 2 phencyclidine, methoxetamine, ketamine and its metabolite) and 5 amphetamines (amphetamine, methamphetamine, MDMA, MDA, 3,4-methylenedioxy-N-ethylamphetamine - MDEA-) by a dynamic multiple reaction monitoring analysis through liquid chromatography - tandem mass spectrometry (LC-MS/MS) is described. This method is very fast, easy to perform and cheap as it only requires the deproteinization of 200μL of blood sample with acetonitrile. The chromatographic separation is achieved with a C18 column. The analysis is very sensitive, with limits of quantification ranging from 0.1 to 0.5ng/mL. The method is linear from 1 to 100ng/mL and the coefficient of determination (R(2)) was always above 0.9900. Precision and accuracy were acceptable at any quality control level and recovery efficiency range was 72-110%. Matrix effects did not negatively affect the analytical sensitivity. This method was successfully applied to three real cases, allowing identification and quantification of: mephedrone and methamphetamine (post-mortem); ketamine, MDMA and MDA (post-mortem); AB-FUBINACA (ante-mortem)., (Copyright © 2016 Elsevier B.V. All rights reserved.)