Fast and Sensitive Chiral Analysis of Amphetamines and Cathinones in Equine Urine and Plasma Using Liquid Chromatography Tandem Mass Spectrometry

A simple, rapid, sensitive and reproducible method for enantiomer analysis of methamphetamine, amphetamine, cathinone and methcathinone was developed and validated. The compounds were extracted from equine plasma and urine using a fast liquid-liquid extraction procedure. Only one milliliter plasma and one hundred microliter urine sample is needed for analysis. The extraction procedure had good recovery (>70%) and the matrix effect was negligible. Enantiomer differentiation and confirmation were achieved using liquid chromatography with chiral stationary phase and mass spectrometry detection. The method demonstrated excellent reproducibility with intra-day and inter-day precision of lower than 5%. The lower limits of detection for all of the compounds studied here were at low pg/mL level for both plasma and urine. This is the first report of the analysis of four chiral compounds in equine plasma and urine. Routine application was demonstrated for (S)- and (R)-enantiomer differentiation.

Raman Optical Activity and Raman Spectra of Amphetamine Species--Quantum Chemical Model Calculations and Experiments

Theoretical calculations and preliminary measurements of vibrational Raman optical activity (ROA) spectra of different species of amphetamine (amphetamine and amphetamine-H+) are reported for the first time. The quantum chemical calculations were carried out as hybrid ab initio DFT-molecular orbital calculations by use of the Gaussian 03W program, based on complete geometry minimizations of the conformational energy of the S-(+)-amphetamine molecule, the S-(+)-amphetamine-H+ ion, and the R-(–)-amphetamine molecule. Following this, harmonic frequency calculations have been made, providing information about the cation vibrational bands, expected in salts of single anions (chlorides) as well as in salts of anions with internal bonds (sulfates, hydrogen phosphates, etc.). It shows that the kind of anion should be given better attention, as so far it has often not been the case, when the spectra are employed for identification purposes. The DFT calculations show that the most stable conformations are those allowing for close contact between the aromatic ring and the amine hydrogen atoms. The internal rotational barrier within the same amphetamine enantiomer has a considerable influence on the Raman and ROA spectra. As predicted the experimental ROA spectra were found to depend on the chirality. Two street samples, provided by the London Police, were also measured and compared to the calculated ROA spectra. The street samples were found to contain different enantiomers of the protonated amphetamine-H+ sulfate. According to the present study the AMPH+ ion in aqueous sulfate solution seems to adopt a conformation in which the phenyl and ammonium groups are in transpositions, similar to what has been found in the solid state.

Drug Seeking Behavior of Amphetamine Addicted Sprague-Dawley Rats Is Eliminated after Nutritional Supplementation

Aim: The effectiveness of nutritional supplementation on drug-seeking behavior of amphetamine-addicted rats during withdrawal was investigated using a biased conditioned place preference (CPP) paradigm. Method: Twenty-four male Sprague-Dawley rats exhibiting baseline preference for the black chamber during a 20-minute pre-conditioning exploration of the CPP box completed the study. On alternate days of an 8-day schedule, twelve rats (Group AMP) were randomly selected, given either amphetamine sulfate (5 mg/ml, i.p.) and confined to the white chamber;or vehicle (1 ml saline, i.p.) and confined to the black chamber. A significant increased percentage time spent and number of entries made by Group AMP to the drug-paired, white chamber on the test day confirmed amphetamine addiction. Group AMP subsequently received increasing doses of amphetamine over 6 days. Following acute drug withdrawal, their CPP performance was compared with that of vehicle treated rats (Group SAL). Groups AMP and SAL were equally divided and randomly assigned to animals fed chow reconstituted with the nutritional supplement (AMP-S and SAL-S) over 8 weeks or standard rat chow (AMP-N and SAL-N). CPP performances for all rats were determined blindly from video recordings following this period. Results: Nutritionally supplemented, amphetamine withdrawn rats (AMP-S) exhibited significantly decreased percentage entries and time spent in the white chamber (p ?ve, control animals. Conclusion: Drug-seeking behavior by amphetamine-addicted animals was eliminated after treatment with a nutritionally supplemented diet.

Amphetamine Alters the Circadian Locomotor Activity Pattern of Adult WKY Female Rats

There are no reports on the effect of amphetamine on female WKY circadian activity pattern. The objective of this study is to investigate whether repeated daily exposure to the psychostimulant amphetamine alters the locomotor circadian rhythm activity patterns of female adult Wistar-Kyoto (WKY) rats. Twenty-four rats were divided into two groups, control (N = 12) and experimental (N = 12), and kept in a 12:12 h light/dark cycle in an open field cage. After 5 to 7 days of acclimation, 11 days of consecutive non-stop recordings began. On experimental day 1, all groups were given an injection of saline. On experimental days 2 to 7, the experimental group was injected with 0.6 mg/kg amphetamine and the control group with saline followed by a washout phase from experimental day 8 to 10, and amphetamine re-challenge or saline on experimental day 11 similar to experimental day 2. Locomotor movements were determined using a computerized animal activity monitoring system, and cosine statistical analysis was used to fit a24-hour curve to the activity pattern. The horizontal activity (HA), total distance (TD), number of stereotypy (NOS), and stereotypical movements (SM) were analyzed for alterations in the circadian rhythm activity patterns. The data demonstrated that chronic amphetamine administration alters the mesor parameter of the circadian rhythm activity patterns, indicating that chronic amphetamine treatment exerts long term effects on these rats.

Amphetamine Conditioned Place Preference in Planarians

Meth- and other amphetamines currently present major drug-abuse concerns. However, the demonstration and study of abuse-related behaviors expressed in animal models is expensive and time-consuming. We previously reported a novel model of conditioned place preference (CPP), which is a standard tool in abuse research, in invertebrates (planarians). In the present study, planarians were tested for light/dark preference, then exposed for 5 min to either d-amphetamine or vehicle (water) in light and then re-tested for place preference (light vs dark). The planarians’ natural strong preference for dark (15 of 16) was significantly altered by amphetamine experience, such that 12 of 16 preferred the unnatural, but amphetamine-associated, light side. These results extend the demonstration of CPP to this invertebrate species and provide further evidence in support of this model to testing/screening amphetamine-like and possibly other drugs of abuse.

Potential therapeutic effects of thymoquinone on treatment of amphetamine abuse

Dear Editor,The prevalence of drug abuse has diverted from traditional drugs such as opioid and cannabis to modern,synthetic and psychoactive drugs such as amphetamine groups,the amphetamine(AT)and methamphetamine[1].Amphetamine is used medically to treat several disorders such as attention deficit hyperactivity disorder,narcolepsy,depression and minor obesity[2].However,AT has also gained

Amphetamine Versus Non Amphetamine-Related First Episode Psychosis in Saudi Arabian Patients

Background: Amphetamines are illicit psychostimulant drugs that can induce psychotic symptoms. Very few studies have been conducted in Kingdom of Saudi Arabia (SA) on amphetamine abuse and related psychosis. Recently, the pattern of amphetamine abuse in SA showed a significant trend of increased frequency. Objectives: To investigate the extent of amphetamine abuse in a sample of Saudi patients hospitalized for first episode of acute psychosis. Also, to compare in that sample between amphetamine psychosis and other psychoses regarding demographic data, premorbid personality and symptoms profile. Method: 106 patients with acute psychosis were hospitalized and screening of urine for amphetamine was conducted for all. Patients’ psychiatric evaluation included interviewing, and ICD-10 criteria for personality disorders. 30 healthy subjects were also included for comparison with patients. Results: 34/106 of psychotic patients (32%) were positive for amphetamine in urine (≥ 1000 ng/ml). The frequency of personality disorders was significantly higher (P < 0.01) in the patients (54/106;51%) compared with healthy subjects (6/30;20%). Also, the incidence of personality disorders was significantly higher (P < 0.01) in amphetamine positive psychosis (25/34;73.5%) compared with amphetamine negative psychosis (29/72;40%). Cluster B personality disorders particularly the antisocial and borderline were significantly higher in amphetamine positive psychosis (13/34;38%) compared with amphetamine negative psychoses (6/72;8%). The symptom profile showed significant difference between amphetamine positive and amphetamine negative psychosis as regards ideas of reference (50% vs. 14%), suspiciousness (44% vs. 11%), suicidal ideation (38% vs. 23%), paranoid delusions (29% vs. 17%) and increased pulse or blood pressure (29% vs. 7%) respectively. Conclusion: Screening of amphetamine in urine among patients with first episode of acute psychosis can help and support the clinical distinction of amphetamine psychosis from other types of psychosis. This is therapeutically critical since the line of treatment may be different between the two types of psychoses.

Polysubstance Use and Overdose Visualized via Maps: Amphetamines and Cocaine

Abuse of drug substances and resultant overdose deaths are no longer very straightforward—viz., attributable to a single chemical entity of known purity. The reality is that most overdose deaths involve polysubstance use (i.e., the use of combinations of substances). Further, the combinations are often of unknown purity, and even of unknown composition. Overdose deaths are at all-time highs. The depressing statistics are monitored and reported by several international and governmental organizations such as the WHO (World Health Organization), CDC (Centers for Disease Control and Prevention), several Institutes of the NIH (National Institutes of Health), Regulators, and Enforcement Agencies (e.g., DEA). The information is disseminated for free for review and use. But it is our observation that although numeric presentation is helpful and adequate for professionals, the non-expert and the visual learner often find a visual representation clearer and compelling. With this in mind, we present the “gestalt” of polysubstance use and overdose using available maps of the data. The previous article in the series considered the opioids. This one considers amphetamines and cocaine, and places the rise in opioid-associated overdose deaths in the context of other abused drugs.

Molecular docking of amphetamine,cathine and cathinone with dihydrofolate reductase:a computational analysis of inhibition of dihydrofolate reductase by khat alkaloids

Interaction of amphetamine,cathine and cathinone with the enzyme dihydrofolate reductase was studied by molecular docking using AutoDock 4.2 as the docking software application.AutoDock 4.2 software serves as a valid and acceptable docking application to study the interactions of small compounds with proteins.Interactions of amphetamine,cathine and cathinone with dihydrofolate reductase were compared to those of methotrexate,a known inhibitor of the enzyme.The calculated free energy of binding(ΔG binding)shows that the three ligands(ΔG=-6.87 to-7.21 kcal/mol;Ki=9.15 to 5.18μM)bind with affinity slightly lower than methotrexate(ΔG=-8.78 kcal/mol;Ki=363 nM).Binding interactions of the three ligands with active site residues of the enzyme are also predicted.All the ligands appear to bind in a similar conformation making extensive VDW contacts in the active site of the enzyme.Hydrogen bonding and pi-pi interaction with key active site residues is also observed.Thus,a probable inhibition of dihydrofolate reductase by khat alkaloids can be explained on the basis of this in silico binding and khat alkaloids can be considered as potential lead compounds in the development of new inhibitors of dihydrofolate reductase which is a potential target of anti-cancer drugs.The results of these studies can serve as a starting point for further computational and experimental studies.

Brain dysfunctions and neurotoxicity induced by psychostimulants in experimental models and humans:an overview of recent findings

Preclinical and clinical studies indicate that psychostimulants,in addition to having abuse potential,may elicit brain dysfunctions and/or neurotoxic effects.Central toxicity induced by psychostimulants may pose serious health risks since the recreational use of these substances is on the rise among young people and adults.The present review provides an overview of recent research,conducted between 2018 and 2023,focusing on brain dysfunctions and neurotoxic effects elicited in experimental models and humans by amphetamine,cocaine,methamphetamine,3,4-methylenedioxymethamphetamine,methylphenidate,caffeine,and nicotine.Detailed elucidation of factors and mechanisms that underlie psychostimulant-induced brain dysfunction and neurotoxicity is crucial for understanding the acute and enduring noxious brain effects that may occur in individuals who use psychostimulants for recreational and/or therapeutic purposes.

Wastewater analysis for nicotine,cocaine,amphetamines,opioids and cannabis in New York City

According to current surveys and overdoses data,there is a drug crisis in the USA.Wastewater-based epidemiology(WBE)is an evolving discipline that analyses wastewater samples to detect drugs and metabolites to estimate drug consumption in a certain community.This study demonstrates how drug relative presence could be tracked by testing wastewater,providing real-time results,in different boroughs in New York City throughout 1 year.We developed and fully validated two analytical methods,one for 21 drugs and metabolites,including nicotine,cocaine,amphetamines,opioids and cannabis markers;and another for the normalization factor creatinine.Both methods were performed by liquid chromatography tandem mass spectrometry(LC-MS/MS)using positive electrospray ionization,achieving a limit of quantification of 5–10 ng/L for drugs and metabolites,and 0.01 mg/L for creatinine.These methods were applied to 48 one-time grab wastewater samples collected from six wastewater treatment plants in New York City(Manhattan,The Bronx,Queens and Brooklyn),eight different times throughout 2016,before and after major holidays,including Memorial Day,4th of July,Labour Day and New Year’s.In this study,the drug group normalized concentrations present in the wastewater samples,in decreasing order,were cocaine,nicotine,opioids,cannabis and amphetamines.When looking at individual compounds,the one with the highest normalized concentration was benzoylecgonine(BE),followed by cotinine,morphine and 11-nor-9-carboxy-tetrahydrocannabinol(THCCOOH).To estimate community use,these concentrations were multiplied by the corresponding correction factor,and the most present were THCCOOH,followed by BE,cotinine and morphine.When comparing the treatment plants by drug group(nicotine,cocaine,amphetamines,opioids and cannabis),samples collected from The Bronx had the highest normalized concentrations for nicotine,cocaine and opioids;The Bronx and Manhattan for cannabis;and Manhattan and Queens for amphetamines.In most of the cases,no effect due to holiday was observed.This study provides the first snapshot of drug use in New York City and how that changes between key calendar dates employing wastewater analysis.

Sex differences in the influence of social context, salient social stimulation and amphetamine on ultrasonic vocalizations in prairie voles

Prairie voles(Microtus ochrogaster)are a socially monogamous rodent species and their cooperative behaviors require extensive communication between conspecifics.Rodents use ultrasonic vocalizations(USVs)to com­municate and because a prairie vole breeder pair must engage in extensive cooperation for successful repro­duction,auditory communication may be critical for this species.Therefore,we sought to characterize USVs in adult male and female prairie voles,and to determine how these calls are influenced by social context,salient social stimuli and the psychostimulant drug of abuse amphetamine(AMPH).Here,we characterize prairie vole USVs by showing the range of frequencies of prairie vole USVs,the proportion of various call types,how these call types compare between males and females,and how they are influenced by social stimulation and AMPH.AMPH caused a robust increase in the number of USVs in both males and females and there was a dramatic sex difference in the complexity of call structures of AMPH-induced USVs,with males emitting more elaborate calls.Moreover,we show that novel(i.e.salient)social cues evoked differential increases in USVs across sex,with males showing a much more robust increase in USV production,both with respect to the frequency and complexity of USV production.Exposure to an estrous female in particular caused an extraordinary increase in USVs in male subjects.These data suggest that USVs may be a useful measure of social motivation in this spe­cies,including how social behaviors can be impacted by drugs of abuse.

Ultrasound-Assisted Low-Density Solvent Dispersive Liquid-Liquid Extraction for the Determination of Amphetamines in Biological Samples Using Gas Chromatography-Mass Spectrometry

In order to control drug crime effectively,it is necessary to develop selective analytical methods suitable for unambiguous identification and determination of drugs in illicit samples and biological matrices.A novel microextraction technique based on ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction,(UA-LDS-DLLME)has been applied to the determination of four amphetamines(methamphetamine,amphetamine,3,4-methylenedioxymethamphetamine,and 3,4-methylenedioxyamphetamine)in urine samples by gas chromatography-mass spectrometry.The parameters affecting extraction efficiency have been investigated and optimized.UA-LDS-DLLME used ultrasound energy to assist in the emulsification process without any disperser solvent.Under the optimized conditions,linearity was observed for all analytes in the 0.15-10 p,g/mL range with correlation coefficients(R)ranging from 0.9886 to 0.9894.The recoveries of75.6-91.4%with relative standard deviations of 2.5-4.0%were obtained.The limits of detection(S/N=3)were estimated to be in the 5-10 ng/mL range.The UA-LDS-DLLME technique had the advantages of shorter extraction time,suitability for simultaneous pretreatments of batches of samples,and the higher extraction efficiency.It was successfully applied to the analysis of amphetamines in real human urine samples.

A prospective longitudinal study shows putamen volume is associated with moderate amphetamine use and resultant cognitive impairments

Background:Amphetamine-type stimulants(ATS)have become a critical public health issue.Animal models have indicated a clear neurotoxic potential of ATSs.In humans,chronic use has been associated with cogni-tive deficits and structural brain abnormalities.However,cross-sectional retrospective designs in chronic users cannot truly determine the causal direction of the effects.Objective:To prospectively determine effects of occasional ATS use on cognitive functioning and brain structure.Methods:In a prospective longitudinal study design,cognitive functioning and brain structure were assessed at baseline and at 12-month follow-up in occasional ATS users(cumulative lifetime use<10 units at baseline).Results:Examination of change scores between the initial examination and follow-up revealed declined verbal memory performance and putamen volume in users with high relative to low interim ATS exposure.In the entire sample,interim ATS use,memory decline,and putamen volume reductions were strongly associated.Conclusions:The present findings support the hypothesis that ATS use is associated with deficient dorsal stri-atal morphology that might reflect alterations in dopaminergic pathways.More importantly,these findings strongly suggest that even occasional,low-dose ATS use disrupts striatal integrity and cognitive functioning.

Stroke recovery enhancing therapies:lessons from recent clinical trials

Poststroke recovery processes include restoration or compensation of function,respectively functions initially lost or new functions acquired after an injury.Therapeutic interventions can enhance these processes and/or reduce processes impeding regeneration.Numerous experimental studies suggest great opportunities for such treatments,but the results from recent large clinical trials using neuromodulators such as dopamine and fluoxetine are disappointing.The reasons for this are manifold affecting forward translation of results from animals models into the human situation.This"translational road block"is defined by differences between animals and humans with regard to the genetic and epigenetic background,size and anatomy of the brain,cerebral vascular anatomy,immune system,as well as clinical function and behavior.Backward blockade includes the incompatible adaption of targets and outcomes in clinical trials with regard to prior preclinical findings.For example,the design of clinical recovery trials varies widely and was characterized by the selection of different clinical endpoints,the inclusion a broad spectrum of stroke subtypes and clinical syndromes as well as different time windows for treatment initiation after infarct onset.This review will discuss these aspects based on the results of the recent stroke recovery trials with the goal to contribute to the currently biggest unmet need in stroke research-the development of a recovery enhancing therapy that improves the functional outcome of a chronic stroke patient.

GroupⅢmetabotropic glutamate receptors and drug addiction

Neuroadaptations of glutamatergic transmission in the limbic reward circuitry are linked to persistent drug addiction.Accumulating data have demonstrated roles of ionotropic glutamate receptors and groupⅠandⅡmetabotropic glutamate receptors(mGluRs)in this event.Emerging evidence also identifies Gαi/o-coupled groupⅢmGluRs(mGluR4/7/8 subtypes enriched in the limbic system)as direct substrates of drugs of abuse and active regulators of drug action.Auto-and heteroreceptors of mGluR4/7/8 reside predominantly on nerve terminals of glutamatergic corticostriatal and GABAergic striatopallidal pathways,respectively.These presynaptic receptors regulate basal and/or phasic release of respective transmitters to maintain basal ganglia homeostasis.In response to operant administration of common addictive drugs,such as psychostimulants(cocaine and amphetamine),alcohol and opiates,limbic groupⅢmGluRs undergo drastic adaptations to contribute to the enduring remodeling of excitatory synapses and to usually suppress drug seeking behavior.As a result,a loss-of-function mutation(knockout)of individual groupⅢreceptor subtypes often promotes drug seeking.This review summarizes the data from recent studies on three groupⅢreceptor subtypes(mGluR4/7/8)expressed in the basal ganglia and analyzes their roles in the regulation of dopamine and glutamate signaling in the striatum and their participation in the addictive properties of three major classes of drugs(psychostimulants,alcohol,and opiates).