MDPV Research Articles

Cyclodextrin-Enabled Enantioselective Complexation Study of Cathinone Analogs.

The characteristic alkaloid component of the leaves of the catnip shrub (Catha edulis) is cathinone, and its synthetic analogs form a major group of recreational drugs. Cathinone derivatives are chiral compounds. In the literature, several chiral methods using cyclodextrins (CDs) have been achieved so far for diverse sets of analogs; however, a comprehensive investigation of the stability of their CD complexes has not been performed yet. To characterize the enantioselective complex formation, a systematic experimental design was developed in which a total number of 40 neutral, positively, and negatively charged CD derivatives were screened by affinity capillary electrophoresis and compared according to their cavity size, substituent type, and location. The functional groups responsible for the favorable interactions were identified in the case of para-substituted cathinone analog mephedrone, flephedrone, and 4-methylethcathinone (4-MEC) and in the case of 3,4-methylendioxy derivative butylone and methylenedioxypyrovalerone (MDPV). The succinylated-β-CD and subetadex exhibited the highest complex stabilities among the studied drugs. The complex stoichiometry was determined using the Job's plot method, and the complex structures were further studied using ROESY NMR measurements. The results of our enantioselective complex formation study can facilitate chiral method development and may lead to evaluate potential CD-based antidotes for cathinone analogs.

Cocaine hydrochloride, cocaine methiodide and methylenedioxypyrovalerone (MDPV) cause distinct alterations in the structure and composition of the gut microbiota

Cocaine is a highly addictive psychostimulant drug of abuse that constitutes an ongoing public health threat. Emerging research is revealing that numerous peripheral effects of this drug may serve as conditioned stimuli for its central reinforcing properties. The gut microbiota is emerging as one of these peripheral sources of input to cocaine reward. The primary objective of the present study was to determine how cocaine HCl and methylenedioxypyrovalerone, both of which powerfully activate central reward pathways, alter the gut microbiota. Cocaine methiodide, a quaternary derivative of cocaine that does not enter the brain, was included to assess peripheral influences on the gut microbiota. Both cocaine congeners caused significant and similar alterations of the gut microbiota after a 10-day course of treatment. Contrary to expectations, the effects of cocaine HCl and MDPV on the gut microbiota were most dissimilar. Functional predictions of metabolic alterations caused by the treatment drugs reaffirmed that the cocaine congeners were similar whereas MDPV was most dissimilar from the other two drugs and controls. It appears that the monoamine transporters in the gut mediate the effects of the treatment drugs. The effects of the cocaine congeners and MDPV on the gut microbiome may form the basis of interoceptive cues that can influence their abuse properties.

Maximum Demand Reduction (MDRed) modelling using MATLAB Stateflow®

Based on renewable energy case studies, the MDRed (Maximum Demand Reduction) Model is created as an optimization apparatus for the solar PV-battery system. In this paper, MATLAB modeling toolbox is used for simulation of the solar PV-BESS design and verification of the MDRed modeling via variations in MD limit and solar PV load. Based on MDRed model, the maximum demand supervisory control algorithm is developed via MATLAB Stateflow® for the battery management system (BMS) to monitor and regulate the load to maintain the net load consumption below MD limit with the solar PV system. In summary, the simulation with the master MD controller shows the effectiveness of the proposed controller on the BMS to cater to MD reductions during solar irradiance and load pattern variations.

Cyanidin prevents MDPV withdrawal-induced anxiety-like effects and dysregulation of cytokine systems in rats

Psychostimulant exposure and withdrawal cause neuroimmune dysregulation and anxiety that contributes to dependence and relapse. Here, we tested the hypothesis that withdrawal from the synthetic cathinone MDPV (methylenedioxypyrovalerone) produces anxiety-like effects and enhanced levels of mesocorticolimbic cytokines that are inhibited by cyanidin, an anti-inflammatory flavonoid and nonselective blocker of IL-17A signaling. For comparison, we tested effects on glutamate transporter systems that are also dysregulated during psychostimulant free period. Rats injected for 9 d with MDPV (1 mg/kg, IP) or saline were pretreated daily with cyanidin (0.5 mg/kg, IP) or saline, followed by behavioral testing on the elevated zero maze (EZM) 72 h after the last MDPV injection. MDPV withdrawal caused a reduction in time spent on the open arm of the EZM that was prevented by cyanidin. Cyanidin itself did not affect locomotor activity or time spent on the open arm, or cause aversive or rewarding effects in place preference experiments. MDPV withdrawal caused enhancement of cytokine levels (IL-17A, IL-1β, IL-6, TNF=α, IL-10, and CCL2) in the ventral tegmental area, but not amygdala, nucleus accumbens, or prefrontal cortex, that was prevented by cyanidin. During MDPV withdrawal, mRNA levels of glutamate aspartate transporter (GLAST) and glutamate transporter subtype 1 (GLT-1) in the amygdala were also elevated but normalized by cyanidin treatment. These results show that MDPV withdrawal induced anxiety, and brain-region specific dysregulation of cytokine and glutamate systems, that are both prevented by cyanidin, thus identifying cyanidin for further investigation in the context of psychostimulant dependence and relapse.

Development and validation of a chiral LC-MS/MS method for the separation and quantification of four synthetic cathinones in human whole blood and its application in stability analysis

Synthetic cathinones, a subclass of new psychoactive substances, have gained high popularity on the recreational drugs market over the past years. These drugs typically have a chiral center, so they may exist as two stereoisomers. Therefore the pharmacological, pharmacokinetic or metabolic properties of their enantiomers are expected to differ. However, these drugs are often synthesized and sold as a racemic mixture, and as a consequence, differentiation of their (R)- and (S)- enantiomers is relevant in clinical and forensic toxicology. Information about single enantiomers of synthetic cathinones is relatively scarce due to challenges of their chiral analysis. Hence, a sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed and validated for the chiral separation and quantification of four synthetic cathinones in human whole blood samples. The method was fully validated in terms of linearity, limit of detection, limit of quantification, bias, precision, carryover, interferences, matrix effects, recovery and processed sample stability and successfully applied to evaluate the stability as well as enantioselective degradation of synthetic cathinones enantiomers under various storage conditions. For most of the analytes, significant enantioselective degradation was observed when stored at room temperature or refrigerated, with the E2-enantiomers observed to more rapidly degrade under both conditions. This is the first report concerning the stability and enantioselective degradation of synthetic cathinone enantiomers in whole blood. Moreover, the inversion study demonstrated enantiomeric inversion of R-(−)- and S-(+)-methylenedioxypyrovalerone (MDPV) in human whole blood and methanolic solution.

Effects of synthetic cathinone naphyrone in the conditioned place preference test – Evidence of its addictive potential

Naphyrone, also known as NRG-1, is a novel psychoactive substance (NPS), a cathinone with stimulatory properties available on the grey/illicit drug market for almost a decade. It is structurally related to infamously known powerful stimulants with the pyrovalerone structure, such as alpha-pyrrolidinovalerophenone (α-PVP) or methylenedioxypyrovalerone (MDPV) that are labeled as a cheap replacement for cocaine and other stimulants. Despite the known addictive potential of α-PVP and MDPV, there are no studies directly evaluating naphyrone’s addictive potential e.g., in conditioned place preference (CPP) test or using self-administration. Therefore, our study was designed to evaluate the addictive potential in a CPP test in male Wistar rats and compare its effect to another powerful stimulant with a high addictive potential – methamphetamine. Naphyrone increased time spent in the drug-paired compartment with 5 and 20 mg/kg s.c. being significant and 10 mg/kg s.c. reaching the threshold (p = 0.07); the effect was comparable to that of methamphetamine 1.5 mg/kg s.c. The lowest dose, naphyrone 1 mg/kg s.c., had no effect on CPP. Interestingly, no dose response effect was detected. Based on these data, we are able to conclude that naphyrone has an addictive potential and may possess a significant risk to users.

Reinforcing Effects of the Synthetic Cathinone α-Pyrrolidinopropiophenone (α-PPP) in a Repeated Extended Access Binge Paradigm.

Synthetic cathinones are designer psychostimulants that are derivatives of the natural alkaloid cathinone, and produce effects similar to more traditional illicit stimulants such as cocaine and methamphetamine. The pyrovalerone cathinones methylenedioxypyrovalerone (MDPV) and α-pyrrolidinopropiophenone (α-PPP) exert their effects via inhibition of presynaptic dopamine and norepinephrine reuptake transporters. While the reinforcing effects of MDPV in rodents are well-established, very few studies have examined self-administration patterns of α-PPP. Users of synthetic cathinones often engage in repeated binge episodes of drug intake that last several days. We therefore sought to determine the reinforcing effects of three doses of α-PPP (0.05, 0.1 and 0.32 mg/kg/infusion) under conditions of prolonged binge-like access conditions, with three 96-h periods of drug access interspersed with 72 h of abstinence. MDPV (0.05 mg/kg/infusion) was used as a comparison drug. Our results show that both MDPV and the high (0.32 mg/kg/infusion) dose of α-PPP are readily self-administered at high levels across all three extended access periods, whereas lower doses of α-PPP produce variable and less robust levels of self-administration. These results indicate that higher doses of α-PPP have reinforcing effects under conditions of extended access, suggesting the potential for abuse and a need for consideration in drug control policies.

Determination of Selected Cathinones in Blood by Solid-Phase Extraction and GC-MS.

The emergence of potentially dangerous new psychoactive substances (NPS) is challenging for forensic laboratories, as well as the ability to develop and validate methods for a rapid and unambiguous monitoring of these compounds. Thereupon, the aim of this work was to establish a methodology for the identification and quantification of four synthetic cathinones already seized in Portugal [4-chloroethcathinone (4-CEC), α-pyrrolidinovalerophenone (α-PVP), 4-chloro-pyrrolidinovalerophenone (4-Cl-PVP) and methylenedioxypyrovalerone (MDPV)] in whole blood samples, using gas chromatography coupled to mass spectrometry (GC-MS). The analytes were extracted from blood by solid-phase extraction (SPE) and derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 5% trimethylchlorosilane (TMCS). For all analytes, linearity was observed from 25 (lower limit of quantification, LLOQ) to 800ng/mL, with coefficients of determination higher than 0.99. The limits of detection (LOD) were 5ng/mL for α-PVP, 4-Cl-PVP and MDPV and 25ng/mL for 4-CEC. The method was selective, precise and accurate, and the extraction efficiency was higher than 85% for all analytes. The target cathinones were stable under different stock conditions, being MDPV the most stable and 4-CEC the least stable compound. The validated analytical method was then applied to real samples that previously tested positive for amphetamines, but no positive samples were found for the cathinones under study. The present method describes for the first time the quantification of 4-CEC and 4-Cl-PVP in whole blood samples by GC-MS, allowing their accurate determination in forensic situations where the compounds are involved.

Detrimental effects of the 'bath salt' methylenedioxypyrovalerone on social play behavior in male rats.

Methylenedioxypyrovalerone (MDPV) is the most popular synthetic cathinone found in products marketed as 'bath salts', widely abused among teenagers and young adults. Synthetic cathinones have pharmacological effects resembling those of psychostimulants, which are known to disrupt a variety of social behaviors. However, despite the popular use of MDPV by young people in social contexts, information about its effects on social behavior is scarce. To investigate the impact of MDPV on social behavior at young age, and the underlying neurobehavioral mechanisms, we focused on social play behavior. Social play behavior is the most characteristic social behavior displayed by young mammals and it is crucial for neurobehavioral development. Treatment with MDPV reduced social play behavior in both juvenile and young adult male rats, and its play-suppressant effect was subject to tolerance but not sensitization. As the behavioral effects of MDPV have been ascribed to dopaminergic and noradrenergic neurotransmission, and given the role of these neurotransmitters in social play, we investigated the involvement of dopamine and noradrenaline in the play-suppressant effects of MDPV. The effects of MDPV on social play were blocked by either the α2 adrenoceptor antagonist RX821002 or the dopamine receptor antagonist flupenthixol, given alone or together at sub-effective doses. In sum, MDPV selectively suppresses the most vigorous social behavior of developing rats through both noradrenergic and dopaminergic mechanisms. This study provides important preclinical evidence of the deleterious effects of MDPV on social behavior, and as such increases our understanding of the neurobehavioral effects of this popular cathinone.

3,4-Methylenedioxypyrovalerone: Neuropharmacological Impact of a Designer Stimulant of Abuse on Monoamine Transporters.

Methylenedioxypyrovalerone (MDPV) is an abused synthetic cathinone, commonly referred to as a "bath salt." Because the dopamine (DA) transporter (DAT) and vesicular monoamine transporter-2 (VMAT-2) are key regulators of both the abuse and neurotoxic potential of structurally and behaviorally related agents, the impact of MDPV on these transporters was investigated. Results revealed that a single in vivo MDPV administration rapidly (within 1 hour) and reversibly increased both rat striatal DAT and VMAT-2 activity, as assessed via [3H]DA uptake in synaptosomes and synaptic vesicles, respectively, prepared from treated rats. There was no evidence of an MDPV-induced increase in plasmalemmal membrane DAT surface expression. Plasma concentrations of MDPV increased dose-dependently as assessed 1 hour after 2.5 and 5.0 mg/kg (s.c.) administration and returned to levels less than 10 ng/ml by 18 hours after 2.5 mg/kg (s.c.). Neither pretreatment with a D1 receptor (SCH23390), a D2 receptor (eticlopride), nor a nicotinic receptor (mecamylamine) antagonist attenuated the MDPV-induced increase in DAT activity. In contrast, eticlopride pretreatment attenuated both the MDPV-induced increase in VMAT-2-mediated DA uptake and an associated increase in cytoplasmic-associated vesicle VMAT-2 immunoreactivity. SCH23390 did not attenuate the MDPV-induced increase in VMAT-2 activity. Repeated MDPV injections did not cause persistent DAergic deficits, as assessed 7 to 8 days later. The impact of MDPV on striatal and hippocampal serotonergic assessments was minimal. Taken together, these data contribute to a growing pharmacological rubric for evaluating the ever-growing list of designer cathinone-related stimulants. The profile of MDPV compared with related psychostimulants is discussed. SIGNIFICANCE STATEMENT: Pharmacological characterization of the synthetic cathinone, 3,4-methylenedioxypyrovalerone (MDPV; commonly referred to as a "bath salt"), is critical for understanding the abuse liability and neurotoxic potential of this and related agents. Accordingly, the impact of MDPV on monoaminergic neurons is described and compared with that of related psychostimulants.

Structure Activity Relationship Studies of Psychostimulant Synthetic Cathinones Reveal Nanomolar Antagonist Potency of α‐Pyrrolidinohexiophenone (α‐PHP) at Human Muscarinic M2Receptors

Synthetic cathinones (SCs), psychostimulant drugs of abuse, are β‐keto analogs of amphetamine that primarily target monoamine transporters as inhibitors or substrates. Certain pyrrolidine‐containing SCs, such as α‐Pyrrolidinohexiophenone (α‐PHP) and methylenedioxypyrovalerone (MDPV), are potent monoamine transporter inhibitors that appear to produce distinct toxic, autonomic—e.g., tachycardia and arrythmia—and neuropsychiatric—e.g., delirium and cognitive impairments—adverse events compared to N‐alkylated SCs and amphetamines, which are monoamine transporter substrates. Although the apparent differences in toxicity could be caused by unique pharmacodynamic effects at monoamine transporters, we hypothesized there is another mechanism. We focused on muscarinic receptors (MRs), as numerous MR antagonists are well‐characterized deliriants that also cause tachycardia. We conducted pharmacological assays assessing human M1, M2, M3, M4, and M5R activity of 10 pyrrolidine‐containing SCs, systematically evaluating the effect of the α‐carbon chain length of methylenedioxy‐containing and unsubstituted α‐propiophenone analogues. [3H]Scopolamine competition binding assays revealed that increasing the α‐carbon chain length of unsubstituted, but not methylenedioxy derivatives led to a stepwise enhancement in affinity (Ki) at all MR subtypes, with highest affinities typically observed at M2Rs. Relative to all other SCs tested, α‐PHP had the highest affinity at M2Rs, with a calculated pKi of 6.28 (Ki= 520 nM). We next assessed the M2R functional effects of α‐PHP using M2R—cAMP and M2R—β‐arrestin recruitment assays; α‐PHP behaved as an M2R competitive antagonist in both assays. α‐PHP antagonized oxotremorine‐elicited cAMP inhibition with an apparent pKb of 7.29 (Kb= 50 nM) and antagonized oxotremorine‐elicited β‐arrestin recruitment with an apparent pKb of 6.22 (Kb= 602 nM). Toxicology studies report that serum concentrations of commonly abused pyrrolidine‐containing SCs, including α‐PHP, can range from 713 to 2,619 nM. Thus, α‐PHP’s potency at M2Rs is physiologically relevant and likely contributes to its toxicity. Given the dominant function of M2Rs to slow heart rate, α‐PHP may cause tachycardia, in part, by inhibition of cardiac M2Rs. This inhibitory effect on the parasympathetic nervous system, in combination with sympathomimetic effects caused by monoamine transporter inhibition could explain the serious adverse cardiovascular events associated with certain pyrrolidine‐containing SCs.Support or Funding InformationFunding: NIDA R21DA040907

Synthetic psychoactive cathinones: hypothermia and reduced lethality compared to methamphetamine and methylenedioxymethamphetamine

RationaleSynthetic psychoactive cathinones (SPCs) are drugs with psychostimulant and entactogenic properties like methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA). Despite clinical reports of human overdose, it remains to be determined if SPCs have greater propensity for adverse effects than MA or MDMA. ObjectivesTo determine whether the SPCs cathinone (CAT), methcathinone (MCAT), mephedrone (MMC), and methylenedioxypyrovalerone (MDPV) have lower LD50 values than MA or MDMA. MethodsMale and female C57Bl/6J mice received single injections of one of 6 doses of a test drug (0–160 mg/kg IP). Temperature and behavioral observations were taken every 20 min for 2 h followed by euthanasia of surviving mice. Organs were weighed and evaluated for histopathological changes. ResultsLD50 values for MA and MDMA, 84.5 and 100.9 mg/kg respectively, were similar to previous observations. The LD50 for MMC was 118.8 mg/kg, but limited lethality was observed for other SPCs (CAT, MCAT, MDPV), so LD50 values could not be calculated. For all drugs, death was associated with seizure, when it was observed. Rather than hyperthermia, dose-dependent hypothermia was observed for MMC, MDPV, CAT, and MCAT. Contrary to initial expectations, none of the SPCs studied here had LD50 values lower than MA or MDMA. ConclusionsThese data indicate that, under the conditions studied here: (1) SPCs exhibit less lethality than MA and MDMA; (2) SPCs impair thermoregulation; (3) effects of SPCs on temperature appear to be independent of effects on lethality.

The effects of acute and repeated methylenedioxypyrovalerone (MDPV) administration on striatal transcriptome networks in male long evans rats

The psychoactive drug methylenedioxypyrovalerone (MDPV) elicits feelings of euphoria and hyperexcitability, but can also result in paranoia, agitation, and depression by unknown mechanisms. We identified molecular networks in the rat striatum that were affected by single or repeated exposure to MDPV. Male Long Evans rats were injected with either saline or MDPV (1 mg/kg) (single or repeated MDPV) over 5 days. To distinguish the effects of repeated MDPV from a single exposure, an additional group received saline over 4 days and then MDPV on the 5th day. Twenty-four hours after the final injection, the left dorsal striatum was processed for transcriptomics. The transcriptome response was subtle after 24 h, and a single gene passed an FDR correction (LOC103691845) following repeated MDPV treatment. Gene set and subnetwork enrichment analyses were conducted to improve data interpretation from a network perspective. Consistent with the mode of action of MDPV, networks related to the nigrostriatal dopaminergic system were altered in the rat striatum. Transcriptional networks related to cognition, short and long-term memory, and synaptic transmission were over-represented in the striatum of rats repeatedly injected with MDPV. This study identifies potential transcriptional networks altered by single or repeated MDPV exposure, which can be interrogated further to elucidate molecular mechanisms underlying cathinone abuse.

Label-Free, Visual Detection of Small Molecules Using Highly Target-Responsive Multimodule Split Aptamer Constructs.

Colorimetric aptamer-based sensors offer a simple means of on-site or point-of-care analyte detection. However, these sensors are largely incapable of achieving naked-eye detection, because of the poor performance of the target-recognition and signal-reporting elements employed. To address this problem, we report a generalizable strategy for engineering novel multimodule split DNA constructs termed "CBSAzymes" that utilize a cooperative binding split aptamer (CBSA) as a highly target-responsive bioreceptor and a new, highly active split DNAzyme as an efficient signal reporter. CBSAzymes consist of two fragments that remain separate in the absence of target, but effectively assemble in the presence of the target to form a complex that catalyzes the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid, developing a dark green color within 5 min. Such assay enables rapid, sensitive, and visual detection of small molecules, which has not been achieved with any previously reported split-aptamer-DNAzyme conjugates. In an initial demonstration, we generate a cocaine-binding CBSAzyme that enables naked-eye detection of cocaine at concentrations as low as 10 μM. Notably, CBSAzyme engineering is straightforward and generalizable. We demonstrate this by developing a methylenedioxypyrovalerone (MDPV)-binding CBSAzyme for visual detection of MDPV and 10 other synthetic cathinones at low micromolar concentrations, even in biological samples. Given that CBSAzyme-based assays are simple, label-free, rapid, robust, and instrument-free, we believe that such assays should be readily applicable for on-site visual detection of various important small molecules such as illicit drugs, medical biomarkers, and toxins in various sample matrices.

LC-MS/MS analysis of 16 synthetic cathinones in hair: Application to postmortem cases

This study aimed the development and validation of a new LC-MS/MS method for detection and quantification of 16 synthetic cathinones (known also as “bath salts”) in human hair and its application to postmortem cases. About 20 mg of hair samples, previously washed and homogenized, were ultrasonicated with 1 mL HCl 0.1M solution. Samples were then extracted using a solid phase extraction procedure (SPE), taken to dryness and reconstituted in 100 μL mobile phase. Finally, they were directly injected into a liquid chromatographic system, coupled with tandem mass spectrometer detector. The method was applied to all the postmortem cases, within the period January 2017–August 2018, involving young people (< 35 years old), where at least one traditional drug of abuse has been detected in biological specimens ( n = 17). The validation criteria parameters were satisfactory according with the international guidelines. A LOQ of 5 pg/mg was obtained for 4-fluoromethcathinone (4-FMC), buphedrone, ethcathinone, methcathinone, mephedrone and naphyrone, while the method proved to be more sensitive for 4-methylethcathinone (4-MEC), methedrone, alpha-pyrrolidinopentiophenone (α-PVP), alpha-pyrrolidinohexiophenone (α-PHP), methylenedioxypyrovalerone (MDPV), butylone, ethylone, 3,4-dimethylmethcathinone (3,4-DMMC), pentedrone and pentylone, reaching a LOQ of 1 pg/mg. Two samples out of 17 cases analyzed provided positive results for synthetic cathinones. One sample has been divided in two segments of 2.5 cm length each. Both segments were positive for 8 different cathinone derivatives, namely: 3,4-DMMC, 4-FMC, 4-MEC, ɑ-PHP, ɑ-PVP, methcathinone, methedrone and pentedrone. The second case provided positive results for ethcathinone. Concentrations are listed in Table 1 . The developed LC-MS/MS method allowed to detect and quantitate 16 different synthetic cathinones in human hair. Nine different synthetic cathinones were detected in two different postmortem cases. The analytical procedure can be easily applied to a limited amount of sample (less than 20 mg hair).

Salting out assisted liquid-liquid extraction for liquid chromatography tandem-mass spectrometry determination of amphetamine-like stimulants in meconium

In the last decade there has been a dramatic increase in the availability and abuse of synthetic cathinones – new amphetamine-like stimulants. Even though their abuse during pregnancy could have serious adverse effects on the fetus, cathinones are not readily included in neonatal toxicological screenings. Meconium (first neonatal stool) is the specimen of choice to reveal long term drug exposure, however as it is a highly complex matrix, the sample preparation is a critical step before the instrumental analysis.The aim of this work was to develop a suitable meconium sample extraction technique using the advantages of salting-out assisted liquid-liquid extraction (SALLE) and using only MS-friendly organic ammonium salts. We further developed and validated liquid chromatography tandem-mass spectrometry method for the determination of ‘traditional’ stimulants (methamphetamine, amphetamine, MDMA) and cathinones (mephedrone, methylenedioxypyrovalerone (MDPV), α-pyrrolidinopentiophenone (α-PVP), methylone, butylone, flephedrone, and naphyrone).Matrix-matched calibration was prepared in the concentration range 10–2000 ng/g. The limits of quantification were determined as 10 ng/g, recoveries ranged from 48.2% to 94.3% and the matrix effect was between 60.2% and 101.4%. Accuracy (86.1–114.5%) and precision (4.9–14.9%) were determined and all validation criteria were met for all analytes except for naphyrone. Finally, our analytical method was tested on a set of real meconium samples, which were found positive for amphetamine, methamphetamine and methylone, thus demonstrating the validity of the method.

Acute Administration of 3,4‐Methylenedioxypyrovalerone (MDPV) Increases Motor Impulsivity in Rats

Synthetic cathinones (SC) are one of the largest categories of emerging novel psychoactive substances, and are typically abused as ‘bath salts’ preparations. 3,4‐ methylenedioxypyrovalerone (MDPV) is a common SC frequently mentioned in both clinical case reports and calls to poison control centers, and has been implicated in increased risky behaviors. Impulsivity is a major factor in risky behaviors, and psychostimulants like cocaine may modulate baseline impulsivity through actions on dopaminergic systems. Like cocaine, the psychoactive effects of MDPV are elicited by selective and potent reuptake inhibition at catecholamine transporters, and studies from our lab have indicated that long‐term administration of MDPV increases impulsive choice in rats. The behavioral construct of impulsivity comprises multiple components, including impulsive choice and motor impulsivity. In the current study, we characterized the effects of acute MDPV on motor impulsivity using a differential reinforcement of low rate of responding (DRL 20 sec) task. Several endpoints were quantified, including number of reinforcers earned (SR), total number of premature responses (PRE), response efficiency (RE, # of reinforcers earned /total premature and reinforced responses), and perseverative responses (PRSV, # of responses following premature responses or reinforced nose pokes until the next available signaled reinforcer). Eighteen drug‐naïve, adult male Sprague‐ Dawley rats were split into three groups of six: a saline negative control (SAL), a cocaine positive control (COC), and an MDPV test group (MDPV). Animals were matched based on RE for the final five days of training (days 45–50), yielding approximately the same RE for each group (MDPV = 20.22%, COC = 19.64%, and SAL = 21.71%). Following stabilization, a satiety challenge was implemented in which animals were fed their daily allotment of food 1 hour prior to the start of their scheduled session. Satiation increased both RE and SR in all groups, while decreasing PRE and PRSV, indicating decreases in motor impulsivity. After a 5 day restabilization, each animal received an acute saline injection, followed by daily injections of either subcutaneous (SQ) saline (SAL group), 1.0 – 30.0 mg/kg intraperitoneal cocaine (COC group), or 0.1 – 3.0mg/kg SQ MDPV (MDPV group). Acute saline injections had no effect on any measure in any group, and further injections of acute saline in the SAL group had no effects on any DRL endpoints. However, escalating acute injections of both cocaine and MDPV dose dependently increased motor impulsivity. At the highest doses tested, RE was reduced to 8.36% in COC animals and to 0.81% in MDPV rats, though operant behavior was completely suppressed in 2 of the 6 MDPV animals. Moreover, the number of PRE and PRSV were increased in both COC and MDPV groups, though MDPV was more potent and effective than cocaine in this regard. Further experiments investigating the effects of long‐term MDPV administration on motor impulsivity are currently underway.Support or Funding InformationThese studies were supported in part by DA039195 and T32DA022981.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Two‐Parameter Exponential‐Demand Formula with a Behavioral Momentum Normalization: A Novel Method to Characterize the Reinforcing Effectiveness of Drugs

Application of behavioral economic principles to the characterization of drugs of abuse has yielded compelling evidence for a relationship between price (P) and consumption (Q). In turn, a quantitative assessment of this relationship provides a metric for comparing the reinforcing effectiveness (α) of various drugs, and demand analysis may provide a screening mechanism to evaluate candidate medications for the management of substance abuse. However, discordance between the α values for some drugs in nonhuman subjects and their reinforcing effectiveness in humans (e.g., high α avalues and high rates of abuse of nicotine) suggests that previous demand equations may require further refinement. In the present work, demand equations were derived to try to more accurately reflect the contribution of behavioral momentum‐ evident in resistance to vehicle extinction‐ to the calculation of reinforcing effectiveness. Resistance to extinction conceivably may influence demand curve elasticity, especially at higher prices. Here, demand curves were derived from intravenous self‐administration data obtained with four psychomotor stimulant drugs using a logarithmic model modified from the Hursh‐Silberberg (2008) exponential equation with a two‐parameter fitting algorithm for consumption versus price. The new algorithm reduces the three free parameter model to a two free parameter model by interpolating data at the minimum fixed ratio (FR) and at the breakpoint pB (i.e., the FR where Q=0). The augmented model takes the form: urn:x-wiley:08926638:media:fsb2fasebj2019331supplement80512:fsb2fasebj2019331supplement80512-math-0001where QFRLdenotes consumption at the lowest evaluated FR and pB denotes the breakpoint price. A strategy was tested for developing a single parameter classification scheme for fitted models using the parameter α as a measure of elasticity of the demand curves. The strategy normalizes behavioral momentum as measured by data for vehicle self‐administration. More specifically, if Q(P) denotes the demand curve for a particular drug, the discounted demand curve, Qd(P), is defined by: urn:x-wiley:08926638:media:fsb2fasebj2019331supplement80512:fsb2fasebj2019331supplement80512-math-0002where Qs(P) denotes the demand curve generated by vehicle. To test this approach, two doses of cocaine, 3,4‐methylenedioxymethamphetamine (MDMA), methylenedioxypyrovalerone (MDPV), and methcathinone (MCAT) were examined in rhesus monkeys (N=4). Using the Hursh‐Silberberg (2008) exponential formula, a rank‐order of reinforcing effectiveness of cocaine&gt;MDPV&gt;MCAT&gt;&gt;MDMA was established, and reinforcing effectiveness did not vary as a function of dose. However, in the Two‐Parameter Exponential‐Demand formula, a rank‐order of cocaine (large dose)&gt;MDMA=MCAT&gt;MDPV (large dose)&gt;MDPV (small dose)&gt;cocaine (small dose) was established. These results suggest that behavioral momentum may influence demand elasticity in a dose‐dependent manner, and α values derived in the augmented model may differentiate drug effects that contribute to price‐consumption relationships in a pharmacologically sensitive manner.Support or Funding InformationDA002519, DA039306, and HMS‐ Livingston AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A multi-analyte LC–MS/MS method for screening and quantification of 16 synthetic cathinones in hair: Application to postmortem cases

A multi-analyte method for detection and quantification of 16 synthetic cathinones (known also as “bath salts”) in human hair has been developed and fully validated using liquid chromatography–tandem mass spectrometry system.About 20 mg of hair samples, previously washed and homogenized, were ultrasonicated with 1 mL HCl 0.1 M solution. Samples were then extracted using a solid phase extraction procedure (SPE), taken to dryness and reconstituted in 100 μL mobile phase. Finally, they were directly injected into a liquid chromatographic system, coupled with tandem mass spectrometer detector.The validation criteria parameters were satisfactory according with the international guidelines. A LOQ of 5 pg/mg was obtained for 4-fluoromethcathinone (4-FMC), buphedrone, ethcathinone, methcathinone, mephedrone and naphyrone, while the method proved to be more sensitive for 4-methylethcathinone (4-MEC), methedrone, alpha-pyrrolidinopentiophenone (α-PVP), alpha-pyrrolidinohexiophenone (α-PHP), methylenedioxypyrovalerone (MDPV), butylone, ethylone, 3,4-dimethylmethcathinone (3,4—DMMC), pentedrone and pentylone, reaching a LOQ of 1 pg/mg.Potential use of bath salts was investigated in postmortem cases of young subjects previously tested positive at least to one traditional drug of abuse. Two samples out of 17 cases analyzed provided positive results for synthetic cathinones. One sample has been divided in two segments of 2.5 cm length each. Both segments were positive for 8 different cathinone derivatives, namely: 3,4-DMMC, 4-FMC, 4-MEC, α-PHP, α-PVP, methcathinone, methedrone and pentedrone. The second case provided positive results for ethcathinone.

LC-High-resolution MS Analysis of the Designer Drugs Methylenedioxy-Pyrovalerone (MDPV) and Methylenedioxy-alpha- pyrolidinobutyrophenone (MDPBP) and Their Main Metabolites in Human Urine

In the present work, LC- Q ExactiveOrbitrap high resolution MS techique in both full-scan MS and targeted MS/MS modes was applied to identify psychoactive substances in human urine samples. Methylenedioxy-pyrovalerone (MDPV), methylenedioxy-alpha-pyrrolidino-butyrophenone(MDPBP) and the main metabolites of both compounds were identified. The preliminary full-scan screening was folowed by targeted ion fragmentation (t-MS2) enabling confirmatory analysis and mass spectral characterization. The presented case is the first confirmation of MDPBP abuse in Romania supported by the toxicological identification of the parent compound and the main metabolites in human urine.