N-ethylpentylone Research Articles

Sex differences in the effects of N-ethylpentylone in young CD1 mice: Insights on behaviour, thermoregulation and early gene expression.

New psychoactive substances such as N-ethylpentylone (NEP) are continuously emerging in the illicit drug market, and knowledge of their effects and risks, which may vary between sexes, is scarce. Our present study compares some key effects of NEP in male and female mice. Psychostimulant, rewarding and reinforcing effects were investigated by tracking locomotor activity, conditioned place preference (CPP) paradigm and through a self-administration (SA) procedure, respectively, in CD1 mice. Moreover, the expression of early genes (C-fos, Arc, Csnk1e, Pdyn, Pp1r1b and Bdnf in addiction-related brain areas) was assessed by qPCR. Finally, serum and brain levels of NEP were determined by UHPLC-MS/MS. NEP-treated males experimented locomotor sensitisation and showed higher and longer increases in locomotion as well as higher hyperthermia after repeated administration than females. Moreover, while preference score in the CPP was similar in both sexes, extinction occurred later, and reinstatement was more easily established for males. Female mice self-administered more NEP than males at a higher dose. Differences in early gene expression (Arc, Bdnf, Csnk1e and Ppp1r1b) were found, but the serum and brain NEP levels did not differ between sexes. Our results suggest that male mice are more sensitive to NEP psychostimulant and rewarding effects. These differences may be attributed to different early gene expression but not to pharmacokinetic factors. Moreover, males appear to be more vulnerable to the hyperthermic effects of NEP, while females might be more prone to NEP abuse.

Comparative Assessment of the Addictive Potential of Synthetic Cathinones by Zebrafish Conditioned Place Preference (CPP) Paradigm.

Synthetic cathinones have gained increasing popularity in the illicit drug market, yet their abuse potential remains poorly understood. In this study, zebrafish were used to compare the addictive potential of three cathinone analogs, namely pentylone, eutylone, and N-ethylpentylone (NEP). The zebrafish received various doses (0 to 60 mg/kg) of the cathinone analogs by oral gavage over two sessions per day for two consecutive days to induce conditioned place preference (CPP). Pentylone, eutylone, and NEP dose-dependently induced CPP, with NEP showing significantly higher CPP than pentylone and eutylone at the dose of 20 mg/kg. The fish that received 60 mg/kg of cathinones underwent extinction, followed by reinstatement triggered by drug priming. NEP required six sessions to meet the criteria of extinction, followed by eutylone, which required four sessions, and pentylone, which required three sessions. Furthermore, NEP and eutylone at a dose of 40 mg/kg could reinstate the extinguished CPP, while 60 mg/kg of pentylone was necessary for CPP reinstatement. The persistence of susceptibility to reinstatement was also assessed at 7 and 14 days after the initial reinstatement. The CPP induced by all three cathinone analogs could be reinstated 7 days after the initial reinstatement, whereas only CPP induced by NEP, but not pentylone and eutylone, could be reinstated again after 14 days. Considering the potency to induce CPP, resistance to extinction, and the propensity for reinstatement, the abuse liability rank order of the cathinone analogs might be as follows: NEP > eutylone > pentylone. These findings suggest that the zebrafish CPP paradigm can serve as a viable model for assessing the relative abuse liability of substances.

A tailored ratiometric fluorescent sensor based on CdTe and MgF2 quantum dots for trace N-ethylpentylone detection.

The development of low-cost and highly sensitive ratiometric fluorescence sensor, CdTe@MIPs/MgF2, for N-Ethylpentylone (NEP) detection in wastewater samples is described. In this system, CdTe@MIPs (λex = 370, λem = 570) are employed as the receptor and response unit for NEP, with MgF2 (λex = 370, λem = 470) as the reference signal to enhance stability. Under optimal conditions, the sensor shows fluorescent quenching response at 570 nm to NEP in linear range of 2-200nM, with LOD of 0.6nM. Thesensor also demonstrates significant selectivity for NEP over other analogues and interferents, making it ideal for practical applications in wastewater analysis. This approach is potentially more cost-effective and sensitive than conventional mass spectrometry in detecting abused substances in sewage. Additionally, the MgF2 fluorescent nano-material was first-ever developed and investigated, which may be significant in future research.

Use of a lab-made screen-printed sensor with chemically deposited boron-doped diamond for simple and selective electrochemical detection of the synthetic cathinone N-ethylpentylone in forensic samples

N-ethylpentylone (NEP), also known as ephylone, is a synthetic cathinone (SC) that has been banned worldwide since 2019 due to alarming cases of intoxication. However, preliminary identification of this drug is of interest in the forensic scenario. Here, a new forensic application using a lab-made screen-printed sensor with chemically deposited boron-doped diamond electrode (LM-SP/BDDE) for the detection of SCs is presented. We present, for the first time, the electrochemical behavior of NEP and its detection in forensic samples using a versatile, robust, low-cost, and simple sensor that requires a small sample volume (30 µL) for the analysis. NEP presented three irreversible and independent redox processes (O1 at +0.88 V, R1 at –1.3 V and R2 at –1.45 V), and a dependent reduction R3 at –1.6 V vs. Ag/AgCl. The proposed method for NEP detection on LM-SP/BDDE was optimized by adsorptive stripping differential pulse voltammetry (AdSDPV). The use of LM-SP/BDDE with AdSDPV provided a good stability of NEP electrochemical responses using the same electrode (n = 5), with RSD < 4.0% for all redox processes. Furthermore, wide linear ranges for NEP determination (1.0 – 100.0 µmol L − 1) with a low LOD (0.66 µmol L − 1) were obtained. Interference studies have been performed for common adulterants and other illicit drugs (semi-synthetics, amphetamines, and other SCs), and a highly selective response for NEP detection was observed. Therefore, LM-SP/BDDE combined with AdSDPV is an alternative tool for selective screening of NEP in on-site forensic analysis, providing rapid and simple preliminary identification.

N, N-Dimethylpentylone (Dipentylone)-A New Synthetic Cathinone Identified in a Postmortem Forensic Toxicology Case Series.

Synthetic cathinones emerged on the novel psychoactive substance (NPS) drug market as alternatives to controlled stimulants and entactogens such as methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA). The majority of synthetic cathinones can be subclassified into two groups: beta-keto amphetamines (i.e., NPS with the suffix "drone") and beta-keto methylenedioxyamphetamines (i.e., NPS with the suffix "lone"). Although a significant number of beta-keto amphetamines have been identified, beta-keto methylenedioxyamphetamines have dominated the NPS market, including notable drugs like methylone, butylone, N-ethyl pentylone (ephylone), eutylone, and now N, N-dimethylpentylone. N, N-Dimethylpentylone, also known as dipentylone or bk-DMBDP, emerged into the illicit drug supply within two months of the international control of eutylone (September 2021). A novel standard addition method was developed and validated for N, N-dimethylpentylone, pentylone, and eutylone, and 18 postmortem cases were quantitated using the method described in this manuscript. The resulting blood concentration range for N, N-dimethylpentylone in this case series was 3.3 to 970 ng/mL (median: 145 ng/mL, mean: 277 ± 283 ng/mL). Pentylone, a metabolite of N, N-dimethylpentylone, was detected in all cases (range: 1.3-420 ng/mL, median: 31 ng/mL, and mean: 88 ± 127 ng/mL). Due to the rise in identifications of N, N-dimethylpentylone in postmortem investigations as well as the potential misidentification of N, N-dimethylpentylone as N-ethyl pentylone, samples testing positive for pentylone should be additionally confirmed for the presence of N, N-dimethylpentylone. Based on prior trends of new synthetic cathinones, it can be theorized that N, N-dimethylpentylone may predominate the U.S. synthetic stimulant market for the next one to two years, however; given the emergence of additional closely related isomeric compounds, it is important to utilize methodology capable of differentiating N, N-dimethylpentylone from its isomers (N-isopropylbutylone, N-ethyl pentylone, N-ethyl N-methyl butylone, hexylone, N-propylbutylone, diethylone, and tertylone).

Fatal Intoxication with a mixture of para-methoxyamphetamine, N-ethylpentylone, and ketamine

New psychoactive substances (NPS) have had a serious impact worldwide, and their determination in postmortem samples has been challenging. Here, we present a fatal case of poisoning involving the combined ingestion of para-methoxyamphetamine (PMA), N-ethylpentylone (NEP), and ketamine, and quantitative analysis of these drugs and para-hydroxyamphetamine (PHA), a primary metabolite of PMA. Case history: A man in his 20s was found dead in his room, wearing only underwear. Pink star-shaped tablets were found adjacent to the decedent. The tablets were submitted to a police forensic science laboratory and identified to contain PMA, NEP, and ketamine. An autopsy was performed in our laboratory, and samples of femoral vein blood and urine were collected for quantitative analysis. Quantitative analyses of PMA, NEP, ketamine, and PHA were performed using an ultra-high-performance liquid chromatograph coupled with a triple quadrupole tandem mass spectrometer (LCMS8030, Shimadzu). Blood samples were extracted using a Micro Volume QuEChERS kit (Shimadzu). Urine samples were prepared by diluting 100-fold with water prior to hydrolysis. Hydrolysis conditions were investigated using β-glucuronidase (Helix pomatia, type HP-2, ≥ 100,000 units/mL) or hydrochloric acid (HCl), as PHA was expected to form conjugates. The method was validated by assessing linearity, intra- and inter-day accuracy and precision, recovery, and matrix effect. Quantitative analyses revealed that the concentrations of PMA, NEP, and ketamine were 2.2 μg/mL, 0.21 μg/mL, and 0.22 μg/mL in femoral vein blood and 133 μg/mL, 9.5 μg/mL, and 13 μg/mL in urine, respectively. The concentration of PHA in urine was 24 μg/mL. PHA concentration was not affected by hydrolysis with β-glucuronidase, but was significantly increased with HCl. This result suggests that PHA is predominantly conjugated as sulfate, rather than glucuronide. The linearity (R2) of the calibration curves was > 0.99, and the accuracy and precision were < 15% for all the target drugs. Based on autopsy findings and toxicology results, the forensic pathologist concluded that the cause of death was intoxication by the simultaneous ingestion of PMA, NEP, and ketamine. Previous reports showed that the toxic blood levels for PMA and NEP ranged above 0.5 μg/mL and 0.1 μg/mL, respectively. The blood levels of PMA and NEP in the present case exceeded the toxic levels, which may have caused acute intoxication and led to death. Ketamine concentration was at the therapeutic level. As in this case, it is not unusual for several substances with different pharmacological effects, such as excitation, sedation, and hallucinations, to be detected simultaneously in a single NPS-related product. Products with multiple pharmacological actions may have unexpected effects on the human body. This report describes a fatal case of poisoning due to PMA, NEP, and ketamine. In addition, PHA, the primary active metabolite of PMA, was quantified in the postmortem samples. There have been no reports examining the hydrolysis conditions for PHA. These validated quantitative values would be helpful in interpreting the cases in which these drugs were ingested.

Behavioural and neurochemical effects after repeated administration of N‐ethylpentylone (ephylone) in mice

N-ethyl-pentylone (NEP), also known as 'ephylone' and N-ethylnorpentylone, has been identified as one of the most recent novel psychostimulants to emerge into the illicit drug market and it has been associated with some intoxications and even fatalities. However, little is known about the consequences of its repeated consumption as well as the role of the monoaminergic system in such consequences. Thus, the aim of our study was to investigate the neurochemical profile and the behavioural effects after both acute and repeated NEP exposure. Male OF1mice were acutely (1, 3, 10mg/kg, i.p.) or repeatedly (1, 3, 10mg/kg, i.p., 5days, twice/day) exposed to NEP, and anxiety-like behaviour, aggressiveness, social interaction, depressive-like symptoms, body temperature, changes in monoaminergic enzymes and neurotransmitters levels as well as ΔFosB in striatum and prefrontal cortex (PFC) from post-mortem tissue were analysed short after drug-exposure or during drug-withdrawal. Acute administration of NEP induced anxiolytic effects but also an aggressive behaviour and social exploration deficits in mice, which persist during NEP-withdrawal. Moreover, NEP induced hyperthermia as well as depressive-like symptoms after repeated administrations that may be related to the decrease in serotonin and noradrenaline levels observed in striatum and PFC. Finally, the long-term increase in ΔFosB levels in striatum after NEP chronic exposure points to a high risk of dependence. Altogether indicates that NEP consumption induces different neurological and neuropsychiatric disorders accompanied by changes in the monoaminergic system, posing a threat to public health.

Structure–Activity Relationship of Novel Second-Generation Synthetic Cathinones: Mechanism of Action, Locomotion, Reward, and Immediate-Early Genes

Several new synthetic cathinones, which mimic the effect of classical psychostimulants such as cocaine or MDMA, have appeared in the global illicit drug market in the last decades. In fact, the illicit drug market is continually evolving by constantly adding small modifications to the common chemical structure of synthetic cathinones. Thus, the aim of this study was to investigate the in vitro and in vivo structure–activity relationship (SAR) of six novel synthetic cathinones currently popular as recreational drugs, pentedrone, pentylone, N-ethyl-pentedrone (NEPD), N-ethyl-pentylone (NEP), 4-methyl-pentedrone (4-MPD), and 4-methyl-ethylaminopentedrone (4-MeAP), which structurally differ in the absence or presence of different aromatic substituents and in their amino terminal group. Human embryonic kidney (HEK293) cells expressing the human isoforms of SERT and DAT were used for the uptake inhibition and release assays. Moreover, Swiss CD-1 mice were used to investigate the psychostimulant effect, rewarding properties (3, 10, and 30 mg/kg, i.p.), and the induction of immediate-early genes (IEGs), such as Arc and c-fos in the dorsal striatum (DS) and ventral striatum (VS) as well as bdnf in the medial prefrontal cortex (mPFC), of the test compounds. Our results demonstrated that all tested synthetic cathinones are potent dopamine (DA) uptake inhibitors, especially the N-ethyl analogs, while the ring-substituted cathinones tested showed higher potency as SERT inhibitors than their no ring-substituted analogs. Moreover, unlike NEP, the remaining test compounds showed clear “hybrid” properties, acting as DAT blockers but SERT substrates. Regarding the locomotion, NEP and NEPD were more efficacious (10 mg/kg) than their N-methyl analogs, which correlates with their higher potency inhibiting the DAT and an overexpression of Arc levels in the DS and VS. Furthermore, all compounds tested induced an increase in c-fos expression in the DS, except for 4-MPD, the least effective compound in inducing hyperlocomotion. Moreover, NEP induced an up-regulation of bdnf in the mPFC that correlates with its 5-HTergic properties. Finally, the present study demonstrated for the first time that NEP, 4-MPD, and 4-MeAP induce reward in mice. Altogether, this study provides valuable information about the mechanism of action and psychostimulant and rewarding properties as well as changes in the expression of IEGs related to addiction induced by novel second-generation synthetic cathinones.

Acute exposure to N-Ethylpentylone induces developmental toxicity and dopaminergic receptor-regulated aberrances in zebrafish larvae

N-Ethylpentylone (NEP) is one of the most recent novel stimulants, and there is limited understanding of its toxicity. Here we employed zebrafish model for analyzing the effects of NEP on early embryos and cardiovascular and nervous systems at late developmental stages. We first observed multi-malformations in early embryos and larvae after NEP administration, together with significant deregulations of brain and heart development-associated genes (neurog1, her6, elavl3, nkx2.5, nppa, nppb, tnnt2a) at transcriptional level. Low-dosed NEP treatment induced an anxiety-like phenotype in zebrafish larvae, while higher doses of NEP exerted an inhibitory effect on locomotion and heart rate. Besides, the expression of th (tyrosine hydroxylase) and th2 (tyrosine hydroxylase 2), identifying dopamine (DA) release, were significantly increased during one-hour free swimming after effective low-dosed NEP administration, along with the upregulation of gene fosab and fosb related to stress and anxiety response. D1R antagonist SCH23390 and D2R antagonist sulpiride partially alleviated the aberrances of locomotion and heart rate, indicating dopaminergic receptors were involved in the bidirectional dosage-dependent pattern of NEP-induced performance. Meanwhile, sulpiride offset the upregulated expression of th, th2 and fosab in the group of 1.5 μM NEP, which highlighted the significant role of D2R in NEP-induced locomotive effects. This study systematically described the developmental, neuronal and cardiac toxicity of NEP in zebrafish, and identified the dopaminergic receptors as one of the downstream effectors of NEP administration.

Evaluating Trends in Novel Psychoactive Substances Using a Sentinel Population of Electronic Dance Music Festival Attendees.

Electronic dance music (EDM) festivals have become a popular venue for recreational drug use, including the use of traditional stimulants like 3,4-methylenendioxymethamphetamine (MDMA) and novel psychoactive substances (NPS). Using this cohort of people who use drugs recreationally, this study sought to collect biological specimens and self-reported drug use data from EDM festival attendees in the United States to monitor regional and temporal trends related to NPS use and turnover between 2014 and 2017. Oral fluid samples were collected at three United States EDM festival locations, including Miami, Florida (2014 to 2017); Tampa, Florida (2017) and Atlanta, Georgia (2017). Samples were screened by liquid chromatography-quadrupole time-of-flight mass spectrometry and confirmed by liquid chromatography-tandem mass spectrometry. Over the 4 years, 1,233 oral fluid samples were collected. With respect to self-reported drug use, 63% of respondents reported medicinal and/or recreational drug use within the last week. When comparing 4 years of data from Miami (2014 to 2017), NPS trends showed the disappearance of alpha-PVP after 2014 followed by a significant increase in ethylone positivity in 2015 and rapid decrease in 2016. Dibutylone was identified for the first time in Miami 2016, and N-ethyl pentylone was identified for the first time in Miami 2017. Additionally, 3,4-methylenendioxymethamphetamine positivity steadily increased from 2014 to 2017. A comparison across study sites (Miami, Tampa and Atlanta) and specific trends with respect to novel simulant use are described within. Using this opportunistic approach of monitoring drug trends, we have found that peak positivity of novel stimulants usually is within a year of their first detection. Understanding the dynamics of NPS drug markets will allow laboratories to plan for resource allocation and scope updates within a timely fashion to assist with the detection and confirmation of these emerging substances in samples submitted for forensic analysis.

Eutylone Intoxications-An Emerging Synthetic Stimulant in Forensic Investigations.

Synthetic stimulants are the largest class of novel psychoactive substances identified each year by forensic laboratories internationally. While hundreds of these drugs appear in drug powders, only a few proliferate in use among forensically relevant populations and eventually emerge in postmortem and clinical investigations. Beta-keto-methylenedioxyamphetamines (i.e., novel psychoactive substances with names ending in "ylone") are currently the most popular subclass of synthetic stimulants. Leading up to its federal scheduling in 2018, N-ethyl pentylone was the most encountered synthetic stimulant. The popularity of N-ethyl pentylone declined once it was scheduled, but it was quickly replaced by eutylone (bk-EBDB), a structurally related analog from the same family. In cases encountered between January 2019 and April 2020, eutylone was quantitatively confirmed in 83 forensic investigations, including postmortem cases and driving under the influence of drugs cases. Matrix types included blood, urine and tissue. Eutylone was identified in cases submitted from 13 states, demonstrating proliferation around the United States; Florida accounted for 60% of the positive cases. The mean concentration of eutylone in postmortem blood was 1,020 ng/mL (standard deviation = ±2,242 ng/mL; median = 110 ng/mL, range = 1.2-11,000 ng/mL, n = 67). The mean concentration of eutylone in blood from driving under the influence of drugs cases was 942 ng/mL (standard deviation = ±1,407 ng/mL; median = 140 ng/mL, range = 17-3,600 ng/mL, n = 7). This report includes cause and manner of death data for 22 postmortem cases. Further analysis of authentic human specimens revealed the presence of three eutylone metabolites, including one unique biomarker and one metabolite in common with butylone. Laboratories should be aware that eutylone may be present in cases of suspected Ecstasy, "Molly" and/or methylenedioxymethamphetamine use, causing or contributing to impairment or death.

Effects of N-ethylpentylone on locomotor activity and anxiety-like behavior in rats.

N-ethylpentylone (NEP), a new synthetic cathinone, has been rising to be one of the most popular cathinone derivatives in recent years. However, research on NEP is rather limited. In this study, locomotor stimulation and sensitization, as well as anxiety-like behavior induced by NEP were studied in Sprague-Dawley rats, using the open field and elevated plus maze respectively. Rats were administered NEP (5, 20 or 50 mg/kg, intraperitoneal), with saline as the negative control and methamphetamine (5 mg/kg) as a positive control. Acute administration of NEP at all the doses tested significantly promoted locomotor activity, presenting an inverted U-shaped dose-effect curve. The highest activity was observed at the 20 mg/kg dose group, with the average distance traveled 18 times higher than the saline group. Repeated administration of NEP enhanced locomotor activity only at the 5 mg/kg dose group. After a week's withdrawal, re-challenge of NEP failed to induce marked behavioral sensitization. In elevated plus maze experiments, both acute and repeated administration of 20 mg/kg NEP induced anxiolytic-like effects, while no significant alteration was observed in the 5 and 50 mg/kg dose groups. In summary, acute administration of NEP caused significantly enhanced locomotor activity in rats at all the tested doses, while repeated NEP administration enhanced locomotor activity only at a low dose (5 mg/kg), while a high dose (20 mg/kg) of NEP induced anxiolytic-like effects after both acute and repeated administration.

Simultaneous determination of N-ethylpentylone, dopamine, 5-hydroxytryptamine and their metabolites in rat brain microdialysis by liquid chromatography tandem mass spectrometry.

N-Ethylpentylone (NEP) is a popular synthetic cathinone abused worldwide. To obtain more information about its pharmacokinetics and pharmacodynamics, a rapid, simple and sensitive liquid chromatography-tandem mass spectrometry method was developed for the determination of NEP, two important neurotransmitters, dopamine and serotonin, and their metabolites, including 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine and 5-hydroxyindole-3-acetic acid, in rat brain microdialysate. The analytes were separated on a Phnomenex Polar C18 column, with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) under gradient elution to shorten the total chromatographic run time. A triple quadruple mass spectrometer coupled with an electrospray ionization source in both positive and negative ion mode was used to detect the analytes. This method showed excellent accuracy (87.4-113.5%) and precision (relative standard deviation <15%) at three quality control levels. The limits of detection were 0.2 ng/mL for NEP and 0.2-50 nm for the others and good linearity was obtained. This study pioneered a method to integrate exogenous drugs and endogenous neurotransmitters as the drugs act on the same determination system, which means that this innovation can provide support for further study of the addictive effects of NEP or other synthetic cathinones on extracellular levels of dopamine and 5-hydroxytryptamine.

Pharmacokinetics of N-ethylpentylone and its effect on increasing levels of dopamine and serotonin in the nucleus accumbens of conscious rats.

N-Ethylpentylone (NEP) is one of the most confiscated synthetic cathinones in the world. However, its pharmacology and pharmacokinetics remain largely unknown. In this study, the pharmacokentics of NEP in rat nucleus accumbens (NAc) was assessed via brain microdialysis after the intraperitoneal (ip) administration of NEP (20 or 50mg/kg). The concentrations of dopamine (DA) and serotonin (5-HT) and their metabolites, including 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), and 5-hydroxyindoleacetic acid (5-HIAA), were simultaneously monitored to elucidate the pharmacological effect of NEP. In addition, the plasma levels of NEP were also assessed. The pharmacokinetics of NEP showed a dose-related pattern, with NEP rapidly passing through the blood-brain barrier and reaching a maximum concentration (Cmax ) at approximately 40-minutes postdose. Approximately 4% of plasma NEP was distributed to the NAc, and considering a homogeneous brain distribution, over 90% of plasma NEP was potentially distributed to the brain. High values of area under curve (AUC) and mean residence time (MRT) of NEP were observed in both the NAc and plasma, indicating large and long-lasting effects. NEP elicited dose-related increases in microdialysate DA and 5-HT and increased the concentration of 3-MT in a dose-related manner. However, the rate of DA converted into 3-MT was unaffected. NEP had a negative effect on the rates of which DA and 5-HT were transformed into DOPAC and 5-HIAA, respectively. In summary, NEP rapidly entered the NAc and showed a long-lasting effect. In addition, DA increased more significantly than 5-HT, indicating a large potential for NEP abuse.

Development and Validation of a Novel All-Inclusive LC-MS-MS Designer Drug Method.

Designer drugs including synthetic cannabinoids and synthetic cathinones are an increasing problem due to the ease of access to these compounds. They present analytical challenges inasmuch as the compound structures are numerous and growing within each class. Typically each class of designer compounds is analyzed separately due to differences in chemistry, desired cut-offs or other reasons. Physicians treating "high-risk" patients typically order tests for all "illicit" substances which can span several test classes. Despite that multiple classes of designer drugs are ordered together, there has not been a comprehensive confirmatory test developed to date. Presented here is a novel comprehensive designer drug LC-MS-MS method that combines synthetic cannabinoids and synthetic cathinones, etizolam, a designer benzodiazepine and mitragynine (kratom), a natural product analgesic. This method improves laboratory throughput with a cycle time of ~4.5 min which affords resolution of crucial isomers, such as ethylone and butylone. Development of this method also provided an opportunity to update the list of compounds within the method. Analytes with fewer than five positive specimens in a year of testing with previous separate methods were removed as old and not current. New analytes were added based on reports from NMS Laboratories and the US Drug Enforcement Administration testing and drug seizures, which included etizolam, its major metabolite α-hydroxyetizolam as well as newer synthetic cannabinoids (5-fluoro ADB metabolite 7, AB-FUBINACA metabolite 3, AB-FUBINACA metabolite 4 and MDMB-FUBINACA metabolite M1) and synthetic cathinones (N-ethyl pentylone). Finally, the impact of the new analytes and cut-off changes are discussed in context with patient results from the first 4 months of testing after implementation of the method in the lab.

The Detection of Novel Stimulants in Oral Fluid from Users Reporting Ecstasy, Molly and MDMA Ingestion.

"Ecstasy" and "Molly" are common drug slang terms used among club and rave cultures to denote preparations believed to contain 3,4-methylenedioxymethamphetamine (MDMA). However, users of Ecstasy and Molly have increasingly tested positive for novel psychoactive substances (NPS), notably novel stimulants. To evaluate hypothesized non-specific and interchangeable use of the terms Ecstasy, Molly and MDMA, self-reported drug use was compared against toxicological findings in biological specimens. Oral fluid specimens were collected from participants attending large multi-day electronic dance music festivals in Miami, FL; Tampa, FL; and Atlanta, GA. Participants additionally completed a structured survey about recent recreational drug use. Collected specimens were screened for therapeutic drugs, common drugs of abuse and NPS using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF). Positive screen results were confirmed by validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods for MDMA, MDA, methylone, dimethylone, ethylone, butylone, dibutylone, eutylone, pentylone, N-ethyl pentylone (ephylone), alpha-PVP and 4-fluoroamphetamine (4-FA). During this 4-year study, 223 participants provided an oral fluid specimen and indicated recent use of Ecstasy, Molly and/or MDMA/MDA. Of these subjects, 203 (91.0%) indicated only one of these drug terms; while 20 (9.0%) participants indicated a combination of multiple terms. Of the 203 participants designating only one drug term, 123 (60.6%) reported Molly use, 55 (27.1%) reported MDMA use and 25 (12.3%) reported Ecstasy use. Seven participants reported the use of MDA, but these responses were paired with MDMA responses due to detection of MDA as a metabolite of MDMA. The results from this study indicate that there are inconsistencies between admission to drug use and toxicological findings in this population. Of the 223 participants who indicated use of Ecstasy, Molly and/or MDMA/MDA, MDMA without a novel stimulant was confirmed in 121 (54.3%) participants, while 66 (29.6%) tested positive for at least one novel stimulant.

N-Ethyl Pentylone (Ephylone) Intoxications: Quantitative Confirmation and Metabolite Identification in Authentic Human Biological Specimens

N-ethyl pentylone (ephylone) has been identified as the most recent novel stimulant to emerge into the arena of evolving novel psychoactive substances (NPS). Due to its novelty, information regarding case reports with associated quantitative confirmations, biotransformation pathways, and identified unique metabolites will assist the scientific community in understanding the implications of the emergence and risks associated with N-ethyl pentylone use. Authentic blood specimens (n = 26) submitted as part of toxicological death investigations or drugged driving casework tested positive for N-ethyl pentylone, and were quantitatively analyzed by liquid chromatography tandem mass spectrometry (LC-MS-MS). N-ethyl pentylone concentrations ranged from 12 to 1,200 ng/mL, with mean (±standard deviation) and median concentrations of 313 (±366) and 125 ng/mL, respectively, excluding one case measured at 50,000 ng/mL. N-ethyl pentylone was often found in combination with other drugs of abuse and NPS, include a variety of novel opioids including fentanyl analogs. Oral fluid specimens (n = 5), collected from recreational drug users at a dance music festival, were quantitatively analyzed using LC-MS-MS. Concentrations ranged from 12.6 to 1,377 ng/mL. Additional analysis was performed to characterize the metabolic profile of N-ethyl pentylone using human liver microsomes (HLM), followed by confirmation of the presence of the proposed metabolites in a subset of the blood specimens and oral fluid specimens. Metabolomic analysis was performed using a liquid chromatograph quadrupole time-of-flight mass spectrometer (LC-QTOF), followed by data processing using MetabolitePilot™ software. In vivo verification of in vitro HLM-generated metabolites resulted in the confirmation of four metabolites. Reduction of the beta-ketone to an alcohol resulted in the most prominent metabolite found in the authentic specimens, and its uniqueness to N-ethyl pentylone leads to this metabolite being an appropriate biomarker to determine N-ethyl pentylone ingestion. This is the first study to report N-ethyl pentylone concentrations and to characterize the metabolic profile of N-ethyl pentylone.