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The μ opioid receptor (MOR) has been an intrinsic target to develop treatment of opioid use disorders (OUD). Herein, we report our efforts on developing centrally acting MOR antagonists by structural modifications of 17-cyclopropylmethyl-3,14-dihydroxy-4,5α-epoxy-6β-[(4′-pyridyl) carboxamido] morphinan (NAP), a peripherally acting MOR-selective antagonist. An isosteric replacement concept was applied and incorporated with physiochemical property predictions in the molecular design. Three analogs, namely, 25, 26, and 31, were identified as potent MOR antagonists in vivo with significantly fewer withdrawal symptoms than naloxone observed at similar doses. Furthermore, brain and plasma drug distribution studies supported the outcomes of our design strategy on these compounds. Taken together, our isosteric replacement of pyridine with pyrrole, furan, and thiophene provided insights into the structure–activity relationships of NAP and aided the understanding of physicochemical requirements of potential CNS acting opioids. These efforts resulted in potent, centrally efficacious MOR antagonists that may be pursued as leads to treat OUD.

Co-users of cannabis and tobacco frequently use cannabis, then tobacco cigarettes, in a sequential pattern within an occasion, that is, they "chase" smoked cannabis with a tobacco cigarette. The objective of this placebo-controlled, double-blind, within-subjects human laboratory study was to gather preliminary data on how smoking active versus placebo cannabis impacts tobacco cigarette smoking behavior, craving, and subjective effects. Adult daily cannabis and tobacco co-users (N = 9) were randomly assigned to two experimental visit orders (i.e., active cannabis (5.2% THC) first visit and placebo cannabis second visit, or vice versa). Participants smoked one cannabis cigarette, and approximately 30 min later were given a 5-min ad libitum period to smoke one of their own brand of tobacco cigarette. As expected, boost in plasma THC levels and cannabis-related subjective effects differed between active and placebo cannabis conditions. Tobacco cigarette puff topography measures and tobacco craving did not differ between cannabis conditions, but there appeared to be between-participants heterogeneity in cumulative total puff volume. After smoking active versus placebo cannabis, the changes in subjective effects of tobacco smoking after adjusting for pretobacco smoking levels were not significant. Results do not support the notion that immediate effects of smoked cannabis change the behavior of tobacco smoking. The strong overlap between cannabis and tobacco smoking may not be explained by primarily pharmacological factors, but may be driven by more nuanced and complex mechanisms involving pharmacological processes as well as learning factors. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Gamma-butyrolactone (GBL), a commonly used industrial solvent, is used recreationally as a central nervous system (CNS) depressant and, therefore, is a United States Drug Enforcement Agency List 1 chemical of the Controlled Substances Act. GBL was identified presumptively in the liquid from JUUL Virginia Tobacco flavored pods during routine untargeted screening analysis of e-cigarette products by gas chromatography–mass spectrometry (GC–MS). Methods for the analysis of GBL were developed for GC–MS and liquid chromatography–tandem mass spectrometry (LC–MS-MS) in the liquids and the aerosol generated from the liquid. Three flavors of JUUL pods available at the time of analysis were obtained by direct purchase from the manufacturer, purchase from a local vape shop and submission from a third party. The only liquid flavor to contain GBL was Virginia Tobacco, with an average of 0.37 mg/mL of GBL, and it was detected in the aerosol. Studies evaluating the pharmacological effects of inhaling GBL do not exist; however, a case report of chronic oral GBL ingestion indicates acute lung injury. The identification of GBL in an e-cigarette product purportedly compliant with federal regulation continues to demonstrate public health and public safety concerns.

Duloxetine, known by its brand name, CymbaltaTM, is a selective serotonin and norepinephrine reuptake inhibitor used to treat major depressive disorders. Determination of patient compliance for duloxetine is typically determined through medication possession ratio (MPR) or plasma concentrations. The purpose of this paper was to characterize normal urinary duloxetine concentrations in patients prescribed duloxetine to monitor patient adherence. Patient data collected from routine screens for duloxetine concentrations in urine were included in this study. Inclusion criteria consisted of patients who were prescribed duloxetine and (i) tested positive for duloxetine, (ii) tested negative for illicit substances and (iii) included creatinine, age and duloxetine dose administered. Of the 5,592 patient urines screened, 2,004 of the results fit into the inclusion criteria. Positive urine concentrations of duloxetine ranged from 50 to 2,722 ng/mL. Duloxetine urine concentrations were normalized to creatinine and dose further characterized by sex, age, body mass index (BMI) and dose in milligrams. Sample distribution included urines collected from 1,487 females and 517 males. The age range of the specimen donors was between 15 and 90 years old with an average age of 52. BMI levels ranged from 13.9 (underweight) to 88.1 (obese), with the average BMI being 33.5. The most common dose of duloxetine prescribed was a daily, oral dose of 60 mg. Analysis of the normalized, transformed creatinine concentrations showed that there was a significant statistical difference (P

Psychedelics, also known as classical hallucinogens, affect processes related to perception, cognition and sensory processing mostly via the serotonin 5-HT(2A) receptor (5-HT(2A)R). This class of psychoactive substances, which includes lysergic acid diethylamide (LSD), psilocybin, mescaline and the substituted amphetamine 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), is receiving renewed attention for their potential therapeutic properties as it relates to psychiatric conditions such as depression and substance use disorders. Current studies focused on the potentially clinical effects of psychedelics on human subjects tend to exclude sex as a biological variable. Much of the understanding of psychedelic pharmacology is derived from rodent models, but most of this preclinical research has only focused on male mice. Here we tested the effects of DOI on head-twitch behavior (HTR) – a mouse behavioral proxy of human psychedelic potential – in male and female mice. DOI elicited more HTR in female as compared to male C57BL/6J mice, a sex-specific exacerbated behavior that was not observed in 129S6/SvEv animals. Volinanserin (or M100907) – a 5-HT(2A)R antagonist – fully prevented DOI-induced HTR in male and female C57BL/6J mice. Accumulation of inositol monophosphate (IP(1)) in the frontal cortex upon DOI administration showed no sex-related effect in C57BL/6J mice. However, the pharmacokinetic properties of DOI differed among sexes – brain and plasma concentrations of DOI were lower 30 and 60 min after drug administration in female as compared to male C57BL/6J mice. Together, these results suggest strain-dependent and sex-related differences in the behavioral and pharmacokinetic profiles of the 5-HT(2A)R agonist DOI in C57BL/6J mice, and support the importance of studying sex as a biological variable in preclinical psychedelic research.

Presented is the analysis of four cannabinoid-based products. These products were part of a case involving visual and auditory hallucinations that precipitated the commission of a felony and subsequent arrest. The products were labeled to contain ∆8-tetrahydrocannabinol (∆8-THC) or THC acetate (THC-O-A). Primary reference materials were not available for ∆8-THC-O-A, ∆10-THC-O-A, cannabidiol di-acetate (CBD-di-O-A) or respective deuterated internal standards. THC-O-A and CBD-di-O-A standards were prepared by derivatizing ∆8-THC, ∆9-THC, ∆10-THC, CBD, ∆9-THC-d3 and CBD-d3 using acetic anhydride. The cannabinoid-based products were determined to contain ∆8-THC, ∆8-THC-O-A, ∆9-THC-O-A and CBD-di-O-A and/or other phytocannabinoids using three different analytical techniques. Direct analysis in real-time–time-of-flight mass spectrometry was used for identifying exact masses. A gas chromatograph–mass spectrometer was used for the identification of compounds and to quantitate THC-O-As in the products. A liquid chromatograph–tandem mass spectrometer was used to identify and quantitate phytocannabinoids and CBD-di-O-A in the products. To the authors’ knowledge, this is the first case report involving the identification of THC-O-As and CBD-di-O-A in commercially available products. Minimal clinical/pharmacological data is available for these emerging synthetic cannabinoids/novel psychoactive substances.

Although cannabinoid receptors (CB) are recognized as targets for renal fibrosis, the roles of endogenous cannabinoid anandamide (AEA) and its primary hydrolytic enzyme, fatty acid amide hydrolase (FAAH), in renal fibrogenesis remain unclear. The present study used a mouse model of post-ischemia-reperfusion renal injury (PIR) to test the hypothesis that FAAH participates in the renal fibrogenesis. Our results demonstrated that PIR showed upregulated expression of FAAH in renal proximal tubules, accompanied with decreased AEA levels in kidneys. Faah knockout mice recovered the reduced AEA levels and ameliorated PIR-triggered increases in blood urea nitrogen, plasma creatinine as well as renal profibrogenic markers and injuries. Correspondingly, a selective FAAH inhibitor, PF-04457845, inhibited the transforming growth factor-beta 1 (TGF-β1)-induced profibrogenic markers in human proximal tubular cell line (HK-2 cells) and mouse primary cultured tubular cells. Knockdown of FAAH by siRNA in HK-2 cells had similar effects as PF-04457845. Tubular cells isolated from Faah

Neonatal drug exposure is currently assessed using meconium, urine, blood, hair, or umbilical cord tissue/blood. Due to the invasiveness, challenges, and limitations of collection, and/or analytical difficulties of these matrices, oral fluid may be a more desirable matrix in diagnosing opioid exposure and risk for opioid withdrawal in neonatal abstinence syndrome. Traditional oral fluid collection devices are not viable options as they are too large for neonates’ mouths and may contain chemicals on the collection pad. Unstimulated and stimulated infant oral fluid samples have been used for therapeutic drug monitoring as an alternative matrix to blood. The objective of this study was to assess the viability of a simple oral fluid collection system using a sterile foam-tipped swab rinsed in phosphate-buffered saline. Two infants were administered fentanyl for post-operative pain relief while hospitalized in the Neonatal Intensive Care Units at the Children’s Hospital of Richmond of Virginia Commonwealth University. Oral fluid samples were collected at 16 h, 2 days, and/or 7 days following the start of intravenous infusion of fentanyl. Samples were analyzed by ultra-high-pressure liquid chromatography–tandem mass spectrometry for fentanyl and norfentanyl after solid-phase extraction. In one of the three samples tested, fentanyl and norfentanyl were detected at concentrations of 28 and 78 ng/mL, respectively. Based on the infusion rate, the theoretical oral fluid fentanyl concentration at steady state was calculated to be 33 ng/mL.

Tramadol is an opioid used in the treatment of moderate to moderately severe pain. Tramadol’s use during pregnancy is generally avoided and may cause some reversible withdrawal effects in neonates, and its use during lactation is not licensed by the manufacturer. A small clinical trial reported infants were exposed to

Opioid usage in the USA has increased over the past decade, with prescriptions increasing from 76 million in 1991 to 207 million in 2013. New regulations have curbed the number of prescriptions, leading to an increase in heroin use. Heroin-related overdoses have quadrupled between 2000 and 2015. The traditional urinary biomarkers for indicating heroin use are a combination of morphine and 6-acetyl morphine (6-AM). Morphine is detectable in urine for several days. 6-AM is detected in urine for 2–8 hours. Papaverine has been proposed as an alternative heroin biomarker. It has been reported to have a 1–2 day detection window. Papaverine metabolites have been reported to have up to a 3-day detection window. Presented is a method for the detection of papaverine and its metabolites, 6-desmethyl papaverine (6-DMP) and 4′, 6-didesmethyl papaverine (4,6-DDMP), in urine using a modified Waters® MCX™ microelution method. An ultra-performance liquid chromatography and tandem mass spectrometry (UPLC–MS-MS), with a Waters’ BEH C18 column, and 20 mM ammonium formate water: 20 mM ammonium formate methanol mobile phase was employed. Calibration curves were linear from 0.1 to 50 ng/mL. No interferences were observed from the analysis of multicomponent therapeutic drug or drugs of abuse control materials; intra- and inter-run precision tests were acceptable. A total of 428 genuine urine specimens where heroin use was suspected were analyzed. These included 101 6-AM and 179 morphine only positive samples as well as 6 morphine-negative samples where papaverine and/or metabolites were detected. The determined concentrations in these samples for papaverine, 6-DMP and 4,6-DDMP ranged from 0.10 to 994, 0.10 to 462 and 0.12 to 218 ng/mL, respectively. The method was rugged and robust for the analysis of papaverine and metabolites, 6-DMP and 4,6-DDMP. The use papaverine and metabolites, 6-DMP and 4,6-DDMP has the potential to increase the detection window of heroin use.

Presented is an ultra-high-pressure liquid chromatographic tandem mass spectrometry (UPLC–MS/MS) method developed for the detection of propylene glycol, glycerol, ethylene glycol and diethylene glycol using isotopically labeled standards in urine as part of ongoing studies to evaluate whether urinary propylene glycol and/or vegetable glycerin concentration are indicators of recent use. Propylene glycol and vegetable glycerol are found in many products that are consumed and used including electronic cigarettes (e-cigarettes). E-cigarettes are battery-powered devices used as an alternative to traditional cigarettes. The liquid formulations aerosolized in these devices largely consist of propylene glycol and/or vegetable glycerol. Published reports regarding the ratio of propylene glycol to glycerol content in these formulations ranged from 50:50 to 100 percent of either. For the analysis of urine specimens from both users and non-users of e-cigarettes, calibrators, controls and specimens were derivatized using benzoyl chloride prior to analysis. They were analyzed using a Waters AcQuity Xevo TQ-S Micro UPLC–MS/MS. Chromatographic separation was performed on an AcQuity UPLC BEH C18 1.7 um, 2.1 × 50 mm, column using a 20 mM ammonium formate in water and 20 mM ammonium formate in methanol as the mobile phase. The method was validated using SWGTOX guidelines for linearity, precision and accuracy, stability, carryover and limit of detection. The linear range was determined using a seven-point calibration curve ranging between 0.5 and 100 mcg/mL. The bias for all validation controls was determined to be ±20% of the expected concentrations with CVs of

Electronic cigarettes (e-cigarettes) are an alternate nicotine delivery system that generate a condensation aerosol to be inhaled by the user. The size of the droplets formed in the aerosol can vary and contributes to drug deposition and ultimate bioavailability in the lung. The growing popularity of e-cigarette products has caused an increase in internet sources promoting the use of drugs other than nicotine (DOTNs) in e-cigarettes. The purpose of this study was to determine the effect of various e-cigarette and e-liquid modifications, such as coil resistance, battery voltage, and glycol and drug formulation, on the aerosol particle size. E-liquids containing 12 mg/mL nicotine prepared in glycol compositions of 100% propylene glycol (PG), 100% vegetable glycerin (VG), or 50:50 PG:VG were aerosolized at three voltages and three coil resistances. Methamphetamine and methadone e-liquids were prepared at 60 mg/mL in 50:50 PG:VG and all e-liquids were aerosolized onto a 10 stage Micro-Orifice Uniform Deposit Impactor. Glycol deposition correlated with drug deposition, and the majority of particles centered between 0.172–0.5 μm in diameter, representing pulmonary deposition. The 100% PG e-liquid produced the largest aerosol particles and the 100% VG and 50:50 PG:VG e-liquids produced ultra-fine particles

Background We describe a case of symptomatic bradycardia resulting from ivabradine toxicity by measurement of ivabradine levels, of which there are limited reports in the literature. Case presentation A 43-year-old White female presented with several days of near syncope and dizziness accompanied by a drop in her heart rate to 50 beats per minute. She was taking ivabradine for inappropriate sinus tachycardia. After excluding several other causes of bradycardia, we made the diagnosis of ivabradine toxicity by measurement of serum ivabradine levels, an approach that is currently not clinically available. Conclusions Measurement of serum ivabradine levels and knowledge of the pharmacokinetic properties of the drug can be utilized to confirm the diagnosis of ivabradine toxicity.

Rationale Natural plant products have been used to promote health, prevent sickness, and treat various ailments. These products often consist of leaves, flowers, bark, roots, seeds, and/or other parts of the plant. Many of the pharmacologically active constituents of these products are known, but the pharmacology of these constituents may not be fully elucidated. Natural plant-based products are also available in various forms other than the raw plant material. A wide array of commercial products such as capsules, powders, extracts, and electronic cigarette (e-cigarette) electronic liquids (e-liquids) are readily available and can be purchased from various outlets, both store-based retailers and online. Newer e-cigarettes are often advertised as "heat not burn" and are used for "vaping" various forms of extracts including "waxes" and "dabs" and raw plant material. Methods A single manufacturer was found online selling "24 different herbs" in powders, extracts, or e-liquids. These were advertised as "legal in the USA" and each product listed multiple effects. Eight e-liquids, six extracts (resins), and four powders from eight different "herbs," namely African dream, areca nut, blue lotus, damiana, kra thum na, kra thum kok, klip dagga, and wild lettuce, were purchased. An advertisement for these products stated, "Most people use the leaves, powder or resin in vaporizers." Direct analysis in real time AccuTOF™ mass spectrometry (DART-MS) was used to identify the psychoactive components in the natural products. Results The psychoactive compounds that were identified in only two of the eight e-liquids, three of the five resins, and three of the four powders were arecaidine, arecoline, coumarin, entadamide, mitragynine, 7-hydroxymitragynine, and nuciferine. Conclusions Psychoactive and potentially harmful substances were present in the powders and resins of the natural products. The newer types of e-cigarettes made for consuming natural products may increase their abuse potential.

Although paclitaxel effectively treats various cancers, its debilitating peripheral neuropathic pain side effects often persist long after treatment has ended. Therefore, a compelling need exists for the identification of novel pharmacologic strategies to mitigate this condition. As inhibitors of monoacylglycerol lipase (MAGL), the primary hydrolytic enzyme of the endogenous cannabinoid, 2-arachidonyolglycerol, produces antinociceptive effects in numerous rodent models of pain, we investigated whether inhibitors of this enzyme (i.e., JZL184 and MJN110) would reverse paclitaxel-induced mechanical allodynia in mice. These drugs dose dependently reversed allodynia with respective ED(50) values (95% confidence limit) of 8.4 (5.2–13.6) and 1.8 (1.0–3.3) mg/kg. Complementary genetic and pharmacologic approaches revealed that the antiallodynic effects of each drug require both cannabinoid receptors, CB(1) and CB(2). MJN110 reduced paclitaxel-mediated increased expression of monocyte chemoattractant protein-1 (MCP-1, CCL2) and phospho-p38 MAPK in dorsal root ganglia as well as MCP-1 in spinal dorsal horn. Whereas the antinociceptive effects of high dose JZL184 (40 mg/kg) underwent tolerance following 6 days of repeated dosing, repeated administration of a threshold dose (i.e., 4 mg/kg) completely reversed paclitaxel-induced allodynia. In addition, we found that the administration of MJN110 to control mice lacked intrinsic rewarding effects in the conditioned place preference (CPP) paradigm. However, it produced a CPP in paclitaxel-treated animals, suggesting a reduced paclitaxel-induced aversive state. Importantly, JZL184 did not alter the antiproliferative and apoptotic effects of paclitaxel in A549 and H460 non-small cell lung cancer cells. Taken together, these data indicate that MAGL inhibitors reverse paclitaxel-induced neuropathic pain without interfering with chemotherapeutic efficacy.

We present the case histories, autopsy findings and toxicology findings of two fatal intoxications involving the designer drug, butyryl fentanyl. The quantitative analysis of butyryl fentanyl in postmortem fluids and tissues was performed by an ultrahigh-performance liquid chromatography tandem mass spectrometry method. In the first case, butyryl fentanyl was the only drug detected with concentrations of 99 ng/mL in peripheral blood, 220 ng/mL in heart blood, 32 ng/mL in vitreous humor, 590 ng/mL in gastric contents, 93 ng/g in brain, 41 ng/g in liver, 260 ng/mL in bile and 64 ng/mL in urine. The cause of death was ruled fatal intoxication by butyryl fentanyl. In the second case, butyryl fentanyl was detected along with acetyl fentanyl, alprazolam and ethanol. The butyryl fentanyl concentrations were 3.7 ng/mL in peripheral blood, 9.2 ng/mL in heart blood, 9.8 ng/mL in vitreous humor, 4,000 ng/mL in gastric contents, 63 ng/g in brain, 39 ng/g in liver, 49 ng/mL in bile and 2 ng/mL in urine. The acetyl fentanyl concentrations were 21 ng/mL in peripheral blood, 95 ng/mL in heart blood, 68 ng/mL in vitreous humor, 28,000 ng/mL in gastric contents, 200 ng/g in brain, 160 ng/g in liver, 330 ng/mL in bile and 8 ng/mL in urine. In addition, the alprazolam concentration was 40 ng/mL and the ethanol concentration was 0.11 g/dL, both measured in peripheral blood. The cause of death in the second case was ruled a mixed drug intoxication. In both cases, the manner of death was accident.

Drug self-administration procedures are the gold standard for laboratory research to study mechanisms of drug use disorders and evaluate candidate medications. However, preclinical-to-clinical translation has been hampered by a lack of coordination. To address this limitation, we previously developed homologous intravenous (IV) cocaine choice self-administration procedures in rhesus monkeys and humans, and then demonstrated their functional equivalence. The present studies sought to determine the sensitivity of these procedures to d-amphetamine maintenance. Three (N = 3) rhesus monkeys with histories of cocaine self-administration and 16 (N = 16) humans with cocaine use disorder completed the studies. Monkeys were maintained on IV d-amphetamine (0, 0.019, 0.037 and 0.074 mg/kg/h), and then completed 7 sessions during each condition in which they completed 9 choice trials to receive 0.14 mg/kg/injection IV cocaine (corresponding to 10 mg/70 kg in humans) or 10 food pellets under independent, concurrent progressive-ratio schedules. Humans were maintained on oral extended release d-amphetamine (0, 30 and 60 mg/day, corresponding to the lowest 3 doses in monkeys) and participated in 12 sessions in which they chose money ($6.00) or IV cocaine (0, 3, 10 and 30 mg/70 kg). Blood samples were taken to compare d-amphetamine plasma levels across species. In monkeys and humans, d-amphetamine reduced the number of cocaine choices and produced comparable blood levels at equivalent daily doses. d-Amphetamine had similar efficacy, though lower potency, at reducing choice for an equivalent cocaine dose in monkeys relative to humans. These coordinated studies support the utility of these procedures as a translational model for cocaine use disorder. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Use of marijuana and cannabinoids has been on the rise in recent years, including in childbearing women. This has resulted in cannabinoids being more frequently identified in breast milk as a result of its high lipid content and cannabinoids having a high lipophilicity, thereby exposing the breastfeeding infant to cannabinoids and other marijuana constituents. Presented is a method for the analysis of Δ9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD) in breast milk. THC, CBN, CBD and their isotopically labeled standards were extracted from breast milk using a modified QuEChERS method and analyzed using ultra-performance liquid chromatography and tandem mass spectrometry. As a result of the high lipid content of breast milk, saponification of the lipids was necessary to improve overall extraction efficiency. The process efficiency percentage for THC, CBD and CBN are 55%, 80% and 25%, respectively. The recovery percentage for THC, CBD and CBN are 95%, 118% and 85%, respectively. The matrix effect percentage for THC, CBD and CBN are 53%, 66% and 26%, respectively. Linearity was assessed from 1 to 100 ng/mL for THC, CBN and CBD and had r2 > 0.996. Validation controls were prepared at 1, 3, 20, 80 and 300 ng/mL (dilution control), and the bias was determined to be less than ±20% with %CVs

'NBOMe' (dimethoxyphenyl-N-[(2-methoxyphenyl)methyl]ethanamine) derivatives are a new class of designer hallucinogenic drugs widely available on the Internet. Currently, 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) is the most popular abused derivative in the USA. There are little published data on the absorption, metabolism and elimination of 25I-NBOMe, or any of the other NBOMe derivatives. Therefore, there are no definitive metabolite biomarkers. We present the identification of fifteen 25I-NBOMe metabolites in phase I and II mouse hepatic microsomal preparations, and analysis of two human urine samples from 25I-NBOMe-intoxicated patients to test the utility of these metabolites as biomarkers of 25I-NBOMe use. The synthesis of two major urinary metabolites, 2-iodo-4-methoxy-5-[2-[(2-methoxyphenyl) methylamino]ethyl]phenol (2-O-desmethyl-5-I-NBOMe, M5) and 5-iodo-4-methoxy-2-[2-[(2-methoxyphenyl)methylamino]ethyl]phenol (5-O-desmethyl-2-I-NBOMe), is also presented. Seven phase II glucuronidated metabolites of the O-desmethyl or the hydroxylated phase I metabolites were identified. One human urine sample contained 25I-NBOMe as well as all 15 metabolites identified in mouse hepatic microsomal preparations. Another human urine sample contained no parent 25I-NBOMe, but was found to contain three O-desmethyl metabolites. We recommend β-glucuronidase enzymatic hydrolysis of urine prior to 25I-NBOMe screening and the use of M5 as the primary biomarker in drug testing.

Podocytopathy and associated nephrotic syndrome (NS) have been reported in a knockout mouse strain (Asah1fl/fl/PodoCre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy of these mice remains unknown. The present study tested whether exosome release from podocytes is enhanced due to Asah1 gene knockout, which may serve as a pathogenic mechanism switching on podocytopathy and associated NS in Asah1fl/fl/PodoCre mice. We first demonstrated the remarkable elevation of urinary exosome excretion in Asah1fl/fl/PodoCre mice compared with WT/WT mice, which was accompanied by significant Annexin-II (an exosome marker) accumulation in glomeruli of Asah1fl/fl/PodoCre mice, as detected by immunohistochemistry. In cell studies, we also confirmed that Asah1 gene knockout enhanced exosome release in the primary cultures of podocyte isolated from Asah1fl/fl/PodoCre mice compared to WT/WT mice. In the podocytes from Asah1fl/fl/PodoCre mice, the interactions of lysosome and multivesicular body (MVB) were demonstrated to be decreased in comparison with those from their control littermates, suggesting reduced MVB degradation that may lead to increase in exosome release. Given the critical role of transient receptor potential mucolipin 1 (TRPML1) channel in Ca2+-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca2+ release through TRPML1 channels is inhibited in the podocytes of Asah1fl/fl/PodoCre mice. By GCaMP3 Ca2+ imaging, it was found that lysosomal Ca2+ release through TRPML1 channels was substantially suppressed in podocytes with Asah1 gene deletion. As an Ac product, sphingosine was found to rescue TRPML1 channel activity and thereby recover lysosome-MVB interaction and reduce exosome release of podocytes from Asah1fl/fl/PodoCre mice. Combination of N, N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, and sphingosine significantly inhibited urinary exosome excretion of Asah1fl/fl/PodoCre mice. Moreover, rescue of Aash1 gene expression in podocytes of Asah1fl/fl/PodoCre mice showed normal ceramide metabolism and exosome secretion. Based on these results, we conclude that the normal expression of Ac importantly contributes to the control of TRPML1 channel activity, lysosome-MVB interaction, and consequent exosome release from podocytes. Asah1 gene defect inhibits TRPML1 channel activity and thereby enhances exosome release, which may contribute to the development of podocytopathy and associated NS.

The NBOMes (N-benzyl-oxy-methyl derivatives of known 2C phenylethylamines) are a new and growing class of potent synthetic stimulants. Case reports provide the bulk of available safety and clinical data for clinicians. We report two cases of NBOMe intoxication with 25C-NBOMe (the first lab-confirmed US case) and 25B-NBOMe, respectively, both confirmed via triple quadrapole mass spectrometry.Case 1: A 16-year-old girl had a generalized seizure after reported use of 25I-NBOMe. She presented with altered mental status, lower extremity rigidity, and elevated CPK (6042 U/L). Despite treatment with benzodiazepines, her lower extremity rigidity persisted and CPK peaked at 47,906 U/L. She was discharged on hospital day 8. Serum and urine specimens confirmed presence of 25C-NBOMe. Case 2: A 15-year-old boy developed bizarre behavior after reported use of 25I-NBOMe. In the ED, he had two generalized seizures and persistent muscle rigidity. CPK peaked at 429 U/L. Seizures were managed with benzodiazepines, and he was discharged within 24 h. Serum specimens revealed 25B-NBOMe.NBOMes are amphetamine derivatives and highly potent 5-HT(2A) receptor agonists. Clinical manifestations are a product of enhanced central sympathetic and serotonergic tone. We report two cases of NBOMe intoxication in patients who believed they used 25I-NBOME, while lab confirmation proved otherwise. Whether unique clinical manifestations are specific to the NBOMe variant, dose, route of administration, or other factors is unknown. Laboratory confirmation may play a role in identifying unexpected NBOMe variants, while contributing to the epidemiologic data on these novel substances.

A new class of synthetic hallucinogens called NBOMe has emerged, and reports of adverse effects are beginning to appear. We report on a case of a suicide attempt after LSD ingestion which was analytically determined to be 25I-NBOMe instead. Clinicians need to have a high index of suspicion for possible NBOMe ingestion in patients reporting the recent use of LSD or other hallucinogens.

The blue lotus flower (Nymphea caerulea) is an Egyptian water lily containing apomorphine and nuciferine. Apomorphine has been described as a psychoactive alkaloid and is a non-selective dopamine agonist primarily used to treat Parkinson's disease as it stimulates dopamine receptors and improves motor function. Nuciferine is an alkaloid associated with dopamine receptor blockade. Today, blue lotus flower is used as a sleep aid and anxiety reliever. The rebuildable dripping atomizer (RDA) is an electronic cigarette that allows direct application of an e-liquid onto the coil in the atomizer for aerosolization, compared to a typical electronic cigarette where the e-liquid is wicked from a storage vessel to the coil. Our laboratory received a dark-brown resin material from a concerned parent. The resin had been confiscated from an adolescent who had a reported history of marijuana use. The resin was later identified as blue lotus flower (N. caerulea). This resin, together with four commercially available blue lotus products, was analyzed for content. Apomorphine was detected in two samples, and nuciferine was detected in all five samples. The confiscated resin was determined to contain no apomorphine and 4300 ng/g of nuciferine. The nuciferine resin was shown to aerosolize using aRDA electric cigarette.

Electronic cigarettes (e-cigarettes) were developed as an alternative method for nicotine delivery and had a significant surge in popularity. E-liquids are formulations used in e-cigarettes, and consist of a ratio of propylene glycol (PG) and vegetable glycerin (VG), a pharmaceutical and/or herbal remedy and, usually, a flavoring agent. Presented is the evaluation of nine cannabidiol (CBD) e-liquids from a single manufacturer for cannabinoids and other psychoactive compounds by Direct Analysis in Real Time Mass Spectrometry (DART-MS) and Gas Chromatography Mass Spectrometry (GC/MS). The analysis of these products resulted in the detection of CBD in all nine produces and the unexpected detections of 5-fluoro MDMB-PINACA (5F-ADB) in four of the products and dextromethorphan (DXM) in one of the products. The analysis of these products illustrates the potential quality control issues that can occur in an unregulated industry. CBD products are believed by many users to offer heath benefits, but the detection of a dangerous cannabimimetic, 5F-ADB, and DXM in these products illustrates the need for oversight.

Cocaine continues to be one of the most widespread abused illicit drugs in the USA. Rapid methods are needed for the identification and quantitation of cocaine and its metabolites, benzoylecgonine (BE), ecgonine methyl ester (EME) and cocaethylene (CE), in biological specimens by clinical and forensic toxicology laboratories. Presented is a differential ion mobility spectrometry–tandem mass spectrometry (DMS–MS-MS) method for the analysis of cocaine and its major metabolites in human serum that requires minimal sample preparation and no column chromatography. A Shimadzu Nexera X2 ultra-high performance liquid chromatography system was used to infuse the samples into the DMS cell at a rate of 30 μL/min. Separation of cocaine and its metabolites were performed in a SelexION DMS component from Sciex coupled to a QTRAP 6500 with an IonDrive Turbo V source for TurbolonSpray® using acetonitrile as a chemical modifier. Analysis consisted of ramping the CoV from −35 V to −6 V while monitoring the multiple reaction monitoring (MRM) transitions of each analyte. The assay was evaluated for linearity, bias, precision, carryover, interferences and stability. Calibration curves ranged from 10 to 1,000 ng/mL with linear regression correlation coefficients (r(2)) of 0.9912 or greater for each analyte. The limit of quantitation was set at 10 ng/mL. Intra-day precision (%CV) ranged from 0% to 15% for cocaine, 1% to 19% for BE, 1% to 17% for EME and 0% to 18% for CE. Inter-day precision ranged from 9% to 14% for cocaine, 2% to 17% for BE, 5% to 11% for EME and 5% to 15% for CE. No carryover or interferences were detected. Bland–Altman analysis of previously analyzed specimens by UPLC–MS-MS showed variability of 30% or less. The method demonstrates the applicability of DMS–MS-MS for high throughout analysis of drugs and their metabolites in clinical and forensic toxicology laboratories.

Synthetic cannabinoids (SCs) are a significant public health concern given their widespread use and severe effects associated with intoxication. However, there is a paucity of controlled human studies investigating the behavioral and physiological effects and pharmacokinetics of these compounds. Designing a reliable method to administer consistent, concentration-dependent synthetic cannabinoids is an integral component of controlled study of these compounds. Further, optimizing methods to assess the parent compounds and metabolites in plasma is critical in order to be able to establish their pharmacokinetics after administration. To develop a reliable method to administer smokable, concentration-dependent SCs, cigarettes were prepared with plant matter adulterated with increasing concentrations of the first generation cannabinoids found in SC products, JWH-018 and JWH-073. Cigarettes were assessed 1-6 months after preparation using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine compound stability over time and concentration consistency throughout the cigarettes. Optimal conditions to detect metabolites in human plasma as a function of storage temperature (-4 °C to -80 °C) and time (24 h - 1 month) were also determined. Analyses verified that the method utilized to develop SC cigarettes yielded consistent, concentration-dependent products within 25% of the expected concentrations. JWH-018, JWH-073 and metabolites in spiked plasma were stable under the time and temperature conditions; concentrations were within ±20% of target values. These studies provide techniques and methods to conduct controlled investigations of the dose-dependent effects of first generation SCs to begin understanding risks associated with use. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'

Abuse of synthetic stimulant compounds resulting in significant toxicity is being increasingly reported by poison centers. Toxicologic assessment is complicated by inconsistent manufacturing processes and limited laboratory testing. We describe a case of self-reported exposure to 25-I (25I-NBOMe), a novel phenethylamine derivative, with subsequent quantification in serum.An 18-year-old male presented to the emergency department (ED) with severe agitation and hallucinations after jumping out of a moving car. He was tachycardiac (150-160 bpm) and hypertensive (150-170 mm Hg systolic and 110 mg Hg diastolic), and required physical restraints and treatment with intravenous lorazepam administration. His symptoms gradually improved and vital signs returned to normal over 48 h, though he continued to have episodes of aggressiveness. An assay was developed by our analytical toxicology laboratory for 25-I, and serum obtained during ED evaluation and treatment was found to contain 0.76 ng/ml of 25-I. Case discussion. For 25I-NBOMe, 25-I is a common abbreviation for 25I-NBOMe, which is a (n-benzyl) phenethylamine in the 2C "family."Initially synthesized for research, cases of self-reported use of 25-I have recently appeared in the literature, some of which contain qualitative urine confirmation. There are no commercially available quantitative assays, and no previous reports have published serum concentrations. 25-I is a potent new synthetic drug with apparent significant behavioral toxicity that can be detected and quantified in serum.

Ethanol is the most widely used and abused drug. While blood is the preferred specimen for analysis, tissue specimens such as brain serve as alternative specimens for alcohol analysis in post-mortem cases where blood is unavailable or contaminated. A method was developed using headspace gas chromatography with flame ionization detection (HS-GC-FID) for the detection and quantification of ethanol, acetone, isopropanol, methanol and n-propanol in brain tissue specimens. Unfixed volatile-free brain tissue specimens were obtained from the Department of Pathology at Virginia Commonwealth University. Calibrators and controls were prepared from 4-fold diluted homogenates of these brain tissue specimens, and were analyzed using t-butanol as the internal standard. The chromatographic separation was performed with a Restek BAC2 column. A linear calibration was generated for all analytes (mean r2 > 0.9992) with the limits of detection and quantification of 100-110 mg/kg. Matrix effect from the brain tissue was determined by comparing the slopes of matrix prepared calibration curves with those of aqueous calibration curves; no significant differences were observed for ethanol, acetone, isopropanol, methanol and n-propanol. The bias and the CVs for all volatile controls were ≤10%. The method was also evaluated for carryover, selectivity, interferences, bench-top stability and freeze-thaw stability. The HS-GC-FID method was determined to be reliable and robust for the analysis of ethanol, acetone, isopropanol, methanol and n-propanol concentrations in brain tissue, effectively expanding the specimen options for post-mortem alcohol analysis.

The disposition in mice of the cannabimimetics JWH-018 and JWH-073 in blood and brain following inhalation of the smoke from the herbal incense product (HIP) “Magic Gold” containing 3.6% JWH-018, 5.7% JWH-073 and less than 0.1% JWH-398 (w/w) is presented. Specimens were analyzed by HPLC/MS/MS. The validation of the method is also presented. Five C57BL6 mice were sacrificed 20 min after exposure to the smoke of 200 mg of “Magic Gold” and a second set of five exposed mice were sacrificed after 20 h. Twenty minutes after exposure to “Magic Gold” smoke, blood concentrations of JWH-018 ranged from 42 to 160 ng/mL (mean: 88 ng/mL ± 42) and those of JWH-073 ranged from 67 to 244 ng/mL (mean: 134 ng/mL ± 62). Brain concentrations 20 min after exposure to “Magic Gold” smoke for JWH-018 ranged from 225 to 453 ng/g (mean: 317 ng/g ± 81) and those of JWH-073 ranged from 412 to 873 ng/g (mean: 584 ng/g ± 163). Twenty hours after exposure to “Magic Gold” smoke, JWH-018 was detected and quantified in only two of the five blood samples. Blood concentrations of JWH-018 were 3.4 ng/mL and 9.4 ng/mL. JWH-073 was detected in only one blood specimen 20 h after exposure at 4.3 ng/mL. Brain concentrations 20 h post exposure for JWH-018 ranged from 7 to 32 ng/g (mean: 19 ng/g ± 9). JWH-073 was not detected in 20 h post exposure brain specimens. JWH-398 was not detected in any of the blood or brain samples. The disposition data presented with the limited data available from human experience provide reasonable expectations for forensic toxicologists in JWH-018 or JWH-073 cases. As with THC after smoking marijuana, blood and brain concentrations of JWH-018 and JWH-073 after HIP smoking can be expected to rise initially to readily detected values, and then drop dramatically over the next few hours to several ng/mL or ng/g, and finally to be at extremely low or undetectable concentrations by 24 h apparently due to extensive biotransformation, and redistribution to body fat.

Sir,Since 2012, dimethoxyphenyl-N-[(2-methoxyphenyl)methyl]ethanamine (NBOMe) derivatives, a new class of designer hallucinogens, have become a threat to public health and safety.[1,2] The most com...

In the last two years, an epidemic of fatal narcotic overdose cases has occurred in the Tampa area of Florida. Fourteen of these deaths involved fentanyl and/or the new designer drug, acetyl fentanyl. Victim demographics, case histories, toxicology findings and causes and manners of death, as well as, disposition of fentanyl derivatives and their nor-metabolites in postmortem heart blood, peripheral blood, bile, brain, liver, urine and vitreous humor are presented. In the cases involving only acetyl fentanyl (without fentanyl, n=4), the average peripheral blood acetyl fentanyl concentration was 0.467 mg/L (range 0.31 to 0.60 mg/L) and average acetyl norfentanyl concentration was 0.053 mg/L (range 0.002 to 0.086 mg/L). In the cases involving fentanyl (without acetyl fentanyl, n=7), the average peripheral blood fentanyl concentration was 0.012 mg/L (range 0.004 to 0.027 mg/L) and average norfentanyl blood concentration was 0.001 mg/L (range 0.0002 to 0.003 mg/L). In the cases involving both acetyl fentanyl and fentanyl (n=3), the average peripheral blood acetyl fentanyl concentration was 0.008 mg/L (range 0.006 to 0.012 mg/L), the average peripheral blood acetyl norfentanyl concentration was 0.001 mg/L (range 0.001 to 0.002 mg/L), the average peripheral blood fentanyl concentration was 0.018 mg/L (range 0.015 to 0.021mg/L) and the average peripheral blood norfentanyl concentration was 0.002 mg/L (range 0.001 mg/L to 0.003 mg/L). Based on the toxicology results, it is evident that when fentanyl and/or acetyl fentanyl were present, they contributed to the cause of death. A novel ultrahigh performance liquid chromatography (UPLC) tandem mass spectrometry (MS/MS) method to identify and quantify acetyl fentanyl, acetyl norfentanyl, fentanyl and norfentanyl in postmortem fluids and tissues is also presented.

A new class of synthetic hallucinogens called NBOMe has emerged as drugs of abuse.Our aim was to conduct a systematic review of published reports of toxicities associated with NBOMe ingestion.We searched PubMed for relevant English-language citations that described adverse effects from analytically confirmed human NBOMe ingestion. Demographic and clinical data were extracted.A total of 10 citations met the criteria for inclusion, representing 20 individual patients. 25I-NBOMe was the most common analogue identified, followed by 25B-NBOMe and 25C-NBOMe. Fatalities were reported in 3 (15%) cases. Of all the patients, 7 (35%) were discharged after a period of observation, whereas 8 (40.0%) required admission to an intensive care unit. The most common adverse effects were agitation (85.0%), tachycardia (85.0%), and hypertension (65.0%). Seizures were reported in 8 (40.0%) patients. The most common abnormalities reported on laboratory tests were elevated level of creatinine kinase (45.0%), leukocytosis (25.0%), and hyperglycemia (20.0%).NBOMe ingestion is associated with severe adverse effects. Clinicians need to have a high index of suspicion for NBOMe ingestion in patients reporting the recent use of hallucinogens.

A new Hydrocodone Enzyme Immunoassay (HEIA; Lin-Zhi International, Inc.) was evaluated for the detection of hydrocodone and its main metabolite, hydromorphone. All specimens were tested with two different cutoff calibrators, 100 and 300 ng/mL, on an ARCHITECT Plus c4000 Clinical Chemistry Analyzer. Controls containing -25% (negative control) and +25% (positive control) of the cutoff calibrators and a drug-free control were analyzed with each batch. All 1,025 urine specimens were previously analyzed by ultra-performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS-MS) for opiates. Approximately, 33% (337/1,019) of the specimens yielded positive results by the HEIA assay at a cutoff concentration of 100 ng/mL and 19% (190/1,025) yielded positive results at the 300 ng/mL cutoff concentration. Of these presumptive positive specimens, UPLC-MS-MS confirmed the presence of hydrocodone and/or hydromorphone >100 ng/mL in 241 specimens and >300 ng/mL in 162 specimens, for each respective cutoff. With the 100 ng/mL cutoff, the HEIA demonstrated a sensitivity of 0.959, a specificity of 0.846 and an overall agreement with the UPLC-MS-MS of 87%. At 300 ng/mL cutoff, the HEIA demonstrated a sensitivity of 0.880, a specificity of 0.966 and an overall agreement of UPLC-MS-MS results of 95%. The Lin-Zhi HEIA 100 ng/mL cutoff assay demonstrated sensitivity for the detection of hydrocodone and hydromorphone in urine. The 300 ng/mL cutoff was less sensitive, but more selective, and should be part of an initial immunoassay screen, particularly in pain management compliance testing.

Poklis and Backer published a survey of the concentrations of fentanyl and norfentanyl that could be expected in urine from patients using Duragesic®, a transdermal fentanyl patch. That study employed a relatively small number of patient data points and analysis by Gas Chromatography/Mass Spectrometry. This work examines a larger population of patient positives for fentanyl and norfentanyl to determine whether more than a decade later the original report remains accurate in predicting the range and median levels of fentanyl and norfentanyl concentrations physicians can expect to see from their patients. Additionally, these data were transformed to develop a model that results in a near Gaussian distribution of urine drug test results. This retrospective approach was developed to transform and normalize urine drug testing results to provide a historical picture of expected patient values for this important analgesic. The resulting near Gaussian distribution is dose independent and as such should be of value to physicians in quickly assessing whether their patient is consistent with this historical population in the broad terms of this model. While this comparison alone is not definitive for adherence with a treatment regimen, together with patient interviews, prescription history and other clinical criteria, it can add an idea of expected patient values from urine drug testing.

We present a high-performance liquid chromatography triple quadrupole mass spectrometry (HPLC-MS-MS) method for the identification and quantification of nine serotonin 5-HT2A receptor agonist hallucinogenic substances from a new class of N-methoxybenzyl derivatives of methoxyphenylethylamine (NBOMe) designer drugs in human urine: 25H-NBOMe, 2CC-NBOMe, 25I-NBF, 25D-NBOMe, 25B-NBOMe, 2CT-NBOMe, 25I-NBMD, 25G-NBOMe and 25I-NBOMe. This assay was developed for the Virginia Commonwealth University Clinical and Forensic Toxicology laboratory to screen emergency department specimens in response to an outbreak of N-benzyl-phenethylamine derivative abuse and overdose cases in Virginia. The NBOMe derivatives were rapidly extracted from the urine specimens by use of FASt™ solid-phase extraction columns. Assay performance was determined as recommended for validation by the Scientific Working Group for Forensic Toxicology (SWGTOX) for linearity, lower limit of quantification, lower limit of detection, accuracy/bias, precision, dilution integrity, carryover, selectivity, absolute recovery, ion suppression and stability. Linearity was verified to be from 1 to 100 ng/mL for each of the nine analytes. The bias determined for the NBOMe derivatives was 86-116% with a

BACKGROUND: We evaluated a new EDDP [2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine] enzyme immunoassay (EDDPI; Lin‐Zhi International, Inc., Sunnyvale, CA) for the detection of this primary methadone urinary metabolite. METHODS: All specimens were tested with two different cutoff calibrators at 150 and 300 ng/ml EDDP on an ADVIA 1200 Chemistry System auto‐analyzer. Controls containing 0, −25% (negative control), and +25% (positive control) of the cutoff calibrators (Lin‐Zhi) were analyzed with each batch. All urine specimens were then analyzed by high‐pressure liquid chromatography/ mass spectrometry/mass spectrometry (HPLC‐MS/MS) for EDDP. RESULTS: Approximately, 42% (151) of the 362 specimens yielded positive results by the EDDP assay at 150 and/or 300 ng/ml cutoff values. Of these specimens, HPLC‐MS/MS confirmed the presence of EDDP > 25 ng/ml in all 151 specimens. No specimen yielding negative EDDPI results contained EDDP by HPLC/MS/MS. At 150 ng/ml cutoff, the EDDPI demonstrated a sensitivity of 1.00, a specificity of 0.986, and an overall agreement of HPLC/MS/MS of >99%. At 300 ng/ml cutoff, the EDDPI demonstrated a sensitivity of 1.00, a specificity of 0.959, and an overall agreement of HPLC/MS/MS results of 97.5%. CONCLUSION: The Lin‐Zhi EDDPI provides a precise, reliable method for the routine detection of methadone metabolite in urine specimens, particularly in pain management compliance testing.

We present a traumatic fatality of a 19-year-old man who had ingested blotter paper containing 25I-NBOMe [2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine]. Postmortem specimens were analyzed by high performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS). Toxicology findings for fluids based upon blood or urine calibrators were as follows: peripheral blood, 405 pg/mL; heart blood, 410 pg/mL; urine, 2.86 ng/mL; and vitreous humor, 99 pg/mL. While findings based upon the method of standard additions were: gastric contents, 7.1 μg total; bile, 10.9 ng/g; brain, 2.54 ng/g and liver, 7.2 ng/g. To our knowledge the presented case is the first postmortem case of 25I-NBOMe intoxication documented by toxicological analysis of tissues and body fluids.

We present a case of 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (25B-NBOMe), an N-benzyl phenethylamines derivative, intoxication and a high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) method for detection and quantification of 25B-NBOMe. A 19-year-old male was found unresponsive with generalized grand mal seizure activity. On the second day of hospitalization, a friend admitted that the patient used 'some unknown drug' called 25B. Serum and urine collected were sent to the Virginia Commonwealth University Medical Center Toxicology Laboratory for analysis. An HPLC-MS/MS method for the identification and quantification of 25B-NBOMe using 2-(2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25H-NBOMe) as the internal standard (ISTD) was developed. As this is a novel, single-case presentation, an assay validation was performed prior to testing to ensure the reliability of the analytical results. The serum and urine specimens were determined to contain 180 pg/ml and1900 pg/ml of 25B-NBOMe, respectively.

2CC-NBOMe (4-chloro-2,5-dimethoxyphenethyl-N-[(2-methoxyphenyl) methyl] ethanamine) and 25I-NBOMe (2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl) methyl] ethanamine) are of a class of N-benzyl phenethylamine derivatives whose synthesis was first reported in the scientific literature in 2011. Recent reports from “personal drug experience websites” and in the popular press indicate these drugs are the latest in a series of designer “Bath Salt” drugs of abuse. The presented high performance liquid chromatography triple quadrupole mass spectrometry (HPLC/MS/MS) method was developed for the detection and quantification of 2CC-NBOMe and 25I-NBOMe in serum of intoxicated emergency department patients. The assay applies 2-(2,5-dimethoxyphenyl)-N-(2-methoxybenzyl) ethanamine (25H-NBOMe) as the internal standard (ISTD). Samples were extracted using solid phase extraction (SPE) columns. The chromatographic separation was performed on a Luna 3μ C8 (2)100Å 100×2.0 mm, column. Detection was accomplished by multiple-reaction monitoring (MRM) via electrospray ionization (ESI) source operating in the positive ionization mode. The calibration curves were linear over the investigated concentration range, 30 to 2000 pg/mL, with a lower limit of detection (LOD) of 10 pg/mL for both 2CC-NBOMe and 25I-NBOMe. The method proved suitable for serum clinical toxicology testing. Two severely intoxicated emergency department patients were determined to have serum concentrations of 250 pg/mL and 2780 pg/mL of 25I-NBOMe using the presented method.

The CB1 receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ(9)-tetrahydrocannabinol and other CB1 orthosteric receptor agonists remain limited because of psychoactive side effects. Positive allosteric modulators (PAMs) offer an alternative approach to enhance CB1 receptor function for therapeutic gain with the promise of reduced side effects. Here we describe the development of the novel synthetic CB1 PAM, 6-methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011), which augments the in vitro and in vivo pharmacological actions of the CB1 orthosteric agonists CP55,940 and N-arachidonoylethanolamine (AEA). ZCZ011 potentiated binding of [(3)H]CP55,940 to the CB1 receptor as well as enhancing AEA-stimulated [(35)S]GTPγS binding in mouse brain membranes and β-arrestin recruitment and ERK phosphorylation in hCB1 cells. In the whole animal, ZCZ011 is brain penetrant, increased the potency of these orthosteric agonists in mouse behavioral assays indicative of cannabimimetic activity, including antinociception, hypothermia, catalepsy, locomotor activity, and in the drug discrimination paradigm. Administration of ZCZ011 alone was devoid of activity in these assays and did not produce a conditioned place preference or aversion, but elicited CB1 receptor-mediated antinociceptive effects in the chronic constriction nerve injury model of neuropathic pain and carrageenan model of inflammatory pain. These data suggest that ZCZ011 acts as a CB1 PAM and provide the first proof of principle that CB1 PAMs offer a promising strategy to treat neuropathic and inflammatory pain with minimal or no cannabimimetic side effects.

Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [(35)S]GTPγS (guanylyl-5'-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [(35)S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist-stimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.

A simple high-performance liquid chromatographic (HPLC) method that allows for the rapid identification and quantification of analgesic and anesthetic solutions typically used in surgical procedures or patient controlled analgesia is presented. The separation of bupivacaine, clonidine, fentanyl, hydromorphone, midazolam, and morphine is complete in less than 20 min. The method allows test solutions to be either directly injected or diluted prior to injection into the HPLC system. The method is useful from the standpoint that pharmaceutical preparations are usually submitted with the known drug of interest and expected concentration. The method is also useful for initial screening of solutions submitted that are either unknown or of questionable identity. The method has been successfully applied as part of hospital-based quality control and quality assurance programs to detect not only errors in the preparation of solutions of scheduled drugs, but also to uncover illegal diversion of drugs of abuse by medical personnel.

We present an evaluation of a new phencyclidine (PCP) enzyme immunoassay (PCPI) for detection in urine. The PCPI was evaluated by testing 523 urine specimens. Controls containing 0 ng/mL of PCP and -25% (negative control) and +25% (positive control) of the 25 ng/mL cutoff calibrator were analyzed with each batch. All urines were analyzed by high-performance liquid chromatography- tandem mass spectrometry (HPLC-MS-MS) for PCP. Of the 523 specimens tested, 218 yielded positive results by the PCP assay. HPLC-MS-MS confirmed the presence of at least 25 ng/mL in 214 specimens, indicating four false-positive results containing 11, 13, 16, and 20 ng/mL PCP. Three specimens yielded a negative result; however, PCP concentrations were within 20% of the 25 ng/mL cutoff value. The overall agreement of PCPI and HPLC-MS-MS results was 98.7%. The sensitivity of the PCPI was 0.982 and the specificity 0.987. Testing at 100 ng/mL of other drugs or their metabolites demonstrated no cross-reactivity. The within-run precision was CV = 2% (n = 12); the between-run precision was CV = < 6% (n = 4). The assay was found linear from -50% to 150% (12.5-37.5 ng/mL) of the cutoff concentration. The Lin-Zhi PCPI provides a precise, reliable method for the routine detection of phencyclidine in urine specimens.

On-site drug tests are becoming increasingly more popular because of their easy test protocols and instantaneous results. This study evaluates the performance of four on-site drug testing devices that use competitive binding immunoassays to qualitatively determine the presence of drugs in urine: Triage | Panel for Drugs of Abuse plus TCA, QuickScreen TM Pro-Multi Drug Screening Tests, Syva | Rapid Test d.a.u. TM 5 and d.a.u. TM 2, and Rapid Drug Screen TM. All devices simultaneously determine the presence of the following drugs of abuse: amphetamine (AMP), benzoylecgonine (BE), 11-nor-9-carboxy-A%tetrahydrocannabinol (THCA), opiates (OPI), and phencyclidine (PCP). Triage and Rapid Drug Screen also simultaneously test for benzodiazepines (BZB) and barbiturates (BRB), whereas QuickScreen and Rapid Test require separate devices for the BZB and BRB analyses. Urine specimens (222) containing drug concentrations around or above cutoff values were screened by ONLINE or EMIT II immunoassays. Of these, 199 yielded positive gas chromatography-mass spectrometry results with at least 17 positive specimens in each drug class. Specimens with the target drugs added at 16.7% above and below the cutoff, 33.3% above and below the cutoff, and 66.7% above the cutoff were also used to evaluate the test devices. Sensitivity and specificity calculations demonstrated that Triage performed most predictably in the donor urine specimens and the drug-added specimens. In addition, it required the least amount of test volume and was the only device in which the appearance of a colored line indicated a positive result. Therefore, of the devices studied, Triage was the most dependable and reproducible on-site drug-screening device. screen for illicit drug usage. Primarily, drug screening eliminates the specimens/individuals who tested negative for certain drugs such that the more complex and time-consuming confirmation process is not overburdened. However, in the emergency room, screening also serves to identify drugs so that a patient will receive the appropriate treatment. Drug rehabilitation programs and justice departments use on-site screening devices to immediately verify compliance, and employers randomly screen employees to curtail absenteeism and workplace accidents. Therefore, on-site urine drug-screening devices need to be reliable and rapid. They must also be cost effective and simple to use because testing may be performed outside the laboratory by individuals with little training. On-site drug-screening devices attempt to fulfill these requirements. The purpose of this study was to evaluate the ability of four on-site drug-screening devices to detect the presence of amphetamine (AMP), benzoylecgonine (BE), 11-nor-9-carboxyA9-tetrahydrocannabinol (THCA), opiates (OPI), phencyclidine (PCP), benzodiazepines (BNZ), and barbiturates (BRB) in urine: Triage Panel for Drugs of Abuse plus TCA, QuickScreen Pro-Multi Drug Screening Tests, Syva Rapid Test d.a.u. 5 and d.a.u. 2, and Rapid Drug Screen. All devices simultaneously determined the presence of five drugs of abuse: AMP, BE, THCA, OPI, and PCP. Triage and Rapid Drug Screen also simultaneously detected the presence of BNZ and BRB. QuickScreen and Rapid Test required separate test cards for BNZs and BRBs. Triage can also determine the presence of tricyclic antidepressants (TCA). However, TCAs were not analyzed in this study (2). All screening results were confirmed by gas chromatography in tandem with mass spectrometry (GC-MS).

Guanfacine (Tenex), an antihypertensive available since 1975, has recently been indicated for the treatment of attention deficit hyperactivity disorder in children (Intuniv). Because of this new usage, a gas chromatography-mass spectrometry method was developed and validated for the determination of guanfacine in urine. Guanfacine and 100 ng of protriptyline (internal standard) were extracted from 1.0 mL urine with 0.5 mL of saturated carbonate/bicarbonate buffer and 2 mL of ethyl acetate. The solvent extract was evaporated and derivatized with heptaflurobutyric anhydride in n-butyl chloride. Chromatographic separation was achieved using a DB-5 capillary column (30 m x 0.32 mm, 0.25 microm). Ions monitored for guanfacine were m/z 86.1, 272.1, and 274.1, and ions monitored for protriptyline were m/z 191.1 and 189.1. Concentrations were determined using calibrators over the range of 0.1-2.0 mg/L. The linear regression for all calibration curves had r2 values > or = 0.99. The limit of detection was 0.05 mg/L; limit of quantitation was 0.1 mg/L; and upper limit of linearity was 10.0 mg/L. Percent recovery of guanfacine at 0.1 and 2.0 mg/L was 93% and 71%, respectively. The method was found acceptable for routine quantitative analysis of guanfacine in urine.

I A diagnostic system to predict the presence of 6-acetylmorphine (6AM) in opiate-positive urines was recently proposed. A twofold criterion based on the total morphine concentration and the total codeine to total morphine concentration ratio was identified. Using relative operating characteristic (ROC) analysis, it was determined that the diagnostic system had a sensitivity of 92%, a specificity of 79%, and an overall accuracy of 73%. We applied similar decision criteria to a study population of 125 opiate- positive urines collected from criminal justice clients of a West Coast reference laboratory. ROC analysis on this population produced very different results: a sensitivity of 91%, a specificity of 49%, and an accuracy of 45%. These data illustrate the importance of choosing a representative study population without any selection biases that may compromise the validity of the accuracy measure. The ROC plot is an important tool for assessing a clinical test's performance, but in order for toxicologists and Medical Review Officers to benefit from the diagnostic test results, they must also know the predictive value (based on test accuracy and the prevalence of heroin use) of the test results. They need to know how well the test predicts the presence of 6AM, and therefore, the illicit use of heroin, in the population of interest whether it be workplace, criminal justice, hospital emergency department clients, or a combination of all populations.

Dehydroepiandrosterone (DHEA) is an endogenous androgenic steroid produced by the ovaries and adrenal glands. Research suggests that DHEA can be converted to testosterone in peripheral tissues. Classified as a nutritional supplement, this compound may be purchased without a prescription. The military and international sports organizations prohibit the use of exogenous androgenic/anabolic steroids. Steroid-screening results are considered "positive" when the urinary ratio of testosterone to epitestosterone (T/E), an inactive synthetic byproduct, exceeds 6:1. Human volunteers ingested the recommended daily dose of 50 mg each morning for 30 days to determine if DHEA causes an adverse effect on this ratio. Urinary samples were collected before ingestion and 2-3 h after ingestion. Urine samples were extracted using solid-phase columns and analyzed using a previously developed gas chromatography-mass spectrometry method. T/E results were compared to an average baseline generated from three urine samples obtained before the study. Mean baseline T/E ratios averaged 0.67 for the seven subjects (range 0.1-1.2). The mean T/E ratio after DHEA ingestion ranged from 0.03 to 2.11. Individual postdose T/E ratios ranged from 0.01 to 3.7. The results from these individuals indicate that the administration of DHEA at this dose, for this period of time, has a minimal effect on urine T/E ratios and would not be expected to result in a positive screen for testosterone abuse. One subject agreed to take a single dose of 250 mg. This acute, high dose caused his T/E ratio to increase by 40% relative to the predose value.