Brain Concentrations of Methylone and Its Metabolites after Systemic Methylone Administration: Relationship to Pharmacodynamic Effects.

3,4-Methylenedioxy-N-methylcathinone (methylone) is a new psychoactive substance with stimulant properties and potential for abuse. Despite its popularity, limited studies have examined relationships between brain concentrations of methylone, its metabolites, and pharmacodynamic effects. The goal of the present study was 2-fold: 1) to determine pharmacokinetics of methylone and its major metabolites-4-hydroxy-3-methoxy-N-methylcathinone (HMMC), 3,4-dihydroxy-N-methylcathinone (HHMC), and 3,4-methylenedioxycathinone (MDC)-in rat brain and plasma and 2) to relate brain pharmacokinetic parameters to pharmacodynamic effects including locomotor behavior and postmortem neurochemistry. Male Sprague-Dawley rats received subcutaneous methylone (6, 12, or 24 mg/kg) or saline vehicle (n = 16/dose), and subgroups were decapitated after 40 or 120 minutes. Plasma and prefrontal cortex were analyzed for concentrations of methylone and its metabolites by liquid chromatography-tandem mass spectrometry. Frontal cortex and dorsal striatum were analyzed for dopamine, 5-HT, and their metabolites by high-performance liquid chromatography-electrochemical detection. Brain and plasma concentrations of methylone and its metabolites rose with increasing methylone dose, but brain methylone and MDC concentrations were greater than dose-proportional. Brain-to-plasma ratios for methylone and MDC were ≥ 3 (range 3-12), whereas those for HHMC and HMMC were ≤ 0.2 (range 0.01-0.2). Locomotor activity score was positively correlated with brain methylone and MDC, whereas cortical 5-HT was negatively correlated with these analytes at 120 minutes. Our findings show that brain concentrations of methylone and MDC display nonlinear accumulation. Behavioral and neurochemical effects of systemically administered methylone are related to brain concentrations of methylone and MDC but not its hydroxylated metabolites, which do not effectively penetrate into the brain. SIGNIFICANCE STATEMENT: Behavioral and neurochemical effects of methylone are related to brain concentrations of methylone and its metabolite MDC but not its hydroxylated metabolites, 4-hydroxy-3-methoxy-N-methylcathinone and 3,4-dihydroxy-N-methylcathinone, which do not effectively penetrate into the brain. Methylone and MDC display nonlinear accumulation in the brain, which could cause untoward effects on serotonin neurons in vulnerable brain regions, including the frontal cortex.

Synthetic cathinone pharmacokinetics, analytical methods, and toxicological findings from human performance and postmortem cases.

Synthetic cathinones are commonly abused novel psychoactive substances (NPS). We present a comprehensive systematic review addressing in vitro and in vivo synthetic cathinone pharmacokinetics, analytical methods for detection and quantification in biological matrices, and toxicological findings from human performance and postmortem toxicology cases. Few preclinical administration studies examined synthetic cathinone pharmacokinetic profiles (absorption, distribution, metabolism, and excretion), and only one investigated metabolite pharmacokinetics. Synthetic cathinone metabolic profiling studies, primarily with human liver microsomes, elucidated metabolite structures and identified suitable biomarkers to extend detection windows beyond those provided by parent compounds. Generally, cathinone derivatives underwent ketone reduction, carbonylation of the pyrrolidine ring, and oxidative reactions, with phase II metabolites also detected. Reliable analytical methods are necessary for cathinone identification in biological matrices to document intake and link adverse events to specific compounds and concentrations. NPS analytical methods are constrained in their ability to detect new emerging synthetic cathinones due to limited commercially available reference standards and continuous development of new analogs. Immunoassay screening methods are especially affected, but also gas-chromatography and liquid-chromatography mass spectrometry confirmation methods. Non-targeted high-resolution-mass spectrometry screening methods are advantageous, as they allow for retrospective data analysis and easier addition of new synthetic cathinones to existing methods. Lack of controlled administration studies in humans complicate interpretation of synthetic cathinones in biological matrices, as dosing information is typically unknown. Furthermore, antemortem and postmortem concentrations often overlap and the presence of other psychoactive substances are typically found in combination with cathinones derivatives, further confounding result interpretation.

Linear pharmacokinetics of 3,4-methylenedioxypyrovalerone (MDPV) and its metabolites in the rat: relationship to pharmacodynamic effects.

3,4-Methylenedioxypyrovalerone (MDPV) is a commonly abused synthetic cathinone in the United States and is associated with dangerous side effects. MDPV is a dopamine transporter blocker that is 10-fold more potent than cocaine as a locomotor stimulant in rats. Previous in vitro and in vivo metabolism studies identified 3,4-dihydroxypyrovalerone (3,4-catechol-PV) and 4-hydroxy-3-methoxypyrovalerone (4-OH-3-MeO-PV) as the two primary MDPV metabolites. This study examined MDPV pharmacokinetics and metabolism, along with associated pharmacodynamic effects in rats receiving 0.5, 1.0 and 2.0 mg/kg subcutaneous (s.c.) MDPV. Blood was collected by an indwelling jugular catheter before dosing and at 10, 20, 30, 60, 120, 240 and 480 minutes thereafter. Plasma specimens were analyzed by liquid chromatography coupled to high-resolution tandem mass spectrometry. Maximum concentrations (Cmax ) and area-under-the-curve (AUC) for MDPV and two metabolites increased proportionally with administered dose, showing linear pharmacokinetics. MDPV exhibited the highest Cmax at all doses (74.2-271.3 µg/l) and 4-OH-3-MeOH-PV the highest AUC (11 366-47 724 minutes per µg/l), being the predominant metabolite. MDPV time to Cmax (Tmax ) was 12.9-18.6 minutes, while 3,4-catechol-PV and 4-OH-3-MeO-PV peaked later with Tmax 188.6-240 minutes after s.c. dosing. Horizontal locomotor activity (HLA) and stereotypy correlated positively with plasma MDPV concentrations, while HLA correlated negatively with MDPV metabolites. These results suggest that the parent compound mediates motor stimulation after systemic MDPV administration, but additionally, metabolites may be inhibitory, may not be active or may not pass the blood brain barrier.

Biochip array technology immunoassay performance and quantitative confirmation of designer piperazines for urine workplace drug testing.

Designer piperazines are emerging novel psychoactive substances (NPS) with few high-throughput screening methods for their identification. We evaluated a biochip array technology (BAT) immunoassay for phenylpiperazines (PNP) and benzylpiperazines (BZP) and analyzed 20,017 randomly collected urine workplace specimens. Immunoassay performance at recommended cutoffs was evaluated for PNPI (5 µg/L), PNPII (7.5 µg/L), and BZP (5 µg/L) antibodies. Eight hundred forty positive and 206 randomly selected presumptive negative specimens were confirmed by liquid chromatography high-resolution mass spectrometry (LC-HRMS). Assay limits of detection for PNPI, PNPII, and BZP were 2.9, 6.3, and 2.1 µg/L, respectively. Calibration curves were linear (R (2) > 0.99) with upper limits of 42 µg/L for PNPI/PNII and 100 µg/L for BZP. Quality control samples demonstrated imprecision <19.3 %CV and accuracies 86.0-94.5 % of target. There were no interferences from 106 non-piperazine substances. Seventy-eight of 840 presumptive positive specimens (9.3 %) were LC-HRMS positive, with 72 positive for 1-(3-chlorophenyl)piperazine (mCPP), a designer piperazine and antidepressant trazodone metabolite. Of 206 presumptive negative specimens, one confirmed positive for mCPP (3.3 µg/L) and one for BZP (3.6 µg/L). BAT specificity (21.1 to 91.4 %) and efficiency (27.0 to 91.6 %) increased, and sensitivity slightly decreased (97.5 to 93.8 %) with optimized cutoffs of 25 µg/L PNPI, 42 µg/L PNPI, and 100 µg/L BZP. A high-throughput screening method is needed to identify piperazine NPS. We evaluated performance of the Randox BAT immunoassay to identify urinary piperazines and documented improved performance when antibody cutoffs were raised. In addition, in randomized workplace urine specimens, all but two positive specimens contained mCPP and/or trazodone, most likely from legitimate medical prescriptions. Graphical Abstract Biochip array technology (BAT) immunoassay for designer piperazines detection in urine. In chemiluminescent immunoassay, the labeled-drug (antigen) competes with the drug in the urine. In the absence of drug, the labeled-drug binds to the antibody releasing an enzyme (horseradish peroxidase) to react with the substrate and producing chemiluminescence. The higher the drug concentration in urine, the weaker the chemiluminescent signal is produced. All presumptive positive specimens and randomly selected presumptive negative specimens were analyzed and confirmed by a liquid chromatography high-resolution mass spectrometry with limit of quantification of 2.5 or 5 µg/L.

Nonlinear pharmacokinetics of (+/-)3,4-methylenedioxymethamphetamine (MDMA) and its pharmacodynamic consequences in the rat.

3,4-Methylenedioxymethamphetamine (MDMA) is a widely abused illicit drug that can cause severe and even fatal adverse effects. However, interest remains for its possible clinical applications in posttraumatic stress disorder and anxiety treatment. Preclinical studies to determine MDMA's safety are needed. We evaluated MDMA's pharmacokinetics and metabolism in male rats receiving 2.5, 5, and 10 mg/kg s.c. MDMA, and the associated pharmacodynamic consequences. Blood was collected via jugular catheter at 0, 0.5, 1, 2, 4, 6, 8, 16, and 24 hours, with simultaneous serotonin (5-HT) behavioral syndrome and core temperature monitoring. Plasma specimens were analyzed for MDMA and the metabolites (±)-3,4-dihydroxymethamphetamine (HHMA), (±)-4-hydroxy-3-methoxymethamphetamine (HMMA), and (±)-3,4-methylenedioxyamphetamine (MDA) by liquid chromatography-tandem mass spectrometry. After 2.5 mg/kg MDMA, mean MDMA Cmax was 164 ± 47.1 ng/ml, HHMA and HMMA were major metabolites, and <20% of MDMA was metabolized to MDA. After 5- and 10-mg/kg doses, MDMA areas under the curve (AUCs) were 3- and 10-fold greater than those after 2.5 mg/kg; HHMA and HMMA AUC values were relatively constant across doses; and MDA AUC values were greater than dose-proportional. Our data provide decisive in vivo evidence that MDMA and MDA display nonlinear accumulation via metabolic autoinhibition in the rat. Importantly, 5-HT syndrome severity correlated with MDMA concentrations (r = 0.8083; P < 0.0001) and core temperature correlated with MDA concentrations (r = 0.7595; P < 0.0001), suggesting that MDMA's behavioral and hyperthermic effects may involve distinct mechanisms. Given key similarities between MDMA pharmacokinetics in rats and humans, data from rats can be useful when provided at clinically relevant doses.

Validation of a novel method to identify in utero ethanol exposure: simultaneous meconium extraction of fatty acid ethyl esters, ethyl glucuronide, and ethyl sulfate followed by LC-MS/MS quantification.

Presence of fatty acid ethyl esters (FAEE), ethyl glucuronide (EtG), and ethyl sulfate (EtS) in meconium, the first neonatal feces, identifies maternal alcohol consumption during pregnancy. Current meconium alcohol marker assays require separate analyses for FAEE and EtG/EtS. We describe development and validation of the first quantitative liquid chromatography tandem mass spectrometry assay for 9 FAEEs, EtG, and EtS in 100 mg meconium. For the first time, these alcohol markers are analyzed in the same meconium aliquot, enabling comparison of the efficiency of gestational ethanol exposure detection. 100 mg meconium was homogenized in methanol and centrifuged. The supernatant was divided, and applied to two different solid phase extraction columns for optimized analyte recovery. Limits of quantification for ethyl laurate, myristate, linolenate, palmitoleate, arachidonate, linoleate, palmitate, oleate, and stearate ranged from 25-50 ng/g, with calibration curves to 2,500-5,000 ng/g. EtG and EtS linear dynamic ranges were 5-1,000 and 2.5-500 ng/g, respectively. Mean bias and between-day imprecision were <15 %. Extraction efficiencies were 51.2-96.5 %. Matrix effects ranged from -84.7 to 16.0 %, but were compensated for by matched deuterated internal standards when available. All analytes were stable (within ±20 % change from baseline) in 3 authentic positive specimens, analyzed in triplicate, after 3 freeze/thaw cycles (-20 °C). Authentic EtG and EtS also were stable after 12 h at room temperature and 72 h at 4 °C; some FAEE showed instability under these conditions, although there was large inter-subject variability. This novel method accurately detects multiple alcohol meconium markers and enables comparison of markers for maternal alcohol consumption.

Quantification of cocaine and metabolites in exhaled breath by liquid chromatography-high-resolution mass spectrometry following controlled administration of intravenous cocaine.

Breath has been investigated as an alternative matrix for detecting recent cocaine intake; however, there are no controlled cocaine administration studies that investigated the drug's disposition into breath. Breath was collected from 10 healthy adult cocaine users by asking them to breathe into a SensAbues device for 3 min before and up to 22 h following 25 mg intravenous (IV) cocaine dosing on days 1, 5, and 10, and assayed with a validated liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method to quantify breath cocaine, benzoylecgonine (BE), ecgonine methyl ester (EME), and norcocaine. The assay was linear from 25 to 1,000 pg/filter, extraction efficiencies were 83.6-126%, intra- and inter-assay imprecision was <10.6%, and bias was between -8.5 and 16.8%. No endogenous or exogenous interferences were observed for more than 75 tested. Analytes were generally stable under short-term storage conditions. Ion suppression was less than 46%. Of breath specimens collected after controlled cocaine administration, 2.6% were positive for cocaine (26.1-66 pg/filter, 1-9.5 h), 0.72% BE (83.3-151 pg/filter, 6.5-12.5 h), and 0.72% EME (50-69.1 pg/filter, 6.5-12.5 h); norcocaine was not detected. Methanolic extraction of the devices themselves, after filters were removed, yielded 19.2% positive cocaine tests (25.2-36.4 pg/device, 10 min-22 h) and 4.3% positive BE tests (26.4-93.7 pg/device, 10 min-22 h), explaining differences between the two extraction techniques. These results suggest that the device reflects the drug in oral fluid as well as lung microparticles, while the filter reflects only drug-laden microparticles. A sensitive and specific method for cocaine, BE, EME, and norcocaine quantification in breath was developed and validated. Cocaine in breath identifies recent cocaine ingestion, but its absence does not preclude recent use.

The Impact of Combined Sewer Overflows on Pharmaceutical and Illicit Drug Levels in New York/New Jersey Waterways.

Pharmaceuticals and drugs of abuse are organic micropollutants of emerging concern in both surface and groundwater worldwide. These compounds are considered to be pseudo-persistent because of their continuous release into water systems. The presence of these compounds in the environment at any concentration poses a potential risk to nontarget organisms. The main sources of these contaminants are wastewater treatment plants (WWTPs) and combined sewer overflows (CSOs). The primary goal of our study was to identify and quantify a panel of 28 commonly prescribed pharmaceuticals (mood-altering drugs, cardiovascular drugs, antacids, antibiotics) and high-prevalence drugs of abuse (cocaine, amphetamines, opioids, cannabis) in river water samples collected from 19 locations in the Hudson and East rivers in New York City. The second goal was to investigate the possible source (WWTP or CSOs) of these micropollutants. Samples were collected weekly from May to August 2021 (n = 224) and May to August 2022 (n = 232), and placed at -20 °C until analysis by liquid chromatography-tandem mass spectrometry. The most frequently detected analytes in 2021 were metoprolol (n = 206, 92%), benzoylecgonine (n = 151, 67%), atenolol (n = 142, 63%), and methamphetamine (n = 118, 53%), and in 2022 the most frequently detected were methamphetamine (n = 194, 84%), atenolol (n = 177, 76%), metoprolol (n = 177, 76%), and 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine (n = 159, 69%). Measured concentrations ranged from the limit of detection (0.50-5.00 ng/L) to 103 ng/L. More drugs and higher concentrations were detected in water contaminated by Enterococci (>60 most probably number) and after rainfall, indicating the influence of CSOs. The presence of drugs in samples with little to no Enterococci and after dry weather events indicates that WWTPs contribute to the presence of these substances in the river, probably due to a low removal rate. Environ Toxicol Chem 2024;43:1592-1603. © 2024 SETAC.

Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method including 25 novel synthetic opioids in hair and subsequent analysis of a Swiss opioid consumer cohort.

Major public health concern is raised by the evidence that common drugs like heroin are now frequently laced or replaced with highly potent novel synthetic opioids (NSOs). The objective of this study was to explore the prevalence and patterns of NSOs in a cohort of Swiss opioid users by hair analysis. Hair analysis is considered an ideal tool for retrospective consumption monitoring. Hair samples from 439 opioid users in Zurich were analyzed. Study inclusion required a previous positive hair test result for heroin metabolites, oxycodone, fentanyl, methadone, or tramadol. The samples were extracted with a two-step extraction procedure, followed by a targeted LC-MS/MS (QTRAP® 6500+) analysis in multiple reaction monitoring mode for a total of 25 NSOs. The method underwent full validation and demonstrated good selectivity and sensitivity with limits of detection (LOD) as low as 0.1 pg/mg. The analyzed sample cohort demonstrated a positivity rate for NSOs of 2.5%, including the following NSOs: butyrylfentanyl, acrylfentanyl, furanylfentanyl, methoxyacetylfentanyl, ocfentanil, U-47700, isobutyrylfentanyl and benzylfentanyl. Furthermore, we were able to identify specific consumption patterns among drug users. The results indicate that hair analysis is a valuable tool for investigating the prevalence of NSOs in drug-using populations, which seems to be low in the case of Swiss opioid users. Nevertheless, the results highlight the need for sensitive analytical detection methods in forensic toxicology to identify and monitor substance distribution in different populations.

Maternal hair analysis for the detection of illicit drugs, medicines, and alcohol exposure during pregnancy.

BACKGROUND/OBJECTIVES: Drug of abuse consumption throughout pregnancy is a serious public health problem and an important economic cost to the health system. The aim of this work was to compare maternal interview and hair analysis to determine drug consumption throughout pregnancy and to study relations among maternal interview, hair results, and neonatal outcomes. METHODS: Two hundred nine mothers agreed to participate. After delivery, they were interviewed and a hair sample collected. Hair samples were segmented in trimesters and analyzed for 35 drugs [opioids, cocaine, amphetamines, Δ-tetrahydrocannabinol (THC), ketamine, methadone, antidepressants, benzodiazepines, and hypnotics; limits of quantification 5-100 pg/mg] and for ethyl glucuronide (limit of quantification 10 pg/mg) by liquid chromatography-tandem mass spectrometry. Statistical analysis was performed with χ test and t test. RESULTS: In the interview, 4.3% mothers declared using illicit drugs during pregnancy (cocaine 1.4%, THC 2.9%, and opiates 1%), 3.3% medicines (methadone 1.9%, benzodiazepines 1.9%, and antidepressants 0.5%), 21.5% tobacco, and 13.7% alcohol. Hair analysis showed 15.4% prevalence in illicit drugs (cocaine 12.4%, THC 3.8%, opiates 1%, and ketamine 1%), 22.5% in medicines (methadone 3.3%, benzodiazepines 11%, antidepressants 9.1%, zopiclone 1%, and fentanyl 1.4%), and 3.9% in alcohol. Neonatal abstinence syndrome was developed in 8.1% newborns, all of them from mothers with high methadone-positive hair results (>926.2 pg/mg). Statistically significant lower newborn weight and length were found in neonates from declared smokers compared with nonsmokers (P < 0.05). CONCLUSIONS: Maternal hair analysis showed to be more sensitive than maternal interview to detect drug use during pregnancy, except for alcohol. In this preliminary study, no statistically significant differences were found between exposed and nonexposed newborns to drugs, except for tobacco consumption.

Alternative matrices for cocaine, heroin, and methadone in utero drug exposure detection.

INTRODUCTION: Drug determination in biological matrices from the mother and the newborn is an objective measure of maternal and fetal drug exposure. The aim of this study was to compare maternal hair, meconium, umbilical cord, and placenta for detecting in utero drug exposure to cocaine, opiates, methadone, and amphetamines. METHOD: Maternal hair, meconium, umbilical cord, and placenta were collected from 175 mother-newborn dyads. Maternal hair (segmented in trimesters) and meconium specimens were analyzed for cocaine, opiates, methadone, and amphetamines. If either maternal hair or meconium tested positive, umbilical cord and placenta were analyzed. Analyses were performed by liquid chromatography tandem mass spectrometry. RESULTS: In hair, 24 participants tested positive; 21 for cocaine [cocaine 20-50,605, benzoylecgonine (BE) 17-46,668 pg/mg], 7 for methadone (76-26,845 pg/mg), 2 for opiates (morphine 298-2398 pg/mg, codeine 65-914 pg/mg, 6-acetylmorphine 1635-15,657 pg/mg), and 1 for amphetamines (amphetamine 1990 pg/mg, 3,4-methylenedioxyamphetamine 30 pg/mg, 3,4-methylenedioxymethamphetamine 294 pg/mg). In meconium, 6 were positive; 5 for methadone [methadone 88-3752, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) 642-25,179 ng/g], 3 for cocaine (cocaine 7, BE 79, hydroxybenzoylecgonine 5-135, ecgonine-methyl ester 2-56 ng/g), and 2 for opiates (morphine 152-1025, morphine-3-glucuronide 22-23, codeine 4-34 ng/g). Placenta and umbilical cord were positive in 5 and 6 cases, respectively; 5 for methadone in placenta (methadone 7-543, EDDP 10-51 ng/g) and cord (methadone 3-183, EDDP 2-109 ng/g); 1 for cocaine in placenta (cocaine 7, BE 2 ng/g) and cord (BE 6 ng/g); and 1 for opiates in placenta (morphine 6, morphine-3-glucuronide 48 ng/g), and 2 in cord (morphine 2, morphine-3-glucuronide 15-38, morphine-6-glucuronide 5 ng/g). Meconium, placenta, and umbilical cord only tested positive if hair concentrations were greater than Society of Hair Testing cutoffs. CONCLUSIONS: Maternal hair is the most sensitive specimen to detect drug consumption during pregnancy. Placenta and umbilical cord could be alternatives to meconium for detecting high in utero drug exposure.

Simultaneous quantification of 28 synthetic cathinones and metabolites in urine by liquid chromatography-high resolution mass spectrometry.

Synthetic cathinones are novel stimulants derived from cathinone, with amphetamines or cocaine-like effects, often labeled "not for human consumption" and considered "legal highs". Emergence of these new designer drugs complicate interpretation of forensic and clinical cases, with introduction of many new analogs designed to circumvent legislation and vary effects and potencies. We developed a method for the simultaneous quantification of 28 synthetic cathinones, including four metabolites, in urine by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). These cathinones include cathinone, methcathinone, and synthetic cathinones position-3'-substituted, N-alkyl-substituted, ring-substituted, methylenedioxy-substituted, and pyrrolidinyl-substituted. One mL phosphate buffer pH 6 and 25 µL IStd solution were combined with 0.25 mL urine, and subjected to solid phase cation exchange extraction (SOLA SCX). The chromatographic reverse-phase separation was achieved with a gradient mobile phase of 0.1 % formic acid in water and in acetonitrile in 20 min. We employed a Q Exactive high resolution mass spectrometer, with compounds identified and quantified by target-MSMS experiments. The assay was linear from 0.5-1 to 100 µg/L, with limits of detection of 0.25-1 µg/L. Imprecision (n = 20) was <15.9 % and accuracy (n = 20) 85.2-118.1 %. Extraction efficiency was 78.9-116.7 % (CV 1.4-16.7 %, n = 5), process efficiency 57.7-104.9 %, and matrix effects from -29.5 % to 1.5 % (CV 1.9-13.1 %, n = 10). Most synthetic cathinones were stable at 4 °C for 72 h (n = 27) and after 3 freeze-thaw cycles (n = 26), but many (n = 19) were not stable at room temperature for 24 h (losses up to -67.6 %). The method was applied to authentic urine specimens from synthetic cathinone users. This method provides a comprehensive confirmation method for 28 synthetic cathinones in urine, with good selectivity and specificity.

Simultaneous determination of opiates, methadone, amphetamines, cocaine, and metabolites in human placenta and umbilical cord by LC-MS/MS.

LC-MS/MS methods for the quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, hydroxybenzoylecgonine, cocaethylene, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), methadone, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in human placenta and umbilical cord were developed and validated. Specimens (1 ± 0.02 g) were homogenized with the Ultra-Turrax T8 disperser and centrifuged, and the supernatant was submitted to solid-phase extraction with Oasis MCX cartridges. Chromatographic separation was performed using an Atlantis T3 analytical column (100 × 2.1 mm, 3 µm) and a gradient of 0.1 % formic acid and acetonitrile. Selectivity was verified in 10 different blank specimens. The method was linear from 1-5 to 100-500 ng/g, depending on the analyte. Limits of detection and quantification ranged from 0.5 to 2.5 ng/g and 1 to 5 ng/g, respectively. Method imprecision was ≤15.3 %, except for MDMA at low quality control (18.1 %); accuracy, 87.1 to 114 %; extraction efficiency, 16.3 to 154.0 % (%CV = 1.8-39.4 %); matrix effect, -75.7 to 449.9 % (%CV = 3.5-50 %); and process efficiency, 8.7 to 316.0 %. The method was applied to authentic placenta and umbilical cord specimens from drug-user pregnant women.

Fast and Sensitive Method for the Determination of 17 Designer Benzodiazepines in Hair by Liquid Chromatography-Tandem Mass Spectrometry.

In recent years, identification and analysis of designer benzodiazepines have become a challenge in forensic toxicology. These substances are analogs of the classic benzodiazepines, but their pharmacology is not well known, and many of them have been associated with overdoses and deaths. As a result, there has been a surge in efforts to develop analytical methods to determine these compounds in different biological samples. Our aim was to develop and validate a fast, sensitive and specific method for determining 17 designer benzodiazepines (adinazolam, clobazam, clonazolam, delorazepam, deschloroetizolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam, flunitrazolam, N-desmethylclobazam, nifoxipam, nitrazolam, meclonazepam, pyrazolam and zolazepam) in hair by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hair samples were decontaminated and pulverized, and a 20 mg aliquot was incubated in methanol in an ultrasound bath (1 h, 25°C). The supernatant was evaporated and reconstituted in 200 µL of mobile phase, and the extracts were filtered (nano-filter vials) before injection into LC-MS-MS. All analytes were eluted from the chromatographic column in 8 min, and two multiple-reaction monitoring (MRM) transitions were used to identify each compound. The limits of quantification were 5 or 25 pg/mg depending on the analyte, and the calibration functions were linear to 200 pg/mg. Imprecision was <19.2% (n = 15), and bias was from -13.7 to 18.3% (n = 15). All the analytes yielded high extraction efficiencies >70% and displayed ion suppression between -62.8% and -23.9% (n = 10). The method was applied to 19 authentic cases. Five samples were positive for flualprazolam ( 200 pg/mg) and/or etizolam (47.4-88.5 pg/mg). In conclusion, the present validated method has proven to be fast, sensitive, specific and capable of determining 17 designer benzodiazepines in hair using LC-MS-MS.

Brain Concentrations of MDPV and its Metabolites in Male Rats: Relationship to Pharmacodynamic Effects.

BACKGROUND: MDPV (3,4-methylenedioxypyrovalerone) is a synthetic stimulant that blocks transmitter uptake at transporters for dopamine and norepinephrine. Less is known about MDPV pharmacokinetics, especially with respect to brain concentrations of the drug and its metabolites. OBJECTIVES: The goal of the present study was: 1) to determine brain concentrations of MDPV and its metabolites, 3,4-dihydroxypyrovalerone (3,4-catechol-PV) and 4-hydroxy-3-methoxy-pyrovalerone (4-OH-3-MeOPV), after administration of MDPV, and 2) to relate brain pharmacokinetic measures to pharmacodynamic endpoints in the same subjects. METHODS: Male Sprague-Dawley rats (300-400 g) received s.c. MDPV injection (1, 2, or 4 mg/kg) or its saline vehicle. Groups of rats were decapitated at 40 min and 240 min postinjection. Locomotor behavior was rated before decapitation, and the core temperature was obtained. Plasma and frontal cortex were analyzed to quantitate MDPV and its metabolites. Striatal samples were analyzed to measure dopamine, serotonin (5-HT), and their metabolites. RESULTS: MDPV displayed brain-to-plasma ratios greater than 1 (range 8.8-12.1), whereas 3,4-catechol-PV and 4-OH-3-MeO-PV showed ratios less than 1 (range 0-0.3). MDPV increased behavioural scores reflective of locomotor stimulation at 40 and 240 min and produced slight hyperthermia at 240 min. MDPV had no effect on striatal dopamine but produced an increase in the metabolite homovanillic acid (HVA). Brain MDPV concentrations were positively correlated with behavioural scores and striatal HVA but not with other endpoints. CONCLUSION: The behavioural effects of MDPV are related to brain concentrations of the parent drug and not its metabolites. The modest effects of MDPV on monoamine systems suggest that other non-monoamine mechanisms may contribute to the effects of the drug in vivo.

Preliminary buprenorphine sublingual tablet pharmacokinetic data in plasma, oral fluid, and sweat during treatment of opioid-dependent pregnant women.

BACKGROUND: Buprenorphine is currently under investigation as a pharmacotherapy to treat pregnant women for opioid dependence. This research evaluates buprenorphine (BUP), norbuprenophine (NBUP), buprenorphine-glucuronide (BUP-Gluc), and norbuprenorphine-glucuronide (NBUP-Gluc) pharmacokinetics after high-dose (14-20 mg) BUP sublingual tablet administration in three opioid-dependent pregnant women. METHODS: Oral fluid and sweat specimens were collected in addition to plasma specimens for 24 hours during gestation weeks 28 or 29 and 34, and 2 months after delivery. Time to maximum concentration was not affected by pregnancy; however, BUP and NBUP maximum concentration and area under the curve at 0 to 24 hours tended to be lower during pregnancy compared with postpartum levels. RESULTS: Statistically significant but weak positive correlations were found for BUP plasma and OF concentrations and BUP/NBUP ratios in plasma and oral fluid. Statistically significant negative correlations were observed for times of specimen collection and BUP and NBUP oral fluid/plasma ratios. BUP-Gluc and NBUP-Gluc were detected in only 5% of oral fluid specimens. In sweat, BUP and NBUP were detected in only four of 25 (12 or 24 hours) specimens in low concentrations (less than 2.4 ng/patch). CONCLUSION: These preliminary data describe BUP and metabolite pharmacokinetics in pregnant women and suggest that, like methadone, upward dose adjustments may be needed with advancing gestation.

Simultaneous analysis of buprenorphine, methadone, cocaine, opiates and nicotine metabolites in sweat by liquid chromatography tandem mass spectrometry.

A liquid chromatography tandem mass spectrometry method for buprenorphine (BUP), norbuprenorphine (NBUP), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine, benzoylecgonine, ecgonine methyl ester (EME), morphine, codeine, 6-acetylmorphine, heroin, 6-acetylcodeine, cotinine, and trans-3'-hydroxycotinine quantification in sweat was developed and comprehensively validated. Sweat patches were mixed with 6 mL acetate buffer at pH 4.5, and supernatant extracted with Strata-XC-cartridges. Reverse-phase separation was achieved with a gradient mobile phase of 0.1% formic acid and acetonitrile in 15 min. Quantification was achieved by multiple reaction monitoring of two transitions per compound. The assay was a linear 1-1,000 ng/patch, except EME 5-1,000 ng/patch. Intra-, inter-day and total imprecision were <10.1%CV, analytical recovery 87.2-107.7%, extraction efficiency 35.3-160.9%, and process efficiency 25.5-91.7%. Ion suppression was detected for EME (-63.3%) and EDDP (-60.4%), and enhancement for NBUP (42.6%). Deuterated internal standards compensated for these effects. No carryover was detected, and all analytes were stable for 24 h at 22 °C, 72 h at 4 °C, and after three freeze/thaw cycles. The method was applied to weekly sweat patches from an opioid-dependent BUP-maintained pregnant woman; 75.0% of sweat patches were positive for BUP, 93.8% for cocaine, 37.5% for opiates, 6.3% for methadone and all for tobacco biomarkers. This method permits a fast and simultaneous quantification of 14 drugs and metabolites in sweat patches, with good selectivity and sensitivity.

Evaluation and applicability of Alere iCup DX 14 for rapid postmortem urine drug screening at autopsy.

Performing point-of-care urine drug screen testing at autopsy by a forensic pathologist may provide an early indication of the presence of analytes of interest during autopsy. An evaluation for the screening of 14 classes of common drugs of abuse in postmortem urine by the point-of-care screening device, Alere iCup DX 14, is presented. One hundred ninety postmortem urine samples were screened with the iCup occurring at autopsy by the forensic pathologist. Positive and negative results obtained from the screening kit were evaluated against confirmatory test results obtained using routine forensic toxicology analyses that employed LC-MS/MS and GC-MS to detect a combination of over 85 common drugs of abuse and medications. Sensitivity for each respective iCup drug class ranged from 66% (buprenorphine) to 100% (methadone, tricyclic antidepressants). Specificity for each respective iCup drug class ranged from 89% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, 3,4-methylenedioxymethamphetamine, methadone). Positive predictive values ranged from 44% (benzodiazepines) to 100% (amphetamines, barbiturates, buprenorphine, methylenedioxymethamphetamine, methadone), while negative predictive values ranged from 96% (methamphetamine) to 100% (barbiturates, methadone, tricyclic antidepressants). A high false-positive rate was yielded by the benzodiazepine class. The lack of fentanyl screening in the point-of-care device is a significant limitation considering its prolific prevalence in forensic casework. The results obtained in the study should be acknowledged when considering the use of the Alere iCup DX 14 in the context of postmortem casework to help indicate potential drug use contemporaneously with autopsy and when requiring such preliminary results prior to the release of a final forensic toxicology report.

Quantification of Classic, Prescription and Synthetic Opioids in Hair by LC-MS-MS.

The current use and misuse of synthetic and prescription opioids in the USA has reached epidemic status. According to the US Department of Health and Human Services, every day more than 130 people in the USA die after overdosing on opioids, and 2.1 million had an opioid use disorder in 2018. Hair is becoming an alternative matrix of increasing interest in forensic toxicology to investigate drug use and abuse patterns due to its long window of detection. The focus of this project was to develop and validate a method that simultaneously detects and quantifies 27 classic, prescription and synthetic opioids in hair by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Hair samples were decontaminated and pulverized in a bead mill. Twenty-five milligrams of hair powder were incubated in a buffer overnight. Mixed mode cation exchange solid phase extraction was carried out before undergoing reversed-phase chromatographic separation, successfully resolving isobaric opioids. We used two multiple reaction monitoring transitions in positive mode to identify each analyte. The linearity range was 1-500 pg/mg for fentanyl and synthetic opioids and 10-500 pg/mg for prescription and classic opioids. Imprecision was <17.5% and bias ranged from -13.6 to 12.0%. Majority of compounds showed extraction efficiency >50%, and ion suppression from -89.2 to -26.6% (CV < 19%, n = 10). This method was applied to 64 authentic cases, identifying 13 compounds from our panel. A sensitive and specific method was developed for the identification and quantification of 27 classic, prescription and synthetic opioids in hair by LC-MS-MS.

Drug testing in biological samples vs. maternal surveys for the detection of substance use during whole pregnancy.

BACKGROUND: Early diagnosis of nicotine, ethanol and drug use during pregnancy is critical in order to provide adequate care. Current screening procedures show limitations in terms of reliability and short windows of detection. OBJECTIVES: To investigate the prevalence and identify biomarkers of substance use and changes in substance use during pregnancy. To compare drug testing results in different types of biological samples (maternal hair, meconium, placenta, umbilical-cord) with self-reported data. PARTICIPANTS AND SETTING: Prospective cohort study using data from pregnant women and their newborns. METHODS: Biological matrices were collected at birth and analyzed by liquid chromatography tandem mass spectrometry. A paper survey was provided to determine substance use habits. RESULTS: 867 mother-newborn pairs were included. According to the analysis of biological samples, 29.1% cases were positive for one or more substances (13.6% nicotine, 8.4% ethanol, 8.3% cocaine, 6.4% cannabis, 5.7% opioids). The profile of the substance-using mother was a single woman, <28 years-old, with no higher education and unemployed. Segmental maternal hair analysis showed a decrease in tobacco, cannabis and cocaine use throughout pregnancy (p < 0.001). The level of concordance between results from interviews and from biological analyses was weak for opioids, cocaine, and cannabis (kappa coefficient < 0.40). Maternal hair detected the highest number of cases, followed by meconium and by placenta and/or umbilical-cord. CONCLUSIONS: Maternal survey was not a reliable screening technique. Analysis of maternal hair detected the highest number of cases with the broadest detection window (whole pregnancy).