Marilyn A. Huestis

25C‐NBOMe and 25I‐NBOMe metabolite studies in human hepatocytes, in vivo mouse and human urine with high‐resolution mass spectrometry

25C-NBOMe and 25I-NBOMe are potent hallucinogenic drugs that recently emerged as new psychoactive substances. To date, a few metabolism studies were conducted for 25I-NBOMe, whereas 25C-NBOMe metabolism data are scarce. Therefore, we investigated the metabolic profile of these compounds in human hepatocytes, an in vivo mouse model and authentic human urine samples from forensic cases. Cryopreserved human hepatocytes were incubated for 3u2009h with 10u2009μM 25C-NBOMe and 25I-NBOMe; samples were analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS) on an Accucore C18 column with a Thermo QExactive; data analysis was performed with Compound Discoverer software (Thermo Scientific). Mice were administered 1.0u2009mg drug/kg body weight intraperitoneally, urine was collected for 24u2009h and analyzed (with or without hydrolysis) by LC-HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF; data were analyzed manually and with WebMetabase software (Molecular Discovery). Human urine samples were analyzed similarly. In vitro and in vivo results matched well. 25C-NBOMe and 25I-NBOMe were predominantly metabolized by O-demethylation, followed by O-di-demethylation and hydroxylation. All methoxy groups could be demethylated; hydroxylation preferably occurred at the NBOMe ring. Phase I metabolites were extensively conjugated in human urine with glucuronic acid and sulfate. Based on these data and a comparison with synthesized reference standards for potential metabolites, specific and abundant 25C-NBOMe urine targets are 5-desmethyl 25C-NBOMe, 25C-NBOMe and 5-hydroxy 25C-NBOMe, and for 25I-NBOMe 2 and 5-desmethyl 25I-NBOMe and hydroxy 25I-NBOMe. These data will help clinical and forensic laboratories to develop analytical methods and to interpret results. Copyright

Approaches, Challenges, and Advances in Metabolism of New Synthetic Cannabinoids and Identification of Optimal Urinary Marker Metabolites

We review approaches for determining metabolism of new synthetic cannabinoids (SCs), and challenges and advances in identifying optimal urinary marker metabolites of SC intake. Metabolic patterns of different SC generations are evaluated, and a practical strategy offered for selecting SC urinary marker metabolites. Novel SCs are incubated with human hepatocytes, the most abundant and characteristic metabolites are identified with high‐resolution mass spectrometry, and proposed hepatocyte marker metabolites are confirmed in authentic positive urine samples.

Cannabinoid disposition in oral fluid after controlled smoked, vaporized, and oral cannabis administration

Oral fluid (OF) is an important matrix for monitoring drugs. Smoking cannabis is common, but vaporization and edible consumption also are popular. OF pharmacokinetics are available for controlled smoked cannabis, but few data exist for vaporized and oral routes. Frequent and occasional cannabis smokers were recruited as participants for four dosing sessions including one active (6.9% Δ9 -tetrahydrocannabinol, THC) or placebo cannabis-containing brownie, followed by one active or placebo cigarette, or one active or placebo vaporized cannabis dose. Only one active dose was administered per session. OF was collected before and up to 54 (occasional) or 72 (frequent) h after dosing from cannabis smokers. THC, 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) were quantified by liquid chromatography-tandem mass spectrometry. OF cannabinoid Cmax occurred during or immediately after cannabis consumption due to oral mucosa contamination. Significantly greater THC Cmax and significantly later THCV, CBD, and CBG tlast were observed after smoked and vaporized cannabis compared to oral cannabis in frequent smokers only. No significant differences in THC, 11-OH-THC, THCV, CBD, or CBG tmax between routes were observed for either group. For occasional smokers, more 11-OH-THC and THCCOOH-positive specimens were observed after oral dosing than after inhaled routes, increasing % positive cannabinoid results and widening metabolite detection windows after oral cannabis consumption. Utilizing 0.3u2009µg/L THCV and CBG cut-offs resulted in detection windows indicative of recent cannabis intake. OF pharmacokinetics after high potency CBD cannabis are not yet available precluding its use currently as a marker of recent use. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Maternal Buprenorphine Maintenance and Lactation

Background: In addition to the well-known benefits of human milk and breastfeeding for the mother and infant, breastfeeding may mitigate neonatal abstinence syndrome severity in prenatally opioid-exposed infants. However, lack of conclusive data regarding the extent of the presence of buprenorphine and active metabolites in human milk makes the recommendation of breastfeeding for buprenorphine-maintained women difficult for many providers. Objective: This study seeks to determine the concentrations of buprenorphine and its active metabolites (norbuprenorphine, buprenorphine-glucuronide, and norbuprenorphine-glucuronide) in human milk, maternal plasma, and infant plasma of buprenorphine-maintained women and their infants. Methods: Up to 10 buprenorphine-maintained women provided paired breast milk and plasma samples at 2, 3, 4, 14, and 30 days postdelivery, and 9 infants provided plasma samples on day 14 of life. All samples were analyzed via liquid chromatography tandem mass spectrometry to determine concentrations of buprenorphine, norbuprenorphine, buprenorphine-glucuronide, and norbuprenorphine-glucuronide by a fully validated method. Results: Concentrations of buprenorphine and metabolites are low in human milk and maternal plasma. Breastfed infant plasma concentrations of buprenorphine were low or undetectable and metabolite concentrations undetectable at 14 days of infant age. There were significant correlations between maternal buprenorphine dose and maternal plasma and human milk buprenorphine concentrations. Conclusion: These data find low concentrations of buprenorphine and metabolites in human milk and lend support to the recommendation for lactation among stable buprenorphine-maintained women. However, the correlation between maternal dose and maternal plasma and human milk buprenorphine concentrations bears further study.

Recommendations for Toxicological Investigation of Drug-Impaired Driving and Motor Vehicle Fatalities—2017 Update

This report describes the outcomes of a process undertaken to review and update the National Safety Councils Alcohol, Drugs and Impairment Divisions recommendations for the toxicological investigation of suspected alcohol and drug-impaired driving cases and motor vehicle fatalities. The updates to the recommendations are made based on a survey of practices in laboratories in the USA and Canada performing testing in these cases, consideration of existing epidemiological crash and arrest data, current drug use patterns, and practical considerations of widely available technology platforms in laboratories performing this work. The final recommendations updates are derived from a consensus meeting of experts recruited from survey respondents and the membership of the National Safety Councils Alcohol, Drug and Impairment Division. The principal changes in this round of recommendations include removal of butalbital, phenobarbital, and phencyclidine from Tier I (mandatory) to Tier II (optional) due to changes in prevalence. In addition, buprenorphine, fentanyl, tramadol, and their metabolites were moved from Tier II to Tier I due to increased prevalence and concerns about their potential for causing impairment. In addition, screening and confirmatory cutoffs for the oral fluid scope were further refined. Other additions were made to the list of Tier II compounds including fentanyl analogs (e.g., acetylfentanyl, butyrylfentanyl, furanylfentanyl, etc), mitragynine, novel opioids (e.g., MT-45, U-47700), atypical antipsychotics, and novel benzodiazepines (e.g., clonazolam, flubromazolam, etc).

Subjective and physiological effects, and expired carbon monoxide concentrations in frequent and occasional cannabis smokers following smoked, vaporized, and oral cannabis administration

BACKGROUNDnAlthough smoking is the most common cannabis administration route, vaporization and consumption of cannabis edibles are common. Few studies directly compare cannabis subjective and physiological effects following multiple administration routes.nnnMETHODSnSubjective and physiological effects, and expired carbon monoxide (CO) were evaluated in frequent and occasional cannabis users following placebo (0.001% Δ9-tetrahydrocannabinol [THC]), smoked, vaporized, and oral cannabis (6.9% THC, ∼54mg).nnnRESULTSnParticipants subjective ratings were significantly elevated compared to placebo after smoking and vaporization, while only occasional smokers ratings were significantly elevated compared to placebo after oral dosing. Frequent smokers maximum ratings were significantly different between inhaled and oral routes, while no differences in occasional smokers maximum ratings between active routes were observed. Additionally, heart rate increases above baseline 0.5h after smoking (mean 12.2bpm) and vaporization (10.7bpm), and at 1.5h (13.0bpm) and 3h (10.2bpm) after oral dosing were significantly greater than changes after placebo, with no differences between frequent and occasional smokers. Finally, smoking produced significantly increased expired CO concentrations 0.25-6h post-dose compared to vaporization.nnnCONCLUSIONSnAll participants had significant elevations in subjective effects after smoking and vaporization, but only occasional smokers after oral cannabis, indicating partial tolerance to subjective effects with frequent exposure. There were no differences in occasional smokers maximum subjective ratings across the three active administration routes. Vaporized cannabis is an attractive alternative for medicinal administrations over smoking or oral routes; effects occur quickly and doses can be titrated with minimal CO exposure. These results have strong implications for safety and abuse liability assessments.

Distinguishing Intake of New Synthetic Cannabinoids ADB-PINACA and 5F-ADB-PINACA with Human Hepatocyte Metabolites and High-Resolution Mass Spectrometry

BACKGROUNDnADB-PINACA and its 5-fluoropentyl analog 5F-ADB-PINACA are among the most potent synthetic cannabinoids tested to date, with several severe intoxication cases. ADB-PINACA and 5F-ADB-PINACA have a different legal status, depending on the country. Synthetic cannabinoid metabolites predominate in urine, making detection of specific metabolites the most reliable way for proving intake in clinical and forensic specimens. However, there are currently no data on ADB-PINACA and 5F-PINACA metabolism. The substitution of a single fluorine atom distinguishes the 2 molecules, which may share common major metabolites. For some legal applications, distinguishing between ADB-PINACA and 5F-PINACA intake is critical. For this reason, we determined the human metabolic fate of the 2 analogs.nnnMETHODSnADB-PINACA and 5F-PINACA were incubated for 3 h with pooled cryopreserved human hepatocytes, followed by liquid chromatography-high-resolution mass spectrometry analysis. Data were processed with Compound Discoverer.nnnRESULTSnWe identified 19 and 12 major ADB-PINACA and 5F-ADB-PINACA metabolites, respectively. Major metabolic reactions included pentyl hydroxylation, hydroxylation followed by oxidation (ketone formation), and glucuronidation of ADB-PINACA, and oxidative defluorination followed by carboxylation of 5F-ADB-PINACA.nnnCONCLUSIONSnWe recommend ADB-PINACA ketopentyl and hydroxypentyl, and ADB-PINACA 5-hydroxypentyl and pentanoic acid, as optimal markers for ADB-PINACA and 5F-ADB-PINACA intake, respectively. Since the 2 compounds present positional isomers as the primary metabolites, monitoring unique product ions and optimized chromatographic conditions are required for a clear distinction between ADB-PINACA and 5F-ADB-PINACA intake.

Smoking in Pregnancy and Fetal Growth: The Case for More Intensive Assessment

IntroductionnMany studies on prenatal tobacco exposure (PTE) effects have relied on single item retrospective measures of PTE. However, it is unclear how these single item measures may relate to more intensive maternal self-reports and to biological markers of maternal use and/or fetal exposure. It is also unclear whether these measures may be more valid predictors of fetal growth (gestational age, birthweight, head circumference, and birth length).nnnMethodsnData were obtained from 258 women during their pregnancy. PTE was assessed by four methods: a single item question, a calendar-based self-report measure from each trimester of pregnancy, maternal salivary cotinine assays, and nicotine and metabolites in infant meconium. We hypothesized that the more intensive measures and biological assays would account for additional variance in birth outcomes, above and beyond the single item measure.nnnResultsnThe single item self-report measure was not related to fetal growth. However, the more intensive calendar based self-report measure and the biological assays of PTE (ie, maternal salivary assays and infant meconium) were significant predictors of poor fetal growth, even with the single item measure in the model.nnnConclusionsnThe negative effects of PTE on important child outcomes may be greatly underestimated in the literature as many studies use single item self-report measures to ascertain PTE. Whereas more intensive self-report measures or biological assays may be cost prohibitive in large scale epidemiological studies, using a combination of measures when possible should be considered given their superiority both identifying prenatal smokers and predicting poor fetal growth.nnnImplicationsnThe present work underscores the importance of measurement issues when assessing associations between PTE and fetal growth. Results suggest that we may be greatly underestimating the negative effects of prenatal smoking on fetal growth and other important child outcomes if we rely solely on restricted single item self-report measures of prenatal smoking. Researchers should consider more intensive prospective self-report measures and biological assays as viable and superior alternatives to single item self-report measures.

Impact of Novel Psychoactive Substances on Clinical and Forensic Toxicology and Global Public Health

Novel psychoactive substances (NPS)6 have been a part of the landscape of clinical and forensic toxicology for over a century, beginning with the introduction of a few new drugs like heroin, lysergic acid diethylamide (LSD), 3,4-methylenedioxymethamphetamine (MDMA), and γ-hydroxybutyric acid (GHB). However, after the appearance of synthetic cannabinoids in the early 2000s, there was a rapid emergence of hundreds of synthetic cathinones, benzodiazepines, and opioids. Toxicology laboratories previously focused on a rather narrow range of compounds, including amphetamines, cannabinoids, cocaine, opioids, antidepressants, salicylate, and acetaminophen. Now, potent fentanyl derivatives are mixed with heroin or substituted entirely, killing unsuspecting drug users at an alarming rate. Toxicology laboratories are challenged with detecting potent drug analogs that are present in the blood for only a short period of time, urinary metabolites whose chemical formulas and structures are initially unknown, and nonavailability of reference standards. Here, 4 international experts discuss what fueled the global NPS market, how toxicology laboratories can best address this challenge, and how public health and law enforcement agencies can help reduce the morbidity and mortality associated with NPS.nnWhat factors contributed to the explosion of NPS onto the recreational drug market? nnSimon D. Brandt: There are important factors that contributed to this phenomenon, but advances in information technology and communication and globalized trade were crucial. Entrepreneurs are now able to instruct contract synthesis laboratories overseas (often in China) to produce any compound of choice on a large scale and in high purity at low cost. From the perspective of a service provider, for example, one specialized in the manufacturing of chemicals, the nature of synthesized compounds does not necessarily make any difference, and some laboratories are well equipped to supply high-quality pharmaceutical drugs and NPS alike. Once the demand is identified, product diversification and expansion of product catalogues follow, …

Cannabis Edibles: Blood and Oral Fluid Cannabinoid Pharmacokinetics and Evaluation of Oral Fluid Screening Devices for Predicting Δ9-Tetrahydrocannabinol in Blood and Oral Fluid following Cannabis Brownie Administration

BACKGROUNDnRoadside oral fluid (OF) Δ9-tetrahydrocannabinol (THC) detection indicates recent cannabis intake. OF and blood THC pharmacokinetic data are limited and there are no on-site OF screening performance evaluations after controlled edible cannabis.nnnCONTENTnWe reviewed OF and blood cannabinoid pharmacokinetics and performance evaluations of the Draeger DrugTest®5000 (DT5000) and Alere™ DDS®2 (DDS2) on-site OF screening devices. We also present data from a controlled oral cannabis administration session.nnnSUMMARYnOF THC maximum concentrations (Cmax) were similar in frequent as compared to occasional smokers, while blood THC Cmax were higher in frequent [mean (range) 17.7 (8.0-36.1) μg/L] smokers compared to occasional [8.2 (3.2-14.3) μg/L] smokers. Minor cannabinoids Δ9-tetrahydrocannabivarin and cannabigerol were never detected in blood, and not in OF by 5 or 8 h, respectively, with 0.3 μg/L cutoffs. Recommended performance (analytical sensitivity, specificity, and efficiency) criteria for screening devices of ≥80% are difficult to meet when maximizing true positive (TP) results with confirmation cutoffs below the screening cutoff. TPs were greatest with OF confirmation cutoffs of THC ≥1 and ≥2 μg/L, but analytical sensitivities were <80% due to false negative tests arising from confirmation cutoffs below the DT5000 and DDS2 screening cutoffs; all criteria were >80% with an OF THC ≥5 μg/L cutoff. Performance criteria also were >80% with a blood THC ≥5 μg/L confirmation cutoff; however, positive OF screening results might not confirm due to the time required to collect blood after a crash or police stop. OF confirmation is recommended for roadside OF screening.ClinicalTrials.gov identification number: NCT02177513.