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Driving under the influence of cannabis (DUIC) is increasing worldwide, and cannabis is the most prevalent drug after alcohol in impaired driving cases, emphasizing the need for a reliable traffic enforcement strategy. ∆ 9 -tetrahydrocannabinol (THC) detection in oral fluid has great potential for identifying recent cannabis use; however, additional data are needed on the sensitivities, specificities, and efficiencies of different oral fluid devices for detecting cannabinoids at the roadside by police during routine traffic safety enforcement efforts. At the roadside, 8945 oral fluid THC screening tests were performed with four devices: AquilaScan®, Dräger DrugTest®, WipeAlyser Reader®, and Druglizer®. A total of 530 samples screened positive for THC (5.9%) and were analyzed by liquid chromatography-tandem mass spectrometry at multiple cutoff concentrations (2 ng/mL, 10 ng/mL, and manufacturers' recommended device cutoffs) to investigate device performance. Results varied substantially, with sensitivities of 0%-96.8%, specificities of 89.8%-98.5%, and efficiencies of 84.3%-97.8%. The Dräger DrugTest® outperformed the other devices with a 96.8% sensitivity, 97.1% specificity, and 97.0% efficiency at a 5-ng/mL LC-MS/MS confirmation cutoff. The WipeAlyser Reader® had good performance with a 91.4% sensitivity, 97.2% specificity, and 96.4% efficiency. AquilaScan® and Druglizer® had unacceptable performance for cannabinoid detection, highlighted by sensitivity <13%. The choice of roadside oral fluid testing device must offer good analytical performance for cannabinoids because of its high prevalence of use and impact on road safety., (© 2024 John Wiley & Sons Ltd.)

N-Ethyl-N-propyltryptamine (EPT), 4-hydroxy-N-ethyl-N-propyltryptamine (4-OH-EPT), and 5-methoxy-N-ethyl-N-propyltryptamine (5-MeO-EPT) are new psychoactive substances classified as tryptamines, sold online. Many tryptamines metabolize rapidly, and identifying the appropriate metabolites to reveal intake is essential. While the metabolism of 4-OH-EPT and 5-MeO-EPT are not previously described, EPT is known to form metabolites by indole ring hydroxylation among others. Based on general knowledge of metabolic patterns, 5-MeO-EPT is also expected to form ring hydroxylated EPT (5-OH-EPT). In the present study, the aim was to characterize the major metabolites of EPT, 4-OH-EPT, and 5-MeO-EPT, to provide markers for substance identification in forensic casework. The tryptamines were incubated with pooled human liver microsomes at 37°C for up to 4 h. The generated metabolites were separated and detected by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. The major in vitro EPT metabolites were formed by hydroxylation, N-dealkylation, and carbonylation. In comparison, 4-OH-EPT metabolism was dominated by double bond formation, N-dealkylation, hydroxylation, and carbonylation in vitro and hydroxylation or carbonylation combined with double bond loss, carbonylation, N-dealkylation, and hydroxylation in vivo. 5-MeO-EPT was metabolized by O-demethylation, hydroxylation, and N-dealkylation in vitro. The usefulness of the characterized metabolites in forensic casework was demonstrated by identification of unique metabolites for 4-OH-EPT in a human postmortem blood sample with suspected EPT or 4-OH-EPT intoxication., (© 2024 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Consumption of hemp products is continuously growing, with an expanding scope of applications. Suppliers operate through different distribution channels, but the Internet is a major retail platform. Hemp products are prepared from cannabis plants and, therefore, might contain a variety of different natural cannabinoids. According to the regulations of the World Anti-Doping Agency, all natural and synthetic cannabinoids are prohibited in-competition, with the explicit exemption of cannabidiol. Therefore, an investigation of 23 hemp products for the presence of cannabinoids was performed to determine the likelihood of unintentional violations of anti-doping regulations. An assay for the detection of 16 cannabinoids in nutritional supplements was developed and validated. The sample preparation consisted of QuEChERS extraction, trimethylsilylation, and analysis by gas chromatography/tandem mass spectrometry. All 23 commercially available hemp products were analyzed, and assay characteristics such as selectivity, limit of detection, limit of identification, limit of quantification, linearity, imprecision, recovery, and accuracy were determined. Twenty of 23 hemp products included a variety of cannabinoids at, occasionally, substantial concentrations, with four products covering the entire spectrum of tested cannabinoids. An ethics committee-approved single-dose administration study was conducted with the commercially available hemp products, investigating the presence of 16 cannabinoids in urine collected pre- and post-consumption. Variable patterns of cannabinoids or their metabolites in urine were observed. In 30% of the urine samples collected 8 h after ingestion, the presence of a prohibited cannabinoid would have resulted in an unintentional violation of anti-doping regulations., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Urine is a common matrix for screening for cannabis use. Urine assays typically measure total 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol (THCCOOH) concentrations after hydrolysis cleaves the glucuronide. Urine THCCOOH concentration is adjusted by urine creatinine concentration or specific gravity, to account for variable hydration states. Therefore, we performed a population pharmacokinetic analysis of the urinary THCCOOH excretion, urinary flow rate, and creatinine excretion rate data. Urine was obtained over 168 h from six subjects who smoked low- (15.8 mg) and high-dose (33.8 mg) Δ 9 -tetrahydrocannabinol (THC) cigarettes on two occasions. Samples were analyzed for THCCOOH concentration by gas chromatography-mass spectrometry (GC-MS) and volume, time, and creatinine concentration measured. A population pharmacokinetic model of the urinary clearance of THCCOOH was created from these data, and potential covariates of urine creatinine concentration and urine creatinine excretion rate were assessed. Elimination clearance of THCCOOH was estimated as 0.104 ± 0.088 L/min, and its urinary clearance was 0.0022 ± 0.0015 L/min. Total urine excretion of THCCOOH was estimated at 2.3%. Urine flow rate and urine creatinine concentrations were significantly correlated, r 2  = 0.35. Creatinine excretion rate was 129.6 ± 71.0 ml/min, and the intrasubject variability was 31%-52% (SD%) during the week. Urinary creatinine excretion rate was a significant covariate for the urinary clearance of THCCOOH. Creatinine clearance is a significant covariate for urinary THCCOOH clearance. Only 2%-3% of bioavailable THC is excreted as THCCOOH and THCCOO-glucuronide via the urine. Correction of urine drug and/or metabolite concentration with urine creatinine concentration or specific gravity may be more problematic than previously appreciated., (© 2022 John Wiley & Sons, Ltd.)

According to the World Anti-Doping Agency (WADA) regulations, cannabinoids use is prohibited in competition except for cannabidiol (CBD) use. For an adverse analytical finding (AAF) in doping control, cannabinoid misuse is based on identification of the pharmacologically inactive metabolite 11-nor-delta-9-carboxy-tetrahydrocannabinol-9-carboxylic acid (carboxy-THC) in urine at a concentration greater than 180 ng/ml. All other (minor) cannabinoids are reported as AAF when identified, except for CBD that has been explicitly excluded from the class of cannabinoids on WADA's Prohibited List since 2018. However, due to the fact that CBD isolated from cannabis plants may contain additional minor cannabinoids, the permissible use of CBD can lead to unintentional violations of antidoping regulations. An assay for the detection of 16 cannabinoids in human urine was established. The sample preparation consisted of enzymatic hydrolysis of glucuronide conjugates, liquid-liquid extraction, trimethylsilylation, and analysis by gas chromatography/tandem mass spectrometry (GC-MS/MS). Spot urine samples from CBD users, as well as specimens obtained from CBD administration studies conducted with 15 commercially available CBD products, were analyzed, and assay characteristics such as selectivity, reproducibility of detection at the minimum required performance level, limit of detection, and limit of identification were determined. An ethical committee approved controlled single dose commercially available CBD products administration study was conducted to identify 16 cannabinoids in urine samples collected after ingestion or application of the CBD products as well as their presence in spot urine samples of habitual CBD users. Variable patterns of cannabinoids or their metabolites were observed in the urine samples, especially when full spectrum CBD products were consumed. The presence of minor cannabinoids or their metabolites in an athlete's in-competition urine sample represents a substantial risk of an antidoping rule violation., (© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Variability in urine dilution complicates urine cannabinoid test interpretation. Normalizing urine cannabinoid concentrations to specific gravity (SG) or creatinine was proposed to account for donors' hydration states. In this study, all urine voids were individually collected from eight frequent and eight occasional cannabis users for up to 85 hours after each received on separate occasions 50.6 mg Δ9-tetrahydrocannabinol (THC) by smoking, vaporization, and oral ingestion in a randomized, within-subject, double-blind, double-dummy, placebo-controlled protocol. Each urine void was analyzed for 11 cannabinoids and phase I and II metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS), SG, and creatinine. Normalized urine concentrations were log 10 transformed to create normal distributions, and Pearson correlation coefficients determined the degree of association between the two normalization methods. Repeated-measures linear regression determined if the degree of association differed by frequent or occasional cannabis use, or route of administration after adjusting for gender and time since dosing. Of 1880 urine samples examined, only 11-nor-9-carboxy-THC (THCCOOH), THCCOOH-glucuronide, THC-glucuronide, and 11-nor-9-carboxy-Δ9-tetrahydrocannabivarin (THCVCOOH) were greater than the method's limits of quantification (LOQs). Associations between SG- and creatinine-normalized concentrations exceeded 0.90. Repeated-measures regression analysis found small but statistically significant differences in the degree of association between normalization methods for THCCOOH and THCCOOH-glucuronide in frequent vs occasional smokers, and in THCVCOOH and THC-glucuronide by route of administration. For the first time, SG- and creatinine-normalized urine cannabinoid concentrations were evaluated in frequent and occasional cannabis users and following oral, smoked, and inhaled cannabis. Both normalization methods reduced variability, improving the interpretation of urine cannabinoid concentrations and methods were strongly correlated., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Prolonged urinary cannabinoid excretion in chronic frequent cannabis users confounds identification of recent cannabis intake that may be important in treatment, workplace, clinical, and forensic testing programs. In addition, differentiation of synthetic Δ9-tetrahydrocannabinol (THC) intake from cannabis plant products might be an important interpretive issue. THC, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) urine concentrations were evaluated during previous controlled cannabis administration studies following tandem alkaline/E. coli β-glucuronidase hydrolysis. We optimized recombinant β-glucuronidase enzymatic urinary hydrolysis before simultaneous liquid chromatography tandem mass spectrometry (LC-MS/MS) quantification of THC, 11-OH-THC, THCCOOH, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), tetrahydrocannabivarin (THCV) and 11-nor-9-carboxy-THCV (THCVCOOH) in urine. Enzyme amount, incubation time and temperature, buffer molarity and pH were optimized using pooled urine samples collected during a National Institute on Drug Abuse, Institutional Review Board-approved clinical study. Optimized cannabinoid hydrolysis with recombinant β-glucuronidase was achieved with 2000 IU enzyme, 2 M pH 6.8 sodium phosphate buffer, and 0.2 mL urine at 37°C for 16 h. The LC-MS/MS quantification method for hydrolyzed urinary cannabinoids was validated per the Scientific Working Group on Toxicology guidelines. Linear ranges were 1-250 μg/L for THC and CBG, 2-250 μg/L for 11-OH-THC, CBD, CBN, THCV and THCVCOOH, and 1-500 μg/L for THCCOOH. Inter-batch analytical bias was 92.4-112.4%, imprecision 4.4-9.3% CV (n = 25), extraction efficiency 44.3-97.1% and matrix effect -29.6 to 1.8% (n = 10). The method was utilized to analyze urine specimens collected during our controlled smoked, vaporized, and edible cannabis administration study to improve interpretation of urine cannabinoid test results., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

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% Δ 9 -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 C max occurred during or immediately after cannabis consumption due to oral mucosa contamination. Significantly greater THC C max and significantly later THCV, CBD, and CBG t last 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 t max 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 0.3 µg/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., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

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 3 h with 10 μM 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 1.0 mg drug/kg body weight intraperitoneally, urine was collected for 24 h 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 © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Cannabinoid stability in oral fluid (OF) is important for assuring accurate results since OF has become a valid alternative matrix of choice for drug testing. We previously published OF cannabinoid stability studies using Quantisal™, Oral-Eze®, and StatSure™ devices stored at room temperature for 1 week, 4 °C for up to 4 weeks, and at -20 °C up to 24 weeks. Extending refrigerated stability up to 3 months would be helpful for clinical and forensic testing, for re-analysis of OF samples and for batching research analyses. Individual authentic OF pools were prepared after controlled smoking of a 6.9% ∆ 9 -tetrahydracannabinol cannabis cigarette; the Quantisal™ device was utilized for OF collection. Fifteen healthy volunteers participated in the Institutional Review Board-approved study. Stability for THC, 11-nor-9-carboxy-THC (THCCOOH), ∆ 9 -tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) were determined after storage at 4 °C for 1, 2, and 3 months. Results within ±20% of baseline concentrations were considered stable. All analytes were stable for up to 2 months at 4 °C for all participants with positive baseline concentrations. Baseline concentrations were highly variable. In total, THC, THCCOOH, THCV, CBD, and CBG were stable for 3 months at 4 °C for pooled positive specimens from 14 of 15, 8 of 9, 7 of 8, 8 of 9, and 9 of 10 participants, respectively. In conclusion, Quantisal™-collected OF specimens should be stored at 4 °C for no more than two months to assure accurate THC, THCCOOH, THCV, CBD, and CBG quantitative results; only one participant's OF was unstable at three months. Published 2016. This article is a U.S. Government work and is in the public domain in the USA., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

AH-7921 (3,4-dichloro-N-[(1-dimethylamino)cyclohexylmethyl]benzamide) is a new synthetic opioid and has led to multiple non-fatal and fatal intoxications. To comprehensively study AH-7921 metabolism, we assessed human liver microsome (HLM) metabolic stability, determined AH-7921's metabolic profile after human hepatocytes incubation, confirmed our findings in a urine case specimen, and compared results to in silico predictions. For metabolic stability, 1 µmol/L AH-7921 was incubated with HLM for up to 1 h; for metabolite profiling, 10 µmol/L was incubated with pooled human hepatocytes for up to 3 h. Hepatocyte samples were analyzed by liquid chromatography quadrupole/time-of-flight high-resolution mass spectrometry (MS). High-resolution full scan MS and information-dependent acquisition MS/MS data were analyzed with MetabolitePilot™ (SCIEX) using multiple data processing algorithms. The presence of AH-7921 and metabolites was confirmed in the urine case specimen. In silico prediction of metabolite structures was performed with MetaSite™ (Molecular Discovery). AH-7921 in vitro half-life was 13.5 ± 0.4 min. We identified 12 AH-7921 metabolites after hepatocyte incubation, predominantly generated by demethylation, less dominantly by hydroxylation, and combinations of different biotransformations. Eleven of 12 metabolites identified in hepatocytes were found in the urine case specimen. One metabolite, proposed to be di-demethylated, N-hydroxylated and glucuronidated, eluted after AH-7921 and was the most abundant metabolite in non-hydrolyzed urine. MetaSite™ correctly predicted the two most abundant metabolites and the majority of observed biotransformations. The two most dominant metabolites after hepatocyte incubation (also identified in the urine case specimen) were desmethyl and di-desmethyl AH-7921. Together with the glucuronidated metabolites, these are likely suitable analytical targets for documenting AH-7921 intake. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Cannabis smoking increases motor vehicle accident risk. Empirically defined cannabinoid detection windows are important to drugged driving legislation. Our aims were to establish plasma cannabinoid detection windows in frequent cannabis smokers and to determine if residual cannabinoid concentrations were correlated with psychomotor performance. Twenty-eight male chronic frequent cannabis smokers resided on a secure research unit for up to 33 days with daily blood collection. Plasma specimens were analyzed for Δ(9) -tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) by gas chromatography-mass spectrometry. Critical tracking and divided attention tasks were administered at baseline (after overnight stay to ensure lack of acute intoxication) and after 1, 2, and 3 weeks of cannabis abstinence. Twenty-seven of the twenty-eight participants were THC-positive at admission (median 4.2 µg/L). THC concentrations significantly decreased 24 h after admission, but were still ≥2 µg/L in 16 of the 28 participants 48 h after admission. THC was detected in 3 of 5 specimens on day 30. The last positive 11-OH-THC specimen was 15 days after admission. THCCOOH was measureable in 4 of 5 participants after 30 days of abstinence. Years of prior cannabis use significantly correlated with THC concentrations on admission, and days 7 and 14. Tracking error, evaluated by the Divided Attention Task, was the only evaluated psychomotor assessment significantly correlated with cannabinoid concentrations at baseline and day 8 (11-OH-THC only). Median THC was 0.3 µg/L in 5 chronic frequent cannabis smokers' plasma samples after 30 days of sustained abstinence. Published 2015. This article is a U.S. Government work and is in the public domain in the USA., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Vaporized cannabis and concurrent cannabis and alcohol intake are commonplace. We evaluated the subjective effects of cannabis, with and without alcohol, relative to blood and oral fluid (OF, advantageous for cannabis exposure screening) cannabinoid concentrations and OF/blood and OF/plasma vaporized-cannabinoid relationships. Healthy adult occasional-to-moderate cannabis smokers received a vaporized placebo or active cannabis (2.9% and 6.7% Δ(9) -tetrahydrocannabinol, THC) with or without oral low-dose alcohol (~0.065g/210L peak breath alcohol concentration [BrAC]) in a within-subjects design. Blood and OF were collected up to 8.3 h post-dose and subjective effects measured at matched time points with visual-analogue scales and 5-point Likert scales. Linear mixed models evaluated subjective effects by THC concentration, BrAC, and interactions. Effects by time point were evaluated by dose-wise analysis of variance (ANOVA). OF versus blood or plasma cannabinoid ratios and correlations were evaluated in paired-positive specimens. Nineteen participants (13 men) completed the study. Blood THC concentration or BrAC significantly associated with subjective effects including 'high', while OF contamination prevented significant OF concentration associations <1.4 h post-dose. Subjective effects persisted through 3.3-4.3 h, with alcohol potentiating the duration of the cannabis effects. Effect-versus-THC concentration and effect-versus-alcohol concentration hystereses were counterclockwise and clockwise, respectively. OF/blood and OF/plasma THC significantly correlated (all Spearman r≥0.71), but variability was high. Vaporized cannabis subjective effects were similar to those previously reported after smoking, with duration extended by concurrent alcohol. Cannabis intake was identified by OF testing, but OF concentration variability limited interpretation. Blood THC concentrations were more consistent across subjects and more accurate at predicting cannabis' subjective effects. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Accurate on-site devices to screen for drug intake are critical for establishing whether an individual is driving under the influence of drugs (DUID); however, on-site oral fluid (OF) cocaine device performance is variable. We evaluated the performance of a newly developed benzoylecgonine (BE) test-strip for the Draeger® DrugTest 5000 device (20 µg/L cut-off) with equivalent cross reactivity for cocaine and BE. Ten cocaine users provided OF, collected with the Draeger cassette and Oral-Eze® and StatSure Saliva Sampler(TM) devices, up to 69 h following 25 mg intravenous cocaine administration. All screening results were confirmed by a validated two-dimensional-gas chromatography-mass spectrometry (2D-GC-MS) method for cocaine and/or BE. Cocaine test-strip median Tlast for screening only results was 6.5 h, and 6.5 h with Oral-Eze® and 4 h for StatSure OF confirmation for cocaine and/or BE at 1, 8, and 10 µg/L; sensitivity, specificity, and efficiency ranged from 85.5 to 100% and 83.3 to 100% for cocaine only confirmation at 8 and 10 µg/L. For the BE test-strip, median Tlast was 12.5 h for screening only and confirmation for cocaine and/or BE at all three cut-offs; sensitivity, specificity, and efficiency ranged from 85.5 to 97.5% and 78.4 to 97.4% with cocaine and/or BE confirmation at 8 and 10 µg/L cut-offs, respectively. The Draeger cocaine test-strip with cocaine only confirmation offers a useful option for monitoring the acute intoxication phase of DUID; additionally the BE test-strip with cocaine and/or BE confirmation increases the length of detection of cocaine intake for workplace drug testing, drug court, parole, pain management, drug treatment programs and both the acute cocaine intoxication and cocaine crash/fatigue phase of DUID. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Methamphetamine is included in drug testing programmes due to its high abuse potential. d-Methamphetamine is a scheduled potent central nervous system stimulant, while l-methamphetamine is the unscheduled active ingredient in the over-the-counter nasal decongestant Vicks® VapoInhaler™. No data are available in oral fluid (OF) and few in plasma after controlled Vicks® VapoInhaler™ administration. We quantified methamphetamine and amphetamine enantiomers in OF collected with two different devices and plasma via a fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Additionally, OF were analyzed with an on-site screening device. Sixteen participants received 7 Vicks® VapoInhaler™ doses according to manufacturer's recommendations. Specimens were collected before and up to 32 h after the first dose. No d-methamphetamine or d-amphetamine was detected in any sample. All participants had measurable OF l-methamphetamine with median maximum concentrations 14.8 and 16.1 μg/L in Quantisal™ and Oral-Eze® devices, respectively, after a median of 5 doses. One participant had measurable OF l-amphetamine with maximum concentrations 3.7 and 5.5 μg/L after 6 doses with the Quantisal™ and Oral-Eze® devices, respectively. There were no positive DrugTest® 5000 results. In the cutoff range 20-50 μg/L methamphetamine with amphetamine ≥limit of detection, 3.1-10.1% of specimens were positive; first positive results were observed after 1-4 doses. Two participants had detectable plasma l-methamphetamine, with maximum observed concentrations 6.3 and 10.0 μg/L after 2 and 5 doses, respectively. Positive OF and plasma methamphetamine results are possible after Vicks® VapoInhaler™ administration. Chiral confirmatory analyses are necessary to rule out VapoInhaler™ intake. Implementing a selective d-methamphetamine screening assay can help eliminate false-positive OF results., (Published 2015. This article is a U.S. Government work and is in the public domain in the USA.)

Δ(9) -Tetrahydrocannabinol (THC) is the most commonly analyzed cannabinoid in oral fluid (OF); however, its metabolite 11-nor-9-carboxy-THC (THCCOOH) offers the advantage of documenting active consumption, as it is not detected in cannabis smoke. Analytical challenges such as low (ng/L) THCCOOH OF concentrations hampered routine OF THCCOOH monitoring. Presence of minor cannabinoids like cannabidiol and cannabinol offer the advantage of identifying recent cannabis intake. Published OF cannabinoids methods have limitations, including few analytes and lengthy derivatization. We developed and validated a sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for THC, its metabolites, 11-hydroxy-THC and THCCOOH quantification, and other natural cannabinoids including tetrahydrocannabivarin (THCV), cannabidiol (CBD), and cannabigerol (CBG) in 1 mL OF collected with the Quantisal device. After solid-phase extraction, chromatography was performed on a Selectra PFPP column with a 0.15% formic acid in water and acetonitrile gradient with a 0.5 mL/min flow rate. All analytes were monitored in positive mode atmospheric pressure chemical ionization (APCI) with multiple reaction monitoring. Limits of quantification were 15 ng/L THCCOOH and 0.2 µg/L for all other analytes. Linear ranges extended to 3750 ng/L THCCOOH, 100 µg/L THC, and 50 µg/L for all other analytes. Inter-day analytical recoveries (bias) and imprecision at low, mid, and high quality control (QC) concentrations were 88.7-107.3% and 2.3-6.7%, respectively (n = 20). Mean extraction efficiencies and matrix effects evaluated at low and high QC were 75.9-86.1% and 8.4-99.4%, respectively. This method will be highly useful for workplace, criminal justice, drug treatment and driving under the influence of cannabis OF testing., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Opiates are an important drug class in drug testing programmes. Ingestion of poppy seeds containing morphine and codeine can yield positive opiate tests and mislead result interpretation in forensic and clinical settings. Multiple publications evaluated urine opiate concentrations following poppy seed ingestion, but only two addressed oral fluid (OF) results; neither provided the ingested morphine and codeine dosage. We administered two 45 g raw poppy seed doses, each containing 15.7 mg morphine and 3.1 mg codeine, 8 h apart to 17 healthy adults. All OF specimens were screened by on-site OF immunoassay Draeger DrugTest 5000, and confirmed with OF collected with Oral-Eze® device and quantified by liquid chromatography-tandem mass spectrometry (1 µg/L morphine and codeine limits of quantification). Specimens (n = 459) were collected before and up to 32 h after the first dose. All specimens screened positive 0.5 h after dosing and remained positive for 0.5-13 h at Draeger 20 µg/L morphine cut-off. Maximum OF morphine and codeine concentrations (Cmax ) were 177 and 32.6 µg/L, with times to Cmax (Tmax ) of 0.5-1 h and 0.5-2.5 h post-dose, respectively. Windows of detection after the second dose extended at least 24 h for morphine and to 18 h for codeine. After both doses, the last morphine positive OF result was 1 h with 40 µg/L 2004 proposed US Substance Abuse and Mental Health Services Administration cut-off, and 0.5 h with 95 µg/L cut-off, recently recommended by the Driving under the Influence of Drugs and Medicines project. Positive OF morphine results are possible 0.5-1 h after ingestion of 15.7 mg of morphine in raw poppy seeds, depending on the cut-off employed., (Copyright © 2014 John Wiley & Sons, Ltd.)

Oral fluid (OF) offers a simple, non-invasive, directly observable sample collection for clinical and forensic drug testing. Given that chronic cannabis smokers often engage in drug administration multiple times daily, evaluating OF cannabinoid pharmacokinetics during ad libitum smoking is important for practical development of analytical methods and informed interpretation of test results. Eleven cannabis smokers resided in a closed research unit for 51 days, and underwent four, 5-day oral delta-9-tetrahydrocannabinol (THC) treatments. Each medication period was separated by 9 days of ad libitum cannabis smoking from 12:00 to 23:00 h daily. Ten OF samples were collected from 9:00-22:00 h on each of the last ad libitum smoking days (Study Days 4, 18, 32, and 46). As the number of cannabis cigarettes smoked increased over the study days, OF THC, cannabinol (CBN), and 11-nor-9-carboxy-THC (THCCOOH) also increased with a significant effect of time since last smoking (Δtime; range, 0.0-17.4 h) and ≥88% detection rates; concentrations on Day 4 were significantly lower than those on Days 32 and 46 but not Day 18. Within 30 min of smoking, median THC, CBN, and THCCOOH concentrations were 689 µg/L, 116 µg/L, and 147 ng/L, respectively, decreasing to 19.4 µg/L, 2.4 µg/L, and 87.6 ng/L after 10 h. Cannabidiol and 11-hydroxy-THC showed overall lower detection rates of 29 and 8.6%, respectively. Cannabinoid disposition in OF was highly influenced by Δtime and composition of smoked cannabis. Furthermore, cannabinoid OF concentrations increased over ad libitum smoking days, in parallel with increased cannabis self-administration, possibly reflecting development of increased cannabis tolerance., (Copyright © 2014 John Wiley & Sons, Ltd.)

Synthetic cannabinoid intake is an ongoing health issue worldwide, with new compounds continually emerging, making drug testing complex. Parent synthetic cannabinoids are rarely detected in urine, the most common matrix employed in workplace drug testing. Optimal identification of synthetic cannabinoid markers in authentic urine specimens and correlation of metabolite concentrations and toxicities would improve synthetic cannabinoid result interpretation. We screened 20 017 randomly collected US military urine specimens between July 2011 and June 2012 with a synthetic cannabinoid immunoassay yielding 1432 presumptive positive specimens. We analyzed all presumptive positive and 1069 negative specimens with our qualitative synthetic cannabinoid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which confirmed 290 positive specimens. All 290 positive and 487 randomly selected negative specimens were quantified with the most comprehensive urine quantitative LC-MS/MS method published to date; 290 specimens confirmed positive for 22 metabolites from 11 parent synthetic cannabinoids. The five most predominant metabolites were JWH-018 pentanoic acid (93%), JWH-N-hydroxypentyl (84%), AM2201 N-hydroxypentyl (69%), JWH-073 butanoic acid (69%), and JWH-122 N-hydroxypentyl (45%) with 11.1 (0.1-2,434), 5.1 (0.1-1,239), 2.0 (0.1-321), 1.1 (0.1-48.6), and 1.1 (0.1-250) µg/L median (range) concentrations, respectively. Alkyl hydroxy and carboxy metabolites provided suitable biomarkers for 11 parent synthetic cannabinoids; although hydroxyindoles were also observed. This is by far the largest data set of synthetic cannabinoid metabolites urine concentrations from randomly collected workplace drug testing specimens rather than acute intoxications or driving under the influence of drugs. These data improve the interpretation of synthetic cannabinoid urine test results and suggest suitable urine markers of synthetic cannabinoid intake., (This article is a U.S. Government work and is in the public domain in the USA.)

Synthetic cannabinoids are marketed as legal alternatives to cannabis, as routine urine cannabinoid immunoassays do not detect synthetic cannabinoids. Laboratories are challenged to identify these new designer drugs that are widely available and represent a major public health and safety problem. Immunoassay testing offers rapid separation of presumptive positive and negative specimens, prior to more costly and time-consuming chromatographic confirmation. The Neogen SPICE ELISA kit targets JWH-018 N-pentanoic acid as a marker for urinary synthetic cannabinoids. Assay performance was evaluated by analyzing 2469 authentic urine samples with the Neogen immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two immunoassay cut-off concentrations, 5 and 10 µg/L, classified samples as presumptive positive or negative, followed by qualitative LC-MS/MS confirmation for 29 synthetic cannabinoids markers with limits of detection of 0.5-10 µg/L to determine the assay's sensitivity, specificity and efficacy. Challenges at ±25% of each cut-off also were investigated to determine performance around the cut-off and intra- and inter-plate imprecision. The immunoassay was linear from 1 to 250 µg/L (r(2)  = 0.992) with intra- and inter-plate imprecision of ≤5.3% and <9%, respectively. Sensitivity, specificity, and efficiency results with the 5 µg/L cut-off were 79.9%, 99.7%, and 97.4% and with the 10 µg/L cut-off 69.3%, 99.8%, and 96.3%, respectively. Cross-reactivity was shown for 18 of 73 synthetic cannabinoids markers evaluated. Good sensitivity, specificity, and efficiency, lack of sample preparation requirements, and rapid semi-automation documented that the Neogen SPICE ELISA kit is a viable method for screening synthetic cannabinoids in urine targeting JWH-018 N-pentanoic acid., (Copyright © 2014 John Wiley & Sons, Ltd.)

N-(Adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135) is a new synthetic cannabinoid in herbal incense products discussed on Internet drug user forums and identified in police seizures. To date, there are no STS-135 clinical or in vitro studies identifying STS-135 metabolites. However, characterizing STS-135 metabolism is critical because synthetic cannabinoid metabolites can possess pharmacological activity and parent compounds are rarely detectable in urine. To characterize the metabolite profile, human hepatocytes were incubated with 10 µmol/L STS-135 for up to 3 h. High-resolution mass spectrometry with software-assisted data mining identified 29 STS-135 metabolites. Less than 25% of STS-135 parent compound remained after 3 h incubation. Primary metabolites were generated by mono-, di- or trihydroxylation with and without ketone formation, dealkylation, and oxidative defluorination of N-fluoropentyl side chain or possible oxidation to carboxylic acid, some of them further glucuronidated. Hydroxylations occurred mainly on the aliphatic adamantane ring and less commonly on the N-pentyl side chain. At 1 h, phase I metabolites predominated, while at 3 h, phase II metabolites were present in higher amounts. The major metabolites were monohydroxy STS-135 (M25) and dihydroxy STS-135 (M21), both hydroxylated on the adamantane system. Moreover, metabolic stability of STS-135 (1 µmol/L) was assessed in human liver microsomes experiments. The in vitro half-life of STS-135 was 3.1 ± 0.2 min and intrinsic clearance (CLint ) was 208.8 mL · min(-1)  · kg(-1) . This is the first report characterizing STS-135 hepatic metabolic pathways. These data provide potential urinary targets to document STS-135 intake in clinical and forensic settings and potential candidates for pharmacological testing., (Copyright © 2014 John Wiley & Sons, Ltd.)

Evaluation of cannabinoid stability in authentic oral fluid (OF) is critical, as most OF stability studies employed fortified or synthetic OF. Participants (n = 16) smoked a 6.8% delta-9-tetrahydrocannabinol (THC) cigarette, and baseline concentrations of THC, 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), and cannabinol (CBN) were determined within 24 h in 16 separate pooled samples (collected 1 h before to 10.5 or 13 h after smoking). OF was collected with the StatSure Saliva Sampler™ and Oral-Eze® devices. Oral-Eze samples were re-analyzed after room temperature (RT) storage for 1 week, and for both devices after 4 °C for 1 and 4 weeks, and -20 °C for 4 and 24 weeks. Concentrations ±20% from initial concentrations were considered stable. With the StatSure device, all cannabinoids were within 80-120% median %baseline for all storage conditions. Individual THC, CBD, CBN and THCCOOH pool concentrations were stable in 100%, 100%, 80-94% and >85%, respectively, across storage conditions. With the Oral-Eze device, at RT or refrigerated storage (for 1 and 4 weeks), THC, CBD and THCCOOH were stable in 94-100%, 78-89%, and 93-100% of samples, respectively, while CBN concentrations were 53-79% stable. However, after 24 weeks at -20 °C, stability decreased, especially for CBD, with a median of 56% stability. Overall, the collection devices' elution/stabilizing buffers provided good stability for OF cannabinoids, with the exception of the more labile CBN. To ensure OF cannabinoid concentration accuracy, these data suggest analysis within 4 weeks at 4 °C storage for Oral-Eze collection and within 4 weeks at 4 °C or 24 weeks at -20 °C for StatSure collection. Published 2014. This article is a U.S. Government work and is in the public domain in the USA., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Oral fluid (OF) is an increasingly popular alternative matrix for drug testing, with cannabinoids being the most commonly identified illicit drug. Quantification of multiple OF cannabinoids and understanding differences in OF cannabinoid pharmacokinetics between frequent and occasional smokers improve test interpretation. The new Oral-Eze® OF collection device has an elution buffer that stabilizes analytes and improves drug recovery from the collection pad; however, its performance has not been independently evaluated. After controlled smoking of a 6.8% Δ(9) -tetrahydrocannabinol (THC) cannabis cigarette by frequent and occasional smokers, OF was collected with the Oral-Eze device for up to 30 h. Samples were analyzed for multiple cannabinoids by a validated 2D-GC-MS method. Frequent smokers had significantly greater OF THCCOOH concentrations than occasional smokers at all times, and showed positive results for a significantly longer time. We evaluated multiple cannabinoid cut-offs; the shortest last detection times were observed when THC ≥ 1 μg/L was combined with CBD or CBN ≥ 1 μg/L. With these cut-offs, last detection times(1-13.5 h) were not significantly different between groups, demonstrating suitability for short-term cannabinoid detection independent of smoking history. Cut-offs utilizing THC alone or combined with THCCOOH showed significantly different last detection times between groups. The widest detection windows were observed with THC ≥ 1 or 2 μg/L or THCCOOH ≥ 20 ng/L. Our data illustrate the effectiveness of the Oral-Eze® device for OF collection, the impact of self-administered smoked cannabis history on OF cannabinoid results, and the ability to improve interpretation and tailor OF cannabinoid cut-offs to fulfill the detection window needs of a given program., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Deterrence of synthetic cathinone abuse is hampered by the lack of a high-throughput immunoassay screen. The Randox Drugs of Abuse V (DOA-V) Biochip Array Technology contains two synthetic cathinone antibodies: Bath Salt I (BSI) targets mephedrone/methcathinone and Bath Salt II (BSII) targets 3',4'-methylenedioxypyrovalerone (MDPV)/3',4'-methylenedioxy-α-pyrrolidinobutiophenone (MDPBP). We evaluated DOA-V synthetic cathinones performance and conducted a full validation on the original assay with calibrators reconstituted in water, and the new assay with calibrators prepared in lyophilized urine; both utilized the same antibodies and were run on the fully automated Evidence® Analyzer. We screened 20 017 authentic military urine specimens and confirmed positives by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 28 synthetic cathinones. Limits of detection (LOD) for the original and new assays were 0.35 and 0.18 (BSI), and 8.5 and 9.2 µg/L (BSII), respectively. Linearity was acceptable (R(2)  >0.98); however, a large negative bias was observed with in-house prepared calibrators. Intra-assay imprecision was <20% BSI-II, while inter-assay imprecision was 18-42% BSI and <22% BSII. Precision was acceptable for Randox controls. Cross-reactivities of many additional synthetic cathinones were determined. Authentic drug-free negative urine pH <4 produced false positive results for BSI (6.3 µg/L) and BSII (473 µg/L). Oxidizing agents reduced BSI and increased BSII results. Sensitivity, specificity, and efficiency of 100%, 52.1%, and 53.0% were obtained at manufacturer's proposed cut-offs (BSI 5 µg/L, BSII 30 µg/L). Performance improved if cut-off concentrations increased (BSI 7.5 µg/L, BSII 40 µg/L); however, there were limited confirmed positive specimens. Currently, this is the first and only fully validated immunoassay for preliminary detection of synthetic cathinones in urine. Published 2014. This article is a U.S. Government work and is in the public domain in the USA., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Oral fluid (OF) is a new biological matrix for clinical and forensic drug testing, offering non-invasive and directly observable sample collection reducing adulteration potential, ease of multiple sample collections, lower biohazard risk during collection, recent exposure identification, and stronger correlation with blood than urine concentrations. Because cannabinoids are usually the most prevalent analytes in illicit drug testing, application of OF drug testing requires sufficient scientific data to support sensitive and specific OF cannabinoid detection. This review presents current knowledge of OF cannabinoids, evaluating pharmacokinetic properties, detection windows, and correlation with other biological matrices and impairment from field applications and controlled drug administration studies. In addition, onsite screening technologies, confirmatory analytical methods, drug stability, and effects of sample collection procedure, adulterants, and passive environmental exposure are reviewed. Delta-9-tetrahydrocannabinol OF concentrations could be >1000 µg/L shortly after smoking, whereas minor cannabinoids are detected at 10-fold and metabolites at 1000-fold lower concentrations. OF research over the past decade demonstrated that appropriate interpretation of test results requires a comprehensive understanding of distinct elimination profiles and detection windows for different cannabinoids, which are influenced by administration route, dose, and drug use history. Thus, each drug testing program should establish cut-off criteria, collection/analysis procedures, and storage conditions tailored to its purposes. Building a scientific basis for OF testing is ongoing, with continuing OF cannabinoids research on passive environmental exposure, drug use history, donor physiological conditions, and oral cavity metabolism needed to better understand mechanisms of cannabinoid OF disposition and expand OF drug testing applicability. Published 2013. This article is a U.S. Government work and is in the public domain in the USA., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA.)