Systematic web monitoring of drug test subversion strategies in the United States.

As negative drug tests are frequently a condition for employment, some people who use drugs will try to subvert the testing. In this study, systematic web monitoring was used to investigate how drug test subversion is discussed online. Posts pertaining to drug test subversion were obtained from public websites and the dark web (n = 634, July-December 2021). Most information from public websites came from Twitter (65%), and 94% of dark web posts were from Reddit. The posts were manually coded to extract quantitative and qualitative information about drug test subversion tactics. Most posts discussed urine drug tests (85%), followed by hair (11%) and oral fluid (2%), and the most discussed drugs were marijuana (72%) and cocaine (7.3%). Urine drug test subversion mainly pertained to specimen substitution, with synthetic urine or urine from another person. Another strategy was to mask diluted urine by ingesting creatine. Urine adulteration was rarely discussed. Hair test subversion involved harsh treatments with products such as bleach, baking soda, and/or detergent. Hair removal was also discussed. Oral fluid test subversion focused on removing drugs from the oral cavity through vigorous brushing of teeth and tongue as well as the use of mouthwash, hydrogen peroxide, gum, and commercial detox products. This study highlights subversion strategies used by donors. Although little evidence was provided as to the effectiveness of these strategies, this information may help guide future studies and development of specimen validity testing to minimize the impact of drug test subversion attempts.

Fentanyl as a marker of illicit drug use in morphine-positive urine specimens from workplace drug testing.

Total morphine is an important urinary marker of heroin use but can also be present from prescriptions or poppy seed ingestion. In specimens with morphine concentrations consistent with poppy seed ingestion (<4,000 ng/mL), 6-acetylmorphine has served as an important marker of illicit drug use. However, as illicit fentanyl has become increasingly prevalent as a contaminant in the drug supply, fentanyl might be an alternative marker of illicit opioid use instead of or in combination with 6-acetylmorphine. The aim of this study was to quantify opiates, 6-acetylmorphine, fentanyl and fentanyl analogs in 504 morphine-positive (immunoassay 2,000 ng/mL cutoff) urine specimens from workplace drug testing. Almost half (43%) of morphine-positive specimens had morphine concentrations below 4,000 ng/mL, illustrating the need for markers to differentiate illicit drug use. In these specimens, fentanyl (22% co-positivity) was more prevalent than 6-acetylmorphine (12%). Co-positivity of 6-acetylmorphine and semi-synthetic opioids increased with morphine concentration, while fentanyl prevalence did not. In 110 fentanyl-positive specimens, the median norfentanyl concentration (1,520 ng/mL) was 9.6× higher than the median fentanyl concentration (159 ng/mL), illustrating the possibility of using norfentanyl as a urinary marker of fentanyl use. The only fentanyl analog identified was para-fluorofentanyl (n = 50), with results from most specimens consistent with para-fluorofentanyl contamination in illicit fentanyl. The results confirm the use of fentanyl by employees subject to workplace drug testing and highlight the potential of fentanyl and/or norfentanyl as important markers of illicit drug use.

Prevalence of ∆8-tetrahydrocannabinol carboxylic acid in workplace drug testing.

∆8-Tetrahydrocannabinol (∆8-THC) recently became widely available as an alternative to cannabis. ∆8-THC is likely impairing and poses a threat to workplace and traffic safety. In the present study, the prevalence of ∆8-THC in workplace drug testing was investigated by analyzing 1,504 urine specimens with a positive immunoassay cannabinoid initial test using a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method quantifying 15 cannabinoid analytes after hydrolysis. ∆8-tetrahydrocannabinol-9-carboxylic acid (∆8-THC-COOH) was detected in 378 urine specimens (15 ng/mL cutoff), compared to 1,144 specimens containing ∆9-THC-COOH. The data could be divided into three general groups. There were 964 (76%) ∆9-THC-COOH-dominant (<10% ∆8-THC-COOH) and 139 (11%) ∆8-THC-COOH-dominant (>90% ∆8-THC-COOH) specimens, with the remaining 164 (13%) specimens showing a mixture of both analytes (>90% ∆8-THC-COOH). Similar concentrations of ∆9-THC-COOH (median 187 ng/mL) and ∆8-THC-COOH (150 ng/mL) as the dominant species support the use of similar cutoffs and decision rules for both analytes. Apart from the carboxylic acid metabolites, 11-hydroxy-∆9-tetrahydrocannabinol (11-OH-∆9-THC, n = 1,282), ∆9-tetrahydrocannabivarin-9-carboxylic acid (∆9-THCV-COOH, n = 1,058), ∆9-THC (n = 746) and 7-hydroxy-cannabidiol (7-OH-CBD, n = 506) were the most prevalent analytes. Two specimens (0.13%) contained ≥140 ng/mL ∆9-THC without ∆9-THC-COOH, which could be due to genetic variability in the drug-metabolizing enzyme CYP2C9 or an adulterant targeting ∆9-THC-COOH. The cannabinoid immunoassay was repeated, and five specimens (0.33%) generated negative initial tests despite ∆9-THC-COOH concentrations of 54-1,000 ng/mL, potentially indicative of adulteration. The use of ∆8-THC is widespread in the US population, and all forensic laboratories should consider adding ∆8-THC and/or ∆8-THC-COOH to their scope of testing. Similar urinary concentrations were observed for both analytes, indicating that the decision rules used for ∆9-THC-COOH are also appropriate for ∆8-THC-COOH.

∆8-THC-COOH cross-reactivity with cannabinoid immunoassay kits and interference in chromatographic testing methods.

Because of structural similarities, the presence of 11-Nor-9-carboxy-∆8-tetrahydrocannabinol (∆8-THC-COOH) in a urine specimen might interfere with testing for 11-Nor-9-carboxy-∆9-tetrahydrocannabinol (∆9-THC-COOH). A set of samples containing ∆8-THC-COOH with concentrations ranging from 10 to 120 ng/mL were tested at cut-offs of 20, 50 and 100 ng/mL using cannabinoid immunoassay reagents from three different manufacturers. Cross-reactivities ranged from 87% to 112% for ∆8-THC-COOH at the cut-off of 50 ng/mL for the three different platforms. Additionally, samples containing both ∆8-THC-COOH and ∆9-THC-COOH were fortified by the National Laboratory Certification Program (NLCP). U.S. Department of Health and Human Services (HHS)-Certified Laboratories tested the samples to determine the interference of ∆8-THC-COOH on confirmatory tests commonly used in workplace drug testing laboratories for the confirmation and quantification of ∆9-THC-COOH. When evaluating confirmation and quantification of ∆9-THC-COOH in the presence of ∆8-THC-COOH, unreportable results for ∆9-THC-COOH were observed because of chromatographic interference or mass ratio failures. However, there were no false-positive ∆9-THC-COOH reports from any HHS-certified laboratory.

Conversion of water-soluble CBD to ∆9-THC in synthetic gastric fluid-An unlikely cause of positive drug tests.

Cannabidiol (CBD) has been shown to convert to ∆9-tetrahydrocannabinol (∆9-THC) in acidic environments, raising a concern of conversion when exposed to gastric fluid after consumption. Using synthetic gastric fluid (SGF), it has been demonstrated that the conversion requires surfactants, such as sodium dodecyl sulfate (SDS), due to limited solubility of CBD. Recently, water-compatible nanoemulsions of CBD have been prepared as a means of fortifying beverages and water-based foods with CBD. Since these emulsions contain surfactants as part of their formulation, it is possible that these preparations might enhance the production of ∆9-THC even in the absence of added surfactants. Three THC-free CBD products, an oil, an anhydrous powder and a water-soluble formulation, were incubated for 3 h in SGF without SDS. The water-soluble CBD product produced a dispersion, while the powder and the oil did not mix with the SGF. No THC was detected with the CBD oil (<0.0006% conversion), and up to 0.063% and 0.0045% conversion to ∆9-THC was observed with the water-soluble CBD and the CBD powder, respectively. No formation of ∆8-THC was observed. In comparison, when the nano-formulated CBD was incubated in SGF with 1% SDS, 33-36% conversion to ∆9-THC was observed. Even though the rate of conversion with the water-soluble CBD was at least 100-fold higher compared to the CBD oil, it was still smaller than ∆9-THC levels reported in CBD products labeled "THC-free" or "<0.3% THC" based on the Agricultural Improvement Act of 2018 (the Farm Bill). Assuming a daily CBD dose of around 30 mg/day, it is unlikely that conversion of CBD to ∆9-THC could produce a positive urinary drug test for 11-Nor-9-carboxy-∆9-THC (15 ng/mL cut-off).

Performance of Hair Testing for Cocaine Use-Comparison of Five Laboratories Using Blind Reference Specimens.

The purpose of this study was to compare results from five commercial hair testing laboratories conducting workplace drug testing with regard to bias, precision, selectivity and decontamination efficiency. Nine blind hair specimens, including cocaine-positive drug user specimens (some contaminated with methamphetamine) and negative specimens contaminated with cocaine, were submitted in up to five replicates to five different laboratories. All laboratories correctly identified cocaine in all specimens from drug users. For an undamaged hair specimen from a cocaine user, within-laboratory Coefficients of Variation (CVs) of 5-22% (median 8%) were reported, showing that it is possible to produce a homogenous proficiency testing sample from drug user hair. Larger CVs were reported for specimens composed of blended hair (up to 29%) and curly/damaged hair (19-67%). Quantitative results appeared to be method-dependent, and the reported cocaine concentrations varied up to 5-fold between the laboratories, making interlaboratory comparisons difficult. All laboratories reported at least one positive result in specimens contaminated with cocaine powder, followed by sweat and shampoo treatments. Benzoylecgonine, norcocaine, cocaethylene and hydroxylated cocaine metabolites were all detected in cocaine powder-contaminated specimens. This indicates that current industry standards for analyzing and reporting positive cocaine results are not completely effective at identifying external contamination. Metabolite ratios between meta- or para-hydroxy-cocaine and cocaine were 6- and 10-fold lower in contaminated specimens compared to those observed in cocaine user specimens, supporting their potential use in distinguishing samples positive due to contamination and drug use.

Update on Urine Adulterants and Synthetic Urine Samples to Subvert Urine Drug Testing.

To avoid a positive urine drug test, donors might try to subvert the test, either by adulterating the specimen with a product designed to interfere with testing or by substituting the specimen for a synthetic urine. A market search conducted in December of 2020 identified 3 adulterants and 32 synthetic urines, and a selection was procured based on specific criteria. Samples prepared with the 3 adulterants and 10 synthetic urines were submitted for testing at five forensic drug testing laboratories to perform immunoassay screening, chromatographic confirmation analysis and specimen validity testing (SVT). One adulterant determined to contain iodate reduced THC-COOH concentrations by 65% and the concentrations of 6-acetylmorphine, morphine, oxycodone, oxymorphone, hydrocodone and hydromorphone by 6-27%. Another adulterant determined to contain nitrite reduced THC-COOH concentrations by 22%, while the third did not affect drug screening or confirmatory testing. Both active adulterants could be identified through positive oxidant screens as well as through signal suppression in cloned enzyme donor immunoassay (CEDIA). The synthetic urines could not be identified either through traditional SVT or by the AdultaCheck10 dipstick. The Synthetic UrineCheck dipstick produced a difference in response between the authentic urine specimen and the synthetic urine samples, but the difference was small and difficult to observe. While most synthetic urines now contain uric acid, magnesium and caffeine, the results indicated that a biomarker panel including endogenous and exogenous markers of authentic urine performed well and clearly demonstrated the absence of biomarkers in the synthetic urines. The SVT assay also offers potential targets for future screening assays.

Prevalence of Cannabidiol, ∆9- and ∆8-Tetrahydrocannabinol and Metabolites in Workplace Drug Testing Urine Specimens.

Given the recent popularity of cannabidiol (CBD) use and the emergence of ∆8-tetrahydrocannabinol (∆8-THC), the prevalence and concentrations of these and other cannabinoids were investigated in 2,000 regulated and 4,000 non-regulated specimens from workplace drug testing. All specimens were screened using liquid chromatography coupled to mass spectrometry (LC-MS-MS) for the presence of 7-hydroxy-CBD (7-OH-CBD) and ∆9-tetrahydrocannabinol-9-carboxylic acid (∆9-THC-COOH), with a cutoff of 2 ng/mL. Specimens screening positive by LC-MS-MS were analyzed by immunoassay at 20, 50 and 100 ng/mL cutoffs and by an LC-MS-MS confirmation method for 11 cannabinoids and metabolites with a 1 ng/mL cutoff. Using a 1 ng/mL cutoff, 98 (4.9%) regulated and 331 (8.3%) non-regulated specimens were positive for ∆9-THC-COOH. Of these, 64% had concentrations below 15 ng/mL. Similarly, 59 (3.0%) regulated and 162 (4.2%) non-regulated specimens were positive for 7-OH-CBD (n = 210), CBD (n = 120) and/or 7-carboxy-cannabidiol (CBD-COOH, n = 120). The median concentrations of 7-OH-CBD, CBD and CBD-COOH in those 221 specimens were 6.3, 1.1 and 1.2 ng/mL, respectively. ∆8-Tetrahydrocannabinol-9-carboxylic acid (∆8-THC-COOH) was identified in 76 (1.3%) specimens. Parent ∆8-THC is a minor cannabinoid in marijuana, which appears to account for the typically low ∆8-THC-COOH concentrations (median 3.4 ng/mL) in most positive specimens. However, elevated concentrations suggested the use of ∆8-THC-containing products in some cases (range 1.0-415 ng/mL). Although 93% agreement was observed between confirmatory LC-MS-MS (15 ng/mL cutoff) and immunoassay (50 ng/mL cutoff), a false-negative specimen (66 ng/mL ∆9-THC-COOH) was identified.

Conversion of 7-Carboxy-Cannabidiol (7-COOH-CBD) to 11-Nor-9-Carboxy-Tetrahydrocannabinol (THC-COOH) during Sample Preparation for GC-MS Analysis.

The growing use of cannabidiol (CBD) products by the general public is expected to result in an increase in the prevalence of CBD and the CBD metabolites in drug testing laboratories. CBD converts into tetrahydrocannabinol (THC) under acid conditions which could produce false-positive results, but little is known about how the presence of the urinary metabolite of CBD, 7-carboxy-cannabidiol (7-COOH-CBD), would affect urine drug testing for 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH). As the operators of the National Laboratory Certification Program (NLCP), we prepared a set of performance testing samples containing 7-COOH-CBD for cannabinoid testing at the laboratories accredited by the NLCP to investigate if 7-COOH-CBD can produce false-positive results for THC-COOH during immunological screening analysis and if 7-COOH-CBD can be converted to THC-COOH. At concentrations up to 2,500 ng/mL, 7-COOH-CBD was not reactive by immunoassay in any of the four different immunoassay kits used. Additionally, we did not observe any significant conversion of 7-COOH-CBD to THC-COOH in assays used by NLCP-certified laboratories. However, we did see conversion when we requested that selected laboratories retest their samples using derivatization with perfluorinated anhydrides in combination with perfluorinated alcohols or when samples containing 7-COOH-CBD were exposed to acid for an extended time.

Urinary Excretion Profile of 11-Nor-9-Carboxy-Δ9-Tetrahydrocannabinol (THCCOOH) Following Smoked and Vaporized Cannabis Administration in Infrequent Cannabis Users.

As cannabis has become more accessible, use of alternative methods for cannabis administration such as vaporizers has become more prevalent. Most prior controlled pharmacokinetic evaluations have examined smoked cannabis in frequent (often daily) cannabis users. This study characterized the urinary excretion profile of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH), the primary analytical outcome for detection of cannabis use, among infrequent cannabis users following controlled administration of both smoked and vaporized cannabis. Healthy adults (N = 17), with a mean of 398 (range 30-1,825) days since last cannabis use, smoked and vaporized cannabis containing 0, 10, and 25 mg of Δ9-tetrahydrocannabinol (THC) across six outpatient sessions. Urinary concentrations of THCCOOH were measured at baseline and for 8 h after cannabis administration. Sensitivity, specificity, and agreement between three immunoassays (IA) for THCCOOH (with cutoffs of 20, 50, and 100 ng/mL) and gas chromatography-mass spectrometry (GC/MS) results (confirmatory concentration of 15 ng/mL) were assessed. THCCOOH concentrations peaked 4-6 h after cannabis administration. Median maximum concentrations (Cmax) for THCCOOH were qualitatively higher after administration of vaporized cannabis compared to equal doses of smoked cannabis. Urine THCCOOH concentrations were substantially lower in this study relative to prior examinations of experienced cannabis users. The highest agreement between IA and GC/MS was observed at the 50 ng/mL IA cutoff while sensitivity and specificity were highest at the 20 and 100 ng/mL IA cutoffs, respectively. Using federal workplace drug-testing criteria (IA cutoff of 50 ng/mL and GC/MS concentration ≥15 ng/mL) urine specimens tested positive in 47% of vaporized sessions and 21% of smoked sessions with active THC doses (N = 68). Urinary concentrations of THCCOOH are dissimilar after administration of smoked and vaporized cannabis, with qualitatively higher concentrations observed after vaporization. Infrequent users of cannabis may excrete relatively low concentrations of THCCOOH following acute inhalation of smoked or vaporized cannabis.

Acute Pharmacokinetic Profile of Smoked and Vaporized Cannabis in Human Blood and Oral Fluid.

Currently, an unprecedented number of individuals can legally access cannabis. Vaporization is increasingly popular as a method to self-administer cannabis, partly due to perception of reduced harm compared with smoking. Few controlled laboratory studies of cannabis have used vaporization as a delivery method or evaluated the acute effects of cannabis among infrequent cannabis users. This study compared the concentrations of cannabinoids in whole blood and oral fluid after administration of smoked and vaporized cannabis in healthy adults who were infrequent users of cannabis. Seventeen healthy adults, with no past-month cannabis use, self-administered smoked or vaporized cannabis containing Δ9-tetrahydrocannabinol (THC) doses of 0, 10 and 25 mg in six double-blind outpatient sessions. Whole blood and oral fluid specimens were obtained at baseline and for 8 h after cannabis administration. Cannabinoid concentrations were assessed with enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods. Sensitivity, specificity and agreement between ELISA and LC-MS-MS results were assessed. Subjective, cognitive performance and cardiovascular effects were assessed. The highest concentrations of cannabinoids in both whole blood and oral fluid were typically observed at the first time point (+10 min) after drug administration. In blood, THC, 11-OH-THC, THCCOOH and THCCOOH-glucuronide concentrations were dose-dependent for both methods of administration, but higher following vaporization compared with smoking. THC was detected longer in oral fluid compared to blood and THCCOOH detection in oral fluid was rare and highly erratic. For whole blood, greater detection sensitivity for ELISA testing was observed in vaporized conditions. Conversely, for oral fluid, greater sensitivity was observed in smoked sessions. Blood and/or oral fluid cannabinoid concentrations were weakly to moderately correlated with pharmacodynamic outcomes. Cannabis pharmacokinetics vary by method of inhalation and biological matrix being tested. Vaporization appears to be a more efficient method of delivery compared with smoking.

Development of a Quantitative LC-MS-MS Assay for Codeine, Morphine, 6-Acetylmorphine, Hydrocodone, Hydromorphone, Oxycodone and Oxymorphone in Neat Oral Fluid.

Recent advances in analytical capabilities allowing for the identification and quantification of drugs and metabolites in small volumes at low concentrations have made oral fluid a viable matrix for drug testing. Oral fluid is an attractive matrix option due to its relative ease of collection, reduced privacy concerns for observed collections and difficulty to adulterate. The work presented here details the development and validation of a liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the quantification of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone and oxymorphone in neat oral fluid. The calibration range is 0.4-150 ng/mL for 6-acetylmorphine and 1.5-350 ng/mL for all other analytes. Within-run and between-run precision were <5% for all analytes except for hydrocodone, which had 6.2 %CV between runs. Matrix effects, while evident, could be controlled using matrix-matched controls and calibrators with deuterated internal standards. The assay was developed in accordance with the proposed mandatory guidelines for opioid confirmation in federally regulated workplace drug testing. The use of neat oral fluid, as opposed to a collection device, enables collection of a single sample that can be split into separate specimens.

Pharmacokinetic Characterization of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in Urine Following Acute Oral Cannabis Ingestion in Healthy Adults.

Understanding the urine excretion profile for Δ9-tetrahydrocannabinol (THC) metabolites is important for accurate detection and interpretation of toxicological testing for cannabis use. Prior literature has primarily evaluated the urinary pharmacokinetics of the non-psychoactive THC metabolite 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) following smoked cannabis administration. The present study examined the urine THCCOOH excretion profile following oral cannabis administration in 18 healthy adults. Following ingestion of a cannabis-containing brownie with 10, 25 or 50 mg of THC (N = 6 per dose), urine specimens were collected on a closed residential research unit for 6 days, followed by three outpatient visits on Days 7-9. Average maximum concentrations (Cmax) of THCCOOH were 107, 335 and 713 ng/mL, and average times to maximum concentration (Tmax) were 8, 6 and 9 h for the 10, 25 and 50 mg THC doses, respectively. Detection windows to first positive and last positive varied as a function of dose; higher doses had shorter time to first positive and longer time to last positive. Considerable inter-subject variability was observed on study outcomes. Gas chromatography/mass spectrometry (GC/MS; 15 ng/mL cutoff) was used as the criterion to assess sensitivity, specificity and agreement for THCCOOH qualitative immunoassay tests using 20, 50 and 100 ng/mL cutoffs. The 50 ng/mL cutoff displayed good sensitivity (92.5%), specificity (92.4%) and overall agreement (92.4%), whereas the 20 ng/mL cutoff demonstrated poor specificity (58.4%), and the 100 ng/mL cutoff exhibited reduced sensitivity (70.9%). Ingestion of cannabis brownies containing the 10 and 25 mg THC doses yielded THCCOOH concentrations that differed in magnitude and time course from those previously reported for the smoked route of administration of comparable doses.

Acute Effects of Smoked and Vaporized Cannabis in Healthy Adults Who Infrequently Use Cannabis: A Crossover Trial.

Importance: Vaporization is an increasingly popular method for cannabis administration, and policy changes have increased adult access to cannabis drastically. Controlled examinations of cannabis vaporization among adults with infrequent current cannabis use patterns (>30 days since last use) are needed. Objective: To evaluate the acute dose effects of smoked and vaporized cannabis using controlled administration methods. Design, Setting, and Participants: This within-participant, double-blind, crossover study was conducted from June 2016 to January 2017 at the Behavioral Pharmacology Research Unit, Johns Hopkins University School of Medicine, and included 17 healthy adults. Six smoked and vaporized outpatient experimental sessions (1-week washout between sessions) were completed in clusters (order counterbalanced across participants); dose order was randomized within each cluster. Interventions: Cannabis containing Δ9-tetrahydrocannabinol (THC) doses of 0 mg, 10 mg, and 25 mg was vaporized and smoked by each participant. Main Outcomes and Measures: Change from baseline scores for subjective drug effects, cognitive and psychomotor performance, vital signs, and blood THC concentration. Results: The sample included 17 healthy adults (mean [SD] age, 27.3 [5.7] years; 9 men and 8 women) with no cannabis use in the prior month (mean [SD] days since last cannabis use, 398 [437] days). Inhalation of cannabis containing 10 mg of THC produced discriminative drug effects (mean [SD] ratings on a 100-point visual analog scale, smoked: 46 [26]; vaporized: 69 [26]) and modest impairment of cognitive functioning. The 25-mg dose produced significant drug effects (mean [SD] ratings, smoked: 66 [29]; vaporized: 78 [24]), increased incidence of adverse effects, and pronounced impairment of cognitive and psychomotor ability (eg, significant decreased task performance compared with placebo in vaporized conditions). Vaporized cannabis resulted in qualitatively stronger drug effects for most pharmacodynamic outcomes and higher peak concentrations of THC in blood, compared with equal doses of smoked cannabis (25-mg dose: smoked, 10.2 ng/mL; vaporized, 14.4 ng/mL). Blood THC concentrations and heart rate peaked within 30 minutes after cannabis administration and returned to baseline within 3 to 4 hours. Several subjective drug effects and observed cognitive and psychomotor impairments persisted for up to 6 hours on average. Conclusions and Relevance: Vaporized and smoked cannabis produced dose-orderly drug effects, which were stronger when vaporized. These data can inform regulatory and clinical decisions surrounding the use of cannabis among adults with little or no prior cannabis exposure. Trial Registration: ClinicalTrials.gov Identifier: NCT03676166.

Detection and quantification of codeine-6-glucuronide, hydromorphone-3-glucuronide, oxymorphone-3-glucuronide, morphine 3-glucuronide and morphine-6-glucuronide in human hair from opioid users by LC-MS-MS.

Current hair testing methods that rely solely on quantification of parent drug compounds are unable to definitively distinguish between drug use and external contamination. One possible solution to this problem is to confirm the presence of unique drug metabolites that cannot be present through contamination, such as phase II glucuronide conjugates. This work demonstrates for the first time that codeine-6-glucuronide, hydromorphone-3-glucuronide, oxymorphone-3-glucuronide, morphine-3-glucuronide and morphine-6-glucuronide are present at sufficient concentrations to be quantifiable in hair of opioid users and that their concentrations generally increase as the concentrations of the corresponding parent compounds increase. Here, we present a validated liquid chromatography tandem mass spectrometry method to quantify codeine-6-glucuronide, dihydrocodeine-6-glucuronide, hydromorphone-3-glucuronide, morphine-3-glucuronide, morphine-6-glucuronide, oxymorphone-3-glucuronide, codeine, dihydrocodeine, dihydromorphine, hydrocodone, hydromorphone, morphine, oxycodone, oxymorphone and 6-acetylmorphine in human hair. The method was used to analyze 46 human hair samples from known drug users that were confirmed positive for opioids by an independent laboratory. Glucuronide concentrations in samples positive for parent analytes ranged from ~1 to 25 pg/mg, and most samples had glucuronide concentrations in the range of ~1 to 5 pg/mg. Relative to the parent concentrations, the average concentrations of the four detected glucuronides were as follows: codeine-6-glucuronide, 2.33%; hydromorphone-3-glucuronide, 0.94%; oxymorphone-3-glucuronide, 0.77%; morphine 3-glucuronide, 0.59%; and morphine-6-glucuronide, 0.93%.

Assessment of the stability of DNA in specimens collected under conditions for drug testing-A pilot study.

For forensic biological sample collections, the specimen donor is linked solidly to his or her specimen through a chain of custody (CoC) sometimes referenced as a chain of evidence. Rarely, a donor may deny that a urine or oral fluid (OF) specimen is his or her specimen even with a patent CoC. The goal of this pilot study was to determine the potential effects of short-term storage on the quality and quantity of DNA in both types of specimen under conditions that may be encountered with employment-related drug testing specimens. Fresh urine and freshly collected oral fluid all produced complete STR profiles. For the "pad" type OF collectors, acceptable DNA was extractable both from the buffer/preservative and the pad. Although fresh urine and OF produced complete STR profiles, partial profiles were obtained after storage for most samples. An exception was the DNA in the Quantisal OF collector, from which a complete profile was obtained for both freshly collected OF and stored OF.

Disruption of uridine homeostasis links liver pyrimidine metabolism to lipid accumulation.

We report in this study an intrinsic link between pyrimidine metabolism and liver lipid accumulation utilizing a uridine phosphorylase 1 transgenic mouse model UPase1-TG. Hepatic microvesicular steatosis is induced by disruption of uridine homeostasis through transgenic overexpression of UPase1, an enzyme of the pyrimidine catabolism and salvage pathway. Microvesicular steatosis is also induced by the inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme of the de novo pyrimidine biosynthesis pathway. Interestingly, uridine supplementation completely suppresses microvesicular steatosis in both scenarios. The effective concentration (EC(50)) for uridine to suppress microvesicular steatosis is approximately 20 µM in primary hepatocytes of UPase1-TG mice. We find that uridine does not have any effect on in vitro DHODH enzymatic activity. On the other hand, uridine supplementation alters the liver NAD(+)/NADH and NADP(+)/NADPH ratios and the acetylation profile of metabolic, oxidation-reduction, and antioxidation enzymes. Protein acetylation is emerging as a key regulatory mechanism for cellular metabolism. Therefore, we propose that uridine suppresses fatty liver by modulating the liver protein acetylation profile. Our findings reveal a novel link between uridine homeostasis, pyrimidine metabolism, and liver lipid metabolism.

Development of an oral form of azacytidine: 2'3'5'triacetyl-5-azacytidine.

Myelodysplastic syndromes (MDSs) represent a group of incurable stem-cell malignancies which are predominantly treated by supportive care. Epigenetic silencing through promoter methylation of a number of genes is present in poor-risk subtypes of MDS and often predicts transformation to acute myelogenous leukemia (AML). Azacitidine and decitabine, two FDA-approved DNA methyltransferase (DNMT) inhibitors, are able to improve overall response although their oral bioavailability complicates their clinical use. This study evaluated 2', 3', 5'-triacetyl-5-azacitidine (TAC) as a potential prodrug for azacitidine. The prodrug demonstrated significant pharmacokinetic improvements in bioavailability, solubility, and stability over the parent compound. In vivo analyses indicated a lack of general toxicity coupled with significantly improved survival. Pharmacodynamic analyses confirmed its ability to suppress global methylation in vivo. These data indicate that esterified nucleoside derivatives may be effective prodrugs for azacitidine and encourages further investigation of TAC into its metabolism, activity, and possible clinical evaluation.

Cannabinoid concentrations in hair from documented cannabis users.

Fifty-three head hair specimens were collected from 38 males with a history of cannabis use documented by questionnaire, urinalysis and controlled, double blind administration of delta9-tetrahydrocannabinol (THC) in an institutional review board approved protocol. The subjects completed a questionnaire indicating daily cannabis use (N=18) or non-daily use, i.e. one to five cannabis cigarettes per week (N=20). Drug use was also documented by a positive cannabinoid urinalysis, a hair specimen was collected from each subject and they were admitted to a closed research unit. Additional hair specimens were collected following smoking of two 2.7% THC cigarettes (N=13) or multiple oral doses totaling 116 mg THC (N=2). Cannabinoid concentrations in all hair specimens were determined by ELISA and GCMSMS. Pre- and post-dose detection rates did not differ statistically, therefore, all 53 specimens were considered as one group for further comparisons. Nineteen specimens (36%) had no detectable THC or 11-nor-9-carboxy-THC (THCCOOH) at the GCMSMS limits of quantification (LOQ) of 1.0 and 0.1 pg/mg hair, respectively. Two specimens (3.8%) had measurable THC only, 14 (26%) THCCOOH only, and 18 (34%) both cannabinoids. Detection rates were significantly different (p<0.05, Fishers' exact test) between daily cannabis users (85%) and non-daily users (52%). There was no difference in detection rates between African-American and Caucasian subjects (p>0.3, Fisher's exact test). For specimens with detectable cannabinoids, concentrations ranged from 3.4 to >100 pg THC/mg and 0.10 to 7.3 pg THCCOOH/mg hair. THC and THCCOOH concentrations were positively correlated (r=0.38, p<0.01, Pearson's product moment correlation). Using an immunoassay cutoff concentration of 5 pg THC equiv./mg hair, 83% of specimens that screened positive were confirmed by GCMSMS at a cutoff concentration of 0.1 pg THCCOOH/mg hair.