Analysis of tetrahydrocannabinol and its metabolite, 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid, in oral fluid using liquid chromatography with tandem mass spectrometry

This paper describes the determination of tetrahydrocannabinol (THC) and its metabolite, 11-nor-Δ?-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid using solid-phase extraction and liquid chromatography with tandem mass spectral detection (LC-MS-MS) and its application to proficiency specimens. The method employs collection of oral fluid with the Quantisal? device, base hydrolysis, solid-phase extraction and LC-MS-MS in positive ion electrospray mode. Because the concentration of the metabolite in oral fluid is quite low, extremely sensitive analytical methods are necessary. The requisite sensitivity was achieved by a simple, rapid derivatization of the compound after extraction. The derivatization conditions did not affect parent THC. The method was fully validated using standard parameters including linearity, sensitivity, accuracy, intra-day and inter-day imprecision, drug recovery from the collection pad, limit of quantitation, limit of detection and matrix effects. The procedure was applied to oral fluid proficiency specimens previously analyzed to assess the stability of THC-COOH.     

Assessment of a natural extraction phase in disposable pipette extraction coupled with the sub-minute determination of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in human urine by fast-GC-FID

The compound 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) consists of the major metabolite of tetrahydrocannabinol (THC) in human urine. Analysis of this compound exhibits an important role in forensic scenario; therefore, several analytical methods have been developed for this purpose. In this study, for the first time, a biosorbent-based disposable pipette extraction was developed for the determination of THC-COOH, using a simple and high-throughput sample preparation technique. In addition, a rapid instrumental analysis was achieved by fast-gas chromatography coupled with flame ionization detection (fast-GC-FID). Different biosorbents were examined, including cork, moringa and bract. The method was optimized through univariate and multivariate designs with the conditions comprised of 15 mg of cork as the extraction phase, pH maintained at 2.5 with 8 cycles of extraction of 60 s each, and 1 cycle of 5 s for the desorption step with a mixture of acetonitrile:methanol (ACN/MeOH). Limit of detection was 0.21 ng mL^?1 and the limit of quantification was 0.73 ng mL^?1, with a linear range from 5 to 100 ng mL^?1 and coefficient of determination (R²) 0.9905. The relative recoveries ranged from 96 to 108%, intraday and interday precisions were lower than 13%. Different samples were subjected to the extractions using the method developed with four positive samples for the analyte.     

Urinary clearance of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol: A detailed pharmacokinetic analysis

Urine is a common matrix for screening for cannabis use. Urine assays typically measure total 11-nor-9-carboxy-Δ⁹-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) Δ⁹-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, r² = 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.     

Δ9-tetrahydrocannabinol and its major metabolite Δ9-tetrahydrocannabinol-11-oic acid as 15-lipoxygenase inhibitors

15-Lipoxygenase (15-LOX) is one of the key enzymes responsible for the formation of oxidized low-density lipoprotein, a major causal factor for atherosclerosis. Δ(9)-Tetrahydrocannabinol (Δ(9)-THC), a major component of marijuana, has suggested to suppress atherosclerosis. Although Δ(9)-THC seems to be attractive for the prevention of atherosclerosis, there is no information about whether or not 15-LOX isoform can be inhibited by Δ(9)-THC. In the present study, Δ(9)-THC was found to be a direct inhibitor for 15-LOX with an IC(50) (50% inhibition concentration) value of 2.42 μM. Furthermore, Δ(9)-THC-11-oic acid, a major and nonpsychoactive metabolite of Δ(9) -THC, but not another Δ(9)-THC metabolite 11-OH-Δ(9)-THC (psychoactive), was revealed to inhibit 15-LOX. Taken together, it is suggested that Δ(9) -THC can abrogate atherosclerosis via direct inhibition of 15-LOX, and that Δ(9)-THC-11-oic acid is shown to be an "active metabolite" of Δ(9) -THC in this case.     

Simultaneous analysis of Δ(9)-tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol in hair without different sample preparation and derivatization by gas chromatography-tandem mass spectrometry

The present study describes a gas chromatography/tandem mass spectrometry-negative ion chemical ionization assay (GC/MS/MS-NCI) for simultaneous analysis of Δ(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) in hair. Each hair sample, of approximately 20mg, was weighed and the sample was dissolved in 1ml of 1M sodium hydroxide (30min at 85°C) in the presence of THC-d(3) and THCCOOH-d(3). For the analysis of THC, hair samples were extracted with n-hexane:ethyl acetate (9:1) two times; acetic acid and sodium acetate buffer were added for the analysis of THCCOOH, and hair samples were re-extracted with n-hexane:ethyl acetate (9:1) two times. The extracts were then derivatized with pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH). This method allowed the analysis of THC and THCCOOH using the GC/MS/MS-NCI assay. This method was also fully validated and applied to hair specimens (n=54) collected from known cannabis users whose urine test results were positive. The concentrations of THC and THCCOOH in hair ranged from 7.52 to 60.41ng/mg and from 0.10 to 11.68pg/mg, respectively. In this paper, we simultaneously measured THC and THCCOOH in human hair using GC/MS/MS-NCI without requiring different sample preparation and derivatization procedures. The analytical sensitivity for THCCOOH in hair was good, while that for THC in hair needs to be improved in further study.     

Are reasons for the first use of drugs and family circumstances predictors of future use patterns?

The objective of this study was to investigate whether the level of drug dependence in adolescents can be predicted by any of the following factors: the reasons to which the first use or non-use of drugs is attributed, living arrangements, economic situation, family history of alcohol use, or school delay. 213 Brazilian adolescents were classified according to DSM-III-R criteria as: 71 non-drug-dependent users (Group 1), 71 lightly/moderately dependent users (Group 2), and 71 severely dependent users (Group 3). Logistic regression identified the following predictors of current drug use patterns: low economic level, school delay, living only with the mother, having a poor/bad family relationship, and "influence of friends," "pleasure seeking," or "curiosity" as reasons for initial drug use. Among Groups 1 and 2, "never felt like trying," "fear of dying from an overdose," and "religious reasons" were the main reasons for not using other drugs. School delay and troubled family relationships were important predictors of current drug dependence, and "pleasure seeking" was a prominent reason for initial drug use. This suggests that drug use prevention should not simply focus on reducing drug availability but also on helping young people to develop good family/peer relationships and finding healthy ways to enjoy themselves.     

Is cannabis a gateway drug? Testing hypotheses about the relationship between cannabis use and the use of other illicit drugs

We outline and evaluate competing explanations of three relationships that have consistently been found between cannabis use and the use of other illicit drugs, namely, (1) that cannabis use typically precedes the use of other illicit drugs; and that (2) the earlier cannabis is used, and (3) the more regularly it is used, the more likely a young person is to use other illicit drugs. We consider three major competing explanations of these patterns: (1) that the relationship is due to the fact that there is a shared illicit market for cannabis and other drugs which makes it more likely that other illicit drugs will be used if cannabis is used; (2) that they are explained by the characteristics of those who use cannabis; and (3) that they reflect a causal relationship in which the pharmacological effects of cannabis on brain function increase the likelihood of using other illicit drugs. These explanations are evaluated in the light of evidence from longitudinal epidemiological studies, simulation studies, discordant twin studies and animal studies. The available evidence indicates that the association reflects in part but is not wholly explained by: (1) the selective recruitment to heavy cannabis use of persons with pre-existing traits (that may be in part genetic) that predispose to the use of a variety of different drugs; (2) the affiliation of cannabis users with drug using peers in settings that provide more opportunities to use other illicit drugs at an earlier age; (3) supported by socialisation into an illicit drug subculture with favourable attitudes towards the use of other illicit drugs. Animal studies have raised the possibility that regular cannabis use may have pharmacological effects on brain function that increase the likelihood of using other drugs. We conclude with suggestions for the type of research studies that will enable a decision to be made about the relative contributions that social context, individual characteristics, and drug effects make to the relationship between cannabis use and the use of other drugs.     

Clinical effects and plasma levels of Δ9-Tetrahydrocannabinol (Δ9-THC) in heavy and light users of cannabis

⁹-Tetrahydrocannabinol (⁹-THC) was administered in a crossover design by smoking and IV injection to groups of heavy and light users of marihuana. Plasma concentrations of ⁹-THC were similar for the groups after IV injection of 5.0 mg ⁹-THC, but the AUC_{0–240 min} showed a trend towards lower values for the heavy user group. To achieve a maximum desired high, both groups smoked similar amounts (about 13 mg) of ⁹-THC. Heavy users tended to have higher plasma levels than light users. The systemic availability of smoked ⁹-THC was significantly higher for the heavy users (heavy users 23±16% vs 10±7% for light users). These results also indicate that heavy cannabis users smoke more efficiently than casual smokers.Both light and heavy users showed more clinical effect following IV administration than after smoking. The response of the heavy users, both with respect to effect on heart and high, was quite comparable to that of light users.The present study does not suggest that tolerance readily develops in heavy users.     

Early-onset cannabis use and cognitive deficits: what is the nature of the association?

BACKGROUND: Individuals who initiate cannabis use at an early age, when the brain is still developing, might be more vulnerable to lasting neuropsychological deficits than individuals who begin use later in life. METHODS: We analyzed neuropsychological test results from 122 long-term heavy cannabis users and 87 comparison subjects with minimal cannabis exposure, all of whom had undergone a 28-day period of abstinence from cannabis, monitored by daily or every-other-day observed urine samples. We compared early-onset cannabis users with late-onset users and with controls, using linear regression controlling for age, sex, ethnicity, and attributes of family of origin. RESULTS: The 69 early-onset users (who began smoking before age 17) differed significantly from both the 53 late-onset users (who began smoking at age 17 or later) and from the 87 controls on several measures, most notably verbal IQ (VIQ). Few differences were found between late-onset users and controls on the test battery. However, when we adjusted for VIQ, virtually all differences between early-onset users and controls on test measures ceased to be significant. CONCLUSIONS: Early-onset cannabis users exhibit poorer cognitive performance than late-onset users or control subjects, especially in VIQ, but the cause of this difference cannot be determined from our data. The difference may reflect (1). innate differences between groups in cognitive ability, antedating first cannabis use; (2). an actual neurotoxic effect of cannabis on the developing brain; or (3). poorer learning of conventional cognitive skills by young cannabis users who have eschewed academics and diverged from the mainstream culture.     

Ovarian hormones and chronic administration during adolescence modify the discriminative stimulus effects of delta-9-tetrahydrocannabinol (Δ⁹-THC) in adult female rats

Marijuana abuse during adolescence may alter its abuse liability during adulthood by modifying the interoceptive (discriminative) stimuli produced, especially in females due to an interaction with ovarian hormones. To examine this possibility, either gonadally intact or ovariectomized (OVX) female rats received 40 intraperitoneal injections of saline or 5.6 mg/kg of Δ⁹-THC daily during adolescence, yielding 4 experimental groups (intact/saline, intact/Δ⁹-THC, OVX/saline, and OVX/Δ⁹-THC). These groups were then trained to discriminate Δ⁹-THC (0.32-3.2 mg/kg) from saline under a fixed-ratio (FR) 20 schedule of food presentation. After a training dose was established for the subjects in each group, varying doses of Δ⁹-THC were substituted for the training dose to obtain dose-effect (generalization) curves for drug-lever responding and response rate. The results showed that: 1) the OVX/saline group had a substantially higher mean response rate under control conditions than the other three groups, 2) both OVX groups had higher percentages of THC-lever responding than the intact groups at doses of Δ⁹-THC lower than the training dose, and 3) the OVX/Δ⁹-THC group was significantly less sensitive to the rate-decreasing effects of Δ⁹-THC compared to other groups. Furthermore, at sacrifice, western blot analyses indicated that chronic Δ⁹-THC in OVX and intact females decreased cannabinoid type-1 receptor (CB1R) levels in the striatum, and decreased phosphorylation of cyclic adenosine monophosphate response element binding protein (p-CREB) in the hippocampus. In contrast to the hippocampus, chronic Δ⁹-THC selectively increased p-CREB in the OVX/saline group in the striatum. Extracellular signal-regulated kinase (ERK) was not significantly affected by either hormone status or chronic Δ⁹-THC. In summary, these data in female rats suggest that cannabinoid abuse by adolescent human females could alter their subsequent responsiveness to cannabinoids as adults and have serious consequences for brain development.     

What is the impact of the 2017 cochrane systematic review and meta-analysis that evaluated the use of PCSK9 inhibitors for lowering cardiovascular disease and mortality?

Introduction: In 2017, Schmidt et al. conducted a Cochrane systematic review and meta-analysis to evaluate the effect of using proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors to reduce low-density-lipoprotein- cholesterol (LDL-C) and cardiovascular disease (CVD). The Cochrane review was a systematic review and meta-analysis of 20 randomized, double-blinded trials that compared the use of PCSK9 inhibitors with statins/ezetimibe, ezetimibe, or placebo for a treatment duration of at least 24 weeks. The use of PCSK9 inhibitors lowered the risk for CVD (OR 0.86 (0.80 to 0.92)) but not mortality (OR 1.02 (0.91 to 1.14)) when compared to placebo.

Areas covered: The following article evaluates the recently published Cochrane review and clarifies the efficacy of PCSK9 inhibitors for improving cardiovascular morbidity and mortality.

Expert opinion: The Cochrane review discussed suggests that PCSK9 inhibitors are effective in lowering LDL-C and the risk of CVD but not the risk of mortality. The higher price of PCSK9 inhibitors is a further deterrent for using them as a substitute for statins – cholesterol lowering medications with history showing they lower mortality. Statins should remain the gold-standard cholesterol-lowering drug class until PCSK9 inhibitors become more affordable and demonstrate consistent efficacy for reducing CVD and mortality.     

Determination of endogenous levels of GHB in human hair. Are there possibilities for the identification of GHB administration through hair analysis in cases of drug-facilitated sexual assault?

We have developed a GC-MS-MS assay for GHB in human hair. Five milligrams of washed hair were hydrolyzed by 1M or 0.01M NaOH before a liquid-liquid extraction with ethyl acetate under acidic conditions. GHB-d(6) was used as the internal standard. TMS derivatives were formed before injection. TBDMS derivatives were used in cases of strong chromatographic interferences or in a confirmatory procedure. Analysis of basal levels of GHB in 61 drug-free donors gave the following results: the mean measured concentration for blond hair was 0.60 ng/mg (n = 12), SD = 0.19 ng/mg, and extreme figures were in the range 0.35-0.95 ng/mg. For brown hair, the mean measured concentration was 0.90 ng/mg (n = 30), SD = 0.42 ng/mg, and extreme figures 0.41-1.86 ng/mg. For black hair, the mean measured concentration was 0.90 ng/mg (n = 19), SD = 0.37 ng/mg, and extreme figures 0.32-1.54 ng/mg, showing no significant differences depending on hair color. Analysis of basal levels of GHB of 12 or more specimens in segmented hair showed a mean concentration of 1.22 ng/mg (0.31-8.4 ng/mg) and a relative standard deviation for each individual ranging from 6.75% to 37.98%. GHB was administered to a healthy 53-year-old white male (light brown hair) at oral dosages of 30, 45, and 60 mg/kg. Beard hair was collected just before administration and 24 h after (and each day for one week for the last dose), and a 7.5-cm scalp hair lock was collected 7 days after the last dose. A rise in GHB concentration was observed in beard hair for the 45 and 60 mg/kg dosages with a maximum at 24 h, whereas no change was observed for the 30 mg/kg dosage. Scalp hair was segmented into 3-mm long segments. The three proximal last segments showed significantly (0.0005 < p < 0.005) different concentrations of GHB (1.22, 1.27, and 1.66 ng/mg, respectively) when compared with the basal physiological level of GHB in this same person (mean = 0.62 ng/mg, SD = 0.15 ng/mg). A case of daily GHB abuse during bodybuilding allowed us to determine a concentration of GHB of 14 ng/mg, in a 2-cm long segment (black hair). A case of rape under the influence of GHB was documented through hair analysis (black hair) and positive analysis of the glass she used. Sampled 7 days after the sexual assault, the three last 3-mm long proximal segments tested for GHB exhibited concentrations of 3.1-5.3 and 4.3 ng/mg, respectively, whereas the mean physiological level determined in this woman was 0.71 ng/mg, SD = 0.17 ng/mg. The authors advise a two-step hair sampling as evidence of GHB consumption: the first sample at the time of exposure to show the contamination by sweat of the proximal segment in case of recent administration with a significant rise of hair level at the root, and the second after at least 3 or 4 weeks to avoid this contamination and determine the levels incorporated in the hair matrix before, during, and after the exposure.     

Assessing cannabis consumption frequency: Is the combined use of free and glucuronidated THCCOOH blood levels of diagnostic utility?

Heavy cannabis consumption is considered incompatible with safe driving. In Swiss traffic policy, drivers suspected of regular cannabis use are therefore required to undergo medical assessment of their long‐term fitness to drive. A whole blood concentration of the cannabis metabolite 11‐nor‐9‐carboxy‐Δ⁹‐tetrahydrocannabinol (THCCOOH) of 40 µg/L is currently used by Swiss forensic experts as the decision limit for regular cannabis consumption. The present study aimed to investigate the suitability of THCCOOH‐glucuronide blood levels as an additional and/or better marker for the frequency of cannabis use. Whole blood samples collected from 23 heavy (≥10 joints/month) and 25 occasional smokers (≥1 joint/month, but ≤ 1 joint/week) enrolled in a placebo‐controlled cannabis smoking study were analyzed for THCCOOH and THCCOOH‐glucuronide. Based on receiver operating characteristic (ROC) curve analysis, concentration thresholds could be established for distinguishing between these two groups. Proposed thresholds for heavy use were THCCOOH‐glucuronide > 52 µg/L (100% specificity; 41% sensitivity) and/or total THCCOOH > 58 µg/L (100% specificity; 43% sensitivity). Optimum thresholds for occasional use were THCCOOH‐glucuronide < 5 µg/L (73% specificity; 97% sensitivity) and/or total THCCOOH < 5 µg/L (62% specificity; 98% sensitivity). Our results indicate that the THCCOOH‐glucuronide whole blood concentration is a useful parameter that complements the free THCCOOH level to assess the frequency of cannabis consumption. The consideration of the blood concentrations of both free and glucuronidated THCCOOH improves the identification of heavy users whose fitness to drive has to be carefully assessed. Copyright © 2016 John Wiley & Sons, Ltd.     

The adverse health effects of cannabis use: What are they,and what are their implications for policy?

Background: The adverse health effects of cannabis are a source of contention in debates about policies towards the drug. Methods: This paper provides a review of epidemiological evidence on the major adverse health effects of cannabis use and considers its implications for policy. Results: The evidence strongly suggests that cannabis can adversely affect some users, especially adolescents who initiate use early and young adults who become regular users. These adverse effects probably include increased risks of: motor vehicle crashes, the development of cannabis dependence, impaired respiratory function, cardiovascular disease, psychotic symptoms, and adverse outcomes of adolescent development, namely, poorer educational outcomes and an increased likelihood of using other illicit drugs. Conclusions: Politically, evidence of adverse health effects favours the status quo in developed countries like Australia where cannabis policy has been framed by the media as a choice between two views: (1) either cannabis use is largely harmless to most users and so we should legalize, or at the very least decriminalize its use; or (2) it harms some of its users so we should continue to prohibit its use.     

Could the WHO model list of essential medicines do more for the safe and appropriate use of injections?

A national drug policy addressing the safe and appropriate use of injections is an important element to prevent overuse and unsafe use of injections. Because the World Health Organization World Health Organization Model List of Essential Medicines is a keystone of national drug policies, the authors examined the way it addresses injection practices. They reviewed the 11th World Health Organization Model List of Essential Medicines to collect information on (1) injectable medicines, (2) diluents, and (3) the recommendations regarding the procurement of injection devices. Of 306 active ingredients on the list, 135 (44%) are mentioned in injectable form. Of these, 41 (30%) need diluents for reconstitution. The list does not mention the need to procure appropriate diluents, injection devices, and safety boxes in quantities that match the quantities of injectable medicines. In addition, the list provides limited information that can be used to forecast the needs of injection devices to administer the injectable medicines that are included in the list. Future revisions of the World Health Organization Model List of Essential Medicines should attempt to reduce the number of injectable formulations on the basis of evidence. In addition, the list should specify that when injectable medicines are being supplied, diluents, single-use syringes, and safety boxes should be supplied. The volume of syringes needed for administration should be specified for each injectable medication on the list to facilitate the forecasting of the needs of injection devices.     

An industry perspective on the use of “atoxigenic” strains of Aspergillus flavus as biological control agents and the significance of cyclopiazonic acid

Several nonaflatoxigenic strains of Aspergillus flavus have been registered in the United States to reduce aflatoxin accumulation in maize and other crops, but there may be unintended negative consequences if these strains produce cyclopiazonic acid (CPA). AF36, a nonaflatoxigenic, CPA-producing strain has been shown to produce CPA in treated maize and peanuts. Alternative strains, including Afla-Guard^® brand biocontrol agent and K49, do not produce CPA and can reduce both aflatoxin and CPA in treated crops. Chronic toxicity of CPA has not been studied, and recent animal studies show significant harmful effects from short-term exposure to CPA at low doses. Grower and industry confidence in this approach must be preserved through transparency.