THC-COOH Research Articles

Cannabis Use and Biomarkers of Inflammation, Immune Activation, and Microbial Translocation in Persons with HIV.

Background: The relationship between cannabis and inflammation among persons with HIV (PWH) remains unclear. We examined whether the cannabis metabolite 11-nor-9-carboxy THC (THC-COOH) is associated with lower levels of plasma biomarkers of inflammation, immune activation, and microbial translocation in PWH. We hypothesized that cannabis use would be associated with lower levels of plasma inflammatory biomarkers than noncannabis use. Methods: We quantified THC-COOH in plasma, with THC-COOH levels between 5.1-69.9 μg/L and ≥70 μg/L being classified as moderate and heavy cannabis use, respectively, with noncannabis use defined as undetected THC-COOH. We measured a panel of plasma biomarkers of inflammation (interleukin [IL]-1-β, tumor necrosis factor-alpha, IL-18, IL-6, and C-reactive protein), immune activation (CD14 and CD163), and microbial translocation (iFABP2 and lipopolysaccharide binding protein [LBP]), with all biomarkers collected on the same day. We used a cross-sectional design and linear regression models to test whether cannabis use is associated with lower biomarker levels. Results: Participants were (N=107) sexual minority men with HIV (median age=32 years, IQR=28, 38), of whom 65% were virally suppressed; 36%, 44%, and 20% were classified as nonuse, moderate, and heavy cannabis, respectively. In linear regression models adjusted for viral suppression, stimulant use, and CD4 counts, heavy cannabis use was significantly associated with lower levels of log10 LBP (β=-0.14, 95% confidence interval: -0.24 to -0.04; false discovery rate=0.0029; partial eta squared=0.07) than noncannabis users. No precise associations were observed for other biomarkers (all p>0.05). Conclusions: Our findings suggest that cannabis use may be associated with lower plasma LBP. Further work is needed to clarify the relationship between cannabis use and biomarkers of microbial translocation in PWH.

Comprehensive Predictions of Cytochrome P450 (P450)-Mediated In Vivo Cannabinoid-Drug Interactions Based on Reversible and Time-Dependent P450 Inhibition in Human Liver Microsomes.

We previously reported the unbound reversible (IC50,u) and time-dependent (KI,u) inhibition potencies of cannabidiol (CBD), delta-9-tetrahydrocannabinol (THC), and THC metabolites 11-hydroxy THC (11-OH THC) and 11-nor-9-carboxy-delta-9-THC (11-COOH THC) against the major cytochrome P450 (P450) enzymes (1A2, 2C9, 2C19, 2D6, and 3A). Here, using human liver microsomes, we determined the CYP2A6, 2B6, and 2C8 IC50,u values of the aforementioned cannabinoids and the IC50,u and KI,u of the circulating CBD metabolites 7-hydroxy CBD (7-OH CBD) and 7-carboxy CBD (7-COOH CBD), against all the P450s listed above. The IC50,u of CBD, 7-OH CBD, THC, and 11-OH THC against CYP2B6 was 0.05, 0.34, 0.40, and 0.32 μM, respectively, and against CYP2C8 was 0.28, 1.02, 0.67, and 3.66 μM, respectively. 7-COOH CBD, but not 11-COOH THC, was a weak inhibitor of CYP2B6 and 2C8. All tested cannabinoids except 11-COOH THC were weak inhibitors of CYP2A6. 7-OH CBD inhibited all P450s examined (IC50,u<2.5 μM) except CYP1A2 and inactivated CYP2C19 and CYP3A, with inactivation efficiencies (kinact/KI,u) of 0.10 and 0.14 minutes-1 μM-1, respectively. Using several different static models, we predicted the following maximum pharmacokinetic interactions (affected P450 probe drug and area under the plasma concentration-time curve ratio) between oral CBD (700 mg) and drugs predominantly metabolized by CYP3A (midazolam, 14.8) > 2C9 (diclofenac, 9.6) > 2C19 (omeprazole, 7.3) > 1A2 (theophylline, 4.0) > 2B6 (ticlopidine, 2.2) > 2D6 (dextromethorphan, 2.1) > 2C8 (repaglinide, 1.6). Oral (130 mg) or inhaled (75 mg) THC was predicted to precipitate interactions with drugs predominately metabolized by CYP2C9 (diclofenac, 6.6 or 2.3, respectively) > 3A (midazolam, 1.8) > 1A2 (theophylline, 1.4). In vivo drug interaction studies are warranted to verify these predictions. SIGNIFICANCE STATEMENT: This study, combined with our previous findings, provides for the first time a comprehensive analysis of the potential for cannabidiol, delta-9-tetrahydrocannabinol, and their metabolites to inhibit cytochrome P450 enzymes in a reversible or time-dependent manner. These analyses enabled us to predict the potential of these cannabinoids to produce drug interactions in vivo at clinical or recreational doses.

Urine drug-testing tampering approaches: Turkish probationers

The growing numbers of individual and social problems associated with drug abuse necessitate new approaches in drug-testing systems. Equally, drug abusers may attempt to invalidate drug testing using different methods such as adulteration, dilution and substitution. This study aims to investigate tampering methods commonly used by Turkish substance-using probationers and evaluate their effects on toxicological drug-testing results. Initially, probationer urinary screening test results and laboratory substitution documents were evaluated to investigate the dilution and substitution attempt. Additionally, an experimental study was carried out by using readily available household products (bleach, vinegar, drain opener, eye drops) for adulteration. The effect of these agents was investigated for 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). It was determined that probationers preferred unbranded products (syringes, nylon bottles, etc.) for urine substitution. To detect dilution, screening test results were evaluated along with creatinine values. The variability of mean creatinine values can change the rate of the before-negative and after-positive ratio. For adulteration method, the high amounts of bleach provided false-negative results for THC-COOH and amphetamine, but spiking in any concentration of bleach affected MDMA results, causing a slight increase. Vinegar did not affect the THC-COOH and amphetamine results. However, false-negative results were observed for MDMA, with high amounts of vinegar-spiked urine samples. Drain opener was added in large quantities, and false-negative results were observed for all analytes. Visine eye drops did not have any effect on THC-COOH or amphetamine, but a high quantity of eye drops had a slight decreasing effect for MDMA.

Fast and sensitive UHPLC-MS/MS analysis of cannabinoids and their acid precursors in pharmaceutical preparations of medical cannabis and their metabolites in conventional and non-conventional biological matrices of treated individual

Monitoring pharmacological active compounds in pharmaceutical preparations of medical cannabis and in conventional and non-conventional biological matrices of treated individuals use requires both a wide linear range and sensitive detection. We have developed and validated a fast and sensitive method using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS) for analysis of Δ-9-tetrahydrocannabinol (THC), cannabidiol (CBD), their acidic precursors Δ-9-tetrahydrocannabinolic acid A (THCA-A) and cannabidiolic acid (CBDA) and some major metabolites of THC such as 11-nor-9-carboxy-THC (THC-COOH), 11-hydroxy-THC (11-OH-THC), Δ-9-THC-Glucuronide (THC-GLUC) and THC–COOH–Glucuronide (THC–COOH–GLUC) in conventional (whole blood and urine) and non-conventional (oral fluid and sweat) of individual treated with medical cannabis preparation. Specifically, THC, THCA-A, CBD and CBD-A were determined in cannabis decoction and oil prepared to treat individuals. The method used positive electrospray ionization (ESI) mode to reach the sensitivity needed to detect minimal amounts of analytes under investigations exposure with limits of quantification ranging from 0.2 to 0.5 ng per milliliter (ng/mL) or ng per patch in case of collected sweat. The validation results indicated this method was accurate (average inter/intra-day error, <10%), precise (inter/intra-day imprecision, <10%), and fast (10 min run time). In addition, time-consuming sample preparation was avoided applying dilute and shoot procedure, meeting the needs for potential large-scale population studies. The analysis of real samples demonstrated a pharmacokinetics of cannabinoids, their precursors and their metabolites dependent from quantity of carboxylated and decarboxylated compounds in pharmaceutical preparations.

Quantitative analysis of 11-nor-9-carboxy-tetrahydrocannbinol (THC–COOH) in urine by LC–MS/MS following a simple filtration

Quantification methods utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS) are common in clinical and forensic toxicology laboratories and the efficiency and rapidity of such methods continues to evolve. In most cases, urine drug confirmation does not require a drug extraction and can quickly and easily be accomplished with a dilution followed by sample filtration. The report describes the validation of a simple confirmation method for 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) and compared two types of filter extraction columns for sample clean-up. The method achieved a linear range of 10-3000ng/mL, acceptable bias (-4.7-2.6%) and precision (0.9-6.9%) and autosampler stability up to 72h. Universal filter columns offered less variable recovery over the linear range and fewer matrix interferences compared to THC-COOH specific filter columns. Authentic specimens testing positive for THC-COOH by LC-MS/MS were in good agreement with typically used GC-MS methods.

Rapid sample pre-treatment prior to GC–MS and GC–MS/MS urinary toxicological screening

Drug screening is an important issue in clinical and forensic toxicology. Gas chromatography coupled to mass spectrometry (GC–MS) remains the gold standard technique for the screening of unknown compounds in urine samples. However, this technique requires substantial sample preparation, which is time consuming. Moreover, some common drugs such as cannabis cannot be easily detected in urine using general procedures. In this work, a sample preparation protocol for treating 200μL of urine in less than 30min is described. The enzymatic hydrolysis of glucuro-conjugates was performed in 5min thanks to the use of microwaves. The use of a deconvolution software allowed reducing the GC–MS run to 10min, without impairing the quality of the compound identifications. Comparing the results from 139 authentic urine samples to those obtained using the current routine analysis indicated this method performed well. Moreover, additional 5-min GC–MS/MS programs are described, enabling a very sensitive target screening of 54 drugs, including THC–COOH or buprenorphine, without further sample preparation. These methods appeared as an interesting alternative to immuno-assays based screening. The analytical strategy presented in this article proved to be a promising approach for systematic toxicological analysis (STA) of drugs in urine.

Cannabis and psychomotor performance: A rational review of the evidence and implications for public policy

A previous US government analysis estimated that among those who acknowledge having driven following the consumption of an illicit substance, a majority of respondents, 70%, affirm having done so following the ingestion of cannabis. [4] While this total is far from negligible, it is far fewer than the number of respondents who acknowledge having driven while under the influence of alcohol. The presence of cannabis’ primary psychoactive ingredient, delta-9-tetrahydrocannabinol (THC), or THC’s primary metabolite, carboxy THC, has frequently been identified in the blood or urine of drivers involved in fatal traffic accidents. [5] Explanations for this result are two-fold. First, cannabis is by far the most widely used illicit drug among the US population, with non-governmental polling data indicating that nearly one out of two Americans admit having tried it. [6] Second, cannabis is the illicit substance most readily detectable by toxicological tests. The presence of THC may be detectable in the blood of occasional cannabis consumers for several hours after past use. [7] In more chronic users, THC may be present at relatively low blood levels for a

Fast liquid chromatography/tandem mass spectrometry determination of cannabinoids in micro volume blood samples after dabsyl derivatization

Due to the non-polar nature and the absence of an ionizable group on the cannabinoids, the ionization efficiency in electrospray is low and leads to poor limits of detection (LOD). The reaction of chloride dabsyl with the phenolic OH group of cannabinoids results in a product containing a tertiary amine, which is easily protonated in positive electrospray mode and can significantly improve the cannabinoids LOD. A rapid, selective and sensitive LC/MS-MS method was developed for quantitative determination of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH–THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC–COOH), cannabinol (CBN) and cannabidiol (CBD), in micro volume blood samples following dabsyl derivatization to enhance signal intensity. The method comprised protein precipitation followed by derivatization with dabsyl chloride and subsequent analysis using liquid chromatography–tandem mass spectrometry (LC/MS-MS). Chromatographic separation was achieved using a 150mm×2.1mm C18 analytical column maintained at 65°C and eluted with a gradient of water and acetonitrile, both containing 0.2% formic acid. The run time was 8min. The assay was successfully validated using the approach based on the accuracy profile. Lower limits of quantification (LOQ) were 0.25ng/mL for THC and THC–COOH, 0.30ng/mL for 11-OH–THC, 0.40ng/mL for CBN and 0.80ng/mL for CBD. A comparative study of cannabinoids in blood and plasma, as determined by the developed LC/MS-MS method or the in-house GC/MS-MS technique, demonstrated an excellent correlation between the two methods. Dabsylation was also tested on-line with a spiral of peek tubing placed in the LC/MS-MS column heater at 65°C before the analytical column. The results obtained with on-line dabsyl derivatization were similar to those observed off-line.

Stability of 11-Nor-delta-9-Tetrahydrocan nabinol-9-carboxylic Acid from Urine Samples Stored in Polypropylene, Polyethylene and Polystyrene Containers at Different Temperatures

The stability of 11-Nor-9-delta-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) from urine specimens stored in polypropylene (PP), polyethylene (PE) and polystyrene (PS) containers was examined. Urinesamples containing 100μg/m L TI-ICCOOH were stored at -20°C, 4°C and 25°C up to 180 days. The samples were analyzed at 0,5, 10, 20, 30, 60, 90, 105, 135, 150 and 180 days, and alteration of the analyte pattern during storage were determined by a validated liquid chromatography-tandem mass (LC-MS/MS). The concentration of THCCOOH significantly decreased to approximately 20% of its initial concentration within 5 days in all three containers at all temperature conditions. All loses of THCCOOH were relatively stabilized after 5 days. The results indicated that THCCOOH was preferred to store in PP and PE containers at 4 °C.

Determination of Drugs of Abuse in Airborne Particles by Pressurized Liquid Extraction and Liquid Chromatography-Electrospray-Tandem Mass Spectrometry

This work describes the first analytical method specifically developed for the multianalyte determination of several drugs of abuse and their metabolites in air. The methodology is based on pressurized liquid extraction (PLE) of atmospheric particles collected by means of high volume sampler equipped with quartz microfiber filters and subsequent analysis of the extracts by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Up to 17 different compounds belonging to five different chemical classes (cocainics, amphetamine-like compounds, opioids, cannabinoids, and lysergic compounds) are determined by means of this methodology. Acquisition is performed in the selected reaction monitoring (SRM) mode recording two transitions per compound (except for amphetamine). Quantitation by the internal standard method is based on the use of surrogated deuterated standards. The method has been validated in terms of linearity, accuracy, repeatability and sensitivity with satisfactory results. Absolute recoveries were above 50% for most investigated compounds. Method precision showed relative standard deviations (RSD) below 13% for all compounds, except for cannabinoids. The method limits of determination ranged from 0.35 pg/m(3) (for 2-oxo-3-hydroxy-LSD) to 22.55 pg/m(3) (for 11-nor-9 carboxy THC). Finally, as a part of the method validation, the optimized procedure was applied to the analysis of ambient air samples (fine grain-size particulates, PM(2.5)) collected at two urban background sites in Barcelona and Madrid (Spain). Results evidenced the presence of cocaine, benzoilecgonine, tetrahydrocannabinol, ecstasy, amphetamine, methamphetamine, and heroin in some or all of the samples investigated. The highest mean daily levels corresponded to cocaine (850 pg/m(3)) followed by heroin (143 pg/m(3)).

Fast confirmation of 11-nor-9-carboxy-Δ 9-tetrahydrocannabinol (THCCOOH) in urine by LC/MS/MS using negative atmospheric-pressure chemical ionisation (APCI)

A fast method using automated solid-phase extraction (SPE) and short-column liquid-chromatography coupled to tandem mass-spectrometry (LC/MS/MS) with negative atmospheric-pressure chemical ionisation (APCI) has been developed for the confirmation of 11-nor-9-carboxy-Δ 9-tetrahydrocannabinol (THCCOOH) in urine samples. This highly specific method which combines chromatographic separation and MS/MS-analysis can be used for the confirmation of positive immunoassay results with a NIDA cut-off of 15 ng/ml. The conjugates of THCCOOH were hydrolysed prior to SPE, and a standard SPE was performed using C18-SPE columns. No derivatisation of the extracts was needed as in GC/MS analysis, and the LC run-time was 6.5 min by gradient elution with a retention time of 2.4 min. Linearity of calibration was obtained in the range between 0 and 500 ng/ml (correlation coefficient R 2=0.998). Using linear regression (0–50 ng/ml) the limit of detection (LOD) was 2.0 ng/ml and the limit of quantitation (LOQ) was 5.1 ng/ml; day-to-day reproducibility and precision were tested at 15 and 250 ng/ml and were 13.4 ng/ml±3.3% and 255.8 ng/ml±4.5%, respectively.

A research note: the outcome of GC/MS/MS confirmation of hair assays on 93 cannabinoid (+) cases

This article reports the outcome of gas chromatography/tandem mass spectrometry confirmations for THC and carboxy-THC on 93 hair samples screened by RIA for cannabinoids. The samples were taken from probationers in Pinellas County, FL, who voluntarily provided the research staff with six hair and six urine specimens, collected at 1-month intervals. There were 40 samples that were RIA (+), urinalysis (−). Samples were selected which had cannabinoid (+) outcomes for hair, urine, or both. The THC and/or the carboxy-THC was (+) on confirmation. Of these 40 samples, 22 were (+) for both THC and carboxy-THC, 15 were (+) for THC but not carboxy-THC, and three were carboxy THC (+), but THC (−). Only one sample had a (+) RIA, but was (−) for both THC and carboxy-THC on confirmation. RIA detection of cannabinoids was confirmed in nearly all cases. Most cases that were RIA (−) but urine (+) were cannabinoid (+) when analyzed by GC/MS/MS.

Simultaneous quantification of opiates, cocaine and cannabinoids in hair

The present paper describes a sensitive method developed in our laboratory for the simultaneous analysis of opiates (morphine, codeine and monoacetylmorphine), cocainics (cocaine and benzoylecgonine) and cannabinoids (Δ 9-tetrahydrocannabinol and 11-nor-Δ 9-tetrahydrocannabinol-9-carboxylic acid) in hair samples. After decontaminating the sample with dichloromethane, two consecutive hydrolyses were performed in order to achieve the best conditions for extracting the three kinds of drugs from the protein matrix. First the opiate and cocainic compounds were extracted by means of a soft acidic hydrolysis with 0.1 N HCl at 50 °C overnight and organic solvent extraction at pH 9.2. The cannabinoids need a stronger basic hydrolysis with 11.8 N KOH for 10 min at laboratory temperature. After adding maleic acid, the cannabinoids were extracted with an organic solvent. The derivatization was carried out with heptafluorobutyric anhydride and hexafluoropropanol. Calibration curves were linear between 0.5–100 ng/mg of hair. Recovery and reproducibility were assured. The quantification limits ranged between 0.04–0.26 ng/mg of hair. Seventy hair samples from known drug abusers were cut into 1-cm segments and analyzed by this method. The ranges of measured concentrations (ng/mg) were 0.31–89 for cocaine, 0.1–5.76 for benzoylecgonine, 0.34–45.79 for morphine, 0.45–39.59 for codeine, 0.09–48.18 for monoacetylmorphine, 0.06–7.63 for THC and 0.06–3.87 for THCCOOH. The results of sectional analyses agreed with the self reported drug histories. The usefulness of this method is in assessing earlier drug consumption, and also at the same time obtaining a chronological profile of the consumption of these three types of drugs.

Confirmation testing of 11-nor-Δ 9-tetrahydrocannabinol- 9-carboxylic acid in urine with micellar electrokinetic capillary chromatography

The major urinary metabolite of the most commonly abused psychotropic drug, Δ 9-tetrahydrocannabiol, is 11-nor-Δ 9-tetrahydrocannabiol-9-carboxylic acid (THC-COOH). With basic hydrolysis, extraction and concentration, this compound can easily be determined using micellar electrokinetic capillary chromatography with on-column multi-wavelength detection. After solid-phase extraction of 5 ml of urine, drug concentrations down to about 10ng/ml can be unambiguously monitored. Peak assignment is achieved through comparison of the retention time and absorption spectrum of the eluting THCCOOH peak with those of computer-stored model runs. The effectiveness of the approach is demostrated with data obtained from urine samples from different patients which tested positively for cannabinoids using a flourescene polarization immunoassay.