THCCOOH Concentrations Research Articles

Hair analysis for Δ9-tetrahydrocannabinolic acid A—New insights into the mechanism of drug incorporation of cannabinoids into hair

Differentiation between external contamination and incorporation of drugs or their metabolites from inside the body via blood, sweat or sebum is a general issue in hair analysis and of high concern when interpreting analytical results. In hair analysis for cannabinoids the most common target is Δ9-tetrahydrocannabinol (THC), sometimes cannabidiol (CBD) and cannabinol (CBN) are determined additionally. After repeated external contamination by cannabis smoke these analytes are known to be found in hair even after performing multiple washing steps. A widely accepted strategy to unequivocally prove active cannabis consumption is the analysis of hair extracts for the oxidative metabolite 11-nor-9-carboxy-THC (THC-COOH). Although the acidic nature of this metabolite suggests a lower rate of incorporation into the hair matrix compared to THC, it is not fully understood up to now why hair concentrations of THC-COOH are generally found to be much lower (mostly <10 pg/mg) than the corresponding THC concentrations. Δ9-Tetrahydrocannabinolic acid A (THCA A) is the preliminary end product of the THC biosynthesis in the cannabis plant. Unlike THC it is non-psychoactive and can be regarded as a ‘precursor’ of THC being largely decarboxylated when heated or smoked. The presented work shows for the first time that THCA A is not only detectable in blood and urine of cannabis consumers but also in THC positive hair samples. A pilot experiment performed within this study showed that after oral intake of THCA A on a regular basis no relevant incorporation into hair occurred. It can be concluded that THCA A in hair almost exclusively derives from external contamination e.g. by side stream smoke. Elevated temperatures during the analytical procedure, particularly under alkaline conditions, can lead to decarboxylation of THCA A and accordingly increase THC concentrations in hair. Additionally, it has to be kept in mind that in hair samples tested positive for THCA A at least a part of the ‘non-artefact’ THC probably derives from external contamination as well, because in condensate of cannabis smoke both THC and THCA A are present in relevant amounts. External contamination by side stream smoke could therefore explain the great differences in THC and THC-COOH hair concentrations commonly found in cannabis users.

Simultaneous quantification of cannabinoids and metabolites in oral fluid by two-dimensional gas chromatography mass spectrometry

Development and validation of a method for simultaneous identification and quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), and metabolites 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) in oral fluid. Simultaneous analysis was problematic due to different physicochemical characteristics and concentration ranges. Neutral analytes, such as THC and CBD, are present in ng/mL, rather than pg/mL concentrations, as observed for the acidic THCCOOH biomarker in oral fluid. THCCOOH is not present in cannabis smoke, definitively differentiating cannabis use from passive smoke exposure. THC, 11-OH-THC, THCCOOH, CBD, and CBN quantification was achieved in a single oral fluid specimen collected with the Quantisal™ device. One mL oral fluid/buffer solution (0.25 mL oral fluid and 0.75 mL buffer) was applied to conditioned CEREX ® Polycrom™ THC solid-phase extraction (SPE) columns. After washing, THC, 11-OH-THC, CBD, and CBN were eluted with hexane/acetone/ethyl acetate (60:30:20, v/v/v), derivatized with N, O-bis-(trimethylsilyl)trifluoroacetamide and quantified by two-dimensional gas chromatography electron ionization mass spectrometry (2D-GCMS) with cold trapping. Acidic THCCOOH was separately eluted with hexane/ethyl acetate/acetic acid (75:25:2.5, v/v/v), derivatized with trifluoroacetic anhydride and hexafluoroisopropanol, and quantified by the more sensitive 2D-GCMS–electron capture negative chemical ionization (NCI-MS). Linearity was 0.5–50 ng/mL for THC, 11-OH-THC, CBD and 1–50 ng/mL for CBN. The linear dynamic range for THCCOOH was 7.5–500 pg/mL. Intra- and inter-assay imprecision as percent RSD at three concentrations across the linear dynamic range were 0.3–6.6%. Analytical recovery was within 13.8% of target. This new SPE 2D-GCMS assay achieved efficient quantification of five cannabinoids in oral fluid, including pg/mL concentrations of THCCOOH by combining differential elution, 2D-GCMS with electron ionization and negative chemical ionization. This method will be applied to quantification of cannabinoids in oral fluid specimens from individuals participating in controlled cannabis and Sativex ® (50% THC and 50% CBD) administration studies, and during cannabis withdrawal.

Analysis of cannabis in oral fluid specimens by GC-MS with automatic SPE

Methamphetamine (MA) is the most commonly abused drug in Korea, followed by cannabis. Traditionally, MA analysis is carried out on both urine and hair samples and cannabis analysis in urine samples only. Despite the fact that oral fluid has become increasingly popular as an alternative specimen in the field of driving under the influence of drugs (DUID) and work place drug testing, its application has not been expanded to drug analysis in Korea. Oral fluid is easy to collect and handle and can provide an indication of recent drug abuse. In this study, we present an analytical method using GC-MS to determine tetrahydrocannabinol (THC) and its main metabolite 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid. The validated method was applied to oral fluid samples collected from drug abuse suspects and the results were compared with those in urine. The stability of THC and THC-COOH in oral fluid stored in different containers was also investigated. Oral fluid specimens from 12 drug abuse suspects, submitted by the police, were collected by direct expectoration. The samples were screened with microplate ELISA. For confirmation they were extracted using automated SPE with mixed-mode cation exchange cartridge, derivatized and analyzed by GC-MS using selective ion monitoring (SIM). The concentrations ofTHC and THC-COOH in oral fluid showed a large variation and the results from oral fluid and urine samples from cannabis abusers did not show any correlation. Thus, detailed information about time interval between drug use and sample collection is needed to interpret the oral fluid results properly. In addition, further investigation about the detection time window ofTHC and THC-COOH in oral fluid is required to substitute oral fluid for urine in drug testing.

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.

Identifying New Cannabis Use with Urine Creatinine-Normalized THCCOOH Concentrations and Time Intervals Between Specimen Collections

A previously recommended method for detecting new cannabis use with creatinine-normalized 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THCCOOH) urine concentrations in periodically collected specimens for treatment, workplace and judicial drug testing applications is refined by considering the time interval between urine collections. All urine specimens were collected from six less-than-daily cannabis users who smoked placebo, 1.75%, and 3.55% THC cigarettes in randomized order, each separated by one week. Ratios (n = 24,322) were calculated by dividing each creatinine-normalized THCCOOH concentration (U2) by that of a previously collected specimen (U1). Maximum, 95% limit, and median U2/U1 ratios with 15 and 6 ng THCCOOH/mL cutoff concentrations, with and without new use between specimens, were calculated for each 24-h interval after smoking up to 168 h and are included in tables. These ratios decreased with increasing interval between collections providing improved decision values for determining new cannabis use. For example, with a 15 ng THCCOOH/mL cutoff concentration and no new use between specimens, the maximum, 95% limit, and median U2/U1 ratios were 3.05, 1.59, and 0.686, respectively, when the collection interval was <or = 24 h and 0.215, 0.135, and 0.085 when it was 96-119.9 h.

Urinary Elimination of 11-Nor-9-Carboxy- 9-tetrahydrocannnabinol in Cannabis Users During Continuously Monitored Abstinence

The time course of 11-nor-9-carboxy-Delta9-tetrahydrocannnabinol (THCCOOH) elimination in urine was characterized in 60 cannabis users during 24 h monitored abstinence on a closed research unit for up to 30 days. Six thousand, one hundred fifty-eight individual urine specimens were screened by immunoassay with values > or = 50 ng/mL classified as positive. Urine specimens were confirmed for THCCOOH by gas chromatography-mass spectrometry following base hydrolysis and liquid-liquid or solid-phase extraction. In 60%, the maximum creatinine normalized concentration occurred in the first urine specimen; in 40%, peaks occurred as long as 2.9 days after admission. Data were divided into three groups, 0-50, 51-150, and > 150 ng/mg, based on the creatinine corrected initial THCCOOH concentration. There were statistically significant correlations between groups and number of days until first negative and last positive urine specimens; mean number of days were 0.6 and 4.3, 3.2 and 9.7, and 4.7 and 15.4 days, respectively, for the three groups. These data provide guidelines for interpreting urine cannabinoid test results and suggest appropriate detection windows for differentiating new cannabis use from residual drug excretion.

The Urinary Disposition of Intravenously Administered 11-Nor-9-carboxy-delta-9-Tetrahydrocannabinol in Humans

The objective of this study was to investigate the fraction of an administered dose of 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THCCOOH) that is actually excreted into urine and to determine its urinary half-life independent of the parent compound. Ten healthy, male marijuana nonusers who were enrolled in the study were administered a single dose of 5 mg THCCOOH by the intravenous route. Urine specimens were collected up to 96 hours after administration. Samples were extracted before and after alkaline hydrolysis. The concentration of unconjugated and total THCCOOH was determined using gas chromatography-mass spectrometry. Most of the THCCOOH found in urine was conjugated, with only 0.14 +/- 0.08% of the dose present as unconjugated THCCOOH. The amount of conjugated THCCOOH ranged from 149.3 to 559.8 (mean +/- SD, 342.8 +/- 117.3) microg, representing a recovery of 3% to 11% of the administered dose. The measured amounts of total THCCOOH were low and highly varied among individuals. Renal excretion does not appear to be the preferred elimination pathway for THCCOOH. Urinary elimination half-life of unconjugated and conjugated THCCOOH ranged from 9.0 to 27.4 (mean +/- SD, 17.3 +/- 5.3) hours and from 10.7 to 27.6 (mean +/- SD, 16.0 +/- 5.0) hours, respectively. Although preliminary in nature, the actual urinary elimination half-life of THCCOOH appears to be significantly shorter than its apparent or terminal half-life reported from single or multiple dosing of delta-9-tetrahydrocannabinol (THC).

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 Δ 9-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.

Estimating Time of Last Oral Ingestion of Cannabis From Plasma THC and THCCOOH Concentrations

Estimating the time of last cannabis use is important in assessing possible impairment of drivers involved in accidents, in verifying accuracy of court testimony and in the future, helpful in therapeutic monitoring of cannabis agonists. In 1992, Huestis et al developed model 1, based on plasma Delta-tetrahydrocannabinol (THC) concentrations, and model 2, on plasma 11-nor-9-carboxy-Delta(9)-tetrahydrocannbinol/THC ratios, that predicted 95% confidence intervals for time of last cannabis use. These models seemed to be valuable when applied to the small amount of data from published studies of oral ingestion, a route of administration more popular with the advent of cannabis therapies. A study was designed to further validate the models after oral ingestion of THC, and to determine whether they could predict last usage after multiple oral doses. Eighteen subjects in IRB-approved studies participated after providing informed consent. Each of 12 subjects in one group received a single 10 mg oral dose of dronabinol (synthetic THC). In another protocol, 6 subjects received 4 different oral daily doses, divided into thirds and administered with meals for 5 consecutive days. There was a 10-day washout period between each dosing regimen. Daily doses were 0.39, 0.47, and 14.8 mg THC in hemp oil and 7.5 mg dronabinol. Blood specimens were collected throughout the study and analyzed for plasma THC and 11-nor-9-carboxy-Delta(9)-tetrahydrocannbinol by gas chromatography/mass spectrometry with limits of quantification (LOQs) of 0.5 and 1.0 ng/mL, respectively. Actual times between ingestion of THC and blood collection spanned 0.5 to 16 hours. All plasma specimens with analyte concentrations >LOQ (n=90) were evaluated. Models 1 and 2 correctly predicted time of last THC ingestion for 74.4% and 90.0% of plasma specimens, respectively. 96.7% of predicted times were correct with one overestimate and 2 underestimates using the time interval defined by the lowest and highest 95% confidence limit of both models. These results provide further evidence of the usefulness of the predictive models in estimating the time of last oral THC ingestion after single or multiple doses.

Effets du cannabis oral et du dronabinol sur la capacité à conduire

Two retrospective epidemiologic studies have shown that cannabis is the main psychoactive substance detected in the blood of drivers suspected of driving under the influence of psychotropic drugs. An oral administration double-blind crossover study was carried out with eight healthy male subjects, aged 22 to 30 years, all occasional cannabis smokers. Three treatments and one placebo were administered to all participants at a two week interval: 20 mg dronabinol, 16.5 mg D9-tétrahydrocannabinol (THC) and 45.7 mg THC as a cannabis milk decoction. Participants were asked to report the subjective drug effects and their willingness to drive under various circumstances on a visual analog scale. Clinical observations, a psychomotor test and a tracking test on a driving simulator were also carried out. Compared to cannabis smoking, THC, 11-OH-THC and THC-COOH blood concentrations remained low through the whole study (<13.1 ng THC/mL,<24.7 ng 11-OH-THC/mL and<99.9 ng THC-COOH/mL). Two subjects experienced deep anxiety symptoms suggesting that this unwanted side-effect may occur when driving under the influence of cannabis or when driving and smoking a joint. No clear association could be found between these adverse reactions and a susceptibility gene to propensity to anxiety and psychotic symptoms (genetic polymorphism of the catechol-O-methyltransferase). The questionnaires have shown that the willingness to drive was lower when the drivers were assigned an insignificant task and was higher when the mission was of crucial importance. The subjects were aware of the effects of cannabis and their performances on the road sign and tracking test were greatly impaired, especially after ingestion of the strongest dose. The Cannabis Influence Factor (CIF) which relies on the molar ratio of active and inactive cannabinoids in blood provided a good estimate of the fitness to drive.

Review of Biologic Matrices (Urine, Blood, Hair) as Indicators of Recent or Ongoing Cannabis Use

Especially for cannabinoids, analytical procedures for the verification of recent use and generally for the assessment of the extent of drug abuse are of interest in clinical and forensic toxicology. For confirmation of abstinence, urine analysis seems to be a useful tool. Serial monitoring of THC-COOH to creatinine ratios can differentiate between recent drug use and residual THC-COOH excretion (THC-COOH/creatinine ratio > or = 0.5 compared with previous specimen ratio). For an assessment of the extent of cannabis use, the determination of free and bound THC-COOH and especially of THC and 11-OH-THC glucuronides are suggested as useful but need further confirmation. Blood analysis is preferred for the interpretation of acute effects after cannabis abuse. The cannabis influence factor (CIF) was demonstrated as a better tool to interpret the concentrations of THC and its metabolites in blood in forensic cases and therefore it was proposed to assume absolute driving inability because of cannabis intoxication from a CIF > or = 10. Additionally, a higher CIF is indicative of a recent cannabis abuse. Also discrimination between occasional use of cannabis and regular drug consumption is possible by analysis of THC-COOH in blood samples because of the long plasma half-life of THC-COOH and its accumulation in the blood of frequent cannabis consumers. In routine tests, blood samples have to be taken within a prescribed 8-day-period, and a THC-COOH concentration >75 ng/mL is assumed to be associated with regular consumption of cannabis products, whereas plasma THC-COOH concentrations <5 ng/mL are associated with occasional consumption. In contrast to other illicit drugs, hair analysis lacks the sensitivity to act as a detector for cannabinoids. THC and especially the main metabolite THC-COOH have a very low incorporation rate into hair and THC is not highly bound to melanin, resulting in much lower concentrations in hair compared with other drugs. Additionally, THC is present in cannabis smoke and also can be incorporated into the hair only by contamination. For the determination of the main metabolite THC-COOH in the picogram or femtogram per milligram range, which indicates an active consumption, special analytical procedures, such as GC/MS/MS techniques, are required.

Distribution of 11-Nor-9-Carboxy-Δ9-tetrahydrocannabinol in Traffic Fatality Cases

11-nor-9-carboxy-Delta9-tetrahydrocannabinol (THC-COOH) distributions in postmortem specimens are rarely reported. Fifty New Jersey State Medical Examiner's cases in which automobile accident deaths suspected of involving marijuana intake were studied for the distributions of THC-COOH in postmortem urine, blood, vitreous humor, and bile specimens. Cases were selected based on immunoassay (TDx) urine test results. If the preliminary urine test indicated the presence of THC-COOH (apparent THC-COOH concentration > or = 20 ng/mL), urine, heart blood, vitreous humor, and bile specimens from the case were analyzed for THC-COOH concentrations by gas chromatography-mass spectrometry. The mean, standard deviation, and range of THC-COOH in heart blood, urine, and bile found in these 50 cases were 0.081, 0.082, and 0.016-0.33 microg/mL; 0.314, 0.415, and 0.044-2.33 microg/mL; and 12.9, 11.4, and 1.03-43.7 microg/mL, respectively. THC-COOH was absent (detection limit, 1 ng/mL) or at low concentration (< 10 ng/mL) in vitreous humor specimens. The mean, standard deviation, and range of the bile-to-blood and urine-to-blood ratios were 242, 196, and 17.2-888 and 4.70, 4.05, and 1.14-19.2, respectively. The highest concentrations of THC-COOH were found in bile and the lowest in vitreous humor. These findings are consistent with the high hydrophobicity nature of THC-COOH and further suggest that bile is the specimen of choice for detecting low level of THC-COOH in postmortem cases.

Urinary excretion profiles of 11-nor-9-carboxy-$Delta;9-tetrahydrocannabinol and 11-hydroxy-$Delta;9-THC: cannabinoid metabolites to creatinine ratio study IV

The objective of this study was to compare urinary excretion patterns of two cannabinoid metabolites in subjects with a history of chronic marijuana use. The first metabolite analyzed was nor-9-carboxy-Δ 9 -tetrahydrocannabinol (Δ 9 -THC-COOH), the major urinary cannabinoid metabolite that is pharmacologically inactive. The second metabolite 11-OH-Δ 9 -THC is an active cannabinoid metabolite and is not routinely measured. Urine specimens were collected from four subjects on 12–20 occasions ≥96 h apart in an uncontrolled clinical setting. Creatinine was analyzed in each urine specimen by the colorimetric modified Jaffé reaction on a SYVA 30R biochemical analyzer. All urine specimens analyzed for 11-OH-Δ 9 -THC had screened positive for cannabinoids with the EMIT II Plus cannabinoids assay (cut-off 50 ng/mL) on a SYVA 30R analyzer and submitted for Δ 9 -THC-COOH confirmation by GC–MS (cut-off concentration 15 ng/mL). Eleven-OH-Δ 9 -THC was measured by GC–MS with a cut-off concentration of 3 ng/mL. Both GC–MS methods for cannabinoid metabolites used deuterated internal standards for quantitative analysis. The mean (range) of urinary Δ 9 -THC-COOH concentration was 1153 ng/mL (78.7–2634) with a cut-off of 15 ng/mL. The mean (range) of Δ 9 -THC-COOH/creatinine ratios (ng/mL Δ 9 -THC-COOH/mmol/L creatinine) was 84.1 (8.1–122.1). The mean (range) urinary of 11-OH-Δ 9 -THC concentration was 387.6 ng/mL (11.9–783) with a cut-off of 3 ng/mL, and the mean (range) of 11-OH-Δ 9 -THC/creatinine ratio (ng/mL 11-OH-Δ 9 -THC/mmol/L creatinine) was 29.7 (1.2–40.7). Of the 63 urine specimens submitted for Δ 9 -THC-COOH confirmation by GC–MS, 59/63 urine specimens (94%) were positive for Δ 9 -THC-COOH and 51/63 (81%) were positive for 11-OH-Δ 9 -THC. Overall, the concentrations of 11-OH-Δ 9 -THC in urine specimens collected ≥96 h apart were lower than Δ 9 -THC-COOH concentrations in 50/51 of the urine specimens in this population. Further urinary cannabinoid excretion studies are needed to assess whether 11-OH-Δ 9 -THC analyses have a role when assessing previous marijuana or hashish use in chronic users whose urine specimens remain positive for Δ 9 -THC-COOH for an extended period of time after last drug use.

An Investigation of the Stability of Free and Glucuronidated 11-Nor-Δ9-Tetrahydrocannabinol-9-carboxylic Acid in Authentic Urine Samples

Preanalytical stability of a drug and its major metabolites is an important consideration in pharmacokinetic studies or whenever the analyte pattern is used to estimate drug habits. Firstly, the stability of free and glucuronidated 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH, THCCOOglu) in authentic urine samples was investigated. Random urine samples of cannabis users (n = 38) were stored at -20, 4, and 20 degrees C up to 15 days and up to 5 days at 40 degrees C, and alterations of the analyte pattern during storage were followed by liquid chromatography-tandem mass spectrometry. Secondly, the influence of pH (range 5.0-8.0) on the stability of the analytes was studied using spiked urine to elucidate the results obtained from authentic samples. In authentic urine samples, the initial pH ranged from 5.1 to 8.8. The glucuronide was found to be highly labile at a storage temperature of 4 degrees C and above. Initially, 18 urine samples tested positive for THCCOOH. After 2 days storage at 20 degrees C, THCCOOH was detectable in a further 4 samples, and 7 more samples tested positive for THCCOOH (5-81 ng/mL) after 15 days. Depending on time and temperature, the glucuronide concentration decreased, resulting in an increase of THCCOOH concentration. However, a loss in mean total THCCOOH concentration was found, which was significantly higher in deteriorated samples than in samples without signs of deterioration after 15 days of storage at 20 degrees C. In the drug-free urine sample separately spiked with THCCOOglu or THCCOOH, the investigations on the stability of the target analytes at various pH values revealed that THCCOOH was stable at pH 5.0. At higher pH values, its concentration slightly decreased with time, and about 69% of the initial THCCOOH concentration was still present at pH 8.0 on day 5. THCCOOglu concentrations rapidly decreased with increasing pH value. For example, only 72% of the initial THCCOOglu concentration could be detected at pH 5.0 on day 1. Degradation of the glucuronide resulted in formation of THCCOOH, which was observed even at pH 5.0. In light of the present findings, advanced forensic interpretations based on the presence of THCCOOH or the pattern of THCCOOH and THCCOOglu in stored urine samples seems questionable.

Stability of 11-Nor-Δ9-carboxy-tetrahydrocannabinol Glucuronide in Plasma and Urine Assessed by Liquid Chromatography-Tandem Mass Spectrometry

Unconjugated 11-nor-Delta(9)-carboxy-tetrahydrocannabinol (THCCOOH) in blood and urine has been proposed as a valuable marker, but the glucuronide (THCCOOglu) is present in considerably higher concentrations than the parent drug. Acyl glucuronides have been shown to be potentially reactive conjugates, which may affect the in vitro metabolite pattern. Extraction procedures and a liquid chromatography-tandem mass spectrometry assay were developed and validated to investigate the stability of THCCOOglu in urine and plasma. Plasma and urine samples with added THCCOOglu were stored at -20, 4, 20, and 40 degrees C up to 10 days. The glucuronide was stable at -20 degrees C in both matrices, whereas THCCOOglu concentrations decreased at all other storage conditions. For a given storage time and temperature, the decrease in plasma was higher than that in urine. At 20 degrees C, a marked change in concentration could be observed within 2 days of storage. Degradation of THCCOOglu followed an apparent first-order process and led to the formation of THCCOOH. The sum of the molar concentrations of both analytes corresponded only to the initial THCCOOglu in plasma and urine samples stored at 4 degrees C. The in vitro degradation of THCCOOglu prevents clinical conclusions based on the metabolite pattern or the concentration of unconjugated THCCOOH in samples stored at > or =4 degrees C for prolonged periods.

Investigation of nitrite adulteration on the immunoassay and GC-MS analysis of cannabinoids in urine specimens.

Nitrite ion has been identified as the active ingredient of two commercial adulterants that could cause discrepant results between the immunoassay screening and gas chromatographic-mass spectrometric (GC-MS) confirmation of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in urine. Procedures to chemically convert the nitrite ion at the beginning of sample preparation for GC-MS analysis may not overcome all nitrite adulteration cases because portions of the THCCOOH might have been lost between the time of sample collection and the time of analysis. This study was conducted to further investigate the influence of both urine sample matrix and the duration of nitrite exposure on nitrite interference of THCCOOH detection. Forty clinical "THC-positive samples" that had been screened and confirmed positive for the presence of THCCOOH were spiked with 0.15M or 0.3M of nitrite. The levels of THCCOOH at various time intervals after nitrite spiking were monitored by instrument-based cannabinoids immunoassays (Syva EMIT d.a.u. and/or Roche Abuscreen ONLINE assays) and by an onsite THC immunoassay (Roche ONTRAK TESTSTIK). Results from this report demonstrate that the two outstanding "urine specimen factors" that dictated the effectiveness of the nitrite adulteration were urinary pH and the original drug concentration before nitrite spiking. Significant decreases in the immunoassay results could be observed within 4 h of nitrite treatment in the majority of samples with acidic urinary pH values. Regardless of their original concentration of THCCOOH (GC-MS ranging from 33 to 488 ng/mL), all of the 20 samples that had acidic pH values gave negative immunoassay results 1 day after nitrite adulteration. In contrast, the immunoassay results of samples with neutral or basic pH values were less affected by nitrite exposure in the same studies. Approximately two-thirds of the samples with pH values greater than 7.0 remained immunoassay-positive 3 days after nitrite spiking. Nevertheless, some of the adulterated urine that showed no change in immunoassay results might exhibit significant decrease in GC-MS recoveries even with bisulfite treatment, collaborating with the observations that a portion of samples screened positive with THC immunoassay in the laboratory could fail to confirm with GC-MS analysis. The decrease or loss of immunoassay detectable cannabinoid cross-reactives in acidic "THC-positive samples" can be attenuated by chemically increasing the pH value of the samples to the basic pH range.

Simultaneous determination of THC-COOH and THC-COOH-glucuronide in urine samples by LC/MS/MS

A fast method using liquid–liquid extraction and HPLC/tandem-mass spectrometry (LC/MS/MS) was developed for the simultaneous detection of 11-Nor-Δ 9-tetrahydrocannabinol-9-carboxylic acid β-glucuronide (THC-COOH-glucuronide) and 11-Nor-Δ 9-tetrahydrocannabinol-9-carboxylic acid (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 even without hydrolysis of the sample or derivatisation of extracts. Liquid–liquid extraction was optimised: with ethylacetate/diethylether (1:1, v/v) THC-COOH-glucuronide and THC-COOH could be extracted in one step. Molecular ions of the glucuronide (MH +, m/ z 521) and THC-COOH (MH +, m/ z 345) were generated using a PE/SCIEX turboionspray source in positive ionisation mode; specific fragmentation was performed in the collision cell of an API 365 triple-quadrupole mass spectrometer and yielded major fragments at m/ z 345 (for THC-COOH-glucuronide) and m/ z 327 as well as m/ z 299 for both cannabinoids. Chromatographic separation was performed using a reversed-phase C8 column and gradient elution with 0.1% formic acid/1 mM ammonium formate and acetonitrile/0.1% formic acid. Retention times were 22.2 min for the glucuronide and 26.8 min for THC-COOH. After enzymatic hydrolysis of urine samples with β-glucuronidase/arylsulfatase (37°C, 5 h), THC-COOH-glucuronide was no longer detectable by LC/MS/MS in urine samples. However, the THC-COOH concentration was increased. For quantitation of THC-COOH, THC-COOH-D 3 was added to the urine samples as internal standard prior to analysis. From the difference of THC-COOH in the native urine and urine after enzymatic hydrolysis, molar concentration ratios of THC-COOH-glucuronide/THC-COOH in urine samples of cannabis users were determined and found to be between 1.3 and 4.5.

Urinary excretion of 11-nor-9-carboxy-delta9-tetrahydrocannabinol and cannabinoids in frequent and infrequent drug users.

Urinary excretion of 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THCCOOH) and cannabinoids was monitored in prison inmates. Urinary specimens were collected up to five times per day. EMIT (cutoff 20 ng/mL; EMIT20) and gas chromatography (GC) (cutoff 10.3 ng/mL, LOD 1.4 ng/mL) were used for cannabinoid screening and THCCOOH confirmation, respectively. Urinary creatinine concentrations were recorded. Of the samples with positive EMIT screens, 78% were confirmed by GC analysis. The plotting of THCCOOH/creatinine ratios (THCCOOH/C) versus time gave smoother excretion curves than THCCOOH concentrations alone. Based on THCCOOH/C the first 5 days after the last reported intake, the mean urinary excretion half-life was 1.3 days in infrequent users, and a median of 1.4 days was found in frequent users. In the latter group, apparent terminal urinary excretion half-lives up to 10.3 days were observed. The last positive specimens were found after 4 days for THCCOOH with cutoff 15.0 ng/mL (NIDA/SAMSHA), 5 days for THCCOOH with cutoff 10.3 ng/mL, and 12 days for cannabinoids (EMIT20) in infrequent users and after 17, 22, and 27 days, respectively, in frequent users. Increases in urinary cannabinoids were sometimes found without concomitant increase in THCCOOH or THCCOOH/C. One subject admitted new cannabis intake, after which marked increases in THCCOOH and THCCOOH/C were observed. In others, new intake was suspected. Considerable variations between consecutive specimens were also observed in THCCOOH concentration and THCCOOH/C ratio without suspicion of a new intake.

Marijuana's impairing effects on driving are moderate when taken alone but severe when combined with alcohol

Previous experimental and epidemiological studies failed to provide unequivocal evidence that marijuana, either alone or in combination with alcohol, impairs a driver's performance to the extent that it will compromise traffic safety. We investigated the effects of marijuana, alone and in combination with alcohol, on actual driving in four, single-blind, randomized, cross-over studies. In Study 1, 24 subjects performed a road-tracking test on a closed segment of a primary highway after smoking marijuana that contained 0, 100, 200 and 300 μg/ kg Δ9-tetrahydrocannabinol (THC). In Study 2, 16 new subjects smoked the same THC doses before they performed a road-tracking and a car-following test; however, this time in the presence of other traffic. In Study 3, two groups of 16 subjects performed a city driving test. One group smoked marijuana delivering 0 and 100 μg/ kg THC prior to driving; the other group drunk orange juice mixed with or without a low dose of alcohol. In Study 4, 18 subjects performed a road-tracking and a car-following test in each of six conditions where they smoked marijuana with 0, 100, or 200 μg/ kg THC after they had consumed orange juice with or without alcohol. In these studies, marijuana alone significantly increased lateral position variability in the road-tracking test and distance variability during deceleration manoeuvres in the car-following test. Reaction times during car-following were not significantly affected, and a THC dose of 100 μg/ kg did not impair city driving performance. Blood plasma concentrations of THC and THC-COOH were not related to the degree of impairment. A low dose of alcohol (i.e. blood alcohol concentrations around 0·04%) impaired performance in all driving tests. Whereas marijuana's effects on driving performance were small (100 μg/ kg THC) or moderate (200 and 300 μg/ kg) when taken alone, they were severe when combined with a low dose of alcohol. In conclusion, marijuana alone impairs driving performance, with the degree of impairment increasing from small to moderate as the THC dose increases from 100 to 300 μg/ kg. However, when low to moderate doses of THC (100 and 200 μg/ kg) are taken in combination with a low dose of alcohol sufficient for attaining a BAC of about 0·04% actual driving is severely impaired. © 1998 John Wiley & Sons, Ltd.

Investigation of the effects of solution composition and container material type on the loss of 11-nor-delta 9-THC-9-carboxylic acid.

The loss of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from solution was studied using fluorescence polarization immunoassay (FPIA) technology and x-ray photoelectron spectroscopy (XPS). Several materials (glass, silylated glass, high density polyethylene, polypropylene, polystyrene, polymethylmethacrylate, Teflon, and Kynar) were studied along with three solvents (water, urine, and Abbott cannabinoids diluent). THC-COOH losses ranging from 0 to 9.7 ng/cm2 and concentration reductions to 46% of starting values were measured. XPS indicated the presence of fluorine-labeled THC-COOH at materials surfaces. A half-life of 10 min was calculated for THC-COOH loss from urine stored in high density polyethylene at room temperature. Sample handling losses during pipetting were determined and ranged from 1.1 to 7.9 ng per aliquot. The effects of sample volume and sample handling on the THC-COOH concentrations of controls were also investigated.