Dried urine spots (DUS) represent a potential alternative sample storage for forensic toxicological analysis. The aim of the current study was to develop and validate a liquid chromatographic tandem mass spectrometric procedure for the detection and quantitative determination of cannabinoids and metabolites in DUS. A two-step extraction was performed on DUS and urine samples. An LC-MS/MS system was operated in multiple reaction monitoring and positive polarization mode. The method was checked for sensitivity, specificity, linearity, accuracy, precision, recovery, matrix effects and carryover. The method was applied to 70 urine samples collected from healthy volunteers and drug addicts undergoing withdrawal treatment. The method was successfully developed for DUS. LODs lower than 2.0 ng/mL were obtained for all the monitored substances. All the validation parameters fulfilled the acceptance criteria either for DUS or urine. Among the real samples, 45 cases provided positive results for at least one compound. A good quali-quantitative agreement was obtained between DUS and urine. A good stability of THC, THCCOOH and THCCOOH-gluc was observed after a 24 h storage, in contrast to previously published results. DUS seems to provide a good alternative storage condition for urine that should be checked for the presence of cannabinoids and metabolites.

Verbal memory deficits are linked to cannabis use. However, self-reported episodic use does not allow for assessment of variance from other factors (e.g., cannabis potency, route of consumption) that are important for assessing brain-behavior relationships. Further, co-occurring nicotine use may moderate the influence of cannabis on cognition. Here we utilized objective urinary measurements to assess the relationship between metabolites of cannabis, 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THCCOOH), and nicotine (cotinine) on verbal memory in young adults.
Adolescents and young adults (n = 103) aged 16-22 completed urinary drug testing and verbal memory assessment (RAVLT). Linear regressions examined the influence of THCCOOH and cotinine quantitative concentrations, and their interaction, on RAVLT scores, controlling for demographics and alcohol. Cannabis intake frequency was also investigated. Secondary analyses examined whether past month or recency of use related to performance, while controlling for THCCOOH and cotinine concentrations.
THCCOOH concentration related to both poorer total learning and long delay recall. Cotinine concentration related to poorer short delay recall. Higher frequency cannabis use status was associated with poorer initial learning and poorer short delay. When comparing to self-report, THCCOOH and cotinine concentrations were negatively related to learning and memory performance, while self-report was not.
Results confirm the negative relationship between verbal memory and cannabis use, extending findings with objective urinary THCCOOH, and cotinine concentration measurements. No moderating relationship with nicotine was found, though cotinine concentration independently associated with negative short delay performance. Findings support the use of both urinary and self-report metrics as complementary methods in substance use research.

Δ9-tetrahydrocannabinol (THC), the primary psychoactive component of cannabis, leads to impaired cognitive and psychomotor function resulting in an increased risk of fatal motor vehicle collisions and other traumas resulting in death. It is important to measure cannabinoids in postmortem cases to improve understanding of this growing public safety issue. However, postmortem concentrations of THC and its primary inactive metabolite, 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH), have not been extensively studied. We aim to further characterize postmortem concentrations of THC and THCCOOH in peripheral blood with and without preservation, central blood, and central \"serum\" to support improved forensic interpretation. Cannabinoids were extracted from blood and \"serum\" from twenty-five decedents using solid phase extraction followed by quantification using gas chromatography - mass spectrometry. We evaluated the impact of sample preservation, reported central blood-to-peripheral blood (CB:PB) ratios and blood-to-\"serum\" ratios, and assessed the relationship of CB:PB and postmortem interval for THC and THCCOOH. Correlations of preserved compared to unpreserved blood were strong with r2 > 0.97. The median CB:PB ratios were 1.1 and 1.3 for THC and THCCOOH, respectively. THCCOOH CB:PB was significantly higher than 1.0 (p-value < 0.001). The CB:PB ratio was only weakly correlated with PMI for both compounds. The median blood-to-\"serum\" ratio was 1.0 for THC and 0.8 for THCCOOH. The blood-to-\"serum\" ratio of THCCOOH was significantly lower than 1.0 (p-value < 0.001). Results demonstrated minimal potential for postmortem redistribution of THC and THCCOOH and that the ratio of blood-to-\"serum\" in postmortem samples differs from the blood-to-plasma ratio established in living humans. Based on these results, it is not recommended to apply a correction factor to THC and THCCOOH concentrations from postmortem blood samples. Our study improves the understanding of postmortem cannabinoid concentrations to support forensic interpretation in cases of fatal motor vehicle accidents.

Despite adolescents and young adults being the most frequent users of cannabis, all information on cannabis drug testing interpretation is based on data from adults.
This study aimed to define the time course of urinary 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) excretion among 70 adolescent and young adult cannabis users during 1 month of biochemically-verified cannabis abstinence.
Urine specimens were collected at non-abstinent baseline and after 2, 3, 8, 15, 21 and 28 days of abstinence. Specimens were tested for THCCOOH with a \'rapid\' immunoassay drug test and a confirmatory assay using liquid chromatography-tandem mass spectrometry, with a 5 ng/mL limit of quantitation. Elimination rate was tested using a population pharmacokinetics model.
Participants had an average of 26 days of abstinence (SD = 6). Initial creatinine-adjusted THCCOOH concentration (CN-THCCOOH) was 148 ng/mg (SD = 157). Half-life was 2 days (SD = 5), with a 10-day window of detection (estimated range: 4-80 days). At the final timepoint and among those with > 25 days of abstinence (n = 62), 40% (n = 25) had THCCOOH concentrations > 5 ng/mL (i.e. detectable on confirmatory assay) and 19% (n = 12) were \'positive\' per federal drug testing guidelines (i.e. values greater than 50 ng/mL on the screening immunoassay and 15 ng/mL on the confirmatory assay). More frequent past month cannabis use was associated with higher baseline CN-THCCOOH concentrations, but not with rate of elimination. Nested five-fold cross-validation suggested high model reliability and predictive validity.
Findings underscore that, as with adults, detectable cannabinoid metabolites do not necessarily indicate recent use in adolescents and young adults. Algorithms that account for THCCOOH levels, assessed longitudinally and time between specimen collections are best equipped to confirm abstinence.
NCT03276221; https://clinicaltrials.gov/ct2/show/NCT03276221?term=Randi+Schuster&rank=1.

BACKGROUND: The acidic forms of cannabinoids, THC-A and CBD-A are naturally present in cannabis plants and preparations and are generally decarboxylated to the active compounds before the use (e.g. thermally decarboxylated through smoking). Hence, the identification of the acidic compounds in urine could be an evidence of cannabis ingestion rather than a passive exposure to smoke. This case report described a 15-month-old child that suffered an acute intoxication by accidental cannabis ingestion. It is important to assess the ingestion and to discriminate it from a passive exposure to better interpret the clinical findings and to establish the correct therapeutic procedure.
METHODS: Urine samples were simply diluted in deionized water and directly injected in the LC-MS/MS system. D3-THCCOOH was used as internal standard. Chromatographic separation of THCCOOH, THC-A and CBD-A was carried out in reversed phase on a c18 column. A triple quad in MRM negative mode was used to monitor the three analytes.
CONCLUSIONS: The developed LC-MS/MS method was simple and fast. A LOD of 3.0ng/mL and a LOQ of 10.0ng/mL were measured for the three compounds. The analytical procedure was validated accordingly to international guidelines. The two urine samples collected from the 15-month-old child at the hospitalization and after three days provided positive results for THCCOOH (130.0 and 10.0ng/mL respectively). THC-A was found only in the urine sample collected at the hospitalization (concentration: 70.0ng/mL).
CONCLUSIONS: THC-A was detected and quantitated in a urine sample of a 15-month-old child.

OBJECTIVE: Generally, urine drug testing for cannabis abuse involves measuring total concentrations of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) obtained by enzymatic and/or alkaline hydrolysis of THCCOOH-glucuronide. As hydrolysis can be inconsistent and incomplete, direct measurement of the two metabolites is preferable. Methodology & results: We developed a high-throughput LC-MS/MS method for simultaneous quantification of free and glucuronidated THCCOOH in urine using coated 96-well plates for analyte extraction and column-switching chromatography. Excellent separation of the two analytes was achieved within 2.5 min, with linear ranges from 5 to 2000 μg/l for THCCOOH and from 10 to 4000 μg/l for THCCOOH-glucuronide.
CONCLUSIONS: The method was successfully validated and applied to authentic urine samples from cannabis consumers, demonstrating its applicability for routine cannabinoid testing.

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.

An isomer of the tetrahydrocannabinol (THC) metabolite 11-nor-9-carboxy-Δ(9)-THC (THCCOOH) had been detected in blood of cannabis users. The present study was initiated to elucidate whether the labile metabolite THCCOOH-glucuronide could be the precursor. THCCOOH-glucuronide was incubated in human serum and albumin (HSA) solution at various temperatures (-18, 4.5, 22 and 37°C) and pH values (pH 7.4 and 8.3) for seven days in the presence or absence of the esterase inhibitor sodium fluoride. Analysis of incubation samples was performed using LC-MS/MS. Marked degradation of THCCOOH-glucuronide was observed at 37°C. It was found that not only THCCOOH, but also the isomer is a degradation product of THCCOOH-glucuronide and its in-vivo production is assumed. Degradation to THCCOOH and the isomer occurred at alkaline pH, in the presence of fluoride-sensitive esterases and of HSA alone. To inhibit isomer formation during sample storage, refrigeration and controlling of the pH are recommended. However, THCCOOH and the isomer exhibit similar properties during incubations in serum, but differ in their interaction with HSA. The present study confirmed the nature of the isomer as degradation product of the abundant THC metabolite THCCOOH-glucuronide. Serum albumin and esterases are obviously involved. The isomer is formed not only during storage, but also under physiological conditions, suggesting that it can be considered an in-vivo metabolite. However, the chemical structure of the isomer remains unknown and further research is necessary.

The concentration of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in whole blood is used as a parameter for assessing the consumption behavior of cannabis consumers. The blood level of THCCOOH-glucuronide might provide additional information about the frequency of cannabis use. To verify this assumption, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and direct quantification of free and glucuronidated THCCOOH in human whole blood was newly developed. The method comprised protein precipitation, followed by injection of the processed sample onto a trapping column and subsequent gradient elution to an analytical column for separation and detection. The total LC run time was 4.5 min. Detection of the analytes was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was fully validated by evaluating the following parameters: linearity, lower limit of quantification, accuracy and imprecision, selectivity, extraction efficiency, matrix effect, carry-over, dilution integrity, analyte stability, and re-injection reproducibility. All acceptance criteria were analyzed and the predefined criteria met. Linearity ranged from 5.0 to 500 μg/L for both analytes. The method was successfully applied to whole blood samples from a large collective of cannabis consumers, demonstrating its applicability in the forensic field.

Urinary 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (Carboxy-THC) concentrations, normalised to creatinine output, have been demonstrated to be a useful tool in the interpretation of the results of a series of urine tests for cannabis. These tests, often termed historical data, can be used to identify potential chronic cannabis users who may present occupational health and safety risks within the workplace. Conversely, the data can also be used to support employee claims of previous regular, rather than recent, cannabis use. This study aimed at examining the mean elimination of Carboxy-THC in 37 chronic users undergoing voluntary abstinence over a 2-week period. Urine specimens were collected prior to the study and after 1 and 2 weeks of abstinence. Carboxy-THC levels in urine were measured by gas chromatography-mass spectrometry (GC-MS) following alkaline hydrolysis, organic solvent extraction and derivatisation to form its pentafluoropropionic derivative. The creatinine-normalised Carboxy-THC concentrations declined rapidly over the 2 weeks of abstinence period and the majority of chronic cannabis users (73%) reduced their urinary Carboxy-THC levels to below the 15-μg/L confirmatory cutoff within that time. The study further highlights the value of historical urinary Carboxy-THC data as a means of identifying potential occupational health and safety risks among chronic cannabis users.