Development and validation of an HPLC-DAD method for the quantification of cannabigerol, cannabidiol, cannabinol and cannabichromene in human plasma and mouse matrices.

Cannabigerol, cannabidiol, cannabinol and cannabichromene are non-psychoactive phytocannabinoids, highly present in Cannabis sativa, for which numerous therapeutical applications have been described. However, additional pre-clinical and clinical data, including toxicopharmacokinetic and pharmacodynamic studies, remain required to support their use in clinical practice and new therapeutic applications. To support these studies, a new high performance liquid chromatography technique (HPLC) with diode-array detection (DAD) was developed and validated to quantify these cannabinoids in human plasma and mouse matrices. Sample extraction was accomplished by protein precipitation and double liquid-liquid extraction. Simvastatin and perampanel were used as internal standards in human and mouse matrices, respectively. Chromatographic separation was achieved in 16 min on an InfinityLab Poroshell® 120 C18 column (4.6 mm × 100 mm, 2.7 µm) at 40 °C. A mobile phase composed of water/acetonitrile was pumped with a gradient elution program at 1.0 mL min-1. The technique revealed linearity in the defined concentration ranges with a determination coefficient of over 0.99. Intra and inter-day accuracy and precision values ranged from -14.83 to 13.97% and 1.08 to 13.74%, respectively. Sample stability was assessed to ensure that handling and storage conditions did not compromise analyte concentrations in different matrices. Carry-over was absent and recoveries were over 77.31%. This technique was successfully applied for the therapeutic monitoring of cannabidiol and preliminary pre-clinical studies with cannabigerol and cannabidiol. All samples were within calibration ranges, with the exception of cannabigerol after intraperitoneal administration. This is the first HPLC-DAD technique that simultaneously quantifies cannabinoids in these biological matrices, supporting future pre-clinical and clinical investigations.

Cannabinoid Stability in Antemortem and Postmortem Blood.

In toxicological testing, drug stability is important when providing quantitative results and interpretation of findings, as well as when collecting correlation data. The goal of this study was to expand on previous stability studies and to evaluate other cannabinoids encountered in forensic toxicology. In this 6-month study, the stability of Δ-9-tetrahydrocannabinol (THC), 11-hydroxy-THC, 11-nor-9-carboxy-THC, Cannabinol and Cannabidiol in antemortem and postmortem blood was evaluated in refrigerated (4°C) and frozen (-4°C) storage conditions. Pooled antemortem and postmortem bloods were fortified at low and high concentrations and stored in untreated glassware. Over 6 months, samples were analyzed by automated extraction and liquid chromatography-mass spectrometry/mass spectrometry to evaluate the change in concentration over time. Samples in each storage condition were analyzed in triplicate 12 times over the 6-month period. Cannabinoids in antemortem blood were more stable in the refrigerated condition than in the frozen condition, with 11-hydroxy-THC, 11-nor-9-carboxy-THC and Cannabinol having more than 80% of the original concentration remaining at the end of the study. Cannabinoids in postmortem blood had improved stability in the frozen storage condition with THC, 11-hydroxy-THC, 11-nor-9-carboxy-THC and Cannabinol in the low concentration pool with more than 80% of the original concentration remaining. These data demonstrated that cannabinoids may decrease in concentration over time when stored in untreated glass vials. To ensure the most accurate determination of drug concentration, samples containing cannabinoids should be analyzed as soon as possible.

The effect of antioxidants on the long-term stability of THC and related cannabinoids in sampled whole blood.

The stability of cannabinoids is complex and crucial for the assessment of impaired driving caused by cannabis. Therefore, the effect of antioxidants on the long-term stability of Δ9 -tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD), 11-hydroxy-Δ9 -tetrahydrocannabinol (THC-OH), and 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THC-COOH) in whole blood samples preserved with fluoride citrate (FC) and fluoride oxalate (FX) mixtures was investigated at different temperatures. The measured concentrations of the cannabinoids in authentic whole blood preserved solely with FC or FX mixtures decreased significantly during prolonged storage at -20°C. On average, less than 5% of the initial concentrations of THC and CBD were recovered after 19 weeks of storage interrupted by 5 thawing/freezing cycles. The rate of decrease was greatest in FC-preserved blood. The repeated thawing/freezing of the samples accelerated the instability progression. At 5°C approximately 60% of the initial concentrations of THC and CBD were recovered after 19 weeks of storage. No significant decrease was observed in samples stored at -80°C during the test period of 5 months. The instability at -20°C was to a great extend avoided by adding 30 mM ascorbic acid (ASC) to the samples before storage. Samples preserved with a combination of the FX mixture and ASC showed no significant decrease in the recovered concentrations during a 5-month storage period interrupted by 6 thawing/freezing cycles. Samples preserved with a combination of the FC mixture and ASC showed almost similar improvements in cannabinoid stability. Other reducing agents such as sodium metabisulfite and glutathione also improved the stability in FX-preserved blood stored at -20°C.

Identification and quantification of psychoactive drugs in whole blood using dried blood spot (DBS) by ultra-performance liquid chromatography tandem mass spectrometry.

A procedure based on ultra-high-pressure liquid chromatography tandem mass spectrometry has been developed for the determination of twenty three psychoactive drugs and metabolites in whole blood using dried blood spot (DBS). Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a linear gradient elution with two solvents: 0.1% formic acid in acetonitrile and 5mM ammonium formate at pH 3. The mass spectrometer was operated in positive ion mode, using multiple reaction monitoring via positive electro-spray ionization. The method was linear from the limit of quantification (5ng/ml for all the analytes apart from 15ng/ml for Δ-9-tetrahydrocannabinol and metabolites) to 500ng/ml, and showed good correlation coefficients (r(2)=0.990) for all substances. Analytical recovery of analytes under investigation was always higher than 75% and intra-assay and inter-assay precision and accuracy always better than 15%. Using the validated method, ten DBS samples, collected at the hospital emergency department in cases of acute drug intoxication, were found positive to one or more psychoactive drugs. Our data support the potential of DBS sampling for non invasive monitoring of exposure/intoxication to psychoactive drugs.

Cannabinoid concentrations detected in fatal road traffic collision victims compared with a population of other postmortem cases.

BACKGROUND: Acute cannabis consumption nearly doubles the risk of motor vehicle collision resulting in injury or death. Limited data have been published regarding the concentrations of cannabinoids associated with fatal road traffic collisions (RTCs), and these have not previously been compared to a population of other postmortem cases. METHODS: We conducted analysis for cannabinoids [Δ(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC, 11-nor-THC-9-carboxylic acid, cannabidiol, and cannabinol], drugs, and alcohol on consecutive fatal RTC cases (100) and non-RTC cases (114) from coroners' jurisdictions in London and southeast England and compared the data. RESULTS: The incidence of cannabinoids detected in non-RTC and RTC cases was similar (25% vs 21%, P = 0.44), but THC was detected more frequently (90% vs 59%, P = 0.01) and at significantly higher concentrations in the cannabinoid-positive RTC cases than the non-RTC cases (P = 0.01). The distribution of non-RTC and RTC cases over 4 categories of THC concentration was significantly different (P = 0.004). There was no significant difference in the concentrations of other cannabinoids detected between the 2 groups. Cannabinoids were detected in more fatal RTC cases (21) than alcohol >80 mg/dL (17). Detection of other drugs was low compared to cannabis and alcohol. CONCLUSIONS: These first data on the concentrations of cannabinoids in the postmortem blood of fatal RTC victims compared with a population of other routine coroners' cases highlight the importance of specifically measuring THC concentrations in the blood to aid interpretation of postmortem cases where cannabis may be implicated.

In vitro stability of free and glucuronidated cannabinoids in blood and plasma following controlled smoked cannabis.

BACKGROUND: Blood and plasma cannabinoid stability is important for test interpretation and is best studied in authentic rather than fortified samples. METHODS: Low and high blood and plasma pools were created for each of 10 participants after they smoked a cannabis cigarette. The stabilities of Δ(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), cannabinol (CBN), THC-glucuronide, and THCCOOH-glucuronide were determined after 1 week at room temperature; 1, 2, 4, 12, and 26 (±2) weeks at 4 °C; and 1, 2, 4, 12, 26 (±2), and 52 (±4) weeks at -20 °C. Stability was assessed by Friedman test. RESULTS: Numbers of THC-glucuronide and CBD-positive blood samples were insufficient to assess stability. In blood, 11-OH-THC and CBN were stable for 1 week at room temperature, whereas THC and THCCOOH-glucuronide decreased and THCCOOH increased. In blood, THC, THCCOOH-glucuronide, THCCOOH, 11-OH-THC, and CBN were stable for 12, 4, 4, 12, and 26 weeks, respectively, at 4 °C and 12, 12, 26, 26, and 52 weeks at -20 °C. In plasma, THC-glucuronide, THC, CBN, and CBD were stable for 1 week at room temperature, whereas THCCOOH-glucuronide and 11-OH-THC decreased and THCCOOH increased. In plasma, THC-glucuronide, THC, THCCOOH-glucuronide, THCCOOH, 11-OH-THC, CBN, and CBD were stable for 26, 26, 2, 2, 26, 12, and 26 weeks, respectively, at 4 °C and 52, 52, 26, 26, 52, 52, and 52 weeks, respectively, at -20 °C. CONCLUSIONS: Blood and plasma samples should be stored at -20 °C for no more than 3 and 6 months, respectively, to assure accurate cannabinoid quantitative results.

Identification of recent cannabis use: whole-blood and plasma free and glucuronidated cannabinoid pharmacokinetics following controlled smoked cannabis administration.

BACKGROUND: Δ9-Tetrahydrocannabinol (THC) is the most frequently observed illicit drug in investigations of accidents and driving under the influence of drugs. THC-glucuronide has been suggested as a marker of recent cannabis use, but there are no blood data following controlled THC administration to test this hypothesis. Furthermore, there are no studies directly examining whole-blood cannabinoid pharmacokinetics, although this matrix is often the only available specimen. METHODS: Participants (9 men, 1 woman) resided on a closed research unit and smoked one 6.8% THC cannabis cigarette ad libitum. We quantified THC, 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), cannabinol (CBN), THC-glucuronide and THCCOOH-glucuronide directly in whole blood and plasma by liquid chromatography/tandem mass spectrometry within 24 h of collection to obviate stability issues. RESULTS: Median whole blood (plasma) observed maximum concentrations (C(max)) were 50 (76), 6.4 (10), 41 (67), 1.3 (2.0), 2.4 (3.6), 89 (190), and 0.7 (1.4) µg/L 0.25 h after starting smoking for THC, 11-OH- THC, THCCOOH, CBD, CBN, and THCCOOH-glucuronide, respectively, and 0.5 h for THC-glucuronide. At observed C(max), whole-blood (plasma) detection rates were 60% (80%), 80% (90%), and 50% (80%) for CBD, CBN, and THC-glucuronide, respectively. CBD and CBN were not detectable after 1 h in either matrix (LOQ 1.0 µg/L). CONCLUSIONS: Human whole-blood cannabinoid data following cannabis smoking will assist whole blood and plasma cannabinoid interpretation, while furthering identification of recent cannabis intake.

Simultaneous and sensitive analysis of THC, 11-OH-THC, THC-COOH, CBD, and CBN by GC-MS in plasma after oral application of small doses of THC and cannabis extract.

Besides the psychoactive Delta(9)-tetrahydrocannabinol (THC), hashish and marijuana as well as cannabis-based medicine extracts contain varying amounts of cannabidiol (CBD) and of the degradation product cannabinol (CBN). The additional determination of these compounds is interesting from forensic and medical points of view because it can be used for further proof of cannabis exposure and because CBD is known to modify the effects of THC. Therefore, a method for the simultaneous quantitative determination of THC, its metabolites 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), CBD and CBN from plasma was developed. The method was based on automatic solid-phase extraction with C(18) ec columns, derivatization with N,O-bistrimethylsilyltrifluoroacetamide (BSTFA), and gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) with deuterated standards. The limits of detection were between 0.15 and 0.29 ng/mL for THC, 11-OH-THC, THC-COOH, and CBD and 1.1 ng/mL for CBN. The method was applied in a prospective pharmacokinetic study after single oral administration of 10 mg THC alone or together with 5.4 mg CBD in cannabis extract. The maximum plasma concentrations after cannabis extract administration ranged between 1.2 and 10.3 ng/mL (mean 4.05 ng/mL) for THC, 1.8 and 12.3 ng/mL (mean 4.9 ng/mL) for 11-OH-THC, 19 and 71 ng/mL (mean 35 ng/mL) for THC-COOH, and 0.2 and 2.6 ng/mL (mean 0.95 ng/mg) for CBD. The peak concentrations (mean values) of THC, 11-OH-THC, THC-COOH, and CBD were observed at 56, 82, 115, and 60 min, respectively, after intake. CBN was not detected. Caused by the strong first-pass metabolism, the concentrations of the metabolites were increased during the first hours after drug administration when compared to literature data for smoking. Therefore, the concentration ratio 11-OH-THC/THC was discussed as a criterion for distinguishing oral from inhalative cannabis consumption.

Isotope effect studies on the dehydrogenation of delta 1-tetrahydrocannabinol in the rat.

Isotope effect studies on the metabolic dehydrogenation of delta 1-tetrahydrocannabinol in rats are described and it is shown that this process is confined to a very short period following i.v. administration. The implications of this finding are discussed.