Wolf_jatsept11.qxd:JATLynneTemplate 7/27/11 10:36 AM Page 1 Journal of Analytical Toxicology, Vol. 35, September 2011 Cannabichromene and Tetrahydrocannabinol Determination in Mouse Blood and Brain by Gas Chromatography–Mass Spectrometry 1 2 2,3 3 Gerald T. DeLong , Carl E. Wolf , Alphonse Poklis , and Aron Lichtman Downloaded from https://academic.oup.com/jat/article/35/7/496/880227 by guest on 24 September 2021 1Occupational and Environmental Health Sciences Division, Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Services, Bethesda, Maryland and Departments of 2Pathology and 3Pharmacology/Toxicology, Virginia Commonwealth University, Richmond, Virginia in marijuana (3). The degree to which these non-THC phyto- Abstract cannabinoids may contribute to marijuana’s overall pharma- Cannabichromene (CBC) is a phytocannabinoid, the second most cological effects, have pharmacological properties of their own, abundant cannabinoid quantitatively in marijuana. CBC has been or modulate the effects of THC remains in question. shown to produce antinociception and anti-inflammatory effects in There are several in vitro and in vivo studies that have uti- rodents. This method is validated for the measurement of THC and lized various mass spectrometry (MS) methods to determine CBC simultaneously after extraction from mouse blood or brain. pharmacokinetic parameters of cannabinoids in humans and Whole brain harvested from mice was homogenized 2:1 (v/w) with laboratory animals (4–7). Often these studies employ methods normal saline. Fifty nanograms of THC-d3 was added to 0.5 mL of that detect THC, 11-OH-THC, and THC-COOH in blood or heparinized mouse blood, brain homogenate, and THC and CBC urine (8). Only a handful of the phytocannabinoids have been fortified blood or brain calibrators, then equilibrated overnight at studied for their pharmacokinetic properties, their possible 5°C. Two milliliters of “ice cold” acetonitrile was added drop-wise while the sample was vortex mixed, and then the sample was modulation of the pharmacokinetics of THC, and ultimately to centrifuged and stored overnight at –30°C. The cannabinoids were correlate these pharmacokinetic parameters to pharmacolog- extracted from the acetonitrile layer with 2 mL of 0.2 N NaOH and ical effects. 4 mL of hexane/ethyl acetate (9:1). The solvent was isolated and Compared to the number of studies that detect cannabi- evaporated to dryness. Trimethylsilyl derivatives were prepared and noids in blood or urine, there are few studies that have uti- then analyzed by gas chromatography–mass spectrometry. lized validated MS methods to measure cannabinoids in brain Linearity in blood and brain of THC and CBC was 2–10,000 ng/mL tissue, which is the site of action for many of the pharmaco- (ng/g). THC and CBC recovery ranged from 56 to 78% in blood logical effects of cannabinoids (9–12). THC and cannabidiol and brain. Precision was demonstrated at 100 ng/mL and 1000 (CBD) have been measured in blood and brain tissue of mice ng/mL with CVs < 15%. The validated method allows for blood to determine possible pharmacokinetic interactions between and brain concentrations of cannabinoids to be quantificated and these cannabinoids (13). In the Varvel et al. study (13), CBD correlated with pharmacological effects produced in mice. significantly increased blood and brain concentrations of THC compared to THC alone and correlated to an increased antinociceptive effect in combination compared to THC Introduction alone. Cannabichromene (CBC) is a phytocannabinoid that has been the second most abundant cannabinoid quantitatively Interest in investigating the pharmacological properties of (behind THC) in some strains of marijuana growing in the Cannabis sativa, or marijuana, is apparent and necessary as United States (14,15). CBC has been shown to produce marijuana is the most commonly used illicit drug in the United antinociception (16) and anti-inflammatory effects in rodents States (1) and remains as one of the most widespread drugs of (17,18). CBC possesses poor affinity to the CB1 receptor (i.e., 9 abuse worldwide. ∆ -Tetrahydrocannabinol (THC) is a proto- Ki value > 10,000 nM) (19). This is consistent with the CB1 typical phytocannabinoid and is recognized as the primary receptor antagonist rimonabant failing to block the tetrad psychoactive constituent in marijuana (2). However, there are activity of CBC, indicating CBC has a non-CB1 receptor mech- an additional 69 phytocannabinoids that have been identified anism of action. In comparison, rimonabant has been reported 496 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission. Wolf_jatsept11.qxd:JATLynneTemplate 7/27/11 10:37 AM Page 2 Journal of Analytical Toxicology, Vol. 35, September 2011 to have a Ki value of 2 nM and and has been shown to block the Reference materials and reagents tetrad effects of THC and other cannabinoids (20–22). Because CBC was obtained from Organix (Woburn, MA) in its oil rimonabant failed to block the tetrad effects of CBC, the mech- form. CBC stock solutions were prepared in methanol at 1, 5, anism by which CBC elicited tetrad effects has yet to be estab- and 70 µg/mL. THC and THC-d3 were obtained from Ceril- lished. liant (Round Rock, TX) as methanolic solutions at 1 mg/mL Whereas past studies have investigated in vitro metabolites and 100 µg/mL, respectively. THC and THC-d3 stock solutions of CBC via MS (14), there are no reported method validations were prepared in methanol at 1, 10, and 100 µg/mL for THC for measuring CBC after extraction from mouse blood or brain and 1 µg/mL for THC-d3 (internal standard). Standards were tissue following CBC in vivo administration. Furthermore, stored at –30°C when not in use. Deionized water was ASTM there is no reported method validation that simultaneously Type I water from the in-house water system. Hexane, ethyl ac- measures THC and CBC concentrations in blood and brain etate, and acetonitrile were HPLC grade or better, and “ice matrices. The goal of this study was to validate an MS method cold” acetonitrile was stored at –30°C in an ice block when not for the quantification of THC and CBC in mouse blood and in use. Sodium hydroxide (NaOH) was ACS grade. The deriva- brain tissue. Determining the upper limit of linearity (ULOL), tizing agent utililized was RC3 [N,O-bis(trimethylsilyl)- limit of detection (LOD), limit of quantification (LOQ), re- trifluoroacetamide (BSTFA) + 10% trimethylchlorosilane] Downloaded from https://academic.oup.com/jat/article/35/7/496/880227 by guest on 24 September 2021 covery, accuracy, and precision comprised the method valida- (Regis Technologies, Morton Grove, IL). tion. Gas chromatography (GC)–MS equipment Samples were analyzed on a Shimadzu QP2010 GC–MS uti- lizing a splitless injection port and DB-5 column (30 m × 0.25- Materials and Methods mm i.d. × 25-µm film thickness, J&W Scientific, Folsom, CA). The carrier gas was helium, and the injection volume of deriva- Animals tized samples was 1 µL. The injection port temperature and This study utilized naïve (mice not used in any previous transfer line temperatures were maintained at 250°C and studies) male ICR mice (Harlan Laboratories, Dublin, VA) 280°C, respectively. The GC oven temperature program was weighing 20–30 g that were housed six to a cage in the animal 190°C, hold 0.1 min, 30°C/min ramp to 330°C, and hold for 1 care facility maintained at 22 ± 2°C on a 12 h light/dark cycle. min. The ion source and quadrupole temperatures were main- Food and water were available ad libitum, and mice were ac- tained at 260°C and 150°C, respectively. Selective ion moni- climated to the room environment (22 ± 2°C) 24 h before sac- toring mode (SIM) was utilized with a dwell time of 10 ms. Re- rifice. All of our animal work was approved under Virginia tention times were 4.14, 3.98, and 4.12 min for THC, CBC, and Commonwealth University’s Institutional Animal Care and Use THC-d3, respectively (Table I). The following quantification Committee (IACUC #AM10312). Mice were intravenously in- (underlined) and qualitative ions (m/z) identified analytes jected with doses of THC or CBC at 0.3 or 10 mg/kg THC and (Table I): THC, 386, 315, 343; CBC, 303, 246, 271; THC-d3, 389, 3.0 or 100 mg/kg CBC (23). 318, 346. Figure 1 contains the structures of CBC and THC. Calibrator preparation Table I. GC–MS Retention Time and Ions Monitored in THC and CBC calibrators were prepared at 0.5, 1, 2, 5, 10, 20, SIM Mode for THC, CBC, and THC-d 3 50, 100, 250, 500, 1000, 2000, 5000, 7500, and 10,000 ng/mL Rt Ions Monitored (ng/g) in each matrix. The 0.5, 1, 2, 5, 10, 20, and 50 ng/mL Analyte (min) (m/z)* (ng/g) calibrators were prepared from the 1.0 µg/mL THC and CBC standards. The 100, 250, and 500 (ng/g) calibrators were THC 4.14 386, 315, 343 prepared from the 10 µg/mL THC and CBC standards, and the CBC 3.98 303, 246, 271 1000, 2000, 5000, 7500, and 10,000 ng/mL (ng/g) calibrators THC-d3 4.12 389, 318, 346 were prepared from the 100 µg/mL THC and 70 µg/mL CBC standards. Blood and brain calibrators were freshly prepared on * Underlined ions were used for quantification. each day the analysis was performed with freshly harvested mouse blood and brain. Sample collection and extraction procedure Naïve ICR mice were decapitated and whole blood was collected in heparinized tubes (Fisher Scientific, Pittsburgh, PA). Whole brains were harvested and stored on ice until the brain tissue was homog- enized.