Cannabichromene and Tetrahydrocannabinol Determination in Mouse Blood and Brain by Gas Chromatography–Mass Spectrometry

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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

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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 utilizing 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 homogenized. Homogenization was completed Figure 1. Structures of CBC and THC. within 2 h following harvest, and the brain tissue was homogenized in a 2:1

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ratio (v/w) of 0.9% unbuffered saline to brain tissue. Aliquots cates divided by the mean peak height of non-extracted repli- (0.5 mL) were prepared from heparinized mouse whole blood cates for respective low and high concentrations. or brain homogenate, and the analytes were added to prepare Accuracy and precision were assessed by adding analytes to calibrators for the calibration curve. THC-d3 (50 µL, 1 µg/mL) a series of six replicates in blood or brain tissue of low (100 was added to all calibrators and samples (minus drug-free sam- ng/mL or ng/g, respectively) and high (1000 ng/mL or ng/g, re- ples), and the mixtures were equilibrated overnight at 5°C. spectively) concentrations of each analyte and determining “Ice cold” acetonitrile (2 mL) was added drop-wise while their concentrations from linear regression of matrix matched the sample was constantly vortex mixed, and then the mixture calibration curves. Accuracy was determined from the mean of was centrifuged at 3000 rpm for 10 min for phase separation. the concentrations of the six replicates (low or high) com- The mixture was stored overnight at –30°C to assist in further pared to the target concentrations [100 or 1000 ng/mL (ng/g in separation of the phases. Two layers are present after centrifu- brain tissue)]. Accuracy was acceptable when means were gation (acetonitrile/aqueous and protein). The aqueous portion within 10% of the target concentrations. Precision was deter- of the sample interferes with further analysis of cannabinoids, mined from the calculated concentrations of the six replicates and must be allowed to separate into a third layer before pro- (low or high) and reported as the percent of the coefficient of

ceeding with the extraction procedure. The top layer (acetoni- variation (%CV). Precision was acceptable when %CV for each Downloaded from https://academic.oup.com/jat/article/35/7/496/880227 by guest on 24 September 2021 trile layer) was isolated, and 2 mL of 0.2 N NaOH was added. concentration was less than 15%. Four milliliters of organic solvent (9:1, hexane/ethyl acetate) was added, and the calibrators were rotated at 30 rpm for 30 min to facilitate mixing. The tubes were then centrifuged at 3000 rpm for 10 min. The organic layer was isolated and evaporated to dryness in a Savant AES1000. Fifty microliters of derivatizing agent

(RC3) was added to each tube, then each Figure 2. Total ion chromatogram of 10 ng/mL CBC, THC-d3, and THC. tube was capped, and the samples were heated at 70°C for 20 min. Samples were then transferred to autosampler vials for GC–MS analysis. Figure 2 contains a total ion chromatogram of 10 ng/mL CBC, THC-d3, and THC.

Validation procedure The method’s linearity was determined by utilizing 15 calibrators of different concentrations in each matrix, prepared and analyzed on 3 separate occasions, then subsequently on 10 separate occasions utilizing 13 calibrators of different concentrations in each matrix. The limits of linearity for the method were determined when the experimental concentration of a calibrator was not within 20% of the expected theoretical concentration, whereas the linearity of the calibration curve was maintained with a satisfactory coefficient of determination (r2 > 0.985). Linearity was determined by linear regression of calibrator concentration versus peak-height ratio of either CBC or THC peak height divided by the peak height of THC-d3. The calibration curve linearity was repeated three times for reproducibility in blood and brain tissue. LOD was defined ad- ministratively as equal to the value of the LOQ. LOQ was defined as the lowest non-zero concentration in which the ion ratios were within 20% of the ion ratios averaged from other calibrators, and the calculated concentration was within 15% of the expected concentration. Recovery was determined by adding analytes to a series of six replicates in blood or brain tissue with low (100 ng/mL or ng/g, respectively) and high (1000 ng/mL or ng/g, respectively) concentrations of each analyte that were extracted and compared to a series of six replicates of low and high concentrations Figure 3. Representative calibration curves (n = 1) of CBC and THC in of each analyte that were not extracted. Recovery was expressed blood (A) and brain (B). as a percentage of the mean peak height of the extracted repli-

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Data analysis THC and CBC (mg/kg), blood concentrations were 0.3 mg/kg Data were collected utilizing Shimadzu GC–MS Solutions THC (2 ng/mL), 10 mg/kg THC (558 ng/mL), 3.0 mg/kg CBC software (version 2.5; Shimadzu Scientific Instruments, (152 ng/mL), and 100 mg/kg CBC (10327 ng/mL). Brain Columbia, MD) and analyzed utilizing Microsoft Office Excel tissue concentrations were 0.3 mg/kg THC (9 ng/g), 10 mg/kg 2003 (Microsoft, Redmond, WA) and Prism 5 (Graphpad Soft- THC (514 ng/g), 3.0 mg/kg CBC (133 ng/g), and 100 mg/kg ware, San Diego, CA). CBC (4411 ng/g).

Results Discussion

For reproducibility of linearity, three linearity curves The MS method employed in this study originated from a were prepared for THC and CBC in blood and brain tissue. collection of methods (4–7). An overnight equilibration period All calibration curves (n = 13) had a correlation of deter- after the addition of internal standard to samples was adopted 2

mination that was acceptable (r ≥ 0.985) (Figure 3, ex- from Goodall and Basteyns (7) and had been previously uti- Downloaded from https://academic.oup.com/jat/article/35/7/496/880227 by guest on 24 September 2021 ample) with a linearity range of 2–10,000 ng/mL or 2– lized in processing samples for an EMIT procedure (25). The 10,000 ng/g in blood and brain tissue respectively for each use of acetonitrile (semi-polar) as an extracting agent fol- analyte (Table II). Ion ratios and calculated concentrations lowed by hexane/ethyl acetate was determined to extract for the 2 ng/mL or 2 ng/g calibrator were acceptable to es- cannabinoids from biological matrices more efficiently than tablish this concentration as the LOD and LOQ for each an- hexane (non-polar) or acetone (6). After the addition of ace- alyte in its respective matrix. tonitrile, the samples were frozen overnight to allow for ef- Results for recovery, accuracy, and precision are presented in fective separation of cannabinoids from biological matrices as Table III. THC recovery, in blood and brain, respectively, at 100 the biological material precipitates at the bottom of the ng/mL (ng/g in brain tissue) was 63% and 55%, and at 1000 sample (4,25). The extraction procedure that was described in ng/mL (ng/g in brain tissue) was 59% and 56%. CBC recovery, our study has been previously utilized to process blood and in blood and brain tissue, respectively, at 100 ng/mL (ng/g in brain samples and ultimately measure concentrations of THC brain tissue) was 65% and 73%, and at 1000 ng/mL (ng/g in and/or CBD in these matrices (26). Varvel and co-workers brain tissue) was 59% and 78%. Accuracy for THC, in blood and (13) used a modification of the extraction procedure that did brain, respectively, at 100 ng/mL was 95 ng/mL and 102 ng/g, not derivatize the samples as they were analyzed by liquid and at 1000 ng/mL, was 1012 ng/mL and 1092 ng/g. Accuracy chromatography–MS. for CBC, in blood and brain tissue, respectively, at 100 ng/mL The MS method that was validated in this study produced was 101 ng/mL and 97 ng/g, and at 1000 ng/mL was 1020 recovery, precision, and accuracy parameters that were con- ng/mL and 1035 ng/g. sistent with other validated GC–MS methods utilized to iden- Precision was demonstrated at 100 ng/mL and 1000 ng/mL tify cannabinoids in biological matrices (27,28). For the (ng/g in brain tissue) as %CV was less than 15% in each ma- cannabinoids that were identified in these previously pub- trix for each analyte (Table III). THC precision (%CV), in lished methods, recovery was generally 50–75%, accuracy was blood and brain, respectively, at 100 ng/mL (ng/g in brain within 10% of the target concentrations, and precision (%CV) tissue) was 14% and 5%, and at 1000 ng/mL (ng/g in brain was within 15%. The linearity range in the previous studies tissue) was 4% and 5%. CBC precision (%CV), in blood and brain tissue, respectively, at 100 ng/mL (ng/g in brain tissue) was 9% and 2%, and at 1000 ng/mL (ng/g in brain tissue) was Table III. Recovery, Accuracy, and Precision for THC and CBC in Blood and Brain Tissue Determined at 100 and 6% and 6%. 1000 ng/mL (ng/g)* The validated method was applied to a research project in- volving exposure of mice to various doses of THC or CBC. The Accuracy and Precision pharmacological effects observed in this research project are Target described in DeLong et al. (23). After intravenous doses of Analyte Matrix Concentration Recovery Mean %CV THC Blood 100 ng/mL 63% 95 14 Table II. LOD, LOQ, and Linearity Range for THC and 1000 ng/mL 59% 1012 4 CBC in Blood and Brain Tissue CBC 100 ng/mL 65% 101 9 1000 ng/mL 59% 1020 6 Analyte Matrix LOD and LOQ Linearity Range THC Brain 100 ng/g 55% 102 5 THC Blood 2 ng/mL 2–10,000 ng/mL 1000 ng/g 56% 1092 5 CBC 2 ng/mL 2–10,000 ng/mL CBC 100 ng/g 73% 102 5 1000 ng/g 78% 1035 6 THC Brain 2 ng/g 2–10,000 ng/g CBC 2 ng/g 2–10,000 ng/g * n = 6, except 100 ng/mL in blood, n = 5.

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