A total of 74 subjects were recruited to perform a study designed to develop a test that confirms recent use of inhaled cannabis within the impairment window as previously described . All subjects received financial compensation for their participation. The study was performed under a clinical protocol approved by the Cancer Immunotherapy Research Institute IRB (assurance #FWA00029851), and all research activities were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects prior to their participation, and a copy of the signed informed consent form was provided to each subject.
To be included, a subject must have been a male or female cannabis user at least 21 years of age. Prior to their scheduled participation, they must have used within the previous 24 hours, but not within the last 12 hours. Upon entry, subjects were asked to complete a questionnaire requesting their age, sex, race, height, weight, cannabis use history (time since last use, number of days used in the last 14 days, how often they use cannabis, number of years of cannabis use), their primary route of cannabis use, whether or not they use tobacco and alcohol, and any medications or supplements they are taking.
Each subject was given a single cannabis cigarette and instructed to smoke as much of it as possible within a 10-min period. Cigarettes containing 500 mg of dried cannabis flower with a Δ9-THC content ranging from 8.5 to 28.4% were prepared immediately before each smoking session. Cannabis supplies were legally obtained from licensed retail establishments in the Sacramento, CA region. A wide variety of chemovars was included to account for the variability in potencies available in numerous cannabis retail establishments in the various U.S. states where recreational and/or medicinal cannabis has been legalized.
Blood Draw Schedule
Blood samples were obtained from all 74 subjects. To establish baseline cannabinoid levels, capillary blood samples were collected prior to smoking. Post-smoking blood samples were collected immediately after smoking and then at 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 min post-smoking. Capillary blood (50-100 μL) was collected into BD Microtainer tubes containing lithium heparin anticoagulant (Thermo Fisher Scientific; Waltham, MA) after pricking subjects’ fingers using 17-gauge lancets (McKesson Medical-Surgical Inc., Richmond, VA). Some capillary blood samples were drawn using automated collection devices from Tasso, Inc. (Seattle, WA) and Seventh Sense Biosystems, Inc. (Medford, MA) equipped with sample reservoirs containing lithium heparin. These devices are designed to draw approximately 100-150 μL of whole blood over a period of 1-3 min.
Breath Collection Schedule
Breath samples were obtained from a total of 44 subjects. The other 30 subjects had only blood samples collected because the original study design was to develop a blood-based cannabis recent use test. Data from these first 30 subjects showed that an additional component, exhaled breath, was needed to more accurately detect recent cannabis use within the impairment window.
To establish baseline cannabinoid levels, breath samples were collected prior to smoking. Post-smoking breath samples were collected immediately after smoking, and then at 10, 20, 30, 40, 50, 60, 80, 120, 180, and 240 min post-smoking in the first 35 subjects. In the last nine subjects, back-to-back breath samples were collected at 20 and 40 min post-smoking. Breath sample collection devices were provided by Sensabues AB (Stockholm, Sweden). These self-contained, single-use devices contain an electrostatic polymer filter and are designed to collect about 20 L of exhaled breath through normal breathing. During sample collection, subjects were seated and instructed to blow through the device until the attached bag was fully inflated. The time required for sample collection was approximately 2-3 minutes. No instances of hyperventilation or other breathing abnormalities were observed. Devices were kept sealed in their original packaging until immediately before use to prevent contamination and used according to the manufacturer’s instructions. The smoking room was well ventilated and allowed to clear for at least 24 hours prior to each subject smoking session. Immediately after sample collection, the devices were resealed, removed from the collection area, and held at room temperature (20-25°C). All samples were extracted and analyzed within 24 hours of collection.
Self-Assessment of Impairment
All 74 subjects were asked to self-assess their level of impairment before smoking and at each designated time point after smoking based on a scale ranging from 0 (no impairment) to 10, which denoted maximal impairment (incapacitation) for that individual. To normalize, impairment data were expressed as a percentage relative to each individual subject’s maximum reported impairment level.
Physical Assessment of Impairment: Horizontal Gaze Nystagmus
In this study, a subset of 44 subjects were evaluated for horizontal gaze nystagmus (HGN) as a physical indicator of impairment. Horizontal gaze nystagmus refers to the involuntary movement or jerking of the eyes as they gaze to either side, and it is a component of standardized field sobriety testing . In this particular test, subjects are asked to keep their head still and follow a slowly moving horizontal object positioned in front of their face using their eyes only. Both eyes are observed for lack of smooth pursuit, nystagmus at maximum eye deviation (45°), and the onset of nystagmus prior to a 45° deviation. The presence or absence of resting nystagmus is also noted.
Chemicals and Reagents
Six of the seven cannabinoid analytes [D9-THC, cannabinol (CBN), cannabigerol (CBG), cannabigerolic acid (CBGA), D9-tetrahydrocannabinolic acid A (D9-THCA), and D9-tetrahydro-cannabivarin (D9-THCV)] and the internal standard (IS; D9-THC-D3) were obtained as certified reference materials (CRMs) manufactured by Cerilliant (Round Rock, TX). Cannabichromene (CBC) was obtained as a CRM from Cayman Chemical (Ann Arbor, MI). When not in use, concentrated stock solutions of these agents and working solutions made therefrom were stored at –20°C.
Acetonitrile, formic acid, methanol, and n-hexane were purchased from Thermo Fisher Scientific and were of LC/MS grade. Ethyl acetate (Acros Organics) was purchased from Thermo Fisher Scientific and was of spectroscopy grade (>99.5%). High purity water (18.2 MΩ) required for preparing the mobile phase and for sample extraction was produced using an EMD Millipore Simplicity water purification system. When not in use, these agents were stored at room temper-ature (20-25°C). Nitrogen (N2), supplied as a cryogenic liquid in a 230L dewar at a purity of 99.998%, or as compressed nitrogen gas at a purity of 99.999% in T-type cylinders, was obtained from Praxair (Danbury, CT).
Analysis of Cannabinoids in Exhaled Breath
A previously validated LC-HRMS analytical method for the quantification of the cannabinoids D9-THC, CBN, CBC, and D9-THCV in exhaled breath was used for the analysis of study samples. Additional cannabinoids analyzed included D9-THCA, CBG, and CBGA. For the preparation of calibration standards, sufficient quantities of the matrix (breath collection devices) were obtained from SensAbues AB. Breath collection devices were kept at room temperature (20-25°C) within their original packaging to prevent contamination.
Concentrated standard calibration solutions were prepared in methanol at 37.5, 75, 150, 375, 750, and 1,500 ng/mL of all cannabinoids combined. Following extraction and reconstitution, final standard concentrations were 2.5, 5.0, 10, 25, 50 and 100 ng/mL, equivalent to approximately 0.2, 0.4, 0.8, 1.9, 3.8, and 7.5 ng/breath filter. The IS solution was prepared in methanol at a concentration of 75 ng/mL. To prepare calibration standards for extraction, 5 µL of the IS working solution and 5 µL of the appropriate calibration standard solution were added directly onto the corresponding filter pad inside the breath collection device. After extraction, the final concentration of the IS was 5 ng/mL (75 µL final volume). Study samples were prepared by spiking with 5 µL IS solution.
Extraction of cannabinoids from breath collection devices was performed as previously described . Briefly, a total of 7 mL methanol were aliquoted through each device and filter housing into glass sample collection tubes. The sample breath collection devices were then removed and the glass tubes were placed in an N-Evap Model 112 analytical nitrogen evaporator (Organomation Associates, Berlin, MA). The eluate was evaporated to dryness under a gentle stream of nitrogen gas, with the water bath temperature set to approximately 50°C. After evaporation, the samples were cooled to room temperature and reconstituted in 75 µL of a solution containing 75% acetonitrile and 25% water with 0.1% formic acid. The samples were then transferred to a glass microinsert-equipped autosampler vial and placed in the autosampler compartment for analysis according to the method. The chromatographic conditions for the analysis of cannabinoids in exhaled breath were the same as previously described .
Analysis of Cannabinoids in Blood
Extraction and analysis of D9-THC and other cannabinoids in whole blood was performed according to a validated method as previously described . Briefly, 50 µL of each sample was mixed with 100 µL of high-purity water in a 1.5-mL microcentrifuge tube and spiked with 5.0 µL of IS solution. To extract, 500 µL of a solution containing 90% n-hexane and 10% ethyl acetate (v/v) was added to each sample, followed by vortexing for 30 sec. Samples were then centrifuged at 9,300 rcf for 10 min. The supernatant was transferred to a 16 mm x 125 mm glass tube and evaporated to dryness under a gentle stream of nitrogen at 50°C. Samples were then reconstituted in 75 µL of a solution composed of 65% acetonitrile, 35% water, and 0.1% formic acid and analyzed by LC-HRMS. Supplies of whole blood needed to prepare calibration standards were obtained from a reliable, cannabis-free donor and kept refrigerated (2-8°C) for up to six weeks.
The LC-HRMS system consisted of a Thermo Scientific Vanquish ultra-high-performance liquid chromatography (UHPLC) system and a Thermo Scientific Q Exactive mass spectrometer. All analytical data were collected and processed using TraceFinder version 4.1 software (Thermo Fisher Scientific). The mass spectrometer and UHPLC system were configured as previously described .