Comparison of Δ9-tetrahydrocannabinol in venous and capillary blood following ad libitum cannabis smoking by occasional and daily users
Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80526, United States
Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Denver, CO 80045, United States
Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA 30322, United States
Department of Anesthesiology and Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States
Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States
Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States
Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States
Abstract
Δ9-Tetrahydrocannabinol (Δ9-THC) is the most prominent and main psychoactive cannabinoid found in cannabis. In forensic matters involving cannabis, such as drugged driving or workplace accident investigations, blood Δ9-THC determination is typically required. Venipuncture by a phlebotomist at a medical facility is often the standard blood collection protocol, but this procedure is time consuming and requires specialized training. Capillary blood collection at the site of a transportation or workplace mishap may provide a collection method that is logistically easier and may better reflect blood cannabinoid concentrations at the time of an incident. This study represents the first temporal comparison of the concentration of Δ9-THC and its primary metabolites in venous and capillary blood obtained from users following ad libitum inhalation of contemporary high-concentration cannabis products. Participants provided their own cannabis from a licensed Colorado dispensary and were instructed to smoke or vape ad libitum the amount most used for the desired effect during a 15-minute period. Capillary blood samples collected at the lateral shoulder using the TAP® II microneedle device and standard venipuncture samples at the forearm were collected contemporaneously at baseline and then 10, 30, 60, 90, and 140 minutes after the last inhalation and were analyzed for Δ9-THC, 11-hydroxy-Δ9-THC, and 11-carboxy-Δ9-THC by liquid chromatography–tandem mass spectrometry. Within-subject Δ9-THC concentrations trended lower, often up to 30 to 40%, in contemporaneous capillary blood samples than in venous blood samples until 140 min after cannabis smoking. Concentrations of the Δ9-THC metabolites 11-hydroxy-Δ9-THC and 11-carboxy-Δ9-THC were equivalent at all but the first timepoint after smoking. Due to logistical advantages, capillary blood collection by microneedle devices may be a viable option for qualitative detection of Δ9-THC and its metabolites soon after an incident or a quantitative determination if the samples are collected at least 2 hours after cannabis inhalation.
Article notes
Untitled section
Received 2025 Feb 3; Revised 2025 Mar 26; Accepted 2025 May 12; Collection date 2025 Sep.
Introduction
Δ9-Tetrahydrocannabinol (Δ9-THC) is the most prominent and main psychoactive cannabinoid found in cannabis. It acts as a partial agonist of the CB1 cannabinoid receptor, causing the characteristic psychotropic effects associated with cannabis, including euphoria and relaxation, as well as potential impairment of psychomotor and cognitive functions [1]. Therefore, cannabis usage has public health relevance for traffic safety, occupational safety, and injury prevention [2]. Δ9-THC is among the drugs most frequently detected in drug-impaired driving cases [3] and in fatally injured drivers in the USA [4]. In forensic matters involving cannabis, such as drugged driving or workplace accident investigations, blood Δ9-THC determination is typically required. Venous puncture by a phlebotomist at a medical facility is often the standard blood collection protocol, although in some jurisdictions a phlebotomist at a law enforcement agency may obtain the sample.
Venous blood is the most common sample collected in forensic situations, as well as in clinical and research applications requiring drug analysis. Although blood collection by venipuncture is readily performed by trained personnel, it is invasive, may be painful, and can cause anxiety in subjects. In pharmacokinetic studies, frequent blood sampling may require repeated venipuncture or insertion of a venous catheter. These aspects of venous blood collection, especially the need of a trained phlebotomist, are not ideal and may prove prohibitive in certain circumstances. Instead, the collection of capillary blood might pose a valid alternative for drug analysis while avoiding these negative aspects of venous blood collection. Compared to venous blood sampling, capillary blood collection is the preferred method of blood collection by patients, with a reduction in anticipatory stress in subjects and lower drop-out rates reported in a clinical study [5]. Applications such as glucose monitoring typically collect capillary blood by fingerpick [6, 7], but capillary blood collection devices that are applied to the upper arm have been developed more recently [8]. In certain studies, capillary blood samples obtained at the fingertip have been found to be comparable to contemporaneous venous samples for drugs [9–14], hormones [15, 16], and blood chemistry panels [17–20]. However, capillary blood sampled at alternative sites, such as the arm or abdomen, may yield different analyte concentrations from blood sampled at more highly perfused fingertip capillaries or venous circulation following drug administration when concentrations in the central compartment are rapidly changing.
Efforts to determine a correlation between cannabinoids in capillary blood vs. venous blood are limited. A few studies utilizing the oral administration of cannabidiol (CBD) products have demonstrated a correlation between capillary and venous blood, suggesting that capillary blood may be a comparable matrix [21–23]. Both Δ9-THC and Δ8-THC have been measured in capillary blood from users smoking cannabis products, but no comparisons with simultaneously collected venous blood have been performed [24, 25]. Following acute use of cannabis, venous blood Δ9-THC concentrations decrease quickly due to tissue distribution [26] such that the more time that elapses between the incident and collection, the less representative the blood sample may be to that present the time of the incident [26, 27]. Capillary blood samples representative of the venous circulation expeditiously obtained without the need for a phlebotomist following a transportation crash or workplace accident might offer a more accurate representation of Δ9-THC at the time of the incident. This study represents the first temporal comparison of the concentration of Δ9-THC and its primary metabolites in venous and capillary blood obtained from users following ad libitum smoking of contemporary high-concentration cannabis products.
Materials and methods
Materials
All cannabinoid standards and deuterium-labeled internal standards were purchased from Cerilliant (Round Rock, TX, USA). Water, methanol, and acetonitrile (LC–MS grade) were purchased from Millipore (Burlington, MA, USA). Sodium fluoride, potassium oxalate, and acetic acid (LC−MS grade) were obtained from Sigma-Aldrich (St Louis, MO, USA). Captiva EMR-Lipid columns (1 mL, 40 mg) were purchased from Agilent Technologies (Santa Clara, CA, USA). Liquid chromatography columns were purchased from Restek Inc. (Bellefonte, PA, USA). TAP® Micro blood collection devices were purchased from YourBio Health (Medford, MA, USA).
Participant recruitment
Participants were identified and recruited from the Denver metro area through participation in prior studies, paid targeted advertising on social media and search engines, in person and online poster distribution, newspaper ads, online referrals, and word of mouth. Interested individuals completed a web-based survey that determined initial study eligibility. A total of 29 healthy adults (aged 21−53) were recruited. Participants were enrolled into one of three cannabis-use categories based on their frequency of cannabis use (daily in the past 30 days vs. at least two times a month but less than 3 times a week) and the form of inhaled cannabis they were familiar with and agreed to use on the day of the study (flower vs. high-concentration concentrate extract products): daily flower (n = 10), daily concentrate (n = 10), and occasional flower (n = 9). Participant demographic characteristics and cannabis use history are shown in Table 1. Study procedures were undertaken with the understanding and written consent of each participant.
| Total | Daily Flower | Daily Concentrate | Occasional Flower | |
|---|---|---|---|---|
| N = 29 | n = 10 | n = 10 | n = 9 | |
| Gender, n (%) | ||||
| Male | 10 (34.5) | 5 (50) | 3 (30) | 2 (22.2) |
| Female | 19 (65.5) | 5 (50) | 7 (70) | 7 (77.8) |
| Age | ||||
| Mean (SD) | 32.8 (7.6) | 34.7 (8.3) | 32.2 (6.5) | 31.4 (7.8) |
| 21−29, n (%) | 12 (41.4) | 4 (40) | 3 (30) | 5 (55.6) |
| 30−39, n (%) | 12 (40) | 3 (30) | 6 (60) | 3 (33.3) |
| 40−49, n (%) | 3 (10) | 2 (20) | 1 (1) | 0 (0) |
| 50−55, n (%) | 2 (6.7) | 1 (10) | 0 (0) | 1 (11.1) |
| Race, n (%) | ||||
| White | 25 (86.2) | 9 (90) | 8 (80) | 8 (88.9) |
| Black/African American | 3 (10.3) | 1 (10) | 2 (20) | 0 (0) |
| Other/No Response | 1 (3.4) | 0 (0) | 0 (0) | 1 (11.1) |
| Ethnicity, n (%) | ||||
| Hispanic/Latino | 5 (17.2) | 1 (10) | 3 (30) | 1 (11.1) |
| Non-Hispanic/Latino | 22 (75.9) | 8 (80) | 7 (70) | 7 (77.8) |
| Declined to respond | 2 (6.9) | 1 (10) | 0 (0) | 1 (11.1) |
| Body mass index (BMI) | ||||
| Mean (SD) | 26.9 (6.2) | 26.6 (6.4) | 27.8 (7.5) | 26.3 (4.5) |
| Cannabis potency (% THC) | ||||
| Mean (SD) | 40.2 (27.0) | 22.0 (4.9) | 72.1 (19.7) | 23.0 (3.9) |
Inclusion and exclusion criteria
Key inclusion criteria included cannabis use on a daily or weekly basis, willingness to consume a minimum of at least two inhalations of cannabis flower or concentrate products (e.g., by dabbing or vaporizing wax, oil, etc) containing less than 2% CBD, and willingness to have venous blood sampled eight times via an indwelling venous catheter and capillary blood sampled up to six times. Participants were excluded if they were taking medication that may strongly interact with components of the cytochrome CYP450 system involved in cannabinoid metabolism (including CYP2C9 and CYP3A/3A5) or using potential recreational drugs (e.g., cocaine or unprescribed amphetamines), as determined by medication history or a urine drug test. Additional exclusion criteria included the following: participants with a history of abuse or addiction to substances other than cannabis (e.g., prescription and nonprescription pharmaceuticals, alcohol, or illicit drugs); participants who expressed an interest in substance abuse treatment within 60 days prior to study enrollment; participants with a past or current diagnosis of schizophrenia, narcolepsy, heart disease, epilepsy, or a traumatic brain injury; participants with untreated bipolar disorder, major depression, sleep apnea, or other uncontrolled medical conditions, as determined by the investigators; and participants with a history of clinically significant adverse event(s) associated with cannabis intoxication.
Cannabis administration
Participants came to the off-campus research site to complete data collection and were asked to abstain from cannabis use (for at least 8 hours for inhaled use and 12 hours for edible use) before the start of their visit. At baseline, participants self-reported significant medical history and medication use and confirmed their cannabis use by recalling the last 30 or 90 (for occasional use groups) days of cannabis use using a Timeline Followback Calendar [28]. Participants were asked to bring their own cannabis labeled with the original dispensary label from a licensed Colorado dispensary, and the product had to contain less than 2% CBD. The product package details, including the Δ9-THC concentration (Table 1), were recorded. Venous and capillary blood samples were taken before and after cannabis consumption. During a 15-minute interval, participants were instructed to smoke or vape ad libitum “the amount you most commonly use for the effect you most commonly desire.” Start time was recorded as the time of the first inhalation. The number of inhalations was counted, and the end time was noted as the time of the last inhalation, which was no more than 15 minutes after the start time.
Capillary blood collection
Capillary blood was collected at baseline (minutes before cannabis use), approximately 10 minutes after the end of cannabis consumption (last inhalation), and 30, 60, 90, and 140 minutes subsequently. Capillary blood was collected using the TAP® II capillary blood collection device purchased in 2022 (Yourbio Health, Medford, MA). Participants were asked to warm their upper outer arm (about three finger widths below the shoulder) with a heat pack to prepare for capillary blood collection. After approximately 5 minutes of warming, the participant’s arm was sanitized with an isopropanol wipe, and a sterile TAP® II blood collection device was placed (with gentle pressure to adhere the sticker underneath) on the participant’s upper outer arm. Capillary blood was collected by clicking the device tab that extended device’s microneedles into the surface of the skin under suction. Collection was considered complete when at least 100µL of blood was collected, or if two minutes had elapsed to prevent coagulation since the blood was not vortexed in the collection tube to thoroughly mix in the lithium heparin coating on the tube. Immediately after collection, samples were transferred with pipettes from the plastic (polyethylene) collection tubes into glass tubes containing 5mg of sodium fluoride and 1mg of potassium oxalate and vortexed. Samples were then labeled and frozen at −16°C prior to analysis. Capillary blood was transferred to the glass vials with sodium fluoride and potassium oxalate for consistency with the venous blood sample collection protocol and storage vessels. Samples that contained less than 50µL of blood were rejected from the analysis.
Venous blood collection
Venous blood samples were collected at baseline (minutes before cannabis use), approximately 10 minutes after the end of cannabis consumption (last inhalation), and 30, 60, 90, and 140 minutes after the start of consumption (first inhalation). The venous and capillary blood collections occurred at the same time. Venous blood was collected approximately 2 minutes (midpoint) after the start of the capillary blood collection. The midpoint of the blood withdrawal time relative to either the first or final inhalation of cannabis was recorded. An upper arm vein was cannulated with an 18-gauge catheter for withdrawal of blood samples. Whole blood samples of approximately 2 mL were collected into gray-top tubes (BD brand vacutainer tubes containing 100mg of sodium fluoride and 20mg of potassium oxalate additive). Samples were placed on a rocker for mixing immediately after collection, rocked for 5min, and stored in a freezer at −16°C. Samples were transferred to the laboratory in a dry ice-cooled carrier for analysis within three weeks of collection.
Cannabinoid analysis by LC−MS/MS
Preparation of calibrators and quality controls
Matrix-matched calibrators and controls were prepared by the addition of appropriate volumes of methanolic stock standard mixtures (0.01, 0.1,1.0, or 10 µg/mL of each cannabinoid) to 100µl of cannabinoid-free whole blood to produce calibrators at 0.5, 1, 5, 10, 50, 100, and 500ng/mL. Quality control samples were prepared at 5ng/mL for each analyte. Quality control samples were run after every 20 subject samples, with an expected accuracy of +/− 20%. Calibrator samples were reanalyzed at the end of the batch, with an expected accuracy of +/− 20%.
Sample preparation
Subject blood samples, matrix-matched standards, and quality control samples were prepared for LC−MS/MS analysis by protein precipitation and lipid removal. Ten microliters of the internal standard solution (0.3 µg/mL Δ9-THC-d3, 0.3 µg/mL 11-hydroxy-Δ9-THC-d3, and 0.8 µg/mL 11-carboxy-Δ9-THC-d9) was added to 100μL of blood sample and vortexed in a polypropylene microcentrifuge tube. Samples that contained less than 50µL of blood were rejected from the analysis. Samples that contained 50−100µL of blood were diluted with cannabinoid-free blood for a final volume of 100µL, and the exact dilution factor was noted for purposes of adjusting final analyte concentrations. A total of 600μL of ice-cold acetonitrile/methanol (85%/15%) was added dropwise while vortexing for 20seconds to precipitate proteins. Samples were centrifuged, and supernatants were transferred to Captiva EMR-Lipid columns for lipid removal. Using a positive-pressure manifold, 3psi of pressure was applied to elute the samples through the columns. Eluents were collected into clean glass test tubes and dried under nitrogen at 45°C. Eluents were reconstituted in 75µL of water/methanol (50%/50%) with 0.1% acetic acid and transferred to autosampler vials with pulled-point inserts for LC−MS/MS analysis.
LC−MS/MS analysis
Samples were analyzed with an Agilent 1290 UHPLC instrument coupled to an Agilent 6460 triple quadrupole mass spectrometer equipped with an Agilent Jet Stream electrospray ionization source (Agilent, Santa Clara, CA). Cannabinoids were chromatographically separated on a Restek Raptor biphenyl column (3.0 × 50mm, 2.7μm) and held at 40°C. A sample volume of 10μL was injected, and a mixture of water with 0.1% acetic acid (A) and methanol with 0.1% acetic acid (B) was introduced at a flow rate of 0.4 mL/min. Gradient elution started at 40% B, which was increased to 70% B over 1 min and subsequently to 75% B over 4min, and ended at 100% B at 6 min. The ionization source conditions used were as follows: positive polarity, nebulizer pressure of 45psi; gas flow of 12L/min at 330°C; sheath gas flow of 12L/min at 390°C; capillary voltage of 3500V; and nozzle voltage of 2000V. The ion transitions monitored can be found in Supplementary data. Analytes were confirmed by the retention time and the product ion ratio (± 20%) correlation between the sample peaks and corresponding standards. Data collection and processing were performed by using Agilent MassHunter quantitative software (v.B.10.01). Quantitation was performed with linear regression using 7-point calibration curves from 0.5ng/mL to 500ng/mL for Δ9-THC, 11-hydroxy-Δ9-THC, and 11-carboxy-Δ9-THC. The limit of detection was 0.25ng/mL for Δ9-THC, 11-hydroxy-Δ9-THC, and 11-carboxy-Δ9-THC, respectively. This method was previously validated following guidelines recommended by the ANSI/ASB Standard 036 Standard Practices for Method Validation in Forensic Toxicology [29] for calibration model, limits of detection and quantitation, accuracy, precision, carryover, dilution integrity, and interferences.
Statistical analysis
Analyzes were conducted separately for each cannabinoid, cannabis-use group, and blood draw time. Absolute and percent differences between the capillary and venous concentrations of Δ9-THC and 11-hydroxy-Δ9-THC and 11-carboxy-Δ9-THC metabolites were calculated for each participant at each blood draw time. Blood samples with concentration levels of “none detected” and “less than the limit of quantitation” were reclassified as 0. This included 6 capillary samples for Δ9-THC, 13 for 11-hydroxy-Δ9-THC, and 1 for 11-carboxy-Δ9-THC. Venous samples reclassified as 0 included 6 for Δ9-THC, 24 for 11-hydroxy-Δ9-THC, and 3 for 11-carboxy-Δ9-THC. Due to the small sample size and non-normality of the cannabinoid distribution, statistical significance of the capillary and venous concentration differences was assessed using the nonparametric Wilcoxon rank sum test, with a P-value <.05 used as the criterion for statistical significance.
Results
Venous and capillary blood collection
Venous samples were collected from 29 participants at all 6 timepoints (n = 174). Collection of capillary samples using the TAP®II blood collection device on the study subjects was occasionally unsuccessful, and ultimately only 148 of 192 attempts (77%) yielded an acceptable capillary sample. Collection failures resulted from insufficient blood volume (< 50µL) (19 samples, 10%), the device failing to adhere to the skin (12 samples, 6%), lack of skin puncture (9 samples, 5%), and blood clotting (4 samples, 2%). A total of 144 matched pairs of venous and capillary blood were available for comparative analysis from 29 independent subjects. The number of matched pair at each time are as follows: 26 at -30 minutes, 23 at 10 minutes, 25 at 30 minutes, 23 at 60 minutes, 24 at 90 minutes, and 23 at 140 minutes. The mean time elapsed from last cannabis inhalation to midpoint of blood draw for capillary and venous samples are displayed in Table 2.
| Capillary | Venous | |
|---|---|---|
| Timepoint | (mins, SD) | (mins, SD) |
| 1 | 8.3 (1.3) | 6.9 (0.7) |
| 2 | 29.8 (4.5) | 24.2 (11.5) |
| 3 | 59.2 (7.4) | 55.5 (8.2) |
| 4 | 88.7 (7.6) | 87 (6.2) |
| 5 | 133.2 (5.3) | 132 (6) |
Δ9-THC concentrations in capillary blood vs venous blood
The median Δ9-THC concentrations in capillary blood were lower than those in venous blood at all time points after cannabis inhalation. Figure 1 displays the median within-subject percent difference between Δ9-THC metabolite concentrations in capillary and venous blood by cannabis-use group. In daily concentrate users, the median Δ9-THC concentrations were 32.4% lower in capillary blood than in venous blood at 10 min, a difference that diminished to 6.9% lower at 140 min. Differences were statistically significant at 10, 30, 60, and 90 min. At 140 min, the Δ9-THC concentrations in capillary and venous blood were not statistically different. Daily flower users had median Δ9-THC concentrations that were 35.7% lower in capillary blood than in venous blood at 30 min, 35.7% lower at 60 min, and 23.2% lower at 140 min. Differences were statistically significant at 30, 60, and 140 min. Occasional flower users had median Δ9-THC concentrations that were 27.9% lower in capillary blood than in venous blood at 10 min and 34.1% lower at 90 min. Differences were statistically significant at 10 and 90 min. At 140 min, the Δ9-THC concentrations were not significantly different in capillary and venous blood for the daily concentrate and occasional flower users but were still significantly lower in capillary blood than in venous blood from daily flower users. Table 3 displays the median Δ9-THC concentrations in capillary and venous blood, median within-subject differences of Δ9-THC, and the median within-subject percent difference of Δ9-THC for each user group at each time point.
| Time (mins) | Capillary blood (ng/ml) | Venous blood (ng/ml) | Median within-subject difference (ng/ml) | Median within-subject difference (%) |
|---|---|---|---|---|
| Daily—concentrate users | ||||
| −30 | 3.0 (1.7, 5.3) | 3.4 (1.6, 5.3) | 0 (−0.6, 0.5) | −1.9 (−18.6, 15.6) |
| 10 | 34.2 (15.8, 52.8) | 61.8 (34.0, 116.8) | −7.8 (−60.2, −4.6)* | −32.4 (−55.5, −19.1)* |
| 30 | 14.2 (8.7, 15.7) | 21.3 (15.1, 23.3) | −5.6 (−14.1, −0.7)* | −33.5 (−47.5, −4.3)* |
| 60 | 9.0 (6.4, 11.5) | 13.8 (8.5, 16.6) | −3.8 (−6.6, −1.8)* | −36.2 (−41.2, −20.3)* |
| 90 | 6.1 (4.1, 7.6) | 9.1 (7.1, 11.1) | −3.0 (−3.7, −0.9)* | −27.1 (−36.6, −16.3)* |
| 140 | 6.6 (4.7, 7.5) | 7.0 (4.7, 8.3) | −0.5 (−1.5, 0.4) | −6.9 (−16.3, 4.9) |
| Daily—flower users | ||||
| −30 | 1.6 (0.9, 3.3) | 2.1 (0.9, 3.6) | 0 (−0.3, 0.1) | 0 (−13.3, 2.6) |
| 10 | 53.2 (29.0, 74.5) | 69.8 (42.6, 81.4) | −7.1 (−19.6, −3.6) | −15.0 (−27.1, −8.8) |
| 30 | 13.5 (13.0, 37.6) | 23.9 (10.3, 45.6) | −7.6 (−15.1, −3.8)* | −35.8 (−39.1, −19.0)* |
| 60 | 7.7 (3.0, 11.5) | 10.5 (6.1, 16.7) | −2.9 (−6.1, −2.4)* | −37.7 (−43.8, −29.9)* |
| 90 | 4.3 (2.8, 8.8) | 5.3 (4.2, 11.1) | −1.9 (−2.3, −1.4) | −31.0 (−39.5, −17.0) |
| 140 | 2.7 (2.0, 7.1) | 4.2 (2.3, 9.0) | −0.7 (−2.3, −0.4)* | −23.2 (−31.3, −16.7)* |
| Occasional—flower users | ||||
| −30 | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) | 0 (0, 0) |
| 10 | 7.7 (5.6, 12.3) | 11.3 (7.6, 18.3) | −3.4 (−6.5, −2.2)* | −29.2 (−34.8, −17.7)* |
| 30 | 2.4 (1.2, 3.3) | 2.1 (1.6, 4.0) | −0.4 (−0.7, −0.1) | −19.3 (−19.9, −6.2) |
| 60 | 0.9 (0.7, 1.4) | 1.3 (0.9, 1.9) | −0.3 (−0.9, 0) | −20.9 (−36.7, 1.7) |
| 90 | 0.7 (0.5, 0.8) | 1.0 (0.6, 1.4) | −0.3 (−0.6, −0.1)* | −34.1 (−37.6, −9.3)* |
| 140 | 0.6 (0.3, 0.8) | 0.6 (0.3, 1.0) | 0 (−0.2, 0) | 0 (−22.6, 0) |
11-Hydroxy-Δ9-THC in capillary blood vs venous blood
The median 11-hydroxy-Δ9-THC concentrations in capillary blood were greater than those in corresponding venous blood to a statistically significant extent 10 min after cannabis inhalation for daily and occasional flower users. For daily concentrate users, the median within-subject percent differences in capillary blood and venous blood 11-hydroxy-Δ9-THC were not statistically different at 10 and 30 min (Fig. 2). Daily concentrate users had median 11-hydroxy-Δ9-THC concentrations in capillary blood that were statistically lower than corresponding venous concentrations at 60, 90, and 140 min. Daily flower users had median 11-hydroxy-Δ9-THC concentrations that were 37.9% higher in capillary blood than in venous blood at 10 min, but this difference diminished and became statistically lower in capillary blood at 90 min. Occasional flower users had median 11-hydroxy-Δ9-THC concentrations that were 42.2% higher in capillary blood than in venous blood at 10 min, but this statistically significant difference was not observed at the other timepoints. At 140 min, the 11-hydroxy-Δ9-THC concentrations in capillary and venous blood were not significantly different for daily flower and occasional flower users but were still significantly lower in capillary blood than in venous blood for daily concentrate users. Note that the absolute value and range of 11-hydroxy-Δ9-THC concentrations encountered in venous and capillary blood were much lower than the measured values of Δ9-THC and 11-carboxy-Δ9-THC, and this may have contributed to diminished statistical power to observe significant differences. All median 11-hydroxy-Δ9-THC concentrations in capillary and venous blood, median within-subject differences of 11-hydroxy-Δ9-THC, and the median within-subject percent difference of 11-hydroxy-Δ9-THC for each user group at each time point can be found in Supplementary data.
11-Carboxy-Δ9-THC in capillary blood vs venous blood
The 11-carboxy-Δ9-THC concentrations in capillary blood exceeded that in venous blood 10 min after cannabis inhalation in all user groups, but these concentrations matched more consistently at the subsequent timepoints. Figure 3 displays the median within-subject percent differences between the 11-carboxy-Δ9-THC metabolite concentrations in capillary and venous blood by cannabis-use group. Daily concentrate users had median 11-carboxy-Δ9-THC concentrations that were 12.6% higher in capillary blood than in venous blood at 10 min, a difference that diminished to less than 5% at 30, 60, 90, and 140 min. Differences were only statistically significant at 10 min. Daily flower users had median 11-carboxy-Δ9-THC concentrations that were 14.2% higher in capillary blood than in venous blood at 10 min, a difference that diminished to less than 8% at 30, 60, 90, and 140 min. Differences were only statistically significant at 10 min. Occasional flower users had median 11-carboxy-Δ9-THC concentrations that were 56% higher in capillary blood than in venous blood at 10 min, a difference that diminished to less than 10% lower at 30, 60, 90, and 140 min. Differences were only statistically significant at 10 min. At 140 min, the 11-carboxy-Δ9-THC concentrations in capillary and venous blood were not significantly different for daily concentrate, daily flower, and occasional flower users. All median 11-carboxy-Δ9-THC concentrations in capillary and venous blood, median within-subject differences of 11-carboxy-Δ9-THC, and the median within-subject percent difference of 11-carboxy-Δ9-THC for each user group at each time point can be found in Supplementary data.
Discussion
This study represents the first temporal comparison of the concentrations of Δ9-THC and its primary metabolites in venous and capillary blood obtained from users following ad libitum smoking of contemporary high-concentration cannabis products. Capillary blood sampled with the TAP®-II device at the shoulder underestimated concurrently measured Δ9-THC concentrations in venous blood, often by considerable percentages, in the minutes to hours after cannabis inhalation for each user group. The biggest differences were seen at the 10-min timepoint shortly after cessation of smoking the cannabis product. These differences in blood sample types, particularly at early timepoints after drug administration, have been seen with other drugs [30]. There could be several explanations for the underestimation of Δ9-THC concentration in the capillary blood samples. Capillary blood is obtained from capillary beds that consist of venules and arterioles joined together. The puncture of the skin results in interstitial and intracellular fluids being mixed with the capillary blood. The small capillary blood volumes collected with the TAP®-II device could contain lower concentrations of Δ9-THC if interstitial or intracellular fluids are diluting the blood. In some of the smaller-volume samples, the sample viscosity was noticeably lower, and the color was less opaque than those of larger-volume capillary samples. Blood panel comparisons of capillary and venous blood have shown reduced or variable protein concentrations in capillary blood [20, 31, 32]. Since Δ9-THC is highly protein bound [33], capillary blood with a reduced or variable protein concentration may transport less Δ9-THC to the capillary collection site, accounting for the lower concentrations. Δ9-THC is very lipophilic, with a large volume of distribution of 10L/kg [1]. It can readily cross capillary membranes, potentially resulting in losses from the capillary blood to the surrounding tissues. The duration of collection is another possible reason for the differences in Δ9-THC concentrations. Capillary blood collection can take up to a few minutes until enough sample volume is available. Therefore, the midpoints of capillary and venous sample collection are not perfectly matched. Due to its rapid tissue distribution, Δ9-THC concentrations rapidly decline early in collection, which could contribute to lower capillary concentrations due to their systematically slightly later midpoint collection times. Notably, by 140 min after the onset of cannabis inhalation, the Δ9-THC concentrations in capillary and venous blood were equivalent for daily concentrate and occasional flower users. The Δ9-THC concentrations in capillary and venous blood for daily flower users were still statistically different but trended toward equivalence.
The fact that blood collection with the TAP®-II device occurred by penetrating skin at the shoulder may also have contributed to the lower concentration of Δ9-THC in capillary blood relative to venous blood. Dermal blood flow, and the quantity of arteriovenous anastomes in the nonglaborous (hairy) dermis of the lateral shoulder are lower relative to that in glaborous (hairless) dermis of sites such as the fingertip. In investigations of site-dependent temporal differences in capillary blood glucose, it was observed that capillary glucose obtained at the nonglaborous skin of the forearm, abdomen, and thigh increased more slowly and to a lesser extent than that at the fingertip in the first two hours following an acute oral glucose challenge or meal [34–36]. The intent of the TAP®-II device (initially targeted to diabetic patients for home measurement of Hemoglobin A1C) for alternate site testing of capillary blood at the shoulder is associated with less pain and a larger skin sampling area than that found at the highly perfused capillary beds at the fingertip. It is conceivable that a modified microneedle device optimized for collection of capillary blood at the fingertip (or other more highly vascularized site such as the lip) might yield capillary blood Δ9-THC concentrations that more closely resemble those in venous blood when sampling occurs soon after cannabis intake.
In most forensic situations, collecting v enous blood within 2 hours of cannabis smoking is not possible [37], so capillary blood may be a viable option in those scenarios. This has been supported by measurements of structurally related cannabidiol, with its concentrations in capillary and venous blood 2 hours after administration [23] and during steady-state dosing [21–22] being equivalent. For clinical or research studies where blood samples are taken less than 2 hours after cannabis administration, the large concentration difference we observed with Δ9-THC indicates that standard venous blood collection may be a better option. In forensics situations where capillary blood was collected shortly after cannabis use, there could be legal implications if a biased low Δ9-THC concentration was compared to per se or permissible inference of intoxication cut-off points such as the 5ng/mL Δ9-THC threshold used in Washington and Colorado respectively.
The primary metabolites of Δ9-THC showed a higher correlation between capillary blood and venous blood for all user groups. The only statistically significant differences were seen at the 10-min timepoint immediately after cessation of cannabis smoking. Since 11-carboxy-Δ9-THC is nonpsychoactive, it is primarily measured as an analyte in urine to document the usage of Δ9-THC in situations such as the workplace, the military, commercial transportation, or athletic competitions that restrict consumption and require drug testing to prove abstinence. Higher correlation for the metabolites suggests that capillary blood might be a viable alternative to urine for documenting the extent of chronic cannabis usage when 11-carboxy-Δ9-THC is the target analyte.
Capillary blood collection is less painful and less stressful than sampling by traditional venous puncture and requires less specialized training. The manufacturers of the TAP®II blood collection device promote it as a one-step solution for capillary blood collection that requires minimal training for users and protects the user from sharps and blood contact [8]. The TAP®II device was chosen for this study of capillary blood collection after preliminary comparison with the Tasso SST blood collection device (Tasso, Inc., Seattle, WA, USA). We were able to obtain more consistent skin punctures and subsequent blood flow with the TAP®II device (data not shown), suggesting that it would be easier to work with in this study. Nonetheless, during preliminary testing, we encountered several challenges in TAP®II device utilization that required deviation from the standard user instructions provided by the manufacturer. Blood flow was sometimes slow, resulting in blood clotting before a sufficient volume was collected. Although the vials were agitated to mix the blood with the lithium heparin anticoagulant in the device, which coated the vial prior to mixing, clotting was not prevented in some samples. Therefore, during the data collection phase of our study protocol, micropipettes were used for the immediate open transfer of capillary blood from the TAP®II device to glass vials containing sodium fluoride and potassium oxalate. The vials were subsequently vortexed to prevent clotting. The transfer of the sample to a new storage vial provided a better matching of the anticoagulants and storage vessel to the gray vacutainers used for venous blood collection. How comparative results would differ if the capillary blood samples were stored in the provided polypropylene vials with lithium heparin is unclear and worthy of investigation in future research. Collection of a sufficient blood volume of at least 50µl for analysis was also challenging with the TAP®II device. A variety of methods were used to promote blood flow, including warming the upper arm with a heat pad, increasing the temperature in the room where blood was being collected, having participants put their feet up during collection, having participants squeeze a stress ball in their hand during collection, and tapping the device to encourage blood flow from the microneedles to the collection tube. Collection failures (19%) still resulted from insufficient blood volume, even with these additional procedures. This issue may be due to the reliance of the device on generation of a vacuum to draw blood from the puncture site [8]. We had 12 instances where the device fell off the arm during collection due to failure of the adhesive. Failure of the adhesive to sufficiently seal the vacuum may also be the reason for the large percentage of samples collected with insufficient volume. If a low-volume sample is collected, problems may arise if analytical methodologies are not validated for low-volume samples or if repeated analysis of a single sample is necessary. Overall, the TAP®II device proved challenging to work with during the study, with success rate of only 77% for collecting an adequate sample for analysis. Capillary blood collection was performed under ideal laboratory-controlled conditions by research staff with experience using the TAP® device, so failure rates may be higher if this specific collection device were utilized under real-world conditions, such as those at a workplace accident scene or roadside during a driving investigation.
Conclusion
Δ9-THC concentrations were significantly lower in contemporaneous capillary blood samples than in venous blood samples, but the concentrations of the Δ9-THC metabolites 11-hydroxy-Δ9-THC and 11-carboxy-Δ9-THC were equivalent at all but the first timepoint after smoking. The Δ9-THC concentrations in capillary blood were closely correlated with those in venous blood 140 min after cannabis smoking. Given its portability, usability by non-phlebotomists, and enhanced subject acceptance compared to venipuncture, capillary blood collection by microneedle devices may be a viable option for determining the concentration of Δ9-THC and its metabolites in situations where blood is collected at least 2-hour after cannabis smoking. However, improvements to current capillary blood collection devices, such as the TAP®II device, must be considered. The low success rate of collecting an adequate volume must be addressed. Future investigations should modify the collection methodology and explore the impact of highly perfused capillary collection sites such as the fingertip or lip on the temporal differences in venous and capillary cannabinoid concentrations, as pharmacokinetics may be influenced by cutaneous capillary density and other site-specific factors.
Supplementary Material
Contributor Information
Gregory Dooley, Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80526, United States.
Suneeta Godbole, Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Denver, CO 80045, United States.
Julia Wrobel, Department of Biostatistics and Bioinformatics, Emory University, Atlanta, GA 30322, United States.
Tom Henthorn, Department of Anesthesiology and Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States.
Ashley Brooks-Russell, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States.
Sarah Limbacher, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States.
Michael Kosnett, Colorado School of Public Health, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, United States.
Supplementary data
Supplementary data are available at Journal of Analytical Toxicology online.
Data availability
Data can be made available upon reasonable request to the corresponding author.
References
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Associated Data
Supplementary Materials
Data Availability Statement
Data can be made available upon reasonable request to the corresponding author.