A Comprehensive Breath Test that Confirms Recent Use of Inhaled Cannabis within the Impairment Window

doi:10.21203/rs.3.rs-821114/v1

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Research Article

A Comprehensive Breath Test that Confirms Recent Use of Inhaled Cannabis within the Impairment Window

https://doi.org/10.21203/rs.3.rs-821114/v1

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published 23 Nov, 2021

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Legalization of cannabis for medicinal and/or recreational use is expanding globally. Although cannabis is being regulated country by country, an accurate recent use test with indisputable results correlated with impairment has yet to be discovered. In the present study, a new approach for determining recent cannabis use within the impairment window after smoking was developed by studying 74 subjects with a mean age of 25 years and average use history of 9 years. Horizontal gaze nystagmus was evaluated along with subject self-assessments of impairment, and blood and breath samples were collected before and after smoking cannabis. Breath and blood pharmaco-kinetic parameters and cannabinoid profiles determined recent use within the impairment window. No subjects were positive for recent use pre-smoking, although all subjects had detectable cannabi-noids in breath samples. We describe an inhaled cannabis recent use test that correlates with impairment and helps protect against wrongful prosecution and workplace discrimination.

Scientific Communication

Legalization of cannabis

Comprehensive Breath Test

Inhaled Cannabis

Impairment Window

Despite several different available testing methods for assessing cannabis use, the ability to effectively establish recent use within the impairment window, generally accepted to be approxi-mately three hours [1, 2], has remained elusive. As a result, inappropriate interpretation of test results by employers, for example, can lead to job applicants not getting hired, or to the wrongful termination of current, non-federal employees who are using cannabis lawfully and responsibly in jurisdictions that allow such use, and where employees are not serving in safety-sensitive positions. As of July 2021, recreational cannabis has been legalized in 19 U.S. states plus Washington, D.C. Some of these states, including Nevada, New Jersey, and New York, have begun to enact laws that ban employment discrimination against current or prospective employees who engage in legal, off-duty recreational cannabis use. Ironically, Colorado, one of the first states to legalize recrea-tional cannabis, provides little legal protection of off-duty cannabis use by employees [3]. At the same time, studies have suggested that the legalization of recreational cannabis has been associated with an increase in fatal traffic collisions [4, 5]. Based on data collected from 2007–2018, a recently published study showed that in U.S. states where recreational cannabis is legal, there has been a 15% increase in fatal collisions and a 16% increase in associated deaths, changes that were sustained beyond the first year following legalization [6]. Clearly, a new type of test that can identify recent cannabis use within the impairment window is needed to help bring an end to cannabis discrimination practices in the workplace while also accurately detecting inappropriate cannabis use, e.g., DUI, to protect public safety.

While it has been known for over 30 years that Δ⁹-tetrahydrocannabinol (Δ⁹-THC) can be detected in exhaled breath [7], only relatively recently has this matrix been explored as a potential means of establishing recent cannabis use within the impairment window [8, 9]. Exhaled breath testing for recent use is predicated on a short period of detection for Δ⁹-THC within the impairment window. A study by Himes et al. suggested that Δ⁹-THC is generally detectable in breath for only about two hours after smoking even in chronic users [8], but more recent studies have shown that Δ⁹-THC remains detectable in breath up to several days since last use [10, 11]. This is a major finding because no meaningful correlation has yet been established between impairment and Δ⁹-THC levels in any matrix tested to date. Because the leading technologies for breath-based testing for recent cannabis use, such as described by Lynch et al. [10] and others [12], rely solely on the detection of Δ⁹-THC, there is a real potential for false positive test results due to the presence of Δ⁹-THC in breath outside of the impairment window.

The complex nature of cannabinoid pharmacokinetics and pharmacodynamics calls for a new approach for effectively determining recent use of cannabis within the impairment window. We hypothesized that incorporating both exhaled breath and blood testing could improve the accuracy of current breath-based testing methods for recent cannabis use, prevent false positive test results, and definitively establish whether a subject is in the impairment window following the use of cannabis through inhalation. We sought to develop a test that incorporates pharmacological changes in Δ⁹-THC and other cannabinoids in breath over time after smoking, which is the most common route of cannabis administration [13]. This approach requires collection of two breath samples separated by a known time interval, which is a critical difference compared to current testing methods that rely on a single sample, as well as a blood sample. The two-sample strategy makes detection of Δ⁹-THC and other cannabinoids possible in their distribution phases, which occur during the first few hours after smoking, and for the evaluation of how cannabinoid levels in breath are changing with time relative to blood. Liquid chromatography high-resolution mass spectrometry (LC-HRMS) bioanalytical methods for the quantification Δ⁹-THC and other cannabinoids in microsamples of whole blood was developed and validated for this purpose [14].

Here we describe the clinical development of a comprehensive breath and blood-based test and its application in determining recent cannabis use and impairment after smoking.

Clinical Study: Subject Demographics

A total of 74 subjects were recruited over a one-year period. There was an approximate 3:1 ratio of males (56) to females (18), and the subjects ranged in age from 21 to 42 years, with an average age of 25.0 ± 4.5 years and a median age of 23 years (see Supplementary Table S1). The subjects reported a mean cannabis use history of 9.0 ± 4.4 years.

Impairment Window Following Cannabis Smoking

The window of impairment following cannabis smoking was evaluated in 74 subjects who self-assessed their impairment level from not impaired to very impaired prior to smoking and at various time points post-smoking using a 10-point scale. Among these subjects, all 74 (100%) reported peak impairment within the first hour after smoking (48 subjects immediately after smoking, 24 subjects at 20 min post-smoking, and two subjects at 60 min post-smoking) (see Supplementary Table S2). The overall window of impairment was found to be approximately three hours after smoking, at which time 68 of 74 subjects (92%) last reported any impairment. There were six subjects (8%) still reporting some degree of impairment outside this window (see Fig. 1). In these six subjects, the percent maximum impairment was an average 23.0 ± 21.7% (range 11.1 to 66.7%). To normalize self-assessed impairment data, the reported impairment levels at each time point for each subject (Supplementary Table S2) were divided by the maximum reported impair-ment level for each subject, with the result expressed as a percentage, as shown in Table 1.

Table 1

Percent maximum self-assessed impairment
	Percent Maximum Self-Assessed Impairment Post-Smoking (minutes)
Subject	Pre-Smoking	0	20	40	60	80	120	140	180	200	240
1	0.0	66.7	100.0	—*	83.3	50.0	16.7	0.0	0.0	0.0	—
2	0.0	33.3	100.0	—	66.7	66.7	0.0	0.0	0.0	0.0	—
3	0.0	66.7	83.3	—	100.0	100.0	83.3	66.7	33.3	0.0	—
4	0.0	100.0	84.6	—	46.2	23.1	7.7	7.7	0.0	0.0	—
5	0.0	60.0	100.0	—	60.0	20.0	0.0	0.0	0.0	0.0	—
6	0.0	100.0	62.5	—	12.5	0.0	0.0	0.0	0.0	0.0	—
7	0.0	100.0	83.3	—	66.7	66.7	50.0	33.3	16.7	0.0	—
8	0.0	66.7	100.0	—	66.7	66.7	33.3	0.0	0.0	0.0	—
9	0.0	100.0	80.0	—	40.0	0.0	0.0	0.0	0.0	0.0	—
10	0.0	100.0	62.5	—	25.0	12.5	0.0	0.0	0.0	0.0	—
11	0.0	100.0	100.0	—	66.7	50.0	33.3	0.0	0.0	0.0	—
12	0.0	100.0	90.0	—	80.0	80.0	60.0	50.0	30.0	20.0	—
13	0.0	100.0	80.0	—	0.0	0.0	0.0	0.0	0.0	0.0	—
14	0.0	100.0	83.3	—	16.7	16.7	16.7	0.0	0.0	0.0	—
15	0.0	100.0	83.3	—	50.0	33.3	0.0	0.0	0.0	0.0	—
16	0.0	100.0	66.7	—	33.3	33.3	0.0	0.0	0.0	0.0	—
17	0.0	75.0	100.0	—	25.0	0.0	0.0	0.0	0.0	0.0	—
18	0.0	87.5	100.0	—	50.0	50.0	37.5	12.5	12.5	0.0	—
19	0.0	66.7	100.0	—	55.6	44.4	22.2	11.1	0.0	0.0	—
20	0.0	20.0	80.0	—	100.0	80.0	60.0	40.0	20.0	0.0	—
21	0.0	100.0	75.0	—	37.5	0.0	0.0	0.0	0.0	0.0	—
22	0.0	100.0	100.0	—	71.4	28.6	0.0	0.0	0.0	0.0	—
23	0.0	100.0	77.8	—	22.2	11.1	0.0	0.0	0.0	0.0	—
24	0.0	100.0	85.7	—	71.4	57.1	42.9	28.6	14.3	0.0	—
25	0.0	100.0	60.0	—	0.0	0.0	0.0	0.0	0.0	0.0	—
26	0.0	75.0	100.0	—	50.0	25.0	0.0	0.0	0.0	0.0	—
27	0.0	100.0	90.0	—	80.0	60.0	50.0	40.0	30.0	15.0	—
28	0.0	60.0	100.0	—	80.0	60.0	20.0	0.0	0.0	0.0	—
29	0.0	100.0	100.0	—	0.0	0.0	0.0	0.0	0.0	0.0	—
30	0.0	100.0	71.4	—	42.9	28.6	14.3	0.0	0.0	0.0	—
31	0.0	100.0	47.1	—	23.5	11.8	0.0	—	0.0	—	—
32	0.0	100.0	100.0	—	33.3	33.3	0.0	—	0.0	—	—
33	0.0	100.0	42.9	—	14.3	0.0	0.0	—	0.0	—	—
34	0.0	100.0	75.0	—	0.0	0.0	0.0	—	0.0	—	—
35	0.0	100.0	68.8	—	43.8	37.5	25.0	—	18.8	—	—
36	0.0	100.0	100.0	—	85.7	71.4	42.9	—	28.6	—	0.0
37	0.0	100.0	66.7	—	33.3	33.3	0.0	—	0.0	—	0.0
38	0.0	100.0	75.0	—	50.0	25.0	12.5	—	0.0	—	0.0
39	0.0	100.0	83.3	—	50.0	16.7	0.0	—	0.0	—	0.0
40	0.0	100.0	100.0	—	75.0	50.0	25.0	—	0.0	—	0.0
41	0.0	66.7	100.0	83.3	16.7	—	0.0	—	0.0	—	—
42	0.0	100.0	90.0	70.0	40.0	—	30.0	—	10.0	—	—
43†	0.0	0.0	0.0	0.0	0.0	—	0.0	—	0.0	—	—
44	0.0	100.0	0.0	0.0	0.0	—	0.0	—	0.0	—	—
45	0.0	100.0	100.0	50.0	0.0	—	0.0	—	0.0	—	—
46	0.0	100.0	60.0	60.0	40.0	—	0.0	—	0.0	—	—
47	0.0	100.0	60.0	30.0	10.0	—	10.0	—	0.0	—	—
48	0.0	100.0	80.0	40.0	20.0	—	0.0	—	0.0	—	—
49	0.0	83.3	100.0	100.0	50.0	—	16.7	—	0.0	—	—
50	0.0	85.7	100.0	42.9	28.6	—	0.0	—	0.0	—	—
51	0.0	100.0	80.0	60.0	40.0	—	20.0	—	0.0	—	—
52	0.0	83.3	100.0	—	—	—	—	—	66.7	66.7	—
53	0.0	100.0	75.0	—	—	—	—	—	25.0	0.0	—
54	0.0	100.0	66.7	—	—	—	—	—	16.7	0.0	—
55	0.0	100.0	83.3	—	—	—	—	—	0.0	0.0	—
56	0.0	100.0	80.0	—	—	—	—	—	0.0	0.0	—
57	0.0	20.0	100.0	—	—	—	—	—	20.0	0.0	—
58	0.0	75.0	100.0	—	—	—	—	—	50.0	0.0	—
59	0.0	100.0	75.0	—	—	—	—	—	37.5	0.0	—
60	0.0	100.0	75.0	—	—	—	—	—	37.5	12.5	—
61	0.0	100.0	70.0	—	—	—	—	—	20.0	0.0	—
62	0.0	100.0	100.0	—	75.0	62.5	—	—	0.0	0.0	—
63	0.0	100.0	77.8	—	55.6	44.4	—	—	22.2	11.1	—
64	0.0	100.0	87.5	—	62.5	37.5	—	—	37.5	0.0	—
65	0.0	100.0	87.5	—	62.5	50.0	—	—	12.5	0.0	—
66	0.0	—	100.0	87.5	87.5	—	—	—	37.5	0.0	—
67	0.0	—	100.0	75.0	50.0	—	—	—	25.0	0.0	—
68	0.0	—	100.0	70.0	40.0	—	—	—	0.0	0.0	—
69	0.0	—	100.0	75.0	62.5	—	—	—	12.5	0.0	—
70	0.0	—	100.0	80.0	70.0	—	—	—	0.0	0.0	—
71	0.0	—	100.0	40.0	0.0	—	—	—	0.0	0.0	—
72	0.0	—	100.0	87.5	87.5	—	—	—	50.0	0.0	—
73	0.0	—	100.0	88.9	77.8	—	—	—	33.3	0.0	—
74	0.0	—	100.0	62.5	50.0	—	—	—	25.0	12.5	—
*Dashes indicate subjects were not sampled at these time points
†Subject failed to complete the self-assessment form

Physical Assessment of Impairment: Horizontal Gaze Nystagmus

Horizontal gaze nystagmus (HGN) was assessed in 44 subjects. Nystagmus, both horizontal and vertical, refers to the involuntary jerking of the eyes as they gaze up or down. Someone experience-ing nystagmus is unaware of its occurrence. Horizontal gaze nystagmus was evaluated prior to smoking cannabis and at various time points up to three hours post-smoking. The results showed that 43 of the 44 subjects (98%) exhibited HGN after smoking cannabis within the three-hour impairment window (Fig. 2A). Within the first 20 min after smoking, 42 of the 44 subjects (95.5%) exhibited HGN (Fig. 2B). These findings corresponded to the self-assessed impairment data, which showed that the average percent maximum impairment peaked within the first 20 min after smoking (Fig. 2C). At three hours post-smoking, the incidence of HGN had fallen to about 23% (10/43 subjects), compared to about 12% prior to smoking (Fig. 2B), which also corresponded well to the self-assessed impairment data showing that the average percent maximum impairment had fallen to approximately 10% three hours after smoking (Fig. 2C).

Baseline Detection of Δ⁹-THC and other Cannabinoids in Exhaled Breath

Out of a total of 34 evaluable subjects, 23 (67.6%) had detectable Δ⁹-THC in their breath at base-line prior to smoking (Table 1), which is consistent with recent reports by Lynch KL et al. [10] and Olla P et al. [11] showing 100% pre-smoking detection rates of D⁹-THC in breath in separate 20-subject and 23-subject studies, respectively. This finding provides further evidence that D⁹-THC cannot be solely relied upon as an indicator of recent cannabis use through inhalation within the impairment window. Other cannabinoids detected in exhaled breath prior to smoking included cannabinol (CBN) in one subject), cannabigerol (CBG) in two subjects, and cannabigerolic acid (CBGA) in four subjects (see Table 2). Notably, none of the 44 subjects were found to have detectable levels of any Δ⁹-THC metabolites in their breath either before or after smoking.

Table 2

Presence of key cannabinoids in exhaled breath before and after smoking
	Percent (%) Positivity*
Cannabinoid Parameters	Baseline (Pre-smoking)	≤ 60 minutes after smoking	> 60 minutes after smoking
D⁹-THC	23/34 (67.6%)	40/40 (100%)	37/40 (92.5%)
CBN	1/34 (2.9%)	37/40 (92.5%)	4/40 (10.0%)
CBC	0/34 (0%)	39/40 (97.5%)	0/40 (0%)
CBG	2/34 (5.9%)	37/40 (92.5%)	1/40 (2.5%)
CBGA	4/34 (11.8%)	18/40 (45.0%)	4/40 (10.0%)
D⁹-THCV	0/34 (0%)	36/40 (90.0%)	0/40 (0%)
*Not all subjects were sampled at all time points.

Detection of Key Cannabinoids in Breath

Table 2 shows the percent positivity of six cannabinoids prior to smoking, within the first hour post-smoking during peak impairment, and more than one hour post-smoking in subjects’ exhaled breath samples. In particular, CBN, cannabichromene (CBC), CBG, and D⁹-tetrahydrocanna-bivarin (D⁹-THCV) all have a much greater incidence in breath during peak impairment compared to pre-smoking. Interestingly, CBC and D⁹-THCV were detected in breath only during the peak impairment window, making these two cannabinoids key indicators of recent cannabis use through inhalation.

Parameters of the Recent Use Breath Test

After analyzing the breath samples from all 44 subjects, 10 pharmacologic parameters were found to be associated with recent cannabis use based on the two-point breath sampling strategy, with additional parameters still being investigated. These included the presence of CBN, CBC, CBG, D⁹-THCV, and CBGA (summarized in Table 2), and short half-lives (< 60 min) for D⁹-THC, CBN, CBC, CBG, and D⁹-THCV (see Table 3). The average Δ⁹-THC half-lives measured from immedi-ately after smoking to 20 min post-smoking (p < 0.0001), and from 20 to 60 min post-smoking (p = 0.0201), were significantly shorter compared to the average Δ⁹-THC half-life measured between 60 and 80 minutes post-smoking (outside of the one-hour peak impairment window post-smoking). Pre-smoking, half-lives were calculable for only D⁹-THC in three subjects. Raw half-life data for each cannabinoid at each time interval are shown in Supplementary Tables S3-S7.

Table 3

Summary of cannabinoid half-lives in breath post-smoking
	Average Half-Life (minutes ± SD)
	Minutes Post-Smoking
Cannabinoid	Pre-Smoking	0–10	0–20	20–40	20–60	40–60	60–80
D⁹-THC	28.8 ± 28.7 (N = 3)	4.4 ± 2.2 (N = 10)	4.2 ± 2.1* (N = 35)	8.1 ± 2.3 (N = 9)	12.8 ± 8.8† (N = 23)	24.4 ± 25.4 (N = 9)	35.0 ± 41.7 (N = 6)
CBN	—‡	3.7 ± 2.1 (N = 11)	3.7 ± 3.0 (N = 20)	—	35.2 (N = 2)	12.8 (N = 1)	—
CBC	—	3.9 ± 2.5 (N = 10)	3.8 ± 2.3 (N = 20)	—	—	—	—
CBG	—	3.6 ± 2.1 (N = 11)	3.7 ± 3.1 (N = 20)	—	—	—	—
D⁹-THCV	—	4.2 ± 3.9 (N = 9)	2.9 ± 0.7 (N = 6)	—	—	—	—
*p < 0.0001 compared to 60–80 min; †p = 0.0201 compared to 60–80 min (two-tailed t-test with Bonferroni’s adjustment for multiple comparisons; a = 0.025).
‡Half-life not calculable.

Breath-Based Test Results

Within the first hour after smoking, which is the period of peak impairment, all 44 subjects (100%) were breath test positive, meaning they all exhibited a short Δ⁹-THC half-life (ranging from 1.0 to 19.1 min), and one or more other indicators of recent use in breath as described above under “Parameters of the Recent Use Breath Test.” Pre-smoking, all 34 subjects sampled were breath test negative, with two subjects exhibiting only short D⁹-THC half-lives. By itself, a short D⁹-THC half-life is insufficient evidence of recent use within the impairment window because D⁹-THC is commonly seen in the breath of non-recent cannabis users. Additional evidence is needed to confirm recent use.

Breath and Blood Test

We hypothesized that the incorporation of both exhaled breath and blood into a comprehensive recent cannabis use test would confirm recent use of inhaled cannabis within the impairment window, improving the accuracy of the two-point breath-based testing method. For this purpose, blood samples were collected from the 44 subjects in addition to breath samples and analyzed for Δ⁹-THC and other cannabinoids. An eleventh recent use parameter that emerged was the breath/blood ratio of Δ⁹-THC, which is a key confirmatory indicator of recent use. This ratio was computed by dividing the Δ⁹-THC peak area ratio to the internal standard (IS) in breath by the corresponding peak area ratio in blood. We found that all of these ratios were < 2 when measured pre-smoking, while all values were > 2 when assessed immediately after smoking. The average breath/blood Δ⁹-THC ratios measured immediately after smoking (p = 0.0015), 20 min post-smoking (p = 0.0108), 60 min post-smoking (p = 0.0107), and 180 min post-smoking (p = 0.0091) remained significantly greater compared to the average pre-smoking ratio (see Fig. 3). In the two subjects who exhibited short D⁹-THC half-lives in breath pre-smoking, their breath/blood D⁹-THC ratios were 0.01 and 0.57, confirming that they had not recently used cannabis within the impairment window.

Breath and Blood Test Results

Within the first hour after smoking, which is the period of peak impairment, all 44 subjects (100%) were breath and blood test positive, meaning they all exhibited a breath/blood Δ⁹-THC ratio ≥ 2, in addition to a short Δ⁹-THC half-life and one or more other indicators of recent use in breath. Overall, using the two-point breath and one-point blood test, 0/34 subjects (0%) tested pre-smoking were positive, indicating no false positive results, and 44/44 subjects (100%) were positive inside the three-hour impairment window. This comprehensive test, which incorporates a breath/blood Δ⁹-THC ratio (Fig. 3) and the presence and half-lives of key cannabinoids in breath (Table 3), definitively established recent cannabis use within the impairment window.

Correlation between Impairment and Recent Use Parameters in Exhaled Breath

All 44 subjects from whom breath samples were collected self-reported peak impairment within the first 20 min after smoking (Table 1), which coincided with the shortest cannabinoid half-lives (Table 3) and peak incidence of horizontal gaze nystagmus (Fig. 2A and 2B).

Feasibility of Back-to-Back Exhaled Breath Sample Collection Strategy

Evidence of recent cannabis use by inhalation dissipates rapidly in breath, and thus allowing 20 min between samplings, which was employed in most of the subjects in this study, may result in loss of valuable data. In nine of the 44 subjects from whom breath samples were collected, a back-to-back sample collection strategy was employed whereby two breath samples were collected in quick succession at 20 and 40 min post-smoking. Approximately 2 min elapsed between the collection of each sample. The very short half-lives observed for Δ⁹-THC, CBN, and other cannabinoids, approximately 6 min or less (see Supplementary Table S8), in exhaled breath would allow for rapid sampling. The pharmacokinetic data from these nine subjects showed that this strategy is feasible. This approach could save a great deal of time and potentially result in more effective testing.

As legalization of medicinal and recreational cannabis continues to expand throughout the U.S and worldwide, so too does the need for an objective means of determining recent cannabis use and impairment, which cannot be established using currently available breath-based or blood-based testing methods. To address this critical unmet need for the benefit of law enforcement, employers, and others for whom determining recent cannabis use and impairment is a necessity, the goal of our study was to devise a comprehensive test based on pharmacological changes in Δ⁹-THC and other cannabinoids that occur with time in exhaled breath and blood, a test that improves the accuracy of breath-based testing by incorporating a confirmatory blood test to prevent false positive results. It was hypothesized that a two-sample testing strategy (collecting two samples separated by a known time interval) could be used to detect cannabinoids in breath during their distribution phases, which occur only during the first few hours after smoking. A confirmatory blood test could be used to compare relative levels of cannabinoids in breath. Higher relative levels of cannabinoids in breath compared to blood would be indicative of very recent use because it is not possible for higher relative levels of cannabinoids to redistribute back into the breath from the blood. Coupled with physical and subject self-assessments of impairment, it was believed that the results of the test could also be tied to recent use within the impairment window.

In the 44 subjects from whom both blood and breath samples were collected, all 44 (100%) tested positive for recent use within the first hour after smoking, which is the time of peak impairment, using the two-point breath and one-point blood test. Pre-smoking, 0% (0/34) of subjects tested positive for recent use, indicating no false positive test results. A positive test result using the two-point breath and one-point blood test indicates that a subject has used cannabis recently through inhalation, i.e., smoking or vaping, and that they are within the three-hour impairment window. Interestingly, approximately 68% of these subjects had detectable levels of Δ⁹-THC in their breath at baseline prior to smoking, in agreement with recent reports by Lynch et al. [10] and Olla et al. [11]. This finding suggests that the mere presence of Δ⁹-THC in breath does not conclusively demonstrate recent use within the impairment window, which could prove to be a major short-coming of the commercial cannabis breathalyzers currently in development.

A potential limitation of the recent use breath test is the possibility for false negative test results. As already pointed out, evidence of recent cannabis use in breath declines rapidly beyond the first hour after smoking, so it is possible that breath samples collected during the second or third hour after cannabis inhalation may no longer exhibit sufficient evidence of recent use, even though the subject may still be impaired. A false negative result obviously favors the test subject, while a false positive result could have potentially serious consequences, including wrongful termination of employment and prosecution. With this test, it is possible to observe short Δ⁹-THC half-lives in breath and breath/blood Δ⁹-THC ratios ≥ 2 when concentrations of Δ⁹-THC are very low, at or near assay limits of detection. In order to avoid false positive test results due to assay variation at very low levels, it is important to ensure that concentrations fall within the validated range of the assay. It may be practical to set a 1.0-ng/mL Δ⁹-THC cutoff level to avoid false positives due to concen-trations near detection limits.

In our study, a wide variation was observed in pre-smoking Δ⁹-THC blood concentrations, which ranged from undetectable to as high as 80 ng/mL, in the absence of impairment, which may be attributable to tolerance and variable usage patterns. After smoking to the desired effect, however, all subjects became impaired, which was correlated with nystagmus. The described two-point breath test was able to accurately detect recent use within the impairment window in subjects with a background of frequent cannabis use. Because of interindividual variation in tolerance, frequency of use, and metabolism among cannabis users, it is possible to observe impairment beyond three hours post-smoking.

As we observed in our study, evidence of recent cannabis use in breath dissipates rapidly. It is thus critical that breath samples are collected as soon as possible following a workplace incident, for example, or once a need for testing arises, in order to prevent the loss of breath evidence. On-site breath collection using a device such as the one employed in this study can easily be performed without specialized training. Likewise, the blood sample can be collected on site using one of the commercially available capillary blood draw devices designed to be used without specialized training. For detecting recent cannabis use by inhalation, a two-point breath test is the most practical application. In the event that a test subject is positive for only a short D⁹-THC half-life in breath, the matching blood sample can optionally be used to confirm recent use if desired. A matching blood sample would always be collected to allow testing for other drugs that can induce impairment, including prescription drugs, and it can be additionally analyzed for cannabinoid content if needed.

While the focus of this test has been on detection of recent inhaled cannabis use, it should be emphasized that the two-point breath and one-point blood test is not limited to just cannabis. The same testing strategy we employed for cannabis in the present study may also prove to be useful for detecting recent use of other impairing drugs such as methamphetamine, phencyclidine, and cocaine that can be administered through vaporization. Exhaled breath testing has already been proven useful for detecting multiple drug types [15, 16], and this test allows the simultaneous testing for cannabis as well as other potentially impairing drugs in both breath and blood. Potential applications include sports medicine, enforcement of workplace drug policy, and law enforcement.

Another limitation of the test as currently designed is that it can detect recent use of cannabis only through inhalation within the impairment window. Further study is needed to detect recent use and impairment following oral consumption of cannabis products. Hypothetically, a similar strategy utilizing two blood samples could be deployed for detecting recent use of orally administered cannabis as well as other orally administered impairing drugs. It is well known that cannabinoid pharmacokinetics differ depending on the route of administration. Because D⁹-THC metabolites cannot be detected in breath, the blood may contain critical information pertaining to recent use, including concentrations of glucuronide metabolites and changes in the ratios of the D⁹-THC metabolites such as 11-hydroxy-D⁹-THC and 11-nor-9-carboxy-D⁹-THC to D⁹-THC and to each other, as previously reported [14, 17].

In conclusion, a new test for recent cannabis use and impairment, based on two-point breath sampling with or without a one-point confirmatory blood test, has been developed that can accurately detect whether a subject has used cannabis through inhalation within the three-hour impairment window, regardless of the potency of the cannabis strain smoked, with no false positive results. The test is based on multiple parameters, including cannabinoid half-lives, which confirm distribution phase kinetics, the presence of key cannabinoids that are observed only after smoking, and a blood test to determine the breath/blood D⁹-THC ratio, which confirms whether the test subject was within the impairment window post-smoking. It is our belief, based on our research, that this test provides an answer to the unmet need of determining recent cannabis use, which in turn protects the public from a safety standpoint by detecting inappropriate use, e.g., DUI, while at the same time protecting responsible cannabis users such as medicinal users from wrongful termination and prosecution. This test may finally help bring an end to cannabis discrimination.

Clinical Study

A total of 74 subjects were recruited to perform a study designed to develop a comprehensive test that confirms recent use of inhaled cannabis within the impairment window. All subjects received financial compensation for their participation. This 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.

Inclusion Criteria

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 supple-ments they are taking.

Cannabis Administration

Each subject was given a single cannabis cigarette and instructed to smoke as much of it as possible within a 10-minute period. Cigarettes containing 500 mg of dried cannabis flower with a Δ⁹-THC content ranging from 8.5–28.4% 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 strains 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 a total of 74 subjects. To establish baseline cannabinoid levels prior to smoking, two capillary blood samples were collected 20 min apart. 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. To establish baseline cannabinoid levels, two breath samples were collected 20 min apart 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 nine of these subjects, back-to-back breath samples were collected at 20 and 40 min post-smoking to evaluate the feasibility of this sampling strategy. 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 approximately 30 L of exhaled breath. Devices were kept sealed in their original packaging until immediately before use to prevent contamination and used according to the manufacturer’s instructions.

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 (not impaired) to 10 (very impaired). 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 [18]. 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.

Analytical Methods

Chemicals and Reagents

Eight of the 10 cannabinoid analytes [Δ⁹-THC, CBN, cannabigerol (CBG), cannabigerolic acid (CBGA), Δ⁹-tetrahydrocannabinolic acid A (Δ⁹-THCA), Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV), 11-OH-D⁹-THC, and 11-nor-9-carboxy-D⁹-THC] and the internal standard (IS; Δ⁹-THC-D₃) were obtained as certified reference materials (CRMs) manufactured by Cerilliant (Round Rock, TX). The 8b,11-diOH-D9-THC metabolite was obtained from ElSohly Laboratories, Inc. (Oxford, MS). 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 analytical method for the quantification of the cannabinoids Δ⁹-THC, CBN, CBC, and Δ⁹-THCV in exhaled breath was used for the analysis of study samples. Additional cannabinoids analyzed included Δ⁹-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. 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.

To extract cannabinoids from the breath collection devices, 2 mL of methanol were aliquoted through each device and filter housing. After adding methanol, approximately 5 min were allowed for all of the solvent to pass through the filter pads. Next, two 2.5-mL aliquots of methanol were passed through the breath devices. Using a 60-mL syringe, approximately 120 cm³ of air was pushed through each device to force all residual methanol through the filter pads. 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. Once evaporation was complete, the samples were allowed to cool to room temperature and reconstituted by adding 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. TraceFinder software performed all required analyses. The chromatographic conditions for the analysis of cannabinoids in exhaled breath were the same as previously described [14].

Analysis of Cannabinoids in Blood

Extraction and analysis of Δ⁹-THC, its major metabolites, and other cannabinoids in whole blood was performed according to a validated method as previously described [14]. 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 borosilicate glass tube and evaporated to dryness under a gentle stream of nitrogen gas 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 [14].

Statistical Methods

Differences between average breath/blood D⁹-THC ratios and cannabinoid half-lives were evaluated using a two-sample equal variance Student’s t-test with a two-tailed distribution and significance level of 0.05. Bonferroni’s adjustment was employed to lessen the likelihood of false positive results, with the significance level being adjusted to a = 0.0125 when comparing the breath/blood D⁹-THC ratios (four tests) and a = 0.0250 when comparing average cannabinoid half-lives (two tests).

Data Availability

All datasets generated and/or analyzed during the present study are either available in the main text and supplementary materials, or can be obtained from the corresponding author on reasonable request.

Acknowledgments

The authors would like to thank Sensabues AB for providing the breath collection devices needed for these studies, and Napa County Deputy Sheriff William MacDonald for his valuable input regarding the physical assessment of drug impairment.

Author Contributions

MWD and CJK conceived and designed the study; MWD, CJK, GTW, and EM acquired, analyzed, and interpreted the data; MWD and GTW drafted the manuscript; MWD, CJK, GTW, and EM reviewed and edited the manuscript. All authors approved the submitted version of the manuscript and agree to be personally accountable for their own contributions.

Competing Interests

MWD, GTW, and CJK are the inventors of the recent cannabis use testing methodology (patents PCT/US2019/028298 and 63/128,547 pending) described in the present work. EM declares no competing interests.

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Competing interest reported. MWD, GTW, and CJK are the inventors of the recent cannabis use testing methodology (patents PCT/US2019/028298 and 63/128,547 pending) described in the present work. EM declares no competing interests.

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A Comprehensive Breath Test that Confirms Recent Use of Inhaled Cannabis within the Impairment Window

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Journal Publication

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Abstract

Figures

Introduction

Results

Clinical Study: Subject Demographics

Impairment Window Following Cannabis Smoking

Physical Assessment of Impairment: Horizontal Gaze Nystagmus

Baseline Detection of Δ9-THC and other Cannabinoids in Exhaled Breath

Detection of Key Cannabinoids in Breath

Parameters of the Recent Use Breath Test

Breath-Based Test Results

Breath and Blood Test

Breath and Blood Test Results

Correlation between Impairment and Recent Use Parameters in Exhaled Breath

Feasibility of Back-to-Back Exhaled Breath Sample Collection Strategy

Discussion

Materials And Methods

Clinical Study

Inclusion Criteria

Cannabis Administration

Blood Draw Schedule

Breath Collection Schedule

Self-Assessment of Impairment

Physical Assessment of Impairment: Horizontal Gaze Nystagmus

Analytical Methods

Chemicals and Reagents

Analysis of Cannabinoids in Exhaled Breath

Analysis of Cannabinoids in Blood

Statistical Methods

Declarations

Data Availability

Acknowledgments

Author Contributions

Competing Interests

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

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Baseline Detection of Δ⁹-THC and other Cannabinoids in Exhaled Breath