Etizolam Blood Concentrations in 191 Forensic Cases in Ontario, Canada (2019–2020)

Abstract

Although not used clinically in North America, etizolam has been identified in forensic samples as an illicit ‘designer’ benzodiazepine. As a central nervous system depressant, analysis for etizolam has probative value in both death investigations and forensic cases where incapacitation or human psychomotor performance is relevant. This report examines toxicological findings and demographic data in a series of authentic forensic cases analyzed between November 2019 and December 2020 in which etizolam was quantified by liquid chromatography-tandem mass spectrometry analysis. Blood concentrations were determined in 191 individuals aged 1–75 years. In living individuals (i.e., impaired driving and sexual assaults), etizolam concentrations ranged from <5 to 767 ng/mL which overlapped with the range of <5 to 260 ng/mL reported in death investigations. In all but one case, other drugs were detected in combination with etizolam. Fentanyl was the most common co-occurring drug and was present in 164 cases (86%). Additional case details are provided for cases of forensic interest: two deaths involving children <3 years of age, two deaths involving body packing and an individual arrested for drug-impaired driving with, to our knowledge, the highest reported etizolam concentration to date.

Introduction

Etizolam is a benzodiazepine analog licensed for use in Japan, Italy and India for the treatment of mood and sleep disorders. When used clinically, it is typically administered in daily doses of 1.0 mg taken in two divided doses (1, 2). Etizolam has been shown to produce both anxiolytic and antidepressant effects and is thought to be well-tolerated with daytime drowsiness being the most reported side effect by patients (1–3). Still, as a central nervous system (CNS) depressant, etizolam has the potential to produce effects such as slurred speech, ataxia, sedation, euphoria, and psychomotor and cognitive impairment. As with other benzodiazepines, the likelihood of adverse effects following administration of etizolam would be greatest for individuals who are naïve to the drug or individuals who have increased their dosage thereby exceeding their level of tolerance. The route of administration and polydrug use must also be considered when assessing potential effects. When benzodiazepines are combined with other CNS depressants such as fentanyl and ethanol, the likelihood of sedation, motor incoordination and respiratory depression increase.

Few studies have examined the effects of etizolam on human performance. In two placebo-controlled studies in which therapeutic doses of etizolam were administered, no significant differences were found in tests of attention, short-term and working memory, psychomotor coordination or speed in decision-making (4, 5). The effects of etizolam following high-dose administration have not been published; however, some concern has been raised with regard to high-dose dependence (6). For example, in a retrospective review of patients admitted to an addiction medicine unit in Italy for benzodiazepine dependence, 11 patients were taking daily dosages of etizolam ranging from 5 to 100 mg (median: 15 mg) (6). These patients were prescribed the drug for either anxiety and/or a sleep disorder; however, these dosages far exceed the recommended maximum daily dose. One patient, taking 15 mg/day etizolam, underwent a neuropsychological evaluation, and decrements were reported in both memory and executive functioning (6).

Data on blood concentrations following therapeutic administration of etizolam are also limited. In one study, healthy young men received 0.5 mg of etizolam twice daily and an average peak plasma concentration of 9.3 ng/mL was determined (7). Meanwhile, multiple blood samples collected between 9.2 and 25.5 h after 1.0 mg chronic daily dosing in a 44-year-old woman produced serum concentrations ranging from 0.56 to 12.21 ng/mL (8). In contrast, blood concentrations measured in forensic cases are significantly greater (9–12). For example, Gevorkyan et al. (13) reported blood concentrations of etizolam ranging from 6.6 to 187 ng/mL (mean: 42 ng/mL) in seven peripheral blood samples from medical examiner cases. Heide (14) and Rohrig (15) reported concentration ranges for living individuals (e.g., suspected impaired driving) of 15–300 ng/mL and 40–330 ng/mL, respectively. These papers demonstrate that when encountered illicitly, etizolam is likely to be present at significantly greater blood concentrations than would be expected if the drug were administered therapeutically. The identification of a blood concentration >300 ng/mL in an ambulatory living individual also suggests that there may be overlap between blood concentrations that contribute to mixed drug intoxication deaths and those that are incidental. Based on the potential effects of etizolam and the increasing prevalence of its use, whether intentionally or as an adulterant, this drug may be toxicologically relevant in many types of forensic cases.

The Centre of Forensic Sciences (CFS) provides toxicology testing in both criminal and noncriminal investigations in the province of Ontario (population of ∼14.5 million people). Because this laboratory performs postmortem toxicology as well as analysis of antemortem samples collected in investigations of impaired driving and sexual assault, it is positioned to provide an overview of etizolam concentrations in most types of forensic casework. This report examines the toxicological findings and demographic data in a series of etizolam-positive cases between 13 November 2019 and 13 December 2020. Concentrations in postmortem blood, antemortem blood and antemortem plasma/serum are discussed in relation to various case types and specific case histories. The prevalence of concurrently detected drugs is emphasized.

Materials and Methods

Case selection

Between 13 November 2019 and 13 December 2020, a total of 10,770 cases were submitted to the CFS for toxicology analysis. Of those cases, etizolam was identified in blood, urine, serum or plasma (by at least one liquid chromatography--tandem mass spectrometry (LC–MS-MS) method and/or an LC–quadrupole time-of-flight-mass spectrometry (QTOF-MS) screen method) 1,231 times. This laboratory only quantifies drugs/metabolites in blood, serum and plasma collected from an intact vessel. Furthermore, the decision to quantify a compound is dependent upon case history, client request and type of investigation. As a result, etizolam was quantified in 16% of the cases in which it was identified (n = 191 cases). These 191 cases were included in this dataset and additional demographic information, case history and other toxicology results were compiled. A variety of case types comprised the dataset including criminal investigations involving living individuals (i.e., sexual assault and suspected impaired driving) and both criminal (homicide) and noncriminal (sudden death) investigations. Legal outcomes were not evaluated in criminal cases, and with rare exception, the cause and/or manner of death were not available in death investigations.

In death investigations with postmortem samples, quantitative analysis was performed on femoral blood except for one case in which femoral blood was not available and heart blood was analyzed. Postmortem blood samples were collected in tubes containing preservative (sodium fluoride) and anticoagulant (sodium citrate). In 13 death investigations, analysis was performed on antemortem samples collected at hospital prior to the death. This included 10 blood samples and 3 plasma/serum samples. In impaired driving and sexual assault cases, where specimens were collected from living individuals, analysis was performed on either whole blood or plasma/serum. All specimens were stored at 4°C prior to analysis.

Comprehensive drug testing was performed in all cases, comprising LC–QTOF-MS screen and at least two different multi-target LC–MS-MS quantitative methods (which together identify and quantify 103 compounds); however, not all cases were subject to the same testing. For example, drug-impaired driving cases were not routinely tested for ethanol and postmortem cases were not routinely tested for cannabinoids (except for homicides, fatal automobile collisions and death investigations subject to a coroner’s inquest).

Analytical methods

In all cases, initial etizolam identification was by a previously validated LC–QTOF-MS screening method (Waters LC–QTOF-MS Forensic Toxicology Screening Application with MS^E technology and a Waters Xevo G2-S QTOF). Quantitation of etizolam was by a multi-target LC–MS-MS quantitative method. This method was previously validated and targets 49 drugs/metabolites with the use of 47 deuterated internal standards; however, only information for etizolam is described here. Etizolam and etizolam-d₃ were obtained from Cerilliant (Round Rock, TX). Briefly, 25 µL of the internal standard (etizolam-d₃) working solution (200 ng/mL) was added to 250 µL of case sample (whole blood, plasma or serum), matrix-matched calibrators and controls to a final matrix concentration of 20 ng/mL. Six calibrators (5, 10, 20, 40, 80 and 160 ng/mL) were prepared and fitted to a quadratic curve with 1/x weighting. The limit of detection (LOD) for the method was 2.5 ng/mL in matrix, and the quantitative range was 5–160 ng/mL. Quality control samples were prepared at 10 ng/mL (low positive control) and 120 ng/mL (high positive control).

Samples were precipitated with 0.75 mL of acetonitrile and centrifuged at 4,000 rpm for 20 min. The supernatant was decanted into 1-mL Phenomenex Phree™ phospholipid removal tubes (P/N 8BS 133-TAK) and centrifuged at 1,500× g (approximately 2,700 rpm) for 8 min at 10°C. Extracts were reconstituted in 1,000 µL of mobile phase (90:10 v/v 10 mM ammonium formate in 0.2% formic acid:0.2% formic acid in acetonitrile).

Extracts were analyzed using a Waters ACQUITY ultra performance liquid chromatography system paired with a Sciex QTRAP 5500 MS-MS or Sciex QTRAP 6500 MS-MS operated in positive mode. Data acquisition was made in multiple reaction monitoring (MRM) mode: MRM1 and MRM2 were m/z transitions 343.0/314.3 and 343.0/206.2 for etizolam and for etizolam-d₃ the m/z transition was 345.9/138.1. Results were reported semi-quantitatively as <5.0 ng/mL where etizolam was detected above the LOD but below the lowest calibrator. For the purpose of this manuscript, the midpoint (3.75 ng/mL) between the LOD and the lowest calibrator was substituted as an estimate of the true concentration. Results obtained above the calibration range of the method were re-extracted using a smaller aliquot of case sample made up to the required volume with drug-free matrix (one case was reported as >160 ng/mL). In some cases, additional drugs were detected by a single LC–QTOF-MS screen but not confirmed or quantified by a second analysis. These unconfirmed findings were included as ‘detected’ drugs for the purposes of tallying the number of concurrent drug findings in each case.

Results and Discussion

Etizolam was first identified by this forensic laboratory in a urine sample submitted in a 2015 death investigation case. A method to quantify the drug was validated in 2016, and its presence in blood and urine samples increased noticeably from that point. Over the time of interest, etizolam was quantified in blood, serum or plasma collected from 191 individuals ranging in age from 1 to 75 years (mean ± SD = 35 ± 12 years); most individuals were male (n = 148) (Table I).

Table I.

Demographic Data for All Cases in Which Etizolam was Quantified (13 November 2019 –13 December 2020)

	Age (years)
	Mean	Median	Range	n
Males	35.5	34.5	1–75	148
Females	32.6	32.0	15–54	43

A summary of etizolam concentrations according to sample type (antemortem vs. postmortem; serum/plasma vs. blood) and case type (i.e., impaired driving, sexual assault and death investigation) is presented in Table II. There was large overlap in concentrations observed in living drivers and in death investigations. Both groups included a small number of cases with etizolam concentrations >160 ng/mL, but the data were skewed toward lower concentrations (Figure 1). For example, 50% of impaired drivers (n = 87) had etizolam concentrations <14 ng/mL and 75% of all death investigations (n = 102) had concentrations <18 ng/mL. Although there is limited published information describing etizolam concentrations following clinical use of the drug, concentrations up to 15 ng/mL in plasma/serum have been associated with a therapeutic range (7, 8). Since most of the concentrations in this case series were measured in whole blood, it should be noted that one limitation when comparing etizolam concentrations from clinical studies to concentrations observed in forensic cases is the potential for relative differences in drug concentration in plasma/serum compared to whole blood. There are no published studies providing a blood-to-plasma ratio for etizolam; however, if etizolam behaves similarly to benzodiazepines such as alprazolam, it could preferentially bind to plasma proteins resulting in whole blood concentrations which are lower than plasma/serum concentrations (16). This could influence the interpretation of concentrations detected in antemortem whole blood of impaired drivers as well as concentrations detected in postmortem death investigations.

Table II.

Etizolam Blood Concentrations (ng/mL) Categorized by Sample Type and Case Category (13 November 2019 –13 December 2020)^*

	Antemortem serum/plasma		Antemortem blood			Postmortem femoral blooda
	Death investigation	Impaired driving	Death investigation	Impaired driving	Sexual assault	Traumatic death	Other
Mean	13.0	16.0	25.2	35.9	12.4	48.5	18.5
Median	6.5	17.2	21.8	13.8	13.0	17.1	7.7
Range	<5–29	8.6–21	<5–60	<5–767	7.9–16	5.7–154	<5–260
n	3	4	10	82b	4	4	85

^*Antemortem samples for death investigations were collected in hospital prior to death.

^aOnly one postmortem blood sample was not femoral blood—a heart blood sample with an etizolam concentration of <5 ng/mL.

^bOne case excluded from statistical analysis because the concentration of etizolam was reported as >160 ng/mL.

Figure 1.

Histogram plots of etizolam concentrations (ng/mL) detected in cases of impaired driving (A) and death investigations (B). Data include blood, plasma and serum samples.

The widest range of etizolam results was observed in suspected impaired drivers where whole blood concentrations ranged from <5 to 767 ng/mL (Table II). The upper limit of this range, a blood etizolam concentration of 767 ng/mL, represents the highest concentration in a living individual in the literature to date and demonstrates that high concentrations of this drug may be tolerated by some users. In this case, etizolam was measured in a whole blood sample collected from an individual stopped by police for erratic driving. Although noted by law enforcement to appear stuporous, have slurred, labored speech and have difficulty walking, the driver was conscious at the time of arrest. This case was one of the few examples in this dataset (n = 27) in which etizolam was detected without concurrent fentanyl; however, several other drugs were detected. Methylenedioxymethamphetamine (210 ng/mL), methylenedioxyamphetamine (21 ng/mL), cocaine (13 ng/mL), benzoylecgonine (340 ng/mL) and amphetamine (360 ng/mL) were present. Phenacetin was identified by LC–QTOF-MS screen but was not confirmed. To our knowledge, the highest previously published concentration of etizolam in a living individual was also a driving impairment case, with an etizolam concentration of 330 ng/mL (15).

Evidence to support the potential for etizolam to cause psychomotor or cognitive decrements comes from case studies on impaired drivers or investigations of other criminal offenses. For example, three living individuals in which etizolam was detected as the only potentially impairing drug in plasma were determined to be impaired by a physician following clinical testing (14). The concentrations of etizolam in these cases ranged from 110 to 210 ng/mL, which is considerably greater than concentrations detected in most of the impaired driving cases in this study, although not dissimilar from the four cases that represent the upper range in this dataset.

Etizolam was quantified in 89 postmortem blood samples with a range of <5 to 260 ng/mL (Table II). Most of these cases were submitted as suspected drug overdoses including the highest case in which 260 ng/mL of etizolam was detected (fentanyl (4.2 ng/mL), furanyl UF-17 (a non-fentanyl, opioid-related drug), and three prescription drugs at therapeutic concentrations were also present). Four of the postmortem cases were traumatic deaths (homicide (n = 2), fatal automobile collision (n = 1) traumatic suicide (n = 1)). The highest etizolam concentration measured among the cases of traumatic death was 154 ng/mL, in a decedent who had been stabbed.

There were two postmortem cases in which the decedent was a child <3 years of age. In the first case, a 30-month-old male was found deceased. Adults present in the home had reported using heroin. Drug paraphernalia seized from the residence tested positive for fentanyl and benzodiazepine(s). The femoral blood concentration of etizolam was 7.7 ng/mL, with fentanyl (18 ng/mL) and amphetamine (290 ng/mL) also detected. Heroin, 6-acetylmorphine and morphine were not detected. The cause of death was determined to be fentanyl and etizolam intoxication. In the second case, the decedent was an 11-month-old male discovered without vital signs. Drug paraphernalia was located at the scene including a purple substance initially believed to be heroin. Femoral blood was not available, and analysis was conducted in heart blood. Heroin, 6-acetylmorphine and morphine were not detected; however, fentanyl and etizolam were present at 7.6 ng/mL and <5 ng/mL, respectively. Traces of benzoylecgonine (<12.5 ng/mL) and methamphetamine (<12.5 ng/mL) were also detected. The cause of death was determined to be fentanyl intoxication.

There were two individuals for whom both postmortem blood and antemortem blood were available for analysis (Table III). The time between collection of antemortem blood at hospital and death of these individuals was 6.6 h for Case A and 35 h for Case B. Although these cases were not related, they had similar case histories. Both decedents were taken to hospital for medical treatment following apparent overdose but later died. At autopsy, ruptured ‘body packs’ suspected to contain drug(s) were in their gastrointestinal tracts. Given the nature of these cases, there are no conclusions that can be drawn about the susceptibility of etizolam to postmortem redistribution.

Table III.

Toxicology Results for Two Death Investigations Where Drug Packages Were Discovered in the Gastrointestinal System of the Decedents at Autopsy

	Case A (Male, 43 years)		Case B (Male, 26 years)
	Antemortem blood (ng/mL)	Femoral blood (ng/mL)	Antemortem blood (ng/mL)	Femoral blood (ng/mL)
Etizolam	14	8.8	34	180
Fentanyl	3.2	15	7.6	>800
Other drugs	Methadone: 60 Carbamazepine: 1,200 Flualprazolama Acetaminophen: 910 Diphenhydramine: nd Naloxone: nd	Methadone: 280 Carbamazepinea Flualprazolama Acetaminophena Diphenhydramine: traces Naloxone: 276	Diphenhydramine: nd Flualprazolam: nd Naloxone: nd THC: 2.6 Carboxy-THC: 57 Hydroxy-THC: det Valproic acid: 20 mg/L Bisoprolola	Diphenhydramine: 150 Flualprazolam: det Naloxone: traces THCb: 1.6 Carboxy-THC: det Hydroxy-THC: det Valproic acid: 29 mg/L Bisoprolol: det

nd = not detected; det = detected, identified and confirmed but not quantified.

^aIdentified but not confirmed or quantified.

^bQuantitation of THC and metabolites was in heart blood.

In this case series, there was only one case in which etizolam was the only drug detected—a suspected impaired driver with 14 ng/mL etizolam measured in serum. At the time of the incident, the driver was noted to be ‘in and out of consciousness’ and sweating. No odor of alcohol was noted on their breath. Although comprehensive drug testing, including LC–QTOF-MS screening and confirmatory LC–MS-MS analysis, was performed in this case, limited sample volume precluded analyses for ethanol, cannabinoids and gamma-hydroxybutyrate (GHB); thus, the presence of these drugs cannot be excluded. Aside from this case, every other individual had least one additional drug detected in their blood.

When parent compounds and metabolites were counted as one drug, and adulterants such as phenacetin, levamisole and lidocaine were included, the median number of other compounds detected in each case was 4 (range: 0–13). The 10 most frequently detected drugs were fentanyl (n = 164), cocaine (n = 66)/benzoylecgonine (n = 98), methamphetamine (n = 67)/amphetamine (n = 52), methadone (n = 51), flualprazolam (n = 47), tetrahydrocannabinol (THC) and/or hydroxy-THC (n = 33)/carboxy-THC (n = 42), naloxone (n = 27), cocaethylene (n = 24), ethanol (n = 19) and flubromazolam (n = 15). The relative frequency of detection for different case types is provided in Figure 2. Deschloroetizolam was detected in one death investigation. Although all cases received comprehensive toxicology testing, including general drug screening by LC–QTOF-MS screen, not all cases were subjected to the same analyses and not all substances were tested for. Accordingly, the data here may be an underestimation of the true number of other substances present with etizolam.

Figure 2.

Frequency of other drugs detected with etizolam in 191 forensic cases in Ontario, Canada.

The high incidence of cases positive for both fentanyl and etizolam (86%) aligns with information that in Ontario benzodiazepines are often present in the illicit fentanyl supply. Testing of drugs and drug paraphernalia by harm reduction initiatives supports this otherwise anecdotal information. For example, a drug-checking service in the city of Toronto operating between October 2019 and March 2020 detected benzodiazepines or benzodiazepine-related drugs in 36% of the 253 samples submitted as ‘expected fentanyl’ (17). Etizolam and flualprazolam were the most frequently detected benzodiazepine in 82 and 36 samples, respectively (17). Notwithstanding the substantial proportion of cases where both fentanyl and etizolam were present, there were 26 cases in which etizolam was present without concurrent fentanyl. The etizolam concentrations in some of these cases were sufficiently high to provide evidence that etizolam can be encountered as a drug on its own in addition to its role as an additive to illicit fentanyl. For example, in one case of impaired driving, an etizolam concentration of 232 ng/mL was measured in a whole blood sample. Fentanyl was not detected, and the only other drugs present were THC (<1 ng/mL) and carboxy-THC (detected qualitatively). This individual had been stopped by police for erratic driving behavior, including striking a median and driving into oncoming traffic. Observations by law enforcement were slurred speech, unsteady balance and ‘wobbly’ legs. Cannabis and related paraphernalia were present in the vehicle. This blood etizolam concentration, in a case where only THC was otherwise detected, suggests etizolam was administered on its own. The availability of etizolam as an illicit drug (as opposed to an adulterant/additive to another drug) aligns with its description as a ‘designer benzodiazepine’ (12). In other published case series, polypharmacy is common and the cause of death in postmortem cases is frequently attributed to multi-drug toxicity (11); however, the strong association between fentanyl and etizolam observed in our data is unique. Although Gevorkyan et al. (13) found concurrent etizolam and fentanyl in 6 of the 10 opioid-related deaths they published, most published case series did not observe fentanyl among etizolam-positive cases (10–12, 14, 15). In 24 postmortem etizolam cases reported by Hilkin et al. (11), pregabalin was the most commonly co-detected drug (64%) and morphine was the most frequently co-detected opioid (54%). In two impaired driving cases, the only other drugs detected besides etizolam were low concentrations of methamphetamine and/or amphetamine, while a third impaired driving case involved only etizolam and THC (15).

Conclusions

With limited prescription use of etizolam worldwide, there is also limited research on the pharmacokinetic and pharmacodynamic profile of etizolam, particularly when taken at doses >1 mg. These missing data limit the ability of a forensic toxicologist to interpret etizolam concentrations in forensic casework. Given the potential for designer benzodiazepines to be of probative value in all types of forensic cases, a review of etizolam cases in Ontario, Canada, was undertaken with a goal to expand the available literature regarding blood concentrations. The resulting data provide etizolam concentrations ranging from <5 to 767 ng/mL in 191 authentic forensic cases of varying etiology. Concentrations in living individuals overlapped with postmortem concentrations, with most blood concentrations approximating a therapeutic range of ≤15 ng/mL. In all but one case, additional drugs were detected with fentanyl the most frequently co-detected drug (86% of individuals). Although these data expand the existing literature regarding etizolam concentrations in forensic cases, gaps remain. For example, there are no studies that comment on postmortem distribution of etizolam and there is limited literature on the nature and extent of psychomotor impairment by this drug. Given the high concentrations of etizolam encountered in suspected cases of impaired driving, this information is particularly relevant. The forensic community would benefit if future research was directed to these topics.

Data availability

The data underlying this article are available in the article and in its online supplementary material.