Develop a method for the analysis of phenazepam, etizolam, and their metabolites in blood and urine; provide epidemiology findings for samples from autopsy and urine-drug-testing cases in Taiwan during the 2019–2021 period.

Now widely abused designer benzodiazepines (DBZDs), phenazepam and etizolam are structural analogues of legal BZDs that are used for the treatment of insomnia, anxiety, muscle spasm, and epilepsy. Illegal uses of phenazepam (a long-acting 1,4-benzodiazepine derivative) and etizolam (a short-acting thienodiazepine derivative) were first reported in 2007 and 2011. An effective UHPLC-MS/MS based methodology was developed for the quantitation of the parent drugs (etizolam, phenazepam)—commonly occurred in low dose (0.5–3mg)—and their metabolites (α-hydroxyetizolam, 3-hydroxyphenazepam) in blood and urine samples at low concentration levels. Epidemiological data of specimens from 6 autopsy and 197 urine-drug-testing cases in Taiwan (during the 2019–2021 period) are reported.

Blood or urine (1mL) samples were extracted under alkaline condition. Extracts were evaporated and reconstituted for injection onto the UHPLC-MS/MS system. Case urine specimens were first hydrolyzed by an enzymatic method prior to the extraction step. With the inclusion of 5- and 50-ng/mL quality controls in all analytical batches, deuterated analogues of the analytes were used as internal standards (ISTD) for the quantitations of phenazepam and etizolam, while α-hydroxytriazolam-d4/lorazepam-d4 were for α-hydroxyetizolam/3-hydroxyphenazepam. The mobile phase included 0.1% formic acid (v/v) in water (A) and methanol (B). Mass spectrometric analysis was performed in positive-ion multiple reaction monitoring (MRM) mode.

Drug-free blood and urine samples (fortified with 10–200ng/mL of the analytes) were used for method validation to achieve the following analytical parameters: (a) average extraction recovery (n=5) was higher than 90%; (b) no matrix effect was observed—was apparently compensated for by respective deuterated ISTDs, if any; (c) intra-/inter-day precision (%CV) and accuracy ranges for blood were 0.56–9.27%/0.94–16.2% and 94–110%/92–109%, while the corresponding ranges for urine were 0.86–6.53%/0.87–13.8% and 86–107%/91–105%; and (d) limits of detection/lower limits of quantitation for the parent drugs and their metabolites were 0.25–5.0/5.0–10ng/mL for urine and 0.25–0.50/1.0–5.0ng/mL for blood.

Among the 6 autopsy cases (4 male and 2 female; all under 40-year old) examined during this period, etizolam (together with methyl-α-pyrrolidinohexiophenone) were identified in only 1 death. For the remaining 5 cases, the range, mean±std, and median of postmortem blood concentrations of phenazepam were 0.020–0.43μg/mL, 0.13±0.17μg/mL, and 0.065μg/mL, respectively. Multiple drugs (such as ketamine, p-methoxymethamphetamine, p-methoxyamphetamine, amphetamines, synthetic cathinones, and nimetazepam) were found in these case specimens, often with levels well over their respective lethal doses. Thus, phenazepam and etizolam were probably not the cause of death. The following statistics were found for the 197 urine-drug-testing cases: (a) etizolam/phenazepam were identified in 141/56 specimens; and (b) the range, mean±std, and median of etizolam/phenazepam found in positive specimens were: 0.014–0.54/0.0050–0.056μg/mL, 0.084±0.14/0.014±0.011μg/mL, and 0.046/0.012μg/mL, respectively. Drugs that are popular in Taiwan (such as ketamine, mephedrone, and metabolites of nimetazepam) were commonly found in these specimens.

This relatively simple protocol was found effective and reliable for routine quantitations of etizolam, phenazepam and their metabolites in blood and urine and successfully applied to the analysis of postmortem and antemortem specimens from forensic cases. Epidemiological data indicated: (a) high positive rates for etizolam/phenazepam in drug-testing specimens (141/56 out of 197); and (b) these drugs were unlikely the cause of death for the 6 autopsy cases hereby reported.