Comparison of Two Immunoassay Screening Methods and a LC-MS/MS in Detecting Traditional and Designer Benzodiazepines in Urine

Sensitive and specific immunoassay screening methods for the detection of benzodiazepines in urine represent an important prerequisite for routine analysis in clinical and forensic toxicology. Moreover, emerging designer benzodiazepines force labs to keep their methodologies updated, in order to evaluate the reliability of the immunochemical techniques. This study aimed at evaluating the sensitivity and specificity of two different immunoassay methods for the detection of benzodiazepines in urine, through a comparison with the results obtained by a newly developed liquid chromatographic tandem mass spectrometric (LC-MS/MS) procedure. A cohort of authentic urine samples (N = 501) were processed, before and after a hydrolysis procedure, through two immunoassays and an LC-MS/MS method. The LC-MS/MS target procedure was optimized for monitoring 25 different molecules, among traditional and designer benzodiazepines, including some metabolites. At least one of the monitored substances was detected in 100 out of the 501 samples. A good specificity was observed for the two immunoassays (>0.99), independently of the cut-offs and the sample hydrolysis. The new kit demonstrated a fairly higher sensitivity, always higher than 0.90; in particular, a high cross-reactivity of the new immunoassay was observed for samples that tested positive for lorazepam and 7-aminoclonazepam. The two immunoassays appeared adequate to monitor not only traditional benzodiazepines but also new designer ones.

Urine Drug Screening in the Era of Designer Benzodiazepines: Comparison of Three Immunoassay Platforms, LC-QTOF-MS, and LC-MS/MS

ABSTRACT This study investigated the presence of designer benzodiazepines in 35 urine specimens obtained from emergency department patients undergoing urine drug screening. All specimens showed apparent false-positive benzodiazepine screening results (i.e., confirmatory testing using a 19-component LC-MS/MS panel showed no prescribed benzodiazepines at detectable levels). The primary aims were to identify the possible presence of designer benzodiazepines, characterize the reactivity of commercially available screening immunoassays with designer benzodiazepines, and evaluate the risk of inappropriately ruling out designer benzodiazepine use when utilizing common urine drug screening and confirmatory tests. Specimens were obtained from emergency departments of a single US Health system. Following clinically ordered drug screening using Abbott ARCHITECT c assays and lab-developed LC-MS/MS confirmatory testing, additional characterization was performed for investigative purposes. Specifically, urine specimens were screened using two additional assays (Roche cobas c502, Siemens Dimension Vista) and LC-QTOF-MS to identify presumptively positive species, including benzodiazepines and non-benzodiazepines. Finally, targeted, qualitative LC-MS/MS was performed to confirm the presence of 12 designer benzodiazepines. Following benzodiazepine detection using the Abbott ARCHITECT, benzodiazepines were subsequently detected in 28/35 and 35/35 urine specimens, respectively, using Siemens and Roche assays. LC-QTOF-MS showed the presumptive presence of at least one non-FDA approved benzodiazepine in 30/35 specimens: flubromazolam (12/35), flualprazolam (11/35), flubromazepam (2/35), clonazolam (4/35), etizolam (9/35), metizolam (5/35), nitrazepam (1/35), and pyrazolam (1/35). Two or three designer benzodiazepines were detected concurrently in 13/35 specimens. Qualitative LC-MS/MS confirmed the presence of at least one designer benzodiazepine or metabolite in 23/35 specimens, with 3 specimens unavailable for confirmatory testing. Urine benzodiazepine screening assays from three manufacturers were cross-reactive with multiple non-US FDA-approved benzodiazepines. Clinical and forensic toxicology laboratories using traditionally designed LC-MS/MS panels may fail to confirm the presence of non-US FDA-approved benzodiazepines detected by screening assays, risking inappropriate interpretation of screening results as false-positives.

Designer Benzodiazepines: A Review of Toxicology and Public Health Risks

The rising use of designer benzodiazepines (DBZD) is a cat-and-mouse game between organized crime and law enforcement. Non-prohibited benzodiazepines are introduced onto the global drug market and scheduled as rapidly as possible by international authorities. In response, DBZD are continuously modified to avoid legal sanctions and drug seizures and generally to increase the abuse potential of the DBZD. This results in an unpredictable fluctuation between the appearance and disappearance of DBZD in the illicit market. Thirty-one DBZD were considered for review after consulting the international early warning database, but only 3-hydroxyphenazepam, adinazolam, clonazolam, etizolam, deschloroetizolam, diclazepam, flualprazolam, flubromazepam, flubromazolam, meclonazepam, phenazepam and pyrazolam had sufficient data to contribute to this scoping review. A total of 49 reports describing 1 drug offense, 2 self-administration studies, 3 outpatient department admissions, 44 emergency department (ED) admissions, 63 driving under the influence of drugs (DUID) and 141 deaths reported between 2008 and 2021 are included in this study. Etizolam, flualprazolam flubromazolam and phenazepam were implicated in the majority of adverse-events, drug offenses and deaths. However, due to a general lack of knowledge of DBZD pharmacokinetics and toxicity, and due to a lack of validated analytical methods, total cases are much likely higher. Between 2019 and April 2020, DBZD were identified in 48% and 83% of postmortem and DUID cases reported to the UNODC, respectively, with flualprazolam, flubromazolam and etizolam as the most frequently detected substances. DBZD toxicology, public health risks and adverse events are reported.

The Development and Validation of a Novel Designer Benzodiazepines Panel by LC–MS-MS

Novel illicit benzodiazepines are among the most active areas of new illicit drug manufacture and use. We describe a method for the detection and quantification of etizolam and its metabolite α-hydroxyetizolam, flubromazolam, clonazolam, diclazepam, delorazepam, bromazepam, flubromazepam, phenazepam, flualprazolam, flunitrazolam, and nitrazolam in human whole blood. After addition of internal standards, samples are buffered and extracted using a liquid–liquid extraction. Analysis is performed using positive-ion electrospray tandem mass spectrometry for detection and quantitation. Calibration ranges were established based on the method performance and differed from compound to compound. Replicates at the lowest calibration point for each compound performed within 5% of CV (Coefficient of Variation). The correlation coefficient was >0.990 for all compounds. Relative standard deviation for all compounds was ≤10% of CV and accuracy was ±10% for both within- and between-run experiments. The maximum average intra- and inter-run imprecision were 5.7%. The maximum average intra- and inter-run imprecision was −8.7%. As part of evaluating the scope for relevancy, samples testing positive in immunoassay but confirmed to be negative in traditional benzodiazepine confirmation method were re-analyzed using this method. The presence of at least one novel benzodiazepine was identified in 70% of these samples. The appearance of these novel “designer” benzodiazepines demonstrates the challenge for toxicology testing and the need for continually updated confirmation methods.