Bioanalytical studies of designer benzodiazepines

The fast appearance of benzodiazepine analogues, referred to as new psychoactive substance (NPS) or designer benzodiazepines, requires the continuous update of detection methods in order to keep up with the latest drugs on the recreational drug market. Moreover, as usually only limited information on toxicity and excretion patterns of these new drugs exists, this needs to be evaluated to report on adverse effects and to determine suitable targets for drug testing.

Urine drug testing usually involves screening using immunoassay followed by confirmation of positive screening results using mass spectrometric (MS) methods. We studied the detectability of designer benzodiazepines in urine using commercial immunoassays and demonstrated that most designer benzodiazepines can be detected by immunoassay. It is thus important to update confirmation methods to include designer benzodiazepines. We also developed a liquid chromatographic–tandem MS (LC–MS/MS) confirmation method for designer benzodiazepines in urine using direct dilution of samples and hydrolysis of conjugates. Subsequently, a further improved screening and confirmation method using LC–high-resolution MS (LC–HRMS(/MS)) was developed. HRMS screening is performed in full scan and is a generic method that can easily include new analytes. LC–HRMS/MS confirmation only requires re-injection of the sample.

Many samples from drug dependent patients with a positive immunoassay screening result for benzodiazepines but not containing prescription medicines detected a designer benzodiazepine instead. Comparable results were obtained for acute intoxication cases from emergency wards in the STRIDA project. In total, 28 designer benzodiazepines were covered by the analytical method and 17 of these were detected in the samples. Classification of a designer benzodiazepine as a narcotic substance generally meant that it was removed from the NPS market and replaced with another novel benzodiazepine. It was further demonstrated that intoxications by designer benzodiazepines might cause central nervous system depression. Studies on metabolic patterns of five designer benzodiazepines using urine samples from confirmed intoxication cases identified suitable analytical targets for urine drug testing, instead of or together with the parent compounds and both with and without hydrolysis of conjugated forms.

In summary, the results demonstrated frequent use of designer benzodiazepines in Sweden and in cases of acute intoxication that they might cause serious adverse effects. This underlines the importance of including designer benzodiazepines and/or metabolites thereof in drug testing. Screening for designer benzodiazepines can be performed by immunoassay or LC–HRMS, and confirmation methods can make use of direct dilution of urine samples followed by hydrolysis and direct injection into LC–MS or LC–HRMS systems.

List of scientific papers

I. Pettersson Bergstrand M, Helander A, Hansson T, Beck O. Detectability of designer benzodiazepines in CEDIA, EMIT II Plus, HEIA, and KIMS II immunochemical screening assays. Drug Test Anal. 2017;9:640-645.
https://doi.org/10.1002/dta.2003

II. Pettersson Bergstrand M, Helander A, Beck O. Development and application of a multi-component LC-MS/MS method for determination of designer benzodiazepines in urine. J Chromatogr B Analyt Technol Biomed Life Sci. 2016;1035:104-10.
https://doi.org/10.1016/j.jchromb.2016.08.047

III. Pettersson Bergstrand M, Beck O, Helander A. Urine analysis of 28 designer benzodiazepines by liquid chromatography–high-resolution mass spectrometry. [Manuscript]

IV. Meyer MR, Bergstrand MP, Helander A, Beck O. Identification of main human urinary metabolites of the designer nitrobenzodiazepines clonazolam, meclonazepam, and nifoxipam by nano-liquid chromatography-high-resolution mass spectrometry for drug testing purposes. Anal Bioanal Chem. 2016;408:3571-91.
https://doi.org/10.1007/s00216-016-9439-6

V. Pettersson Bergstrand M, Meyer MR, Beck O, Helander A. Human urinary metabolic patterns of the designer benzodiazepines flubromazolam and pyrazolam studied by liquid chromatography-high resolution mass spectrometry. Drug Test Anal. 2018;10:496-506.
https://doi.org/10.1002/dta.2243

VI. Bäckberg M, Pettersson Bergstrand M, Beck O, Helander A. Clinical characteristics of new designer benzodiazepines – intoxication cases from the Swedish STRIDA project. [Manuscript]