Development and application of a multi-component LC–MS/MS method for determination of designer benzodiazepines in urine

doi:10.1016/j.jchromb.2016.08.047

Journal of Chromatography B

Volume 1035, 1 November 2016, Pages 104-110

https://doi.org/10.1016/j.jchromb.2016.08.047 Get rights and content

Highlights

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A multi-component method for detection of designer benzodiazepines in urine has been developed and validated.
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The method includes hydrolysis of glucuronide conjugates and a simple direct injection into the LC–MS/MS system.
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When applied to authentic urine samples, the method detected at least one of the analytes in 40% of the collected samples.

Abstract

New psychoactive substances (NPS) have become an increasing drug problem in the past decade. For detection of NPS, new analytical methods have to be developed, and the methods also have to be updated regularly. This study aimed at developing a multi-component LC–MS/MS method for detection and quantification of 11 NPS of the benzodiazepine sub-class (“designer benzodiazepines”) in urine specimens. The method involves dilution of urine with internal standard and hydrolysis of any glucuronide conjugated forms. Separation of the compounds was achieved on a BEH Phenyl column, followed by MS/MS detection in positive electrospray mode. Method validation was performed following the EMA guideline. The method was applied to study the occurrence of designer benzodiazepines in Sweden in 2014–2015, by analysis of 390 samples retrieved from a routine drug testing laboratory. In 40% of these samples, selected based on a positive immunoassay benzodiazepine screening but a negative MS confirmation for the standard set of prescription benzodiazepines, intake of designer benzodiazepines was revealed. These results stress the importance of using and updating confirmation methods to include the increasing number of designer benzodiazepines appearing on the NPS market.

Introduction

In a joint report from the European Monitoring Center for Drugs and Drug Addiction (EMCDDA) and Europol published in 2016, it was noted that a sub-class of the new psychoactive substances (NPS), benzodiazepines, has become an increasing drug problem [1]. EMCDDA first reported these substances as a separate class in a report from 2014 [2] and they are now generally referred to as “designer benzodiazepines”. The first benzodiazepine reported as an NPS to the EMCDDA was phenazepam in 2007 [3] and the first substance reported that was not already a pharmaceutical product was pyrazolam [4], which was reported in 2012 [5].

Therapeutically, benzodiazepines are prescribed to treat several disorders, such as anxiety, insomnia and epilepsy [6]. However, it is well-known that caution must be taken since they can induce drug dependence [6]. Benzodiazepines are commonly misused together with other psychoactive drugs, e.g. opioids [7]. Patients undergoing substitution therapy for opioid dependence have been reported to co-administer benzodiazepines with methadone or buprenorphine, to achieve a heroin-like effect [7].

Abuse of the designer benzodiazepine etizolam has been reported as an increasing problem in Japan [8], Europe, and the USA [9]. Additionally, designer benzodiazepines have been reported in cases concerning driving under the influence of drugs [10] and in drug-related deaths [11], [12], [13], and classical benzodiazepines are also known to be used in drug related crimes [14]. Use of several designer benzodiazepines has been reported from the Swedish STRIDA project, which is aimed at investigating the occurrence of NPS use in the country and to assess the associated clinical symptoms and health risks [15].

For analysis of classical benzodiazepines, a common approach is to use initial screening by immunochemical assays followed by confirmation using methods based on mass spectrometry (MS). It was recently shown that also several designer benzodiazepines can be detected using commonly used immunoassays [16]. Furthermore, MS, and other techniques such as electron-capture detection, nitrogen-phosphorus detection, and UV detection, coupled to separation techniques such as gas and liquid chromatography (LC), have been used for analysis of designer benzodiazepines [4], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33]. However, most methods were designed to measure only one or a few substances. In routine clinical toxicology, multi-component methods are necessary since a large number of analytes must be screened for in a short time.

The aim of this study was to develop and validate a multi-component LC–MS/MS confirmation method for detection and quantification of 11 designer benzodiazepines in urine; clonazolam, deschloroetizolam, diclazepam, etizolam, flubromazepam, flubromazolam, flutazolam, meclonazepam, nifoxipam, phenazepam, and pyrazolam. In addition, another aim was to apply the method for analysis of urine samples from a routine drug testing laboratory to obtain an indication of their use in Sweden.

Section snippets

Chemicals

Methanol of LC–MS grade and ammonium acetate of ≥98% purity were obtained from Thermo Fisher Scientific (Waltham, MA, USA), formic acid for MS and E. coli ß-glucuronidase from Sigma-Aldrich (St. Louis, MO, USA), and acetonitrile of HPLC gradient grade, acetic acid and sodium hydroxide of pro analysis grade, and 25% ammonia solution from VWR (Radnor, PA, USA). Ultra-pure water (>18 MΩ/cm) was prepared in-house using a Milli-Q water purification system (Millipore, Bedford, MA, USA).

Clonazolam,

Method development

The starting point during the method development was to modify a previously published and routinely used method [36]. However, initial experiments revealed that the chromatography system was not suitable for the benzodiazepine analytes covered by this study. Therefore, a column change from BEH C18 to BEH Phenyl and a gradient modification was necessary to improve chromatographic performance resulting in a final method using two IS with a run time of 3.1 min (Fig. 1) and a total analysis time of

Conclusion

In conclusion, a fast multi-component method for determination of 11 designer benzodiazepines in urine has been successfully developed and validated. When the method was applied for analysis of clinical samples submitted for routine drug testing, the results revealed that 40% of the samples screening positive but later confirmed negative for the classical set of prescription benzodiazepines instead contained designer benzodiazepines. These results indicate a wide-spread use of designer

Ethical permits

The urine samples obtained from routine analysis (No. 00-230) and the STRIDA project (No. 2013/116–31/2) were approved by the regional ethical review board.

Acknowledgement

This work was in part founded by grants from the Public Health Agency of Sweden.

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Development and application of a multi-component LC–MS/MS method for determination of designer benzodiazepines in urine

Highlights

Abstract

Introduction

Section snippets

Chemicals

Method development

Conclusion

Ethical permits

Acknowledgement

Drug Alcohol Depend.

Forensic Sci. Int.

Forensic Sci. Int.

Forensic Sci. Int.

Forensic Sci. Int.

Forensic Sci. Int.

J. Chromatogr.

J. Chromatogr. B

J. Chromatogr. B

Anal. Chim. Acta

Forensic Sci. Int.

J. Chromatogr. B

J. Chromatogr. B

J. Pharm. Sci.

Forensic Sci. Int.

EU Drug Markets Report: In-Depth Analysis

Characterization of the designer benzodiazepine pyrazolam and its detectability in human serum and urine

Forensic Toxicol.

Rang and Dalés Pharmacology