Login Register Cart 0
Generic placeholder image

Current Pharmaceutical Design

Editor-in-Chief

ISSN (Print): 1381-6128
ISSN (Online): 1873-4286

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Determination of Traditional and Designer Benzodiazepines in Urine through LC-MS/MS

Author(s): Brian Rossi, Francesca Freni, Claudia Carelli, Matteo Moretti, Dario Galatone, Claudia Vignali and Luca Morini*

Volume 28, Issue 32, 2022

Published on: 14 September, 2022

Page: [2622 - 2638] Pages: 17

DOI: 10.2174/1381612828666220831103224

Price: $65

Abstract

Background: The detection of new designer benzodiazepines in biological fluids and tissues, together with the traditional ones, could represent an important analytical update for laboratories performing clinical and forensic toxicological analysis.

Objective: A liquid chromatography tandem mass spectrometry method (LC-MS/MS) has been developed, fully validated, and applied to a cohort of real urine samples collected from patients under withdrawal treatment and from intoxication cases.

Methods: 100 μL urines were added to a buffer solution containing deuterated internal standards; the samples were then extracted through a liquid/liquid procedure, dried under a nitrogen stream, and reconstituted in mobile phase. The chromatographic separation was performed in reverse phase through a C18 column with gradient elution. Mass spectrometry operated in positive polarization and multiple reaction monitoring mode.

Results: 25 molecules were optimized for instrumental analysis: 9 designer benzodiazepines and 16 traditional compounds (parent drugs and main metabolites). Sensitivity, specificity, linearity, accuracy, imprecision, recovery, matrix effects, and carry-over have been evaluated for all molecules. Only cinazepam did not satisfy all acceptance criteria for validation. 10 among the 50 analyzed samples tested positive for at least one of the monitored molecules. In particular, two different samples collected from the same case provided positive results for flubromazepam, a designer benzodiazepine.

Conclusion: The method was proven to be useful in detecting not only traditional benzodiazepines but also new designer ones. The identification of a New Psychoactive Substance in real samples confirmed that analytical procedures should be updated to include as many substances as possible.

Keywords: Benzodiazepines, NPS, urine, LC-MS/MS, flubromazepam, designer benzodiazepines, forensic toxicology.

« Previous Next »
[1]
Karch SB. Drug Abuse Handbook. 2nd ed. CRC Press BoCa Raton 2007.
[2]
Olsen RW, Yang J, King RG, Dilber A, Stauber GB, Ransom RW. Barbiturate and benzodiazepine modulation of GABA receptor binding and function. Life Sci 1986; 39(21): 1969-76.
[http://dx.doi.org/10.1016/0024-3205(86)90320-6] [PMID: 2431244]
[3]
de Jager AD, Bailey NL. Online extraction LC–MS/MS method for the simultaneous quantitative confirmation of urine drugs of abuse and metabolites: Amphetamines, opiates, cocaine, cannabis, benzodiazepines and methadone. J Chromatogr B Analyt Technol Biomed Life Sci 2011; 879(25): 2642-52.
[http://dx.doi.org/10.1016/j.jchromb.2011.07.029] [PMID: 21839693]
[4]
ElSohly MA, Gul W, Murphy TP, Avula B, Khan IALC. -(TOF) MS analysis of benzodiazepines in urine from alleged victims of drug-facilitated sexual assault. J Anal Toxicol 2007; 31(8): 505-14.
[http://dx.doi.org/10.1093/jat/31.8.505] [PMID: 17988465]
[5]
Jeong YD, Kim MK, Suh SI, In MK, Kim JY, Paeng KJ. Rapid determination of benzodiazepines, zolpidem and their metabolites in urine using direct injection liquid chromatography–tandem mass spectrometry. Forensic Sci Int 2015; 257: 84-92.
[http://dx.doi.org/10.1016/j.forsciint.2015.07.043] [PMID: 26282513]
[6]
Karampela S, Vardakou I, Papoutsis I, et al. Direct urine analysis for the identification and quantification of selected benzodiazepines for toxicology screening. J Chromatogr B Analyt Technol Biomed Life Sci 2012; 902: 42-6.
[http://dx.doi.org/10.1016/j.jchromb.2012.06.012] [PMID: 22790390]
[7]
Maurer H, Pfleger K. Identification and differentiation of benzodiazepines and their metabolites in urine by computerized gas chromatog-raphy—mass spectrometry. J Chromatogr, Biomed Appl 1987; 422: 85-101.
[http://dx.doi.org/10.1016/0378-4347(87)80442-5] [PMID: 3437028]
[8]
Salomone A, Gerace E, Brizio P, Gennaro MC, Vincenti M. A fast liquid chromatography–tandem mass spectrometry method for deter-mining benzodiazepines and analogues in urine. Validation and application to real cases of forensic interest. J Pharm Biomed Anal 2011; 56(3): 582-91.
[http://dx.doi.org/10.1016/j.jpba.2011.06.007] [PMID: 21737221]
[9]
Xiong L, Wang R, Liang C, et al. Determination of co-administrated opioids and benzodiazepines in urine using column-switching solid-phase extraction and liquid chromatography–tandem mass spectrometry. J Chromatogr A 2015; 1395: 99-108.
[http://dx.doi.org/10.1016/j.chroma.2015.03.064] [PMID: 25888098]
[10]
Nakamura M. Analyses of benzodiazepines and their metabolites in various biological matrices by LC-MS(/MS). Biomed Chromatogr 2011; 25(12): 1283-307.
[http://dx.doi.org/10.1002/bmc.1598] [PMID: 21400551]
[11]
Lee JH, Park HN, Kim NS, Park S, Lee YM, Kang H. Development of a specific fragmentation pattern-based quadrupole-Orbitrap™ mass spectrometry method to screen drugs in illicit products. Sci Justice 2020; 60(1): 86-94.
[http://dx.doi.org/10.1016/j.scijus.2019.08.001] [PMID: 31924293]
[12]
Ameline A, Richeval C, Gaulier JM, Raul JS, Kintz P. Detection of the designer benzodiazepine flunitrazolam in urine and preliminary data on its metabolism. Drug Test Anal 2019; 11(2): 223-9.
[http://dx.doi.org/10.1002/dta.2480] [PMID: 30109775]
[13]
Garland JM, Hull JD, Bender CL, Marshall L, Holt AC. Evidence of designer benzodiazepine use in routine healthcare urine drug speci-mens. J Addict Med 2022; 16(3): 354-6.
[PMID: 34172626]
[14]
Huppertz LM, Moosmann B, Auwärter V. Flubromazolam - Basic pharmacokinetic evaluation of a highly potent designer benzodiaze-pine. Drug Test Anal 2018; 10(1): 206-11.
[http://dx.doi.org/10.1002/dta.2203] [PMID: 28378533]
[15]
Moosmann B, Huppertz LM, Hutter M, Buchwald A, Ferlaino S, Auwärter V. Detection and identification of the designer benzodiazepine flubromazepam and preliminary data on its metabolism and pharmacokinetics. J Mass Spectrom 2013; 48(11): 1150-9.
[http://dx.doi.org/10.1002/jms.3279] [PMID: 24259203]
[16]
Moosmann B, Auwärter V. Designer benzodiazepines: Another class of new psychoactive substances. Handb Exp Pharmacol 2018; 252: 383-410.
[http://dx.doi.org/10.1007/164_2018_154] [PMID: 30367253]
[17]
Moosmann B, Bisel P, Auwärter V. Characterization of the designer benzodiazepine diclazepam and preliminary data on its metabolism and pharmacokinetics. Drug Test Anal 2014; 6(7-8): 757-63.
[http://dx.doi.org/10.1002/dta.1628] [PMID: 24604775]
[18]
Moosmann B, Bisel P, Franz F, Huppertz LM, Auwärter V. Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines - an update comprising adinazolam, cloniprazepam, fonazepam, 3-hydroxyphenazepam, metizolam and nitrazolam. J Mass Spectrom 2016; 51(11): 1080-9.
[http://dx.doi.org/10.1002/jms.3840] [PMID: 27535017]
[19]
Moosmann B, Bisel P, Westphal F, et al. Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines: An update comprising flunitrazolam, norflurazepam, and 4′‐chlorodiazepam (Ro5-4864). Drug Test Anal 2019; 11(3): 541-9.
[http://dx.doi.org/10.1002/dta.2561] [PMID: 30578721]
[20]
Moosmann B, King LA, Auwärter V. Designer benzodiazepines: A new challenge. World Psychiatry 2015; 14(2): 248.
[http://dx.doi.org/10.1002/wps.20236] [PMID: 26043347]
[21]
Bäckberg M, Pettersson Bergstrand M, Beck O, Helander A. Occurrence and time course of NPS benzodiazepines in Sweden – results from intoxication cases in the STRIDA project. Clin Toxicol 2019; 57(3): 203-12.
[http://dx.doi.org/10.1080/15563650.2018.1506130] [PMID: 30348014]
[22]
Bergstrand PM, Helander A, Beck O. Development and application of a multi-component LC–MS/MS method for determination of de-signer benzodiazepines in urine. J Chromatogr B Analyt Technol Biomed Life Sci 2016; 1035: 104-10.
[http://dx.doi.org/10.1016/j.jchromb.2016.08.047] [PMID: 27697727]
[23]
Scientific Working Group for Forensic Toxicology. Scientific Working Group for Forensic Toxicology (SWGTOX) standard practices for method validation in forensic toxicology. J Anal Toxicol 2013; 37(7): 452-74.
[http://dx.doi.org/10.1093/jat/bkt054] [PMID: 23934984]
[24]
Morini L, Pozzi F, Groppi A. Stability of benzodiazepines in hair after prolonged exposure to chlorinated water. Forensic Sci Int 2017; 278: 217-20.
[http://dx.doi.org/10.1016/j.forsciint.2017.07.003] [PMID: 28750304]
[25]
Moretti M, Andrello L, Visonà S, et al. Evaluation of benzodiazepines and zolpidem in nails and their stability after prolonged exposure to chlorinated water. J Pharm Biomed Anal 2018; 152: 137-42.
[http://dx.doi.org/10.1016/j.jpba.2018.01.051] [PMID: 29414005]
[26]
Bergstrand PM, Richter LHJ, Maurer HH, Wagmann L, Meyer MR. In vitro glucuronidation of designer benzodiazepines by human UDP-glucuronyltransferases. Drug Test Anal 2019; 11(1): 45-50.
[http://dx.doi.org/10.1002/dta.2463] [PMID: 29996009]

Rights & Permissions Print Cite
Article Metrics
3
© 2024 Bentham Science Publishers | Privacy Policy