MSACL 2024 Abstract

Unmasking Designer Benzodiazepines: Comprehensive Analysis Reveals Evasion from Targeted Detection Methods in Substance Abuse Users

Raymond T. Suhandynata(1,2), Melissa A. Hoffman(3), Alec Saitman(4), Robert L. Fitzgerald(1), Amadeo Pesce(1,5)
(1) Department of Pathology, University of California San Diego Health, San Diego, CA, 92121 (2) Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, 92093 (3) Vividion Therapeutics, San Diego, CA, 92121 (4) LabCorp West Division, Portland, OR, 97213 (5) Precision Diagnostics, San Diego, CA, 92121

Raymond Suhandynata, PhD DABCC (Presenter) University of California, San Diego

Presenter Bio: Dr. Suhandynata is an Assistant Professor at the University of California San Diego with appointments in the Skaggs School of Pharmacy and Department of Pathology. He serves as the Associate Laboratory Director for the CMCR reference laboratory and the Associate Director of the UCSD ComACC clinical chemistry fellowship. He completed his Clinical Chemistry fellowship training at the UC San Diego Center for Advanced Laboratory Medicine, under the direction of Dr. Robert Fitzgerald. He has extensive experience with applications of mass spectrometry in research, pre-clinical, and clinical laboratories. Areas of interest include phospho-proteomics to identify novel kinase targets by LC-MS/MS, SUMO proteomics to identify cellular signals involved in chromosome segregation, utilization of MALDI-TOF MS in to identify antibiotic resistant bacteria in the clinical specimens, and development of targeted LC-MRM/PRM assays for small molecules and peptides. Addtionally, he as made significant contributions during the COVID-19 pandemic, validating several COVID-19 serology LDTs at UCSD Health.

Relevant Financial Disclosures (within past 24 months, reported on Apr 22, 2023) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction:
Designer benzodiazepines belong to the synthetic depressant class designer drugs and pose a significant health risk to substance users. Standard practice for drugs of abuse testing in clinical laboratories typically rely on targeted LC-MS/MS analysis of urine specimens, predominantly focusing on commonly prescribed benzodiazepines and their metabolites, omitting designer benzodiazepines. These limitations have prompted some laboratories to incorporate broad-spectrum high-resolution LC-MS/MS analysis into their testing protocols to facilitate the identification of designer benzodiazepines and other designer drugs.

Methods:
A total of 23,747 urine specimens, originating from pain clinics and drug rehabilitation centers, underwent testing for benzodiazepines using a clinically validated benzodiazepine immunoassay and targeted LC-MS/MS benzodiazepine panel. Out of these, 945 were positive by immunoassay (Thermo Fisher DRI Benzodiazepine), while 886 were confirmed positive by LC-MS/MS. Specimens with discordant results (positive by immunoassay but negative by targeted LC-MS/MS) underwent further investigation using a broad-spectrum LC-QTOF-MS approach. MS spectra were acquired in positive MSE mode and matched to a Waters UNIFI toxicology spectral library containing over 1,500 compounds and significant library matches were confirmed by purchasing standard or certified reference material of the suspected analyte when available Additionally, immunoassay cross-reactivity of four designer benzodiazepines was evaluated using three separate immunoassays.

Results:
Among the 945 benzodiazepine immunoassay positive urine specimens tested, 6.2% (n=59) were not positive for benzodiazepines when tested by the targeted LC-MS/MS panel. Further analysis of 51 of these specimens (8 specimens could not be sequestered) by LC-QTOF-MS revealed designer benzodiazepines in 66.7% (n=34) of them. The most prevalent designer benzodiazepines detected were flualprazolam, etizolam, and flubromazolam.

Conclusion:
This study underscores the limitations of traditional targeted LC-MS/MS analysis in detecting designer benzodiazepines among substance abuse users. The adoption of a comprehensive approach involving immunoassay screening followed by both targeted and broad-spectrum LC-MS/MS methods is crucial for developing an accurate understanding of designer benzodiazepines usage in the population.