Poster Presentations for Hannah Lusk

LC-MS/MS Analysis of Bromazolam and Its Metabolites in Clinical Specimens from a San Francisco Patient Cohort Hannah Lusk (Presenter) University of California San Francisco

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Poster #5c View Map

This poster will be attended on Thursday at 12:15 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION:
Benzodiazepines are central nervous system (CNS) depressants commonly used to treat anxiety, insomnia, seizures, and muscle spasms. Sedative effects induce relaxation and reduce anxiety but can also cause side effects such as drowsiness, altered mental status, dizziness, respiratory depression, and bradycardia. Due to their anxiolytic properties, benzodiazepines are frequently misused and carry a high risk of overdose, particularly when combined with other depressants. While several FDA-approved benzodiazepines, including alprazolam, lorazepam, and diazepam, are legally available by prescription in the United States, the illicit drug supply increasingly contains non-FDA-approved designer benzodiazepines. These compounds pose a significant public health concern due to their highly variable potency and poorly characterized pharmacokinetic profiles, increasing the risk of unintentional overdose. Bromazolam is one such designer benzodiazepine. Our laboratory conducts biosurveillance in San Francisco (SF) using a comprehensive mass spectrometry (MS)-based drug testing method, which has detected a recent rise in bromazolam-positive cases. In 2024, we identified 19 positive cases compared to 4 in 2023. To date, limited data exist on bromazolam’s pharmacokinetics, with only one study evaluating its metabolism using in vitro pooled human liver S9 fractions and in vivo analysis of serum and urine from two patients. To address this knowledge gap, we analyzed remnant serum and urine samples from SF patients to quantify bromazolam and characterize its metabolites. This study provides real-world data on bromazolam exposure and metabolism, offering insights into its pharmacokinetics and potential health risks.

OBJECTIVES:
The objective of this study is to characterize the metabolism of bromazolam in patient urine and serum samples. By analyzing bromazolam and its metabolites in real clinical samples, we aim to improve our understanding of its major metabolic pathways, thereby supporting toxicological and forensic investigations.

METHODS:
Ten remnant serum and 18 remnant urine samples were collected following a positive bromazolam result from our clinically validated comprehensive drug test using untargeted LC-QTOF-MS. Chromatographic separation was achieved with a C-18 column using a 10-minute gradient (2%-100% organic). Samples were analyzed on a SCIEX ZenoTOF® 7600 in positive mode with a TOF-MS survey scan and SWATH-triggered acquisition of high-resolution product ion spectra. Urine metabolites were identified through untargeted analysis. Serum concentrations of bromazolam and alpha-hydroxy bromazolam were quantified using 13 calibrators, a double blank, a blank, and four quality controls, all prepared in drug-free human serum to cover a dynamic range from 0.1 ng/mL to 1 µg/mL. Bromazolam-D5, at a concentration of 100 ng/mL, served as the internal standard. Quantification was performed using multiple reaction monitoring (MRM) on a SCIEX 4500 triple quadrupole MS. Chromatographic separation was carried out with a C-18 column using a 2-minute gradient (2%-100% organic). Sample preparation involved protein precipitation, centrifugation, evaporation under a steady stream of nitrogen gas, and reconstitution in the initial mobile phase. Data analysis was performed using PeakView software, and quantification was conducted using MultiQuant software.

RESULTS:
Bromazolam, bromazolam glucuronide, and hydroxy-bromazolam glucuronide were detected in all urine specimens analyzed using LC-QTOF-MS. Other metabolites identified in the original study that used human liver S9 fractions in vitro were not detected. Hydroxy-bromazolam glucuronide was the predominant metabolite, with an area under the curve (AUC) an average of 40% higher than the bromazolam peak. The ratios of bromazolam glucuronide and hydroxy-bromazolam glucuronide to bromazolam varied across samples, suggesting inter-individual differences in metabolic clearance. Concentrations in serum samples covered the calibration range and varied significantly between patients. IRB-approval was recently obtained to allow for medical chart review of positive bromazolam cases to evaluate if clinical symptoms and outcomes are associated with serum concentrations.

CONCLUSIONS:
The consistent detection of bromazolam glucuronide metabolites in patient urine samples underscores the significance of phase II metabolism in bromazolam clearance. In several samples, bromazolam was detected at lower levels than glucuronidated metabolites, indicating that failing to screen for these metabolites may lead to an underestimation of bromazolam-positive urine. This suggests that hydrolyzing samples before MS analysis or directly monitoring the glucuronide metabolite could extend the detection window and improve sensitivity. Variability in metabolite-to-parent drug ratios highlights inter-individual differences that may impact bromazolam’s pharmacologic effects. However, correlating bromazolam concentrations in serum with clinical symptoms is challenging due to poly-drug exposure, which complicates the attribution of symptoms to bromazolam versus other substances. Notably, no published clinical cases of bromazolam exist, with current literature primarily focusing on postmortem analyses. Our findings enhance the understanding of bromazolam pharmacokinetics, confirming its glucuronidation and metabolism to hydroxy-bromazolam.