A Rapid and Comprehensive UHPLC-MS/MS Clinical Toxicology Research Method for the Analysis of Benzodiazepines in Urine

Importance of Topic

Benzodiazepines are widely prescribed for neurological and psychiatric disorders but also subject to significant abuse. The emergence of novel designer analogues has increased public health risks due to unknown potency and toxicity. A rapid, reliable, and comprehensive analytical method is essential for clinical toxicology, forensic investigations, and quality control to detect both traditional and emerging benzodiazepine compounds in urine.

Objectives and Study Overview

The primary goal was to develop and validate a confirmatory UHPLC-MS/MS research method capable of simultaneous quantification of 26 traditional and designer benzodiazepines in urine. The study focused on simplifying sample preparation, achieving robust chromatographic separation, and ensuring accurate and precise quantitation across a wide dynamic range.

Methodology

Sample preparation was performed using a mixed-mode solid phase extraction in-well protocol. Urine samples (100 µL) underwent enzymatic hydrolysis with β-glucuronidase, acidification, washing, and elution directly in Oasis MCX µElution plates, eliminating transfer steps and reducing manual handling.

Chromatographic separation was achieved on an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm) at 40 °C with a 0.6 mL/min gradient of 0.1% formic acid in water and acetonitrile. The total run time was four minutes, with baseline separation for all analytes.

Mass spectrometric detection employed a Xevo TQ-S micro system in electrospray positive ion mode, monitoring two MRM transitions per analyte and one for each deuterated internal standard. Calibration curves were constructed over 5–1000 ng/mL (10–1000 ng/mL for low-abundance compounds) with 1/x weighting and quadratic fitting.

Used Instrumentation

• Oasis MCX µElution Plate (mixed-mode SPE)
• ACQUITY UPLC I-Class FTN system
• ACQUITY UPLC BEH C18 Column (1.7 µm, 2.1 × 100 mm)
• Xevo TQ-S micro Triple Quadrupole Mass Spectrometer
• MassLynx and TargetLynx XS (or waters_connect QUAN Review) software

Main Results and Discussion

Chromatographic profiling demonstrated baseline separation of 26 benzodiazepines within 3.5 minutes. Mean extraction recoveries across six urine lots averaged 86% (range 79–102%) with CVs <10%. Matrix effects ranged from 30% suppression to 35% enhancement but were consistently corrected to <20% bias using internal standards for all but two compounds lacking isotopically labelled surrogates.

Validation over five days showed calibration linearity (R2 ≥ 0.990), limits of quantitation at 5 ng/mL (10 ng/mL for selected amine metabolites), and QC accuracy within ±15% (±20% at the lowest level). External UTAK quality controls achieved 71% of values within ±20% bias and 93% within ±30%. An independent proficiency test confirmed satisfactory performance (z-scores ≤ 2) for diazepam and nordiazepam.

Benefits and Practical Applications

• Rapid in-well SPE workflow with minimal manual steps
• High and consistent recoveries across multiple matrices
• Simultaneous analysis of 26 traditional and designer benzodiazepines in four minutes
• Wide dynamic range (5–1000 ng/mL) suitable for clinical and forensic samples
• Robust data review via integrated software, reducing turnaround times

Future Trends and Applications

• Extension to additional emerging benzodiazepine analogues
• Automation of in-well sample preparation for high-throughput laboratories
• Integration with laboratory information management systems (LIMS)
• Application in clinical pharmacokinetic studies and workplace drug testing
• Adaptation to other biological matrices, such as plasma or oral fluid

Conclusion

The described UHPLC-MS/MS method offers a rapid, accurate, and comprehensive tool for the quantification of a broad panel of benzodiazepines in urine. Its streamlined sample preparation, fast chromatographic separation, and reliable mass spectrometric detection support its application in clinical toxicology research and forensic laboratories.

References

1. Brunetti A. et al. Designer Benzodiazepines: A Review of Toxicology and Public Health Risks. Pharmaceuticals. 14:560, 2021.
2. European Monitoring Centre for Drugs and Drug Addiction. New benzodiazepines in Europe – a review. June 2021.
3. Danaceau J.P. et al. A Comprehensive Method for Analysis of Pain Management Drugs and Drugs of Abuse. Waters Application Note 720006187, 2019.