Quantification of 35 benzodiazepines in human plasma by LC-HRAM-MS for clinical research

Significance of the topic

Benzodiazepines are among the most prescribed psychoactive drugs worldwide, used for anxiety, insomnia and seizure management. Due to potential misuse, narrow therapeutic windows and the need for accurate dosing, sensitive and selective quantification in human plasma is critical for clinical research and therapeutic drug monitoring.

Objectives and overview

This work presents the development and validation of a rapid LC-HRAM-MS method for simultaneous quantification of 35 benzodiazepines in human plasma. The target was to complete chromatographic separation and mass spectrometric detection within 7 minutes, using Thermo Scientific Vanquish Flex UHPLC and Orbitrap Exploris 120 instrumentation together with ClinMass calibrators and controls.

Methodology and instrumentation

Samples (50 µL plasma) were processed via simple offline protein precipitation with addition of internal standards. The supernatant was injected (2 µL) onto a Vanquish Flex Binary UHPLC system equipped with a RECIPE-supplied column and mobile phases. Chromatographic separation was achieved in 7.0 min at 0.6 mL/min. Detection employed an Orbitrap Exploris 120 mass spectrometer operated in positive heated electrospray ionization (HESI) with full-scan data-dependent MS2 (resolution 60 000, scan range m/z 70–450). Quantitation and data processing were conducted in TraceFinder 5.1.

• Sample preparation: 100 µL precipitating solution with IS
• Chromatography: 7 min binary gradient, column temperature 40 °C
• Mass spectrometry: positive HESI, full scan – ddMS2
• Calibration: ClinMass LC-MS/MS calibrators covering relevant concentration ranges

Main results and discussion

Linearity was excellent across all analytes (R² ≥ 0.998, 1/x weighting). Lower limits of quantitation ranged from 0.173 to 36.6 µg/L. No significant carryover was observed in blank injections. Accuracy evaluations showed bias within ±15% for all controls. Intra-assay precision was below 7.1% CV and inter-assay precision below 9.7% CV. Representative chromatograms and calibration curves confirmed high selectivity and sensitivity.

Benefits and practical applications

This method offers a streamlined workflow with rapid sample preparation and high-resolution detection, delivering reliable results suited for clinical research, therapeutic drug monitoring and QA/QC in pharmaceutical and forensic toxicology laboratories.

Future trends and potential applications

Future developments may include automation of sample preparation, expansion to additional drug classes or metabolites, higher throughput configurations and integration with real-time data analysis platforms. HRAM-MS selectivity will support multiplex assays and exploration of novel benzodiazepine analogs in clinical and forensic contexts.

Conclusion

A robust, reproducible and sensitive 7-minute LC-HRAM-MS method for quantifying 35 benzodiazepines in human plasma has been established. The combination of simple protein precipitation, a comprehensive internal standard kit and high-resolution mass spectrometry meets the stringent requirements of clinical research and therapeutic drug monitoring.