Determination of Benzodiazepines in Urine by CE-MS/MS

Significance of the Topic

The widespread use of benzodiazepines for anxiety and sleep disorders has led to their frequent involvement in drug intoxications, traffic incidents, and criminal activities. In forensic toxicology, accurate and rapid methods to detect and quantify these compounds at trace levels in biological fluids are essential for legal investigations and public safety.

Objectives and Study Overview

This study aimed to establish a capillary electrophoresis–tandem mass spectrometry (CE-MS/MS) protocol for the simultaneous determination of six benzodiazepines—lorazepam, clonazepam, temazepam, oxazepam, diazepam, and nitrazepam—in urine. A modified QuEChERS extraction was applied for sample cleanup, and matrix-matched calibration covered concentrations from 10 to 500 ng/mL.

Methodology and Instrumentation

The analytical workflow combined an Agilent 7100 CE system with an Agilent 6430 triple quadrupole MS. Key parameters included:

• Background electrolyte: 0.1 M formic acid, pH 2.4
• Applied voltage: 28 kV
• Capillary: PVA-coated silica, 50 µm i.d., 58 cm effective length
• Sample injection: 12 s at 50 mbar, temperature 25 °C
• Sheath liquid: 0.01 M formic acid/methanol (50:50, v/v) at 5 µL/min
• Ionization: ESI positive mode, MRM transitions for quantification and confirmation

Sample preparation used a high-pH (12.4) acetonitrile extraction with MgSO₄/NaCl partitioning and 0.2 µm filtration, eliminating the need for dispersive SPE cleanup.

Main Results and Discussion

• Complete separation of all six benzodiazepines in under 10 minutes with baseline resolution using the PVA-coated capillary.
• Linear calibration curves over 10–500 ng/mL with R² > 0.998 for each analyte.
• Limits of detection between 0.9 and 1.4 ng/mL; limits of quantification from 2.9 to 4.6 ng/mL.
• Recoveries in spiked urine (10–200 ng/mL) ranged from 89 to 121% with RSDs below 6% (n = 3).

Benefits and Practical Applications

• High sample throughput: analysis time under 10 minutes per injection.
• Minimal sample and reagent consumption, generating low chemical waste.
• Excellent sensitivity and selectivity for forensic confirmation and routine screening.
• Simplified sample preparation adaptable to diverse toxicology workflows.

Future Trends and Possibilities

• Adaptation of the CE-MS/MS platform for additional drug classes and biological matrices.
• Coupling with high-resolution MS for comprehensive screening of unknown compounds.
• Further miniaturization and automation of sample handling and data processing.
• Development of new capillary coatings to enhance separation of highly polar metabolites.

Conclusion

The developed CE-MS/MS method delivers a rapid, sensitive, and robust approach for simultaneous detection of six benzodiazepines in urine. Its streamlined workflow, strong analytical performance, and low resource requirements position it as a valuable tool for forensic and clinical toxicology laboratories.

References

1. Szatkowska, P.; et al. Analytical methods for determination of benzodiazepines. Central European Journal of Chemistry, 2014, 12(10), 994–1007.
2. Tomita, M.; Okuyama, T. Application of capillary electrophoresis to the simultaneous screening and quantitation of benzodiazepines. Journal of Chromatography B, 1996, 678, 331–337.
3. Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) recommendations. U.S. Department of Justice – Drug Enforcement Administration, Third Edition, 2007, pp. 14–16.
4. Hudson, J.; et al. Benzodiazepine and Z-drug quantitation using an Agilent 6430 LC/MS/MS. Agilent Technologies Application Note 5991-2291EN, 2013.