Extraction of Benzodiazepines from Whole Blood Using ISOLUTE® SLE+ Prior to GC/MS Analysis

Significance of the Topic

The reliable extraction of benzodiazepines from whole blood is essential for forensic toxicology, clinical diagnostics and workplace drug testing. High-purity sample preparation enhances sensitivity and reproducibility in GC/MS analyses, enabling accurate quantitation of low-level drug residues.

Study Objectives and Overview

This application note presents a protocol for isolating a panel of 20 benzodiazepines and their metabolites from human whole blood using ISOLUTE SLE+ supported liquid extraction (SLE+) prior to GC/MS. The method targets compounds such as diazepam, midazolam, alprazolam and their analogues, and evaluates recovery, precision, limits of quantitation and calibration performance.

Methodology and Instrumentation

Sample Preparation:

• Add internal standard to 1 mL whole blood.
• Adjust pH with 1% ammonium hydroxide and dilute with aqueous buffer.
• Load 750 µL of treated sample onto a 1 mL SLE+ column.
• Elute analytes with two 2.5 mL aliquots of dichloromethane (DCM) or methyl tert-butyl ether (MTBE).
• Evaporate under nitrogen and reconstitute in ethyl acetate for derivatization with MTBSTFA.

Instrumentation:

• GC: Agilent 7890A with DB-5 column, splitless injection.
• MS: Agilent 5975C in Selected Ion Monitoring (SIM) mode targeting specific quantifier and qualifier ions.
• Evaporation: TurboVap LV or SPE Dry concentrator at 40 °C.

Main Results and Discussion

Recoveries for 100 ng/mL spiked blood ranged between 80 % and 110 % for most benzodiazepines with RSDs below 10 % using either DCM or MTBE. Calibration curves from 10 to 500 ng/mL showed excellent linearity (r2 > 0.992). Lower limits of quantitation (LLOQs) varied by analyte, with values as low as 10 ng/mL under optimal conditions.

Benefits and Practical Applications

This SLE+ approach eliminates protein precipitation and emulsion issues, reduces hands-on time and solvent usage compared to traditional liquid-liquid extraction. The method delivers clean extracts suitable for high-throughput bioanalysis in forensic and clinical laboratories.

Future Trends and Potential Applications

Further integration with automated platforms and miniaturized SLE formats can increase throughput. Expanding the panel to include novel designer benzodiazepines and concurrent extraction of other drug classes will broaden clinical and forensic utility. Coupling with high-resolution mass spectrometry may improve specificity for complex matrices.

Conclusion

The ISOLUTE SLE+ procedure provides a robust, reproducible and scalable workflow for benzodiazepine extraction from whole blood, delivering low LLOQs and excellent linearity for routine GC/MS analysis.

Reference

Biotage Application Note AN854, 2015