Extraction of Benzodiazepines From Human Urine Using ISOLUTE® SLE+ in Column Format Prior to GC-MS analysis

Significance of the Topic

Monitoring benzodiazepine levels in biological fluids is critical for clinical diagnostics, therapeutic drug monitoring, forensic investigations and quality control in pharmaceutical and toxicology laboratories. A reliable sample preparation method that delivers high recoveries, low limits of quantification and supports green chemistry principles can improve throughput and data quality.

Study Objectives and Overview

The aim of this application note was to develop and validate a streamlined supported liquid extraction (SLE) protocol using ISOLUTE® SLE+ 1 mL plates for the isolation of 17 common benzodiazepines from human urine. The extracted analytes were derivatized and quantified by GC-MS in selective ion monitoring (SIM) mode. Two elution solvents (dichloromethane and ethyl acetate) were compared to demonstrate compatibility with non-halogenated, environmentally friendlier options.

Methodology and Instrumentation

Sample Preparation and Extraction

• Dilution: 500 µL urine mixed with 500 µL sodium acetate buffer (100 mM, pH 5).
• Enzymatic hydrolysis: β-glucuronidase from H. pomatia (approx. 4500 U/mL) at 60 °C for 2 h, followed by pH adjustment to 7 with 25 % ammonium hydroxide.
• SLE Loading: 1 mL pretreated urine applied to ISOLUTE SLE+ column; allow 5 min equilibration under light vacuum.
• Elution: Two 2.5 mL aliquots of dichloromethane (or ethyl acetate) with adsorption pauses; final vacuum pulse to complete elution.
• Post-extraction: Evaporation under 80 L/min air at 35 °C, reconstitution in 50 µL ethyl acetate.
• Derivatization: 1:1 addition of TBDMSTFA with 1 % TBMSCl, 70 °C for 20 min.

GC-MS Analysis

• Instrument: Agilent 7890A GC coupled to 5975A MSD.
• Column: Zebron ZB-5MSi, 30 m × 0.25 mm, 0.25 µm film, with 5 m pre-column.
• Carrier gas: Helium at 1.2 mL/min constant flow.
• Inlet: Pulsed splitless (9.8 psi at 250 °C); pressure pulse to 20 psi at 0.75 min.
• Oven program: 50 °C for 1.5 min, 25 °C/min to 150 °C, 10 °C/min to 320 °C (6 min hold).
• MSD: Source 230 °C, quadrupole 150 °C, SIM mode with seven time segments for target ions.

Main Results and Discussion

Recoveries of all 17 benzodiazepines ranged above 90 % with both dichloromethane and ethyl acetate, demonstrating comparable performance of a greener elution alternative. Limits of quantification of 40 ng/mL (500 µL urine) were achieved for each analyte, with peak-to-peak signal-to-noise ratios exceeding 14:1 and RSDs below 10 % (n = 3). Overlaid SIM chromatograms confirmed clear separation and detection across the 17–28 min window.

Benefits and Practical Applications

• High throughput: rapid 1 mL extraction format eliminates emulsion issues common to LLE.
• Green chemistry: validated use of ethyl acetate reduces halogenated solvent consumption.
• Robust quantification: sensitive SIM detection with low LOQs and consistent RSDs.
• Flexible workflow: compatible with routine clinical, forensic or pharmaceutical screening.

Future Trends and Applications

Advances in sample preparation and detection will further enhance benzodiazepine analysis:

• Micro-SLE formats and on-line automated workflows to boost throughput.
• Integration with high-resolution MS for broader screening panels.
• Development of novel sorbent chemistries for improved matrix cleanup.
• Extended adoption of green solvents and miniaturized extraction to reduce environmental impact.

Conclusion

The optimized ISOLUTE SLE+ protocol provides a rapid, reliable and environmentally considerate method for extracting a comprehensive panel of benzodiazepines from urine. Combined with GC-MS SIM detection, it delivers excellent recoveries, low quantification limits and high analytical precision, making it an attractive approach for clinical, forensic and quality control laboratories.

Reference

No specific literature references were listed in the original application note.