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

Significance of the Topic

The reliable detection of barbiturates in whole blood is critical for forensic toxicology, clinical diagnostics and therapeutic drug monitoring. Rapid, reproducible extraction methods that minimize sample handling and avoid protein precipitation are essential to support high-throughput laboratories and maintain data quality.

Objectives and Overview

This study describes a streamlined supported liquid extraction (SLE) procedure using ISOLUTE SLE+ 1 mL columns to isolate seven barbiturate analytes from whole blood ahead of gas chromatography–mass spectrometry (GC/MS) analysis. The protocol also accommodates simultaneous recovery of other drug classes, including amphetamines, benzodiazepines and opiates.

Methodology and Instrumentation

Sample Preparation and Extraction

• Spike 1 mL whole blood with 10 µL internal standard mixture (100 ng/mL).
• Add 1 mL of 1 % ammonium hydroxide solution, vortex to mix.
• Load 750 µL of the treated sample onto an ISOLUTE SLE+ column and allow absorption for 5 min.
• Elute analytes with two 2.5 mL portions of dichloromethane (DCM) under gravity, followed by a brief vacuum pulse.
• Alternative solvent: methyl tert-butyl ether (MTBE) for non-chlorinated extraction.
• Evaporate extracts under a gentle nitrogen stream at 40 °C and reconstitute in ethyl acetate before transfer to GC vials.

Instrumentation

• Gas Chromatograph: Agilent 7890A with Agilent J&W DB-5 column (30 m × 0.25 mm ID, 0.25 µm).
• Carrier Gas: Helium at 1.2 mL/min constant flow.
• Injector: 150 °C splitless mode, 1 µL injection volume.
• Oven Program: 120 °C (1 min), ramp 12 °C/min to 192 °C, then 100 °C/min to 330 °C, with 1.6 min backflush.
• Mass Spectrometer: Agilent 5975C in SIM mode; source 230 °C, quadrupole 150 °C.
• Analyte ions monitored include quantifier and qualifier masses for each barbiturate.

Main Results and Discussion

Recovery and Precision

• Typical recoveries at 100 ng/mL exceeded 85 % for all barbiturates with both DCM and MTBE, with RSD values below 10 %.

Calibration and Sensitivity

• Calibration curves (10–500 ng/mL) produced coefficients of determination (r2) between 0.9983 and 0.9990.
• Lower limits of quantitation ranged from 20 to 100 ng/mL, depending on the analyte and extraction solvent.

Benefits and Practical Applications

The ISOLUTE SLE+ protocol eliminates the need for protein precipitation and reduces emulsion formation, speeding up sample preparation and improving laboratory throughput. High and consistent recoveries support accurate quantitation in forensic and clinical toxicology settings.

Future Trends and Possibilities of Use

Emerging directions include:

• Automation of SLE workflows to further increase sample throughput.
• Extension of the method to additional drug classes and metabolites.
• Evaluation of greener solvents and miniaturized extraction formats.
• Coupling with high-resolution mass spectrometry for enhanced selectivity.

Conclusion

The described ISOLUTE SLE+ extraction procedure delivers clean, reproducible barbiturate extracts from whole blood with low limits of quantitation. Its simplicity and adaptability make it well suited for routine forensic, clinical and research laboratories.