Extraction of Barbiturates From Human Urine Using ISOLUTE® SLE+ Columns with GC-MS Analysis

Importance of the Topic

Barbiturates remain critical targets in clinical toxicology and forensic analysis due to their narrow therapeutic index and potential for misuse. Reliable urinary screening ensures patient safety and supports law enforcement investigations.

Objectives and Study Overview

This study evaluates a streamlined extraction of seven barbiturate drugs from human urine using ISOLUTE SLE+ supported liquid extraction columns coupled with GC-MS detection. The goal is to achieve high recoveries, low limits of quantification, and reproducible performance in a simplified workflow.

Methodology and Instrumentation

The workflow comprises:

• Sample pretreatment: Mix 500 μL urine with 500 μL 100 mM ammonium acetate buffer (pH 5).
• Loading: Apply the 1 mL pretreated sample to an ISOLUTE SLE+ 1 mL column and allow absorption for 5 minutes.
• Extraction: Elute barbiturates with two 2.5 mL aliquots of dichloromethane under gravity, followed by a brief vacuum step.
• Post-extraction: Evaporate eluate to dryness at room temperature under a nitrogen stream and reconstitute in 200 μL ethyl acetate.

Instrumentation parameters include:

• Gas chromatography: Helium carrier at 2 mL/min; splitless inlet at 150 °C; oven ramp from 120 to 290 °C at 15 °C/min; in-port flash alkylation with 1 μL sample and 1 μL 0.2 M TMAH in methanol.
• Mass spectrometry: Source at 230 °C, quadrupole at 150 °C, solvent delay 7 minutes, SIM mode across six time windows targeting characteristic ions for each barbiturate.

Main Results and Discussion

The method delivered quantitative recoveries between 103 and 108% at 10 ng/mL for all seven analytes, with relative standard deviations below 10% (n=3). Chromatograms showed clear separation of compounds within 7.5 to 11 minutes without matrix interferences or emulsion issues.

Benefits and Practical Applications

• High analyte recovery and precision ensure reliable quantification at low ng/mL levels.
• Supported liquid extraction eliminates emulsion formation and reduces hands-on time.
• Simple evaporation and reconstitution streamline sample preparation in high-throughput laboratories.

Future Trends and Possibilities

Emerging opportunities include automation of the SLE+ workflow, extension to other drug classes, integration with LC–MS platforms for complementary data, and adoption of greener solvents to enhance sustainability in bioanalytical laboratories.

Conclusion

The ISOLUTE SLE+ GC-MS method offers a robust, efficient, and reproducible solution for the extraction and analysis of barbiturates in human urine, meeting stringent requirements for forensic and clinical toxicology applications.