Extraction of gamma-Hydroxybutyric Acid (GHB) from Urine Using ISOLUTE® SLE+ Prior to GC/MS Analysis

Significance of the topic

The accurate extraction and quantification of gamma-hydroxybutyric acid in urine is critical for clinical toxicology and forensic investigations. Conventional liquid–liquid extraction methods can be labor intensive, prone to emulsions, and time consuming. Supported liquid extraction (SLE) offers a streamlined alternative, improving throughput and consistency.

Objectives and Study Overview

This study evaluates the performance of ISOLUTE SLE+ plates (200 µL capacity) and columns (400 µL capacity) for isolating GHB and its deuterated internal standard from human urine, followed by GC/MS analysis. Key aims include assessing recovery, precision, linearity, and baseline cleanliness.

Methodology

Sample Preparation

• Mix 200 µL of urine with 200 µL of 0.2% aqueous formic acid and add GHB-D6 internal standard.
• Load onto SLE+ plate or column; allow absorption for 5 minutes.
• Elute twice with 1 mL ethyl acetate under gravity, followed by a brief vacuum pull.
• Dry eluate under air or nitrogen stream or TurboVap for 30 minutes at ambient temperature.
• Reconstitute in 50 µL ethyl acetate and 50 µL BSTFA/1% TMCS; vortex and transfer to GC vial.

Instrumentation

GC Conditions

• System: Agilent 7890A with QuickSwap
• Column: Zebron ZB-35, 30 m × 0.25 mm × 0.25 µm
• Carrier gas: He at 1.2 mL/min
• Inlet: 250 °C split 10:1; injection volume 1 µL
• Oven program: 70 °C start; 10 °C/min to 140 °C; 120 °C/min to 350 °C; 1.6 min back-flush

MS Conditions

• Interface: Agilent 5975C
• Ion source 230 °C; quadrupole 150 °C
• Acquisition: SIM mode; GHB quantifier ion m/z 233, qualifier m/z 117; GHB-D6 ions m/z 239 and 240

Key Results and Discussion

Recoveries ranged from 52 % to 69 % across three donors. Precision was good, with RSDs below 9 % for the plate and below 4 % for the column. Calibration curves for both formats showed excellent linearity (r2 > 0.999) over 2.5 to 50 µg/mL. Compared to traditional SPE, the SLE+ method delivered lower baseline noise in the GC/MS chromatogram, enhancing confidence in trace-level GHB detection.

Benefits and Practical Applications

The SLE+ protocol simplifies sample preparation by eliminating emulsions and multiple transfer steps. Reduced handling accelerates throughput in forensic and clinical laboratories. Compatibility with existing GC/MS platforms makes adoption straightforward without compromising assay sensitivity or specificity.

Future Trends and Applications

Integration of supported liquid extraction with automated and high-throughput workflows is anticipated. Expanding the technique to other small polar drug analytes and exploring greener solvent systems will further enhance sustainability. Miniaturized SLE cartridges could reduce solvent volumes and waste in routine testing.

Conclusion

Supported liquid extraction using ISOLUTE SLE+ plates and columns offers a robust, reproducible, and efficient workflow for the extraction of GHB from urine. High linearity, acceptable recoveries, and low matrix interference demonstrate its suitability for forensic and clinical applications.

References

Biotage Application Note AN828 Extraction of gamma-Hydroxybutyric Acid from Urine Using ISOLUTE SLE+ prior to GC/MS Analysis (2014)