A Reduced Workflow Solution for the Analysis of Gamma-Hydroxybutyrate (GHB) in Human Hair Samples Via an Automated Bead Mill as a Precursor to GCxGC-TOFMS and GC-HRT

Importance of the Topic

Gamma-hydroxybutyrate (GHB) is both an endogenous molecule and an illicit drug often encountered in forensic and clinical settings. Its rapid elimination from blood and urine limits traditional detection windows, whereas hair analysis enables retrospective monitoring of GHB exposure over weeks to months. Reliable and efficient workflows that overcome the complexity of hair matrices and low analyte levels are essential for accurate forensic and anti-doping investigations.

Objectives and Study Overview

This study aimed to streamline sample preparation for hair analysis of GHB by introducing an automated bead-mill homogenization step ahead of two high-resolution gas chromatography–mass spectrometry platforms (GCxGC-TOFMS and GC-HRT). The work compared traditional cutting methods with bead-based homogenization to assess extraction efficiency, chromatographic separation, spectral quality, and quantitation performance.

Methodology and Instrumentation

Sample Preparation

• Head hair from two volunteers (A and B) was washed sequentially with water and dichloromethane under sonication, then air-dried.
• Aliquots were divided for two pre-treatments: bead-mill homogenization (Biotage Bead Ruptor 24) versus manual cutting into 1–2 mm segments.
• Extraction used methanol with GHB-d6 internal standard, incubated overnight at 40 °C, followed by nitrogen drying.
• Derivatization employed BSTFA at 60 °C for 1 hour prior to GC analysis.

Instrumentation Used

• GCxGC-TOFMS (LECO Pegasus 4D): dual GC column setup (Rxi-5MS and Rtx-200), modulated 4 s, helium carrier gas at 1.5 mL/min, pulsed splitless injection.
• GC-HRT (LECO Pegasus GC-HRT): single-dimensional GC with high resolving power (~50 000), full mass range 45–510 m/z, acquisition at 10 spectra/s.
• Data processing: ChromaTOF-HRT software for deconvolution, library searching (NIST, Wiley), and accurate-mass quantitation.

Main Results and Discussion

Chromatographic Separation and Spectral Quality

• GCxGC improved resolution of GHB and co-eluting urea, as evidenced by distinct surface and contour plots (m/z 147).
• GC-HRT delivered high-resolution mass spectra with mass accuracy <1 ppm, enabling confident identification and formula assignment.

Quantitation Performance

• Calibration curves for GHB (m/z 233.1024 and 239.1400) were linear (r>0.997) across 0.15–5.0 ng/mg.
• Bead-based homogenization yielded up to 35% higher GHB recovery compared to manual cutting.
• Volunteer B showed consistently higher GHB incorporation than volunteer A.

Complex Matrix Profiling
The high-resolution platforms enabled detection of multiple endogenous and exogenous compounds (e.g., sunscreen ingredients, insect repellents) in hair extracts, illustrating comprehensive sample characterization beyond target analytes.

Benefits and Practical Applications

• Automated bead-mill homogenization reduces manual labor and improves analyte recovery and reproducibility.
• GCxGC-TOFMS and GC-HRT combine enhanced chromatographic resolution and mass accuracy to minimize interferences in complex matrices.
• The workflow supports robust forensic and anti-doping testing by extending detection windows and increasing confidence in low-level GHB quantitation.

Future Trends and Possibilities

Further automation of hair sample processing, integration with data-driven screening algorithms, and expansion to multiplexed drug panels are anticipated. Emerging hyphenated techniques (e.g., LCxGC, ion mobility) and machine-learning-based spectral deconvolution will likely enhance throughput and selectivity in forensic toxicology and clinical monitoring.

Conclusion

This study demonstrates that automated bead-mill homogenization coupled with GCxGC-TOFMS and GC-HRT delivers a streamlined, high-resolution workflow for GHB analysis in human hair. The approach maximizes extraction efficiency, spectral quality, and quantitation accuracy, offering a powerful tool for forensic and clinical laboratories.

Reference

Watt BM, Alonso DE, Binkley J, Patrick J, Kero FA, Vandell V, Gairloch E, Nolt B, Enzweller T, Jones R, Williams L, Scott KS. A Reduced Workflow Solution for the Analysis of Gamma-Hydroxybutyrate (GHB) in Human Hair Samples Via an Automated Bead Mill as a Precursor to GCxGC-TOFMS and GC-HRT.