Simultaneous determination of γ-hydroxybutyrate and its precursor substances γ-butyrolactone and 1,4-butanediol in beverages using UHPLC-MS/MS

Importance of the Topic

γ-Hydroxybutyrate (GHB) and its precursors γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are central nervous system depressants often misused in drug-facilitated crimes. Their colorless, odorless properties allow surreptitious addition to beverages, posing significant public health and forensic challenges. Reliable, rapid analytical methods are essential for screening and quantifying these substances in complex drink matrices to support law enforcement, food safety monitoring, and quality control in the beverage industry.

Objectives and Study Overview

This work presents the development and validation of a UHPLC-MS/MS method enabling simultaneous determination of GHB, GBL, and 1,4-BD in various beverage types. Key goals included:

• Achieving complete chromatographic separation to avoid in-source conversion interferences.
• Establishing robust sample preparation protocols for dairy, juice, carbonated, coffee, tea, vinegar, and alcoholic drinks.
• Demonstrating method performance metrics (linearity, sensitivity, accuracy, precision) suitable for regulatory and forensic applications.

Methodology and Instrumentation

Sample preparation varied by beverage type: protein precipitation with acetonitrile for dairy and coffee; direct dilution and filtration for juices; degassing and dilution for carbonated drinks. Chromatographic separation used an Agilent Eclipse Plus C18 column (150×3.0 mm, 1.8 µm) at 40 °C with 0.02% formic acid in water (A) and methanol (B) under a gradient (5%–95% B). The flow rate was 0.4 mL/min and injection volume 2 µL. Mass detection employed an Agilent 6470 triple quadrupole with ESI in positive mode, monitoring specific MRM transitions for each analyte to ensure both quantitation and confirmation.

Main Results and Discussion

The optimized method achieved baseline separation of GHB, GBL, and 1,4-BD within 10 min, with resolution >5 for critical peak pairs. Calibration curves in solvent showed excellent linearity (R²≥0.999). Limits of quantitation met low µg/kg levels. Recovery experiments across eight beverage matrices yielded 88.9–108.2% recoveries, with intra-day precision RSDs between 1.1–9.8%. The method effectively distinguished genuine GBL from in-source dehydration products of GHB, avoiding false positives.

Benefits and Practical Applications

With rapid analysis times and minimal sample handling, the method supports high-throughput screening of beverages in forensic casework and quality control laboratories. Its sensitivity and selectivity meet stringent regulatory requirements for food and beverage safety. The approach can be integrated into routine monitoring of illicit adulteration and forensic investigations of drug-facilitated incidents.

Future Trends and Applications

Advancements may include coupling with automated sample preparation platforms and expanding the target list to additional emerging psychoactive substances. Miniaturized or portable UHPLC-MS/MS systems could enable on-site screening at points of sale or during field investigations. Data-driven approaches using high-resolution MS and machine learning hold promise for enhanced pattern recognition and discovery of novel adulterants.

Conclusion

The described UHPLC-MS/MS protocol offers a fast, accurate, and reproducible tool for simultaneous determination of GHB and its precursors in diverse beverages. Its robust performance and adaptability make it valuable for forensic toxicology, food safety testing, and industrial quality assurance.

Reference

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