Introducing Semi-Automated GC/Q-TOF Screening with the AssayMAP Bravo Sample Prep Platform for Antidoping Control

Significance of the Topic

Analytical toxicology for anti-doping requires ever higher sensitivity, selectivity, reproducibility and throughput. Traditional manual liquid-liquid extraction limits sample capacity, carries risk of analyte losses and hinders retrospective data mining. Automated micro-solid phase extraction combined with high-resolution mass spectrometry offers a powerful alternative, enabling lower detection limits, improved efficiency and the ability to re-interrogate full-scan data for newly discovered metabolites.

Study Objectives and Overview

This work presents a semi-automated workflow integrating the Agilent AssayMAP Bravo sample preparation platform with both GC quadrupole time-of-flight (GC/Q-TOF) and GC triple quadrupole (GC/TQ) mass spectrometers for routine doping control. The aims are to reduce hands-on time, decrease limits of detection across diverse compound classes, enable robust quantitation of endogenous steroids and facilitate retrospective screening of nonhydrolyzed sulfated metabolites.

Methodology and Instrumentation Used

The sample preparation uses Agilent AssayMAP Bravo with 25 µL reversed-phase resin cartridges for automated SPE. Urine samples are spiked with deuterated internal standards, enzymatically hydrolyzed or left intact for sulfated metabolite analysis, then loaded, washed and eluted with methanol/acetonitrile. Extracts are dried, derivatized with MSTFA/NH4I/ethanethiol, and injected onto:

• Agilent 7250 GC/Q-TOF with single-taper SSL inlet and J&W HP-1ms column
• Agilent 7000C GC/TQ with PTV inlet and DB-35MS column

Data acquisition covers full-scan high-resolution accurate mass (50–750 m/z) and dynamic MRM modes. An accurate mass Personal Compound Database and Library (PCDL) of 320 WADA-prohibited compounds supports screening.

Main Results and Discussion

The automated SPE workflow reduced evaporation and transfer time from over 75 min (manual LLE) to less than 10 min with minimal supervision. Limits of detection improved by up to four-fold for nonpolar steroids and over 200-fold for polar diuretics such as furosemide. GC/Q-TOF calibration curves for key steroids demonstrated wide dynamic ranges and coefficients of determination (R2) above 0.997. Mass axis stability remained within ±1.5 ppm after automated calibration every five injections. Automated compound verification in MassHunter screener dramatically reduced the manual review burden by flagging only outliers. Additionally, the workflow enabled detection of nonhydrolyzed sulfated metabolites of mesterolone and metenolone, extending detection windows from days to over a week. Full-scan acquisition allows retrospective interrogation for newly identified marker compounds.

Benefits and Practical Applications of the Method

• High throughput: capacity for >20 000 samples per year
• Lower detection limits across steroids, stimulants, diuretics and hormone modulators
• Reduced hands-on time and error risk via automated SPE
• Cleaner extracts and reduced organic solvent waste
• Simultaneous targeted quantitation and full-scan screening
• Retrospective data mining for emerging metabolites
• Automated data review for unbiased, consistent results

Future Trends and Potential Applications

Further expansion of accurate mass libraries will enable broader suspect screening across new substance classes. Integration of miniaturized and multiplexed automated prep systems will boost lab productivity while reducing environmental impact. Coupling high-resolution screening with AI-driven data evaluation promises real-time anti-doping intelligence. Ongoing research on long-term steroid metabolites will benefit from retrospective mining of archived full-scan datasets.

Conclusion

The combination of Agilent AssayMAP Bravo automated SPE with GC/Q-TOF and GC/TQ instrumentation establishes a robust, high-throughput workflow for modern anti-doping analysis. This approach delivers superior sensitivity, broad dynamic range, streamlined sample handling and powerful retrospective screening capability, meeting evolving WADA requirements.

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