Fast Detection of Derivatized Drug Samples (Methyl Derivatives) in Spiked Horse Urine Samples Using GC-TOFMS

Significance of the Topic

The integrity of competitive horse racing hinges on reliable, high-throughput detection of illicit performance enhancers. Non-steroidal anti-inflammatory drugs and appetite stimulants often fight unfair advantages by affecting recovery and performance. Implementing a robust analytical workflow using methyl derivatization combined with fast gas chromatography time-of-flight mass spectrometry (GC-TOFMS) ensures trace-level identification and quantitation, safeguarding industry standards and animal welfare.

Study Objectives and Overview

This study aimed to develop a rapid, sensitive method for detecting six methylated drug derivatives spiked into horse urine at four concentration levels (high, medium, low, very low). Target compounds included ibuprofen, diclofenac, eltenac, vedaprofen, clanobutin, and indomethacin. Key goals were to:

• Achieve complete separation and identification within a total run time under 8 minutes
• Establish limits of detection below 10 ng/mL
• Demonstrate linearity of response for quantitation using calibration curves
• Validate the workflow for routine screening at a centralized doping laboratory

Methodology and Instrumentation

Samples provided by the National Horse Racing Authority were derivatized with diazomethane (methylation) prior to analysis. Key instrumentation and conditions:

• System: LECO Pegasus III GC-TOFMS with ChromaTOF software and True Signal Deconvolution
• Column: 10 m×0.18 mm×0.20 µm Rtx-5
• GC parameters: splitless injection at 200 °C, 50 °C (1 min) to 300 °C at 50 °C/min, total runtime 7.2 min
• Carrier gas: helium at 1.0 mL/min constant flow
• TOFMS: source at 200 °C, acquisition 10 spectra/s, mass range 120–380 u

Results and Discussion

All six methyl derivatives were detected in samples down to very low levels (<10 ng/mL) with similarity scores above 460 (out of 1000) and up to 937 for eltenac. Two derivatives absent from the NIST library (eltenac, vedaprofen) were added to a custom user library, enabling confident identification. True Signal Deconvolution effectively resolved low-level peaks overlapped by matrix components, producing high-quality spectra matching user library entries. Calibration curves constructed from medium, low, and very low spiked samples exhibited excellent linearity (r=0.99998 for eltenac), demonstrating quantitative capability.

Benefits and Practical Applications

This workflow offers:

• Fast turnaround (<8 min per sample) for high sample throughput
• Sub-10 ppb detection limits for regulatory compliance
• Accurate identification in complex urine matrices without extensive cleanup
• Flexibility to extend the user library for new or rare analytes
• Avoidance of SIM-based interferences through full-spectrum deconvolution

Future Trends and Opportunities

Advances may include automated on-line derivatization, integration with high-resolution mass analyzers for broader untargeted screening, and machine-learning algorithms to refine deconvolution and library matching. Expanding user libraries to encompass novel designer drugs will further enhance anti-doping capabilities. Miniaturized, field-deployable GC-TOFMS systems could enable on-site preliminary testing at race venues.

Conclusion

A fast GC-TOFMS method was successfully developed for the detection and quantitation of six methylated drugs in horse urine. Sub-10 ppb sensitivity, rapid runtime, and robust deconvolution make this approach suitable for routine anti-doping laboratories. The customizable user library and excellent linearity support both qualitative screening and quantitative assessments.