Analysis of Urine SRMs Using Solid Phase Micro Extraction, Dynamic Headspace and Liquid Injection with Comprehensive Two-Dimensional Gas Chromatography (GCxGC) High Resolution Time-of-Flight Mass Spectrometry

Importance of the Topic

Urinalysis remains a fundamental tool in clinical diagnostics and biochemical research due to urine’s noninvasive collection, low protein and lipid interference, and its capacity to store metabolites and drugs over time. Advances in gas chromatography coupled with high-resolution mass spectrometry open new opportunities for detailed metabolomic profiling and biomarker discovery in health and disease.

Objectives and Study Overview

• Assess three sample-introduction strategies—solid-phase microextraction (SPME), dynamic headspace (DHS), and liquid injection with chemical derivatization—for comprehensive volatile and semi-volatile analysis in urine.
• Deploy two-dimensional gas chromatography (GCxGC) with LECO Pegasus® high-resolution time-of-flight MS (GC-HRT 4D) to separate, identify, and quantify urinary compounds from smoking and non-smoking Standard Reference Materials (SRM 3673 and SRM 3672).
• Demonstrate the benefits of enhanced chromatographic resolution and accurate mass detection for confident compound annotation.

Methodology

Sample preparation involved:

• SPME: Polyacrylate fiber extraction at 80 °C with agitation cycles, desorption in a programmable injector.
• DHS: Trapping volatiles on Tenax® tubes over 1 hour, thermal desorption into GC.
• Twister™ stir-bar sorptive extraction for extended volatile capture.
• Liquid injection: Urease treatment, protein precipitation, evaporation, and two-step derivatization (methoximation and silylation) to broaden analyte coverage.

Instrumental Setup

• Chromatograph: Agilent 7890B with Gerstel autosampler.
• Columns: Rxi-5 Sil MS (30 m × 0.25 mm × 0.25 µm) for first dimension; Rxi-17 Sil MS (0.6 m × 0.25 mm × 0.25 µm) for GCxGC second dimension.
• Modulation: Three-second thermal modulator with temperature offsets.
• Mass spectrometer: LECO Pegasus HRT; EI ionization; mass range 35–510 m/z; resolving power up to 50 000; acquisition 200 spectra/s for GCxGC.

Main Results and Discussion

GCxGC-HRT 4D provided improved separation, revealing over two dozen characteristic peaks including aromatic phenols, lactones, fatty acids, steroids, and sugar derivatives. SPME and DHS efficiently captured volatiles such as p-cresol and furaldehydes, while derivatized liquid injections uncovered monosaccharides, disaccharides, and hydrophilic metabolites. High-resolution accurate mass deconvolution enabled library matching with similarity scores typically above 900 and mass errors below 1 ppm.

Benefits and Practical Applications

• Rapid, confident identification of a broad chemical space in urine for clinical diagnostics and exposomics.
• Noninvasive monitoring of metabolic changes in smokers versus non-smokers.
• Flexible sample-introduction options tailored to analyte volatility and polarity.
• High-throughput profiling applicable to quality control, toxicology, and biomarker research.

Future Trends and Potential Applications

Emerging directions include automation of multicomponent sampling workflows, integration with data-mining and machine learning for pattern discovery, and coupling GCxGC-HRT with stable isotope labeling to trace metabolic pathways. Expansion into broader biofluids and environmental matrices will further extend the utility of this platform.

Conclusion

This study demonstrates that combining SPME, DHS, and derivatized liquid injection with GCxGC-HRT 4D provides a powerful, reproducible approach for in-depth urinary metabolite profiling. Enhanced chromatographic resolution, high mass accuracy, and rapid data processing yield confident compound identification across diverse chemical classes, paving the way for advanced clinical and research applications.