Smoker’s and Non-Smoker’s Urine Comparis on Using Comprehensive Two-Dimensional Gas Chromatography High Performance Time-of-Flight Mass Spectrometry

Significance of the Topic

Urine is a readily accessible, non-invasive biofluid that accumulates a wide range of endogenous metabolites, xenobiotics and drug residues over extended periods. Its relative simplicity (low protein and lipid content) and high analyte concentrations make it ideal for diagnostic testing and metabolic profiling. Comprehensive two-dimensional gas chromatography coupled with high-performance time-of-flight mass spectrometry (GCxGC-TOFMS) offers enhanced separation power and confident compound identification, addressing the complexity of urinary matrices.

Objectives and Study Overview

This study aimed to:

• Implement GCxGC-TOFMS for detailed separation of urinary compounds.
• Apply rapid data processing tools to identify metabolites and xenobiotics.
• Compare the chemical profiles of smoker’s and non-smoker’s urine reference materials (NIST).

Methodology and Instrumentation

Sample preparation involved urease treatment, centrifugation, drying and a two-step derivatization: methoximation (MEOX) followed by silylation (MSTFA). GCxGC separations used a dual-stage quad jet modulator with a non-polar primary column and a mid-polar secondary column. TOFMS data were acquired in electron ionization mode and processed using ChromaTOF® software.

Used Instrumentation

• Gas Chromatograph: Agilent 7890 with LECO L-PAL 3 autosampler.
• Modulator: LECO Dual Stage Quad Jet (4 s modulation).
• Columns: Rxi-5 MS (30 m × 0.25 mm × 0.25 µm) and Rxi-17 Sil MS (0.6 m × 0.25 mm × 0.25 µm).
• Mass Spectrometer: LECO Pegasus® BT 4D TOFMS (EI, 45–600 m/z, up to 200 spectra/s).
• Software: ChromaTOF® Brand for peak finding, library matching, retention index filtering and Target Analyte Finding (TAF).

Main Results and Discussion

GCxGC-TOFMS resolved over 200 compounds in both matrices. Key findings included:

• Non-smoker’s urine contained a diverse array of organic acids, amino acids, sugars and diacids with high spectral similarity and consistent retention indices.
• Smoker’s urine exhibited additional tobacco-related markers (cotinine, trans-3′-hydroxycotinine, 3-pyridinol) and elevated levels of aromatic compounds and drugs (nicotine metabolites, caffeine, analgesics).
• TAF processing rapidly quantified differences, showing significant area increases for tobacco markers in smoker’s samples.

Benefits and Practical Applications

The enhanced chromatographic resolution and high-speed TOFMS allow:

• Comprehensive non-targeted profiling of complex biofluids.
• Rapid targeted screening for specific biomarkers or xenobiotics.
• Reliable compound identification via combined spectral matching, retention index filtering and mass delta assessments.

This approach supports clinical diagnostics, forensic toxicology, pharmacokinetic studies and quality control in environmental and occupational health.

Future Trends and Potential Applications

Emerging directions include:

• Integration with machine learning for automated pattern recognition and anomaly detection.
• Development of streamlined workflows for high-throughput clinical screening.
• Expansion of spectral libraries with novel biomarkers and distinct population cohorts.
• Coupling with complementary detectors (e.g., high-resolution Orbitrap) for enhanced mass accuracy.

Conclusion

This study demonstrates that GCxGC-TOFMS, combined with robust data processing, provides a powerful platform for detailed urinary metabolite and xenobiotic profiling. It distinguishes smoker’s from non-smoker’s chemical signatures with high confidence and paves the way for advanced applications in biomedical research, diagnostics and toxicology.