GCxGC-TOFMS of the Cooling Agent WS-3 in Mint Oils and Cinnamon Flavor

Importance of the Topic

Cooling agents such as WS-3 are widely used to enhance the freshness and sensory profile of mint oils, cinnamon flavors and other consumer products. Regulatory requirements and customer specifications in regions like Europe and Japan demand reliable certification that WS-3 is absent or below trace levels (e.g. 50 ppb) in mint oils. The complexity of natural flavor matrices poses a significant analytical challenge for conventional one-dimensional GC-MS, leading to the development of more advanced techniques.

Objectives and Study Overview

The primary goal of this study was to develop and validate a comprehensive two-dimensional GC method coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) for sensitive and accurate determination of WS-3 in peppermint oil, spearmint oil and cinnamon flavor. Specific objectives included:

• Demonstrate chromatographic separation and spectral deconvolution of WS-3 in complex matrices.
• Generate matrix-matched calibration curves down to 20 ppb (2 pg on-column) and evaluate linearity.
• Compare performance against traditional GC-quadrupole MS.

Methodology and Instrumentation

Sample Preparation

• Mint oils and cinnamon flavor diluted ten-fold in acetone to minimize viscosity and chromatographic overload.
• Matrix-matched standards prepared by spiking WS-3 into diluted samples at levels from 2 to 54.5 pg on-column, using tetra-bromothiophene as internal standard.

GC-quadrupole MS Conditions

• Column: 60 m × 0.25 mm × 0.25 µm DB-5.
• Carrier gas: Helium at 1.0 mL/min constant flow.
• Injection: 2 µL split (150:1) at 270 °C.
• Oven program: 80 °C (1 min) to 300 °C at 10 °C/min.
• Mass spectrometer operated in selected ion recording (SIR) mode targeting m/z 211.

GC×GC-TOFMS Conditions

• Primary GC: Agilent 6890 with LECO thermal modulator and secondary oven.
• Column 1: 30 m × 0.25 mm × 0.25 µm Rtx-1.
• Column 2: 1 m × 0.18 mm × 0.18 µm Rtx-200.
• Carrier gas: Helium at 1.0 mL/min.
• Injection: 1 µL splitless at 250 °C, valve open 60 s.
• Oven 1 program: 150 °C (1 min) to 165 °C at 0.5 °C/min, then to 280 °C at 38 °C/min.
• Oven 2 offset: +5 °C; modulation time: 4 s; total run time ~45 min.
• TOFMS: LECO Pegasus, EI 70 eV, source 225 °C, mass range 45–450 u, acquisition 200 spectra/s.
• Data processed with LECO ChromaTOF software featuring automated peak find and spectral deconvolution.

Main Results and Discussion

One-dimensional GC-TOFMS chromatograms showed overwhelming matrix background at the expected WS-3 retention time, preventing accurate quantification at low levels. In contrast, GC×GC contour and surface plots provided a second-dimension separation that resolved WS-3 from co-eluting interferences on the Rtx-200 column. Key findings:

• WS-3 eluted at a distinct location in the second-dimension, highlighted in contour plots for each matrix.
• Calibration curves for peppermint oil, cinnamon flavor and spearmint oil were linear over ~20–500 ppb with percent differences < 10% for most points.
• Spearmint oil sample contained native WS-3 (~55 ppb), confirmed by calibration and unspiked analysis.
• Thermal modulation sliced peaks into < 200 ms windows, yielding enhanced sensitivity and enabling detection of sub-pg on-column levels.
• Spectral deconvolution in ChromaTOF separated WS-3 spectra from large co-eluting peaks, achieving high match scores (> 0.89) against reference spectra.

Benefits and Practical Applications

GC×GC-TOFMS offers significant advantages for flavor analysis:

• Enhanced peak capacity and orthogonal separation improve resolution in complex matrices.
• Rapid full-scan acquisition supports narrow GC×GC peaks and automated deconvolution.
• Accurate quantification down to regulatory thresholds (20–50 ppb) for WS-3 and similar compounds.
• Robust workflow suitable for QA/QC laboratories, flavor companies and compliance testing.

Future Trends and Opportunities

Further developments may include:

• Lower detection limits via optimized modulation and injection techniques, enabling sub-ppb analyses.
• Application of GC×GC-TOFMS to a broader range of flavor and fragrance compounds, including natural and synthetic additives.
• Integration with chemometric and machine-learning tools for rapid screening and profiling of complex samples.
• Advances in miniaturized or portable GC×GC-TOFMS systems for on-site flavor quality control.

Conclusion

Comprehensive two-dimensional GC coupled with time-of-flight MS is a powerful approach for the sensitive and reliable determination of WS-3 in complex mint oils and flavors. The combination of increased chromatographic resolution, rapid full-scan data acquisition and automated spectral deconvolution enables quantification at or below 20 ppb. This method addresses industry demands for trace-level certification and quality assurance in flavor applications.

Reference

• Leffingwell & Associates web site: http://www.leffingwell.com/cooler_than_menthol.htm