Off-line LC-GC×GC-MS: A Powerful Approach for Highly Detailed Analysis of Essential Oils

Significance of the Topic

Essential oils are complex mixtures of volatile compounds of great economic and industrial value, used in food, cosmetics, pharmaceuticals and perfumery. Traditional one-dimensional gas chromatography coupled to mass spectrometry (GC–MS) often fails to resolve all components in highly complex matrices. The combination of liquid chromatography and comprehensive two-dimensional gas chromatography with quadrupole mass spectrometry (LC–GC×GC–quadMS) addresses these limitations by fractionating the sample and enhancing separation power and sensitivity.

Objectives and Study Overview

This study demonstrates an off-line LC–GC×GC–quadMS workflow applied to cold-pressed orange essential oil. The main goals were to:

• Separate hydrocarbons and oxygenated constituents via silica-based HPLC fractionation.
• Perform highly detailed qualitative profiling of each fraction by cryogenically modulated GC×GC–quadMS.
• Compare the number and confidence of compound identifications versus conventional one-dimensional GC–quadMS.

Instrumentation Used

The analytical platform comprised:

• LC System: Shimadzu CBM-20A communication module, LC-30AD dual-plunger pumps, DGU-20A degasser, SPD-M20A PDA detector, CTO-20A oven, SIL-30AC autosampler.
• Fraction collection: Manual disconnect of transfer line; collected hydrocarbon and oxygenated fractions, concentrated under nitrogen.
• GC×GC–quadMS: Shimadzu GC2010 with QP2010 Ultra quadrupole MS; first column SLB-5ms, second column Supelcowax-10 polyethylene glycol; Zoex loop-type cryogenic modulator, modulation every 5 s with 400 ms hot pulse; GC program 50–250 °C at 3 °C/min; helium carrier gas.
• Conventional GC–quadMS: Shimadzu GC2010 with QP2010 Plus; SLB-5ms capillary; same temperature program; helium carrier; split injections.

Methodology

HPLC fractionation employed a 100 mm×3 mm silica column with a hexane–MTBE gradient. Hydrocarbon fraction (1.5–3 min) and oxygenated fraction (7.3–14 min) were collected and reduced to 100 µL. In GC×GC, each fraction was injected with optimized split ratios for monoterpenes, sesquiterpenes and oxygenates. Mass spectra were acquired in full-scan electron ionization mode (40–360 m/z) at 33 Hz frequency. Compound identification criteria were based on MS library match (≥75 %) and linear retention index agreement within ±15 units, categorized as reliable, presumed or tentative.

Main Results and Discussion

The off-line LC–GC×GC–quadMS method identified a total of 219 analytes in orange oil, compared to 50 compounds by one-dimensional GC–quadMS. Key findings include:

• Hydrocarbons: 56 named by GC×GC–quadMS (vs. 27 by GC–quadMS), with 18 first reports in orange oil.
• Oxygenated compounds: 162 named by GC×GC–quadMS (vs. 23), including many aldehydes, alcohols, ketones and esters, 91 of which were previously unreported.
• Enhanced resolution prevented modulator overload from dominant limonene and allowed observation of approx. 300 components in the 2D chromatogram.
• Improved sensitivity and selectivity led to reliable identifications even for low-abundance analytes.

Benefits and Practical Applications

The combined LC–GC×GC–quadMS approach offers:

• Superior separation of complex matrices by removing major interferences.
• Increased detection sensitivity through band compression in GC×GC.
• Comprehensive profiling for quality control, authenticity testing and research in essential oil characterization.

Future Trends and Potential Applications

Potential developments include:

• On-line coupling of LC and GC×GC–MS for automated, high-throughput analysis.
• Extension to other natural product classes, environmental samples and complex industrial mixtures.
• Integration with advanced data-processing tools and machine learning for pattern recognition and biomarker discovery.

Conclusion

The off-line LC–GC×GC–quadMS workflow provides unprecedented depth in essential oil analysis, identifying over four times more components than conventional GC–MS. This strategy opens new avenues for comprehensive profiling and quality assessment of volatile mixtures.

References

• G. Dugo et al., in: Citrus Oils, G. Dugo & L. Mondello (Eds.), CRC, 2011.
• G. Dugo et al., in: Citrus, G. Dugo & A. Di Giacomo (Eds.), Taylor & Francis, 2002.