Determination of heterocyclic oxygen compounds in Citrus essential oils by Supercritical fluid chromatography-tandem mass spectrometry

Significance of the Topic

Oxygen heterocyclic compounds (OHCs) such as coumarins, furocoumarins and polymethoxyflavones are bioactive secondary metabolites in the non-volatile fraction of cold-pressed Citrus essential oils. While they exhibit beneficial properties, excessive consumption of coumarins and photoactivated furocoumarins can pose health risks. Regulatory bodies including the European Commission and IFRA impose strict limits on furocoumarin levels in cosmetics and food, creating demand for sensitive, rapid and environmentally friendly analytical methods for routine quality control.

Objectives and Study Overview

This work aimed to develop and validate a green analytical approach based on supercritical fluid chromatography coupled to tandem mass spectrometry (SFC-QqQ-MS/MS) for simultaneous separation and quantification of 28 OHCs in cold-pressed Citrus essential oils. The method targeted a fast analysis time, low organic solvent consumption and achievement of stringent performance criteria defined by EURACHEM guidelines.

Methodology

Five cold-pressed Citrus oils (lemon, bergamot, bitter orange, sweet orange, mandarin) were diluted (1:1 000 and 1:20 000 v/v) in ethanol without further pretreatment. Calibration curves were constructed by spiking distilled lemon oil at ten concentration levels (0.001–3 mg L⁻¹) in triplicate. Validation metrics included linearity, limits of detection and quantification (LoD, LoQ), accuracy, intra- and inter-day precision.

Instrumental Setup

An SFC system (Shimadzu Nexera-UC) with a backpressure regulator and APCI-equipped LCMS-8050 triple quadrupole detector was employed. Column screening of eight stationary phases identified a pentafluorophenyl core-shell column (150×2.1 mm, 2.7 μm) as optimal. Mobile phase comprised CO₂ (solvent A) and methanol (solvent B) with a gradient from 2 to 10 % B over 10 min at 1.0 mL min⁻¹, 40 °C and 120 bar. MRM acquisition provided selective detection of each OHC.

Main Results and Discussion

The optimized method achieved baseline separation of all 28 compounds within 8 min, using <10 % methanol. Calibration curves showed R² values >0.9952. LoQs ranged from 0.0015 to 0.1536 mg L⁻¹. Accuracy (80–119 %) and precision (CV<8 %) met validation criteria. Solvent consumption per run was reduced by ~35 % versus HPLC-MS/MS. Quantitative profiling revealed lime oil as richest in OHCs and mandarin oil as poorest. Specific marker compounds varied by oil type.

Benefits and Practical Applications

The SFC-QqQ-MS/MS method offers a rapid, sensitive and eco-friendly alternative to traditional HPLC approaches. Low solvent use, high throughput and robust performance make it suitable for routine quality control of OHCs in Citrus essential oils, cosmetics and food ingredients.

Future Trends and Potential Applications

Further development may integrate online supercritical fluid extraction (SFE) with SFC-QqQ-MS/MS for automated, direct extraction and analysis. Expansion to finished cosmetic and food matrices could enhance monitoring workflows and reduce laboratory turnaround times.

Conclusion

The validated SFC-QqQ-MS/MS method delivers fast, accurate and environmentally responsible analysis of 28 OHCs in Citrus essential oils. Its performance and green credentials support adoption in industrial quality control and regulatory compliance.

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