Profiling of Citrus Oils and Determination of Furocoumarins in Citrus Oils Using the Agilent 1290 Infinity 2D-LC Solution

Significance of the Topic

Citrus essential oils are widely used in cosmetics, food flavoring and consumer products. Their complex composition includes psoralens and methoxylated flavonoids that influence safety and quality. Certain furocoumarins such as 5 methoxypsoralen are phototoxic and classified as probable or known carcinogens. Regulatory limits require sensitive and selective analytical methods for accurate profiling and quantification of these compounds in citrus oils.

Objectives and Study Overview

This study applied comprehensive two dimensional liquid chromatography with diode array and mass spectrometric detection to profile a range of citrus oil samples and to determine furocoumarin levels. A normal phase first dimension was coupled with a reversed phase second dimension to achieve high orthogonality and peak capacity. The method was evaluated on lemon, orange and bergamot oils and spiked mixtures to assess separation performance and quantitative accuracy for target analytes.

Instrumentation

An Agilent 1290 Infinity two dimensional LC solution was used with the following modules and accessories:

• Two binary pumps for first and second dimension gradients
• Autosampler with temperature control
• Thermostatted column compartment
• Diode array detector set at 315 nm for routine profiling and at 254 nm for furocoumarin analysis
• Two position four port valve with two 20 microliter loops for modulation
• Agilent single quadrupole LC MSD with APCI source for identity confirmation

Methodology

First dimension employed a silica based column with a hexane ethyl acetate gradient to separate nonpolar coumarins and psoralens. Fractionation occurred every 30 seconds into modulation loops. The second dimension used a C18 reversed phase column with rapid water acetonitrile gradients to resolve coeluting first dimension fractions. Injection volumes of 0.4 to 0.8 microliters ensured sharp peaks. Mass spectrometer settings were optimized for APCI ionization in positive mode across 150 to 700 m/z.

Main Results and Discussion

The two dimensional approach successfully resolved over a dozen coumarin and psoralen analogues in lemon and orange oil mixtures. Distinct profiles highlighted compositional differences among three lemon oils and one bergamot sample. Quantitative analysis focused on 5 methoxypsoralen, which was detected in three of four oils at concentrations of approximately 656, 138, 56 and 51 micrograms per milliliter. Comprehensive orthogonality enabled separation of target furocoumarins from complex matrix peaks that coelute in one dimensional methods.

Benefits and Practical Applications

The developed 2D LC method offers significant advantages for food and cosmetic quality control:

• Enhanced separation capacity for complex essential oil matrices
• Reliable detection and quantification of phototoxic furocoumarins
• Rapid profiling of multiple citrus varieties in a single run
• Automated data processing with comprehensive software integration

Future Trends and Applications

Advances may include coupling two dimensional LC with high resolution mass spectrometry for improved structural elucidation, miniaturized modulation devices for higher throughput, and application of machine learning for automated pattern recognition in oil authentication. Expanding the platform to other botanical extracts will further enhance quality and safety monitoring in natural product industries.

Conclusion

The Agilent 1290 Infinity 2D LC solution proved effective for detailed profiling of citrus oils and accurate determination of carcinogenic furocoumarins. Orthogonal separation and mass spectrometric confirmation enable robust quality assessment in compliance with regulatory guidelines.

References

1. Commission Directive 95/34/EC adapting Council Directive 76/768/EEC on cosmetic products, Official Journal L 186, 1995.
2. Scientific Committee on Consumer Products, Opinion on furocoumarins in cosmetic products SCCP/0942/05, 2005.
3. Frérot E and Decorzant E, Journal of Agricultural and Food Chemistry 52, 6879–6886, 2004.
4. Vanhoenacker G et al. Agilent Technologies Application Note 5990-4033EN, 2009.
5. Dugo P et al. Analytical Chemistry 76, 2525–2530, 2004.
6. François I et al. Journal of Chromatography A 1178, 33–42, 2008.