Ultra high pressure comprehensive two-dimensional liquid chromatography combined with hybrid mass spectrometry for the elucidation of carotenoids in red chili peppers

Importance of the Topic

Carotenoids are key bioactive compounds in red chili peppers, contributing to color, antioxidant properties, and nutritional value. However, their structural similarity and wide polarity range pose analytical challenges. Ultra high pressure comprehensive two-dimensional liquid chromatography (UHPLC×LC) coupled with hybrid mass spectrometry offers enhanced separation and identification power, critical for accurate profiling in food quality control and nutritional research.

Study Objectives and Overview

This study aimed to develop and evaluate a novel normal-phase (NP) × reversed-phase (RP) UHPLC comprehensive two-dimensional method with hybrid LCMS-IT-TOF detection for elucidating carotenoids in Capsicum annuum L. The performance of three instrumental setups was compared in terms of peak capacity, orthogonality, under-sampling, and identification capabilities versus a conventional NP-LC×RP-LC system.

Methodology and Instrumentation

Red chili pepper extracts were prepared by solvent extraction with methanol, ethyl acetate, and petroleum ether (1:1:1). Separation employed a micro-bore cyano column in the first dimension (1D) and serially coupled C18 fused-core columns in the second dimension (2D). Three distinct modulation times (0.75, 1.50, and 1.00 min) with tailored gradients were tested under 54 to 85 MPa back pressure.

• UHPLC System: Shimadzu Nexera LC-30A with SPD-M20A PDA detector (250–550 nm)
• Interface: High-speed, high-pressure 6-port valves controlled by LCMSsolution software
• Mass Spectrometer: Shimadzu LCMS-IT-TOF with APCI source (positive/negative switching, 200–1200 m/z)
• Data Processing: Chromsquare® software for 2D map reconstruction and peak capacity calculations

Main Results and Discussion

The NP-LC×RP-LC benchmark achieved a theoretical peak capacity of 990, which decreased after orthogonality and undersampling corrections. Transitioning to UHPLC×LC improved theoretical capacity up to 1395 (setup #2) and 1125 (setup #3), with corrected and effective values demonstrating superior separation efficiency, especially for structurally similar diol-mono-keto esters.
Hybrid MS detection resolved co-eluting isomers by selective fragmentation patterns. For example, capsanthin diesters with overlapping UV spectra were distinguished via characteristic [M+H−FA] fragments. Up to 33 carotenoids, free and esterified, were confidently identified without reference standards. Fast polarity switching enabled simultaneous trace-level quantification and structural elucidation.

Benefits and Practical Applications

• Enhanced resolution of complex carotenoid mixtures and isomeric species
• Improved confidence in compound identification through MSn fragmentation
• Higher peak capacity and orthogonality for comprehensive profiling
• Automated workflow suitable for food quality control and natural product research
• Reduced analysis time with robust, reproducible results

Future Trends and Potential Uses

Advances in column packing, instrumentation speed, and data processing algorithms will further enhance 2D-LC×LC-MS workflows. Applications may extend to metabolomics, lipidomics, and screening of bioactive compounds in various food matrices. Integration with machine learning will facilitate automated feature recognition and quantitative prediction in complex samples.

Conclusion

The developed NP-LC×RP-UHPLC×LCMS-IT-TOF method delivers significant improvements over conventional 2D-LC approaches for carotenoid analysis in red chili peppers. Enhanced peak capacity, orthogonality, and MS-based identification afford a powerful tool for high-confidence profiling, supporting quality control and research applications.

Reference

Shimadzu application note PO-CON1230E, IMSC 2012.