Mass spectrometric elucidation of carotenoids in red chili peppers using high pressure comprehensive two-dimensional liquid chromatography

Importance of Topic

This work addresses the comprehensive profiling of carotenoids in red chili pepper, a key group of bioactive pigments with significant roles in food quality, nutritional value, and natural colorant applications. Accurate identification and quantification of individual carotenoids and their esterified forms are essential for food authentication, quality control and health‐related research. Two‐dimensional liquid chromatography (LC×LC) coupled with mass spectrometry (MS) offers a powerful approach to resolve complex mixtures and overcome co‐elution issues encountered in one‐dimensional separations.

Objectives and Study Overview

The primary goal was to develop and optimize a normal phase (NP) LC× reversed phase (RP) UHPLC method combined with diode array detection (PDA) and ion trap time‐of‐flight MS (LCMS-IT-TOF) for detailed carotenoid fingerprinting in Capsicum annuum L. The study compared three system configurations differing in 2D column arrangements and modulation times (0.75, 1.50 and 1.00 min). Key performance metrics such as peak capacity, orthogonality and undersampling were evaluated to determine the best compromise between separation power and analysis speed.

Methodology and Instrumentation

Sample Preparation:

• Extraction solvent: methanol/ethyl acetate/petroleum ether (1:1:1, v/v/v).
• Sample matrix: red chili pepper (Capsicum annuum L.).

Instrumentation:

• LC platform: Shimadzu Nexera LC-30A with LC×LC interface (high‐pressure, 6-port switching valves).
• PDA detection: SPD-M20A, 250–550 nm.
• Mass spectrometer: Shimadzu LCMS-IT-TOF with APCI source (positive/negative), mass range 200–1200 m/z.
• Software: LCMSsolution® for system control; Chromsquare® for 2D data visualization.

Chromatographic Conditions:

• 1D column: Ascentis ES Cyano, 250×1.0 mm, 5 µm; mobile phases hexane and hexane/butyl acetate/acetone; gradient 0–65 min from 100% to 0% hexane; flow 10 µL/min.
• 2D column (Set-up 1): Single Ascentis Express C18, 30×4.6 mm, 2.7 µm; IPA/water-ACN gradient; modulation time 0.75 min.
• 2D column (Set-ups 2 and 3): Two serially coupled Ascentis Express C18 columns; modulation times 1.50 and 1.00 min respectively; optimized gradient steps for sharper fractionation.

Main Results and Discussion

Separation Performance:

• Set-up 3 (1.00 min modulation) achieved the highest effective peak capacity (984) by combining improved fractionation and reduced undersampling.
• Orthogonality increased when two C18 columns were serially connected, enhancing separation of structurally similar carotenoids.

Structural Elucidation:

• LCMS-IT-TOF discriminated isobaric diol-monoketo-diesters (e.g. compounds 11 and 12) by accurate mass (error <5 ppm) and MS/MS fragmentation patterns indicating fatty acid losses.
• Fast polarity switching enabled simultaneous detection of positive and negative ions, supporting trace‐level analysis and confident identification.

Comprehensive Profiling:
Up to 33 carotenoids, both free and esterified, were annotated without external standards. Contour plots illustrated the grouping of carotenoids into ten classes by polarity in 1D and hydrophobicity in 2D, with elution order reflecting fatty acid chain length.

Benefits and Practical Applications

The optimized NP-LC×RP-UHPLC/MS workflow offers:

• Superior resolution for complex carotenoid mixtures.
• High mass accuracy and MSn fragmentation for unambiguous structure assignment.
• Robust quantification of low‐abundance pigments.
• Potential for quality control in food processing, authentication of chili pepper varieties and nutritional profiling.

Future Trends and Opportunities

Further enhancements and applications may include:

• Integration with ultrahigh‐resolution MS (Orbitrap or FT-ICR) for isotope fine‐structure analysis.
• Miniaturized 2D systems to reduce solvent consumption and enhance sensitivity.
• Automated data processing using machine learning for rapid peak annotation.
• Expansion to other botanicals, functional foods and breeding programs targeting carotenoid-rich cultivars.

Conclusion

The refined NP-LC×RP-UHPLC coupled with LCMS-IT-TOF provides a powerful platform for in‐depth carotenoid fingerprinting in red chili pepper. Optimized modulation strategies and serial 2D column configurations achieved high peak capacity and orthogonality, enabling reliable identification of 33 compounds. This approach supports advanced quality control, nutritional studies and research into plant pigment diversity.