Multiple Analytical Techniques For Carotenoid Analysis In Capsicum Cultivars

Importance of the Topic

Carotenoids are essential natural pigments and antioxidants with complex structural diversity, including free and esterified forms. Precise profiling of these compounds is critical for food quality control, nutritional evaluation, authenticity verification, and assessment of crop ripeness and oxidative stability.

Objectives and Study Overview

This study compares and develops three advanced analytical platforms for the direct analysis of native carotenoids in Capsicum cultivars, avoiding saponification: 1) an on-line supercritical fluid extraction–supercritical fluid chromatography–mass spectrometry (SFE-SFC-MS) system, 2) reversed-phase HPLC with serially coupled C30 columns, and 3) innovative normal-phase×reversed-phase two-dimensional (NP-LC×RP-LC and NP-LC×UHPRP-LC) separations.

Methodology and Instrumentation

Three complementary approaches were implemented:

• SFE-SFC-MS: Shimadzu Nexera UC with SFE-30A extraction vessel, CO₂/methanol gradient, Core‐Shell C30 SFC column, APCI-triple quadrupole mass spectrometer (positive/negative modes, MRM).
• RP-HPLC: Shimadzu HPLC with two serial YMC C30 reversed-phase columns (250×4.6 mm, 5 µm), PDA detection at 450 nm (250–600 nm full scan), APCI-APCI-MS.
• NP-LC×RP-LC & NP-LC×UHPRP-LC: Shimadzu Nexera-e system with cyano 1D microbore column (250×1.0 mm, 5 µm), fused-core C18 2D column(s) (30×4.6 mm, 2.7 µm), two high-pressure six-port switching valves, PDA and IT-TOF MS (APCI) detection.

Main Results and Discussion

The SFE-SFC-MS workflow completed extraction and chromatographic separation in 18 min, identifying over 52 native carotenoids and chlorophyll derivatives, including mono- and di-acyl esters. The RP-HPLC method with serial C30 columns doubled peak capacity (from 73 to 95) and resolved critical isomeric pairs. The NP-LC×RP-LC system achieved a theoretical 2D peak capacity of 1395; its UHPLC-based variant with 1.00 min modulation afforded a practical peak capacity of 984, enabling separation of 33 carotenoids in 10 chemical classes (hydrocarbons, mono-ols, di-ols, epoxides, ketones, mono- and di-esters, and polyoxygenated xanthophylls).

Benefits and Practical Applications

These methods deliver rapid, high-precision, and low-contamination workflows for comprehensive carotenoid profiling in food matrices. They support quality assurance, authenticity testing, ripeness evaluation, and stability studies without laborious saponification steps.

Future Trends and Opportunities

Future developments may include integration with high-resolution mass spectrometry for structural elucidation, automated data processing using machine learning, expanded multidimensional separation strategies, enhanced reference libraries, and applications to broader biological and industrial samples.

Conclusion

A multimodal analytical strategy combining SFE-SFC, high-resolution RP-HPLC, and comprehensive LC×LC provides robust, high-throughput, and detailed characterization of native carotenoids in Capsicum, facilitating diverse research and industrial applications.

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