Characterization of 16 Capsicum Varieties by Evaluation of Their Carotenoid Profle by SFE-SFC-MS/MS

Significance of the topic

The accurate profiling of carotenoids in Capsicum species has wide implications for food science, agriculture and health. Carotenoids are responsible for the vibrant colors of peppers and possess antioxidant properties that contribute to nutritional value. Rapid and solvent-efficient analytical methods are needed to support breeding programs, quality control, authenticity assessment and nutritional studies.

Objectives and study overview

This work aimed to develop a fully online coupling of supercritical fluid extraction and chromatography with triple quadrupole mass spectrometry (SFE-SFC-MS/MS) for the direct characterization of native carotenoids in 16 distinct Capsicum varieties. The goal was to achieve comprehensive identification of free carotenoids, monoesters, diesters and chlorophylls in one run lasting under 20 minutes, enabling varietal discrimination based on their pigment profiles.

Methodology and instrumentation

Sample preparation involved homogenizing 1 g of fresh pepper with an adsorbent powder, loading 50 mg equivalent into a 0.2 mL extraction vessel and performing static and dynamic supercritical CO2 extraction with methanol as co-solvent. Chromatographic separation used a C30 column (150×4.6 mm, 2.7 μm) with CO2/methanol gradient at 2 mL/min and 35 °C. Detection employed a Shimadzu NEXERA-UC platform combining SFE-30A, SFC-30A back pressure regulators and an LCMS-8050 with APCI source, controlled by LabSolutions 5.80. Multiple scan modes (SCAN, SIM, MRM) in positive and negative APCI captured structural and ester fragmentation patterns.

Main results and discussion

Using the optimized SFE-SFC-MS/MS workflow, 41 carotenoids and chlorophylls were identified across the 16 pepper cultivars. The number of detected compounds varied from 4 in Aji Capsicum baccatum to 23 in Erotico Capsicum baccatum. Capsanthin was ubiquitous except in the yellow-fruited Aji sample, while specific mono- and diesters served as markers for individual varieties. Naga Chocolate Capsicum chinense displayed the richest diester content, highlighting the method’s power to reveal subtle compositional differences.

Benefits and practical applications

• Complete native profiling without saponification preserves carotenoid esters and chlorophyll derivatives.
• Run times below 20 minutes support high throughput screening in breeding and quality control.
• Reduced organic solvent use aligns with green analytical chemistry principles.
• Detailed pigment fingerprints enable cultivar authentication and nutritional assessment.

Future trends and potential uses

Expansion of online SFE-SFC-MS/MS to other classes of phytochemicals (e.g., phenolics, terpenes) can further enhance comprehensive food profiling. Integration with chemometric and machine-learning tools will support rapid classification, authenticity verification and breeding selection. Advances in greener co-solvents and miniaturized instrumentation may further lower ecological impact and enable on-site analysis in agricultural settings.

Conclusion

The developed SFE-SFC-MS/MS method offers a fast, efficient and environmentally friendly platform for detailed native carotenoid characterization in Capsicum varieties. It successfully differentiated 16 cultivars based on their pigment profiles and identified 41 compounds in under 20 minutes, demonstrating its suitability for diverse applications in food research, quality control and agricultural development.