Qualitative and Quantitative Analysis of β-carotene Using UPC2

Importance of the Topic

The accurate and rapid analysis of carotenoids such as β-carotene, lutein and lycopene is critical in food quality control, dietary supplement validation and clinical research. These fat-soluble pigments act as antioxidants and precursors to vitamin A in humans. As regulatory requirements tighten and demand rises, efficient methods that reduce analysis time and solvent usage are in high demand.

Objectives and Study Overview

This study aimed to develop and validate an UltraPerformance Convergence Chromatography™ (UPC2) method for the qualitative separation of three major carotenoids and the quantitative analysis of β-carotene in dietary supplement capsules. The goals included minimizing run times, reducing organic solvent consumption and demonstrating accuracy and precision in real samples.

Methodology and Instrumentation

A Waters ACQUITY UPC2 System equipped with a photodiode array detector was used. Key chromatographic conditions were:

• Column: ACQUITY UPC2 HSS C18 SB, 3.0 × 100 mm, 1.8 µm
• Mobile phase: carbon dioxide and ethanol (25% co-solvent) isocratic
• Flow rate: 1.5 mL/min; Column temperature: 40 °C; Back pressure: 2190 psi
• Injection volume: 1 µL; Detection wavelength: 440 nm with 550–600 nm reference

Sample preparation involved dissolving standards or capsule contents directly in methyl tert-butyl ether (MTBE), requiring no evaporation or reconstitution.

Key Results and Discussion

Column screening identified the HSS C18 SB phase as providing baseline resolution of lycopene, β-carotene and lutein in under two minutes. A 25% ethanol isocratic method optimized run time and peak shape. Calibration of β-carotene in MTBE was linear over 0.0001–0.1 mg/mL (R² > 0.9999). Limits of detection and quantitation were 50 ng/mL and 100 ng/mL, respectively. Capsule assays showed recoveries consistent with the 15 mg label claim and demonstrated inter- and intra-assay relative standard deviations below 1%.

Benefits and Practical Applications

• Four-fold faster than conventional reversed-phase HPLC methods
• 85% reduction in organic solvent consumption
• Direct injection of non-polar extracts simplifies workflow
• High sensitivity and reproducibility support routine quality control and compliance testing

Future Trends and Potential Applications

UPC2 could be extended to profile a wider array of carotenoids and other non-polar micronutrients. Coupling UPC2 with mass spectrometry may enhance structural characterization of isomers and degradation products. The method’s speed and green credentials make it promising for high-throughput screening in food, nutraceutical and clinical laboratories.

Conclusion

This work demonstrates that UPC2 provides rapid, sensitive and environmentally sustainable analysis of β-carotene and related carotenoids. The optimized isocratic method delivers high resolution in under two minutes, simplifies sample preparation and maintains excellent accuracy and precision, making it ideal for regulatory and research applications.

Reference

1. Rivera SM and Canela-Garayoa R. J. Chromatogr. A. 2012;1224:1–10.
2. Plozza T, Trenerry VC and Caridi D. Food Chem. 2012;134:559–563.
3. Blake CJ. J. AOAC Int. 2007;90(4):897–910.
4. Hung PV and Hatcher DW. Food Chem. 2011;125:1510–1516.
5. Mitrowska K, Vincent U and von Holst C. J. Chromatogr. A. 2012;1233:44–53.
6. Chauveau-Duriot B et al. Anal. Bioanal. Chem. 2010;397:777.
7. Granado-Lorencio F et al. Anal. Bioanal. Chem. 2010;397:1389–1393.
8. AOAC Official Method 2005.07. Reversed-Phase HPLC Method.