Analysis of Flavanones in Citrus

Significance of the Topic

Flavanones are plant‐derived compounds with notable antioxidant, anti‐inflammatory, and health‐promoting properties. Accurate quantification of these bioactive molecules in citrus fruits is essential for food quality control, nutritional assessment, and research into their physiological effects. A rapid and reliable analytical method enhances throughput in both research laboratories and industrial QA/QC settings.

Objectives and Study Overview

This study aims to develop and validate a high‐throughput reversed‐phase liquid chromatography method for the separation and quantification of five key flavanones—narirutin, naringin, hesperidin, naringenin, and hesperetin B—in citrus samples. The objectives include optimizing chromatographic conditions to achieve baseline resolution within a short run time and demonstrating method robustness and reproducibility.

Methodology and Instrumentation

A Shim-pack XR-ODS column (75 mm × 3.0 mm I.D., 2.2 μm) was used for reversed‐phase separation. The mobile phase consisted of two solvents: (A) 10 mmol/L ammonium formate buffer at pH 3.7 mixed with acetonitrile (9:1, v/v), and (B) the same buffer mixed with acetonitrile (2:8, v/v). A gradient program ramped from 0% B at 0 min to 50% B at 5 min, then to 100% B at 5.01 min (held to 6 min), followed by re-equilibration to 0% B by 9 min. Chromatographic parameters included a flow rate of 1.0 mL/min, column temperature of 35 °C, injection volume of 2 μL, and UV detection at 258 nm using a semi-micro flow cell.

Used Instrumentation

• Shimadzu LC system equipped with Shim-pack XR-ODS column (75 mm × 3.0 mm, 2.2 μm)
• UV detector set at 258 nm with semi-micro flow cell
• Data acquisition and control software for gradient programming

Results and Discussion

The optimized gradient provided baseline separation of all five flavanones within a total runtime of 9 minutes. Retention times were well‐resolved, demonstrating sharp peak shapes and minimal carryover. The method showed high precision, with relative standard deviations below 2% for peak area and retention time across replicate injections. Detection limits were sufficient for typical concentrations found in citrus extracts, supporting quantitative analysis without sample concentration steps.

Benefits and Practical Applications

• Rapid analysis increases sample throughput in research and quality control laboratories.
• High resolution and reproducibility support reliable quantification of flavanones in complex matrices.
• Method simplicity facilitates routine implementation without extensive user training.
• Applicability to nutritional studies, product labeling, and regulatory compliance in the food industry.

Future Trends and Possibilities

Emerging directions include coupling this LC method with mass spectrometry for enhanced sensitivity and specificity, implementing green solvent alternatives to reduce environmental impact, and integrating automated sample preparation to further boost throughput. Advances in high‐pressure UHPLC columns may allow even shorter runtimes and improved separation efficiency.

Conclusion

This study presents a fast, robust, and reproducible LC‐UV method for the analysis of key flavanones in citrus fruits. The optimized gradient on a Shim-pack XR-ODS column achieves clear separation within minutes, offering a practical tool for food science research and industrial quality control.