Analysis of Naringin and Neohesperidin in Fructus Aurantii

Significance of the Topic

Flavanone glycosides such as naringin and neohesperidin are key bioactive compounds in citrus‐derived herbal medicines. Reliable quantification of these markers is essential for quality control, standardization of Fructus Aurantii preparations and ensuring consistency in pharmaceutical, nutraceutical and food applications.

Objectives and Study Overview

The goal of this study was to develop and validate a reversed‐phase HPLC method for simultaneous determination of naringin and neohesperidin in Fructus Aurantii extracts. The method employs a Shimadzu Shim-pack VP-ODS column and UV detection, aiming for simple sample preparation, good resolution and reproducible quantitation.

Methodology and Instrumentation

Sample Preparation:

1. Weigh 0.20 g of powdered Fructus Aurantii and transfer to a stoppered flask.
2. Add 50 mL of 50 % methanol, close and reweigh to account for solvent loss.
3. Reflux at 80 °C for 1.5 h, cool and reweigh.
4. Adjust volume to original weight with 50 % methanol, shake and filter.
5. Transfer 10 mL filtrate to a 25 mL volumetric flask, dilute to volume with methanol and mix.

Instrumentation and Chromatographic Conditions:

• Column: Shim-pack VP-ODS (250 × 4.6 mm i.d., 5 μm)
• Mobile Phase: Acetonitrile–Water (20:80) with phosphoric acid adjusted to pH 3.0
• Flow Rate: 1.0 mL/min
• Column Temperature: 40 °C
• Injection Volume: 10 μL
• Detection: UV at 283 nm
• Vial: LabTotal™ vial for LC/LCMS

Main Results and Discussion

Baseline separation of naringin and neohesperidin was achieved within a single run. Retention times were reproducible with relative standard deviations below 1 %. Calibration curves were linear over the tested range (10–100 μg/mL) with correlation coefficients (r2) exceeding 0.999. Recovery studies showed accuracies between 98 % and 102 %, indicating minimal matrix interference. Chromatograms of standard mixtures and Fructus Aurantii extracts demonstrated clear peak identity and absence of coeluting impurities.

Benefits and Practical Applications

This method offers:

• Rapid and robust quantification of key flavanone glycosides in herbal matrices
• Simple sample preparation suitable for routine quality control laboratories
• High reproducibility and sensitivity without the need for complex derivatization

It can be directly applied in pharmaceutical QC departments, herbal supplement manufacturing and regulatory compliance testing.

Future Trends and Potential Applications

Advances may include adoption of ultra-high-performance liquid chromatography (UHPLC) for faster analysis times, coupling with mass spectrometry for enhanced specificity, and implementation of green extraction techniques to reduce solvent consumption. Integration with online sample preparation and automated data processing will further streamline workflows.

Conclusion

The described RP-HPLC method using a Shim-pack VP-ODS column provides precise, accurate and efficient determination of naringin and neohesperidin in Fructus Aurantii. Its operational simplicity and strong performance characteristics make it well suited for widespread use in quality control and research settings.

Reference

Shimadzu Corporation. ERAS-1000-0066, First Edition March 2023.