Analysis of Free Anthraquinone in Rhubarb

Importance of Topic

Anthraquinones are key bioactive compounds in rhubarb, widely used in traditional herbal medicines and modern formulations. Accurate quantification of free anthraquinones supports quality control, standardization of herbal extracts and ensures safety and efficacy in phytopharmaceutical applications.

Objectives and Study Overview

The study presents a reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous analysis of five free anthraquinones in rhubarb: aloe-emodin, rhein, emodin, chrysophanol and emodin methyl ether. The aim is to establish a rapid, reliable and reproducible protocol suitable for routine quality assurance in herbal laboratories.

Methodology and Used Instrumentation

A Shim-pack GIS C18 column (250 mm × 4.6 mm I.D., 3 μm) was employed under the following chromatographic conditions:

• Mobile phase: 0.1% phosphoric acid : methanol (15 : 85, v/v)
• Flow rate: 0.8 mL/min
• Column temperature: 40 °C
• Injection volume: 20 μL
• Detection: UV at 254 nm

Sample preparation involved reflux extraction of powdered rhubarb (0.15 g) in 25 mL methanol for one hour, followed by volume adjustment, filtration and direct injection.

Main Results and Discussion

The method achieved baseline separation of all five anthraquinones within a single run. Retention times were consistent and peak shapes sharp, indicating high resolution and minimal coelution. The calibration curves for each analyte displayed excellent linearity over the tested concentration ranges, and the extraction procedure provided satisfactory recovery rates, demonstrating the method’s accuracy and precision.

Benefits and Practical Applications

This HPLC protocol offers several advantages for analytical laboratories:

• High throughput suitability for batch analysis of herbal samples
• Robust performance with reproducible retention times and peak areas
• Simple sample preparation using standard laboratory equipment
• Applicability to quality control, compliance testing and formulation development

Future Trends and Potential Applications

Emerging directions include coupling the method with mass spectrometry for structural confirmation, miniaturization in micro-LC formats to reduce solvent usage, and adoption of greener mobile phases. Integration into automated workflows and digital data management systems will further enhance throughput and traceability in industrial QC settings.

Conclusion

The described reversed-phase HPLC method with Shim-pack GIS C18 provides a reliable, efficient and easily implementable approach for quantifying free anthraquinones in rhubarb. Its strong performance characteristics make it well suited for routine quality assessment in herbal medicine research and production.

Reference

Shimadzu Corporation. First Edition: Dec. 2021, ERAS-1000-0295.