Analysis of Kakkontousing Comprehensive Two-Dimensional LC Nexera-e

Importance of the Topic

Kakkonto is a complex herbal formulation widely used in traditional Chinese medicine for its anti‐inflammatory and analgesic properties. Its multi‐constituent nature presents analytical challenges when determining active marker compounds such as glycyrrhizic acid. High‐resolution two‐dimensional liquid chromatography (2D‐LC) offers enhanced separation power to resolve coeluting components and deliver accurate quantitation in quality control and research settings.

Objectives and Study Overview

This study aimed to demonstrate the capability of the Shimadzu Nexera-e system for comprehensive 2D‐LC analysis of Kakkonto extracts. Specifically, the work focused on (1) establishing separation conditions that maximize resolution of glycyrrhizic acid in a complex matrix, (2) validating the method’s repeatability and linearity for glycyrrhizic acid quantitation, and (3) applying the protocol to three commercial extract granule products to compare their glycyrrhizic acid content.

Methodology and Instrumentation

The analytical setup employed the Nexera-e comprehensive 2D‐LC system configured with:

• First-dimension column: Shim-pack XR-ODSⅡ, 100 mm×1.5 mm, 2.2 µm, neutral phosphate buffer (pH 6.9)/acetonitrile gradient at 0.05 mL/min.
• Second-dimension column: Shim-pack Velox C18, 50 mm×2.1 mm, 2.7 µm, acidic phosphate buffer (pH 2.7)/acetonitrile gradient at 1.5 mL/min.
• Modulation by two 50 µL loops with 1-minute cycle, column temperature 40 °C, UV detection at 254 nm.

The auto-gradient function in LabSolutions software synchronized 2D gradients with the 1D elution to counteract solvent‐strength effects. Sample preparation involved extracting 1.5 g granule in 50% methanol, centrifugation, filtration, and 2 µL injection.

Main Results and Discussion

Repeatability testing of a 50 mg/L glycyrrhizic acid standard yielded %RSD of 0.01 for 1D retention time, <0.01 for 2D retention time, and 3.6% for blob area. Calibration over 50–1000 mg/L was highly linear (r2=0.9996). Two-dimensional contour plots clearly isolated the glycyrrhizic acid peak from other matrix components. Application to three commercial Kakkonto samples showed distinct chromatographic fingerprints and quantified glycyrrhizic acid at approximately 529 mg/L, 558 mg/L, and 983 mg/L after sample preparation.

Benefits and Practical Applications

The Nexera-e 2D‐LC approach offers:

• Superior separation of overlapping herbal constituents compared to 1D‐LC.
• High precision and accuracy in glycyrrhizic acid quantitation suitable for QC laboratories.
• Rapid screening of multiple product formulations, facilitating comparative quality assessments.

Future Trends and Possibilities

Emerging developments in two‐dimensional LC include integration with high‐resolution mass spectrometry for structural elucidation of unknown compounds, miniaturized 2D platforms for reduced solvent consumption, and AI‐driven method optimization to streamline complex gradient programming. Advances in green chromatography may further reduce the environmental footprint of herbal analysis.

Conclusion

The Shimadzu Nexera-e system demonstrated robust performance for comprehensive 2D‐LC analysis of Kakkonto extracts. Method validation confirmed excellent repeatability and linearity for glycyrrhizic acid quantitation, and application to commercial products highlighted the technique’s utility in herbal quality control.

References

No additional literature was provided in the source document.