Authentication of Traditional Chinese Prescriptions Using Comprehensive 2D-LC

Significance of the topic

Authentication of traditional Chinese herbal prescriptions is critical to ensure safety and efficacy. The complex composition and natural variability of plant-based formulations make them susceptible to adulteration and counterfeiting. Comprehensive chromatographic fingerprinting offers a holistic quality control strategy by detecting multiple characteristic compounds simultaneously.

Objectives and study overview

This study develops and demonstrates a comprehensive two-dimensional liquid chromatography (2D-LC) approach for authenticating Si-Wu-Tang, a four-herb prescription used in female health. Individual herbs are analyzed separately to build retention time and mass spectral templates of their key constituents. These templates are then matched against the complete prescription to confirm each herb’s presence. The methodology’s sensitivity is further evaluated by omitting or replacing one herb.

Methodology

Decoctions of Si-Wu-Tang and its individual herbs are prepared by cold soak, boiling, centrifugation, and filtration. A comprehensive 2D-LC system uses a reversed-phase SB-Aq column in the first dimension and a Poroshell Bonus-RP column in the second. A 2-position/4-port valve alternates fractions every 30 seconds. UV detection at 254 nm is combined with high-resolution mass spectrometry (Agilent 6530 Q-TOF) operated in positive and negative electrospray modes.

Applied instrumentation

• Agilent 1290 Infinity 2D-LC (binary pumps, autosampler, thermostatted column compartment, diode array detector)
• Agilent 6530 Accurate-Mass Q-TOF LC/MS with Jet Stream ESI source
• Software: OpenLAB CDS ChemStation, 2D-LC Acquisition, MassHunter Workstation, LCxLC-HRMS Edition

Main results and discussion

• Key constituents such as senkyunolide A, Z-ligustilide, ferulic acid, gallic acid, and paeoniflorin were tentatively identified in the individual herbs.
• Retention time–mass spectral templates were generated for each herb.
• In authentic Si-Wu-Tang, over 75 % of template peaks per herb matched in both ionization modes, confirming each component’s presence.
• Omission or substitution of Rhizoma Chuanxiong reduced matched peaks by more than half, demonstrating the method’s specificity for detecting adulteration.
• Nonmatching peaks were mainly due to compound coelution affecting base peak intensities in the complex matrix.

Benefits and practical applications of the method

• Comprehensive 2D-LC fingerprinting enables simultaneous detection of multiple herb-specific markers.
• Enhanced peak capacity reduces coelution, improving selectivity over one-dimensional methods.
• Template matching facilitates rapid authentication and adulteration screening of multi-component herbal formulations.
• The workflow is adaptable to pharmaceutical, nutraceutical, and regulatory quality control environments.

Future trends and potential applications

• Integration with multivariate analysis and machine learning could automate pattern recognition and improve discrimination power.
• Application to other complex botanical and natural product mixtures for comprehensive quality assessment.
• Development of miniaturized or portable 2D-LC platforms for field deployment.
• Coupling with orthogonal detection technologies such as ion mobility spectrometry may further enhance peak capacity and compound identification.

Conclusion

Comprehensive 2D-LC combined with high-resolution mass spectrometry provides a powerful platform for authenticating complex herbal prescriptions like Si-Wu-Tang. The approach of constructing and matching retention time–mass spectral templates of individual herb constituents enables precise verification of formulation composition and sensitive detection of adulteration, supporting routine quality control in traditional medicine industries.

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