Simultaneous quantification of multiple marker compounds in herbal medicine by high performance liquid chromatography (HPLC) analysis is still a challenge due to the complexity in various parameters to be considered and co-existing multi-components. As a case study, a reliable HPLC method for simultaneous quantification of paeoniflorin from Paeoniae Radix and decursin from Angelicae Gigantis Radix in various commercial herbal medicine was developed based on analytical quality by design (AQbD) strategy. As a first step, risk assessment was performed to select the critical method parameters (CMPs) which were decided as organic mobile phase ratio and column oven temperature. In order to evaluate the effect of the CMPs on critical method attributes (CMAs) of peak resolution and tailing, central composite design (CCD) was employed. The final chromatographic conditions were optimized as follows: column- C 18 , 4.6 × 250 mm, 5 μm particle size; mobile phase- A: acetonitrile, B: 0.1% acetic acid water; detection wavelength- 235 nm for paeoniflorin, 325 nm for decursin; column oven temperature- 25 o C; flow rate- 1.0 mL/min; gradient mobile phase system as Time (min) : % A, 0:14, 25:14, 30:50, 60:50, 61:100, 65:100, 66:14, 75:14. The method was successfully validated according to the International Conference on Harmonization (ICH) guidelines and piloted for ten commercial herbal medicines.

In this study, we have successfully established a high-performance thin-layer chromatography (HPTLC) method for the quality assessment of Actinidiae Fructus Vermicultus, known as Mokcheonryo(ja) in Korea. This is the dried vermiculate fruit of Actinidia polygama and A. kolomikta, as stipulated by the Korean Herbal Pharmacopoeia (KHP). However, the Korean herbal market often witnesses the inclusion and distribution of ‘Mihudo’, an alternative herbal product sourced from the dried fruits of A. arguta, belonging to the same botanical genus. This confluence has raised substantial apprehensions concerning the veracity of quality. In response to this concern, we have meticulously developed an HPTLC analytical methodology capable of differentiation between Mokcheonryo and Mihudo by exploiting their distinct chemical profiles. We identified umbelliferone as a key marker compound for Mokcheonryo and quantified the content of umbelliferone in each sample using a TLC scanner. Throughout this study, we confirmed distinct fingerprints for Mokcheonryo and Mihudo, providing a reliable means to differentiate between these two herbal medicines. Furthermore, the presence of umbelliferone in Mokcheonryo serves as an indicator compound for quality assessment. The proposed HPTLC method offers a practical and effective tool for ensuring the quality and authenticity of Mokcheonryo in the herbal market.