Development and Validation of Analytical Method for Quatitative Analysis of Andrographolide from Androdraphis paniculata Products by High Perfomance Liquid Chromatography

Abstract

Introduction and Objective: Due to the outbreak of coronavirus disease 2019 (COVID-19), Andrographis
paniculata, or Fa Thalai Chon in Thai, has been used to treat this disease, leading to more varieties of A. paniculata
products available in the market. Therefore, an analytical method was developed to determine the andrographolide
content in three variants of A. paniculata products: A. paniculata capsules, A. paniculata extract capsules, and
A. paniculata extract tablets. The method will be used for the quality control of A. paniculata products in the
market.
Methods: The study was divided into two parts: (1) development of a method for extraction of
A. paniculata in products using methanol as a solvent and ultrasonic sonication and (2) validation of the analytical
method according to the ICH Guidelines using High Performance Liquid Chromatography (HPLC) technique with a Zorbax C18 column (4.6 5 150 mm, 5-μm particle size). The mobile phase was a mixture of water and methanol (50:50), with a flow rate of 1.0 mL/min, and detection under UV light at the 224 nm wavelength.
Results: The 5-minute methanol extraction of A. paniculata followed by a 15-minute extraction with 50% methanol, coupled with ultrasonic sonication, yielded an andrographolide content comparable to that achieved
using the Thai Herbal Pharmacopoeia 2021 extraction method. This method demonstrated specificity and a linear
relationship for the concentrations ranging from 0.02 to 0.14 mg/mL, with a correlation coefficient (r) of ≥ 0.9999.
Precision, represented by %RSD, ranged from 0.2% to 1.1%, while accuracy, measured by percentage recovery
fell within the 95% to 99% range. Moreover, the method exhibited robustness against variations in mobile phase
ratio, flow rate, and column temperature.
Discussion: The comparison between the reflux method and ultrasonic sonication method revealed that the latter is a viable alternative, offering ease of use, convenience, and elimination of carcinogenic chemicals like dichloromethane. Additionally, it reduced both the analytical time and extraction steps for andrographolide
extraction.
Conclusion: The developed HPLC method is suitable for analyzing andrographolide content in three variations
of A. paniculata products (capsules, extract capsules and extract tablets), encompassing all products found in the market