Development of the HPLC-DAD method for chromone determination and standardization in Harrisonia perforata (Blanco) Merr. and Thai herbal Ya-Ha-Rak formulation

Harrisonia perforata (Blanco) Merr. (H. perforata) is a plant material used in the Ya-Ha-Rak formula for the treatment of fever in COVID-19 patients in Thailand and contains two chromones as major active compounds, namely O-methylalloptaeroxylin (OML) and peucinin-7-methyl ether (P7ME). In Thailand, however, there is currently no standard quality control method available for this plant. This study aims to develop and validate analytical methods to measure levels of OML and P7ME in samples including branches, roots, and extracts of H. perforata, and Ya-Ha-Rak formulations. In the development process, a number of columns and elution conditions to determine OML and P7ME in the Ya-Ha-Rak formulations and H. perforata raw materials were investigated. Ideal conditions, including C18, highpurity silica at 4.6x150 mm, 3.5 mu m particle size, controlling the column oven at 40 degrees C, and eluting with an acidic mobile phase, significantly improved the separation of OML and P7ME from other elutes in the chromatogram with good suitability parameters. The validation method gave good values for the correlation coefficient (R-2 = 0.9999) for both compounds. The limits of detection (LOD) and quantification (LOQ) were in the ranges of 1.31 and 1.03 mu g/ml and 4.37 and 3.45 mu g/ml, respectively. The recovery percentage fell between 99.97 and 109.75%, and the RSD value of inter-day and intra-day determinations was under 2.0%. The validated method was successfully applied to the analysis of real Ya-Ha-Rak and H. perforata raw material samples from various areas in Thailand.