Quantitative Analysis of Rosmarinic Acid in Dry Extract of Orthosiphon aristatus (Blume) Miq. Leaves utilizing Ultra-High Performance Liquid Chromatography.

Abstract

      This study aimed to determine rosmarinic acid content in the dry extract of Orthosiphon aristatus (Blume) Miq leaves utilizing Ultra-High Performance Liquid Chromatography (UHPLC). The research was divided into three parts: 1) development of an extraction method of the dry extract from O. aristatus leaves using sonication and reflux extraction with ethanol; 2) validation of the analytical method using Ultra-High Performance Liquid Chromatography (UHPLC) in accordance with ICH guideline; 3) determination of rosmarinic acid content in the dry extract.The optimal extraction condition of preparation of the dry extract to obtain high rosmarinic acid content was determined to be sonication extraction with 70% v/v ethanol for 70 minutes. The optimized chromatographic system utilized a C18 column (4.6 x 150 mm, 5 µm) as the stationary phase. The mobile phase consisted of an isocratic mixture of acetonitrile and 0.1% formic acid in water (30:70), with a flow rate of 0.5 ml/min and detection at 330 nm. Under these conditions, the peak for rosmarinic acid appeared at a retention time of 9.6–9.7 minutes. The method demonstrated high specificity and excellent linearity within the concentration range of 20–160 µg/ml.The analysis of 12 samples of the extract revealed a rosmarinic acid content ranging from 1.34 % to 4.13 % w/w and the mean was 2.09+0.83 %w/w. The developed analytical method offers significant advantages over previously reported methods due to its simplicity and reduced time required for both sample and mobile phase preparation.