Development and Validation of Analytical Method for Quantitative Analysis of Rosmarinic Acid from Orthosiphon aristatus (Blume) Miq. Leaves and Its Finished Products by Ultra-High Performance Liquid Chromatography
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Abstract
Introduction and Objectives: Orthosiphon aristatus (Blume) Miq., commonly known as Java tea, is currently listed in the Thai National List of Essential Medicines in the form of herbal tea and is also commercially available as capsule. However, the current Thai Herbal Pharmacopoeia (THP) lacks an official content specification for the quantitative determination of active marker in both the raw material and its finished products. This study aimed to develop and validate an analytical method for rosmarinic acid content in this medicinal plant and its finished products, namely, capsule and tea.
Methods: This research was divided into three parts: 1) development of an extraction method for rosmarinic acid from O. aristatus using reflux extraction with 50% v/v 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 raw material and its finished products.
Results: The optimal extraction condition was determined to be reflux extraction with 50% v/v ethanol for 30 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 UV 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 10–140 µg/ml, with a correlation coefficient (r) ≥ 0.9999. Precision studies showed %RSD values between 0.6–1.5, while accuracy was confirmed with % recovery ranging from 96–101. Furthermore, the method exhibited robustness against variations in mobile phase proportions, flow rate, and column temperature. The analysis of 14 samples of Java Tea revealed a rosmarinic acid content ranging from 0.35% to 2.30% w/w and the mean was 0.60+0.16 %w/w. The rosmarinic acid contents of the capsule and the tea ranged from 0.37 % to 0.44 %w/w and from 0.22 % to 0.61 %w/w, respectively.
Discussion: 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. It is applicable for the quantification of rosmarinic acid in both raw material and its commercial products.
Conclusion: This validated method serves as a valuable tool for not only determining the rosmarinic acid content but also standardizing the rosmarinic acid content in the O. aristatus monograph including its commercial products for future publication of the Thai Herbal Pharmacopoeia.
Keywords: Rosmarinic acid, Orthosiphon aristatus (Blume) Miq., Ultra-High Performance Liquid Chromatography
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