Abstract

VALIDATION OF HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY DENSITOMETRY METHOD FOR QUANTITATIVE DETERMINATION OF USNIC ACID IN ETHYL ACETATE EXTRACTS OF USNEA SP

Regina Andayani*, Amri Bakhtiar, Friardi and Aulia Dwi Putri

ABSTRACT

This research aims to determine usnic acid content in ethyl acetate extracts of six usnea sp (Usnea mekista (Stirt.) G. Awasthi, Usnea baileyi (Stirt.) Zahlbr. Usnea articulata (L.) Hoffm, Usnea longissima Ach. Usnea perplexans Stirt., Usnea molliuscula Stirt.) using validated High Performance Thin Layer Chromatography Densitometric Method. The thallus of lichen was extracted by maceration with ethyl acetate. Ethyl acetate extracts were evaporated by rotary evaporator. Separation was carried out on HPTLC silica gel 60 F254 plate as a stationary phase using a mixture of ethyl acetate-hexane (7:3 v/v) as the mobile phase. Detection was performed densitometrically in absorbance mode at 282 nm. Usnic acid standard solution in the concentration range of 50-500 μg/mL shows linearity with a correlation coefficient (r) = 0.9999. Limit of detection and limit of quantitation obtained were 5.20 μg/mL and 17.32 μg/mL, respectively. Precision of the method was evaluated by intraday and interday relative standard deviation (RSD). The results were intraday RSD of peak response 0.06-0.70% and interday RSD of peak response 0.25-1.16%. Accuracy was determined by calculating the percentage recovery at three concentration levels. The results were 98.37%, 97.54% and 99.22% respectively. Usnic acid contents found in Usnea mekista (Stirt.) G. Awasthi, Usnea baileyi (Stirt.) Zahlbr. Usnea articulata (L.) Hoffm, Usnea longissima Ach. Usnea perplexans Stirt., Usnea molliuscula Stirt. were 51.26%, 10.40%, 16.56%, 38.64%, 12.13%, and 50.92% respectively. The highest usnic acid content was found in the Usnea mekista species with a level of 51.26%. This HPTLC method was validated in accordance with International Conference on Harmonization(ICH) guideline requirements and proven to provide appropriate results.

Keywords: Usnic Acid; Usnea sp; Validation; HPTLC Densitometry.