Simultaneous estimation of Artemisinin and its precursor Artemisinic Acid in Artemisia annua L. by HPTLC

Artemisinin (AN) and artemisinic acid (AA), valuable phyto-pharmaceutical molecules, are well known anti-malarials but now their activities against diseases like cancer, schistosomiasis, HIV, Hepatitis B, and Leishmaniasis are also reported. For the simultaneous estimation of AN and AA in the callus and leaf extract of A. annua L. plants, we embarked on a simple, rapid, selective, reliable and fairly economical HPTLC method. Experimental conditions such as band size, chamber saturation time, migration of solvent front, slit width etc. were critically studied and the optimum conditions were selected. The separations were achieved using toluene: ethyl acetate 9:1 (v/v) as mobile phase on pre-coated silica gel plates G 60F₂₅₄. Good resolution was achieved with R_f 0.35 ± 0.02 and 0.26 ± 0.02 at 536nm for AN and 626nm for AA, respectively in absorption-reflectance mode. The method displayed a linear relationship with r² value 0.997 and 0.995 for AN and AA, respectively, in the concentration range of 100 – 1500 ng for AN and 100 – 1000 ng for AA. The method was validated for specificity by obtaining UV in-situ overlay spectra and sensitivity by estimating LOD (30 ng for AN and 15 ng for AA) and LOQ (80 ng for AN and 45 ng for AA) values. The accuracy was checked by recovery studies conducted at three different levels (25, 50, and 75%) and the average percentage recovery was found to be 101.99% for AN and 103.84% for AA. The precision was analysed by inter-day and intra-day precision and was found to be 5.64 and 3.25% RSD for AN and 7.03 and 3.66% RSD for AA. The analysis of statistical data substantiate that this HPTLC method can be used for the simultaneous estimation of AN and AA in biological samples.