High-performance thin-layer chromatographic method for quantitative determination of 4alpha-methyl-24beta-ethyl-5alpha-cholesta-14,25-dien-3beta-ol, 24beta-ethylcholesta-5,9(11),22E-trien-3beta-ol, and betulinic acid in Clerodendrum inerme

A sensitive, selective, precise, and robust high-performance thin-layer chromatography method was developed and validated for analysis of two new recently isolated sterols, 4alpha-methyl-24beta-ethyl-5alpha-cholesta-14,25-dien-3beta-ol (1) and 24beta-ethylcholesta-5,9(11),22E-trien-3beta-ol (2), and a triterpene, betulinic acid (3), in Clerodendrum inerme extract. The method employed HPTLC plates precoated with silica gel 60F(254 )as the stationary phase. To achieve good separation, an optimised mobile phase consisting of toluene-acetone (94:06, v/v) was used (R(f )0.48, 0.34, and 0.22 for compounds 1, 2, and 3, respectively). Densitometric determination of the above compounds was carried out in reflection/absorption mode at 620 nm. Optimised chromatographic conditions provide well separated compact spots for the compounds 1, 2, and 3. The calibration curves were linear in the concentration range of 100-2500 ng/spot. The method was validated for precision, robustness, and recovery. The limits of detection and quantitation were 5, 6, and 10 microg/mL and 14, 18, and 29 microg/mL, respectively, for 1, 2, and 3. The method reported here is reproducible and convenient for quantitative analysis of these compounds in the aerial parts of C. inerme.