High‐performance thin‐layer chromatographic method for quantitative determination of 4α‐methyl‐24β‐ethyl‐5α‐cholesta‐14,25‐dien‐3β‐ol, 24β‐ethylcholesta‐5,9(11),22 E ‐trien‐3β‐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, 4α‐methyl‐24β‐ethyl‐5α‐cholesta‐14,25‐dien‐3β‐ol ( 1 ) and 24β‐ethylcholesta‐5,9(11),22 E ‐trien‐3β‐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 μg/mL and 14, 18, and 29 μg/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 .