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),22E-trien-3β-ol, and betulinic acid in Clerodendrum inerme

• Published in: Journal of separation science 2007-08, Vol.30 (13), p.2086-2091
• Main Authors: Pandey, Richa, Verma, Ram K, Gupta, Madan M
• Format: Article
• Language: English
• Subjects: absorption; aerial parts; betulinic acid; Clerodendrum inerme; densitometry; detection limit; HPTLC; quantitative analysis; silica gel; Sterols; thin layer chromatography; Triterpene
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.200700073

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),22E-trien-3β-ol (2), and a triterpene, betulinic acid (3), in Clerodendrum inerme extract. The method employed HPTLC plates precoated with silica gel 60F₂₅₄ as the stationary phase. To achieve good separation, an optimised mobile phase consisting of toluene-acetone (94:06, v/v) was used (Rf 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.