HPTLC method for the simultaneous determination of four indole alkaloids in Rauwolfia tetraphylla: a study of organic/green solvent and continuous/pulse sonication.

A new validated high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitation of four antipsychotic indole alkaloids (IAs), reserpiline (RP, 1), α-yohimbine (YH, 2), isoreserpiline (IRP, 3) and 10-methoxy tetrahydroalstonine (MTHA, 4) as markers in the leaves of Rauwolfia tetraphylla. Extraction efficiency of the targeted IAs from the leaf matrix with organic and ecofriendly (green) solvents using percolation, ultrasonication and microwave techniques were studied. Non-ionic surfactants, viz. Triton X-100, Triton X-114 and Genapol X-80 were used for extraction and no back-extraction or liquid chromatographic steps were used to remove the targeted IAs from the surfactant-rich extractant phase. The optimized cloud point extraction was found a potentially useful methodology for the preconcentration of the targeted IAs. The separation was achieved on silica gel 60F(254) HPTLC plates using hexane-ethylacetate-methanol (5:4:1, v/v/v) as mobile phase. The quantitation of IAs (1-4) was carried out using the densitometric reflection/absorption mode at 520 nm after post chromatographic derivatization using Dragendorff's reagent. The method was validated for peak purity, precision, accuracy, robustness, limit of detection (LOD) and quantitation (LOQ). Method specificity was confirmed using retention factor (R(f)) and visible spectral (post chromatographic scan) correlation of marker compounds in the samples and standard tracks.