Simultaneous HPTLC identification of Fig, Senna and P. hybridus using Ion Exchange Chromatography

The commercial product fig film coated tablets with Senna contains fig powder and dry extracts of Senna and P. hybridus as active pharmaceutical ingredients. Until now each active ingredient was identified in a separate analysis. Therefore, a new HPTLC technique for simultaneous identification for fig, Senna and P. hybridus was developed for fig film coated tablets with Senna. Fructose, sennosides A/B and cichoric acid were chosen as suitable markers for the corresponding active ingredients fig, Senna and P. hybridus. Due to the fact that these three markers have different chemical characteristics, a pre-purification step must be performed to separate the active compounds. After extraction using a basic methanol solution, the resulting solution is applied to a cation exchange column. Using pH defined solutions, the three markers are eluted into two different fractions. After application of the fractions onto an HPTLC plate and development of the plate (mobile phase: formic acid:water:2-butanone:ethyl acetate (10:10:40:40) (V:V:V:V)), three different staining solutions are used for visualization. During the first staining, the plate is immersed in natural product reagent followed by PEG400. This step visualizes cichoric acid, which is the marker of P. hybridus, at 366nm. The second immersion step into thymol reagent visualizes the markers fructose and sennosides A/B of fig and Senna, respectively, at Vis light and 366nm. With this method we are, therefore, able to analyze all relevant markers on one HPTLC-Plate. These markers only elute in the corresponding fractions and no co-elution is observed. The technique allows identification of all chosen markers in newly manufactured fig film coated tablets as well as stability samples of the product. Validation of this analytical procedure shows that it is selective, specific and robust.