Evaluation of TLC-densitometry assay validation applied to complex matrix

Considering the need to have a fast, cheap and effective method for quality control routine, thin layer chromatography/densitometry has been a chosen method to comply those requests. However, this technique has its limitations when used to determination assays for substances in complex matrices, such as vegetal extracts. Considering this chromatographic technique restriction during the drug development assay, a new procedure was developed to overcome the difficulty in obtaining the validation parameters within the international acceptance criteria, mainly selectivity and repeatability parameters. The developed method was applied in the betulinic acid (BA) quantification from extracts of natural products. All separations were obtained on plates Si60 F254 with n-hexane:ethyl acetate:acetic acid (7:3:0.3) at 560nm, after derivatization with vanillin-sulphuric reagent. The system gave compact spots for BA (Rf. 0.5) and exhibited linearity (R2= 0.999). The methodology was compared with a validated gas chromatography methodology and no statistical difference was observed.