Development of HPLC/fluorescence detection method for chiral resolution of dansylated benzimidazoles derivatives.

The development of high performance liquid chromatography method on amylose-based stationary phase (Chiralpak AD) with n-hexane-2-propanol (90:10, v/v) as mobile phase allowed the enantioseparation of non-dansylated and dansylated benzimidazole derivatives, prepared from potent-AMPK activators, to be achieved. Using both fluorescence and ultraviolet detection, limits of detection and quantification were determined. Fluorescence detection seems to be the most appropriate technique since the first enantiomer of dansylated benzimidazole 8 eluted with a limit of quantification of 2.25 nm. The quantification limit was improved 10-fold by fluorescence detection compared with a previous report describing mass spectrometry detection. Linearity and repeatability parameters were validated. These lower limits obtained by fluorescence detection, associated with good resolution observed for the dansylated derivatives, make this chiral methodology convenient for the use of these fluorescent potent-AMPK activators as probes to elucidate their cellular localization and their mechanism of action.