[Separation of levonorgestrel, progesterone and testosterone by micellar electrokinetic capillary chromatography]

Separation of levonorgestrel, progesterone and testosterone was achieved by using micellar electrokinetic capillary chromatography (MECC). A column of 53 cm (to the detector) x 50 microns i.d. uncoated fused-silica capillary (totally 68 cm in length) and an ultra-violet detector with fixed wavelength at 254 nm were used throughout all the experiments. MECC analysis was optimized by evaluating three different micelle-forming agents, the concentration of SDS and several of organic additives in a 10 mmol/L borate running buffer (pH 9.2). Complete separations were obtained with either 10%-20% acetonitrile or 20 mmol/L 2,6-dimethyl-beta-cyclodextrin (DM-beta-CD) as the modifier in buffer. When the acetonitrile volume fraction was in the range of 0-15%, the migration times of the three steroids increased with the acetonitrile volume fraction, but in the range of 15%-20% acetonitrile concentrations, the situation was opposite. This behavior of the steroids was attributed to the interaction of two opposite effects, an increased mobility due to decreased partition coefficient and a decreased electroosmotic flow (EOF). Both beta-cyclodextrin (beta-CD) and gamma-cyclodextrin (gamma-CD) were found to be inadequate for a satisfactory separation.