Optimization of a Solid-Phase Extraction Protocol for Fractionation of Selected Steroids Using Retention Data from Micro Thin-layer Chromatography

In this paper a simple protocol is described for estimating of solid-phase extraction (SPE) elution volumes of steroids based on retention data generated from micro-planar chromatography. Particularly, the retention of selected steroids, including estrogens and progestagens, was studied on wettable with water octadecylsilica HPTLC plates and mobile phases composed of methanol:water mixtures ranging from 20 to 100% (v/v). It was found that TLC retention data can be linearized by plotting RM values of steroids against a reciprocal form of the organic modifier molar fraction (1/Xs). Using such a mathematical approach, the retention parameter of steroids investigated could be easily back-calculated for a wide range of mobile-phase compositions, using few initial experimental data points. The hold-up time of SPE cartridges filled with 0.5 g of C-18 adsorbent was determined experimentally, and appropriate retention factor values (kSPE) for components of interest studied were calculated. Using an appropriate slope and intercept coefficients of the linear-regression equation formed as log kSPE = aRM + b, the steroids SPE elution volumes were predicted beyond the experimental data range that was available for a solid-phase extraction experiment, particularly for mobile phases that contained a high level of water.