Ultra-fast separation of estrogen steroids using subcritical fluid chromatography on sub-2-micron particles

Estrogen steroids, represented by estradiol and its related substances, include both structurally very close and simultaneously different analogs. Their separation still remains an analytical challenge. Subcritical fluid chromatography (SbFC) on sub-2-micron particles was found to be an appropriate tool to obtain fast and efficient separation of nine target analytes. Among the four tested stationary phases charged hybrid modified with PFP (pentafluorophenyl) moiety was found to be the most convenient providing the fastest separation within 1.6 min using quick gradient elution with carbon dioxide and methanol as an organic modifier. However, complete separation was obtained also on other tested phases including bare hybrid stationary phase, hybrid stationary phase modified with 2-EP (2-ethylpyridine) and also C18, which is less typical in SbFC. The baseline separation on the latter columns was achieved by means of a temperature increase, a change in organic modifier type and gradient time increase respectively. Quantitative performance was evaluated at optimized conditions and method validation was accomplished. Excellent repeatability of both retention times (RSD<0.15%) and peak areas (RSD<1%) was observed. The method was linear in the range of 1.0-1000.0 μg/ml for all steroids with the lowest calibration point being an LOQ, except for Δ-derivatives, that provided better sensitivity and thus LOQ of 0.5 μg/ml. The sensitivity was sufficient for the analysis of real samples although it was still five times lower compared to UHPLC-UV experiments.