Separation of urinary estrogens by micellar electrokinetic chromatography

Urinary estrogen levels are important for monitoring the normal pregnancy process as well as for the diagnosis of reproductive diseases. 17 beta-Estradiol and estrone are maintained at very low concentrations in urine and, therefore, are difficult to determine using standard chromatography with UV detection. In the present study, we describe a potential method for the determination of urinary estrogens (estrone, estradiol and estriol) using a solid-phase extraction and rapid capillary electrophoretic (CE) separations. Micellar electrokinetic chromatographic (MEKC) analysis was optimized by evaluating the number of surfactants in a 5 mM borate-5 mM phosphate separation buffer, of which sodium cholate (75-90 mM) was found to be optimal. Changing the hydrophobic character of the separation buffer with organic additives had a significant effect on the resolution of the three estrogens and an internal standard (d-equilenin). The addition of an organic additive (20% acetonitrile) was found to be necessary for the resolution of all components of the mixture. Substitution with 20% methanol provided a similar separation with better resolution but at the cost of increased analysis time. Analysis of two extracted urine samples from 18-weeks and 21-weeks pregnant women showed that, with the present technology, CE can provide adequate resolution and superior speed, but the sensitivity limits attainable with the existing technology may limit its utility to the measurement of estriol and estrone.