Reversed-phase liquid chromatographic method for estrogen determination in equine biological samples

Equine unsaturated estrogens are the main components of brand formulations indicated for hormonal replacement therapy in both hypogonadic and postmenopausal women. These hormones are produced by the fetoplacental unit during equine gestation. A method is described for the quantitative determination of equilenin (EL), equilin (EQ), 17alpha-dihydroequilin (17dEQ), and estrone (El) in the plasma of a pregnant mare. Blood samples are obtained weekly during pregnancy by jugular venipuncture using sodium ethylenediaminetetracetic as the anticoagulant. For the quantitation of these estrogens, plasma is submitted to enzymatic hydrolysis followed by liquid-liquid extraction. A high-performance liquid chromatographic system equipped with a UV detector set at 220 nm and an ODS Hypersil column is used. The method met precision, specificity, and accuracy requirements. The hormonal levels determined in one target mare throughout pregnancy were 97.91 to 449.13, 116.47 to 266.02, 74.92 to 235.54, and 84.26 to 300.03 ng/mL, reaching a maximum towards the 25th, 20th, 33rd, and 27th weeks, respectively, for E1, EL, EQ, and 17dEQ. The method was successfully tested by quantitating these estrogens in the plasma from a pregnant mare. Its applicability to the study of estrogen bioavailability and bioequivalence is suggested.