Development of a HPLC-ECD method for the simultaneous determination of three synthetic estrogens in milk

Abstract

A simple, accurate and inexpensive high performance liquid chromatography with electrochemical detection (HPLC-ECD) method for the simultaneous separation and determination of the three synthetic estrogens including diethylstilbestrol, dienestrol, and hexestrol in milk was established. For the sample treatment procedure, acetonitrile was added to the milk sample, the mixture was vortex mixed, centrifuged, concentrated by evaporation and determined by HPLC-ECD. The detection and quantification limits of the three estrogens obtained with ECD were lower than those obtained by diode array detection (DAD). Calibration curves, limit of detection and recoveries of diethylstilbestrol, dienestrol, and hexestrol with ECD ranged from 0.04 to 24 μg ml⁻¹, 4.7 × 10⁻³ to 7.1 × 10⁻³ μg ml⁻¹, and 83 to 103%, respectively. Meanwhile, the effect of thermal treatment on the stability of the estrogens was investigated, and the results showed that these compounds initially decreased by 26% for diethylstilbestrol, 32% for dienestrol, and 41% for hexestrol after five minutes of heat treatment, then remained stable with increasing duration of heat treatment. The method was applied to the determination of the three synthetic estrogens in authentic milk samples.