Determination of Estrogens in Milk by HPLC using Salting-Out Extraction

Abstract

A simple salting-out assisted liquid–liquid extraction (SALLE) method with acetonitrile combined with high-performance liquid-chromatography was developed and applied for the determination four kinds of estrogens (estradiol (βE2), diethylstilbestrol (DES), estrone (E1) and progesterone (PES)) in milk. The influences of effective parameters such as salt (type and amount), water-miscible organic solvent, phase volume, equilibration time, centrifugation time and pH of sample solution were all studied and optimized. Under optimized experimental conditions, calibration plots were found to be linear in the range of 0.01–5 mg/l for E1, DES and βE2, and 0.02-8 mg/l for PES, with coefficient of determinations more than 0.996, preconcentration factors of 85-89 for those estrogens in milk. The relative recoveries ranged over 95.1-102.5%, the relative standard deviations (RSD) were 0.3-4.4% (n =5). And the limits of quantification achieved with this method were 0.4×10⁻³ mg/l for βE2, 0.7×10⁻³ mg/l for E1, 0.65×10⁻³mg/l for DES and 2.4×10⁻³ mg/l for PES, respectively.