Hollow-fiber liquid-phase microextraction for the determination of natural and synthetic estrogens in milk samples

• A separation of nine natural and synthetic estrogens by HPLC–DAD/FD is proposed.
• A HF-LPME method is suggested for the extraction of these estrogens from milk.
• Deproteinization and parameters affecting HF-LPME were studied and optimized.
• Calibration of the whole method was done for skimmed, semi-skimmed and whole milk.
• A precision and accuracy study for further validation was made for each matrix.

In this work, a group of nine estrogens, four of them being natural (estriol, 17β-estradiol, 17α-estradiol and estrone), four being synthetic (17α-ethynylestradiol, diethylstibestrol, dienestrol and hexestrol) and one metabolite (2-hydroxyestradiol) have been extracted and preconcentrated from milk samples with different fat content (whole, semi-skimmed and skimmed). After protein precipitation with acetonitrile containing acetic acid, evaporation of the supernatant and reconstitution of the residue in water, hollow-fiber liquid-phase microextraction (HF-LPME) using 1-octanol as extraction solvent was applied to further preconcentrate the analytes. Separation, determination and quantification were achieved by high-performance liquid chromatography coupled to a diode array detector and a fluorescence detector set in series. Deproteinization conditions, as well as parameters affecting the extraction efficiency in HF-LPME (pH of the sample, ionic strength, extraction time, stirring speed, temperature and desorption conditions) were investigated and optimized. Calibration, precision and accuracy studies were carried out to validate the methodology in different types of milk providing LODs in the low μg/L range.