A green extraction method based on carbon nitride sorbent for the simultaneous determination of free and conjugated estrogens in milk

This study proposes a quick and green method based on carbon nitride sorbent to extract six free and five conjugated estrogens from milk samples simultaneously. The adsorption process was studied in detail via adsorption isotherms, while the rate-limiting steps were investigated using adsorption kinetics. An experimental design was carried out for the elution step optimization regarding elution time, pH, and percentage of organic solvent. The best elution condition was 40 min at alkaline pH without the use of dichloromethane. After the matrix solid phase extraction optimization, extraction was carried out using this magnetic material after a previous deproteinization step. Separation, determination, and quantification of the target analytes were achieved by ultra-high-performance liquid chromatography coupled with triple quadrupole-tandem mass spectrometry. The methodology was validated in 6 milk samples. The LODs and LOQs for free and conjugated estrogens investigated were in the range of 0.01–0.1 ng mL−1. The recoveries of estrogens (concentration range of 0.5–10 ng mL−1) from milk samples were in the range of 89–100%, with standard deviations ranging between 1 and 3%. The method was successfully applied to milk samples leading to the identification of estrone, 17-α-estradiol, and 17-β-estradiol and four forms of conjugated estrogens (17β-estradiol-3-glucuronide; estrone-3-sulfate; 17β-estradiol-3,17β-disulfate; 17β-estradiol- 3-sulfate).