Nanostructured solvent based microextraction followed by a novel strategy for online phase separation coupled with HPLC for determination of ethinyl estradiol Behnam Ebrahimpour, a Yadollah Yamini, * a Shahram Seidi b and Fatemeh Rezaei a Supramolecular solvent microextraction coupled with high performance liquid chromatography based on a new strategy for phase separation was developed for determination of ethinyl estradiol in water samples. Ethinyl estradiol was extracted from 10 mL of a water sample into a supramolecular solvent, a nanostructured solvent, which was formed in situ from reverse micelles of a biosurfactant in a THF– water solution. After extraction, the supramolecular solvent was retained in an inline filter and was separated from the aqueous solution and then, it was eluted and transferred to an HPLC column. Experimental design and response surface methodology (RSM) were used for optimization of different parameters that influence the extraction efficiency of the method. Under optimal conditions, ethinyl estradiol was effectively extracted and a preconcentration factor of 237 was obtained. The calibration curve was investigated in the concentration range of 0.1–200 mgL 1 and good linearity was achieved with a coefficient of determination better than 0.995. A detection limit of 0.1 mgL 1 and suitable precision with RSD ¼ 5.1% (n ¼ 3) were obtained. Finally, the proposed method was applied to determine the concentrations of ethinyl estradiol in different water samples and provided acceptable recoveries. Introduction Endocrine disruptors are any externally originating chemical compounds, either natural or synthetic, that interfere with normal endocrine functions. These compounds are thought to affect the binding, synthesis, signaling, or decomposition of essential hormones. 1,2 Among the wide range of substances with endocrine-disrupt- ing properties, estrogens (female hormones such as 17b-estradiol, estrone, ethinyl estradiol and estriol) are of particular interest due to their high estrogenic potency. 3 Urinary excretion of natural estrogens (e.g. estrone, estradiol and estriol) and synthetic compounds used in medicine, as contraceptives and in some hormonal therapies (e.g. ethinyl estradiol, mestranol), or in veterinary practice, as growth promoters of farm animals (e.g. diethylstilbestrol), together with their incomplete removal in sewage treatment plants, have caused the presence of several estrogens and related compounds in the aquatic environment. 4–7 Synthetic estrogens, such as the potent estrogen ethinyl estradiol (it was rst synthesized in 1938 (ref. 8) and has since then been well characterized), are used extensively for contraceptive and therapeutic purposes. Ethinyl estradiol can act as an endocrine disruptor in aquatic organisms and mammals through environ- mental exposure or the food chain and induce reproductive disorders. 9,10 Therefore, analytical chemists must provide the most accurate, sensitive and analytically robust methods for the isolation, identication, and quantication of EDCs, which can be present in trace amounts in aqueous systems. During the last few years, numerous efforts have been devoted to the development of analytical methodologies sensitive enough to allow the determi- nation of estrogens in environmental samples. Different analyt- ical protocols based on gas chromatography-mass spectrometry (GC-MS), 11–13 liquid chromatography-mass spectrometry (LC- MS), 14,15 and HPLC-tandem mass spectrometry (HPLC-MS/MS) 16,17 have been reported. However, although these methods are highly sensitive and specic they are quite laborious, and, most oen, quite expensive. The main aim of sample preparation is to clean up and concentrate the analytes of interest, while rendering them in a form that is compatible with the analytical instruments. 18 Today, the miniaturization and automation of conventional sample preparation techniques is one of the main tasks in analytical chemistry, 19,20 as evidenced by the number of articles published on this subject in the last two decades. Among different methods used for pretreatment of samples, microextraction techniques a Department of Chemistry, Tarbiat Modares University, P. O. Box 14115-175, Tehran, Iran. E-mail: yyamini@modares.ac.ir; Fax: +98-21-82883455; Tel: +98-21-82883417 b Department of Analytical Chemistry, Faculty of Chemistry, K. N. Toosi University of Technology, Tehran, Iran Cite this: Anal. Methods, 2014, 6, 2936 Received 3rd December 2013 Accepted 14th February 2014 DOI: 10.1039/c3ay42155d www.rsc.org/methods 2936 | Anal. Methods, 2014, 6, 2936–2942 This journal is © The Royal Society of Chemistry 2014 Analytical Methods PAPER