Validation of HPLC Method for the Determination of 17α-Ethynylestradiol (EE2) in Aqueous Phase

To cite this article: Validation of HPLC method for the determination of 17α-ethynylestradiol (EE2) in aqueous phase. (2018). J. Phys. Sci., 29(Supp. 3), 87–94,


Significant impact of estrogen pollution in the ecological system has drawn the attention of researchers as estrogenic pollution causes the change in female gonadal phenotype, decrease in fertility and fish feminisation, that eventually lead to depopulation. Major sources of estrogenic pollution are hormone treatments and discharges of humans and animals. As vital as it is to investigate the presence of estrogenic pollution in water bodies, there is a lack of simple validated method to determine the level of estrogenic pollution in water bodies. The only determination method currently being used is highresolution gas chromatography/mass spectrometry (HRGC/HRMS) under the Clean Water Act. However, this method is not widely applied due to its unavailability in most common laboratories. Thus, this current research investigates and validates a rapid and accurate estimation of 17α-ethynylestradiol (EE2) using a high-performance liquid chromatography (HPLC), an equipment commonly available in most research laboratories. This method validation is done in accordance with the guidelines for pharmaceutical drug detection, which include system suitability, system sensitivity, system linearity, accuracy and precision. The suitable wavelength of the HPLC was detected at 280 nm, with the limit of detection and quantification at the differential height of Δ0.0465 mAU and Δ0.1550 mAU each. The accuracy and precision of the system were validated at coefficient of variance in the range of 0.01% to 0.09%, which is much lower than the accepted value of 5%. Lastly, the validation of system linearity gives a regression value of 0.9993. Thus, this method is deemed valid for the detection of EE2 in aqueous solution.


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