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Abstract
In the present study, a simple and rapid reversed phase HPLC/diode‐array UV spectroscopy method has been developed for the determination of cyproterone acetate, norgestrel, and ethynyl estradiol in human breast milk. The method was based on the use of an isocratic elution system consisting of 0.015 M propane‐1‐sulfonic acid sodium salt and acetonitrile (60:40 v/v). The analytical column, Hypersil BDS C‐18 (150×4.6 mm) 3 µm, was operating at 25°C with a flow rate 1 mL/min. Good quantitation was obtained in the concentration range of 7.2–115.2 ng/mL for ethynyl estradiol, 4.9–155.6 ng/mL for norgestrel and 4.4–116.2 ng/mL for cyproterone acetate by the use of UV detection at 210, 246, and 284 nm, respectively. The statistical evaluation of the method was examined performing intra‐ and inter‐day validation and was found to be satisfactory, with high accuracy and precision results (R.S.D.<10.7%). Limits of detection (LOD) and limits of quantitation (LOQ) were 1.0 ng/mL and 4.4 ng/mL for cyproterone acetate, 1.1 ng/mL and 4.9 ng/mL for norgestrel, and 1.8 ng/mL, 7.2 ng/mL for ethynyl estradiol, respectively. The biological fluid (breast milk) was treated using solid phase extraction cartridges, SPE, to remove all endogenous interferences from sample matrix. The solid phase extraction protocol was optimized in terms of retention and elution. The mean absolute recoveries were 93.8% for cyproterone acetate, 93.5% for norgestrel, and 99.7% for ethynyl estradiol.