Direct quantitative thin-layer chromatographic determination of levonorgestrel and ethinyloestradiol in oral contraceptives by diffuse reflection and fluorescence methods

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Abstract
Two thin-layer chromatographic methods are described for the determination of levonorgestrel (D-norgestrel) and ethinyloestradiol in oral contraceptives. Development was carried out on both silica gel 60 F254 pre-coated commercial plates and high-performance thin-layer chromatography silica gel 60 F254 pre-coated commercial plates. By this means the two substances can be determined together directly, by means of a chromatogram-spectrophotometer, using either the diffuse reflection or the fluorescence method. In the diffuse reflection method, the levonorgestrel spots are measured in the ultraviolet range on the silica gel plate at 248 nm. The ethinyloestradiol spots can be measured only after the plate has been sprayed with 5% methanolic sulphuric acid and after heating for 10 min at 120 °C (red spots appear in the visible range at 530 nm). Using the fluorescence method, the spots are sprayed separately on a silica gel plate with 0.5% methanolic sulphuric acid and heated at 120 °C for 10 min. Fluorescent spots appear for both substances, which are excited at a wavelength of 365 nm and measured at λmax. 485 nm for levonorgestrel and 560 nm for ethinyloestradiol. These methods for the determination are suitable for control and stability tests and for tests on content uniformity of these substances in drug forms. They are rapid and specific, and can be readily reproduced. The relative standard deviation for the diffuse reflection method is 0.44–2.6% and for the fluorescence method 3.4–5.2%, depending on the type of plates used.

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