Measurement of ethynylestradiol and levonorgestrel incorporated into sustained release contraceptive formulations

High performance liquid chromatography was used to measure the concentrations of ethynylestradiol (EE) and levonorgestrel (LNG) released from contraceptive devices into aqueous medium containing the cationic detergent, benzalkonium chloride. Most of the detergent was removed after solvent extraction of the steroid, although small amounts of it remained in the steroid phase. Over the course of many injections into octasilyl (C8) or octadecylsilyl (C18) reversed-phase columns, the chromatographic profile of EE was gradually altered with multiple peaks emerging. The profile of LNG was not changed. EE chromatographed as a double peak on a fully end-capped C18 column. Rechromatography of each peak yielded a mixture of the two. Mass spectral analysis showed that the peaks differed only in the gain or loss of an equivalent of water. Introduction of a cation exchange column before the analytical column removed residual benzalkonium ions, and by thus preventing deterioration of the column, permitted EE to consistently emerge as a single, symmetrical peak.