Multiple Optimization for Extraction of Ethinylestradiol, Levonorgestrel, and Their Main Impurities using Mixture Design

In pharmaceutical analysis, the development of extraction procedures focuses mainly on the optimization of active pharmaceutical ingredients. However, this may lead to low recovery values for impurities, which is a disadvantage for the development of stability-indicating methods to assess the expiration time of drugs in stability studies. Here, we have proposed a multiple optimization for the extraction of ethinylestradiol, levonorgestrel, and their main impurities aiming to develop a stability-indicating chromatographic procedure. A mixture experimental design was adopted to optimize the recovery of ethinylestradiol, levonorgestrel, and 32 impurities. Mixtures of acetonitrile (0 to 60%), methanol (0 to 60%), and water (40 to 80%) were used to extract the analytes from samples subjected to dry thermal degradation. Recovery values for ethinylestradiol and levonorgestrel ranged between 12.5–100.0% and 47.4–100.0%, respectively. Optimal recovery values were achieved using a mixture of methanol (60%) and water (40%). The optimized extraction mixture provided significantly higher recoveries for 18 substances than the initial extraction mixture, including 10 unknown impurities. Most of these unknown impurities did not have a good recoverability. Thus, the multiple optimization of the extraction procedure exhibits great potential for application to optimize sample preparation for stability-indicating analytical procedures.