Abstract Given the growing interest in steroid hormone research in milk, it is essential to monitor the quality of this product and its derivatives using robust methods. A simple method involving extraction and analysis by RP-HPLC-DAD was optimized and validated for the analysis of progesterone and estradiol in milk. The quality of separation and classic validation parameters were evaluated. The robustness of the method was assessed by monitoring hormones in intentionally added milk (1 µg/mL) and in its processed products, such as cheese, yogurt, whey, and ricotta. The method proved suitable for the proposed purpose, showing a linearity coefficient R > 0.999 for the range of 0.5 to 5.0 µg/mL, with detection limits of 15 and 31 ng/mL and quantification limits of 41 and 91 ng/mL for progesterone and estradiol, respectively. It showed good precision (CV% ≤ 4.08) and recovery from 98.2 to 103.6% in milk. In the robustness assessment, the method proved to be suitable for monitoring progesterone and estradiol in milk and dairy products. The behavior of these hormones in the products showed that the pasteurization and fermentation processes did not impact the substances. A high concentration was present in the product: cheese > ricotta > yogurt > whey, with the cheese concentration increasing sevenfold compared to the fortified milk.
Pereira et al. (Tue,) studied this question.