UPLC®-MS/MS, an analytical tool for the accurate and rapid quantification of phytoestrogen metabolites in milk

The term ‘phytoestrogen’ is used to define a wide variety of nonsteroidal compounds that occur naturally in many plants. When they are absorbed by cows, these polyphenolic compounds undergo biotransformation by specific bacteria present in the gastrointestinal tract. The resulting metabolites are absorbed in the organism and some are excreted in milk. The impact of these compounds on human health divides opinion. Some scientists believe that they might have adverse health effects. Others believe the opposite, especially with regard to their microbial metabolites, such as equol. Whatever one's view on the subject, scientists need accurate, sensitive and rapid analytical methods in order to continue the research on clarifying the phytoestrogen issue. Cow's milk is an animal product that is common in the human diet, and it is therefore important to evaluate its content of phytoestrogen metabolites. In order to study the human intake of equol via commercial milk, an analytical method was developed and validated following EMA/CVMP/VICH/463202/2009 guidelines. Enzymatic hydrolysis was used to release the equol. It was then extracted using double liquid/liquid extraction and analyzed using UPLC®-MS/MS, with an analysis runtime of only 5 min. This analytical method produced a linear calibration curve with a high correlation coefficient (R2≥0.996) between 5 and 1,000 ng mL−1. Good intra- and inter-day precision (RSDs≤5.3% and ≤5.2%, respectively) and accuracy (bias≤8.6%) were achieved. The recovery rate differed slightly among the different types of milk, ranging between 60.6±1.09% and 82.3±5.21%. Good method repeatability was observed (RSDs