[en] 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<15%). There was neither a matrix effect nor a carry-over effect, and the sample extracts were stable during storage for at least 7 days at −21°C and 5°C. In order to apply the proposed method and obtain an initial estimate of equol concentration in cow’s milk in Belgium, 44 samples of various brands found in several supermarkets and 5 raw milk samples from Walloon farms were analyzed. Equol was found in all the samples analyzed, with a concentration ranging from 10 to 50 ng mL−1 for conventional milk and from 70 to 130 ng mL−1 for organic milk. These results were compared with other European studies and the same trend was observed.
The results obtained show the specificity, sensitivity and precision of this method for analyzing oestrogenic metabolite-equol in cow’s milk. This study should be extended to include a greater number of samples and be implemented over a longer period to see if there is a correlation between livestock practice and/or sample origins with the equol content in milk. The addition of other phytoestrogen compounds with this method, using UPLC®-MS/MS technology, could also lead to more reliable monitoring of these compounds in dairy production.
