Cow's milk can be used as a potential source of equol in the human diet. In order to study human intake, however, it is necessary to develop a reliable and sensitive analytical method. This paper reports on the validation of an analytical method using ultra-performance liquid chromatography coupled with a tandem mass spectrometry detector to quantify the equol in commercial milks (raw, whole, semi-skimmed, and skimmed milk). The equol was initially released using enzymatic hydrolysis, and it was then extracted using a double liquid/liquid extraction. The analytical method produced a linear calibration curve with a high correlation coefficient (R 2 ≥0.996) between 5 and 1,000 ng.mL -1 . Good intra-and inter-day precision (≤5.3% and≤5.2%, respectively) and accuracy (≤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 (≤15%). There was neither matrix effect nor carry-over effect, and the sample extracts were stable for at least 7 days of storage at -21 °C and 5 °C. The method proved to be specific, sensitive, precise, and accurate and was used for the first time to quantify the equol content in Belgian commercial cow's milk. In all the samples analyzed, equol was present at a concentration ≥10 ng.mL -1 and had a significantly higher content in organic than in conventional milk. The study also found that the mean concentrations of equol were similar for each type of commercial conventional cow's milk.

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