The ratio between free and conjugated estrogens constitutes important information about the presence of these analytes in milk matrices. In most works, it was demonstrated that 80-90%
of total estrogens were present as methylated, hydroxylated, glucuronated or sulfated metabolites. due to the high propotion of these hydrophilic conjugated molecules,estrogens must be present in the aqueous fraction of milk; they demonstrated this by analyzing milk with different fat proportions. Regarding this,demonstrated that the percentage of glucuronated and sulfated forns of E1 and E2 was very important in milk samples. They also found higher concentrations of total E2 than in previous studies, which they justified by there having been insufficient hydrolysis in previous works(where the total amount of E2 was determined after hydrolysis), possible oxidation of E2 to E1 or studies carried out in different gestation periods.