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The hydrolysis of the conjugates of oestrone, oestradiol-17β and oestriol present in human urine has been investigated using acids and the enzymes β-glucuronidase and phenol sulphatase derived from Patella vulgata.

Using acid hydrolysis, maximum yields were obtained from the majority of urines by boiling 60 min with 15 vol. % concentrated hydrochloric acid. Under these conditions losses amounting to approx. 20% of the oestrogens present occur in concentrated urine specimens. These losses can be diminished by diluting the urine with water before hydrolysis.

Using enzymic hydrolysis, maximum yields were obtained by incubating the urine for 96 hr at 37° C and pH 4·7 with approx. 600 u./ml. β-glucuronidase.

When allowance was made for the losses which occur during acid hydrolysis, the yields from pregnancy and non-pregnancy urine were approximately the same by the two hydrolytic procedures. From this, it is inferred that no unknown major error exists in either procedure.