Environmental endocrine disruptors such as estrone (E1)and
-estradiol (E2) are excreted in human urine primarily as water-soluble glucuronides and sulfates that candissociate in wastewater treatment systems to the moreactive free
estrogens. Measurement of the distribution andfate of the steroid conjugates and the corresponding free
estrogens in treatment plants and receiving waters iscritical for understanding the reproductive and developmental effects of these substances on aquatic organisms.A sensitive method to measure steroid
estrogen conjugates in matrix-rich sewage influents and effluents (methoddetection limits ranged from 0.04 to 0.28 ng/L) has beendeveloped using HPLC tandem mass spectrometry withelectrospray ionization. The method employs extensivesample purification by selective extraction from an OasisHLB solid-phase cartridge followed by separation by anionexchange chromatography. This purification scheme,combined with a stable isotope dilution approach, wasused to overcome problems of matrix suppression ofionization and permitted selective and sensitive detectionof six target conjugates of E1 and E2. Accurate quantitation was highly dependent on the method of samplepreservation. Acidification of each sample (pH 2.0) waseffective in preventing enzymatic or chemical
decomposition of steroid conjugates in all sample types, whereasglucuronide conjugates were hydrolyzed in the presenceof mercury and formalin preservatives. Measured concentrations of steroid sulfates in the influent to a sewagetreatment plant were ~100 times greater than that of therespective steroid glucuronides, suggesting that the preponderance of glucuronides had dissociated prior toreaching the treatment plant. A small percentage of thesteroid sulfates persisted through biological treatment ofsewage and was measured in the effluent. Steroid conjugates that survive
decomposition or bypass biologicaltreatment of municipal wastewater are released intosurface waters and may serve as a source of free steroids.