Confirmation of a novel, stable estrogen metabolite, as 5a,6a-epoxy-estrone sulfate in stallion blood by LC-MS/MS

Mass spectrometry (MS) has become pivotal for accurately delineating intricate molecular structures for steroids present in minute quantities within biological samples. This study utilized liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) to identify and characterize a 'new' estrogen metabolite, 5α,6α-epoxy-estrone sulfate, in stallion serum from three animals. The estrogen structure was predicted previously using radiolabeled steroids. HRMS/MS, in combination with a seamless sample preparation involving liquid-liquid extraction and chromatographic separation, enabled accurate mass spectrometric identification of the target metabolite. A distinct chromatographic peak corresponding to the metabolite displayed a fragmentation pattern consistent with its predicted structure. Fragment ions at m/z 79.9 and 285.1 resulting from precursor ion m/z 365.5 [M-H]^- suggested the presence of a sulfated group and epoxy form of estrone, with an additional oxygen atom when compared with those for a reference standard of estrone sulfate. The assignment of other fragment ions from the target ion further elucidated the predicted structure. Evidence for a structure unique from any other estrogen metabolite on record was demonstrated on two different LC-QTOF instruments. Its identification in the blood circulation ensures distribution throughout the body. The potential significance for future physiological/pathological investigations is discussed.