Analysis Of Dicarbonyl Steroids In Human Serum Based On LC-MS/MS

Steroids,with cyclopentane polyphenanthrene,are a sort of tetracyclic aliphatic compounds.Most of the steroids contain carbonyl groups,and the disorder of steroids is closely related to the occurrence and development of a variety of diseases.Due to high structural similarity,low concentration,poor ionization efficiency and easy interference caused by endogenous substances,these components are usually derivatized,then analyzed by LC-MS/MS.In order to study the derivatization behavior of dicarbonyl steroids,the classic carbonyl derivatization reagent Gillard T was used to derivatize dicarbonyl steroids in this study.The influence of the amount of derivatization reagent on the formation of single and double derivatized species was studied using LC-QTRAP-MS/MS.This results showed that excessive amount of derivatization reagent will cause the conversion of single-derivatized species to double-derivatized species.Thus,the quantification of dicarbonyl steroids based on single-derivated species in conventional analysis may be inaccurate,while the quantification of dicarbonyl steroids using double-derivatived products has lower sensitivity but better accuracy.Therefore,it is necessary to ensure that the conversion reactions will not occur after the derivatization reaction reaches the plateau when quantitating polycarbonyl steroids.To quantify the dicarbonyl steroids in blood,the fragmentation rules of single and double derivated dicarbonyl steroid standards by GT were summarized in this study.Subsequently,chemical derivatization,UPLC-MS/MS,and hydrogen/deuterium exchange technology were combined to qualitatively analyze the dicarbonyl steroids in human serum.Since hydrogen/deuterium exchange mass spectrometry technology can distinguish hydroxyl steroids containing monocarbonyl and dicarbonyl steroids,dicarbonyl steroids in human serum were accurately identified by this method.Finally92 carbonyl-containing steroids were identified from human serum samples,and 30 of them were determined to be dicarbonyl steroids using hydrogen/deuterium exchange mass spectrometry technology.This study revealed the rules of reaction between dicarbonyl steroids and derivatization reagents,and provided guidance for the accurate quantification of those components.The proposed strategy which was based on chemical derivatization method,hydrogen/substitution exchange,and UPLC-MS/MS technology provides a new analytical method for the identification of dicarbonyl steroids in complex systems.The identified dicarbonyl steroids is significant for the discovery of biomarkers.