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Comprehensive profiling of free and conjugated estrogens by capillary electrophoresis-time of flight/mass spectrometry.

The biological activity of estrogens is tightly regulated by regioselective phase I/II metabolic transformations that are critical to human health. Current methods for analysis of urinary estrogens are limited by complicated sample pretreatment and/or inadequate specificity for free estrogens and their glucuronide/sulfate conjugates that vary widely in their intrinsic polarity. In this work, direct speciation of intact estrogen conjugates and their regioisomers is demonstrated using capillary electrophoresis-time-of-flight/mass spectrometry (CE-TOF/MS) when using an alkaline buffer system with negative ion mode detection. This method allows for resolution of weakly acidic native estrogens, anionic estrogen conjugates and their positional isomers without significant matrix-induced ion suppression effects in human urine. Identification of unknown estrogen metabolites using CE-TOF/MS is supported by accurate mass together with their characteristic relative migration times, which can be predicted based on two intrinsic physicochemical properties of an ion. CE-TOF/MS offers a promising strategy for comprehensive profiling of estrogens and other classes of steroid conjugates that is needed for deeper insight into the etiology and treatment of chronic disorders associated with impaired estrogen metabolism.