Liquid Chromatography-Mass Spectrometric Analysis of Dehydroepiandrosterone and Related Steroids Utilizing a Temperature-Responsive Stationary Phase

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A new method was developed for the analysis of age-related steroids, such as dehydroepiandrosterone (DHEA), androstenedione (ASD), and testosterone (TES), utilizing a temperature-responsive polymer of poly(N-isopropylacrylamide) (PNIPAAm) as the stationary phase of LC-MS. A temperature-dependent separation of the steroids was achieved using an aqueous mobile phase. The efficacy of MeOH as an organic modifier in temperature-responsive chromatography (TRC) was also evaluated by LC-MS analysis of steroids. Although the MeOH content in the mobile phase did not significantly influence the resolution, MeOH enhanced the peak intensities of most of the steroids with an atmospheric pressure chemical ionization (APCI) interface. Well-resolved chromatograms of three steroids in plasma and saliva were obtained at 50 °C with a 5% MeOH aqueous solution as the eluent. To increase the detection sensitivity for MS, DHEA was derivatized with isonicotinoyl azide (INA), and subjected to LC-ESI-MS/MS.