Simultaneous Analysis of Free and Sulfated Steroids by Liquid Chromatography/Mass Spectrometry with Selective Mass Spectrometric Scan Modes and Polarity Switching

• Published in: Analytical chemistry (Washington) Vol. 88; no. 23; pp. 11624 - 11630
• Main Authors: Lee, Su Hyeon, Lee, Nanhee, Hong, Youngmin, Chung, Bong Chul, Choi, Man Ho
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06-12-2016
• Subjects: Analytical chemistry; Chromatography; Extraction; Flow rate; Liquid chromatography; Mass spectrometry; Obesity; Phenyls; Steroids; Sulfates
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.6b03183

Sulfated steroids can act as a latent form of active free steroids, coexisting with them in biological specimens. To evaluate the metabolic significance of free and sulfated steroid species, a simultaneous analysis of eight free steroids [cholesterol, pregnenolone, 17α-hydroxypregnenolone, progesterone, 17α-hydroxyprogesterone, dehydroepiandrosterone (DHEA), testosterone, and 17β-estradiol] and four biologically relevant sulfated steroids was developed and validated, using selected-ion and multiple-reaction monitoring modes coupled to polarity-switching liquid chromatography/mass spectrometry (LC/MS). All steroids were separated on a reversed-phase phenyl column (50 mm × 2 mm, 3 μm) at a flow rate of 0.5 mL/min. The limits of quantification ranged from 0.1 to 50 ng/mL at extraction recoveries of 94.1–105.5%, while the precision and accuracy were 2.5–9.3% and 92.4–105.9%, respectively. Quantitative results obtained for samples from obese girls showed that the serum levels of DHEA sulfate were significantly increased (P = 0.004), along with the metabolic ratio representing DHEA sulfotransferase (P < 0.02). The developed novel LC/MS method can quantitatively profile both free and sulfated steroids in a single analytical run.