title: Profiling intact steroid sulfates and unconjugated steroids in biological fluids by liquid chromatography-tandem mass spectrometry (LC-MS-MS).
abstract: Within the combined DFG research project "Sulfated Steroids in Reproduction" an analytical method was needed for determining sulfated and unconjugated steroids with highest specificity out of different biological matrices such as aqueous solution, cell lysate and serum. With regard to this analytical challenge, LC-MS-MS presents the technique of choice because it permits (1) analysis of the intact steroid conjugate, (2) allows for simultaneous determination of multiple analytes (profiling, targeted metabolomics approach) and (3) is independent of phenomena such as cross-reactivity. Sample work up consisted of incubation of sample with internal standards (deuterium labeled steroids) followed by solid phase extraction. Only serum samples required a protein precipitation step prior to solid phase extraction. The extract was divided in two parts: six steroid sulfates (E1S, E2S, AS, 16-OH-DHEAS, PREGS, DHEAS) were analyzed by C18aQ-ESI-MS-MS in negative ion mode and eleven unconjugated steroids (E3, 16-OH-DHEA, E1, E2, (4)A, DHEA, T, 17-OH-PREG, Prog, An, PREG) were analyzed by C18-APCI-MS-MS in positive ion mode. For steroid sulfates, we found high sensitivities with LoQ values ranging from 0.08 to 1 ng mL(-1). Unconjugated steroids showed LoQ values between 0.5 and 10 ng mL(-1). Calibration plots showed excellent linearity. Mean intra- and inter-assay CVs were 2.4% for steroid sulfates and 6.4% for unconjugated steroids. Accuracy - determined in a two-level spike experiment - showed mean relative errors of 5.9% for steroid sulfates and 6.1% for unconjugated steroids. In summary, we describe a novel LC-MS-MS procedure capable of profiling six steroid sulfates and eleven unconjugated steroids from various biological matrices.
authors.affiliation: Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetlogy, Center of Child and Adolescent Medicine, Justus-Liebig-University, Feulgenstrasse 12, 35392 Giessen, Germany.
authors.forename: Christina E
authors.initials: CE
authors.lastname: Galuska
authors.forename: Michaela F
authors.initials: MF
authors.lastname: Hartmann
authors.forename: Alberto
authors.initials: A
authors.lastname: Sánchez-Guijo
authors.forename: Katharina
authors.initials: K
authors.lastname: Bakhaus
authors.forename: Joachim
authors.initials: J
authors.lastname: Geyer
authors.forename: Gerhard
authors.initials: G
authors.lastname: Schuler
authors.forename: Klaus-Peter
authors.initials: KP
authors.lastname: Zimmer
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Wudy
mesh_terms: D001774:Blood Chemical Analysis / Q000379:methods*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D015203:Reproducibility of Results; D013256:Steroids / Q000097:blood* / Q000737:chemistry*; D013431:Sulfates / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D013256:Steroids; D013431:Sulfates