title: Validation of highly sensitive simultaneous targeted and untargeted analysis of keto-steroids by Girard P derivatization and stable isotope dilution-liquid chromatography-high resolution mass spectrometry.
abstract: A multiplexed quantitative method for the analysis of three major unconjugated steroids in human serum by stable isotope dilution liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed and validated on a Q Exactive Plus hybrid quadrupole/Orbitrap mass spectrometer. This quantification utilized isotope dilution and Girard P derivatization on the keto-groups of testosterone (T), androstenedione (AD) and dehydroepiandrosterone (DHEA) to improve ionization efficiency using electrospray ionization. Major isomeric compounds to T and DHEA; the inactive epimer of testosterone (epiT), and the metabolite of AD, 5α-androstanedione (5α-AD) were completely resolved on a biphenyl column within an 18min method. Inter- and intra-day method validation using LC-HRMS with qualifying product ions was performed and acceptable analytical performance was achieved. The method was further validated by comparing steroid levels from 100μL of serum from young vs older subjects. Since this approach provides high-dimensional HRMS data, untargeted analysis by age group was performed. DHEA and T were detected among the top analytes most significantly different across the two groups after untargeted LC-HRMS analysis, as well as a number of other still unknown metabolites, indicating the potential for combined targeted/untargeted analysis in steroid analysis.
authors.affiliation: A.J. Drexel Autism Institute, Drexel University, USA. Electronic address: ajf354@drexel.edu.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Frey
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, USA. Electronic address: qingqing@mail.med.upenn.edu.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, USA. Electronic address: cbusch@mail.med.upenn.edu.
authors.forename: Christine
authors.initials: C
authors.lastname: Busch
authors.affiliation: Drexel School of Medicine, Drexel University, USA. Electronic address: drf49@drexel.edu.
authors.forename: Daniel
authors.initials: D
authors.lastname: Feldman
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania, USA. Electronic address: bottlisa@mail.med.upenn.edu.
authors.forename: Lisa
authors.initials: L
authors.lastname: Bottalico
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, USA. Electronic address: mesaros@upenn.edu.
authors.forename: Clementina A
authors.initials: CA
authors.lastname: Mesaros
authors.affiliation: Penn SRP Center and Center of Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, USA. Electronic address: ianblair@exchange.upenn.edu.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Blair
authors.affiliation: Division of Pulmonary, Allergy and Critical Care, University of Pennsylvania, USA. Electronic address: avachani@mail.med.upenn.edu.
authors.forename: Anil
authors.initials: A
authors.lastname: Vachani
authors.affiliation: A.J. Drexel Autism Institute, Drexel University, Center of Excellence in Environmental Toxicology, University of Pennsylvania, USA. Electronic address: nws28@drexel.edu.
authors.forename: Nathaniel W
authors.initials: NW
authors.lastname: Snyder
mesh_terms: D000735:Androstenedione / Q000032:analysis / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D003687:Dehydroepiandrosterone / Q000032:analysis / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D044967:Serum / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013256:Steroids / Q000032:analysis* / Q000737:chemistry; D013739:Testosterone / Q000032:analysis / Q000737:chemistry
chemical_list: D013256:Steroids; D013739:Testosterone; D000735:Androstenedione; D003687:Dehydroepiandrosterone
keywords: Girard P; High resolution/accurate mass; Liquid chromatography; Mass spectrometry; Steroid; Testosterone