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Journal article
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
Steroids, Vol.116
Dec 2016
PMCID: PMC5127713
PMID: 27743906
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
[Display omitted]
•Keto-steroids, including testosterone, are analytes of biological interest.•LC-high resolution MS offers unique potential for steroid assays.•Quantitative validation of a Girard P based keto-steroid assay was conducted.•Simultaneous untargeted steroid profiling and targeted quantitation is possible.
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.
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Details
- 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
- Creators
- Alexander J. Frey - Drexel UniversityQingqing Wang - Translational TherapeuticsChristine Busch - Translational TherapeuticsDaniel Feldman - Drexel UniversityLisa Bottalico - University of PennsylvaniaClementina A. Mesaros - Translational TherapeuticsIan A. Blair - Translational TherapeuticsAnil Vachani - University of PennsylvaniaNathaniel W. Snyder - Drexel University
- Publication Details
- Steroids, Vol.116
- Publisher
- Elsevier
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- A.J. Drexel Autism Institute
- Identifiers
- 991019167741704721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemistry & Molecular Biology
- Endocrinology & Metabolism