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Development of a multi-class steroid hormone screening method using Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS)

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Abstract

Monitoring complex endocrine pathways is often limited by indirect measurement or measurement of a single hormone class per analysis. There is a burgeoning need to develop specific direct-detection methods capable of providing simultaneous measurement of biologically relevant concentrations of multiple classes of hormones (estrogens, androgens, progestogens, and corticosteroids). The objectives of this study were to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for multi-class steroid hormone detection using biologically relevant concentrations, then test limits of detection (LOD) in a high-background matrix by spiking charcoal-stripped fetal bovine serum (FBS) extract. Accuracy was tested with National Institute of Standards and Technology Standard Reference Materials (SRMs) with certified concentrations of cortisol, testosterone, and progesterone. 11-Deoxycorticosterone, 11-deoxycortisol, 17-hydroxypregnenolone, 17-hydroxyprogesterone, adrenosterone, androstenedione, cortisol, corticosterone, dehydroepiandrosterone, dihydrotestosterone, estradiol, estriol, estrone, equilin, pregnenolone, progesterone, and testosterone were also measured using isotopic dilution. Dansyl chloride (DC) derivatization was investigated maintaining the same method to improve and expedite estrogen analysis. Biologically relevant LODs were determined for 15 hormones. DC derivatization improved estrogen response two- to eight-fold, and improved chromatographic separation. All measurements had an accuracy ≤14 % difference from certified values (not accounting for uncertainty) and relative standard deviation ≤14 %. This method chromatographically separated and quantified biologically relevant concentrations of four hormone classes using highly specific fragmentation patterns and measured certified values of hormones that were previously split into three separate chromatographic methods.

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Acknowledgments

For partial funding, we acknowledge the National Oceanographic and Atmospheric Administration (NOAA) and the Smart State Center of Economic Excellence for Marine Genomics. We extend thanks to Lori Schwacke of NOAA for being the impetus to develop this method. Also, we thank Kevin Huncik for performing instrument maintenance and troubleshooting assistance. Finally we would like to thank the NIST/National Research Council postdoctoral program for financial support during this research.

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Authors and Affiliations

  1. National Institute of Standards and Technology, Hollings Marine Laboratory, 331 Fort Johnson Rd, Charleston, SC, 29412, USA

    Ashley S. P. Boggs, John A. Bowden & John R. Kucklick

  2. Medical University of South Carolina, Hollings Marine Laboratory, 331 Fort Johnson Rd, Charleston, SC, 29412, USA

    Thomas M. Galligan & Louis J. Guillette Jr.

Authors
  1. Ashley S. P. Boggs
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  2. John A. Bowden
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  3. Thomas M. Galligan
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  4. Louis J. Guillette Jr.
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  5. John R. Kucklick
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Correspondence to Ashley S. P. Boggs.

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Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. The authors declare that they have no conflict of interest.

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Boggs, A.S.P., Bowden, J.A., Galligan, T.M. et al. Development of a multi-class steroid hormone screening method using Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS). Anal Bioanal Chem 408, 4179–4190 (2016). https://doi.org/10.1007/s00216-016-9512-1

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  • DOI: https://doi.org/10.1007/s00216-016-9512-1

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