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Rapid and reliable steroid hormone profiling in Tursiops truncatus blubber using liquid chromatography tandem mass spectrometry (LC-MS/MS)

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Abstract

Monitoring of marine mammal steroid hormone status using matrices alternative to blood is desirable due to the ability to remotely collect samples, which minimizes stress to the animal. However, measurement techniques in alternative matrices such as blubber described to date are limited in the number and types of hormones measured. Therefore, a new method using bead homogenization to QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction, C18 post extraction cleanup and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and applied to the measurement of hormone suites in bottlenose dolphin blubber. Validations were conducted in blubber from fresh dead stranded bottlenose dolphin. The final method consisting of two LC separations and garnet bead homogenization was tested for extraction efficiencies. Steroids were separated using a biphenyl column for reproductive hormones and C18 column for corticosteroids. Three hormones previously noted in blubber, testosterone, progesterone, and cortisol, were quantified in addition to previously unmeasured androstenedione, 17-hydroxyprogesterone, 11-deoxycortisol, 11-deoxycorticosterone, and cortisone in a single sample (0.4 g blubber). Extraction efficiencies of all hormones from blubber ranged from 84% to 112% and all RSDs were comparable to those reported using immunoassay methods (< 15%). The method was successfully applied to remote biopsied blubber samples to measure baseline hormone concentrations. Through this method, increased coverage of steroid hormone pathways from a single remotely collected sample potentially enhances the ability to interpret biological phenomena such as reproduction and stress in wild dolphin populations.

The steroid hormone profile is quantifiable from a single sample of bottlenose dolphin blubber using liquid chromatography tandem mass spectrometry. This profile can be applied to remotely collected dart biopsies and be used to determine reproductive or stress status of a wild-living dolphin.

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Acknowledgements

We would like to thank Louis J. Guillette Jr. for being the uniting force that made this collaborative project possible. Also, we would like to thank those who contributed their hard work in the coordination and collection of these samples including the South Carolina Marine Mammal Stranding Network, Brian Balmer, and Eric Zolman. We thank Patricia Rosel from the National Marine Fisheries Service, Southeast Fisheries Science Center for genetic analysis of the dart biopsies for sex determination. This research was made possible through a grant from the Office of Naval Research Marine Mammals and Biology Program and additional funding was provided by the National Institute of Standards and Technology, the National Oceanic and Atmospheric Administration, Marine Mammal Health and Stranding Response Program, and the National Academies National Research Council Research Associateship Program for a postdoctoral fellowship from 2013 to 2015.

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Authors and Affiliations

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

    Ashley S. P. Boggs, Tracey B. Schock & John R. Kucklick

  2. National Oceanic and Atmospheric Administration, National Centers for Coastal Ocean Science, 219 Fort Johnson Rd, Charleston, SC, 29412, USA

    Lori H. Schwacke & Wayne E. McFee

  3. National Marine Mammal Foundation, Charleston, SC, 29412, USA

    Lori H. Schwacke

  4. College of Graduate Studies, Medical University of South Carolina, 68 President Street, Charleston, SC, 29425, USA

    Thomas M. Galligan

  5. JHT, Inc. under contract to National Oceanic and Atmospheric Administration, National Centers for Coastal Ocean Science, Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, SC, 29412, USA

    Thomas M. Galligan & Jeanine S. Morey

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  1. Ashley S. P. Boggs
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  2. Tracey B. Schock
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  3. Lori H. Schwacke
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  4. Thomas M. Galligan
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  6. Wayne E. McFee
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Correspondence to Ashley S. P. Boggs.

Ethics declarations

All research protocols used were approved by a NOAA Institutional Animal Care and Use Committee. Collections were conducted in an ethical manner in concordance with ethical standard guidelines provided by the Office of Protected Resources, Marine Mammal Health and Stranding Response Program and Animal Welfare Act and under the NOAA authorization 109(h) of the Marine Mammal Protection Act.

This research was partially funded by the Office of Naval Research (ONR) under grant award numbers N0001412IP20053, N0001411IP20085, and N000141110542. The authors declare that they have no conflict of interest in the publication of this manuscript. Commercial equipment, instruments, or materials are identified to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology nor the National Oceanographic and Atmospheric Administration, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

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Boggs, A.S.P., Schock, T.B., Schwacke, L.H. et al. Rapid and reliable steroid hormone profiling in Tursiops truncatus blubber using liquid chromatography tandem mass spectrometry (LC-MS/MS). Anal Bioanal Chem 409, 5019–5029 (2017). https://doi.org/10.1007/s00216-017-0446-z

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  • DOI: https://doi.org/10.1007/s00216-017-0446-z

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