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Reproductive and Stress Steroid Hormone Analysis in Cetacean Blubber by Liquid Chromatography - Tandem Mass Spectrometry

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Mary Hayden, David Klein, Seenivasan Subbiah, Christiana Wittmaack, Veronique Lesage, Yves Morin, Jorge Urban Ramirez, Carlos Lopez Montalvo, John Bickham and Celine Godard-Codding

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International Whaling Commission


The profiling of reproductive and stress steroid hormones in cetaceans provides vital information for both conservation and management decisions. This is especially the case for endangered species, such as the western gray whale (Eschrichtius robustus) subpopulation, as stated by the IWC scientific community. We previously validated reproductive hormone profiling in gray whales using ELISA (Gendron et al. 2015) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Hayden et al. 2016, Hayden et al. 2017b) and subsequently reported on advancements of the LC-MS/MS methodology (Hayden et al. 2017b). Here we report on (1) further optimization of the LC-MS/MS methodology (both positive and negative ion modes), (2) the quantitation of progesterone, testosterone, cortisol, and estradiol in small (50 mg w/w) samples in both the gray whale and the beluga, and, (3) to our knowledge, the first detection of pregnancy in cetaceans by LC-MS/MS. Steroid hormones were extracted, separated from lipids using gel permeation chromatography, and then quantified using a Thermo Scientific TSQ Quantum Access Max MS mass spectrometry system in heated electrospray positive ionization mode and atmospheric-pressure chemical negative ionization mode through selected reaction monitoring. Quantitation of endogenous steroid hormones was performed with the use of isotopically labeled internal standards. Limits of quantitation and detection were compatible with reported levels of steroid hormones in cetacean blubber and extraction efficiencies were comparable to liquid-liquid extraction efficiencies. LC-MS/MS has the capability of quantitating multiple steroid hormones simultaneously, which can be invaluable to assess pregnancy, health and fitness in a single sample. Therefore, this methodology is ideal for species or samples where tissue weight is limited, such as the IUCN-listed critically endangered Western gray whale population. Future directions of this research will involve the inclusion of other biologically relevant steroid hormones in the analysis panel and the establishment of baselines indicative of reproductive maturity using additional samples from animals of known sex and reproductive status.

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