Sensitive LC-ESI-MS/MS Chromatography for Multisteroid Profiling in Low-Mass Whale Blubber Samples

While steroid hormones can give an indication of stress, physiological health and reproductive status in whales, analysis of these hormones is often limited by sample mass. In response, Norwegian researchers have developed a sensitive analytical method using solid phase extraction-liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for the identification and quantification of eight steroid hormones (cortisol, cortisone, testosterone, androstenedione, progesterone, 11-deoxycortisol, 11-deoxycorticosterone and 17-hydroxyprogesterone) in small amounts of whale blubber (≤50 mg). A paper based on their efforts was published in Conservation Physiology.¹

Why are Steroid Studies Concerning Whales Important?

Corticosteroids, such as cortisol and its precursors cortisone and 11-deoxycortisol, and 11-deoxycorticosterone, a precursor to aldosterone, can provide an indication of acute and chronic stress in whales, as well as metabolic regulation, because of their role in lipid metabolism and gluconeogenesis.^2,3 Reproductive steroid hormones, such as progesterone, 17-hydroxyprogesterone, testosterone and testosterone’s precursor androstenedione, can reveal the sexual maturity and pregnancy status of the whale providing the sample.^4,5 Therefore, the data compiled from steroid hormone are important for conservation efforts and ecological studies, as well as to assess effects of human activities.^6-8

How Did the Researchers Conduct Their Study?

Blubber biopsies were collected for this research from stranded killer whales (Orcinus orca; n = 7), harbor porpoises (Phocoena phocoena; n = 3) and sperm whales (Physeter macrocephalus; n = 4), harvested common minke whales (n = 10; Balaenoptera acutorostrata) and free-living killer whales (n = 2) in Norway and the Barents Sea. Steroid hormones were separated using pentafluorophenyl propyl stationary phase and an optimized gradient program with a total 20-min run. All samples (n = 26) were analyzed at 50-mg sample mass, and a subset (n = 11) at 25 mg or lower.¹

The researchers reported that low limit of detection and limit of quantification values resulted in high detection rates of hormones, and similar hormone concentrations between parallel samples and at the lowest tested sample mass (14 mg) were found. Cortisol concentrations in sperm whales (4.3 ± 1.6 ng/g) were four times higher than stranded killer whales (1.1 ± 0.96 ng/g), which in turn were up to four times higher than free-living killer whales (0.23 ± 0.12 ng/g), harbor porpoise (0.64 ± 0.38 ng/g) and harvested minke whales (0.22 ± 0.13), possibly due to fatality type and duration of stress prior to death.¹ 

“By minimizing sample mass without compromising precision or sensitivity,” wrote the authors of the paper,¹ “our method allows for steroid hormone analysis even on samples with limited availability, strengthening our ability to monitor and conserve marine mammal populations.”

“Our study,” the authors continue,¹ “is the first quantification of steroid hormones in killer whale, harbor porpoise and sperm whale blubber, and of a hormone other than progesterone in minke whale blubber. Application of our method can enable species comparisons and responses to various stressors, as well as indications of pregnancy and sexual maturity, to allow for effective conservation and management of these species.”

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References

1. Dulgheriu, D.; Andvik, C.; Jourdain, E. et al. A Sensitive Liquid Chromatography-Tandem Mass Spectrometry Analytical Method of Steroid Hormones in Small Blubber Samples from Four Whale Species. Conserv Physiol. 2026, 14 (1), coag023. DOI: 10.1093/conphys/coag023
2. 2.Peckett, A. J.; Wright, D. C.; Riddell, M. C. The Effects of Glucocorticoids on Adipose Tissue Lipid Metabolism. Metabolism 2011, 60 (11), 1500-1510. DOI: 10.1016/j.metabol.2011.06.012
3. 3.Cappello, V.; Marchetti, L.; Parlanti, P. et al. Ultrastructural Characterization of the Lower Motor System in a Mouse Model of Krabbe Disease. Sci Rep. 2016, 6 (1), 1. DOI: 10.1038/s41598-016-0001-8
4. 4.Dalle Luche, G.; Boggs, A. S. P.; Kucklick, J. R. et al. Androstenedione and Testosterone but Not Progesterone are Potential Biomarkers of Pregnancy in Humpback Whales (Megaptera novaeangliae) Approaching Parturition. Sci Rep. 2020, 10 (1), 2954. DOI: 10.1038/s41598-020-58933-4
5. Boggs, A. S. P.; Ragland, J. M.; Zolman, E. S. et al. Remote Blubber Sampling Paired with Liquid Chromatography Tandem Mass Spectrometry for Steroidal Endocrinology in Free-Ranging Bottlenose Dolphins (Tursiops truncatus). Gen Comp Endocrinol 2019, 281, 164–172. DOI: 10.1016/j.ygcen.2019.06.006
6. 6.Wikelski, M.; Cooke, S. J. Conservation Physiology. Trends Ecol Evol. 2006, 21 (1), 38-46. DOI: 10.1016/j.tree.2005.10.018
7. Parsons, E. C. M.; Baulch, S.; Bechshoft, T. et al. Key Research Questions of Global Importance for Cetacean Conservation. Endanger Species Res 2015, 27, 113–118. DOI: 10.3354/esr00655
8. Kowalska, M.; Woźniak, M.; Kijek, M. et al. Management of Validation of HPLC Method for Determination of Acetylsalicylic Acid Impurities in a New Pharmaceutical Product. Sci Rep. 2022, 12 (1), 1. DOI: 10.1038/s41598-021-99269-x