Introduction
Steroid hormones mediate physiology and behavior; without these compounds, animals would be unable to reproduce or survive long enough to do so. The lipophilic nature of these small molecules aids in their transport into many tissues, enabling scientists to conduct wildlife studies through the collection of feces, hair, blubber, and other methods less invasive than capture. Whale and dolphin (i.e., cetacean) blubber, can be collected at sea by remote blubber biopsy and contains adrenal and gonadal steroids (1, 2).
Scientists often use enzyme immunoassay (EIA) to quantify steroid hormones in wildlife samples. EIAs are sensitive, affordable, and simple to use; however, they are subject to antibody cross-reactivity (3, 4) and are limited to one hormone per analysis. In contrast, mass spectrometry offers high specificity and the ability to concurrently measure multiple compounds (5). Enabled by efficient blubber extraction methods, e.g., Boggs et al. (6), liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed for quantifying steroid hormones in cetacean blubber (6, 7).
LC-MS/MS methods have been employed to reliably quantify corticosteroids, androgens, progestogens, and estrogens in blubber from dolphins (6, 8) and baleen whales (7, 9, 10). Good chromatographic separation has been achieved by running corticosteroids on a C18 column and separately running androgens and progestogens on a Biphenyl column (6). Because many steroids have identical precursor and product ions, using different chromatographic sorbents can reliably separate multiple classes of steroids from one sample extraction.
Though studies have successfully employed these methods for assessments of stress and reproductive hormones in blubber from free-ranging populations, the inter-specific variability of blubber tissue (11) makes it necessary to test for matrix effects before applying existing methods to new species. In this study, we tested and adapted methods established in Boggs et al. (6) for use with blubber from short-finned pilot whales (Globicephala macrorhynchus ). We conducted a spike recovery experiment with blubber samples from stranded individuals to assess this method’s accuracy and precision for application in this species’ blubber tissue.
This study aimed to expand existing methods by adding three steroid hormones connected to stress in cetaceans: aldosterone, dehydroepiandrosterone (DHEA), and dehydroepiandrosterone-sulfate (DHEAS). Aldosterone, a mineralocorticoid, regulates sodium balance and increases in response to stressors in marine mammal species (12, 13). Aldosterone has been measured in cetacean serum and feces using immunoassay (14) and recently detected in the blubber of a stranded gray whale (10). DHEA and its sulfonated version, DHEAS are prohormones primarily synthesized in the adrenal cortex and secreted in response to adrenocorticotropic hormone (ACTH). However, they are also synthesized in the gonads and are categorized as androgens. Changes in DHEAS and DHEA concentrations have been connected to chronic stress (15) and disease (16), but neither of these hormones have been measured in cetacean blubber.
We added these steroid hormones through the optimization of an LC-MS/MS method using a C18 column. Isotopically labeled internal standards (IS) were added to samples to enable accurate analyte quantification by controlling for sample loss throughout processing. Ideally, methods include a matched IS for each analyte which helps verify peak identity and reduce matrix interference in measurements, but the expense of these compounds impedes universal use. To guide decisions around which ISs should be used in future methods, we assessed and compared the performance of 10 isotopically labeled ISs for 11 analytes.