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.