Discussion
The Boggs et al. (2017) methods for quantifying steroid hormones in
whale blubber can be applied to blubber samples from short-finned pilot
whales. As the spike-recovery experiment demonstrated, this tissue
matrix does not interfere with the reliable measurement of hormones when
assessed with appropriate internal standards. When using matched ISs,
accuracy and RSD were acceptable for all 11 hormones measured, except B.
This is rectified by substituting B-d 4 with an
appropriate IS, like 11DOC-13C3.
Accuracy was similar to Boggs 2017 (84 % -112 %) and showed increased
precision for some analytes when compared with Boggs et al. 2017 and
Dalle Luche et al. 2019 (6, 9). This improvement was likely due to the
inclusion of additional matched ISs for adrenal analytes. Differences in
T and P4 concentrations between adult male and
known-pregnant female samples demonstrate the biological validity of
this method, which is promising for the future application of this
method to blubber biopsy samples from free-swimming individuals.
The C18 method optimized in this study is among the first to validate
the measurement of aldosterone and DHEA in whale blubber. Though
endogenous aldosterone was not present in measurable quantities in the
pilot whale blubber assessed, this is the first LC-MS/MS method to
quantify DHEA in cetacean blubber. We expect adrenal steroids to be
significantly higher in blubber from live-stranded whales than
free-swimming individuals. Even so, analyte concentrations were below RL
in some samples. What this method gains from specificity, it loses in
sensitivity. This is especially true for ALD, which had a relatively
high RL of 3.54 ng/g, which is similar to the LOD published in Wittmaack
et al. 2022 (10), 3.72 ng/g. Future studies should explore this further
to determine whether resolution can be improved and whether ALD
metabolites are abundant and measurable in this tissue.
The IS assessment conducted in this study highlights the necessity of
careful IS selection when developing LC-MS/MS methods. Not all analytes
from this study required matched ISs. In most cases, nearby13C-labeled IS peaks could be used as substitutes
without sacrificing significant accuracy and precision. However,
chromatographic proximity is not the sole indicator of a suitable IS
substitution. Even using IS substitutions of nearby13C-labeled peaks affected performance and
concentrations measured (see
S/11DOC-13C3 in Table 4). Matrix
interferences at the retention time of the analyte could be responsible
for this effect. ALD-d 4 and
B-d 4 standards performed poorly compared to13C-labeled standards. This could stem from
hydrogen/deuterium exchange occurring during extraction or measurement,
or may reflect the relatively low sensitivity of the method to these
analytes. If using ALD-d 4 or
B-d 4 as ISs, researchers should consider using
concentrations well above endogenous levels or reliable IS
substitutions. Further studies should assess the use of13C-labeled ISs for ALD and B.
This is the first study to validate the measurement of multi-class
steroid hormone profiling in short-finned pilot whales. Where prior
studies used immunoassay to measure progesterone in only females (18) or
testosterone in only males (19), we present a comprehensive method to
concurrently measure hormones regardless of sex. Comprehensive studies
like this can maximize the information gleaned from small and
difficult-to-obtain samples, such as blubber biopsies. Aside from
testosterone, this is the first study to quantify steroid hormones in
male short-finned pilot whales. This method did not prove effective in
the measurement of aldosterone but was able to reliably detect DHEA,
which has not been previously measured in short-finned pilot whales.
While corticosterone is the dominant glucocorticoid in some smaller
animals, like mice and birds, cortisol was the most abundant
glucocorticoid observed in these samples. The predominance of cortisol
echoes the patterns seen in cetaceans and other large mammals. The use
of LC-MS/MS for highly-specific multiclass steroid profiling can inform
which analytes researchers select for studies with single-target
methods, like immunoassay.