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.