Rapid and reliable steroid hormone profiling in Tursiops truncatus blubber using liquid chromatography tandem mass spectrometry (LC-MS/MS)

Abstract

Monitoring of marine mammal steroid hormone status using matrices alternative to blood is desirable due to the ability to remotely collect samples, which minimizes stress to the animal. However, measurement techniques in alternative matrices such as blubber described to date are limited in the number and types of hormones measured. Therefore, a new method using bead homogenization to QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction, C18 post extraction cleanup and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed and applied to the measurement of hormone suites in bottlenose dolphin blubber. Validations were conducted in blubber from fresh dead stranded bottlenose dolphin. The final method consisting of two LC separations and garnet bead homogenization was tested for extraction efficiencies. Steroids were separated using a biphenyl column for reproductive hormones and C18 column for corticosteroids. Three hormones previously noted in blubber, testosterone, progesterone, and cortisol, were quantified in addition to previously unmeasured androstenedione, 17-hydroxyprogesterone, 11-deoxycortisol, 11-deoxycorticosterone, and cortisone in a single sample (0.4 g blubber). Extraction efficiencies of all hormones from blubber ranged from 84% to 112% and all RSDs were comparable to those reported using immunoassay methods (< 15%). The method was successfully applied to remote biopsied blubber samples to measure baseline hormone concentrations. Through this method, increased coverage of steroid hormone pathways from a single remotely collected sample potentially enhances the ability to interpret biological phenomena such as reproduction and stress in wild dolphin populations.

A novel method to measure steroid hormone concentrations in walrus bone from archaeological, historical, and modern time periods using liquid chromatography/tandem mass spectrometry

“…Where expected concentration (EC) is divided by mean actual concentration measured in spiked bone tissue (MAC) and then multiplied by 100 (Table ) . Steroid hormone concentrations in marine mammal serum and blubber have been previously validated using LC/MS/MS methods …”

“…Immunoassay techniques are beneficial when sample mass is abundant, and they generally lower the cost of analysis . However, immunoassays require relatively large sample masses and multiple assays for multi‐hormone analyses, which increases required lab time . In addition, cross‐reactivity with target steroid hormone metabolites can lead to inflated hormone concentrations .…”

“…Furthermore, due to complicated logistics of collecting tissue samples from free‐ranging marine mammals and animal care standards for managed populations, marine mammal biopsies, blow samples, fecal samples, etc., once obtained, are relatively small and are slated for multiple different analyses, (e.g., contaminants, fatty acids, disease) . Thus, researchers need to efficiently analyze tissue samples and have been transitioning from using immunoassays to more sophisticated analyses, like liquid chromatography/tandem mass spectrometry (LC/MS/MS) …”

“…LC/MS/MS analysis allows for greater utility of samples collected from rarely encountered species, such as marine mammals. For example, eight different hormones have been analyzed in a single 0.40 g blubber sample using LC/MS/MS compared with a single hormone being measured in 0.15 g using ELISAs . LC/MS/MS measures the amount of the actual target analyte, resulting in low cross‐reactivity of metabolites and greater accuracy of actual steroid hormone concentrations in samples .…”

“…LC/MS/MS measures the amount of the actual target analyte, resulting in low cross‐reactivity of metabolites and greater accuracy of actual steroid hormone concentrations in samples . Recently, a variety of LC/MS/MS methods have been developed to measure multiple hormones in a single sample of marine mammal blubber, whale blow, and serum . Serum represents circulating hormone concentrations (short‐term), while blubber represents approximately weekly to monthly hormone concentrations .…”