A sensitive liquid chromatography-tandem mass spectrometry analytical method of steroid hormones in small blubber samples from four whale species.

Abstract

Steroid hormones can give an indication of stress, physiological health and reproductive status in whales; however, analysis is often limited by sample mass. We developed a sensitive analytical method using solid phase extraction-liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) for the identification and quantification of eight steroid hormones (cortisol, cortisone, testosterone, androstenedione, progesterone, 11-deoxycortisol, 11-deoxycorticosterone and 17-hydroxyprogesterone) in small amounts of whale blubber (≤50 mg). Blubber biopsies were collected from stranded killer whales (Orcinus orca; n = 7), harbour porpoises (Phocoena phocoena; n = 3) and sperm whales (Physeter macrocephalus; n = 4), harvested common minke whales (n = 10; Balaenoptera acutorostrata) and free-living killer whales (n = 2) in Norway and the Barents Sea. Steroid hormones were separated using pentafluorophenyl propyl stationary phase and an optimized gradient programme with a total 20-min run. All samples (n = 26) were analysed at 50-mg sample mass, and a subset (n = 11) at 25 mg or lower. Low limit of detection and limit of quantification values resulted in high detection rates of hormones, and we found similar hormone concentrations between parallel samples and at the lowest tested sample mass (14 mg). Cortisol concentrations in sperm whales (4.3 ± 1.6 ng/g) were four times higher than stranded killer whales (1.1 ± 0.96 ng/g), which in turn were up to four times higher than free-living killer whales (0.23 ± 0.12 ng/g), harbour porpoise (0.64 ± 0.38 ng/g) and harvested minke whales (0.22 ± 0.13), possibly due to fatality type and duration of stress prior to death. We provide the first quantification of blubber steroid hormones in killer whale, minke whale, sperm whale and harbour porpoise, and in less than 50-mg sample mass. By minimizing sample mass without compromising precision or sensitivity, our method allows for steroid hormone analysis even on samples with limited availability, strengthening our ability to monitor and conserve marine mammal populations.