Analysis of progesterone and estrone-sulfate in feces of American Bison using liquid chromatography coupled to mass spectrometry: Technical validation and correlation with blood levels

Abstract

American Bison's wild nature limits blood sample availability to study its endocrinology. This report describes progesterone (P4) and estrone-sulfate (E1S) assays in American Bison feces using Liquid Chromatography coupled with Mass Spectrometry (LC-MS). In 2 ranches, samples of feces (n = 73) and serum (n = 93) were collected in pregnant and nonpregnant American Bison. Feces samples (250 mg) were extracted with methanol, purified, and concentrated. Then, feces and serum samples were assayed using LC-MS, according to our previously described technique. Fecal matrix homogeneity was determined by measuring steroids in different areas of the sample and concentration evolutions were evaluated after storage at room temperature. During the validation process, lower limits of quantification were 20 pg/g (E1S) and 4 ng/g (P4) by meeting the following criteria: relative standard deviation <15% and relative bias <15%. By measuring hormones in different spots from the same sample, a moderate variability for E1S (coefficient of variation [CV] up to 21.3%) and a high variability for P4 (CV up to 85.5%) were highlighted. Correlation between concentrations in feces and in serum was higher for E1S (r = 0.77) than for P4 (r = 0.65) and P4 could be assayed in pregnant and nonpregnant animals whereas E1S was only present in pregnant. Feces storage at room temperature induced modification of steroid concentrations. The quantification of E1S and, at a lower level, of P4 in feces is an interesting alternative to serum assay to describe the pregnancy-related evolution of these steroids in American Bisons, with feces ideally stored frozen and mixed before the LC-MS procedures.