Development and validation of a liquid chromatography coupled to mass spectrometer (LC-MS) method for the simultaneous quantification of estrone-3-sulfate, progesterone, estrone and estradiol in serum of mares and American bisons

Abstract

Steroid concentrations in serum are fluctuating during pregnancy of many mammal species. The current knowledge about endocrinology of gestation is mainly based on immunoassays. However, the lack of specificity of these assays hampers the reliability of the results. In the present work, we developed and validated a methodology associating liquid chromatography (LC) and mass spectrometry (MS) to simultaneously quantify, with high specificity and accuracy, estrone-3-sulfate (E3S), progesterone (PRO), estrone (E1) and estradiol (E2) in serum of two different mammal species.The sample preparation procedure is based on a simple protein precipitation and a derivatization with dansyl chloride. After the chromatographical separation, compounds were analyzed with a triple-quadrupole mass spectrometer operating in multiple reaction monitoring. Mare and American bison serum samples were analyzed with the validated method and results were compared with concentrations measured with commercial radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA) and chemiluminescent microparticle immunoassay (CMIA).Following these criterions: relative standard deviation <15% and relative bias <15%, lower limits of quantification of 0.5 ng/mL (E3S), 0.1 ng/mL (PRO) and 2 pg/mL (E1 and E2) were achieved. Most of the comparison between immunoassays and LC-MS showed poor correlation and proportional differences.Our LC-MS method is able to simultaneously quantify several steroid hormones with high specificity, accuracy and sensitivity in serum of two different mammal species. Our method constitutes a useful and performant tool for veterinary clinicians and LC-MS should thus be used to update and refine the current knowledge about the endocrinology of pregnancy in mammals.