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.

The research article describes a study where a method using liquid chromatography and mass spectrometry (LC-MS) was developed to accurately measure levels of specific hormones in the blood of pregnant mammals, in this case mares and American bisons. The new method showed higher specificity and accuracy compared to existing methods, suggesting it could be a valuable tool for understanding hormone fluctuations during gestation in mammals.

Objective of the Study

• The aim of the research was to develop and validate a new method that can simultaneously quantify, with high specificity and accuracy, the levels of four hormones relevant to pregnancy – estrone-3-sulfate (E3S), progesterone (PRO), estrone (E1) and estradiol (E2) – in the blood serum of mares and American bisons.

Methodology of the Study

• The proposed method combined liquid chromatography (LC) and mass spectrometry (MS) – a versatile analytical technology with a wide range of applications. As part of the sample preparation procedure, a protein precipitation and a derivatization with dansyl chloride were performed.
• Following chromatographical separation, the compounds were analyzed using a triple-quadrupole mass spectrometer operating in multiple reaction monitoring mode.

Results and Findings

• This research yielded significant results with the new method successfully quantifying the targeted hormones with relative standard deviation less than 15% and relative bias less than 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.
• When comparing these results with concentrations measured by the commonly used methods such as commercial radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and chemiluminescent microparticle immunoassay (CMIA), there was poor correlation and proportional differences – indicating the superior performance of the newly devised LC-MS method.

Conclusion

• Conclusively, the newly developed LC-MS method provided a more specific, accurate, and sensitive measurement for the quantification of various steroid hormones related to pregnancy in mares and American bisons. This demonstrates that LC-MS could be an effective tool for veterinary clinicians and should be used to explore and refine the current understanding of the endocrinology of gestation in mammals.