The development and validation of a turbulent flow chromatography-tandem mass spectrometry method for the endogenous steroid profiling of equine serum.
Abstract: A method for the detection and quantitation of 35 endogenous steroids in equine serum was developed and validated. Androgens, estrogens, progestins and their metabolites potentially present in serum were simultaneously monitored in one method using on-line sample extraction by turbulent flow chromatography (TFC) on a 2-dimensional liquid chromatography system and detected on a triple-stage quadrupole mass spectrometer by electrospray ionization. Analytes were detected and quantitated by single-reaction monitoring or selected-ion monitoring. Limits of detection (range 0.025-10 ng mL(-1)) and quantitation (range 0.125-25 ng mL(-1)) along with recovery and matrix effects were determined for each analyte. Inter- and intra-day accuracy and precision was assessed for with the majority of analytes having %CV less than 20% and accuracy within 20% of the expected concentrations. Eight of the 35 analytes were unable to meet these guidelines across all of the quality control concentrations monitored for each analyte. This method was used to determine the endogenous steroid profiles of Thoroughbred horses and has been modified for use in non-human primates and cell culture.
Copyright © 2012 Elsevier B.V. All rights reserved.
Publication Date: 2012-06-26 PubMed ID: 22902915DOI: 10.1016/j.jchromb.2012.06.021Google Scholar: Lookup
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- Journal Article
Summary
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The researchers developed and validated a method to analyze a range of naturally occurring steroids in horse blood serum, including androgens, estrogens, and progestins, and their metabolites. This technique used turbulent flow chromatography and mass spectrometry. They assessed its accuracy and precision, and found it appropriate for use with majority of the monitored steroids.
Method Development
- The research team prepared a method to simultaneously detect and measure 35 naturally occurring steroids in horse blood serum, including androgens, estrogens, and progestins, and their metabolites.
- This procedure used a technique called turbulent flow chromatography (TFC) on a 2-dimensional liquid chromatography system. TFC is a form of rapid online extraction method used to separate analytes (the components of interest in a sample) from bulk matrix.
- The separated steroids were then identified using a device called a triple-stage quadrupole mass spectrometer, by applying an electrically charged mist (or “electrospray ionization”).
Method Validation
- The researchers validated their method by determining for each steroid, the smallest amount that can be detected (limit of detection), the smallest amount that can be accurately measured (limit of quantitation), as well as the recovery and matrix-effects, which refer to the efficiency of extracting the steroids and the potential influences of other compounds in the serum, respectively.
- They also tested the technique for its consistency (precision), and the closeness of the measurements to the actual values (accuracy).
- The majority of the steroids met the predefined criteria of having a coefficient of variation (a measure of relative variability) of less than 20% and an accuracy within 20% of the expected concentrations. However, 8 out of the 35 steroids monitored could not meet these guidelines.
Applications
- This method was employed to establish the naturally occurring steroid profile of Thoroughbred horses.
- It has also been adapted for use with non-human primates and cell cultures, demonstrating its versatility in a variety of biological contexts.
Cite This Article
APA
Moeller BC, Stanley SD.
(2012).
The development and validation of a turbulent flow chromatography-tandem mass spectrometry method for the endogenous steroid profiling of equine serum.
J Chromatogr B Analyt Technol Biomed Life Sci, 905, 1-9.
https://doi.org/10.1016/j.jchromb.2012.06.021 Publication
Researcher Affiliations
- California Animal Health and Food Safety Laboratory, School of Veterinary Medicine, University of California at Davis, Davis, CA 95616, USA. benmoeller@gmail.com
MeSH Terms
- Androgens / blood
- Animals
- Chromatography, Liquid / methods
- Doping in Sports
- Estrogens / blood
- Horses
- Limit of Detection
- Male
- Progestins / blood
- Reproducibility of Results
- Steroids / blood
- Tandem Mass Spectrometry / methods
Citations
This article has been cited 3 times.- Zhang J, Tang C, Oberly PJ, Minnigh MB, Achilles SL, Poloyac SM. A sensitive and robust UPLC-MS/MS method for quantitation of estrogens and progestogens in human serum. Contraception 2019 Apr;99(4):244-250.
- Scholtz EL, Krishnan S, Ball BA, Corbin CJ, Moeller BC, Stanley SD, McDowell KJ, Hughes AL, McDonnell DP, Conley AJ. Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5α-dihydroprogesterone. Proc Natl Acad Sci U S A 2014 Mar 4;111(9):3365-70.
- Mosburg M, Li Y, Helmes E, Falt TD, Trott JF, Solomon G, Hovey RC, Moeller BC. Determination of Hormonal Growth Promotants in Beef Using Liquid Chromatography-Tandem Mass Spectrometry. Drug Test Anal 2025 Aug;17(8):1237-1251.
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