Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry.
Abstract: Anabolic androgenic steroids are related to the male sex hormones and are abused in equine sports. In an effort to deter the abuse of anabolic steroids, a sensitive LC-MS/MS method was developed for detection, quantification and confirmation of eight major anabolic steroids (testosterone, normethandrolone, nandrolone, boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, and stanozolol) in equine plasma. Formation of solvent adduct ions of the analytes was observed under electrospray ionization (ESI) conditions, and desolvation of the solvent adduct ions by source collision-induced decomposition (CID) increased the abundance of the [M+H]+ ions as well as the multiple-reaction monitoring (MRM) signals. ESI (+) and APCI (+) were compared with respect to sensitivity for the analytes and the former provided better sensitivity. The matrix effect on ion suppression or enhancement was evaluated, and was negligible. Confirmation of the analytes was performed using criteria of three ion transitions and LC retention time of each analyte. The limit of detection (LOD) and quantification (LOQ) was 25 pg/mL. The limit of confirmation (LOC) was 25 pg/mL for boldenone; 50 pg/mL for normethandrolone, nandrolone, and methandrostenolone; and 100 pg/mL for testosterone, THG, trenbolone, and stanozolol. The analytes were evaluated for stability and found to be stable in plasma for 24h at room temperature, 13 days at 4 degrees C, and 34 days at -20 and -70 degrees C. The method was successfully applied to analyses of equine plasma samples for pharmacokinetics study. This method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in equine plasma.
Publication Date: 2005-11-10 PubMed ID: 16289956DOI: 10.1016/j.jchromb.2005.09.045Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research developed a sensitive method for detecting and measuring eight major anabolic steroids, which are often illegally used in horse sports, in horse plasma. The method was found effective for differentiating these compounds and assessing their amounts.
Research Methodology
- The researchers developed a method based on liquid chromatography and tandem mass spectrometry (LC-MS/MS), specifically designed to detect, measure, and confirm the presence of eight main anabolic steroids (testosterone, normethandrolone, nandrolone, boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, and stanozolol) in horse blood plasma.
- They observed the formation of solvent adduct ions under electrospray ionisation (ESI) that, upon collision-induced decomposition (CID), increased the [M+H]+ ions abundance and the multiple-reaction monitoring (MRM) signals.
- One aspect of the research was comparing ESI and Atmospheric Pressure Chemical Ionization (APCI) sensitivity. The findings showed that ESI had superior sensitivity in this context.
- The researchers also looked into how the ion suppression or enhancement of the matrix could impact the results, but ultimately found its effects to be insignificant.
Analyte Confirmation and Sensitivity
- The confirmation of these substances (analytes) relied on three ion transitions and the LC retention time of each analyte.
- The detection and quantification limit (LOD and LOQ) was 25 pg/mL, while the confirmation limit varied between 25 pg/mL for boldenone, up to 100 pg/mL for testosterone, THG, trenbolone, and stanozolol.
Stability and Application
- It was crucial to establish the stability of the analytes. The researchers found that these anabolic steroids remained steady in plasma under various conditions: room temperature for 24 hours, 4 degrees Celsius for 13 days, and -20 and -70 degrees Celsius for 34 days.
- The successful application of this method occurred during an analysis of horse plasma samples during a pharmacokinetics study, confirming the method’s usefulness and sensitivity for detecting, quantifying, and confirmation of these anabolic steroids in horse plasma.
Cite This Article
APA
Guan F, Uboh CE, Soma LR, Luo Y, Rudy J, Tobin T.
(2005).
Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid chromatography and tandem mass spectrometry.
J Chromatogr B Analyt Technol Biomed Life Sci, 829(1-2), 56-68.
https://doi.org/10.1016/j.jchromb.2005.09.045 Publication
Researcher Affiliations
- University of Pennsylvania, School of Veterinary Medicine, Department of Clinical Studies, New Bolton Center Campus, 382 West Street Road, Kennett Square, PA 19348, USA.
MeSH Terms
- Anabolic Agents / blood
- Animals
- Doping in Sports
- Female
- Horses
- Male
- Reproducibility of Results
- Sensitivity and Specificity
- Spectrometry, Mass, Electrospray Ionization / methods
Citations
This article has been cited 6 times.- Zhang Q, Du H, Zhang Y. Recent progress on the detection of animal-derived food stimulants using mass spectrometry-based techniques.. Front Nutr 2023;10:1226530.
- Caprioli G, Genangeli M, Mustafa AM, Petrelli R, Ricciutelli M, Sagratini G, Sartori S, Laus F, Vittori S, Cortese M. Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS.. Pharmaceuticals (Basel) 2021 Apr 21;14(5).
- Koutsoumanis K, Allende A, Alvarez-Ordóñez A, Bolton D, Bover-Cid S, Chemaly M, Davies R, De Cesare A, Herman L, Lindqvist R, Nauta M, Peixe L, Ru G, Simmons M, Skandamis P, Suffredini E, Sánchez JÁ, Blagojevic B, Fürst P, Garin-Bastuji B, Jensen HE, Paulsen P, Baert K, Barrucci F, Broglia A, Georgiadis M, Hempen M, Hilbert F. Evaluation of public and animal health risks in case of a delayed post-mortem inspection in ungulates.. EFSA J 2020 Dec;18(12):e06307.
- Goel R, Muthusamy B, Pandey A, Prasad TS. Human protein reference database and human proteinpedia as discovery resources for molecular biotechnology.. Mol Biotechnol 2011 May;48(1):87-95.
- Gavinelli M, Arioli F, Fracchiolla ML, Casati A, Pompa G. Simultaneous measurement of boldenone (alpha and beta), ADD, testosterone, epitestosterone and AED in bovine faeces.. Vet Res Commun 2008 Sep;32 Suppl 1:S295-8.
- Wolf-Yadlin A, Hautaniemi S, Lauffenburger DA, White FM. Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks.. Proc Natl Acad Sci U S A 2007 Apr 3;104(14):5860-5.
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