Rapid screening of anabolic steroids in horse urine with ultra-high-performance liquid chromatography/tandem mass spectrometry after chemical derivatisation.
Abstract: Liquid chromatography/mass spectrometry (LC/MS) has been successfully applied to the detection of anabolic steroids in biological samples. However, the sensitive detection of saturated hydroxysteroids, such as androstanediols, by electrospray ionisation (ESI) is difficult because of their poor ability to ionise. In view of this, chemical derivatisation has been used to enhance the detection sensitivity of hydroxysteroids by LC/MS. This paper describes the development of a sensitive ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) method for the screening of anabolic steroids in horse urine by incorporating a chemical derivatisation step, using picolinic acid as the derivatisation reagent. The method involved solid-phase extraction (SPE) of both free and conjugated anabolic steroids in horse urine using a polymer-based SPE cartridge (Abs Elut Nexus). The conjugated steroids in the eluate were hydrolysed by methanolysis and the resulting extract was further cleaned up by liquid-liquid extraction. The resulting free steroids in the extract were derivatised with picolinic acid to form the corresponding picolinoyl esters and analysed by UHPLC/MS/MS in the positive ESI mode with selected-reaction-monitoring. Separation of the targeted steroids was performed on a C18 UHPLC column. The instrument turnaround time was 10.5 min inclusive of post-run equilibration. A total of thirty-three anabolic steroids (including 17β-estradiol, 5(10)-estrene-3β,17α-diol, 5α-estrane-3β,17α-diol, 17α-ethyl-5α-estran-3α,17β-diol, 17α-methyl-5α-androstan-3,17β-diols, androstanediols, nandrolone and testosterone) spiked in negative horse urine at the QC levels (ranging from 0.75 to 30 ng/mL) could be consistently detected. The intra-day and inter-day precisions (% RSD) for the peak area ratios were around 7-51% and around 1-72%, respectively. The intra-day and inter-day precisions (% RSD) for the relative retention times were both less than 1% for all analytes, except the inter-day precision for boldione at 1.2%. The extraction recoveries for all targets were not less than 48%. With exceptional separation achieved by the UHPLC system, matrix interferences were minimal at the expected retention times of the selected transitions. As detection was performed with an UHPLC system coupled to a fast-scanning triple quadrupole mass spectrometer, the method could easily be expanded to accommodate additional steroid targets. This method has been validated for recovery and precision, and could be used regularly for doping control testing of anabolic steroids in horse urine samples.
Copyright © 2012 Elsevier B.V. All rights reserved.
Publication Date: 2012-01-10 PubMed ID: 22265177DOI: 10.1016/j.chroma.2011.12.095Google Scholar: Lookup
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Summary
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The article describes the development of a sensitive method for detecting anabolic steroids in horse urine using a combination of liquid chromatography and mass spectrometry. It particularly explains how they enhanced this detection process by incorporating a chemical derivation step.
Testing Methodology
- The researchers used ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS), which is a more sensitive method for detecting the presence of anabolic steroids, particularly in horse urine samples. The sensitivity is even further increased by introducing a derivatisation step using picolinic acid.
- The process begins with the solid-phase extraction of both free and conjugated anabolic steroids from horse urine. This is carried out with the help of an Abs Elut Nexus, a polymer-based SPE cartridge.
- Following this, the conjugated steroids are processed further through a method referred to as methanolysis, and then cleaned up further by a liquid-liquid extraction process.
- The resulting free steroids are then treated (derivatised) with picolinic acid to form picolinoyl esters. These esters are then analysed through UHPLC/MS/MS in positive ESI mode using selected-reaction-monitoring.
Precision and Recovery
- The researchers were able to consistently detect the presence of thirty-three different anabolic steroids in the horse urine samples when added at various concentrations (0.75 to 30 ng/mL). These steroids included testosterone, nandrolone, 17β-estradiol, and a variety of estrenes, estranes, and androstandiols.
- The precision of the method was evaluated via intra-day and inter-day precisions for peak area ratios and relative retention times. The peak area ratios showed a precision range of around 7-51% intra-day and 1-72% inter-day, while the relative retention times precision was less than 1% for almost all analytes.
- The recoveries of all targets after extraction were at least 48%, demonstrating reasonable efficiency in the detection process.
Conclusion
- Under optimal conditions, the UHPLC system provided minimal matrix interferences at the expected retention times of the selected transitions. Also, the method would allow for expansion to include additional steroid targets because of the high-speed scanning capabilities of the coupled triple quadrupole mass spectrometer.
- Therefore, this method provides enhanced sensitivity and precision for the detection of anabolic steroids in horse urine, making it a valuable technique for regular doping control testing in the equine industry.
Cite This Article
APA
Wong CH, Leung DK, Tang FP, Wong JK, Yu NH, Wan TS.
(2012).
Rapid screening of anabolic steroids in horse urine with ultra-high-performance liquid chromatography/tandem mass spectrometry after chemical derivatisation.
J Chromatogr A, 1232, 257-265.
https://doi.org/10.1016/j.chroma.2011.12.095 Publication
Researcher Affiliations
- Racing Laboratory, The Hong Kong Jockey Club, Sha Tin Racecourse, Sha Tin, NT, Hong Kong, China. colton.hf.wong@hkjc.org.hk
MeSH Terms
- Anabolic Agents / chemistry
- Anabolic Agents / urine
- Animals
- Chromatography, High Pressure Liquid / methods
- Doping in Sports
- Horses
- Hydroxysteroids / chemistry
- Hydroxysteroids / urine
- Male
- Methanol
- Picolinic Acids
- Reproducibility of Results
- Sensitivity and Specificity
- Solid Phase Extraction
- Tandem Mass Spectrometry / methods
Citations
This article has been cited 10 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.
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- 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).
- Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Kusano K, Nagata SI. Microfluidic Quantitative PCR Detection of 12 Transgenes from Horse Plasma for Gene Doping Control. Genes (Basel) 2020 Apr 23;11(4).
- Tozaki T, Ohnuma A, Takasu M, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI. Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control. Genes (Basel) 2019 Mar 21;10(3).
- Tozaki T, Karasawa K, Minamijima Y, Ishii H, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI. Detection of phosphorothioated (PS) oligonucleotides in horse plasma using a product ion (m/z 94.9362) derived from the PS moiety for doping control. BMC Res Notes 2018 Oct 29;11(1):770.
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