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Analysis of glucuronide and sulfate steroids in urine by ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry

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Abstract

Profiling conjugated urinary steroids to detect anabolic-steroid misuse is recognized as an efficient analytical strategy in both chemical-food-safety and anti-doping fields. The relevance and robustness of such profiling rely on the analysis of glucuronide and sulfate steroids, which is expected to have properties including accuracy, specificity, sensitivity, and, if possible, rapidity. In this context, the ability of ultra-high-performance supercritical-fluid chromatography (UHPSFC) hyphenated tandem mass spectrometry (MS–MS) to provide reliable and accurate phase II analysis of steroids was assessed. Four stationary phases with sub-2 μm particles (BEH, BEH 2-ethyl-pyridine, HSS C18 SB, and CSH fluorophenyl) were screened for their capacity to separate several conjugated steroid isomers. Analytical conditions including stationary phase, modifier composition and percentage, back pressure, column temperature, and composition and flow rate of make-up solvent were investigated to improve the separation and/or the sensitivity. Thus, an analytical procedure enabling the analysis of eight glucuronide and 12 sulfate steroids by two different methods in 12 and 15 min, respectively, was optimized. The two procedures were evaluated, and UHPSFC–MS–MS analysis revealed its ability to provide sensitive (limits of quantification: 0.1 ng mL−1 and 0.5 ng mL−1 for sulfate and glucuronide steroids, respectively) and reliable quantitative performance (R 2 > 0.995, RSD < 20 %, and bias < 30 %) through the use of suitable labeled internal standards. Comparison with UHPLC–MS–MS was performed, and UHPSFC–MS–MS obtained better performance in terms of sensitivity. Finally, as a proof of concept, this so-called green technology was used in a chemical-food-safety context to profile steroid conjugates in urine samples from bovines treated with estradiol.

Glucuronide and sulfate steroids analysis in urine by ultra-high performance supercritical fluid chromatography hyphenated tandem mass spectrometry

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Acknowledgments

Mickael Doué was a fellowship recipient of the French Region Pays de la Loire and the French Ministry of Agriculture (contract n°2011-09551). We gratefully thank Waters (Milford, MA, USA) for providing this study with Acquity UPC2 device and stationary phases.

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Authors and Affiliations

  1. École Nationale Vétérinaire, Agroalimentaire et de l’alimentation Nantes-Atlantique, Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), LUNAM Université, Oniris, Atlanpole-La Chantrerie, CS 50707, 44307, Nantes, France

    Mickael Doué, Gaud Dervilly-Pinel, Karinne Pouponneau, Fabrice Monteau & Bruno Le Bizec

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  1. Mickael Doué
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  2. Gaud Dervilly-Pinel
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  3. Karinne Pouponneau
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  4. Fabrice Monteau
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  5. Bruno Le Bizec
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Corresponding author

Correspondence to Gaud Dervilly-Pinel.

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Published in the topical collection on Hormone and Veterinary Drug Residue Analysis with guest editors Siska Croubels, Els Daeseleire, Sarah De Saeger, Peter Van Eenoo, and Lynn Vanhaecke.

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Doué, M., Dervilly-Pinel, G., Pouponneau, K. et al. Analysis of glucuronide and sulfate steroids in urine by ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry. Anal Bioanal Chem 407, 4473–4484 (2015). https://doi.org/10.1007/s00216-015-8573-x

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  • DOI: https://doi.org/10.1007/s00216-015-8573-x

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