Screening for anabolic steroids in sports: analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry.
In order to improve the detection capabilities of anabolic androgenic steroids (AAS) in sports, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for the simultaneous detection of AAS phase I and phase II intact urinary metabolites (glucuronides and sulfates) was developed. A total of 36 metabolites (7 unconjugated; 19 glucuronides and 10 sulfates) corresponding to 15 of the most reported AAS were included. Analytes were extracted from urine using C18 cartridges. LC and MS conditions were studied in-depth to determine the most sensitive and selective conditions for each analyte. A selected reaction monitoring method was set up. The optimization of the experimental parameters for 13 metabolites not available as standards was performed using excretion study urines. Extraction recoveries were above 77% for all 23 validated analytes. Intra-day precision was lower than 21%, and LODs were in the range 0.25-4ng/mL for 18 of the 23 analytes. Matrix effect was evaluated using post column infusion and ranged from 92 to 147%. The method was successfully applied to excretion study urines of different exogenous AAS. The suitability of the strategy was demonstrated with methyltestosterone and stanozolol excretion study urines by achieving detection times of 22 and 21 days, respectively. The method is compliant with the World Antidoping Agency requirements for most of the studied compounds. It represents a cost-effective approach that improves the detection capabilities of AAS by increasing the sensitivity for some metabolites and by including recently described phase II long-term metabolites not detectable using the current screening strategy.
Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003 Barcelona, Spain.
Georgina
G
Balcells
Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003 Barcelona, Spain.
Oscar J
OJ
Pozo
Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003 Barcelona, Spain.
Argitxu
A
Esquivel
Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003 Barcelona, Spain; Department of Biological Chemistry and Molecular Modeling, Instituto de Química Avanzada de Cataluña (IQAC), Consejo Superior de Investigaciones Científicas (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain.
Aristotelis
A
Kotronoulas
Department of Biological Chemistry and Molecular Modeling, Instituto de Química Avanzada de Cataluña (IQAC), Consejo Superior de Investigaciones Científicas (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain.
Jesús
J
Joglar
Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003 Barcelona, Spain; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003 Barcelona, Spain.
Jordi
J
Segura
Bioanalysis Research Group, IMIM, Hospital del Mar Medical Research Institute, Doctor Aiguader 88, 08003 Barcelona, Spain; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Doctor Aiguader 88, 08003 Barcelona, Spain. Electronic address: rventura@imim.es.
Rosa
R
Ventura
D045930:Anabolic Agents / Q000652:urine*; D002851:Chromatography, High Pressure Liquid; D004300:Doping in Sports / Q000517:prevention & control*; D020719:Glucuronides / Q000652:urine; D057230:Limit of Detection; D013431:Sulfates / Q000652:urine; D053719:Tandem Mass Spectrometry; D045165:Testosterone Congeners / Q000652:urine; D016482:Urinalysis / Q000191:economics / Q000379:methods*
D045930:Anabolic Agents; D020719:Glucuronides; D013431:Sulfates; D045165:Testosterone Congeners
Anabolic steroids; Doping; Glucuronides; LC–MS/MS; Sulfates; Urine