title: Complementing the characterization of in vivo generated N-glucuronic acid conjugates of stanozolol by collision cross section computation and analysis.
abstract: Detailed structural information on metabolites serving as target analytes in clinical, forensic, and sports drug testing programmes is of paramount importance to ensure unequivocal test results. In the present study, the utility of collision cross section (CCS) analysis by travelling wave ion mobility measurements to support drug metabolite characterization efforts was tested concerning recently identified glucuronic acid conjugates of the anabolic-androgenic steroid stanozolol. Employing travelling-wave ion mobility spectrometry/quadrupole-time-of-flight mass spectrometry, drift times of five synthetically derived and fully characterized steroid glucuronides were measured and subsequently correlated to respective CCSs as obtained in silico to form an analyte-tailored calibration curve. The CCSs were calculated by equilibrium structure minimization (density functional theory) using the programmes ORCA with the data set B3LYP/6-31G and MOBCAL utilizing the trajectory method (TM) with nitrogen as drift gas. Under identical experimental conditions, synthesized and/or urinary stanozolol-N and O-glucuronides were analyzed to provide complementary information on the location of glucuronidation. Finally, the obtained data were compared to CCS results generated by the system's internal algorithm based on a calibration employing a polyalanine analyte mixture. The CCSs ΩN2 calculated for the five steroid glucuronide calibrants were found between 180 and 208 Å(2) , thus largely covering the observed and computed CCSs for stanozolol-N1'-, stanozolol-N2'-, and stanozolol-O-glucuronide found at values between 195.1 and 212.4 Å(2) . The obtained data corroborated the earlier suggested N- and O-glucuronidation of stanozolol, and demonstrate the exploit of ion mobility and CCS computation in structure characterization of phase-II metabolic products; however, despite reproducibly measurable differences in ion mobility of stanozolol-N1'-, N2'-, and O-glucuronides, the discriminatory power of the chosen CCS computation algorithm was found to be not appropriate to allow for accurate assignments of the two N-conjugated structures. Using polyalanine-based calibrations, significantly different absolute values were obtained for all CCSs, but due to a constant offset of approximately 45 Å(2) an excellent correlation (R(2)  = 0.9997) between both approaches was observed. This suggests a substantially accelerated protocol when patterns of computed and polyalanine-based experimental data can be used for structure elucidations instead of creating individual analyte-specific calibration curves.
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.|European Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/Bonn, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Dib
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Höppner
authors.affiliation: Bayer CropScience AG, Alfred-Nobel-Str. 50, 40789, Monheim, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Lagojda
authors.affiliation: Bayer CropScience AG, Alfred-Nobel-Str. 50, 40789, Monheim, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Kuehne
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Sander
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Opfermann
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
mesh_terms: D000465:Algorithms; D045930:Anabolic Agents / Q000652:urine*; D000728:Androgens / Q000652:urine*; D002138:Calibration; D002853:Chromatography, Liquid; D003198:Computer Simulation; D004300:Doping in Sports; D020719:Glucuronides / Q000652:urine*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D050217:Metabolic Detoxication, Phase II; D058573:Performance-Enhancing Substances / Q000652:urine*; D011237:Predictive Value of Tests; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013197:Stanozolol / Q000652:urine*; D015813:Substance Abuse Detection / Q000379:methods* / Q000592:standards; D053719:Tandem Mass Spectrometry; D016482:Urinalysis
chemical_list: D045930:Anabolic Agents; D000728:Androgens; D020719:Glucuronides; D058573:Performance-Enhancing Substances; D013197:Stanozolol
keywords: anabolic steroids; doping; ion mobility; mass spectrometry; sport