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.

Detection of stanozolol O‐ and N‐sulfate metabolites and their evaluation as additional markers in doping control

“…[20] The phase II metabolites of 4STAN and 16STAN are still not completely characterized (position of the glucuronide moiety is not known) nor commercially available ( Figures 1,[7][8]. [22] Up to now, studies have focused in glucuronoconjugated metabolites obtained using both, hydrolysis with β-glucuronidase enzymes and direct detection. [21] Characterization of these metabolites has been recently achieved by ion mobility and nuclear magnetic resonance (NMR) spectroscopy.…”

“…[21] Characterization of these metabolites has been recently achieved by ion mobility and nuclear magnetic resonance (NMR) spectroscopy. [22] Up to now, studies have focused in glucuronoconjugated metabolites obtained using both, hydrolysis with β-glucuronidase enzymes and direct detection. Sulfate metabolites have not been previously studied.…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…Characterizing metabolic products and elucidating their biotransformation pathways from the administered drug to renally excreted urinary metabolites have been complex yet important aspects in doping controls. Hence, the utility of ion mobility and collision cross section (CCS) computation in facilitating and contributing to urinary metabolite characterization has been assessed in the case of recently identified N ‐glucuronides of stanozolol . Measuring and computing the CCS values of potential metabolites readily allowed for differentiating stanozolol N ‐ and O ‐glucuronides, while unequivocally assigning structures to N 1’‐ and N 2’‐stanozolol glucuronides was only accomplished after chemical synthesis and nuclear magnetic resonance spectroscopy of the analytes.…”

“…Hence, the utility of ion mobility and collision cross section (CCS) computation in facilitating and contributing to urinary metabolite characterization has been assessed in the case of recently identified N-glucuronides of stanozolol. [71] Measuring and computing the CCS values of potential metabolites readily allowed for differentiating stanozolol N-and O-glucuronides, while unequivocally assigning structures to N1'-and N2'-stanozolol glucuronides was only accomplished after chemical synthesis and nuclear magnetic resonance spectroscopy of the analytes. However, reducing the number of potential structures of steroid metabolites to be synthesized for analyte confirmation purposes is of considerable help and can be supported by the presented approach.…”

The 33rd Manfred Donike Workshop on Doping Analysis

“…[5] Moreover, established phase-II metabolites of stanozolol (N-glucuronides) were subjected to orthogonal characterization methods, including ion mobility and nuclear magnetic resonance (NMR) spectroscopy, corroborating existing doping control procedures. [6] Glucocorticoids of both natural/endogenous and synthetic origin were also studied by isotope-ratio mass spectrometry, providing a complementary tool to uncover the misuse of cortisol and related metabolites. [7] Furthermore, the relevance of studies into the prevalence of meldonium (mildronate) were underlined by findings of up to 2% of routine doping control samples containing meldonium as demonstrated by an isotope-dilution liquid chromatographymass spectrometry (LC-MS) based approach.…”