Stanozolol‐N‐glucuronide metabolites in human urine samples as suitable targets in terms of routine anti‐doping analysis

Abstract

The exogenous anabolic-androgenic steroid (AAS) stanozolol stays one of the most detected substances in professional sports. Its detection is a fundamental part of doping analysis, and the analysis of this steroid has been intensively investigated for a long time. This contribution to the detection of stanozolol doping describes for the first time the unambiguous proof for the existence of 17-epistanozolol-1 0 Nglucuronide and 17-epistanozolol-2 0 N-glucuronide in stanozolol-positive human urine samples due to the access to high-quality reference standards. Examination of excretion study samples shows large detection windows for the phase-II metabolites stanozolol-1 0 N-glucuronide and 17-epistanozolol-1 0 N-glucuronide up to 12 days and respectively up to almost 28 days. In addition, we present appropriate validation parameters for the analysis of these metabolites using a fully automatic method online solid-phase extraction (SPE) method already published before. Limits of identification (LOIs) as low as 100 pg/ml and other validation parameters like accuracy, precision, sensitivity, robustness, and linearity are given.

HepG2 as promising cell‐based model for biosynthesis of long‐term metabolites: Exemplified for metandienone

“…Though Westerink and Schoonen as well as Gerets et al showed phase II enzyme expression in HepG2 cells, previous studies focusing steroid biotransformation investigated testosterone phase I metabolism solely 10–14 . In recent years, the diagnostic relevance of intact phase II metabolites was increasing, for example, shown for exogenous AASs like stanozolol and clostebol as well as for epiandrosterone‐sulfate as a marker for misuse of testosterone and related endogenous steroids 15–23 …”

“…[10][11][12][13][14] In recent years, the diagnostic relevance of intact phase II metabolites was increasing, for example, shown for exogenous AASs like stanozolol and clostebol as well as for epiandrosterone-sulfate as a marker for misuse of testosterone and related endogenous steroids. [15][16][17][18][19][20][21][22][23] The investigation of the HepG2 cells as an adequate in vitro model to study and generate metabolites for doping analysis was the aim of the present study. The exogenous AAS metandienone, for which the biotransformation has been well studied over the last decades, was chosen as model substance.…”

Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry

“…The employed LC-HRMS/MS-based method also allowed for the detection of known phase II metabolites [ 30 ]. Such phase II metabolites of steroids commonly exhibit an ionization behavior similar to that of the unconjugated analytes [ 31 ], which facilitated the detection of three different hydroxylated and glucurono-conjugated metabolites (at m / z 521.2857) and one hydroxylated and sulfo-conjugated species (at m / z 425.2105).…”

Annual banned‐substance review: Analytical approaches in human sports drug testing 2020/2021

“…The characterization of such metabolites and options of mimicking the biotransformation of drugs and drug candidates into analytes that represent appropriate targets for enhancing the comprehensiveness and retrospectivity of ITPs (vide infra) has been pursued further. A prime example of how research into the long‐term elimination of AAS can support anti‐doping efforts has been stanozolol, for which the presence and structure of N ‐glucuronides of stanozolol itself and its 17‐epimer were comprehensively confirmed by chemical synthesis and employed in a confirmatory test method based on LC‐MS/MS 44 . Urine samples were subjected to an online‐SPE using a phenyl‐hexyl trapping column (10 × 3 mm, 2.6 μm particle size), which was interfaced to a C‐18 analytical column (100 × 2.1 mm, 2.6 μm particle size) and, subsequently, via positive ESI to a quadrupole/orbitrap mass analyzer.…”

“…A prime example of how research into the long-term elimination of AAS can support anti-doping efforts has been stanozolol, for which the presence and structure of N-glucuronides of stanozolol itself and its 17-epimer were comprehensively confirmed by chemical synthesis and employed in a confirmatory test method based on LC-MS/MS. 44 Urine samples were subjected to an online-SPE using a phenyl-hexyl trapping column (10 Â 3 mm, 2.6 μm particle size), which was interfaced to a C-18 analytical column (100 Â 2.1 mm, 2.6 μm particle size) and, subsequently, via positive ESI to a quadrupole/orbitrap mass analyzer. Gradient elution with 0.2% formic acid (solvent A) and methanol containing 0.1% formic acid (solvent B) and targeted MS/MS experiments at a resolution of 70,000 (@m/z 200) allowed for separating and identifying all four stanozolol N-glucuronide isomers within 13 min with a limit of identification (LOI) of 100 pg/ml.…”