Detecting the abuse of 19‐norsteroids in doping controls: A new gas chromatography coupled to isotope ratio mass spectrometry method for the analysis of 19‐norandrosterone and 19‐noretiocholanolone

Abstract

The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography-tandem mass spectrometry (GC-MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/ml and not originated from pregnant female athletes or female treated with 19-norethisterone.19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TCs), whereas androsterone (A), pregnanediol (PD), and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone-based formulations.Before the instrumental analysis, urine samples (<25 ml) were hydrolyzed with β-glucuronidase from Escherichia coli and extracted with n-pentane. Compounds of interest were purified through a single (for PT) or double (for 19-NE, 19-NA, A, and PD) liquid chromatographic step, to reduce the background noise and eliminate interferences that could have affect the accuracy of δ 13 C values. The limit of quantification (LOQ) of 2 ng/ml was ensured for both 19-NA and 19-NE. The 19-NE determination could be helpful in case of "unstable" urine samples, in late excretion phases or when coadministration with 5α-reductase inhibitors occur.

5α‐reductase inhibitors: Evaluation of their potential confounding effect on GC‐C‐IRMS doping analysis

“…Firstly, the GC–MS/MS quantification of anabolic steroids and their metabolites as trimethylsilyl derivatives was carried out on a volume of 2 mL of urine ( procedure A ) 34–36 . Therefore, variable volume of urine from 3 to 21 ml, depending on the ERC and TC urinary levels previously estimated by GC–MS/MS, were processed to perform the GC‐C‐IRMS analyses and determine the δ 13 C values of the urinary steroids profile markers ( procedure B ) 33 and the other target analytes (19‐NA and 19‐NE, by procedure C ) 29,32 …”

“…In both B and C protocols , the dried residues were dissolved in 50 μl of an ISTDLC solution at 100 μg/ml in water:methanol (50:50) to undergo the next HPLC purification step; no derivatization process was included. HPLC purification is necessary to get urinary extracts lacking in interferences able to impact the accuracy and reliability of the δ 13 C values measured during the IRMS analyses: one ( procedure B ) or two ( procedure C ) HPLC sequential steps were conducted, 29,32,33 as shown below.…”

“…Similarly, the combined administration of 5‐ARIs and 19‐norsteroids could mask the forbidden intake of nandrolone and precursors. Urinary concentration of 19‐norandrosterone (19‐NA), selected as the key target metabolite, could fall below the threshold value of 2.5 ng/ml, 23,26–28 and the supporting analysis of 19‐noretiocholanolone (19‐NE) might be crucial to avoid false negative results 29,30 …”

“…We herein carried out a preliminary research on three male subjects after the administration of 19‐norandrostenedione, a nandrolone precursor, whose end glucuronated metabolites are 19‐NA and 19‐NE, with the combined use of a single or repeated dose of finasteride at low dosage. δ 13 C values of the 5α‐ and 5β‐metabolites commonly determined in the GC‐C‐IRMS procedure for detecting the abuse of pseudo‐endogenous steroids 30,31 were measured by following a previously published procedure based on GC‐C‐IRMS 29,32,33 . 19‐NE, recently added by WADA (World Anti‐Doping Agency) in the TD2021NA, 30 has been considered as an auxiliary target compound (TC): it is typically excreted in smaller amounts, but it could become the main metabolite due to the suppressive effect of 5‐ARIs on the production of 19‐NA.…”

“…Urinary concentrations of the analytes of interest have been estimated by GC–MS/MS (gas chromatography coupled to tandem mass spectrometry) following the analytical procedure currently in use in our laboratory in the daily routine doping controls 34–36 . The resulting values were compared with those already presented after the sole oral intake of 19‐norandrostenedione to provide a baseline urinary steroid and isotopic profile 29 …”