Sensitive detection of 3′-hydroxy-stanozolol glucuronide by liquid chromatography–tandem mass spectrometry

Tudela, Eva; Deventer, Koen; Eenoo, Peter Van

doi:10.1016/j.chroma.2013.01.001

Cited by 39 publications

(52 citation statements)

References 28 publications

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“…As described for methyltestosterone, the detection capabilities for stanozolol have been significantly improved compared with current screening methods through the monitoring of recently reported long-term metabolites. For stanozolol metabolites, a more selective sample preparation procedure based on cation exchange solid phase extraction taking advantage of its basic nitrogen may be used to obtain cleaner extracts for confirmation purposes as previously described [37].…”

Section: Excretion Studies Of Methyltestosterone and Stanozololmentioning

confidence: 99%

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

Balcells

Pozo

Esquivel

et al. 2015

Journal of Chromatography A

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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.

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Section: Excretion Studies Of Methyltestosterone and Stanozololmentioning

confidence: 99%

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

Balcells

Pozo

Esquivel

et al. 2015

Journal of Chromatography A

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“…These metabolites showed considerable greater concentrations in some athlete's samples. [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]. [17][18][19] The utility of direct analysis of glucuronides together with the commercialization of 3'-hydroxy-stanozolol glucuronide (3STAN-G) (Figure 1, 6) allowed the development of a method that permitted limits of detection of 25-50 pg/mL.…”

Section: Introductionmentioning

confidence: 99%

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

Balcells

Matabosch

Ventura

2016

Drug Testing and Analysis

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Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC-MS/MS to establish potential long-term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M-I to M-XI) were detected and characterized by LC-MS/MS. This paper provides valuable data on the ionization and fragmentation of O-sulfates and N-sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long-term metabolite (epistanozolol-N-glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse. Copyright © 2016 John Wiley & Sons, Ltd.

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“…In 1986, the analysis of stanozolol abuse by means of a major urinary metabolite referred to as 3'-OH-stanozolol (Figure 1, 2) as accomplished by Schänzer and Donike, [2] initiating in-depth investigations into the metabolic fate of this frequently observed AAS [3][4][5] and options to improve detection times after cessation of its use by elite athletes. [13] In the present study, the use of high resolution/high accuracy mass spectrometry for the detection of 3'-OHstanozolol glucuronide is outlined, and complementary information on N-conjugated glucuronide metabolites of stanozolol and 17-epistanozolol and the use of these in routine doping controls is provided. [13] In the present study, the use of high resolution/high accuracy mass spectrometry for the detection of 3'-OHstanozolol glucuronide is outlined, and complementary information on N-conjugated glucuronide metabolites of stanozolol and 17-epistanozolol and the use of these in routine doping controls is provided.…”

Section: Introductionmentioning

confidence: 99%

Expanding analytical possibilities concerning the detection of stanozolol misuse by means of high resolution/high accuracy mass spectrometric detection of stanozolol glucuronides in human sports drug testing

Schänzer

Guddat

Thomas

et al. 2013

Drug Testing and Analysis

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Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3'-OH-stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to stanozolol-N-glucuronide and 17-epistanozolol-N-glucuronide. By means of chemical synthesis, stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3'-OH-stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3'-OH-stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epistanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established methodology over a period of six months to 659 routine sports drug testing samples, a total of 85 adverse analytical findings was uncovered, 72 of which would have remained undetected using earlier employed GC-MS/MS approaches.

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Sensitive detection of 3′-hydroxy-stanozolol glucuronide by liquid chromatography–tandem mass spectrometry

Cited by 39 publications

References 28 publications

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

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

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

Expanding analytical possibilities concerning the detection of stanozolol misuse by means of high resolution/high accuracy mass spectrometric detection of stanozolol glucuronides in human sports drug testing

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