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Área de trabajo: Control de Dopaje.

Título: Detección de Stanozolol Glucuronida en test de drogas deportivas  por Espectrometría de Masa-Masa de Alta Resolución y Masa Exacta.

Título original: Detection of Stanozolol Glucuronides in Human Sports Drug Testing by Meansof High-Resolution, Accurate-MassMass Spectrometry

Autor: Wilhelm Schänzer1, Sven Guddat1, Andreas Thomas1, Georg Opfermann1, Hans Geyer1, and Mario Thevis1,2

1Institute of Biochemistry – Center for Preventive Doping Research, German Sport University Cologne, Cologne, Germany; 2European Monitoring Center for Emerging Doping Agents, Cologne/Bonn, Germany

Introduction

The analysis of the anabolic steroid stanozolol (Figure 1a) has proved to be challenging for gas chromatography mass spectrometry (GC-MS) methods due to stanozolol’s peculiar physicochemical properties. The uncovering of stanozolol abuse by means of its major urinary metabolite 3’-OH-stanozolol (Figure 1b) as accomplished by Schänzer and Donike1 initiated investigations into the metabolic fate of this anabolic agent. The molecular features of stanozolol and its metabolites demand sophisticated derivatization and separation steps for GC/MS-based methodologies. Methods based on liquid chromatography with electrospray-ionization tandem mass spectrometry (LC-MS/MS), on the other hand, provide benefits such as lower limits of detection (LODs) and detection windows with expanded metabolite identification. 3’-OH-stanozolol glucuronide (Figure 1c) is the latest metabolite analyzed at 25–50 pg/mL in human urine. In the present study, the use of high-resolution, accurate-mass mass spectrometry for the detection of 3’-OH-stanozolol glucuronide is outlined. Complementary information on N-conjugated glucuronide metabolites of stanozolol and 17-epistanozolol and the use of these in routine doping controls is provided.

 

Experimental

Administration Samples

Two healthy male volunteers (56 and 61 years of age) received a single oral dose of 5 mg of stanozolol (Winstrol®). Urine samples were collected prior to (blank) and up to 28 days post administration of the drug. The urine specimens were stored at -20 °C until preparation and analysis. The study was approved by the local ethical committee and written consent was obtained from both participants.

 

Sample Preparation

Ninety microliters of urine were enriched with 10 µL of an acetonitrile solution containing the internal standard methyltestosterone (1 µg/mL). The samples were vortexed for 10 s and subjected to LC-MS/MS analysis. Confirmatory analyses were conducted by applying 1 mL of urine to a solid-phase extraction (SPE) cartridge preconditioned with 2 mL of water and 2 mL of methanol. After the sample had passed through, the resin was washed with 2 mL of water and the analytes eluted with 2 mL of methanol. The organic phase was evaporated to dryness and reconstituted in 100 µL of solvents A (0.1% formic acid) and B (acetonitrile) (1:1, v/v) for LC-MS/MS analysis.

 

LC-MS/MS

The analyses were conducted using a Thermo Scientific™ Accela™ 1250 liquid chromatograph interfaced via a heated electrospray ionization (HESI-II) source to a Thermo Scientific™ Q Exactive™ Focus mass spectrometer. The LC was equipped with a Nucleodur® C18 Pyramid analytical column, 50 x 2 mm, particle size 1.8 µm, (Macherey-Nagel, Düren, Germany) and a corresponding precolumn (4 x 2 mm, particle size 3 µm). The mobile phases 0.1% formic acid (A) and acetonitrile (B) were used to perform a gradient elution at 200 µL/min from 99% of A to 100% of B in 7 min, followed by re-equilibration for 3 min.

 

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