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

Stanozolol is one of the most frequently detected anabolic steroids in doping control samples. This compound is metabolized to a large extent and its metabolites can be detected in urine much longer than the parent compound. The main stanozolol metabolites are excreted in urine as glucuronide conjugates and 3′-hydroxy-stanozolol glucuronide (3STANG) is one of the most important in human urine. Therefore enzymatic hydrolysis is usually applied prior to extraction.In this article a method for the sensitive detection of intact 3′-hydroxy-stanozolol glucuronide, by liquid chromatography tandem mass spectrometry, is described.The method takes advantage of an easy and fast sample preparation based on a single solid-phase extraction avoiding enzymatic hydrolysis or derivatization. It allows to detect stanozolol abuse in human urine at 25 pg mL−1. The method was validated according to Eurachem guidelines. The matrix effect, expressed as ion enhancement was +14%. The extraction recovery of the method was 93%. The limit of detection (LOD), whereby all WADA-criteria in chromatography and mass spectrometry are fulfilled, was determined at 50 pg mL−1. Application of the method to an excretion study revealed that the 3′-hydroxy-stanozolol glucuronide could be confirmed for 10 days after oral administration of 2 mg of stanozolol, prolonging detection times compared to other metabolites and methodologies by almost 50%.

► A simple and fast confirmation method by LC–MS of stanozolol misuse is presented.
► 3′-hydroxy-stanozolol glucuronide is used as target compound.
► Solid-phase extraction is used for sample preparation and no hydrolysis is required.
► The limit of detection is 50 pg mL−1 and this is lower than previous described methods.
► This method permits prolongation of the detection times by almost 50%.