Screening of anabolic steroids in horse urine by liquid chromatography-tandem mass spectrometry.

The study presents a method for identifying anabolic steroids in horse urine using liquid chromatography-tandem mass spectrometry (HPLC-MS-MS), which stands as an improvement over the traditional gas chromatography-mass spectrometry technique due to its high precision and lower sensitivity to certain steroids.

Introduction and Problem Statement

• Anabolic steroids, known for boosting athletic performance, are prohibited substances in sports such as the Olympic games and horse racing.
• Conventional methods to monitor steroid abuse involve identifying anabolic steroids or their metabolites in urine samples using gas chromatography-mass spectrometry (GC-MS).
• Despite their usage, these techniques have downsides; they frequently involve complex sample preparation and chemical derivatisation and they might not be sensitive enough in detecting certain steroids.

Proposed Approach and Methodology

• This study proposes a higher-performance method based on liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) aimed at identifying anabolic steroids that are not adequately detected by GC-MS.
• A detailed process, comprising enzyme-treated urine, solid-phase extraction (SPE), and further cleanup via a base wash, was followed.
• Steroid separation is performed using a reversed-phase DB-8 column with acetic acid and methanol serving as the mobile phase in a gradient elution programme.
• The mass spectrometer, utilized to detect the steroids, was operated in the positive electrospray ionisation mode with multiple reaction monitoring (MRM).

Validation of the Method

• The sensitivity of the method was demonstrated successfully by consistently detecting urine samples fortified with lower concentration levels of 15 different anabolic steroids.
• No significant matrix interferences, which could negatively affect the detection, were observed at the targeted ion mass’ retention time in blank urine samples.
• The method’s effectiveness was further evaluated on parameters like specificity, sensitivity, precision, recovery rates and the performance of the enzyme hydrolysis step.

Outcome and Importance of the Study

• The method proved to be successful in analysing urine samples for methenolone acetate, thereby indicating it could be an effective tool in identifying anabolic steroids and their metabolites in horse urine.
• Ultimately, this study affirms the potential utility of HPLC-MS-MS in the context of horse racing performance regulation, offering a more robust and efficient detection method than the traditional GC-MS.