Online turbulent flow extraction coupled with liquid chromatography-tandem mass spectrometry for high throughput screening of anabolic steroids in horse urine.

The research presents a novel method for rapid screening of anabolic steroids and their metabolites in horse urine using online turbulent flow extraction coupled with liquid chromatography-tandem mass spectrometry.

Method Development

• The researchers developed a high throughput method for simultaneous screening of anabolic steroids and their metabolites in equine urine.
• To overcome the challenges posed by the complex matrices and high viscosity of horse urine, the researchers used turbulent flow chromatography, a technique that can simplify urine pretreatment.
• The urine was initially treated with mixed-mode cation exchange solid phase extraction and then hydrolyzed using β-glucuronidase/arylsulfatase. These steps helped to break down the complex substances in the urine into simpler substances.
• Then, the processed sample was loaded onto the TurboFlow Cyclone extraction column to eliminate further matrix. This process helps remove additional unnecessary components from the urine sample, further simplifying the contents before analysis.
• Following this, the stripped sample was run through a fused-core C18 column (a type of high performance liquid chromatography column) before undergoing detection using tandem mass spectrometry (MS/MS).

Validation and Results

• The method’s optimization and validation were extensively discussed. All analytes (substances being measured) were detected rapidly (<10min) with high sensitivity (on the picogram to nanogram per milliliter level).
• No interference was observed during detection, meaning that the results of the screening were pure and reliable for each targeted steroid.
• The method’s linearity range was between 0.1-10ng/mL for nine steroids and 1.0-50ng/mL for the others, with correlation of coefficient values above 0.995. Linearity range refers to the range within which a particular test will give accurate results, while the correlation coefficient values indicate a very strong correlation between the actual concentration levels and the levels as detected by the screening method.
• Precision and accuracy of the method ranged from 0.1 to 14.5% and 1.7 to 12.4%, respectively, indicating a comparatively high level of accuracy and consistency in the test results.
• The method was then successfully applied to analyze anabolic steroids in horse urine following administration of a model drug, demonstrating its effectiveness and practical application.