Screening of over 100 drugs in horse urine using automated on-line solid-phase extraction coupled to liquid chromatography-high resolution mass spectrometry for doping control.

The study presents a quick method for the analysis of horse urine samples to detect over 100 types of drugs. The technique employs automated on-line solid-phase extraction coupled with liquid-chromatography/high resolution mass spectrometry, allowing for faster, accurate detection of potential doping drugs in horse urine at very low levels.

Research Methodology

• The urine sample initially undergoes hydrolysis using β-glucuronidase, an enzyme that releases drugs that are bound or ‘conjugated’. This is crucial because many drugs and their metabolites are conjugated, meaning they are joined with other compounds to aid their excretion from the body. The enzyme used here cleaves these linkages, freeing up the drugs for analysis.
• After hydrolysis, the sample is subjected to centrifugal filtration. Essentially, it involves spinning the test sample at high speeds to separate its components. The end result of this step is a filtrate, which contains the substances that need to be analysed.
• The filtrate is then injected into an online solid-phase extraction (SPE) system. This system consists of a pre-column filter and a SPE cartridge column designed for analyte and matrix component separation. During this step, unwanted matrix components are sent to waste, leaving behind only the analytes – the substances of interest – for further evaluation.

Process and Detection

• The analytes are subjected to ‘refocusing’ and chromatographic separation using a C analytical column. This allows for the dispersion of the different components within the sample according to how they react to the column’s properties. This separation method is important for reliable and accurate detection.
• Detections are done via a full-scan High-Resolution Mass Spectrometry (HRMS) in both positive and negative electrospray ionisation modes. This is a type of mass spectrometry that generates ions from the sample by expelling it through a small orifice at high voltage. The time for each full cycle of this method, also including the time to clean up the sample and prepare for the next one, is 16 minutes.

Key Findings and Conclusion

• The researchers found no considerable matrix interference at the expected retention times of the targeted masses. This indicates that the method was successful in isolating the analytes from other urine components that could hamper the analysis.
• More than 90% of the studied drugs showed limits of detection (LoDs) at or below 5ng/mL, with some reaching as low as 0.05ng/mL. This high sensitivity is significant for doping controls where substances are often present in extremely low quantities.
• Data-dependent acquisition (DDA) provided product-ion scan data to further verify the presence of detected analytes. In other words, it made it possible to double-check the ions detected in the first round of mass spectrometry against a library of known values.
• The method’s viability was demonstrated by successful detection of drugs in samples taken for doping control.