A broad-spectrum equine urine screening method for free and enzyme-hydrolysed conjugated drugs with ultra performance liquid chromatography/tandem mass spectrometry.

The research article centers on a newly developed method using ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) for more efficient and effective screening of a multitude of drugs in equine urine. With this new method, the simultaneous detection of 140 drugs can be achieved in less than 13 minutes, greatly reducing labor and instrumental workload.

Original Screening Methods and their Limitations

• The authors’ laboratory formerly used four different liquid chromatography/mass spectrometric (LC/MS) methods to detect a wide range of drugs in equine urine, including anti-diabetic drugs, sedatives, steroids, diuretics, and more.
• Despite their range, these methods were labor-intensive and time-consuming, and they did not cover some target analytes, limiting their effectiveness.

Development and Mechanism of the New UPLC/MS/MS Method

• In response to these limitations, the authors developed a new method utilizing UPLC/MS/MS that consolidates all target analytes detected by the original four LC/MS methods, resulting in improved detection limits and coverage.
• The new method uses a solid-phase extraction (SPE) of two aliquots (portions) of equine urine with two Abs Elut Nexus cartridges.
• One aliquot of the urine sample undergoes treatment with β-glucuronidase prior to SPE. This enzyme breaks down glucuronic acid in the urine, facilitating drug detection.
• The second aliquot is processed directly, allowing preservation of drugs that may decompose during enzyme hydrolysis.
• The combined eluate, which is the dissolved drug portion, is analyzed by UPLC/MS/MS using positive and negative electrospray ionisation in the selected-reaction-monitoring mode, an optimized detection scheme.
• These steps altogether allow simultaneous detection of 140 drugs at ppb (parts per billion) to sub-ppb levels in equine urine in less than 13 minutes.

Benefits and Results of the New Method

• The new method drastically reduces matrix interference, or background noise, during the detection process due to the superior chromatographic separation achieved with the UPLC system, ensuring more accurate measurements.
• Moreover, the method has been validated for recovery (how well the extraction process works) and precision, making it a reliable resource for drug detection.
• Given its efficiency and effectiveness, the authors’ lab now uses this method as an important part of their array of screening techniques for doping control analysis in equine urine samples.