Direct injection liquid chromatography/electrospray ionization mass spectrometric horse urine analysis for the quantification and confirmation of threshold substances for doping control. II. Determination of theobromine.

This study presents methods for the detection and measurement of theobromine — a banned substance in horse sports — in equine urine using liquid chromatography and mass spectrometry.

Research Goals and Methods

• The goal of this research was to create reliable, efficient methods for detecting and quantifying theobromine, a substance prohibited in equine sports due to its use in doping. Theobromine has a threshold level of 2 micrograms per milliliter (µg mL^-1) in horse urine.
• The study developed two liquid chromatography/mass spectrometry (LC/MS) techniques: Ion Trap Mass Spectrometry (ITMS) and Time-of-Flight Mass Spectrometry (TOFMS). These methods were designed to be simple and quick as they bypass the typically slow extraction step by directly injecting diluted urine samples into the LC/MS system.
• An internal standard, 3-propylxanthine, was used to improve the accuracy of the measurements. The methods were validated across a linear range of 0.75-15 µg mL^-1.

Outcomes and Implications

• The study discovered significant variance in the signals of theobromine and the internal standard in different matrices. To mitigate these effects, a standard additions calibration method was used.
• Both the ITMS and TOFMS methods achieved acceptable consistency and accuracy, with relative standard deviations for intra- and inter-day analysis below 8.6% and 7.2% (ITMS) or 5.7% and 5.8% (TOFMS), respectively. The bias was also less than 8.7% in both techniques.
• These techniques have practical applications in sports doping control. They were successfully applied to two real case samples, demonstrating their simplicity, accuracy, and selectivity. This means the method can accurately identify and measure theobromine in samples with potential interference.