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

In equine sport, theobromine is prohibited with a threshold level of 2 µg mL −1 in urine, hence doping control laboratories have to establish quantitative and qualitative methods for its determination. Two simple liquid chromatography/mass spectrometry (LC/MS) methods for the identification and quantification of theobromine were developed and validated using the same sample preparation procedure but different mass spectrometric systems: ion trap mass spectrometry (ITMS) and time‐of‐flight mass spectrometry (TOFMS). Particle‐free diluted urine samples were directly injected into the LC/MS systems, avoiding the time‐consuming extraction step. 3‐Propylxanthine was used as the internal standard. The tested linear range was 0.75–15 µg mL −1 . Matrix effects were evaluated analyzing calibration curves in water and different fortified horse urine samples. A great variation in the signal of theobromine and the internal standard was observed in different matrices. To overcome matrix effects, a standard additions calibration method was applied. The relative standard deviations of intra‐ and inter‐day analysis were lower than 8.6 and 7.2%, respectively, for the LC/ITMS method and lower than 5.7 and 5.8%, respectively, for the LC/TOFMS method. The bias was less than 8.7% for both methods. The methods were applied to two case samples, demonstrating simplicity, accuracy and selectivity. Copyright © 2009 John Wiley & Sons, Ltd.