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 microg 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 microg 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.
Vonaparti
A
A
Doping Control Laboratory of Athens, Olympic Athletic Center of Athens 'Spiros Louis', 37 Kifissias Ave., 151 23 Maroussi, Greece.
Lyris
E
E
Panderi
I
I
Koupparis
M
M
Georgakopoulos
C
C
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004300:Doping in Sports; D006736:Horses; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015813:Substance Abuse Detection / Q000379:methods*; D013805:Theobromine / Q000652:urine*; D016482:Urinalysis / Q000379:methods*; D014665:Vasodilator Agents / Q000652:urine*
D014665:Vasodilator Agents; D013805:Theobromine