Determination quali-quantitative of feed contaminants in complementary feedingstuffs for horses

Caffeine, theobromine, morphine, theophylline, atropine, scopolamine, codeine, bufotenine, dimethyltryptamine and tebaine, are widespread natural environmental substances. These compounds, regarded as doping agents by equestrian and racing regulators, can naturally contaminate horse feed affecting the performance of the animal and producing positive results in the anti-doping controls of urine and blood. They are infact well absorbed when administered by oral intake. For this purpose an LC-MS method for the simultaneous determination of all these substances has been developed and validated to analyze the complementary feedingstuffs for horses. A simple and fast liquid-liquid extraction on 0,5 mg of product is used. Chromatographic separation, using caffeine C13 like Internal Standard, was developed on a C18 Synergie Polar® column, with Methanol and 0.1% and Formic acid in water as mobile phases. Triple quadrupole mass spectrometer, equipped with an ESI source was operated in positive ion mode. All the analytes were determined by a Multiple Reaction Monitoring (MRM). For the validation of the method the following parameters were evaluated: limit of detection (LOD), limit of quantification (LOQ), intrainter assay accuracy and precision. The same method was also validated like quantitative method for caffeine and codeine due to their frequent presence found in different complementary feeds. The developed method is routinely used to control the complementary feedingstuffs for horses.