Determination of ketosteroids in meat trough derivatization chemistry and liquid chromatography tandem Mass spectrometry.

Method reliable through the derivatization chemistry. Stable isotope dilution analysis without the costs of labeled analytes. Ketosteroids have important effects on the human body, for example, sexual differentiation, bone homeostasis, cardiovascular system, brain or liver. For their anabolic effect, some ketosteroids are used as growth promoters in animal husbandry. Are administered to cattle breeding to increase their muscle and reduce fat content. The European Union has categorically banned the use of anabolic steroids in farm animal. The Directive 2003/74/EC specifically prohibits the use of sevenhormones used as growth promoters: 17β-estradiol, progesterone, testosterone, melengestrol acetate, trenbolone acetate and zeranol acetate. Different methods have been reported for the assay , including liquid chromatography/tandem mass spectrometry (LC/MSMS). Here we present a fast and reliable method of assay of ketosteroids based on UPLC-MSMS and derivatization chemistry. Eight steroids (progesterone, testosterone, cortisone, hydrocortisone, Melengestrolo acetato, Trenbolone acetato, pregnenolone, androsterone, were assayed. The meat was crushed and extracted with methanol, the methanol was washed with n-hexane and evaporated, the crude extrcted was purified by SPE cartridge (C18). The derivatization step was performed by mixing the latter solution, with a 1 M solution of methoxyamine, and heated at 68° for 1hr. The reaction residue was dried under a gentle nitrogen flow and reconstituted with an appropriate solution of ketosteroids standards derivatized with d3-methoxyamine,previously prepared. The final solution was injected into the UHPLC/MSMS. Two transitions were monitored for each derivatized steroid; the most abundant was used for the assay. The method here shown is based on the derivatization of the ketonic moiety of steroids by methoxyamine. The quantitative analysis is performed by MRM approach monitoring two transition for each analytes and for the labelled internal standards (ketosteroids standards derivatized with d3-methoxyamine). The calibration curve obtained is built using triplicate samples of d0- derivatized hormones, whose concentrations range from 0.5 to 20 ppb. The deuterated internal standards are used at a concentration of 5 ppb. Good linearity is achieved in this range for each analyte. To check the accuracy of the method, a spiked sample are prepared and submitted to analysis. The accuracy values were in all cases in the range of 80-110% confirming the goodness of the method. Some Recovery tests have been performed using blank matrix samples spiked with synthetic steroids such as trenbolone and melengstrol acetate. The latter experiments have shown that the yield of extraction processes were around 60%. The derivatization process test performed using standard solution of hormones was complete. Finally, good values of LOQ and LOD were achieved demonstrating that this method is competitive against the usual assay of hormones.