Title: DEVELOPMENT, OPTIMIZATION AND APPLICATION OF AN ANALYTICAL METHODOLOGY BY ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY FOR DETERMINATION OF AMANITINS IN URINE AND LIVER SAMPLES

27 Amanitins, highly toxic cyclopeptides isolated from various Amanita species, are the 28 most potent poisons accounting for the hazardous effects on intestinal epithelium cells and 29 hepatocytes, and probably the sole cause of fatal human poisoning. 30 The present study was focused on the development, optimization and application of an 31 analytical methodology by ultra performance liquid chromatography-tandem mass 32 spectrometry (UPLC-MS/MS), following urine and liver sample preparation by protein 33 precipitation with organic solvents, and solid phase extraction (SPE) procedure, for the 34 determination of the amatoxins, αand β-amanitin. Linearity, detection and quantification 35 limits, selectivity, sensitivity, intra and inter-assay precision and recovery were studied, in 36 order to guarantee reliability in the analytical results. The developed method proved to be 37 specific and selective, with LOD (Limit of Detection) values for αand β-amanitin of 0.22 38 and 0.20 ng mL in urine and 10.9 and 9.7 ng g in liver, respectively. LOQ (Limit of 39 Quantification) values ranged from 0.46-0.57 ng mLin urine and 12.3-14.7 ng g in tissue, 40 for both amanitins. Linearity, in the range of 10.0 to 200.0 ng mLor ng g, shows that 41 coefficients of correlation were greater than 0.997 for α-amanitin and 0.993 for β-amanitin. 42 Precision was checked at three levels during three consecutive days with intra-day and inter43 day coefficients of variation not greater than 15.2%. The extraction recovery presents good 44 results for the concentrations analyzed, with values ranging from 90.2-112.9% for both 45 matrices. 46 Thus, the proposed analytical method is innovative, presents a high potential in the 47 identification, detection and determination of αand β-amanitins in urine and tissue samples, 48 as well as in other biological samples, such as kidney and mushrooms. 49