2.6. Validation procedureThe method

2.6. Validation procedure
The method was validated according to internationally accepted recommendations [25–28]. The parameters studied were selectivity, limits of detection (LOD), limits of quantification (LOQ), linearity, repeatability, intermediate precision, accuracy, robustness and carryover.
Selectivity/specificity was tested by analysing 10 pools of different sets of blank samples of each matrix: whole blood, urine and vitreous humor. Two groups (n = 10) were prepared for each body fluid in vials of 10 mL. The first group was prepared by adding 100 mL of the sample with 1 mL of internal standard, while the second group of the same sample was further supplemented with the substances under study, at a concentration of 50 mg/L. Ten negative and ten positive samples were obtained for each matrix, which were analyzed using the above mentioned procedure.
Linearity was studied by analyzing 10 samples in uniformly distributed concentrations, using the procedure already described. Thus, the calibrators were assigned according to the working concentration range selected for each mixture, as well as for chloroform.
Linearity was analysed by studying the regression of the calibration curves obtained from the peak area ratio between analyte and internal standard vs analyte concentration. The criteria for acceptance included a R2 value of at least 0.99.