Seite - (000106) - in Biomedical Chemistry: Current Trends and Developments

decomposition products or even co-prescribed drugs include the most common interferences. Thus, it is necessary to establish that the chromatographic signal produced is only due to the analyte and not as a result of the co-elution of other compounds. A bioanalytical method is considered to be selective if the lack of response in the blank biological matrix at the retention time of the analytes is demonstrated at the lower limit of quantification (LLOQ). The determination of method sensitivity, linearity, precision and accuracy is required by the three guidelines and usually performed at three concentration levels in several replicates (typically three to five). Linearity should be demonstrated within the defined calibration range with the quality of the calibration curve as an aspect of the greatest importance in bioanalytical method validation. In fact, as Almeida et al. (2002) stated, the quality of bioanalytical data is highly dependent on the quality of the calibration curve used to extrapolate the analyte concentrations in unknown samples. The calibration curve is the relationship between instrumental response and the concentrations of the analyte and it must be generated for each analyte, using a sufficient number of calibration standards (Table 2.1.5). Precision, which expresses the closeness degree among individual measures of an analyte when the same procedure is applied repeatedly to multiple aliquots of a homogenous matrix, is frequently performed on the same day (intra-day precision or repeatability) and over different days (intermediate precision or inter-day precision), both expressed by the relative standard deviation (RSD), which is the absolute value of coefficient of variation (CV). Accuracy, which describes the closeness between the mean experimental data obtained by the method and the corresponding nominal concentration, is also determined intra- and inter-daily and is expressed by the relative error (RE), as a percentage, which is the ratio between experimental and nominal concentrations, or by bias, which is the deviation from the nominal value as a percentage. Matrix effects, carry-over and dilution integrity have been recently integrated in the guideline issued by EMA as well as stability evaluation. From a practical point of view, these parameters can be estimated from the analysis of QC samples, which represent the future real samples. Table 2.1.5: The ICH, FDA and EMA validation parameters and respective limits instituted.