**7. Validation parameters of the methods used to analyze feed additives**

A significant element in verifying a chemical method, including chromatography, is its validation. Validation is a confirmation through examining and presenting some objective evidence that some particular requirements regarding the intended application have been fulfilled. The basic validation parameters include: calibration linearity, the limit of detection and quantitative determination, precision (repeatability, indirect precision, reproducibility), recovery and uncertainty.

Calibration linearity is defined as a relationship between the readings of the measuring device and the concentration of a particular component, in conformity with the regression equation: bx + a = y. The measure of linearity is Pearson's linear correlation coefficient (r) for parameters with regular distribution. The scale presented below is adopted to estimate the correlation coefficient: 0.0-0.2: very weak relationship; 0.2-0.4: weak relationship; 0.4-0.6: moderate relationship; 0.6-0.8: strong relationship; 0.8-1.0: very strong relationship.

In case of feed additives discussed in the present chapter, determined with HPLC methods, external calibration was used.

In case of chromatographic methods the value of limit of detection (LOD) may be determined on the basis of the obtained chromatogram of blanc sample, as the threefold value of a noise signal. To do this, it is necessary to determine the level of noise, by measuring on the chromatogram the range of signal change near retention time of examined

analyte. With chromatographic methods, the bottom limit of the method's application may be also regarded as the content of the analyzed component, which is equal to the lowest concentration of the standard used for calibration.

Using High Performance Liquid Chromatography (HPLC) for Analyzing Feed Additives 181

coefficient (for two replications d = 1.128) makes the standard deviation of within laboratory reproducibility. In order to verify the method's precision the Horwitz ratio named HorRat (H), may be used which is the ratio of the relative standard deviation of reproducibility SDRt calculated from the Horwitz formula SDRt =2 C-0.15, where C stands for concentration expressed as a dimentionless mass fraction (e.g. 1 mg/kg = 10-6). In order to adjust it to the conditions of repeatability, target standard deviation SDRt is multiplied by 0.50 (RSDr = 0.5 RSDR), [3]. Satisfactory values of the HorRat making the measurement of precision are included in the range of 0.5 < H < 2 [3]. In case of participating in interlaboratory tests and obtaining satisfactory results, it is possible to include the precision parameters obtained in these analyses. The accuracy of a method may be determined by

Each laboratory should possess a program of quality assurance of its analyses within good laboratory practice. In case of chromatographic methods steering the quality may be



Control material may be provided by certified reference material, CRM (matrix + analyzed substance), material from proficiency testing with a value assigned, enriched material prepared in the laboratory (fortified sample) and control material with recognized content of

In compliance with the recommendations of the EN ISO/IEC 17025:2005 [24] standard and requirements defined in some regulations, in order to assess and interpret the result of a test, it is necessary to use the uncertainty of measurement. We hardly ever know the real content of the analyte and the result of the test is biased with an error. Hence, the idea of "uncertainty of measurement" has been introduced which is defined as "a parameter associated with the result of a measurement, that characterises the dispersion of the values that could reasonably be attributed to the measurand" [26]. The EN ISO/IEC 17025:2005 standard recommends at point 5.4.6.2 that testing laboratories should possess and make use of procedures for assessing the uncertainty of measurement. To assess the uncertainty of methods used to analyze feeds the most frequently used approach is the model, consistent with the GUM [26] guidebook, which consists of finding the components of uncertainty and uses the law of error propagation. Using this particular approach to assess uncertainty, it is

the tested and stable in time component, previously determined in the laboratory.

calculating the recovery degree or examining certified reference material, CRM.

implemented through performing one or more of the activities listed below:

**8. Quality assurance and an uncertainty of result** 



prepared and analyzed on the previous day), - analyzing the blanc sample and fortified sample,


feed components,


CVr – coefficient of variation; CVip – intermediate precision; rec. - recovery

**Table 7.** Validation parameters obtained for selected feed additives – vitamins in feeds


**Table 8.** Validation parameters obtained for other feeds

During the validation process in a laboratory the precision of a method is determined through examining such parameters as repeatability and within-laboratory reproducibility (intermediate precision). Within-laboratory reproducibility may be calculated on the basis of control charts or from the range between parallel results of an analysis (replications) of a feed additive, in compliance with the Nordtest [23] handbook. For two or more replications for the analyses of an analyte in each sample it is necessary to calculate the mean value, the difference between measurements (range), relative difference in % and next mean relative difference (%) for all samples of a particular type of feed. The mean range divided by the coefficient (for two replications d = 1.128) makes the standard deviation of within laboratory reproducibility. In order to verify the method's precision the Horwitz ratio named HorRat (H), may be used which is the ratio of the relative standard deviation of reproducibility SDRt calculated from the Horwitz formula SDRt =2 C-0.15, where C stands for concentration expressed as a dimentionless mass fraction (e.g. 1 mg/kg = 10-6). In order to adjust it to the conditions of repeatability, target standard deviation SDRt is multiplied by 0.50 (RSDr = 0.5 RSDR), [3]. Satisfactory values of the HorRat making the measurement of precision are included in the range of 0.5 < H < 2 [3]. In case of participating in interlaboratory tests and obtaining satisfactory results, it is possible to include the precision parameters obtained in these analyses. The accuracy of a method may be determined by calculating the recovery degree or examining certified reference material, CRM.
