**4. Extraction and extract purification**

A sample for a quantitative analysis should be prepared in such a way that the isolation of a selected analyte and removal of interfering substances is possible. The condition necessary for adequate quantitative determination in liquid chromatography is eliminating any possibility of coelution and minimization the drift of a basic line. Extractions of the analyzed substances were done classically by shaking the sample with a solvent. In case of vitamins B1 and B2 and carotenoids, such as canthaxanthin and apocarotenic acid ester, extraction was performed in an ultrasonic bath. In order to purify the extract, aluminum oxide (e.g. canthaxanthin), celite and anhydrous sodium sulphate (e.g. vitamin K3) were used and PTFE and Nylon (PA) (0.45 μm or 0.20 μm) syringe filters were applied before injecting on the chromatographic column. Syringe (hydrophobic) PTFE filters are used in case of solutions with high acid and base content, whereas nylon (hydrophilic) filters are used with aqueous and organic solutions. Filtration of extracts is necessary as it prolongs durability of a column due to eliminating permanent contamination which blocks the column's intake and increases back pressure. If needed, the analyte may be concentrated by evaporating the solvent. When there is a risk that the studied analyte becomes oxidized, evaporation is done in neutral gas, e.g. nitrogen or argon.

Using High Performance Liquid Chromatography (HPLC) for Analyzing Feed Additives 175

used. The identification and content of analyte was examined with the method of absolute calibration (with external model), analyzing separately the sample and the model and identifying the peaks with the help of retention values, comparing retention time of the identified substance with the retention time of a standard, chromatographed in identical conditions. Examples of chromatograms for the standard extract and the sample of the examined analyte (riboflavin) with the use of fluorescent detector are presented below

**Figure 1.** Characteristic chromatograms of riboflavin: chromatogram of standard solution

**Figure 2.** Characteristic chromatograms of riboflavin: chromatogram of premix extract

Selected methods of testing feed additives presented below were validated with the help of a high pressure liquid chromatograph (Dionex P-680) with fluorescence detector (Dionex RF

**6. Selected methods of testing feed additives** 

2000) or with a diode array.

(Fig.1, Fig.2).

The removal of gelatin-and-sugar beadlets protecting vitamins A and D3 is done at the stage of saponification and transforming the vitamins into alcoholic forms. Douša & Břicháč [16] demonstrated that saponification in standard conditions did not affect the results of analyses. In case of canthaxanthin enzymatic hydrolysis through adding trypsin and pepsin is used. While determining the total content of thiamine and/or riboflavin in feedingstuffs (endogenic and added) at the stage of preliminary preparation an ultrasonic bath and also enzymatic hydrolysis (taka-diastase) are applied.
