**1. Introduction**

164 Chromatography – The Most Versatile Method of Chemical Analysis

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Feed additives have been widely used in animal nutrition. Recommendations concerning using feed additives, their categories and the description of requirements related to such additives can be found in the regulation (EU) No 1831/2003 [1]. Detailed regulations oblige the entities launching feed additives on the market to specify the methods used to analyse active substances of additives for the needs of official feed control. Official feed control is implemented in order to monitor adequate and safe use of feed additives in animal nutrition. Moreover, controlling feed production in this respect results in improving the quality and safety of animal products for consumers.

HPLC methods have been widely used in the analyses of feed additives, such as vitamins, feed colorants, antioxidants, amino acids and coccidiostats in preparations, premixes and feed mixtures. It is relatively simple to analyse preparations as they are usually composed of a particular active substance and a carrier. A premix is more complex feed consisting of a combination of a dozen different feed additives on a mineral (calcium carbonate) or organic (wheat bran) carrier. Complete feed mixtures used in animal nutrition, produced by combining premixes with feed materials are often greased and subjected to further hydro- or barothermal processing, e.g. pelleting, extrusion or expanding. In order to counteract decomposition of the active substance, feed additives are secured by protective coating, e.g. vitamin A, canthaxanthin, which enhances their durability in the feed matrix. A specific protection of feed additives by protective coating, thermal processing, greasing the feed, varied composition of feed materials in mixtures may hinder the transfer of the active substance into a solution during extraction and purification of the extract.

The key issue becomes selection of a chromatographic column (in a normal phase or reversed phase), mobile phase, detector, as well as optimization the conditions of

© 2012 Rubaj et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Rubaj et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

chromatographic separation. In examining feed additives with the use of HPLC methods the most frequently used types are spectrophotometric detection (UV-VIS), detection with the help of diode array and fluorescent detection. The choice of optimal parameters for chromatographic separation is done during validation of the method. The analyses in this respect should be accompanied by an assessment of the method's robustness [2]. A practical way to verify the precision of a method in a laboratory (repeatability) and in interlaboratory studies (reproducibility) is taking advantage of Horwitz equation [3].

Using High Performance Liquid Chromatography (HPLC) for Analyzing Feed Additives 167

prevent invasive diseases (e.g. coccidiosis – coccidiostats), to reduce oxidation processes of feed's components (antioxidants), to enhance the dietary value and quality in food products

In case of some vitamins and antioxidants their maximum content in feed mixtures was determined, e.g. for vitamins A and D3, antioxidants (ethoxyquin, BHA, BHT), carotenoids (canthaxanthin, apocarotenoic acid ester and others). Maximum contents are subject to official control in reference to their conformity with the requirements related to the safety of feedingstuffs. Additionally, a feed manufacturer is obliged to declare the content of feed additives on the label of a premix or feed mixture. Thus, it is necessary to have access to analytical methods for testing the content of feed additives in a wide range of concentrations

Table 1 presents examples of well-known HPLC methods for examining fat-soluble vitamins in feedingstuffs, including the official methods accepted by the European Commission. A commonly used method of preparing a sample for analysing the content of vitamin A is alkaline hydrolysis during which gelatin/sugar cross-linked beadlets which protect vitamin A in the form of retinol acetate are solved and then purified by liquid-liquid extraction. An interesting option for purifying vitamin A extracts from feed mixture with the use of the SPE technique was presented by Fedder & Ploger [7]. The step of alkaline hydrolysis is also used while determining vitamins E and D3. Chromatographic separation of vitamins, except for vitamin D3 where preliminary separation and fraction collection are necessary [5], does not present any serious problems. However, a problem may be posed by the quality and durability of a standard, as well as poor precision resulting from a too low of analytical weight [16]. It is necessary to verify vitamin A standards with the use of a

The official methods of determining the content of water-soluble vitamins, such as B1, B2 and B6 are based on spectrophotometric or fluorometric methods [8,13]. The results of analyses using these methods may be biased with errors due to some interferences from other substances in the variable feed matrix. Recently HPLC methods to determine vitamins B1, B2, B6, nicotinic acid and nicotinamide in mineral preparations and mixtures [8], as well as vitamin B1 in feed mixtures and premixes [9] were published (Table 2). Due to the high limit of quantification for vitamin B1 amounting to 5 mg/kg according to Italian Official Method [9], it cannot be used for analysing vitamin B1 in typical feed mixtures to which it is normally added at the amount of 2-4 mg/kg. Moreover, the method quoted above makes it possible to examine vitamin B1 added to feedingstuffs but not the total content of this vitamin, regarding its presence in feed materials. It is thus necessary to have access to chromatographic methods enabling the examination of water-soluble vitamins present in feed materials and added in the form of feed additives. The procedures of HPLC methods of vitamins B1 and B2 developed during the authors' own studies are presented later in the

of animal origin (amino acids, carotenoids– egg yolk coloration).

in the preparations containing additives, premixes and feed mixtures.

spectrophotometric method [4].

chapter [17,18].

Using HPLC methods for examining feed additives was the subject matter of numerous studies on the basis of which official methods of analysing certain feed additives were developed. The studies presented the basic validation parameters for the methods of examining the content of fat-soluble vitamins [4-7], water-soluble vitamins [8,9], coccidiostats [4,10-12], and other feed additives, amino acids, methionine hydroxy analog and antioxidants [13,4,8]. However, in case of carotenoids such as canthaxanthin or apocarotenoic acid ester official methods of examining these additives are still based on spectrophotometric measurement rather than on HPLC methods [13,8].

New requirements have been introduced regarding the validation parameters for the methods of analysing feed additives, e.g. those listed in the regulation No 882/2004 [14], taking into consideration, among others, the uncertainty of measurement. It is necessary to determine the uncertainty of measurement with a particular method in order to interpret adequately the result of examining feed additives in feedingstuffs and to assess acceptable tolerance in compliance with the requirements of the regulation No 939/2010 [15]. The new requirements in this area should be taken into account while validating the methods of testing feed additives in order to solve the problem of interpreting the results.

The aim of the present work was to offer a review of HPLC methods used for analysing active substances in certain feed additives, with regard to current requirements defined in the regulations. In some justified cases the results of the authors' own studies were presented, as well as the procedures for determining vitamins B1 and B2, canthaxanthin and methionine hydroxy analog (MHA). Some validation parameters were presented, such as the limit of the method's quantification (LOQ), linearity of the calibration curve, repeatability, within-laboratory reproducibility (intermediate precision), recovery and the uncertainty of measurement. Also, the results of verifying the developed methods and laboratories participating in proficiency testing (PT) were demonstrated. The ways of quality assurance of the tests in reference to HPLC methods were discussed. The work presents the method of assessing combined standard uncertainty of measurement with the use of experimental approaches based on within-laboratory reproducibility and calculations for the bias of the method on the basis of CRM studies or PT results.
