Validation of a multi-analyte HPLC method for the determination of carotenoids used as feed additives in fish and poultry feed: results of an interlaboratory study

ABSTRACT

The performance characteristics of a multi-analyte method for the determination of all 10 carotenoids authorised as feed additives within the EU were assessed via an interlaboratory study. The analytical method is based on reversed phase high performance liquid chromatography (RP-HPLC) coupled to an optical detector set at 410 nm. The analysis is particularly challenging due to the presence of various stereoisomers of each carotenoid, and the use of these compounds via natural or synthetic formulations, requiring a special sample preparation. EU regulations specifying the conditions of use set legal limits for these substances in compound feedingstuffs ranging from 6 mg kg⁻¹ to 138 mg kg⁻¹, depending on the individual carotenoid and the target animal for which the feed is supplemented with this carotenoid. The purpose of the multi-analyte method validated in this paper is to facilitate the monitoring of carotenoids at relevant levels when used as feed additives in compound feedingstuffs and pre-mixtures. The interlaboratory study delivered precision data for 43 different analyte/mass fraction/matrix combinations, covering a mass fraction range of the target analytes from 2.6 mg kg⁻¹ to 3861 mg kg⁻¹. The relative standard deviations for repeatability (RSD_r) varied from 2.2 to 16.2 % with a mean value of 6 %, while the relative standard deviations for reproducibility (RSD_R) varied from 6.8 to 39 % with a mean value of 21 %. Given the broad scope of the method covering 10 carotenoids added to compound feedingstuffs and pre-mixtures via different formulations, this multi-analyte method is considered fit for the intended purpose.

KEYWORDS:

• Feed additives
• carotenoids
• multi-analyte method
• interlaboratory study

Acknowledgments

The authors are grateful to Joseph Schierle and Andre Duesterloh (DSM Nutritional Products, Kaiseraugst, Switzerland) and to the members of CEN/TC 327/WG 3 (Animal feedingstuffs - Methods of sampling and analysis, Feed additives and drugs) for their continuous scientific support during the discussion of the results of the study.

The authors would like to thank the laboratories for their participation in this interlaboratory study: Österreichische Agentur für Gesundheit und Ernährungssicherheit GmbH, Inst. f. Tierernährung und Futtermittel/Abt. Zusatzstoffanalytik und Prüfung – AGES (Vienna, Austria), National Reference Laboratory of the Central Institute for Supervising and Testing in Agriculture, Ústřední kontrolní a zkušební ústav zemědělský,– ÚKZÚZ (Prague, Czech Republic), Finnish Food Safety Authority Evira Research and Laboratory Department, Chemistry and Toxicology Unit (Helsinki, Finland), BASF (Ludwigshafen, Germany), LUFA-ITL (Kiel, Germany), SGS Institut Fresenius GmbH (Germany), Federal Institute for Risk Assessment (BfR), National Reference Laboratory for Feed Additives, Unit Contaminants, Department Safety in the Food Chain (Berlin, Germany), Landesbetrieb Hessisches Landeslabor (Kassel, Germany), National Food Chain Safety Office, Food and Feed Safety Directorate, Feed Investigation Reference Laboratory (Budapest, Hungary), Ministero politiche agricole alimentari e forestali (Modena, Italy), National Institute of Nutrition and Seafood Research (Bergen, Norway), Nofima AS (Bergen, Norway), Laboratori Agroalimentari (DAAM) de Cabrils –Generalitat de Catalunya (Cabrils, Spain), DSM Nutritional Products (Kaiseraugst, Switzerland), Nutrition Analytical Service (Stirling, United Kingdom), Novus International (St. Charles MO, USA).

Declaration of interest statement

No potential conflict of interest was reported by the authors.