Development and validation of a multi-analyte method for the regulatory control of carotenoids used as feed additives in fish and poultry feed.

Carotenoids are used in animal nutrition mainly as sensory additives that favourably affect the colour of fish, birds and food of animal origin. Various analytical methods exist for their quantification in compound feed, reflecting the different physico-chemical characteristics of the carotenoid and the corresponding feed additives. They may be natural products or specific formulations containing the target carotenoids produced by chemical synthesis. In this study a multi-analyte method was developed that can be applied to the determination of all 10 carotenoids currently authorised within the European Union for compound feedingstuffs. The method functions regardless of whether the carotenoids have been added to the compound feed via natural products or specific formulations. It is comprised of three steps: (1) digestion of the feed sample with an enzyme; (2) pressurised liquid extraction; and (3) quantification of the analytes by reversed-phase HPLC coupled to a photodiode array detector in the visible range. The method was single-laboratory validated for poultry and fish feed covering a mass fraction range of the target analyte from 2.5 to 300 mg kg. The following method performance characteristics were obtained: the recovery rate varied from 82% to 129% and precision expressed as the relative standard deviation of intermediate precision varied from 1.6% to 15%. Based on the acceptable performance obtained in the validation study, the multi-analyte method is considered fit for the intended purpose.