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.

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 (RSDr) varied from 2.2 to 16.2 % with a mean value of 6 %, while the relative standard deviations for reproducibility (RSDR) 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.

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-1. 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.

Interlaboratory comparison of an analytical method for the determination of semduramicin in poultry feed at the authorized level using high-performance liquid chromatography coupled to postcolumn derivatization and spectrophotometric detection.

The performance characteristics of a method based on HPLC with postcolumn derivatization and spectrophotometric detection for the quantification of semduramicin in poultry feedingstuffs have been determined via a collaborative study. Semduramicin is a feed additive that is authorized for fattening chickens within the European Union at a minimum and maximum content of 20 and 25 mg/kg in feedingstuffs, respectively. The target concentration of semduramicin in the test samples ranged from 11.5 to 45.0 mg/kg. The study has been conducted with two different types of test material, namely, feedingstuff samples that have been previously ground in our laboratory and pelleted feedingstuffs. In the latter case, the laboratories participating in the study had to grind the samples prior to analysis. The obtained RSD for repeatability (RSD(r)) ranged from 2 to 10% for the ground materials, and from 2 and 7% for the pelleted materials. The RSD for reproducibility (RSDR) varied between 11 and 16% for the ground materials, and between 12 and 15% for the pelleted materials. These data indicated that grinding as an additional step in the analytical procedure did not influence the precision profile of the method. In addition, the HorRat values for all test materials were below or equal to 1.5, thus demonstrating that the obtained precision data were acceptable for the purpose of the method. Furthermore, an estimation of trueness based on statistical treatment of the results reported from the laboratories for spiked samples revealed acceptable mean recovery values of 88 +/- 4%. Based on the obtained performance profile, the method can be considered fully validated and transferable to control laboratories to be used within the framework of official control.

Determination of semduramicin in poultry feed additive, premixture and compound feed by liquid chromatography and UV spectrophotometric detection after post-column derivatisation.

A new, simple and fit for purpose method based on liquid chromatography with UV spectrophotometric detection and post-column derivatisation (LC-UV-PCD) for the determination of semduramicin in poultry compound feed, premixtures and feed additive as well as its discrimination from other coccidiostats in poultry compound feed has been developed and single-laboratory validated. The concentration levels of the target analyte at which the validation experiments have been carried out varied between 12.8 and 51.3 mg kg(-1) in compound feed, covering the authorised levels of semduramicin according to European Union legislation. Furthermore, the method has been validated for a premixture sample containing semduramicin at 3 g kg(-1) and the feed additive containing semduramicin at 51 g kg(-1). The method developed involved a simple extraction of the coccidiostats with acetonitrile from the feed samples followed by a filtration of the supernatants. The resulting supernatants were submitted to chromatographic analysis. When analysing the feed additive and the premixture samples, the extraction solution was appropriately diluted prior to LC-UV-PCD analysis. The analytes were quantified through an external calibration curve prepared with pure semduramicin standards. The relative standard deviations for repeatability and for intermediate precision varied from 2.4 to 8.8% and from 2.6 to 8.8%, respectively, and the values for the relative recovery rate ranged from 89 to 95%. The limit of detection (LOD) and limit of quantification (LOQ) were estimated to be below 1 mg kg(-1) and 3 mg kg(-1), respectively. Moreover, the results showed a comparable performance profile, when using methyl isobutyl ketone instead of acetonitrile as extraction solvent.Based on the obtained method performance characteristics, the method is considered suitable for the determination of semduramicin in poultry compound feed at authorised level, in premixtures and in the feed additive, hence allowing the enforcement of the European Union legislation regarding the control and the monitoring in feedingstuffs.