European union reference laboratory for feed additives: experience from 11 years organising proficiency testing

Figures & data

Table 1. Analytical methods for feed additives established by EU regulation, ISO, CEN (EN standards) and used by the laboratories participating in the proficiency tests included in this publication. Reversed phase high performance liquid chromatography: RP-HPLC. UV: Ultraviolet. MS: single quadrupole mass spectrometry. MS/MS: tandem mass spectrometry. ⁺: the EN standards are also included in Commission Implementing Regulation (EU) 2024/771.

Reference	Target analyte and principle of the method
Regulation (EC) No 771/2024, annex IV, A:	Vitamin A by RP-HPLC using a UV or a fluorescence detector
Regulation (EC) No 771/2024, annex IV, B:	Vitamin E by (RP-HPLC using a UV or a fluorescence detector
Regulation (EC) No 771/2024, annex IV, D	Halofuginone by RP-HPLC using an UV detector
Regulation (EC) No 771/2024, annex IV, E	Robenidine by RP-HPLC using an UV detector
Regulation (EC) No 771/2024, annex IV, F:	Diclaruril by RP-HPLC using a UV detector
Regulation (EC) No 771/2024, annex IV, G	Lasalocid sodium by RP-HPLC using a spectrofluorometric detector
EN 15782	Nicarbazin by RP-HPLC using an UV detector^+
EN 15781	Maduramicin ammonium by RP-HPLC using an UV detector^+
EN 16158	Semduramicin by RP-HPLC using an UV detector or MS^+
EN 17550	Carotenoids, in this study exclusively: astaxanthin, canthaxanthin, adonirubin by RP-HPLC using an UV detector^+
EN 17299	Coccidiostats (halofuginone hydrobromide, robenidine hydrochloride, nicarbazin, diclazuril, decoquinate, monensin sodium, salinomycin sodium, narasin, lasalocid A sodium, semduramicin (sodium) and maduramicin ammonium alpha) by RP-HPLC-MS/MS^+
EN 16159	Selenium by hydride generation atomic absorption spectrometry (HGAAS)^+
EN 15510	Cobalt by ICP-AES^+
EN 17547	Vitamin A, E and D3 by high performance liquid- chromatography - UV detection (RP-HPLC-UV)^+
ISO 14183	Monensin, narasin, salinomycin-RP-HPLC and post-column derivatisation
EN 12821	Vitamin D3 by high performance liquid chromatography - UV detection (RP-HPLC-UV)*

* The target matrix

of this EN standard is food, but an applicant for vitamin D₃ demonstrated by experiments that this method is also applicable to compound feed.

Table 2. Details of the proficiency tests (PTs) and corresponding feed additives included in the four case studies. In all cases, the test material was compound feed containing the feed additives. For PTs with several analytes the number of laboratories reporting results differed amongst these analytes. RP-HPLC: Reversed-phase high performance liquid chromatography. NP: Normal phase. UV: Ultraviolet detection. There the following four methods for establishing the assigned values of the PTs. Method A: by formulation based on spiking blank compound feed with solutions containing known amount of the target analytes. Method B: by formulation based on spiking blank compound feed with solid feed additive preparations containing known amount of the target analytes. Method C: by calculating the mean value from measurements conducted by expert laboratories that did not participate in the PTs. Method D: by calculating the robust mean of the results from a Sub-group of laboratories participating in the PTs and applying an official analytical method.

Year of PT	Number of case study	Analytes included in the PTs	Assigned values of the feed additive	Way of determining the assigned value	Analytical methods used	Number of laboratories	Report* identification number
2012	1	Material 1: Narasin and diclazuril Material 2: Lasalocid, monensin and salinomycin	Carry-over level ranging from 0.01 to 1.2 mg kg^-1	Method A	Different methods based on RP- HPLC coupled to mass spectrometry	23–30	JRC77456
2013	1	Material 1: Narasin, maduramicin and monensin Material 2: Lasalocid, nicarbazin, diclazuril and halofuginone	Carry-over level ranging from 0.01 to 1.3 mg kg^-1	Method A	Different methods based on RP-HPLC coupled to mass spectrometry	19–30	JRC87389
2014	1	Material 1: Monensin, narasin, diclazuril and robenidine Materal 2: Lasalocid, decoquinate, semduramicin and halofuginone Materal 3: Diclazuril	Mat 1,2: carry-over level ranging from 0.01 to 1.2 mg kg^-1 Mat 3: additive level: 1.5 mg kg^-1	Method A	Different methods based on RP-HPLC coupled to mass spectrometry or UV detection	29–38	JRC95756
2015	2 and 4	Material 1: Preparation containing canthaxanthin Material 2: Natural product containing canthaxanthin, astaxanthin and adonirubin	Mat 1: 19.3 mg kg^-1 Mat 2: 5.9-58 mg kg^-1	Method B	Different methods based on RP-HPLC or NP-HPLC coupled to visual absorption spectrophotometry	8	JRC100872
2016	3	Vitamin A	10558 IU kg^-1	Method D	EU Official and other methods based on RP-HPLC coupled to UV detection	11	JRC106472
2017	1	Cobalt	1.2 mg kg^-1	Method C	Various methods applying different digestion and detection methods	23	JRC110506
2017	1	Monensin, narasin and diclazuril	Carry-over level ranging from 0.01 to 1.2 mg kg^-1	Method A	Different methods based on RP- HPLC coupled to mass spectrometry or UV detection	25	JRC106472
2018	1	Selenium	0.47 mg kg^-1	Method C	Various methods applying different digestion and detection methods	25	JRC113627
2019	4	Material 1: Natural product containing canthaxanthin, astaxanthin and adornirubin Material 2: Preparation containing canthaxanthin	Mat 2: 5.0-50 mg kg^-1 Mat 2: 10 mg kg^-1	Method B	Different methods based on RP-HPLC or NP-HPLC coupled to visual absorption spectrophotometry	14–16	JRC118500
2019	1	Material 1: Diclazuril (authorised level) Material 2: Diclazuril (carry over level) Material 3: Monensin, narasin (authorised levels)	Carry-over and authorisation level ranging from 0.01 to 37 mg kg^-1	Method A: Mat 2 Method B: Mat 1 and 3	Different methods based on RP- HPLC coupled to mass spectrometry or UV detection	18–22	JRC118421
2021	1	Vitamin D3	2643 IU kg^-1	Method B	Different methods based on RP-HPLC coupled to mass spectrometry or UV detection	17	JRC126805
2022	3	Vitamin A	4535 IU kg^-1	Method D	EU Official and other standardised methods based on RP-HPLC coupled to UV detection	17	JRC134613

* The reports are available on the EURL-FA website (https://joint-research-centre.ec.europa.eu/eurl-fa-eurl-feed-additives_en) or on request to the EURL-FA ([e-mail]).

Figure 1. Measurement capability of laboratories assessed by proficiency tests (PTs) expressed as relative differences (Eq. 2(2), $$\mathit{\text{Rel}}\_\mathit{\text{Diff}}\left(\mathrm{\%}\right)=\frac{100\times (x_{\mathit{\text{Rob}}}-x_{\mathit{\text{Pt}}})}{x_{\mathit{\text{PT}}}}$$(2), ): first, the robust means of the analytical results submitted by the laboratories were calculated individually for each analyte. Subsequently, these values were compared with the corresponding assigned values established by formulation (coccidiostats and vitamin D₃) or by reference values set by external expert laboratories (trace elements) according to Eq. (2)(2), $$\mathit{\text{Rel}}\_\mathit{\text{Diff}}\left(\mathrm{\%}\right)=\frac{100\times (x_{\mathit{\text{Rob}}}-x_{\mathit{\text{Pt}}})}{x_{\mathit{\text{PT}}}}$$(2), . The green line for the two trace elements shows the mean value of the corresponding relative differences calculated for cobalt and selenium, respectively.

Table 3. Conditions for mixing canthaxanthin produced by chemical synthesis (identification number 2a161g) into compound feed. Given the known instability of carotenoids, this feed additive is exclusively added via preparations.

Parameter	Value
Sample amount of compound feed for analysis	20 g
Mass fraction of canthaxanthin in the preparation	100 g kg^−1
Target mass fraction of canthaxanthin in compound feed	20 mg kg^-1
Density of the feed additive	0.7 g cm^−3
Absolute mass of the preparation in 20 g sample	4 mg
Particle size range of the preparation	150 to 600 µm

Figure 2. Relation between the relative standard deviation (RSD) of the number of particles and the corresponding particle size. First, the number of particles were calculated separately for each particle size using the information from Table 3. Subsequently, the RSD was determined from the poisson distribution for the different particle numbers. Given the acceptable RSD of about 3%, the target particle size should be between 150 and 250 µm.

Figure 3. Box plots of the relative differences between the laboratories’ results and the labelled content of vitamin A in the commercial compound feed used as test material (Eq. 4(4), $$\mathit{\text{Rel}}\_\mathit{\text{Diff}}\left(\mathrm{\%}\right)=\frac{100\times (x_{\mathit{\text{lab}}}-x_{\mathit{\text{label}}})}{x_{\mathit{\text{label}}}}$$(4), ). All laboratories of the 2022 PT exercise reported values clearly below the labelled values, while these differences were much less in the 2016 PT. This also applied to the assigned values in the PT exercise.

Figure 4. Box plots of the relative differences between the laboratories’ results and the assigned value of three carotenoids (canthaxanthin, astaxanthin and adonirubin) in the commercial compound feeds used as test materials (Eq. 5(5) $$\mathit{\text{Rel}}\_\mathit{\text{Diff}}\left(\mathrm{\%}\right)=\frac{100\times (x_{\mathit{\text{lab}}}-x_{\mathit{\text{pt}}})}{x_{\mathit{\text{pt}}}}$$(5) ). Large deviations of the results from the assigned values were observed in the proficiency tests (PTs) from 2015. After a training exercise organised by the EURL-FA for the laboratories concerned, the results of a PT from 2019 showed significant less variations, thus confirming overall improvement of the laboratories’ performance.

European Union. 2024. Commission Implementing Regulation (EU) 2024/771 of 29 February 2024 amending Regulation (EC) No 152/2009 laying down the methods of sampling and analysis for the official control of feed. Off J Eur Union L 15.3. 2024:1–129.

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Data availability statement

The data presented in this article are freely available from cited published articles.