L-threonine (3c410)

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2016/1220

For the determination of L-threonine in the feed additive:

- Food Chemical Codex ‘L-threonine monograph’ and

- Ion exchange chromatography method with post-column derivatisation and photometric detection (IEC-UV/FD) - EN ISO 17180

For the determination of threonine in premixtures:

- ion exchange chromatography coupled with post-column derivatisation and photometric detection (IEC-UV/FD) - EN ISO 17180 and

- ion exchange chromatography coupled with post-column derivatisation and photometric detection (IEC-UV), Commission Regulation (EC) No 152/2009 (Annex III, F)

For the determination of threonine in premixtures, compound feed, feed materials and water:

- Ion exchange chromatography method with post-column derivatisation and photometric detection (IEC- UV): Regulation (EC) No 152/2009 (Annex III, F)

Executive Summary

In the current application authorisation is sought for L-Threonine under Articles 4(1) and 10(2), category 'nutritional additives' and functional group 3(c) 'amino acids, their salts and analogues' according to Annex I of Regulation (EC) No 1831/2003. Specifically, authorisation is sought for the use of L-Threonine for all animal species and categories. The feed additive is intended to be mixed either in premixtures or added directly to complete feedingstuffs or water. The two Applicants (FAD-2010-0058 and FAD-2010-0081) suggested no minimum or maximum L-Threonine concentrations in premixtures, feedingstuffs and water. For the determination of L-Threonine in premixtures and feedingstuffs the Applicants submitted the ring-trial validated Community method for amino acids. The method applies for the determination of free (synthetic and natural) and total (peptide-bound and free) amino acids, using an amino acid analyzer or High Performance Liquid Chromatography (HPLC) equipment. However, only performance characteristics for the determination of total LThreonine are reported: a relative standard deviation for repeatability (RSD_r) ranging from 1.9 to 4.1% and a relative standard deviation for reproducibility (RSD_R) ranging from 3.8 to 11.7%. Based on the performance characteristics presented, the EURL recommends for official control, the ring-trial validated Community method based on ion exchange chromatography coupled with post-column derivatisation to determine L-Threonine in premixtures and feedingstuffs. For the determination of the active substance in the feed additive, both Applicants submitted the abovementioned ring trial validated Community method designed for the analysis of premixtures and feedingstuffs. One Applicant (FAD-2010-0081) identified an alternative ring-trial validated method developed by the “Association of Official Agricultural Chemists” "Lysine, Methionine and Threonine in Feed Grade Amino Acids and Premixes" (AOAC 999:13 - 2004). Additionally the EURL identified a ring-trial validated method developed by the “Association of German Agricultural Analytical and Research Institutes” (VDLUFA, Germany – Method 4.11.6). Both the methods are based on the same principle of the Community method (i.e. extraction of the sample with diluted hydrochloric acid and measuring the target analyte with HPLC coupled with post-column derivatisation system) and are explicitly designed to determine free L-Threonine in feed additive and premixtures at amino acid contents higher than 100 g/kg. The following performance characteristics covering both methods were reported: a RSD_r ranging from 0.5 to 2.4%; and RSD_R ranging from 0.9 to 4.7%. Based on the performance characteristics presented, the EURL recommends for official control, the ring trial validated AOAC 999:13 and VDLUFA 4.11.6 methods based on ion exchange chromatography coupled with post-column derivatisation to determine the free L-Threonine in the feed additive. The Applicants provided no experimental data for the identification of L-Threonine in water. Therefore, the EURL cannot evaluate nor recommend a method for the official control to determine L-Threonine in water. Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005) is not considered necessary.

- PLEASE NOTE THAT THE ORIGINAL REPORT HAS BEEN AMENDED -