L-isoleucine

In the current application an authorisation is sought under Article 4(1) for L-isoleucine produced by fermentation with Corynebacterium glutamicum KCCM80185, under the category/functional group 3(c) 'nutritional additives'/'amino acids, their salts and analogues', according to Annex I of Regulation (EC) No 1831/2003. The authorisation is sought for all animal species. According to the Applicant L-isoleucine has a minimum purity (mass fraction) of 90 %. The feed additive is intended to be added directly into feedingstuffs (or through premixtures) and water for drinking. However, the Applicant did not propose any minimum or maximum content of L-isoleucine in feedingstuffs. For the quantification of isoleucine in the feed additive, premixtures and feedingstuffs the Applicant submitted an in-house validated analytical method based on high performance liquid chromatography coupled to fluorescence detection (HPLC-FLD). The Applicant did not present a verification study and therefore, the EURL cannot recommend the method for official control purposes. For the quantification of isoleucine in feed a ring-trial validated European Union (EU) method (Commission Regulation (EC) No 152/2009) based on ion-exchange chromatography (IEC) coupled to photometric detection exists (IEC-VIS). This method, designed for the analysis of amino acids in premixtures and feedingstuffs, does not distinguish between the salts and the amino acid enantiomers. This method was further ring-trial validated resulting in the EN ISO 13903:2005 method. The following performance characteristics were reported for the quantification of total isoleucine: relative standard deviation for repeatability (RSD_r) ranging from 2.0 to 5.4 % and relative standard deviation for reproducibility (RSD_R) ranging from 6.8 to 14.3 %. The Applicant did not provide experimental data to determine isoleucine in water. Nevertheless, as concluded in previous EURL reports, the EURL recommends the EU method for official control for the quantification of isoleucine in the feed additive and water. Furthermore, for the determination of L-isoleucine in the feed additive and premixtures the ring-trial validated method EN ISO 17180:2013 - "Animal feeding stuffs – Determination of lysine, methionine and threonine in commercial amino acid products and premixtures" is also applicable. In addition, the EURL found the "L-isoleucine monograph" of the Food Chemical Codex (FCC) for the identification of 
L-isoleucine in the feed additive. In the frame of the present request of authorisation the EURL recommends for official control (i) the "L-isoleucine monograph" of the Food Chemical Codex (FCC) for the identification of L-isoleucine in the feed additive; (ii) the ring-trial validated method EN ISO 17180:2013 based on ion-exchange chromatography coupled to visible or fluorescence detection (IEC-VIS/FLD) to quantify free isoleucine in the feed additive and premixtures; and (iii) the ringtrial validated European Union method based on IEC-VIS for the quantification of isoleucine in the feed additive, premixtures, feedingstuffs and 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), as last amended by Regulation (EU) 2015/1761) is not considered necessary.

Recommended Method

as of:

-Commission Implementing Regulation (EU) 2023/2583

For the identification of L-isoleucine in the feed additive:

• Food Chemical Codex ‘L-isoleucine monograph’

   

For the quantification of isoleucine in the feed additive:

• Ion-exchange chromatography coupled with post-column derivatisation and optical detection (IEC-VIS/FLD) or
• Ion-exchange chromatography coupled to post-column derivatisation and photometric detection (IEC-VIS)

For the quantification of isoleucine in premixtures:

• Ion-exchange chromatography coupled with post-column derivatisation and optical detection (IEC-VIS/FLD) or

• Ion-exchange chromatography coupled to post-column derivatisation and photometric detection (IEC-VIS) – Commission Regulation (EC) No 152/2009

   

For the quantification of isoleucine in compound feed:

• Ion-exchange chromatography coupled to post-column derivatisation and photometric detection (IEC-VIS) – Commission Regulation (EC) No 152/2009

For the quantification of isoleucine in water:

• Ion-exchange chromatography coupled to post-column derivatisation and photometric detection (IEC-VIS)