Pyridoxine hydrochloride or Vitamin B6 - 3a831

In the current application authorisation is sought under articles 4(1) (new use in water) and 10(2) (re-evaluation of the already authorised additive under council directive 70/524/EEC) for pyridoxine hydrochloride (Vitamin B₆) under the category/functional group 3(a) ‘nutritional additives’/‘vitamins, pro-vitamins and chemically well defined substances having similar effect’ according to Annex I of Regulation (EC) No 1831/2003. Authorisation is sought for the use of the feed additive for all animal species and categories. According to the Applicant the product is a white to yellowish powder containing a minimum of 98.5 % pyridoxine hydrochloride. The feed additive is intended to be incorporated in feedingstuffs (including complete feedingstuffs or complementary feedingstuffs) through premixtures or directly in water. However, the Applicant did not specify any maximum or minimum concentration of Vitamin B₆ in feedingstuffs or water, similarly to what was set in previous regulation.

For the determination of the Vitamin B₆ in the feed additive the Applicant proposes the titration method described in the European Pharmacopoeia (Ph.Eur.6^th, monograph 0245). The EURL considers this method suitable to be used within the frame of official control. 

For the determination of Vitamin B₆ in premixtures the Applicant proposes a single-laboratory validated method, based on ion pair reversed phase High Performance Liquid Chromatograpy (RP-HPLC) coupled to an UV detector. This method provided satisfactory results when compared to the collaborative trial organised by the Association of German Agricultural Analytical and Research Institutes (VDLUFA, Germany). The following performance characteristics were reported for the VDLUFA ring-trial validated UV-HPLC method on premixtures containing from 627 to 11530 mg Vitamin B6 /kg of premixture: 

- a relative standard deviation for repeatability (RSD_r) ranging from 2.6 to 3.4 %, and

- a relative standard deviation for reproducibility (RSD_R) ranging from 4.0 to 5.1 %. 

Based on these acceptable performance characteristics, the EURL recommends for official control the ring-trial validated VDLUFA method based on UV-HPLC for the determination of Vitamin B₆ in premixtures within the concentration range covered by the collaborative study. 

For the determination of Vitamin B₆ in feedingstuffs and in water, the Applicant submitted a simplified method based on the European Standard (EN 14164:2008, "Foodstuffs – Determination of vitamin B₆ by HPLC"). The Applicant demonstrated the suitability of this method for the determination of Vitamin B₆ in feedingstuffs and water, for Vitamin B₆ concentrations ranging from 0.6 to 400 mg/kg feedingstuffs and water.

The Applicant determined the following performance characteristics, which are in agreement with those reported in the standard method: 

- a relative standard deviation for repeatability (RSD_r) ranging from 1.3 to 7.3 %, and

- a relative standard deviation for intermediate precision (RSD_ip) ranging from 5.0 to 17.8 %. 

Based on these acceptable performance characteristics the EURL recommends for official control the simplified method based on the European Standard method (EN 14164:2008) to determine Vitamin B₆ in feedingstuffs and water, within the concentration range covered by the experimental data.

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.

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2021/507

For the determination of pyridoxine hydrochloride (Vitamin B6) in the feed additive: 

Titration with perchloric acid (Ph. Eur. 10th edition, monograph 0245) 

For the determination of pyridoxine hydrochloride (Vitamin B6) in premixtures: 

Reversed phase High Performance Liquid Chromatography coupled to UV detector (RP-HPLC-UV) - VDLUFA Bd.III, 13.9.1 method 

For the determination of pyridoxine hydrochloride (Vitamin B6) in feedingstuffs and water: 

Reversed phase High Performance Liquid Chromatography coupled to fluorescence detector (RP-HPLC-FLD) – method based on EN14164:2008