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Thiamine hydrochloride or Vitamin B1 (3a820), Thiamine mononitrate or Vitamin B1 (3a821)

Thiamine hydrochloride, Thiamine mononitrate

Details

Publication date
13 May 2014 (Last updated on: 6 June 2016)

Description

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2015/897

For the characterisation of thiamine hydrochloride in the feed additive:

- high performance liquid chromatography with UV detection (HPLC-UV) — US Pharmacopeia 32 (‘thiamine hydrochloride’ monograph)

For the quantification of thiamine hydrochloride/thiamine mononitrate in premixtures:

- ion-exchange high performance liquid chromatography coupled to UV detector (HPLC- UV) - VDLUFA Bd. III, 13.9.1, or

—reversed phase high performance liquid chromatography coupled to fluorescence detection (HPLC-FL) - decree 20.2.2006, Official Italian Journal No 50, 1.3.2006

For the quantification of thiamine hydrochloride/thiamine mononitrate in feedingstuffs:

- reversed phase high performance liquid chromatography coupled to fluorescence detection (HPLC-FL) - decree 20.2.2006, Official Italian Journal No 50, 1.3.2006

For the quantification of thiamine hydrochloride/thiamine mononitrate in water:

- reversed phase high performance liquid chromatography (HPLC) with post-column derivatisation and fluorescence detection

m1-w-3a820_3a821.pdf
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For the characterisation of thiamine mononitrate in the feed additive:

- high performance liquid chromatography with UV detection (HPLC-UV) — US Pharmacopeia 32 (‘thiamine mononitrate’ monograph)

Executive Summary

In the current applications1,2,3 authorisation is sought under articles 4(1) and 10(2) for Vitamin B1 (thiamine mononitrate1,2,3 and thiamine hydrochloride1,3) 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. Specifically, authorisation is sought for the use of the feed additives for all categories and species.

According to the Applicants, the feed additives are crystalline powders with a minimum purity of 93.5 % and 96 % for thiamine hydrochloride and thiamine mononitrate, respectively. The feed additives are intended to be incorporated in feedingstuffs (including complete feedingstuffs or complementary feedingstuffs) through premixtures or directly in water. However, the Applicants did not specify any maximum or minimum concentrations of thiamine mononitrate and thiamine hydrochloride in feedingstuffs or water.

For the characterisation of thiamine mononitrate and thiamine hydrochloride in the feed additives the EURL identified US Pharmacopoeia monographs where quantification is based on high performance liquid chromatography (HPLC) with UV detection at 254 nm. Even though no performance characteristics are provided, the EURL recommends for official control these HPLC-UV methods to characterisation of thiamine mononitrate and thiamine hydrochloride in the feed additives.

For the quantification of Vitamin B1 in premixtures, Applicants1,2 submitted the ring-trial validated VDLUFA method (Bd.III,13.9.1), based on ion-exchange high performance liquid chromatography coupled to UV detector (HPLC-UV). The following performance characteristics were reported for premixtures samples containing Vitamin B1 levels ranging from 1570 to 7940 mg/kg: - a relative standard deviation for repeatability (RSDr) ranging from 2.1 to 2.6 %; and - a relative standard deviation for reproducibility (RSDR) ranging from 4.2 to 7.4 %. Based on these performance characteristics, the EURL recommends for official control this ring-trial validated HPLC-UV method to quantify Vitamin B1 (thiamine mononitrate and thiamine hydrochloride) in premixtures containing high Vitamin B1 levels ranging from 1500 to 8000 mg/kg.

Additionally, the EURL identified an alternative ring-trial validated method for the quantification of Vitamin B1 in premixtures and feedingstuffs, based on reversed phase high performance liquid chromatography coupled to fluorescence detector (HPLC-FL) - published in the Italian Official Journal (decree 20/02/2006, Official Italian Journal n. 50 1/3/2006). The following performance characteristics were reported for premixtures and feedingstuffs samples containing Vitamin B1 levels ranging from 7 to 484 mg/kg: - a RSDr ranging from 4.2 to 4.7 %; - a RSDR ranging from 5 to 13 %; - a recovery rate (RRec) ranging from 88 to 97 %; and - a limit of quantification (LOQ) of 5 mg/kg. Based on these performance characteristics the EURL recommends for official control this alternative ring-trial validated HPLC-FL method to quantify thiamine mononitrate and thiamine hydrochloride in premixtures and feedingstuffs, containing Vitamin B1 levels ranging from 7 to 500 mg/kg.

For the quantification of Vitamin B1 in water Applicant (FAD-2010-0140) submitted a reversed phase high performance liquid chromatography coupled to fluorescence detector after post- column derivatisation derived from the ring-trial validated CEN method (EN 14122:2003). The Applicant reported for water samples containing Vitamin B1 levels ranging from 3.6 to 4.4 mg/L the following performance characteristics: - RSDr ranging from of 1.0 to 2.8 %; - a relative standard deviation of intermediate precision (RSDip) equal to 10.5 %; and - a limit of quantification (LOQ) of 0.06 mg/L water. Additionally recovery rates (RRec) ranging from 98 to 100 % were reported for Vitamin B1 content ranging from 0.5 to 368 mg/L. These performance characteristics are in good agreement with those reported for the CEN method EN 14122:2003. Based on experimental evidence available, the EURL recommends for official control the adapted/modified CEN method based on HPLC-FL to quantify thiamine mononitrate and thiamine hydrochloride 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.

Note: 1 FAD-2010-0040, 2 FAD-2010-0052, 3 FAD-2010-0140

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finrep-fad-vitb1_group.pdf
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