Ref.

Ares(2014)1520266 - 13/05/2014

EUROPEAN COMMISSION
DIRECTORATE GENERAL
JOINT RESEARCH CENTRE
Directorate D: Institute for Reference Materials and Measurements
European Union Reference Laboratory for Feed Additives

JRC.D.5/SFB/CvH/ZE/mds/Ares

Evaluation Report on the Analytical Methods submitted

in connection with the Application for Authorisation of a Feed
Additive according to Regulation (EC) No 1831/2003

Vitamin B1
(FAD-2010-0040; CRL/100032)
(FAD-2010-0052; CRL/100035)
(FAD-2010-0140; CRL/100184)
 Evaluation Report on the Analytical Methods submitted
in connection with the Application for Authorisation of a Feed
Additive according to Regulation (EC) No 1831/2003

Dossier related to: FAD-2010-0040 - CRL/100032

FAD-2010-0052 - CRL/100035
FAD-2010-0140 - CRL/100184

Name of Feed Additive: Vitamin B1

Active Agent (s): Thiamine mononitrate

Thiamine hydrochloride

Rapporteur Laboratory: European Union Reference Laboratory for

Feed Additives (EURL-FA)
Geel, Belgium

Report prepared by: Zigmas Ezerskis and Rebeca Fernandez

Orozco

Report checked by: Piotr Robouch (EURL-FA)

Date: 13/05/2014

Report approved by: Christoph von Holst

Date: 13/05/2014
 EURL Evaluation Report “Vitamin B1”

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

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

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 EURL Evaluation Report “Vitamin B1”

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.

KEYWORDS
Vitamin B1, thiamine mononitrate, thiamine hydrochloride, nutritional additives, vitamins, all
animal species and categories

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1. BACKGROUND
In the current applications1,2,3 authorisation is sought under articles 4(1) (new use in water)
and 10(2) (re-evaluation of the additive already authorised under Council Directive
70/524/EEC) 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 [1]. Specifically, authorisation is sought for the use of the feed additives
for all categories and species [1].
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
[2,3].
The feed additives are intended to be incorporated in feedingstuffs (including complete
feedingstuffs or complementary feedingstuffs) through premixtures or directly in water [3].
However, the Applicants did not specify any maximum or minimum concentration of
thiamine mononitrate and thiamine hydrochloride in feedingstuffs or water [2].
The EURL previously evaluated the analytical methods for the quantification of Vitamin B1 in the
frame of the FAD-2010-0040, FAD-2010-0052 and FAD-2010-0140 dossiers [4-6]. Based on two
requests from Commission [7,8] to re-evaluate analytical methods of Vitamin B1 in premixtures
and water, the EURL decided to amend the above mentioned EURL reports taking into account
the supplementary information provided by the Applicants. This EURL report presents the
amendment of the scientific evaluation of the analytical methods of Vitamin B1 related to the
dossiers: FAD-2010-0040, FAD-2010-0052 and FAD-2010-0140.

2. TERMS OF REFERENCE
In accordance with Article 5 of Regulation (EC) No 378/2005, as last amended by Regulation
(EC) No 885/2009, on detailed rules for the implementation of Regulation (EC) No
1831/2003 of the European Parliament and of the Council as regards the duties and the tasks
of the European Union Reference Laboratory concerning applications for authorisations of
feed additives, the EURL is requested to submit a full evaluation report to the European Food
Safety Authority for each application or group of applications. For this particular dossier, the
methods of analysis submitted in connection with Vitamin B1 (thiamine mononitrate and
thiamine hydrochloride) and their suitability to be used for official controls in the frame of the
authorisation were evaluated.

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

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3. EVALUATION
Identification /Characterisation of the feed additive
Qualitative and quantitative composition of impurities in the additive
When required by EU legislation, analytical methods for official control of undesirable
substances in the additive (e.g. arsenic, cadmium, lead, mercury, aflatoxin B1 and dioxins) are
available from the respective European Union Reference Laboratories [9].

Description of the analytical methods for the determination of the active substance in feed
additive, premixtures, feedingstuffs and water
For the characterisation of thiamine mononitrate and thiamine hydrochloride in the feed
additives the Applicants proposed European Pharmacopoeia monographs [10,11] where the
quantification assay is based on potentiometric titration. However, the EURL identified
instead the US Pharmacopoeia monographs [12,13] where quantification is based on high
performance liquid chromatography with UV detection at 254 nm.
Even though no performance characteristics are provided, the EURL recommends for official
control the HPLC-UV methods described in the US Pharmacopoeia monographs, to
characterise thiamine mononitrate and thiamine hydrochloride in the feed additives.
For the quantification of Vitamin B1 in premixtures, Applicant (FAD-2010-0140) proposed a
single-laboratory validated method [14], similar to the official ring-trial validated method by
the Association of German Agricultural Analytical Research Institutes (VDLUFA) [15], while
Applicants (FAD-2010-0040 and FAD-2010-0052) proposed the VDLUFA method as such.
According to the VDLUFA method, the premixtures sample (1 to 3 g) is treated with
methanolic titriplex-V (diethylenetriaminepentaacetic acid) solution in an ultrasonic bath for 1
to 2 minutes. An aliquot of the solution is then diluted with water and titriplex-V solution,
filtered through a membrane filter and subjected to the chromatographic analysis. The
analysis is performed using ion-exchange high performance liquid chromatography coupled to
UV detector (HPLC-UV). When adjusted at 275 nm it allows the simultaneous detection of
Vitamin B1, B2, B6, Nicotinic acid and Nicotinamide; while 246 nm is used to detect
Vitamin B1 alone. The target analytes are quantified using external standards calibration [15].
Based on the satisfactory performance characteristics presented in Table 1, the EURL
recommends for official control the ring-trial validated VDLUFA method to quantify
thiamine mononitrate and thiamine hydrochloride in premixtures containing Vitamin B1
concentration levels ranging from 1500 to 8000 mg/kg.

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 EURL Evaluation Report “Vitamin B1”

Table 1: Performance characteristics of ring-trail validated (RTV) or single-laboratory

validated (SLV) methods for the quantification of Vitamin B1 in various matrices,
including premixtures (PM), feedingstuffs (FS) and water.

Matrix Content, mg/kg RSDr, % RSDR, % RRec, % Ref.

PM 1570 - 7940 2.1 - 2.6 4.2 - 7.4 RTV, [15]

PM & FS 7 - 484 4.2 - 4.7 5 - 13 88 - 97 RTV, [18]

Water 3.6 - 4.4 1.0 - 2.8 10.5 * SLV,[17]

0.5 - 368 98 - 100

Food CRM (a) 4.5 - 8.1 3.2 – 4.2 12 - 23 RTV, [19]

Foodstuffs (b) 1.1 - 13.1 7.0 - 9.0 13 - 32

RSDr and RSDR: relative standard deviation for repeatability and reproducibility;
(*) RSDip: relative standard deviation for intermediate precision;
(a) CRM 121 (whole meal flour), CRM 421 (milk powder), CRM 485 (mixed vegetables), CRM 487 (pig's liver);
(b) tube feeding solution, baby food, powdered milk, meal with fruits and yeast.

For the quantification of Vitamin B1 in feedingstuffs, Applicant (FAD-2010-0040) did not

submit any method; Applicant (FAD-2010-0052) proposed a method published in the
literature by Rubaj et al. (2008) [16], while Applicant (FAD-2010-0140) suggested using a
slightly modified CEN method (EN 14122:2003) [17]. However, the EURL identified instead
the ring-trial validated method for premixtures and feedingstuffs, published in the Italian
Official Journal [18].
According to this method, the sample (5g) is treated with warm diluted hydrochloric acid.
After cooling to room temperature, the extract is filtered and an aliquot is oxidised to
transform the Vitamin B1 into thiochrome. The thiochrome solution is placed on a solid phase
extraction (SPE) cartridge, which is washed with a solution of methanol in phosphate buffer.
Finally the target analyte is eluted with methanol. The analysis is performed using the
reversed phase high performance liquid chromatography (HPLC) coupled to fluorescence
detector at excitation wavelength of 360 nm and emission wavelength of 430 nm [18].
Vitamin B1 is very sensitive to light; temperature and oxygen, therefore the light protected
glasses (brown glass) have to be used during analysis. The Applicant reported a limit of
quantification (LOQ) of 5 mg/kg feedingstuffs [17].
Based on the satisfactory performance characteristics presented in Table 1 the EURL
recommends for official control the ring-trial validated HPLC-UV method published in the

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 EURL Evaluation Report “Vitamin B1”

Italian Official Journal to quantify thiamine mononitrate and thiamine hydrochloride in

premixtures and feedingstuffs, containing Vitamin B1 concentration levels ranging from 7 to
500 mg/kg.
For the quantification of Vitamin B1 in water Applicant (FAD-2010-0140) submitted a
slightly adapted/modified CEN method EN 14122 to be applied to water samples [17].
According to this method, a water sample (2 to 5 ml) is treated with 50 ml sulphuric acid
(0.05 M) at 50 oC for 20 min, then let to cool to room temperature. The extract is adjusted
with the mobile phase to yield approximately 0.5 μg/ml of Vitamin B1 in the solution. An
aliquot of the sample is centrifuged for 5 min and 20 μl of the supernatant are injected into
HPLC system. The sample is oxidised to thiochrome through post-column derivatisation
using alkaline hexacyanoferrate (III) solution and further detected by fluorescence detector at
excitation wavelength of 370 nm and emission wavelength of 435 nm. The Applicant reported
a LOQ of 0.06 mg/L [17].
The performance characteristics presented in Table 1 for the quantification of Vitamin B1 in
water are in good agreement with those published in the EN 14122 standard [19]. Based on
the satisfactory performance characteristics provided, the EURL recommends for official
control the adapted/modified CEN method, based on reversed phase high performance liquid
chromatography coupled to fluorescence detector after post- column derivatisation (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.

4. CONCLUSIONS AND RECOMMENDATIONS

In the frame of this authorisation the EURL recommends for official control:

− the US Pharmacopoeia 32 monographs: 'thiamine mononitrate' and 'thiamine

hydrochloride', based on High-Performance Liquid Chromatography with UV
detection at 254 nm (HPLC-UV), to characterise thiamine mononitrate and thiamine
hydrochloride in the feed additives;

− the VDLUFA Bd. III, 13.9.1 method, based on ion-exchange high performance liquid
chromatography coupled to UV detector (HPLC-UV), to quantify Vitamin B1
(thiamine mononitrate and thiamine hydrochloride) in premixtures containing Vitamin
B1 concentration levels ranging from 1500 to 8000 mg/kg.

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− the Italian official method, based on reversed phase high performance liquid
chromatography coupled to fluorescence detector (HPLC-FL), to quantify Vitamin B1
(thiamine mononitrate and thiamine hydrochloride) in feedingstuffs and in premixtures
containing Vitamin B1 concentration levels ranging from 7 to 500 mg/kg.

− the adapted/modified CEN method (EN 14122:2003) based on reversed phase high
performance liquid chromatography coupled to fluorescence detector after post-
column derivatisation (HPLC-FL) to quantify thiamine mononitrate and thiamine
hydrochloride in water.

Recommended text for the register entry (analytical method)

For the characterisation of thiamine mononitrate in feed additive:
− high performance liquid chromatography with UV detection (HPLC-UV) -
US Pharmacopeia 32 ('thiamine mononitrate' monograph)
For the characterisation of thiamine hydrochloride in feed additive:
− high performance liquid chromatography with UV detection (HPLC-UV) -
US Pharmacopeia 32 ('thiamine hydrochloride' monograph)
For the quantification of thiamine mononitrate and thiamine hydrochloride 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 20/02/2006, Official Italian Journal n. 50 1/3/2006
For the quantification of thiamine mononitrate and thiamine hydrochloride in feedingstuffs:
− reversed phase high performance liquid chromatography coupled to fluorescence
detection (HPLC-FL) – decree 20/02/2006, Official Italian Journal n. 50 1/3/2006
For the quantification of thiamine mononitrate and thiamine hydrochloride in water:
− reversed phase high performance liquid chromatography (HPLC) with post- column
derivatisation and fluorescence detection

5. DOCUMENTATION AND SAMPLES PROVIDED TO EURL

In accordance with the requirements of Regulation (EC) No 1831/2003, reference samples of

thiamine mononitrate and thiamine hydrochloride have been sent to the European Union
Reference Laboratory for Feed Additives. The dossier has been made available to the EURL
by EFSA.

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6. REFERENCES
a,b,c
[1] Application, Reference SANCO/D2:
Forw. Appl. 1831/0039/2010; 1831/0034/2010; 1831/0088/2010
a,b,c
[2] Application, Proposal for Register Entry – Annex A
a,b,c
[3] Technical dossier, Section II: Identity, characterisation and conditions of use of the
feed additive; methods of analysis
#
[4] Evaluation report, FAD-2010-0040 – JRC.DG.D6/CvH/RMO/ag/ARES(2011)72601
#
[5] Evaluation report, FAD-2010-0052 – JRC.DG.D6/CvH/RMO/ag/ARES(2010)787557
[6] NFAD letter, FAD2010-0140 – JRC.DG.D6/FSQ/CvH/RM/MdS/Ares(2011)762748
[7] Request letter, Ref. Ares (2014)1209965 - 16/04/2014
[8] Request letter, Ref. Ares (2014)1209919 - 16/04/2014
[9] Commission Regulation (EC) No 776/2006 amending Annex VII to Regulation (EC) No
882/2004 of the European Parliament and of the Council as regards to Community
Reference Laboratories
a
[10] Technical Dossier, Section II – Annex 2.01_Ph.Eur 6.0, 01/2008:0531
a
[11] Technical Dossier, Section II – Annex 2.02_Ph.Eur 6.0, 01/2008:0303
[12] US Pharmacopeia 32, 'thiamine mononitrate' monograph
http://www.uspbpep.com/usp32/pub/data/v32270/xref_usp32nf27s0_m82530.html
[13] US Pharmacopeia 32, 'thiamine hydrochloride' monograph
http://www.uspbpep.com/usp32/pub/data/v32270/xref_usp32nf27s0_m82470.html
c
[14] Technical Dossier, Section II – Annex_II_29
a
[15] Technical Dossier, Section II – Annex 2.09_VDLUFA Methodenbuch Bd.III, 13.9.1
b
[16] Technical Dossier, Section II – Ref. II 2.2
c
[17] Technical Dossier, Supplementary information – Annex_II_37 (Revised version)
[18] Decreto 20 febbraio 2006. Approvazione del metodo ufficiale di analisi per la
determinazione della vitamina B1 negli alimenti per animali – Supplemento n. 19
c
[19] Technical Dossier, Section II – Annex_II_33_EN 14122:2003, Foodstuffs -
Determination of Vitamin B1 by HPLC

#
http://irmm.jrc.ec.europa.eu/EURLs/EURL_feed_additives/authorisation/evaluation_reports/
a
Refers to Dossier no: FAD-2010-0040
b
Refers to Dossier no: FAD-2010-0052
c
Refers to Dossier no: FAD-2010-0140

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7. RAPPORTEUR LABORATORY & NATIONAL REFERENCE LABORATORIES

The Rapporteur Laboratory for this evaluation was European Union Reference Laboratory for
Feed Additives, IRMM, Geel, Belgium. This report is in accordance with the opinion of the
consortium of National Reference Laboratories as referred to in Article 6(2) of Commission
Regulation (EC) No 378/2005, as last amended by Regulation (EC) No 885/2009.

8. ACKNOWLEDGEMENTS
The following National Reference Laboratories contributed to this and/or previous (FAD-
2010-0040 and FAD-2010-0052) reports:
- Centro di referenza nazionale per la sorveglianza ed il controllo degli alimenti per gli
animali (CReAA), Torino (IT)
- Schwerpunktlabor Futtermittel des Bayerischen Landesamtes für Gesundheit und
Lebensmittelsicherheit (LGL), Oberschleißheim (DE)
- Thüringer Landesanstalt für Landwirtschaft (TLL), Abteilung Untersuchungswesen,
Jena (DE)
- Państwowy Instytut Weterynaryjny, Puławy (PL)
- Staatliche Betriebsgesellschaft für Umwelt und Landwirtschaft, Labore Landwirtschaft,
Nossen (DE)1
- Skúšobné laboratórium – Oddelenie analýzy krmív, Ústredný kontrolný a skúšobný
ústav poľnohospodársky, Bratislava (SL)
- Univerza v Ljubljani, Veterinarska fakulteta. Nacionalni veterinarski inštitut, Enota za
patologijo prehrane in higieno okolja, Ljubljana (SL)
- Fødevarestyrelsen, Ringsted (DK)2
- Instytut Zootechniki w Krakowie, Krajowe Laboratorium Pasz, Lublin (PL)
- Ústřední kontrolní a zkušební ústav zemědělský (ÚKZÚZ), Praha (CZ)
- Österreichische Agentur für Gesundheit und Ernährungssicherheit (AGES), Wien (AT)

1
Name and address according to Regulation (EC) No 885/2009: Sächsische Landesanstalt für Landwirtschaft.
Fachbereich 8 – Landwirtschaftliches Untersuchungswesen, Leipzig
2
Name and address according to Regulation (EC) No 885/2009: Plantedirektoratet, Laboratorium for Foder og
Gødning, Lyngby

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