Vitamin B 1 (FAD ; CRL/100032) (FAD ; CRL/100035) (FAD ; CRL/100184)

Transcription

1 Ref. Ares(2014) /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 B 1 (FAD ; CRL/100032) (FAD ; CRL/100035) (FAD ; CRL/100184)

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3 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 CRL/ FAD CRL/ FAD CRL/ Name of Feed Additive: Vitamin B 1 Active Agent (s): Thiamine mononitrate Thiamine hydrochloride Rapporteur Laboratory: Report prepared by: Report checked by: Date: Report approved by: Date: European Union Reference Laboratory for Feed Additives (EURL-FA) Geel, Belgium Zigmas Ezerskis and Rebeca Fernandez Orozco Piotr Robouch (EURL-FA) 13/05/2014 Christoph von Holst 13/05/2014

4 EXECUTIVE SUMMARY In the current applications 1,2,3 authorisation is sought under articles 4(1) and 10(2) for Vitamin B 1 (thiamine mononitrate 1,2,3 and thiamine hydrochloride 1,3 ) 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 B 1 in premixtures, Applicants 1,2 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 B 1 levels ranging from 1570 to 7940 mg/kg: - a relative standard deviation for repeatability (RSD r ) ranging from 2.1 to 2.6 %; and - a relative standard deviation for reproducibility (RSD R ) 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 B 1 (thiamine mononitrate and thiamine hydrochloride) in premixtures containing high Vitamin B 1 levels ranging from 1500 to 8000 mg/kg. Additionally, the EURL identified an alternative ring-trial validated method for the quantification of Vitamin B 1 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 , 2 FAD , 3 FAD /9

5 samples containing Vitamin B 1 levels ranging from 7 to 484 mg/kg: - a RSD r ranging from 4.2 to 4.7 %; - a RSD R ranging from 5 to 13 %; - a recovery rate (R Rec ) 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 B 1 levels ranging from 7 to 500 mg/kg. For the quantification of Vitamin B 1 in water Applicant (FAD ) 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 B 1 levels ranging from 3.6 to 4.4 mg/l the following performance characteristics: - RSD r ranging from of 1.0 to 2.8 %; - a relative standard deviation of intermediate precision (RSD ip ) equal to 10.5 %; and - a limit of quantification (LOQ) of 0.06 mg/l water. Additionally recovery rates (R Rec ) ranging from 98 to 100% were reported for Vitamin B 1 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 B 1, thiamine mononitrate, thiamine hydrochloride, nutritional additives, vitamins, all animal species and categories 2/9

6 1. BACKGROUND In the current applications 1,2,3 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 B 1 (thiamine mononitrate 1,2,3 and thiamine hydrochloride 1,3 ) 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 B 1 in the frame of the FAD , FAD and FAD dossiers [4-6]. Based on two requests from Commission [7,8] to re-evaluate analytical methods of Vitamin B 1 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 B 1 related to the dossiers: FAD , FAD and FAD 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 B 1 (thiamine mononitrate and thiamine hydrochloride) and their suitability to be used for official controls in the frame of the authorisation were evaluated. 1 FAD , 2 FAD , 3 FAD /9

7 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 B 1 in premixtures, Applicant (FAD ) 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 and FAD ) 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 B 1, B 2, B 6, Nicotinic acid and Nicotinamide; while 246 nm is used to detect Vitamin B 1 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 B 1 concentration levels ranging from 1500 to 8000 mg/kg. 4/9

8 Table 1: Performance characteristics of ring-trail validated (RTV) or single-laboratory validated (SLV) methods for the quantification of Vitamin B 1 in various matrices, including premixtures (PM), feedingstuffs (FS) and water. Matrix Content, mg/kg RSD r, % RSD R, % R Rec, % Ref. PM RTV, [15] PM & FS RTV, [18] Water * SLV,[17] Food CRM (a) RTV, [19] Foodstuffs (b) RSD r and RSD R : relative standard deviation for repeatability and reproducibility; (*) RSD ip : 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 B 1 in feedingstuffs, Applicant (FAD ) did not submit any method; Applicant (FAD ) proposed a method published in the literature by Rubaj et al. (2008) [16], while Applicant (FAD ) 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 B 1 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 B 1 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 5/9

9 Italian Official Journal to quantify thiamine mononitrate and thiamine hydrochloride in premixtures and feedingstuffs, containing Vitamin B 1 concentration levels ranging from 7 to 500 mg/kg. For the quantification of Vitamin B 1 in water Applicant (FAD ) submitted a slightly adapted/modified CEN method EN 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 o C 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 B 1 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 B 1 in water are in good agreement with those published in the EN 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, method, based on ion-exchange high performance liquid chromatography coupled to UV detector (HPLC-UV), to quantify Vitamin B 1 (thiamine mononitrate and thiamine hydrochloride) in premixtures containing Vitamin B 1 concentration levels ranging from 1500 to 8000 mg/kg. 6/9

10 the Italian official method, based on reversed phase high performance liquid chromatography coupled to fluorescence detector (HPLC-FL), to quantify Vitamin B 1 (thiamine mononitrate and thiamine hydrochloride) in feedingstuffs and in premixtures containing Vitamin B 1 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 postcolumn 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, 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. 7/9

11 6. REFERENCES [1] a,b,c Application, Reference SANCO/D2: Forw. Appl. 1831/0039/2010; 1831/0034/2010; 1831/0088/2010 [2] a,b,c Application, Proposal for Register Entry Annex A [3] a,b,c Technical dossier, Section II: Identity, characterisation and conditions of use of the feed additive; methods of analysis [4] # Evaluation report, FAD JRC.DG.D6/CvH/RMO/ag/ARES(2011)72601 [5] # Evaluation report, FAD JRC.DG.D6/CvH/RMO/ag/ARES(2010) [6] NFAD letter, FAD JRC.DG.D6/FSQ/CvH/RM/MdS/Ares(2011) [7] Request letter, Ref. Ares (2014) /04/2014 [8] Request letter, Ref. Ares (2014) /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 [10] a Technical Dossier, Section II Annex 2.01_Ph.Eur 6.0, 01/2008:0531 [11] a Technical Dossier, Section II Annex 2.02_Ph.Eur 6.0, 01/2008:0303 [12] US Pharmacopeia 32, 'thiamine mononitrate' monograph [13] US Pharmacopeia 32, 'thiamine hydrochloride' monograph [14] c Technical Dossier, Section II Annex_II_29 [15] a Technical Dossier, Section II Annex 2.09_VDLUFA Methodenbuch Bd.III, [16] b Technical Dossier, Section II Ref. II 2.2 [17] c Technical Dossier, Supplementary information Annex_II_37 (Revised version) [18] Decreto 20 febbraio Approvazione del metodo ufficiale di analisi per la determinazione della vitamina B 1 negli alimenti per animali Supplemento n. 19 [19] c Technical Dossier, Section II Annex_II_33_EN 14122:2003, Foodstuffs - Determination of Vitamin B 1 by HPLC # a Refers to Dossier no: FAD b Refers to Dossier no: FAD c Refers to Dossier no: FAD /9

12 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/ ACKNOWLEDGEMENTS The following National Reference Laboratories contributed to this and/or previous (FAD and FAD ) 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 9/9