EN 15607:2009

Foodstuffs - Determination of d-biotin by HPLC

This European Standard specifies a method for the determination of the mass fraction of d-biotin by high performance liquid chromatography (HPLC). The method has been validated in an inter-laboratory test on fortified and non-fortifed samples such as cereal breakfast powder, infant milk powder, lyophilized green peas with ham, lyophilized chicken soup and on nutritive orange juice, at levels from 16 μg/100 g to 200 μg/100 g. For further information on the validation data, see Annex B. NOTE 1 d-biocytin can also be estimated by this method. But none of the samples used for the validation step contained d-biocytin. Nonetheless the recovery rate is more than 90 % for d-biotin and d-biocytin, see [2] and [3]. NOTE 2 The method underestimates the real biotin content when used for samples containing egg.

Lebensmittel - Bestimmung von D-Biotin mit HPLC

Diese Europäische Norm legt ein Verfahren zur Bestimmung des Massenanteils an D Biotin in Lebensmitteln mit Hochleistungsflüssigchromatographie (en: high performance liquid chromatography, HPLC) fest. Das Verfahren wurde in einem Ringversuch an angereicherten und naturbelassenen Proben wie pulverisierten Frühstückscerealien, Milchpulver für Säuglinge, grünen Erbsen mit Schinken (lyophilisiert), Hühnersuppe (lyophilisiert) und an angereichertem Orangensaft validiert, die 16 µg/100 g bis 200 µg/100 g an D Biotin enthielten. Weitere Informationen, siehe Anhang B.
ANMERKUNG 1   Mit diesem Verfahren kann auch D Biocytin bewertet werden, obwohl keine der Proben, die im Ringversuch verwendet wurden, D Biocytin enthielt. Dennoch beträgt die Wiederfindungsrate für D Biotin und D Biocytin mehr als 90 %, siehe [2] und [3].
ANMERKUNG 2   Bei der Untersuchung von Proben, die Ei enthalten, ergibt das Verfahren zu kleine Werte für den Biotin Gehalt.

Produits alimentaires - Dosage de la d-biotine par CLHP

La présente Norme européenne spécifie une méthode de détermination de la fraction massique de la d biotine par chromatographie liquide haute performance (CLHP). La méthode a été validée dans un essai interlaboratoire sur des échantillons enrichis et non enrichis tels que poudre de céréales pour petits déjeuners, lait en poudre pour nourrissons, petits pois au jambon lyophilisés, soupe au poulet lyophilisée et sur du jus d'orange nutritif, à des niveaux allant de 16 µg/100 g à 200 µg/100 g. Pour de plus amples informations sur les données de validation, voir l'Annexe B.
NOTE 1   La d-biocytine peut également être estimée par la présente méthode. Toutefois, aucun des échantillons utilisés dans l'étape de validation ne contenait de d-biocytine. Néanmoins, le taux de récupération est supérieur à 90 % pour la d-biotine et la d-biocytine (voir [2] et [3]).
NOTE 2   La méthode sous-estime la teneur réelle en biotine lorsqu'elle est utilisée avec des échantillons contenant de l'oeuf.

Živila - Določevanje d-biotina s HPLC

General Information

• Status: Published
• Publication Date: 26-May-2009
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 9 - Vitamins and Carotenoids
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Completion Date: 28-Oct-2020
• Ref Project: SIST EN 15607:2009 - Foodstuffs - Determination of d-biotin by HPLC

Standards Content (Sample)

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Lebensmittel - Bestimmung von d-Biotin mit HPLCProduits alimentaires - Dosage de la d-biotine par CLHPFoodstuffs - Determination of d-biotin by HPLC67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 15607:2009SIST EN 15607:2009en,fr,de01-september-2009SIST EN 15607:2009SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15607May 2009ICS 67.050 English VersionFoodstuffs - Determination of d-biotin by HPLCProduits alimentaires - Dosage de la d-biotine par CLHPLebensmittel - Bestimmung von d-Biotin mit HPLCThis European Standard was approved by CEN on 23 April 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:
Avenue Marnix 17,
B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15607:2009: ESIST EN 15607:2009

Typical chromatogram . 11Annex B (informative)
Precision data . 13Bibliography . 14 SIST EN 15607:2009

1 Katal (symbol ''kat'') is a derived SI unit of enzyme activity. One katal is that catalytic activity which will raise the rate of reaction by one mol/s in a specified assay system. SIST EN 15607:2009

has been used 2. 4.2.19 Avidin fluoresceine isothiocyanate (Avidin-FITC), labelled, 80 % protein, 2 mol to 4 mol FITC per mol of avidin 4.2.20 Stock solution reagent for post-column binding reaction, (avidin-FITC) = 50 mg/ml Dissolve 2,5 mg of avidin-FITC (4.2.19) in 50 ml of phosphate buffer solution pH = 7,0 (4.2.16). This solution is stable for 2 weeks at 4 °C. 4.2.21 Reagent for post-column binding reaction, (avidin-FITC) = 2 mg/ml Add 600 ml of phosphate buffer solution pH = 7,0 (4.2.16) to 25 ml of the stock solution (4.2.20). Filter this solution through a 0,45 µm filter (5.5). This solution is stable for 8 hours, screened from light. 4.2.22 HPLC mobile phase Mix 80 parts per volume of phosphate buffer solution pH = 6,0 (4.2.15) with 20 parts per volume of methanol (4.2.1). Filter this solution through a 0,45 µm filter (5.5). 4.2.23 Taka-diastase from Aspergillus Oryzae, enzyme activity is 1 500 nkat/mg (1 500 nkat/mg corresponds to 100 U/mg), suitable for samples with a high starch content. 4.3 Standard substances 4.3.1 General D-biotin and d-biocytin can be obtained from various suppliers. The baseline separation of d-biotin and d-biocytin shall be verified. So it is necessary to prepare a standard solution. The biotin content of the standard can be confirmed according to the European Pharmacopoeia procedure [6]. 4.3.2 d-biotin, w(C10H16N2O3S) ≥ 99 % 4.3.3 d-biocytin, w(C16H28N4O4S) ≥ 98 % 4.4 Stock solutions 4.4.1 d-biotin, (C10H16N2O3S)
= 100 µg/ml Dissolve an amount of the d-biotin standard substance (4.3.2), approximately 10 mg to the nearest 0,1 mg in 100 ml of distilled water. It may take 4 h to 5 h to dissolve. This solution is stable for 2 months at –18 °C.
2 This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 15607:2009

= 100 µg/ml Dissolve an amount of the d-biocytin standard substance (4.3.3), approximately 10 mg to the nearest 0,1 mg in 100 ml of distilled water. This solution is stable for 2 months at –18 °C. 4.5 Standard solutions 4.5.1 d-biotin solution, (C10H16N2O3S) = 0,05 µg/ml to 0,30 µg/ml Prepare for example a solution with 1 ml of the stock solution (4.4.1) in 10 ml of distilled water. Then prepare six calibration solutions (0,5 ml, 1,0 ml, 1,5 ml, 2,0 ml, 2,5 ml and 3 ml) in 100 ml of distilled water. These solutions are stable for 1 day. 4.5.2 d-biocytin solution, (C16H28N4O4S) = 0,30
µg/ml Prepare for example a solution with 1 ml of the stock solution (4.4.2) in 10 ml of distilled water. Then prepare a standard solution with 3 ml in 100 ml of distilled water. Solution is stable for 1 day. 5 Apparatus 5.1 General Usual laboratory apparatus and glassware, and the following. 5.2 Oven Capable of maintaining a temperature of 37 °C ± 2 °C. 5.3 HPLC system Consisting of a pump, sample injecting device, fluorescence detector with excitation wavelength set at 490 nm and emission wavelength set at 520 nm, and an evaluation system such as an integrator. 5.4 Analytical reverse-phase separating column, e.g. LiChrospher® 100 RP-18 endcapped 3 The column shall ensure a baseline resolution of the analytes concerned with the following characteristics: a) a length of 250 mm; b) an inner diameter of 4,0 mm; c) a particle size of 5 µm. Other particle sizes or column dimensions than specified in this European Standard may be used. Separation parameters shall be adapted to such other materials to guarantee equivalent results.
3 LiChrospher® 100 RP-18 endcapped is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 15607:2009

Filtering of the mobile phase as well as of the sample test solution through a membrane filter prior to use or injection can increase longevity of the columns. 5.6 Post-column reactor derivatization system Consisting of a suita
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