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STANDARD
SIST EN 15607:2009
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
EN 15607:2009 (E) 2 Contents Page Foreword .31Scope .42Normative references .43Principle .44Reagents .45Apparatus .76Procedure .87Calculation .98Precision .99Test report . 10Annex A (informative)
Typical chromatogram . 11Annex B (informative)
Precision data . 13Bibliography . 14 SIST EN 15607:2009
EN 15607:2009 (E) 3 Foreword This document (EN 15607:2009) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by November 2009, and conflicting national standards shall be withdrawn at the latest by November 2009. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. WARNING — The use of this standard may involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: 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 the United Kingdom. SIST EN 15607:2009
EN 15607:2009 (E) 4 1 Scope 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. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use – Specification and test methods (ISO 3696:1987) 3 Principle D-biotin is extracted from food after an enzymatic treatment and quantified by HPLC with post-column binding reaction, see [2] and [3]. The complexion of d-biotin with avidin appears to be very specific. On that account, this protein, covalently bound to a fluorescent marker, fluorescein 5-isothiocyanate, can be used as a reagent for a post-column binding of d-biotin, see [4] and [5]. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and water of at least grade 1 according to EN ISO 3696:1995, or double distilled water. 4.2 Chemicals and solutions 4.2.1 Methanol, HPLC grade, mass fraction w(CH3OH) ≥ 99,8 % 4.2.2 Sulfuric acid solution, substance concentration c(H2SO4) = 1 mol/l 4.2.3 Sulfuric acid solution, c(H2SO4) = 1,5 mol/l 4.2.4 Citric acid monohydrate, w(C6H8O7·H2O) ≥ 99,7 % 4.2.5 Sodium monohydrogen phosphate dihydrate, w(Na2HPO4·2H2O) ≥ 99,8 %
SIST EN 15607:2009
EN 15607:2009 (E) 5 4.2.6 Gluthatione, w(C10H17N3O6S) ≥ 98 % 4.2.7 EDTA sodium salt dihydrate, w(C10H14N2Na2O8·2H2O) ≥ 99 % 4.2.8 Potassium monohydrogen phosphate, w(K2HPO4) ≥ 96 % 4.2.9 Potassium dihydrogen phosphate, w(KH2PO4) ≥ 99,5 % 4.2.10 Citrate buffer solution Dissolve 0,462 g of citric acid monohydrate (4.2.4) and 1,05 g of sodium monohydrogen phosphate dihydrate (4.2.5) in 450 ml of distilled water. Adjust the solution to pH = 5,7 with sulfuric acid solution (4.2.3), and then dilute to 500 ml with distilled water. This solution is stable for 1 day. 4.2.11 Gluthatione solution, mass concentration (C10H17N3O6S) = 10 g/l Dissolve 30 mg of gluthatione (4.2.6) in 3 ml of distilled water. This solution is stable for 1 day. 4.2.12 EDTA solution, (C10H14N2Na2O8·2H2O) = 10 g/l Dissolve 0,1 g of EDTA (4.2.7) in 10 ml of distilled water. This solution is stable for 1 day. 4.2.13 Potassium monohydrogen phosphate solution, c(K2HPO4) = 0,1 mol/l Dissolve 17,4 g of potassium monohydrogen phosphate (4.2.8) in 1000 ml of water. This solution is stable for 2 days. 4.2.14 Potassium dihydrogen phosphate solution, c(KH2PO4) = 0,1 mol/l Dissolve 13,6 g of potassium dihydrogen phosphate (4.2.9) in 1000 ml of water. This solution is stable for 2 days. 4.2.15 Phosphate buffer solution pH = 6,0 Mix 4.2.13 and 4.2.14 in such a proportion that the final solution has a pH of 6,0 (e.g. 30 parts per volume of 4.2.13 and 70 parts per volume of 4.2.14). This solution is stable for 1 week at room temperature. 4.2.16 Phosphate buffer solution pH = 7,0 Mix 4.2.13 and 4.2.14 in such a proportion that the final solution has a pH of 7,0 (e.g. 40 parts per volume of 4.2.13 and 60 parts per volume of 4.2.14). This solution is stable for 1 week at room temperature. 4.2.17 Papain powder, (CAS 9001-73-4), enzyme activity is 15 nkat/mg1 with substrate N-benzoyl-L-arginine ethyl ester (BAEE) at pH = 6,2 and t = 25 oC. 15 nkat/mg corresponds to 1 U/mg. 4.2.18 Papain solution, (papain) = 20 g/l 4.2.18.1 General Dissolve 1 g of papain powder (4.2.17) in 50 ml of citrate buffer solution (4.2.10). This solution is stable for 5 days at 4 °C.
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
EN 15607:2009 (E) 6 4.2.18.2 Activity check of papain The activity of papain can be checked by making a second extract (see 6.2) with a double amount of enzyme. Verify that the level of d-biotin calculated is the same and not higher. NOTE For the interlaboratory study, the papain powder from VWR International GmbH, Hilpertstraße 20a, 64295 Darmstadt, ref. nr. 26.146.180
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 t
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