1、September 2009DEUTSCHE NORM English price group 10No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.050!$Yq“1547856ww
2、w.din.deDDIN EN 15607Foodstuffs Determination of d-biotin by HPLCEnglish version of DIN EN 15607:2009-09Lebensmittel Bestimmung von D-Biotin mit HPLCEnglische Fassung DIN EN 15607:2009-09www.beuth.deDocument comprises 16 pagesDIN EN 15607:2009-09 National foreword This standard has been prepared by
3、Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). Preliminary work was done by Working Group WG 9 “Vitamins and Carotenoids”. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Fo
4、od and Agricultural Products Standards Committee), Technical Committee NA 057-01-13 AA Vitamine und Carotinoide. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 ISO 5725-2 DIN ISO 5725-2 National Annex NA (informative)
5、 Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725-2, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method 2 EUROP
6、EAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15607 May 2009 ICS 67.050 English Version Foodstuffs Determination of d-biotin by HPLC Produits alimentaires Dosage de la d-biotine par CLHP Lebensmittel Bestimmung von D-Biotin mit HPLC This European Standard was approved by CEN on 23 April 2009. CEN
7、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 national standards may be obtained on applic
8、ation 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 translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre ha
9、s 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, Port
10、ugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means
11、reserved worldwide for CEN national Members. Ref. No. EN 15607:2009: EEN 15607:2009 (E) 2 Contents PageForeword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76 Procedure .87 Calculation 98 Precision .99 Test report . 10Annex A (informative) Typical chromatogram 11Annex B
12、(informative) Precision data . 13Bibliography . 14DIN EN 15607:2009-09 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 t
13、he 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 b
14、e 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 wit
15、h 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 count
16、ries 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, Spai
17、n, Sweden, Switzerland and the United Kingdom. DIN EN 15607:2009-09 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
18、 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 g/100 g to 200 g/100 g. For further information on the validation data, see Annex B. NOTE 1 d-biocyti
19、n 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.
20、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 fo
21、r 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 accou
22、nt, 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
23、 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
24、 monohydrate, w(C6H8O7H2O) 99,7 % 4.2.5 Sodium monohydrogen phosphate dihydrate, w(Na2HPO42H2O) 99,8 % DIN EN 15607:2009-09 EN 15607:2009 (E) 5 4.2.6 Gluthatione, w(C10H17N3O6S) 98 % 4.2.7 EDTA sodium salt dihydrate, w(C10H14N2Na2O82H2O) 99 % 4.2.8 Potassium monohydrogen phosphate, w(K2HPO4) 96 % 4.
25、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
26、 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, (C10H14N2Na2O82H2O) = 10 g
27、/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 Potass
28、ium 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.
29、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 p
30、er 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/mg1with 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
31、 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
32、in a specified assay system. DIN EN 15607:2009-09 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 int
33、erlaboratory study, the papain powder from VWR International GmbH, Hilpertstrae 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-col
34、umn 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
35、 pH = 7,0 (4.2.16) to 25 ml of the stock solution (4.2.20). Filter this solution through a 0,45 m 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 methan
36、ol (4.2.1). Filter this solution through a 0,45 m 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 b
37、e 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
38、.3 d-biocytin, w(C16H28N4O4S) 98 % 4.4 Stock solutions 4.4.1 d-biotin, (C10H16N2O3S) = 100 g/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 mon
39、ths 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. DIN EN 15607:2009-09 EN 15607:2009 (E) 7 4.4.2 d-biocy
40、tin, (C16H28N4O4S) = 100 g/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 g/ml to 0,30 g/ml
41、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) =
42、0,30 g/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.
43、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 s
44、eparating column, e.g. LiChrospher100 RP-18 endcapped 3The 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 m. Other particle sizes or column dimensions than specifie
45、d in this European Standard may be used. Separation parameters shall be adapted to such other materials to guarantee equivalent results. 3 LiChrospher100 RP-18 endcapped is an example of a suitable product available commercially. This information is given for the convenience of users of this Europea
46、n 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. DIN EN 15607:2009-09 EN 15607:2009 (E) 8 5.5 Filter device Large and small scale filter devices to filter HPLC mobile phases and sample sol
47、utions respectively, e.g. of 0,45 m pore size is appropriate. NOTE 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 suitable re
48、agent delivery system, a T-type connecting tube followed by a knitted open-tubular (KOT) reactor with a length of 10 m made of polytetrafluoroethylene (PTFE) tubing with an inner diameter of 0,5 mm and a helix diameter of 14 mm prepared according to for example 7, (KOT2 model). Knitted open-tubular
49、reactors can commercially be obtained from for example Supelco 4)or MicroSolv Tech 4. 6 Procedure 6.1 Sample preparation Homogenize the test sample. Grind coarse material with an appropriate mill and mix again. Measures such as pre-cooling shall be taken to avoid exposing to high temperature for long periods of time. 6.2 Extraction Weigh an appropriate amount of the test sample to the nearest mg, e.g. 0,5 g to 10 g (equivalent from 2 g to 15 g
