1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12148 : 1997 The Eu
2、ropean Standard EN 12148 : 1996 has the status of a British Standard ICS 67.160.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Fruit and vegetable juices Determination of hesperidin and naringin in citrus juices Method using high performance liquid chromatographyBS EN 1214
3、8 : 1997 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 May 1997 BSI 1997 The following BSI references relate to the work on this standard: Commit
4、tee reference AW/21 Draft for comment 91/53941 DC ISBN 0 580 27057 2 Amendments issued since publication Amd. No. Date Text affected Committees responsible for this British Standard The preparation of this British Standard was entrusted to Technical Committee AW/21, Fruit and vegetable juices, upon
5、which the following bodies were represented: British Fruit Juice Importers Association British Retail Consortium British Soft Drinks Association Limited Campden and Chorleywood Food Research Association Laboratory of the Government Chemist Leatherhead Food Research Association Ministry of Agricultur
6、e, Fisheries and Food Royal Society of ChemistryBS EN 12148 : 1997 BSI 1997 i Contents Page Committees responsible Inside front cover National foreword ii Foreword 2 Text of EN 12148 3ii BSI 1997 BS EN 12148 : 1997 National foreword This British Standard has been prepared by Technical Committee AW/2
7、1, and is the English language version of EN 12148 : 1996 Fruit and vegetable juices Determination of hesperidin and naringin in citrus juices Method using high performance liquid chromatography, published by the European Committee for Standardization (CEN). EN 12148 was produced as a result of inte
8、rnational discussions in which the United Kingdom took an active part. Cross-references Publication referred to Corresponding British Standard ISO 3696 : 1987 BS EN ISO 3696 : 1995 Specification for water for laboratory use ISO 5725 : 1986, to which informative reference is made in the text, has bee
9、n superseded by ISO 5725-1 : 1994, ISO 5725-2 : 1994, ISO 5725-3 : 1994, ISO 5725-4 : 1994 and ISO 5725-6 : 1994 which are identical with BS ISO 5725 Accuracy (trueness and precision) of measurement methods and results, BS ISO 5725-1 : 1994 General principles and definitions, BS ISO 5725-2 : 1994 Ba
10、sic method for the determination of repeatability and reproducibility of a standard measurement method, BS ISO 5725-3 : 1994 Intermediate measures of the precision of a standard measurement method, BS ISO 5725-4 : 1994 Basic method for the determination of the trueness of a standard measurement meth
11、od, and BS ISO 5725-6 : 1994 Use in practice of accuracy values. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the EN title page, pages 2 to 10, an inside ba
12、ck cover and a back cover.CEN European Committee for Standardization Comite Europe en de Normalisation Europa isches Komitee fu r Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1996 Copyright reserved to CEN members Ref. No. EN 12148 : 1996 E EUROPEAN STANDARD EN 12148 NORME EUROPE
13、 ENNE EUROPA ISCHE NORM September 1996 ICS 67.160.20 Descriptors: Food products, beverages, fruit and vegetable juices, chemical analysis, determination, high performance, liquid chromatography English version Fruit and vegetable juices Determination of hesperidin and naringin in citrus juices Metho
14、d using high performance liquid chromatography Jus de fruits et de le gumes De termination de la teneur en hespe ridine et en naringine dans les jus dagrumes Me thode utilisant la chromatographie liquide a haute performance (HPLC) Frucht- und Gemu sesa fte Bestimmung von Hesperidin und Naringin in C
15、itrussa ften Hochleistungs-flu ssigkeitschromatographisches Verfahren This European Standard was approved by CEN on 1996-05-09. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard
16、 without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other langu
17、age made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland,
18、 Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12148 : 1996 BSI 1997 Foreword This European Standard has been prepared by Technical Committee CEN/TC 174, Fruit and vegetable juices Method of analysis, of which the Secretariat is held by AFN
19、OR. 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 March 1997, and conflicting national standards shall be withdrawn at the latest by March 1997. According to the CEN/CENELEC Internal Regulations
20、, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. Contents Pa
21、ge Foreword 2 1 Scope 3 2 Normative references 3 3 Symbols and abbreviations 3 4 Principle 3 5 Reagents 3 6 Apparatus 3 7 Procedure 4 8 Calculation 4 9 Precision 5 10 Test report 5 Annexes A (informative) Bibliography 6 B (informative) Statistical results of the interlaboratory test 6 C (informative
22、) Examples of chromatograms 7Page 3 EN 12148 : 1996 BSI 1997 1 Scope This European Standard specifies a method for the determination of the hesperidin and naringin contents, using high performance liquid chromatography (HPLC), in fruit and vegetable juices and related products. 2 Normative reference
23、s This European Standard incorporates by dated or undated references, provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these
24、publications apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. EN ISO 3696 : 1995 Water for analytical laboratory use Specification and test methods ISO 5725 : 1986 Precision of tes
25、t methods Determination of repeatability and reproducibility for a standard test method by inter-laboratory tests 3 Symbols and abbreviations 3.1 Symbols For the purposes of this standard the following symbols apply: c Substance concentration; r Content of hesperidin or naringin respectively in samp
26、les (mass concentration in milligrams per litre of juice). 3.2 Abbreviations For the purposes of this standard the following abbreviations apply: hesperidin hesperidin-7-rutinoside naringin naringenin-7-neohesperidoside HPLC high performance liquid chromatography UV ultraviolet AUFS absorption units
27、 full scale 4 Principle Hesperidin and naringin are extracted into a dimethylformamide solution. After heating at 90 C a membrane filtered aliquot portion of the sample is separated by reversed phase HPLC using UV-detection and calculation is by the external standard method. 5 Reagents 5.1 General U
28、se only reagents of recognized analytical grade and only water in accordance with at least grade 1 of EN ISO 3696 : 1995. 5.2 Acetic acid. 5.3 Acetic acid, c (CH 3 COOH) = 0,01 mol/l. 5.4 Acetonitrile, HPLC-grade. 5.5 Di-ammonium oxalate monohydrate, (NH 4 ) 2 C 2 O 4 H 2 O. 5.6 Dimethylformamide. 5
29、.7 Hesperidin (3.2). 5.8 Naringin (3.2). 5.9 Standard solution of naringin and hesperidin. For preparing the stock solution, 120 mg of naringin (5.8) and hesperidin (5.7) are dissolved in 20 ml of dimethylformamide (5.6) in a 100 ml volumetric flask. After the solids have dissolved, the solution is
30、made up to volume with acetic acid solution (5.3). To prepare the hesperidin/naringin working standard solution (120 mg/l of each compound), the stock solution (1 200 mg/l) is diluted 1 in 10 with a mixture of acetic acid solution (5.3) and dimethylformamide (5.6) (8 to 2 volumes respectively). The
31、working standard solution is stable for 30 days when stored at 4 C. From the stock solution (1 200 mg/l) prepare further standards by dilution of the stock solution with a mixture of acetic acid solution (5.3) and dimethylformamide (5.6) (8 to 2 volumes) to give solutions containing 60 mg/l, 30 mg/l
32、 and 15 mg/l of both hesperidin and naringin. 5.10 Ammonium oxalate solution. c (NH 4 ) 2 C 2 O 4 H 2 O) = 0,025 mol/l. The ammonium oxalate solution is prepared by dissolving 3,55 g of di-ammonium-oxalate monohydrate (5.5) in 1000 ml water. 5.11 Mobile phase for the HPLC-analysis. The mobile phase
33、for the determination of hesperidin and naringin is prepared by mixing 200 ml of acetonitrile (5.4), 800 ml of water and 0,5 ml of acetic acid (5.2). The acetonitrile and water shall be measured separately and the composition of the mobile phase shall be prepared exactly. 6 Apparatus Usual laborator
34、y apparatus and, in particular, the following: 6.1 Volumetric flask,5 0m l . 6.2 Volumetric pipette,1 0m l .Page 4 EN 12148 : 1996 BSI 1997 6.3 Disposable filter, non-sterile, hydrophilic (of pore size 0,45mm) for the filtration of sample solutions. 6.4 HPLC-column: HPLC-column, C18, 250 mm3 4 mm, 5
35、mm; HPLC-column, C18, 250 mm34,6 mm, 5mm. 6.5 HPLC-equipment: pump, column (6.4) and UV detector. 6.6 Water bath, capable of reaching and maintaining 90 C. 7 Procedure 7.1 Preparation of the test sample Normally products shall not be pre-treated and their analysis by this method shall be on a volume
36、tric basis, results being expressed per litre of the sample. The analysis of concentrated products may also be carried out on a volumetric basis, after dilution to a known relative density. In this case, the relative density shall be indicated. Based on a weighed sample and taking the dilution facto
37、r for analysis into account, the results may also be expressed per kilogram of product. In products with high viscosity and/or very high content of cells (for example pulp), determination on the basis of a weighed test sample is the usual procedure. Mix cloudy samples well before dilution. 7.2 Test
38、procedure 7.2.1 Sample preparation Keep the sample at ambient temperature. Mix samples well before opening and take the test sample (7.1) immediately. Pipette 10 ml of the test sample or diluted concentrated products (7.1) into the 50 ml-volumetric flask (6.1). Then add 10 ml of ammonium oxalate sol
39、ution (0,025 mol/l) (5.10) and 10 ml of dimethylformamide (5.6). Mix the diluted samples thoroughly and dilute to volume (50 ml) with water. Heat the samples in a temperature controlled water bath (6.6) at 90 C for 10 min. After cooling to room temperature, filter an aliquot portion of the solution
40、through a membrane filter (6.3) prior to analysis by HPLC. 7.2.2 HPLC measurement After equilibration of the HPLC-system, analyse the samples (HPLC-equipment (6.5), column (6.4), mobile phase (5.11). When using a fixed wavelength detector, measurement is at a wavelength of 280 nm or, in the case of
41、variable wavelength detector, 287 nm is used. Detection is at sensitivities between 0,05 to 0,2 AUFS. Flow rate : Approximately 1 ml/min Injection volume : 20ml The standard and sample solutions are injected in successive runs. As the stability of the sample solutions is limited, the test shall be c
42、ompleted within 24 h. 8 Calculation The calculation is made according to the external standard method by integration of the peak areas or by measuring the peak heights taking into account the retention times and the linearity of the calibration curve. During calculation, take into account any diluti
43、on factor and the relationship of the value to mass or volume. If a concentrated product has been diluted to single strength, report the relative density of the single strength sample. Calculate the hesperidin and naringin content,r H and r N of each sample using the following formula: r H = 3 XF (1
44、) A HP RF H r N = 3 XF (2) A NP R FN where: A HP is the peak area for the hesperidin content of the sample; A NP is the peak area for the naringin content of the sample; XF is the dilution factor (5 for juices, for diluted concentrated samples this dilution factor has to be calculated); RF H is the
45、response factor for hesperidin; RF N is the response factor for naringin. Calculate the response factor using the following formula: RF H = A NS r HS and RF N = A NS r NS Where: A HS is the peak area for the hesperidin content of the standard solution (5.9); A NS is the peak area for the naringin co
46、ntent of the standard solution (5.9); r HS is the hesperidin content of the standard solution (5.9); r NS is the naringin content of the standard solution (5.9). Express the naringin and hesperidin results in milligrams per litre.Page 5 EN 12148 : 1996 BSI 1997 9 Precision Details of the interlabora
47、tory test on the precision of the method are summarized in annex B. The values derived from the interlaboratory test may not be applicable to analyte concentration ranges and matrices other than given in annex B. 9.1 Repeatability The absolute difference between two single test results found on iden
48、tical test material by one operator using the same apparatus within the shortest feasible time interval will exceed the repeatability limit r in not more than 5 % of the cases. The values are : hesperidin r = 19 mg/l; naringin r = 27 mg/l. 9.2 Reproducibility The absolute difference between two sing
49、le test results on identical test material reported by two laboratories will exceed the reproducibility limit R in not more than 5 % of the cases. The values are : hesperidin R = 61 mg/l; naringin R = 72 mg/l. 10 Test report The test report shall contain the following data: all information necessary for the determination of the sample (kind of sample, origin of sample, designation); a reference to this European Standard; the date and type of sampling procedure (if known); the date of receipt; the date of
