1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12857:1999 The Euro
2、pean Standard EN 12857:1999 has the status of a British Standard ICS 67.040 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Foodstuffs Determination of cyclamate High performance liquid chromatographic methodThis British Standard, having been prepared under the direction of th
3、e Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 September 1999 BSI 09-1999 ISBN 0 580 32595 4 BS EN 12857:1999 Amendments issued since publication Amd. No. Date Comments National foreword This British Standar
4、d is the English language version of EN 12857:1999. The UK participation in its preparation was entrusted to Technical Panel AW/-/3, Food analysis Horizontal methods, which has the responsibility to: aid enquirers to understand the text; present to the responsible European committee any enquiries on
5、 the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Sta
6、ndards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Stand
7、ard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover,
8、an inside front cover, the EN title page, pages 2 to 13 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued.EUROPEAN STANDARD EN 12857 NORME EUROPENNE EUROPISCHE NORM April 1999 ICS 67.050 English version Foodstuffs Determination of cycla
9、mate High performance liquid chromatographic method Produits alimentaires Dosage du cyclamate Mthode par chromatographie liquide haute performance Lebensmittel Bestimmung von Cyclamat Hochleistungs flssigchromatographisches Verfahren This European Standard was approved by CEN on 16 April 1999. CEN m
10、embers 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 without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on applic
11、ation 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 language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has th
12、e same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Commit
13、tee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stassart, 36 B-1050 Brussels 1999 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members Ref. No. EN 12857:1999 EPage 2 EN 12857:1999 BSI
14、 09-1999 Contents Page Foreword 2 1 Scope 3 2 Normative references 3 3 Principle 3 4 Reagents 3 5 Apparatus and equipment 4 6 Procedure 5 7 Calculation 7 8 Precision 8 9 Test report 9 Annex A (informative) Determination of the chlorine content in hypochlorite solutions 10 Annex B (informative) Figur
15、es 11 Annex C (informative) Precision data 13 Annex D (informative) Bibliography 13 Foreword This European Standard 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 na
16、tional standard, either by publication of an identical text or by endorsement, at the latest by October 1999, and conflicting national standards shall be withdrawn at the latest by October 1999. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
17、countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom.Page 3 EN 12857:1999 BSI 09-1999 1 Scope This Eu
18、ropean Standard specifies a high performance liquid chromatographic (HPLC) method for the determination of sodium cyclamate in foodstuffs 1, 2, 3. The method has been validated in an inter-laboratory test according to ISO 5725:1986 4 which has been carried out with lemonade, orange juice beverage, f
19、ruit yogurt and spray cream. In addition to the matrices investigated in the inter-laboratory test, experiences have shown that the method is also applicable to various foodstuffs such as gherkins, canned morello cherries, pineapple, orange nectar, apricot jam, blackberry jam, cherry nectar, hard ca
20、ndy, mixed fruit yogurt, strawberry yogurt, fruit quark, rice-pudding-apple-raisins, chocolate custard powder, cream dessert vanilla powder, vanilla ice and passion fruit-orange ice cream 2, 3. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from
21、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 publications apply to this draft European Standard only when incorporated in it by ame
22、ndment or revision. For undated references the latest edition of the publication referred to applies. EN ISO 3696, Water for analytical laboratory use Specification and test methods. (ISO 3696:1987) 3 Principle Sodium cyclamate is extracted from the sample with water, converted to N,N-dichlorocycloh
23、exylamine and determined by HPLC on a reversed-phase column using UV detection at a wavelength of 314 nm. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at least grade 1 as defined in EN ISO 3696. When preparing solutions, the p
24、urity of the substances shall be taken into account. 4.1 Methanol, for HPLC. 4.2 n-Heptane, for HPLC. 4.3 Light petroleum, having a boiling range from 40 C to 60 C. 4.4 Sodium sulfate, anhydrous. If necessary, wash with n-heptane to remove lipophilic contaminants. 4.5 Sodium carbonate solution, r (N
25、a 2 CO 3 ) = 50 g/l 1) . 4.6 Sodium hypochlorite solution (1,7 % active chlorine) Dilute commercially available sodium hypochlorite solution containing more than 1,7 % active chlorine with water to get a mass fraction of 1,7 % of active chlorine. Check the content of active chlorine of the sodium hy
26、pochlorite solution regularly, e.g. using the procedure as described in annex A. 4.7 Sulfuric acid, w(H 2 SO 4 ) = 50 % 2)1)r is the mass concentration. 2)w is the mass fraction.Page 4 EN 12857:1999 BSI 09-1999 4.8 Sodium cyclamate standard solutions Weigh, to the nearest 0,1 mg, about 898 mg of sod
27、ium cyclamate in a 200 ml volumetric flask and dilute to the mark with water, r (cyclohexylsulfamic acid) = 800 mg/200 ml 3) . Pipette 0,25 ml, 1 ml, 2,5 ml, 5 ml, 10 ml and 20 ml of this solution in 100 ml volumetric flasks and dilute to the mark with water. The cyclohexylsulfamic acid concentratio
28、n of these solutions is 10 mg/l, 40 mg/l, 100 mg/l, 200 mg/l, 400 mg/l and 800 mg/l. 4.9 Carrez solution No. 1 Dissolve 15 g of potassium hexacyanoferrate (II) (K 4 Fe(CN) 6 3H 2 O) with a mass fraction of at least 99 % in water and dilute to 100 ml. 4.10 Carrez solution No. 2 Dissolve 30 g zinc sul
29、fate (ZnSO 4 7H 2 O) with a mass fraction of at least 99,5 % in water and dilute to 100 ml. 4.11 Mobile phase Mix 80 parts per volume methanol (4.1) with 20 parts per volume water, filter through suitable membrane filters, e.g. of pore size 0,45 mm and degas, e.g. for 5 min in an ultrasonic bath. If
30、 neccessary, the proportion of methanol to water in the mobile phase may have to be modified slightly, to achieve adequate results with the column used. 4.12 Conditioning solution for HPLC, EDTA solution, r (EDTA) = 10 g/l, filter through suitable membrane filters, e.g. of pore size 0,45 mm and dega
31、s, e.g. for 5 min in an ultrasonic bath 4.13 Cellulose, powdered, with a mass fraction of at least 99,9 %, acid washed. 5 Apparatus and equipment Usual laboratory apparatus and, in particular, the following. 5.1 HPLC apparatus, comprising the following. 5.1.1 High performance liquid chromatograph, c
32、onsisting of a pump, a sample injector, an ultraviolet (UV) spectrometer (capable of operating at a wavelength of 314 nm, preferably a diode array detector), with a recorder and/or integrator which allows the measurement of peak heights and peak areas. 5.1.2 Analytical reversed phase separating colu
33、mn, e.g. with: a RP C 18 stationary phase of 5 mm; a length of 250 mm; internal diameter of 4 mm; a guard column, RP C 18 (optional, but recommended especially for all solid sample materials). Performance criterion for suitable analytical columns is the baseline resolution of the cyclamate peak. Whe
34、never interferences are identified with a diode array detector or by measurement at a second wavelength, an alternative chromatographic condition shall be chosen for the determination of cyclamate. 5.2 Ultrasonic bath. 5.3 Membrane filter, of suitable pore size, e.g. 0,45 mm.3)The conversion factor
35、from sodium cyclamate to cyclohexylsulfamic acid is 0,890 9.Page 5 EN 12857:1999 BSI 09-1999 5.4 Filtration unit, with filter holder for membrane filter for filtering and degassing mobile phase (4.11) and conditioning solution (4.12). 5.5 Homogenizer. 5.6 Water bath, capable of being maintained at 6
36、0 C. 5.7 Centrifuge, capable of producing a centrifugal acceleration of at least 1 400g at the base of the centrifuge tubes (5.8). 5.8 Centrifuge tubes, preferably made of glass, of suitable capacity, e.g 50 ml, sealable. 5.9 Separating funnels, of suitable capacity, e.g. 50 ml and 100 ml. 5.10 Flut
37、ed filter paper, medium fast, qualitative. 5.11 Phase separation filters, (optional). 6 Procedure 6.1 Preparation of the sample solutions 6.1.1 Liquid products and products giving clear solutions (e.g. clear fruit juices, filtered gherkin brine, hard candy) Dilute liquid products, filtered if necess
38、ary, with water to give a cyclamate content of approximately 400 mg/l or take liquid products directly for derivatization. Dissolve solid products in water to give a clear solution having a cyclamate content of approximately 400 mg/l. 6.1.2 Semi solid samples, (e.g. dairy products, desserts, spray c
39、ream, cloudy juices, jams, marmalades) Homogenize the sample thoroughly for 1 min. Weigh, to the nearest 1 mg, about 15 g of the sample into a 100 ml volumetric flask, add 80 ml of water and place the flask for approximately 10 min in an ultrasonic bath (5.2). Any other mass of the sample may be cho
40、sen yielding a cyclamate content of not more than 400 mg/l in the 100 ml volumetric flask. Add 1 ml to 2 ml of Carrez solution No. 1 (4.9), mix, and add the same volume of Carrez solution No. 2 (4.10). Mix, dilute to the mark with water. Filter through a fluted filter paper, discarding the first 10
41、ml of the filtrate. To make allowance for the volume of any precipitate, if the fat-free insoluble matter in the initial sample mass exceeds approximately 3 g, it is advisable to centrifuge the clarified sample mixture for 10 min at a centrifugal acceleration of at least 1 400g before filtering it q
42、uantitatively into the 100 ml volumetric flask. Wash the settled matter twice with water and centrifuge again, collect each of the supernatants in the 100 ml volumetric flask and then dilute the solution to the mark with water. This procedure may also be followed when the amount of insoluble matter
43、is less than 3 g. Take 20 ml of this solution for derivatization (see 6.2). 6.1.3 Chocolate and related products Weigh, to the nearest 1 mg, about 15 g of the sample into a centrifuge tube (5.8) and stand the centrifuge tube in a water bath at 60 C until the sample melts completely. Carefully and sl
44、owly add 25 ml of light petroleum (4.3), mix thoroughly, seal the centrifuge tube and place it in an ultrasonic bath for 30 s and mix again. Centrifuge the sealed centrifuge tubes for 10 min at at least 1 400g. Decant the light petroleum layer and repeat the extraction with 25 ml of light petroleum,
45、 again decanting the light petroleum layer. Evaporate the remaining light petroleum by placing the centrifuge tube for 15 min in a water bath at 60 C and mixing. Add 30 ml of water and mix thoroughly. Stand the centrifuge tube for 5 min in the ultrasonic bath. Transfer the solution with approximatel
46、y 40 ml of water quantitatively into a 100 ml volumetric flask, add 1 ml of Carrez solution No. 1 (4.9), mix, add 1 ml of Carrez solution No. 2 (4.10) and mix thoroughly. Bring the solution to a temperature of 20 C, dilute to the mark with water. Filter through a fluted filter paper, discarding the
47、first 10 ml of the filtrate.Page 6 EN 12857:1999 BSI 09-1999 To make allowance for the volume of any precipitate, if the fat-free insoluble matter in the initial sample mass exceeds approximately 3 g, it is advisable to centrifuge the clarified sample mixture for 10 min at a centrifugal acceleration
48、 of at least 1 400g before filtering it quantitatively into the 100 ml volumetric flask. Wash the settled matter twice with water and centrifuge again, collect each of the supernatants in the 100 ml volumetric flask and then dilute the solution to the mark with water. This procedure may also be foll
49、owed when the amount of insoluble matter is less than 3 g. Take 20 ml of this solution for derivatization (see 6.2). 6.1.4 Fat emulsions and products containing those (e.g. mayonnaise) Homogenize the sample thoroughly. Weigh, to the nearest 1 mg, about 15 g of homogenized mayonnaise into the centrifuge tube (5.8). Add 2,5 g of powdered cellulose (4.13) and mix. Add 25 ml of light petroleum (4.3) and mix. Seal the centrifuge tube and place it in an ultrasonic bath for 30 s and mix again. Centrifuge for 10 mi
