1、 Reference number ISO 22855:2008(E) ISO 2008INTERNATIONAL STANDARD ISO 22855 First edition 2008-01-15 Fruit and vegetable products Determination of benzoic acid and sorbic acid concentrations High-performance liquid chromatography method Fruits, lgumes et produits drivs Dtermination des teneurs en a
2、cides benzoque et sorbique Mthode par chromatographie liquide haute performance ISO 22855:2008(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedde
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7、in Switzerland ii ISO 2008 All rights reservedISO 22855:2008(E) ISO 2008 All rights reserved iii Contents Page Foreword iv 1 Scope. 1 2 Principle. 1 3 Reagents and materials . 1 4 Apparatus 2 5 Sample. 2 6 Procedure 2 7 Calculation. 4 8 Precision 4 9 Test report. 6 Annex A (informative) Chromatogram
8、 . 7 Annex B (informative) Statistical results of the interlaboratory test 8 Bibliography . 10 ISO 22855:2008(E) iv ISO 2008 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of pr
9、eparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, i
10、n liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main tas
11、k of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attentio
12、n is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 22855 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 3, Fruit and vegetabl
13、e products. INTERNATIONAL STANDARD ISO 22855:2008(E) ISO 2008 All rights reserved 1 Fruit and vegetable products Determination of benzoic acid and sorbic acid concentrations High-performance liquid chromatography method 1 Scope This International Standard specifies a method using high-performance li
14、quid chromatography for the determination of the concentration of benzoic and sorbic acids in fruit and vegetable juices. NOTE This method is based on IFU method 63 2 . 2 Principle Extraction of benzoic acid and/or sorbic acid from a test portion using a mixture of ammonium acetate buffer solution a
15、nd methanol, under acidic conditions. The concentration of benzoic and/or sorbic acid is determined by means of high-performance liquid chromatography (HPLC) using a reverse phase column and ultraviolet (UV) detector. 3 Reagents and materials Use only reagents of recognized analytical grade, unless
16、otherwise specified, and water of HPLC grade. 3.1 Acetic acid (CH 3 COOH), glacial. 3.2 Methanol (CH 3 OH), for HPLC. 3.3 Ammonium acetate (CH 3 COONH 4 ), 0,01 mol/l solution. Dissolve 0,771 g of ammonium acetate in 1 l of water. 3.4 Ammonium acetate/acetic acid (CH 3 COONH 4 /CH 3 COOH), buffer so
17、lution. Mix 1 000 volume parts of ammonium acetate solution (3.3) with 1,2 volume parts of acetic acid (3.1). 3.5 Benzoic acid (C 6 H 5 COOH), stock solution. Dissolve benzoic acid to obtain stock solution (C 6 H 5 COOH) = 100 mg/ml in 40 ml of methanol (3.2) and dilute to the mark with water in a 1
18、00 ml volumetric flask. 3.6 Sorbic acid CH 3 (CH:CH) 2 COOH, stock solution. Dissolve sorbic acid to obtain stock solution, CH 3 (CH:CH) 2 COOH = 100 mg/ml, in 40 ml of methanol (3.2) and dilute to the mark with water in a 100 ml volumetric flask. 3.7 Potassium hexacyanoferrate(II), trihydrate, K 4
19、Fe(CN) 6 3H 2 O. ISO 22855:2008(E) 2 ISO 2008 All rights reserved3.8 Zinc sulfate, heptahydrate, (ZnSO 4 7H 2 O), 300 g/l solution. 3.9 Extraction solution Mix 60 volume parts of ammonium acetate/acetic acid buffer solution (3.4) with 40 volume parts of methanol (3.2). 3.10 Eluent for HPLC Mix 50 vo
20、lume parts of ammonium acetate solution (3.4) with 40 volume parts of methanol for HPLC (3.2) and adjust to a pH of 4,5 to 4,6 with acetic acid (3.1). Filter the eluent over a membrane filter (4.2). 3.11 Carrez solution I Dissolve 150 g of potassium hexacyanoferrate(II) (3.7) in water in a 1 000 ml
21、volumetric flask. Dilute to the mark with water and mix the solution. 3.12 Carrez solution II Dissolve 300 g of zinc sulfate (3.8) in water in a 100 ml volumetric flask. Dilute to the mark with water and mix the solution. 3.13 Pleated filter paper, hard. 4 Apparatus Usual laboratory apparatus and, i
22、n particular, the following. 4.1 Ultrasonic bath. 4.2 Membrane filters, of pore size 0,45 m, for aqueous solutions (e.g. cellulose acetate); diameter dependent on the filter holder. 4.3 Filter holder, for membrane filters with suitable aspirating and collection vessels. 4.4 High-performance liquid c
23、hromatograph, equipped with a UV-detector (variable wavelength) and recorder and/or integrator or computer with the appropriate integrating programme. 4.5 Reverse phase separation column, e.g. reverse phase C8, 250 mm 4,6 mm, particle size 5 m. 5 Sample A representative sample should have been sent
24、to the laboratory. It should not have been damaged or changed during transport or storage. 6 Procedure 6.1 Preparation of test solution Homogenize or mix the sample carefully. Concentrated juice should be diluted to single strength. ISO 22855:2008(E) ISO 2008 All rights reserved 3 6.1.1 Clear sample
25、s Dilute 5,00 ml to 10,00 ml (V 1 ) of a sample in approximately 75 ml of extraction solution (3.9) in a 100 ml volumetric flask. Put the flask in the ultrasonic bath (4.1), mix the contents for at least 10 min and then dilute to the mark with extraction solution (3.9) at 20 C. Filter the solution t
26、hrough a membrane filter (4.2). 6.1.2 Cloudy samples Dilute 5,00 ml to 10,00 ml (V 1 ) of a sample in approximately 75 ml of extraction solution (3.9) in a 100 ml volumetric flask. Put the flask in the ultrasonic bath (4.1) and mix the contents for at least 10 min. Then add 1,0 ml of Carrez solution
27、 I (3.11) and 1,0 ml of Carrez solution II (3.12) for clarification. Mix the solution carefully after each addition and dilute to the mark with the extraction solution (3.9) at 20 C. Filter the solution over a paper filter (3.13); discard the first millilitres of filtrate. Filter the clear solution
28、through a membrane filter (4.2). 6.2 Preparation of the calibration curves Dilute benzoic acid stock solution (3.5) and/or sorbic acid stock solution (3.6) with extraction solution (3.9) at 20C to obtain standard solutions I, II and III with benzoic acid and/or sorbic acid concentrations of 10 mg/l,
29、 25 mg/l and 50 mg/l, respectively. Inject 10 l of each of the calibration solutions into the chromatograph (4.4), under the following conditions: flow rate: approximately 1,2 ml/min; wavelength for UV-detection: 235 nm (0,08 AUFS - absorbance unit full scale). Prepare the calibration curves by plot
30、ting the peak areas against benzoic acid and/or sorbic acid concentration, in milligrams per litre. 6.3 Determination Inject 10 l of the test solution (6.1) into the chromatograph using the same conditions as for the preparation of the calibration graph. Identify the benzoic acid and/or sorbic acid
31、peaks of the test solution by comparison with the peaks of the calibration solutions. NOTE 1 For optimal separation of benzoic and/or sorbic acid, a slight change in the composition of the eluent may be necessary. NOTE 2 Under the conditions described in this procedure, it is possible to determine t
32、he methyl, ethyl, and propyl esters of 4-hydroxybenzoic acid as well (see chromatogram in Annex A). NOTE 3 Matrix peaks can cause interference with the analysis of benzoic acid in orange juice. In such a case, a suitable clean-up step is necessary. NOTE 4 Identification of benzoic and sorbic acids i
33、n a sample is performed by comparing with the retention time of the standard solutions. It is possible to identify the analysed acids by using other methods of identification: spiking with single substances, viewing of absorption spectra at required wavelengths and measuring the absorption at differ
34、ent wavelengths. NOTE 5 Quantification is carried by the external standard method with integration of peak area or measurement of peak heights. It is necessary to check linearity of the calibration function, e.g. with standard solutions I, II and III. ISO 22855:2008(E) 4 ISO 2008 All rights reserved
35、7 Calculation Determine the concentration of benzoic and/or sorbic acid in the test solution directly from the calibration curve (6.2). Calculate the benzoic acid concentration of the sample, Ain milligrams per litre, using the following equation (external standard method): 1s t A 21 100 A AV = wher
36、e A 1is the peak area or peak height of benzoic acid or sorbic acid in the test sample, expressed in area or length counts, respectively; A 2is the peak area or peak height of benzoic acid or sorbic acid in the test standard solution, expressed in area or length counts, respectively; stis the concen
37、tration of the standard solution, in milligrams per litre; V 1is the volume of the test sample solution, in millilitres. The result(s) is(are) expressed, in milligrams per litre, to one decimal place. 8 Precision The precision of the method was established by an interlaboratory test organized by the
38、 analytical commission of the International Fruit Union (IFU). In this test, samples of orange and grape juices were investigated. The statistical treatment was performed by the Max von Pettenkofer Institut of the German Federal Office of Public Health. See Annex B for a summary of the statistical r
39、esults of this test. Statistical parameters are expressed in accordance with ISO 5725-2 1 . 8.1 Repeatability Benzoic acid Orange juice: r = 3,5 s r= 1,25 Grape juice: r = 3,5 s r= 1,25 Sorbic acid Orange juice: r = 2,8 s r= 1,00 Grape juice: r = 2,3 s r= 0,88 4-Hydroxybenzoic acid methyl ester Oran
40、ge juice: r = 4,5 s r= 1,60 Grape juice: r = 3,8 s r= 1,37 4-Hydroxybenzoic acid ethyl ester Orange juice: r = 4,5 s r= 1,59 Grape juice: r = 4,8 s r= 1,70 ISO 22855:2008(E) ISO 2008 All rights reserved 5 4-Hydroxybenzoic acid propyl ester Orange juice: r = 5,3 s r= 1,88 Grape juice: r = 5,4 s r= 1,
41、93 where r is the repeatability limit; s ris the standard deviation of repeatability. 8.2 Reproducibility Benzoic acid Orange juice: R = 13,9 s R= 4,96 Grape juice: R = 8,9 s R= 3,18 Sorbic acid Orange juice: R = 11,0 s R= 3,93 Grape juice: R = 7,3 s R= 2,61 4-Hydroxybenzoic acid methyl ester Orange
42、 juice: R = 19,0 s R= 6,79 Grape juice: R = 11,7 s R= 4,17 4-Hydroxybenzoic acid ethyl ester Orange juice: R = 9,8 s R= 3,5 Grape juice: R = 8,8 s R= 3,14 4-Hydroxybenzoic acid propyl ester Orange juice: R = 16,8 s R= 6,00 Grape juice: R = 17,7 s R= 6,32 where R is the reproducibility limit; s Ris t
43、he standard deviation of reproducibility. ISO 22855:2008(E) 6 ISO 2008 All rights reserved9 Test report The test report shall specify: a) all information necessary for the complete identification of the sample; b) the sampling method used, if known; c) the test method used, with reference to this In
44、ternational Standard; d) all operating details not specified in this International Standard, or regarded as optional, together with details of any incidents which may have influenced the test result(s); e) the test result(s) obtained, and, if the repeatability has been checked, the final quoted resu
45、lt obtained. ISO 22855:2008(E) ISO 2008 All rights reserved 7 Annex A (informative) Chromatogram Column Diameter 4,6 mm, length 250 mm Ultrasphere-octyl (RP 8), particle size 5 m Eluent Ammonium acetate solution/Methanol for HPLC (50 + 40 volume parts)+ Acetic acid (pH = 4.5) Flow rate 1,2 ml/min UV
46、-detection 235 nm (0,08 AUFS) Recorder speed 3 mm/min Injection volume 10 l Standard substances: 1. Benzoic acid (RT:8,13) a2. Sorbic acid (RT:9,33) 3. 4-Hydroxybenzoic acid methyl ester (RT:10,86) 4. 4-Hydroxybenzoic acid ethyl ester (RT:20,34) 5. 4-Hydroxybenzoic acid propyl ester (RT:42,36) aRT =
47、 Retention time, minutes. bInject. (standard) = 25 mg/l in each case Figure A.1 HPLC-separation of standard solution II (6.2) ISO 22855:2008(E) 8 ISO 2008 All rights reservedAnnex B (informative) Statistical results of the interlaboratory test Table B.1 Benzoic acid Sample Parameters Orange juice Gr
48、ape juice Mean benzoic acid concentration (mg/l) 72,8 57,0 Repeatability standard deviation s r(mg/l) 1,25 1,25 Repeatability coefficient of variation CV r(%) 1,71 2,19 Repeatability limit r = 2,8 s r(mg/l) 3,5 3,5 Reproducibility standard deviation s R(mg/l) 4,96 3,18 Reproducibility coefficient of
49、 variation CV R(%) 6,81 5,57 Reproducibility limit R = 2,8 s R(mg/l) 13,9 8,9 Table B.2 Sorbic acid Sample Parameters Orange juice Grape juice Mean sorbic acid concentration (mg/l) 91,1 65,7 Repeatability standard deviation s r(mg/l) 1,0 0,88 Repeatability coefficient of variation CV r(%) 1,10 1,34 Repeatability limit r = 2,8 s r(mg/l) 2,8 2,3 Reproducibility standard deviation s R(mg/l) 6,79 2,61 Reproducibility coefficient of variation CV R(%) 7,45 3,97 Reproduci
