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1、INTERNATIONAL STANDARD IS0 8128-1 First edition 1993-07-01 Apple juice, apple juice concentrates and drinks containing apple juice - Determination of patulin content - Part 1: Method using high-performance liquid chromatography Jus de pommes, concenti base de jus de pommes - Dgtermina tion de la ten
2、eur en patuline - Partie 7: Mi haute performance Reference number - - IS0 8128-1:1993(E) Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 03:02:55 MDTNo reproduction or networking
3、 permitted without license from IHS -,-,- IS0 8128=1:1993(E) Foreword IS0 (the International Organization for Standardization) is a worldwide federation of national standards bodies (IS0 member bodies). The work of preparing International Standards is normally carried out through IS0 technical commi
4、ttees. 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, in liaison with ISO, also take part in the work. IS0 collaborates closely with the I
5、nternational Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. 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 memb
6、er bodies casting a vote. International Standard IS0 8128-1 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Sub-Committee SC 3, fruit and vegetable products. IS0 8128 consists of the following parts, under the general title Apple juice, apple juice concentrates and drinks
7、containing apple juice - Deter- mination of patulin content: - Part 1: Method using high-performance liquid chromatography - Part 2: Method using thin-layer chromatography Annex A of this part of IS0 8128 is for information only. 0 IS0 1993 All rights reserved. No part of this publication may be rep
8、roduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without per- mission in writing from the publisher. I International Organization for Standardization Case Postale 56 l CH-1211 Geneve 20 l Switzerland Printed in Switzerland ii Copyright
9、International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 03:02:55 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD IS0 8128=1:1993(E) Apple juice, ap
10、ple juice concentrates and drinks containing apple juice - Determination of patulin content - Part 1: Method using high-performance liquid chromatography 1 Scope This part of IS0 8128 specifies a method using high- performance liquid chromatography for the determi- nation of the patulin content of a
11、pple juice, apple juice concentrates and drinks containing apple juice. The limit of detection of the method is IO kg/l, based on 5 ml of ready-to-drink apple juice. NOTE I IS0 8128-2 specifies a method using thin-layer chromatography. 2 Principle Extraction of patulin from a test portion using ethy
12、l acetate, followed by partitioning of the extract with aqueous sodium carbonate solution. Qualitative and quantitative determination of the patulin content by means of high-performance liquid chromatography (HPLC) using an ultraviolet (UV) detector. 3 Reagents Use only reagents of recognized analyt
13、ical grade, and water of HPLC grade. 3.1 Solvent, ethyl acetate. 3.2 Mobile phase, acetonitrile, 10 % (VW) solution. 3.3 Extraction solution, 14 g/l aqueous solution of anhydrous sodium carbonate. 3.4 Acetate buffer, pH 4. Mix 16,4 ml of dilute acetic acid c(CH A276 is the molecular absorbance of th
14、e patulin solution at the maximum (276 nm) of the absorption spectrum (see note 3); C is the apparatus constant (usually 1). is the relative molecular mass of patulin; NOTE 3 The molecular absorbance coefficient of patulin measured in ethanol at 276 nm is equal to 14 600. 4. Apparatus Rinse the labo
15、ratory apparatus before use with a 10 g/l sodium hypochlorite solution. Usual laboratory apparatus and, in particular, the fol- lowing. 4.1 High-performance liquid chromatograph, equipped with a UV detector (capable of operating at 276 nm) and equipped with a recorder and/or integrator. 4.2 ODS) rev
16、erse-phase equivalent column, with - an efficiency of at least per metre; - length of 250 mm; column, or any other 35 000 theoretical plates - inner diameter of 4,6 mm; - a stationary phase*) with particle size of 5 pm. 5 Sampling . It is important that the laboratory receive a sample which is truly
17、 representative and has not been dam- aged or changed during transport or storage. 6 Procedure 6.1 Preparation of test solution Dilute apple juice concentrates with water, using a I:5 mixture of concentrate to water, by volume. Then proceed as for the other products, as follows. 1) ODS = octadecylsi
18、lane 2) Bonded Octadecyl Cl8 is an example of a suitable product available commercially. This information is given for the con- venience of users of this International Standard and does not constitute an endorsement by IS0 of this product. 3) HMF = 5-hydroxymethylfurfural 6.1.1 Extract 5,0 ml of the
19、 laboratory sample (diluted with water if necessary) with 5,0 ml of ethyl acetate (3.1) for at least 1 min. Repeat the extraction twice more using 5,0 ml portions of ethyl acetate. Combine the three ethyl acetate phases and extract with 2,0 ml of sodium carbonate solution (3.3). CAUTION - After comb
20、ining the phases, carry out the extraction with sodium carbonate as quickly as possible, i.e. 1 min to 2 min, since patulin is not stable in alkaline media. 6.1.2 Extract the carbonate phase (6.1 .I) with an- other 5,0 ml portion of ethyl acetate and combine with the preceding portions. Reject the c
21、arbonate phase. Add 5 drops of acetic acid (3.5), mix and evaporate using a vacuum evaporator until 1 ml to 2 ml remain. 6.1.3 Transfer quantitatively the solution thus ob- tained to a vial of about 5 ml capacity, using several portions (about 1 ml each) of ethyl acetate, and evap- orate to dryness
22、under a stream of nitrogen at about 40 “C. Dissolve the residue in 500 1 of either the mobile-phase solvent (3.2) or acetate buffer (3.4). 6.2 Preparation of the calibration curve Using a pipette, transfer 1,O ml, 2,0 ml, 3,0 ml, 5,0 ml and 7,5 ml of the patulin standard solution (3.6.1) to a series
23、 of five 10 ml one-mark volumetric flasks and make up to the mark with the acetate buffer (3.4). Adjust the flow rate applied to the HPLC column to about 1 mI/min and adjust the sensitivity so that an absorbance of 0,Ol gives a full-scale deflection. Inject 10 1 to 30 1 of each of the calibration so
24、lutions into the chromatograph (4.1). Prepare the calibration curve by plotting the peak heights (or areas) against the patulin concentration in micrograms per millilitre. 6.3 Determination Inject 10 1 to 30 1 of the test solution (6.1.3) into the chromatograph using the same conditions as for the p
25、reparation of the calibration graph. Identify the patulin peak of the test solution by com- parison with the peaks of the calibration solutions. It is important to distinguish the patulin peak from the HMF peak.31 2 Copyright International Organization for Standardization Provided by IHS under licen
26、se with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 03:02:55 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 8128=1:1993(E) 7 Calculation Determine the concentration of patulin in the test solution directly from the calibration curve (6
27、.2). Cal- culate the patulin content of the sample, pp, in micro- grams per litre, as follows: Pp = IOOPpt where PPt is the concentration of patulin in the test solution, read from the calibration curve, expressed in micrograms per millilitre. 8 Precision The precision of the method has been checked
28、 by collaborative studies41. Statistical parameters are expressed in accordance with IS0 5725W 8.1 Repeatability Y = 41,9; s, = 14,9 where 8.2 Reproducibility R = 47,5;. sR = 22,6 where R is the reproducibility limit; SR is the standard deviation of reproducibility. 9 Test report The test report sha
29、ll specify - the method used, - the test result obtained, and - if the repeatability has been checked, the final quoted result obtained. It shall also mention all operating details not specified in this part of IS0 8128, or regarded as optional, to- gether with details of any incidents which may hav
30、e influenced the test result. The test report shall include all information necessary for the complete identification of the sample. is the repeatability limit; Sr is the standard deviation of repeatability. Copyright International Organization for Standardization Provided by IHS under license with
31、ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 03:02:55 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 8128=1:1993E). Annex A (informative) Bibliography l IS0 5725:1986, Precision of test methods - Determination of repeatability and repro
32、ducibility for a standard test method by inter-laboratory tests. 2 IS0 8128-2:1993, Apple juice, apple juice con- centrates and drinks containing apple juice - Determination of patulin content - Part 2: Method using thin-layer chromatography. 3 Official Methods of Analysis, Vol. 12, Washington DC: A
33、OAC, 1975, p. 463. 4 KUBACKI, S.J. and Goszcz, H. IUPAC Collab- orative Study of HPLC methods for the deter- mination of patulin in apple juice. J. Pure Appl. Chem., 60 (6): 871-876 (1988). 5 TANNER, H. and ZANIER, C. Ueber eine neue Patulin Bestimmung in Fruchtssften und Konzentraten. Schvveiz. Z.
34、Obst Weinbau, 112: 656-662 (1976). 6 TANNER, H. and ZANIER, C. Amtliche Methoden zur Bestimmung von Patulin. Mitt. Gebiete Labsm. Hyg., 75: 506-513 (1984). 7 POHLAND, A.E. et a/. Physicochemical data for some selected mycotoxins. J. Pure Appl. Chem., 54: 2219-2284 (1982). Copyright International Org
35、anization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 03:02:55 MDTNo reproduction or networking permitted without license from IHS -,-,- This page intentionally left blank Copyright International Organization fo
36、r Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 03:02:55 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 8128=1:1993(E) UDC 663.813:634.11:543.544:632.4 Descriptors: agricultljral product
37、s, plant products, food products, beverag determination of content, patulin, high performance liquid chromatography. es, apples, fruit and vegetabl e juices, chemical analysis, Price based on 4 pages Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 03:02:55 MDTNo reproduction or networking permitted without license from IHS -,-,-