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1、IS0 8128 PT*2 93 = 4853903 0538801 005 I NTERNATI ON AL S TA N DA R D IS0 8128-2 First edition 1993-07-01 Apple juice, apple juice concentrates and drinks containing apple juice - Determination of patulin content - Part 2: Method using thin-layer chromatography Jus de pommes, concentrs de jus de pom
2、mes et boissons base de jus de pommes - Dtermination de la teneur en patuline - Partie 2: Mthode par chromatographie sur couche mince - - Reference number IS0 81 28-211 993(E) Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Stan
3、dards 1/9972545001 Not for Resale, 04/26/2007 03:02:55 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 8328 PT*2 93 4853903 0538802 T43 m IS0 8128-2: 1993(E) Foreword IS0 (the International Organization for Standardization) is a worldwide federation of national standard
4、s bodies (IS0 member bodies). The work of preparing International Standards is normally carried out through IS0 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 organiz
5、ations, governmental and non-governmental, in liaison with ISO, also take part in the work. IS0 collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circ
6、ulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard IS0 81 28-2 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Sub-Committee SC 3, Fruit and vegetab
7、le products. IS0 8128 consists of the following parts, under the general title Apple juice, apple juice concentrates and drinks containing apple juice - Deter- mination of patulin content: - Part I: Method using high-performance liquid chromatography - Part 2: Method using thin-layer chromatography
8、Annex A of this part of IS0 8128 is for information only. O IS0 1993 All rights reserved. No part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without per- mission in writing from the publisher. Interna
9、tional Organization for Standardization Case Postale 56 CH-1 21 1 Genve 20 Switzerland Printed in Switzerland II 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 re
10、production or networking permitted without license from IHS -,-,- IS0 8328 PT*2 93 4853903 0538803 988 INTERNATIONAL STANDARD IS0 8128-2:1993(E) Apple juice, apple juice concentrates and drinks containing apple juice - Determination of patulin content - Part 2: Method using thin-layer chromatography
11、 1 Scope This part of IS0 8128 specifies a method using thin- layer chromatography for the determination of the patulin content of apple juice, apple juice concentrates and drinks containing apple juice. The limit of detection of the method is 25 pg/i, based on 50 ml of ready-to-drink apple juice. N
12、OTE 1 For more precise analyses or in case of a dis- pute, the HPLC method specified in IS0 8128-1 should be used. 3.2 Developing solvents, for two-directional TLC: benzene/methanol/acetic acid (80 % by mass) mixture (1 9 2 1 by volume); toluene/ethyl acetatelformic acid (90 % by mass) mixture (5:4:
13、1 by volume). 3.3 Silica gel, for column chromatography, of 0,063 mm to 0,2 mm particle size. 3.4 Eluting solution, toluenelethyl acetate mixture (75:25 by volume). 2 Principle 3.5 Patulin standard solution ( C , H , O , ) . Extraction of patulin in a mixture of ethyl acetate and chloroform (32 by v
14、olume). Filtration of the extract on a silica-gel column and qualitative and semi- quantitative determination by means of two- directional thin-layer chromatography (TLC). The spots are developed using a 3-methyl-2-benzothiazoline hy- drazone (MBTH) hydrochloride solution. 3 Reagents Use only reagen
15、ts of recognized analytical grade and water of the purity required for chromatography. WARNING - Special attention should be paid when using benzene or chloroform, which are toxic and may cause explosions. 3.1 Solvents, ethyl acetate, chloroform and toluene. 3.5.1 Preparation Weigh, to the nearest 0
16、.1 mg, 10,O mg of patulin in a 100 ml one-mark volumetric flask and dissolve it in ethyl acetate (3.1). Make up to the mark with ethyl acetate. Pipette 10,O ml of this solution into another 100 ml one-mark volumetric flask and make up to the mark with ethyl acetate. The patulin content of this stand
17、ard solution is 1 O pg/ml approximately. Measure the absorbance at 276 nm of this standard solution on an appropriate spectrometer using quartz cells of optical path length 10 mm. NOTE 2 The preparation of the standard solution and the control of its purity are based on reference a. 1 Copyright Inte
18、rnational 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 -,-,- IS0 8128 PT*Z 93 W 4851703 0538804 814 IS0 8128-2: 1993(E) 3.
19、5.2 Calculation of the concentration Calculate the concentration pp, expressed in micro- grams per millilitre, of the patulin solution (3.5.1) using the formula A x M , x 1 O O O x C A276 Pps = where A A276 is the absorbance of the patulin standard solution; is the molecular absorbance of the patuli
20、n solution at the maximum (276 nm) of the absorption spectrum (see note 3). is the relative molecular mass of patulin; is the apparatus constant (usually 1). Mr C NOTE 3 measured in ethanol at 276 nm is equal to 14 600. The molecular absorbance coefficient of patulin 3.6 MBTH hydrochloride solution
21、Dissolve 0.5 g of 3-methyl-2-benzothiazoline hydra- zone (MBTH) hydrochloride monohydrate in 100 ml of water. Store in a refrigerator and prepare a fresh solution at least every 3 days. 4 Apparatus Rinse the laboratory apparatus before use with a 1 O g/I sodium hypochlorite solution. Usual laborator
22、y apparatus and, in particular, the fol- lowing. 4.1 Chromatographic columns,3 of dimensions 300 mm x 22 mm, with a 250 cm reservoir and a stopcock, equipped at one end with a sintered glass disc. 4.2 TLC equipment, glass developing tanks, long- wavelength ultraviolet (UV) lamp (360 nm) and spray- i
23、ng device. 4.3 Fluorodensitometer 4.4 TLC plates, 20 cm x 20 cm, coated with silica gel (3.3) (layer thickness 0,25 mm), without a fluor- escent indicator. 4.5 Oven, ventilated, capable of operating at 130 “C f 1 “C. 5 Sampling It is important that the laboratory receive a sample which is truly repr
24、esentative 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 1 :5 mixture of concentrate to water, by volume. Then proceed as for the other products, as follows. Extract 50 mi of the lab
25、oratory sample (diluted with water if necessary) with a 50 ml portion of ethyl acetate/chloroform mixture (3:2 by volume) for at least 1 min. Repeat the extraction with two more 50 ml portions of ethyl acetate/chloroform mixture and filter each portion through a sintered glass funnel, containing a 1
26、 cm layer of anhydrous sodium sulfate, collecting the filtrate directly in a 250 ml evaporation flask. Evaporate to dryness under vacuum using a rotary evaporator and transfer quantitatively the residue thus obtained into a 100 ml graduated cylinder, rinsing the flask with four 5 mI portions of ethy
27、l acetate. Dilute to 25 ml with ethyl acetate, and then to 100 ml with toluene. 6.2 Column chromatography Place a small plug of glass wool in the bottom of the column (4.11, then add 25 ml of toluene. Add a slurry comprising 15 g of silica gel (3.3) in 40 ml of toluene to the column, and then add 15
28、 g of anhydrous sodium sulfate and drain off the solvent until the sur- face of the solvent is level with the top of the packing. Add the extract of the test portion to the column and allow to drain until its surface is level with the top of the packing. Discard the eluate. Add 200 mI of eluting sol
29、ution (3.4) to the column and collect the eluate in a graduated cylinder, using a flow rate of about 5 ml/min. Evaporate the eluate until about 2 mI re- mains. Transfer quantitatively the concentrated eluate to a 20 ml tube, using four portions (of about 4 ml each) of ethyl acetate and evaporate to
30、dryness under a stream of nitrogen. Immediately dissolve the residue in 500 pI of ethyl acetate since patulin is not stable. 6.3 Thin-layer chromatography Activate the pre-coated TLC plates (4.4) at 11 O “C for 2 h. Spot, using a capillary pipette or microsyringe, a 20 pi aliquot portion of the puri
31、fied extract of the sample (6.2) onto the TLC plate at a distance of 2 cm from its left-hand edge and 2 cm from its lower edge. Spot a 5 pI aliquot portion of the patulin stan- 2 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical S
32、tandards 1/9972545001 Not for Resale, 04/26/2007 03:02:55 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 8128 PT*2 93 = 4851903 0538805 750 W dard solution (3.5) onto the TLC plate at a distance of 2 cm from its right-hand edge and 2 cm from its lower edge. Spot a 5 pI
33、 aliquot portion of the patulin stan- dard solution 2 cm from the left-hand edge and 2 cm from the upper edge. Spot a 10 FI aliquot portion of the patulin standard solution 2 cm from the left-hand edge and 4 cm from the upper edge (see figure 1). Develop the chromatogram in direction I using the ben
34、zene/methanol/acetic acid mixture (3.2) until the solvent front reaches a distance of 14 cm. Remove the plate from the tank, dry in air and then develop the chromatogram in direction II using the toluene/ethyl acetatelformic acid mixture (3.2) until the solvent front reaches a distance of 14 cm. Rem
35、ove the plate from the tank and allow to dry at ambient temperature. Spray the TLC plate with the MBTH hydrochloride solution (3.6) and then dry for 15 min in the oven (4.5) set at 130 “C. 6.4 Determination Determine the quantity of patulin in the extract by comparing the intensity of fluorescence o
36、f the yellow-brownish spot from the extract with those of the standard reference spots under UV long- wavelength irradiation. Look for the patulin spot from the extract at the intersection of the perpendicular lines originating from the standard reference spots. If the intensity of fluorescence give
37、n by the 20 p I of extract is greater than those of the standard reference spots, dilute the extract with ethyl acetate and repeat the TLC procedure specified in 6.3. If the intensity of fluorescence of the spot from the extract is compar- able with one of those of the standard reference spots, it m
38、eans that the concentration of patulin in the sample analysed is either 25 pg/l or 50 pg/l respect- ively. NOTE 4 When using the method specified, the separ- ation of patulin from HMFlJ is complete, and therefore there is no risk of interference. VI V2 is the volume, in microlitres, of the spot of t
39、he test portion; is the volume, in microlitres, of the spots of the patulin standard solution (3.5) giving an intensity of fluorescence equal to that given by the spot of the test solution; is the concentration, in micrograms per millilitre, of the patulin standard solution. PPS 8 Precision Statisti
40、cal parameters are expressed in accordance with IS0 572511. 8.1 Repeatability r = 33,4; s , = 29.4 where r is the repeatability limit; s, is the standard deviation of repeatability. 8.2 Reproducibility R = 41 ,O; SR = 35,8 where R is the reproducibility limit; S , is the standard deviation of reprod
41、ucibility. 9 Test report The test report shall specify - the method used. 7 Calculation - the test result obtained, and Calculate the patulin content of the sample, pp, in micrograms per litre, as follows: v2 Ppc x vo 50Vi Pp = where V, is the volume, in microlitres, of the final dilution of the pur
42、ified extract of the test portion; IS0 81 28-2: 1993( E) - if the repeatability has been checked, the final 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 have influenced the test result.
43、 quoted result obtained. The test report shall include all information necessary for the complete identification of the sample. 1) HMF = 5-hydroxymethylfurfural 3 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/99725
44、45001 Not for Resale, 04/26/2007 03:02:55 MDTNo reproduction or networking permitted without license from IHS -,-,- IS0 8128 PT*2 93 4853903 0538806 697 r IS0 8128-2: 1993(E) I Dimensions in centimetres Dlrectlon II 1 Figure 1 - Two-directional thin-layer chromatography 4 Copyright International Org
45、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 -,-,- IS0 8328 PT*2 73 4851703 0538807 523 IS0 81 28-2: 1993( E) Annex A (i n f
46、o r m a t ive) Bibliography l IS0 5725:1986, Precision of test methods - Method using high-performance liquid chro- Determination of repeatability and reproducibility for a standard test method by inter-laboratory tests. 3 Official Methods of Analysis, Vol. 12, 2 IS0 8128-1:1993, Apple juice, apple
47、juice con- centrates and drinks containing apple juice - NISKANWN, A. J. Food Sci., ma tograph y. Washington DC: AOAC, 1975, p. 463. 4 LINDROTH, S. and Determination of patulin content - Part I: 43, 446-448 (1 978). 5 Copyright International Organization for Standardization Provided by IHS under lic
48、ense 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 PT+2 93 4851903 0538808 4hT IS0 81 28-2: 1993( E) UDC 663.813:634.11:543.544:632.4 Descriptors: agricultural products, plant products, food products, beverages, apples, fruit and vegetable juices, chemical analysis, determination of content, patulin, thin layer chromatography. Price based on 5 pages Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Te