ISO-8128-2-1993.pdf
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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
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