UGANDA STANDARD
 
       US ISO 8128-1
First Edition
2009-mm-dd
 
Reference numbe r 
     US ISO 8128-1:1993
 © UNBS 2009
  
Apple juice, apple juice concentrates and drinks containing 
apple juice — Determination of patulin content — Part 1: 
Method using high-performance liquid chromatography 
 US ISO 8128-1: 1993 
 
© UNBS  2009 – All rights reserved ii Compliance with this standard does not, of itself confer immunity from legal obligations 
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct application  
© UNBS 2009 
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form or by any means, electronic or mechanical, in cluding photocopying and microfilm, without prior written 
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     US ISO 8128-1: 1993  
© UNBS  2009 - All rights rese rved                iii National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry  of Tourism, Trade and 
Industry established under Cap 327, of the Laws of U ganda. UNBS is mandated to co-ordinate the elaboration 
of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex A limentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT/SPS Agr eements of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a gi ven field or area and consists of representatives of 
consumers, traders, academician s, manufacturers, governme nt and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committ ee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council. 
This Uganda Standard, US ISO 8128-1, Apple juice, apple juice concentrates and drinks containing
apple juice — Determination of patulin content — Part 1: Method using high-performance liquid 
chromatography,  is identical with and has been reproduced fr om an International Standard, ISO 8128-1:1993, 
Apple juice, apple juice concentrates and drinks cont aining apple juice — Determination of patulin content — 
Part 1: Method using high-performance liquid chromatography, and adopted as a Uganda Standard.  
This standard was developed under the subcommittee on fruits and vegetables by the Food and agriculture 
Standards Technical Committee (UNBS/TC 2). 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." 
 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, concent& de jus de pommes et boissons ;i base de jus 
de pommes - Dgtermina tion de la teneur en patuline - 
Partie 7: M&hode par chromatographie en phase liquide ;i haute 
performance 
Reference number 
- - IS0 8128-1:1993(E) 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 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, 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 
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. 
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 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 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. I 
International Organization for Standardization 
Case Postale 56 l CH-1211 Geneve 20 l Switzerland 
Printed in Switzerland 
ii INTERNATIONAL STANDARD IS0 8128=1:1993(E) 
Apple juice, apple 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 apple 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 ethyl 
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 analytical 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&OOH) = 0,2 mol/l] with 3,6 ml of sodium 
acetate [c(CH,COONa) = 0,2 mol/l]. 
3.5 Acetic acid, glacial. 
3.6 Patulin standard solution (C,H&). 
3.6.1 Preparation 
Weigh, to the nearest 0,l mg, IO,0 mg of patulin in 
a 100 ml one-mark volumetric flask and dissolve it in 
the acetate buffer (3.4). Make up to the mark with the 
acetate buffer. 
Pipette 10,O ml of this solution into another 100 ml 
one-mark volumetric flask and make up to the mark 
with the acetate buffer. 
The patulin content of this standard solution is 
IO kg/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 [3]. 
3.6.2 Calculation of the concentration 
Calculate the concentration pp,, expressed in micro- 
grams per millilitre, of the patulin solution (3.6.1) using 
the formula 
Pps = A x Mr x 1 000 x C 
A276 IS0 81284:1993(E) 
A is the absorbance of the patulin standard 
solution; 
A276 is the molecular absorbance of the 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 laboratory 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) reverse-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 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 = octadecylsilane 
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 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 combining 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 carbonate 
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 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 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 solutions 
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 
preparation 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 IS0 8128=1:1993(E) 
7 Calculation 
Determine the concentration of patulin in the test 
solution directly from the calibration curve (6.2). 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 by 
collaborative studies[41. 
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 shall 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 have 
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. IS0 8128=1:1993[E). 
Annex A 
(informative) 
Bibliography 
[l] IS0 5725:1986, Precision of test methods - 
Determination of repeatability and reproducibility 
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: AOAC, 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. 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). This page intentionally left blank IS0 8128=1:1993(E) 
UDC 663.813:634.11:543.544:632.4 
Descriptors: agricultljral products, plant products, food products, beverag 
determination of content, patulin, high performance liquid chromatography. es, apples, fruit and vegetabl e juices, chemical analysis, 
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