UGANDA STANDARD
 
        US ISO 22855
First Edition
2009-mm-dd
 
Reference numbe r
    US ISO 22855:2008
 © UNBS 2009
  
Fruit and vegetable products — Determination of benzoic acid 
and sorbic acid concentrations — High-performance liquid 
chromatography method 
 US ISO 22855: 2008 
 
© 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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Requests for permission to reproduce this document should be addressed to 
The Executive Director 
Uganda National Bureau of Standards 
P.O. Box 6329 
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Uganda 
Tel: 256 41 505 995 
Fax: 256 41 286 123 
E-mail: unbs@infocom.co.ug
Web: www.unbs.go.ug  
      US ISO 22855:2008  
© 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 22855, Fruit and vegetable products — Determination of benzoic acid
and sorbic acid concentrations — High-performance liquid chromatography method,  is identical with and
has been reproduced from an International Standard, ISO 22855:2008, Fruit and vegetable products — 
Determination of benzoic acid and sorbic acid concentrations — High-performance liquid chromatography 
method, 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." 
  
 
  
 Reference number
ISO 22855:2008(E)
© ISO 2008
  
 INTERNATIONAL 
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, légumes et produits dérivés — Détermination des teneurs en 
acides benzoïque et sorbique — Méthode par chromatographie liquide 
à haute performance 
 ISO 22855:2008(E) 
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ii © ISO 2008 – All rights reserved
  ISO 22855:2008(E) 
© ISO 2008 – All rights reserved  iiiContents Page 
Foreword....................................................................................................................... ..................................... iv  
1 Scope ........................................................................................................................ ............................. 1  
2 Principle.................................................................................................................... ............................. 1  
3 Reagents a nd materi als ....................................................................................................... ................ 1  
4 Apparatus .................................................................................................................... .......................... 2  
5 Sample ....................................................................................................................... ............................ 2  
6 Procedure .................................................................................................................... .......................... 2  
7 Calculation.................................................................................................................. ........................... 4  
8 Precision.................................................................................................................... ............................ 4  
9 Test report .................................................................................................................. ........................... 6  
Annex A  (informative)  Chromatogram ............................................................................................................. 7  
Annex B  (informative)  Statistical results of th e interlaboratory test ............................................................ 8  
Bibliography ................................................................................................................... .................................. 10  
 ISO 22855:2008(E) 
iv © ISO 2008 – All rights reserved
 Foreword 
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies 
(ISO member bodies). The work of preparing 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, in 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 task 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. 
Attention 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 
vegetable products . 
 INTERNATIONAL STANDARD ISO 22855:2008(E)
 
© ISO 2008 – All rights reserved  1Fruit 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 liquid 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 and 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 otherwise specified, and water of HPLC grade. 
3.1 Acetic acid  (CH3COOH), glacial. 
3.2 Methanol  (CH3OH), for HPLC. 
3.3 Ammonium acetate  (CH3COONH4), 0,01 mol/l solution. 
Dissolve 0,771 g of ammonium acetate in 1 l of water. 
3.4 Ammonium acetate/acetic acid  (CH3COONH4/CH3COOH), buffer solution. 
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
6H5COOH), stock solution. 
Dissolve benzoic acid to obtain stock solution [ ρ(C6H5COOH) = 100 mg/ml] in 40 ml of methanol (3.2) and 
dilute to the mark with water in a 100 ml volumetric flask. 
3.6 Sorbic acid  [CH3(CH:CH)2COOH], stock solution. 
Dissolve sorbic acid to obtain stock solution, ρ[CH3(CH:CH)2COOH] = 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, K4[Fe(CN)6]⋅3H2O. ISO 22855:2008(E) 
2 © ISO 2008 – All rights reserved
 3.8 Zinc sulfate , heptahydrate, (ZnSO4⋅7H2O), 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 volume 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 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, in particular, the following. 
4.1 Ultrasonic bath . 
4.2 Membrane filters , of pore size 0,45 µm, 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 chromatograph , 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 µm. 
5 Sample 
A representative sample should have been sent 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  36.1.1 Clear samples 
Dilute 5,00 ml to 10,00 ml ( V1) 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 through 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 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 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 20 °C to obtain standard solutions I, II and III with benzoic acid and/or sorbic acid concentrations of 10 mg/l, 
25 mg/l and 50 mg/l, respectively. 
Inject 10 µl 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 plotting the peak areas against benzoic acid and/or sorbic acid 
concentration, in milligrams per litre. 
6.3 Determination 
Inject 10 µl 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 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 the 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 in 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 different 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
 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, ρA in milligrams per litre, using the 
following equation (external standard method): 
1s t
A
21100 A
AVρρ⋅×=⋅ 
where 
A1 is the peak area or peak height of benzoic acid or sorbic acid in the test sample, expressed in area or 
length counts, respectively; 
A2 is the peak area or peak height of benzoic acid or sorbic acid in the test standard solution, expressed 
in area or length counts, respectively; 
ρst is the concentration of the standard solution, in milligrams per litre; 
V1 is 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 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 results of this test. 
Statistical parameters are expressed in accordance with ISO 5725-2 [1]. 
8.1 Repeatability 
Benzoic acid 
Orange juice: r = 3,5 sr = 1,25 
Grape juice: r = 3,5 sr = 1,25 
Sorbic acid 
Orange juice: r = 2,8 sr = 1,00 
Grape juice: r = 2,3 sr = 0,88 
4-Hydroxybenzoic acid methyl ester 
Orange juice: r = 4,5 sr = 1,60 
Grape juice: r = 3,8 sr = 1,37  
4-Hydroxybenzoic acid ethyl ester  
Orange juice: r = 4,5 sr = 1,59 
Grape juice: r = 4,8 sr = 1,70 ISO 22855:2008(E) 
© ISO 2008 – All rights reserved  54-Hydroxybenzoic acid propyl ester  
Orange juice: r = 5,3 sr = 1,88 
Grape juice: r = 5,4 sr = 1,93 
where 
r is the repeatability limit; 
sr is the standard deviation of repeatability. 
8.2 Reproducibility 
Benzoic acid 
Orange juice: R = 13,9 sR = 4,96 
Grape juice: R = 8,9 sR = 3,18 
Sorbic acid 
Orange juice: R = 11,0 sR = 3,93 
Grape juice: R = 7,3 sR = 2,61 
4-Hydroxybenzoic acid methyl ester 
Orange juice: R = 19,0 sR = 6,79 
Grape juice: R = 11,7 sR = 4,17 
4-Hydroxybenzoic acid ethyl ester 
Orange juice: R = 9,8 sR = 3,5 
Grape juice: R = 8,8 sR = 3,14 
4-Hydroxybenzoic acid propyl ester 
Orange juice: R = 16,8 sR = 6,00  
Grape juice: R = 17,7 sR = 6,32 
where 
R is the reproducibility limit; 
sR is the standard deviation of reproducibility. ISO 22855:2008(E) 
6 © ISO 2008 – All rights reserved
 9 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 International 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 result obtained.  ISO 22855:2008(E) 
© ISO 2008 – All rights reserved  7Annex A  
(informative) 
 
Chromatogram 
 
Column Diameter 4,6 mm, length 250 mm 
 Ultrasphere-octyl (RP 8), particle size 5 µm 
Eluent Ammonium acetate solution/Methanol for HPLC (50 + 40 volume parts)+ Acetic acid (pH = 4.5) 
Flow rate 1,2 ml/min 
UV-detection 235 nm (0,08 AUFS) 
Recorder speed 3 mm/min 
Injection volume 10 µl 
Standard substances: 1. Benzoic acid (RT:8,13) a 
 2. 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) 
a RT = Retention time, minutes. 
b Inject. 
ρ(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 reserved
 Annex B  
(informative) 
 
Statistical results of the interlaboratory test 
Table B.1 — Benzoic acid 
Sample 
Parameters 
Orange juice Grape juice 
Mean benzoic acid concentration (mg/l) 72,8 57,0 
Repeatability standard deviation sr (mg/l) 1,25 1,25 
Repeatability coefficient of variation  CVr (%) 1,71 2,19 
Repeatability limit r = 2,8 × sr (mg/l) 3,5 3,5 
Reproducibility standard deviation sR (mg/l) 4,96 3,18 
Reproducibility coefficient of variation  CVR (%) 6,81 5,57 
Reproducibility limit R = 2,8 × sR (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 sr (mg/l) 1,0 0,88 
Repeatability coefficient of variation  CVr (%) 1,10 1,34 
Repeatability limit r = 2,8 × sr (mg/l) 2,8 2,3 
Reproducibility standard deviation sR (mg/l) 6,79 2,61 
Reproducibility coefficient of variation  CVR (%) 7,45 3,97 
Reproducibility limit R = 2,8 × sR (mg/l) 11,0 7,3 
 
Table B.3 — 4-Hydroxybenzoic acid methyl ester 
Sample 
Parameters 
Orange juice Grape juice 
Mean 4-hydroxybenzoic acid methyl ester concentration (mg/l) 83,4 75,5 
Repeatability standard deviation sr (mg/l) 1,60 1,37 
Repeatability coefficient of variation  CVr (%) 1,92 1,81 
Repeatability limit r = 2,8 × sr (mg/l) 4,5 3,8 
Reproducibility standard deviation  sR (mg/l) 6,79 4,17 
Reproducibility coefficient of variation  CVR (%) 8,141 5,52 
Reproducibility limit R = 2,8 × sR (mg/l) 19,0 11,7 
 ISO 22855:2008(E) 
© ISO 2008 – All rights reserved  9Table B.4 — 4-Hydroxybenzoic acid ethyl ester 
Sample 
Parameters 
Orange juice Grape juice 
Mean 4-hydroxybenzoic acid ethyl ester concentration (mg/l) 57,4 81,5 
Repeatability standard deviation sr (mg/l) 1,59 1,70 
Repeatability coefficient of variation  CVr (%) 2,77 2,09 
Repeatability limit r = 2,8 × sr (mg/l) 4,5 4,8 
Reproducibility standard deviation sR (mg/l) 3,5 3,14 
Reproducibility coefficient of variation  CVR (%) 6,10 3,85 
Reproducibility limit R = 2,8 × sR (mg/l) 9,8 8,8 
 
Table B.5 — 4-Hydroxybenzoic acid propyl ester 
Sample 
Parameters 
Orange juice Grape juice 
Mean 4-hydroxybenzoic acid propyl ester concentration (mg/l) 51,0 87,6 
Repeatability standard deviation sr (mg/l) 1,88 1,93 
Repeatability coefficient of variation  CVr (%) 3,69 2,20 
Repeatability limit r = 2,8 × sr mg/l 5,3 5,4 
Reproducibility standard deviation sR (mg/l) 6,00 6,32 
Reproducibility coefficient of variation  CVR (%) 11,76 7,21 
Reproducibility limit R = 2,8 × sR (mg/l) 16,8 17,7 
 
 ISO 22855:2008(E) 
10 © ISO 2008 – All rights reserved
 Bibliography 
[1] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results — Part 2: 
Basic method for the determination of repeatability and reproducibility of a standard measurement method  
[2] IFU Method 63:1995, Preservatives (HPLC) . International Federation of Fruit Juice Producers 
 ISO 22855:2008(E) 
ICS  67.080.01 
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© ISO 2008 – All rights reserved