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                                                                                                               ICS.67.200.10  
  
 
 
 
 
 
  DRAFT  TANZANIA STANDARD  
          
                      Edible cotton seed oil – Specification  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                   TANZANIA BUREAU OF STANDARDS  
 
    ©TBS 2019  – All rights reserved  
 
                                                                                                             TBS/AFDC 4(5975)P 3 
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                                                                                                               ICS.67.200.10  
 Edible cotton seed oil – Specification  
 
0 Foreword  
Cottonseed  oil is extracted from the seeds of cotton plant of various species.Cottonseed  have similar 
structures to other oil seeds such as sunflower seeds, having an oil bearing kernel surrounded by a 
hard outer hull; during processing, the oil is extracted from the kernel. Edible Cottonseed  oil is used for 
salad oil, mayonnaise, salad dressing, and similar products because of stability of its flavour .It is one 
of traded edible vegetable oils in Tanzania . 
 
This Tanzania Standard  has been prepare d to ensure the safety and quality of edible cottonseed  oil 
produced in,exported  or imported into the country.  
 
In preparation of this  Tanzania  standard considerable help was derived from : 
CODEX STAN 210 -1999 (Amended 2015) Codex standard for named vegeta ble oil s 
published by the Codex Alimentarius Commission  
 
ISO 15141 -2018  Cereals and cereal products  — Determination of ochratoxin A — High 
performance liquid chromatographic method with immunoaffinity column cleanup and 
fluorescence detection  published by International Organization for Standardization.  
 
In reporting the results of a test or analysis made in accordance with this Tanzania Standard, if the 
final value, observed or calculated is to be rounded off, it shall be done in accordance with TZS 4.  
 
1 Scope  
This Tanzania Standard prescribes the requiremen ts and methods of samp ling and test s for edible 
cotton seed oil.  
 
2 Normative r eference s 
The following referenced documents are indispensable for the application of this document. For dated 
references,  only the edition cited applies. For undated references, the latest edition of the referenced 
document (including any amendments) applies ; 
 
CODEX STAN 192 , General standards for food additives               
TZS 4 ,  Rounding off numerical values  
TZS 54 , Animal and Vegetable fats and oils – Sampling  
TZS 76 ,  Methods for determination of arsenic  
TZS 109, Food processing units – Code of hygiene — General  
TZS 268 , General atomic absorption – Spectro – Photometric method for determination of lead in 
food stuffs   
TZS 288 -1, Animal and vegetable fats and oils  Preparation of methyl esters of fatty acids  
TZS 288 -2, Animal and vegetable fats and oils  Analysis by gas chromatography of methyl ester of 
fatty acids  
TZS 538  , Packaging and labeling of foods  
TZS 799 , Foodstuffs – Determination of aflatoxin B1, and the total content of aflatoxins B1, B2, G1 
and G2 in cereals, nuts and derived products – High\-performance liquid chromatographic method  
TZS 1313 , Fortified edible oils and fats — Specification  
TZS 1322, Oils and fats Sampling and test methods – Purity test  
TZS 1324 , Animal and vegetable fats and oils – Determination of peroxide value -Iodometric (visual) 
end point determination                                                                                                               TBS/AFDC 4(5975)P 3 
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 TZS 1325 ,  Animal and vegetable fats and oils - Determination of saponification value  
TZS 1326 ,  Animal and vegetable fats and oils – Determination of moisture and volatile matter  
TZS 1327 ,  Animal and vegetable fats and oils – Determination of iodine value  
TZS 1328 ,  Essential oils – Determination of relative density at 20  °C – Reference method  
TZS 1329 ,  Animal and vegetable fats and oils – Determination of refractive index  
TZS 1330 ,  Animal and vegetable fats and oils – Determination of lovibond colour  
TZS 1331 ,  Animal and vegetable fats and oils – Determination of acid value  and acidity  
TZS 1332 ,  Animal and vegetable fats and oils – Determination of unsaponifiable matter -method using 
diethyl ether extraction  
TZS 1335 ,  Animal and vegetable fats and oils – Determination of copper, iron and nickel content -
graphite furnace at omic absorption  
TZS 1336 ,  Animal and vegetable fats and oils – Determination of insoluble impurities content  
TZS 1369,  Animal and Vegetable fats and oils – Determination of Butylhydroxyanisole (BHA) and 
Butylhydroxytoluene (BHT) – Gas liquid chromatograp hic method  
TZS 1370,  Animal and Vegetable fats and oils  – Determination of tocopherol and tocotrienol content 
by High Performance Liquid Chromatography  
TZS 1371 Animal and vegetable fats and oils — Determination of individual and total sterol contents -
Gas chromatographic method  
TZS 1775, Animal and Vegetable fats and oils  - Detection and identification of antioxidants  - Thin-layer 
chromatographic method  
 
3 Term s and definitions  
For the purpose of this Tanzania Standard the definition s below shall apply;   
 
3.1 edible oils  
food stuffs which are composed of glycerides of fatty acids of vegetable, animal or marine origin which 
is suitable for human consumption. They may contain small amounts of other lipids such as 
phosphatides, of unsaponifiable constituents and of free fatty acids naturally present  in the oil.  
 
3.2 vegetable oil  
any of a large group of oils that are esters of fatty acids and glycerol, obtained from the leaves, fruit, or 
seeds of plants.  
 
3.3 edible cottonseed oil  
edible oil derived from the seeds of various cultivated species of Gossypium spp . 
 
3.4 virgin cotton seed oil 
cotton seed oil obtained, without altering the nature of the oil, by mechanical procedures, for example, 
expelling or pressing, and the application of heat only. It may have be en purified by washing with 
water, settling, filtering and centrifuging only.  
 
3.5 refined cotton seed oil  
cotton seed oil obtained, by mechanical procedures and/or solvent extraction and subjected to refining 
processes.  
 
4.Requirements  
4.1 General require ments  
Edible cotton seed  oil shall   
a) be fit for human consumption;                                                                                                               TBS/AFDC 4(5975)P 3 
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 b) be free from  foreign and  rancid odour and taste;  
c) have colour  characte ristic of designated product  ; 
d) be clear and free from adulterants,  sediment s, suspended or foreign matter and separated 
water and;  
e) be free from admixture with mineral or other oils of vegetable or animal origin when tested in 
accordance with the methods prescribed in TZS 1322.  
f) be free from gossypol when t ested according to the method prescribed in  Annex B . 
 
4.2  The clarit y of the oil shall be judged by the absence of turbidity after keeping the filtered sample at 
30 0C for 24 hours.  
 
4.3 Specific requirements  
Edible cotton seed  oil shall comply with  specific requirements  given in table  1 when tested in 
accordance with the  methods specified therein ; 
  Table  1-Specific  require ments for edible cotton seed  oil 
S. No.  Parameter  Requirement  Test method   
i.  Relative density( at 20 oC/water at 20  oC) 0.918 -0.926  TZS 1328  
  
ii.  Refractive index, (ND 40 o C ) 1.458 - 1.466  TZS 1329  
 
iii.  Saponification value, mg KOH/g oil  189 - 198 TZS 1325  
 
iv.  Iodine value  (Wijs) , g/100  100– 123 TZS 1327  
v.  Unsaponifiable matter, g/kg, max.   15 
 TZS 1332  
vi.  Fatty acids composition,%of total fatty 
acids  As in Annex A  TZS 288 (Part 
1&2)  
vii.  Moisture and matter vol atile at 105 o C, % 
m/m, max.  0.2 TZS 1326  
viii.  Insoluble impurities, % m/m, max.  0.05 TZS 1336  
ix.  Free Fatty Acid(FFA) as oleic acid , %max Refined  oils: 0.3 
Cold pressed and 
virgin oil s: 2 TZS 1331  
x.  Peroxide value, mEq peroxide - oxygen/kg 
oil, max.  Refined  oils :10  
Cold pressed and 
virgin oil s: 15 TZS 1324  
xi.  Colour,  in a 0.635 c m cell in  Lovibond  
unit, expressed as (Y+5R) not deeper 
than 10 TZS 1330  
 
xii.  Total sterols(mg/kg)  2700 -6400 TZS 1371  
xiii.  Halphen test  Positive  TZS 1322  
 
4.3 Food additives  
 
4.3.1  General  
 
4.3.1.1  Food additives may be used during processing of edible cottonseed oil and shall be in 
accordance to Codex Stan 192.                                                                                                               TBS/AFDC 4(5975)P 3 
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 4.3.1.2  In addition f ood additives shall not be used in virgin or cold pressed oils. In Refined  the 
additives may be used sub ject to tables 2, 3, and 4.  
4.3.2  Flavours  
Natural flavours and their identical synthetic equivalents, and other synthetic flavours may be used, 
except those which are known to be hazardous.  
4.3.4  Antioxidants  
4.3.4.1  Antioxidants specified in Table 2 ma y be used ; 
            Table 2– Requirements for antioxidant in edible co ttonseed  oil 
INS 
No. Antioxidant  Requirements  Test method  
304 Ascorbyl palmitate, mg/kg, max  
500 (singly or 
in 
combination)  TZS 1775  
305 Ascorbyl stearate , mg/kg, max  
307a  Toco pherol, d -alpha - ,  mg/kg, max  
300 (singly or 
in 
combination)  TZS 1370  307b  Tocopherol concentrate, mixed , 
mg/kg, max  
307c  Tocopherol, dl -alpha , mg/kg, max  
310 Propyl gallate , mg/kg, max  100  TZS 1775  
319 Tertiary butyl hydroquinone (TBHQ) , 
mg/kg, max  120  
320 Butylated hydroxyanisole (BHA) , 
mg/kg, max  175  TZS 1369  
 
321 Butylated hydroxytoluene 
(BHT) ,mg/kg, max   75  
Any combination of gallates, BHA, BHT, and/or 
TBHQ , mg/kg, max  200 within 
individual 
limits  TZS 1775  
389 Dilauryl thiodipropionate , mg/kg, max  200  
 
4.3.4.2  Antioxidant  synergists specified in Table 3  may be used.  
              Table 3  — Antioxidant synergists  
INS 
No. Antioxidant synergist  Requirements  Test method  
330 Citric acid , mg/kg, max  
GMP  TZS 1775  
 331(i)  Sodium dihydrogen citrate, 
mg/kg, max  
331(iii)  Trisodium citrate , mg/kg, 
max 
384 Isopropyl citrates , mg/kg, 
max 100 (Singly or in 
combination)  472c  Citric and fatty acid esters 
of glycerol , mg/kg, max                                                                                                               TBS/AFDC 4(5975)P 3 
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4.3.5  Antifoaming agents (deep frying oi l) 
Antifoaming agents s pecified in Table 4 may be used;  
Table 4 — Antifoaming agents  
INS 
No. Antifoaming agent  Requirements  Test method  
900a  Polydimethylsiloxane, mg/kg, 
max 10  TZS  1775  
 
4.4 Fortification  
Edible cottonseed oil m ay be fortified in accor dance with TZS 1313.  
 
5 Hygiene  
Edible cottonseed oil  shall be produced, processed, handled and stored in accordance with TZS 109  . 
 
6  Contaminants  
 
6.1 Pesticide residues  
Edible co ttonseed  oil shall comply with relevant maximum pesticide residue limits established by the 
Codex Alimentarius Commission.  
6.2 Soap content  
Edible co ttonseed  oil shall have the soap content not exceed 0.005%  when tested according to TZS 
1322  
 
6.3. Metals  contaminants  
Edible co ttonseed  oil shall comply with maximum limits establ ished by Codex Alimentarius 
Commission as specified in Table 5.  
 
Table 5– Acceptable levels of metal contaminants in edible co ttonseed  oil 
 
Characteristics  Maximum level  Method of test  
Iron (Fe) mg/kg   
 Virgin                5.0 
Refined              1.5 
 TZS 1335  
Copper (Cu) mg/kg  
 Virgin               0.4 
Refined            0.1 
 TZS 1335  
Lead (Pb) mg/kg  
 0.1 TZS 268  
Arsenic (As) mg/ kg  
 0.1 TZS 76  
 
6.4 Mycotoxins  
 
6.4.1 Total aflatoxin shall not exceed 10 µg/kg while aflatoxin B1 shall not exceed 5 µg/kg when tested 
in accordance with TZS 799 . 
 
6.4.2  Ochratoxin A level shall not exceed 5µg/kg when tested in accordance with the method specified 
in Annex C.  
 
6.4.3  Edible cottonseed  oil shall b e free from  Poly-Hydroxy alkanoates (PHA).  
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7  Sampling a nd Tests  
 
7.1 Sampling  
Edible cottonseed oil shall be sampled as prescribed in TZS 54.  
 
7.2 Tests  
7.2.1  Testing shall be in accordance with TZS 1322 and as provided in the respective tables and 
Annexes of this Tanzania Standard.  
7.2.2 Quality of reagent s  
Unless specified otherwise, analytical grade chemicals and distill ed water shall be used in tests.  
 
 
 
8  Packaging, Marking And Labeling  
Edible cottonseed oil shall be packed, marked and labeled in accordance with TZS 538 .  
 
8.1  Packaging  
Edible cotto nseed  oil shall be packaged in food grade containers and sealed in manner to ensure the 
safety and quality requirements specified in this standard are maintained throughout the shelf life of 
the product.  
8.2    Marking and labeling  
8.2.1 In addition each C ontainer of product shall be legibly and indelibly marked with the following 
information:  
a) Name of the product  
b) The words virgin or refined shall be declared on the label to indicate the type of oil  
c) Name, physical and postal address of the manufacturer and/o r packer  
d) Date of manufacture and expiry  date 
e) Language -Kiswahili/English or Kiswahili and English  
f) A complete list of ingredients in descending order of proportion  
g) Net content  
h) Batch number  
i) Manufacturers registered trade mark  
j) Country of origin  
k) The phrase “Pl ace the products away from direct sunlight”.  
l) Nutritional information(option)  
 
8.2.2  The containers may also be marked with the TBS Mark of Quality.  
 
NOTE  - The TBS Mark of Quality may be used by the packers only under licence from TBS. Particulars of 
conditions under which the licenses are granted, may be obtained from TBS.  
 
 
 
 
 
 
 
 
 
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 ANNEX A  
  (normative)  
                                             Free fatty acids composition  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                             
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
Carbon configuration   
Fatty acid 
composition(%)  Test Methods  
 
 
C6:0  ND   TZS 288 (Part 1&2)  
 
C8:0  ND  
C10:0  ND  
C12:0  ND-0.2  
C14:0  0.6-1.0  
C16:0                 21.4-26.4  
C16:1  ND-1.2  
C17:0  ND-0.1  
C17:1  ND-0.1  
C18:0  2.1-3.3  
C18.1  14.7-21.7  
C18:2  46.7-58.2  
C18:3  ND-0.4  
C 20:0  0.2-0.5  
C 20:1  ND-0.1  
C20:2  ND-0.1  
C22:0  ND-0.6  
C22:1  ND-0.3  
C22:2  ND-0.1  
C24:0  ND-0.1  
C24:1  ND  
NOTE - Free fatty acid composition is expressed as % of total fatty acids  
                              ND-none detectable,defined as ≤0.05                                                                                                               TBS/AFDC 4(5975)P 3 
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 ANNEX B  
(normative)  
                                     Determination of Free and Total Gossypol  
 
1. Introduction  
Free gossypol refers  to gossypol and gossypol derivatives in cottonseed products which are soluble in 
aqueou s acetone under the conditions of this method.  
 
2. Apparatus  
 
2.1 Mechanical shaker  
2.2 Spectrophotometer  
2.3 Glass beads about 6 mm diameter  
2.4 Erlenmeyer flasks 250 ml  
2.5 Pipettes,  
2.6 Filter paper, ( Whatman No 2 or equivalent)   
2.7 Volumetric flasks 25, 200 ,250 ml  
2.8 Water b ath for operation at 100°C .  
 
3. Reagents  
        Solvents  
3.1 Aqueous acetone  – Mix 700 ml acetone with 300 ml distilled water  
3.2 Aqueous Isopropyl alcohol  (2 – propanol )  – Mix 800 isopropyl alcohol with 200 ml water.  
3.3 Aniline  – distilled over a small amount of  zinc dust. Redistill when the reagent blank exceeds 
0.022 absorbance ( 95 % transmittance)  
3.4 Thiourea solution  – Dissolve 10 gm thiourea in water and make up to 100 ml  
3.5 HCl – 1.2 N Dilute 106 ml conc. HCl (35 -37%) to 1 litre with water.  
3.6 Gossypol - Primary  standard or gossypol acetic acid (89.61 % gossypol by wt) to be used for 
calibration.  
3.7 Standard Gossypol solution  – Prepare by accurately weighing 25 mg primary standard 
gossypol or 27.9 mg gossypol acetic acid and transferring quantitatively to a 250 ml volumetric 
flask using 100 ml of acetone. Add 1 ml glacial acetic acid, 75 ml water, dilute to volume with 
acetone and mix well. Pipette  50 ml of this solution into a 200 ml volumetric flask, add 100 ml 
acetone, 60 ml water and dilute to volume with aceton e. Mix well. This standard gossypol 
solution contains 0.025 mg of gossypol per ml if exactly 25 mg gossypol or 27.9 mg of 
gossypol acetate were weighed. It is stable for 24 hrs when protected from light.  
 
      4. Procedure  
4.1  The weight of the sample and the aliquot of the acetone extract to be taken for test shall 
depend on the gossypol content but sample size should not exceed 2 -5 gm if the free 
gossypol is expected to be between 0.2 – 0. 5 % and the aliquot of extract to be taken for test 
should be 10 m l.  
4.2  Transfer the accurately weighed sample to a 250 ml Erlenmeyer flask  Add a few glass beads 
and 50 ml aqueous acetone, stopper and shake vigorously on a mec hanical shaker for 1 
hour.  
4.3  Filter through a dry filter paper discarding the first 5 ml and colle ct filterate in a small flask.  
4.4  Pipette duplicate aliquots into 25ml volumetric flasks  
4.5  To one sample solution designated as solution A, add 2 drops of 10 % aqueous thiourea, 1 
drop of  1.2 N HCl and dilute to volume with aqueous isopropyl alcohol.  
4.6  To the second sample designated as solution B, add 2 drops of 10 %,aqueous thiourea, 1 
drop of 1.2 N HCl and 2 ml of redistilled aniline. A rapid delivery pipette may be used for 
dispensing aniline.                                                                                                               TBS/AFDC 4(5975)P 3 
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 4.7  Prepare a reagent blank containing a volume of aqueous aceto ne solution equal to that of the 
sample aliquot and add 2 drops of 10% thiourea and 2 ml of anil ine( do not add any 1.2 N 
HCl).  
4.8  Heat the sample aliquot B and the reagent blank in a boi ling water bath for 30 minutes.  
4.9  Remove the solutions from the  bath, ad d about 10 ml of aqueous isopropyl alcohol; to effect 
homogeneous solutio n and cool to room temperature.  
4.10 Dilute to volume  with aqueous isopropyl alcohol.  
4.11 Determine the absorbance of sample aliquot A at 440 nm using aqueous isopropyl alcohol to   
set the in strument at zero absorbance (100% transmittance).  
4.12 With the instrument at zero absorbance with aqueous isopropyl alcohol, determine the 
absorbance of reagent blank. If the reagent blank exceeds 0.022 absorbance units, the 
analysis must be repeated u sing fre shly distilled aniline.  
4.13 Determine the absorbance of sample aliquot B at 440 nm using the reagent blank to set 
instrument at 0 absorbance.  
 
Calculate the corrected absorbance of the aliquot as follows ; 
Corrected absorbance = (absorbance of B – absorbance o f A)  
 
4.14 From the corrected absorbance of the sample, determine the mg of gossypol in the sample 
aliquot by reference to a calibration graph prepared by taking1 , 2 , 3, 4, 5, 7 , 8, 10 ml aliquot 
of standard gossypol solution ( 0.025 mg / ml) into 25 ml vol umetric  flask.  
4.15 To one set of aliquots designated C add 2 drops of 10 % aqueous thiourea, 1 drop of 1.2 N 
HCl and dilute to volume with aqueous isopropyl alcoho l and determine its absorbance.  
4.16 To the other set of aliquots designated D add 2 drops of aqueous thiourea, 2 drops of 1.2 N    
HCl a nd 2 ml of redistilled aniline.  
4.17 Prepare a reagent blank containing 10 ml of aqueous acetone, 2 drops of aqueous thiourea 
and 2 ml of aniline (do not add HCl). Heat the standards and the reagent blank in boiling water 
bath for 30 minutes, cool and dilute to volume with aqueous isopropyl alcohol and determine 
their absorbance.  
4.18 Determine corrected absorbance. Plot the corrected absorbance for each gossypol standard 
against mg of gossypol in 25 ml volume to obtain the calibrat ion graph . 
 
 
                                                     Total Gossypol  
 
1. Introduction  
Total gossypol refers to gossypol and gossypol derivatives both free and bound in cottonseed 
products which are capable of reacting with 3 - amino -1 propanol in  dimethylformamide solution to 
form diaminopropanel complex which then reacts with aniline to form dianilinogossypol under the 
conditions specified in this method  
 
2. Apparatus  
(As described in determination of free gossypol)  
 
     3   Reagents  
      3.1 Solv ents   
 Isopropyl alcohol  (n – propanol),  
 n – hexane  (B.P 68 -69 0C), dimethyl formamide,  
 3  amino 1 propanol  (propanolamine), free of colour,  
 glacial acetic acid  and  
 aniline , the aniline should be redistilled over zinc dust using water cooled condenser.  
3.2  Isopropyl alcohol - hexane mixture  ( 60 + 40 )                                                                                                               TBS/AFDC 4(5975)P 3 
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 3.3  Complexing reagent  prepared by pipetting 2 ml of 3 amino -1 propanol and 10 ml glacial 
acetic acid into a 100 ml volumetric flask, cooling to room temperature and diluting to volume 
with dimethyl formamide. P repare reagent weekly and store in a refrigerator when not in use.   
3.4 Gossypol or Gossypol acetic acid  as primary standard.  
3.5  Standard Gossypol solution  prepared by weighing 25 mg of primary standard gossypol or 
27.9 mg of gossypol acetic acid into a 50 ml vo lumetric flask. Dissolve in and make up to 
volume with complexing reagent. Solution is stable for 1 week if stored in refrigerator. The 
solution contains 0.50 mg gossypol per ml .Multiply gossypol acetic acid with 0.8962 to obtain 
mg of gossypol  
 
 
 
4 Procedu re  
 4.1  Weigh 0.5 – 0.75 gm sample accurately and transf er to a 50 ml volumetric flask  
4.2 Add 10 ml complexing reagent. Prepare reagent blank containing 10 ml of complexing reagent                                                     
in a 50 ml volumetric fl ask.  
4.3 Heat sample and blank in a water bath at 100°C for 30 minutes, cool, dilute to volume with 
isopropyl alcohol - hexane mixture.  
4.4 Filter through 11 cm filter paper into a 50 ml glass stoppered Erlenmeyer flask discarding first 5 
ml of the filterate . 
4.5  Pipette 2 ml of duplicate sample extract into 25 ml volumetric flasks. Pipette duplicate blank 
aliquots of same volume as sample aliquot into 25 ml volumetric flasks.  
4.6 Dilute one set of sample and blank aliquots with isopropyl – hexane mixture and reserve as  
reference solutions for absorption measurement. 4.7  
4.7 Add 2 ml of aniline by pipette to the other set of samples and reagent blank aliquots, heat in a 
water bath for 30 minutes, cool, dilute to volume with isopropyl – hexane mixture and mix well. 
Allow to s tand for 1 hour.  
4.8 Measure the absorbance at 440 nm of reagent blank treated with aniline using blank aliquot 
without aniline as reference solution.  
4.9 Determine absorbance of sample aliquot reacted with aniline using diluted sample aliquot without 
aniline as reference solution.  
4.10 Subtract absorbance of reagent blank from that of sample aliquot treated with aniline to obtain 
corrected absorbance.  
4.11 From corrected absorbance of sample aliquot determine mg gossypol in sample aliquot  
reference to a calibration graph  prepared  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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ANNEX  C 
(normative ) 
 Determination of ochratoxin A — High performance liquid chromatographic 
method with immunoaffinity column cleanup and fluorescence detection  
1.Principle  
Ochratoxin A (OTA) is extracted by  acetonitrile -water. The extract is purified using an immunoaffinity 
column and ochratoxin A is determined by high performance liquid chromatography (HPLC) on a 
reverse -phase column and fluorescence detection. The result is verified, if required, by deriva tization 
with boron trifluoride in methanolic solution.  
2.Reagents  
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and 
only distilled water or water of grade 1. Solvents shall be of quality for HPLC analysis.  
2.1 Acetonitrile . 
2.2 Methanol . 
2.3 Sodium chloride (NaCl) . 
2.4 Glacial acetic acid, φ(CH 3COOH) ≥ 98 %.  
2.5 Tween -20. 
2.6 Sodium bicarbonate (NaHCO 3). 
2.7 Disodium hydrogen phosphate (Na 2HPO 4). 
2.8 Potassium dihydrogen phosphate (KH 2PO 4). 
2.9 Potassium chl oride (KCl) . 
2.10 Hydrochloric acid , c(HCl) = 12 mol/l.  
2.11 Ochratoxin A , in crystal form or as a film in ampoules.  
2.12 Extraction solvent , mix 60 volume parts of acetonitrile ( 2.1) and 40 volume parts of water.  
2.13 Phosphate buffered saline (PBS) , diss olve 8 g NaCl ( 2.3), 1.2 g Na 2HPO 4 (2.7), 0.2 g KH 2PO 4 
(2.8) and 0. 2 g KCl ( 2.9) in about 990 ml water. Adjust pH to 7 with HCl ( 2.10) and dilute to 1 l with 
water.  
2.14 Washing solution , dissolve 25 g NaCl ( 2.3), 5 g NaHCO 3 (2.6) and 0. 1 ml Tween -20 (2.5) in 1 l 
water.  
2.15 Mobile phase , mix 48 volume parts of acetonitrile ( 2.1) with 51 volume parts of water and 1 
volume parts of glacial acetic acid ( 2.4) and degas this solution before use.  
2.16 Toluene . 
2.17 Solvent mixture , mix 99 volume parts of toluene  (2.16) with 1 volume parts of glacial acetic acid 
(2.4). 
2.18 Ochratoxin A stock solution . 
Dissolve 1 mg of the ochratoxin A (crystals) ( 2.11) or the contents of 1 ampoule (if ochratoxin A has 
been obtained as a film) ( 2.11) in solvent mixture ( 2.17) to g ive a soluti on containing approximately 20 
µg/ml to 30 µ g/ml of ochratoxin A.                                                                                                               TBS/AFDC 4(5975)P 3 
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 To determine the exact concentration, record the absorption curve between a wavelength of 300 nm 
and 370 nm in 5 nm steps in a 1 cm quartz cell ( 3.12) with solvent mixture ( 2.17) as reference. Identify 
the wavelength for maximum absorption by recording in 1 nm steps around the maximum as 
reference . 
Calculate the mass concentration of ochratoxin A, ρOTA, in micrograms per millilitre of solution using 
Formula (1) : 
                                                                            (1) 
where  
                 Amax is the absorption determined at the maximum of the absorption 
curve(here: at 333  nm);  
                 M is the relative molecular mass of ochratoxin A ( M = 403 g/mol);  
                 κ is the molar absorption coefficient of ochratoxin A, in solvent 
mixture(here: 544 m2/mol);  
                 δ is the path length of the cell i n centimetres.  
 
Store this solution at approximately −18 °C. A solution stored in this way is usually stable for 12 
months. Check the concentration of the solution if it is older than 6 months.  
2.19 Ochratoxin A standard solution , ρOTA = 1 µ g/ml.  
Evaporat e under a nitrogen flow 1 ml of the stock solution ( 2.18) or the aliquot portion which is 
equivalen t to an absolute amount of 100 µ g of ochratoxin A to dryness and dilute to 100 ml with the 
mobile phase ( 2.15). 
This solution can be stored in a refrigerator  at 4 °C. Stability shall be checked.  
2.20 Ochratoxin A calibration solutions.  
Pipette suitable volumes of ochratoxin A standard solution ( 2.19), e.g. 0.05 ml, 0.1 ml, 0.25 ml, 0. 5 ml 
and 1 ml into, for example, a 100 ml volumetric flask ( 3.15) and dilute to the mark with the mobile 
phase ( 2.15). The amount of ochratoxin A in the calibration soluti ons should cover the range of 0.05 
ng to 1.0 ng per 100 µ l injection volume. The calibration solutions should be freshly prepared from 
ochratoxin A standard solut ion (2.19) before each HPLC analysis.  
2.21 Sodium hypochlorite solution, ρ(NaOCl) = 4 g/100 ml.  
2.22 Boron trifluoride . 
2.23 Boron trifluoride in methanol solution , ρ(BF 3) = 14 g/100 ml.  
2.24 Dichloromethane . 
2.25 Sodium sulfate, anhydrous . 
2.26 Elution solvent , mix 98 volume parts of methanol ( 2.2) and 2 volume parts of glacia l acetic acid 
(2.4). 
WARNING — Use a well maintained fume hood. Avoid contact with skin, eyes, and respiratory 
tract.  
3.Apparatus and equipment  
Usual laboratory equipment and, in particular, the following.                                                                                                               TBS/AFDC 4(5975)P 3 
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 3.1 Analytical balance , accurate to 10 mg.  
3.2 Blender , 1 l jar and cover, explosion -proof.  
3.3 Filter paper , 
a) folded filter paper, or  
b) glass microfibre filter.  
3.4 Centrifuge tube , 50 ml.  
3.5 Membrane filter for aqueous solutions , made of polytetrafluoroethylene (PTFE), with a diameter 
of 25 mm and  a pore size of 0.2 µ m. 
3.6 Immunoaffinity column , which shall contain antibodies raised against ochratoxin A, ToxinFast ® 
Ochratoxin A Immunoaffinity Column (Huaan Magnech) 1) or equivalent.  
3.7 Glass syringe , 10 ml.  
3.8 Vacuum pump . 
3.9 Rotary evaporator , with a water bath capable of being controlled between 20 °C and 50 °C.  
3.10 Laboratory mill , suitable to grind to 1 mm.  
3.11 UV -Spectrometer , suitable for measurement at wavelengths of 300 nm up to 370 nm, having a 
spectral band width of not more than ± 2 nm. 
3.12 Quartz cells , with 1 cm optical path length and no significant absorption between wavelengths of 
300 nm and 370 nm.  
3.13 Conical flask , 150 ml.  
3.14 Sieve , with an aperture size of not more than 1 mm.  
3.15 Volumetric flask , 100 ml.  
3.16 Microsyrin ge, of capacity 500 µ l. 
3.17 HPLC apparatus , comprising  
a) high performance liquid chromatograph , eluent reservoir, a pump, an injection system, a 
fluorescence detector with variable wavelength setting and a data processing, e.g. an integrator 
with plotter , and  
b) analytical reverse -phase HPLC separating column , C18, which ensures a baseline resolved 
resolution of the ochratoxin A peak from all other peaks.  
length:  150 mm  
internal diameter:  4.6 mm  
spherical particles of size:  5 µm  
4. Procedure  
4.1 Gen eral 
The whole analytical procedure should be performed in one working day. If several samples are 
processed at the same time all samples should be analysed during the following night using an 
automatic sample injector.  
4.4 Extraction of ochratoxin A from the sample                                                                                                               TBS/AFDC 4(5975)P 3 
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 4.4.1 Extraction  
Place 25 g ( m), weighed to the nearest 0. 1 g, of the sample into a conical flask or a blender ( 3.2), add 
100 ml extraction solvent ( 2.12) (V1). Cover and shake for 30 min or blend for 3 min. The extract is 
centrifuged at 8 000 g for 5 min or filtered through folded filter paper [ 3.3 a)]. 
4.4.2 Dilution  
Pipet e 4.0 ml ( V2) filtered extract into 50 ml centrifuge tube ( 3.4), and dilute with 26. 0 ml ( V3) PBS 
solution ( 2.13). The diluted extract is centrifuged at 8000 g for 5min, and th en collected as extract A.  
Alternatively,  pipet 6. 0 ml ( V2) filtered extract into 50 ml centrifuge tube ( 3.4), and dilute with 39. 0 ml 
(V3) PBS solution ( 2.13). The diluted extract is filtered through a glass microfibre filter [ 3.3 b)], and 
then collected as extract B.  
4.5 Immunoaffnity column cleanup  
Pass all extract A or 30. 0 ml extract B ( V4) through the OTA immunoaffinity column at a flow -rate of 
about 1 to 2 drops per second, followed by 10 ml washing solution ( 2.14) and 10 ml distilled water at 2 
drops per second. Elute OTA with 1. 5 ml elution solvent ( 2.26). Evaporate eluate to dryness over 
steam bath under N 2 cautiously without exceeding 40°C. Redissolve in 0. 5 ml ( V5) mobile phase 
(2.15). Transfer to liquid chromatography (LC) vial. If necessary, th e sample can be filtered through 
PTFE membrane ( 3.5) before analysis by LC. Elution of ochratoxin A and the subsequent steps in the 
procedure described in this clause can depend on the type of immunoaffinity columns used. The 
elution volume, for example, s hould be checked to ensure it is appropriate for the type of column used.  
Be careful not to overload the immunoaffinity column . 
4.6 HPLC operating conditions  
When the column [ 3.17 b)] and the mobile phase ( 2.15) are used the following settings have been 
found to be appropriate.  
Flow rate:  1 ml/min  
Fluorescence detection:  Excitation wavelength:  333 nm  
Emission wavelength:  460 nm   
Injection volume:  50 µl  
Column temperature:  35 °C  
4.7 Calibration graph  
Prepare a calibration graph at the beginning of the analysis and whenever the chromatographic 
conditions are changed.  
Inject at least four calibration solutions of different sui table concentrations (see 2.20). Plot the 
fluorescence values (peak height or peak area) of the ochratoxin A calibration solution s (2.20) against 
the ochratoxin A mass concentrations in nanograms. Ensure that the linearity check is carried out  [5]. 
4.8 Ide ntification  
Identify ochratoxin A by comparing the retention time of the sample with that of the standard 
substance. Sometimes it can be necessary to identify the ochratoxin A peak by simultaneous injection 
of sample test solution and standard solution.  
4.9 Determination  
Chromatograph the sample. To carry out the determination by the external standard method, integrate 
the peak area or determine the peak height, and compare the results with the corresponding values 
for the standard substance with the neares t peak area/height, or use a calibration graph. In the case of 
a calibration graph, additional solutions with concentrations within the linear range may be prepared 
for the calibration graph.                                                                                                               TBS/AFDC 4(5975)P 3 
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 Inject equal volumes of sample test solution and standard soluti on used for the calibration graph.  
Read off the mass of ochratoxin A, in nanograms, corresponding to the fluorescence of the sample 
test solution from the calibration graph.  
If the ochratoxin A response of the sample is outside the calibration graph, adjus t the amount of 
sample injected by concentrating or diluting the sample test solution.  
4.10 Confirmation  
If necessary, confirm the identity by disappearance of the peak at the retention time for ochratoxin A 
and appearance of a new peak at the same retenti on time as that of standard  methyl ester of 
ochratoxin A.  
Take 200 µl of the sample test solution prepared as in 4.5, transfer into a pear -shaped flask and 
evaporate to dryness in a rotary evaporator ( 3.9). Take up the residue in 0.5 ml of dichloromethane  
(2.24), and add 1 ml of boron trifluoride methanol solution ( 2.23). 
Stopper the flask tightly and heat it in a water bath at 50 °C to 60 °C for 15 min. After cooling, transfer 
the solution into a 50 ml separating funnel containing 15 ml of water, shake th ree times with 5 ml of 
dichloromethane each time for 30 s. Combine the organic phases in a second 50 ml separating funnel, 
add 10 ml of water for washing and shake for 30 s.  
Subsequently filter the dichloromethane phase through sodium sulfate ( 2.25) into a  pear-shaped flask, 
evaporate to dryness, take up in 500 µl of mobile phase ( 2.15) and subject this solution to 
chromatographic separation under the conditions as described in 4.6. The completeness of 
derivatization can be checked from the chromatograms. I t is possible with this procedure to verify 
mass fractions of ochratoxin A of not less than 0.4 µg/kg.  
An adequate standard solution ( 2.20) should be treated separately to check the retention times of the 
ochratoxin A methyl ester and the completeness of t he derivatization.  
9 Calculation  
Calculate the mass fraction X1 of ochratoxin A in micrograms per kilogram using Formula (2) (external 
standard method):  
                                                                                                   (2) 
where  
V1 is the volume of the solvent used for extraction, in millilitres ( V1 = 100 ml);  
V2 is the volume of the extract used for dilution, in millilitres (extract A V2 = 4 ml,  or 
extract B V2 = 6 ml);  
V3 is the volume of the PBS solution used for di lution, in millilitres(extract A V3 = 26 
ml, or extract B V3 = 39 ml);  
V4 is the volume of the diluted extract used for cleanup, in millilitres ( V4 = 30 ml);  
V5 is the volume of the diluted extract used for cleanup, in millilitres ( V4 = 30 ml);  
c is the concentration of OTA in final solution  used for LC determination, in µ g/l;  
m is the mass of the test portion, in grams.  
Report the result in the relevant format and after rounding to two decimal places.                                                                                                               TBS/AFDC 4(5975)P 3 
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 NOTE Relevant legislation can apply.  
Indicate whether or not a correction for recovery has been applied.