TBS/AFDC4 (5976)P 3 
                                                                                                        REV TZS 52:2010   
  
  
  
  
 
           
  
               DRAFT  TANZANIA STANDARD  
          
                      Edible  coconut oil – Specification  
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
             TANZANIA BUREAU OF STANDARDS  
 
    ©TBS 2019  – All rights reserved  
  TBS/AFDC4 (5976)P 3 
                                                                                                        REV TZS 52:2010   
 Edible  coconut oil – Specification  
 
0 Foreword  
Coconut oil is extracted from the kernel or meat of mature coconuts  harvested from the coconut palm 
(Cocos nucifera  L.).It has various applications including  cooking  since the higher saturated fat content 
allows for greater stability  at higher temperatures and better shelf life.  It is one of traded edible 
vegetable oils in Tanzania.  
 
This Tanzania Standard  has been prepared to ensure the safety and quality of  edible coconut oil 
produced in, exported  or imported into the country.  
 
In preparation of this Tanzania standard con siderable help was derived from :  
CODEX STAN 210 -1999 (Amend ed 2015) Codex standard for named  vegetable oils  
published by Codex Alimentarius Commission   
 
ISO 15141 -2018  Cereals and cereal products — Determination of ochratoxin A — High 
performance liquid chromatographic method with immunoaffinity column cleanup an d 
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 b e done in accordance with TZS 4 .  
 
1 Scope  
This Tanzania Standard prescribes the requirements and the methods of sampling and test for edible  
coconut oil.  
 
2 Reference  
The following referenced documents are indispensable for the application of this Tanz ania Standard. 
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 numeri cal 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 o f 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 fats and oils Specifica tion  
TZS 1322, Oils and fats Sampling and test methods – Purity tests  
TZS 1324 , Animal and vegetable fats and oils – Determination of peroxide value – Iodometric (visual) 
end point determination  
TZS 1325, Animal and vegetable fats and oils – Determinati on of saponification value    TBS/AFDC4 (5976)P 3 
                                                                                                        REV TZS 52:2010   
 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 de nsity at 20  ° C– Reference method  
TZS 1329, Animal and vegetable fats and oils – Determination of refractive index  
TZS 1331,  Animal and vegetable fats and oils – Determination of acid value and acidity  
TZS 1332,  Animal and vegetable fats and oils – Deter mination 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 atomic absorption  
TZS 1336,  Animal and vegetable fats and oils – Dete rmination of insoluble impurities content  
TZS 1369,  Animal and Vegetable fats and oils – Determination of Butylhydroxyanisole (BHA) and 
Butylhydroxytoluene (BHT) – Gas liquid chromatographic method  
TZS 1370 , Animal and Vegetable fats and oils – Determinati on 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 oil s  Detection and identification of antioxidants - Thin-
layer chromatographic method  
 
3 Terms and definitions  
For the purpose of this Tanzania Standard, the following  terms and  definitions below shall apply:  
 
3.1 edible oils  
food stuffs which are compose d 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 acid s naturally present in t he 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  coconut oil  
edible oil derived fro m the kernel of Cocos nucifera Linn . 
 
3.4 virgin coconu t oil 
coconut 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 been purified by washing with 
water, settling, filtering and centrifuging only.  
 
3.5 refined coconut oil  
coconut oil obtained, by mechanical procedures and/or solvent extraction and subjected to refining 
processes  
 
4 Requirements  
4.1General  requirements  
Edible  coconut oil shall  
a) be fit for human consumption;  
b) be free from  foreign and   rancid odour and taste;  
c) have colour  characte ristic of designated product  ;   TBS/AFDC4 (5976)P 3 
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 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 origi n when tested in 
accordance with the methods prescribed in TZS 1322.  
 
4.2 Specific requirements  
Edible coconut  oil shall comply with  specific requirements  given in table  1 when tested in 
accordance with the methods specified therein . 
Table  1-Specific  requ irements for edible coconut  oil 
S. No.  Parameter  Requirement  Test method  
i.  Relative density(  at 40 oC/water at 20  oC) 0.908 -0.921  TZS 1328  
  
ii.  Refractive index, ( ND 40 o C ) 1.448 - 1.450  TZS 1329  
 
iii.  Saponification value, mg KOH/g oil  248 - 265 TZS 1325  
 
iv.  Iodine value  (Wij’s) , g/100  6.3– 10.6 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 volatile at 105 o C, % 
m/m, max.  0.2 TZS 1326  
viii.  Insol uble impurities, % m/m, max.  0.05 TZS 1336  
ix.  Free Fatty Acids (FFA)  as lauric acid,  % 
max.  Refined oils: 0. 2 
Cold pressed and 
virgin oils: 1.5 
 TZS 1331  
x.  Peroxide value, mEq peroxide - oxygen/kg 
oil, max  Refined oils :10  
Cold pressed and 
virgin oils: 15  
 TZS 1324  
xi.  Total sterols(mg/kg)  400-1200 TZS 1371  
xii.  Reichert values  6  -   8.5 See Annex B  
xiii.  Polenske values  13  -  18 See Annex B  
 
4.3 Food additives  
4.3.1 General  
4.3.1.1  Food additives may be used during processing of edible coconut oil and shall be in accordance 
to Codex Stan 192.  
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 subject to tables 2, 3, and 4 .   TBS/AFDC4 (5976)P 3 
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 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.  
             Table 2  – Requirements for antioxidant in  edible  coconut oil  
 
INS 
No. Antioxidant  Requirements  Test method  
304 Ascorbyl palmitate, mg/kg, max  
500 (sing ly or 
in 
combination)  TZS 1775  
305 Ascorbyl stearate , mg/kg, max  
307a  Tocopherol, 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  
 
 
Antioxidant  synergists specified in Table 3  may be used.  
                              Table 3  — Antioxidant synergists  
INS 
No. Antioxidant synergist  Requ irements  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 e sters 
of glycerol , mg/kg, max  
 
4.3.4 Antifoaming agents (deep frying oil)  
Antifoaming agents specified in Table 4 may be used.    TBS/AFDC4 (5976)P 3 
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 Table 4 — Antifoaming agents  
INS 
No. Antifoaming agent  Requirements  Test method  
900a  Polydimethylsiloxane, mg/kg, 
max 10  TZS  1775  
 
4.4 Fortification  
Edible coconut oil may be fortified in accordance with TZS 1313.  
 
5    Hygiene  
 
Edible coconut oil  shall be produced, processed, handled and stored in accordance n TZS 109.  
 
6 Contaminants  
 
6.1 Pesticide residues  
Edible coconut  oil shall comply with relevant maximum pesticide residue limits established by th e 
Codex Alimentarius Commission.  
6.2 Soap content  
Edible coconut  oil shall have the soap content not exceed 0.005%  when tested according to TZS 
1322  
 
6.3. Metals  contaminants  
Edible coconut  oil shall comply with  maximum limits established by Codex Alimentarius Commission 
as specified in Table 5 . 
Table 5– Acceptable levels of metal contaminants in edible  coconut 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 coconut  oil shall be free from Poly -Hydroxy alkanoates (PHA).  
 
 
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 7    Sampling and Tests  
 
7.1 Sampling  
Edible  coconut 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 
Anne xes of this Tanzania St andard.  
7.2.2 Quality of reagents  
Unless specified otherwise, pure analytical grade chemicals and distill ed water shall be used in tests.  
 
 
8 Packaging, marking and labeling  
Edible  coconut oil shall be packed, marked and la beled in accordance with TZS 538. 
 
8.1 Packaging  
Edible  coconut oil shall be packaged in containers made from food grade packaging material and 
sealed in a manner that will safeguard the hygienic, nutritional and organoleptic properties of the 
product throughout the shelf life of the product . 
 
8.2 Marking and labeling  
8.2.1 Containers of edible  coconut oil 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/or 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) Manufacturer s registered trade mark  
j) Country of origin  
k) The phrase “Place the products away from direct sunlight”.  
l) Nutritional Information(optional)  
 
8.2.2 The containers shall also be marked with the TBS Standards Mark of Quality.  
 
NOTE  - The TBS Mark of Quality may b e 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  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                             
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                            
Carb on configuration   
Fatty acid 
composition(%)  Test Methods  
 
 
C6:0  ND-0.7       TZS 288(Part 1 
&2) 
 
C8:0  4.6-10.0  
C10:0  5.0-8.0  
C12:0  45.1-53.2  
C14:0  16.8-21.0  
C16:0                 7.5-10.2  
C16:1  ND  
C17:0  1.0-3.0  
C17:1  12.0-19.0  
C18:0  1.0-3.5  
C18.1  ND-0.2  
C18:2  ND-0.2  
C18:3  ND-0.2  
C 20:0  ND-0.2  
C 20:1  ND-0.2  
C20:2  ND  
C22:0  ND  
C22:1  ND  
C22:2  ND  
C24:0  ND  
C24:1  ND  
NOTE - Free fatty acid composition is expressed as % of total fatty acids  
                              ND-none detectable,defined as ≤0.05    TBS/AFDC4 (5976)P 3 
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                                                           ANNEX B  
                                                                       (normative)  
                                        Determination of Reichert -Meissl and Polenske Value  
Principle  
The material is saponified by heating with glycerol sodium hydroxide solution and then split by 
treatment with dilute sulfuric acid. The volatile acids are immediately steam distilled. The soluble 
volatile acid in the distillate are filtered out and estimated by titration with standard sodium hydroxide 
solution.  
 Analytical Importance  
These determinatio ns have been used principally for analysis of butter and margarines. Butter fat 
contains mainly butyric acid glycerides. Butyric acid is volatile and soluble in water.  
No other fat contains butyric acid glycerides, and therefore, the Reichert -Meissl value  of the butter fat 
is higher than that for any other fat. Coconut oil and palm kernel oil contain appreciable quantities of 
caprylic capric and lauric acid glycerides. These fatty acids are steam volatile but not soluble in water, 
and hence give high Polen ske value.  
Apparatus  
a. An all-glass distillation  assembly conforming to specifications given in AOCS Official Methods 
Cd 5 -40 or Methods of Analysis, AOAC - 17th Edn.,2000 Figure 925.41, chapter 41 page 14 or 
distillation apparatus as shown in the diagram bel ow; 
b. 25 ml beaker   
c. 100 ml graduated cylinder   
d. 100 ml pipette  
e. Graduated burette  
f. Asbestos board with a hole about 65 mm dia  for supporting the flask over the burner. During  
distillation the flask shall fit snugly into the hole of the board to prevent the fl ame from impinging 
on the surface of the flask above the hole.  
g. Bunsen burner  sufficiently large to allow completion of distillation in the prescribed time.  
 
 
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                                          Figure 1:    Reichert -Meissl Distillation Apparatus  
 
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Reagents  
a. Glycerine  
b. Concentrated sodium hydroxide solution : 50 % (w /w) Dissolve Sodium Hydroxide in equal wt 
of water and store solution in a bottle. Use clear solution free from deposit.  
c. Pumice stone grains   
d. Dilute sulfuric acid solution : Approximately  1.0 N  
e. Sodium hydroxide solution : 0.1N solution in water, accurately standardised  
f. Phenolpthalein indicator : Dissolve 0.1 g of phenolpthalein in 100 ml of ethyl alcohol  
g. Ethyl alcohol : 90% by volume and neutral to phenolphthalein  
 
 Procedure  
1. Weigh accurate ly 5 ±0.1g of filtered oil or fat sample into a clean, dry, 300ml distilling flask.  
 
2. Add 20 ml of glycerine and 2 ml of concentrated sodium hydroxide solution, and heat with swirling 
over a flame until completely saponified, as shown by the mixture becomi ng perfectly clear.  
 
3. Cool the contents slightly and add 90 ml of boiling distilled water, which has been vigorously 
boiled for about 15 min. After thorough mixing the solution should remain clear. If the solution is 
not clear (indicating incomplete saponif ication) or is darker than light yellow (indicating over -
heating), repeat the saponification with a fresh sample of the oil or fat. If the sample is old, the 
solution may sometimes be dark and not clear.  
 
4. Add about 0.6 - 0.7 gm of pumice stone grains, and 50 ml of dilute sulfuric acid solution.  
 
5. Immediately connect the flask to the distillation apparatus. Place the flask on asbestos board so 
that it fits snugly into the aperture. This will prevent the flame from impinging on the surface of the 
flask above the level of the liquid and avoid super heating.  
 
6. Heat very gently until the liberated fatty acids melt and separate. Then set the flame so that 110 
ml of distillate shall be collected within 19 to 21 min. The beginning of the distillation is to be taken 
as the moment when the first drop of the distillate falls from the condenser in the receiving flask. 
Keep the water in the condenser flowing at a sufficient speed to maintain the temperature of the 
outgoing water from the condenser between 15 and 20°C. Col lect the distillate in a graduated 
flask.  
 
7. When the distillate exactly reaches the 110 ml mark on the flask, remove the flame and  quickly 
replace the flask by a 25 ml measuring cylinder. Stopper the graduated flask and without mixing 
placed it in a water bath maintained at 15ºC for 10 min so that the 110 ml graduation mark is 1 cm 
below the water level in the bath. Swirl round the contents of the flask from time to time.  
 
8. Remove the graduated flask from the cold water bath, dry the outside and mix the con tent gently 
by inverting the flask 4 to 5 times without shaking. Avoid wetting the stopper with the insoluble 
acids.  
 
 
9. Filter the liquid through a dry, 9 cm Whatman No. 4 filter paper. Reject the first 2 -3 ml of the 
filterate and collect the rest in a dry  flask. The filtrate should be clear.  
 
10. Pipette 100 ml of the filtrate and add 5 drops of the phenolphthalein solution, and titrate against 
standard 0.1N sodium hydroxide solution.  
 
11. Run a Blank Test without the fat, but using the same quantities of the rea gents.  
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Calculation  
 Reichert -Meissl Value = (A – B) x N x 11  
 where,  
A = Volume in ml of standard sodium hydroxide solution required for the the test;  
B = Volume in ml in standard sodium hydroxide solution required for the blank; and  
N = Normality of s tandard sodium hydroxide solution.  
 
Determination of Polenske Value  
After titrating the soluble volatile acids, detach the still head and rinse t the condenser with three 
successive 15 ml portions of cold distilled water passing each washing separately th rough the 
measuring cylinder, 110 ml graduated flask and the filter paper and allow all of it to pass through.  
Discard all the washings. Place the funnel on a clean conical flask. Dissolve the insoluble fatty acids 
by three similar washings of the condens er, the measuring cylinder, the 110 ml flask with stopper, and 
the filter paper with 15 ml portions of ethyl alcohol.  
Combine the alcoholic washings in a clean flask, add 5 drops of phenolphthalein indicator solution, 
and titrate with standard (0.1N) sodi um hydroxide solution.  
                           Polenske value = 10 x V x N  
where,  
V = Volume in ml of standard sodium hydroxide solution required for the test; and  
N = Normality of the standard sodium hydroxide solution.  
Note: - Unless the directions are followed in every detail reproducible results cannot be obtained.  
 
 
 
 
 
 
 
 
 
 
 
 
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ANNEX  C 
(Normative ) 
 Determination of ochratoxin A — High performance liquid chromatographic 
method with immunoaffinity column cleanup and fluorescence detection  
1.Principl e 
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 derivatization 
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 sha ll 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 chloride (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) , dissolve 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 a bout 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 v olume 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 sol ution . 
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 give a soluti on containing approximately 20 
µg/ml to 30 µ g/ml of ochratoxin A.    TBS/AFDC4 (5976)P 3 
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 To dete rmine 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 st eps 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.  
3.1 Analytical balance , accurate to 10 mg.    TBS/AFDC4 (5976)P 3 
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 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  
4.4.1 Extraction    TBS/AFDC4 (5976)P 3 
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 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 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 the n 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 a s 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, the  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, sh ould 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.  
Inject equal volumes of sample test solution and standard soluti on used for the calibration graph.    TBS/AFDC4 (5976)P 3 
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 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.  
NOTE Relevant legislation can apply.  Indicate whether or not a correction for recovery has been applied.    TBS/AFDC4 (5976)P 3 
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