TBS/AFDC 4(5978)P 3 
                        
 
                              DRAFT  TANZANIA STANDARD  
 
                       Edible  Palm Kernel  Oil– Specification  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  TANZANIA BUREAU OF STANDARDS                                                                                                                                                                                              
TBS/AFDC 4(5978)P 3 
  
Edible Palm Kernel Oil  – Specification  
 
0 Foreword   
Palm kernel oil is obtained from t he kernel of the oil palm fruit. Its composition and properties differ 
significantly from palm oil.  It contains more saturated fats than palm oil and is very commonly used in 
commercial 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 palm kernel oil 
produced in , exported  or imported into the country.  
 
In preparation of this standard considerable help was derived from : 
 
CODEX STAN 210  -1999  (Amended 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 and 
fluorescence detection  published by International Organization for Standardization.  
 
In reporting the results of a test  or analysis made in accordance with this 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 specifies  the requirement s and methods of sampling and test for  edible  palm 
kernel oil. 
 
2  Reference  
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 (i ncluding 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, Anim al and vegetable fats and oils - Preparation of methyl esters of  fatty acids  
 TZS 288 -2, Anima l 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 -Specification  
 TZS 1322, Oils and fats Sampling and test methods – Purity tests                                                                                                                                                                                              
TBS/AFDC 4(5978)P 3 
  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 – 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 0c – Reference method  
 TZS 1329, Animal and vegetable fats and oils – Determination of ref ractive 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 unsaponif iable 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 – Determination of insolubl e 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 – 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 ide ntification of antioxidants - Thin-layer 
chromatographic method  
 
3 Terms and definitions  
For the purpose of this Tanzania standard the following terms and definitions shall apply:  
 
3.1 edible oils  
food stuffs which are composed of glycerides of fatty acid s 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 palm kernel oil   
edible plant oil  derived from the  kernel  of the fruit of the oil  palm  Elaeis guineensis .  
 
3.4 virgin palm kernel  oil 
palm kernel 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 palm kernel  oil 
palm kernel  oil obtained, by mechanical procedures and/or solvent extraction and subjected to refining 
processes .                                                                                                                                                                                             
TBS/AFDC 4(5978)P 3 
 3.6 cold pressed palm kernel  oil 
palm kernel  oil obtained, without altering the nature of the  oil by mechanical procedures, for example, 
expelling or pressing, without the application of heat. It may have been purified by washing with water, 
settling, filtering and centrifuging only . 
 
4 Requirements  
4.1 General  requirements  
Edible palm kernel oil shall; 
a) be fit for human consumption;  
b) be free from foreign and  rancid odour and taste;  
c) have colour  characteristic of designated product ;  
d) be clear and free from adulterants, sediments, suspended or foreign matter and separated 
water and;  
e) be free from admi xture with mineral or other oils of vegetable or animal origin when tested in 
accordance with the methods prescribed in TZS 1322.  
 
4.2 Specific requirements  
Edible palm kernel 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 palm kernel  oil 
S. No.  Parameter  Requirement  Test method   
i.  Relative density( at 40oC/water at 20oC) 0.899 -0.914  TZS 1328  
ii.  Refractive index,  (ND 40 o C ) 1.448 - 1.452  TZS 1329  
iii.  Saponification value, mg KOH/g oil  230 - 254 TZS 1325  
iv.  Iodine value  (Wij’s) , g/100  14.1 – 21.0 TZS 1327  
v.  Unsaponifiable matter, g/kg, max.   10
 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.  Insoluble impurities, % m/m, max.  0.05 TZS 1336  
ix.  Free Fatty Acid(FFA) as lauric acid , 
%max.  Refined :      0.2 
Cold pressed and 
virgin oil s: 1.5 TZS 1331  
x.  Peroxide value, mEq peroxide oxygen/kg 
oil, max.  Refined :10  
Cold pressed and 
virgin oil s: 15 TZS 1324  
xi.  Colour, units in a 0.25in ch cell Lovibond, 
max.   Red          1.5 
Yellow         20 TZS 1330  
 
xii.  Total sterols(mg/kg)  700-1400  TZS 1371  
xiii.  Reichert values  4  -   7 See Annex B  
xiv.  Polenske values  8  -  12 See Annex  B                                                                                                                                                                                             
TBS/AFDC 4(5978)P 3 
 4.3 Food additives  
4.3.1 General  
4.3.1.1  Food  additives may be used during processing of edible palm kernel  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 non -virgin the 
additives may be used subject to tables 2, 3, and 4 . 
4.3.2 Flavours  
Natural flavours and their identical synthetic equivalent s, and other synthetic flavours may be used, 
except those which are known t o be hazardous .  
4.3.4 Antioxidants  
Antioxidants specified in Table 2 may be used.  
                 Table  2-Antioxidant  
INS 
No. Antioxidant  Requirements  Test method  
304 Ascorbyl pa lmitate, mg/kg, max  
500 (singly 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 galla tes, BHA, BHT, and/or 
TBHQ , mg/kg, max  200 within 
individual 
limits  TZS 1775  
389 Dilauryl thiodipropionate , mg/kg, max  200  
 
4.3.4 Antioxidant synergists  
Antioxidant  synergists specified in Table 3  may be used.  
             
                                                                                                                                                                                             
TBS/AFDC 4(5978)P 3 
                      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  
 
4.3.5 Antifoaming agents (deep frying oil)  
Antifoaming agents specified in Table 4 may be used.  
Table  4 — Antifoaming agents  
INS 
No. Antifoaming agent  Requirem ents Test method  
900a  Polydimethylsiloxane, mg/kg, 
max 10  TZS  1775  
 
4.4 Fortification  
Edible palm kernel  oil may be fortif ied in accordance with TZS 1313.  
 
5  Hygiene  
 
Edible palm kernel oil  shall be produced, processed, handled and stored in accordanc e with TZS 109.  
6 Contaminants  
6.1 Pesticide residues  
Edible palm kernel oil shall comply with relevant  maximum pesticide residue limits established by th e 
Codex Alimentarius Commission.  
6.2   Soap content  
Edible palm kernel oil shall have the soap content  not exceed 0.005%  when tested according to TZS 
1322  
6.3     Metals  contaminants  
Edible palm kernel oil shall comply with  maximum limits being established by Codex Alimentarius 
Commission as specified in Table 5 . 
 
 
 
                                                                                                                                                                                             
TBS/AFDC 4(5978)P 3 
 Table 5 — Requirements  for metal contam inants in edible palm kernel  oil 
S. No.  Contaminant  Requirements  Test method  
i ) Copper (Cu) mg/kg,max  
Virgin          0. 4 
Refined       0.1 TZS 1335  
ii) Iron (Fe) mg/kg ,max   
 Virgin          5.0 
Refined        1.5 
 TZS 1335  
    iii) Lead, mg/kg , max  0.1 
 TZS 268 
iv) Arsenic, mg/kg , max  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 test ed in accordance with the method specified 
in Annex C   
 
6.4.3  Edible palm kernel oil shall b e free from  Poly-Hydroxy alkanoates (PHA) . 
 
7  Sampling and Tests  
 
7.1 Sampling  
Edible palm kernel  oil shall be sampled as prescribed in TZS 54 . 
 
7.2 Test s  
7.2.1 Testing shall be done as per TZS 1322 and as provided in the respective Tables and Annex es of 
this standard.  
 
7.2.2 Quality of reagents  
Unless specified otherwise, analytical grade chemicals and distill ed water shall be used in tests  
 
 
8  Packaging, mark ing and labeling  
Edible palm kernel  oil shall be packed, marked and labeled in accordance with TZS 538 . 
 
8.1  Packaging  
Edible palm kernel  oil shall be packaged in food grade containers and sealed in manner to ensure the 
safety and quality requirements spe cified in this standard are maintained throughout the shelf life of 
the product.  
8.2 Marking and labeling  
8.2.1  In addition each Container 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/or packer  
d) Date of manufacture and expiry  date 
e) Language -Kiswahili /English or Kiswahili  and English  
f) A complete list of ingredie nts in descending order of proportion  
g) Net content  
h) Batch number                                                                                                                                                                                              
TBS/AFDC 4(5978)P 3 
 i) Manufacturers 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 may also be marked wi th 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.  
 
                                                                                                                                                                                             
TBS/AFDC 4(5978)P 3 
  
ANNEX A  
(normative)  
                                              Free fatty acids composition  
 
 
 
 
 
 
 
 
 
 
 
                    
 
 
 
 
                      
 
               NOTE -Free fatty acid composition is expressed as % of total fatty acids  
                               ND-none detectable,defined as ≤0.05%  
 
 
 
 
 
 
 
 
  
Carbon configuration   
Range  Method of test  
 
             C6:0  ND-0.8  
             C8:0  2.4-6.2  
             C10:0  2.6-5.0  
C12:0  45.0-55.0  
C14:0  14.0-18.0  
C16:0  6.5-10.0  
C16:1  
C17:0  
C17:1  ND-0.2 
                              ND 
                              ND TZS 288(Part 
1&2 
 
C18:0  1.0-3.0  
C18:1  12.0-19.0  
C18:2  1.0-3.5  
C18.3  ND-0.2  
C20:0  ND-0.2  
C20:1  ND-0.2  
 C 22:1  
C 22:2  
C 24:0  
C24:1  ND 
ND 
ND 
ND  
   
                                                                                                                                                                                               
TBS/AFDC 4(5978)P 3 
                                                              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 filter ed out and estimated by titration with standard sodium hydroxide 
solution.  
 Analytical Importance  
These determinations have been used principally for analysis of butter and margarines. Butter fat 
contains mainly butyric acid glycerides. Butyric acid is vol atile 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 Polenske value.  
Apparatus  
a. An all-glass distillation  assembly conforming to specifications given in AOCS Official Methods 
Cd 5 -40 or Methods of An alysis, AOAC - 17th Edn.,2000 Figure 925.41, chapter 41 page 14 or 
distillation apparatus as shown in the diagram below;  
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 flame 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.                                                                                                                                                                                              
TBS/AFDC 4(5978)P 3 
  
 
                                              
                        Fig 1.  Reichert -Meissl Distillation Apparatus  
 
                                                                                                                                                                                             
TBS/AFDC 4(5978)P 3 
  
Reagents  
a. Glycerine  
b. Concentrated sodium hydroxide solution : 50 % (w /w) Dissolve Sodium Hydroxide in equal wt 
of wat er 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 accurately 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 o f concentrated sodium hydroxide solution, and heat with swirling 
over a flame until completely saponified, as shown by the mixture becoming 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 saponification) 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. Collect the distillate in a graduated 
flask.  
 
7. When the distillate exactly reaches the 110 ml mark on the flask, remove th e 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 content 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 reagents.  
                                                                                                                                                                                             
TBS/AFDC 4(5978)P 3 
 Calculation  
 Reichert -Meissl Value = (A – B) x N x 11  
 where,  
A = Volume in ml of standard sodium hydroxide so lution required for the the test;  
B = Volume in ml in standard sodium hydroxide solution required for the blank; and  
N = Normality of stand ard 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 through 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 condenser, 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) sodium hydroxide solution.  
                           Polenske value = 10 x V x N  
where,  
V = Volume in ml of standard sodi um 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.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                                                                                                                                                                             
TBS/AFDC 4(5978)P 3 
 ANNEX C  
(Normative ) 
 Determinatio n 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 o chratoxin 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 ana lysis, 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 ac id, 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.  
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                                                                                                                                                                                             
TBS/AFDC 4(5978)P 3 
 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.  
3.2 Blender , 1 l jar and cover, explosion -proof.                                                                                                                                                                                              
TBS/AFDC 4(5978)P 3 
 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/AFDC 4(5978)P 3 
 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/AFDC 4(5978)P 3 
 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.                                                                                                                                                                                              
TBS/AFDC 4(5978)P 3 
 Indicate whether or not a correction for recovery has been applied.