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 DRAFT  TANZANIA STANDARD  
          
                      Edible sesame seed oil – Specification  
 
 
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
                 TANZANIA BUREAU OF STANDARDS  
 
    ©TBS 2019  – All rights  reserved  
                                                                                                                                  TBS/AFDC4 (5977 )P3 
                                                                                                  REV TZS 51:2010   
 
Edible sesame seed oil – Specification  
 
0  Foreword  
Sesame seed oil is derived from the seed of the sesame plant . Sesame seeds are pressed and 
crushed to release the oils. There are many processing methods for this oil, which either involve 
manually intensive techniques or chemical extraction methods . It is a popular edible oil that is 
commonly used as a food ingredient and condiment . Edible sesame seed  oil is one of traded edible 
vegetable oils in Tanzania . 
 
This Tanzania Standard  has been prepared to ensure the safety and quality of edible sesame seed 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 (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 
sesame seed oil.  
 
2  Normative r eference s 
The following referenced documents are indispensable for the application of this document. For dated 
references, only th e 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 Ve getable  fats and oils - Sampling.  
TZS 76, Methods for determination of arsenic  
TZS 109 , Food processing units –Code of hygiene  
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 – Determinatio n 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 te st 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 -Determination of saponification value.                                                                                                                                    TBS/AFDC4 (5977 )P3 
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 TZS 1326 , Animal and veg etable 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 vegeta ble 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 atomic absorption.  
 TZS 1336 , Animal and vegetable fats and oil s-Determination 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 – Dete rmination of tocopherol and tocotrienol content 
by High Performance Liquid Chromatography  
 TZS 1775 , Animal and Vegetable fats and oils - Detection and identification of antioxidants - Thin-layer 
chromatographic method  
 TZS 1371, Animal and vegetable fats an d oils — Determination of individual and total sterol contents -
Gas chromatographic method  
3 Terms and d efinitions  
For the purpose of this Tanzania Standard, the following terms and definitions below shall apply:  
 
3.1 edible oils  
food stuffs which are com posed 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 sesame seed oil (sesame oil ) 
edible  oil derived from sesame seeds (seeds of Sesamum i ndicum  L.). 
 
3.4 virgin sesame seed oil   
sesame 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 sesame seed oil  
sesame oil obtained, by mechanical procedures and/or solvent extraction and subjected to refining 
processes  
 
3.6 cold pressed sesame seed oil  
sesame 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.  
 
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4 Requirements  
4.1 General requirements  
Edible  sesame se ed 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  ; 
d) be clear and free from adulterants,  sediment s, suspended or foreign matter and separated 
water and;  
e) be free fr om admixture 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 sesame seed oil shall comply with  specific requirements  given in table  1 when teste d in 
accordance with the methods specified therein  
Table 1 —Specific  requirements of edible sesame  seed  oil 
S. No.  Characteristic  Requirement  Test method  
i) Moisture and matter volatile at 105 o C, % 
m/m, max.  0.2 TZS 1326  
ii) Insoluble impurities, % m/m , max.  0.05 TZS 1336  
iv) Relative density ( at 20 o C/ water at  
20 o C)  0.915 - 0.924  TZS 1329  
v) Refractive index,  (ND 40 o C) 1.465 - 1.469  TZS 1328  
vi) Saponification value, mg KOH/g oil  186 - 195 TZS 1325  
vii) Iodine value  (Wij’s) ,g/100  104 - 120 TZS 1327  
viii) Colour, units in a 0.25  inch cell Lovibond, 
max.  Refined  oil: 2R  
Virgin oil: 20R  TZS 1330  
ix) Unsaponifiable matter, g /kg, max . 20 TZS 1332  
x) Free Fatty Acid(FFA) as oleic acid , %max Refined : 0.3 
Virgin oil:   2  TZS 1331  
xi) Peroxide value, mEq peroxide oxygen/kg 
oil, max.  10  TZS 1324  
   xii) Fatty acids composition,%of total fatty 
acids  As in Annex A  TZS 288 (Part 
1&2)  
   xiii) Total sterols , (mg/kg)  4500 -19000  TZS 1371  
xiv) Presence of sesame seed oil  Positive  Annex B  
4.3 Food ad ditives  
4.3.1  General  
4.3.1.1  Food additives may be used during processing of edible sesame seed 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 (5977 )P3 
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4.3.2  Flavours  
Natural flavours and their identical synthetic equivalents, and other synthetic flavours may be used, 
except those wh ich are known to be hazardous.  
4.3.4  Antioxidants  
Antioxidants specified in Tab le 2 may be used.  
Table 2 — Antioxidants  
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  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  
 
4.3.5 Antioxidant synerg ists 
Antioxidant synergists specified in Table 3 may be used.  
                       Table3 — 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/AFDC4 (5977 )P3 
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4.3.6  Antifoaming agents (deep frying oil)  
Antifoaming agents specified  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 sesame seed  oil may be fortifi ed in accordance with TZS 1313  
 
5 Hygiene  
 
Edible sesame seed oil  shall be produced, processed, handled and stored in accordance with TZS 
109. 
 
6 Contaminants  
 
6.1 Pesticide residues  
Edible sesame  seed  oil shall comply with relevant  maximum pesticide residue limits established by the 
Codex Alimen tarius Commission for this commodity.  
 
6.2   Soap content  
Edible sesame seed oil shall have the soap content not exceed 0.005%  when tested according to 
TZS 1322  
6.3   Metal contaminants  
Edible sesame seed oil shall comply with maximum limits established by  Codex Alimentarius 
Commission as specified in Table 5.  
           Table 5  — Requirements for metal contaminants in edible sesame  seed  oil 
S. No.  Contaminant  Requirements  Test method  
i) Iron, mg/kg, max  Virgin:   5.0 
Refined :1.5 
  
 
TZS 1335  
ii) Copper, mg/kg, max  Virgin:     0.4 
Refined : 0.1 
iv) Lead, mg/kg, max  0.1 TZS 268 
 
v) 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 tested in accordance with the method specified 
in Annex C.  
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6.4.3  Edible sesame seed oil shall b e free from  Poly-Hydroxy alkanoates (PHA).  
 
7  Sampling a nd Tests  
 
7.1 Sampling  
Edible sesame seed 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 
Annexe s of this Tanzania Standard.  
7.2.2 Quality of reagents  
Unless specified otherwise,  analytical  grade chemicals and distill ed water shall be used in tests  
 
8  Packaging, Marking and Labeling  
Edible sesame seed oil shall be packed, marked and labeled in accordance with TZS 538 . 
 
8.1  Packaging  
Edible sesame  seed  oil shall be packaged in food grade c ontainers 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 container of product shall be legibly and indel ibly 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) 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 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 grant ed, 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  
C14:0  ND-0.1  
C16:0                 7.9-12.0  
C16:1  ND-0.2  
C17:0  ND-0.2  
C17:1  ND-0.1  
C18:0  4.5-6.7  
C18.1  34.4-45.5  
C18:2  36.9-47.9  
C18:3  0.2-1.0  
C 20:0  0.3-0.7  
C 20:1  ND-0.3  
C20:2  ND  
C22:0  ND-1.1  
C22:1  ND  
C22:2  ND  
C24:0  ND-0.3  
C24:1  ND  
NOTE - Free fatty acid  composition is expressed as % of total fatty acids  
                              ND-none detectable,defined as ≤0.05                                                                                                                                    TBS/AFDC4 (5977 )P3 
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                                                                       ANNEX B  
(normative)  
 
Test for the presence of sesame seed oil (mo dified baudiouin test)  
 
A.1 General   
 
The development of a permanent pink colour with fur fural solution in the presence of hydrochloric acid 
indicates the presence of sesame seed oil.  
 
A.2 Reagents   
 
The reagents shall be of recognized analytical qualit y.  
 
A.2.1 Hydrochloric acid (36-37%) , specific gravity 1.19  
 
A.2.2  Furfural solution   
 
Two per cent (2%) of furfural distilled not earlier than 24 hours prior to the test in 90% (v/v) ethanol 
(specific gravity 0.832 to 0 835). The reagent is stable up to  3 months if kept in a refrigerator  
 
A.3 Procedure  
 
A.3.1  Take 5 ml of the oil or melted fat in a 25 ml measuring cylinder (or test tube) provided with a 
glass stopper, and add 5 ml of hydrochloric acid and 0.4 ml of furfural solution.  
 
A.3.2  Insert the glass stopper and shake vigorously for two minutes.  
 
A.3.3  Allow the mixture to separate.  
 
A.3.4  The development of a pink or red colour in the acid layer indicates presence of sesame seed oil.  
 
A.3.5  Confirm by adding 5 ml of water and shaking again.  
 
A.3.6  If the colour persists, sesame seed oil is present. If the colour disappears, sesame seed oil is 
absent.  
 
 
 
 
 
 
 
 
 
 
 
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ANNEX C 
(Normative ) 
 
 Determination of ochratoxin A — High performance liquid chromatographic 
method with immunoaffinity column cle anup 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 col umn 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 distill ed 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 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 (5977 )P3 
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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.                                                                                                                                    TBS/AFDC4 (5977 )P3 
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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/AFDC4 (5977 )P3 
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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 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 t hen 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, t he 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, should 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                                                                                                                                   TBS/AFDC4 (5977 )P3 
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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.  
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 se parating funnel containing 15 ml of water, shake three 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 dichlor omethane 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. It 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 the 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 dilution, 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 use d for cleanup, in millilitres ( V4 = 30 ml);                                                                                                                                    TBS/AFDC4 (5977 )P3 
                                                                                                  REV TZS 51:2010   
 
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 pla ces. 
NOTE Relevant legislation can apply.  
Indicate whether or not a correction for recovery has been applied.