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            DRAFT TANZANIA STANDARD  
 
 
Edible olive oil and edible  olive pomace oil — Specification  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
               
 
 
TANZANIA BUREAU OF STANDARDS  
 
 
 
TBS/AFDC 4(5974)P 3 
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 Edible  olive oil and edible  olive pomace oil — Specification  
 
0 Fore word  
Olive oil is produced by pressing whole olives. Once the mechanical oil extraction of olive oil  is complete,  
approximately 5 -8% of the oil remains in the pulp, which then needs to be extracted with the help of 
solvents . Olive pomace oil  is extracted from olive pulp after the first press . Edible oli ve oil and edible 
olive pomace oil are  commonly used in cooking, whether for frying or as a salad dressing . They are one 
of the traded edible vegetable oils in Tanzania .  
 
This Tanza nia Standard has been prepared to ensure the safety and quality of edible olive oil and edible 
olive pomace oil  produced in , exported or imported into the country . 
 
In the preparation of this Tanzania Standard considerable help was derived from:  
Codex Stan  33-1981  Amended in 2013 , Codex standard for olive oils and olive pomace oils  
published by the Codex Alimentarius Commission , 
  
International  Olive Counci l COI/T.20/Doc. No 11 /Rev. 3 of 2017,  method for the  determination 
of stigmastidienes in vegetable oi ls published by International Olive Council   
 
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  the  International Organization for Standardization.  
 
In reporting the results of a test or analysis made in accordance with this  Tanzania Standard, if the 
final value, observ ed or calculated is to be rounded off, it shall be done in accordance with 
TZS 4.  
 
1. Scop e 
This Tanzania Standard prescribes the require ments  and methods of sampling and test for 
edible  olive o il and edible olive pomace oil intended for direct human consumption.  
 
2. Normative reference s 
 
The following referenced standards are indispensable fo r the application of this standard. For 
dated references, only the edition cited applies. For undated references, the latest edition of 
the referenced standard (in cluding 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 spectrophotometric 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 esters of 
fatty acids  
 
TZS 538, Packaging and labeling of foods  
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 TZS 799, Foodstuffs – Determination of aflatoxin B1, and the total content of aflatoxins B1, B2, G1 and 
G2 in cereals, nuts and derived products – High\-performance liquid chromatographic method  
 
TZS 1313 , Fortified edible oils and fats — Specification  
 
TZS 1322, Oils and fats — Sampling and test methods — Purity 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  
 
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 ºC — Reference method  
 
TZS 1329, Animal and vegetable fats and oils — Determination of refractive index  
 
TZS 1330, Animal and vegetable fats and oils — Dete rmination 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 oils — Determination of insoluble impurities content  
 
TZS 1368, Animal and vegetable fats and o ils — Determination of ultraviolet absorbance expressed 
as specific UV extinction  
 
TZS 1370, Animal and vegetable fats and oils — Determination tocopherol and tocotrienol content by 
HPLC  
 
TZS 1371, Animal and vegetable fats and oils — Determination of ind ividual and total sterol contents  
-Gas chromatographic method  
 
TZS 1372, Olive oils and olive pomace oils — Determination of wax content by capillary gas 
chromatography   
 
TBS/ AFDC 4( 6108 ), Oils and Fats  — Determination of the difference between actual and  
theoretical content of triacyglycerols with ECN 42  in olive oils  
 
TBS/ AFDC 4( 6109 ), Animal and vegetable fats and oils — Determination of the composition of fatty 
acids in the 2 -position of the triglyceride molecules  
 
TBS/AFDC 25(5981) /ISO 5496,  Sensory a nalysis -- Methodology -- Initiation and training of assessors 
in the detection and recognition of odours  
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 TBS/AFDC 25(5982) /ISO 11037,  Sensory analysis -- Guidelines for sensory assessment of the colour 
of products  
 
TBS/AFDC 25(6121)/ISO 3972,  Sensory an alysis -- Methodology -- Method of investigating sensitivity 
of taste  
 
3. Term s 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 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 veget able 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  olive oil  
edible o il obtained solely from the fruit of the olive tree ( Olea europaea L.), to the exclusion o f oils 
obtained using solvents or re -esterification processes and of any mixture with oils of other kinds.  
  
3.4 extra virgin olive oil  
virgin olive oil with a free acidity, expressed as oleic acid, of not more than 0.8 grams per 100 grams 
and whose other characteristics correspond to those laid down for this category  
 
3.5 virgin olive oil s 
oils obtained from the fruit of the olive tree ( Olea europaea L.) solely by mechanical or other physical 
means under conditions, particularly thermal conditions, that do  not lead to alterations in the oil, and 
which have not undergone any treatment other than washing, decanting, centrifuging and filtration.  
 
3.6 olive-pomace oil  
olive oil that is extracted from olive pulp after the first press treated with solvents or ot her physical 
treatments, to the exclusion of oils obtained by re -esterification processes and of any mixture with oils 
of other kinds.  
 
3.7 refined olive oil  
olive oil obtained from virgin olive oils by refining methods which do not lead to alterations in the initial 
glyceridic structure.  
 
3.8 refined olive -pomace oil  
oil obtained from crude olive -pomace oil by refining methods which do not lead to alterations in the initial 
glyceridic structure.  
 
 
4. Requirements  
 
4.1 General requirements  
Edible  olive oil and edible  olive pomace 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 from admixture with mineral or other oils of vegetable or animal origin when tested in 
accordance with the methods prescribed in TZS 1322.  
 
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 4.1.1 Edible  olive oil and edible  olive pomace oil shall have sensory characteristics as shown in Table 
1. 
Table 1Sensory characteristics  
S/N Characteristic  Refined olive 
oil Olive oil  Refined olive -
pomace oil  Olive -
pomace oil  
 Test 
method  
1 Odour  Acceptable  Good  Acceptable  Acceptable  TBS/AFDC 
25(5981  
2 Taste  Acceptable  Good  Acceptable  Acce ptable  TBS/AFDC 
25(6121)  
3 Colour  light yellow  light, 
yellow to 
green  light, yellow to 
brownish yellow  light, yellow 
to green  TBS/AFDC 
25(5982)  
 
 
4.1.2  The clarity of the product  shall be judged by the absence of turbidity after keeping the filtered 
sample at 20 ºC for 24 h.  
 
 
4.2 Specific requirements  
4.2.1  Edible  olive oil and edible  olive pomace oil shall have specific requirements as shown in Table 2. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 Table 2 — Specific requirements  of edible  olive oil and edible  olive - pomace oil  
 
S/N Characteristic  Requirements for types  Test 
method  
Olive pomace 
oil Refined olive 
pomace oil  Virgin olive 
oil  Refined olive 
oil  Olive oil  
1 Refractive index 
(ND 20 ºC)  
1.4680 -1.4707  
  
1.4677 -1.4705  
 
 
 TZS 1329  
2 Relative de nsity 
at 20 ºC/Water 
20ºC  0.910 - 0.916  
 TZS 1328  
 
3 Saponification 
value , mg KOH/g  
 182-193 
 184-196 
 TZS 1325  
 
4 Iodine value 
(Wijs ), g/100  
 75-92 
 75-94 
 TZS 1327  
 
5 Unsaponifiable 
matter , g/kg,  max  
30 
  
15 
 TZS 1332  
6 Moisture and 
volatile matter at 
105 o C, % m/m, 
max.   
0.1 
  
0.2 
  
0.1 
  
0.1 
 TZS 1326  
7 Insoluble 
impurities, % 
m/m, max   
0.05 
  
0.1 
  
0.05 
 TZS 1336  
 
8 Peroxide value , 
mEq  oxygen/ kg, 
max.  15 5 20 5 15 TZS 1324  
9 Free fatty acid as 
(oleic acid ) 
,%max.  0.5 
 0.15 
 1.0 0.15 
 0.5 
 TZS 1331  
 
10 Test for presence 
of olive residue 
(pomace) oil in 
olive oil   
Positive  
  
Not applicable  
  
Negative  
 ANNEX A  
 
11 Total sterols , 
mg/kg, min.  
 1600  
 1800  
 1000  
 TZS 1371  
 
12 Erythrodiol and 
uvaol (% total 
sterols), max.   
Not applicable  
  
4.5 
 TZS 1371  
13 Wax content, 
mg/kg  
  
>350  
  
≤250  
  
≤350  
 TZS 1372  
14 Absorbency in UV 
at 270nm, max.  
 1.70 2.00 0.25 1.10 0.90 TZS 1368  
 
15 Soap content, % 
m/m, max.   
0.005  
 TZS 1322  
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 16 Stigmastadiene 
content, mg/kg,  
max 
 Not applicable  Not applicable  0.15 Not applicable  Not 
applicable  ANNEX B  
17 Difference 
between actual 
and theoretical 
ECN 42 
Triglyceride 
content, max.  
  
 
0.5 
  
      
  0.2  
 
0.3 
 TBS/  
AFDC 
4(6108)  
 
4.2.2 Absorbency in ultra -violet at 232 nm as determined by TZS 1368  shall be 2.504 max f or Extra 
virgin olive oil and 2.604 max for Virgin olive oil                         
 
4.2.3 Absorbency in ultra -violet at 270 nm for Extra virgin olive oil  shall be 0.22 max  as determined 
by TZS 1368  
 
4.2.4 Fatty acid composition as determined by TZS 288 (Part 1& 2) (% total fatty acids) shall be as 
shown in Table 3.  
 
Table 3  Fatty acid composition  
Fatty acid  Virgin olive oils  Olive oil and  
refined  olive oil  Olive -pomace oil 
and refined  olive 
pomace oil  
C14:0  0.0 – 0.05 0.0 – 0.05 0.0 – 0.05 
C16:0  7.5 – 20.0 7.5 – 20.0 7.5 – 20.0 
C16:1  0.3 – 3.5 0.3 – 3.5 0.3 – 3.5 
C17:0  0.0 – 0.3 0.0 – 0.3 0.0 – 0.3 
C17:1  0.0 – 0.3 0.0 – 0.3 0.0 – 0.3 
C18:0  0.5 – 5.0 0.5 – 5.0 0.5 – 5.0 
C18:1  55.0 – 83.0 55.0 – 83.0 55.0 – 83.0 
C18:2  3.5 – 21.0 3.5 – 21.0 3.5 - 21.0 
C18:33  - - - 
C20:0  0.0 – 0.6 0.0 – 0.6 0.0 - 0.6 
C20:1  0.0 – 0.4 0.0 – 0.4 0.0 - 0.4 
C22:0  0.0 – 0.2 0.0 – 0.2 0.0 - 0.3 
C24:0  0.0 – 0.2 0.0 – 0.2 0.0 - 0.2 
 
4.2.5  Saturated fatty acids at the 2 -position in the triglyceride (sum of pa lmitic & stearic acid s) as 
determined by TBS/AFDC4( 6109 ) shall be as shown in the table 4 . 
                   
  Table 4  Composition Characteristics  
Type of oil  Requirement ( % Max)  
 
Virgin olive oils  1.5  
 
Refined olive oil  1.8   
 
Olive oil  1.8   
 
Refined olive-pomace oil  2.2  
 
Olive -pomace oil  2.2  
 
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 4.3 Food additives  
 
4.3.1 Edible  olive oil and edible  olive-pomace oil may contain alpha -tocopherols d -alpha tocopherol, 
mixed tocopherol concentrate, dl -alpha -tocopherol, to restore natural tocoph erol lost during the refining 
process. The concentration of alpha -tocopherol in the final product shall not exceed 200 mg/kg when 
tested in accordance with  TZS 1370.  
 
4.3.2 Edible  olive oil and edible  olive-pomace oil may also contain food additives which are prescribed 
in Codex  Stan 192.  In addition, food additives shall not be used in virgin or cold pressed oils.  
4.4 Fortification  
Edible olive oil and edible olive pomace oil may be fortified in accordance with TZS 1313.  
 
5. Hygiene  
 
Edible  olive oil and edible  olive pomace o il shall be produced, processed and  handled in accordance 
with TZS 109 .  
 
6. Contaminants  
 
6.1 Metal contaminants  
The level of  metal  contaminants in edible  olive oil and edible  olive pomace oil shall conform to the limits 
specified in Table 5 . 
 
Table  5 — Limits for metal contaminants in  edible  olive oil and edible  olive pomace oil  
 
S/N Contaminant  Maximum level  Test method  
1 Iron, mg/kg  3 
 TZS 1335  
2 Copper, mg/kg  0.1 
 TZS 1335  
3 Lead, mg/kg  0.1 
 TZS 268  
4 Arsenic, mg/kg  0.1 
 TZS 76  
5 Nickel, mg/kg  0.1 
 TZS 1335  
 
6.2 Pesticide residues  
The maximum allowable pesticide residue limits in edible  olive oil and edible  olive pomace oil shall be 
as prescribed in the relevant Codex Alimentarius Standard.  
 
6.3 Mycotoxins  
 
6.3.1  Total aflatoxi n shall not exceed 10 µg/kg while aflatoxin B1 shall not exceed 5 µg/kg when  tested 
in accordance with TZS 799 . 
 
6.3.2 Ochratoxin A level shall not exceed 5µg/kg when tested in accordance with the method specified 
in Annex C.  
 
6.4.3  Edible olive oil and edible olive pomace oil shall b e free from  Poly-Hydroxy alkanoates (PHA).  
 
 
 
 
 
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 7. Sampling and test methods  
 
7.1 Sampling  
Edible  olive oil and edible  olive pomace oil shall be sampled as prescribed in TZS 54 .  
 
7.2 Test method  
7.2.1  Unless specified otherwi se, analytical grade chemicals and distill ed water shall be used 
in tests.  
 
7.2.2  Testing shall be in accordance with TZS 1322 and as provided in the respective tables and 
Annex es of this Tanzania Standard.  
 
8 Packaging, marking and labeling  
 
8.1 Edible  olive oil and edible  olive pomace oil shall be supplied in suitably sealed and closed food grade 
containers of material protecting the product from spoilage or contamination without adversely affecting 
the physical, chemical and sensory quality of the prod uct. 
 
8.2 Edible  olive oil and edible  olive pomace oil shall be marked and labeled in accorda nce with TZS 
538. In addition, each container of edible  olive oil and edible  olive pomace  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) Manufacturers registered trade mark  
j) Country of origin  
k) The phrase “Place the products away from direct sunlight”.  
l) Nutritional Information(optional)  
 
8.3 The containers shall also be marked with the TBS Standards Mark of Quality.  
 
NOTE — The TBS Standards Mark of Quality may be used by manufacturers only under licence from TBS. 
Particulars of conditions under which the licences are granted, may be obtained from  TBS.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 ANNEX  A 
(Normative)  
Test for presence of olive residue (pomace) oil in olive oil  
 
 
A.1 Principle  
 
The test is based on the temperature of precipitation of salts of fatty acids after saponification.  
 
 
A.2 Preparation of sample  
 
The sa mple is filtered through paper at a temperature slightly above the melting point of certain solid 
constituents which could separate from the fluid fatty matter.  
 
 
A.3 Procedure  
 
A.3.1. Saponify 1 g of oil by boiling for 10 min with 5 ml alcoholic KOH  (42.5  gm KOH in 72 ml water 
made up to 500 ml with 95 % ethyl alcohol).  
 
A.3.2.  After cooling add 1.5 ml aqueous acetic acid ( 1+ 2 by volume such that 1.5 ml exactly neutralizes 
5 ml of aqueous alcoholic KOH)  and 50 ml of 70 % ethanol  warmed to 50 ºC.  
 
A.3.4 Mix, insert a thermometer and allow to cool.  
 
 
A.4 Results  
 
If a precipitate form s above 40 ºC, the test for the presence of olive residue oil is positive. Allow to cool 
to ambient temperature for 12 h. Observe solution again. The formation of a flocculen t precipitate floating 
in the middle of the liquid also indicates that the test is positive. A cloudiness not forming into flakes 
does not indicate the presence of olive residue oil.  
 
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                                                                         ANNEX B  
(Normative)  
                               Determination of stigmastadienes in vegetable oils  
1. Principle  
Isolation of unsaponifiable matter. Separation of st eroidal hydrocarbon fraction by column 
chromatography on silica gel and analysis by cap illary gas chromatography.  
 
2. Apparatus  
 
2.1. 250 ml flat-bottomed flasks   
 
2.2 Reflux condenser . 
 
2.3. 500 ml separating funnels . 
 
2.4. 100 ml round -bottomed flasks . 
 
2.5. Rotary evaporator . 
 
2.6. Glass chromatography column  (1.5 cm i.d. by 50 cm l ength)  with Teflon stopcock and a plug of 
glass wool fibre at the bottom. To prepare the silica gel column, pour hexane into the chromatography 
column to a depth of approxim ately 5 cm and then fill with a slurry of silica gel in hexane (15 g in 40 
ml) with the h elp of hexane portions. Allow to settle and finish settling by applying slight vibration. Add 
anhydrous sodium sulphate to a height of 0.5 cm, finally elute the excess hexane.  
 
2.7 Gas chromatograph  with flame ionisation detector, split  or on -column inject or and oven 
programmable to within ± 1ºC. 
 
2.8. Fused silica capillary columns  for gas chromatograph y (0.25 or 0.32 mm i.d. by 25 m length) 
coated with 5% ph enylmethylsilicone phase, 0.25 µ m film thickness.  
 
Note 1   Other columns of similar or lower polar ity can be used.  
 
2.9. Integrator -recorder  with possibility of valley -valley integration mode.  
 
2.10. 5-10 µl microsyringe  for gas chromatography with hardened needle.  
 
 
3. Reagents  
All reagents should be of analytical grade unless otherwise specified. The  water used  
should be distilled water, or water of at least equivalent purity.  
 
3.1. Hexane or mixture of alkanes of b.p. interval 65 -70oC, distilled with rectifying  
column.  
 
Note 2   The hexane must be distilled to remove impurities.  
 
3.2. 96 v/v  ethanol.  
 
3.3. Anhydrous sodium sulphate . 
 
3.4. Alcoholic potassium hydroxide solution  at 10% w/v. Ad d 10 ml of water to 50 g potassium 
hydroxide, stir, and then dissolve the mixture in ethanol to 500 ml.  
 
Note 3    Alcoholic potash turns brown on standing. It sho uld b e prepared freshly each day and kept in well -
stoppered dark glass bottles.  
 
3.5. Silica gel 60 for column chromatography, 70 -230 mesh (Merck ref. 7734 or similar).  
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 Note 4   Usually, silica gel can be used directly from the original container without any treatment. However, 
some batches of silica may sho w low activity resulting in bad chromatographic separations. Under these 
circumst ances, the silica gel should be treated in the following way: deactivate the silica gel by heating for a 
minimum of four hours at 550  ºC. After heating, place the silica gel i n a desiccator while the gel is cooling and 
then transfer the silica gel to a stopp ered flask. Add 2% of water and shake until no lumps can be se en and the 
powder flows freely.  
 
3.6. Stock solution (20 0 ppm) of cholesta -3,5-diene (Sigm a, 99% purity) in hexane (10 mg in 50 
ml). 
 
3.7. Standard solution of cholesta -3,5-diene in hexan e at a concentration of 20 ppm, obtained by 
dilution of the stock solution (5.6).  
 
Note 5   If kept at under 4  ºC, solutions  3.6 and 3 .7 will not deteri orate over a period of at least 4 months.  
 
3.8. Carrier gas for chromatography: N-50 helium or hydrogen.  
 
3.9. Auxiliary gases  for flame ionisation detector: N -50 hydrogen and purified air.  
 
3.10 Solution of n -nonacosane in hexa ne at a concentration of approx. 100 ppm.  
 
 
4. Procedure  
 
4.1. Preparation of unsaponifiable matter:  
 
4.1.1. Weigh 20 ± 0.1 g of oil into a 250 -ml flask, add 1 ml of t he standard solution of cholesta -3,5 
diene (20 mg) and 75 ml of alcoholic potas h at 10%, fit reflux condenser, and heat to slight boiling for 
30 minutes. Remove the flask containing the sample from the heat and allow the solution to cool 
slightly (do not a llow to cool completely as the sample will set). Add 100 ml of water and transfer the 
solution to a separating funnel with the aid of 100 ml of hexane. Shake the mi xture vigorously for 30 
seconds and leave to stratify.  
 
Note 6   If an emulsion is produced, wait as it disappears rap idly or add small quantities of ethanol.  
 
4.1.2. Transfer the a queous phase beneath to a second sepa rating funnel and extract again with 100 
ml of hexane. Once more run off the lower phase and wash the hexane extracts (combined in another 
separating funnel) three  times with 100 ml each time of a mixture of ethanol -water (1:1) until neutral 
pH is reached.  
 
4.1.3. Pass the hexane solution through anhydrous sodium s ulphate (50 g), wash with 20 ml hexane 
and evaporate in a rotary evaporator at 30  ºC and low pressure until dryness.  
 
4.2. Separation of steroidal hydrocarbon fraction:  
 
4.2.1. Take the residue to the fractionating column with the aid of two 1 -ml portions of hexane, run the 
sample onto the column by allowing the solution level  to drop to the top of the sodium sulphate and 
start chromatographi c elution with hexan e at a flow rate of 1 ml/min. approx. Discard the first 25 -30 ml 
of the elution and then collect the following 40 ml fraction  
 
Note 7   The first fraction contains saturated hydrocarbons (Figu re 1 a) and the second fraction steroidal 
hydrocarbons. Further  elution provides squ alene and related compounds. To achieve a good separation between 
the saturated and steroidal hydrocarbons, the  fraction volumes have to be optimised. To do so, t he volume of the 
first fraction should be adjusted so that when the secon d fraction is analysed the peaks representing the saturated 
hydrocarbons are low (see Figure 1c) ; if they do not appear but the intensity of the standard peak is low, the 
volume should be reduced. Anyway, a complete separation between the components of th e first and second 
fractions is unnecessary, as there is no overlapping of peaks durin g GC analysis. The optimisation of the volume 
of the second fraction is generally not needed as a good separation exists with the further components. 
Nevertheless, th e pre sence of a large peak at a retention time approx. 1.5 min lower than the standar d is due to 
squalene, and it is indicative of a bad separation.  TBS/AFDC 4(5974)P 3 
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 4.2.2. Evaporate the second fraction in an evaporator at 30  ºC and low pressure until dryness, and 
immediately d issolve the residue in 0.2 ml of h exane. Keep the solution in the refrigerator until 
analysis.  
 
Note 8   Residues 4.1.3. and 4 .2.2 should not be kept dry or at room tempera ture. As soon as they are obtained, 
the solvent should be added and the solutions sh ould be kept in the refrigerator.  
 
4.3. Gas chromatography  
 
4.3.1. Working conditi ons 
 Injector temperature: 300  o C. 
 Column temperature: 320  o C. 
 Integrator -recorder: The parameters for integration should be fixed so  as to give a correct 
assessment of the areas.  
Valley -valley integration mode is recommended.  
 Sensitivity: About 16 times the minimum attenuation.  
 Amount of solution injected: 1 ml.  
 Oven programming temperatures: Initial 235  o C for 6 min. and  then rising at 2  o C/min. up to   
285 o C. 
 Injector with 1:15 flow divider.  
 Carrier: Helium or hydrogen at about 120 kPa pressure.  
 
These conditions may be adjusted in accordance with the characteristics of the chromatograph and 
the column to give chromatograms meet ing the following requirements: internal s tandard peak within 
approx. 5 min. of the time given in 4.3.2; the internal standard peak should be at least 80% of the full 
scale.  
 
The gas chromatographic system must be checked by injecting a mixture of the stock solution of 
cholestadiene (3.6) and n -nonacosane solution (3 .10). The  cholesta -3,5-diene peak has to appear 
before the n -nonacosane (Fig. 1c); if this does not oc cur, two steps can be taken: to bring down the 
initial oven temperature or to replace the GC -column by a less polar one.  
 
4.3.2 Peak i dentification  
The internal standard peak appears at approx. 19 min. a nd the stigmasta -3,5-diene at a relative 
retention time of 1.29 (see Figure 1b).  
 
The stigmasta -3,5-diene goes with small quantities  of an isomer, and usually both originate a single 
chromatographic peak. Nevertheless , if the column is too polar or shows a great resolving power, the 
isomer can appear a s a small peak before and close to that of stigmasta -3,5-diene. In this case, the 
two areas have to be summed (Figure 2). It is advisable to  elimin ate the stigmastadiene split by 
replacing the column by one which is a less polar or has a wider internal diameter.  
 
Note 9   Stigmastadienes for reference can be obtained from the analysis of a refined vegetable oil by applying 
the method for the d etermina tion of steroidal hydrocarbons. Stigmastadienes ori ginate a significant and easily 
identifiable peak.  
 
Note 10   Care must be taken that there is no overlap between the interna l standard and stigmastadienes and 
any of the peaks which appear in the  first fraction eluted from the silica gel column . 
 
4.3.3 Quantitative analysis  
 
The stigmastadienes content is determined according to the formula:  
                                                 
                                                                                As x Mc  
                   mg/kg of sti gmastadienes = ----------------  
                                                                   Ac x Mo  
 
                  
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 where: As  = area of stigmastadienes peak ( if the peak is split up, su m of the areas of the two isomers)  
                    
                    Ac= area of internal standard  (cholestadiene)  
                   
                    Mc = mass of standard added, in micrograms  
                     
                    Mo = mass of  oil taken, in grams.  
       
                    Detection limit: About 0.01 mg/kg.  
 
Note 11   When stigmastadienes appear in concentrations of more than 4 mg/kg, i f quantifying is required, the 
method for determination of steroidal hydrocarbons must be ap plied. 
 
Figure 1 . Gas chromatograms obtained from olive oil sam ples analysed on a fused silica capillary 
column (0.25 mm i.d. by 25 m length) coated with 5% - phenylmethylsilicone, 0.25 mm film thickness.  
 
a) First fraction (30 ml) from a virgin oil, spiked wi th standard.  
b) Second fraction (40 ml) fr om an olive oil containing 0.10 mg/kg of stigmastadienes.  
c) Second fraction (40 ml) conta ining a small proportion of the first fraction.  
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TBS/AFDC 4(5974)P 3 
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 Annex  C 
(normative ) 
 
 Determination of O chratoxin A — High performance liq uid chromatographic 
method with immunoaffinity column cleanup and fluorescence detection  
 
1.Principle  
Ochratoxin A (OTA) is extracted by acetonitrile -water. The extract is purified using an immunoaffinity 
column and ochratoxin A is determined by high perfo rmance 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 onl y reagents of recognized analytical grade and only 
distilled water or water of grade 1. Solvents shall be of quality for HPLC analysis.  
2.1 Acetonitrile . 
2.2 Methanol . 
2.3 Sodium chloride (NaCl) . 
2.4 Glacial acetic acid, φ(CH 3COOH) ≥ 98 %.  
2.5 Tween -20. 
2.6 Sodium bicarbonate (NaHCO 3). 
2.7 Disodium hydrogen phosphate (Na 2HPO 4). 
2.8 Potassium dihydrogen phosphate (KH 2PO 4). 
2.9 Potassium 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/AFDC 4(5974)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.  
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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/AFDC 4(5974)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/AFDC 4(5974)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 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);  
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 . 
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 Indicate whether or not a correction for recovery has been applied.