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Milk fat – Detection of foreign fat by gas chromatographic analyses of

triglycerides (reference method)

Milk fat — Detection of foreign fats by gas

chromatographic analysis of triglycerides
(Reference method)
Contents
1 Scope
2 Normative references
3 Term and definition
4 Principle
5 Reagents
6 Apparatus
7 Sampling
8 Procedure
8.1 Preparation of column (silanization)
8.2 Filling of column
8.3 Preparation of test samples
8.4 Preparation of sample solution
8.5 Chromatographic triglyceride determination
9 Integration, evaluation and control of the measuring conditions
10 Qualitative foreign fat detection
11 Quantitative foreign fat determination
11.1 Calculation
11.2 Expression of test results
12 Range of application of the detection method
13 Precision
13.1 Interlaboratory test
13.2 Repeatability
13.3 Reproducibility
14 Range of application of the detection method
15 Accuracy
15.1 Critical difference
15.2 Acceptability of results
16 Test report

Foreword
ISO (the International Organization for Standardisation) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee has
been established has the right to be represented on that committee. International organisations,
governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO ……| IDF ….. was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk
and milk products and the International Dairy Federation (IDF), in collaboration with AOAC International. It is
being published jointly by ISO and IDF and separately by AOAC International.

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Foreword
IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National
Committee in every member country. Every National Committee has the right to be represented on the IDF
Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in
the development of standard methods of analysis and sampling for milk and milk products.
Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the
National Committees for voting. Publication as an International Standard requires approval by at least 50% of
IDF National Committees casting a vote.
ISO …. | IDF … , was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk
and milk products and the International Dairy Federation (IDF), in collaboration with AOAC International. It is
being published jointly by ISO and IDF and separately by AOAC International.
All work was carried out by the Joint ISO/IDF/AOAC Action Team Fat of the Standing Committee on Main
components of milk under the aegis of its project leader, Dr. Joachim Molkentin (DE).

Milk fat — Detection of foreign fats by gas chromatographic

analysis of triglycerides (Reference method)
1 Scope
This International Standard specifies a reference method for the detection of foreign fats, of both vegetable
fats and animal fats such as beef tallow and lard in milk fat of milk products using gas chromatographic
analysis of triglycerides. Using defined triglyceride formulae, vegetable and animal fats are qualitatively and
quantitatively detected in pure milk fat irrespective of feeding or lactation conditions.
NOTE 1 Butyric acid (C4) occurs exclusively in milk fat and enables quantitative estimations of low to
mean amounts of milk fat in vegetable fats to be made. Despite that qualitative and quantitative information
can hardly be provided in the addition range of up to mass fraction of at least 20 % foreign fat to pure milk
fat. The reasoning for that is the large variation of C4 approximately ranging between a mass fraction of 3,5
% and 4,5 %.
NOTE 2 Practically, quantitative results can only be obtained by triglyceride analyses, because the sterol
content of vegetable fats is different as a function of production and treatment conditions.

2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use – Specification and test methods.
ISO 1211 (IDF 1), Milk – Determination of fat content - Gravimetric method (Reference method)
ISO 2450 (IDF 16), Cream – Determination of fat content - Gravimetric method (Reference method)
ISO 7328 (IDF 116), Milk-Based edible ices and ice mixes. Determination of fat content - Gravimetric method
(Reference method)
ISO 7208 (IDF 22), Skimmed milk, whey & buttermilk. Determination of fat content - Gravimetric method
(Reference method)

3 Term and definition

For the purposes of this document, the following term and definition applies.

3.1
foreign fat in milk fat
mass fraction of foreign fat determined by the procedure specified in this International Standard.
NOTE The foreign fat content, being all vegetable and animal fats except milk fat, is expressed as a
percentage by
mass.

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4 Principle
After extraction a stock solution of the milk fat is prepared. From that stock solution the triglycerides (total
carbon numbers) are determined by gas chromatography using packed columns. By inserting the weight
percentage of the fat molecules of different size (C24 – C54, using the even C numbers only) in the
triglyceride formula, the foreign fats are either qualitatively detected or quantitatively determined.
NOTE Observing the evaluation described here capillary gas chromatography can be used, if it is guaranteed
that
comparable results are achieved. Suited methods have already been described [18].

5 Reagents
5.1 General
All reagents shall be of recognized analytical grade. Use water complying with the requirements of ISO
3696, grade 2.

5.2 Carrier gas

nitrogen, of purity degree ³ 99,996 %.

5.3 Fat standards

for standardizing standard milk fat according to section 6.5.4.
5.3.1 Triglyceride standard
saturated, suited products are available commercially.
5.3.2 Cholesterol standard.

5.4 Methanol
(CH3OH), free of water.

5.5 n-Hexane
(CH3(CH2)4CH3).

5.6 n-Heptane
(CH3(CH2)5CH3).

5.7 Toluene
(C6H5CH3).

5.8 Dimethylchlorosilane solution.

Dissolve 50 ml dimethylchlorosilane in 283 ml toluene (5.7).

5.9 Combustion gas

hydrogen and synthetic air.

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Stationary phase
packed with 3-% OV-1 on 125/150 μm (100/120 mesh) Gas ChromQ.
NOTE The indication of grain was transferred to the SI-unit μm according to BS 410:1988 “British Standard
Specification for test sieves”.

5.11 Cocoa butter solution

with a mass fraction of 10 % cacoa.

6 Apparatus
Usual laboratory equipment and, in particular, the following.

6.1 High temperature gas chromatograph

The high temperature gas chromatograph shall be suited for temperatures of at least between 400 °C and
450 °C, equipped with a flame ionization detector (FID) and constant mass flow controller for the carrier gas.
The flow of the combustion gas should be set on 30 ml/min H2 and 270 ml/min synthetic air. Given the high
carrier gas flow, the flame jet shall be particularly large.
NOTE 1 Because of the high temperatures occurring during triglyceride analyses, the glass inserts in the
FID or in the injector system should be frequently cleaned.
The gas chromatograph shall be equipped with septa, withstanding high temperatures, which can be
frequently used and exhibit generally a very low degree of “bleeding”. Exchange the septa at regular intervals,
e.g. after roughly 100 injections or as soon as the resolution deteriorates (see figure 4).
NOTE 2 Suited are Chromblue (tm) septa (Chrompack).

6.2 Chromatography column

Use a U-shaped glass column, of inside diameter 2 mm and of length 500 mm, which is first silanized
according to section 6.1 with dimethylchlorosilane in order to deactivate the glass surface.
NOTE Suited columns are also somewhat longer (of lenght 80 mm – 200 mm) packed columns. With them
a slightly better reproducibility of the results can be achieved. On the other hand, the stationary phase
exhibits occasionally fractures after operation, which may lead, in turn, to worse quantitative results. Further,
the FID flame is easily extinguished as a result of the required extremely high carrier gas flow of 75 ml to 85
ml/min.

6.3 Arrangement for filling the column (see figure 1).

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6.3.1 Plastic column

with screwed-on end caps, provided with a mark up to which the required quantity of stationary phase can
be filled.
6.3.2 Fine sieve
with mesh size of approximately 100 mm, with screw cap suited for sealing the glass column according to
figure 1.
6.3.3 Silanized glass wool
deactivated.
6.3.4 Vibrator
for uniform distribution of the stationary phase during filling.

6.4 Extrelut column

of capacity 1 ml to 3 ml, filled with silica gel. Alternatively, the extrelut column can be used for the
extraction to obtain milk fat.

6.5 Graphite seal

of diameter 6,4 mm (1/4"), with 6 mm bore.

6.6 Silanizing devices

for silanizing the glass surface of the column according to clause 6.1.
6.6.1 Woulff bottle.
6.6.2 Water suction pump.

6.7 Water bath

capable of maintaining a temperature of 50 °C ± 2 °C.

6.8 Drying cabinet

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capable of operating at 50 °C ± 2 °C and 100 °C ± 2 °C.

6.9 Microlite pipette.

6.10 Graduated pipette

of capacity 5 ml.

6.11 Round-bottomed flask

of capacity 50 ml.

6.12 Erlenmeyer flask

of nominal capacity 50 ml.

6.13 Funnel

6.14 Fine-pored filter.

6.15 Rotary evaporator.

6.16 Ampoules
of nominal capacity 1 ml, to be sealed with an aluminium cap, with a septum in the interior.

6.17 Injection syringe

with syringe plunger not reaching into the tip of the needle.
NOTE With these syringes better reproducibility of the results will be obtained. In order to avoid
deterioration of the septum, the tip of the needle should be checked at regular intervals (e.g. with a
stereomicroscope).

7 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling
method is given in ISO 707 (IDF 50).
It is important that the laboratory receive a sample, which is truly representative and has not been damaged
or changed during transport or storage.

8 Procedure
8.1 Preparation of column (silanization)
After connecting the Woulff bottle (5.6.1) (see figure 2) with the water suction pump, dip tube 2 into the
dimethylchlorosilane solution 5.8. The column is filled with solution by closing the stopcock. Open the
stopcock again and subsequently remove the two tubes.
Fix the column on a stand and completely fill it with the dimethyldichlorosilane solution (5.8) using a pipette.
Let the column stand for 20 min to 30 min and then replace the Woulff bottle by a filter flask. Empty the
column by connecting it with the water suction pump (6.6.2) (see figure 3).

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8.2 Filling of column

Rinse the emptied column (8.1) successive using 75 ml toluene (5.7) and 50 ml methanol (5.4). Dry the
rinsed column in the drying cabinet (6.8) set at 100 °C for approximately 30 min.

Use the arrangement as represented in figure 1 to fill the column. Fill the stationary phase according to
5.10 in the plastic column up to the mark. Seal the lower end of the glass column to be filled with an
approximately 1 cm long plug of silanized glass wool (6.3.3) and pressed using a steel rod. Close the end of
the column with the fine sieve according to 6.3.2.
Fill the column under pressure (3 bar and a flow of N2) with the stationary phase. To obtain uniform,
continuous and firm packing, move a vibrator up and down the glass column during filling. After filling, press a
solid plug of silanized glass wool (6.3.3) into the other end of the packed column. Cut off the protruding ends
and press the plug into the column for a few millimetres with a spatula.

8.3 Preparation of test samples

Use for test sample preparation one of the three following methods.
8.3.1 Isolation of milk fat from butter.
Weigh 5 g to 10 g test sample in a suitable vessel. To melt the sample, place the vessel in a water bath
(6.7) set at 50 °C. Preheat a 50 ml Erlenmeyer flask (6.12) and a funnel (6.13) with inserted the fine-pored
filter (6.14) in the drying cabinet (5.8) set at 50 °C. Filter the fat layer of the molten sample using the
preheated device.
NOTE  The obtained milk fat will almost be phospholipid-free.
8.3.2 Extraction of the fat fraction according to the Röse-Gottlieb gravimetric method
Extract the fat fraction from the test sample by using the gravimetric method described either in ISO 1211,
ISO 2450, ISO 7208 or ISO 7328.
If phospholipids are present in the obtained milk fat, a cholesterol peak will be obtained which is increased
by approximately 0,1 %.
The triglyceride spectrum standardized to 100 with cholesterol (5.3.2) is thereby influenced only to a

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negligible extent.
8.3.3 Extraction from milk using silica gel columns
0,7 ml test sample tempered to 20 °C are applied to a 1 ml to 3 ml Extrelut column (6.4) with a microlitre
pipette (6.9). Allow to distribute uniformly on the silica gel for approximately five min.
For denaturing the protein-lipid complexes, use the graduated pipette (6.10) to add 1,5 ml of methanol
(5.4). Subsequently, extract the fat fraction from the test sample with 20 ml n-hexane (5.5). Add the n-
hexane slowly in small amounts. Collect the solvent draining off in a 50 ml round-bottomed flask (6.11)
previously dried to a constant, known weight.
After extraction led the column drain until empty. Distill from the eluate the solvents off on a rotatory
evaporator (6.15) with the round-bottom flask in its water bath set at between 40 °C and 50 °C.
The flask is dried and the fat yield determined by weighing.
NOTE Fat extractions according to Gerber, Weibull-Berntrop, Schmid-Bondzynski-Ratzlaff or isolation of
milk fat using detergents (BDI-method) are not suited for triglyceride analysis With these methods more or
less large quantities of partial glycerides or phosholipids can pass into the fat phase.

8.4 Preparation of sample solution

For gas chromatography a 5 % solution of the fat in n-heptane obtained according to clause 8.3 is used.
For preparing the sample solution corresponding amounts of the sample material obtained in 8.3.1 and 8.3.2
are weighed and dissolved in corresponding amounts of n-heptane (5.6).
Use the sample preparation in 8.3.3 to calculate the amount of n-heptane (5.6) to be added to the test
sample material in the flask on the basis of weighing and the remainer dissolved in it. Approximately add 1
ml of the test sample solution in an ampoule (6.16).

8.5 Chromatographic triglyceride determination

With high temperatures of up to 350 °C, to be able to elute the long-chain triglycerides C53-C56, an
increase in baseline can easily occur. Particularly, if the columns have not adequately been conditioned in the
beginning. The rise of the baseline at high temperatures can be avoided by either combining two columns or a
baseline subtraction.
When using the compensating mode or an procedure with single columns, as well as for the glass inserts in
the injector and the detector, use graphite seals (6.5).

8.5.1 Baseline correction

To avoid a baseline rising, one of the following four methods can be used.
8.5.1.1 Combination of columns
Use two packed columns in a compensating mode.
8.5.1.2   Baseline correction by the gas chromatograph
Avoid rising of the baseline by the application of a run of the gas chromatograph without injection of a fat
solution and subsequent subtraction of the stored baseline.
8.5.1.3   Baseline correction by integration software
Avoid rising of the baseline by the application of a run of the integration system without injection of a fat
solution and subsequent subtraction of the stored baseline.

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8.5.1.4 Baseline correction by adequate conditioning

NOTE With adequate initial conditioning of the column and approximately 20 injections with milk fat
solutions baseline rising at high temperatures is frequently so low that baseline correction is not necessary.
8.5.2 Injection technique
To avoid discrimination effects, apply the “hot injection” technique to achieve better quantitative results
with the high-boiling triglyceride components. Here, the fat solution is drawn up in the syringe and the cold
needle of the syringe warmed up prior to injection for approximately three seconds in the injector head. Then,
rapidly inject the syringe content.
NOTE With this injection technique the risk of fractionation phenomena inside the syringe or the injection
block is reduced. “On-column” direct injection in the upper, extended heated part of the column is not
applied, because the fragments of the septum, which accumulate here, as well as contaminations can be
easily eliminated with the used technique by regularly changing an injector insert without dismounting the
column.
Bending of the tip of the needle caused by touching the bottom of the sample beaker (even if it is hardly
visible to the eye) shall be absolutely avoided in order not to damage the septum.
8.5.3 Conditioning of a packed column
During steps (a) to (c) do not connect the top of the column to the detector to avoid contamination.
Condition the filled column (6.3) as follows:

a. Flow the column with N2 , with the flow speed set at 40 ml/min and at 50 °C for 15 min;
b. Heat the column with 1 K/min up to 355 °C, with the flow speed set at 10 ml N2/min;
c. Hold the column at 355 °C for 12 h to 15 h;
d. Inject two times 1 ml of the cocoa butter solution (5.11) using the respective temperature
program;
e. Inject 20 times 0,5 mm of a milk fat solution over two to three days according to 7.4.

Use the pair of columns with the best quantitative results (the response factors should be almost 1) for the
following procedure. Do not use the column, if the obtained response factors are more then 1,20.
8.5.4 Calibration
For calibration, determine the response factors of the corresponding triglycerides, as well as of the
cholesterol of a milk fat (standardized fat) using the standardized triglycerides (at least the saturated
triglycerides C24, C30, C36, C42, C48 and C54, as well as cholesterol; better still additionally C50 and C52).
Intermediate response factors can be calculated by mathematical interpolation.
Using the standardized milk fat, perform two to three calibrations every day. If almost identical results are
obtained, well reproducible quantitative results are achieved with triglyceride analysis of test samples.
The standardized milk fat has a stock life of several months if stored at maximally –18 °C. This milk fat can
be used as a standard.
NOTE The response factor of each constituent may also be determined using a standardised fat with a
certified triglyceride composition, such as CRM 519 (anhydrous milk fat) obtainable from the Institute for
Reference Materials and Measurements, Geel, Belgium.
8.5.5 Temperature programme, carrier gas and other conditions for triglyceride analysis

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a. Temperature programme: Set the initial column temperature at 210 °C and hold for one min. Then
increase with 6 °C/min to 350 °C and hold at that (final) temperature for five min.
b. Detector, injector and oven temperatures: Set at 370 °C respectively. Maintain the detector,
injector, and oven temperature (initial temperature) at a constant level (also overnight, during
weekends and holidays).
c. Carrier gas: Use nitrogen and a flow rate of 40 ml/min. If 80 cm columns are used, set the flow
speed at least at 75 ml/min N2. The carrier gas flow shall be constantly maintained (also
overnight, as well as during weekends and holidays). Adjust the exact carrier gas flow in a manner
that independent of column length C54 is eluted at 341 °C.
d. Duration of analysis: Completed in 29,3 min.
e. Injection volume: Inject 0,5 ml. Rinse the syringe with pure heptane (5.6) several times after each
injection.
f. FID conditions: See 6.1. Ignite the flame ionization detector at the beginning of each working day.

9 Integration, evaluation and control of the measuring

conditions
Triglycerides with odd acyl-c number (2n + 1) are combined with the preceding even-numbered
triglyceride (2n). The less reproducible low C56 contents are not taken into account. The remaining
triglycerides (peak area) in the chromatogram, including cholesterol (peak near to C24) are multiplied by the
respective response factors of the standard fat (last calibration) and altogether normalized to 100. Besides
free cholesterol the triglycerides (C24, C26, C28, C30, C32, C34, C36, C38, C40, C42, C44, C46, C48, C50,
C52 and C54) are, thus, evaluated. Results are given in percent (mass fraction).
Evaluate the chromatogram peaks with an integrator with which the baseline can be plotted. A
reintegration with optimized integration parameters should be possible. Figures 5 and 6 demonstrate two
examples of triglyceride chromatograms. Figure 5 shows a chromatogram which can be well evaluated, whilst
figure 6 represents a sporadic error in the C50 to C54 range, the baseline running incorrectly compared with
figure 5. Such typical errors can be detected with a high degree of certainty and avoided only by use of an
integrator with which the baseline is plotted.

To control measuring conditions, use the relative standard deviations (RSD: coefficient of variation x 100)
given in Table 1 for the different triglycerides. They were calculated from 19 consecutive analyses of the same

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milk fat sample.

Table 1 — Relative standard deviations (RSD) of triglyceride contents (n=19)
Tryglyceride RSD (%)

C24 10,00

C26 2,69

C28 3,03

C30 1,76

C32 1,03

C34 0,79

C36 0,25

C38 0,42

C40 0,20

C42 0,26

C44 0,34

C46 0,37

C48 0,53

C50 0,38

C52 0,54

C54 0,60
If the RSDs are considerably higher than the values in Table 1, the chromatographic conditions are not
appropriate. If so verify the septa or the carrier gas flow rate. Small particles of the septum may also have
formed deposits on the glass wool at the entrance of the column or the column might have become unsuited
for use as a result of ageing, temperature influences, etc. (see Figure 3).
NOTE  The values given in Table 1 are not mandatory, but are indicative for quality control purposes.
However, if higher RSD values are accepted, the repeatability and reproducibility limits given in point 11 must
nevertheless be complied with.

10 Qualitative foreign fat detection

For the detection of foreign fats triglyceride formulae (Table 2) with limits S (Table 3) have been
developed, in which the S-values of pure milk fats can fluctuate. If these limits are transgressed, the presence
of foreign fat can be assumed. The most sensitive formula for the detection of lard addition is, e.g.:
S = 6,5125 - C26 + 1,2052 - C32 +1,7336 - C34 +1,7557 - C36 + 2,2325 - C42 + 2,8006 - C46 +
2,5432 - C52 + 0,9892 - C54
NOTE  Using 755 different milk fat samples a 99 % confidence range of S = 97,96 – 102,04 was
established for pure milk fat samples with a standard deviation for all S-values SD = 0,39897.
Starting from the triglyceride composition of an unknown fat sample such a formula allows, without using a
computer, to verify in a simple manner whether the sum of the triglyceride contents stated here with the
corresponding factors falls outside the range of 97,96 – 102,04. If outside, it is most probably that one has to
do with foreign fat addition.
For the detection of different foreign fats, Table 2 shows a different triglyceride formulae. For the detection
of foreign fats like soybean oil, sunflower oil, olive oil, rape-seed oil, linseed oil, wheat germ oil, maize germ
oil, cotton seed oil and hydrogenized fish oil but also for vegetable fats like coconut- and palm kernel fat, as
well as for palm oil and beef tallow, a common formula can be used respectively.
Since the triglyceride composition of the foreign fats is also subjected to fluctuations, up to four different
experimentally measured foreign fat triglyceride data of the same type were used. (With the same foreign fat
types the least favourable limit has been considered, respectively (see Table 4)). With the following “Total
formula” similarly good results can be obtained for all foreign fats:
S = -2,7575 - C26 + 6,4077 - C28 + 5,5437 - C30 -15,3247 - C32 + 6,2600 - C34 + 8,0108 - C40 -

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5,0336 - C42 + 0,6356 - C44 + 6,0171 - C46

Calculations for the detection of any foreign fat combination in milk fat have shown that, e.g., although
with the formula for lard given in Table 2 the limit for this foreign fat in low, namely 2,7 %. Other fats such
as coconut fat, palm oil or palm kernel fat with detection limits of 26,8 %, 12,5 % and 19,3 % respectively,
can with this formula only be detected if extremely high amounts have been added to milk fat. This applies
also to the other formula in Table 2.

Table 2 — Triglyceride formulae for detecting various foreign fats in milk fat,
indicating the standarddeviations SD for S

Formula for soybean, sunflower, olive, rape-seed, linseed, wheat germ, maize germ,
cotton seed and fish oil
S = 2,0983 – C30 + 0,7288 – C34 + 0,6927 – C36 + 0,6353 – C38 + 3,7452 – C40 – 1,2929 C 42 +
1,3544 – C44 + 1,7013 – C46 + 2,5283 – C50;
SD = 0,38157
Formula for coconut and palm kernel fat
S = 3,7453 – C32 + 1,1134 – C36 + 1,3648 – C38 + 2,1544 – C42 + 0,4273 – C44 + 0,5809 – C46 +
1,2926 C48 + 1,0306 – C50 + 0,9953 – C52 + 1,2396 – C54;
SD =   0,11323
Formula for palm oil and beef tallow
S = 3,6644 – C28 + 5,2297 – C30 – 12,5073 – C32 + 4,4285 – C34 – 0,2010 – C36 + 1,2791 – C38
+ 6,7433 C40 – 4,2714 – C42 + 6,3739 – C46;
SD =   0,81094
Formula for lard
S = 6,5125 – C26 + 1,2052 – C32 + 1,7336 – C34 + 1,7557 – C36 + 2,2325 – C42 + 2,8006 – C46 +
2,5432 C52 + 0,9892 – C54;
SD =   0,39897
Therefore, use for checking of an unknown fat sample all formula given in Table 2 and the Total formula, if
the sample is likely to be a mixture of milk fat and one of the 14 different foreign fats or a combination of
these foreign fats. If, by inserting the triglyceride of a fat sample to be analysed an S-value is obtained,
which falls outside the S-ranges of only one of the five formulae as shown in Table 3, then the sample is
most likely a modified milk fat. Detection of a foreign fat in milk fat by means of one of the four formulas in
Table 2 does not allow conclusions to be drawn on the type of the foreign fat admixture.

Table 3 — Limits for milk fats

Foreign Fat S-range

Soybean, sunflower, olive, rape-seed, linseed, wheat, germ, maize germ, cotton, 98,05 – 101,95
fish oil

Coconut and palm kernel fat 99,42 – 100,58

Palm oil and beef tallow 95,90 – 104,10

Lard 97,96 – 102,04

Total formula 95,68 – 104,32

In table 4 the detection limits for the different foreign fats with a 99 % confidence are given. The first
column shows the minimal detection limits for the best milk fat formulae in Table 2.
In the second column the detection limits for the total formula are given. Although the limits are
somewhat higher, only this formula is necessary to detect a little bit higher amounts of foreign fats. With all
formulae also combinations of the different foreign fats can be detected. The ranges of variation of the
triglycerides of different foreign fats of one type have no considerable influence on the detection limits.

Table 4 — 99 % limits of detection by addition of foreign fat to milk fat in %

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Foreign Fat Individual formula Total formula

Soybean oil 2,1 4,4

Sunflower oil 2,3 4,8

Olive oil 2,4 4,7

Coconut oil 3,5 4,3

Palm oil 4,4 4,7

Palm kernel fat 4,6 5,9

Rape-seed oil 2,0 4,4

Linseed oil 2,0 4,0

What germ oil 2,7 6,4

Maize germ oil 2,2 4,5

Cotton seed oil 3,3 4,4

Lard 2,7 4,7

Beef tallow 5,2 5,4

Hydrogenised fish oil 5,4 6,1

NOTE The S-ranges are calculated in that way, that a foreign fat is only assumed, if the limits of the
individual formula are exceeded (see Table 4).

11 Quantitative foreign fat determination

11.1 Calculation
In order to obtain quantitative information on foreign fat content, calculate the foreign fat content, wf, in
the test sample using the following equation:

where

wf is the mass fraction, in percent, of an unknown foreign fat or foreign fat mixture in an unknown
milk fat sample;
S is the results from addition of unknown foreign fat by inserting the triglycerides of the foreign
fat/milk fat mixture in the above total triglyceride formula;
Sf is the mean S-value obtained by inserting the triglyceride data of the pure foreign fats in this
formula and by calculating a mean value ( Sf = 7,56).

NOTE If an unknown foreign fat is added to milk fat, the mean S-value of the different foreign fats for the
total formula is chosen for SF / This mean S-value is obtained by inserting the triglyceride data of the pure
foreign fats in this formula and by calculating a mean value (SF = 7,56). Good quantitative results concerning
any foreign fat additions are also obtained using the palm oil/beef tallow formula (Table 2) and a mean SF -
value of 10,57.
With known foreign fat types, insert the following SF-values in the above mentioned formula. Chose the
respective foreign fat formula from Table 2.

Table 5 — Sr-values of various foreign fats

Foreign Fat Sr

Soybean oil 8,18

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Sunflower oil 9,43

Olive oil 12,75

Coconut oil 118,13

Palm oil 7,55

Palm kernel oil 112,32

Rape-seed oil 3,30

Linseed oil 4,44

Wheat germ oil 27,45

Maize germ oil 9,29

Cotton seed oil 41,18

Lard 177,55

Beef tallow 17,56

Fish oil 64,12

11.2 Expression of test results

Express the test results to two decimal places

12 Range of application of the detection method

The method described in this International Standard applies to bulk milks and is based on the
representatives of milk fat samples. Highly specific detection would be possible, if for a representative number
of milk fats the formula described in 11.1 were derived for different countries. There could be particularly
suited possibilities of detection obtained, if in the different countries formula described here, were set up of a
representative number of milk fats. In this case, the use of complex computer programmes is not required, if
the triglyceride combinations used in Table 2 are applied and the factors redetermined by using the method of
least squares.
By applying the S-ranges as shown in Table 3 the formula is, under particular feeding conditions as, for
instance, underfeeding or feeding of cows with feed yeast or Ca-soaps, generally applicable. Only in case of
extreme feeding conditions (e.g. high uptake of pure feed oils, high administration of Ca-soaps combined with
feed fat etc.) the formula will partly indicate a modified milk fat.
NOTE Fractionated milk fats are generally recognized as unmodified milk fat, if a modification is assumed
only, when the limits are exceeded. Only with fractionated milk fats with unusual milk fat composition, as,
e.g. in case with a hard fraction, obtained with fractionation by physical methods at high temperatures of
approximately 30 °C with low yields of a few percent or with fractionation with over critical CO 2, the formulae
indicate a modification. Milk fat fractionation may, however, be detected using other procedures e.g. a
differential scanning calorimetric method.

13 Precision
13.1 Interlaboratory test
The values for the repeatability and reproducibility were derived from the result of an interlaboratory test
carried out in accordance with ISO 57252). The values derived from this interlaboratory test were determined
using milk fat on the basis of the formula from Table 2 and the S-ranges in Table 3 and may not be
applicable to concentration ranges and matrices other than those given.

13.2 Repeatability
The absolute difference between two independent single test results, obtained using the same method on
identical test material in the same laboratory by the same operator using the same equipment within a short

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interval of time, will in not more than 5 % of cases be greater than the limits mentioned in table 6.

Table 6 — Repeatability limits (r) for the different formulae

Foreign Fat Limit for r

Soyabean, sunflower, olive, rape-seed, linseed, wheat germ, maize germ, cotton, fish oil 0,67

Coconut and palm kernel fat 0,12

Palm oil and beef tallow 1,20

Lard 0,58

Total formula 1,49

13.3 Reproducibility
The absolute difference between two independent single test results, obtained using the same method on
identical test material in different laboratories with different operators using different equipment, will in not
more than 5 % of cases be greater than the limits mentioned in table 7.

Table 7 — Reproducibility limits (R) for the different formulae

Foreign Fat Limit for R

Soyabean, sunflower, olive, rape-seed, linseed, wheat germ, maize germ, cotton, fish 1,08
oil

Coconut and palm kernel fat 0,40

Palm oil and beef tallow 1,81

Lard 0,60

Total formula 2,07

14 Range of application of the detection method

The described method applies to bulk milks and is based on the representatives of milk fat samples.
Highly specific detection would be possible, if for a representative number of milk fats, formulae as
described above were derived for different countries.
There could be particularly suited possibilities of detection obtained, if in the different countries formulae,
as have been described here, were set up of a representative number of milk fats. In this case, the use of
complex computer programmes is not required, if the triglyceride combinations used in Table 2 are applied
and the factors redetermined by using the method of least squares.
By applying the S-ranges as shown in Table 3 the formulae are, under particular feeding conditions as, for
instance, underfeeding or feeding of cows with feed yeast or Ca-soaps, generally applicable. Only in the case
of extreme feeding conditions (e.g. high uptake of pure feed oils, high administration of Ca-soaps combined
with feed fat etc.) the formulae partly indicate modified milk fat.
NOTE Fractionated milk fats are generally recognized as unmodified milk fat, if a modification is assumed
only, when the limits are exceeded. Only with fractionated milk fats with unusual milk fat composition, as it
is, e.g., the case with a
hard fraction, obtained with fractionation by physical methods at high temperatures of approximately 30 °C
with low yields of a few percent or with fractionation with overcritical CO 2, the formulae indicate a
modification.
Milk fat fractionation may, however, be detected using other procedures e.g.  a differential scanning-
calorimetric method.

15 Accuracy

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15.1 Critical difference

With the repeatability (r) and the reproducibility (R) the critical differences for all S-ranges of Table 3 can
be calculated (duplicate analyses). The respective values are given in Table 8.

Table 8 — Critical differences for all triglyceride formulae

Foreign Fat range

Soyabean, sunflower, olive, rape-seed, linseed, wheat germ, maize germ, cotton, fish 97,43 –
oil 102,57

Coconut and palm kernel fat 99,14 –

100,86

Palm oil and beef tallow 94,94 –

105,09

Lard 97,65 –
102,35

Total formula 94,58 –

105,42

15.2 Acceptability of results

All calibrated with two rounded decimals calculated triglyceride contents of C24, C26, C28 to C54 as well
as cholesterol shall be normalized to exactly 100.
Use the results of the duplicate analysis as check on the repeatability. If the absolute difference between the
two S-results for all five triglyceride formulae do not transgress the repeatability limits r in Table 6, then the
repeatability requirement is met.
NOTE For control of optimal gas chromatographic conditions and especially of the quality of the column it
should be guaranteed that with 10 repetition runs the difference of the maximum and minimum S-values of
all five triglyceride formulae do not transgress the range x – r, with x = 1,58 (for 10 runs, see literature
(17)), and the repeatability limits r for the different formulae in Table 6.

16 Test report
The test report shall specify:

a. all information necessary for the complete identification of the sample;

b. the sampling method used, if known;
c. the test method used, with reference to this International Standard;
d. all operational details not specified in this International Standard, or regarded as optional, together
with details of any incidents which may have influenced the test result(s);
e. the test result(s) obtained, and, if the repeatability has been checked, the final quoted result
obtained.

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