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.

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.

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.

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).

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

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).

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 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.

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:

1. Flow the column with N2 , with the flow speed set at 40 ml/min and at 50 °C for 15 min;
2. Heat the column with 1 K/min up to 355 °C, with the flow speed set at 10 ml N2/min;
3. Hold the column at 355 °C for 12 h to 15 h;
4. Inject two times 1 ml of the cocoa butter solution (5.11) using the respective temperature program;
5. 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

1. 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.
2. 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).
3. 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.
4. Duration of analysis: Completed in 29,3 min.
5. Injection volume: Inject 0,5 ml. Rinse the syringe with pure heptane (5.6) several times after each injection.
6. 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 milk fat sample.

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

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 - 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

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

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 %

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

• w_f 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;
• S_f 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 (S_f= 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 S_F/ 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 (S_F = 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 S_F-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

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 CO₂, 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 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

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

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 CO₂, the formulae indicate a modification.

Milk fat fractionation may, however, be detected using other procedures e.g.  a differential scanning-calorimetric method.

15 Accuracy

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

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:

1. all information necessary for the complete identification of the sample;
2. the sampling method used, if known;
3. the test method used, with reference to this International Standard;
4. 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);
5. the test result(s) obtained, and, if the repeatability has been checked, the final quoted result obtained.