Joachim Molkentin, Dietz Precht Institute for Chemistry and Physics, Federal Dairy Research Centre, Kiel, Germany © WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000 0931-5985/2000/0303-0194 $17.50+.50/0 194 Eur. J. Lipid Sci. Technol. 2000, 194–201 Research Paper 1 Introduction According to an EC-regulation [1] mixed spreadable fats are allowed to contain 10–80% of milk fat related to their total fat content. However, up to now no analytical method has officially been specified for the control of the declared milk fat content in these products. A well-known principle of milk fat quantitation in mixed fats is the gas-chromato- graphic analysis of butyric acid (C4) [2, 3], that exclusive- ly occurs in milk fat. Thus, the milk fat proportion can be calculated from the C4 contents in the mixed fat and in the underlying pure milk fat. Alternatively, the calculation is based on a mean C4 content for the pure milk fat, requir- ing only the analysis of the mixed then. As there are nu- merous analytical standards for butyric acid analysis [4–6], a comparison of such methods was performed [7] leading to the development of an improved draft standard for quantitation of milk fat in mixed fats [8]. While this draft was successfully checked in the authors’ laboratory using mixtures of milk fat with foreign fats of animal or vegetable origin [9] as well as with partially hydrogenated vegetable fats [10], three ring tests were performed within the EU experts group “Milk and milk products” of the European Commission (DG VI), Brussels. The work presented here describes the limits of the draft method as well as the progress achieved during the three ring tests, finally en- abling the calculation of precision data. 2 Materials and methods 2.1 Samples The base milk fats were obtained from butter resulting from different feeding conditions by melting and passing the fat layer through a folded filter MN 615ff 1/4 (Macherey-Nagel, Düren, Germany) at 50 °C in an oven. Beef tallow was purified in a similar way at 80 °C. Soy- bean oil and coconut fat were purchased at retail stores. Mixtures of milk fat with non-milk fats were prepared in different ratios as outlined later. 2.2 Preparation of samples for gas chromatography 100 ± 20 mg of fat were weighed to the nearest 0.1 mg and dissolved in 5 ml methyl valerate standard (n-hep- tane, containing 0.4 mg/ml methyl valerate). 1 ml of this solution was mixed with 20 μl sodium methylate solution (2 N in methanol) in a sample vial, shaken vigorously for 3 min (Vortex mixer) and centrifuged for 1 min (2000 min –1 ). After addition of 100 mg sodium hydrogen sulphate- monohydrate the vial was recapped, mixed again for Validation of a gas-chromatographic method for the determination of milk fat contents in mixed fats by butyric acid analysis Gas-chromatographic analysis of butyric acid (C4) in mixed spreadable fats can be used to calculate the milk fat proportion, either by additional analysis of the underlying pure milk fat (component sample) or by using a mean C4 content instead. After com- parison of several analytical variants and subsequent development of an improved draft standard for quantitation of milk fat in mixed fats, this draft was checked for accu- racy in three EU-wide ring tests performed with 26 participating laboratories. Due to the natural variation of the butyric acid content in European milk fats, deviations from the actual milk fat content may amount up to ± 10% in addition to analytical errors when calculating with a mean C4 content instead of the C4 content of a component sample. Though the reproducibility could be improved by application of correction factors de- rived from a reference fat, only the calculation based on the additional analysis of a component sample of the pure milk fat led to an acceptable accuracy concerning the trueness of the results in combination with good repeatability (r) and reproducibility (R). Finally, several further improvements of the draft standard led to a precision of r = 4.3% and R = 6.0% relative to the mean of two milk fat contents determined. Thus, the test- ed procedure proved its suitability for a control method of the declared milk fat content in mixed fat spreads. Keywords: Mixed fat, milk fat content, butyric acid, gas chromatography, accuracy. Correspondence: Joachim Molkentin, Institute for Chemistry and Physics, Federal Dairy Research Centre, Hermann-Weig- mann-Str. 1, 24103 Kiel, Germany. Phone:+49 431-6092257, Fax: +49 431-6092300; e-mail: molkentin@bafm.de