In the present study a novel approach for Fourier transform infrared (FT-IR) characterization of the fatty acid composition of milk based on dried film measurements has been presented and compared to a standard FT-IR approach based on liquid milk measurements. Two hundred and sixty-two (262) milk samples were obtained from a feeding experiment, and the samples were measured with FT-IR as dried films as well as liquid samples. Calibrations against the most abundant fatty acids, CLA (i.e., 18:2<i>cis</i>-9, <i>trans</i>-11), 18:3<i>cis</i>-9, <i>cis</i>-12, <i>cis</i>-15, and summed fatty acid parameters were obtained for both approaches. The estimation errors obtained in the dried film calibrations were overall lower than the corresponding liquid sample calibrations. Similar and good calibrations (i.e., <i>R</i><sup>2</sup> ranges from 0.82 to 0.94 (liquid samples) and from 0.88 to 0.97 (dried films)) for short-chain fatty acids (6:0–14:0), 18:1<i>cis</i>-9, SAT, MUFA, and iodine value were obtained by both approaches. However, the dried film approach was the only approach for which feasible calibrations (i.e., <i>R</i><sup>2</sup> ranges from 0.78 to 0.93) were obtained for the major saturated fatty acids 16:0 and 18:0, the minor fatty acid features 4:0, CLA (i.e., 18:2<i>cis</i>-9, <i>trans</i>-11), PUFA, and the summed 18:1 <i>trans</i> isomers. For the dried film approach, logical spectral features were found to dominate the respective fatty acid calibration models. The preconcentration step of the dried film approach could be expected to account for a major part of the prediction improvements going from predictions in liquid milk to predictions in dried films. The dried film approach has a significant potential for use in high-throughput applications in industrial environments and might also serve as a valuable supplement for determination of genetic and breeding factors within research communities.

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