In the present study, a novel approach for mid-infrared (IR)-based prediction of bovine milk fatty acid composition is introduced. A rapid, solvent-free, two-step centrifugation method was applied in order to obtain representative milk fat fractions. IR spectra of pure milk lipids were recorded with attenuated total reflection Fourier-transform infrared (ATR-FT-IR) spectroscopy. Comparison to the IR transmission spectra of whole milk revealed a higher amount of significant spectral information for fatty acid analysis. Partial least squares (PLS) regression models were calculated to relate the IR spectra to gas chromatography/mass spectrometry (GC/MS) reference values, providing particularly good predictions for fatty acid sum parameters as well as for the following individual fatty acids: C10:0 (R2P = 0.99), C12:0 (R2P = 0.97), C14:0 (R2P = 0.88), C16:0 (R2P = 0.81), C18:0 (R2P = 0.93), and C18:1cis (R2P = 0.95). The IR wavenumber ranges for the individual regression models were optimized and validated by calculation of the PLS selectivity ratio. Based on a set of 45 milk samples, the obtained PLS figures of merit are significantly better than those reported in literature using whole milk transmission spectra and larger datasets. In this context, direct IR measurement of the milk fat fraction inherently eliminates covariation structures between fatty acids and total fat content, which poses a common problem in IR-based milk fat profiling. The combination of solvent-free lipid separation and ATR-FT-IR spectroscopy represents a novel approach for fast fatty acid prediction, with the potential for high-throughput application in routine lab operation.

This study introduces the first mid-infrared (IR)–based method for determining the fatty acid composition of human milk. A representative milk lipid fraction was obtained by applying a rapid and solvent-free two-step centrifugation method. Attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy was applied to record absorbance spectra of pure milk fat. The obtained spectra were compared to whole human milk transmission spectra, revealing the significantly higher degree of fatty acid–related spectral features in ATR FT-IR spectra. Partial least squares (PLS)–based multivariate regression equations were established by relating ATR FT-IR spectra to fatty acid reference concentrations, obtained with gas chromatography–mass spectrometry (GC-MS). Good predictions were achieved for the most important fatty acid sum parameters: saturated fatty acids (SAT, R2CV = 0.94), monounsaturated fatty acids (MONO, R2CV = 0.85), polyunsaturated fatty acids (PUFA, R2CV = 0.87), unsaturated fatty acids (UNSAT, R2CV = 0.91), short-chain fatty acids (SCFA, R2CV = 0.79), medium-chain fatty acids (MCFA, R2CV = 0.97), and long-chain fatty acids (LCFA, R2CV = 0.88). The PLS selectivity ratio (SR) was calculated in order to optimize and verify each individual calibration model. All mid-IR regions with high SR could be assigned to absorbances from fatty acids, indicating high validity of the obtained models.