Fatty Acid Determination in Human Milk Using Attenuated Total Reflection Infrared Spectroscopy and Solvent-Free Lipid Separation

Abstract

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, R²_CV = 0.94), monounsaturated fatty acids (MONO, R²_CV = 0.85), polyunsaturated fatty acids (PUFA, R²_CV = 0.87), unsaturated fatty acids (UNSAT, R²_CV = 0.91), short-chain fatty acids (SCFA, R²_CV = 0.79), medium-chain fatty acids (MCFA, R²_CV = 0.97), and long-chain fatty acids (LCFA, R²_CV = 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.

Graphical Abstract