In order to predict omega-6 and omega-3 fatty acids in the diet of humans, seventy-three pork back fat adipose tissue samples were measured with Raman spectroscopy directly on adipose tissue and on melted fat. Melted fat samples were, in addition, measured with Fourier transform infrared (FT-IR) spectroscopy. Gas chromatography analyses were conducted as the reference analysis. Partial least squares regression (PLSR) was used to calibrate and validate all models predicting omega-3 and omega-6 fatty acids contents from spectra. Omega-6 fatty acids in melted fat measured with FT-IR was predicted with a correlation coefficient (<i>R</i>) of 0.93 and a root mean square error of cross-validation (RMSECV) of 1.61% of the total amount of fatty acids. Raman spectra measured on melted fat gave a prediction of omega-6 fatty acids with <i>R</i> = 0.97, and RMSECV = 0.99% of total amount of fatty acids. Omega-6 fatty acids were predicted with <i>R</i> = 0.94, and RMSECV = 1.50% of the total amount of fatty acids using Raman spectra recorded on adipose tissue. For omega-3 fatty acids, the highest <i>R</i> = 0.91, and lowest RMSECV = 0.23% of the total amount of fatty acids were obtained from Raman spectra acquired on melted fat. FT-IR and Raman spectroscopy may be used as rapid, nondestructive methods to determine omega-6 and omega-3 fatty acids in melted fat. Raman spectroscopy can also be used directly on adipose tissue.

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