Effect of Ethanol Purity and Sample Preparation Strategy on Free Fatty Acid Determination in Fish Oil by Alkalimetric Titration

Abstract

The accuracy of free fatty acid (FFA) determination in fish oil is strongly influenced by solvent purity and sample preparation strategy. This study evaluated the effect of ethanol grade on FFA determination using an alkalimetric titration method and assessed the homogeneity and repeatability of individual sample analysis in comparison with large-scale solution homogenization. Titrations were performed using standardized potassium hydroxide solution (0.1 N) with phenolphthalein as the endpoint indicator, and each analytical condition was evaluated in triplicate. Pro-analysis grade ethanol and technical-grade ethanol were employed, with prior solvent neutralization to correct background acidity. For bulk homogenization, 17.5 g of fish oil was dissolved in 175 mL of ethanol (1:10, w/v) and heated at 40 ± 2 °C under magnetic stirring at a constant moderate speed for 15 minutes. Aliquots equivalent to 2.5 g were withdrawn sequentially under continuous agitation and immediately analyzed. Individual sample analysis demonstrated excellent repeatability for both solvent grades, as indicated by identical analytical results across replicates, whereas technical-grade ethanol produced systematically higher FFA values. In contrast, large-scale solution homogenization resulted in poor repeatability for both solvents, reflected by high relative standard deviation values, indicating inadequate homogeneity of the fish oil–ethanol mixtures. Statistical evaluation confirmed that the observed differences were analytically significant and primarily attributed to solvent purity rather than random analytical error. These findings highlight the critical role of solvent quality and sample handling in ensuring reliable free fatty acid determination in fish oil by alkalimetric titration.