Comparison and validation of 2 analytical methods for the determination of free fatty acids in dairy products by gas chromatography with flame ionization detection

Abstract

Accurate quantification of free fatty acids (FFA) in dairy products is important for quality control, nutritional, antimicrobial, authenticity, legislative, and flavor purposes. In this study, the performance of 2 widely used gas chromatographic flame ionization detection methods for determination of FFA in dairy products differing in lipid content and degree of lipolysis were evaluated. We used a direct on-column approach where the isolated FFA extract was injected directly and a derivatization approach where the FFA were esterified in the injector to methyl esters using tetramethylammonium hydroxide as a catalyst. A comprehensive validation was undertaken to establish method linearity, limits of detection, limits of quantification, accuracy, and precision. Linear calibrations of 3 to 700mg/L (R2>0.999) and 20 to 700mg/L (R2>0.997), and limits of detection and limits of quantification of 0.7 and 3mg/L and 5 and 20mg/L were obtained for the direct injection on-column and the derivatization method, respectively. Intraday precision of 1.5 to 7.2% was obtained for both methods. The direct injection on-column method had the lower levels of limits of detection and quantification, because FFA are directly injected onto the GC as opposed to the split injection used in the derivatization method. However, the direct injection on-column method experienced accumulative column phase deterioration and irreversible FFA absorption because of the acidic nature of the injection extract, which adversely affected method robustness and the quantification of some longer chain FFA. The derivatization method experienced issues with quantification of butyric acid at low concentrations because of coelution with the injection solvent peak, loss of polyunsaturated FFA due to degradation by tetramethylammonium hydroxide, and the periodic emergence of by-product peaks of the tetramethylammonium hydroxide reaction that interfered with the quantification of some short-chain FFA. The derivatization method is more robust, and because the derivatization step can be automated, it is more suitable for routine analysis of FFA in dairy products. However, considerable scope exists to develop an alternative gas chromatography with flame ionization detection method to quantify FFA in dairy products without any limitations that is robust and accurate.