Validation of the sensitive and accurate quantitation of the fatty acid distribution in bovine milk

A method for the precise analysis of the complex mixture of fatty acids in milk has been developed and validated. The triacylglycerol of nonanoic acid was applied as the internal standard (ISTD) for absolute quantification. Milk lipids were extracted by miniaturised ultrasonication and methylated with trimethylsulfonium hydroxide. Resulting fatty acid methyl esters were determined by gas chromatography with flame ionisation detection giving excellent resolution, including separation of several 18:1 isomers. The low quantitation limit (0.01 mg mL−1 milk) indicates that the sensitivity of the method is sufficient to quantify up to 50 fatty acids, from 4:0 to 23:0. Measurements of precision provided excellent results for different bovine milk samples of different fat content (coefficient of variance: 1.9% and 9.8% for intra- and interday precision, respectively). Recovery averaged 108 ± 3.5%. Evaluation of methods for determining the total fat content showed that gravimetry is no longer needed when using the ISTD.