Determination of docosahexaenoic acid in milk using affinity solid-phase purification with argentous ions and modified thin layer chromatography

This paper reports a simple method for docosahexaenoic acid determination in bovine milk. The method consists of three steps: (1) one-step DHA methylation in bovine milk without lipid extraction, (2) concentration of DHA methyl ester by AgNO₃-modified silica gel column chromatography, and (3) detection of DHA methyl ester using spot focused-high performance thin layer chromatography. DHA was directly methylated in bovine milk and selectively concentrated in accordance with its intrinsic affinity to argentous ions. Purified DHA methyl ester was developed on newly designed thin layer chromatographic plates with focusing slits and determined densitometrically using conventional software, following visualisation with iodine staining. In addition to the densitometrical analysis, it is also possible for practical, semiquantitative DHA determination to be visually performed on site in manufacturing industries. Unlike conventional methods, the method described needs neither lipid extraction procedures that use a large amount of organic solvent, nor a gas chromatograph system.