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• Two new methods for fatty acid methylation without lipid extraction were validated. • Dried milk can be reliably used for in situ alkali-catalysed transesterification. • Acidic methylation of fresh milk is feasible with optimised reaction parameters. • The new methods are applicable to both homogenised and raw milk samples. Determination of the fatty acid (FA) profile of milk fat generally involves total lipid extraction from liquid milk, transesterification and GC analysis. The lipid extraction step is time consuming and often employs toxic solvents such as chloroform. Two alternative methods are presented here that skip the lipid extraction step and allow the determination of FA composition via direct transesterification of dried milk and liquid milk respectively. We have shown that dried milk can be used directly in alkaline-catalysed methylation, whereas direct transesterification of both dried milk and fresh milk is feasible with acidic methanol. Both methods generate similar results as compared to the classical two-step method (i.e. lipid extraction and FA methylation) when optimised methylation parameters (temperature, time, milk and reagent volume) are followed. By omitting the lipid extraction step, these simplified one-step methods offer a much higher throughput and a reduced cost in FA composition analysis of milk samples. [ABSTRACT FROM AUTHOR]