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Dried blood spots (DBS) by fingertip prick collection for fatty acid profiling are becoming increasingly popular due to ease of collection, minimal invasiveness and its amenability to high‐throughput analyses. Herein, we assess a microwave‐assisted direct transesterification method for the production of fatty acid methyl esters (FAME) from DBS. Technical replicates of human whole blood were collected and 25‐μL aliquots were applied to chromatography strips prior to analysis by a standard 3‐h transesterification method or microwave‐assisted direct transesterification method under various power (variable vs constant), time (1–5 min) and reagent (1–10 % H2SO4 in methanol) conditions. In addition, a standard method was compared to a 5‐min, 30‐W power microwave in 1 % H2SO4 method for FAME yield from whole blood sphingomyelin, and sphingomyelin standards alone and spiked in whole blood. Microwave‐assisted direct transesterification yielded no significant differences in both quantitative (nmol/100 µL) and qualitative (mol%) fatty acid assessments after as little as 1.5‐ and 1‐min reaction times, respectively, using the variable power method and 5 % H2SO4 in methanol. However, 30‐W power for 5 min increased total FAME yield of the technical replicates by 14 %. This increase appears largely due to higher sphingomyelin‐derived FAME yield of up to 109 and 399 % compared to the standard method when determined from whole blood or pure standards, respectively. In conclusion, microwave‐assisted direct transesterification of DBS achieved in as little as 1‐min, and 5‐min reaction times increase total fatty acids primarily by significantly improving sphingomyelin‐derived fatty acid yield.

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