An efficient fast gas chromatographic method for simultaneous determination of elaidic acid, vaccenic acid and rumenic acid contents in human plasma phospholipids and human milk was optimized and validated. Two capillary columns, RTX-2330 and SP-2560, both of high polarity but with different dimensions (40 m × 0.18 mm I.D. and 0.10 μm film thickness, and 75 m × 0.18 mm I.D. and 0.14 μm film thickness, respectively), were compared for the separation of these fatty acids within a complete fatty acid profile. Separation with the SP-2560 column gave the best results. In comparison with the commonly used 100 m × 0.25 mm × 0.20 μm columns, this new type of fast column allowed the separation of fatty acid methyl esters with the same resolution but in less time, 32.2 min. In addition, separation of the phospholipid fraction in human plasma samples was optimized by using 96-well extraction plates filled with an aminopropyl phase. Recoveries ranged between 95.8% and 103.7%. Intra-assay and inter-assay precision ranged between 0.76% and 8.87%. Application of this method showed that it is a rapid and reliable method for quick and correct identification and quantification of these fatty acids in routine analysis.