Measurement of Hydroxy and Hydroperoxy Fatty Acids by a High Pressure Liquid Chromatography with a Column-switching System

Hydroxy and hydroperoxy fatty acids were labeled with 9-bromomethylacridine at room temperature. They were separated from the degradation products and less polar fatty acid derivatives on an octyl silicagel column, and put on an octadecyl silicagel column by on-line column switching. By this method, picomolar levels of the derivatives were measured with good reproducibility. The detection limit of 16-hydroxy-hexadecanoic acid as a representative was 0.9 pmol (S/N =3) and the relative standard deviation of its peak areas was 2.5% (18.5 pmol, n = 7). The method was used for the measurement of hydroxy fatty acids derived from hydroperoxy fatty acids and phosphatidylcholine (PC) hydroperoxides spiked in human plasma. By incubation at 37°C for 4h with human plasma, the hydroperoxy fatty acid was reduced to the corresponding hydroxy fatty acid. In this condition, the PC hydroperoxides showed a considerable decrease, however, a small portion (2.5–3%) of PC hydroperoxides decomposed gave the corresponding hydroxy fatty acids.