AbstractFor determination of the double‐bond position in polyunsaturated C24–30 fatty acids from marine organisms, methoxy derivatives were prepared. Diagnostic mass spectral fragment as well as molecular ion intensities were obtained by adjusting the ion source optics in the presence of ammonia at a lower source pressure than used conventionally. A lower detection limit was observed compared to conventional methane chemical ionization, which is a more favorable condition for capillary gas chromatography. Analysis of fatty acids from the spongeCalyx niceaensis showed the double‐bond position of 8 unsaturated fatty acids, including two new ones. In addition, structural proof is provided for the presence of a new cyclopropane‐containing fatty acid: 19,20‐methylene‐hexacosanoic acid.