A mixture of methyl ester derivatives of fatty acids from the oils of pine nuts was well resolved to five fractions differing by degree of unsaturation by silver ion solid-phase extraction column chromatography ($Ag^{+}$-SEC). Polyunsaturated fatty acid with non-methylene interrupted conjugated double bond (NMiDB) radical held more strongly to silver ions in the column than methylene interrupted conjugated double bond (MiDB) one when they had the same number of double bonds. Although both the picolinyl ester and DMOX derivative provided clear mass ion species powerful enough to elucidate the structure of the polyunsaturated fatty acid (PUFA) with NMiDB and/or methylene interrupted conjugated double bond (MiDB) radical in the oils, the picolinyl ester of PUFA with NMiDB radical did not provide a cluster of mass ions neighboring diagnostic mass ions induced by the double bond in the proximal to the carboxyl group. However, the DMOX derivative of PUFA with NMiDB group as well as MiDB showed abundant mass ion species differing by gaps of 12 amu, which made it possible with greater ease to locate the double bonds in the molecule. The oil contained $C_{18:2{omega}6}$ (46.2 %) and $C_{18:1{omega}9}$ (25.4 %) as main components, and considerable amounts of PUFAs with NMiDB radical such as ${Delta}^{5.;9.;12}-C_{18:3}$ (16.0 %), ${Delta}^{5.;9}-C_{18:2}$ (2.3 %) and ${Delta}^{5.;11.;14}-C_{20:3}$ (0.8 %).