ANALYSIS OF OXYLIPINS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH EVAPORATIVE LIGHT-SCATTERING DETECTION AND PARTICLE-BEAM MASS-SPECTROMETRY

The metabolism of 13S-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid was investigated in a crude enzyme extract from mung bean seedlings (Phas eolus radiatus L.). Hy droperoxide-metabolizing activity was mainly du e to a hydroperoxide lyase and, to a lesser extent, to an allene oxide synthase and a peroxygenase. Oxylipins originating from hydrolysis an d cyclization of the allene oxide synthase product 12,13-epoxy-9Z,11,1 5Z-octadecatrienoic acid and from peroxy-genase catalysis were identif ied by high-performance liquid chromatography (HPLC) particle beam-mas s spectrometry (PB MS) and quantified by normal-phase HPLC with an eva porative light-scattering detector (ELSD). An advantage of this method ology was the possibility to avoid extensive derivatization procedures commonly used for the gas chromatographic analysis of oxylipins. Owin g to a comparable sample inlet system, the ELSD served an important an alytical pilot function for the PB-MS: Qualitatively identical chromat ographic patterns were obtained with both detection systems. The HPLC system enabled the separation of methyl 12-oxo-phytodienoate, methyl 1 1-hydroxy-12-oxo-9Z,15Z-octadecadienoate, methyl 12-oxo-13-hydroxy-9Z, 15Z-octadecadienoate, methyl 9-hydroxy-12-oxo-10E,15Z-octadecadienoate , methyl 13-hydroxy-9Z,11E,15Z-octadecatrienoate, methyl 15,16-epoxy-1 3-hydroxy-9Z,11E-octadecadienoate, and methyl 13-hydroperoxy-9Z,11E,15 Z-octadecatrienoate on a Lichrospher DIOL column within 33 min. Compar ed with a diode array detector, the ELSD proved to be more sensitive, in the case of methyl 12-oxo-13-hydroxy-9Z,15Z-octadecadienoate by a f actor of about 15. In addition, volatile metabolites were analyzed by capillary gas chromatography. The yield of the hydroperoxide lyase pro duct 2E-hexenal was 49%, whereas the sum of oxylipins reached about 15 %.