Analysis of oxylipins by high‐performance liquid chromatography with evaporative light‐scattering detection and particle beam‐mass spectrometry

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Abstract
The metabolism of 13 S‐hydroperoxy‐9Z,11E,15Z‐octadecatrienoic acid was investigated in a crude enzyme extract from mung bean seedlings (Phaseolus radiatus L.). Hydroperoxide‐metabolizing activity was mainly due to a hydroperoxide lyase and, to a lesser extent, to an allene oxide synthase and a peroxygenase. Oxylipins originating from hydrolysis and cyclization of the allene oxide synthase product 12,13‐epoxy‐9Z,11,15Z‐octadecatrienoic acid and from peroxygenase catalysis were identified by high‐performance liquid chromatography (HPLC) particle beam‐mass spectrometry (PB‐MS) and quantified by normal‐phase HPLC with an evaporative light‐scattering detector (ELSD). An advantage of this methodology was the possibility to avoid extensive derivatization procedures commonly used for the gas chromatographic analysis of oxylipins. Owing to a comparable sample inlet system, the ELSD served an important analytical pilot function for the PB‐MS: Qualitatively identical chromatographic patterns were obtained with both detection systems. The HPLC system enabled the separation of methyl 12‐oxo‐phytodienoate, methyl 11‐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‐13‐hydroxy‐9Z,11E,15Z‐octadecatrienoate, and methyl 13‐hydroperoxy‐9Z,11E,15Z‐octadecatrienoate on a Lichrospher DIOL column within 33 min. Compared 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 factor of about 15. In addition, volatile metabolites were analyzed by capillary gas chromatography. The yield of the hydroperoxide lyase product 2E‐hexenal was 49%, whereas the sum of oxylipins reached about 15%.

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