Simultaneous Analysis of Phytohormones and Lipid Peroxidation Products in Plant Seeds by LC-MS/MS after Molecular Weight Cutoff Filtration

Abstract

Investigation of the phytohormone and lipid peroxidation product levels in plant seeds can provide an insight into their germination potential and vigor. Simultaneous analysis of these diverse classes of compounds presents a significant challenge for a sample preparation method tailored to accommodate their distinct chemical properties. In this study, an analytical method consisting of simple extraction, molecular weight cutoff filtration, and subsequent liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis was developed for accurate quantification of 11 phytohormones including 2 auxins, 4 cytokinins, 2 gibberellins, abscisic acid, salicylic acid, and jasmonic acid in plant seeds. Additionally, the new method was used for the analysis of 4 lipid peroxidation products like polyunsaturated fatty acids and malondialdehyde. All 15 target plant biochemicals in corn seed tissue were simultaneously analyzed using as little as 50 mg of sample amount. The method detection limits were low, in the range of 0.08–8 ng/mg of corn seed samples. The sample spike recoveries (77–125%) and reproducibility (3–18% relative standard deviation, RSD) were good for all analytes at ng to mg concentration range using minimum amount of seed samples. To our knowledge, this is the first method combining simple sample preparation with LC-MS/MS detection for simultaneous analysis of such a large panel of phytochemicals using very small amount of seed tissue. The method is expected to have broad applications in the seed quality assessment for germination potential and vigor, as well as in the investigation of phytohormone and lipid peroxidation metabolites.