Rapid Analysis for α-Tocopherol and Its Oxidative Products in the Pisum sativum L. Leaf Using Supercritical Fluid Chromatography-Medium Vacuum Chemical Ionization Tandem Mass Spectrometry.

Abstract

A method for the rapid determination of α-tocopherol (α-T) and its oxidative products in plant tissue has been developed using supercritical fluid extraction (SFE) coupled with supercritical fluid chromatography (SFC) and medium vacuum chemical ionization (MVCI) with tandem mass spectrometry. The method is designed to study changes in levels for α-T and its oxidative products in plant cells during photosynthesis, aiming to observe the light response curves. α-T oxidation is a non-enzymatic self-defense mechanism in plant cells. Unlike enzyme-involved reactions, it cannot be stopped, so the oxidation continues in crude extracts even after extraction. Therefore, a real-time in-situ method is essential for tracking the light response curves. To optimize the selective reaction monitoring method, the reaction mixture of α-T and singlet oxygen (¹O₂), generated by rose Bengal under light illumination, was used as the source of oxidative products. The relative abundance changes in α-tocopherylquinone and 8a-hydroperoxy tocopherone in Pisum sativum L. (Pea) leaves under excessive light illumination have been preliminarily analyzed as part of the light response curve study. The method archives a throughput of 10-15 minutes for analyzing duplicate leaf samples. This process includes cutting off the leaf, sectioning it, placing the sample in a frozen SFE vessel, and conducting SFE/SFC analysis. Consequently, the average throughput is approximately 5-7 minutes per sample.