Development of a rapid LC-MS/MS method for the simultaneous quantification of various flavonoids and phytohormones extracted from Medicago Truncatula leaves

Abstract

Flavonoids are small metabolites of plants, which are involved in the regulation of plant development as well as defence against pathogens. Quantitation of these flavonoids in plant samples is highly important and essential in the food and herbal industry. Hence, robust, reliable and sensitive methods are required for the analysis of these compounds in plant samples. In the present study, a high performance liquid chromatographic-tandem mass spectrometric (HPLC- ESI-MS/MS) method was developed and validated for the determination of nine flavonoids, including Liquiritigenin, Naringenin, Genistein, Daidzein, Formononetin, Biochanin A, 2-hydroxy formononetin, 2-methoxyformononetin, Medicarpin, and two phytohormones, Salicylic acid and Jasmonic acid, in Medicago truncatula. The analytes were separated by means of C-18, reversed-phase chromatography and detected using QTRAP mass spectrometer. Molecules were quantified using different transitions in positive and negative ion modes simultaneously in 12 minutes. The on-column limit of detection of all the analytes was as low as 0.03 pg, whereas the limit of quantification of all the compounds was observed upto 0.1 pg levels. Further the method was also validated in terms of specificity, linearity (r2> 0.99), average recovery (90.6 - 110.3%), accuracy (RE% [≤] 3%) and precision (RSD% [≤] 3%). As a proof of concept, the developed method was successfully used for the quantitation of these flavonoids and phytohormones from leaf extract of Medicago truncatula.