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

Abstract

Flavonoids, isoflavonoids, and phytohormones are small metabolites of plants, which are involved in the regulation of plant development and/or defense against pathogens. Quantitation of these metabolites in plants is important to basic research on crop protection and in the food and herbal industry. Hence, a robust, reliable, and sensitive method is 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 isoflavonoids including genistein, daidzein, formononetin, biochanin A, 2′-hydroxyformononetin, 2′-methoxyformononetin, medicarpin, flavonoids; liquiritigenin, naringenin, and 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. 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 up to 0.1 pg levels. Further the method was also validated in terms of selectivity, linearity (r² > 0.99), 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 metabolites from leaf extract of M. truncatula.

Graphical Abstract

Keywords:

• HPLC–ESI-MS/MS
• jasmonic acid
• Medicago truncatula
• medicarpin
• QTRAP

Disclosure statement

The authors do not have any conflicts of interest to declare.

Additional information

Funding

This research work was carried out at the Mass spectrometry facility of the Advanced Technology Platform Centre (ATPC), managed by the Regional Centre for Biotechnology (RCB), Faridabad (HR)-India. The authors NS and NSh are highly thankful to the Department of Biotechnology, Ministry of Science and Technology, New Delhi - India (Grant No. BT.MED-II/ATPC/BSC/01/2010) for their financial support to the facility.PA acknowledges PhD fellowship grant from the Council of Scientific and Industrial Research. DC thanks Innovative Young Biotechnologist Award from DBT, Govt. of India, [BT/09/IYBA/2015/12] and intramural funds from RCB.