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 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 (r 2 > 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.

Characterization of Forage Quality, Phenolic Profiles, and Antioxidant Activity in Alfalfa (Medicago sativa L.)

“…The highest MYR, as the most abundant flavonoid in bound extract, had foreign cultivar Florida 66 (502.2 μg/g dm), which is 4.3 times more than the lowest value in OS 101 (117.1 μg/g dm) ( Figure 3 b; Table S4 ). In alfalfa samples, in addition to the flavonoids found in our research, some authors also mentioned glabridin, naringin, and luteolin as major flavonoids [ 13 , 36 , 40 , 41 , 42 ].…”

“…In contrast to the extensively studied triterpene saponins and the phytoestrogen compounds of Medicago species [ 5 , 12 , 13 ], Medicago polyphenols are less genus-specific and generally encountered in many legumes [ 34 ]. As we mentioned before, most plant phenolics are present in soluble and insoluble-bound forms.…”

“…In addition to the extensive research of Medicago species triterpene saponins and phytoestrogens [ 12 , 13 , 14 ], alfalfa contains potentially valuable phenolics with an excellent ability to capture oxidative free radicals [ 12 , 15 ]. Several classes of alfalfa phenolics were reported in the literature, and among them, phenolic acids and flavonoids are widely present [ 9 , 16 , 17 ].…”

Comprehensive Metabolite Fingerprinting of Australian Black and Green Olives and Their Antioxidant and Pharmacokinetics Properties

“…2'-Hydroxyformononetin is well-known for reducing oxidative stress and inflammation, improving bone health and density, and potentially reducing the risk of certain cancers. It may also benefit cardiovascular health, including lowering blood pressure and improving cholesterol levels [52]. Dihydroformononetin has been found to have anti-diabetic effects, including improving insulin sensitivity, and reducing blood sugar levels.…”