Determination of Flavonoids in Stellaria by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Abstract

The genus Stellaria (Caryophyllaceae) presents widely distributed plants often used in traditional medicine. Flavonoids are highly active plant secondary metabolites that may be involved in some of the effects of Stellaria plants. In this study, two new high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS-MS) methods were developed for the determination of flavonoids and isoflavonoids in plant material. The separations were performed on a reverse-phase C₁₈ column with gradient elution using methanol and water with 0.5% acetic acid as the mobile phase. Multiple reaction monitoring was used for the tandem mass spectrometry detection and the two most intensive transitions were chosen for the identification of each analyte. The limits of detection and the limits of quantification were between 0.2 and 15.0 ng/mL and 0.6 and 50.0 ng/mL. The developed methods were successfully used for the analyses of four representatives of the genus Stellaria. All the studied herbs contained luteolin and its 7-O-glycosides, naringenin, kaempferol, quercetin, its glycoside rutin, apigenin, and its 7-O-glycoside. One coumarin, scopoletin, was also found. Isoflavones were primarily represented by genistein, genistin, and ononin. Some of the analytes were detected for the first time in Stellaria sp. The findings support that these methods are suitable for analyses of plant material.

Keywords:

• Caryophyllaceae
• Flavonoid
• High-performance liquid chromatography–electrospray ionization tandem mass spectrometry
• HPLC–MS
• Isoflavonoid
• Stellaria

Notes

^a Solvent A: 40% (v/v) methanol/water with 0.5% acetic acid.

^b Solvent B: 100% methanol with 0.5% acetic acid.

^a The intraday-precision was evaluated by repeated injection (n = 6) of a mixture of standards at a concentration of 0.5 µg/mL performed on one day.

^b The inter-day precision was evaluated by repeated injection (n = 15) of a mixture of standards at a concentration of 0.5 µg/mL performed on five days.

^c Limit of detection (S/N = 3).

^d Limit of quantification (S/N = 10).

^e Slopes and intercepts of calibration curves ± standard deviation.

^f Regression coefficient.

^a The intraday-precision was evaluated by repeated injection (n = 6) of a mixture of standards at a concentration of 0.5 µg/mL performed on one day.

^b The inter-day precision was evaluated by repeated injection (n = 15) of a mixture of standards at a concentration of 0.5 µg/mL performed on five days.

^c Limit of detection (S/N = 3).

^d Limit of quantification (S/N = 10).

^e Slopes and intercepts of calibration curves ± standard deviation.

^f Regression coefficient.

^a Sample collected in year 2012.

^b Sample collected in year 2009.

^a Sample collected in year 2012.

^b Sample collected in year 2009.