Simultaneous Determination of Organic Acids and Flavonoids in Lonicerae flos by Liquid Chromatography-Tandem Mass Spectrometry
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Abstract:
In this paper, an analytical method for the determination of 4 organic acids (chlorogenic acid, caffeic acid, isochlorogenic acid A and isochlorogenic acid C) and 14 flavonoids (quercetin, rutin, luteolin, cynaroside, kaempferol, astragalin, apigenin, rhoifolin, baicalein, baicalin, quercetin-3-methylether, diosmetin, chrysin and kaempferide) in Lonicerae flos were developed and validated by solid phase extraction and liquid chromatography with tandem mass spectrometry (LC-MS/MS). In Lonicerae flos samples, the organic acids and flavonoids were extracted by methanol solution (concentration: 50%), ultrasonication for 40 min, and cleaned up with HLB column by solid phase extraction. The methanol eluent was ?ltered through nylon membrane and analysis by LC-MS/MS. The solution was separated by Mightysil RP-18 column (150 mm × 4.6 mm (i.d), 3 μm) and the quantitative detection was performed on LC-MS/MS by multiple reaction monitoring (MRM) mode under negative electrospray ionization (ESI-). External standard method was used for quantitation analysis. The limits of quantification (LOQs, S/N>10) were 0.005 g/kg~0.05 g/kg. Single-laboratory method validation data were determined, the calibration standard curves concentrations were 0.05~1.00 g/kg and the correlation coefficient of calibration standard curves was more than 0.993. The recoveries were found in the range of 69.2%~116% and the relative standard deviations were 3.3%~12.0% at the respiked levels (10.0, 20.0 and 40.0 g/kg for chlorogenic acid; 1.0, 2.0 and 4.0 g/kg for caffeic acid; 5.00, 10.0 and 20.0 g/kg for isochlorogenic acid A and isochlorogenic acid C; 0.05, 0.10 and 0.20 g/kg for other compounds). This method was suitable for simultaneous determination and confirmation of 4 organic acid and 14 flavonoids in Lonicerae flos. Key words: Lonicerae flos; organic acid; flavonoids; high performance liquid chromatography with tandem mass spectrometry of quantification (LOQs, S/N>10) were 0.005 g/kg~0.05 g/kg. Single-laboratory method validation data were determined, the calibration standard curves concentrations were 0.05~1.00 g/kg and the correlation coefficient of calibration standard curves was more than 0.993. The recoveries were found in the range of 69.2%~116% and the relative standard deviations were 3.3%~12.0% at the respiked levels (10.0, 20.0 and 40.0 g/kg for chlorogenic acid; 1.0, 2.0 and 4.0 g/kg for caffeic acid; 5.00, 10.0 and 20.0 g/kg for isochlorogenic acid A and isochlorogenic acid C; 0.05, 0.10 and 0.20 g/kg for other compounds). This method was suitable for simultaneous determination and confirmation of 4 organic acid and 14 flavonoids in Lonicerae flos.
