Simultaneous quantification of polymethoxylated flavones and coumarins in Fructus aurantii and Fructus aurantii immaturus using HPLC–ESI-MS/MS

The major lipid-soluble constituents in Fructus aurantii (zhiqiao) and Fructus aurantii immaturus (zhishi) are polymethoxylated flavones (PMFs) and coumarins. In the present study, a high-performance liquid chromatography with electrospray ionization tandem mass spectrometry method was developed to quantify PMFs (nobiletin, tangeretin, 5-hydroxy-6,7,8,4′-tetramethoxyflavone, and natsudaidai) and coumarins (marmin, meranzin hydrate, and auraptene) simultaneously. PMFs and coumarins were detected by electrospray ionization tandem mass spectrometry in positive ion mode and quantified with multiple reaction monitor. Samples were separated on a Diamonsil C18 (150 mm × 4.6 mm, 5 μm) column using acetonitrile and formic acid–water solution as a mobile phase in gradient mode with a flow rate at 0.5 mL/min. All calibration curves showed good linearity (r2 > 0.9977) within the test ranges. Variations of the intraday and interday precisions were less than 4.07%. The recoveries of the components were within the range of 95.79%–105.04% and the relative standard deviations were less than 3.82%. The method developed was validated with acceptable accuracy, precision, and extraction recoveries and can be applied for the identification and quantification of four PMFs and three coumarins in citrus herbs.

► Seven lipid-soluble bioactive constituents (PMFs and coumarins) in Fructus aurantii and Fructus aurantii immaturus were simultaneously analyzed.
► 5-Hydroxy-6,7,8,4′-tetramethoxyflavone, natsudaidai and coumarin (meranzin hydrate, marmin and auraptene) were first quantified.
► The total content of seven constituents in zhiqiao and zhishi varied dramatically with the variety and the production region.
► The HPLC–ESI-MS method was simple and convenient for the qualitative and quantitative analysis of PMFs and coumarins.