HPLC-ESI-MS/MS QUANTITATIVE METHOD FOR SIMULTANEOUS ANALYSIS OF FIVE BIOACTIVE CONSTITUENTS OF FORSYTHIA SUSPENSA IN RAT BILE AFTER ORAL ADMINISTRATION OF FORSYTHIA SUSPENSA EXTRACT

Abstract

The compounds (+)-pinoresinol-β-D-glucoside (1), matai-resinol- 4′-O-glucoside (2), hyperin (3), phillyrin (4), and phillygenin (5) are the primary bioactive ingredients in Forsythia suspensa, which possesses many pharmacological activities, such as heat-clearing and detoxifying, free radical clearing, antioxidative, liver-protection, antibacterial, and so on. In the present study, we employed a sensitive and selective high-performance liquid chromatography electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/MS) method for the quantification of the five bioactive constituents of Forsythia suspensa in rat bile. Acetaminophen was used as the internal standard (IS). The bile samples were pretreated by liquid–liquid extraction with ethyl acetate (EtOAc). The chromatographic separation was carried out on a C₁₈ column with gradient elution. The detection of analytes was performed on a tandem mass system equipped with a turbo ion spray interface in negative mode using multiple-reaction monitoring (MRM). The specificity, linearity, accuracy, precision, recovery, matrix effect, and several stabilities were validated for the five bioactive constituents in rat bile samples. The proposed method showed appropriate accuracy and repeatability and was suitable for pharmacokinetic studies of the five bioactive constituents after oral administration of Forsythia suspensa extract.

Keywords:

• bile
• bioactive constituent
• Forsythia suspense
• HPLC-ESI-MS
• quantification
• simultaneous analysis

ACKNOWLEDGMENT

The authors are thankful for the financial support of the Natural Science Foundation of Hebei Province – CSPC Pharmaceutical Joint Research Fund, China (H2012206079) and Technology Research and Development Planning of Shijiazhuang, Hebei Province of China (10146513A).

Notes

^a Means ± standard deviation.