Screening of epidermal growth factor receptor inhibitors in natural products by capillary electrophoresis combined with high performance liquid chromatography–tandem mass spectrometry

• A method for screening of EGFR inhibitors from natural extracts is established.
• High quality screening data can be obtained.
• The method enables chemists to discover new bioactive natural compounds.
• Quercetin and rutin are verified to be the EGFR inhibitors.

A method for screening of inhibitors to epidermal growth factor receptor (EGFR) in natural product extracts with capillary electrophoresis (CE) in conjunction with high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) is reported. The method was established by employing 5-carboxyfluorescein labeled substrate peptide, two commercially available EGFR inhibitors OSI-744 and ZD1839, and a small chemical library consisted of 39 natural product extracts derived from the Traditional Chinese Medicines. Biochemical assay of crude natural product extracts was carried out by using CE equipped with a laser induced fluorescence detector. The CE separation allowed an accurately quantitative measurement of the phosphorylated product, hence the measurement of the enzymatic activity as well as the inhibition kinetics. The hits are identified if the peak area of the phosphorylated product is reduced in comparison with the negative control. The active constituents in the natural product extract were then identified by an assay-guided isolation with HPLC–MS/MS system. With the method, the flavonoids component of the Lycopus lucidus extract, namely quercetin and rutin were identified to be the active ingredients. Their IC50 values were determined as 0.88 μM and 10.1 μM, respectively. This result demonstrated a significant merit of our method in the identification of the bioactive compounds in natural products.