Rapid magnetic solid-phase extraction combined with ultra-high performance liquid chromatography and quadrupole-time-of-flight mass spectrometry for analysis of thrombin binders from a crude extract and injection of Erigeron breviscapus†
Thrombin, which acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, plays crucial roles in the blood coagulation pathway. Inhibition of thrombin leading to the decrease of thrombus formation may be used for treating cerebral ischemia and infarction. In the present study, a new approach of thrombin immobilized magnetic nanoparticles coupled with ultra-high performance liquid chromatography and quadrupole-time-of-flight mass spectrometry was established for rapidly extraction of thrombin binders from a crude extract of Erigeron breviscapus and its injection. Scutellarin, a model inhibitor of thrombin, was chosen to interrogate the optimal extraction conditions, including wash times, ion strength, temperature, pH, and incubation time. Meanwhile, the specificity of the approach was investigated by using denatured thrombin and the inactive compound specnuezhenide. The optimal screening conditions were as follows: wash times: 4, wash solvent: 50% acetonitrile–water, incubation time: 70 min, temperature: 37 °C, ion strength: 10 mM, pH: 6.8. Moreover, the linearity, accuracy, precision, and matrix effect of the approach were well validated. Finally, thrombin binders were extracted from the crude extract and injection of Erigeron breviscapus by applying the established approach, and were subsequently sent to be validated by a thrombin inhibitory assay. Eleven thrombin inhibitors were identified, and ten of which, including erigoster B, scutellarin A, erigeroside, neochlorogenic acid, chlorogenic acid, 4-caffeoylquinic acid, luteolin 7-O-β-D-glucuronide, 4,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid and 3,5-di-O-caffeoylquinic acid, were reported for the first time.