Rapid, high-throughput, and sensitive screening of herbal product metabolites in blood plasma using 2D carbon microfiber fractionation coupled with quadrupole time-of-flight high-resolution mass spectrometry: Huangkui capsule insights.

Drug metabolite characterization from complex endogenous matrices such as plasma is a challenging task, especially for herbal product compounds. The analyses are generally carried out by flow-injection mass spectrometry (FI-MS) or combined with liquid chromatography (LC-MS), but the first one suffers from significant matrix effects, while the second is time-consuming and unsuitable for high-throughput analysis. In this study, a rapid method combining 2D-carbon microfiber fractionation (2DµCFs) with quadrupole time-of-flight high-resolution mass spectrometry (QTOF-HRMS) is proposed for the comprehensive characterization of herbal metabolites in plasma. Samples are separated into three fractions depending on their polarity (strong, medium, weak) and sequentially injected into MS, reducing matrix effects and enhancing metabolite coverage. Each 2DµCFs-QTOF-HRMS run takes only 5 min, enabling the detection of approximately 10,000 m/z features per injection-representing a 4-fold increase in the number of detected features compared to FI-MS. The proposed method maintains the high sensitivity offered by LC-MS, greatly simplifying the analytical procedure. By this approach, 14 potential effective compounds of Huangkui capsule (HKC), from dried flowers of Abelmoschus manihot (A. manihot), were characterized in blood plasma. Furthermore, the comparison of chemical components among different parts of A. manihot was performed, providing valuable insights into its active ingredients and creating a bridge between phytochemistry and clinical applications.