Development and validation of a simple ultra‐high‐performance‐tandem mass spectrometry method for the simultaneous determination of five bioactive components in rat plasma of Hedysari Radix

Hedysari Radix is a commonly used traditional Chinese medicine that improves immunity; formononetin, ononin, calycosin, medicarpin, and vanillic acid play an important role in achieving this effect. Herein, a precise and sensitive ultra‐high‐performance‐tandem mass spectrometry method was developed and validated for the determination of five bioactive components that were extracted from plasma using a protein precipitation method. A Waters CORTES C18 (4.6 × 50 mm, 2.7 µm) and a mobile phase composed of methanol and water (containing 0.2% formic acid) were used for the separation. The method was linear within the concentration range of 19.53–625.00 ng/mL for formononetin; 0.01–3.13 ng/mL for ononin; 0.01–3.13 ng/mL for calycosin; 0.16–5.00 ng/mL for medicarpin; and 19.53–625.00 ng/mL for vanillic acid. This method has a lower limit of quantification, is simple, and has a short analysis time and a high degree of separation. The intra‐day precisions and inter‐day precisions of quality control samples were traced to be below 8.58% and 12.64%, respectively. Fidelity intervened from −8.61% to 10%. Finally, the developed method was used to determine the concentrations of the five bioactive components in rat plasma after the administration of rubbing‐ and non‐rubbing processed Hedysari Radix.