Pyungwi-san has been used to treat the digestive system diseases, physconia, nausea, anorexia, and dyspepsia in Korea. In this study, an ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS/MS) method was optimized for simultaneous determination of the 14 marker components, spinosin, liquiritirn apioside, liquiritin, narirutin, 6″′-feruloylspinosin, hesperidin, liquiritigenin, glycyrrhizin, 6-gingerol, atractylenolide III, honokiol, atractylenolide II, magnolol, and atractylenolide I in Pyungwi-san extract. All analytes were separated on a Waters Acquity UPLC BEH C18 analytical column (2.1×100 mm, 1.7 μm) with maintained at 45° C. The mobile phase consisted of 0.1% (v/v) aqueous formic acid and acetonitrile. The MS conditions were as follows: capillary voltage 3.3 kV, extractor voltage 3.0 V, RF lens voltage 0.3 V, source temperature 120℃, desolvation temperature 300℃, desolvation gas 600 L/h, cone gas 50 L/h and collision gas 0.14 mL/min. The coefficient of determination of 14 analytes was 0.9989-1.0000. The limits of detection and quantification values of the all analytes were 0.04-2.56 and 0.13-7.69 ng/mL, respectively. As a result of the analysis using the established LC-ESI-MS/MS method, the 5 components, spinosin, 6'''-feruloylspinosin, atractylenolide III, II, and I derived from Zizyphi Fructus and Atractylodis Rhizoma, were not detected in this extract. On the other hand, the 9 components except for the 5 components were 4.15-498.87 mg/kg in lyophilized Pyungwi-san extract. Among these components, glycyrrhizin, marker compound of Glycyrrhizae Radix et Rhizoma, was detected the most amount as a 498.87 mg/kg.
