Establishment of Non‐Target Screening and Quantitative Analysis Strategy Based on UHPLC Coupled With Orbitrap Exploris 240 and QqQ Mass Spectrometry to Improve the Quality Control of Ziyu Ointment

Ziyu ointment (ZYO) is a commonly used, approved ingredient in traditional Chinese medicine (TCM) medication for treating superficial swelling and ulcers. However, due to limited methodologies, more accurate and selective qualitative and quantitative analysis methods for its active ingredients have not yet been established. This impedes both the full clinical development and application of ZYO as well as the clarification of its pharmacological mechanism. In this research, a rapid and sensitive UHPLC-Orbitrap Exploris MS method with higher-energy collisional dissociation (HCD) was established for identifying ZYO's chemical components. A total of 167 chemical components were found, including 34 carboxylic acids and esters, 32 phenylpropanoids, 27 terpenoids, 18 flavonoids, 12 naphthoquinones, 11 phthalides, 8 phenolic acids, 5 phenols, 3 steroids, 2 quinones, 2 alkaloids, and 13 miscellaneous compounds. Among these, UHPLC-QqQ-MS/MS was used to simultaneously determine 12 analytes (either with high contents or significant bioactivities), namely, (1) loganin, (2) sweroside, (3) ferulic acid, (4) senkyunolide I, (5) bergapten, (6) ziyuglycoside II, (7) imperatorin, (8) shikonin, (9) ligustilide, (10) isoimperatorin, (11) β-hydroxyisovalerylshikonin, and (12) levistilide A. The foundation for future use and quality assurance of the active ingredients in ZYO was established by this study.