The rapid discovery and identification of physalins in the calyx of Physalis alkekengi L.var.franchetii (Mast.) Makino using ultra-high performance liquid chromatography–quadrupole time of flight tandem mass spectrometry together with a novel three-step d

• Fragmentation pathways and rules of physalins were obtained.
• Diagnostic ions for rapid screening of physalins were determined.
• Diagnostic ions for classification of physalins were determined.
• A novel three-step data mining strategy for analysis of MS/MS data was established.
• A UHPLC–QTOF-MS/MS method was established for efficient identification of physalins.

Physalins, uniquely discovered from genus physalis, showed significant bioactivities in many aspects. It is therefore very important for the exploration of natural resources rich of physalins. However, there is no efficient approach for rapid discovery and identification of this class of compounds due to their structural complexity. To address the issue, the fragmentation pathways and correspondingly fragmentation rules of physalins in negative MS/MS mode were thoroughly investigated in this study using seven physalin standards. As a result, diagnostic ions for the rapid screening of physalins and classification of different types of physalins were determined based on their MS/MS fragmentation patterns. On top of that, an integrated approach using UHPLC–QTOF-MS/MS together with a novel three-step data mining strategy was developed for the systematic analysis of physalins in complex samples. Consequently, 46 physalins including 20 novel ones were efficiently discovered and identified from the crude extracts of Ph. alkekengi calyx. The present study laid a foundation for future study of different parts of Ph. alkekengi and other physalis species with regard to rapid discovery of novel physalins. In addition, this study provided a base for establishing a quality control method of the raw materials of Ph. alkekengi according to the profile of physalins.