The concept of a Quality marker (Q-marker) has emerged as a crucial tool for ensuring the safety and efficacy of Traditional Chinese Medicine (TCM) formulas. However, significant challenges remained in the identification and practical application of Q-marker, particularly due to the scarcity of reference standards. This study aimed to achieve a multidimensional integration of chemical profiling, target tissue distribution and in vivo high-throughput screening model to effectively identify the Q-marker of Shensong Yangxin Capsule (SSYX) and propose a linear substitution strategy for the quantification of multiple components. First, the chemical constituents of SSYX were detected and systematically characterized using UHPLC/Q-TOF MS. Next, through heart distribution study, high-exposure components in vivo were identified after the oral administration of SSYX. Third, a high-throughput arrhythmia zebrafish model was employed to further screen for key constituents. Finally, potential Q-marker were selected by integrating the aforementioned studies, and a quantification method for the Q-marker was developed using UHPLC-TQ-MS. The results of chemical profiling, heart tissue distribution and anti-arrhythmic activities were integrated into four properties: specificity, traceability from prescription to in vivo, effectiveness and prescription compatibility, which led to the identification of 30 ingredients as potential Q-marker of SSYX. Subsequently, an external standard method (ESM) was developed for these 30 components and applied to the analysis of 10 commercial batches of SSYX. In addition, the feasibility of multi-marker detection via a linear substitution method (LSM) was explored for the first time using SSYX as a case study for method development, based on the stability of linear equations of the compounds in single standard solutions and multi-component mixed standard solutions. The simultaneous quantification of 30 components in SSYX was achieved by employing two linearly stable substances, greatly reducing the amount of standard substances used while maintaining measurability and convenience. A comparison of LSM and ESM revealed no significant difference in the component contents calculated by the two methods, with relative errors within ± 4 %. Our results suggested that 30 ingredients, including six key elements, could be considered as Q-marker of SSYX. Moreover, the LSM strategy offered a novel approach for developing environmentally friendly and convenient methods for the quality control of multi-index components in TCM formulas.
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