Comprehensive chemical profiling and quality marker discovery in Shenxiao granules via orthogonal HILIC×RP 2D-LC/MS...

Shenxiao granules (SXG) are an effective traditional Chinese medicine (TCM) used in clinical settings in China for the treatment of diabetic nephropathy; however, the quality control of SXG faces several challenges due to its complex multi-herb composition and the wide polarity range of bioactive constituents. To address these challenges, we developed a novel and comprehensive analytical platform that integrated offline orthogonal HILIC×RP two-dimensional liquid chromatography (with a theoretical peak capacity of 7161), high-resolution quadrupole time-of-flight mass spectrometry, and a mass defect filtering (MDF)-based data acquisition algorithm. This strategy enabled the systematic characterization of 260 compounds, of which 36 were authenticated by standards, representing the most comprehensive chemical profile of SXG to date. Twelve critical quality markers (Q-markers) were further identified through a three-dimensional evaluation framework that encompasses pharmacopoeial compliance, pharmacological relevance to nephroprotection, and consensus fingerprint peaks. Rigorous validation demonstrated the method's robustness, with a relative standard deviation of less than 5% for precision, limits of detection ranging from 0.02 to 6.56 ng/mL, and recoveries between 84 and 119%. In conclusion, the established "multidimensional separation-computational annotation-hierarchical Q-marker screening" approach not only provides comprehensive chemical profiling and Q-marker discovery for SXG but also offers an excellent blueprint for the quality standardization of complex TCM formulations.