LC-MS/MS multi-component quantification and PCA-based quality evaluation of Guipi decoction freeze-dried powder with...

Guipi Decoction (GPD) is a traditional Chinese medicine formula used for insomnia and fatigue, but comprehensive quality evaluation of its freeze-dried powder (GDFP) remains challenging due to the lack of a fully validated multi-component analytical method. In this study, an LC-MS/MS method was developed and validated according to ICH Q2(R2) for simultaneous quantification of 11 markers covering seven herbal materials in GDFP. Target analytes were screened by TOF-MS using a literature-curated in-house ion pair database. The method showed excellent linearity (R² ≥ 0.9935), low LODs (0.026-0.61 ng mL^-1), a uniform LOQ of 4 ng mL^-1, negligible matrix effects (|ME%| < 15%), and acceptable spike recoveries (86.5-115.1% with RSD < 17.8%). The method was successfully applied to six independently prepared GDFP batches and a commercial Guipi mixture after lyophilization. Principal component analysis (PCA) of eight reliably quantified markers explained 83.2% of total variance, identifying ginsenoside Rg1 (19.3%), ferulic acid (18.6%), and spinosin (17.1%) as the primary contributors to batch-to-batch variation. One-way ANOVA confirmed significant inter-batch differences for ten compounds, with spinosin showing the largest variation (F = 123.46 and p < 0.001). Based on a multi-criteria assessment combining PCA contribution, analytical performance, and reported pharmacological activities, these three compounds are statistically prioritized as candidate quality markers warranting future pharmacodynamic validation. This work provides a chemically robust and statistically validated analytical framework for GDFP quality evaluation.