Simultaneous Quantification of Twelve Endogenous HPA Axis Compounds by Protein Precipitation and HILIC‐UPLC–MS/MS in a Mouse Model of Insomnia

RATIONALE: Accurate quantification of neurotransmitters and hormones within the hypothalamic-pituitary-adrenal (HPA) axis is essential for elucidating their pathological roles in neurodegenerative diseases. METHODS: In this study, a solid-phase extraction (SPE) combined with hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-UPLC-MS/MS) method was developed for the simultaneous determination of 12 endogenous compounds in the HPA axis. The method was fully validated according to FDA standards. RESULTS: All analytes showed good linearity over the concentration range of 0.19-19.53 ng/mL, with lower limits of quantification (LLOQs) ranging from 0.19 to 19.53 ng/mL. Accuracy (relative bias) and precision (relative standard deviation) were within ±15%. Matrix effects were evaluated (85.7%-114.3%), and extraction efficiencies were found to be satisfactory (89.1%-108.9%). Compared with other LC-MS-based techniques, this method effectively eliminates interference from endogenous substances by utilizing SPE columns. CONCLUSIONS: The proposed method exhibited excellent performance in terms of linearity, precision, accuracy, stability, and matrix effects, demonstrating superior practicality and reliability for the analysis of complex biological samples.