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.