Simultaneous Quantification of Twelve Endogenous HPA Axis Compounds by Protein Precipitation and HILIC‐UPLC–MS/MS in a Mouse Model of Insomnia
Copyright policy )Simultaneous Quantification of Twelve Endogenous HPA Axis Compounds by Protein Precipitation and HILIC‐UPLC–MS/MS in a Mouse Model of Insomnia
ABSTRACT 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.
Male, Hypothalamo-Hypophyseal System, Liquid Chromatography-Mass Spectrometry, Solid Phase Extraction, Reproducibility of Results, Pituitary-Adrenal System, Mice, Disease Models, Animal, Tandem Mass Spectrometry, Limit of Detection, Sleep Initiation and Maintenance Disorders, Linear Models, Animals, Hydrophobic and Hydrophilic Interactions, Chromatography, High Pressure Liquid
Male, Hypothalamo-Hypophyseal System, Liquid Chromatography-Mass Spectrometry, Solid Phase Extraction, Reproducibility of Results, Pituitary-Adrenal System, Mice, Disease Models, Animal, Tandem Mass Spectrometry, Limit of Detection, Sleep Initiation and Maintenance Disorders, Linear Models, Animals, Hydrophobic and Hydrophilic Interactions, Chromatography, High Pressure Liquid
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