Comparation of the Determination of 5-Hydroxytryptamin and 2 Related Compounds in Mouse Hippocampus by HPLC with Fluorescence Detector and UHPLC-Q-Obitrap-Mass Spectrometry

Two methods (i.e. HPLC with FLD and UHPLC-Q-Obitrap-MS) for determination of 5-hydroxytryptamine (5-HT), tryptophan (TRP) and 5-hydroxyindole acetic acid (5-HIAA) in hippocampus of mouse affected by depression were studied comparatively. In the pretreatment of sample, 0.4 mol·L^-1 HClO₄ solution was added to the homogenate of mouse hippocampus by the ratio of 1 to 5 (g∶mL) to precipitate the protein. After swirling and centrafuging, the supernatants obtained by each of the 2 pretreatment method were used separately for analysis by the 2 methods. Separation on C₁₈ column was adopted in both methods. As shown by the experimental results, strong matrix interference was encountered in the method of Q-Obitrap-MS, while symmetrical chromatographic peaks with narrow peak with for the 3 objective analytes were obtained and no significant matrix effect was observed by the method of HPLC-FLD, the standard curves of which showed linearity in the concentration range within 500.0 μg·L^-1. Test for recovery was made by addition of standards at 3 concentration levels, giving results of recovery of 86.2%, 92.1%, and 91.0%. Detection limits (3S/N) found for the 3 analytes were 1.0, 1.0, 0.5 μg·L^-1, respectively.