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  • Title: LC-MS/MS-based quantification of tryptophan metabolites and neurotransmitters in the serum and brain of mice.
    Author: Wang LS, Zhang MD, Tao X, Zhou YF, Liu XM, Pan RL, Liao YH, Chang Q.
    Journal: J Chromatogr B Analyt Technol Biomed Life Sci; 2019 Apr 01; 1112():24-32. PubMed ID: 30836315.
    Abstract:
    l-Tryptophan (Trp) metabolites and related neurotransmitters play crucial roles in physiological functions, and their imbalances are implicated in the pathology of depression, Alzheimer's disease and other diseases. Measurement of Trp metabolites and related neurotransmitters possesses a great potential to elucidate the disease mechanisms and evaluate the outcomes of therapeutic interventions. A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of Trp, l-kynurenine (Kyn), kynurenic acid (Kyna), 3-hydroxykynurenine (3-HK), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE), l-glutamic acid (Glu), γ-aminobutyric acid (GABA) and acetylcholine (ACh) in mice serum and the brain tissues in a single chromatographic run. Samples were spiked with the internal standard, mixed with trifluoroacetic acid to precipitate protein and analyzed by LC-MS/MS. Chromatographic separation was achieved using a Restek Ultra Aqueous C18 column in combination with a gradient elution within 8 min. Mass spectrometric detection was performed using multiple reaction monitoring with electrospray ionization source in positive mode. The method exhibited good selectivity and correlation coefficient values for the calibration curves of each analyte were >0.99. The limit of detection and quantification ranged from 0.96 to 24.48 nmol/L and 3.42 to 244.82 nmol/L, respectively. The intra- and inter-day precision were ≤13.92%. All analytes were stable in prepared samples at room temperature in the autosampler for 24 h. This method was successfully applied to the analysis of biological samples from control and chronic mild stress (CMS) induced depression mice. It was found that Kyn and 3-HK pathways were enhanced by CMS, while the levels of Trp, Kyna, 5-HIAA, Glu, GABA and ACh were significantly reduced. The changes in 5-HT and NE levels were not uniform in the periphery and the brain. This method can therefore be applied to analyze Trp metabolites and related neurotransmitters levels to monitor disease states, study the mechanisms and outcomes of therapeutic interventions.
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