Research On HPLC-HRMS Method For The Detection Of Small Amine Molecules In Brain Tissue And Fermented Snacks

High performance liquid chromatography-mass spectrometry coupling technology(HPLC-MS)combines the advantages of good separation ability of HPLC and high selectivity and sensitivity of MS,which can eliminate the problems of fragment overlap and background interference in the chromatographic separation and significantly improve the separation efficiency and signal identification capacity,thus enabling the separation and detection of a variety of compounds and fulfilling the needs of qualitative and quantitative analysis in complex samples.Now,HPLC-MS has been extensively used in various fields such as pharmaceutical,food,environmental,proteomics and metabolomics analysis.Both monoamine neurotransmitters and biogenic amines are small amine molecules.Metabolic imbalance of neurotransmitters has been shown to be directly related to various diseases.Excessive intake of biogenic amines in food can produce various toxic effects on human body.A variety of analytical methods such as gas chromatography-mass spectrometry,ion chromatography,capillary electrophoresis,and high performance liquid chromatography-ultraviolet detector,fluorescence detector and triple quadrupole mass spectrometry have been used for the detection of neurotransmitters and biogenic amines.Compared with the low-resolution mass spectrometry equipped with triple quadrupole detector,the high-resolution mass spectrometry(HRMS)can provide more accurate ion mass and has higher identification and analysis capacity.After being combined with HPLC,HPLC-HRMS technology has the advantages of strong reliability,high specificity and sensitivity.Therefore,the establishment of HPLC-HRMS methods for the detection of small amine molecules in brain tissue and fermented snacks is of great significance for disease research and food safety.Based on this,this dissertation carries out the research work mainly embracing the development of the HPLC-HRMS methods for the separation and detection of small amine molecules in brain tissue and fermented snacks.This dissertation is divided into five chapters as follows:Chapter 1: The basic principles and main applications of HPLC-MS are summerized;the classification,functions and main analytical tools of neurotransmitters and the toxicity,quantitative criteria and detection methods of biogenic amines are briefly introduced;the principle and application of the detection of neurotransmitters and bioamines by HPLC-MS were summarized in detail.Chapter 2: In this work,a HPLC-HRMS method was developed for the simultaneous separation and analysis of eight target neurotransmitters,and the electrospray ionization parameters of the mass spectrometry,including sheath gas rate,ion transport tube temperature and spray voltage were optimized.Under the optimal condition,the detection of the eight neurotransmitters was completed within 7 min in the Full-MS scan mode.The sensitivity and reproducibility of the proposed method were also investigated.The intra-day and inter-day relative standard deviations(RSD)of migration time were 0.10~0.73% and 0.13~0.73%,respectively;the intra-day and inter-day RSD of peak areas were 1.43~4.37% and 1.14~7.54%,respectively;the limits of detection(LOD)for each analyte were 1~50 n M and the limits of quantification(LOQ)were 10~100 n M.The method has been applied to the detection of eight neurotransmitters in rat brain tissue,providing a rapid and reliable analytical method to better investigate the relationship between neurotransmitter content and disease.Chapter 3: To further improve the sensitivity of the eight target neurotransmitters,in this work,the benzoyl chloride derivatization was combined with HPLC-HRMS(in Full-MS mode)to establish an analytical method with high sensitivity,good reproducibility and wide linear range,that can complete the detection of eight benzoyled neurotransmitters within 12.5 min.The proposed method has a detection limit of 0.1~20 n M and a quantification limit of 1~100 n M for the target analytes.The intra-day and inter-day RSD for peak area and migration time for each analyte was less than 12%.Clomipramine(CLO)was injected in young rats to sleep deprivation,and the sleep-waking phase analysis and depression-related behavioral test on rats were conducted,showing the depression model in rats was successfully established.The HPLC-HRMS method has been successfully applied to the detection of eight target neurotransmitters in the brain tissue of saline control and CLO depressed rats.The proposed method provides a basis for in-depth investigation into the neural mechanisms and clinical diagnosis of depression and sleep disorders.Chapter 4: In this work,combining benzoyl chloride derivatization with HPLC-HRMS technology,an analytical method with high sensitivity,good reproducibility and wide linear range has been developed for the simultaneous detection of biogenic amines.The HPLC-HRMS method was completed in 12 minutes for the separation and detection of eight biogenic amines with a linear range of 3-6 orders of magnitude,linear regression coefficients of greater than 0.9967,LOD and LOQ at the pg/m L level,and recoveries of 82.48~122.13%.The proposed method has been successfully used to detect the content of biogenic amines in several fermented snacks.And the changes of content of biogenic amines in samples stored under room temperature for different time have been investigated,which provides a reliable guarantee for food safety.Chapter 5: Conclusion and prospects.