Small molecular phenolic substance detection method and application thereof

Abstract

The invention discloses a micromolecular phenolic substance detection method and application thereof, particularly relates to a high-sensitivity detection technology used for detecting trace endogenous micromolecular phenolic substances in a living body, and belongs to the technical field of pharmacy. The invention relates to a small molecular phenolic substance detection method, which is characterized in that a sample containing small molecular phenolic substances is subjected to derivatization method pretreatment by dansyl chloride chemical derivatization, and the detection sensitivity of the small molecular phenolic substances is increased through a sensitizing group. According to the invention, the concept of endogenous phenolomics is put forward for the first time, and important small molecular metabolites of catecholamine neurotransmitters are focused to participate in emotion regulation and attention and cognitive functions. A set of method for comprehensively detecting 31 ESMPs is established, and the UPLC-QqQ-MS / MS technology is combined to ensure high sensitivity and specificity. In the future, key biomarkers can be identified by comparing the ESMPs levels of mental disease patients and healthy people, early diagnosis and individualized treatment strategies are supported, and the important role of the ESMPs in mental diseases is revealed.

Description

Technical Field

[0001] The invention relates to a method for detecting small-molecule phenolic substances and an application thereof, in particular to a high-sensitivity detection technology for detecting trace amounts of endogenous small-molecule phenolic substances in organisms, and belongs to the field of pharmaceutical technology. Background Art

[0002] As of 2022, the global coverage of mental illness has exceeded one billion people, becoming one of the most burdensome health problems in the world. With the deepening of understanding of mental illness, accurate diagnosis and development of related therapeutic drugs have gradually become research hotspots. There are many methods for detecting mental illness, among which chemical detection methods have received widespread attention due to their efficiency and objectivity.

[0003] Commonly used techniques for detecting mental illness include: clinical observation and interviews, psychological tests, neuroimaging examinations, neuroelectrophysiological examinations, laboratory tests, etc. Among them, liquid chromatography-mass spectrometry (LC-MS) is an analytical technique currently used in laboratory tests to quantify and identify small molecule compounds in biological samples. This technology can detect trace components in samples with high sensitivity and is widely used in the detection of biomarkers for mental illness. By comparing samples from patients and healthy people, specific compounds related to mental illness and their concentration changes can be revealed.

[0004] Small-molecule phenols (molecular weight less than 300) containing only three elements, C, H, and O, are widely found in animals and plants and are called natural small-molecule phenols (NSMPs). There are two sources of NSMPs in humans and some animals. One is endogenous small-molecule phenols (ESMPs) that exist in the metabolic pathways of phenylalanine and tyrosine and are mainly produced by catecholamine neurotransmitters. They can be found in body fluids such as plasma, cerebrospinal fluid, and urine. The other is exogenous, including exogenous small-molecule phenols that are directly absorbed and transported from food to the body, and small-molecule phenol metabolites that are absorbed and transported to the body by the gastrointestinal tract after food is decomposed by intestinal microorganisms.

[0005] Through in-depth literature research and experimental studies in the early stage, it was found that NSMPs have some common pharmacological activities, such as sedation, anxiolysis, smooth muscle relaxation, etc., which are called phenolic phase (Phenolism); therefore, it is believed that the phenolic hydroxyl group is an active functional group that can exert its effects by affecting the nervous system. As important small molecule metabolites of catecholamine neurotransmitters, the relationship between ESMPs and the nervous system and mental illnesses deserves further exploration, and they may become potential biomarkers and drug development targets for diagnosing mental illnesses in the future.

[0006] Based on the above background, effective detection of ESMPs in organisms will help to further explore the interaction between exogenous NSMPs and ESMPs, reveal the important role of ESMPs in living organisms, and provide new targets for the development of drugs to treat mental illnesses. However, due to its structure, ESMPs have a small molecular weight, and only phenolic hydroxyl groups in the skeleton can be used for fluorescence and ionization. The response signals to fluorescence and mass spectrometry are weak, and its chemical properties are unstable. It is easy to undergo oxidative decomposition under light and alkaline conditions. In addition, their content in organisms such as humans and animals is low, and the matrix in biological samples is complex and there are many endogenous interfering substances, which have an impact on the analysis and detection of ESMPs. At present, there are few studies on the systematic analysis and detection of ESMPs and their use as potential biomarkers for diagnosing mental illnesses. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for detecting small molecule phenolic substances and its application, to establish a new sample pretreatment and analytical detection method that is more systematic, effective and sensitive than ordinary detection, to detect endogenous small molecule phenols (NSMPs) in organisms and to construct endogenous phenolomics.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for detecting small molecule phenolic substances comprises the following steps:

[0010] S1: Preparation of solutions, including preparation of standard stock solution, preparation of standard working solution, preparation of standard curve working solution and quality control sample solution, and preparation of internal standard solution;

[0011] S2: Sample pretreatment: using dansyl chloride to chemically derivatize samples containing small molecule phenols to increase the detection sensitivity of small molecule phenols through sensitizing groups;

[0012] S3: Chromatography-mass spectrometry is used to determine the content of small molecular phenolic substances in the sample under chromatographic conditions and mass spectrometry conditions.

[0013] The small molecule phenolic substances are endogenous small molecule phenolic substances, which are small molecule metabolites produced by catecholamine neurotransmitters.

[0014] The small molecule phenols include 3-methoxy-4-hydroxyphenyl glycol (MHPG), 4-hydroxy-3-methoxyphenylacetic acid (VMA), 4-hydroxyphenyl lactic acid (HDPL), 4-hydroxyphenylpyruvic acid (HPPA), 2-hydroxyphenylacetic acid (HPAT), 3-methoxy-4-hydroxyphenylacetic acid (HVA), 3-hydroxyphenylacetic acid (3-HPAA), 4-hydroxyphenylacetic acid (HPAA), 3-hydroxybenzoic acid (3-HBA), 4-hydroxyphenylethanol (TYR), 2-hydroxycinnamic acid (HCM), 4-hydroxybenzoic acid (HBA), 3-(3-hydroxyphenyl) propionic acid (HPPT), 3-(4-hydroxyphenyl) propionic acid (3,4-HPHPA), 3-hydroxycinnamic acid (3-HCM), trans-3-hydroxycinnamic acid (THCM), 4-hydroxy Cinnamic acid (PHCA), trans-4-coumaric acid (PCA), 2-(4-hydroxyphenyl)propionic acid (2,4-HPHPA), 4-hydroxy-3-methoxybenzaldehyde (HMBH), 3,4-dihydroxyphenylacetic acid (DOMA), 3,4-dihydroxyphenylglycol (DHPG), 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylpropionic acid (DHPP), 3,4-dihydroxyphenylethanol (DOPET), 3,4-dihydroxybenzoic acid (3,4-DHBA), 2,5-dihydroxybenzoic acid (DHBA), caffeic acid (CA), 2,5-dihydroxyphenylacetic acid (HGA), 3,4-dihydroxyphenylacetaldehyde (DOPAL), 3,4-dihydroxybenzaldehyde (3,4-DHBAD), 2,5-dihydroxybenzaldehyde (DHBD).

[0015] The step S1 of preparing the solution refers to:

[0016] (1) Preparation of standard stock solution: Accurately weigh an appropriate amount of small molecule phenol standard and dissolve it in 1% acetic acid-methanol solution to prepare a 1 mmol / L standard stock solution; all standard stock solutions are packaged and stored in a -20°C refrigerator for later use;

[0017] (2) Preparation of standard working solutions: Accurately measure appropriate amounts of the above-mentioned small molecule phenol standard stock solutions and place them in 10 mL volumetric flasks. Add 1% acetic acid-methanol solution to make the volume to 10 mL. Prepare mixed standard working solutions with concentrations of 4 μmol / L and 30 μmol / L, respectively, and place them in a 4°C refrigerator for later use;

[0018] (3) Preparation of standard curve working solution and quality control sample solution: Accurately measure an appropriate amount of the mixed standard working solution, dilute it step by step with 1% acetic acid-methanol solution to a series of standard curve working solutions of the required concentration, and store it in a 4°C refrigerator for later use;

[0019] The preparation of quality control sample solution means: accurately measure an appropriate amount of mixed standard working solution, use 1% acetic acid-methanol solution to prepare three mixed standard QC solutions of low, medium and high mass concentrations, and place them in a 4°C refrigerator for later use.

[0020] (4) Preparation of internal standard solution means: accurately weigh an appropriate amount of internal standard CA, add 1% acetic acid-methanol solution to dissolve it and prepare a 1 mmol / L internal standard stock solution. Accurately weigh an appropriate amount of the above internal standard stock solution, dilute it with 1% acetic acid-methanol solution to a concentration of 1 μmol / L (internal standard working solution A) and 10 μmol / L (internal standard working solution B), which are used as internal standard working solutions for brain tissue homogenate matrix and plasma matrix, respectively.

[0021] The sample in step S2 includes a sample pre-treatment of a brain tissue homogenate matrix or a sample pre-treatment of a plasma matrix.

[0022] The sample pretreatment of the brain tissue homogenate matrix refers to: adding a corresponding volume of 1% acetic acid-methanol solution to the brain tissue according to its own weight and then homogenizing to obtain 100 mg / mL brain tissue homogenate; after centrifugation at 4°C and 12000rpm for 10 minutes, accurately aspirating 100 μL of the brain homogenate supernatant, adding 100 μL of internal standard working solution A, vortex mixing for 1 minute, and blowing dry with N2; adding 350 μL of 4mmol / L dansyl chloride-acetonitrile solution and 350 μL of 0.1mol / L, pH=11 Na2CO3-NaHCO3 buffer solution to the residual dry matter, vortex mixing for 1 minute, and then heating in a metal bath at 80°C for derivatization for 20 minutes; finally, adding 300 μL of 15% formic acid-water solution to the mixed derivatization system to adjust the pH, vortex mixing for 1 minute, centrifuging at 4°C and 12000rpm for 10 minutes, and then aspirating the supernatant for UPLC-QqQ-MS / MS injection analysis, and all operations must be performed under light-proof conditions;

[0023] The sample pretreatment of the plasma matrix refers to: adding an appropriate amount of 1% acetic acid-methanol solution to the plasma to obtain undiluted and 100-fold diluted plasma samples; after centrifugation at 4°C and 12000rpm for 10 minutes, accurately aspirating 100 μL of the plasma supernatant, adding 100 μL of the internal standard working solution B, vortex mixing for 1 minute, and blowing dry with N2; adding 350 μL of 4mmol / L dansyl chloride-acetonitrile solution and 350 μL of 0.1mol / L, pH=11 Na2CO3-NaHCO3 buffer solution to the residual dry matter, vortex mixing for 1 minute, and then heating and derivatizing in an 80°C metal bath for 20 minutes; finally, adding 300 μL of 15% formic acid-water solution to the mixed derivatization system to adjust the pH, vortex mixing for 1 minute, and centrifuging at 4°C and 12000rpm for 10 minutes, and then aspirating the supernatant for UPLC-QqQ-MS / MS injection analysis; all operations must be performed under light-proof conditions.

[0024] The chromatographic conditions optimized in step S3 include chromatographic columns (ACQUITY UPLC BEH C18, ACQUITY UPLC HSS T3, ACQUITY UPLC BEH HILIC and ACQUITY UPLC BEH Amide), mobile phases (methanol-water, acetonitrile-water), elution gradients of mobile phases (9 min, 13 min, 18 min, 20 min), mobile phase additives (formic acid, acetic acid, ammonium formate, ammonium acetate), volume fractions of mobile phase additives (0.1% formic acid, 0.01% acetic acid, 5 mM ammonium formate, 5 mM ammonium acetate), flow rate (0.2-0.5 mL / min), and column temperature (25-35 ° C).

[0025] The optimized mass spectrometry conditions included 31 ESMPs and internal standard CA for quantitative analysis in MRM mode monitoring ion pairs, cone voltage (2-70 V), and collision voltage (10-50 V).

[0026] The optimal chromatographic conditions are: using ACQUITY UPLC HSS T3 chromatographic column (2.1×100mm, 1.8μm); flow rate: 0.3mL / min; column temperature: 35°C; injection volume: 2μL; mobile phase is 0.1% formic acid aqueous solution (A)-acetonitrile (B). Gradient elution is used, and the elution conditions are 0-0.5min, 40%-55% B; 0.5-5.5min, 55%-70% B; 5.5-6min, 70%-83% B; 6-7.5min, 83%-95% B; 7.5-8.5min, 95% B; 8.5-9min, 95%-40% B;

[0027] The optimal mass spectrometry conditions were as follows: using a Waters UPLC-Xevo TQ-S micro triple quadrupole liquid-mass spectrometry system, an electrospray ion source (ESI source), positive ion mode scanning, multiple reaction monitoring mode (MRM) detection, collision gas argon (Ar), purge gas nitrogen (N2), capillary voltage 3kV, ion source temperature 150°C, desolvation temperature 350°C, and desolvation flow rate 650L / h. In order to avoid contamination of the ion source, the first 1min of mobile phase directly entered the waste liquid; 31 ESMPs and internal standard CA were used for quantitative analysis of MRM mode monitoring ion pairs using corresponding parameters.

[0028] The corresponding parameters are:

[0029] (1) The retention time of MHPG is 2.47 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 23 NO6S, parent ion m / z 418.8590, product ion m / z 171.3964, cone voltage 4V, collision voltage 28V;

[0030] (2) The retention time of VMA is 2.50 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO7S, parent ion m / z 431.8165, product ion m / z 170.9891, cone voltage 66V, collision voltage 26V;

[0031] (3) The retention time of HDPL is 2.89 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO6S, parent ion m / z 415.8216, product ion m / z 170.9874, cone voltage 38V, collision voltage 30V;

[0032] (4) The retention times of HPPA are 3.04 and 4.10 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO6S, parent ion m / z 413.8059, product ion m / z 170.9258, cone voltage 32V, collision voltage 28V;

[0033] (5) The retention time of HPAT is 3.52 minutes, the number of dansyl groups is 1, and the molecular formula is C 20 H 19 NO5S, parent ion m / z 385.7472, product ion m / z 170.0688, cone voltage 6V, collision voltage 28V;

[0034] (6) The retention time of HVA is 3.52 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO6S, parent ion m / z 415.8216, product ion m / z 170.9856, cone voltage 46V, collision voltage 22V;

[0035] (7) The retention time of 3-HPAA is 3.60 minutes, the number of dansyl groups is 1, and the molecular formula is C 20 H 19 NO5S, parent ion m / z 386.8966, product ion m / z 171.3098, cone voltage 28V, collision voltage 22V;

[0036] (8) The retention time of HPAA is 3.63 minutes, the number of dansyl groups is 1, and the molecular formula is C 20 H 19 NO5S, parent ion m / z 385.7472, product ion m / z 171.0062, cone voltage 8V, collision voltage 22V;

[0037] (9) The retention time of 3-HBA is 3.81 minutes, the number of dansyl groups is 1, and the molecular formula is C 19 H 17 NO5S, parent ion m / z 372.0508, product ion m / z 171.0125, cone voltage 70V, collision voltage 28V;

[0038] (10) The retention time of TYR is 3.85 minutes, the number of dansyl groups is 1, and the molecular formula is C 20 H 21 NO4S, parent ion m / z 371.9595, product ion m / z 171.0015, cone voltage 34V, collision voltage 24V;

[0039] (11) The retention time of HCM is 3.90 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO5S, parent ion m / z 398.0665, product ion m / z 170.0973, cone voltage 60V, collision voltage 14V;

[0040] (12) The retention time of HBA is 3.92 minutes, the number of dansyl groups is 1, and the molecular formula is C 19 H 17 NO5S, parent ion m / z 371.7954, product ion m / z 170.9853, cone voltage 38V, collision voltage 26V;

[0041] (13) The retention time of HPPT is 4.02 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO5S, parent ion m / z 400.6568, product ion m / z 171.0337, cone voltage 10 V, collision voltage 37 V;

[0042] (14) The retention time of 3,4-HPHPA is 4.04 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO5S, parent ion m / z 399.8267, product ion m / z 170.9957, cone voltage 34V, collision voltage 28V;

[0043] (15) The retention time of 3-HCM is 4.16 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO5S, parent ion m / z 398.0026, product ion m / z 170.8724, cone voltage 30 V, collision voltage 26 V;

[0044] (16) The retention time of THCM is 4.16 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO5S, parent ion m / z 398.0665, product ion m / z 170.9413, cone voltage 30V, collision voltage 28V;

[0045] (17) The retention time of PHCA is 4.23 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO5S, parent ion m / z 398.0026, product ion m / z 170.9855, cone voltage 38V, collision voltage 20V;

[0046] (18) The retention time of PCA is 4.22 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO5S, parent ion m / z 398.0026, product ion m / z 170.9872, cone voltage 36V, collision voltage 24V;

[0047] (19) The retention time of 2,4-HPHPA is 4.27 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO5S, parent ion m / z 399.9544, product ion m / z 170.9985, cone voltage 38V, collision voltage 22V;

[0048] (20) The retention time of HMBH is 5.55 min, the number of dansyl groups is 1, and the molecular formula is C 20 H 19 NO5S, parent ion m / z 385.7472, product ion m / z 170.0688, cone voltage 6V, collision voltage 24V;

[0049] (21) The retention time of DOMA is 5.71 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 30 N2O9S2, parent ion m / z 650.9796, product ion m / z 170.0444, cone voltage 2V, collision voltage 20V;

[0050] (22) The retention time of DHPG is 5.85 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 32 N2O8S2, parent ion m / z 635.6592, product ion m / z 170.0748, cone voltage 62V, collision voltage 40V;

[0051] (23) The retention time of DOPAC is 6.90 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 30 N2O8S2, parent ion m / z 635.8149, product ion m / z 170.8625, cone voltage 60V, collision voltage 48V;

[0052] (24) The retention time of DHPP is 7.07 min, the number of dansyl groups is 2, and the molecular formula is C 33 H 32 N2O8S2, parent ion m / z 648.8726, product ion m / z 171.2429, cone voltage 50V, collision voltage 48V;

[0053] (25) The retention time of DOPET is 7.17 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 32 N2O7S2, parent ion m / z 620.9416, product ion m / z 169.7701, cone voltage 24V, collision voltage 36V;

[0054] (26) The retention time of 3,4-DHBA is 7.18 min, the number of dansyl groups is 2, and the molecular formula is C 31 H 28N2O8S2, parent ion m / z 620.9690, product ion m / z 170.3245, cone voltage 50V, collision voltage 44V;

[0055] (27) The retention time of DHBA is 7.18 min, the number of dansyl groups is 2, and the molecular formula is C 31 H 28 N2O8S2, parent ion m / z 620.9690, product ion m / z 170.9897, cone voltage 50V, collision voltage 40V;

[0056] (28) The retention time of CA is 7.23 min, the number of dansyl groups is 2, and the molecular formula is C 33 H 30 N2O8S2, parent ion m / z 647.2402, product ion m / z 170.2648, cone voltage 56V, collision voltage 40V;

[0057] (29) The retention time of HGA is 7.26 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 30 N2O8S2, parent ion m / z 635.8149, product ion m / z 170.0015, cone voltage 64V, collision voltage 40V;

[0058] (30) The retention time of DOPAL is 7.67 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 30 N2O7S2, parent ion m / z 618.9808, product ion m / z 170.0973, cone voltage 32V, collision voltage 10V;

[0059] (31) The retention time of 3,4-DHBAD is 8.03 min, the number of dansyl groups is 2, and the molecular formula is C 31 H 28 N2O7S2, parent ion m / z 604.7825, product ion m / z 170.0461, cone voltage 52V, collision voltage 40V;

[0060] (32) The retention time of DHBD is 8.47 min, the number of dansyl groups is 2, and the molecular formula is C 31 H 28 N2O7S2, parent ion m / z is 604.7825, product ion m / z is 169.9528, cone voltage is 48V, collision voltage is 36V.

[0061] The small molecule phenolic substance detection method described in the present invention is used for developing targets or drugs for mental illnesses, and the mental illnesses are anxiety, depression, and sleep disorders.

[0062] The innovative research of the present invention is:

[0063] Chemical derivatization is a method of indirectly determining the content of the analyte by chemically reacting the analyte with a derivatization reagent containing a specific functional group to generate a derivatization product containing a specific group. The modified sensitizing group in the derivatization reagent can increase the signal intensity of the analyte.

[0064] Dansyl chloride (DNSCl) is a strong fluorescent agent. It was originally used as a fluorescent derivatization reagent for amino acids. In recent years, it has been often used as a pre-column derivatization reagent for biogenic amines. As a pre-column derivatization reagent, dansyl chloride has the characteristics of simple derivatization operation, good derivative stability and high sensitivity.

[0065] The present invention utilizes the characteristic that dansyl chloride can combine with phenolic hydroxyl groups under alkaline conditions, greatly improving the ionization efficiency of ESMPs derivatives and significantly enhancing the detection sensitivity of ESMPs. Then, using the high sensitivity and multiple reaction ion monitoring mode of UPLC-QqQ-MS / MS, a new endogenous phenolomics method capable of simultaneously analyzing and detecting 31 ESMPs was established.

[0066] The advantages of the present invention are as follows:

[0067] 1. Concept of endogenous phenolics

[0068] This paper first proposed the concept of "endogenous phenolics", a framework that focuses on important small molecule metabolites produced by catecholamine neurotransmitters (such as dopamine and norepinephrine). These small molecule compounds play a key role in the nervous system and are involved in many complex physiological processes, including emotion regulation, attention maintenance, and cognitive function.

[0069] 2. In vivo ESMPs detection method

[0070] The present invention has established a comprehensive detection method to identify and quantify 31 different ESMPs in the body. This method combines ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS / MS) to ensure high sensitivity and high specificity, and can detect trace endogenous small molecule phenols in biological samples. Through this method, we can accurately understand the content of different types of ESMPs in different tissues and their dynamic changes.

[0071] 3. Role in mental illness

[0072] The present invention is of great significance for revealing the role of ESMPs in mental illness. By comparing the ESMPs levels of people with mental illness and normal people, we can find some key biomarkers. These markers can not only serve as early diagnostic indicators, but also guide the formulation of individualized treatment strategies.

[0073] The present invention is of great significance for revealing the role of ESMPs in mental illnesses. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0075] Figure 1A MRM patterns of 31 ESMPs

[0076] Figure 1B MRM patterns of 31 ESMPs

[0077] Figure 1C MRM patterns of 31 ESMPs

[0078] Figure 1D MRM patterns of 31 ESMPs

[0079] FIG. 2A to FIG. 2E Optimization of derivatization conditions for 31 ESMPs:

[0080] Figure 2A Optimization of derivatization conditions for 31 ESMPs - pH value

[0081] Figure 2B Optimization of derivatization conditions for 31 ESMPs - buffer volume

[0082] Figure 2C Optimization of derivatization conditions for 31 ESMPs - Derivatization reagent volume

[0083] Figure 2D Optimization of derivatization conditions for 31 ESMPs - Derivatization temperature

[0084] Figure 2E Optimization of derivatization conditions for 31 ESMPs - Derivatization time

[0085] FIG. 3A to FIG. 3E Optimization of derivatization conditions for internal standard CA:

[0086] Figure 3A Optimization of derivatization conditions for internal standard CA-pH

[0087] Figure 3B Optimization of derivatization conditions for internal standard CA - buffer solution volume

[0088] Figure 3C Optimization of derivatization conditions for internal standard CA - derivatization reagent volume

[0089] Figure 3D Optimization of derivatization conditions for internal standard CA - derivatization temperature

[0090] Figure 3E Optimization of derivatization conditions for internal standard CA - derivatization time

[0091] Figure 4 Derivatization efficiency of 31 ESMPs and internal standard CA

[0092] Figure 5A This is the TIC image of the blank mouse brain tissue homogenate sample matrix

[0093] Figure 5B TIC diagram of blank mouse brain tissue homogenate plus LLOQ sample matrix

[0094] Fig. 6A TIC diagram of blank mouse plasma sample matrix

[0095] Figure 6B TIC diagram of blank mouse plasma spiked with LLOQ sample matrix

[0096] Figure 7 Comparison of chromatograms before and after derivatization of 31 ESMPs

[0097] Figure 8 Anxiolytic pharmacodynamic study process for DISS and SA

[0098] 9A to 9D The OFT experimental results of anxiety-like behavior mice. Control is the blank control group, Model is the CUMS model group, DZP is the diazepam group, DISS-L is the DISS low-dose group, DISS-H is the DISS high-dose group, SA-L is the SA low-dose group, and SA-H is the SA high-dose group; compared with the blank group, ns means no significant difference, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001; ns indicates no significant difference compared with the model group. * indicates P < 0.05, ** indicates P < 0.01, *** Indicates P < 0.001:

[0099] Fig. 9A OFT experimental results of anxiety-like behavior mice - latency of first entry into the central area

[0100] Fig. 9BOFT experimental results of anxiety-like behavior mice - the number of times they entered the central area

[0101] Fig. 9C OFT experimental results of anxiety-like behavior mice - time spent in the central area

[0102] Fig.9D OFT results for anxiety-like behavior mice - total distance moved in the central area

[0103] Fig. 10A and Fig. 10B The results of the EPM experiment on anxiety-like behavior mice. Control is the blank control group, Model is the CUMS model group, DZP is the diazepam group, DISS-L is the DISS low-dose group, DISS-H is the DISS high-dose group, SA-L is the SA low-dose group, and SA-H is the SA high-dose group; compared with the blank group, ns means no significant difference. # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001; ns indicates no significant difference compared with the model group. * indicates P < 0.05, ** indicates P < 0.01, *** Indicates P < 0.001:

[0104] Fig. 10A Results of the EPM experiment on anxiety-like behavior mice - the number of times they entered the open arm

[0105] Fig. 10B Results of the EPM experiment on anxiety-like behavior mice - time spent in the open arm

[0106] FIG. 11A to FIG. 11C The results of the NSFT experiment on anxiety-like behavior mice. Control is the blank control group, Model is the CUMS model group, DZP is the diazepam group, DISS-L is the DISS low-dose group, DISS-H is the DISS high-dose group, SA-L is the SA low-dose group, and SA-H is the SA high-dose group; compared with the blank group, ns means no significant difference. # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001; ns indicates no significant difference compared with the model group. * indicates P < 0.05, ** indicates P < 0.01, *** Indicates P < 0.001:

[0107] Fig.11A Results of NSFT experiment on anxiety-like behavior mice - latency to first food intake

[0108] Fig. 11B Results of NSFT experiment on anxiety-like behavior mice - total number of food intake

[0109] Fig. 11C Results of NSFT experiment on anxiety-like behavior mice - eating time

[0110] FIG. 12A to FIG. 12X The ESMPs content in the brain tissue of mice with anxiety-like behavior after administration of DISS and SA. Compared with the blank group, ns means no significant difference. # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001; ns indicates no significant difference compared with the model group. * indicates P < 0.05, ** indicates P < 0.01, *** Indicates P < 0.001:

[0111] Fig. 12A ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-HBA

[0112] Fig. 12B ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA

[0113] Fig. 12C ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA

[0114] Fig.12D ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-HPAA

[0115] Fig.12E The content of ESMPs in the brain tissue of mice with anxiety-like behavior after administration of DISS and SA

[0116] Fig.12F ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA

[0117] Figure 12G ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-HMBH

[0118] Fig.12H ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-3-HCM

[0119] Fig.12IESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-PHCA

[0120] Fig.12J PCA to investigate the content of ESMPs in the brain tissue of mice with anxiety-like behavior after administration of DISS and SA

[0121] Figure 12K ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-THCM

[0122] Figure 12L ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-3,4-DHBAD

[0123] Figure 12M To investigate the content of ESMPs in the brain tissue of mice with anxiety-like behavior after administration of DISS and SA-DOPAC

[0124] Fig.12N ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-3,4-DHBA

[0125] Fig.12O DHBA was used to investigate the content of ESMPs in the brain tissue of mice with anxiety-like behavior after administration of DISS and SA.

[0126] Figure 12P The content of ESMPs in brain tissue of mice with anxiety-like behavior after administration of DISS and SA

[0127] Figure 12Q To investigate the content of ESMPs in the brain tissue of mice with anxiety-like behavior after administration of DISS and SA-DOPAL

[0128] Figure 12R ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA

[0129] Figure 12S ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-HCM

[0130] Figure 12T ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-3,4-HPHPA

[0131] Figure 12U ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-2,4-HPHPA

[0132] Figure 12VMHPG to evaluate the content of ESMPs in the brain tissue of mice with anxiety-like behavior after administration of DISS and SA

[0133] Figure 12W To investigate the content of ESMPs in the brain tissue of mice with anxiety-like behavior after administration of DISS and SA-VMA

[0134] Figure 12X ESMPs content in brain tissue of mice with anxiety-like behavior after administration of DISS and SA-DHBD

[0135] FIG. 13A to FIG. 13Z The ESMPs content in the plasma of mice with anxiety-like behavior after administration of DISS and SA. Compared with the blank group, ns means no significant difference. # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001; ns indicates no significant difference compared with the model group. * indicates P < 0.05, ** indicates P < 0.01, *** Indicates P < 0.001:

[0136] Fig.13A ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0137] Fig. 13B ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0138] Fig. 13C ESMPs levels in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0139] Fig.13D PCA to investigate the ESMPs levels in the plasma of mice with anxiety-like behavior after administration of DISS and SA

[0140] Fig.13E ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA-THCM

[0141] Fig.13F ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0142] Figure 13G To investigate the ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA.

[0143] Fig.13HTo investigate the content of ESMPs in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0144] Fig.13I ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA -3,4-HPHPA

[0145] Fig.13J ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA -2,4-HPHPA

[0146] Figure 13K The content of ESMPs in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0147] Figure 13L To investigate the ESMPs content in the plasma of mice with anxiety-like behavior after administration of DISS and SA-HBA

[0148] Figure 13M ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA-HMBH

[0149] Fig.13N The levels of ESMPs in the plasma of mice with anxiety-like behavior after administration of DISS and SA

[0150] Fig.13O ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0151] Figure 13P ESMPs levels in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0152] Figure 13Q ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA-3,4-DHBA

[0153] Figure 13R DHBA-ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0154] Figure 13S MHPG to evaluate the plasma ESMPs content in mice with anxiety-like behavior after administration of DISS and SA

[0155] Figure 13T To investigate the ESMPs content in the plasma of mice with anxiety-like behavior after administration of DISS and SA-VMA

[0156] Figure 13U DHBA-ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0157] Figure 13V The levels of ESMPs in the plasma of mice with anxiety-like behavior after administration of DISS and SA

[0158] Figure 13W To investigate the ESMPs content in the plasma of mice with anxiety-like behavior after administration of DISS and SA-DOPAC

[0159] Figure 13X DHPP-ESMPs content in plasma of mice with anxiety-like behavior after administration of DISS and SA

[0160] Figure 13Y The content of ESMPs in plasma of mice with anxiety-like behavior after administration of DISS and SA-HPAT

[0161] Figure 13Z HPPT to investigate the ESMPs content in the plasma of mice with anxiety-like behavior after administration of DISS and SA DETAILED DESCRIPTION

[0162] Example 1: Optimization of Dansyl Chloride Derivatization Conditions

[0163] 1. Experimental reagents and instruments

[0164] 3-Methoxy-4-hydroxyphenyl glycol (MHPG, CAS: 534-82-7), 4-hydroxy-3-methoxyphenylacetic acid (VMA, CAS: 55-10-7), 4-hydroxyphenyl lactic acid (HDPL, CAS: 306-23-0), 4-hydroxyphenylpyruvic acid (HPPA, CAS: 156-39-8), 2-hydroxyphenylacetic acid (HPAT, CAS: 614-75-5), 3-methoxy-4-hydroxyphenylacetic acid (HVA, CAS: 306-08-1), 3-hydroxyphenylacetic acid (3-HPAA, CAS: 621-37-4), 4-hydroxyphenylacetic acid (HPAA, CAS: 156-38 -7), 3-hydroxybenzoic acid (3-HBA, CAS: 99-06-9), 4-hydroxyphenylethanol (TYR, CAS: 501-94-0), 2-hydroxycinnamic acid (HCM, CAS: 614-60-8), 4-hydroxybenzoic acid (HBA, CAS: 99-96-7), 3-(3-hydroxyphenyl)propionic acid (HPPT, CAS: 621-54-5), 3-(4-hydroxyphenyl)propionic acid (3,4-HPHPA, CAS: 501-97-3), 3-hydroxycinnamic acid (3-HCM, CAS: 588-30-7), trans-3-hydroxycinnamic acid (THCM, CAS: 14755-02- 3), 4-hydroxycinnamic acid (PHCA, CAS: 7400-08-0), trans-4-coumaric acid (PCA, CAS: 501-98-4), 2-(4-hydroxyphenyl)propionic acid (2,4-HPHPA, CAS: 938-96-5), 4-hydroxy-3-methoxybenzaldehyde (HMBH, CAS: 121-33-5), 3,4-dihydroxyphenylacetic acid (DOMA, CAS: 775-01-9), 3,4-dihydroxyphenyl glycol (DHPG, CAS: 28822-73-3), 3,4-dihydroxyphenylacetic acid (DOPAC, CAS: 102-32-9), 3,4-dihydroxyphenylpropionic acid (D HPP, CAS: 1078-61-1), 3,4-dihydroxyphenylethanol (DOPET, CAS: 10597-60-1), 3,4-dihydroxybenzoic acid (3,4-DHBA, CAS: 99-50-3), 2,5-dihydroxybenzoic acid (DHBA, CAS: 490-79-9), caffeic acid (CA, CAS: 331-39-5), 2,5-dihydroxyphenylacetic acid (HGA, CAS: 451-13-8), 3,4-dihydroxyphenylacetaldehyde (DOPAL, CAS: 5707-55-1), 3,4-dihydroxybenzaldehyde (3,4-DHBAD, CAS: 139-85-5), 2,5-Dihydroxybenzaldehyde (DHBD, CAS: 1194-98-5) and dansyl chloride (CAS: 605-65-2) were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and the purity of each standard product was ≥98%; Na2CO3-NaHCO3 buffer solution (0.1 mol / L, pH = 11) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; mass spectrometry grade acetonitrile, methanol and formic acid were purchased from Thermo Fisher Scientific (China) Co., Ltd.; mass spectrometry grade acetic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; ultrapure water was purchased from Guangzhou Watsons Food and Beverage Co., Ltd.,

[0165] Waters UPLC-Xevo TQ-S micro triple quadrupole liquid-mass spectrometer (including autosampler, binary gradient pump, column oven, vacuum degasser, Masslynx 4.1 mass spectrometer workstation) (Waters, USA); high-speed low-temperature refrigerated centrifuge (TGL-16aR, Shanghai Anting Scientific Instrument Factory); CNC ultrasonic cleaner (KQ-300DB, Kunshan Ultrasonic Instrument Co., Ltd.); electronic analytical balance (ME104E / 02, Shanghai Mettler-Toledo Instrument Co., Ltd.); vortex mixer (VORTEX 3, IKA, Germany); nitrogen blowdown instrument (N-EVAP 116, Organomation, USA); constant temperature metal bath (DKT200-4, Hangzhou Miou Instrument Co., Ltd.).

[0166] 2. Experimental Methods

[0167] 2.1 Determination of optimal derivatization conditions

[0168] In order to obtain good derivatization efficiency, various conditions for the derivatization of ESMPs with dansyl chloride were optimized. In this experiment, the optimized conditions included derivatization solvents (methanol, acetonitrile and acetone), buffer solution pH (9.0-11.0), buffer solution volume (50-500 μL), derivatization reagent volume (10-500 μL), reaction temperature (30-100 °C) and reaction time (5-35 min). After UPLC-QqQ-MS / MS analysis, the optimal derivatization conditions were selected based on the obtained peak area and mass spectrometry response values ​​for the establishment of a new method.

[0169] 2.2 Chromatographic conditions

[0170] Optimize the chromatographic conditions. Select the chromatographic column that can better separate the 31 ESMPs among ACQUITY UPLC BEH C18 column, ACQUITY UPLC HSS T3 column, ACQUITY UPLC BEH HILIC column and ACQUITY UPLC BEH Amide column; select the appropriate mobile phase (methanol-water, acetonitrile-water), optimize the elution gradient of the mobile phase (9min, 13min, 18min, 20min); select the appropriate mobile phase additives (formic acid, acetic acid, ammonium formate and ammonium acetate), optimize the volume fraction of mobile phase additives (0.1% formic acid, 0.01% acetic acid, 5mM ammonium formate, 5mM ammonium acetate); select the appropriate flow rate (0.2-0.5mL / min) and column temperature (25-35℃).

[0171] The optimal chromatographic conditions finally determined are as follows. ACQUITY UPLC HSS T3 column (2.1×100mm, 1.8μm) was used; flow rate: 0.3mL / min; column temperature: 35℃; injection volume: 2μL; mobile phase was 0.1% formic acid aqueous solution (A)-acetonitrile (B). Gradient elution was used, and the elution conditions were 0-0.5min, 40%-55%B; 0.5-5.5min, 55%-70%B; 5.5-6min, 70%-83%B; 6-7.5min, 83%-95%B; 7.5-8.5min, 95%B; 8.5-9min, 95%-40%B.

[0172] 2.3 Mass spectrometry conditions

[0173] A Waters UPLC-Xevo TQ-S micro triple quadrupole liquid-mass spectrometry system was used, with an electrospray ion source (ESI source), positive ion mode scanning, multiple reaction monitoring mode (MRM) detection, collision gas argon (Ar), purge gas nitrogen (N2), capillary voltage 3kV, ion source temperature 150°C, desolvation gas temperature 350°C, and desolvation gas flow rate 650L / h. In order to avoid contamination of the ion source, the mobile phase of the first 1min directly enters the waste liquid. The MRM mode monitoring ion pairs and other optimal parameters for quantitative analysis of 31 ESMPs and internal standard CA are shown in Table 1, and the MRM diagram is shown in Figure 1.

[0174] Table 1: Detailed information on the optimal MRM parameters for 31 ESMPs and internal standards after CA derivatization

[0175]

[0176]

[0177] 3. Experimental results

[0178] Three common solvents for dansyl chloride were selected in this experiment, including methanol, acetonitrile and acetone. The experimental results showed that dansyl chloride has the best solubility in acetonitrile and the highest mass spectrometry response. Therefore, acetonitrile was selected as the solvent for the derivatization reagent dansyl chloride.

[0179] The optimal pH range of derivatization is 9.0-11.0. In the Na2CO3-NaHCO3 buffer solution at pH=11, the peak area of ​​ESMPs derivatives is the largest ( Figure 2A ). At a buffer solution volume of 350 μL, the peak area of ​​the ESMPs derivative reached its maximum value. Then, as the volume of the buffer solution increased, the peak area gradually decreased. Therefore, adding 350 μL of Na2CO3-NaHCO3 buffer solution with pH = 11 to the derivatization system is the optimal condition ( Figure 2B ).

[0180] Optimize the volume of the derivatization reagent dansyl chloride-acetonitrile solution within 10 to 500 μL. Figure 2C As shown in Figure 2, the optimal volume of 4 mmol / L dansyl chloride-acetonitrile solution is 350 μL, at which the complete derivatization of the analyte can be ensured. When the derivatization volume exceeds 350 μL, the peak area of ​​the ESMPs derivative no longer changes significantly ( Figure 2C ).

[0181] The reaction temperature optimization range is 30-100℃, and the reaction time optimization range is 5-35min. The results show that the maximum peak area ( FIG. 2D to FIG. 2E ).

[0182] Combined with the above experimental results, the optimal derivatization conditions when using dansyl chloride chemical derivatization as a sample pretreatment method for detecting ESMPs are: the buffer solution is Na2CO3-NaHCO3 buffer solution, the concentration is 0.1mol / L, the pH is 11, and the volume is 350μL; the derivatization reagent is dansyl chloride, the solvent is acetonitrile, the concentration is 4mmol / L, and the volume is 350μL; the reaction temperature is 80℃; the reaction time is 20min. This optimal derivatization condition is consistent with the experimental conclusion obtained by the internal standard, and the results are reliable ( FIG. 3A to FIG. 3E ).

[0183] Example 2: Investigation of the Degree of Derivatization of Dansyl Chloride under Optimal Derivatization Conditions

[0184] 1. Experimental reagents and instruments

[0185] Same as Example 1.

[0186] 2. Experimental Methods

[0187] 2.1 Solution preparation

[0188] Appropriate amounts of 31 ESMPs standards were accurately weighed respectively, and diluted with 1% acetic acid-methanol solution to prepare a mixed standard solution with a concentration of 500 ng / mL, which was then placed in a 4°C condition module and stored for later use.

[0189] 2.2 Chemical derivatization with dansyl chloride

[0190] Accurately weigh an appropriate amount of dansyl chloride and add it to acetonitrile to obtain a 4mmol / L dansyl chloride-acetonitrile solution. Take 200μL of the mixed standard solution with a concentration of 500ng / mL, add 350μL Na2CO3-NaHCO3 buffer solution (0.1mol / L, pH=11) and 350μL dansyl chloride-acetonitrile solution. Vortex mix for 1min and react at 80℃ in the dark for 20min, add 15% formic acid-water solution to adjust the pH of the mixed solution to 7. Vortex mix for 1min and centrifuge at 4℃ and 12000rpm for 10min. Take the supernatant for UPLC-QqQ-MS / MS analysis.

[0191] 2.3 Chromatographic conditions

[0192] Same as Example 1.

[0193] 2.4 Mass spectrometry conditions

[0194] Same as Example 1.

[0195] 3 Experimental results

[0196] The derivatization efficiency of 31 ESMPs was calculated according to formula (1). The results showed that the derivatization efficiency of 31 ESMPs exceeded 95%, indicating that the dansyl chloride chemical derivatization method was stable and reliable under the optimal derivatization conditions ( Figure 4 ).

[0197]

[0198] Example 3: Establishment of a new method for endogenous phenolomics and methodological investigation

[0199] 1. Experimental reagents and instruments

[0200] Same as Example 1.

[0201] 2. Experimental Methods

[0202] 2.1 Solution preparation

[0203] 2.1.1 Preparation of standard stock solution

[0204] Accurately weigh appropriate amounts of 31 ESMPs standards and dissolve them in 1% acetic acid-methanol solution to prepare 1 mmol / L standard stock solutions. All standard stock solutions were packaged and stored in a -20°C refrigerator for later use.

[0205] 2.1.2 Preparation of standard working solution

[0206] Accurately measure appropriate amounts of the above 31 ESMPs standard stock solutions, place them in a 10 mL volumetric flask, add 1% acetic acid-methanol solution to make up to 10 mL, prepare mixed standard working solutions with concentrations of 4 μmol / L and 30 μmol / L, respectively, and place them in a 4°C refrigerator for later use.

[0207] 2.1.3 Preparation of standard curve working solution and quality control sample solution (QC)

[0208] Accurately measure an appropriate amount of the mixed standard working solution, dilute it step by step with 1% acetic acid-methanol solution to the required concentration of the series of standard curve working solutions, and place it in a 4°C refrigerator for later use. The mass concentration range in the brain tissue homogenate matrix is: HPPA, HPPT, DOMA, DHPG, DOPET and HGA are 0.005-20nmol / L, DHPP and DHBD are 0.05-10nmol / L, MHPG, VMA, HPAT, 3-HPAA, HCM, 3,4-HPHPA, 2,4-HPHPA are 0.1-20nmol / L, DOPAL is 0.2-50nmol / L, 3,4-DHBA and DHBA are 0.5-100nmol / L, 3-HCM, THCM, PHCA, PCA, HMBH, DOPAC and 3,4-DHBAD are 1-80nmol / L, HDPL, HVA, HPAA, 3-HBA, TYR and H BA is 1-200nmol / L; the mass concentration range in plasma matrix: MHPG, VMA, HPAT, HPPT, DOMA, DHPG, DOPAC, DHPP, DOPET, HGA and DHBD are 0.05-20nmol / L, 3-HPAA, HCM, 3,4-DHBA and DHBA are 0.1-50nmol / L, HVA, HBA and HMBH are 2-200nmol / L, HDPL and 2,4-HPHPA are 5-500nmol / L, HPAA, TYR and 3,4-HPHPA are 8-800nmol / L, 3-HBA, 3-HCM, THCM, PHCA, PCA and 3,4-DHBAD are 20-2000nmol / L.

[0209] The quality control samples (QC) were selected as low-concentration quality control samples (QC-L) not higher than 3 times the lower limit of quantitation (LLOQ) in the linear range, medium-concentration quality control samples (QC-M) near the middle of the standard curve range, and high-concentration quality control samples (QC-H) at about 75% of the upper limit of the standard curve range. Accurately measure an appropriate amount of the mixed standard working solution, prepare low, medium, and high mass concentrations of mixed standard QC solutions with 1% acetic acid-methanol solution, and place them in a 4°C refrigerator for later use. Quality control samples of HPPA, HPPT, DOMA, DHPG, DOPET and HGA with concentrations of 0.01, 0.2 and 15 nmol / L, quality control samples of DHPP and DHBD with concentrations of 0.1, 1 and 8 nmol / L, quality control samples of MHPG, VMA, HPAT, 3-HPAA, HCM, 3,4-HPHPA and 2,4-HPHPA with concentrations of 0.2, 2 and 15 nmol / L, quality control samples of DOPAL with concentrations of 0.5, 2 and 40 nmol / L, quality control samples of 3,4-DHBA and DHBA with concentrations of 1, 10 and 60 nmol / L, quality control samples of 3-HCM, THCM, PHCA, PCA, HMBH, DOPAC and 3,4-DHBAD with concentrations of 2, 20 and 150 nmol / L, quality control samples of HDPL, HVA, HPAA, 3-HBA , TYR and HBA quality control samples; MHPG, VMA, HPAT, HPPT, DOMA, DHPG, DOPAC, DHPP, DOPET, HGA and DHBD quality control samples with concentrations of 0.1, 2, and 15 nmol / L, 3-HPAA, HCM, 3,4-DHBA and DHBA quality control samples with concentrations of 0.2, 10, and 40 nmol / L, HVA, HBA and HMBH quality control samples with concentrations of 5, 50, and 150 nmol / L, HDPL and 2,4-HPHPA quality control samples with concentrations of 10, 100, and 400 nmol / L, HPAA, TYR and 3,4-HPHPA quality control samples with concentrations of 20, 200, and 600 nmol / L, 3-HBA, 3-HCM, THCM, PHCA, PCA and 3,4-DHBAD quality control samples with concentrations of 50, 500, and 1500 nmol / L in plasma matrix.

[0210] 2.1.4 Preparation of internal standard solution

[0211] Accurately weigh an appropriate amount of internal standard CA, add 1% acetic acid-methanol solution to dissolve it to prepare a 1mmol / L internal standard stock solution. Accurately weigh an appropriate amount of the above internal standard stock solution, dilute it with 1% acetic acid-methanol solution to a concentration of 1μmol / L (internal standard working solution A) and 10μmol / L (internal standard working solution B), which are used as internal standard working solutions for brain tissue homogenate matrix and plasma matrix, respectively.

[0212] 2.2 Sample pretreatment

[0213] 2.2.1 Sample preparation of brain tissue homogenate matrix

[0214] Brain tissue was homogenized by adding the corresponding volume of 1% acetic acid-methanol solution according to its own weight to obtain 100 mg / mL brain tissue homogenate. After centrifugation at 4°C and 12000 rpm for 10 min, 100 μL of the brain homogenate supernatant was accurately aspirated, 100 μL of internal standard working solution A was added, vortexed for 1 min, and blown dry with N2. 350 μL of 4 mmol / L dansyl chloride-acetonitrile solution and 350 μL of 0.1 mol / L, pH=11 Na2CO3-NaHCO3 buffer solution were added to the residual dry matter, vortexed for 1 min, and then heated in an 80°C metal bath for derivatization for 20 min. Finally, 300 μL of 15% formic acid-water solution was added to the mixed derivatization system to adjust the pH, vortexed for 1 min, and centrifuged at 4°C and 12000 rpm for 10 min. The supernatant was aspirated for UPLC-QqQ-MS / MS injection analysis. All operations must be performed under light-proof conditions.

[0215] 2.2.2 Sample pretreatment of plasma matrix

[0216] An appropriate amount of 1% acetic acid-methanol solution was added to the plasma to obtain undiluted and 100-fold diluted plasma samples. After centrifugation at 4°C and 12000rpm for 10min, 100μL of plasma supernatant was precisely aspirated, 100μL of internal standard working solution B was added, vortexed for 1min, and blown dry with N2. 350μL of 4mmol / L dansyl chloride-acetonitrile solution and 350μL of 0.1mol / L, pH=11 Na2CO3-NaHCO3 buffer solution were added to the residual dry matter, vortexed for 1min, and then heated in an 80°C metal bath for 20min. Finally, 300μL of 15% formic acid-water solution was added to the mixed derivatization system to adjust the pH, vortexed for 1min, and centrifuged at 4°C and 12000rpm for 10min. The supernatant was aspirated for UPLC-QqQ-MS / MS injection analysis. All operations must be performed under light-proof conditions.

[0217] 2.3 Chromatographic conditions

[0218] Same as Example 1.

[0219] 2.4 Mass spectrometry conditions

[0220] Same as Example 1.

[0221] 2.5 Methodological Review

[0222] This part conducts methodological validation of the established analytical method according to the relevant provisions in the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part IV, Principles for Validation of Quantitative Analysis Methods for Biological Samples (9012). The validation content includes selectivity, standard curve and linear range, lower limit of quantification, precision and accuracy, matrix effect, residue and stability. All operations must be performed under light-proof conditions.

[0223] 2.5.1 Selectivity

[0224] Since the analyte in this experiment is an endogenous component, a certain concentration of mixed standard solution is added to the blank matrix to examine the selectivity of the method. According to the method described in "2.1.3 Preparation of standard curve working solution and quality control sample solution (QC)" and "2.2 Sample pretreatment" of this embodiment, 6 different batches of blank matrix samples and 6 different batches of LLOQ standard added samples of blank matrix were prepared. The response value of the interfering component should be lower than 20% of the ESMPs LLOQ response and lower than 5% of the internal standard response.

[0225] 2.5.2 Standard curve and linear range

[0226] Accurately pipette 100 μL of 31 ESMPs standard solutions of series concentrations under "2.1.3 Preparation of standard curve working solution and quality control sample solution (QC)", and add 100 μL of blank mouse brain homogenate or plasma matrix sample and 100 μL of internal standard working solution respectively. Then, according to the method under "2.2 Sample pretreatment", prepare the blank matrix standard sample solution of series concentrations for injection analysis. Each concentration level is measured in parallel 3 times, and the peak areas of ESMPs and internal standards are recorded. The average response of ESMPs in 6 blank samples is set as A, and the response of ESMPs in blank matrix standard sample solution is set as B. The concentration of ESMPs standard added to the blank matrix (nmol / L) is used as the horizontal axis (x), and the value of B minus A is used as the vertical axis (y). Linear regression calculation is performed using weighted least squares method to obtain the corresponding linear regression equation, linear range and correlation coefficient. Substitute the average response value into the linear regression equation to calculate the actual concentration. The RE% between the calculated value and the labeled value at each concentration point is generally required to be within ±15%, and the LLOQ is within ±20%.

[0227] 2.5.3 Lower limit of quantification

[0228] LLOQ, as the lowest point of the standard curve, can be used to describe the detection ability of the test compound. According to the method described in "2.2 Sample Pretreatment", 6 solutions of the lowest concentration point of the 31 ESMPs standard curve working solutions were prepared. Parallel measurements were performed 3 times within one working day, and the test was carried out for 3 consecutive working days. The precision was expressed as RSD% and the accuracy was expressed as RE%. The intra-day precision and accuracy as well as the inter-day precision and accuracy were calculated respectively. Near the LLOQ, both RSD% and RE% should not exceed ±20%.

[0229] 2.5.4 Precision and Accuracy

[0230] Precision is used to describe the closeness of repeated measurements of the substance to be tested, and accuracy is used to reflect the closeness of the measured value of the substance to be tested under this method to the labeled concentration of the analyte. Precision and accuracy are generally evaluated using QC samples of low, medium, and high concentrations.

[0231] Accurately pipette 100 μL of brain homogenate or plasma matrix samples from 6 blank mice, add 100 μL of mixed standard QC solution of low, medium and high mass concentrations under "2.1.3" and 100 μL of internal standard working solution, treat the blank matrix samples according to the method described in "2.2 Sample Pretreatment", and obtain QC-L, QC-M, and QC-H samples of 31 ESMPs with different mass concentrations. Prepare 6 copies of each concentration in parallel, prepare 3 analytical batches in total, and repeat the measurement 3 times for each sample. Record the response value of ESMPs and substitute it into the accompanying standard curve to calculate the corresponding concentration. RSD% is used to represent precision, and RE% is used to represent accuracy. The same batch of QC samples are tested for intra-batch precision and accuracy, and different batches of QC samples are tested for inter-batch precision and accuracy. Both RSD% and RE% are required to be no more than ±15%.

[0232] 2.5.5 Matrix Effects

[0233] The matrix effect is a necessary consideration for mass spectrometry detection. The matrix refers to other components in the sample except the target analyte, which often interferes with the detection of the analyte and affects the accuracy of the test results. These interferences and effects are called matrix effects. Since ESMPs are endogenous components, it is difficult to obtain blank samples without ESMPs in the experiment. Therefore, this experiment uses the matrix effect to deduct the response value of ESMPs in the blank sample. The specific method is as follows:

[0234] (1) Take 100 μL of 6 blank mouse brain homogenate or plasma matrix samples from different sources, add 100 μL of the mixed standard QC solution of low, medium and high mass concentrations under "2.1.3" and 100 μL of the internal standard working solution, treat according to the method described in "2.2 Sample Pretreatment" and inject for analysis. Prepare 6 replicates for each mass concentration and record the ESMPs response value as A.

[0235] (2) Replace the blank mouse brain homogenate or plasma matrix sample with 1% acetic acid-methanol solution, add 100 μL of the mixed standard QC solution of low, medium and high mass concentrations under "2.1.3" and 100 μL of the internal standard working solution, treat and analyze according to the method described in "2.2 Sample Pretreatment", prepare 6 replicates for each mass concentration, and record the ESMPs response value as B.

[0236] (3) After adding 100 μL of the internal standard working solution to 100 μL of blank mouse brain homogenate or plasma matrix sample, the sample was processed according to the method described in “2.2 Sample Pretreatment” and injected for analysis. Six blank samples were prepared in parallel, and the average response value of ESMPs was recorded as C.

[0237] The matrix effect was calculated using formula (2), and it was required to be within the range of 85% to 115% to be considered unaffected by the matrix.

[0238]

[0239] 2.5.6 Residue

[0240] Residue investigation refers to investigating the residue in the entire system after the test and minimizing it without affecting the precision and accuracy of the test. Samples were injected in the order of blank matrix sample, upper limit of quantification (ULOQ) sample, blank matrix sample, and LLOQ sample, and the measurement was repeated 3 times to investigate the residual effect of the sample. The response value of ESMPs in the blank matrix sample should not exceed 20% of the LLOQ and should not exceed 5% of the internal standard response.

[0241] 2.5.7 Stability

[0242] The stability test of this study includes short-term stability, room temperature stability, long-term stability and repeated freeze-thaw stability. 100 μL of brain homogenate or plasma matrix samples from 6 blank mice were accurately aspirated, and 100 μL of mixed standard QC solution and 100 μL of internal standard working solution of low, medium and high mass concentrations under "2.1.3" were added. Six copies of each mass concentration were prepared in parallel. After being treated according to the method described in "2.2 Sample Pretreatment", the samples were placed in an automatic sampler at 4°C for 24 hours, placed at room temperature for 4 hours, placed in a -80°C refrigerator for half a month, and placed in a -80°C refrigerator and then taken out to thaw at room temperature. The stability after repeated freeze-thaw cycles was tested 3 times. RSD% and RE% were calculated based on the accompanying standard curve, and both should not exceed ±15%.

[0243] 2.6 Data Statistical Analysis

[0244] Masslynx Version 4.1 mass spectrometry workstation (Waters, USA) was used for data acquisition and raw data processing. A peak list including compound retention time, peak area, response value and other information under each channel in MRM mode was obtained and input into Excel for subsequent data processing.

[0245] 3. Experimental results

[0246] 3.1 Methodological results of brain tissue homogenate matrix

[0247] 3.1.1 Selectivity

[0248] Under the above experimental conditions, blank mouse brain tissue homogenate samples and LLOQ standard spiked samples were sampled and analyzed after pretreatment. The TIC diagrams of 31 ESMPs are shown in Figure 5A and Figure 5B The results showed that the peak shapes of 31 ESMPs in blank mouse brain tissue homogenate samples and LLOQ standard spiked samples were good and the retention times were consistent. In addition, the peak areas and response values ​​of the chromatographic peaks increased after adding a certain concentration of mixed standard solution, indicating that the 31 ESMPs were not interfered by other endogenous components or impurities in the brain tissue homogenate matrix, and met the relevant requirements for biological sample analysis.

[0249] 3.1.2 Standard curve and linear range

[0250] In this experiment, the corresponding standard was added to the blank brain tissue homogenate containing ESMPs. After deducting the background response of the blank brain tissue homogenate matrix, a standard curve was established with the added concentration of the standard. Three of the 31 ESMPs were not linear during fitting. The linear regression equation, linear range and correlation coefficient of the remaining 28 ESMPs are shown in Table 2. The standard curve has a total of 8 concentration points. The results showed that these 28 ESMPs showed good linear relationships within the linear range in the table, with R 2 All of them were greater than 0.99, which met the relevant requirements for biological sample analysis. The obtained response values ​​were substituted into the standard curve to calculate the actual concentration and RE%. The results are shown in Table 3. The RE% of 28 ESMPs at LLOQ was less than 20%, and the RE% of the other standard curve concentration points was within 15%, which met the requirements.

[0251] Table 2: Linear relationship study of 28 ESMPs in brain tissue homogenate matrix (n=6)

[0252]

[0253]

[0254] Table 3: Relative deviations between the calculated values ​​and the indicated values ​​of the concentration points of the standard curve of brain tissue homogenate of 28 ESMPs (n=6)

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263] 3.1.3 Lower limit of quantification

[0264] The results are shown in Table 4. The intra-day and inter-day precision RSD% of the quantitative lower limit of 28 ESMPs were less than 20%, and the intra-day and inter-day accuracy RE% were less than 20%, which met the measurement requirements for biological sample analysis.

[0265] 3.1.4 Matrix Effects

[0266] The results are shown in Table 5. Among the 28 ESMPs, the matrix effects of 24 ESMPs in mouse brain tissue homogenate matrix were between 85.20% and 112.68%, and the RSD% was between 2.67% and 13.97%, which met the determination requirements for biological sample analysis. The matrix effect results of the remaining 4 ESMPs were not within the acceptable range. The experimental results show that under this pretreatment method, the matrix effects of these 24 ESMPs are relatively stable, and the influence of the matrix effect on their quantification is not significant.

[0267] 3.1.5 Precision and Accuracy

[0268] The results of intra-batch and inter-batch precision and intra-batch and inter-batch accuracy of 24 ESMPs in brain tissue matrix are shown in Table 6. The intra-batch accuracy RE% of 24 ESMPs at low, medium and high concentrations was between -10.28% and 10.35%, and the intra-batch precision RSD% was between 1.27% and 10.52%; the inter-batch accuracy RE% was between -6.77% and 7.20%, and the inter-batch precision RSD% was between 0.25% and 6.97%. The above results were all within the specified range, indicating that the method has good precision and accuracy and meets the relevant requirements for biological sample analysis.

[0269] 3.1.6 Residue

[0270] The residue investigation results are shown in Table 7. After three cycles of high concentration-blank sample determination, the residues of 24 ESMPs in blank brain tissue homogenate samples were all lower than 20% of the LLOQ response and did not exceed 5% of the internal standard response, indicating that the pretreatment method had a small residue and met the standard.

[0271] 3.1.7 Stability

[0272] The results of the stability study of 24 ESMPs in brain tissue matrix are shown in Table 8. The short-term sampler stability of 24 ESMPs placed in an autosampler at 4°C for 24 hours was between -7.87% and 9.33% RE%, and RSD% was between 1.27% and 10.52%; the short-term room temperature stability of 24 hours placed at room temperature was between -9.06% and 12.22% RE%, and RSD% was between 1.90% and 11.67%; the long-term stability of 24 hours placed in a -80°C refrigerator was between -9.11% and 10.69% RE%, and RSD% was between 1.60% and 11.39%. This shows that the ESMPs in brain tissue samples have good stability under the above storage conditions and meet the requirements of biological sample analysis.

[0273] In the freeze-thaw stability of DOPAL, MHPG and DHBD after three cycles of freeze-thaw at -80℃ and room temperature, the RE% and RSD% in the low-concentration quality control samples exceeded 15%, indicating that attention should be paid to the preservation of biological samples when measuring ESMPs, and it is not appropriate to measure after repeated freeze-thaw.

[0274]

[0275]

[0276] Table 5: Matrix effect results of 24 ESMPs in brain tissue homogenate matrix (n=6)

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284] Table 7: Results of residual investigation of 24 ESMPs in brain tissue homogenate matrix (n=3)

[0285]

[0286]

[0287]

[0288]

[0289]

[0290] 3.2 Methodological results of plasma matrix

[0291] 3.2.1 Selectivity

[0292] TIC graphs of blank mouse plasma samples and LLOQ standard spiked samples of 31 ESMPs are shown in Fig. 6A and Figure 6BThe results showed that the peak shapes of the 31 ESMPs in blank mouse plasma samples and LLOQ standard spiked samples were good and the retention times were consistent. In addition, the peak areas and responses of the chromatographic peaks increased after the addition of a certain concentration of mixed standard solution, indicating that the 31 ESMPs were not interfered by other endogenous components or impurities in the plasma matrix, and met the relevant requirements for biological sample analysis.

[0293] 3.2.2 Standard curve and linear range

[0294] The corresponding standard was added to the blank plasma, and after deducting the background response of the blank plasma matrix, a standard curve was established with the added concentration of the standard. Five of the 31 ESMPs were not linearly related during fitting, and a linear regression equation could not be established. The linear regression equation, linear range and correlation coefficient of the remaining 26 ESMPs are shown in Table 9. The standard curve has a total of 10 concentration points. The results showed that the 26 ESMPs showed good linear relationships within the linear range in the table, and R2 was greater than 0.99, which met the relevant requirements for biological sample analysis. The obtained response value was substituted into the standard curve to calculate the actual concentration and RE%. The results are shown in Table 10. The RE% of the 26 ESMPs at the LLOQ was less than 20%, and the RE% of the remaining standard curve concentration points was within 15%, which met the requirements.

[0295] Table 9: Linear relationship study of 26 ESMPs in plasma matrix (n=6)

[0296]

[0297]

[0298] Table 10: Relative deviations between the calculated values ​​and the indicated values ​​of the concentration points of the 26 ESMPs plasma standard curves (n=6)

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307] 3.2.3 Lower limit of quantification

[0308] The results are shown in Table 11. The intra-day and inter-day precision RSD% of the quantitative lower limit of 26 ESMPs were less than 20%, and the intra-day and inter-day accuracy RE% were less than 20%, which met the measurement requirements for biological sample analysis.

[0309]

[0310]

[0311] 3.2.4 Matrix Effects

[0312] The results are shown in Table 12. The matrix effects of 26 ESMPs in mouse plasma matrix were between 87.46% and 113.98%, and the RSD% was between 1.94% and 13.51%. The matrix effects were relatively stable and met the requirements for biological sample analysis.

[0313] Table 12: Matrix effect results of 26 ESMPs in plasma matrix (n=6)

[0314]

[0315]

[0316]

[0317] 3.2.5 Precision and Accuracy

[0318] The results of intra-batch and inter-batch precision and intra-batch and inter-batch accuracy of 26 ESMPs in plasma matrix are shown in Table 13. The intra-batch accuracy RE% of 26 ESMPs at low, medium and high concentrations was between -10.70% and 13.76%, and the intra-batch precision RSD% was between 0.75% and 13.49%; the inter-batch accuracy RE% was between -7.65% and 11.89%, and the inter-batch precision RSD% was between 0.10% and 6.64%. The above results were all within the specified range, indicating that the method had good precision and accuracy and met the relevant requirements for biological sample analysis.

[0319]

[0320]

[0321]

[0322]

[0323] 3.2.6 Residue

[0324] The residue investigation results are shown in Table 14. After three cycles of high concentration-blank sample determination, the residues of 26 ESMPs in blank plasma samples were all lower than 20% of the LLOQ response and did not exceed 5% of the internal standard response, meeting the standards for biological sample analysis.

[0325] Table 14: Residual effect results of 26 ESMPs in plasma matrix (n=3)

[0326]

[0327] 3.2.7 Stability

[0328] The results of the stability study of 26 ESMPs in plasma matrix are shown in Table 15. The short-term sampler stability of 26 ESMPs placed in an autosampler at 4°C for 24 hours was between -10.70% and 13.76% RE%, and RSD% was between 1.08% and 10.84%; the short-term room temperature stability of 26 ESMPs placed at room temperature for 24 hours was between -11.49% and 13.67% RE%, and RSD% was between 1.20% and 13.39%; the long-term stability of 26 ESMPs placed in a -80°C refrigerator for half a month was between -9.63% and 13.75% RE%, and RSD% was between 1.75% and 11.83%. This shows that the ESMPs in plasma samples have good stability under the above storage conditions and meet the requirements of biological sample analysis.

[0329] In the freeze-thaw stability of 3-HPAA, DHBA, MHPG, DHBD, HGA, DOPAC and DHPP in the low-concentration quality control samples after three cycles of freeze-thaw at -80°C and room temperature, the RE% and RSD% exceeded 15%. This indicates that attention should be paid to the preservation of biological samples when measuring ESMPs, and it is not appropriate to measure after repeated freeze-thaw cycles.

[0330]

[0331]

[0332]

[0333]

[0334]

[0335] Example 4: Comparison

[0336] 1 Experimental reagents and instruments

[0337] Same as Example 1.

[0338] 2 Experimental methods

[0339] 2.1 Solution preparation

[0340] Appropriate amounts of 31 ESMPs standards were accurately weighed respectively, and diluted with 1% acetic acid-methanol solution to prepare a mixed standard solution with a concentration of 500 ng / mL, which was stored at 4°C for future use.

[0341] 2.2.2 Chemical derivatization with dansyl chloride

[0342] Same as Experiment 2.

[0343] 2.2.3 Comparison of sample pretreatment methods

[0344] The peak area and mass spectrometry response value obtained after injection of the common prepared solution and the dansyl chloride chemical derivatization method into UPLC-QqQ-MS / MS were compared.

[0345] 2.4 Chromatographic conditions

[0346] Chromatographic column: ACQUITY UPLC HSS T3 chromatographic column (2.1×100 mm, 1.8 μm); flow rate: 0.3 mL / min; column temperature: 35°C; injection volume: 2 μL; the mobile phase was 0.01% acetic acid aqueous solution (A)-acetonitrile (B), and gradient elution was adopted, and the elution conditions were 0-1 min, 2% B; 1-4 min, 2%-17% B; 4-6 min, 17%-35% B; 6-7 min, 35%-80% B; 7-8 min, 80%-2% B.

[0347] 2.5 Mass spectrometry conditions

[0348] A Waters UPLC-Xevo TQ-S micro triple quadrupole liquid-mass spectrometry system was used, with an electrospray ion source (ESI source), negative ion mode scanning, multiple reaction monitoring mode (MRM) detection, capillary voltage of 3 kV, ion source temperature of 150 °C, desolvation temperature of 350 °C, and desolvation flow rate of 650 L / h. MRM mode monitoring ion pairs and other optimal parameters are shown in Table 16.

[0349] Table 16: Detailed information on optimal parameters for 31 ESMPs MRM modes

[0350]

[0351]

[0352] 3. Results

[0353] After the introduction of dansyl chloride as a derivatization agent, the enhancement of detection sensitivity was evaluated by comparing the peak areas of ESMPs before and after derivatization. The experiment found that the peak areas of 31 ESMPs after derivatization increased significantly by 5 to 1161 times (Table 17, Figure 7 ). This indicates that the derivatization process significantly enhances the detection sensitivity of ESMPs, and this method can currently be used as a better sample pretreatment method for detecting ESMPs.

[0354] Table 17: Comparison of 31 ESMPs before and after derivation

[0355]

[0356]

[0357] Example 5: Application of the new endogenous phenolics method in mice with anxiety-like behavior

[0358] 1. Experimental animals, reagents and instruments

[0359] 77 SPF healthy male C57BL / 6J mice, 8 months old, weighing 30±2g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., with the experimental animal production license number: SCXK (Beijing) 2019-0010. The animal experimental procedures followed the guidelines of EU Directive 2010 / 63 / EU on animal experiments and the Regulations of the People's Republic of China on the Administration of Laboratory Animals. All animal experiments were approved by the Medical and Laboratory Animal Ethics Committee of Beijing University of Chinese Medicine (No. BUCM-2023031607-1134). During the experiment, the animals were kept in the animal room of Liangxiang Campus of Beijing University of Chinese Medicine, with an ambient temperature of (25±2)℃, a humidity of 45±5%, and a circadian light rhythm of 12h / 12h (8:00-20:00). During the feeding period, the mice were free to eat and drink.

[0360] Diazepam injection was purchased from Tianjin Jinyao Pharmaceutical Co., Ltd. (National Medicine Standard H12020957, Batch No.: 2101301), DISS was purchased from Chengdu Ruifensidedan Biotechnology Co., Ltd. (CAS: 530-59-6, Batch No.: RFS-J05002210024, HPLC>98%), SA was purchased from Chengdu Ruifensidedan Biotechnology Co., Ltd. (CAS: 13981-98-8, Batch No.: RFS-F01302210024, HPLC>98%), and Uletose was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (CAS: 51-79-6, Batch No.: C15887472, purity>98%). The rest is the same as in Example 1.

[0361] EthoVision XT 9 image acquisition and analysis system (Noldus, Netherlands). The rest is the same as in Example 1.

[0362] 2. Experimental Methods

[0363] 2.1 Animal grouping, modeling and drug administration

[0364] After 3 days of adaptive feeding, 77 C57BL / 6J mice were randomly divided into blank control group, model group, diazepam group, DISS low-dose group, DISS high-dose group, SA low-dose group and SA high-dose group, with 11 mice in each group.

[0365] In this experiment, a multifactorial approach was used to establish a chronic unpredictable mild stress (CUMS) model in mice. A total of seven stimuli were used, including a modified water platform for 20 hours (20:00 to 16:00 the next day), restraint + ultrasound for 4 hours (9:00 to 11:00, 14:00 to 16:00), tail clamping for 1 minute / time, tilting the cage 45° overnight (20:00 to 8:00 the next day), strobe for 24 hours (8:00 to 8:00 the next day), day and night reversal (lights off from 8:00 to 20:00, lights on from 20:00 to 8:00), and no bedding for 24 hours (8:00 to 8:00 the next day). The water platform and one other random stimulation method were selected every day for 8 consecutive weeks. Drug administration began from the 6th week of modeling and continued for 3 weeks. The drugs were administered orally twice a day, with deionized water in the morning and the corresponding drugs in the evening. The control group and the model group were given equal volumes of deionized water in the morning and evening. Behavioral experiments were performed on the fourth day of drug administration. Figure 8 and Table 18.

[0366] Table 18: Animal grouping scheme for DISS and SA anxiolytic pharmacodynamics studies

[0367]

[0368]

[0369] 2.2 Open field experiment

[0370] The open field test (OFT) was conducted in an open field test box for mice. The size of the test box (length × width × height) was 40 cm × 40 cm × 40 cm, and the bottom was a square. The bottom was divided into 16 squares (10 cm × 10 cm) of equal area by a video recording system. The central area of ​​20 cm × 20 cm was defined as the central area, and the rest were peripheral areas. The mice were fasted overnight the day before the experiment, and only drinking water was provided during the period. The mice were placed in the laboratory to adapt to the environment 1 hour before the experiment. On the fourth day, 30 minutes after the drug administration, the mice were placed in the center of the test box and allowed to explore freely in the box for 5 minutes. The behavioral state was tracked in real time by a camera directly above the test box, and the latency of the mouse's first entry into the central area, the number of times it entered the central area, the time it stayed in the central area, and the total distance moved were recorded. After the experiment of each mouse, 75% ethanol was used to clean the feces and urine left at the bottom of the test box to eliminate the interference of odor on the experiment. Each group of mice was cross-tested to ensure that the experiment was carried out in a quiet environment.

[0371] 2.3 Elevated plus maze test

[0372] The elevated plus-maze test (EPM) was conducted using an elevated plus-maze apparatus for mice. The apparatus consists of two relatively open arms (35cm×5cm), two relatively closed arms (35cm×5cm×15cm), and a central platform (5cm×5cm) connecting the four arms to form a "cross"-shaped maze, which is fixed on a bracket. This apparatus is made of black polypropylene material, and the contact surface is non-reflective. The maze apparatus is about 50cm higher than the floor of the room, and the top is usually unobstructed and surrounded by curtains. The mice were fasted overnight the day before the experiment, and only drinking water was provided during the period. The mice were placed in the laboratory 1h before the experiment to adapt to the environment. 30 minutes after administration on the 8th day, the mouse was placed on the central platform of the elevated plus maze with its head facing the open arm on one side and its back facing the experimenter. After being placed, the timer was started and the number of times the mouse entered the open arm (Open arm entry, OE), the number of times the mouse entered the closed arm (Cosed arm entry, CE), the time it stayed in the open arm (Open arm time, OT) and the time it stayed in the closed arm (Closed arm time, CT) within 5 minutes were recorded. According to formula (3) and formula (4), the percentage of the number of times the mouse entered the open arm to the total number of times the mouse moved (OE%) and the percentage of the time it stayed in the open arm to the total time it stayed in the wall (OT%) were calculated respectively. During the experiment, the mouse was considered to have entered any arm when both its front and rear feet entered the arm. If any foot exited the arm during the experiment, it indicated that the indicator was over. After the experiment of each mouse, 75% ethanol was used to clean the feces and urine left in the plus maze to eliminate the influence of odor on the experiment. Each group of mice was cross-tested to ensure that the experiment was carried out in a dark and quiet environment.

[0373]

[0374]

[0375] 2.4 Novelty-suppressed feeding experiment

[0376] Novelty suppressed feeding test (NSFT) was conducted overnight fasting the day before the experiment, during which only drinking water was provided. Mice were placed in the laboratory to adapt to the environment 1 hour before the experiment. On the 14th day, 30 minutes after administration, the mice were placed in an autonomous activity box, and high-fat feeds of equal size and quantity were placed in the central container of the box. After being placed in the box, the timing was started and the latency of the first feeding within 5 minutes (starting to bite the feed instead of just sniffing or playing with the feed), the total number of feedings and time were recorded. After the experiment, each mouse was required to use 75% ethanol to clean up the feces and urine left in the cross maze to eliminate the influence of odor on the experiment. Each group of mice was cross-tested to ensure that the experiment was carried out in a quiet environment.

[0377] 2.5 Endogenous phenolic analysis

[0378] 2.5.1 Collection of biological samples

[0379] Three days after the last behavioral experiment, the mice were fasted overnight, with only drinking water provided. On the second day, the corresponding drugs were given and 30 minutes later, 20% ulanose solution was injected intraperitoneally for anesthesia. The eyeballs were removed for blood collection, and the blood was collected in EP tubes coated with sodium heparin. After standing at room temperature for 30 minutes, it was centrifuged at 4°C and 3500rpm for 15 minutes, and the supernatant was aspirated as a plasma sample. After the blood was collected, the mouse chest cavity was quickly opened, the syringe needle was inserted into the apex of the left ventricle of the heart, and the right atrial appendage was cut. The heart was perfused with 0.9% saline solution until the outflowing liquid was clear. After the perfusion, the head was immediately cut off, and the whole brain of the mouse was quickly peeled off on an ice box and placed in liquid nitrogen. After all the samples were collected, the plasma and brain tissue samples were stored in a -80°C refrigerator.

[0380] 2.5.2 Sample pretreatment

[0381] 2.5.2.1 Brain tissue sample pretreatment

[0382] Same as Example 3.

[0383] 2.5.2.2 Plasma sample pretreatment

[0384] Same as Example 3.

[0385] 2.5.3 Chromatographic conditions

[0386] Same as Example 1.

[0387] 2.5.4 Mass spectrometry conditions

[0388] Same as Example 1.

[0389] 2.6 Data Statistical Analysis

[0390] Masslynx Version 4.1 mass spectrometry workstation (Waters, USA) was used for data acquisition and raw data processing to obtain a peak list including compound retention time, peak area, response value and other information under each channel in MRM mode, which was input into Excel for subsequent processing and substituted into the accompanying standard curve to calculate the corresponding concentration.

[0391] 3.1 Behavioral Experimental Results

[0392] 3.1.1 Open field test results

[0393] This study used OFT behavioral experiments to explore the anxiety state of mice. 9A to 9DAs shown. Compared with the blank control group, the model group showed significant differences in all four evaluation indicators, which can indicate that the mice in the model group have anxiety-like behavior, and the CUMS model established in this study is reliable. Compared with the model group, diazepam can significantly reduce the latency of the mice with anxiety-like behavior to enter the central area for the first time, and significantly increase the number of mice entering the central area, the time they stay in the central area, and the total distance they move (P<0.001); although the effects of DISS and SA on improving the anxiety state of mice are not as good as diazepam, they have significant differences in all indicators compared with the model group. Among them, the difference in the low-dose SA group is the most significant, and the ability to increase the number of mice entering the central area and the total distance they move is equivalent to diazepam (P<0.001).

[0394] 3.1.2 Elevated plus maze test results

[0395] In this study, the anti-anxiety effects of DISS and SA on mice with anxiety-like behavior were evaluated by EPM behavioral experiments. Fig. 10A and Fig. 10B As shown. Compared with the blank control group, the time and number of times the model group mice entered the open arm were significantly reduced, indicating that the model group mice were in an anxious state. Compared with the model group, diazepam could significantly improve the anxiety-like behavior of mice (P<0.01); the OE% and OT% of mice in the DISS and SA administration groups showed an increasing trend, among which the DISS high-dose group and the SA low-dose group showed a significant increasing trend.

[0396] 3.1.3 Results of novelty-induced food intake suppression experiment

[0397] In this study, the NSFT behavioral experiment was used to observe the behavior of mice ingesting novel foods in a new environment to infer their anxiety level. The results are shown in Figure 11. Compared with the blank control group, the total number and time of food intake in the model group were significantly reduced (P<0.05). Although the latency of the first food intake showed an increasing trend, there was no significant difference. Compared with the model group, diazepam can significantly increase the number and time of food intake in mice with anxiety-like behavior; although the difference in indicators between the DISS and SA administration groups is not as significant as that of diazepam, it can significantly alleviate the anxiety state of mice, among which the DISS high-dose group and the SA low-dose group had the best improvement effect.

[0398] 3.2 Results of endogenous phenolomics in brain tissue

[0399] The results of quantitative analysis of endogenous phenolics in brain tissue samples of mice with anxiety-like behavior are shown in Tables 19 and FIG. 12A to FIG. 12X As shown, a total of 24 ESMPs were detected in brain tissue samples.

[0400] Compared with the blank control group, the model group had significant differences in the contents of 15 ESMPs, of which 12 ESMPs were significantly upregulated, namely HPAA, HDPL, HMBH, HVA, DOPAC, 3,4-DHBA, DHBA, DOPAL, HCM, 2,4-HPHPA, MHPG and VMA; and 3 ESMPs were significantly downregulated, namely TYR, 3,4-DHBAD and HPAT. This indicates that there is fluctuation in the metabolism of ESMPs in the brain tissue of mice with anxiety-like behavior.

[0401] Compared with the model group, diazepam can significantly change the levels of 21 ESMPs, and can adjust 18 of them to the level of the blank control group. The levels of ESMPs in the brain tissue of mice in the DISS and SA administration groups were basically consistent with the diazepam group. The DISS low-dose group can adjust 15 ESMPs, the DISS high-dose group can adjust 14 ESMPs, the SA low-dose group can adjust 12 ESMPs, and the SA high-dose group can adjust 10 ESMPs.

[0402] 3.3 Results of plasma endogenous phenolics

[0403] The results of quantitative analysis of endogenous phenolics in plasma samples of mice with anxiety-like behavior are shown in Table 20 and Figure 13. A total of 26 ESMPs were detected in the plasma samples.

[0404] Compared with the blank control group, there were 8 ESMPs with significant differences in the content in the model group, among which only MHPG content was significantly upregulated, and the remaining 7 ESMPs contents were significantly downregulated, namely 3,4-DHBAD, HDPL, HVA, 3-HPAA, DHBD, HGA and DOPAC, indicating that there were fluctuations in ESMPs metabolism in the plasma of mice with anxiety-like behavior.

[0405] Compared with the model group, diazepam can significantly change the levels of 13 ESMPs, and can adjust the levels of 8 of them. DISS has a better ability to adjust the levels of mouse plasma ESMPs than diazepam. The low-dose and high-dose DISS groups can adjust 13 and 12 ESMPs to the level of the blank control group, respectively, the low-dose SA group can adjust 8 ESMPs, and the high-dose SA group can adjust 6 ESMPs.

[0406]

[0407]

[0408]

[0409]

[0410] The series of detailed descriptions listed above are only specific descriptions of feasible implementation methods of the present invention. They are not intended to limit the scope of protection of the present invention. Those skilled in the art can design many other modifications and implementation methods, which will fall within the scope and spirit of the principles disclosed in this application.

Claims

1. A method for detecting small molecule phenolic substances, characterized in that The steps include: S1: Preparation of solutions, including preparation of standard stock solution, preparation of standard working solution, preparation of standard curve working solution and quality control sample solution, and preparation of internal standard solution; S2: Sample pretreatment: using dansyl chloride to chemically derivatize samples containing small molecule phenols to increase the detection sensitivity of small molecule phenols through sensitizing groups; S3: Chromatography-mass spectrometry is used to determine the content of small molecular phenolic substances in the sample under chromatographic conditions and mass spectrometry conditions.

2. A method for detecting small molecule phenolic substances according to claim 1, characterized in that: The small molecule phenolic substances are endogenous small molecule phenolic substances, which are small molecule metabolites produced by catecholamine neurotransmitters.

3. A method for detecting small molecule phenolic substances according to claim 1 or 2, characterized in that: The small molecule phenols include 3-methoxy-4-hydroxyphenyl glycol (MHPG), 4-hydroxy-3-methoxyphenylacetic acid (VMA), 4-hydroxyphenyl lactic acid (HDPL), 4-hydroxyphenylpyruvic acid (HPPA), 2-hydroxyphenylacetic acid (HPAT), 3-methoxy-4-hydroxyphenylacetic acid (HVA), 3-hydroxyphenylacetic acid (3-HPAA), 4-hydroxyphenylacetic acid (HPAA), 3-hydroxybenzoic acid (3-HBA), 4-hydroxyphenylethanol (TYR), 2-hydroxycinnamic acid (HCM), 4-hydroxybenzoic acid (HBA), 3-(3-hydroxyphenyl) propionic acid (HPPT), 3-(4-hydroxyphenyl) propionic acid (3,4-HPHPA), 3-hydroxycinnamic acid (3-HCM), trans-3-hydroxycinnamic acid (THCM), 4-hydroxy Cinnamic acid (PHCA), trans-4-coumaric acid (PCA), 2-(4-hydroxyphenyl)propionic acid (2,4-HPHPA), 4-hydroxy-3-methoxybenzaldehyde (HMBH), 3,4-dihydroxyphenylacetic acid (DOMA), 3,4-dihydroxyphenylglycol (DHPG), 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylpropionic acid (DHPP), 3,4-dihydroxyphenylethanol (DOPET), 3,4-dihydroxybenzoic acid (3,4-DHBA), 2,5-dihydroxybenzoic acid (DHBA), caffeic acid (CA), 2,5-dihydroxyphenylacetic acid (HGA), 3,4-dihydroxyphenylacetaldehyde (DOPAL), 3,4-dihydroxybenzaldehyde (3,4-DHBAD), 2,5-dihydroxybenzaldehyde (DHBD).

4. A method for detecting small molecule phenolic substances according to claim 1, characterized in that: The step S1 of preparing the solution refers to: (1) Preparation of standard stock solution: Accurately weigh an appropriate amount of small molecule phenol standard and dissolve it in 1% acetic acid-methanol solution to prepare a 1 mmol / L standard stock solution; all standard stock solutions are packaged and stored in a -20°C refrigerator for later use; (2) Preparation of standard working solutions: Accurately measure appropriate amounts of the above-mentioned small molecule phenol standard stock solutions and place them in 10 mL volumetric flasks. Add 1% acetic acid-methanol solution to make the volume to 10 mL. Prepare mixed standard working solutions with concentrations of 4 μmol / L and 30 μmol / L, respectively, and place them in a 4°C refrigerator for later use; (3) Preparation of standard curve working solution and quality control sample solution: Accurately measure an appropriate amount of the mixed standard working solution, dilute it step by step with 1% acetic acid-methanol solution to a series of standard curve working solutions of the required concentration, and store it in a 4°C refrigerator for later use; Preparation of quality control sample solution means: accurately measure an appropriate amount of mixed standard working solution, use 1% acetic acid-methanol solution to prepare three mixed standard QC solutions of low, medium and high mass concentrations, and place them in a 4°C refrigerator for later use; (4) Preparation of internal standard solution means: accurately weigh an appropriate amount of internal standard CA, add 1% acetic acid-methanol solution to dissolve it and prepare a 1 mmol / L internal standard stock solution. Accurately weigh an appropriate amount of the above internal standard stock solution, dilute it with 1% acetic acid-methanol solution to a concentration of 1 μmol / L (internal standard working solution A) and 10 μmol / L (internal standard working solution B), which are used as internal standard working solutions for brain tissue homogenate matrix and plasma matrix, respectively.

5. A method for detecting small molecule phenolic substances according to claim 1, characterized in that The sample in step S2 includes a sample pre-treatment of a brain tissue homogenate matrix or a sample pre-treatment of a plasma matrix.

6. A method for detecting small molecule phenolic substances according to claim 5, characterized in that The sample pretreatment of the brain tissue homogenate matrix refers to: adding a corresponding volume of 1% acetic acid-methanol solution to the brain tissue according to its own weight and then homogenizing to obtain 100 mg / mL brain tissue homogenate; after centrifugation at 4°C and 12000rpm for 10 minutes, accurately aspirating 100 μL of the brain homogenate supernatant, adding 100 μL of internal standard working solution A, vortex mixing for 1 minute, and blowing dry with N2; adding 350 μL of 4mmol / L dansyl chloride-acetonitrile solution and 350 μL of 0.1mol / L, pH=11 Na2CO3-NaHCO3 buffer solution to the residual dry matter, vortex mixing for 1 minute, and then heating in an 80°C metal bath for derivatization for 20 minutes; finally, adding 300 μL of 15% formic acid-water solution to the mixed derivatization system to adjust the pH, vortex mixing for 1 minute, centrifuging at 4°C and 12000rpm for 10 minutes, and then aspirating the supernatant for UPLC-QqQ-MS / MS injection analysis, and all operations must be performed under light-proof conditions; The sample pretreatment of the plasma matrix refers to: adding an appropriate amount of 1% acetic acid-methanol solution to the plasma to obtain undiluted and 100-fold diluted plasma samples; after centrifugation at 4°C and 12000rpm for 10 minutes, accurately aspirating 100 μL of the plasma supernatant, adding 100 μL of the internal standard working solution B, vortex mixing for 1 minute, and blowing dry with N2; adding 350 μL of 4mmol / L dansyl chloride-acetonitrile solution and 350 μL of 0.1mol / L, pH=11 Na2CO3-NaHCO3 buffer solution to the residual dry matter, vortex mixing for 1 minute, and then heating and derivatizing in an 80°C metal bath for 20 minutes; finally, adding 300 μL of 15% formic acid-water solution to the mixed derivatization system to adjust the pH, vortex mixing for 1 minute, and centrifuging at 4°C and 12000rpm for 10 minutes, and then aspirating the supernatant for UPLC-QqQ-MS / MS injection analysis; all operations must be performed under light-proof conditions.

7. A method for detecting small molecule phenolic substances according to claim 1, characterized in that The chromatographic conditions optimized in step S3 include chromatographic columns (ACQUITY UPLC BEH C18, ACQUITY UPLC HSS T3, ACQUITY UPLCBEH HILIC and ACQUITY UPLC BEH Amide), mobile phases (methanol-water, acetonitrile-water), elution gradients of mobile phases (9 min, 13 min, 18 min, 20 min), mobile phase additives (formic acid, acetic acid, ammonium formate, ammonium acetate), volume fractions of mobile phase additives (0.1% formic acid, 0.01% acetic acid, 5 mM ammonium formate, 5 mM ammonium acetate), flow rate (0.2-0.5 mL / min), and column temperature (25-35 ° C). The optimized mass spectrometry conditions included 31 ESMPs and internal standard CA for quantitative analysis in MRM mode monitoring ion pairs, cone voltage (2-70V), and collision voltage (10-50V).

8. A method for detecting small molecule phenolic substances according to claim 7, characterized in that The optimal chromatographic conditions are: using ACQUITY UPLC HSS T3 chromatographic column (2.1×100mm, 1.8μm); flow rate: 0.3mL / min; column temperature: 35°C; injection volume: 2μL; mobile phase is 0.1% formic acid aqueous solution (A)-acetonitrile (B). Gradient elution is used, and the elution conditions are 0-0.5min, 40%-55% B; 0.5-5.5min, 55%-70% B; 5.5~6min, 70%~83%B; 6~7.5min, 83%~95%B; 7.5~8.5min, 95%B; 8.5~9min, 95%~40%B; The optimal mass spectrometry conditions were as follows: using a Waters UPLC-Xevo TQ-S micro triple quadrupole liquid-mass spectrometry system, an electrospray ion source (ESI source), positive ion mode scanning, multiple reaction monitoring mode (MRM) detection, collision gas argon (Ar), purge gas nitrogen (N2), capillary voltage 3kV, ion source temperature 150°C, desolvation temperature 350°C, and desolvation flow rate 650L / h. In order to avoid contamination of the ion source, the first 1min of mobile phase directly entered the waste liquid; 31 ESMPs and internal standard CA were used for quantitative analysis of MRM mode monitoring ion pairs using corresponding parameters.

9. A method for detecting small molecule phenolic substances according to claim 8, characterized in that The corresponding parameters are: (1) The retention time of MHPG is 2.47 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 23 NO6S, parent ion m / z 418.8590, product ion m / z 171.3964, cone voltage 4V, collision voltage 28V; (2) The retention time of VMA is 2.50 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO7S, parent ion m / z 431.8165, product ion m / z 170.9891, cone voltage 66V, collision voltage 26V; (3) The retention time of HDPL is 2.89 min, the number of dansyl groups is 1, the molecular formula is C21H21NO6S, the parent ion m / z is 415.8216, the product ion m / z is 170.9874, the cone voltage is 38 V, and the collision voltage is 30 V; (4) The retention time of HPPA is 3.04 and 4.10 min, the number of dansyl groups is 1, the molecular formula is C21H19NO6S, the parent ion m / z is 413.8059, the product ion m / z is 170.9258, the cone voltage is 32 V, and the collision voltage is 28 V; (5) The retention time of HPAT is 3.52 minutes, the number of dansyl groups is 1, and the molecular formula is C 20 H 19 NO5S, parent ion m / z 385.7472, product ion m / z 170.0688, cone voltage 6V, collision voltage 28V; (6) The retention time of HVA is 3.52 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO6S, parent ion m / z 415.8216, product ion m / z 170.9856, cone voltage 46V, collision voltage 22V; (7) The retention time of 3-HPAA is 3.60 minutes, the number of dansyl groups is 1, and the molecular formula is C 20 H 19 NO5S, parent ion m / z 386.8966, product ion m / z 171.3098, cone voltage 28V, collision voltage 22V; (8) The retention time of HPAA is 3.63 minutes, the number of dansyl groups is 1, and the molecular formula is C 20 H 19 NO5S, parent ion m / z 385.7472, product ion m / z 171.0062, cone voltage 8V, collision voltage 22V; (9) The retention time of 3-HBA is 3.81 minutes, the number of dansyl groups is 1, and the molecular formula is C 19 H 17 NO5S, parent ion m / z 372.0508, product ion m / z 171.0125, cone voltage 70V, collision voltage 28V; (10) The retention time of TYR is 3.85 minutes, the number of dansyl groups is 1, and the molecular formula is C 20 H 21 NO4S, parent ion m / z 371.9595, product ion m / z 171.0015, cone voltage 34V, collision voltage 24V; (11) The retention time of HCM is 3.90 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO5S, parent ion m / z 398.0665, product ion m / z 170.0973, cone voltage 60V, collision voltage 14V; (12) The retention time of HBA is 3.92 minutes, the number of dansyl groups is 1, and the molecular formula is C 19 H 17 NO5S, parent ion m / z 371.7954, product ion m / z 170.9853, cone voltage 38V, collision voltage 26V; (13) The retention time of HPPT is 4.02 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO5S, parent ion m / z 400.6568, product ion m / z 171.0337, cone voltage 10 V, collision voltage 37 V; (14) The retention time of 3,4-HPHPA is 4.04 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO5S, parent ion m / z 399.8267, product ion m / z 170.9957, cone voltage 34V, collision voltage 28V; (15) The retention time of 3-HCM is 4.16 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO5S, parent ion m / z 398.0026, product ion m / z 170.8724, cone voltage 30V, collision voltage 26V; (16) The retention time of THCM is 4.16 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO5S, parent ion m / z 398.0665, product ion m / z 170.9413, cone voltage 30V, collision voltage 28V; (17) The retention time of PHCA is 4.23 minutes, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO5S, parent ion m / z 398.0026, product ion m / z 170.9855, cone voltage 38V, collision voltage 20V; (18) The retention time of PCA is 4.22 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 19 NO5S, parent ion m / z 398.0026, product ion m / z 170.9872, cone voltage 36V, collision voltage 24V; (19) The retention time of 2,4-HPHPA is 4.27 min, the number of dansyl groups is 1, and the molecular formula is C 21 H 21 NO5S, parent ion m / z 399.9544, product ion m / z 170.9985, cone voltage 38V, collision voltage 22V; (20) The retention time of HMBH is 5.55 min, the number of dansyl groups is 1, and the molecular formula is C 20 H 19 NO5S, parent ion m / z 385.7472, product ion m / z 170.0688, cone voltage 6V, collision voltage 24V; (21) The retention time of DOMA is 5.71 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 30 N2O9S2, parent ion m / z 650.9796, product ion m / z 170.0444, cone voltage 2V, collision voltage 20V; (22) The retention time of DHPG is 5.85 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 32 N2O8S2, parent ion m / z 635.6592, product ion m / z 170.0748, cone voltage 62V, collision voltage 40V; (23) The retention time of DOPAC is 6.90 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 30 N2O8S2, parent ion m / z 635.8149, product ion m / z 170.8625, cone voltage 60V, collision voltage 48V; (24) The retention time of DHPP is 7.07 min, the number of dansyl groups is 2, and the molecular formula is C 33 H 32 N2O8S2, parent ion m / z 648.8726, product ion m / z 171.2429, cone voltage 50V, collision voltage 48V; (25) The retention time of DOPET is 7.17 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 32 N2O7S2, parent ion m / z 620.9416, product ion m / z 169.7701, cone voltage 24V, collision voltage 36V; (26) The retention time of 3,4-DHBA is 7.18 min, the number of dansyl groups is 2, and the molecular formula is C 31 H 28 N2O8S2, parent ion m / z 620.9690, product ion m / z 170.3245, cone voltage 50V, collision voltage 44V; (27) The retention time of DHBA is 7.18 min, the number of dansyl groups is 2, and the molecular formula is C 31 H 28 N2O8S2, parent ion m / z 620.9690, product ion m / z 170.9897, cone voltage 50V, collision voltage 40V; (28) The retention time of CA is 7.23 min, the number of dansyl groups is 2, and the molecular formula is C 33 H 30 N2O8S2, parent ion m / z 647.2402, product ion m / z 170.2648, cone voltage 56V, collision voltage 40V; (29) The retention time of HGA is 7.26 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 30 N2O8S2, parent ion m / z 635.8149, product ion m / z 170.0015, cone voltage 64V, collision voltage 40V; (30) The retention time of DOPAL is 7.67 min, the number of dansyl groups is 2, and the molecular formula is C 32 H 30 N2O7S2, parent ion m / z 618.9808, product ion m / z 170.0973, cone voltage 32V, collision voltage 10V; (31) The retention time of 3,4-DHBAD is 8.03 min, the number of dansyl groups is 2, and the molecular formula is C 31 H 28 N2O7S2, parent ion m / z 604.7825, product ion m / z 170.0461, cone voltage 52V, collision voltage 40V; (32) The retention time of DHBD is 8.47 min, the number of dansyl groups is 2, and the molecular formula is C 31 H 28 N2O7S2, parent ion m / z is 604.7825, product ion m / z is 169.9528, cone voltage is 48V, collision voltage is 36V.

10. Use of a small molecule phenolic substance detection method according to any one of claims 1 to 9 in developing targets or drugs for mental illnesses, wherein the mental illnesses are anxiety, depression, and sleep disorders.

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