Detection method for targeted determination of neurotransmitter and metabolite thereof

Abstract

The invention relates to the technical field of detection of endogenous substances in organisms, in particular to a detection method for targeted determination of neurotransmitters and metabolites thereof. According to the detection method for targeted determination of the neurotransmitters and the metabolites thereof, the amine neurotransmitters and the phenol neurotransmitters are subjected to derivatization reaction to obtain stable derivatization products, then derivatization parts and non-derivatization parts are mixed, global analysis of neurotransmitter metabolite groups can be achieved, and the analysis method is reliable and has precision, accuracy and stability.

Description

Technical Field

[0001] The present invention relates to the technical field of detection of endogenous substances in organisms, and particularly relates to a detection method for targeted determination of neurotransmitters and their metabolites. Background Art

[0002] Neurotransmitters (NT) are chemical substances that act as messengers released from the presynaptic membrane. By binding to specific receptors, they mediate the excitatory or inhibitory functions of neurons and play important roles in aspects such as movement, emotion, and immune response. The neurotransmitters that complete the signal transmission task will be metabolized or re-uptaken into synaptic vesicles to participate in the next release. Each link of this process occurs in a strict temporal and spatial order, and neurotransmitter metabolism disorders can lead to the occurrence of various neuropsychiatric diseases. Existing studies have shown that dopamine metabolism disorders can lead to phenylketonuria and Parkinson's disease; the level of 5-hydroxytryptamine is closely related to depression; GABA (γ-aminobutyric acid) has functions such as anti-anxiety, anti-depression, and anti-epilepsy. It can be known from the Chinese invention patent with the application number 202010658243.X that targeted metabolic analysis of the NT metabolome can be used as a clinical indicator for the diagnosis and treatment of neuropsychiatric diseases, for predicting outcomes in clinical studies, and providing insights into the pathophysiology of the disease process.

[0003] The NT metabolome is mainly composed of small molecule metabolites, including amino acids, cholines, amines, and their metabolites. Due to the large differences in the physicochemical properties and concentration levels of the NT metabolome, and it is easily interfered by matrix molecules or co-eluted compounds and ion suppression in the ESI (electrospray ionization source) mode. There are research reports that pre-column derivatization can effectively improve the detection sensitivity of metabolites such as amino acids. From the perspective of chemical structure, chemical derivatization methods based on LC-MS / MS (liquid chromatography-tandem mass spectrometry) can detect amine (primary amine, secondary amine) and phenolic NT. However, due to the diverse chemical groups of compounds in the neurotransmitter metabolome, there is currently a lack of a universal labeling reagent, and it is difficult to achieve global analysis of the neurotransmitter metabolome based on MS (mass spectrometry) technology. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: to provide a detection method for targeted determination of neurotransmitters and their metabolites.

[0005] To solve the above technical problem, the technical solution adopted by the present invention is: a detection method for targeted determination of neurotransmitters and their metabolites, comprising the following steps: S1. Dissolve 2-chloro-L-phenylalanine in an acetonitrile solution, and then divide it into two portions. One portion serves as the non-derivatized internal standard solution, and dansyl chloride solution is added to the other portion for derivatization reaction, followed by re-dissolving with an acetonitrile solution to obtain the derivatized internal standard solution. Mix the non-derivatized internal standard solution and the derivatized internal standard solution, and dilute with an acetonitrile solution to obtain the internal standard solution. Divide the blank matrix diluent into two portions. One portion serves as the derivatized test solution, and the other portion serves as the non-derivatized test solution. Add dansyl chloride solution to the derivatized test solution for derivatization reaction to obtain the derivatized portion. Add the non-derivatized test solution to the derivatized portion, dry it under nitrogen, add the internal standard solution for re-dissolving, and take the supernatant after centrifugation to obtain the quality control sample. S2. Treat the pretreated animal sample using the treatment method of the blank matrix diluent in S1 to obtain the test solution. S3. Perform UHPLC-MS / MS analysis on the internal standard solution, the quality control sample, and the test solution respectively, establish the matrix standard curve, and calculate the content of neurotransmitters and their metabolites in the biological sample.

[0006] The beneficial effects of the present invention are as follows: The detection method for targeted determination of neurotransmitters and their metabolites in the present invention performs derivatization reaction on amine and phenolic neurotransmitters to obtain stable derivatized products, and then mixes the derivatized portion and the non-derivatized portion, which can achieve the global analysis of the neurotransmitter metabolome. The analysis method is reliable and has precision, accuracy, and stability. Description of the Drawings

[0007] Figure 1 The mass spectrometry chromatogram of the blank matrix in Example 1 of the present invention is shown. Figure 2 The mass spectrometry chromatogram of the quality control sample in Example 1 of the present invention is shown. Figure 3 The mass spectrometry chromatogram of the test solution in Example 1 of the present invention is shown. Description of reference numerals: 1. Choline; 2. Quinolinic acid; 3. Kynurenic acid; 4. N - arachidonoylethanolamine; 5. 2 - arachidonoylglycerol; 6. Arachidonic acid; 7. Histidine; 8. Arginine; 9. Histamine; 10. 1 - Methylhistamine; 11. Glutamine; 12. Taurine; 13. Serine; 14. Aspartic acid; 15. Glutamic acid; 16. Glycine; 17. γ - Aminobutyric acid; 18. Kynurenine; 19. Tryptophan; 20. 3 - Methoxy - 4 - hydroxyphenylethylene glycol; 21. Phenylalanine; 22. 5 - Hydroxyindole - 3 - acetic acid; 23. N - Acetyl - 5 - hydroxytryptamine; 24. Homovanillic acid; 25. 3 - Hydroxy - 2 - aminobenzoic acid; 26. Tryptamine; 27. Tyrosine; 28. 3,4 - Dihydroxyphenylacetic acid; 29. 5 - Hydroxytryptamine; 30. 3 - Methoxytyramine; 31. Tyramine; 32. Norepinephrine; 33. Dopamine. Detailed implementation mode

[0008] To describe in detail the technical content, achieved objectives and effects of the present invention, the following is described in conjunction with the implementation modes and with reference to the accompanying drawings.

[0009] A detection method for targeted determination of neurotransmitters and their metabolites, comprising the following steps: S1. Dissolve 2 - chloro - L - phenylalanine in an acetonitrile solution, and then divide it into two portions. One portion is used as a non - derivatized internal standard solution, and dansyl chloride solution is added to the other portion for derivatization reaction and then re - dissolved with an acetonitrile solution to obtain a derivatized internal standard solution; Mix the non - derivatized internal standard solution and the derivatized internal standard solution, and dilute with an acetonitrile solution to obtain an internal standard solution; Divide the blank matrix dilution solution into two portions. One portion is used as a derivatized test solution, and the other portion is used as a non - derivatized test solution; Add dansyl chloride solution to the derivatized test solution for derivatization reaction to obtain a derivatized part; Add the non - derivatized test solution to the derivatized part, dry it with nitrogen, add the internal standard solution for re - dissolution, and take the supernatant after centrifugation to obtain a quality control sample; S2. Process the pretreated animal sample using the treatment method of the blank matrix dilution solution in S1 to obtain a test solution; S3. Perform UHPLC - MS / MS analysis on the internal standard solution, quality control sample, and test solution respectively, establish a matrix standard curve, and calculate the contents of neurotransmitters and their metabolites in the biological sample.

[0010] As can be seen from the above description, the beneficial effects of the present invention are as follows: The chemical groups of the compounds in the neurotransmitter metabolome are diverse, and there is currently a lack of a universal labeling reagent. In response to this, the present invention proposes a dual pretreatment method that combines derivatization and non-derivatization. Dansyl chloride is used to label amine (primary amine, secondary amine) and phenolic neurotransmitters, effectively improving the detection sensitivity of amine and phenolic neurotransmitters. At the same time, in order to specifically analyze other compounds such as tertiary amines and choline that cannot be labeled by dansyl chloride, the unlabeled aliquot is mixed with the dansyl chloride derivatization product to achieve the simultaneous determination of various structural types of compounds in the neurotransmitter metabolome. The derivatized internal standard and the derivatized analyte solution, as well as the non-derivatized internal standard and the non-derivatized analyte solution, each have the same key chemical structure and functional groups between them, which can avoid the differences in ionization efficiency and extraction recovery rate between the analyte and the internal standard due to structural differences, reduce the influence of the signal fluctuation of the mass spectrometry instrument on the detection result, and further improve the accuracy of the detection result.

[0011] Further, the preparation method of the blank matrix diluent includes the following steps: Pretreat the animal sample to obtain a blank matrix, and dilute the blank matrix with the reference substance stock solution to obtain the blank matrix diluent.

[0012] Further, the animal sample is a brain tissue.

[0013] Further, the preparation method of the blank matrix includes the following steps: Step 1: Mix at least two biological samples in equal mass, add a solvent and grind, vortex and centrifuge in sequence, then take the supernatant and dry it with nitrogen to obtain a primary freeze-dried powder; Step 2: Repeat the grinding to drying steps in Step 1 for the primary freeze-dried powder at least twice to obtain a matrix freeze-dried powder; Step 3: Re-dissolve the matrix freeze-dried powder with an acetonitrile solution to obtain a blank matrix.

[0014] Further, the preparation method of the blank matrix includes the following steps: Weigh 30 mg of brain tissue from each of 6 mice for mixing, add 1 - 5 mL of physiological saline pre-cooled to 4 °C for grinding, then add 3 - 15 mL of acetonitrile pre-cooled to 4 °C, and centrifuge at 12000 rpm and 4 °C for 5 - 15 min; After centrifugation, take the supernatant and add physiological saline for homogenization, repeat the grinding to drying steps 3 times to obtain a matrix freeze-dried powder; Add 1 - 5 mL of 50% v / v acetonitrile to the matrix freeze-dried powder for re-dissolution to obtain the blank matrix.

[0015] As can be seen from the above description, the blank matrix can be extracted more efficiently by using the preparation method of the present invention.

[0016] Further, the preparation method of the reference stock solution comprises the following steps: Dissolve the standards of neurotransmitters and their metabolites in an acetonitrile solution to obtain the reference stock solution.

[0017] Further, S2 comprises the following steps: Divide the pretreated animal samples into two parts, one part as the derivatized test solution and the other part as the non-derivatized test solution; Add dansyl chloride solution to the derivatized test solution for derivatization reaction to obtain the derivatized part; Add the non-derivatized test solution to the derivatized part, dry it under nitrogen, re-dissolve it with the internal standard solution, and take the supernatant after centrifugation to obtain the test sample solution.

[0018] Further, the pretreatment in S2 comprises the following steps: Add the animal sample to precooled physiological saline for grinding, then add acetonitrile solution and perform vortex and centrifugation in sequence, and take the supernatant.

[0019] Further, the derivatization reaction in S1 comprises the following steps: Add 50% v / v acetonitrile solution and sodium carbonate / sodium bicarbonate buffer to the reactants for mixing, then add dansyl chloride solution for mixing and incubation. After incubation, add sodium hydroxide solution and formic acid solution in sequence, then add ethyl acetate for extraction, and evaporate the extracted product to dryness under nitrogen.

[0020] As can be seen from the above description, sodium hydroxide is added in the derivatization reaction to quench the excessive dansyl chloride, and formic acid solution is added to neutralize the excessive sodium hydroxide and make the mixture acidic.

[0021] Further, the volume ratio of the reactants, 50% v / v acetonitrile solution and sodium carbonate / sodium bicarbonate buffer is: 1 - 3:1 - 3:10 - 30.

[0022] Further, the incubation temperature is 45 - 60 °C.

[0023] Further, the derivatization reaction in S1 comprises the following steps: Take 10 - 30 μL of the reactants, add 10 - 30 μL of 50% v / v acetonitrile solution and 100 - 300 μL of sodium carbonate / sodium bicarbonate buffer, mix well, then add 100 - 300 μL of freshly prepared dansyl chloride solution, vortex and mix well. Incubate in a water bath at 45 - 60 °C for 50 - 100 min, then add 50 - 100 μL of sodium hydroxide solution, and incubate in a water bath at 45 - 60 °C for 5 - 15 min; Then add 50 - 100 μL of 10% v / v formic acid, add twice the volume of ethyl acetate for extraction, and evaporate the extracted product to dryness under nitrogen.

[0024] As can be seen from the above description, the dansyl chloride derivatization method of the present invention can make amines (primary amines, secondary amines) and phenolic neurotransmitters react to form stable derivatized products, and the derivatized products have good responses in mass spectrometry, ensuring the smooth progress of detection.

[0025] Further, the particle size of the packing material of the chromatographic column in the UHPLC-MS / MS analysis of S3 is less than 5 μm.

[0026] As can be seen from the above description, the chromatographic column selected in the present invention effectively increases the chromatographic retention and separation effect of the analyte.

[0027] Further, in the UHPLC-MS / MS analysis of S3, an aqueous solution of ammonium formate containing 0.1 wt% v / v formic acid and 10 mM ammonium formate is used as mobile phase A, and acetonitrile is used as mobile phase B.

[0028] As can be seen from the above description, the mobile phase selected in the present invention not only helps to separate isomers but also gives better peak shapes to each component.

[0029] Example 1 of the present invention is: a detection method for targeted determination of neurotransmitters and their metabolites, and the specific steps are as follows: 1. Instruments and reagents 1.1. Experimental reagents Choline (product number: E38650) was purchased from Shanghai Macklin Biochemical Co., Ltd. N-Arachidonoylethanolamine (product number: A274932) was purchased from Shanghai Merck Chemical Technology Co., Ltd. Quinolinic acid (product number: P71030), Kynurenic acid (product number: K94411), 2-Arachidonoylglycerol (product number: A47350), Arachidonic acid (product number: Z96181), 2-Chloro-L-phenylalanine (product number: C63461), Histidine (product number: L88031), Arginine (product number: L24551), Histamine (product number: H99632), Glutamine (product number: L51431), Taurine (product number: T58631), Serine (product number: L10371), Aspartic acid (product number: L53401), Glutamic acid (product number: L65861), Glycine (product number: G12762), γ-Aminobutyric acid (product number: 56-12-2), Kynurenine (product number: D75970), Tryptophan (product number: L65231), Phenylalanine (product number: L42551), 5-Hydroxyindole-3-acetic acid (product number: H35450), Homovanillic acid (product number: H20310), N-Acetyl-5-hydroxytryptamine (product number: A41636018), 3-Hydroxy-2-aminobenzoic acid (product number: A91436017), Tryptamine (product number: T46251), Tyrosine (product number: L91721), 3,4-Dihydroxyphenylacetic acid (product number: D26390), 5-Hydroxytryptamine (product number: H69440), Tyramine (product number: 51-67-2), Norepinephrine (product number: R30850), Dopamine (product number: A54861), Dansyl chloride (product number: D47040) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 1-Methylhistamine (product number: M11468) was purchased from BOC Sciences, USA. 3-Methoxy-4-hydroxyphenylethylene glycol (product number: H946340) was purchased from Toronto Research Chemicals, Canada. 3-Methoxytyramine (product number: A917315) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Methanol, formic acid, ammonium formate (all of mass spectrometry grade) were purchased from Merck, Germany. Ultrapure water was prepared in the laboratory.

[0030] 1.2, Experimental instruments LCMS-8045 liquid-mass spectrometer (Shimadzu, Japan), KB-3 vortex mixer (Qilin Bell Instrument Co., Ltd., Haimen City), H1650R desktop refrigerated small high-speed centrifuge (Hunan Xiangyi Experimental Development Co., Ltd.), LUKYM-I sample freezing grinder (Guangzhou LUKA Sequencing Instrument Co., Ltd.), HH.S21-6 digital display constant temperature water bath (Shanghai Boxun Industrial Co., Ltd.), EP 225SM-DR electronic analytical balance (Plissex, Switzerland), KQ5200DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), dry nitrogen blower (Shanghai Lichen Bangsi Instrument Technology).

[0031] 1.3 Brain tissue samples Six healthy male ICR mice weighing 18-22 g were selected for this study and provided by Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd. The brain tissue of each mouse was collected in EP tubes and placed in a -80°C refrigerator for later use.

[0032] 2. Experimental methods 2.1. Liquid phase conditions: Chromatographic column: Thermo Scientific™ Hypersil GOLD™ C18 Column (2.1×100 mm, 1.9 μm); mobile phase A: water (containing 0.1% v / v formic acid and 10 mM ammonium formate), B: acetonitrile; gradient elution: 0~2.0 min, 10% v / v B; 2.0~4.0 min, 10% v / v~30% v / v B; 4.0~10.0 min, 30% v / v~80% v / v B; 10.0~15.0 min, 80% v / v B; 15.0~16.0 min, 80% v / v~10% v / v B; 16.0~26.0 min, 10% v / v B; column temperature 55℃; flow rate 0.2 mL / min; injection volume 2 μL.

[0033] 2.2. Mass spectrometry conditions: The triple quadrupole mass spectrometer was equipped with an ESI source and multiple reaction monitoring (MRM) detection was used in positive and negative ion modes. The optimized mass spectrometry parameters were as follows: interface temperature: 300°C; DL temperature: 250°C; heating block temperature: 400°C; nebulizer gas flow rate: 3 L / min; drying gas flow rate: 10 L / min; heating gas flow rate: 10 L / min. The monitoring information of neurotransmitters and their metabolites is shown in Table 1.

[0034] Table 1

[0035] 2.3 Sample preparation 2.3.1. Configure each derivatization reagent according to Table 2.

[0036] Table 2

[0037] 2.3.2. Preparation of reference substance stock solution Take appropriate amounts of choline, quinolinic acid, kynurenic acid, N - arachidonoylethanolamine, 2 - arachidonyl glycerol, arachidonic acid, histidine, arginine, histamine, 1 - methylhistamine, glutamine, taurine, serine, aspartic acid, glutamic acid, glycine, γ - aminobutyric acid, kynurenine, tryptophan, 3 - methoxy - 4 - hydroxyphenylethylene glycol, phenylalanine, 5 - hydroxyindole - 3 - acetic acid, N - acetyl - 5 - hydroxytryptamine, homovanillic acid, 3 - hydroxy - 2 - aminobenzoic acid, tryptamine, tyrosine, 3,4 - dihydroxyphenylacetic acid, 5 - hydroxytryptamine, 3 - methoxytyramine, tyramine, norepinephrine, dopamine and dissolve them with 50% v / v acetonitrile to prepare a reference substance solution containing 1 g of choline, 1 g of N - arachidonoylethanolamine, 1 g of 2 - arachidonyl glycerol, 1 g of arachidonic acid, 1 g of histidine, 1 g of arginine, 1 g of histamine, 1 g of 1 - methylhistamine, 1 g of glutamine, 1 g of taurine, 1 g of serine, 0.4 g of aspartic acid, 1 g of glutamic acid, 1 g of glycine, 1 g of γ - aminobutyric acid, 0.5 g of kynurenine, 1 g of tryptophan, 1 g of 3 - methoxy - 4 - hydroxyphenylethylene glycol, 1 g of phenylalanine, 1 g of 5 - hydroxyindole - 3 - acetic acid, 1 g of homovanillic acid, 1 g of tryptamine, 0.4 g of tyrosine, 1 g of 3,4 - dihydroxyphenylacetic acid, 1 g of 5 - hydroxytryptamine, 1 g of 3 - methoxytyramine, 1 g of tyramine, 1 g of norepinephrine, 1 g of dopamine in 1 L of 50% v / v acetonitrile. Store it in a refrigerator at 4°C.

[0038] 2.3.3. Preparation of internal standard solution Take an appropriate amount of 2-chloro-L-phenylalanine and dissolve it in 50% v / v acetonitrile to prepare a solution containing 1 g of 2-chloro-L-phenylalanine per 1 L. Then divide it into two portions. One portion is used as the non-derivatized internal standard solution and stored in a refrigerator at 4 °C for later use. Another 25 μL of the 2-chloro-L-phenylalanine solution is transferred to a clean EP tube, add an equal volume of 50% v / v acetonitrile, add 100 μL of sodium carbonate / sodium bicarbonate buffer (pH = 9.4), add 100 μL of freshly prepared dansyl chloride solution (18 mg / mL), vortex for 20 s, and react at 60 °C for 1 h. After 1 h, add 60 μL of sodium hydroxide solution (20 mg / mL) to the mixture and incubate at 60 °C for another 10 min to quench the excess dansyl chloride. Then add 60 μL of 10% v / v formic acid to neutralize the excess sodium hydroxide and make the mixture acidic. Add twice the volume of ethyl acetate to the mixture to extract the dansyl chloride-labeled 2-chloro-L-phenylalanine and evaporate to dryness under nitrogen. Add 25 μL of 50% v / v acetonitrile to redissolve, centrifuge at 13000 rpm for 10 min at 4 °C, take the supernatant, and obtain the derivatized internal standard solution, which is stored in a refrigerator at 4 °C for later use. Take appropriate amounts of the non-derivatized internal standard solution and the derivatized internal standard solution, dilute with 30% v / v acetonitrile solution to prepare an internal standard solution containing 1 mg of non-derivatized internal standard and 2 mg of derivatized internal standard per 1 L.

[0039] 2.3.4 Preparation of Test Solution Precisely weigh 50 mg of mouse brain tissue, add 200 μL of physiological saline pre-cooled to 4 °C and homogenize. Subsequently, add 600 μL of acetonitrile pre-cooled to 4 °C to it. The mixture is vortexed for 2 min, ultrasonically extracted for 10 min, and centrifuged at 15000 rpm for 10 min. After centrifugation, take an equal amount of the supernatant and dry it under nitrogen. Add 100 μL of 50% v / v acetonitrile, vortex for 1 min, and ultrasonically dissolve for 5 min to obtain the pretreated product.

[0040] Take 25 μL of the pretreated product, add an equal volume of 50% v / v acetonitrile, add 100 μL of sodium carbonate / sodium bicarbonate buffer (pH = 9.4), add 100 μL of freshly prepared dansyl chloride solution (18 mg / mL), vortex for 20 s, and react at 60 °C for 1 h. After 1 h, add 60 μL of sodium hydroxide solution (20 mg / mL) to the mixture and incubate at 60 °C for another 10 min to quench the excess dansyl chloride. Then add 60 μL of 10% v / v formic acid to neutralize the excess sodium hydroxide and make the mixture acidic. Add twice the volume of ethyl acetate to the mixture to extract the dansyl chloride-labeled metabolite and evaporate to dryness under nitrogen. Then add 40 μL of the non-derivatized test solution and dry it under nitrogen. Add 80 μL of the internal standard solution, vortex for 1 min, ultrasonically dissolve for 5 min, centrifuge at 13000 rpm for 10 min at 4 °C, take the supernatant, and prepare the test solution.

[0041] 2.3.5 Preparation of Quality Control Samples Precisely weigh 50 mg of brain tissue from each mouse, mix them, add 1.2 mL of physiological saline pre-cooled to 4 °C for homogenization, then add 3.6 mL of acetonitrile pre-cooled to 4 °C to the mixture. Vortex the mixture for 2 min, perform ultrasonic extraction for 10 min, centrifuge at 15000 rpm for 10 min, transfer the supernatant, dry it under nitrogen, add physiological saline for homogenization, and perform the extraction three times using the same treatment method as above. Take the supernatant of the last extraction, dry it under nitrogen to obtain the matrix freeze-dried powder. Reconstitute the matrix freeze-dried powder with 50% v / v acetonitrile to obtain the blank matrix, and store it at 4 °C for later use.

[0042] Take the blank matrix and serially dilute it with the reference stock solution to prepare blank matrix dilutions with different concentrations in the range of 0.001 - 200 μg·mL -1 within the range.

[0043] Take 25 μL of blank matrix dilutions with different concentrations respectively. One portion is used as the derivatized sample to be measured; the other portion is added with an equal volume of 50% v / v acetonitrile, 100 μL of sodium carbonate / sodium bicarbonate buffer solution (pH = 9.4), 100 μL of freshly prepared dansyl chloride solution (18 mg / mL), vortex for 20 s, and react at 60 °C for 1 h. The chemical reaction process is shown in Equation 1. After 1 h, add 60 μL of sodium hydroxide solution (20 mg / mL) to the mixture, and incubate at 60 °C for another 10 min to quench the excess dansyl chloride. Then add 60 μL of 10% v / v formic acid to neutralize the excess sodium hydroxide and make the mixture acidic. Add twice the volume of ethyl acetate to the mixture to extract the dansyl chloride-labeled metabolites and evaporate to dryness under nitrogen. Then add 40 μL of the non-derivatized sample to be measured and dry it under nitrogen. Add 80 µL of the internal standard solution, vortex for 1 min, reconstitute by ultrasonic treatment for 5 min, centrifuge at 13000 rpm for 10 min at 4 °C, take the supernatant, and prepare quality control samples with different concentrations.

[0044] (Equation 1).

[0045] 2.4 UHPLC-MS / MS Analysis 2.4.1 Linear Relationship 2.4.1.1 Preparation of Matrix Standard Curve Analyze quality control samples with different concentrations on the instrument to obtain the matrix standard curve. Among them, the dilution concentration points of the test solution are consistent with those of the reference solution.

[0046] 2.4.1.2 Plotting of Matrix Standard Curve The matrix standard curve was drawn by the internal standard method, and the ion pairs monitored by the analytes in the mass spectrometry are shown in Table 1. Taking the mass concentration (x, ng / mL) of the reference substance as the abscissa and the ratio (y) of the peak area of each reference substance to the peak area of the internal standard as the ordinate, the standard curve was drawn and linear regression was performed by the least square method to obtain the corresponding linear regression equation. The results showed that the linear relationships of each component were good within the corresponding linear ranges. At the same time, the injection concentration with a signal-to-noise ratio of 10 times (S / N = 10) of the peak area in the matrix standard curve was used as the lower limit of quantification. The results are shown in Table 3, and Table 3 is the inspection result table of the linear range and lower limit of quantification of the matrix standard curve.

[0047] Table 3

[0048] 2.4.2 Results of sample determination The test solutions of 6 mice were analyzed on the machine respectively. The respective area ratios were substituted into the matrix standard curve for calculation. The contents of neurotransmitters and their metabolites in the brain tissues of each mouse are shown in Table 4 (μg·g -1 ). The mass chromatograms of the quality control samples and test solutions are as shown in Figure 2 , Figure 3 ( Figures 1 - 3 in which IS-1 is the non-derivatized internal standard, and IS-2 is the derivatized internal standard).

[0049] Table 4

[0050] The detection examples of the present invention are as follows: 1. Investigation of blank matrix Take 25 μL of blank matrix, add an equal volume of 50% v / v acetonitrile, add 100 μL of sodium carbonate / sodium bicarbonate buffer solution (pH = 9.4), add 100 μL of freshly prepared dansyl chloride solution (18 mg / mL), vortex for 20 s, and react at 60 °C for 1 h. After 1 h, add 60 μL of sodium hydroxide solution (20 mg / mL) to the mixture and incubate at 60 °C for another 10 min to quench the excess dansyl chloride. Then add 60 μL of 10% v / v formic acid to neutralize the excess sodium hydroxide and make the mixture acidic. Add twice the volume of ethyl acetate to the mixture to extract the dansyl chloride-labeled metabolites and evaporate to dryness under nitrogen. After drying the derivatization site under nitrogen, add 40 μL of non-derivatized test solution and dry under nitrogen. Add 80 µL of internal standard solution, vortex for 1 min, redissolve by ultrasonic treatment for 5 min, centrifuge at 13000 rpm at 4 °C for 10 min, and take the supernatant for analysis on the machine. No peaks appear in the analyte channel and the internal standard channel of the blank matrix. The mass chromatogram of the blank matrix is as shown in Figure 1 as shown.

[0051] 2. Influence of the analyte on the internal standard channel Take 25 μL of the diluted control stock solution at the highest concentration point of the matrix standard curve, add an equal volume of 50% v / v acetonitrile, add 100 μL of sodium carbonate / sodium bicarbonate buffer (pH = 9.4), add 100 μL of freshly prepared dansyl chloride solution (18 mg / mL), vortex for 20 s, and react at 60 °C for 1 h. After 1 h, add 60 μL of sodium hydroxide solution (20 mg / mL) to the mixture, and incubate at 60 °C for another 10 min to quench the excess dansyl chloride. Then add 60 μL of 10% v / v formic acid to neutralize the excess sodium hydroxide and make the mixture acidic. Add twice the volume of ethyl acetate to the mixture to extract the dansyl chloride-labeled metabolites and evaporate to dryness under nitrogen. Add 40 μL of the non-derivatized test solution and dry under nitrogen. Add 80 µL of the internal standard solution, vortex for 1 min, redissolve by ultrasonic treatment for 5 min, centrifuge at 13,000 rpm at 4 °C for 10 min, and take the supernatant for instrumental analysis. The analyte does not peak in the internal standard channel, indicating that the analyte has no effect on the internal standard.

[0052] 3. Influence of the internal standard on the analyte Take the internal standard solution for instrumental analysis. The internal standard does not peak in the analyte channel, indicating that the internal standard has no effect on the analyte.

[0053] 4. Investigation of system residue After the liquid chromatography-mass spectrometry runs to the end of the highest point of the matrix standard curve, analyze the blank sample to investigate the influence of system residue. The blank sample does not peak in either the analyte channel or the internal standard channel, indicating that there is no system residue.

[0054] 5. Accuracy and precision According to the matrix standard curve range, three points of the matrix standard curve were respectively selected as the low-concentration quality control point (LQC), medium-concentration quality control point (MQC), and high-concentration quality control point (HQC). Prepare QCs with 3 concentrations using blank matrix, with 5 replicates for each concentration. Take 25 μL of the test sample, add an equal volume of 50% v / v acetonitrile, add 100 μL of sodium carbonate / sodium bicarbonate buffer (pH = 9.4), add 100 μL of freshly prepared dansyl chloride solution (18 mg / mL), vortex for 20 s, and react at 60 °C for 1 h. After 1 h, add 60 μL of sodium hydroxide solution (20 mg / mL) to the mixture and incubate at 60 °C for another 10 min to quench the excess dansyl chloride. Then add 60 μL of 10% v / v formic acid to neutralize the excess sodium hydroxide and make the mixture acidic. Add twice the volume of ethyl acetate to the mixture to extract the dansyl chloride-labeled metabolites and evaporate to dryness under nitrogen. Add 40 μL of the non-derivatized test solution and dry it under nitrogen. Add 80 μL of the internal standard solution, vortex for 1 min, redissolve by ultrasonic treatment for 5 min, centrifuge at 13000 rpm for 10 min at 4 °C, and take the supernatant for instrumental analysis. Calculate the precision and accuracy, and the results are shown in Table 5 and Table 6. Table 5 and Table 6 are the tables of the precision and accuracy test results. The RSDs of the within-day and between-day precision tests of the analytes are all less than 15% v / v, indicating good instrument precision. The relative error (Relative error, RE) is between -14.68% v / v and 13.22% v / v, indicating good instrument accuracy.

[0055] Table 5

[0056] Table 6

[0057] 6. Extraction Recovery Take a brain tissue sample with a known content. The content of each neurotransmitter and its metabolite is Cs. Weigh 3 mg precisely and add 10 μL of the internal standard solution (LQC), medium-quality control solution (MQC), and high-quality control solution (HQC) to it respectively. Mix well, then add 90 μL of 50% v / v acetonitrile and vortex for 2 min. Centrifuge at 12000 rpm at 4 °C for 10 min. Transfer 25 μL of the supernatant, add an equal volume of 50% v / v acetonitrile, add 100 μL of sodium carbonate / sodium bicarbonate buffer solution (pH = 9.4), add 100 μL of freshly prepared dansyl chloride solution (18 mg / mL), vortex for 20 s, and react at 60 °C for 1 h. After 1 h, add 60 μL of sodium hydroxide solution (20 mg / mL) to the mixture and incubate at 60 °C for another 10 min to quench the excess dansyl chloride. Then add 60 μL of 10% v / v formic acid to neutralize the excess sodium hydroxide and make the mixture acidic. Add twice the volume of ethyl acetate to the mixture to extract the dansyl chloride-labeled metabolites and evaporate to dryness under nitrogen. After drying the derivatization site under nitrogen, add 40 μL of the non-derivatized test solution and dry it under nitrogen. Add 80 μL of the internal standard solution, vortex for 1 min, dissolve it by ultrasonic treatment for 5 min, centrifuge at 13000 rpm at 4 °C for 10 min, and take the supernatant for analysis by machine. Record and calculate the content of each neurotransmitter and its metabolite as Cn, and the theoretical content as Ct.

[0058] ; The results are shown in Table 7, which is the result table of the recovery rate investigation. The extraction recovery rates of each analyte are between 85.03% v / v and 113.61% v / v, and the RSD < 15% v / v, meeting the requirements for the determination of biological samples.

[0059] Table 7

[0060] In summary, the detection method for targeted determination of neurotransmitters and their metabolites provided by the present invention has the following advantages: 1. Based on a dual pretreatment method combining derivatization and non-derivatization, the content of 33 components in the neurotransmitter metabolite group is determined under the same conditions, which can be used for targeted analysis of the changes in the content of neurotransmitters and their metabolites in biological samples with diseases. It has the characteristics of simple pre-treatment and pretreatment steps of biological samples, and provides a feasible determination method for studying the relationship between neurotransmitters and their metabolites and diseases.

[0061] 2. By adopting the dansyl chloride derivatization method, stable derivatization products are generated for amines (primary amines, secondary amines) and phenolic neurotransmitters in the neurotransmitter metabolite group, and the derivatization products have good responses in mass spectrometry.

[0062] 3. By adopting the UHPLC-MS / MS technology to improve the resolution ability of the neurotransmitter metabolome, the detection targeting, detection sensitivity and detection accuracy of neurotransmitters and their metabolites are further improved.

[0063] 4. Adjusting the specific sample pretreatment method, derivatization method and chromatographic method according to neurotransmitters and their metabolites fills the blank of the method for simultaneously determining neurotransmitters and their metabolites in biological samples.

[0064] The above are only the embodiments of the present invention, and thus do not limit the patent scope of the present invention. Any equivalent transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in the related technical field, shall be equally included in the patent protection scope of the present invention.

Claims

1. A method for detecting neurotransmitters and their metabolites, characterized in that: The steps include: S1. Dissolve 2-chloro-L-phenylalanine in acetonitrile solution, and then divide it into two parts, one part is used as a non-derivatized internal standard solution, and add dansyl chloride solution to the other part for derivatization reaction, and then use acetonitrile solution to redissolve it to prepare a derivatized internal standard solution; mix the non-derivatized internal standard solution and the derivatized internal standard solution, and dilute it with acetonitrile solution to prepare an internal standard solution; The blank matrix dilution solution was divided into two parts, one as the derivatized test solution and the other as the non-derivatized test solution; Adding dansyl chloride solution to the derivatization test solution to carry out a derivatization reaction to obtain a derivatization site; Add the non-derivatized test solution to the derivatized part, blow dry with nitrogen, add the internal standard solution to re-dissolve, centrifuge and take the supernatant to prepare the quality control sample; S2, treating the pretreated animal sample using the blank matrix diluent treatment method in S1 to prepare a test solution; S3. Perform UHPLC-MS / MS analysis on the internal standard solution, quality control sample, and test solution, respectively, establish a matrix standard curve, and calculate the content of neurotransmitters and their metabolites in the biological sample.

2. The method for detecting targeted neurotransmitters and their metabolites according to claim 1, characterized in that: The method for preparing the blank matrix diluent comprises the following steps: pre-treating an animal sample to obtain a blank matrix, and diluting the blank matrix with a reference substance stock solution to obtain the blank matrix diluent.

3. The method for detecting targeted neurotransmitters and their metabolites according to claim 2, characterized in that: The animal sample is brain tissue.

4. The method for detecting targeted neurotransmitters and their metabolites according to claim 2, characterized in that: The preparation method of the blank matrix comprises the following steps: Step 1, mixing at least two biological samples of equal mass, adding a solvent, grinding, vortexing and centrifuging in sequence, and then taking the supernatant and drying it with nitrogen to obtain a primary lyophilized powder; Step 2, repeating the step of grinding the primary freeze-dried powder to dryness in step 1 at least twice to obtain a base freeze-dried powder; Step 3: Dissolve the matrix lyophilized powder in acetonitrile solution to prepare a blank matrix.

5. The method for detecting targeted neurotransmitters and their metabolites according to claim 2, characterized in that: The method for preparing the reference substance stock solution comprises the following steps: dissolving the standard substance of the neurotransmitter and its metabolites in an acetonitrile solution to prepare the reference substance stock solution.

6. The method for detecting targeted neurotransmitters and their metabolites according to claim 1, characterized in that: The pretreatment in S2 comprises the following steps: adding pre-cooled physiological saline to the animal sample for grinding, then adding acetonitrile solution for vortexing and centrifugation in sequence, and taking the supernatant.

7. The method for detecting targeted neurotransmitters and their metabolites according to claim 1, characterized in that: The derivatization reaction in S1 comprises the following steps: adding 50% v / v acetonitrile solution and sodium carbonate / sodium bicarbonate buffer to the reactant, mixing, then adding dansyl chloride solution, mixing and incubating, adding sodium hydroxide solution and formic acid solution in sequence after the incubation, and then adding ethyl acetate for extraction, and evaporating the extracted product to dryness under nitrogen.

8. The method for detecting targeted neurotransmitters and their metabolites according to claim 7, characterized in that: The volume ratio of the reactant, 50% v / v acetonitrile solution and sodium carbonate / sodium bicarbonate buffer is: 1-3:1-3:10-30.

9. The method for detecting targeted neurotransmitters and their metabolites according to claim 7, characterized in that: The incubation temperature is 45-60°C.

10. The method for targeted determination of neurotransmitters and their metabolites according to claim 1, characterized in that: The filler particle size of the chromatographic column in the UHPLC-MS / MS analysis of S3 is less than 5 μm.

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