High-sensitivity DPJ-CE-MS method for detecting neurotransmitter and histamine metabolite, kit and application

Abstract

The invention discloses a high-sensitivity DPJ-CE-MS method for detecting neurotransmitters and histamine metabolites, a kit and application, and belongs to the technical field of life analysis and detection. According to the method, a dynamic pH linkage on-line enrichment technology is adopted, alkaline ammonia water and a sample are sequentially injected, and effective pre-concentration of various target objects is achieved in a capillary tube. The background electrolyte and sheath fluid taking formic acid-methanol as a system, and key conditions such as separation voltage, electrospray voltage and the like are optimized. The method combines the advantages of high-efficiency separation of capillary electrophoresis and high-sensitivity and high-specificity detection of mass spectrum, and simultaneous analysis of 11 trace substances such as noradrenaline, glutamic acid, gamma-aminobutyric acid, 5-hydroxytryptamine, histamine and metabolites thereof in the cerebrospinal fluid is realized. Methodology verification shows that the method has excellent sensitivity, precision and accuracy, the detection limit is as low as 0.05-5 ng / mL, and a reliable analysis tool is provided for neuroscience basic research and discovery of biomarkers of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of life analysis and detection technology, specifically relating to a highly sensitive DPJ-CE-MS method, kit, and application for detecting neurotransmitters and histamine metabolites. Background Technology

[0002] Neurotransmitters are chemical substances that transmit signals between neurons or between neurons and effectors in the nervous system. As the "chemical language" of the nervous system, neurotransmitters play a crucial role in regulating the human nervous, immune, and cardiovascular systems. Disruptions in neurotransmitter balance can trigger numerous clinical diseases, such as motor disorders, mental illnesses, and cognitive problems. The metabolic pathways of neurotransmitters permeate multiple fields, including basic neuroscience, disease mechanism analysis, drug development, and clinical diagnosis. Pathway abnormalities are a key trigger for the vast majority of neurological and psychiatric diseases. An excess of excitatory neurotransmitters (such as glutamate, acetylcholine, and norepinephrine) or a deficiency of inhibitory neurotransmitters (such as gamma-aminobutyric acid and serotonin) can lead to neuronal overactivation (e.g., epilepsy, anxiety disorders); conversely, an imbalance can result in weakened signal transmission (e.g., Parkinson's disease, depression). However, metabolic pathways also exhibit mutual constraints and synergistic effects. For example, the synthesis of serotonin depends on tryptophan hydroxylase (TPH), while tryptophan is also a substrate of the kynurenine pathway; their competitive relationship directly affects serotonin levels. The synthesis of γ-aminobutyric acid (GABA) originates from the decarboxylation of glutamate, and metabolic imbalance in GABA disrupts the excitation-inhibition balance, leading to dysfunction of neural circuits. These connections not only provide key targets for elucidating disease mechanisms but also offer important evidence for drug development, clinical diagnosis, and the discovery of disease biomarkers. Therefore, developing efficient and highly sensitive analytical detection technologies to monitor neurotransmitter metabolic pathways holds promise for driving groundbreaking progress in neuroscience.

[0003] Traditional methods for neurotransmitter detection include fluorescence, electrochemical, and immunoassay methods. However, these methods suffer from drawbacks such as poor specificity, narrow analytical range, and cumbersome operation, limiting their practical application. High-performance liquid chromatography-mass spectrometry (HPLC-MS) is currently an important method for neurotransmitter detection, offering advantages such as high sensitivity and good selectivity. However, chromatographic columns are easily contaminated by biological matrices, requiring complex pretreatment and purification steps. Furthermore, the mobile phase often contains organic solvents, resulting in weak retention of highly polar neurotransmitters and limited separation efficiency. Specific chromatographic columns or pre-column derivatization of neurotransmitters are needed to improve this. Recent advancements in gene-encoded optical probe technology, nanosensing technology, and mass spectrometry imaging technology have also provided diverse means for neurotransmitter detection. However, the first two technologies generally suffer from poor selectivity, only able to detect a limited number of neurotransmitters; while mass spectrometry imaging technology has poor detection sensitivity and cumbersome pretreatment steps.

[0004] Capillary electrophoresis-mass spectrometry (CE-MS), as a highly efficient separation and analysis technique, combines the high separation efficiency of CE with the high sensitivity and specificity of MS. It offers significant advantages for detecting neurotransmitters in biological samples with strong polarity, low concentration, and complex matrices. Neurotransmitter metabolic pathways are complex networks involving precursors, neurotransmitters themselves, metabolic intermediates and end products, and regulatory molecules. These substances exhibit significant differences in pKa and charge states, allowing for wide dynamic range and simultaneous multi-component analysis on CE-MS. Furthermore, CE significantly outperforms other analytical methods in terms of sample consumption, sample compatibility, and environmental friendliness. It can also be combined with simple and efficient in-column online enrichment techniques to achieve pre-concentration of low-concentration neurotransmitters, compensating for the insufficient sensitivity of MS. Therefore, the technical characteristics of CE-MS perfectly match the chemical nature of neurotransmitters and their metabolites and the analytical needs of biological samples. It not only provides accurate data for fundamental research on metabolic pathway mechanisms but also offers efficient and reliable technical support for the discovery of disease biomarkers and drug development.

[0005] Based on the limitations of existing analytical methods and the outstanding advantages of CE-MS, a highly sensitive DPJ-CE-MS method for detecting neurotransmitters and histamine metabolites was developed. This method combines a simple dynamic pH-linked (DPJ) capillary column in-line pre-enrichment technique with optimization of the CE-MS interface, enrichment conditions, separation and detection conditions, significantly improving the detection sensitivity of the method and achieving highly sensitive detection of analytes. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a highly sensitive DPJ-CE-MS method for detecting neurotransmitters and histamine metabolites, for the analysis of trace targets in cerebrospinal fluid samples.

[0007] To achieve the above objectives, the present invention adopts the following technical solution;

[0008] A highly sensitive DPJ-CE-MS method for detecting neurotransmitters and histamine metabolites, the method comprising the following steps:

[0009] Provide a pretreated fused silica capillary and fill the capillary with a background electrolyte solution;

[0010] Alkaline solution and sample solution containing analyte and internal standard are sequentially injected through the capillary injection end, and the analyte is pre-enriched online using the dynamic pH linkage principle.

[0011] A separation voltage is applied in BGE to cause the enriched analytes to be separated by electrophoresis in the background electrolyte solution and migrate toward the outlet of the capillary.

[0012] The analytes that migrate to the capillary outlet are introduced into a high-resolution mass spectrometer for detection in the presence of sheath fluid via a capillary electrophoresis-mass spectrometry interface.

[0013] The alkaline solution is ammonia water, the background electrolyte (BGE) is an aqueous solution of formic acid and methanol, and the sheath fluid (SL) is an aqueous solution of formic acid and methanol.

[0014] As a further aspect of the present invention, the capillary pretreatment includes sequentially rinsing and activating with methanol, ultrapure water, 1M NaOH, ultrapure water, 0.1M NaOH, ultrapure water, and BGE; and rinsing the capillary with background electrolyte (BGE) between two analytical runs.

[0015] As a further embodiment of the present invention, the ammonia solution is 2% (v / v) and is injected at a pressure of 4 psi for 5 seconds; and the sample solution is injected at a pressure of 2 psi for 40 seconds.

[0016] As a further embodiment of the present invention, the separation voltage is +25kV, and an auxiliary pressure of 0.5psi is applied, with a total separation time of 30 minutes.

[0017] As a further aspect of the present invention, the background electrolyte (BGE) comprises 0.05% (v / v) formic acid and 5% (v / v) methanol, and the sheath fluid comprises 0.1% (v / v) formic acid and 8% (v / v) methanol.

[0018] As a further aspect of the present invention, the electrospray voltage of the high-resolution mass spectrometer is +2.2kV, and the ion transmission tube temperature is 320℃.

[0019] As a further aspect of the present invention, the analyte is selected from at least one of the following 11 substances: norepinephrine (NE), glutamic acid (Glu), aspartic acid (Asp), γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT), histamine (Hist), 1-methylhistamine (1-MH), methylimidazolium acetic acid (1-MeIAA), imidazolium-4-acetic acid (Im-4-AA), dopamine (DA), and tryptophan (Trp); and the internal standard is deuterated histamine (D~4~-Hist).

[0020] As a further aspect of the present invention, the mixed concentration of the 11 analytes entering the capillary electrophoresis detection is 50 ng / mL.

[0021] As a further aspect of the present invention, the high-resolution mass spectrometer employs an electrospray ionization source and performs qualitative and quantitative analysis by extracting ion chromatograms.

[0022] As a further embodiment of the present invention, the capillary electrophoresis-mass spectrometry interface includes: a fused silica capillary that has been etched with hydrofluoric acid and whose outlet outer diameter has been reduced to about 60 μm, and a borosilicate glass nozzle that has been drawn and prepared with a tip inner diameter of 10-20 μm; the distance between the capillary outlet and the nozzle tip is about 400 μm.

[0023] As a further aspect of the present invention, the etching process involves etching the capillary tip with a 48% (v / v) hydrofluoric acid solution for 90 minutes. During etching, the capillary tip is immersed in hydrofluoric acid to a depth of approximately 500 μm, and octanol is applied to the interface to form a meniscus.

[0024] On the other hand, the present invention also provides a kit for detecting neurotransmitters and histamine metabolites, comprising: 0.05% (v / v) formic acid and 5% (v / v) methanol for preparing a background electrolyte solution; 2% (v / v) ammonia for online pre-concentration; 0.1% (v / v) formic acid and 8% (v / v) methanol for preparing a sheath fluid; and standards for the analyte and deuterated histamine.

[0025] Thirdly, the present invention also provides the application of a kit for detecting neurotransmitters and histamine metabolites in the preparation of products for detecting neurotransmitters and histamine metabolites.

[0026] Fourthly, the present invention also provides a kit for detecting neurotransmitters and histamine metabolites in biological samples.

[0027] In summary, due to the adoption of the above technical solution, the beneficial technical effects of the invention are as follows:

[0028] High sensitivity and low detection limits: Dynamic pH linkage (DPJ) online pre-enrichment technology effectively overcomes the inherent limitation of low sample loading in CE-MS coupling, achieving highly efficient concentration of trace neurotransmitters and histamine metabolites. The limits of detection (LOD) for target analytes reach 0.05-5 ng / mL, and the limits of quantitation (LOQ) reach 0.05-8 ng / mL. The enrichment factor (FE) for many substances can reach up to 37-fold, significantly improving detection sensitivity.

[0029] Excellent separation efficiency and selectivity: The optimized background electrolyte (BGE) and sheath fluid (SL) system, combined with specific separation voltage and auxiliary pressure, provides good capillary electrophoresis separation and peak shape for the target analysis of neurotransmitters and their metabolites with significant differences in physicochemical properties, ensuring the accuracy of simultaneous analysis of multiple components in complex biological matrices.

[0030] High precision and accuracy: Method validation results showed that the intra-day and inter-day relative standard deviations (RSDs) of peak area and migration time for all analytes were below 12.6% and 5.8%, respectively. Spiked recovery experiments in cerebrospinal fluid matrix showed recoveries ranging from 77.5% to 130.9% with RSDs ranging from 0.6% to 10.1%, demonstrating the method's good reproducibility and accuracy, making it suitable for practical biological sample analysis.

[0031] Dedicated interface enhances performance: The optimized capillary electrophoresis-mass spectrometry interface, including specially etched capillaries and pulled nozzles, and precise control of the distance between the capillary outlet and the nozzle tip, as well as between the nozzle tip and the ion transmission tube inlet, effectively reduces dead volume and sample dilution effect, improves ionization efficiency and signal stability, and is a key hardware guarantee for the method to achieve high-sensitivity detection.

[0032] Simple to operate and sample-friendly: The method has relatively simple pretreatment steps and low sample consumption, making it particularly suitable for high-throughput, multi-index analysis of precious and limited clinical samples such as cerebrospinal fluid.

[0033] The method has broad application prospects: It provides an efficient and reliable analytical tool for the study of the pathophysiological mechanisms of neurological diseases, mental diseases and allergic diseases, the discovery and validation of potential biomarkers, and the study of the pharmacokinetics of related drugs. Attached Figure Description

[0034] Figure 1 The structural diagrams (A) and (B) of eight neurotransmitters, three histamine metabolites, and the internal standard deuterated histamine are shown.

[0035] Figure 2 This is a schematic diagram of a device for etching capillary tips;

[0036] Figure 3 A schematic diagram showing the relationship between the capillary, nozzle, and ion transport tube at the CE-MS interface;

[0037] Figure 4 This is a schematic diagram illustrating the dynamic pH connection principle.

[0038] Figure 5 The dynamic pH-linked three-factor optimization diagram is as follows: (A) NH3·H2O concentration; (B) NH3·H2O injection amount; (C) Analyte injection amount;

[0039] Figure 6 Optimization diagram for CE separation conditions: (A) Formic acid concentration in BGE; (B) Methanol concentration in BGE; (C) Auxiliary pressure applied during separation; (D) Separation voltage;

[0040] Figure 7MS condition optimization diagram: (A) Formic acid concentration in SL; (B) Methanol concentration in SL; (C) Electrospray voltage; (D) Ion transport tube temperature;

[0041] Figure 8 The extracted ion chromatograms are shown for the analyte and internal standard (20 ng / mL) under optimal conditions. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0044] The underlying logic and technical approach of this method:

[0045] The core objective of this invention is to establish a DPJ-CE-MS analytical method integrating online enrichment, efficient separation, and highly sensitive detection for the simultaneous detection of multiple key neurotransmitters and histamine metabolites in cerebrospinal fluid. Its underlying technical approach follows this logic:

[0046] Hardware optimization lays the foundation: First, by performing fine physical processing and spatial alignment optimization on the capillary and nozzle of the CE-MS sheath flow interface, a dedicated interface with small dead volume and high ionization efficiency is constructed, providing a stable hardware platform for subsequent high-sensitivity detection.

[0047] Online enrichment enhances sensitivity: To address the issue of extremely low target analyte concentrations, dynamic pH linkage (DPJ) technology is introduced. By sequentially creating acid-base zones within the capillary, effective accumulation and pre-concentration are achieved by utilizing the abrupt change in the charge state of the analyte at the interface. This transforms large-volume injections into sharp detection peaks, directly improving the signal-to-noise ratio.

[0048] Separation conditions are optimized to ensure specificity: Based on the differences in charge and polarity of target substances (including acidic amino acids, monoamines, etc.), the composition of BGE (formic acid to methanol ratio), separation voltage and auxiliary pressure are systematically optimized to achieve baseline separation and avoid signal interference during mass spectrometry detection.

[0049] Optimization of detection conditions ensures optimal response: Finally, the composition of the sheath fluid, electrospray voltage, and ion transmission tube temperature at the mass spectrometer end are synergistically optimized to ensure that all target analytes can be stably and efficiently ionized, thereby obtaining the optimal mass spectrometry response.

[0050] The experimental instruments and reagents used in this experiment were as follows: All capillary electrophoresis experiments were performed on a Beckman PA800plus CE analysis system (Beckman, Fullerton, CA, USA) equipped with a CMPEMASS-IICE-MS ion source (CMPScientific, Brooklyn, NY), and coupled with a Q-Exactive mass spectrometer (ThermoFisherScientific) to acquire analyte signals. The data were analyzed using ThermoXcalibur software.

[0051] Histamine (Hist), 1-Methylhistamine (1-MH), γ-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT), norepinephrine (NE), imidazole-4-aceticacid (Im-4-AA), 1-(1-methyl-1H-imidazole-4-yl)acetic acid (1-Methylimidazoleaceticacid (1-MeIAA), tryptophan (Trp), L-glutamic acid (Glu), and L-aspartic acid (Asp) were all purchased from Alta Scientific Co., Ltd. (Tianjin, China), with a purity ≥98%. Their structural formulas are shown below. Figure 1 A. Methanol (MeOH) was HPLC grade and purchased from Merck (Darmstadt, Germany); formic acid (FA) and ammonia (NH3·H2O) were HPLC grade and purchased from Sigma-Aldrich (Shanghai, China); the experimental water was commercially available Wahaha® purified water (Hangzhou Wahaha Group Co., Ltd., conforming to GB / T6682 Class I water standard).

[0052] Fused silica capillary tubes (360 μm outer diameter, 50 μm inner diameter) were purchased from Polymicro Technologies (Phoenix, AZ, USA). Borosilicate glass capillary tubes (1.0 mm outer diameter, 0.75 mm inner diameter) were purchased from Sutter Instrument (Novato, USA).

[0053] Example 1: Establishment and optimization of a highly sensitive DPJ-CE-MS method for detecting neurotransmitters and histamine metabolites.

[0054] Design of the CE-MS sheath flow interface:

[0055] The capillary tip and the nozzle are independent and separate components, precisely aligned and tightly fitted in space, together forming the core of the CE-MS sheath flow interface. To fabricate the capillary tip, a 48% HF solution was used for etching; a schematic diagram of the etching process is shown below. Figure 2 The capillary was first filled with water using a siphon effect, and the polyacrylamide coating about 1 cm from the capillary tip was burned off. It was then inserted into HF coated with octanol until the capillary tip just touched the HF (approximately 500 μm deep into the HF). The octanol was used to form a meniscus at the interface between the capillary and HF, resulting in different degrees of etching. The total etching time was 90 minutes, ultimately forming a capillary tip with an unchanged inner diameter and a reduced outer diameter to approximately 60 μm. For the nozzle, a P-97 microelectrode pulling instrument was used for pulling. Five programs were set with heating current values ​​(HEAT) of 481, 515, 530, 540, and 560, pulling strength values ​​(PULL) of 13, 23, 53, 70, and 80, pulling speed (VELOCITY) of 5, and cooling time (TIME) of 100. Under these conditions, a cycle of pulling apart the borosilicate glass capillary was performed to prepare a nozzle tip sealed with a fine filament. Before each use, the filament was gently removed with a ceramic cutting disc, ensuring the nozzle tip's inner diameter was 10–20 μm. A schematic diagram of the capillary-nozzle-ion transport tube relationship at the CE-MS interface is shown below. Figure 3 The distance from the capillary outlet to the tip of the nozzle is approximately 400 μm, and the distance between the tip of the nozzle and the MS ion transport tube is controlled to be 1.8 mm.

[0056] DPJ condition optimization:

[0057] In this method, the analyzed substances are all monoamine or amino acid neurotransmitters. Based on their pKa values, amino acid neurotransmitters are completely protonated in acidic BGE and reverse their charge in alkaline solution, migrating in the opposite direction and accumulating. Monoamine neurotransmitters, on the other hand, are deprotonated in alkaline solution, becoming electroneutrally neutral and accumulating in abundance. Accumulation continues until the injected alkaline solution is completely neutralized, at which point normal electrophoretic separation begins. See the DPJ schematic diagram. Figure 4 .

[0058] The effect of ammonia concentration on DPJ was investigated within the concentration range of 0-5% NH3·H2O. Figure 5As shown in Figure A, most analytes reached their maximum response at an ammonia concentration of 2%. With increasing concentration, the resolution deteriorated, and the signal intensity of some analytes tended to stabilize. However, the signal intensity of several key analytes, such as DA, NE, and 5-HT, decreased, and some even became undetectable. Regarding the injection volume of NH3·H2O, the effect of different injection pressures (1–5 psi) was investigated at a fixed injection time (5 s). With increasing injection pressure, the signal intensity of each analyte increased, and tended to stabilize above 4 psi. Therefore, an injection pressure of 4 psi was chosen for ammonia (…). Figure 5 B). The effect of different injection times (5–50 s) was investigated at an injection pressure of 2 psi. The signal intensity of the analyte increased with increasing injection time. Figure 5 C). While achieving better enrichment folds and maintaining peak shape, a sample injection time of 40 s was chosen; longer injection times lead to peak tailing and incomplete enrichment. It can be seen that after applying DPJ, injecting a large volume of analyte still maintains a sharp peak shape, confirming the high effectiveness of the studied capillary pre-concentration method.

[0059] Optimization of CE separation conditions:

[0060] This method selects a formic acid-methanol-water system as the BGE (Bipolar Genomic Analysis) system. Formic acid provides an acidic environment, while methanol improves peak shape, increases resolution, and facilitates MS ionization. The concentration of FA (Fluorine A) is optimized within the range of 0–0.30%. Figure 6 As shown in Figure A, when the formic acid concentration is 0.05%, the peak areas of most analytes reach their maximum values. Excessively high formic acid concentrations lead to a decrease in the signal intensity and resolution of the analytes, possibly due to excessive Joule heating. The concentration of MeOH should be optimized within the range of 0–15%, as follows: Figure 6 As shown in Figure B, the addition of MeOH had no significant effect on the peak area, but it effectively improved the peak shape symmetry and resolution of the analytes. Furthermore, it was found that increasing the methanol concentration led to a longer analyte migration time, with a better response at a concentration of 5%. The auxiliary pressure applied during separation was optimized within the range of 0.1–1 psi to stabilize the electroosmotic flow and shorten the analysis time. Figure 6 As shown in Figure C, the analyte signal decreases with increasing applied pressure, and the resolution deteriorates; however, the lower the applied pressure, the longer the analyte elution time. Ultimately, an applied pressure of 0.5 psi was selected. For the separation voltage, optimization was performed within the range of 20–28 kV. It was found that changes in voltage had no significant impact on the analyte signal strength and resolution, but the migration time shortened with increasing separation voltage. Considering the Joule heating effect caused by excessively high voltage, a separation voltage of +25 kV was finally chosen. Figure 6 D).

[0061] Optimization of MS detection conditions:

[0062] The composition of the sheath fluid affects the ionization efficiency of the analyte and the intensity of the mass spectrometry signal. To match the composition of BGE and obtain ionization efficiency and electrospray stability, a formic acid-methanol-water system was also selected as the sheath fluid. Figure 7 As shown in Figure A, the concentration of FA was optimized within the range of 0.05% to 0.3%, and the best response was observed for all analytes when the FA concentration was 0.1%. Figure 7 As shown in B, the concentration of the organic additive MeOH was optimized within the range of 5-10%, and 8% MeOH was selected for subsequent studies. The electrospray voltage and ion transmission tube temperature affect ionization efficiency and detection reliability; therefore, these factors were optimized. Figure 7 As shown in C and 7D, the 11 analytes showed the best response when the electrospray voltage was +2.2kV and the ion transfer tube temperature was 320℃.

[0063] DPJ-CE-MS analysis:

[0064] First, 2% (v / v) NH3·H2O was injected at a pressure of 4 psi (5 s), followed by injection of the analyte at a pressure of 2 psi (40 s). Finally, a separation voltage of +25 kV and an additional pressure of 0.5 psi were applied, and the mixture was run for 30 min. BGE was 0.05% (v / v) formic acid + 5% (v / v) methanol, and SL was 0.1% (v / v) formic acid + 8% (v / v) methanol; the electrospray voltage was +2.2 kV, and the ion transfer tube temperature was 320 °C. Under optimal conditions, the extracted ion chromatograms of the 11 analytes and the internal standard (20 ng / mL) are shown below. Figure 8 As shown.

[0065] Example 2

[0066] Validation and application of a highly sensitive DPJ-CE-MS method for detecting neurotransmitters and histamine metabolites;

[0067] Processing of cerebrospinal fluid samples:

[0068] Take 30 μL of cerebrospinal fluid sample, add 2 times the volume of pre-cooled MeOH, vortex mix for 30 s, then transfer to a low-temperature high-speed centrifuge and centrifuge at 4 °C and 13000 rpm for 20 min. Take the supernatant into a new 1.5 mL centrifuge tube, evaporate the solvent by vacuum centrifugation, then add an equal volume of 0.1% (v / v) formic acid water containing internal standard to reconstitute the supernatant. Transfer the processed sample to a micro-volume vial for CE-MS analysis.

[0069] Calibration curve, limit of detection (LOD), limit of quantitation (LOQ):

[0070] Calibration curves were established for 11 analyte standards within a concentration range of 0.05–1000 ng / mL, as shown in Table 1. A good linear relationship was observed between the logarithm of the concentration and the logarithm of the peak area for the 11 neurotransmitters and their metabolites within the linear range, with a linear correlation coefficient R² > 0.99. The limits of detection (LODs) for the 11 analytes were defined as the lowest concentration detectable by CE-MS, ranging from 0.05–5 ng / mL; the limits of quantitation (LOQs) were defined as the lowest concentration within the linear range, ranging from 0.05–8 ng / mL. The peak areas, LODs, and LOQs of enriched and unenriched DPJ samples were compared at the same analyte concentrations to assess the enrichment factor (FE). Based on peak area, the FE reached a maximum of 37-fold; based on LOD, the maximum increase was 25-fold; and based on LOQ, the maximum increase was 25-fold.

[0071] Table 1

[0072]

[0073] Precision analysis:

[0074] Eleven standard solutions were injected three times a day at low, medium, and high concentrations (10, 50, and 100 ng / mL) for three consecutive days. The relative standard deviation (RSD) of the peak area and migration time in the extracted ion chromatograms of the analytes was calculated to assess the precision of the method. As shown in Table 2, the intra-day RSD values ​​(n=3) for peak area and migration time of all analytes were below 10.5% and 1.2%, respectively, while the inter-day RSD values ​​(n=3) were below 12.6% and 5.8%, respectively. This indicates that the established method has good reproducibility at low, medium, and high concentrations, meeting the detection requirements of practical samples.

[0075] Table 2

[0076]

[0077] Accuracy analysis:

[0078] Under optimal conditions, cerebrospinal fluid samples were used as the matrix for spiked recovery experiments at three concentration levels: low, medium, and high (10-200 ng / mL). GABA, Trp, Glu, and Asp were spiked for recovery after a ten-fold dilution of the sample, while the remaining substances were spiked for recovery after a three-fold dilution. The results are shown in Table 3. The recoveries of the 11 analytes ranged from 77.5% to 130.9%, and the RSD values ​​of three parallel assays were between 0.6% and 10.1%, indicating that the established method has good accuracy and can be used for practical sample analysis.

[0079] Table 3

[0080]

[0081] The sequence number of each step in the above embodiments does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0082] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A DPJ-CE-MS method for high sensitive detection of neurotransmitters and histamine metabolites, characterized in that, Includes the following steps: Provide a pretreated fused silica capillary and fill the capillary with a background electrolyte solution; Alkaline solution and sample solution containing analyte and internal standard are sequentially injected through the capillary injection end, and the analyte is pre-enriched online using the dynamic pH linkage principle. A separation voltage is applied in BGE to cause the enriched analytes to be separated by electrophoresis in the background electrolyte solution and migrate toward the outlet of the capillary. The analytes that migrate to the capillary outlet are introduced into a high-resolution mass spectrometer for detection in the presence of sheath fluid via a capillary electrophoresis-mass spectrometry interface. The alkaline solution is ammonia, the background electrolyte is an aqueous solution of formic acid and methanol, and the sheath fluid is an aqueous solution of formic acid and methanol.

2. The DPJ-CE-MS method for high sensitive detection of neurotransmitters and histamine metabolites according to claim 1, characterized in that, The capillary pretreatment includes sequential rinsing and activation with methanol, ultrapure water, 1 M NaOH, ultrapure water, 0.1 M NaOH, ultrapure water, and BGE; between two analytical runs, the capillary is rinsed with background electrolyte.

3. The DPJ-CE-MS method for high sensitive detection of neurotransmitters and histamine metabolites according to claim 1, characterized in that, The ammonia solution had a volume percentage concentration of 2% and was injected at a pressure of 4 psi for 5 seconds; the sample solution was injected at a pressure of 2 psi for 40 seconds.

4. The DPJ-CE-MS method for high sensitive detection of neurotransmitters and histamine metabolites according to claim 1, characterized in that, The separation voltage was +25 kV, and an auxiliary pressure of 0.5 psi was applied. The total separation time was 30 minutes. The background electrolyte contained 0.05% formic acid and 5% methanol by volume, and the sheath fluid contained 0.1% formic acid and 8% methanol by volume.

5. The DPJ-CE-MS method for highly sensitive detection of neurotransmitters and histamine metabolites according to claim 1, characterized in that, The high-resolution mass spectrometer uses an electrospray ionization source and performs qualitative and quantitative analysis by extracting ion chromatograms; the electrospray voltage of the high-resolution mass spectrometer is +2.2 kV and the ion transmission tube temperature is 320 ℃.

6. The DPJ-CE-MS method for highly sensitive detection of neurotransmitters and histamine metabolites according to claim 1, characterized in that, The analytes to be tested are selected from at least one of the following 11 substances: norepinephrine, glutamic acid, aspartic acid, γ-aminobutyric acid, 5-hydroxytryptamine, histamine, 1-methylhistamine, methylimidazolium acetic acid, imidazolium-4-acetic acid, dopamine, and tryptophan; and the internal standard is deuterated histamine. The mixed concentration of the 11 analytes to be tested in capillary electrophoresis is 50 ng / mL.

7. The DPJ-CE-MS method for high sensitive detection of neurotransmitters and histamine metabolites according to claim 1, characterized in that, The capillary electrophoresis-mass spectrometry interface includes: a fused silica capillary that has been etched with hydrofluoric acid and has its outlet outer diameter reduced to 60 μm, and a borosilicate glass nozzle that has been drawn and has a tip inner diameter of 10-20 μm; the distance between the capillary outlet and the nozzle tip is 400 μm.

8. The DPJ-CE-MS method for high sensitive detection of neurotransmitters and histamine metabolites according to claim 7, characterized in that, The hydrofluoric acid etching process involves etching the capillary tip with a 48% (by volume) hydrofluoric acid solution for 90 minutes. During etching, the capillary tip is immersed in hydrofluoric acid to a depth of 500 μm, and octanol is applied to the interface to form a meniscus.

9. A kit for detecting neurotransmitters and histamine metabolites, characterized by, Include: The background electrolyte was prepared using 0.05% formic acid and 5% methanol by volume. The volume percentage concentration of ammonia water used for online pre-enrichment is 2%. Formic acid with a volume percentage concentration of 0.1% and methanol with a volume percentage concentration of 8% were used to prepare the sheath fluid; As well as analytes and standards or mixtures of standards for deuterated histamine.

10. Use of the kit for detecting neurotransmitters and histamine metabolites according to claim 9 in the preparation of a detection product for studying nervous system diseases, mental diseases, allergic diseases or digestive system diseases, which comprises detecting the contents of neurotransmitters and histamine metabolites in a cerebrospinal fluid sample.

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