A highly sensitive method for selective enrichment of important biomarkers in brain tissue

Abstract

The application discloses a kind of high-sensitivity analysis methods for selectively enriching important biomarkers of brain tissue, and belongs to the technical field of biomarker detection.The method disclosed in the application uses 3-(trimethoxysilyl) methyl propyl methacrylate modified capillary column inner wall to obtain the modified capillary column with double bond on the surface, then uses specific monomer, crosslinking agent, pore-forming agent and initiator to react, to obtain poly(GMA-co-EDMA) organic polymer capillary monolithic column, then hydrochloric acid and triethylamine solution are sequentially introduced to react, to obtain TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column;The method establishes a new method for effectively enriching and high-sensitivity analyzing important biomarkers NAA, NAAG and NAG in brain tissue, and the quaternary ammonium salt group on the TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column can produce strong anion exchange with NAA, NAAG and NAG, to achieve the purpose of selectively enriching NAA, NAAG and NAG.

Description

Technical Field

[0001] This invention belongs to the field of biomarker detection technology, specifically relating to a highly sensitive analytical method for selectively enriching important biomarkers in brain tissue. Background Technology

[0002] Biomarkers are objectively measurable and evaluable indicators used to reflect different physiological or pathological states of an organism, playing important roles within the body. N-acetylaspartate (NAA) is synthesized from aspartate and acetyl-CoA and is primarily found in neurons. Studies have shown that changes in endogenous NAA levels are associated with brain injury and various neurological diseases (such as Alzheimer's disease and Parkinson's disease), making it an important biomarker for assessing neuronal state, quantity, and activity. Another biomarker, N-acetylaspartate (NAAG), is present in the brain and plays a crucial role in traumatic brain injury, stroke, epilepsy, schizophrenia, and age-related neurodegenerative diseases. Furthermore, decreased NAG levels can lead to hyperammonemia, which in turn can cause central nervous system dysfunction, resulting in cerebral edema, altered mental status, seizures, coma, and even death. Given the importance of NAA, NAAG, and NAG in the human brain, and considering their extremely low concentrations in brain tissue samples, conventional detection techniques face new challenges. Therefore, there is an urgent need to establish a highly sensitive and selective method for the simultaneous extraction, enrichment, and tracking detection of endogenous NAA, NAAG, and NAG in brain tissue.

[0003] Due to the complexity of actual sample matrices and the low concentration of target analytes, accurate quantification of target analytes from actual samples (such as brain tissue) remains a significant challenge. Current sample pretreatment methods for NAA, NAAG, and NAG determinations include liquid-liquid extraction, derivatization, and resin extraction. However, these methods all have limitations. Liquid-liquid extraction consumes large amounts of organic solvents; derivatization is quite complex and time-consuming, and sometimes suffers from incomplete derivatization and the generation of byproducts. Resin extraction utilizes commercially available Diaion SK-1 (cation exchanger) and Diaion SA-1 (anion exchanger) resins in a two-step solid-phase extraction process to purify the target analyte. However, the two-step solid-phase extraction process is labor-intensive, time-consuming, and unsuitable for rapid routine analysis; furthermore, Diaion SK-1 resin requires regeneration for reuse. Therefore, a novel, cost-effective, low-solvent-consumption, and simple-to-operate sample pretreatment method is urgently needed for NAA, NAAG, and NAG analysis. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide a highly sensitive analytical method for selectively enriching important biomarkers in brain tissue, so as to solve the technical problems of high testing cost, large raw material consumption and complex operation of existing detection methods.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention discloses a highly sensitive analytical method for selectively enriching important biomarkers in brain tissue, comprising the following steps:

[0007] S1: The inner wall of the capillary column was modified with 3-(trimethoxysilyl)propyl methacrylate to obtain the modified capillary column; then the monomer, crosslinking agent, pore-forming agent and initiator were mixed and injected into the modified capillary column to carry out the polymerization reaction. After the reaction was completed, a poly(GMA-co-EDMA) organic polymer capillary monolithic column was obtained.

[0008] S2: After passing hydrochloric acid into the poly(GMA-co-EDMA) organic polymer capillary monolithic column, the column is rinsed until the washing solution is neutral, and then triethylamine solution is injected to carry out the reaction. After the reaction is completed, TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column is obtained.

[0009] S3: An extraction device was prepared using a TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column. The target analytes in the brain tissue sample solution were then analyzed using the extraction device. Elution was performed using an elution solvent, and the eluent was collected for content determination.

[0010] Further, in S1, the monomer is glycidyl methacrylate; the crosslinking agent is ethylene glycol methacrylate; the porogen is n-propanol / 1,4-butanediol / water; and the initiator is azobisisobutyronitrile.

[0011] Further, the mass ratio of the monomer, crosslinking agent, porogen and initiator is (100-140):(20-60):(170-260):(1.2-2).

[0012] Further, the weight ratio of n-propanol, 1,4-butanediol and water is (100-140):(60-100):(10-20).

[0013] Furthermore, in S1, the polymerization reaction is carried out at a temperature of 50–70°C for a duration of 18–32 h.

[0014] Furthermore, in S2, after hydrochloric acid is introduced into the poly(GMA-co-EDMA) organic polymer capillary monolithic column, it is sealed at room temperature for 12-20 hours, rinsed with water until the washing solution is neutral, and then rinsed with acetonitrile.

[0015] Furthermore, in S2, the ratio of the amount of hydrochloric acid, triethylamine solution and monomer in S1 is (1-3) mL: (1-3) mL: (100-140) mg;

[0016] The reaction temperature was 80℃ and the reaction time was 12 hours.

[0017] Furthermore, in S3, the pH value of the brain tissue sample solution to be tested is 6.0; and acetonitrile with a volume fraction of 10% is added to the brain tissue sample solution to be tested.

[0018] Furthermore, in S3, the volume of the brain tissue sample solution to be tested is 1.0 mL.

[0019] Furthermore, in S3, the elution solvent is acetone containing 10% formic acid.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention discloses a highly sensitive analytical method for selectively enriching important biomarkers in brain tissue. The method involves modifying the inner wall of a capillary column with 3-(trimethoxysilyl)methacrylate to obtain a modified capillary column with double bonds on its surface. Subsequently, a reaction is carried out using specific monomers, crosslinking agents, pore-forming agents, and initiators to obtain a monolithic poly(GMA-co-EDMA) organic polymer capillary column. This column is then sequentially purged with hydrochloric acid and triethylamine solution to obtain a TEA@poly(GMA-co-EDMA) organic polymer capillary column. The synthesized monolithic organic polymer capillary column containing quaternary ammonium salts is connected to a microinjection pump to achieve in-vitro injection-specimen electrophoresis (IT-SPME), selectively enriching the target analyte through strong anion exchange. This method establishes an effective method for enriching and highly sensitively analyzing important biomarkers in brain tissue. A novel method for detecting NAA, NAAG, and NAG has been developed. The quaternary ammonium salt groups on a TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column can generate strong anion exchange with NAA, NAAG, and NAG, achieving selective enrichment of these substances. This method is also the most sensitive method for detecting NAA, NAAG, and NAG to date. According to relevant experimental results, this method is the first to detect endogenous NAA, NAG, and NAAG in a 5mg brain tissue sample. Compared with previously reported methods requiring 40mg to 1g of brain tissue sample, this method significantly reduces sample consumption, which is beneficial for pathophysiological and therapeutic research on brain diseases. Under optimized conditions, this method exhibits a wide linear range, good precision, and low detection limit, showing broad application prospects. Attached Figure Description

[0022] Figure 1 Scanning electron microscope (SEM) images of the morphology of poly(GMA-co-EDMA) organic polymer capillary monolithic columns (a, b) and TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic columns (c, d);

[0023] Where: a, c - overall view; b, d - close-up view;

[0024] Figure 2 This is a schematic diagram of the extraction device prepared using a TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column;

[0025] Figure 3 The effect of elution solvent type on IT-SPME;

[0026] Figure 4 Comparison of extraction effects of brain tissue sample solutions at different pH values;

[0027] Figure 5The effect of different salt concentrations of the brain tissue sample solution on IT-SPME results;

[0028] Figure 6 The results show the effect of different acetonitrile contents in the brain tissue sample solution on the extraction efficiency.

[0029] Figure 7 The results show the effect of the volume of the brain tissue sample solution on the extraction efficiency.

[0030] Figure 8 Multiple reaction monitoring chromatograms of NAA, NAG, and NAAG;

[0031] Among them: a-direct injection analysis; b-injection analysis after extraction with TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column.

[0032] Figure 9 The levels of endogenous NAA, NAAG, and NAG in different brain tissue locations. Detailed Implementation

[0033] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0034] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0035] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0036] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0037] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0038] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0039] This invention discloses a highly sensitive analytical method for selectively enriching important biomarkers in brain tissue, mainly comprising the following steps:

[0040] 1) Preparation of monolithic organic polymer capillary columns

[0041] Synthesis of poly(GMA-co-EDMA) organic polymer capillary monolithic column: The inner wall of the capillary was modified with 3-(trimethoxysilyl)methacrylate to give it double bonds on the surface, thus obtaining the modified capillary column.

[0042] Subsequently, 120 mg of GMA glycidyl methacrylate monomer, 40 mg of EDMA ethylene glycol methacrylate crosslinking agent, 140 mg of n-propanol / 80 mg of 1,4-butanediol / 20 mg of water porogen, and 1.6 mg of AIBN azobisisobutyronitrile initiator were mixed evenly and ultrasonically degassed for 10 min. The mixture was then injected into the modified capillary column, and both ends of the capillary were sealed with silicone rubber. The column was placed in a 60 °C oven for polymerization reaction for 24 h. After the polymerization reaction was completed, the capillary was connected to a micro-injection pump, and unreacted monomers, crosslinking agents, and porogens were removed by rinsing with methanol to obtain a poly(GMA-co-EDMA) organic polymer capillary monolithic column.

[0043] 2) Synthesis of TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column

[0044] 2 mL of 10% hydrochloric acid was introduced into a poly(GMA-co-EDMA) organic polymer capillary monolithic column using a micro-injection pump. The column was sealed and left to stand overnight at room temperature. After rinsing with water until neutral, the column was rinsed with acetonitrile. Subsequently, 2 mL of triethylamine (TEA) solution was passed through the poly(GMA-co-EDMA) organic polymer capillary monolithic column. The column was filled with TEA and sealed with silicone rubber. After reacting in an 80°C oven for 12 h, the monolithic material was continuously rinsed with large amounts of water and methanol to complete the preparation of the TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column.

[0045] 3) IT-SPME

[0046] The IT-SPME extraction device is a modified 1mL disposable syringe, that is, a 4cm long capillary column replaces the original needle. Figure 2 As shown, the entire process includes four steps: activation, sample loading, washing, and elution. The TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column was activated sequentially with 1 mL of methanol, 0.5 mL of water, and 0.5 mL of sample solvent (10 mM ammonium acetate containing 10% acetonitrile, pH 6.0). Then, 1 mL of the target brain tissue sample solution was passed through the monolithic column at a flow rate of 2.0 mL / h. Subsequently, the capillary monolithic column was washed with 0.5 mL of water to remove unbound impurities. Finally, the target analyte extracted onto the monolithic column was eluted with 0.5 mL of acetone containing 10% formic acid at a flow rate of 1.0 mL / h.

[0047] Subsequently, high-performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) analysis was performed: the eluent was dried under nitrogen, redissolved in 100 μL of mobile phase, and analyzed by UHPLC-MS / MS. Chromatographic conditions: mobile phases were 0.1% formic acid / aqueous solution (A) and 0.1% formic acid / acetonitrile solution (B); flow rate was 0.2 mL / min; injection volume was 1 μL. The gradient elution program was set as follows: solution B 0–3 min, 2–4%; 3–4 min, 4–96%; 4–4.1 min, 96–2%; 4.1–6 min, 2%. Multiple reaction monitoring (MRM) scanning mode was used. Ion source parameters were as follows: ion transfer tube voltage set to 2.8 kV (negative ion mode); ion source temperature set to 500 °C; curtain gas pressure set to 45 psi; nebulizer pressure set to 45 psi. The deprotonated quasi-molecular ion of the target compound was selected as the parent ion, and the most abundant ion in the secondary mass spectrometer was selected as the daughter ion. Quantitative analysis was performed by detecting the m / z channels 174.1→88.1 (NAA), 303.0→285.2 (NAAG), 188.1→102.1 (NAG), and 177.1→58.1 (NAA-D3, N-acetyl-DL-aspartic acid-2,3,3-d3) of the target analyte and internal standard.

[0048] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0049] To investigate the extraction performance of TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic columns, researchers used NAA, NAAG, and NAG standards as model compounds for IT-SPME and systematically optimized factors affecting extraction efficiency, including: the type of elution solvent, the pH of the sample solution, the salt concentration of the sample solution, the organic solvent content of the sample solution, and the volume of the sample solution. To obtain more reliable experimental conditions, three replicates were established for each condition.

[0050] I. The Influence of Elution Solvent Type on IT-SPME

[0051] Choosing a suitable elution solvent is crucial for eluting the target analyte. This application compares the elution effects of acetonitrile, acetonitrile containing 10% formic acid, acetone, acetone containing 10% formic acid, methanol, and methanol containing 10% formic acid. Figure 3As shown, adding formic acid to the elution solvent can improve elution efficiency. Acetone containing 10% formic acid and methanol containing 10% formic acid have relatively good elution effects. Considering that acetone is more volatile than methanol and can shorten the drying time, acetone containing 10% formic acid was ultimately chosen as the elution solvent.

[0052] II. The effect of pH value of the brain tissue sample solution on IT-SPME

[0053] like Figure 4 As shown, this application investigated the effect of sample substrate pH in the range of 3.0–9.0 on the extraction efficiency. The results showed that the extraction efficiency was best when the sample pH was 6.0. Therefore, the pH of the sample solution was controlled at 6.0 for subsequent studies.

[0054] III. The effect of salt concentration of the brain tissue sample solution on IT-SPME

[0055] NAA, NAAG, and NAG are polar chemicals; therefore, the salt concentration in the sample solution affects the extraction efficiency of IT-SPME. To assess the effect of sample solution salt concentration on IT-SPME, different concentrations of NaCl (0–100 mM) were added to the sample solution. Figure 5 As shown, the addition of NaCl did not improve the extraction and enrichment of the target analyte, indicating that salting out was not significant in this study. Therefore, no salt was added when preparing the sample solution.

[0056] IV. The effect of organic solvent content in the brain tissue sample solution on IT-SPME

[0057] like Figure 6 As shown, adding a small amount of acetonitrile to the brain tissue sample solution is beneficial for the extraction and enrichment of the target analyte. However, when the acetonitrile content in the brain tissue sample solution exceeds 10%, the extraction efficiency of the target analyte decreases. Therefore, 10% acetonitrile was selected for further research.

[0058] V. Effect of the volume of the brain tissue sample solution on IT-SPME

[0059] The volume of the brain tissue sample solution to be tested not only affects the IT-SPME process but also changes the total sample pretreatment time. For example... Figure 7 As shown, the extraction efficiency increases linearly with increasing sample loading volume, indicating that the prepared organic polymer capillary monolithic column has a high sample loading capacity for the target analyte. Considering the total sample preparation time, the final volume of the brain tissue sample solution to be tested was set to 1.0 mL.

[0060] Example 1

[0061] A highly sensitive analytical method for selectively enriching important biomarkers in brain tissue includes the following steps:

[0062] S1: The inner wall of the capillary was modified with 3-(trimethoxysilyl)propyl methacrylate to obtain a modified capillary column; then 120 mg GMA, 40 mg EDMA, 140 mg n-propanol / 80 mg 1,4-butanediol / 20 mg water and 1.6 mg AIBN were mixed evenly, ultrasonically degassed for 10 min, and then injected into the modified capillary column. The two ends of the capillary were sealed with silicone rubber and placed in a 60 °C oven for polymerization reaction for 24 h. After the polymerization reaction was completed, the capillary was connected to a micro-injection pump and rinsed with methanol to remove unreacted monomers, crosslinking agents and pore-forming agents to obtain a poly(GMA-co-EDMA) organic polymer capillary monolithic column;

[0063] S2: Using a micro-injection pump, 2 mL of 10% hydrochloric acid was introduced into the poly(GMA-co-EDMA) organic polymer capillary monolithic column. The column was sealed and left to stand overnight at room temperature. After rinsing with water until neutral, it was rinsed with acetonitrile. Then, 2 mL of TEA solution was passed through the poly(GMA-co-EDMA) organic polymer capillary monolithic column. The monolithic column was filled with TEA and sealed with silicone rubber. After reacting in an 80°C oven for 12 h, the monolithic material was continuously rinsed with large amounts of water and methanol to obtain the TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column.

[0064] S3: An extraction device was prepared using a TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column. 5 mg of mouse cerebral cortex was weighed and added to 400 μL of cold extraction buffer (80% acetonitrile containing 0.1% formic acid). After homogenization and centrifugation, 10 μL of the supernatant was collected and diluted to 10 mL with the sample solvent. Internal standard NAA-D3 was added to achieve a final concentration of 0.4 nM, thus preparing a mouse cerebral cortex sample solution. The mouse cerebral cortex sample solution was subjected to IT-SPME, and the eluent was collected for analysis by UHPLC-MS / MS to determine the concentrations of endogenous NAA, NAAG, and NAG in the mouse cerebral cortex.

[0065] Example 2

[0066] Unlike Example 1, this example uses a cerebellar sample solution, while the remaining steps are the same as in Example 1, measuring the concentrations of endogenous NAA, NAAG, and NAG in the cerebellum.

[0067] Example 3

[0068] Unlike Example 1, this example uses a hippocampal sample solution, while the remaining steps are the same as in Example 1, measuring the concentrations of endogenous NAA, NAAG, and NAG in the hippocampus.

[0069] Example 4

[0070] A highly sensitive analytical method for selectively enriching important biomarkers in brain tissue includes the following steps:

[0071] S1: The inner wall of the capillary was modified with 3-(trimethoxysilyl)propyl methacrylate to obtain a modified capillary column; then 100 mg GMA, 60 mg EDMA, 100 mg n-propanol / 60 mg 1,4-butanediol / 10 mg water and 2 mg AIBN were mixed evenly, ultrasonically degassed for 10 min, and injected into the modified capillary column. The two ends of the capillary were sealed with silicone rubber and placed in a 50℃ oven for polymerization reaction for 32 h. After the polymerization reaction was completed, the capillary was connected to a micro-injection pump and rinsed with methanol to remove unreacted monomers, crosslinking agents and pore-forming agents to obtain a poly(GMA-co-EDMA) organic polymer capillary monolithic column;

[0072] S2: Using a micro-injection pump, 3 mL of 10% hydrochloric acid was introduced into the poly(GMA-co-EDMA) organic polymer capillary monolithic column. The column was sealed at room temperature for 20 h. After rinsing with water until neutral, it was rinsed with acetonitrile. Then, 3 mL of TEA solution was flowed through the poly(GMA-co-EDMA) organic polymer capillary monolithic column. The monolithic column was filled with TEA and sealed with silicone rubber. After reacting in an 80℃ oven for 12 h, the monolithic material was continuously rinsed with large amounts of water and methanol to obtain the TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column.

[0073] S3: An extraction device was prepared using a TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column. 5 mg of mouse cerebral cortex was weighed and added to 400 μL of cold extraction buffer (80% acetonitrile containing 0.1% formic acid). After homogenization and centrifugation, 10 μL of the supernatant was collected and diluted to 10 mL with the sample solvent. Internal standard NAA-D3 was added to achieve a final concentration of 0.4 nM, thus preparing a mouse cerebral cortex sample solution. The mouse cerebral cortex sample solution was subjected to IT-SPME, and the eluent was collected for analysis by UHPLC-MS / MS to determine the concentrations of endogenous NAA, NAAG, and NAG in the mouse cerebral cortex.

[0074] Example 5

[0075] A highly sensitive analytical method for selectively enriching important biomarkers in brain tissue includes the following steps:

[0076] S1: The inner wall of the capillary was modified with 3-(trimethoxysilyl)propyl methacrylate to obtain a modified capillary column; then 140 mg GMA, 20 mg EDMA, 140 mg n-propanol / 60 mg 1,4-butanediol / 20 mg water and 2 mg AIBN were mixed evenly, ultrasonically degassed for 10 min, and injected into the modified capillary column. The two ends of the capillary were sealed with silicone rubber and placed in a 70℃ oven for polymerization reaction for 18 h. After the polymerization reaction was completed, the capillary was connected to a micro-injection pump and rinsed with methanol to remove unreacted monomers, crosslinking agents and pore-forming agents to obtain a poly(GMA-co-EDMA) organic polymer capillary monolithic column;

[0077] S2: 1 mL of 10% hydrochloric acid was introduced into the poly(GMA-co-EDMA) organic polymer capillary monolithic column using a micro-injection pump. The column was sealed at room temperature for 20 h. After rinsing with water until neutral, it was rinsed with acetonitrile. Then, 1 mL of TEA solution was passed through the poly(GMA-co-EDMA) organic polymer capillary monolithic column. The monolithic column was filled with TEA and sealed with silicone rubber. After reacting in an 80℃ oven for 12 h, the monolithic material was continuously rinsed with large amounts of water and methanol to obtain the TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column.

[0078] S3: An extraction device was prepared using a TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column. 5 mg of mouse cerebral cortex was weighed and added to 400 μL of cold extraction buffer (80% acetonitrile containing 0.1% formic acid). After homogenization and centrifugation, 10 μL of the supernatant was collected and diluted to 10 mL with the sample solvent. Internal standard NAA-D3 was added to achieve a final concentration of 0.4 nM, thus preparing a mouse cerebral cortex sample solution. The mouse cerebral cortex sample solution was subjected to IT-SPME, and the eluent was collected for analysis by UHPLC-MS / MS to determine the concentrations of endogenous NAA, NAAG, and NAG in the mouse cerebral cortex.

[0079] In the above embodiments, the sample solutions can be selected from the optimal conditions of pH 6.0, the addition of 10% acetonitrile, a volume of 1.0 mL, and acetone containing 10% formic acid as the elution solvent.

[0080] Figure 9The study showed the levels of endogenous NAA, NAAG, and NAG in different brain tissues. The concentrations of endogenous NAA in the cerebral cortex, cerebellum, and hippocampus of healthy 6-8 week old mice were 9.34 μmol / g, 8.11 μmol / g, and 7.76 μmol / g, respectively; the concentrations of NAG were 0.70 μmol / g, 0.65 μmol / g, and 0.89 μmol / g, respectively; and the concentrations of NAAG were 0.78 μmol / g, 1.02 μmol / g, and 0.93 μmol / g, respectively.

[0081] To investigate the morphology of the TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column, scanning electron microscopy (SEM) was used to characterize both the first-stage synthesized poly(GMA-co-EDMA) organic polymer capillary monolithic column and the final synthesized TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column. Figure 1 As shown in the image, the SEM images show that the monolithic material fills the entire capillary and is tightly bonded to the inner wall of the capillary, and has a porous structure, which ensures that the resulting monolithic column has good permeability and low column pressure.

[0082] Based on the developed IT-SPME technology, researchers further established a highly sensitive analytical method combining IT-SPME and LC-MS for the quantitative detection of NAA, NAAG, and NAG. The linear range, limit of detection, limit of quantitation, precision, accuracy, recovery, and matrix effects of this method were investigated in detail. The results are shown in Table 1. NAA and NAG showed good linearity in the range of 0.1–80 nM, and NAAG in the range of 0.2–160 nM; R 2 The values ​​ranged from 0.9950 to 0.9965. The limits of detection (SNR = 3) for the three target analytes were 0.019–0.052 nM, and the limits of quantitation (SNR = 10) were 0.064–0.173 nM, making it the most sensitive method developed to date for the detection of endogenous NAA, NAAG, and NAG. The intra-day and inter-day precision at low, medium, and high concentrations were all less than 15%, and the method accuracy ranged from 94.9% to 113.5%. In summary, this method exhibits high sensitivity and good precision, making it highly suitable for the quantitative detection of target analytes in real biological samples.

[0083] Next, the researchers evaluated the matrix effect and recovery rate of the aforementioned IT-SPME coupled with LC-MS method. Spiking experiments were conducted at low, medium, and high concentrations using mouse hippocampal tissue as the matrix, and the target analytes were determined using the newly developed method. Calculations showed that the recoveries of NAA ranged from 89.7% to 100.4%, NAAG from 105.7% to 109.1%, and NAG from 95.5% to 104.7%. The matrix effect was assessed using the post-extraction method, and the results showed a matrix effect of 94.5% to 103.5%. Therefore, this method does not exhibit significant matrix effect interference and can be used for the analysis of endogenous NAA, NAAG, and NAG in brain tissue samples.

[0084] Table 1. Methodological data for the combined IT-SPME and LC-MS analysis of NAA, NAAG, and NAG.

[0085]

[0086] Among them: low concentration solutions: containing 0.2 nM NAA, 0.2 nM NAG and 0.4 nM NAAG

[0087] Medium concentration solution: containing 1 nM NAA, 1 nM NAG and 2 nM NAAG

[0088] High-concentration solution: contains 50 nM NAA, 50 nM NAG and 100 nM NAAG.

[0089] To verify the selective enrichment ability of the newly developed IT-SPME / LC-MS method for NAA, NAAG, and NAG, the MRM spectra of the target analytes after direct injection and extraction enrichment using a TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column were compared. Figure 8 As shown, compared with direct UHPLC-MS / MS analysis, the signals of NAA, NAAG, and NAG significantly increased after extraction using the TEA@poly(GMA-co-EDMA) organic polymer capillary monolithic column. The enrichment factors (the ratio of the peak area of ​​the analyte after IT-SPME to the peak area before IT-SPME) for NAA, NAAG, and NAG were 10.1, 8.9, and 10.4, respectively, indicating that the TEA@poly(GMA-co-EDMA) monolithic column had ideal extraction efficiency for all three targets.

[0090] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. An analytical method for selectively enriching target analytes in brain tissue, characterized in that, The target analytes are N-acetylaspartic acid, N-acetylaspartylglutamic acid, and N-acetylaspartic acid, and the steps include: S1: The inner wall of a capillary column was modified with 3-(trimethoxysilyl)propyl methacrylate to obtain a modified capillary column; subsequently, monomers, crosslinking agents, porogens, and initiators were mixed and injected into the modified capillary column for polymerization. After the reaction, poly(GMA- co -EDMA) organic polymer capillary monolithic column; the monomer is glycidyl methacrylate; the crosslinking agent is ethylene glycol methacrylate; the porogen is n-propanol / 1,4-butanediol / water; the initiator is azobisisobutyronitrile; S2: In poly(GMA- co After passing hydrochloric acid through the EDMA organic polymer capillary monolithic column and rinsing until the washing solution is neutral, triethylamine solution is injected to initiate the reaction. After the reaction is complete, TEA@poly(GMA- co -EDMA) organic polymer capillary monolithic column; S3: Using TEA@poly(GMA- co An EDMA (Enhanced Mesotherapy) organic polymer capillary monolithic column extraction device was used to prepare the extraction apparatus. The target analyte in the brain tissue sample solution was then analyzed using the extraction apparatus. Elution was performed using an elution solvent, and the eluent was collected for content determination. The pH value of the brain tissue sample solution was 6.

0. The brain tissue sample solution contained 10% acetonitrile (by volume). The elution solvent was acetone containing 10% formic acid.

2. The analytical method for selectively enriching target analytes in brain tissue according to claim 1, characterized in that, The mass ratio of the monomer, crosslinking agent, porogen and initiator is (100~140):(20~60):(170~260):(1.2~2).

3. The analytical method for selectively enriching target analytes in brain tissue according to claim 2, characterized in that, The weight ratio of n-propanol, 1,4-butanediol and water is (100~140):(60~100):(10~20).

4. The analytical method for selectively enriching target analytes in brain tissue according to claim 1, characterized in that, In S1, the polymerization reaction is carried out at a temperature of 50-70 °C for 18-32 h.

5. The analytical method for selectively enriching target analytes in brain tissue according to claim 1, characterized in that, In S2, in poly(GMA- co After passing hydrochloric acid through the EDMA organic polymer capillary monolithic column, it is sealed at room temperature for 12-20 hours, rinsed with water until the washing solution is neutral, and then rinsed with acetonitrile.

6. The analytical method for selectively enriching target analytes in brain tissue according to claim 1, characterized in that, In S2, the ratio of the amount of hydrochloric acid, triethylamine solution and monomer in S1 is (1~3) mL: (1~3) mL: (100~140) mg; The reaction was carried out at a temperature of 80 °C for 12 h.

7. The analytical method for selectively enriching target analytes in brain tissue according to claim 1, characterized in that, In S3, the volume of the brain tissue sample solution to be tested is 1.0 mL.

Images