Method and kit for extracting and detecting concentration of monoamine neurotransmitters in saliva based on magnetic solid-phase extraction method

Abstract

The invention belongs to the technical field of monoamine neurotransmitter detection, and particularly relates to a method and kit for extracting and detecting the concentration of monoamine neurotransmitters in saliva based on a magnetic solid-phase extraction method, and the method comprises the following steps: taking a saliva sample, and adding magnetic solid-phase extraction magnetic beads with a weak cation exchange function and a methanol-containing aqueous solution as internal standards; the steps of activation, balancing, sample loading, leaching and elution are sequentially carried out, and the composition of an eluent is compatible with that of a high performance liquid chromatography mobile phase; carrying out separation and detection by adopting a liquid chromatography-electrochemical detector; and calculating the content of the monoamine neurotransmitter in the saliva sample according to a standard curve established by the internal standard. According to the invention, the extraction of epinephrine, noradrenaline, dopamine and serotonin can be automatically and quickly realized. A saliva sample is not filtered, and the phenomenon that the sample cannot flow out due to large particulate matter blocking the sieve plate is avoided. The operation difficulty is reduced, the eluent does not need to be subjected to nitrogen blowing treatment again, and the loss of a target object and the fluctuation of the recovery rate are avoided.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of monoamine neurotransmitter detection, and particularly relates to a method and a kit for extracting and detecting the concentration of monoamine neurotransmitters in saliva based on magnetic solid-phase extraction. BACKGROUND

[0002] As a non-invasive biological sample, saliva has significant clinical medical, neuroscientific, and psychological health research value in the detection of neurotransmitters such as adrenaline, norepinephrine, dopamine, and serotonin.

[0003] Adrenaline is the core hormone of the "fight or flight" response, and its saliva level can reflect acute stress states (such as trauma, anxiety), helping to assess psychological stress or post-traumatic stress disorder (PTSD). The saliva adrenaline level of patients with pheochromocytoma is significantly elevated, which can be used as an auxiliary diagnostic and postoperative recurrence monitoring indicator. In addition, abnormal adrenaline secretion may affect blood glucose regulation and be related to the early identification of diabetic ketoacidosis.

[0004] Elevated norepinephrine levels are closely related to chronic stress and anxiety, and saliva detection can reflect the state of overactivation of the autonomic nervous system. Persistent elevation of norepinephrine can induce hypertension or arrhythmia, and combined with blood indicators, it can be used to assess cardiovascular risk. In neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, the degeneration of norepinephrine neurons leads to a decrease in saliva levels, which can be used as a potential early biomarker.

[0005] Reduced dopamine levels are associated with depression and anhedonia, while elevated levels can induce mania or addictive behavior (such as drug dependence). Parkinson's disease patients have significantly reduced saliva dopamine, and detection can assist in diagnosis and monitoring of levodopa treatment effectiveness. Dopamine regulates attention and decision-making in the prefrontal cortex, and saliva detection provides a new perspective for the study of attention deficit hyperactivity disorder (ADHD).

[0006] Reduced serotonin levels are closely related to depression and anxiety, and saliva detection can be used to assess the therapeutic effect of drugs such as selective serotonin reuptake inhibitors (SSRIs). Serotonin is involved in circadian rhythm regulation, and its saliva level fluctuations can reflect the risk of sleep disorders or binge eating. Patients with irritable bowel syndrome (IBS) have abnormal saliva 5-HT levels, suggesting dysfunction of the brain-gut axis.

[0007] Currently, perchloric acid protein precipitation, dansyl chloride derivatization and other methods are commonly used for extraction and derivatization. Due to the extremely low content of adrenaline, norepinephrine, dopamine, serotonin in saliva, and the presence of mucin, various active enzymes and various complex interferents such as food residues in saliva, the traditional detection method is limited by low sensitivity, high complexity of pretreatment, long detection time, and cannot efficiently detect monoamine substances in saliva. There is no report on simultaneous detection of adrenaline, norepinephrine, dopamine and serotonin in saliva. The purpose of the present application is to provide a method for simultaneously detecting the concentrations of adrenaline, norepinephrine, dopamine and serotonin in saliva based on the prior art. Due to the key role of adrenaline, norepinephrine, dopamine and serotonin in nerve conduction regulation, the detection of related substances in saliva can help clinicians better diagnose, treat and evaluate the efficacy of patients with depression, mental illness, Parkinson's disease and Alzheimer's disease, which has important clinical significance.

[0008] The utility model patent with application number 202210524154.5 provided by Kangpuwei (Wuhan) Medical Technology Co., Ltd. provides a method for detecting neurotransmitters, which requires the use of PWCX solid-phase extraction column for pretreatment, the addition of various reagents such as methanol and ammonium acetate, and the sequential cleaning and activation of the solid-phase extraction column before the enrichment of the sample. After enrichment, the sample needs to be washed complexly, the eluent needs to be blown dry with nitrogen, and then redissolved with a solution. After a series of treatments, LC-MS / MS detection is performed, which is extremely complex and time-consuming and labor-intensive. The use of a mass spectrometer with high operation difficulty also poses a challenge to personnel pretreatment and instrument operation.

[0009] The invention patent with application number 202510098448.X provided by Shenzhen Hospital of Traditional Chinese Medicine provides a method for detecting neurotransmitters, which requires the use of derivatization for extraction and treatment. Derivatization requires the addition of highly toxic derivatizing reagents and precise selection of derivatization reaction conditions, such as the ratio of derivatizing reagents, the temperature of derivatization reaction, and the pH value of derivatization reaction. The sample after derivatization reaction needs to be extracted with ethyl acetate, the volume of the extract is large and needs to be blown dry with nitrogen, and then redissolved with a redissolution solution. The operation steps require precise addition of derivatizing reagents and precise control of pH and temperature. Ethyl acetate solvent is toxic to experimental personnel, and long-term manual processing will inevitably cause health problems. At the same time, the derivatization experimental conditions depend on the experience of the experimenter, and the method conversion is difficult, which is not conducive to the promotion in the clinic.

[0010] The application number 201510076850.4 Chinese People's Liberation Army Military Medical College Institute of Toxicology and Drug Research provides a kind of neurotransmitter substance detection kit, and online derivatization is used with the method of online solid phase extraction for pre-treatment.Online derivatization is used with online solid phase extraction, and complex liquid pipeline system and liquid phase control software system need to be built.The sample after derivatization needs to be accurately controlled to pass through online solid phase extraction column for enrichment, but with the detection of sample, nonlinear pollutant accumulation will occur in chromatographic column, which will cause abnormal attenuation of chromatographic separation capacity of chromatographic column, reduce system repeatability and cause abnormal analysis performance.It is difficult to apply to the detection of clinical sample with high repeatability and reliability. SUMMARY

[0011] The purpose of the present application is to solve the problems in the prior art, such as extremely complex operation steps, time-consuming and laborious, and difficulty in application for clinical sample detection with high repeatability and reliability.

[0012] In order to achieve the above purpose, the present application provides a saliva monoamine neurotransmitter extraction and detection method based on magnetic solid phase extraction, characterized in that it comprises the following steps:

[0013] S1. Take saliva sample, add magnetic solid phase extraction magnetic beads with weak cation exchange function and methanol aqueous solution containing 1-5 ng / mL 3,5-dihydroxybenzoic acid as internal standard;

[0014] S2. Perform activation, balance, sample loading, elution and elution steps in sequence, wherein the volume ratio of elution liquid to elution liquid in elution and elution steps is 6:1, and the composition of the elution liquid is compatible with high performance liquid chromatography mobile phase, so that the obtained elution liquid can be directly injected for detection without nitrogen blowing;

[0015] S3. Separate and detect the elution liquid by liquid chromatography-electrochemical detector;

[0016] S4. Calculate the content of monoamine neurotransmitter in the saliva sample according to the standard curve established by the internal standard.

[0017] Preferably, the volume fraction of methanol in the methanol aqueous solution in S1 is 10%-50%.

[0018] Preferably, the methanol aqueous solution in S1 can be replaced by a buffer solution, and the buffer solution is ammonium acetate solution with a concentration of 10-50 mmol / L.

[0019] Preferably, the shock stirring time of activation, balance and elution in S2 is 1 min, and the shock stirring time of sample loading and elution is 5 min, and the standing and magnetic absorption time is 1 min.

[0020] Preferably, the mobile phase in the high performance liquid chromatography separation in S2 is 10mmol / L-50mmol / L sodium dihydrogen phosphate, 1mmol / L-5mmol / L sodium octane sulfonate and 100μmmol / L-500μmmol / L disodium ethylenediaminetetraacetate.

[0021] Preferably, the monoamine neurotransmitter comprises at least one of adrenaline, norepinephrine, dopamine and serotonin.

[0022] Preferably, the analysis condition of the high performance liquid chromatography-electrochemical detector in S3 is: mobile phase 50mmol / L sodium dihydrogen phosphate, 200μmol / L disodium ethylenediaminetetraacetate, 1mmol / L sodium octane sulfonate, 10% acetonitrile; flow rate 1000μL / min; column temperature 35℃; electrochemical detector voltage +750mV vs.Ag / AgCl.

[0023] Preferably, the liquid chromatography-electrochemical detector in S3 adopts isocratic gradient, the chromatographic column is C18 column, 4.6*250mm, 5μm, and the single needle analysis time is 30min.

[0024] The application also provides a saliva monoamine neurotransmitter detection kit for the method, characterized by comprising:

[0025] Magnetic solid phase extraction magnetic beads with weak cation exchange function;

[0026] Internal standard of 10%-50% methanol aqueous solution containing 1-5ng / mL 3,5-dihydroxybenzoic acid;

[0027] Activation solution, equilibrium solution, elution solution and elution solution compatible with high performance liquid chromatography mobile phase;

[0028] Automatic magnetic bead extraction plate or adaptive container.

[0029] Compared with the prior art, the application has the advantages of:

[0030] 1. The application can automatically and quickly extract adrenaline, norepinephrine, dopamine and serotonin in saliva samples through weak cation solid phase extraction magnetic beads. The saliva sample is not filtered, and the problem of large particle blocking the screen plate in solid phase extraction, which leads to the sample unable to flow out, is avoided.

[0031] 2. The use of automatic magnetic bead extraction equipment effectively reduces the operation difficulty of experiment personnel. Only the sample needs to be added to the automatic processing plate and placed in the machine for automatic processing. The whole processing process only needs 22min, and personnel do not need to intervene at all, avoiding the need for personnel to operate and add various reagents throughout the process in the traditional solid phase extraction column method.

[0032] 3. The reasonable proportion of elution and washing components can effectively remove the sticky protein, food residue and other interference substances in the saliva. The eluent does not need to be treated by nitrogen blowing again, so as to avoid the loss of target substances and the fluctuation of recovery rate in the nitrogen blowing process. The eluent components are completely compatible with the mobile phase sample.

[0033] 4. In the detection method, the isocratic gradient is used for the mobile phase, and the single needle analysis time is about 30 minutes. The HPLC-ECD detector is matched. The ECD detector has specific sensitivity to adrenaline, norepinephrine, dopamine and serotonin. When detecting redox substances, the detection limit matches the sensitivity of mass spectrometry. The daily use and maintenance cost of the ECD detector is much lower than that of the mass spectrometer, and the economy is better. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 , which is the linear curve of norepinephrine standard;

[0035] Figure 2 , which is the linear curve of adrenaline standard;

[0036] Figure 3 , which is the linear curve of dopamine standard

[0037] Figure 4 , which is the linear curve of serotonin standard

[0038] Figure 5 , which is the detection spectrum of adrenaline, norepinephrine, dopamine and serotonin and internal standard DHBA; wherein 1) is adrenaline, 2) is norepinephrine, 3) is dopamine, 4) is serotonin, and 5) is internal standard DHBA. DETAILED DESCRIPTION

[0039] The terms used in the present application have the meanings generally understood by those of ordinary skill in the art, unless otherwise specified.

[0040] In the following examples, various processes and methods not specifically described are conventional methods in the art.

[0041] The reagents used in the following examples are commercially available, and the experimental operations and experimental conditions not mentioned are referred to the conventional operations and conventional conditions in the art.

[0042] The specific embodiments of the present application are illustrated in the following examples.

[0043] The present embodiment provides a method for extracting and detecting monoamine neurotransmitters in saliva based on magnetic solid phase extraction.

[0044] I. Reagent preparation

[0045] Internal standard solution

[0046] Internal standard solution is a solution containing 2 ng / ml DHBA (3,5-dihydroxybenzoic acid) in 10% methanol water.

[0047] Quality control

[0048] Adrenaline, norepinephrine, dopamine, and serotonin were respectively configured as stock solutions using 0.1M hydrochloric acid, and then diluted to obtain working solutions, the working solution concentrations of norepinephrine, dopamine, and serotonin were all 1ug / ml. Using 0.1% PBSA (polybutylene succinate adipate), each working solution was diluted and mixed to prepare three quality controls with different concentrations, and the specific concentrations of the quality controls are shown in Table 1 below:

[0049] Table 1

[0050]

[0051] Standard

[0052] Adrenaline, norepinephrine, dopamine, and serotonin were respectively configured as stock solutions using 0.1M hydrochloric acid, and then diluted to obtain working solutions, the working solution concentrations of norepinephrine, dopamine, and serotonin were all 1ug / ml. Using 10% methanol water, each working solution was diluted and mixed to prepare three quality controls with different concentrations, and the specific concentrations of the standard are shown in Table 2 below:

[0053] Table 2

[0054]

[0055]

[0056] The prepared standard and quality control were respectively sub-packed in cryogenic tubes and stored at -20 degrees Celsius. When used, the cryogenic tubes were placed in a backlit cool place for 20 minutes, and then used after vortex mixing for 5 minutes.

[0057] II. The following steps were taken to extract the test substance:

[0058] S1. Take 500ul of standard, quality control or saliva sample in a 1.5mL centrifuge tube, add 20uL of internal standard solution, shake and mix, then add 500ul of 50mmol / L ammonium acetate solution, vortex and shake for 5min;

[0059] S2. Turn on the automatic magnetic bead extractor, and after the instrument self-check is completed, select the corresponding detection program;

[0060] S3. Take an automatic magnetic bead extraction plate, remove the aluminum film cover on the surface of the automatic magnetic bead extraction plate, mix the standard, quality control or saliva sample with the internal standard solution and buffer solution in the sample slot position (3, 9 position), and then add it into the sample slot. The loading of samples in the automatic magnetic bead extraction plate is shown in Table 3 below;

[0061] S4. Place the automatic magnetic bead extraction plate in the extraction instrument, select monoamine sample analysis in the control panel, and the specific program of the automatic extraction instrument is shown in Table 4 below;

[0062] S5. After waiting for the sample processing to be completed, use a pipette to remove the processed samples in slots 6 and 12, and add them into the sample bottle for HPLC-ECD detection.

[0063] Table 3

[0064]

[0065] Table 4

[0066]

[0067]

[0068] HPLC-ECD detection

[0069] Chromatographic conditions:

[0070] The chromatographic column is ENM-Miracle C18 120A5um 4.6X250mm

[0071] The mobile phase is 50mmol / L NaH2PO4, 200μmmol / L EDTA·2Na, 1mmol / L sodium octane sulfonate, 10% acetonitrile

[0072] Column temperature: 35℃

[0073] The sampling isocratic gradient condition, and the specific liquid chromatography conditions are shown in Table 5 below:

[0074] Table 5

[0075]

[0076] The parameters of electrochemical detection are shown in Table 6 below:

[0077] Table 6

[0078] Item Analysis Condition Flow Rate 1000 μL / min Column Temp. 35℃ Applied potential +750 mV vs. Ag / AgCl Time Constant 3.0 sec Analysis time 30 min Injection volume 40ul

[0079] III. Effect comparison

[0080] Effect comparison 1 (linear curve fitting degree)

[0081] According to the method of the above embodiment, the weak cation exchange magnetic solid phase extraction magnetic beads are used to extract and detect the standard samples with DHBA as the internal standard. The standard curve is established by taking the concentration ratio of the standard sample as the x-axis and the peak area ratio of the standard sample and the internal standard as the y-axis. The results show that the linear fitting equation of the monoamine neurotransmitter substances in their respective concentration ranges is good, and the correlation coefficient is above 0.99. The obtained standard curve, linear equation and linear range are shown in Table 7, and the detection spectrum of adrenaline, norepinephrine, dopamine, serotonin and the internal standard DHBA is shown in Table 8. Figure 1 - Figure 4 Figure 5

[0082] Table 7

[0083]

[0084]

[0085] Effect comparison 2 (linear point deviation)

[0086] According to the actual concentration value of the standard sample detected in effect 1, the deviation from the theoretical value is calculated, and the results are shown in the table. According to Table 8, the deviation of the standard sample detection results is within 15%. It shows that the method of extracting monoamine neurotransmitters in saliva based on weak cation exchange magnetic solid phase extraction magnetic beads is feasible and reliable.

[0087] Table 8

[0088] Compound / Linear point Norepinephrine Epinephrine Dopamine Serotonin Cal 1 -8.06％ -7.34％ -5.94％ -2.16％ Cal 2 -7.25％ -6.53％ -5.40％ -2.25％ Cal 3 -1.38％ -1.79％ -1.58％ -4.93％ Cal 4 2.06％ 2.17％ 1.82％ 9.65％ Cal 5 1.07％ 0.99％ 0.84％ -0.07％ Cal 6 -0.65％ -0.62％ -0.53％ -0.88％

[0089] Effect comparison 3 (extraction recovery rate and precision)

[0090] According to the method of the embodiment, the weak cation exchange magnetic solid phase extraction magnetic beads are used to extract and detect the three concentration points of the quality control sample.

[0091] The concentration ratio of the measured quality control sample and the corresponding standard solution theoretical concentration is calculated to calculate the extraction recovery rate of the weak cation exchange magnetic solid phase extraction magnetic beads. The calculated detection extraction recovery rate is shown in Table 9. The extraction recovery rate is between 85% and 115% (as shown in Table 9). It shows that the method of extracting monoamine neurotransmitters in saliva based on weak cation exchange magnetic solid phase extraction magnetic beads is feasible and reliable.

[0092] Table 9

[0093] Recovery Norepinephrine Epinephrine Dopamine Serotonin LQC 92.56％ 98.55％ 99.18％ 88.42％ MQC 94.38％ 96.12％ 103.54％ 86.75％ HQC 95.43％ 93.71％ 95.75％ 89.72％

[0094] As can be seen from the above, the calculated extraction recovery rates of the four compounds are between 85% and 115%, which meet the detection requirements.

[0095] ​​At the same time, each quality control sample was determined in parallel for 5 times, and determined continuously for 3 days, and the intra-day and inter-day precision was calculated. The calculated intra-day and inter-day precision of the detected is shown in Table 10, according to the results, the intra-day precision of the quality control sample CV is between 15%, and the inter-day precision of the quality control sample CV is between 15%. It is shown that the extraction of monoamine neurotransmitters in saliva based on weak cation exchange magnetic solid phase extraction magnetic beads is feasible and reliable.

[0096] Table 10

[0097]

[0098]

[0099] The calculated intra-day precision of the quality control sample CV of the four compounds is less than 15%, and the inter-day precision of the quality control sample CV is less than 15%, which meets the detection requirements.

[0100] The above is a detailed description of the embodiments, which facilitates the correct understanding and use of the present application by those skilled in the art. Any improvement or modification technical solution obtained by those skilled in the art on the basis of the prior art without innovative labor, only through analysis, analogy or limited enumeration and other methods, should be within the protection scope determined by the claims.

Claims

1. A method for extracting and detecting monoamine neurotransmitters in saliva based on magnetic solid-phase extraction, characterized in that, Includes the following steps: S2. Perform activation, equilibration, sample loading, rinsing and elution steps in sequence, wherein the volume ratio of rinsing solution to eluent in the rinsing and elution steps is 6:1, and the composition of the eluent is compatible with the high performance liquid chromatography mobile phase so that the obtained eluent can be directly injected for detection without nitrogen drying. S3. The eluent was separated and detected using liquid chromatography-electrochemical detector; S4. Calculate the content of monoamine neurotransmitters in the saliva sample based on the standard curve established by the internal standard.

2. The method for extraction and detection of monoamine neurotransmitters in saliva based on magnetic solid-phase extraction as described in claim 1, characterized in that, The methanol volume fraction in the methanol aqueous solution in S1 is 10% to 50%.

3. The method for extraction and detection of monoamine neurotransmitters in saliva based on magnetic solid-phase extraction as described in claim 1, characterized in that, The methanol-water solution in S1 can also be replaced with a buffer solution, wherein the buffer solution is an ammonium acetate solution with a concentration of 10 mmol / L-50 mmol / L.

4. The method for extraction and detection of monoamine neurotransmitters in saliva based on magnetic solid-phase extraction as described in claim 1, characterized in that, The stirring time for activation, equilibration, and rinsing in S2 is 1 min, the stirring time for sample loading and elution is 5 min, and the static magnetic absorption time is 1 min.

5. The method for extraction and detection of monoamine neurotransmitters in saliva based on magnetic solid-phase extraction as described in claim 1, characterized in that, The mobile phase used in S2 for high-performance liquid chromatography separation contains 10 mmol / L-50 mmol / L sodium dihydrogen phosphate, 1 mmol / L-5 mmol / L sodium octanesulfonate, and 100 μmmol / L-500 μmmol / L disodium ethylenediaminetetraacetate.

6. The method for extraction and detection of monoamine neurotransmitters in saliva based on magnetic solid-phase extraction as described in claim 1, characterized in that, The monoamine neurotransmitters include at least one of adrenaline, noradrenaline, dopamine, and serotonin.

7. The method for extraction and detection of monoamine neurotransmitters in saliva based on magnetic solid-phase extraction as described in claim 1, characterized in that, The analytical conditions for high performance liquid chromatography-electrochemical detector in S3 are as follows: mobile phase: 50 mmol / L sodium dihydrogen phosphate, 200 μmol / L disodium ethylenediaminetetraacetate, 1 mmol / L sodium octanesulfonate, 10% acetonitrile; flow rate: 1000 μL / min; column temperature: 35 ℃; electrochemical detector voltage: +750 mV vs. Ag / AgCl.

8. The method for extraction and detection of monoamine neurotransmitters in saliva based on magnetic solid-phase extraction as described in claim 1, characterized in that, The liquid chromatography-electrochemical detector in S3 uses an isocratic gradient, the chromatographic column is a C18 column, 4.6×250mm, 5μm, and the single-needle analysis time is 30min.

9. A salivary monoamine neurotransmitter detection kit for use in the method according to any one of claims 1 to 8, characterized in that, include: Magnetic solid-phase extraction beads with weak cation exchange function; Internal standard: 10%–50% methanol aqueous solution containing 1–5 ng / mL 3,5-dihydroxybenzoic acid; Activation solution, equilibration solution, rinsing solution, and elution solution, wherein the elution solution is compatible with the mobile phase of high performance liquid chromatography; Automatic magnetic bead extraction plate or compatible container.

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