Pretreatment method for sample to be detected, and method and kit for simultaneously detecting multiple neurotransmitters

Abstract

The invention discloses a to-be-detected sample pretreatment method, a method for simultaneously detecting multiple neurotransmitters and a kit, and the to-be-detected sample pretreatment method comprises the following steps: carrying out pretreatment on a to-be-detected sample by adopting WCX solid-phase extraction magnetic beads; the to-be-detected sample is treated and then is used for detection by adopting an isotope internal standard method and a liquid chromatography-tandem mass spectrometry method at the same time. According to the technical scheme, multiple neurotransmitters, especially one or more of dopamine, noradrenaline, 5-hydroxytryptamine, gamma-aminobutyric acid and melatonin can be accurately detected at the same time, the method has very high sensitivity, the to-be-detected substance in the sample is enriched while automatic sample treatment is achieved, the detection sensitivity is high, and the detection accuracy is high. Complex operation is not needed, the pretreatment time is greatly shortened, combined detection of metabolites of different concentration levels is achieved, interference of complex matrixes on target detection is eliminated, the method is rapid and stable, and the kit can be stable for 2 years at the temperature of 2-8 DEG C.

Description

Technical Field

[0001] The present invention belongs to the technical field of blood testing, and in particular relates to a method for pre-treating a sample to be tested, a method for simultaneously detecting multiple neurotransmitters, and a kit. Background Art

[0002] With the accelerating pace of modern life, more and more people are experiencing sleep disorders. Neurotransmitters play a key role in maintaining chemical balance in the brain. In recent years, numerous studies have demonstrated that neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and melatonin are closely linked to sleep regulation. Dopamine and norepinephrine are excitatory neurotransmitters. Excessive levels of these neurotransmitters can lead to difficulty falling asleep or staying asleep, while low levels can cause hypersomnia. Serotonin, GABA, and melatonin are inhibitory neurotransmitters. Low levels of these neurotransmitters can lead to sleep difficulties and poor sleep quality in patients with sleep disorders. GABA and melatonin, as medications, also have therapeutic effects on insomnia. By measuring the levels of these neurotransmitters, doctors can more accurately determine the type of sleep disorder a patient has and its potential causes, thereby developing more effective treatment plans and monitoring blood drug concentrations and therapeutic efficacy.

[0003] At present, the clinical detection methods of neurotransmitters mainly include enzyme-linked immunosorbent assay, chemiluminescence, high-performance liquid chromatography and liquid chromatography-mass spectrometry. Among them, enzyme-linked immunosorbent assay and chemiluminescence are easy to operate and have a short detection time, but because they are based on antibody-antigen reaction, the detection specificity is poor and it is impossible to detect multiple metabolites simultaneously. High-performance liquid chromatography can separate multiple metabolites through liquid phase, but the sensitivity is low. When performing multi-index combined detection, it cannot meet the detection requirements of indicators at different concentration levels. Liquid chromatography-mass spectrometry (LC-MS / MS) has very high sensitivity, specificity and accuracy, and can detect multiple metabolites at a time, providing richer information for clinical diagnosis, and is suitable for the detection of neurotransmitters.

[0004] In the study "Study on the Effects of Bisphenol Exposure on Neurotransmitter Metabolism Based on UHPLC-MS / MS Technology", Nanjing Medical University established a liquid chromatography-tandem mass spectrometry detection method for 27 neurotransmitters including dopamine, norepinephrine, 5-hydroxytryptamine, γ-aminobutyric acid, and melatonin. However, the sample processing process involves complex operations such as extraction, volatilization, and derivatization, which cannot be automated. In addition, the derivatization process is easily affected by multiple factors such as pH, temperature, and time, and the reaction needs to be carried out under high temperature conditions, which is not suitable for clinical high-throughput detection.

[0005] Therefore, the existing methods need to be improved, and it is hoped to obtain a method that is fast, simple to operate, and suitable for high-throughput detection. Summary of the Invention

[0006] In view of the defects in the prior art, the purpose of the present invention is to provide a method for pre-treatment of samples to be tested, a method and a kit for simultaneously detecting multiple neurotransmitters. In the technical solution described in the present invention, by innovatively adopting WCX solid-phase extraction magnetic bead technology, combined with the LC-MS / MS (high performance liquid chromatography) method of optimized gradient elution, the detection of multiple neurotransmitters can be completed simultaneously in a short time. It should be pointed out that in the technical solution described in the present invention, the pre-treatment process has achieved a breakthrough in fully automated operation, shortening the processing time by 80% compared with the traditional solid-phase extraction method, and avoiding the derivatization step, and effectively eliminating matrix interference through selective enrichment of magnetic beads.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] In a first aspect, the present invention provides a method for pre-treating a sample to be tested, the method comprising the steps of: pre-treating the sample to be tested using WCX solid phase extraction magnetic beads;

[0009] The sample to be tested is processed and used for detection using both the isotope internal standard method and the liquid chromatography-tandem mass spectrometry method.

[0010] Preferably, the sample to be tested is serum or plasma.

[0011] Preferably, the specific operation of the pre-treatment using WCX solid phase extraction magnetic beads is as follows:

[0012] Step S11: taking 500 μL of the sample to be tested, adding 50 μL of internal standard solution and 400 μL of buffer solution thereto, and mixing to obtain a first treatment solution;

[0013] Step S12: Take WCX solid phase extraction magnetic beads and add them to 400 μL of equilibration solution, and shake them for equilibrium;

[0014] Step S13: mixing the magnetic beads after oscillation equilibrium in step S12 and the first treatment solution obtained in step S11 to obtain a second treatment solution and adsorbed magnetic beads;

[0015] Step S14: taking the adsorbed magnetic beads and washing them with 400 μL of eluent and 100 μL of elution solution in sequence, and the eluted liquid is the third treatment liquid;

[0016] Step S15: 2 μL of the second treatment liquid is added to the third treatment liquid, and the mixture is mixed to obtain a pre-treated sample to be tested.

[0017] Preferably, the buffer is ammonium acetate buffer with a pH of 8 to 10;

[0018] and / or, the equilibrium solution is an aqueous solution containing 0-10 vol% methanol;

[0019] and / or, the eluent is an aqueous solution containing 0-10 vol% methanol;

[0020] and / or, the eluent is a 50 vol% methanol aqueous solution containing 1-5 vol% formic acid;

[0021] And / or, the internal standard solution is one or more of the following: 3 ng / mL dopamine-d4, 30 ng / mL norepinephrine-d6, 200 ng / mL serotonin-d4, 1600 ng / mL γ-aminobutyric acid-d6, 0.6 ng / mL melatonin-d4.

[0022] Preferably, in step S12, before adding the WCX solid phase extraction magnetic beads to 400 μL of the equilibration solution, the WCX solid phase extraction magnetic beads are pre-activated with methanol to obtain activated magnetic beads, and the amount of the activated magnetic beads added is 1 to 5 mg.

[0023] In a second aspect, the present invention provides a method for simultaneously detecting multiple neurotransmitters, the method comprising the following steps:

[0024] Step S1: treating the sample to be tested using the above-mentioned sample pretreatment method, and subjecting the treated sample to be tested to high performance liquid chromatography to separate the target analyte from the interfering components in the sample to determine the target analyte;

[0025] Step S2: detecting the mass-to-charge ratio response of the target analyte and its corresponding isotope internal standard by a mass spectrometer;

[0026] Step S3: quantification using the isotope internal standard method, with the concentration ratio of the target analyte to the internal standard as the X-axis and the peak area ratio of the target analyte to the internal standard as the Y-axis, to establish a calibration curve, and calculate the content of the target analyte based on the standard curve;

[0027] The target analyte is a neurotransmitter and / or its metabolite, and the neurotransmitter is one or more of the following: dopamine, norepinephrine, 5-hydroxytryptamine, gamma-aminobutyric acid and melatonin.

[0028] Preferably, in step S1, the high performance liquid chromatography adopts all conditions:

[0029] Condition A: Mobile phase A was 0.05 vol% formic acid in water containing 2 mM ammonium acetate;

[0030] Condition B: Mobile phase B was 0.05 vol% formic acid in methanol containing 2 mM ammonium acetate;

[0031] Condition C: The column was Phenomenex F5, with a particle size of 2.6 μm and a size of 3 × 100 mm;

[0032] Condition D: Use a flow rate of 0.6-0.8 mL / min;

[0033] Condition E: column temperature 40-45°C;

[0034] Condition F: injection volume is 5-7 μL.

[0035] Preferably, when the high performance liquid chromatography performs gradient elution, the specific operation is as follows:

[0036] The initial ratio of mobile phase A to mobile phase B was 98:2;

[0037] The gradient elution process is as follows: within 0-1 minute, the volume ratio of mobile phase A to mobile phase B is gradually changed from 98:2 to 35:65 at a constant speed;

[0038] The volume ratio of mobile phase A to mobile phase B was gradually changed from 35:65 to 5:95 at a constant speed within 1-2 minutes;

[0039] The volume ratio of mobile phase A to mobile phase B was maintained at 5:95 for 2–3 min;

[0040] During 3.1-4 minutes, the ratio of mobile phase A to mobile phase B was maintained at the initial ratio.

[0041] Preferably, in step S2, the mass spectrometer is specifically operated as follows: in electrospray ionization mode, multiple reaction monitoring is used to perform positive ion mode scanning; the electrospray voltage is 4500V, the ion source temperature is 550°C, the curtain gas is 25psi, the collision gas is 8unit, and the ion source gases 1 and 2 are both 55psi.

[0042] In a third aspect, the present invention proposes a kit comprising a test sample pretreatment reagent, a standard curve, an internal standard solution, and a quality control product, characterized in that the kit uses the above-mentioned test sample pretreatment method to treat the test sample and / or uses the above-mentioned method to detect the test sample.

[0043] In some preferred embodiments, the calibration curve may be one or more of the following:

[0044] The six dopamine concentrations were: 5 pg / mL, 15 pg / mL, 50 pg / mL, 150 pg / mL, 500 pg / mL, and 1250 pg / mL;

[0045] The six norepinephrine concentrations were: 60 pg / mL, 180 pg / mL, 600 pg / mL, 1800 pg / mL, 6000 pg / mL, and 15000 pg / mL;

[0046] The six concentrations of 5-HT were: 4 ng / mL, 12 ng / mL, 40 ng / mL, 120 ng / mL, 400 ng / mL, and 1000 ng / mL;

[0047] The six concentrations of γ-aminobutyric acid were: 5 ng / mL, 15 ng / mL, 50 ng / mL, 150 ng / mL, 500 ng / mL, and 1250 ng / mL;

[0048] The seven concentrations of melatonin are: 0.5pg / mL, 4pg / mL, 12pg / mL, 40pg / mL, 120pg / mL, 400pg / mL and 1000pg / mL.

[0049] In some preferred embodiments, the internal standard solution can be one or more of the following:

[0050] 3ng / mL dopamine-d4, 30ng / mL norepinephrine-d6, 200ng / mL serotonin-d4, 1600ng / mL gamma-aminobutyric acid-d6, 0.6ng / mL melatonin-d4;

[0051] In some preferred embodiments, the quality control product may be one or more of the following:

[0052] The two concentrations of dopamine were: 25 pg / mL and 750 pg / mL;

[0053] The two concentrations of norepinephrine were: 300 pg / mL and 9000 pg / mL;

[0054] The two concentrations of 5-HT were: 20 ng / mL and 600 ng / mL;

[0055] The two concentrations of γ-aminobutyric acid were: 25 ng / mL and 750 ng / mL;

[0056] The two concentrations of melatonin are: 20pg / mL and 600pg / mL.

[0057] In addition, in some embodiments, the pretreatment reagents for the sample to be tested include a magnetic bead liquid, an eluent, a buffer solution, a balancing liquid, and a rinse solution, wherein the magnetic bead liquid can be, for example, a methanol solution of 10 mg / mL WCX solid phase extraction magnetic beads; the eluent can be, for example, a 50 vol% methanol aqueous solution containing 2 vol% formic acid; the buffer solution can be, for example, an ammonium acetate buffer solution with a pH of 9; the balancing liquid can be, for example, an aqueous solution containing 5 vol% methanol; and the rinse solution can be, for example, an aqueous solution containing 5 vol% methanol.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] (1) The technical solution described in the present invention provides a method for simultaneously detecting dopamine, norepinephrine, 5-hydroxytryptamine, γ-aminobutyric acid, and melatonin in human serum or plasma. The method has very high sensitivity. Specifically, the detection sensitivity of dopamine can reach 5 pg / mL, the detection sensitivity of norepinephrine can reach 60 pg / mL, the detection sensitivity of 5-hydroxytryptamine can reach 4 ng / mL, the detection sensitivity of γ-aminobutyric acid can reach 5 ng / mL, and the detection sensitivity of melatonin can reach 0.5 pg / mL.

[0060] (2) The technical solution described in the present invention uses WCX solid-phase extraction magnetic beads to selectively extract the analyte from the blood sample, purifying and enriching the analyte in the sample while realizing automated sample processing. Therefore, the technical solution described in the present invention does not require complex operations such as vacuum drying and heated nitrogen blowing, and greatly shortens the pre-processing time.

[0061] (3) The technical solution described in the present invention achieves the joint detection of five metabolites at different concentration levels through optimization of pretreatment and liquid chromatography-mass spectrometry conditions, eliminating the interference of complex matrices on target detection. In some preferred embodiments, the simultaneous detection of five neurotransmitters can be achieved within 4 minutes.

[0062] The kit prepared by the technical solution of the present invention can be stable for 2 years at 2-8°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The chromatograms of the neurotransmitters to be detected and their internal standards in the standard curve, where dopamine (DA), norepinephrine (NE), serotonin (5-HT), melatonin (MT) and gamma-aminobutyric acid (GABA) are the analytes, and their corresponding isotopic internal standards are the internal standards, namely DA-d4, NE-d6, GABA-d6, 5-HT-d4 and MT-d4;

[0064] Figure 2The figure shows the chromatograms of the neurotransmitters to be tested and their internal standards in clinical samples. Among them, dopamine (DA), norepinephrine (NE), serotonin (5-HT), melatonin (MT), and gamma-aminobutyric acid (GABA) are the analytes, and their corresponding isotopic internal standards are internal standards, namely DA-d4, NE-d6, GABA-d6, 5-HT-d4, and MT-d4. DETAILED DESCRIPTION

[0065] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0066] Example 1

[0067] Step 1: Sample preparation

[0068] 1. Preparation of standard curve, internal standard solution and quality control

[0069] (1) Standards and internal standards of dopamine, norepinephrine, 5-hydroxytryptamine, γ-aminobutyric acid, and melatonin were prepared into primary stock solutions according to Table 1, and further prepared into secondary stock solutions according to Table 2.

[0070] Table 1. Standards and internal standard primary stock solutions

[0071]

[0072] Table 2. Secondary stock solutions of standards and internal standards

[0073]

[0074] (2) Prepare the mixed working solution by mixing the stock solutions in Table 1 and Table 2 according to Table 3.

[0075] Table 3. Standard mixed working solution

[0076]

[0077] (3) The mixed working solution in Table 3 was prepared into a standard working solution and a quality control working solution according to the concentrations shown in Table 4 and Table 5 below, using a 50 vol% methanol aqueous solution containing 0.1 vol% formic acid as the solvent.

[0078] Table 4. Calibration curve working solution

[0079]

[0080] Table 5. Quality Control Working Solution

[0081]

[0082] (4) Preparation of standard and quality control samples: 4 g of bovine serum albumin was added to 96 mL of purified water. After vortex mixing, 2 mL of 100 mg / mL ascorbic acid and 2 mL of 100 mg / mL citric acid were added to prepare the matrix. The matrix and each standard working solution / quality control working solution were mixed at a volume ratio of 49:1 to prepare standard and quality control samples of different concentrations. The concentrations of the standard and quality control samples are shown in Table 6.

[0083] Table 6. Standard curves and quality control samples at different concentrations

[0084]

[0085] (5) Prepare the internal standard stock solutions in Tables 1 and 2 into mixed internal standard working solutions according to Table 7 below.

[0086] Table 7. Mixed internal standard working solution

[0087]

[0088] (6) Prepare the internal standard solution by mixing the internal standard working solution in Table 7 according to Table 8 below.

[0089] Table 8. Internal standard solution

[0090]

[0091] Step 2: Sample pretreatment

[0092] WCX solid-phase extraction magnetic beads were purchased from Biosepur biology, and magnetic bead extractor (model: NE-02-H-96) was purchased from Guangzhou Bay Area Biotechnology Co., Ltd.

[0093] (1) Take 500 μL of the sample to be tested, add 50 μL of internal standard solution and 400 μL of pH 9 ammonium acetate buffer, and vortex to mix to obtain the first treatment solution;

[0094] (2) 2 mg of methanol-activated WCX solid phase extraction magnetic beads were placed in a 96-well plate, placed in a magnetic bead extractor, added to 400 μL of 5 vol% methanol aqueous solution, and shaken for 1 min;

[0095] (3) adding the magnetic beads after oscillation equilibrium in step (2) to the first treatment solution and shaking for 2.5 minutes to obtain the second treatment solution and the adsorbed magnetic beads;

[0096] (4) The adsorbed magnetic beads were first washed with 400 μL of 5 vol% methanol aqueous solution for 1 min, and then eluted with 100 μL of 50 vol% methanol aqueous solution containing 2 vol% formic acid for 2.5 min to obtain a third treatment solution;

[0097] (5) Take 2 μL of the second treatment solution and add it to the third treatment solution, mix well to obtain a pretreated sample for LC-MS / MS detection.

[0098] Step 3: LC-MS / MS detection

[0099] In this embodiment, the instrument used is a high performance liquid chromatography-tandem mass spectrometer, model AB SCIEX6500.

[0100] (1) Chromatographic conditions are:

[0101] Mobile phase A: 0.05 vol% formic acid in water containing 2 mM ammonium acetate;

[0102] Mobile phase B: 0.05 vol% formic acid in methanol containing 2 mM ammonium acetate;

[0103] Chromatographic column: Phenomenex F5, 2.6 μm, 3*100 mm;

[0104] The flow rate was 0.6 mL / min, the column temperature was 40 °C, and the injection volume was 5 μL;

[0105] The gradient elution parameters are shown in Table 9 below.

[0106] Table 9. Gradient elution table

[0107] Time (min) Mobile phase B (%) B.Curve 0 2 0 1 65 0 2 95 0 3 95 0 3.1 2 0 4 2 0

[0108] (2) Mass spectrometry conditions are:

[0109] In electrospray ionization mode, multiple reaction monitoring was used for positive ion mode scanning; the electrospray voltage was 4500 V, the ion source temperature was 550°C, the curtain gas was 25 psi, the collision gas was 8 units, and the ion source gases 1 and 2 were both 55 psi.

[0110] The mass spectrometry acquisition parameters used to detect five neurotransmitters and their internal standards are shown in Table 10 below.

[0111] Table 10. Mass spectrometry acquisition parameters for five neurotransmitters and their internal standards

[0112]

[0113]

[0114] (3) Calculation of measurement results

[0115] Based on retention time and ion pairs, read the mass spectrometric response signals of the standard curve and the analyte in the sample, as well as the corresponding internal standard peak area values. Plot a fitting curve using the ratio of the standard curve peak area to the internal standard peak area as the Y value and the standard curve concentration as the X value. Substitute the peak area ratio of the analyte in the sample to the internal standard into the corresponding curve equation to calculate the concentration of the analyte in the corresponding blood sample.

[0116] Step 4: Methodological Validation

[0117] (1) Specificity

[0118] The specificity of the detection method of the present invention was studied by examining the separation of the analyte and its interfering peaks in the sample. The results are shown in Figure 1 and Figure 2 .in, Figure 1 and Figure 2 The chromatograms of the standard curve and the analyte / internal standard in the clinical sample are respectively. Figure 1 and Figure 2 It can be seen from the figure that the retention times of the five neurotransmitters and their internal standards in the standard curve and clinical samples are consistent, and there is no interference near each peak, indicating good specificity.

[0119] (2) Marking the song

[0120] The isotope internal standard quantification method was used to establish a calibration curve and calculate the concentration of the analyte. Table 11 shows the linear fitting results of the five compounds in their respective concentration ranges. The linearity was good and the correlation coefficient was above 0.990.

[0121] Table 11. Linearity of five compounds

[0122] Serial number Compound Linear range Correlation coefficient 1 DA 5-1250 pg / mL r＝0.99901 2 NE 60-15000 pg / mL r＝0.99813 3 5-HT 4-1000 ng / mL r＝0.99786 4 MT 0.5-1000 pg / mL r＝0.99926 5 GABA 5-1250 ng / mL r＝0.99881

[0123] (3) Accuracy

[0124] Working solution of known concentration was added to plasma samples to prepare spiked samples with high and low concentrations. Six samples were processed in parallel and the recovery results were calculated. See Tables 12 to 16.

[0125] Table 12. DA spike recovery results

[0126]

[0127]

[0128] Table 13. NE spike recovery results

[0129]

[0130] Table 14.5-HT spike recovery results

[0131]

[0132] Table 15. MT spike recovery results

[0133]

[0134] Table 16. GABA spike recovery results

[0135]

[0136] The results in Tables 12 to 16 show that the recoveries of the five neurotransmitters were between 90% and 110%, which all met the requirements.

[0137] (4) Precision

[0138] Plasma samples at two concentrations, high and low, were processed in parallel for 6 times per day for 3 consecutive days. The intra-assay precision for each day and the inter-assay precision for each day were calculated. The results are shown in Tables 17 to 21 below:

[0139] Table 17. DA intra-batch and inter-batch precision results

[0140]

[0141]

[0142] Table 18. NE intra-batch and inter-batch precision results

[0143]

[0144] Table 19.5-HT intra-batch and inter-batch precision results

[0145]

[0146] Table 20. MT intra-batch and inter-batch precision results

[0147]

[0148] Table 21 GABA intra-batch and inter-batch precision results

[0149]

[0150] The results in Tables 17 to 21 above show that the intra-batch and inter-batch precision test results of the five compounds were all within 10%, which met the requirements.

[0151] Comparative Example 1

[0152] HLB solid phase extraction magnetic beads were purchased from Biosepur biology.

[0153] The WCX solid-phase extraction magnetic beads in Example 1 were replaced with HLB solid-phase extraction magnetic beads, and the remaining procedures were the same as in Example 1. Compared with the WCX solid-phase extraction magnetic beads, the HLB solid-phase extraction magnetic beads reduced the extraction efficiency of DA and NE by more than 90% and the extraction efficiency of 5-HT by approximately 50%, as shown in Table 22.

[0154] Table 22. Comparison of peak areas of samples in Example 1 and Comparative Example 1

[0155]

[0156] Comparative Example 2

[0157] The mobile phase in Example 1 was changed to an aqueous solution containing 0.05 vol% formic acid and a methanol solution containing 0.05 vol% formic acid, and the remaining procedures were the same as in Example 1. For low-concentration clinical samples (near the limit of quantification), the mobile phase system containing buffered salts significantly improved the sensitivity of DA, NE, MT, and 5-HT, as shown in Table 23.

[0158] Table 23. Peak areas of low concentration samples under two mobile phase conditions

[0159]

[0160] Comparative Example 3

[0161] The matrix in Example 1 was replaced with 4 wt% BSA. All other procedures were the same as in Example 1, and the standard curve / quality control were stored at 30°C for 3 days. After 3 days of storage at 30°C, samples prepared with 4 wt% BSA matrix showed varying degrees of degradation of DA, NE, and 5-HT, with degradation of DA and NE exceeding 70%. Samples prepared with 4 wt% BSA matrix supplemented with ascorbic acid and citric acid showed a deviation within 10%, demonstrating good stability. See Tables 24 and 25 for details.

[0162] Table 24. Stability results of high and low concentration samples prepared using the matrix (using Example 1)

[0163]

[0164]

[0165] Table 25. Stability results of high and low concentration samples prepared with matrix (using Comparative Example 3)

[0166]

[0167] Comparative Example 4

[0168] The sample pretreatment process in Example 1 was changed to a solid phase extraction plate method, wherein the WCX solid phase extraction plate was purchased from Biosepur Biology, and the positive pressure solid phase extraction device (model Cleanert M96) was purchased from Tianjin Bona Aijieer Technology Co., Ltd. The specific pretreatment process is as follows:

[0169] (1) Take 500 μL of the sample to be tested, add 50 μL of internal standard solution and 400 μL of pH 9 ammonium acetate buffer, and vortex to mix to obtain the first treatment solution;

[0170] (2) Add 200 μL of methanol and 400 μL of 5 vol% methanol aqueous solution to the solid phase extraction plate to activate and equilibrate the solid phase extraction plate and keep the filler moist;

[0171] (3) Add the first treatment liquid to the solid phase extraction plate, let it stand for 5 minutes, then start positive pressure, control the liquid outflow rate to 1-2 drops / s, and collect the outflow liquid as the second treatment liquid;

[0172] (4) Rinse the solid phase extraction plate with 400 μL of a solution containing 5 vol% methanol in water, drain and discard the effluent;

[0173] (5) Add 100 μL of 50 vol% methanol aqueous solution containing 2 vol% formic acid, let it stand for 5 minutes, then start positive pressure, control the liquid outflow rate to 1-2 drops / s, and collect the outflow liquid as the third treatment liquid.

[0174] (6) Take 2 μL of the second treatment solution and add it to the third treatment solution and mix well to obtain a pretreated sample.

[0175] It can be seen from this comparative example that it takes 2 hours to complete a sample pre-treatment using a solid phase extraction plate and a solid phase extraction device. The pre-treatment process is cumbersome, involves multiple manual liquid additions, and requires manual adjustment of the liquid outflow rate. It is also prone to filler clogging, resulting in incomplete extraction of some pores. However, it only takes 20 minutes to perform a sample pre-treatment using the WCX solid phase extraction magnetic bead method of Example 1 of this case. There is no filler clogging problem, and it can be combined with a magnetic bead extractor to achieve automated processing, greatly shortening the processing time. In addition, the solid phase extraction method of Comparative Example 4 requires a gas generator to achieve positive pressure. The WCX solid phase extraction magnetic bead method of Example 1 has no gas supply requirements and has lower requirements for the experimental environment.

[0176] Comparative Example 5

[0177] Take the standard curve and quality control products of different concentrations in Example 1, freeze-dry them, and form a test kit together with the sample pretreatment reagent and internal standard solution. Take 1 box of reagents and accelerate aging at 55°C for 6 days as a stability investigation reagent (i.e., the aging reagent in Table 26); take another box of reagents that have not been accelerated aged as a control reagent. The same group of plasma samples were quantitatively analyzed using the stability investigation reagent and the control reagent respectively. Compared with the test results of the control reagent, the content deviation of the analyte in the sample measured by the stability investigation reagent was within the range of ±10%, as shown in Table 26. According to the Arrhenius equation, the accelerated aging of the test kit at 55°C for 6 days is equivalent to the stability of storage at 4°C for 750 days. Therefore, it can be seen from Table 26 that the neurotransmitter detection kit of the present invention can be stable at 4°C for at least 2 years.

[0178] Table 26. Results of accelerated stability test of the kit

[0179]

[0180]

[0181] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for pre-processing a sample to be tested, characterized in that: The method for pre-treating the sample to be tested comprises the steps of: pre-treating the sample to be tested using WCX solid phase extraction magnetic beads; The sample to be tested is processed and used for detection using both the isotope internal standard method and the liquid chromatography-tandem mass spectrometry method.

2. The method for pre-treating a sample to be tested according to claim 1, wherein: The sample to be tested is serum or plasma.

3. The method for pre-treating a sample to be tested according to claim 1 or 2, wherein: The specific operation of the pre-treatment using WCX solid phase extraction magnetic beads is as follows: Step S11: taking 500 μL of the sample to be tested, adding 50 μL of internal standard solution and 400 μL of buffer solution thereto, and mixing to obtain a first treatment solution; Step S12: Take WCX solid phase extraction magnetic beads and add them to 400 μL of equilibration solution, and shake them for equilibrium; Step S13: mixing the magnetic beads after oscillation equilibrium in step S12 and the first treatment solution obtained in step S11 to obtain a second treatment solution and adsorbed magnetic beads; Step S14: taking the adsorbed magnetic beads and washing them with 400 μL of eluent and 100 μL of elution solution in sequence, and the eluted liquid is the third treatment liquid; Step S15: 2 μL of the second treatment liquid is added to the third treatment liquid, and the mixture is mixed to obtain a pre-treated sample to be tested.

4. The method for pre-treating a sample to be tested according to claim 3, wherein: The buffer solution is ammonium acetate buffer solution with a pH of 8 to 10; and / or, the equilibrium solution is an aqueous solution containing 0-10 vol% methanol; and / or, the eluent is an aqueous solution containing 0-10 vol% methanol; and / or, the eluent is a 50 vol% methanol aqueous solution containing 1-5 vol% formic acid; And / or, the internal standard solution is one or more of the following: 3 ng / mL dopamine-d4, 30 ng / mL norepinephrine-d6, 200 ng / mL serotonin-d4, 1600 ng / mL γ-aminobutyric acid-d6, 0.6 ng / mL melatonin-d4.

5. The method for pre-treating a sample to be tested according to claim 3, wherein: In step S12, before adding the WCX solid phase extraction magnetic beads to 400 μL of the equilibration solution, the WCX solid phase extraction magnetic beads are pre-activated with methanol to obtain activated magnetic beads, wherein the amount of the activated magnetic beads added is 1 to 5 mg.

6. A method for simultaneously detecting multiple neurotransmitters, characterized in that: The method comprises the following steps: Step S1: treating the sample to be tested by the sample pretreatment method according to any one of claims 1 to 5, and subjecting the treated sample to be tested to high performance liquid chromatography to separate the target analyte from the interfering components in the sample to determine the target analyte; Step S2: detecting the mass-to-charge ratio response of the target analyte and its corresponding isotope internal standard by a mass spectrometer; Step S3: quantification using the isotope internal standard method, with the concentration ratio of the target analyte to the internal standard as the X-axis and the peak area ratio of the target analyte to the internal standard as the Y-axis, to establish a calibration curve, and calculate the content of the target analyte based on the standard curve; The target analyte is a neurotransmitter and / or its metabolite, and the neurotransmitter is one or more of the following: dopamine, norepinephrine, 5-hydroxytryptamine, gamma-aminobutyric acid and melatonin.

7. The method according to claim 6, characterized in that In step S1, the high performance liquid chromatography adopts all of the following conditions: Condition A: Mobile phase A was 0.05 vol% formic acid in water containing 2 mM ammonium acetate; Condition B: Mobile phase B was 0.05 vol% formic acid in methanol containing 2 mM ammonium acetate; Condition C: The column was Phenomenex F5, with a particle size of 2.6 μm and a size of 3 × 100 mm; Condition D: Use a flow rate of 0.6-0.8 mL / min; Condition E: column temperature 40-45°C; Condition F: injection volume is 5-7 μL.

8. The method according to claim 6, characterized in that When the high performance liquid chromatography performs gradient elution, the specific operation is as follows: The initial ratio of mobile phase A to mobile phase B was 98:2; The gradient elution process is as follows: within 0-1 minute, the volume ratio of mobile phase A to mobile phase B is gradually changed from 98:2 to 35:65 at a constant speed; The volume ratio of mobile phase A to mobile phase B was gradually changed from 35:65 to 5:95 at a constant speed within 1-2 minutes; The volume ratio of mobile phase A to mobile phase B was maintained at 5:95 for 2–3 min; During 3.1-4 minutes, the ratio of mobile phase A to mobile phase B was maintained at the initial ratio.

9. The method according to claim 6, characterized in that In step S2, the mass spectrometer is specifically operated as follows: in electrospray ionization mode, multiple reaction monitoring is used to perform positive ion mode scanning; the electrospray voltage is 4500V, the ion source temperature is 550°C, the curtain gas is 25psi, the collision gas is 8unit, and the ion source gases 1 and 2 are both 55psi.

10. A kit, characterized in that The kit includes a sample pretreatment reagent, a standard curve, an internal standard solution and a quality control product. The kit uses the sample pretreatment method according to any one of claims 1 to 5 to treat the sample and / or uses the method according to any one of claims 6 to 9 to detect the sample.

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