Mental and psychological disorder metabolic index detection kit and application thereof

Abstract

The invention provides a mental and psychological disorder metabolic index detection kit and application thereof, and belongs to the technical field of biological disease diagnosis. According to the present invention, the sample and the mixed internal standard working solution are mixed, the formic acid acetonitrile solution is added, the centrifugation is performed, and the supernatant is taken, such that the sample pretreatment method can effectively extract a variety of mental and psychological disorder metabolism indexes, and provides the high-resolution, high-specificity and high-sensitivity detection for the liquid chromatography-mass spectrometry method. When the kit and the detection method are used for verifying serum, plasma or urine samples, it is ensured that all parameters meet the requirements, and the test result is good in intra-batch and inter-batch precision and high in accuracy. The kit and the detection method are used for detecting metabolic indexes of mental and psychological disorders in serum, plasma or urine, accurate quantification is provided for clinical projects, and clinical requirements can be met.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological disease diagnosis, and in particular relates to a detection kit for metabolic indexes of mental and psychological disorders and an application thereof. Background Art

[0002] There is a close correlation between mental and psychological disorders (such as depression, anxiety, schizophrenia, etc.) and metabolic indicators. Studies have shown that these disorders not only affect the individual's mental state, but may also cause metabolic abnormalities, thereby increasing the risk of cardiovascular disease, diabetes, etc. Mental and psychological disorders can assist in the diagnosis of diseases, assessment of conditions and guidance of treatment by detecting metabolite levels. Therefore, the detection of metabolic indicators of mental and psychological disorders can assess the patient's physical condition. It is of great significance to the monitoring and treatment of patients, combined with psychotherapy, lifestyle adjustments and drug treatment to improve the overall health of patients.

[0003] Traditional detection methods for detecting metabolic indicators of mental and psychological disorders include colorimetry, ultraviolet inactivation spectrophotometry, fluorescence, and microbiological methods. These methods can only determine the content of one or two of a certain mental or psychological disorder detection indicator. The operation steps are complicated, the blood sample volume is large, and the results are unstable and there are many interfering factors. At present, there are reports in the prior art on methods for judging mental and psychological disorders by detecting metabolites using liquid chromatography-mass spectrometry, but the pretreatment methods are too simple or too complicated, and there is a lack of quantitative data. The sample matrix is ​​single, and it is impossible to explain whether the quantitative results meet the requirements. The data is not complete and lacks credibility. Some substances used in sample pretreatment cause contamination to the instrument. Therefore, the development of a mental and psychological disorder metabolic indicator detection kit and detection method that is simpler to operate, more efficient, more accurate, and more sensitive is a technical problem that needs to be solved at present. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a metabolic index detection kit for mental and psychological disorders and its application, which can effectively extract metabolic indexes of mental and psychological disorders and provide high-resolution, high-specificity and high-sensitivity detection for liquid chromatography-mass spectrometry methods.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a sample pretreatment method for detecting metabolic indicators of mental and psychological disorders, comprising the following steps: mixing a sample with a mixed internal standard working solution, adding a formic acid acetonitrile solution, centrifuging, and taking a supernatant; the sample comprises serum, plasma, or urine; and the mixed internal standard working solution comprises one or more of 3-MT-d4, MN-d3, NMN-d3, 5-HIAA-d6, MLN-d4, 5-HT-d4, Trp-d3, Phe-d5, Met-d3, Arg-13C6, Glu-d3, COR-d4, BMAA-d3, HPHPA-d4, VMA-d3, HVA-d5, and 3-HHA-13C2,15N.

[0007] Preferably, the volume ratio of the mixed internal standard working solution to the sample is 1:1-5.

[0008] Preferably, the volume percentage concentration of the formic acid acetonitrile solution is 0.01-1%.

[0009] Preferably, the volume ratio of the sample to the formic acid-acetonitrile solution is 1:1-5.

[0010] Preferably, the centrifugal speed is 8000-14000 rpm and the time is 3-7 min.

[0011] The present invention provides a sample pretreatment kit for detecting metabolic indicators of mental and psychological disorders, comprising the mixed internal standard working solution and a formic acid acetonitrile solution.

[0012] The present invention provides a kit for detecting metabolic indicators of mental and psychological disorders, comprising the sample pretreatment kit and reagents used in chromatography and mass spectrometry detection.

[0013] The present invention provides a method for detecting metabolic indicators of mental and psychological disorders for non-disease diagnosis purposes, comprising the following steps: detecting and analyzing the supernatant obtained by the sample pretreatment method using a liquid chromatography tandem mass spectrometry detection system.

[0014] Preferably, the chromatographic conditions in the liquid chromatography tandem mass spectrometry detection system include: mobile phase A 0.05-0.3% formic acid aqueous solution, mobile phase B 0.05-0.3% formic acid acetonitrile solution, flow rate 0.4-0.8 mL / min, injection volume 2-10 μL, injection temperature 5-7°C, chromatographic column Agilent Eclipse XDB-C18, column oven 35-45°C, run time 4.5-6.5 min; mass spectrometry source parameter settings include: ion source ESI+ / ESI-, curtain gas 25 psi, collision gas 10 psi, spray voltage 5500 / -4500 V, ion source temperature 550°C, nebulizer gas 50 psi, auxiliary heating gas 50 psi.

[0015] The present invention provides the sample pretreatment kit or the use of the kit in preparing a product for detecting metabolic indicators of mental and psychological disorders.

[0016] In the present invention, "LOD" refers to the limit of detection, also known as the detection limit, which refers to the corresponding amount of 3 times the value of the instrument background signal generated by the matrix blank, or the average value of the background signal generated by the matrix blank plus 3 times the standard deviation of the mean. It is one of the important indicators of sensitivity. "LLMI" specifies the lower limit of quantity, which refers to the lowest concentration or content of the substance to be measured that can be accurately quantitatively determined using a specific method under the premise that the limited error can meet the predetermined requirements. "CV" refers to the coefficient of variation, which is the ratio of the standard deviation to the mean, expressed as a percentage (%). "QC1" refers to a low-concentration quality control product. In the embodiment, low concentration refers to a relatively low concentration within a certain range. It does not have a fixed range. For different detection objects, the range it refers to is different. "QC2" refers to a high-concentration quality control product. In the embodiment, high concentration refers to a relatively high concentration within a certain range. It does not have a fixed range. For different detection objects, the range it refers to is different.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The sample pretreatment method described in this invention utilizes a protein precipitation (PPT) method, which effectively extracts a variety of metabolic markers of mental and psychological disorders, providing high-resolution, high-specificity, and high-sensitivity detection for liquid chromatography-mass spectrometry (LC-MS). Furthermore, the sample pretreatment method avoids lyophilization and derivatization, is simple, time-saving, and can effectively increase detection throughput.

[0019] Furthermore, the detection method or test kit described herein is applicable to samples including serum, plasma, or urine, encompassing a wide variety of sample matrices. By validating the serum, plasma, or urine matrix, various parameters are ensured to meet the required standards. The test kit exhibits excellent intra- and inter-batch precision and high accuracy. This detection method or test kit is used to detect metabolic markers of mental and psychological disorders in serum, plasma, or urine, providing accurate quantification for clinical projects and meeting clinical needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The standard curve and chromatogram of 3-MT detection are shown.

[0021] Figure 2 Standard curve and MN chromatogram for MN detection.

[0022] Figure 3 Standard curve and NMN chromatogram for NMN detection.

[0023] Figure 4The standard curve and chromatogram of 5-HIAA detection are shown.

[0024] Figure 5 The standard curve and MLN chromatogram are for MLN detection.

[0025] Figure 6 5-HT detection standard curve and 5-HT chromatogram.

[0026] Figure 7 This is the standard curve and Trp chromatogram for Trp detection.

[0027] Figure 8 This is the standard curve and Phe chromatogram for Phe detection.

[0028] Figure 9 Standard curve and Met chromatogram for Met detection.

[0029] Figure 10 This is the standard curve and Arg chromatogram for Arg detection.

[0030] Figure 11 This is the standard curve and Glu chromatogram for Glu detection.

[0031] Figure 12 This is the COR detection standard curve and COR chromatogram.

[0032] Figure 13 Figure 3 is the standard curve and BMAA chromatogram for BMAA detection.

[0033] Figure 14 This is the standard curve and HPHPA chromatogram for HPHPA detection.

[0034] Figure 15 The standard curve and VMA chromatogram are for VMA detection.

[0035] Figure 16 This is the HVA detection standard curve and HVA chromatogram.

[0036] Figure 17 The standard curve and chromatogram of 3-HHA detection are shown.

[0037] Figure 18 3-HPP detection standard curve and 3-HPP chromatogram.

[0038] Figure 19 The graphs show the effect of sample pretreatment release agents on COR detection. The left graph shows a 0.1% formic acid in methanol solution, and the right graph shows a 0.1% formic acid in acetonitrile solution.

[0039] Figure 20The left graph shows the effect of sample pretreatment release agents on BMAA detection. The left graph shows a 0.1% formic acid in methanol solution, and the right graph shows a 0.1% formic acid in acetonitrile solution.

[0040] Figure 21 The left graph shows the effect of sample pretreatment release agents on HVA detection. The left graph shows a 0.1% formic acid in methanol solution, and the right graph shows a 0.1% formic acid in acetonitrile solution.

[0041] Figure 22 The figure below shows the effect of mobile phase on 3-MT detection. The left figure shows mobile phase B with 0.2% formic acid in methanol, and the right figure shows mobile phase B with 0.2% formic acid in acetonitrile.

[0042] Figure 23 The figure below shows the effect of mobile phase on MN detection. The left figure shows mobile phase B with 0.2% formic acid in methanol, and the right figure shows mobile phase B with 0.2% formic acid in acetonitrile.

[0043] Figure 24 The left figure shows the effect of mobile phase B on 5-HT detection. The right figure shows mobile phase B with 0.2% formic acid in methanol, and the left figure shows mobile phase B with 0.2% formic acid in acetonitrile.

[0044] illustrate: Figure 1-18 The horizontal axis of the standard curve is in ng / mL, and the vertical axis is in ratio. DETAILED DESCRIPTION

[0045] The present invention provides a sample pretreatment method for detecting metabolic indicators of mental and psychological disorders, comprising the following steps: mixing a sample with a mixed internal standard working solution, adding a formic acid acetonitrile solution, centrifuging, and collecting the supernatant; the sample comprises serum, plasma, or urine; and the mixed internal standard working solution comprises one or more of 3-MT-d4, MN-d3, NMN-d3, 5-HIAA-d6, MLN-d4, 5-HT-d4, Trp-d3, Phe-d5, Met-d3, Arg-13C6, Glu-d3, COR-d4, BMAA-d3, HPHPA-d4, VMA-d3, HVA-d5, and 3-HHA-13C2,15N. The present invention uses a blood collection tube (anticoagulant tube) to collect venous blood, centrifuges the whole blood, collects the supernatant, and obtains plasma. The present invention uses a vacuum negative pressure blood collection tube (coagulant tube) to collect venous blood, centrifuges the whole blood, collects the supernatant, and obtains serum. Before collecting urine in the present invention, acetic acid is added to the urine collection container (for the purpose of maintaining the pH value between about 2 and 4), and urine is collected for 24 hours for subsequent processing.

[0046] In the present invention, the volume ratio of the mixed internal standard working solution to the sample is 1:1-5, preferably 1:1.5-4.5, and more preferably 1:2, 1:3 or 1:4. The purpose of mixing the mixed internal standard working solution with the sample in the present invention is to allow the internal standard and the sample to be fully mixed. By adding the mixed internal standard working solution, the matrix effect is reduced, and the test results are more accurate; at the same time, the role of the mixed internal standard working solution is to correct the loss caused by the pretreatment process of the target (or the target cannot be completely extracted), and the test results are more accurate.

[0047] In the present invention, the volume percentage concentration of the formic acid acetonitrile solution is 0.01-1%, preferably 0.02-0.9%, and more preferably 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0048] In the present invention, the volume ratio of the sample to the formic acid-acetonitrile solution is 1:1-5, preferably 1:2-4, and more preferably 1:3. The formic acid-acetonitrile solution used herein is also referred to as a releasing agent. Its purpose is to release the target compound from the sample and remove impurities via protein precipitation. The type of releasing agent affects the shape of the chromatographic peak in instrumental detection.

[0049] In the present invention, the centrifugal speed is 8000-14000 rpm, and the time is 3-7 min. As an embodiment, the centrifugal speed after adding the formic acid acetonitrile solution is preferably 9000-12000 rpm, more preferably 10000 rpm, and the time is preferably 4-5.5 min, more preferably 4.5 min or 5 min.

[0050] The present invention also provides a sample pretreatment kit for detecting metabolic indicators of mental and psychological disorders, comprising the mixed internal standard working solution and a formic acid acetonitrile solution. The volume percent concentration of the formic acid acetonitrile solution is 0.01-1%, preferably 0.02-0.9%, and more preferably 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%. The preparation of the mixed internal standard working solution of the present invention comprises the following steps: accurately pipetting 2.2 mL of ultrapure water into each internal standard solution bottle, mixing for 1 minute until each internal standard lyophilized powder is completely dissolved; then mixing each dissolved internal standard solution to obtain a mixed internal standard working solution, wherein the final concentration of each internal standard in the prepared mixed internal standard working solution is as follows: HPHA-d4 6500-12000 ng / mL, 3-MT-d4 65-120 ng / mL, VMA-d3 6500-12000 ng / mL, HVA-d5 6500-12000 ng / mL, MN-d3 650-1200 ng / mL, NMN-d3 200-500 ng / mL, 5-HIAA-d6 7500-18000 ng / mL, 3-HHA-13C2,15N 650-1200 ng / mL, MLN-d4 100-220ng / mL, Trp-d3 3500-5000ng / mL, 5-HT-d4 1000-2500ng / mL, Phe-d5 6500-12000ng / mL, Met-d3 2500-4500ng / mL, Arg-13C6 6500-12000ng / mL, Glu-d3 10000-25000ng / mL, COR-d4 100-250ng / mL, BMAA-d3 2000-4500ng / mL.

[0051] In the present invention, the volume percentage concentration of the formic acid-acetonitrile solution in the sample pretreatment kit is 0.01-1%, preferably 0.02-0.9%, and more preferably 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%. The preparation of the 0.1% by volume formic acid-acetonitrile solution of the present invention comprises the following steps: adding 1 mL of formic acid to 1000 mL of acetonitrile and mixing.

[0052] The present invention also provides a kit for detecting metabolic indicators of mental and psychological disorders, comprising the sample pretreatment kit and reagents used for chromatography and mass spectrometry detection. The reagents used for chromatography detection in the present invention include an aqueous formic acid solution and a formic acid-acetonitrile solution; the formic acid aqueous solution has a volume ratio of formic acid to water of 1:400-1:1000, preferably 1:450-1:800, and more preferably 1:500; the formic acid-acetonitrile solution has a volume ratio of formic acid to acetonitrile of 1:400-1:1000, preferably 1:450-1:800, and more preferably 1:500.

[0053] The present invention also provides a method for detecting metabolic indicators of mental and psychological disorders for non-disease diagnosis purposes, comprising the following steps: detecting and analyzing the supernatant obtained by the sample pretreatment method using a liquid chromatography tandem mass spectrometry detection system, and automatically displaying the detection results on the instrument end.

[0054] In the present invention, the chromatographic conditions in the liquid chromatography-tandem mass spectrometry detection system include: mobile phase A 0.05-0.3% formic acid aqueous solution, mobile phase B 0.05-0.3% formic acid acetonitrile solution, flow rate 0.4-0.8 mL / min, injection volume 2-10 μL, injection temperature 5-7°C, chromatographic column Agilent Eclipse XDB-C18 (4.6×150 mm, 5 μm), column oven 35-45°C, run time 4.5-6.5 min; mass spectrometry source parameter settings include: ion source ESI+ / ESI-, curtain gas (CUR) 25 psi, collision gas (CAD) 10 psi, spray voltage (IS) 5500 / -4500 V, ion source temperature (TEM) 550°C, nebulizer gas (Gas1) 50 psi, auxiliary heating gas (Gas2) 50 psi. The chromatographic conditions and mass spectrometry source parameters set in the present invention are obtained by evaluating 18 detection indicators. On the one hand, the characteristics of each detection indicator under these parameter conditions, such as strong specificity, good reproducibility, and strong stability, are taken into account. On the other hand, good separation is ensured between the detection indicator to be tested and the interference, so that clinical samples can be accurately quantified.

[0055] The present invention also provides the sample pretreatment kit or the use of the kit in preparing a product for detecting metabolic indicators of mental and psychological disorders.

[0056] In the present invention, unless otherwise specified, all components or reagents are commercially available products well known to those skilled in the art.

[0057] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0058] In the following examples, the instruments used include:

[0059] AB SCIEX Triple Quad TM 4500MD liquid chromatography tandem mass spectrometry detection system (AB SCIEX, USA); Thermo Scientific Fresco21 high-speed refrigerated centrifuge (USA); multi-tube vortex mixer (VortexGenie2, USA); positive pressure device (Biotage, Sweden); adjustable pipettes (Eppendorf 0.5-10 μL, 10-100 μL, 100-1000 μL).

[0060] In the following examples, the reagents and consumables used include:

[0061] Ultrapure water (Milli-Q IQ7000, USA); methanol (Merk, USA); acetonitrile (Fisher, USA); isopropyl alcohol (Fisher, USA); bovine serum albumin (Sigma, Germany); polybutylene succinate (Sigma, Germany); ethylenediaminetetraacetic acid disodium salt (Shanghai Test, China); D-isoascorbic acid (McLean, China); sodium metabisulfite (Sigma, Germany); ProClin TM 950 (Sigma, Germany).

[0062] In the following examples, the internal standards used are as follows:

[0063] 3-(3-Hydroxyphenyl)-3-hydroxypropionic acid internal standard (HPHPA-d4) (IsoReag, China); 3-methoxytyramine-d4 hydrochloride (3-MT-d4) (PuFen, China); vanillylmandelic acid-d3 (VMA-d3) (PuFen, China); homovanillic acid (HVA-d5) (BePure, China); metanephrine hydrochloride-[d3] (MN-d3) (BePure, China); normetanephrine-[d3] hydrochloride (NMN-d3) (BePure, China); 5-hydroxyindoleacetic acid-d6 (5-HIAA-d6) (PuFen, China); [13C2,15N]-3-hydroxyhippuric acid ([13C2,15N](3-HHA-d3) (IsoReag, China) China); [2H4]-melatonin (MLN-d4) (IsoReag, China); L-tryptophan-d3 (Trp-d3) (PuFen, China); [2H4]-5-hydroxytryptamine hydrochloride (5-HT-d4) (IsoReag, China); phenylalanine-d5 (Phe-d5) (PuFen, China); methionine-d3 (Met-d3) (IsoReag, China); L-arginine-[13C6] hydrochloride (Arg-d6) (BePure, China); glutamate-d3 (Glu-d3) (PuFen, China); cortisol-[d4] (COR-d4) (BePure, China); 3-(N-methylamino)-L-alanine-d3 (BMAA-d3) (Ruipuyuan, China).

[0064] The calibrators or quality control products used in the following examples are as follows:

[0065] 3-Methoxytyramine (3-MT) (BePure, China); metanephrine (MN) (BePure, China); methylnorepinephrine (NMN) (BePure, China); 5-hydroxyindoleacetic acid (5-HIAA) (PuFen, China); melatonin (MLN) (China National Institute for Food and Drug Control, China); 5-hydroxytryptamine (5-HT) (China National Institute for Food and Drug Control, China); tryptophan (Trp) (Dr.E, Germany); phenylalanine (Phe) (Tanmo Quality Control, China); methionine (Met) (Dr.E, Germany); arginine (Arg) (B ePure, China); glutamate (Glu) (BePure, China); cortisol (COR) (China National Institute for Food and Drug Control, China); β-methylamino-L-alanine (BMAA) (Sigma, Germany); 3-(3-hydroxyphenyl)-3-hydroxypropionic acid (HPHPA) (Sigma, Germany); vanillylmandelic acid (VMA) (BePure, China); homovanillic acid (HVA) (BePure, China); 3-hydroxyhippuric acid (3-HHA) (CATO, China); 3-hydroxyphenylpropionic acid (3-HPP) (CATO, China).

[0066] Unless otherwise specified, the preparation methods of some reagents in the following examples are as follows:

[0067] Preparation of calibrator solutions: Accurately pipette 0.3 mL of ultrapure water into each bottle of calibrator and mix for 1 min until the lyophilized powder is completely dissolved. Prepare the calibrator solutions of the required concentrations (see Table 6) and set aside.

[0068] Preparation of quality control solution: Accurately pipette 0.3 mL of ultrapure water into each bottle of quality control solution and mix for 1 min until the lyophilized powder is completely dissolved. Prepare the required concentration of each quality control solution as shown in Table 6 and set aside.

[0069] Preparation of mixed internal standard working solution: Accurately pipette 2.2 mL of ultrapure water into each bottle of internal standard, mix for 1 min until each internal standard lyophilized powder is completely dissolved, and then mix according to the final concentration of each internal standard shown in Table 1 to obtain the mixed internal standard working solution.

[0070] Mobile phase A is 0.2% formic acid in water. Prepare by adding 500 mL of ultrapure water and 1 mL of formic acid to a reagent bottle, mixing thoroughly. Ultrasonicate for 10 minutes. Label the solution "Mobile Phase A-" and store at room temperature (expiration date: 7 days).

[0071] Mobile phase B is a 0.2% formic acid in acetonitrile solution. Prepare by adding 500 mL of acetonitrile and 1 mL of formic acid to a reagent bottle, mixing thoroughly. Ultrasonicate for 10 minutes. Label the solution "Mobile Phase B" and store at room temperature (expiration date: 30 days).

[0072] Needle Wash Solution, methanol: ultrapure water = 1:1 (V:V). Preparation: Add 250 mL of methanol, then 250 mL of ultrapure water to a reagent bottle; mix thoroughly and sonicate for 10 minutes. Label as Needle Wash Solution and store at room temperature (expiration date: 1 month).

[0073] Primer seal cleaning solution (isopropyl alcohol:ultrapure water = 1:4 (v:v)). Prepare by adding 100 mL of isopropyl alcohol and 400 mL of ultrapure water to a reagent bottle, mixing thoroughly. Ultrasonicate for 10 minutes. Label the solution "Primer Seal Cleaner" and store at room temperature (expiration date: 1 month).

[0074] Example 1

[0075] The pre-treatment method of plasma or serum samples is as follows:

[0076] (1) The mixed internal standard working solution contains 17 internal standards, namely 3-MT-d4, MN-d3, NMN-d3, 5-HIAA-d6, MLN-d4, 5-HT-d4, Trp-d3, Phe-d5, Met-d3, Arg-13C6, Glu-d3, COR-d4, BMAA-d3, HPHPA-d4, VMA-d3, HVA-d5, 3-HHA-13C2,15N.

[0077] The preparation steps of the mixed internal standard working solution are as follows: accurately pipette 2.2 mL of ultrapure water into each internal standard solution bottle, mix for 1 min until the lyophilized powder of each internal standard is completely dissolved, and then mix the 17 completely dissolved internal standard solutions to obtain the mixed internal standard working solution. The final concentration of each internal standard in the mixed internal standard working solution is shown in Table 1.

[0078] Table 1 Final concentration of each internal standard in the mixed internal standard working solution

[0079]

[0080] (2) Pipette 60 μL of plasma or serum sample into a 1.5 mL EP tube, then add 20 μL of mixed internal standard working solution, vortex mix for 1 min, and then centrifuge at high speed (12000 rpm, 20 s). Pour the solution on the EP tube cap into the tube to prevent splashing when opening the cap.

[0081] (3) Then, 180 μL of 0.1% (V / V) formic acid acetonitrile solution was added to the EP tube after centrifugation in step (2), vortexed for 5 minutes, and then centrifuged at high speed (12000 pm) for 5 minutes.

[0082] (4) Take 120 μL of the supernatant after centrifugation in step (3) and place it in a sampling bottle or a 96-well plate to complete the sample pretreatment step.

[0083] Example 2

[0084] The urine sample pre-treatment method is as follows:

[0085] The urine was diluted 5 times with ultrapure water, and the other steps were the same as in Example 1.

[0086] Example 3

[0087] A method for detecting metabolic indicators of mental and psychological disorders in human serum / plasma / urine, comprising the following steps:

[0088] (1) The plasma or serum sample pretreatment method is the same as in Example 1. The urine sample pretreatment method is the same as in Example 2.

[0089] (2) Place the sample vial or 96-well plate containing the processed sample into the AB SCIEX Triple Quad TM Sample loading and analysis on the 4500MD liquid chromatography-tandem mass spectrometry detection system: Enter the parameters in Tables 2-5 and equilibrate the instrument. Place the sample (processed sample, calibrators, and quality control materials listed in Table 6) into the autosampler and program the injection sequence (e.g., C1-C5, blank sample, QC1 and QC2, samples 1, 2, and 3). After the injections are complete, the instrument automatically processes the data. Enter the concentration according to the target value for each test indicator, and the instrument automatically analyzes and detects the sample results.

[0090] The chromatographic conditions are shown in Table 2. The liquid phase gradient elution parameters are shown in Table 3.

[0091] Table 2 Chromatographic conditions

[0092]

[0093] Table 3 Liquid phase gradient elution parameters

[0094]

[0095]

[0096] The mass spectrometry source parameters in the mass spectrometry method are shown in Table 4. The target compound and internal standard acquisition parameters are shown in Table 5.

[0097] Table 4 Mass spectrometry source parameters

[0098] parameter value Ion source ESI Curtain air (CUR) (psi) 25 Collision gas (CAD) (psi) 10 Spray voltage (IS) (V) 5500 / -4500 Ion source temperature (TEM) (℃) 550 Atomizing gas (Gas1) (psi) 50 Auxiliary heating gas (Gas2) (psi) 50

[0099] Table 5 Target compounds and internal standard acquisition parameters

[0100]

[0101]

[0102] Note: The ions marked with * are quantitative ion pairs.

[0103] Example 4

[0104] A sample pretreatment kit for detecting metabolic indicators of mental and psychological disorders in human serum / plasma / urine, comprising the following reagents: 20 μL of the mixed internal standard working solution prepared in Example 1 and 180 μL of a 0.1% (V / V) formic acid acetonitrile solution.

[0105] Example 5

[0106] A sample pretreatment kit for detecting metabolic indicators of mental and psychological disorders in human serum / plasma / urine, comprising the following reagents: 10 μL of the mixed internal standard working solution prepared in Example 1 and 170 μL of a 0.05% (V / V) formic acid acetonitrile solution.

[0107] Example 6

[0108] A sample pretreatment kit for detecting metabolic indicators of mental and psychological disorders in human serum / plasma / urine, comprising the following reagents: 30 μL of the mixed internal standard working solution prepared in Example 1 and 190 μL of a 0.15% (V / V) formic acid acetonitrile solution.

[0109] Example 7

[0110] A kit for detecting metabolic indicators of mental and psychological disorders in human serum / plasma / urine, comprising the following reagents: the reagents in the sample pretreatment kit of Example 4, the calibrators and quality control substances shown in Table 6, 0.2% formic acid aqueous solution, and 0.2% formic acid acetonitrile solution.

[0111] Table 6 Calibrators (C1-C5) and quality control products (low concentration QC1 and high concentration QC2), unit ng / mL

[0112]

[0113]

[0114] Comparative Example 1

[0115] The method of Example 3 was used, and the pretreatment release agent in step (1) was 0.1% formic acid methanol solution or 0.1% formic acid acetonitrile solution to detect the calibrants COR (5 ng / mL), BMAA (30 ng / mL) and HVA (200 ng / mL). The results are shown in Figure 19-21 .

[0116] When the releasing agent is 0.1% formic acid methanol solution, the COR peak shape is poor and the peak area (2439) is low; when the releasing agent is 0.1% formic acid acetonitrile solution, the COR peak shape is better and the peak area (9468) is high, so the COR project chooses 0.1% formic acid acetonitrile solution as the protein precipitant.

[0117] When the releasing agent is 0.1% formic acid methanol solution, the BMAA peak shape is poor and the baseline is high; when the releasing agent is 0.1% formic acid acetonitrile solution, the BMAA peak shape is better and the baseline is low, so the BMAA project chooses 0.1% formic acid acetonitrile solution as the protein precipitant.

[0118] When the releasing agent is 0.1% formic acid methanol solution, the HVA peak shape is poor and the area (1298) is low; when the releasing agent is 0.1% formic acid acetonitrile solution, the HVA peak shape is better and the area (11900) is high, so the HVA project chooses 0.1% formic acid acetonitrile solution as the protein precipitant.

[0119] Comparative Example 2

[0120] The method described in Example 3 was used, with mobile phase A consisting of 0.2% formic acid in methanol and mobile phase B consisting of 0.2% formic acid in acetonitrile or 2% formic acid in methanol. Calibrators 3-MT (2 ng / mL), MN (2 ng / mL), and 5-HT (2 ng / mL) were tested. The results are shown in Table 1. Figure 22-24 .

[0121] When mobile phase B is 2% formic acid in methanol, the 3-MT peak shape is poor and the peak area (7164) is low. When mobile phase B is 0.2% formic acid in acetonitrile, the 3-MT peak shape is better and the peak area (32480) is high. Therefore, 0.2% formic acid in acetonitrile is selected as mobile phase B for the 3-MT project.

[0122] When mobile phase B is 0.2% formic acid in methanol solution, the MN peak shape is poor and the peak area (22890) is low; when mobile phase B is 0.2% formic acid in acetonitrile solution, the MN peak shape is better and the peak area (43730) is high, so 0.2% formic acid in acetonitrile solution is selected as mobile phase B for the MN project.

[0123] When mobile phase B is 0.2% formic acid in methanol solution, the 5-HT peak shape is poor and the peak area (14530) is low; when mobile phase B is 0.2% formic acid in acetonitrile solution, the 5-HT peak shape is better and the peak area (195800) is high, so 0.2% formic acid in acetonitrile solution is selected as mobile phase B for the 5-HT project.

[0124] In summary, when mobile phase A is 0.2% formic acid aqueous solution and mobile phase B is 0.2% formic acid acetonitrile solution as the liquid phase detection conditions, the peak shape of the project is better and the response is higher.

[0125] Experimental Example 1

[0126] The performance test was performed using the kit described in Example 7 and the detection method described in Example 3, as follows:

[0127] 1. Limit of detection (LOD)

[0128] Evaluation criteria: The signal-to-noise ratio of each sample can be obtained in AnalystMD Software. The sample concentration with an average signal-to-noise ratio ≥ 3:1 is considered the LOD.

[0129] (1) Screen low-concentration serum / plasma / urine samples and select suitable samples

[0130] Based on clinical experience and needs, the minimum target concentration of each detection index in serum / plasma / urine is determined as shown in Tables 7-9. The kit is used to test 30 samples of serum / plasma / urine. According to the target concentration of each detection index, suitable serum / plasma / urine samples are screened for detection limit determination. The specific detection and quantification methods are the same as those in Example 3.

[0131] (2) Experimental method: Each sample was tested once. Injection order of the samples to be tested: random injection.

[0132] The LODs of serum samples measured in this experiment are shown in Table 7, all of which meet the evaluation requirements.

[0133] Table 7 LOD values ​​of each test index for serum samples, unit ng / mL

[0134]

[0135] The LODs of plasma determination in this experiment are shown in Table 8 below, all of which meet the evaluation requirements.

[0136] Table 8 LOD values ​​of various test indicators for plasma samples, unit ng / mL

[0137]

[0138]

[0139] The LODs of urine determination in this experiment are shown in Table 9 below, and all meet the evaluation requirements.

[0140] Table 9 LOD values ​​of various test indicators for urine samples, unit ng / mL

[0141]

[0142] 2. Lower limit of quantification (LLMI)

[0143] Evaluation criteria: The deviation between the mean and theoretical values ​​of the detected concentration of LLMI was ≤±15% and the CV was ≤20%.

[0144] Based on clinical experience and needs, the theoretical values ​​of each detection index in serum are determined as shown in Table 10. 30 serum samples were tested, and suitable serum samples were screened according to the theoretical concentration of each detection index for quantitative limit determination. The specific detection and quantification process was the same as in Example 3.

[0145] Experimental Method: Each serum was tested 10 times. Sample injection order: Random injection. The statistical results are shown in Table 10 below, all meeting the evaluation requirements.

[0146] Table 10 Detection results of 18 substances in serum, unit ng / mL

[0147]

[0148]

[0149] 3. Intra-batch and inter-batch precision

[0150] Evaluation criteria: The concentrations of QC1 and QC2 based on Table 6 are theoretical values, with a CV ≤ 15%.

[0151] Experimental methods:

[0152] 1) QC1 and QC2 were tested 10 times daily using the kit of the present invention for 3 days. The injection order was from low to high, with low-concentration samples first and high-concentration samples last. The specific detection and quantification steps were the same as in Example 3.

[0153] 2) The data of the first, second and third days are the intra-batch results; the data of the three days of testing are analyzed together as the inter-batch results.

[0154] The test results are shown in Table 11, indicating that the intra-assay and inter-assay precision CVs of all test indicators in QC1 and QC2 were ≤ 15%, and the method met the evaluation criteria.

[0155] Table 11 Intra-batch and inter-batch precision test results of quality control products QC1 and QC2

[0156]

[0157]

[0158] 4. Accuracy

[0159] Evaluation criteria: Recovery rate is 85-115%.

[0160] (1) An internationally recognized certified reference material (NIST 1950) was used to verify the accuracy of the kit. The detection index was Met (ng / mL), and the detection method was the same as in Example 3. The results are shown in Table 12. The results show that the detection method and detection kit of the present invention have high accuracy.

[0161] Table 12. Detection results of Met in NIST 1950, unit: ng / mL

[0162]

[0163] (2) Experimental methods:

[0164] Sample selection: Select appropriate serum samples based on the results of detection limit and quantification limit.

[0165] Prepare spiked samples: Add high and low concentrations of standard solution to each serum sample using the external spike method to obtain two spiked samples. Specific spike concentrations are shown in Table 13.

[0166] Experimental procedure: The kit was used to test serum samples (basic samples) and spiked samples. The specific detection and quantification steps were the same as in Example 3. The calculation results are shown in Table 13.

[0167] Referring to the concentrations of the detection indicators C1-C5 shown in Table 6, the detection method of the present invention was used to obtain the standard curves and chromatograms of 3-MT, MN, NMN, 5-HIAA, MLN, 5-HT, Trp, Phe, Met, Arg, Glu, COR, BMAA, HPHPA, VMA, HVA, 3-HHA, and 3-HPP. Figures 1-18 .

[0168] The results in Table 13 show that the recovery rates of serum samples for all test indicators were within 85-115%, which met the evaluation criteria.

[0169] Table 13 Recovery test results of various indicators in serum samples

[0170]

[0171] 5. Linear range

[0172] Evaluation criteria: correlation coefficient R ≥ 0.99, and target value bias ≤ ± 15%.

[0173] Experimental method: The kit was used to test the C1-C5 calibrators in Table 6, and the test was repeated 3 times. The specific detection and quantification steps were the same as those in Example 3.

[0174] The experimental results-regression curve is shown in Table 14. The results show that the correlation coefficient R of all detection indicators is ≥0.99, and the target value bias of all detection indicators is ≤±15%.

[0175] Table 14 Linear range test results of each test index

[0176]

[0177]

[0178]

[0179] Experimental Example 2 Clinical Sample Testing

[0180] Fifty clinical plasma samples and 20 clinical urine samples were tested using the kit described in Example 7 and the detection method described in Example 3. All clinical samples were obtained from the Shanghai Mental Health Center. The results are shown in Tables 15 and 16. Based on the test results of the clinical samples in the tables, hospital physicians made clinical diagnoses.

[0181] Table 1550 clinical plasma sample test results

[0182]

[0183]

[0184] Table 1620 clinical urine samples

[0185]

[0186]

[0187] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A sample pre-processing method for detecting metabolic indicators of mental and psychological disorders, characterized in that: The steps include: Mix the sample with the mixed internal standard working solution, add formic acid acetonitrile solution, centrifuge, and take the supernatant; The sample includes serum, plasma or urine; The mixed internal standard working solution includes one or more of 3-MT-d4, MN-d3, NMN-d3, 5-HIAA-d6, MLN-d4, 5-HT-d4, Trp-d3, Phe-d5, Met-d3, Arg-13C6, Glu-d3, COR-d4, BMAA-d3, HPHPA-d4, VMA-d3, HVA-d5, and 3-HHA-13C2,15N.

2. The sample pretreatment method according to claim 1, wherein: The volume ratio of the mixed internal standard working solution to the sample is 1:1-5.

3. The sample pretreatment method according to claim 1, wherein: The volume percentage concentration of the formic acid acetonitrile solution is 0.01-1%.

4. The sample pretreatment method according to claim 1, wherein: The volume ratio of the sample to the formic acid acetonitrile solution is 1:1-5.

5. The sample pretreatment method according to claim 1, wherein: The centrifugal speed is 8000-14000 rpm, and the time is 3-7 minutes.

6. A sample pretreatment kit for detecting metabolic indicators of mental and psychological disorders, characterized in that: The method comprises the mixed internal standard working solution and the formic acid acetonitrile solution described in claim 1.

7. A kit for detecting metabolic indicators of mental and psychological disorders, characterized in that: The method comprises the sample pretreatment kit according to claim 6, and reagents used for chromatography and mass spectrometry detection.

8. A method for detecting metabolic indicators of mental and psychological disorders for non-disease diagnosis purposes, characterized in that: The method comprises the following steps: detecting and analyzing the supernatant obtained by the sample pretreatment method according to claim 1 using a liquid chromatography tandem mass spectrometry detection system.

9. The method according to claim 8, wherein The chromatographic conditions in the liquid chromatography tandem mass spectrometry detection system include: mobile phase A 0.05-0.3% formic acid in water, mobile phase B 0.05-0.3% formic acid in acetonitrile, flow rate 0.4-0.8 mL / min, injection volume 2-10 μL, injection temperature 5-7°C, chromatographic column Agilent Eclipse XDB-C18, column oven 35-45°C, run time 4.5-6.5 min; The mass spectrometer source parameter settings include: ion source ESI+ / ESI-, curtain gas 25 psi, collision gas 10 psi, spray voltage 5500 / -4500 V, ion source temperature 550°C, nebulizer gas 50 psi, and auxiliary heating gas 50 psi.

10. Use of the sample pretreatment kit according to claim 6 or the kit according to claim 7 in preparing a product for detecting metabolic indicators of mental and psychological disorders.

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