Method for detecting sleep-related neurotransmitter marker in urine and marker

Abstract

The invention provides a method for synchronously detecting neurotransmitter related biomarkers and a marker, the method comprises the following steps: (1) sample pretreatment: adding a stable isotope labeled internal standard substance mixed solution into a to-be-detected sample, adding a protein precipitant, centrifugally precipitating protein, centrifuging, taking supernate, and performing solid-phase extraction by using a solid-phase extraction column to obtain a sample solution; then eluting a target object by using an eluent, collecting the target object, concentrating and dissolving the target object in a reconstitution fluid to prepare a to-be-detected solution; the filler of the solid phase extraction column comprises MAX (maleic anhydride) and PrimeHLB (Hydrophile Lipophile Balance) resin; and (2) detecting the components and content of the sleep-related neurotransmitter in the sample solution by adopting high performance liquid chromatography-tandem mass spectrometry. According to the method, the sleep-related neurotransmitters in human urine can be quickly and accurately screened and analyzed at one time in a short time, and the requirements of actual detection are met.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and particularly to a method for detecting sleep-related neurotransmitter markers in urine. Background Art

[0002] A variety of neurotransmitters play important physiological roles in the human body and are closely related to the sleep-wake cycle, sleep quality, and sleep disorders. Detection of these neurotransmitters can be used to evaluate sleep problems, assist in diagnosis, and monitor the changes in the condition as early as possible. Among them, melatonin is secreted by the pineal gland and has sedative and hypnotic effects, and can improve insomnia caused by jet lag, sleep phase delay, and circadian rhythm disorders; 6-sulfatoxymelatonin is the metabolite of melatonin in the liver; 5-hydroxytryptamine plays an important regulatory role in sleep, mood, and cognitive function, and low levels may lead to sleep disorders; γ-aminobutyric acid can inhibit central nervous excitement, play a role in controlling epilepsy, and can also improve the sleep quality of patients and increase the time of deep sleep; cortisol is a type of glucocorticoid that can resist external stress, and low levels may lead to emotions such as anxiety and fear in the body, resulting in sleep fragmentation, shortened sleep time, and insomnia; dehydroepiandrosterone can regulate the endocrine level in the brain and relieve insomnia symptoms; too high testosterone levels may interfere with the normal sleep-wake rhythm; 17-hydroxyprogesterone is also related to the endocrine system; abnormalities of these neurotransmitters may all affect sleep, so the detection of the concentration levels of these substances in the human body is of great significance in the treatment of sleep disorders.

[0003] Compared with blood samples, urine samples are more convenient to collect, have high repeatability, and can be sampled multiple times, which makes it possible to perform continuous monitoring through urine analysis. The urine matrix is complex, containing a variety of interfering substances, and the concentrations of these biomolecules in urine are low, which brings difficulties to accurate and sensitive detection. Currently, commonly used detection methods include gas chromatography-mass spectrometry (GC-MS), immunoassay, etc. However, traditional immunoassays have low sensitivity. When the sample concentration is very low, a large sample volume needs to be enriched and purified; they have poor applicability, and the antibodies of the analytes are prone to cross-react with endogenous substances or metabolites with similar structures, resulting in false positive results; the analysis efficiency is low, usually only one substance can be measured each time, and qualitative and quantitative analysis of multiple hormones cannot be performed simultaneously. The gas chromatography-mass spectrometry technique has high sensitivity, but often requires cumbersome derivatization steps and a long analysis time (generally ≥30 min), and currently lacks a derivatization reagent suitable for universality. Therefore, there is an urgent need to develop a method that is simple to operate, has a low cost, and can detect multiple sleep-related neurotransmitters in urine at one time to meet the needs of clinical and scientific research.

[0004] This study aims to establish a new detection method. By optimizing the sample pretreatment process and detection means, it realizes the rapid, sensitive, and accurate quantitative detection of 8 sleep-related neurotransmitters in human urine. Compared with existing studies, this method has the significant advantages of simpler operation, lower cost, and the ability to detect multiple target substances at one time, which will greatly improve the detection efficiency, reduce the research cost, and provide strong technical support for in-depth research on the interactions of these biomolecules under physiological and pathological conditions, as well as the diagnosis and treatment of clinical diseases. Summary of the Invention

[0005] In view of this, the present invention provides a combination of sleep-related neurotransmitter markers and a detection method. To achieve the above object, the technical solution of the present invention is as follows:

[0006] <First aspect>

[0007] A method for simultaneously detecting neurotransmitter-related biomarkers, the method comprising the following steps:

[0008] (1) Sample pretreatment: Add a stable isotope-labeled internal standard mixture to the sample to be tested, add a protein precipitant (including methanol or acetonitrile), centrifuge (centrifuge at 10,000 - 12,000 revolutions, for 5 - 10 minutes) to precipitate proteins, take the supernatant after centrifugation and perform solid-phase extraction using a solid-phase extraction column, then use an eluent to elute the target substance, collect the target substance, concentrate and dissolve it in a reconstitution solution (a methanol - aqueous solution of 10:90 - 20:80) to prepare a test solution;

[0009] The internal standard includes melatonin, 5-hydroxytryptamine, 6-sulfatoxymelatonin, γ-aminobutyric acid, cortisol, dehydroepiandrosterone, testosterone, 17-hydroxyprogesterone;

[0010] The packing material of the solid-phase extraction column includes MAX and PrimeHLB resin;

[0011] (2) Use high-performance liquid chromatography - tandem mass spectrometry to detect the components and contents of sleep-related neurotransmitters in the sample solution.

[0012] In step (1), the volume ratio of the sample to be tested to the protein precipitant is 1:2 - 1:3.

[0013] In step (1), the elution method is to elute with an eluent containing 0.1% trifluoroacetic acid in acetonitrile - methanol (7:3, v / v), and the flow rate is 1 - 2 drops / s.

[0014] The HPLC conditions in step (2) include:

[0015] Mobile phase A: 0.1% formic acid in water;

[0016] Mobile phase B: methanol;

[0017] Chromatographic column: Phenomenex C18 (2.1×100 mm, 2.6 μm)

[0018] Gradient elution was adopted: injection volume was 3 μL, column temperature was 40 °C, and flow rate was 0.3 mL / min;

[0019] The gradient elution conditions were as follows:

[0020] Time (min) A(％) B(％) Flow rate (mL / min) 0.00 90 10 0.3 3 90 10 0.3 4 5 95 0.3 7 5 95 0.3 7.10 90 10 0.3 8 90 10 0.3 。

[0021] In step 1, in the internal standard mixture, the concentration of each internal standard was 800 - 1000 ng / mL;

[0022] In step (1), the volume ratio of the sample to be tested to the protein precipitant was 1:1 - 1:3.

[0023] The volume ratio of the sample to be tested to the internal standard mixture was 200:1 - 400:1.

[0024] In step (2), the mass spectrometry conditions included:

[0025] SCIEX Triple Quad 4500 mass spectrometer, the ion source voltage was 5500 V, the ion source temperature was 500 °C, and the electrospray ionization - positive ion mode was adopted; the detection method was multiple reaction monitoring; the auxiliary gas was 55 psi, the collision gas was 9 psi, and the nebulizing gas was 50 psi, all of which were nitrogen; the mass spectrometry parameters were as follows:

[0026]

[0027]

[0028] In step (1), the ratio of MAX to Prime HLB resin was 1 - 2:1 - 2 in mass ratio; preferably 2:1.

[0029] In step (1), the elution method was acetonitrile - methanol (5:5 - 7:3, v / v) containing 0.1% trifluoroacetic acid, and the flow rate was controlled at 1 - 2 drops / s.

[0030] <Second aspect>

[0031] A combination of sleep - related neurotransmitter markers in urine, the markers including melatonin (MEL), 5 - hydroxytryptamine (5 - HT), 6 - sulfatoxymelatonin (aMT6s), γ - aminobutyric acid (GABA), cortisol (CRL), dehydroepiandrosterone (DHEA), testosterone (T), 17 - hydroxyprogesterone (17α - OHP).

[0032] The present invention also provides a kit containing the biomarker as described above.

[0033] When using the solid-phase extraction column, the solid-phase extraction column is activated successively with a polar organic solvent (methanol) and an aqueous solvent (ultrapure water).

[0034] The beneficial effects of the present invention are:

[0035] The present invention discloses a method for detecting 8 sleep-related neurotransmitters in human urine, including the steps of: preparing a novel solid-phase extraction column with a mixture of MAX and PRiME HLB resin as the adsorption packing layer; adding an internal standard to a human urine sample, adding methanol or acetonitrile, centrifuging to precipitate proteins, taking the supernatant, diluting it with ultrapure water, and performing solid-phase extraction using the novel solid-phase extraction column, eluting the solid-phase extraction column with an eluent, nitrogen blowing, re-dissolving with a reconstitution solution, and centrifuging twice to obtain a human urine sample solution to be detected; using high performance liquid chromatography-tandem mass spectrometry to detect the components and contents of sleep-related neurotransmitters in the human urine sample solution.

[0036] The method for detecting sleep disorder hormone biomarkers in human urine disclosed in the present invention can quickly and accurately complete the screening and analysis of 8 neurotransmitters in human urine at one time in a short period, meeting the requirements of actual detection; meanwhile, the method of the present invention improves the detection sensitivity, saves the sample processing time and detection time, and can efficiently and accurately complete the screening and analysis of multiple target neurotransmitters in human urine through one experiment, which has important significance for further understanding and studying the influence of neurotransmitters on sleep. Description of the Drawings

[0037] By reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings, other features, objects, and advantages of the present invention will become more apparent:

[0038] Figure 1 It is the chromatograms of two substances, melatonin MEL and testosterone T, in Example 1 of the present invention; wherein a is the chromatogram of melatonin MEL, and b is the chromatogram of testosterone T;

[0039] Figure 2 It is the standard curves of two substances, melatonin MEL and 17α-hydroxyprogesterone 17α-OHP, detected in Example 2 of the present invention; wherein a is the standard curve of melatonin MEL, and b is the standard curve of testosterone T;

[0040] Figure 3 It is the comparison chart of the detection results of 8 neurotransmitters extracted by the novel solid-phase extraction column with three different mass ratios of MAX and PRiME HLB mixed resins in Example 3 of the present invention;

[0041] Figure 4Comparison chart of the detection results of three solid-phase extraction elution methods in Example 3 of the present invention (Group A eluent: acetonitrile-methanol (7:3, v / v) solution containing 0.1% trifluoroacetic acid; Group B eluent: 5 mL of methanol (0.1% formic acid); Group C eluent: 5 mL of acetonitrile (0.5% ammonia)). Detailed implementation manners

[0042] The present invention will be described in detail below with reference to the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several adjustments and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0043] In the following examples:

[0044] Example 1 Mass spectrometry parameter optimization and liquid phase condition screening

[0045] (1) Mass spectrometry condition screening

[0046] For mass spectrometry condition screening, SCIEX Triple Quad 4500 mass spectrometer was used, the ion source voltage was 5500 V, the ion source temperature was 500 °C, and the electrospray ionization-positive ion mode was adopted; the detection method was multiple reaction monitoring; the auxiliary gas was 55 psi, the collision gas was 9 psi, and the nebulizing gas was 50 psi, all of which were nitrogen. The mass spectrometry parameter optimization is shown in Table 1.

[0047] Table 1 Mass spectrometry parameters of neurotransmitters

[0048]

[0049]

[0050] (2) Mobile phase screening

[0051] The selection of the mobile phase is very important for the accurate quantification of the target compound. Therefore, to screen out the mobile phase that can give the highest response of the target compound, it is necessary to compare the response values of different mobile phases and the corresponding target compounds (taking melatonin, 5-hydroxytryptamine, and cortisol as examples). The results are shown in Table 2:[[]]

[0052] Table 2 Mobile phase screening result table

[0053]

[0054] As can be seen from Table 2, for mobile phase A: 0.1% formic acid in water, and mobile phase B: methanol, the response values of the target compounds are the highest. Therefore, this mobile phase condition is used for liquid chromatography.

[0055] (3) The liquid chromatography conditions are as follows:

[0056] Mobile phase A: 0.1% formic acid in water;

[0057] Mobile phase B: methanol;

[0058] Chromatographic column: Phenomenex C18 (2.1×100 mm, 2.6 μm)

[0059] Gradient elution was adopted: injection volume 3 μL, column temperature 40 °C, flow rate 0.3 mL / min;

[0060] The gradient elution conditions are shown in Table 3 below:

[0061] Table 3 Mobile phase gradient elution conditions

[0062] Time (min) A(％) B(％) Flow rate (mL / min) 0 95 5 0.3 2 95 5 0.3 5 5 95 0.3 7 5 95 0.3 7.10 95 5 0.3 8 95 5 0.3

[0063] Figure 1 For example of the chromatogram, among them, Figure 1 as shown in a is the chromatogram of melatonin MEL substance, Figure 1 as shown in b is the chromatogram of 17-hydroxyprogesterone 17α-OHP substance.

[0064] Example 2 Preparation of standard curve and quality control samples

[0065] 1. Obtain the standard curves of 8 sleep-related neurotransmitters:

[0066] 1). Preparation of high-concentration standard stock solutions:

[0067] Preparation of high-concentration single-standard stock solutions: Accurately weigh 10.0 mg of each of the 8 neurotransmitter standards, and dissolve them in methanol and make up to 10 mL respectively to obtain 8 high-concentration single-standard stock solutions with a concentration of 1000 μg / mL.

[0068] Then prepare an appropriate amount of 1:9 (v / v) methanol-water as the diluent for the subsequent working solutions and standard curve preparation.

[0069] In order to ensure that different analytes are quantitatively analyzed within their respective optimal concentration ranges, thereby improving the accuracy, precision and sensitivity of the quantitative results, it is necessary to prepare standard curves with different linear ranges. In the present invention, we control the concentration ranges of CRL and aMT6s standard samples at 20 - 1000 ng / mL, the concentration ranges of GABA, 5-HT and DHEA standard samples at 2 - 100 ng / mL, and the concentration ranges of 17α-OHP, T and MEL standard samples at 0.2 - 10 ng / mL.

[0070] 2). Preparation of internal standard mixture

[0071] 100 μL of each of the 8 neurotransmitter internal standard stock solutions at 100 μg / mL were separately dissolved in 10 mL of methanol to prepare a mixed internal standard solution SIS with a concentration of 1000 ng / mL.

[0072] 3), The volume of the standard solution is 200 μL, and the added internal standard solution is 20 μL

[0073] The concentrations (ng / mL) of the obtained standard solutions are shown in Table 4 below:

[0074] Table 4

[0075] Compound STD 1 STD 2 STD 3 STD 4 STD 5 STD 6 STD 7 STD 8 CRL, aMT6s 20 50 100 200 400 500 750 1000 GABA, 5-HT, DHEA 2 5 10 20 40 50 75 100 17α-OHP, T, MEL 0.2 0.5 1 2 4 5 7.5 10

[0076] 4), Using the ratio of the peak area of the analyte in the standard solution to the peak areas of melatonin and steroid internal standards as the ordinate, and the standard solution concentration as the abscissa, a standard curve was plotted;

[0077] The prepared sample solution was measured, the ratio of the peak areas of the analyte to the internal standard in the sample was measured, and it was substituted into the linear regression equation of the standard curve to calculate the concentration of the analyte;

[0078] The linear equations and correlation coefficients of the standard curves for 8 sleep-related neurotransmitters are shown in Table 5, and the standard curves for two steroid substances, melatonin MEL and testosterone T, are as Figure 2 shown, where Figure 2 a in Figure 2 is the standard curve of MEL,

[0079] Table 5 Standard Curve and Correlation Coefficient Equation

[0080]

[0081]

[0082] Example 3 Optimization of Sample Pretreatment Method

[0083] (1) Preparation of Solid Phase Extraction Column

[0084] By adjusting the ratio of PRiME HLB to MAX resin powder, the optimal packing ratio of the solid phase extraction column was found to obtain the best enrichment effect.

[0085] ① Preparation of Packing

[0086] MAX resin powder: Select commercially available Waters MAX resin (30 μm particle size);

[0087] Prime HLB: Select commercially available Waters PRiME HLB resin containing phenyl groups (30 μm particle size);

[0088] Mixing: Mix the MAX resin and the PRiME HLB resin in different mass ratios to prepare a mixed filler. Three different ratios of mixed fillers are prepared as follows:

[0089] Solid-phase extraction column 1 (filler 1): 60 mg MAX + 60 mg PRiME HLB (mass ratio 1:1)

[0090] Solid-phase extraction column 2 (filler 2): 40 mg MAX + 80 mg PRiME HLB (mass ratio 1:2)

[0091] Solid-phase extraction column 3 (filler 3): 80 mg MAX + 40 mg PRiME HLB (mass ratio 2:1)

[0092] ② Preparation of solid-phase extraction columns

[0093] Fill the prepared mixed fillers with different ratios into empty solid-phase extraction column tubes. The diameter of the adsorbed filler layer is 0.8 ± 0.1 cm, the thickness is 0.5 ± 0.1 cm, and the mass is 120 ± 5 mg.

[0094] ③ Screening of solid-phase extraction columns

[0095] Use the above three solid-phase extraction columns to conduct urine sample detection tests. The specific steps are as follows:

[0096] Note: The following artificial urine is purchased from Pickering Laboratories and is designed to simulate the real human urine environment.

[0097] (a) Add 8 sleep-related neurotransmitter standards to the artificial urine to make the concentrations of melatonin, 5-hydroxytryptamine, 6-sulfatoxymelatonin, γ-aminobutyric acid, cortisol, dehydroepiandrosterone, testosterone, and 17-hydroxyprogesterone 10 ng / mL.

[0098] (b) Add 5 mL of artificial urine sample, 20 μL of internal standard, and 5 mL of methanol solution to three 50 mL centrifuge tubes respectively; place the 3 centrifuge tubes in a centrifuge and centrifuge at 12,000 rpm for 10 min, and take the supernatant.

[0099] (c) Select three solid-phase extraction columns 1, 2, and 3 with the above-prepared three different mass ratios, and number them A, B, and C. Activate them with 3 mL of methanol and 3 mL of ultrapure water, and slowly load 10 mL of ultrapure water-diluted samples into the solid-phase extraction columns for extraction.

[0100] (d) Elute the samples in groups A, B, and C with 5 mL of eluent (acetonitrile-methanol (7:3, v / v) containing 0.1% trifluoroacetic acid), and control the elution rate at 1 drop / s.

[0101] (e) The eluents of each group were collected in 5 mL cryotubes, and concentrated to nearly dry by nitrogen blowing at 40 °C. They were re-dissolved with 1 mL of methanol-water solution with a volume ratio of methanol to water of 1:9, and then centrifuged again in a centrifuge at 14,000 rpm for 5 min. After centrifugation, the samples were transferred to chromatographic vials for measuring the concentration of the target neurotransmitter substances;

[0102] They were detected on the machine under the chromatographic and mass spectrometric conditions in Example 1, and the concentrations of various substances were calculated by the internal standard method using the linear equation obtained in Example 2. The test results are as Figure 3 shown. It can be seen from the figure that the new solid-phase extraction column with a mass ratio of MAX to PRiME HLB of 1:2 has a better adsorption effect on 8 neurotransmitters.

[0103] (2) Solid-phase extraction method screening test

[0104] The aim was to screen an efficient solid-phase extraction method for enriching and purifying sleep-related neurotransmitter substances in human urine sample solutions, and to provide a reliable sample pretreatment method for subsequent quantitative analysis.

[0105] The specific steps refer to the solid-phase extraction column screening method, with the difference that in this example, all solid-phase extraction columns used were the newly screened solid-phase extraction columns (MAX and PRiME HLB resins, mass ratio of 1:2), and three different eluents were selected for the sample detection rate test. The three groups of eluents are as follows:

[0106] Group A eluent: 5 mL of acetonitrile-methanol (7:3, v / v) containing 0.1% trifluoroacetic acid

[0107] Group B eluent: 5 mL of methanol (0.1% formic acid)

[0108] Group C eluent: 5 mL of acetonitrile (0.5% ammonia water)

[0109] The test results are as Figure 4 shown. By comparing the results obtained by the three different elution methods of A, B, and C, it can be concluded that when eluting with eluent A (acetonitrile-methanol (7:3, v / v) solution containing 0.1% trifluoroacetic acid), the concentrations of the 8 substances obtained are generally higher than those of groups B and C, indicating that the treatment with group A has a higher recovery rate.

[0110] In summary, using a solid-phase extraction column with a mass ratio of MAX to PRiME HLB of 1:2 and using 5 mL of acetonitrile-methanol (7:3, v / v) solution containing 0.1% trifluoroacetic acid as the eluent for solid-phase extraction can more efficiently enrich and purify neurotransmitters in human urine, providing a reliable sample pretreatment method for subsequent quantitative analysis.

[0111] Example 4 Application effect test

[0112] (1) Recovery rate

[0113] Prepare quality control products with three different concentrations of high, medium, and low in artificial urine. Add 20 μL of internal standard solution to each quality control product and conduct a spike recovery experiment. Each sample is continuously measured three times to calculate its average recovery rate and the relative standard deviation within the batch, as shown in Table 6:

[0114] Table 6

[0115]

[0116]

[0117] (2) Accuracy and precision

[0118] Use artificial urine to prepare quality control samples with high, medium, and low concentration levels on the same day and for three consecutive days to measure the accuracy and precision of within-batch and between-batch determinations. The results are shown in Table 7:

[0119] Table 7

[0120]

[0121] (3) Matrix effect

[0122] Add 20 μL of internal standard to 5 portions of real urine (collected at the same time). After processing according to the steps of Example 3, compare the matrix and pure solvent signals to evaluate the matrix effect. The results are shown in Table 8:

[0123] Table 8 Matrix effect of 8 sleep-related neurotransmitter markers

[0124] Substance Matrix effect (%) Melatonin 93.72±4.23 5-Hydroxytryptamine 105.56±6.98 6-Sulphatoxymelatonin 98.28±3.31 γ-Aminobutyric acid 107.91±5.05 Cortisol 99.62±3.19 Dehydroepiandrosterone 102.36±7.89 Testosterone 94.47±4.28 17-Hydroxyprogesterone 100.58±7.12

[0125] Detection of 8 neurotransmitters in real human urine samples in Example 5

[0126] This example aims to verify the application effect of the detection method established by the present invention in real human urine samples (collected at the same time), including the feasibility, accuracy, and sensitivity of the method.

[0127] Detecting human urine samples includes the following steps:

[0128] 1) Add 5 mL of each of the 3 urine samples to three 50 mL centrifuge tubes and number them X, Y, and Z respectively;

[0129] 2) Pretreat the three groups of samples with the optimized pretreatment method in Example 3, nitrogen blow and concentrate, redissolve, and then centrifuge twice to obtain the samples to be tested.

[0130] (3) Detection was performed on the chromatograph and mass spectrometer under the conditions in Example 3, and the concentrations of various substances were calculated by the internal standard method using the linear equation obtained in Example 2. The test results are shown in Table 9.

[0131] Table 9 Detection Results of 8 Neurotransmitter Substances in Human Urine Samples (unit: ng / mL)

[0132] Number MEL 5-HT aMT6s GABA CRL DHEA T 17α-OHP X 0.015 24.62 17.54 0.12 37.5 2.52 0.65 2.36 Y 0.025 29.79 24.33 0.24 53.5 8.91 1.27 1.05 Z 0.016 31.43 41.32 0.071 30.6 7.37 1.62 1.13

[0133] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for synchronously detecting neurotransmitter-related biomarkers, characterized in that, The method includes the following steps: (1) Sample pretreatment: A stable isotope-labeled internal standard mixture is added to the sample to be tested, a protein precipitant is added, the protein is centrifuged and precipitated, and after centrifugation, the supernatant is taken for solid-phase extraction using a solid-phase extraction column. Then, the target substance is eluted using an eluent, the target substance is collected, concentrated, and dissolved in a reconstitution solution to prepare a test solution; The internal standards include melatonin, 5-hydroxytryptamine, 6-sulfatoxymelatonin, γ-aminobutyric acid, cortisol, dehydroepiandrosterone, testosterone, and 17-hydroxyprogesterone; the packing material of the solid-phase extraction column includes MAX and PrimeHLB resin; (2) Using high performance liquid chromatography-tandem mass spectrometry to detect the components and contents of sleep-related neurotransmitters in the sample solution.

2. The method according to claim 1, characterized in that, In step (1), the volume ratio of the sample to be tested to the protein precipitant is 1:2 - 1:

3.

3. The method according to claim 1, wherein In step (2), the HPLC conditions include: Mobile phase A: 0.1% formic acid in water; Mobile phase B: methanol; Chromatographic column: Phenomenex C18 (2.1×100 mm, 2.6 μm) Gradient elution is adopted: injection volume is 3 μL, column temperature is 40 °C, and flow rate is 0.3 mL / min; The gradient elution conditions are as follows: 。 4. The method according to claim 1, wherein In step (2), the mass spectrometry conditions include: SCIEX Triple Quad 4500 mass spectrometer, ion source voltage is 5500 V, ion source temperature is 500 °C, and electrospray ionization-positive ion mode is adopted; the detection method is multiple reaction monitoring; the auxiliary gas is 55 psi, the collision gas is 9 psi, and the nebulizing gas is 50 psi, all of which are nitrogen; the mass spectrometry parameters are as follows:

5. The method according to claim 1, wherein In step (1), the sample is human urine.

6. The method according to claim 1, wherein In step (1), the mass ratio of MAX to Prime HLB resin is 1 - 2:1 - 2.

7. The method according to claim 1, characterized in that, In step (1), the elution method is acetonitrile-methanol (5:5 - 7:3, v / v) containing 0.1% trifluoroacetic acid, and the flow rate is controlled at 1 - 2 drops / s.

8. A combination of sleep-related neurotransmitter markers in urine, characterized in that, The markers include melatonin (MEL), 5-hydroxytryptamine (5-HT), 6-sulfatoxymelatonin (aMT6s), γ-aminobutyric acid (GABA), cortisol (CRL), dehydroepiandrosterone (DHEA), testosterone (T), and 17-hydroxyprogesterone (17α-OHP).

9. A kit containing the markers described in claim 8.

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