A method and kit for extracting and detecting amino acid neurotransmitters and melatonin in plasma based on solid phase extraction

Abstract

The invention discloses a method and a kit for extracting and detecting the concentration of amino acid neurotransmitters and melatonin in plasma based on solid phase extraction, and the detection method comprises the following steps: S1, extracting and enriching amino acid neurotransmitters and melatonin in calibrants, quality control products or plasma samples using a mixed anion exchange solid phase extraction plate; S2, sample separation by ultra-high performance liquid chromatography; S3, mass spectrometry detection; S4, calculating the content of melatonin and amino acid neurotransmitters in the sample based on a standard curve. The detection method and the kit of the present invention can effectively remove proteins and inorganic salts in plasma, quickly realize the extraction of γ-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan, and melatonin, and the concentration of the organic reagent is low, which can better wash out water-soluble impurities, reduce the interference of impurities, and thus improve the detection sensitivity of the target compound.

Description

Technical Field

[0001] The present invention relates to the technical field of melatonin detection, and in particular to a method and a kit for extracting and detecting the concentrations of amino acid neurotransmitters and melatonin in plasma based on a solid phase extraction method. Background Art

[0002] Amino acid neurotransmitters and melatonin mainly include γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT, serotonin), 5-hydroxytryptophan (5-HTP) and melatonin (MT), which can be used clinically to assist in the diagnosis of sleep disorders and endocrine-related diseases.

[0003] Sleep is a complex physiological process. During sleep, the body repairs itself, which is beneficial for physical recovery and growth. Lack of sleep can lead to emotional instability and increase the risk of anxiety and depression. People spend about one-third of their lives sleeping. Lack of high-quality sleep not only affects people's daytime activities, but also damages the function of the immune system, and is prone to sleep disorders in the long run. The incidence of sleep disorders in modern society is very high. It is estimated that sleep disorders affect at least 20-40% of adults. Similarly, various types of sleep difficulties also affect 25-62% of children, affecting their developmental status.

[0004] GABA is an important inhibitory neurotransmitter with a sedative effect in the human body. Studies have shown that it plays an important role in the energy metabolism of the human brain, improves sleep, and can be used as a sleep inducer. Studies have shown that abnormal levels of GABA in the body can easily cause neurological disorders, such as Alzheimer's disease, Parkinson's disease, schizophrenia, autism, and acute post-traumatic stress disorder. When GABA is lacking, people are prone to anxiety and other emotions.

[0005] Melatonin is secreted by the pituitary pineal gland and has a hypnotic and sedative effect. Its task is to regulate circadian rhythms and sleep patterns. Low melatonin levels are one of the important causes of insomnia, and supplementing melatonin and its related precursors can help improve sleep. Studies have shown that a combination of a tryptophan-rich breakfast, regular morning light, and evening supplementary light can improve the secretion of melatonin at night, thereby improving sleep quality and reducing the time required to fall asleep. Treatment with 5-hydroxytryptophan can also positively affect the level of wakefulness, thereby reducing children's sleep night terrors. 5-hydroxytryptamine (5-HT), also known as serotonin, is a precursor of melatonin. Through different neural pathways, it plays an important role in regulating sleep-wake rhythms and central fatigue. Studies have shown that when the 5-HT content in the brain of animals is reduced, experimental animals will suffer from insomnia. Increasing the level of 5-HT can effectively improve the clinical symptoms of patients with insomnia.

[0006] At present, protein precipitation, dansyl chloride derivatization and other methods are commonly used for extraction and derivatization. Since γ-aminobutyric acid is an amphoteric compound, 5-hydroxytryptamine and 5-hydroxytryptophan are acidic substances, and melatonin is a neutral substance, it is difficult to detect them together. There is no report on the simultaneous detection of γ-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan and melatonin in plasma. The purpose of the present invention is to provide a method for simultaneously detecting the concentrations of γ-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan and melatonin on the basis of the prior art. Due to the key role of γ-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan and melatonin in sleep regulation, the detection of related substances in serum can understand their concentration levels in the body, thereby helping clinicians to better diagnose, treat and evaluate the efficacy of patients with sleep disorders-insomnia, which has important clinical significance.

[0007] Patent application CN118759076A of Pluo (Wuhan) Medical Biotechnology Co., Ltd. provides a method for detecting neurotransmitters related to tic disorders, which is mainly a neurotransmitter detection method. The detection indicators that are the same as those of the present invention only include two compounds (5-hydroxytryptamine and γ-aminobutyric acid). The precipitant is acid-containing isopropanol, which needs to be precooled at a temperature of -20°C to -60°C for 5-20 minutes, and needs to be incubated at 50°C for 10 minutes before detection. LC-MS / MS detection is performed after a series of treatments such as low-temperature treatment of the precipitant, adjustment of pH, and high-temperature derivatization. The operation steps are extremely complicated, time-consuming and labor-intensive, and there is incomplete derivatization, which affects the sensitivity detection of the compound.

[0008] Patent application CN118226045A of Hefei Xinzhi Medical Device Co., Ltd. provides a detection method, reagent and detection system for detecting more than ten markers at the same time, mainly biomarkers, including neurotransmitter detection methods. The detection indicators same as the present invention include two compounds (γ-aminobutyric acid and 5-hydroxytryptophan), and LC-MS / MS detection is performed by protein precipitation, pH adjustment, centrifugation, etc., which is complicated and time-consuming. In addition, it is reported in the literature that the concentration of γ-aminobutyric acid in healthy human plasma is 13.40±2.75ng / mL, and the linear low point of the patent detection is 4nmol / L (38ng / mL), which greatly exceeds the concentration range of γ-aminobutyric acid in healthy human plasma, and has little reference value for the clinical detection of γ-aminobutyric acid in plasma.

[0009] Patent application CN118817928A of Yujing Technology (Shanghai) Co., Ltd. discloses a method for simultaneously and highly sensitively detecting melatonin and polar alkaloid compounds based on WCX solid phase extraction. It is mainly a method for detecting melatonin and serotonin. The detection indicators that are the same as those of the present invention include two compounds. The patented method cannot simultaneously extract γ-aminobutyric acid in plasma and has no reference significance for the present invention. γ-aminobutyric acid, as an endogenous substance in the human body, has a small molecular weight (only 103.12), a simple structure, no ultraviolet absorption, and its polarity is very strong (logP = -3.17). At the same time, the reference range given by the melatonin Quest diagnosis is 2.0-80pg / mL during the day; 18.5-180.0pg / mL at night, so it is required to have sufficient sensitivity to detect melatonin in normal human plasma. In addition, the existing literature reports that the Log P of serotonin is also very low. Compounds such as γ-aminobutyric acid and serotonin cannot be extracted by SPE with a C18 matrix. All public reports on the enrichment of melatonin use a C18 matrix SPE method, so the solid phase extraction plate needs to use a mixed ion exchange mixed mode adsorbent. The patent application uses a WCX SPE plate with a cation exchange matrix, which is suitable for the extraction of quaternary ammonium salt compounds and for the extraction of strong base compounds. γ-aminobutyric acid (PI = 7.295) has carboxyl and hydroxyl groups in its compound structural groups, and melatonin is a neutral compound. The present invention uses a mixed anion solid phase extraction plate filler with the characteristics of a C18 matrix and a cation exchange matrix, which has a better effect on the joint detection of γ-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan, and melatonin. Summary of the invention

[0010] The object of the present invention is to provide a method and a kit for extracting and detecting the concentration of amino acid neurotransmitters and melatonin in plasma based on solid phase extraction. Specifically, the present invention discloses a method for extracting γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT), 5-hydroxytryptophan (5-HTP), and melatonin (MT) in plasma based on a mixed anion exchange solid phase extraction plate (MAX SPE). The present invention comprises the following steps: firstly, γ-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan and melatonin in plasma are extracted and enriched using a mixed anion exchange solid phase extraction plate, then the samples are separated by ultra-high performance liquid chromatography, and finally mass spectrometry is performed. The present invention uses a mixed anion exchange solid phase extraction plate to extract four kinds of standard substances and internal standards with known concentrations, and finally detects them by liquid chromatography tandem mass spectrometry to establish a standard curve, thereby calculating the concentrations of four kinds of neurotransmitter substances in human plasma. This method can be used to assist in the diagnosis of endocrine-related diseases such as schizophrenia, sleep disorders, and psychological medical diseases, as well as the development process, efficacy, and prognosis of the disease, thereby providing patients with personalized diagnostic and treatment information.

[0011] The objective of the present invention is achieved through the following technical solutions:

[0012] In a first aspect, the present invention provides a method for extracting and detecting the concentration of amino acid neurotransmitters and melatonin in plasma based on solid phase extraction, comprising the following steps:

[0013] S1. Extract and enrich amino acid neurotransmitters and melatonin in plasma using a mixed anion exchange solid phase extraction plate;

[0014] S2, sample separation by ultra-high performance liquid chromatography;

[0015] S3, performing mass spectrometry detection;

[0016] S4. Calculate the content of melatonin and amino acid neurotransmitters in the sample based on the standard curve method.

[0017] As some specific embodiments of the present invention, the amino acid neurotransmitter includes at least one of γ-aminobutyric acid, 5-hydroxytryptamine (serotonin) and 5-hydroxytryptophan.

[0018] As some specific implementations of the present invention, step S1 specifically includes:

[0019] S11, using methanol to activate the mixed anion exchange solid phase extraction plate;

[0020] S12, adding ultrapure water to balance the solid phase extraction plate;

[0021] S13, mixing the standard, quality control or plasma sample with the internal standard solution and the ammonia solution, loading the sample onto a solid phase extraction plate, pressurizing and enriching the sample (using a pressurizing device), and discarding the effluent;

[0022] S14, (under the action of the positive pressure device) using an ammonia solution as eluent I to elute the target substance trapped on the solid phase extraction plate, remove impurities, and discard the effluent;

[0023] S15, using methanol aqueous solution as eluent II, performing secondary elution, eluting the target substance trapped on the solid phase extraction plate again, removing impurities, and discarding the effluent;

[0024] S16, eluting the target substance trapped on the solid phase extraction plate with methanol containing acetic acid;

[0025] S17. Blow dry with nitrogen, add ultrapure water to dissolve, and shake gently to mix.

[0026] As some specific embodiments of the present invention, in step S13, the internal standard solution is a 80%-100% (v / v) methanol solution containing 10-50 ng / mL γ-aminobutyric acid-D2, 5-20 ng / mL 5-hydroxytryptamine-D4, 5-20 ng / mL 5-hydroxytryptophan-D3, and 10-50 pg / mL melatonin-D3.

[0027] As some specific embodiments of the present invention, in step S13, the ammonia water is an ammonia water solution with a mass fraction of 1%-5%.

[0028] As some specific embodiments of the present invention, in step S14, the eluent I is an ammonia solution with a mass fraction of 1-5%;

[0029] And / or, in step S15, the eluent II is a 40%-50% (v / v) methanol aqueous solution.

[0030] As some specific embodiments of the present invention, in step S16, the mass percentage content of acetic acid in the methanol containing acetic acid is 2%-5%.

[0031] As some specific embodiments of the present invention, in step S2, when liquid chromatography is performed, mobile phase A is an aqueous solution containing 5-20 mM ammonium acetate and 0.1% (v / v) formic acid, and mobile phase B is acetonitrile containing 0.1% (v / v) formic acid.

[0032] As some specific embodiments of the present invention, in step S3, when liquid chromatography is performed, the elution gradient of the mobile phase is:

[0033]

[0034] As some specific embodiments of the present invention, in step S3, the mass spectrometry conditions are:

[0035]

[0036]

[0037] As some specific embodiments of the present invention, in step S3, the mass spectrometry parameter table is:

[0038] Compound Precursor ion Product ions Impact voltage (v) GABA 104.0 45.0 21 GABA 104.0 87.0 10 GABA_D2 106.1 89.0 10 5-HT 177.1 115.0 28 5-HT 177.1 160.1 10 5-HT_D4 181.1 164.1 11 5-HTP 221.2 162.0 18 5-HTP 221.2 204.0 9 5-HTP_D3 224.2 206.1 10 MT 233.2 130.0 46. MT 233.2 174.1 14 MT_D3 236.2 174.1 13

[0039] In a second aspect, the present invention provides a kit for extracting and detecting amino acid neurotransmitters and melatonin in plasma based on solid phase extraction, wherein the amino acid neurotransmitters and melatonin in plasma are extracted and detected by any of the methods described above.

[0040] The method and kit of the present invention are helpful for the diagnosis, treatment and evaluation of therapeutic effect of patients with sleep disorder-insomnia, and have important clinical significance.

[0041] The inventor's research and development project at the same time, "A method and kit for extracting and detecting melatonin in plasma and related substances in its synthesis pathway based on magnetic adsorbents", has three detection indicators that are the same as those of the present invention. The method in the project, i.e., a magnetic mixed strong cation exchange adsorbent (MCX), is used to extract and enrich the γ-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan and melatonin of the present invention. First, 4% (v / v) phosphoric acid is used to acidify the sample to be tested. The γ-aminobutyric acid in the sample to be tested can be exchanged and adsorbed on the magnetic adsorption solid phase extraction plate under the action of 4% phosphoric acid, and then an organic phase solvent containing ammonia water is used for elution. The test results show that both γ-aminobutyric acid and its internal standard have interfering peaks near the retention time, thereby affecting the quantification of γ-aminobutyric acid. Therefore, the method of the project is not suitable for extracting, enriching and detecting the hungry γ-aminobutyric acid in the present application.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1) The mixed anion exchange solid phase extraction plate can quickly extract γ-aminobutyric acid, 5-hydroxytryptamine, 5-hydroxytryptophan, and melatonin, providing an experimental scheme for the rapid extraction and detection of amino acid neurotransmitters and melatonin;

[0044] 2) Processing samples through solid phase extraction plates can effectively remove proteins and inorganic salts in plasma, which is beneficial for instrument detection and has important significance for the detection of large-throughput samples;

[0045] 3) The concentration of organic reagent in the eluent is low, which can better elute water-soluble impurities and reduce the interference of impurities, thereby improving the detection sensitivity of the target compound;

[0046] 4) During solid phase extraction, 1% ammonia solution and 50% methanol solution were used for two-step elution, 1% ammonia solution was used to remove impurities adsorbed on the solid phase extraction plate, and 50% methanol solution was used based on the characteristics of the compounds of the present application. Suitable organic reagents were selected to further remove impurities on the plate, reduce interference from impurities, and improve the detection sensitivity of the target compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0048] Figure 1 is the standard curve of γ-aminobutyric acid (GABA);

[0049] Figure 2 is the standard curve of 5-hydroxytryptamine (5-HT);

[0050] Figure 3 is a standard curve diagram of 5-hydroxytryptophan (5-HTP);

[0051] Figure 4 is the standard curve of melatonin (MT);

[0052] Figure 5 This is the detection spectrum of γ-aminobutyric acid (GABA) at the lowest point of the standard, Cal 1;

[0053] Figure 6 This is the 5-hydroxytryptamine (5-HT) detection spectrum of the standard substance, Cal 1, the lowest point;

[0054] Figure 7 This is the 5-hydroxytryptophan (5-HTP) detection spectrum of the standard substance, Cal 1, the lowest point;

[0055] Figure 8 This is the melatonin (MT) detection spectrum of the standard substance, the lowest point Cal 1;

[0056] Fig. 9 The graph shows the comparison results of the peak areas detected after the plasma samples of the same concentration were extracted using different volume fractions of methanol solutions as eluent II in Example 3. DETAILED DESCRIPTION

[0057] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0058] Example 1

[0059] 1. Reagent Preparation

[0060] 1. Internal standard solution

[0061] The internal standard solution is an aqueous solution containing 20 ng / mL γ-aminobutyric acid-D2 (from Bepure), 20 ng / mL 5-hydroxytryptamine-D4 (from Bepure), 20 ng / mL 5-hydroxytryptophan-D3 (from Bepure), and 20 pg / mL melatonin-D3 (from Shanghai Pufen Biotechnology) in 80% methanol by volume;

[0062] 2. Quality control products

[0063] Methanol / water (volume ratio 1 / 1, containing 1% ascorbic acid by mass) was used to prepare γ-aminobutyric acid (γ-GABA, from zzstandard), 5-hydroxytryptamine (5-HT, from China Food and Drug Inspection Institute), 5-hydroxytryptophan (5-HTP, from Shanghai Anpu Biological) and melatonin (MT, from China Food and Drug Inspection Institute) into stock solutions, which were then diluted to obtain working solutions. The concentrations of γ-GABA, 5HT, 5HTP and MT working solutions were 10μg / mL, 10μg / mL, 4μg / mL and 4ng / mL, respectively. Human plasma (from GoldenWest) was used to dilute and mix the working solutions to form three different concentrations of quality control products. The concentrations of the quality control products are shown in Table 1.

[0064] Table 1 Quality control product concentration table

[0065]

[0066] 3. Standard products

[0067] γ-aminobutyric acid (γ-GABA), 5-hydroxytryptamine (5-HT), 5-hydroxytryptophan (5-HTP) and melatonin (MT) were prepared into stock solutions using methanol / water (volume ratio 1 / 1, containing 1% ascorbic acid by mass), and then diluted to obtain working solutions. The concentrations of γ-GABA, 5-HT, 5-HTP and MT working solutions were 10 μg / mL, 10 μg / mL, 4 μg / mL and 4 ng / mL, respectively. Each working solution was diluted and mixed with a diluent containing 1% (w / w) bovine serum albumin (source: Sigma) diluted with phosphate buffer to form a series of standard products with a concentration gradient. The concentrations of each standard product are shown in Table 2.

[0068] Table 2 Standard concentration table

[0069]

[0070]

[0071] Freeze-dry the prepared standard and quality control products to make freeze-dried powders. When using, use a calibrated 1000μL pipette to accurately transfer 0.5mL of ultrapure water to the freeze-dried standard or quality control product, vortex for 5 minutes, and use after fully dissolving.

[0072] 2. Take the following steps to extract the analyte:

[0073] 1. Take out 400 μL of standard, quality control or plasma sample into a 1.5 mL centrifuge tube, add 50 μL of internal standard solution and shake to mix, then add 400 μL of 1% (w / w) ammonia solution and vortex for 5 minutes.

[0074] 2. Use 500 μL methanol to activate the MAX SPE plate used for solid phase extraction, with a specification of 5 mg / 1 mL;

[0075] 3. After activation, continue to add 500 μL of ultrapure water to the plate to balance the solid phase extraction plate;

[0076] 4. Transfer 850 μL of the mixed sample obtained in step 1 to the solid phase extraction plate, pressurize it with a positive pressure device, and maintain the rate of 2-3 seconds per drop to enrich it through the solid phase extraction plate until the effluent is completely discarded;

[0077] 5. Under the action of the positive pressure device, add 500 μL of eluent I (1% (w / w) ammonia solution) to elute the target substance trapped on the solid phase extraction plate, maintaining a rate of 2-3 seconds per drop to remove impurities adsorbed on the solid phase extraction plate, and discard the effluent;

[0078] 6. Continue to add 500 μL of eluent II (aqueous solution containing 50% (v / v) methanol) to the solid phase extraction plate for the second eluent;

[0079] 7. Under the action of the positive pressure device, maintain a rate of 2-3 seconds per drop, and add 200 μL of methanol containing 2% (w / w) acetic acid to elute the target substance trapped on the solid phase extraction plate;

[0080] 8. Blow the eluate containing the target substance obtained in step 7 to dryness under a nitrogen flow, then add 50 μL of ultrapure water to dissolve it, gently shake it on an oscillating plate for 60 seconds to mix it, and then transfer all the solution into an injection vial or a 96-well injection plate for LC-MS / MS detection.

[0081] 3. LC-MS / MS Detection

[0082] 1. Chromatographic conditions:

[0083] The chromatographic column was Acquity UPLC HSS C18 column (3.0 × 50 mm, 3 μm, Waters);

[0084] Mobile phase A was an aqueous solution containing 20 mM ammonium acetate + 0.1% (v / v) formic acid, and mobile phase B was acetonitrile containing 0.1% (v / v) formic acid;

[0085] Column temperature: 35°C.

[0086] The mobile phase gradient conditions used are shown in Table 3.

[0087] Table 3 Mobile phase gradient conditions

[0088]

[0089]

[0090] 2. Mass spectrometry conditions:

[0091] The mass spectrometer parameters are shown in Table 4, and the compound detection mass spectrometer parameters are shown in Table 5.

[0092] Table 4 Mass spectrometer parameters are as follows

[0093] project parameter Ion Source Type H-ESI Mode Positive,3500 Sheath Gas(Arb) 50 Aux Gas(Arb) 25 Sweep Gas(Arb) 0 Ion Transfer Tub Temp(℃) 350 Vaporizer Temp(℃) 400

[0094] Table 5 Mass spectrometry parameters

[0095] Compound Precursor ion Product ions Impact voltage (v) γ-GABA 104.0 45.0 21 γ-GABA 104.0 87.0 10 GABA_D2 106.1 89.0 10 5-HT 177.1 115.0 28 5-HT 177.1 160.1 10 5-HT_D4 181.1 164.1 11 5-HTP 221.2 162.0 18 5-HTP 221.2 204.0 9 5-HTP_D3 224.2 206.1 10 MT 233.2 130.0 46. MT 233.2 174.1 14 MT_D3 236.2 174.1 13

[0096] Example 2

[0097] In this embodiment, a detailed optimization and comparison experiment was conducted on the plasma sample eluent I, and the composition of the eluent I was determined by comprehensively considering the overall detection method.

[0098] In Example 1, when eluting in step 5 of extracting the analyte, the eluent I used was a 1% (w / w) ammonia solution. In this example, the composition of the eluent I was adjusted. Except for the composition of the eluent I, the remaining steps were consistent with Example 1. The plasma samples of the same concentration (quality control product MQC, γ-GABA concentration of 80 ng / mL, 5-HT concentration of 80 ng / mL, 5-HTP concentration of 30 ng / mL, MT concentration of 30 pg / mL) were extracted and detected, and the peak area data of each analyte were compared, as shown in Table 6 below.

[0099] Table 6 Peak area display of the test results of different eluents I for plasma samples of the same concentration

[0100]

[0101] In this example, different reagents and different ratios of eluent I components were tried to compare the elution effects of samples of the same concentration under the same conditions. The experiment found that when 1% (w / w) ammonia solution was used as the loading eluent I, the elution effect of the four targets was the best. Therefore, in the experiment, using 1% (w / w) ammonia solution as eluent I can achieve good results.

[0102] Example 3

[0103] In this embodiment, a detailed optimization and comparison experiment was conducted on the plasma sample eluent II, and the composition of the eluent II was determined by comprehensively considering the overall detection method.

[0104] In Example 1, when performing elution in step 6 of extracting the analyte, the eluent II used is an aqueous solution containing 50% (v / v) methanol. In this example, the composition of the eluent II is adjusted. Except for the composition of the eluent II, the remaining steps are consistent with Example 1. The plasma samples of the same concentration (quality control product MQC, γ-GABA concentration is 80 ng / mL, 5-HT concentration is 80 ng / mL, 5-HTP concentration is 30 ng / mL, MT concentration is 30 pg / mL) are extracted and detected, and the peak area data of each analyte are compared. The results are as follows: Fig. 9 shown.

[0105] according to Fig. 9As shown in the results, in this embodiment, different proportions of methanol solution were tried as eluent II component, and then elution and detection were performed, and the elution effect of samples of the same concentration under the same conditions was compared. The results showed that when the eluent II component was between 50% (v / v) methanol solution and 90% (v / v) methanol solution, as the proportion of methanol increased, the difference in the elution effect of γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT) and 5-hydroxytryptophan (5-HTP) target compounds was not obvious, while the elution effect trend of melatonin (MT) decreased significantly. At the same time, 100% methanol and 100% acetonitrile were investigated as eluent II, and it was found that when the solvents were methanol and acetonitrile, respectively, the elution effect of γ-aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT) and 5-hydroxytryptophan (5-HTP) was not much different, but the elution effect of melatonin was also significantly inferior to 50% (v / v) methanol. Therefore, 50% (v / v) methanol solution as eluent II can achieve good elution effect for the four compounds.

[0106] Effect Example 1 - Calibration Curve and Linear Point Deviation

[0107] According to the method of Example 1, each standard was extracted and tested by isotope internal standard quantitative method through a mixed anion exchange solid phase extraction plate, and a standard curve was established with the concentration ratio of the standard as the x-axis and the peak area ratio of the standard to the internal standard as the y-axis. The results showed that the linear fitting equations of amino acid neurotransmitters and melatonin within their respective concentration ranges had good linearity, and the correlation coefficient was above 0.99. The obtained standard curve is shown in Figure 1-4 The linear equation and linear range are shown in Table 7.

[0108] Table 7 Linear range and linear relationship of target compounds

[0109] Compound Linear range <![CDATA[Correlation coefficient (R 2 )]]> Linear fitting curve graph GABA 2-500ng / mL 0.9998 Figure 1 Serotonin 1-500ng / mL 0.9999 Figure 2 5-Hydroxytryptophan 0.2-200ng / mL 0.9998 Figure 3 Melatonin 0.5-200pg / mL 0.9959 Figure 4

[0110] Effect Example 2--Linear Point Deviation

[0111] According to the actual concentration value of the standard detected in Example 1, the deviation from the theoretical value was calculated, and the results are shown in Table 8. According to Table 8, it can be seen that the deviation of the standard test results is within 15%. This shows that the method for extracting amino acid neurotransmitters and melatonin from plasma based on mixed anion exchange solid phase extraction is feasible and reliable.

[0112] Table 8 Relative deviation between actual concentration value and theoretical value of standard

[0113] Compound / Linear Point GABA 5-HT 5-HTP MT Cal 1 11.18％ 1.98％ 6.36％ 12.88％ Cal 2 -9.43％ 1.24％ -0.95％ 3.69％ Cal 3 -3.35％ -3.62％ -7.77％ -5.34％ Cal 4 -1.01％ -0.04％ 0.73％ -14.50％ Cal 5 4.00％ 0.21％ 2.22％ -6.31％ Cal 6 -1.39％ 0.22％ -0.52％ 6.54％

[0114] Cal1-6 correspond to the standards S1-S6 in Example 1, respectively, and when the lowest point of the standard Cal 1 is detected, the signal-to-noise ratios of the four compounds are all greater than 10 (e.g. Figure 5-8 As shown), it can meet the detection requirements. Figure 5 This is the detection spectrum of γ-aminobutyric acid (GABA) at the lowest point of the standard, Cal 1; Figure 6 This is the 5-hydroxytryptamine (5-HT) detection spectrum of the standard substance, Cal 1, the lowest point; Figure 7 This is the 5-hydroxytryptophan (5-HTP) detection spectrum of the standard substance, Cal 1, the lowest point; Figure 8 This is the melatonin (MT) detection spectrum of the standard substance, Cal 1, the lowest point.

[0115] Effect Example 3 - Extraction Recovery and Precision

[0116] According to the method of Example 1, quality control samples at three concentration points were extracted and detected using a mixed anion exchange solid phase extraction plate.

[0117] The ratio of the measured concentration of the quality control sample to the theoretical concentration of the corresponding standard solution is used to calculate the solid phase extraction plate extraction recovery rate. The calculated extraction recovery rate is shown in Table 9: The measured extraction recovery rate is between 85% and 115%. This shows that the method of extracting amino acid neurotransmitters and melatonin from plasma based on mixed anion exchange solid phase extraction is feasible and reliable.

[0118] Table 9 Calculation of extraction recovery

[0119] Recovery rate GABA 5-HT 5-HTP MT LQC 98.23％ 95.02％ 94.87％ 100.78％ MQC 91.89％ 92.45％ 85.42％ 102.32％ HQC 97.88％ 93.88％ 102.10％ 98.77％

[0120] The calculated extraction recoveries of the four compounds were between 85% and 115%, meeting the detection requirements.

[0121] At the same time, each quality control sample was measured in parallel 5 times for 3 consecutive days to calculate the intra-day and inter-day precision. The calculated intra-day and inter-day precisions are shown in Table 10: The intra-day precision quality control sample CV was between 15% and the inter-day precision quality control sample CV was between 15%. This shows that the method of extracting amino acid neurotransmitters and melatonin from plasma based on mixed anion exchange solid phase extraction is feasible and reliable.

[0122] Table 10 Calculation of intra-day and inter-day precision

[0123]

[0124] The calculated CV of intra-day precision quality control samples of the four compounds was less than 15%, and the CV of inter-day precision quality control samples was less than 15%, which met the detection requirements.

[0125] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for extracting and detecting the concentration of amino acid neurotransmitters and melatonin in plasma based on solid phase extraction, characterized in that: The steps include: S1. Extract and enrich amino acid neurotransmitters and melatonin in plasma using a mixed anion exchange solid phase extraction plate; S2, sample separation by ultra-high performance liquid chromatography; S3, enter mass spectrometry detection; S4. Calculate the content of melatonin and amino acid neurotransmitters in the sample based on the standard curve method.

2. The method according to claim 1, characterized in that: The amino acid neurotransmitter includes at least one of gamma-aminobutyric acid, 5-hydroxytryptamine (serotonin), and 5-hydroxytryptophan.

3. The method according to claim 1, characterized in that Step S1 specifically includes: S11, using methanol to activate the mixed anion exchange solid phase extraction plate; S12, adding ultrapure water to balance the solid phase extraction plate; S13, mixing the standard, quality control or plasma sample with the internal standard solution and ammonia water, loading the sample onto the equilibrated solid phase extraction plate, pressurizing and enriching, and discarding the effluent; S14, using an ammonia solution as eluent I to elute the target substance trapped on the solid phase extraction plate, remove impurities, and discard the effluent; S15, using methanol aqueous solution as eluent II, performing secondary elution, eluting the target substance trapped on the solid phase extraction plate again, removing impurities, and discarding the effluent; S16, eluting the target substance trapped on the solid phase extraction plate with methanol containing acetic acid; S17. Blow dry with nitrogen, add ultrapure water to dissolve, and shake to mix.

4. The method according to claim 3, characterized in that In step S13, the internal standard solution is a methanol solution containing 10-50 ng / mL γ-aminobutyric acid-D2, 5-20 ng / mL 5-hydroxytryptamine-D4, 5-20 ng / mL 5-hydroxytryptophan-D3, and 10-50 pg / mL melatonin-D3 with a volume fraction of 80%-100%.

5. The method according to claim 3, characterized in that: In step S13, the ammonia water is an ammonia water solution with a mass fraction of 1%-5%.

6. The method according to claim 3, characterized in that In step S14, the eluent I is an ammonia solution with a mass fraction of 1%-5%; And / or, in step S15, the eluent II is a methanol aqueous solution with a volume fraction of 40%-50%.

7. The method according to claim 3, characterized in that In step S16, the mass percentage content of acetic acid in the methanol containing acetic acid is 2%-5%.

8. The method according to claim 1, characterized in that In step S2, when liquid chromatography is performed, mobile phase A is an aqueous solution containing 5-20 mM ammonium acetate and 0.1% by volume formic acid, and mobile phase B is acetonitrile containing 0.1% by volume formic acid.

9. The method according to claim 1, characterized in that: In step S2, when liquid chromatography is performed, the mobile phase gradient is:

10. A kit for detecting amino acid neurotransmitters and melatonin in plasma based on solid phase extraction, characterized in that: The amino acid neurotransmitters and melatonin in plasma are extracted and detected using the method described in any one of claims 1 to 9.

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