A method and kit for extracting and detecting melatonin in plasma and related substances in its synthesis pathway based on magnetic adsorbent

Abstract

The present invention discloses a detection method and a kit for extracting melatonin and related substances in its synthesis pathway from plasma based on a magnetic adsorbent. The detection method comprises: S1, extracting and enriching tryptophan, serotonin, 5-hydroxytryptophan and melatonin from plasma samples or standard products with a magnetic adsorbent; S2, separating samples by ultra-high performance liquid chromatography, and then performing mass spectrometry detection; S3, establishing a calibration curve according to the detection results of the standard products, and calculating the concentrations of tryptophan, serotonin, 5-hydroxytryptophan and melatonin in the plasma samples according to the calibration curve. A kit for detecting melatonin and related substances in its synthesis pathway in plasma is also disclosed. The automated operation of the experimental process is realized by extracting compounds based on a magnetic solid phase extractor, which has the advantages of simple operation, short extraction time, small amount of organic solvent used, fast solid-liquid phase separation speed, etc., and efficiently captures the target analytes of the sample matrix, making the separation and enrichment process simple, fast and efficient.

Description

Technical Field

[0001] The invention relates to a melatonin detection method, in particular to a method and a kit for extracting and detecting melatonin in plasma and related substances in its synthesis pathway based on a magnetic adsorbent. Background Art

[0002] Melatonin and related substances in its synthesis pathway mainly include tryptophan (Trp), serotonin (5-HT), 5-hydroxytryptophan (5-HTP), and melatonin (MT). These compounds play a key role in the nervous system.

[0003] Tryptophan (β-indolylalanine) is a precursor of the important neurotransmitter 5-hydroxytryptamine in the human body. Tryptophan plays a vital role in human health and disease and is involved in the biosynthesis of serotonin and melatonin. Figure 1 The figure shows the 5-hydroxytryptamine pathway of tryptophan metabolism. Tryptophan is converted into 5-hydroxytryptophan by tryptophan hydroxylase, which is further decarboxylated to produce 5-hydroxytryptamine. Melatonin is a metabolite of 5-hydroxytryptamine and can be converted into melatonin by 5-hydroxytryptamine N-acetyltransferase and 5-hydroxytryptamine N-acetylmethoxytransferase.

[0004] Serotonin is a neurotransmitter that is mainly found in the central nervous system and intestines. It plays an important role in mood regulation, appetite, digestion, sleep and social behavior. Low levels of serotonin are associated with depression, anxiety and other mood disorders. 5-HTP has multiple biological activities such as regulating biological rhythms, emotions, cognition, scavenging free radicals, promoting inflammatory signal transduction, and also has an important impact on bone metabolism. Melatonin is the main hormone secreted by the pineal gland. Its most common function is to regulate biological rhythms. In addition, it can also act as a free radical scavenger and a strong antioxidant, and has anti-inflammatory effects. It participates in the regulation of the immune system and has a direct immune-enhancing effect on the human body. In addition, it also has protective and anti-cancer effects in brain and gastrointestinal diseases, mental illnesses and cardiovascular diseases. In the future, it is very likely to be developed into a clinical drug or dietary health product with low dosage, mild efficacy, low toxicity and side effects, and good tolerance.

[0005] Detecting and understanding melatonin and related substances in its synthesis pathway can help: 1) Evaluate sleep disorders. For insomnia patients, detecting melatonin synthesis levels can help doctors determine whether the patient's biological clock is disordered. For example, patients with long-term insomnia and insufficient melatonin synthesis may have circadian rhythm disordered sleep disorders. 2) It can assist in the detection of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. In the early diagnosis, it is meaningful to detect melatonin levels. Because these diseases are often accompanied by increased oxidative stress and reduced melatonin synthesis, detecting its synthesis can help to determine the progression of the disease to a certain extent. 3) Melatonin itself is an endocrine hormone, and detecting its synthesis can help diagnose diseases related to endocrine disorders. Abnormal melatonin synthesis may be related to pineal lesions. By detecting its synthesis, it can provide clues for the diagnosis of pineal diseases (such as pinealoma, etc.). 4) In cancer treatment, the detection of melatonin synthesis is also of certain value. Some studies have found that melatonin may have an inhibitory effect on tumor cells, and its abnormal synthesis may be related to the occurrence of certain tumors, such as breast cancer. Detecting its level is helpful in studying the pathogenesis of cancer.

[0006] So far, the analysis methods of melatonin and related substances in its synthesis pathway and single compounds mostly use protein precipitation, liquid-liquid extraction and solid phase extraction plate extraction, and the pre-treatment of these methods is done manually without automation. The low level of automation in sample pre-treatment is currently a pain point in clinical mass spectrometry analysis technology, which restricts the improvement of mass spectrometry efficiency. There is no report on the simultaneous detection method for these compounds. The present invention uses magnetic bead automated extraction, which has good prospects, is simple to operate and takes less time.

[0007] Patent CN113588804B of Nanjing Pinsheng Medical Testing Laboratory Co., Ltd. discloses a method for detecting the concentration of serotonin and melatonin in serum. This patent only detects serotonin and melatonin, but its pretreatment is complicated and time-consuming. It uses a liquid-liquid extraction method and requires the addition of a large amount of toxic and harmful organic solvents. The steps are cumbersome, time-consuming, labor-intensive, and environmentally polluting, and the efficiency is low.

[0008] Patent application CN118777490A of Shanghai Mental Health Center (Shanghai Psychological Counseling Training Center) discloses a method for detecting and analyzing 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan, kynurenine, and melatonin in serum, which is mainly a neurotransmitter detection method. The detection indexes identical to those of the present invention include three compounds, and the target compound is mainly extracted by protein precipitation. The protein precipitation operation steps are simple and low in cost, but nitrogen blowing and redissolution are required after precipitation, which is time-consuming. In addition, some of the biological samples treated by this method will not be completely precipitated, so they are susceptible to the influence of matrix effects when performing mass spectrometry detection, resulting in ion suppression or enhancement.

[0009] Patent CN115902039B of Dalian Boyuan Medical Technology Co., Ltd. discloses a method for detecting melatonin concentration in plasma, which only involves a melatonin detection method. The pretreatment is performed by protein precipitation, SPE plate solid phase extraction, etc. to extract melatonin for LC-MS / MS detection. The steps are cumbersome and a large number of solutions are prepared. The use of solid phase extraction plates is not convenient for experimenters to extract and operate, and increases the cost of the experiment.

[0010] 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 a method for detecting melatonin and serotonin, mainly through solid phase extraction of target compounds by SPE plate. The SPE sample pretreatment scheme adopted in the scheme is complicated to operate, and the time and labor cost required for operation are high, and it is not easy to realize the automation of pretreatment and detection process. In comparison, the scheme disclosed in the present invention is based on magnetic beads to extract melatonin and related substances in its synthetic pathway in plasma, with simple pretreatment and easier automation. Summary of the invention

[0011] The purpose of the present invention is to provide a method and kit for extracting and detecting melatonin in plasma and related substances in its synthesis pathway based on a magnetic adsorbent. Specifically, the present invention discloses a method for extracting and detecting tryptophan (Trp), serotonin (5-HT), 5-hydroxytryptophan (5-HTP), and melatonin (MT) in plasma based on a magnetic hybrid strong cation exchange adsorbent (MCX). The detection method of the present invention comprises the following steps: first, tryptophan, serotonin, 5-hydroxytryptophan, and melatonin in plasma are extracted and enriched using a magnetic hybrid strong cation adsorbent, and then the samples are separated by ultra-high performance liquid chromatography, and finally mass spectrometry is performed. The present invention uses a magnetic hybrid strong cation exchange adsorbent to extract four substance standards and internal standards of known concentrations, and finally detects them by liquid chromatography tandem mass spectrometry to establish a standard curve, thereby calculating the concentrations of the four substances in human plasma. This method can remove impurities and enrich melatonin and related substances in its synthesis pathway during the extraction process, which is of great significance for the diagnosis and differential diagnosis of sleep disorders, early diagnosis of Alzheimer's disease and Parkinson's disease, pineal tumors, etc., as well as the development process, efficacy and prognosis of the disease, thereby providing patients with personalized diagnostic and treatment information.

[0012] The objective of the present invention is achieved through the following technical solutions:

[0013] In a first aspect, the present invention provides a method for extracting melatonin and related substances in its synthesis pathway from plasma based on a magnetic adsorbent, comprising the following steps:

[0014] S1. Add internal standard solution and phosphoric acid solution to standard products, quality control products or plasma samples to prepare samples to be tested;

[0015] S2, adding the magnetic adsorbent to methanol for activation, and then transferring it to water for equilibrium;

[0016] S3, transferring the magnetic adsorbent treated in step S2 to the sample to be tested in step S1;

[0017] S4, washing with water and methanol aqueous solution in sequence;

[0018] S5. Use acetonitrile containing ammonia water as an eluent to elute the sample adsorbed on the magnetic adsorbent to obtain the product.

[0019] As some specific embodiments of the present invention, in step S1, the phosphoric acid solution contains phosphoric acid with a volume fraction of 4%-10%.

[0020] As some specific embodiments of the present invention, in step S1, the internal standard solution is a solution containing 0.1-0.5 μg / mL tryptophan-D5, 2-10 ng / mL 5-hydroxytryptamine-D4, 2-10 ng / mL 5-hydroxytryptophan-D3, and 2-20 pg / mL melatonin-D3 with a volume fraction of 80%-100% methanol.

[0021] As some specific embodiments of the present invention, in step S2, the magnetic adsorbent is prepared by mixing magnetic adsorption dry powder with ethanol; the dosage ratio of the magnetic adsorption dry powder to ethanol is 30-50 mg:1 mL.

[0022] As some specific embodiments of the present invention, the magnetic adsorption dry powder is a magnetic mixed strong cationic adsorbent.

[0023] As some specific embodiments of the present invention, in step S4, the volume fraction of methanol in the methanol aqueous solution is 40%.

[0024] As some specific embodiments of the present invention, in step S5, the volume fraction of ammonia water in the eluent is 1.5-3%.

[0025] Preferably, the volume fraction of the ammonia water is 2%.

[0026] As some specific embodiments of the present invention, the method for extracting melatonin and related substances in its synthesis pathway from plasma based on a magnetic adsorbent is carried out in a fully automatic extraction instrument, and the extraction method is set as follows:

[0027] Ladies and Gentlemen name Add liquid Volume (μL) Stirring time(s) Static magnetic absorption time (s) 1 activation Methanol 400 30 30 2 balance water 400 30 30 3 Loading Samples to be tested 800 120 60 4 Rinse I water 800 30 30 5 Rinse II 40% (v / v) methanol in water 800 120 30 6 Elution Acetonitrile containing 2% (v / v) ammonia 100 120 60 7 Waste Discharge / 400 30 0 .

[0028] In a second aspect, the present invention provides a method for detecting melatonin in plasma and related substances in its synthesis pathway, comprising the following steps:

[0029] A1. Using any of the methods described above, extract and enrich tryptophan, serotonin, 5-hydroxytryptophan, and melatonin in standard products, quality control products, or plasma samples using a magnetic adsorbent;

[0030] A2, sample separation by ultra-high performance liquid chromatography followed by mass spectrometry detection;

[0031] A3. Establish a standard curve based on the test results of the standard substance, and calculate the concentrations of tryptophan, serotonin, 5-hydroxytryptophan, and melatonin in the plasma sample based on the standard curve.

[0032] As some specific embodiments of the present invention, in step A2, when chromatographic separation 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.

[0033] As some specific embodiments of the present invention, in step A2, the chromatographic conditions include:

[0034] The chromatographic column was Acquity UPLC HSS C18 column (3.0 × 50 mm, 3 μm, Waters);

[0035] Mobile phase A was an aqueous solution containing 5 mM ammonium acetate + 0.1% (v / v) formic acid, and mobile phase B was acetonitrile containing 0.1% (v / v) formic acid;

[0036] The column temperature was 35°C.

[0037] As some specific embodiments of the present invention, in step A2, the mobile phase gradient of the chromatography is:

[0038]

[0039]

[0041] As some specific embodiments of the present invention, in step A3, the mass spectrometry conditions include:

[0042] project parameter Ion Source Type H-ESI Mode Positive,3500 Sheath Gas(Arb) 50 Aux Gas(Arb) 20 Sweep Gas(Arb) 2 Ion Transfer Tub Temp(℃) 350 Vaporizer Temp(℃) 400

[0043] The Q1 / Q3 ion channels are selected as:

[0044] Compound Precursor ion Product ions Impact voltage (v) Trp 205.1 146.1 17 Trp 205.1 188.1 25 Trp-D5 210.3 150.1 18 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 .

[0045] In a third aspect, the present invention provides a kit for extracting or detecting melatonin in plasma and related substances in its synthesis pathway, and any of the methods described above is used to extract or detect melatonin in plasma and related substances in its synthesis pathway.

[0046] The kit can extract and detect melatonin and related substances in its synthesis pathway in plasma based on a magnetic adsorbent, which is helpful for the early diagnosis of clinical sleep disorders, Alzheimer's disease and Parkinson's disease, and the diagnosis and differentiation of patients with pineal tumors, etc.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1) Rapidly extract melatonin and related substances in its synthesis pathway from plasma through a magnetic mixed strong cation exchange adsorbent and improve its stability, providing an experimental scheme for the rapid extraction and detection of melatonin and related substances in its synthesis pathway;

[0049] 2) The extraction process can be completed within 10 minutes, and the entire experimental operation can be completed within 1 hour. The current existing technology takes no less than 3 hours to operate on the same sample;

[0050] 3) This experiment uses automated instruments for extraction, which is of great significance for the detection of a large number of samples. The operation is convenient, fast and cost-saving;

[0051] 4) Melatonin and related substances in its synthesis pathway are of great significance in disease diagnosis, but the melatonin content is low and needs to be extracted in a dark environment. It degrades quickly at room temperature. Currently, protein precipitation, liquid-liquid extraction, solid-phase extraction and other methods are commonly used for extraction and enrichment. This process is time-consuming and complicated. The automated extraction method based on magnetic powder is simple and time-saving, which is conducive to the detection of melatonin and related substances;

[0052] 5) Using 4% phosphoric acid solution as the loading solution, together with the sample, to prepare the sample to be tested, and extracting and enriching, the 4% phosphoric acid solution is used to acidify the sample to ensure that the target analyte has a charge, which is conducive to adsorption and extraction by the activated solid phase extraction filler through ionic force, thereby intercepting the target analyte to the greatest extent;

[0053] 6) Using acetonitrile containing 2% (v / v) ammonia as an eluent for extraction, enrichment and detection, using 2% ammonia to neutralize the charge of the target compound adsorbed on the magnetic adsorbent, and completely eluting it under the action of an organic solvent, thereby improving the detection sensitivity of the target compound and facilitating the quantification of the target compound in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] 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:

[0055] Figure 1 Schematic diagram of the 5-hydroxytryptamine pathway for tryptophan metabolism;

[0056] Figure 2 is the standard curve of tryptophan (Trp);

[0057] Figure 3 is the standard curve of serotonin (5-HT);

[0058] Figure 4 is a standard curve diagram of 5-hydroxytryptophan (5-HTP);

[0059] Figure 5 is the standard curve of melatonin (MT);

[0060] Figure 6 This is the detection spectrum of the tryptophan (Trp) detection limit (0.05 μg / mL);

[0061] Figure 7 This is the detection spectrum of the detection limit (2ng / mL) of serotonin (5-HT);

[0062] Figure 8 It is the detection spectrum of the detection limit (1 ng / mL) of 5-hydroxytryptophan (5-HTP);

[0063] Fig. 9 This is the detection spectrum of melatonin (MT) detection limit (0.5pg / mL). DETAILED DESCRIPTION

[0064] 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.

[0065] Example 1

[0066] Step 1: Reagent preparation

[0067] 1. Reagent preparation:

[0068] (1) Internal standard solution: a solution containing 0.5 μg / mL tryptophan-D5 (purchased from Toronto Research Chemical), 10 ng / mL 5-hydroxytryptamine-D4 (purchased from Bepure), 10 ng / mL 5-hydroxytryptophan-D3 (purchased from Bepure), and 10 pg / mL melatonin-D3 (purchased from Shanghai PuFen Biotechnology) in 80% (v / v) methanol.

[0069] (2) Magnetic adsorbent: Take out the glass bottle containing magnetic adsorption dry powder (magnetic mixed strong cationic adsorbent), add 1 mL of ethanol for every 50 mg to prepare a suspension to obtain a magnetic adsorbent for standby use.

[0070] (3) Phosphoric acid aqueous solution: an aqueous solution containing 4% (v / v) phosphoric acid.

[0071] 2. Preparation of quality control products:

[0072] Tryptophan (Trp, from China Food and Drug Inspection Institute), serotonin (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) 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 Trp, 5HT, 5-HTP, and MT working solutions were 10 mg / mL, 10 μg / mL, 4 μg / mL, and 4 ng / mL, respectively. Human plasma (purchased from oldenWest) was used to dilute and mix the working solutions to obtain three concentrations of quality control products. The concentrations of the quality control products are shown in Table 1.

[0073] Table 1 Quality control product concentration table

[0074] Concentration of quality control products Trp (μg / mL) 5HT (ng / mL) 5-HTP (ng / mL) MT(pg / mL) LQC 0.15 6 3 1.5 MQC 7.5 80 30 30 HQC 40 400 160 160

[0075] 3. Preparation of standard products:

[0076] Tryptophan (Trp), serotonin (5HT), 5-hydroxytryptophan (5-HTP), and melatonin (MT) were prepared into stock solutions using methanol / water (volume ratio 1 / 1, containing 1% ascorbic acid), and then diluted to obtain working solutions. The concentrations of Trp, 5HT, 5-HTP, and MT working solutions were 10 μg / mL, 10 μg / mL, 4 μg / mL, and 4 ng / mL. 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 the standard products are shown in Table 2.

[0077] Table 2 Standard concentration table

[0078] Standard concentration Trp (μg / mL) 5HT (ng / mL) 5-HTP (ng / mL) MT(pg / mL) Cal 1 0.05 2 1 0.5 Cal 2 0.2 5 2.5 2 Cal 3 1 20 10 10 Cal 4 5 50 20 25 Cal 5 10 100 50 50 Cal 6 25 250 100 100 Cal 7 50 500 200 200

[0079] 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.

[0080] Step 2: Extract and enrich the analyte with a magnetic adsorbent

[0081] 1. Take out 400 μL of the standard, quality control or sample to be tested and put it into a 1.5 mL centrifuge tube. Add 50 μL of internal standard solution and shake to mix. Then add 400 μL of phosphoric acid aqueous solution and vortex for 5 minutes to prepare the sample to be tested.

[0082] 2. As shown in Table 3 below, transfer the solution corresponding to each column in the 96-well plate, set the extraction method shown in Table 3 on the Auto M32 fully automatic extractor (source: Suzhou Aijebya Bioelectronics Technology Co., Ltd.), and add 10 μL of magnetic adsorbent to the first column.

[0083] Table 3 List of liquid addition methods and fully automatic extraction method settings

[0084] Ladies and Gentlemen name Add liquid Volume (μL) Stirring time(s) Static magnetic absorption time (s) 1 activation Methanol 400 30 30 2 balance water 400 30 30 3 Loading Samples to be tested 800 120 60 4 Rinse I water 800 30 30 5 Rinse II 40% (v / v) methanol in water 800 120 30 6 Elution Acetonitrile containing 2% (v / v) ammonia 100 120 60 7 Waste Discharge / 400 30 0

[0085] After the extraction, gently shake on the shaking plate for 60 seconds to mix, then transfer all the solutions into a new 96-well sample plate, blow dry with nitrogen, take 30μL of the solution for re-dissolution, and perform LC-MS / MS detection on the machine. Nitrogen blowing and re-dissolution takes 20 minutes, which greatly shortens the processing time compared to the conventional protein precipitation method, which takes 2 hours to blow dry with nitrogen after extraction, thus greatly shortening the processing time.

[0086] Step 3: Test on the machine

[0087] After the above treatment, the calibrator and the plasma sample to be tested were respectively tested by high performance liquid chromatography tandem mass spectrometry.

[0088] 1. Chromatographic conditions:

[0089] Chromatographic column: Acquity UPLC HSS C18 column (3.0×50 mm, 3 μm, Waters);

[0090] Mobile phase A: containing 5 mM ammonium acetate + 0.1% formic acid in water;

[0091] Mobile phase B: acetonitrile solution containing 0.1% formic acid;

[0092] Column temperature: 35°C.

[0093] 2. Liquid phase elution gradient is shown in Table 4:

[0094] Table 4 Chromatographic gradient table

[0095] Time (min) Flow rate (mL / min) Ratio of mobile phase A (%) Ratio of mobile phase B (%) 0.00 0.5 99 1 0.50 0.5 99 1 2.50 0.5 30 70 3.50 0.5 5 95 4.00 0.5 5 95 4.10 0.5 99 1 5.00 0.5 99 1

[0096] 3. Mass spectrometry conditions:

[0097] The mass spectrometry parameters are shown in Tables 5 and 6.

[0098] Table 5 Mass spectrometry parameters

[0099] project parameter Ion Source Type H-ESI Mode Positive,3500 Sheath Gas(Arb) 50 Aux Gas(Arb) 20 Sweep Gas(Arb) 2 Ion Transfer Tub Temp(℃) 350 Vaporizer Temp(℃) 400

[0100] Table 6 Q1 / Q3 ion channel selection table

[0101] Compound Precursor ion Product ions Impact voltage (v) Trp 205.1 146.1 17 Trp 205.1 188.1 25 Trp-D5 210.3 150.1 18 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

[0102] Step 4: According to the test results of the standard, a standard curve is established with the concentration ratio of the standard as the x-axis and the ratio of the peak area of ​​the standard to the internal standard as the y-axis. The concentrations of tryptophan, serotonin, 5-hydroxytryptophan, and melatonin in the plasma sample are calculated according to the standard curve.

[0103] Example 2

[0104] This example conducted a detailed optimization and comparison experiment on the plasma sample loading solution, and comprehensively considered the overall detection method to determine the composition of the optimal loading solution.

[0105] Example 1 When preparing the sample to be tested in step 2, 400 μL of 4% (v / v) phosphoric acid aqueous solution is used as the loading solution, and mixed with the internal standard solution and the standard / quality control product / sample for preparation. This example conducts a comparative experiment on different loading solution compositions, and dilutes it 1:1 with a plasma sample of the same concentration (i.e., the quality control product MQC, Trp concentration is 7.5 μg / mL, 5-HT concentration is 80 ng / mL, 5-HTP concentration is 30 ng / mL, and MT concentration is 30 pg / mL). Except for adjusting the 4% phosphoric acid aqueous solution to the loading solution of other components, the remaining steps are carried out according to Example 1. The peak area data of each target analyte in the test results are shown in Table 7 below:

[0106] Table 7 Peak area display results of different loading solutions

[0107]

[0108] In this example, different types and proportions of loading solution components were tried to compare the elution effects of samples of the same concentration under the same conditions. The results showed that 4% (v / v) phosphoric acid aqueous solution was the best for eluting the four targets.

[0109] Example 3

[0110] In this embodiment, a detailed optimization and comparison experiment was conducted on the components of different eluents, and the overall detection method was comprehensively considered to determine the best eluent formula.

[0111] In the fully automatic extraction of step 2 of Example 1, acetonitrile containing 2% (v / v) ammonia water is used for elution. In this embodiment, different solvents are used as eluents to elute the target. Except that the eluent composition is different from that of Example 1, the remaining steps are carried out according to Example 1, and the same concentration of plasma samples (quality control product MQC, Trp concentration is 7.5 μg / mL, 5-HT concentration is 80 ng / mL, 5-HTP concentration is 30 ng / mL, MT concentration is 30 pg / mL) are tested. The peak area data of different eluents are shown in Table 8 below:

[0112] Table 8 Peak area display results of different eluents

[0113]

[0114] In this embodiment, the elution effects of two reagents, acetonitrile and methanol, and the corresponding mixed reagents were tried. The experiment found that when acetonitrile was used as the elution solvent, 2% (v / v) ammonia water had the best elution effect on the target. When comparing acetonitrile, methanol, methanol acetonitrile in different proportions, and ammonia water with the same concentration as the eluent, pure acetonitrile had a better elution effect as the solvent, so in the experiment, 2% (v / v) ammonia water acetonitrile was used as the eluent for elution, and good results were obtained.

[0115] Effect Example 1--Linear Range Verification

[0116] According to the method of Example 1, each standard product was extracted and tested by a magnetic mixed strong cation exchange adsorbent. The results showed that the linear fitting equation of melatonin and related substances in its synthesis pathway had good linearity within their respective concentration ranges, and the correlation coefficient was above 0.99. The obtained standard curve is as follows: Figure 2-5 The linear fitting equation and linear range are shown in Table 9 below.

[0117] Table 9 Linear range and equations of four compounds

[0118]

[0119] Effect Example 2--Linear Point Deviation

[0120] According to the detection value of the standard substance calculated in Example 1, the deviation between it and the theoretical value is compared, and the results are shown in Table 10. According to the results in Table 10, it can be seen that the deviation between the detection result of each standard substance to be tested and the theoretical value is within 15%, indicating that the method for extracting melatonin and related substances in its synthetic pathway in plasma based on a magnetic mixed strong cation exchange adsorbent is feasible and reliable.

[0121] Table 10 Relative deviation between actual test value and theoretical value of standard products

[0122]

[0123]

[0124] When testing the lowest point of the calibration product, Cal 1, the signal-to-noise ratios of the three compounds were all greater than 10 ( Figure 6-9 ), which can meet the detection sensitivity 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 melatonin and related substances in its synthesis pathway from plasma based on a magnetic adsorbent, characterized in that: The steps include: S1. Add internal standard solution and phosphoric acid solution to standard products, quality control products or plasma samples to prepare samples to be tested; S2, adding the magnetic adsorbent to methanol for activation, and then transferring it to water for equilibrium; S3, transferring the magnetic adsorbent treated in step S2 to the sample to be tested in step S1; S4, washing with water and methanol aqueous solution in sequence; S5. Use acetonitrile containing ammonia water as an eluent to elute the sample adsorbed on the magnetic adsorbent to obtain the product.

2. The method according to claim 1, characterized in that In step S1, the phosphoric acid solution contains 4%-10% phosphoric acid by volume.

3. The method according to claim 1, characterized in that In step S1, the internal standard solution is a methanol solution containing 0.1-0.5 μg / mL tryptophan-D5, 2-10 ng / mL 5-hydroxytryptamine-D4, 2-10 ng / mL 5-hydroxytryptophan-D3, and 2-20 pg / mL melatonin-D3 with a volume fraction of 80%-100%.

4. The method according to claim 1, characterized in that: In step S2, the magnetic adsorbent is prepared by mixing magnetic adsorption dry powder with ethanol; the dosage ratio of the magnetic adsorption dry powder to ethanol is 30-50 mg:1 mL.

5. The method according to claim 4, characterized in that The magnetic adsorption dry powder is a magnetic mixed type strong cationic adsorbent.

6. The method according to claim 1, characterized in that In step S5, the volume fraction of ammonia water in the eluent is 1.5-3%.

7. A method for detecting melatonin in plasma and related substances in its synthesis pathway, characterized in that: The steps include: A1. Using the method described in any one of claims 1 to 6, using a magnetic adsorbent to extract and enrich tryptophan, serotonin, 5-hydroxytryptophan, and melatonin in a standard, a quality control, or a plasma sample; A2, sample separation by ultra-high performance liquid chromatography followed by mass spectrometry detection; A3. Establish a standard curve based on the test results of the standard substance, and calculate the concentrations of tryptophan, serotonin, 5-hydroxytryptophan, and melatonin in the plasma sample based on the standard curve.

8. The detection method according to claim 7, characterized in that: In step A2, when chromatographic separation 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 detection method according to claim 7, characterized in that: In step A2, the mobile phase gradient of the chromatography include: 。 10. A kit for extracting or detecting melatonin in plasma and related substances in its synthesis pathway, characterized in that: Melatonin in plasma and related substances in its synthesis pathway are extracted by the method described in any one of claims 1 to 6, or melatonin in plasma and related substances in its synthesis pathway are detected by the method described in any one of claims 7 to 9.

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