Method for detecting multiple neurotransmitter compounds based on high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to a method for detecting multiple neurotransmitter compounds based on high performance liquid chromatography-tandem mass spectrometry. The method comprises the following steps: S1, preparing a standard curve solution of each neurotransmitter and drawing a standard curve; s2, precipitating protein in the plasma / serum sample by using a protein precipitant, and taking supernate; s3, adding a derivatization reagent into the supernate for derivatization reaction to obtain a to-be-detected solution; s4, simultaneously loading the to-be-detected solution and the standard curve solution to high performance liquid chromatography-tandem mass spectrometry for joint detection to obtain the peak area value of each neurotransmitter compound in the to-be-detected solution; s5, calculating the concentration of each neurotransmitter compound in the liquid to be detected according to the standard curve of each neurotransmitter compound in combination with the peak area value obtained in the step S3; wherein the neurotransmitter compound comprises a derivatization product of a neurotransmitter containing a primary amine group and / or a phenolic hydroxyl group and a neurotransmitter containing a secondary amine group, and the derivatization reagent is a benzoyl chloride-acetonitrile solution.

Description

Technical Field

[0001] This disclosure relates to the field of detection technology, specifically to a method for detecting multiple neurotransmitter compounds based on high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0002] With the rapid development of science and technology and society, insomnia has become one of the global public health problems, and its incidence rate is showing an increasing trend year by year as social pressure increases.

[0003] Modern medicine has shown that neurotransmitter imbalance is one of the core pathologies of insomnia. Neurotransmitters are classified into amino acids, monoamines, and polypeptides, involving a variety of neurotransmitters such as γ-aminobutyric acid (GABA), glutamate (Glu), serotonin (5-HT), norepinephrine (NE), dopamine (DA), and melatonin (MT).

[0004] GABA, an important inhibitory neurotransmitter, is widely found in vertebrates, plants, and microorganisms, and has various physiological functions, including promoting glucose metabolism, acetylcholine synthesis, growth hormone secretion, and stabilizing mood. If GABA levels are low or its transmission function is impaired, diseases such as epilepsy, Parkinson's disease, and sleep disorders may occur.

[0005] Glutamate (Glutamate) plays a crucial role in protein metabolism and is the most abundant excitatory neurotransmitter in the vertebrate nervous system. Glutamate also plays a vital role in the central nervous system, regulating synaptic activity, maintaining neural network stability, and serving as a pivotal point for neuronal damage and inflammatory responses. Glutamate participates in cortical activation and behavioral arousal via AMPA and NMDA receptors, and is converted to GABA under the catalysis of glutamate decarboxylase (GAD). Imbalance between GABA and Glu homeostasis is considered a major neurobiological basis for sleep-wake rhythm disorders. Therefore, clinical recommendations suggest that simultaneous testing of GABA and Glu is necessary.

[0006] 5-HT is a monoamine neurotransmitter widely distributed in the central nervous system and intestines, primarily involved in mood regulation, sleep, and appetite. It plays a role in regulating sleep and emotional responses, maintaining slow-wave sleep, shortening the sleep onset and light sleep stages, thus achieving a sedative-hypnotic effect.

[0007] Norepinephrine (NE) is a neurotransmitter primarily synthesized and secreted by sympathetic neurons and noradrenergic neurons in the brain. Noradrenergic neurons constitute the majority of the locus coeruleus in the anterior dorsal region of the pons, a key nucleus in the arousal system. Studies have shown that elderly patients with primary insomnia have lower 24-hour plasma NE levels than those with good sleep.

[0008] Dopamine (DA) is a monoamine neurotransmitter that can regulate the extrapyramidal system, mood, cognition, and physical activity. It has a behavioral excitatory effect, and its elevated levels can increase the number of awakenings and induce insomnia. Regulating abnormally elevated DA levels can play a role in treating primary insomnia.

[0009] MT is a hormone secreted by the pineal gland in the brain. MT secretion exhibits a distinct diurnal rhythm, with secretion suppressed during the day and active at night, earning it the nickname "sleep hormone." Secretion levels are highest in infancy and early childhood, gradually decreasing after adolescence, and may significantly decline in the elderly. Long-term insufficient MT secretion may increase the risk of insomnia, mood disorders, and even cardiovascular disease; therefore, individuals with persistent sleep problems should monitor their MT levels.

[0010] GABA often exists in solution as zwitterions (a negatively charged carboxyl group and a positively charged amino group). Glu is an acidic amino acid with two carboxyl groups, and MT is a neutral substance. These compounds have very different acidity and alkalinity, making it difficult to extract and purify them using the principle of ion exchange. At the same time, due to their extremely different polarities, it is also difficult to use the same detection method for detection and analysis.

[0011] Existing technologies, such as the detection method for targeted determination of neurotransmitters and their metabolites disclosed in CN120064483A, use dansyl chloride solution for derivatization reaction. However, the derivatization conditions for this method are cumbersome, requiring temperature incubation, and the derivatization efficiency is low. CN118961986A discloses a detection method using benzoyl chloride as a derivatizing reagent, but it cannot determine highly polar glutamate compounds. CN119757594A utilizes a mixed-type anion exchange solid-phase extraction plate to extract and enrich amino acid neurotransmitters and melatonin in plasma, but solid-phase extraction significantly increases detection costs and cannot meet the requirements for glutamate determination. Summary of the Invention

[0012] Therefore, the purpose of this disclosure is to enable the detection of highly polar neurotransmitters, improve the convenience of neurotransmitter detection, and reduce detection costs.

[0013] To achieve the above objectives, the present disclosure adopts the following technical solution: A method for detecting multiple neurotransmitter compounds based on high performance liquid chromatography-tandem mass spectrometry includes the following steps: S1. Prepare standard curve solutions for each neurotransmitter and plot the standard curves for each neurotransmitter; S2. Take a plasma / serum sample, precipitate the proteins in the plasma / serum sample with a protein precipitant, and take the supernatant; S3. Add a derivatizing reagent to the supernatant to carry out a derivatization reaction and obtain the test solution; S4. The test solution and the standard curve solution are simultaneously loaded onto high performance liquid chromatography-tandem mass spectrometry for joint detection to obtain the peak area values ​​of each neurotransmitter compound in the test solution; S5. Based on the standard curves of each neurotransmitter compound and the peak area values ​​obtained in step S4, calculate the concentration of each neurotransmitter compound in the test solution. The neurotransmitter compound includes derivatized products of neurotransmitters containing primary amine groups and / or phenolic hydroxyl groups and neurotransmitters containing secondary amine groups, wherein the derivatizing reagent is a benzoyl chloride-acetonitrile solution.

[0014] Preferably, the neurotransmitter containing primary amine groups and / or phenolic hydroxyl groups includes one or more of γ-aminobutyric acid (GABA), glutamate (Glu), serotonin (5-HT), norepinephrine (NE), and dopamine (DA); The neurotransmitter containing a secondary amine group is melatonin (MT).

[0015] More preferably, the concentration points C1~C6 of the standard curve solutions for each neurotransmitter are as follows: compound C1 (ng / mL) C2 (ng / mL) C3 (ng / mL) C4 (ng / mL) C5 (ng / mL) C6 (ng / mL) DA 0.2 0.4 1 2 4 10 NE 0.2 0.4 1 2 4 10 GABA 10 20 50 100 200 500 MT 0.2 0.4 1 2 4 10 Glu 10 20 50 100 200 500 5-HT 10 20 50 100 200 500

[0016] Preferably, in step S2, the protein precipitant is one or more of methanol, acetonitrile, and isopropanol.

[0017] More preferably, the protein precipitant is a mixture of methanol and acetonitrile, wherein the volume ratio of methanol to acetonitrile is 1:4.

[0018] Preferably, in step S3, before carrying out the derivatization reaction, the step further includes adjusting the pH of the supernatant.

[0019] More preferably, the pH of the supernatant is adjusted using a buffer solution, the buffer solution comprising a mixed buffer solution of sodium carbonate solution and sodium bicarbonate solution; The volume ratio of the sodium carbonate solution to the sodium bicarbonate solution is 1:1 to 1:5, and the concentrations of the sodium carbonate solution and the sodium bicarbonate solution are 0.01 mol / L to 1 mol / L, respectively.

[0020] Preferably, in step S3, the volume ratio of benzoyl chloride to acetonitrile solution in the benzoyl chloride-acetonitrile solution is 1% to 10%.

[0021] More preferably, in step S4, the mobile phase A of the high performance liquid chromatography is an aqueous solution containing 0.1~2mM ammonium acetate and 0.1%~1% by volume formic acid, and the mobile phase B is a methanol or acetonitrile solution.

[0022] Preferably, the gradient elution program for high-performance liquid chromatography is as follows: T(min) Mobile phase A (%) Mobile phase B (%) 0 100 0 1.0 100 0 2.0 40 60 3.0 10 90 3.7 10 90 4.1 100 0 6 100 0

[0023] The technical solution claimed in this disclosure achieves the following beneficial effects: 1. The benzoyl chloride derivatization method is very simple, requiring no incubation or heating. Derivatization is completed by shaking for 5-10 minutes, and the derivatization efficiency is higher than that of conventional derivatizing agents such as dansyl chloride and 2,4-dinitrofluorobenzene.

[0024] 2. By precipitating proteins in the sample, high-abundance proteins in blood samples can be removed, thereby reducing the interference of the protein matrix on the target analyte and improving detection sensitivity. Simultaneously, it reduces the reaction between benzoyl chloride and proteins, improving derivatization efficiency. This pretreatment method eliminates the need for cumbersome purification techniques such as solid-phase extraction, further reducing operation time and procedures.

[0025] 3. Compared with the existing benzoyl chloride derivatization method, the protein precipitation pretreatment combined with benzoyl chloride derivatization method provided in this disclosure can determine highly polar glutamate compounds, thereby providing more diagnostic indicators for clinical diagnosis. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 This is a derivative mechanism of GABA.

[0028] Figure 2 This is the derivation mechanism of Glu.

[0029] Figure 3 This is a derivative mechanism of 5-HT.

[0030] Figure 4 This is the derivation mechanism of NE.

[0031] Figure 5 This is the derivation mechanism of DA.

[0032] Figure 6 The image shows a chromatogram of GABA in a serum sample.

[0033] Figure 7 The image shows a chromatogram of Glu in a serum sample.

[0034] Figure 8 The chromatogram shows 5-HT in a serum sample.

[0035] Figure 9 The image shows a chromatogram of NE in a serum sample.

[0036] Figure 10 This is a chromatogram of DA in a serum sample.

[0037] Figure 11 The chromatogram shows MT in a serum sample.

[0038] Figure 12 The image shows the separation chromatograms of neurotransmitter standards (DA, 5-TH, MT) on an amino chromatography column.

[0039] Figure 13 The image shows the separation chromatograms of neurotransmitter standards (GABA, Glu, NE) on an amino chromatography column.

[0040] Figure 14 Chromatograms showing the separation of neurotransmitter standards (DA, 5-TH, MT) on a HILIC column.

[0041] Figure 15 The chromatograms show the separation of neurotransmitter standards (GABA, Glu, NE) on a HILIC column. Detailed Implementation

[0042] To make the objectives, technical solutions, and beneficial effects of the embodiments in this disclosure clearer, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0043] Example 1 This embodiment provides a method for plotting standard curves.

[0044] Since glutamate is present in almost all biological samples, PBS (phosphate-buffered saline) was used as the substrate for preparing the standard curve. The specific steps are as follows: 1. Solution preparation 1.1 Preparation of single-standard stock solution Weigh 10 mg of γ-aminobutyric acid (GABA), glutamic acid (Glu), 5-hydroxytryptamine (5-HT), norepinephrine (NE), dopamine (DA), and melatonin (MT) standards respectively, and dilute to 10 ml with 0.1% formic acid aqueous solution to prepare a 1000 μg / mL single standard stock solution.

[0045] 1.2 Preparation of mixed standard solutions A certain volume of the above single-standard stock solution was transferred to the same volumetric flask and diluted to volume with an aqueous solution containing 0.1% formic acid to obtain a mixed standard solution. The concentrations of DA, NE, and MT in the mixed standard solution were 1 μg / mL, and the concentrations of GABA, Glu, and 5-TH were 50 μg / mL.

[0046] 1.3 Preparation of internal standard solution Weigh 1 mg each of DA-D4 (deuterated dopamine), NE-D6 (deuterated norepinephrine), GABA-D2 (deuterated γ-aminobutyric acid), MT-D4 (deuterated melatonin), Glu-D3 (deuterated glutamate), and 5-HT-D4 (deuterated serotonin) into six 1 mL volumetric flasks, and dilute to volume with 70% methanol-water solution to prepare internal standard stock solutions. Then, pipette a certain volume of each internal standard stock solution into the same volumetric flask and dilute to volume with ultrapure water to obtain internal standard working solutions with concentrations of 0.2 μg / mL for DA-D4, NE-D6, and MT-D4, and 20 μg / mL for GABA-D2, Glu-D3, and 5-HT-D4, respectively.

[0047] 1.4 Preparation of Alternative Matrices in Standard Curves Transfer 1 mL of 10•PBS phosphate buffer to a 10 mL volumetric flask and dilute to volume with ultrapure water to obtain 1•PBS phosphate buffer, which can be used as an alternative matrix in establishing the standard curve.

[0048] 2. Establish the standard curve 2.1 Establishment of Standard Curve Concentration Points and Quality Control Samples Transfer 0.05 mL of the mixed standard solution to a 10 mL volumetric flask, add 1•PBS buffer to dilute to the mark, and obtain the C6 concentration point of the standard curve; then transfer 0.4 mL, 0.2 mL, 0.1 mL, 0.04 mL and 0.02 mL of the solution at the C6 concentration point to 1 mL volumetric flasks, and dilute to the mark with 1•PBS buffer to obtain the C5~C1 concentration points of the standard curves for each compound.

[0049] Transfer 0.3 mL and 0.05 mL of the solution at concentration point C6 to 1 mL volumetric flasks, respectively, and dilute to volume with 1-PBS buffer to obtain the HQC (high-quality control) and LQC (low-quality control) concentration points for the standard curves of each compound. The concentrations of each compound are shown in Table 1.

[0050] Table 1. Concentrations at C1-C6 points of the standard curves for each compound

[0051] 2.2 Plotting and Verification of Calibration Curves Following the above preparation method, the standards were derivatized (refer to Example 3 for this operation), and quantified using the internal standard method. A standard curve was plotted using the ratio of the target analyte peak area to the internal standard peak area after derivatization as the ordinate and the compound concentration as the abscissa, employing the instrument's built-in software. The concentration of each compound in the sample was then determined. The limit of quantitation (LOQ) was the lowest point of the curve. The table below shows the linearity results of the standard curves for each standard:

[0052] Based on the above results, each compound exhibits good linearity within its respective linear range, and the correlation coefficients meet the requirements.

[0053] 2.3 Limit of Detection Five PBS matrix samples close to the limit of quantitation were selected. Each sample was pretreated according to the sample pretreatment method, and each sample was repeated five times. The treated samples were injected into high performance liquid chromatography-tandem mass spectrometry. The concentration point with a signal-to-noise ratio (S / N or SNR, which is the core indicator for measuring the "effective signal quality" of the detection system, essentially the ratio of the intensity of the target detection signal to the intensity of background interference (noise)) of not less than 3:1 was selected as the limit of detection. The results of the limit of detection are shown in the table below.

[0054] Example 2 This embodiment provides the establishment of a high-performance liquid chromatography-tandem mass spectrometry method.

[0055] 1. High Performance Liquid Chromatography Conditions The chromatographic column was: Shanghai Anpu C18-WP 2.1•100mm, 3μm; Mobile phase A is a 0.2 mM ammonium acetate and 0.1% formic acid aqueous solution; mobile phase B is a pure methanol or pure acetonitrile solution; column temperature: 40℃; Injection volume: 20 μL.

[0056] The elution gradient of the mobile phase in liquid chromatography is shown in Table 2.

[0057] Table 2. Elution gradient of mobile phase in liquid chromatography T(min) Mobile phase A (%) Mobile phase B (%) 0 100 0 1.0 100 0 2.0 40 60 3.0 10 90 3.7 10 90 4.1 100 0 6 100 0 2. Mass spectrometry conditions The parameters of the mass spectrometry ion source are shown in Table 3.

[0058] Table 3. Mass spectrometry ion source parameters

[0059] Among them, MRM (Multiple Reaction Monitoring) is the most commonly used targeted quantification mode in tandem mass spectrometry (especially triple quadrupole mass spectrometry QqQ MS). Its core is to specifically capture the characteristic ion signals of target compounds through "two mass screenings" to achieve high sensitivity and high accuracy quantification of trace substances in complex matrices.

[0060] The mass spectrometry ion pair parameters are shown in Table 4.

[0061] Table 4. Mass spectrometry ion pair parameters

[0062] Example 3 This embodiment provides the pretreatment, derivatization, and detection of the sample to be tested.

[0063] 1. Preprocessing Proteins are major interfering substances in serum / plasma. This study screened different protein precipitants to determine their types and proportions.

[0064] Since the compounds to be tested have different polarities, the responses of each compound after protein precipitation using methanol, acetonitrile, isopropanol and their mixed solvents were investigated. The results are shown in Table 5.

[0065] Table 5. Responses of each test compound to different protein precipitants

[0066] Based on the area of ​​each compound after treatment with different protein precipitants in Table 5 above, and considering the quantitative requirements of each compound, it can be seen that the compounds respond more strongly under the methanol:acetonitrile (1:4) condition. Therefore, methanol:acetonitrile (1:4) was selected as the protein precipitant. The specific operation of protein precipitation is as follows: 1) Take 100 μL of serum or plasma sample, standard, and quality control into a 1 mL 96-well plate, and add 10 μL of internal standard; 2) Add 300 μL of a methanol:acetonitrile (1:4) protein precipitant to the above sample, vortex to mix, then centrifuge and retain the supernatant. Protein precipitation removes high-abundance proteins from blood samples, thereby reducing interference from the protein matrix and improving detection sensitivity. Simultaneously, it reduces the reaction between benzoyl chloride and proteins, improving the efficiency of the subsequent derivatization step.

[0067] 2. Derivative Because Glu is highly polar, it is difficult to retain in reversed-phase systems. Furthermore, NE and DA are present in low concentrations in the human body. Therefore, derivatization is used to improve the polarity of compounds, increasing the retention of each polar compound in the chromatographic column. Derivatization can also enhance the response of compounds with lower sensitivity. Specifically: 1) Prepare 1M sodium carbonate solution and 1M sodium bicarbonate solution separately. Transfer 1mL of sodium carbonate solution and 1mL of sodium bicarbonate solution into 10mL centrifuge tubes, and dilute to volume with ultrapure water to obtain a 0.1M buffer solution; 2) Adjust the pH of the sample solution Transfer 200 μL of the supernatant after the protein precipitation to a new 96-well plate, add 50 μL of the above buffer solution, and mix for 5 min. 3) Preparation of derivatization reagents Take 0.5 mL of benzoyl chloride solution and place it in a 10 mL volumetric flask. Dilute to volume with acetonitrile solution to obtain a 5% benzoyl chloride derivatizing reagent. 4) Add 50 μL of derivatizing reagent to the pH-adjusted sample solution and mix thoroughly.

[0068] Appendix Figures 1 to 5 The derivation mechanisms of GABA, Glu, 5-HT, NE and DA are shown respectively.

[0069] 3. Testing The pre-treated test samples, standards, and quality control samples were analyzed under the conditions described in Example 2. Samples were injected into a high-performance liquid chromatography-tandem mass spectrometer (HPLC-MS / MS). A standard curve was plotted using the ratio of the target analyte peak area to the internal standard peak area as the ordinate and the compound concentration as the abscissa, employing the instrument's built-in software. The concentrations of each compound in the sample were then determined. The chromatograms of each compound in the obtained human serum sample are shown below. Figure 6-11 Highly polar glutamate compounds can also be detected.

[0070] Compared with existing non-derivative methods, the peak conditions of non-derivative samples cannot be kept consistent because the strong polarity of glutamate in them differs significantly from that of other neurotransmitters. Figure 12-13 and Figure 14-15 The effectiveness of directly separating neurotransmitters from the aforementioned underivatized standards using amino chromatography and HILIC chromatography columns is demonstrated, respectively. Figure 12-15 It is known that HILIC columns and conventional amino columns used for separating amino acids do not achieve good retention of most neurotransmitters.

[0071] In existing technologies, CN119757594A uses a mixed anion exchange solid-phase extraction plate to extract and enrich amino acid neurotransmitters and melatonin in plasma, followed by sample separation by ultra-high performance liquid chromatography, then mass spectrometry detection, and finally calculation of the content of melatonin and amino acid neurotransmitters in the sample based on the standard curve method. CN118961986A uses a combination of benzoyl chloride derivatization and solid-phase extraction to detect melatonin, serotonin, histamine, and dopamine. In response to the above two existing technologies, the applicant conducted a spiked experiment based on CN119757594A and CN118961986A. The results showed that glutamate could not be effectively retained in the C18 matrix packing material of CN118961986A and the mixed anion (MAX) solid-phase extraction packing material of CN119757594A. The experimental results are shown in the table below, indicating that neither can detect glutamate.

[0072] This invention utilizes protein precipitation technology combined with simple and easy-to-operate derivatization techniques to achieve the detection and analysis of six neurotransmitter compounds, while simultaneously achieving the separation and content detection of highly polar glutamate, which can assist in the symptomatic treatment of insomnia patients in clinical practice.

[0073] The embodiments and application examples described above are merely illustrative descriptions of this disclosure and are not intended to limit the scope of this disclosure. Any modifications and improvements made by those skilled in the art to the technical solutions of this disclosure without departing from the spirit of this disclosure should fall within the protection scope defined by this disclosure.

Claims

1. A method for detecting multiple neurotransmitter compounds based on high performance liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: S1. Prepare standard curve solutions for each neurotransmitter and plot the standard curves for each neurotransmitter; S2. Take a plasma / serum sample, precipitate the proteins in the plasma / serum sample with a protein precipitant, and take the supernatant; S3. Add a derivatizing reagent to the supernatant to carry out a derivatization reaction and obtain the test solution; S4. The test solution and the standard curve solution are simultaneously loaded onto high performance liquid chromatography-tandem mass spectrometry for joint detection to obtain the peak area values ​​of each neurotransmitter compound in the test solution; S5. Based on the standard curves of each neurotransmitter compound and the peak area values ​​obtained in step S4, calculate the concentration of each neurotransmitter compound in the test solution. The neurotransmitter compound includes derivatized products of neurotransmitters containing primary amine groups and / or phenolic hydroxyl groups and neurotransmitters containing secondary amine groups, wherein the derivatizing reagent is a benzoyl chloride-acetonitrile solution.

2. The method according to claim 1, characterized in that, The neurotransmitters containing primary amine groups and / or phenolic hydroxyl groups include one or more of γ-aminobutyric acid (GABA), glutamate (Glu), serotonin (5-HT), norepinephrine (NE), and dopamine (DA). The neurotransmitter containing a secondary amine group is melatonin (MT).

3. The method according to claim 2, characterized in that, The concentration points C1~C6 of the standard curve solutions for each neurotransmitter are as follows: 。 4. The method according to claim 1, characterized in that, In step S2, the protein precipitant is one or more of methanol, acetonitrile, and isopropanol.

5. The method according to claim 4, characterized in that, The protein precipitant is a mixture of methanol and acetonitrile, wherein the volume ratio of methanol to acetonitrile is 1:

4.

6. The method according to claim 1, characterized in that, In step S3, before the derivatization reaction is carried out, the pH of the supernatant is adjusted.

7. The method according to claim 6, characterized in that, The pH of the supernatant is adjusted using a buffer solution, the buffer solution comprising a mixed buffer of sodium carbonate solution and sodium bicarbonate solution; The volume ratio of the sodium carbonate solution to the sodium bicarbonate solution is 1:1 to 1:5, and the concentrations of the sodium carbonate solution and the sodium bicarbonate solution are 0.01 mol / L to 1 mol / L, respectively.

8. The method according to claim 1, characterized in that, In step S3, the volume of benzoyl chloride in the benzoyl chloride-acetonitrile solution is 1% to 10% of the volume of the acetonitrile solution.

9. The method according to claim 1, characterized in that, In step S4, the mobile phase A of the high performance liquid chromatography is an aqueous solution containing 0.1~2mM ammonium acetate and 0.1%~1% formic acid by volume, and the mobile phase B is a pure methanol or pure acetonitrile solution.

10. The method according to claim 1, characterized in that, The high-performance liquid chromatography gradient elution program is as follows: 。

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