A kind of analysis and detection method of phenylhydrazone compounds

Abstract

The invention provides an analysis and detection method for phenylhydrazone compounds and related substances thereof, and relates to the technical field of chemical drug analysis methods. The method uses argon as a deoxygenation means of the mobile phase, combines a solution containing ascorbic acid to flush the HPLC system, and simultaneously adds a small amount of ascorbic acid to the diluent, and uses liquid chromatography to detect the phenylhydrazone compounds. A stable and repeatable method for detecting phenylhydrazone compounds is obtained. Under the analysis and detection method of the invention, the related substances of the phenylhydrazone compounds are stable, and the entire analysis and detection system can maintain a low-oxide environment for a long time, meeting the verification needs. Under the method, the detection sensitivity of the phenylhydrazone compounds can reach 0.10 μg / ml, and the linear range is 0.10 μg / ml to 0.30 mg / ml.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical drug analysis methods, and specifically relates to an analysis and detection method for phenylhydrazone compounds. Background Art

[0002] Phenylhydrazone is a compound containing -C=N-NH-phenyl structural unit in the molecule. Phenylhydrazone is usually used as an important intermediate for constructing nitrogen-containing five-membered, six-membered, and seven-membered heterocyclic compounds. Many drugs containing N heterocyclic rings must use hydrazone or phenylhydrazone as starting materials or intermediates. Phenylhydrazone is active because it contains C=N double bonds and is prone to oxidation and Diels-Alder addition reactions, resulting in impurities that are not reproducible. Therefore, it is necessary to establish an analytical method for phenylhydrazone that can be analyzed stably.

[0003] Take indomethacin as an example. Indomethacin is a non-steroidal anti-inflammatory drug suitable for antipyretic and analgesic purposes. It is clinically used to treat soft tissue inflammations such as arthritis, cancer pain, gout, bursitis, tendonitis, etc. It can also be used for symptomatic antipyretic of high fever, fever caused by malignant tumors or other uncontrollable fever. Compound C (1-benzylidene-2-(4-methoxyphenyl)hydrazine) is a phenylhydrazone compound and is an important starting material in the synthesis of indomethacin. The chemical structure of compound C is shown below:

[0004]

[0005] Starting from compound C, indomethacin is synthesized with a short route and low cost. The solid phase of compound C is relatively stable. After a long-term stability test of 6 months (2-8°C), the related substances have not changed significantly; however, compound C is extremely unstable in solution, the C=N double bond is easily oxidized, the oxidation process is complex, there is secondary degradation, and the monomer of the oxidized impurity is unavailable. In HPLC, the related substances are not repeated. Even if they are prepared and added, a series of oxidized impurities will appear, and the impurities are large and small, and the number of impurities is also changing. Because of the poor stability of phenylhydrazone, compound C lacks a stable analytical method for quality control, which limits the application of compound C as the starting material of indomethacin. In the synthesis route, compound C must be bypassed as a starting material, and the synthesis steps must be increased, which will increase the cost.

[0006] At present, there are few studies on the detection of phenylhydrazone compounds, but most of the studies are on several HPLC detection and analysis methods, among which. Chinese invention patent CN108828112B discloses a method for pre-treatment and content determination of samples containing m-anisaldehyde, wherein anisaldehyde-2,4-dinitrophenylhydrazone in the sample is mixed with acetonitrile-propylene glycol-water solution to obtain an extract, and the extract is subjected to HPLC detection, the chromatographic column is a C18 reverse phase chromatographic column, the mobile phase A is a 0.3-10% ammonium acetate aqueous solution, the mobile phase B is acetonitrile, and the elution is performed using a gradient elution program. However, the above detection method does not effectively eliminate the problem of oxidation of the C=N double bond, and there is currently no public analysis and detection research on compound C.

[0007] Therefore, there is a need for an analytical detection method that can accurately detect compound C and has good antioxidant protection during the detection process, so that the repeatability and accuracy of the detection results can be improved without interfering with the detection process. Summary of the invention

[0008] The present invention aims at the problems existing in the prior art and provides an analytical detection method for phenylhydrazone compounds. HPLC is used to pass argon gas into the mobile phase as a deoxygenation method, and then the reducing property of ascorbic acid is utilized to flush the HPLC system with an ascorbic acid solution, and a small amount of ascorbic acid is added to the sample diluent for deoxygenation, so as to obtain an analytical detection result of compound C with good accuracy and repeatability.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] A method for analyzing and detecting phenylhydrazone compounds comprises the steps of:

[0011] S1. Instrument pretreatment, flushing of HPLC and chromatographic column:

[0012] S101, connect the two channels, first flush the four channels with acetonitrile, then flush with pure water;

[0013] S102, connect the chromatographic column, and rinse the four channels with acetonitrile-water-ascorbic acid solution;

[0014] S103, immerse the four channels in pure water and rinse for 5-10 minutes;

[0015] S104, connect the chromatographic column to the instrument, first flush the chromatographic column with a mixture of mobile phase A and mobile phase B at 1 mL / min: mobile phase A: mobile phase B = 20:80, v:v, for 10-20 min, and then flush the column with 1 mL / min of mobile phase B for 35-50 min;

[0016] S2. preparing a sample solution of a phenylhydrazone compound: using a diluent to prepare a phenylhydrazone compound; the diluent is an acetonitrile-water-ascorbic acid solution; and using an HPLC method to analyze and detect the phenylhydrazone compound;

[0017] The analytical detection conditions of the HPLC method are:

[0018] Chromatographic column: Waters X-Bridge RP18 (150×4.6-mm, 3.5 μm);

[0019] Mobile phase A: 0.05-0.2% trifluoroacetic acid solution treated with argon;

[0020] Mobile phase B: acetonitrile treated with argon;

[0021] Elution: Gradient elution, the gradient elution program is:

[0022] Time (min) % Mobile phase A (v / v) % Mobile phase B (v / v) 0 90 10 20 10 90 25 10 90 25.1 90 10 30 90 10

[0023] Flow rate: 0.8-1.2mL / min;

[0024] Column temperature: 10°C;

[0025] Sample plate temperature: 2-10°C;

[0026] Injection volume: 5-10 μL;

[0027] Detection wavelength: 235nm.

[0028] Preferably, in step S101, the flushing flow rate is 1-4 mL / min, and the flushing time is 3-10 min.

[0029] Further preferably, the flushing flow rate is 2 mL / min, and the flushing time is 5 min; the purpose of the flushing is to remove salt and organic impurities.

[0030] Preferably, in step S102, the flushing flow rate is 1.0-4 mL / min, and the flushing time is 8-16 h.

[0031] More preferably, the flushing flow rate is 1.2-2 mL / min, and the flushing time is 10-14 h.

[0032] Preferably, in steps S101 and S102, the flow ratio of the four channels is 20-30:20-30:20-30:20-30.

[0033] Further preferably, the flow ratio of the four channels is 25:25:25:25.

[0034] Preferably, in step S102, the acetonitrile-water-ascorbic acid (V C ) solution, water: acetonitrile: V C =800-1000:50-150:0.05-0.1(v:v:m).

[0035] Further preferably, the acetonitrile-water-ascorbic acid (V C ) solution, water: acetonitrile: V C =850-950:80-120:0.05-0.1(v:v:m).

[0036] More preferably, the acetonitrile-water-ascorbic acid (V C ) solution, water: acetonitrile: V C =900:100:0.1(v:v:m).

[0037] Preferably, in step S102, during the flushing period, the needle and the quantitative loop may be washed 1-4 times.

[0038] In step S102, the purpose of flushing is to remove oxidizing substances that may exist in the instrument and the chromatographic column.

[0039] In step S103, the purpose of immersing in pure water is to remove V C .

[0040] Preferably, in step S104, the needle washing solution is a 50% acetonitrile solution.

[0041] Preferably, in step S104, the time for flushing the chromatographic column with the mixed solution of mobile phase A and mobile phase B is 15 minutes, and the time for flushing with mobile phase B is 40 minutes.

[0042] Preferably, in step S104, the flushing flow rate is 0.8-1.2 mL / min.

[0043] Further preferably, the flushing flow rate is 1 mL / min.

[0044] In step S104, the purpose of flushing is to remove V C .

[0045] Preferably, in step S104, during the flushing period, the needle and the quantitative loop may be washed 1-4 times.

[0046] Preferably, in step S2, the diluent is an acetonitrile-water-ascorbic acid solution, water: acetonitrile: V C =300-500:50-150:0.01-0.02(v:v:m).

[0047] Further preferably, the diluent is acetonitrile-water-ascorbic acid solution, water: acetonitrile: V C =350-450:80-120:0.01(v:v:m).

[0048] More preferably, the diluent is acetonitrile-water-ascorbic acid solution, water: acetonitrile: V C =400:100:0.01(v:v:m).

[0049] Preferably, the preparation method of the diluent is: acetonitrile, water and ascorbic acid are mixed in a certain proportion, argon is introduced for bubbling for 2-4 hours, cooled to 2-8°C, sealed and stored at low temperature; if stored for more than 24 hours, re-bubbling is required.

[0050] Preferably, in step S2, the phenylhydrazone compound is compound C (1-benzylidene-2-(4-methoxyphenyl)hydrazine).

[0051] Further preferably, the chemical structural formula of the compound C is as follows:

[0052]

[0053] Preferably, the mobile phase A is a 0.1% trifluoroacetic acid solution treated with argon.

[0054] Preferably, the preparation method of the mobile phase A is: trifluoroacetic acid and water are mixed evenly, ultrasonically treated for 20-40 minutes, and argon gas is introduced for bubbling treatment for 2-4 hours.

[0055] Preferably, the preparation method of the mobile phase B is: ultrasonic treatment of acetonitrile for 20-40 minutes, and bubbling treatment with argon for 2-4 hours; the bubbling validity period of the mobile phase A and mobile phase B is 24 hours. If it exceeds 24 hours, argon needs to be re-introduced for bubbling treatment.

[0056] Preferably, the flow rate is 1.0 mL / min; the injection plate temperature is 5° C.; and the injection volume is 10 μL.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The analytical detection method of compound C provided by the present invention adopts a combined method of filling the mobile phase with argon, adding ascorbic acid to the diluent, and washing HPLC with ascorbic acid, which can effectively perform analytical detection of phenylhydrazone compounds (compound C) without oxidation interference, and the detection precision of compound C is high and the repeatability is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is the HPLC spectrum of the blank solution of the analytical detection method of the present invention;

[0060] Figure 2 is the HPLC spectrum of the LOQ solution of the analytical detection method of the present invention;

[0061] Figure 3 It is the HPLC spectrum of the separation solution of the analysis and detection method of the present invention;

[0062] Figure 4 It is the HPLC spectrum of 0.4 μg / mL STD solution of the analytical detection method of the present invention;

[0063] Figure 5 It is the HPLC spectrum of 0.2 mg / mL STD solution of the analytical detection method of the present invention;

[0064] Figure 6 It is the HPLC spectrum of the sample solution of 0.2 mg / mL compound C in the analysis and detection method of the present invention;

[0065] Figure 7 is a linear data graph of compound C in the analytical detection method of the present invention;

[0066] Figure 8 is a linear residual distribution diagram of compound C in the analytical detection method of the present invention;

[0067] Fig. 9 It is a linear data graph of compound IM02 of the analytical detection method of the present invention;

[0068] Fig.10 is the linear residual distribution diagram of compound IM02 of the analytical detection method of the present invention;

[0069] Fig.11 It is the detection limit HPLC spectrum of compound C and IM02 of the analytical detection method of the present invention;

[0070] Fig.12 It is the quantitative limit HPLC spectrum of compound C and IM02 of the analytical detection method of the present invention;

[0071] Fig.13 It is the HPLC spectrum of compound C by the analysis and detection method of the embodiment and comparative example of the present invention. DETAILED DESCRIPTION

[0072] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of protection claimed in this application, and those skilled in the art can make various changes and modifications to the invention of this application based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0073] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels.

[0074] In the following examples, the ascorbic acid was purchased from Aladdin with a product number of A103533; the IM02 (benzaldehyde) standard was purchased from Maclean with a product number of B802738.

[0075] The standard of compound C was prepared in-house. The preparation method was as follows: benzaldehyde and p-methoxyphenylhydrazine were chemically synthesized in dichloromethane under the action of a base. The HPLC characterization chart of compound C (concentration 0.2 mg / mL) is shown in Figure 6 , the content of compound C is 99.8%.

[0076] Example 1 Analysis and Detection Method

[0077] 1.1 Reagents: acetonitrile, trifluoroacetic acid, ascorbic acid, C standard, IM02 (benzaldehyde) standard.

[0078] 1.2 Instrument pretreatment, flushing of HPLC and chromatographic column:

[0079] Phenylhydrazone compound samples are particularly sensitive to oxidants. All possible oxidants in the system need to be removed in advance. The specific steps are as follows:

[0080] S101, connect two channels, and four channels (ratio is 25:25:25:25) are first rinsed with acetonitrile for 5 min, and then with pure water for 5 min, with a flushing flow rate of 2 mL / min to remove salt and organic impurities;

[0081] S102, connect the chromatographic column, and rinse the four channels (ratio 25:25:25:25) with acetonitrile-water-ascorbic acid solution overnight, water: acetonitrile: V C =900:100:0.1 (v:v:m), flushing flow rate is 1mL / min; remove possible oxidizing substances in the instrument and chromatographic column, during which the needle and quantitative loop can be washed 1-2 times.

[0082] S103, immerse the four channels in pure water purge for 5-10 minutes to remove V in the channel pipelines C ;

[0083] S104, four channels are respectively introduced into four different solutions, namely mobile phase A, mobile phase B, pure water, and needle washing solution; firstly, a mixture of mobile phase A and mobile phase B with a volume ratio of 20:80 is introduced to wash the chromatographic column for 15 minutes; then mobile phase B is introduced to wash for 40 minutes; the washing flow rate is 1mL / min; the purpose of washing is to remove the residual V in the chromatographic column C During this period, the needle and quantitative loop can be washed 1-2 times.

[0084] 1.3HPLC analysis chromatographic detection conditions are:

[0085]

[0086]

[0087] 1.4 Solution preparation

[0088] 1.4.1 Blank solution (diluent): 400 mL acetonitrile, 100 mL water, 10 mg ascorbic acid (V C ) and mix evenly until ascorbic acid is completely dissolved, then pass argon gas into the bottom of the bottle and bubble for 2 hours, cool to 2-8°C before use, seal and store at low temperature; if stored for more than 24 hours, re-bubble.

[0089] 1.4.2 Standard solution (STD solution): Weigh 40mg±4.0mg of compound C standard, accurately weigh, place in a 20mL brown volumetric flask, dissolve with diluent and dilute to volume, shake well. Transfer 1mL of the above solution to a 10mL brown volumetric flask, dilute with diluent to volume, shake well. If it is insoluble, it can be treated with ultrasound. Prepare two portions in the same way, mark STD1 and STD2 (compound C: 0.2mg / mL), prepare before use, store at 2-8 degrees, use within 6 hours, otherwise it needs to be re-prepared.

[0090] 1.4.3 LOQ solution (limit of quantitation solution): Accurately pipette 1 ml of STD1 solution into a 100 mL volumetric flask, dilute to volume with diluent, and shake well. Then pipette 1 ml into a 20 mL volumetric flask, dilute to volume with diluent, and shake well. (Compound C: 0.1 μg / mL, 0.05%)

[0091] 1.4.4 Resolution stock solution: Weigh about 2.0 mg of IM02 standard into a 100 ml brown volumetric flask, dissolve and dilute to scale with diluent, and shake well. (IM02: 20 μg / mL).

[0092] 1.4.5 Resolution solution: Accurately pipette 1 ml of the resolution stock solution into a 100 ml volumetric flask, dissolve and dilute to the mark with STD1, and shake well (IM02 concentration is 0.2 μg / mL; compound C concentration is 0.2 mg / mL).

[0093] 1.4.6 Sample solution (TS solution): Weigh 40.0 mg ± 4.0 mg of compound C sample, weigh accurately, place in a 20 mL brown volumetric flask, dissolve with diluent and dilute to volume, shake well. Transfer 1 ml of the above solution to a 10 mL brown volumetric flask, dilute with diluent to volume, shake well. If it is insoluble, it can be treated with ultrasound. Prepare two portions in the same way, mark them as TS1 and TS2, and prepare them before use. (Compound C: 0.2 mg / mL).

[0094] The sample solution of compound C (concentration of 0.2 mg / mL) was analyzed and tested, and the HPLC spectrum is shown in Figure 6 .

[0095] 1.5 Detection process

[0096] After the instrument system is stable, inject samples according to the following sequence:

[0097]

[0098]

[0099] *: The last stitch is used as system blank, and the others are used as balanced system.

[0100] When testing, ensure that the system meets the following parameters:

[0101] (1) The blank has no interference (the area of ​​the interfering peak in the blank is less than 50% of the LOQ area);

[0102] (2) LOQ peak area signal-to-noise ratio greater than 10;

[0103] (3) In the resolution solution, the resolution between IM02 and the main peak is greater than 2.0;

[0104] (4) The ratio of the means of the respective response factors of STD1 and STD2 was 98.0-102.0%;

[0105] (5) The response factors RSD of all STD1 were ≤ 2.0%.

[0106] 1.6 Calculation

[0107] Purity = 100% - total impurities;

[0108] Total impurities = IM02+ (greater than 0.05% of the sum of unknown single impurities);

[0109] IM02 and unknown impurities were calculated using the area normalization method with correction factor: impurity = area% × CF; where: Area%: area percentage of impurities in the sample solution, %; CF: correction factor for impurities.

[0110] name Retention time min Relative retention time Correction Factor CF Compound C 16.7 1.00 1.00 IM02 (Benzaldehyde) 9.2 0.55 0.89 ascorbic acid 1.8 0.11 NA Unknown single impurity NA NA 1.0

[0111] Result requirements:

[0112] (1) Report the average of the two samples.

[0113] (2) If the impurity is greater than 0.05%, report the actual measured value; if it is less than 0.05%, report <0.05%.

[0114] Example 2 Analytical Detection Method Verification

[0115] Summary of verification results of related substances: Methodological verification was carried out on the related substances in the analysis and detection method. The detection and analysis method was the method of Example 1. The verification results are shown in Table 1.

[0116] Table 1

[0117]

[0118]

[0119] 2.1 System Applicability

[0120] The methodological-system suitability verification of the analytical detection method of compound C was performed. The specific data of the system suitability results are shown in Tables 2 and 3:

[0121] The HPLC spectrum of LOQ solution is shown in Figure 2 , the HPLC spectrum of STD solution is shown in Figure 4-5 .

[0122] Table 2

[0123] Table 3

[0124]

[0125] 2.2 Exclusivity

[0126] The methodological specificity of the analytical detection method for compound C was verified. The specific spectra of the specificity results of each solution are shown in Figure 1 and Figure 3 .

[0127] 2.3 Accuracy and precision

[0128] The analytical detection method of compound C was validated for methodology-accuracy and precision. The specific data of the accuracy and precision results are shown in Tables 4 and 5:

[0129] Table 4

[0130]

[0131]

[0132] Table 5

[0133]

[0134] 2.4 Linearity

[0135] The methodological linearity of the analytical detection method of compound C was verified. The specific data of the linearity results are shown in Tables 6, 7 and Figure 7-10 :

[0136] Table 6

[0137]

[0138]

[0139] Table 7

[0140]

[0141]

[0142] 2.5 Limit of Detection and Limit of Quantitation The analytical detection method of compound C was validated in terms of methodology - limit of detection and limit of quantitation. The specific data of the limit of detection (LOD - limit of detection solution) and limit of quantitation (LOQ - limit of quantitation solution) are shown in Tables 8-9 (Compound C) and 10-11 (IM02). Figure 11-Figure 12 :

[0143] Table 8

[0144]

[0145] Table 9

[0146]

[0147] Table 10

[0148]

[0149] Table 11

[0150]

[0151] 2.6 Solution stability

[0152] The analytical detection method of compound C was validated for solution stability. The specific data of the solution stability results are shown in Tables 12-13 (STD solution) and Tables 14-15 (TS solution):

[0153] Table 12

[0154]

[0155] Table 13

[0156]

[0157] Table 14

[0158]

[0159] Table 15

[0160]

[0161] Comparative Example 1

[0162] Different from Example 1, no antioxidant protection was performed on the test sample and the test method, that is, no argon was passed through the mobile phase (no argon bubbling treatment was performed), no ascorbic acid was added to the diluent, and no ascorbic acid was used to rinse the high performance liquid chromatograph and the chromatographic column. The remaining steps and chromatographic test conditions were the same.

[0163] The sample solution of compound C (concentration of 0.2 mg / mL) was analyzed and tested, and the HPLC spectrum is shown in Fig.13 (The spectrum line is number 1).

[0164] The HPLC spectrum of the sample solution of compound C of Example 1 is shown in Fig.13 (The spectrum line is number 4).

[0165] Comparative Example 2

[0166] The difference from Example 1 is that in the detection method, only the mobile phase is bubbled with argon gas, and no ascorbic acid is added to the diluent, and no ascorbic acid is used to rinse the HPLC and the chromatographic column. The remaining steps and chromatographic detection conditions are the same.

[0167] The sample solution of compound C (concentration of 0.2 mg / mL) was analyzed and tested, and the HPLC spectrum is shown in Fig.13 (The spectrum line is number 2).

[0168] Comparative Example 3

[0169] The difference from Example 1 is that in the detection method, ascorbic acid is not used to rinse the HPLC and the chromatographic column. The remaining steps and chromatographic detection conditions are the same.

[0170] The sample solution of compound C (concentration of 0.2 mg / mL) was analyzed and tested, and the HPLC spectrum is shown in Fig.13 (The spectrum line is number 3).

[0171] The results of the analysis and detection of the purity of phenylhydrazone compounds, the relative retention time (RRT) of impurities, and the proportion of impurities in Example 1 and Comparative Examples 1-3 are shown in Table 16.

[0172] Table 16

[0173]

[0174] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for analyzing and detecting phenylhydrazone compounds, characterized in that: Includes steps: S1. Instrument pretreatment, flushing of HPLC and chromatographic column: S101, connect the two channels, first flush the four channels with acetonitrile, then flush with pure water; S102, connect the chromatographic column, and rinse the four channels with acetonitrile-water-ascorbic acid solution; S103, immerse the four channels in pure water and rinse for 5-10 minutes; S104, connect the chromatographic column to the instrument, first flush the chromatographic column with a mixture of mobile phase A and mobile phase B at 1 mL / min: mobile phase A: mobile phase B = 20:80, v:v, for 10-20 min, and then flush the column with 1 mL / min of mobile phase B for 35-50 min; S2. Prepare a phenylhydrazone compound sample solution: prepare the phenylhydrazone compound using a diluent; the diluent is an acetonitrile-water-ascorbic acid solution, and analyze and detect the phenylhydrazone compound using HPLC; The analytical detection conditions of the HPLC method are: Column: Waters X-Bridge RP18, 150 × 4.6-mm, 3.5 μm; Mobile phase A: 0.05-0.2% trifluoroacetic acid solution treated with argon; Mobile phase B: acetonitrile treated with argon; Elution: Gradient elution, the gradient elution program is: Flow rate: 0.8-1.2mL / min; Column temperature: 10°C; Sample plate temperature: 2-10℃; Injection volume: 5-10 μL; Detection wavelength: 235nm; The chemical structural formula of the phenylhydrazone compound is: ； The preparation method of the phenylhydrazone compound is as follows: benzaldehyde and p-methoxyphenylhydrazine are chemically synthesized in dichloromethane under the action of a base.

2. The analysis and detection method according to claim 1, characterized in that: In step S101, the flushing flow rate is 1.0-4 mL / min, and the flushing time is 3-10 min.

3. The analysis and detection method according to claim 1, characterized in that: In step S102, the flushing flow rate is 1.0-4 mL / min, and the flushing time is 8-16 h.

4. The analysis and detection method according to claim 1, characterized in that: In steps S101 and S102, the flow ratio of the four channels is 20-30:20-30:20-30:20-30:20-30.

5. The analysis and detection method according to claim 1, characterized in that: In step S102, the acetonitrile-water-ascorbic acid solution comprises water: acetonitrile: V C =800-1000:50-150:0.05-0.1, v:v:m.

6. The analysis and detection method according to claim 1, characterized in that: In step S104, the time for flushing the chromatographic column with the mixed solution of mobile phase A and mobile phase B is 15 minutes, and the time for flushing with mobile phase B is 40 minutes; the flushing flow rate is 0.8-1.2 mL / min.

7. The analysis and detection method according to claim 1, characterized in that: In step S2, the diluent is acetonitrile-water-ascorbic acid solution, water: acetonitrile: ascorbic acid solution = 300-500: 50-150: 0.01-0.02, v:v:m; the preparation method of the diluent is: after mixing acetonitrile, water and ascorbic acid, argon is introduced for bubbling for 2-4h, and cooled to 2-8°C.

8. The analysis and detection method according to claim 1, characterized in that: In step S2, the mobile phase A is a 0.1% trifluoroacetic acid solution treated with argon.

9. The analysis and detection method according to claim 1, characterized in that: The preparation method of the mobile phase A is: trifluoroacetic acid and water are mixed evenly, ultrasonically treated for 20-40 minutes, and argon gas is introduced for bubbling for 2-4 hours; the preparation method of the mobile phase B is: acetonitrile is ultrasonically treated for 20-40 minutes, and argon gas is introduced for bubbling for 2-4 hours.

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