CN115494184B - Method for simultaneously detecting methyl cumene sulfonate and ethyl cumene sulfonate in medicine - Google Patents
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Abstract
The invention provides a method for simultaneously detecting methyl cumene sulfonate and ethyl cumene sulfonate in a medicine, which adopts an ultra-high performance liquid chromatography-mass spectrometer to detect a reference substance solution and a sample solution respectively, and calculates according to an external standard method to determine the content of the methyl cumene sulfonate and the ethyl cumene sulfonate in a sample. According to the invention, ultra-high performance liquid chromatography is adopted for separation, high selectivity and high sensitivity mass spectrum are adopted for simultaneously detecting the methyl cumene sulfonate and the ethyl cumene sulfonate, the sample pretreatment is simple, the sample matrix has no interference to the target peak determination, and the method has good durability and high detection efficiency.
Description
Technical Field
The invention relates to the field of medicine analysis, in particular to a method for simultaneously detecting methyl cumene sulfonate and ethyl cumene sulfonate in a medicine.
Background
Cumene sulfonic acid or cumene sulfonate is an important raw material in organic synthesis, and can form genotoxic impurities, namely methyl cumene sulfonate and ethyl cumene sulfonate, with trace amounts of methanol and ethanol in the process, and the substances can react with DNA in an alkylation way, so that the substances can become causes for causing cancers, and therefore, the control of the toxicological attention threshold (TTC) level of the impurities in the medicine is very important. The European Medicine Evaluation Agency (EMEA), the United states Food and Drug Administration (FDA), and the International conference on drug registration (ICH) all make limit regulations on genotoxic impurities that must be controlled below the toxicological attention threshold (TTC) level. Wherein the structure of the methyl cumene sulfonate and the ethyl cumene sulfonate is as follows:
at present, a detection method of cumene sulfonate is not found, and a method for simultaneously detecting methyl cumene sulfonate and ethyl cumene sulfonate in a medicine is necessary to be developed.
Disclosure of Invention
The invention provides a method for simultaneously detecting methyl cumene sulfonate and ethyl cumene sulfonate in a medicine, which adopts ultra-high performance liquid chromatography-mass spectrometry to simultaneously detect the methyl cumene sulfonate and the ethyl cumene sulfonate, and has the advantages of high selectivity and high sensitivity, simple sample pretreatment, no interference to the determination of a target peak caused by a sample matrix, and good durability, and the method comprises the following steps:
step 1: adding a diluent into the cumene sulfonate methyl ester and cumene sulfonate ethyl ester reference substance to prepare a reference substance solution;
step 2: adding a diluent into a sample to be tested to prepare a sample solution to be tested;
step 3: detecting the reference substance solution in the step 1 and the sample solution in the step 2 by adopting an ultra-high performance liquid chromatography-mass spectrometer, and determining the contents of methyl cumene sulfonate and ethyl cumene sulfonate in a sample according to an external standard method;
wherein, the chromatographic conditions of the ultra performance liquid chromatography-mass spectrometer in the step 3 are as follows:
chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
mobile phase: mobile phase a is a volatile aqueous ammonium salt solution, preferably an aqueous ammonium formate solution or an aqueous ammonium acetate solution, most preferably an aqueous ammonium formate solution; the concentration of the mobile phase A is 0.002-0.05 mol/L, preferably 0.005-0.05 mol/L, and most preferably 0.01mol/L;
mobile phase B is methanol or acetonitrile, preferably methanol;
the elution mode of the mobile phase is gradient elution, and the gradient elution is carried out according to the following procedures:
| time (min) | Mobile phase A% | Mobile phase B% |
| 0.0 | 70 | 30 |
| 4.0 | 20 | 80 |
| 5.0 | 20 | 80 |
| 5.1 | 70 | 30 |
| 7.0 | 70 | 30 |
Preferably, the drug is selected from the group consisting of drugs in which cumene sulphonic acid or cumene sulphonic acid salt is used in the process and methanol or ethanol is used, preferably acarbose or micafungin sodium.
Preferably, the diluent in step 1 or step 2 is methanol, acetonitrile, water or a mixture thereof, preferably an aqueous methanol solution or an aqueous acetonitrile solution, most preferably an aqueous methanol solution.
Preferably, the diluent in step 1 or step 2 is 10% to 80% aqueous methanol or acetonitrile (V/V), preferably 10% to 60% aqueous methanol or acetonitrile (V/V), more preferably 20% to 50% aqueous methanol or acetonitrile (V/V), even more preferably 25% to 50% aqueous methanol or acetonitrile (V/V), most preferably 30% aqueous methanol or acetonitrile (V/V).
Preferably, the column diameter of the octadecylsilane chemically bonded silica chromatographic column in the step 3 is 2.1-4.6 mm, the column length is 50-100 mm, and the particle size is 1.6-3.5 μm; preferably, the column diameter of the octadecylsilane chemically bonded silica chromatographic column is 2.1 mm-3.5 mm, the column length is 50 mm-100 mm, and the particle diameter is 1.6 mu m-2.5 mu m; more preferably, the column diameter of the octadecylsilane chemically bonded silica chromatographic column is 2.1 mm-3.0 mm, the column length is 50 mm-100 mm, and the particle diameter is 1.7 mu m-2.5 mu m; most preferably octadecylsilane chemically bonded silica chromatographic column Agilent Eclipse Plus C 18 RRHD, column diameter of 2.1mm, column length of 50mm, and particle size of 1.8 μm.
Preferably, the column temperature of the chromatographic column in step 3 is from 25 ℃ to 45 ℃, preferably from 30 ℃ to 45 ℃, more preferably from 30 ℃ to 40 ℃, most preferably 35 ℃.
Preferably, the flow rate of the mobile phase in step 3 is from 0.2mL/min to 0.6mL/min, preferably from 0.2mL/min to 0.5mL/min, more preferably from 0.2mL/min to 0.4mL/min, and most preferably 0.3mL/min.
Preferably, the parameters of the mass spectrum in step 3 are:
scanning mode: monitoring mass spectrum multiple reactions;
ion source: an electrospray ion source;
ion source mode: a positive mode;
capillary voltage: 1.0-4.0 KV, preferably 2.0-4.0 KV, more preferably 3.5KV;
drying gas temperature: 300 to 650 ℃, preferably 350 to 600 ℃, more preferably 400 to 550 ℃, most preferably 500 ℃;
source temperature: 150 ℃.
Preferably, the ultra performance liquid chromatography-mass spectrometer used in the step 3 is Waters ACQUITY I UPLC Class-TQ-S micro or Agilent 1290-6470.
Compared with the prior art, the invention has the beneficial effects that:
the content of the methyl cumene sulfonate and the ethyl cumene sulfonate in the sample can be detected simultaneously; the pretreatment of the sample is simple, the sample adopts a direct dissolution mode, and the derivatization treatment is not needed; the separation degree is good, and the methyl cumene sulfonate and the ethyl cumene sulfonate are completely and effectively separated from the sample within 7 minutes; the linear correlation coefficient is good, and the linear correlation coefficient r of the methyl cumene sulfonate and the ethyl cumene sulfonate in the respective linear ranges is more than or equal to 0.999; the sensitivity is high, the lowest quantitative limit of the instrument detection of the methyl cumene sulfonate is 5.7350ng/mL, the lowest quantitative limit of the instrument detection of the ethyl cumene sulfonate is 4.9500ng/mL, the lowest detection limit of the methyl cumene sulfonate is 1.7205ng/mL, and the lowest detection limit of the ethyl cumene sulfonate is 1.4850ng/mL; the recovery rate is high, and the standard adding accuracy recovery rate of the methyl cumenesulfonate and the ethyl cumenesulfonate is 90-115%. The method simultaneously detects two cumene sulfonates, improves the detection efficiency, adopts high-selectivity and high-sensitivity mass spectrum for detection, has simple sample pretreatment and no interference to the measurement of a target peak by a sample matrix; the method has good durability and high detection efficiency, and can well detect the contents of the methyl cumene sulfonate and the ethyl cumene sulfonate in the sample so as to ensure the product quality.
Drawings
FIG. 1 is a diagram showing the ultra-high performance liquid-mass spectrum combination of cumene sulfonate and ethyl cumene sulfonate blank solvent.
Fig. 2 is a ultra-high performance liquid-mass spectrum combined spectrum of the cumene sulfonate and the ethyl cumene sulfonate reference substance solution of the invention.
FIG. 3 is a linear diagram of methyl cumene sulfonate of the present invention.
FIG. 4 is a linear graph of ethyl cumene sulfonate of the present invention.
Detailed Description
The following examples further illustrate the invention, but it is necessary to point out that the following examples are given for the purpose of illustration only and are not to be construed as limiting the scope of the invention, which is defined by the claims.
In the following examples, all temperatures are degrees celsius unless otherwise indicated; unless otherwise indicated, the various starting materials and reagents were all from commercial sources and were used without further purification; unless otherwise indicated, each solvent was an industrial grade solvent and was used without further treatment. Methanol is commercially available chromatographic purity, ammonium formate is commercially available mass spectrometry purity, water is self-made ultrapure water, and the purity of the isopropyl benzene sulfonic acid methyl ester and the purity of the isopropyl benzene sulfonic acid ethyl ester reference are 98 percent (all purchased from Shanghai Limited company of Mordisia medical technology).
Preparation of test solutions
The acarbose or micafungin sodium sample is weighed and dissolved and diluted into a solution containing 4mg/mL of acarbose or micafungin sodium by 30% methanol water solution (V/V) respectively to be used as a test solution.
Preparation of control stock solutions
And respectively precisely weighing a proper amount of the reference substances of the methyl cumene sulfonate and the ethyl cumene sulfonate, and dissolving and diluting the reference substances into reference substance stock solutions with the concentration of the methyl cumene sulfonate and the ethyl cumene sulfonate of 50ng/mL by using a diluent.
Example 1
Ultra-high performance liquid-mass spectrometry detection condition
Instrument: wo-superfine ultra-high performance liquid-triple quadrupole mass spectrometer (Waters ACQUITY I UPLC Class-TQ-Smmicro)
Chromatographic conditions:
chromatographic column: agilent Eclipse Plus C 18 RRHD 50*2.1mm 1.8μm
Flow rate: 0.3mL/min
Column temperature: 35 DEG C
Sample injection amount: 5.0 mu L
Mobile phase a:0.01mol/L ammonium formate aqueous solution
Mobile phase B: methanol
A diluent: 30% aqueous methanol (V/V)
Blank solvent: same diluent
Gradient:
mass spectrometry conditions:
(1) Specificity experiments
a) Blank solvent: 30% aqueous methanol (V/V), wherein the ion chart is shown in FIG. 1;
b) Control solution: transferring 4.0mL of the control stock solution into a 10mL volumetric flask, diluting to scale with a diluent, and mixing uniformly, wherein the characteristic ion diagram is shown in figure 2;
c) Adding a labeled test sample solution: 40mg of acarbose sample is weighed into a 10mL volumetric flask, 4.0mL of reference substance stock solution is added, a proper amount of diluent is used for dissolving, then the diluent is used for diluting to a scale, and the mixture is uniformly mixed.
Weighing 40mg of micafungin sodium sample in a 10mL volumetric flask, adding 4.0mL of control stock solution, dissolving with a proper amount of diluent, diluting to scale with the diluent, and mixing well.
According to the method, the special solutions of the blank solvent, the reference substance solution and the labeled test sample solution are detected, the detection results are shown in table 1, and from the experimental results, the blank solvent and the sample solution have no interference on the detection of the methyl cumene sulfonate and the ethyl cumene sulfonate, and the specificity of the ultra-high performance liquid-mass spectrometry combined detection method is good.
TABLE 1 results of determination of specific solutions for example 1
(2) Limit of detection and limit of quantification
a) Quantitative limiting solution: transferring 1.0mL of the reference substance stock solution into 6 10mL volumetric flasks, diluting to scale with a diluent, and uniformly mixing;
b) Detection limit solution: transferring 3.0mL of the first quantitative limiting solution into a 10mL volumetric flask, diluting to a scale with a diluent, and uniformly mixing;
the quantitative limit solution and the detection limit solution are detected according to the method, the signal to noise ratio (S/N) of the quantitative limit solution is more than or equal to 10, the detection limit solution (S/N) is more than or equal to 3, and the detection results are shown in the table 2 and the table 3:
table 2 example 1 ultra high performance liquid-mass spectrometry quantitation limit
TABLE 3 detection limits for ultra high performance liquid chromatography-mass spectrometry of example 1
(3) Linearity of
Linear solution: and (3) transferring 1.0mL, 2.0mL, 4.0mL, 6.0mL and 8.0mL of stock solution of methyl cumenesulfonate and ethyl cumenesulfonate into 5 volumetric flasks of 10mL respectively, diluting to scale with a diluent, uniformly mixing, and preparing two parts of stock solution of ethyl cumenesulfonate and linear concentration in parallel.
According to the above method, a needle was introduced into each linear solution, and a linear equation and a linear correlation coefficient were calculated, and the results are shown in Table 4.
Table 4 example 1 ultra high performance liquid-mass spectrometer detection method linear regression equation, linear correlation coefficient r
From the experimental results, the high performance liquid-mass spectrometer detection method has good linear relation in the detection range, and the linear correlation coefficient r is 0.9999, so that the contents of the methyl cumene sulfonate and the ethyl cumene sulfonate can be accurately quantified.
(4) Accuracy and repeatability experiments
a) Accuracy experiment
Test solutions of acarbose or micafungin sodium are prepared, and three reference solutions of methyl cumene sulfonate and ethyl cumene sulfonate with different concentration levels are respectively added, and three parts of each solution are prepared in parallel, wherein the preparation concentrations are shown in tables 5 and 6. Each of the labeled test sample solutions was subjected to sample injection analysis once according to the method described above, and the recovery rate of each solution and the average recovery rate of each concentration level were calculated, and the results are shown in tables 5 and 6.
TABLE 5 accuracy of ultra high Performance liquid chromatography-Mass Spectrometry (HPLC-MS) detection method in example 1 micafungin sodium
TABLE 6 accuracy of ultra high performance liquid chromatography-mass spectrometry detection method in acarbose of example 1
b) Repeatability experiments
Preparing 6 parts of marked acarbose or marked micafungin sodium solution with medium concentration level, carrying out sample injection analysis on each part of solution according to the method, and calculating RSD of the recovery rate of 6 parts of marked solution. The results are shown in tables 7 and 8.
TABLE 7 example 1 ultra high Performance liquid chromatography mass spectrometry detection method repeatability in micafungin sodium
Table 8 example 1 repeatability of ultra high performance liquid chromatography-mass spectrometry detection method in acarbose
According to the experimental results, the accuracy and the repeatability of the detection method of the high performance liquid-mass spectrometer are 90-115%, the RSD is less than 5%, and the accuracy and the repeatability are good.
(5) Sample detection of methyl cumene sulfonate and ethyl cumene sulfonate in acarbose or micafungin sodium
Weighing acarbose sample 40mg in a 10mL volumetric flask, dissolving with a proper amount of diluent, diluting to scale with the diluent, and mixing uniformly; 40mg of micafungin sodium sample is weighed into a 10mL volumetric flask, dissolved with a proper amount of diluent, diluted to a scale with the diluent, uniformly mixed, and the detection results are shown in tables 9 and 10.
Table 9 example 1 micafungin sodium sample detection results
TABLE 10 acarbose sample detection results example 1
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From the above experimental results, no methyl cumene sulfonate or ethyl cumene sulfonate was detected in both acarbose and micafungin sodium samples, and both reported to be less than the detection limit.
Example 2
Ultra-high performance liquid-mass spectrometry detection condition
Instrument: agilent 1290-6470
Chromatographic conditions:
chromatographic column: agilent Eclipse Plus C 18 RRHD 50*2.1mm 1.8μm
Flow rate: 0.3mL/min
Column temperature: 35 DEG C
Sample injection amount: 5.0 mu L
Mobile phase a:0.01moL/L ammonium formate aqueous solution
Mobile phase B: methanol
A diluent: 30% aqueous methanol (V/V)
Blank solvent: same diluent
Gradient:
| time (min) | Mobile phase A% | Mobile phase B% |
| 0.0 | 70 | 30 |
| 4.0 | 20 | 80 |
| 5.0 | 20 | 80 |
| 5.1 | 70 | 30 |
| 7.0 | 70 | 30 |
Mass spectrometry conditions:
/>
(1) Specificity experiments
a) Blank solvent: 30% aqueous methanol (V/V);
b) Control solution: transferring 4.0mL of the control stock solution into a 10mL volumetric flask, diluting to a scale with a diluent, and uniformly mixing;
c) Adding a labeled test sample solution: 40mg of acarbose sample is weighed into a 10mL volumetric flask, 4.0mL of reference substance stock solution is added, a proper amount of diluent is used for dissolving, then the diluent is used for diluting to a scale, and the mixture is uniformly mixed.
Weighing 40mg of micafungin sodium sample in a 10mL volumetric flask, adding 4.0mL of control stock solution, dissolving with a proper amount of diluent, diluting to scale with the diluent, and mixing well.
According to the method, the special solutions of the blank solvent, the reference substance solution and the labeled test sample solution are detected, the detection results are shown in table 11, and from the experimental results, the blank solvent and the sample solution have no interference on the detection of the methyl cumene sulfonate and the ethyl cumene sulfonate, and the specificity of the ultra-high performance liquid-mass spectrometry combined detection method is good.
TABLE 11 results of determination of specific solutions of example 2
| Project | Isopropyl benzene sulfonic acid methyl ester | Isopropylbenzene sulfonic acid ethyl ester |
| Blank solvent retention time (min) | ND | ND |
| Control solution retention time (min) | 2.72 | 3.95 |
| Time (min) remaining in the labeling solution | 2.72 | 3.95 |
| Retention time (min) in sample solution | ND | ND |
(2) Limit of detection and limit of quantification
a) Quantitative limiting solution: transferring 1.0mL of the reference substance stock solution into 6 10mL volumetric flasks, diluting to scale with a diluent, and uniformly mixing;
b) Detection limit solution: transferring 3.0mL of the first quantitative limiting solution into a 10mL volumetric flask, diluting to a scale with a diluent, and uniformly mixing;
the quantitative limit solution and the detection limit solution are detected according to the method, the signal to noise ratio (S/N) of the quantitative limit solution is more than or equal to 10, the detection limit solution (S/N) is more than or equal to 3, and the detection results are shown in the table 12 and the table 13:
table 12 example 2 ultra performance liquid-mass spectrometry quantitation limit
Table 13 example 2 ultra performance liquid-mass spectrometry detection limits
(3) Linearity of
Linear solution: and (3) transferring 1.0mL, 2.0mL, 4.0mL, 6.0mL and 8.0mL of stock solution of methyl cumenesulfonate and ethyl cumenesulfonate into 5 volumetric flasks of 10mL respectively, diluting to scale with a diluent, uniformly mixing, and preparing two parts of stock solution of ethyl cumenesulfonate and linear concentration in parallel.
According to the above method, a needle was introduced into each linear solution, and a linear equation and a linear correlation coefficient were calculated, and the results are shown in Table 14.
Table 14 example 2 ultra performance liquid-mass spectrometer detection method linear regression equation, linear correlation coefficient r
From the experimental results, the high performance liquid-mass spectrometer detection method has good linear relation in the detection range, and the linear correlation coefficients are 0.9998 and 0.9999, so that the contents of the methyl cumene sulfonate and the ethyl cumene sulfonate can be accurately quantified.
(4) Accuracy and repeatability experiments
a) Accuracy experiment
Test solutions of acarbose or micafungin sodium were prepared, and three different concentration levels of methyl cumene sulfonate and ethyl cumene sulfonate reference solutions were added, respectively, and three portions of each concentration were prepared in parallel, and the prepared concentrations are shown in tables 15 and 16. Each aliquot of the labeled test sample solution was analyzed by sample injection according to the method described above, and the recovery rate for each aliquot and the average recovery rate for each concentration level were calculated. The results are shown in tables 15 and 16.
TABLE 15 accuracy of ultra high Performance liquid chromatography-Mass Spectrometry (HPLC-MS) detection method in example 2 micafungin sodium
TABLE 16 accuracy of ultra high performance liquid chromatography-mass spectrometry detection method in acarbose example 2
b) Repeatability experiments
Preparing 6 parts of marked acarbose or marked micafungin sodium solution with medium concentration level, carrying out sample injection analysis on each part of solution according to the method, and calculating RSD of the recovery rate of 6 parts of marked solution. The results are shown in tables 17 and 18.
Table 17 example 2 repeatability of ultra high performance liquid-mass spectrometer detection method in micafungin sodium
Table 18 example 2 repeatability of ultra high performance liquid chromatography-mass spectrometry detection method in acarbose
According to the experimental results, the accuracy and the repeatability of the detection method of the high performance liquid-mass spectrometer are 95-105%, the RSD is less than 5%, and the accuracy and the repeatability are good.
(5) Sample detection of methyl cumene sulfonate and ethyl cumene sulfonate in acarbose or micafungin sodium
Weighing acarbose sample 40mg in a 10mL volumetric flask, dissolving with a proper amount of diluent, diluting to scale with the diluent, and mixing uniformly; 40mg of micafungin sodium sample was weighed into a 10mL volumetric flask, dissolved with a proper amount of diluent, diluted to the scale with the diluent, and mixed uniformly, and the detection results are shown in tables 19 and 20.
Table 19 example 2 micafungin sodium sample detection results
TABLE 20 acarbose sample detection results example 2
From the above experimental results, no methyl cumene sulfonate or ethyl cumene sulfonate was detected in both acarbose and micafungin sodium samples, and both reported to be less than the detection limit.
Comparative example 1 Mobile phase A was 0.1% (V/V) aqueous formic acid solution and mobile phase B was methanol
The 0.01moL/L aqueous ammonium formate solution in example 1 was replaced with 0.1% (V/V) aqueous formic acid solution, and the other chromatographic mass spectrometry conditions (example 1) were unchanged. 3 parts of the medium concentration level of the labeled acarbose solution were prepared, each solution was sampled and analyzed once, and the chromatogram was recorded, and the results are shown in Table 21. The results show that when 0.1% (V/V) formic acid aqueous solution is used as the mobile phase, the sample labeling recovery rate is increased with time, the recovery rate is lower and lower, and the laboratory recovery rate requirement (50% -150% of required recovery rate) is exceeded.
TABLE 21 recovery of 0.1% (V/V) formic acid aqueous solution as mobile phase Shi Aka wave sugar concentration addition standard solution
Comparative example 2 chromatographic column length 150mm
The chromatographic column is replaced by Waters ACQUITY UPLC BEH C 18 150mm x 2.1mm 1.8 μm, other chromatographic mass spectrometry conditions (example 1) were unchanged, and isopropyl benzene sulfonate and ethyl cumene sulfonate control solutions were sampled, and no isopropyl benzene sulfonate and ethyl cumene sulfonate target peaks were found, indicating that the isopropyl benzene sulfonate and ethyl cumene sulfonate target peaks were enhanced in retention and did not elute.
Claims (33)
1. A method for simultaneously detecting methyl cumene sulfonate and ethyl cumene sulfonate in a medicament, which is characterized by comprising the following steps:
step 1: adding a diluent into the cumene sulfonate methyl ester and cumene sulfonate ethyl ester reference substance to prepare a reference substance solution;
step 2: adding a diluent into a sample to be tested to prepare a sample solution to be tested;
step 3: detecting the reference substance solution in the step 1 and the sample solution in the step 2 by adopting an ultra-high performance liquid chromatography-mass spectrometer, and determining the contents of methyl cumene sulfonate and ethyl cumene sulfonate in a sample according to an external standard method;
wherein, the chromatographic conditions of the ultra performance liquid chromatography-mass spectrometer in the step 3 are as follows:
chromatographic column: octadecylsilane chemically bonded silica chromatographic column;
mobile phase: the mobile phase A is ammonium formate aqueous solution or ammonium acetate aqueous solution; the concentration of the mobile phase A is 0.002-0.05 mol/L;
mobile phase B is methanol;
the elution mode of the mobile phase is gradient elution, and the gradient elution is carried out according to the following procedures:
。
2. The method of claim 1, wherein the mobile phase a is an aqueous ammonium formate solution.
3. The method according to claim 1, wherein the concentration of mobile phase a is 0.005-0.05 mol/L.
4. The method according to claim 1, wherein the concentration of mobile phase a is 0.01mol/L.
5. The method of claim 1, wherein the drug is selected from the group consisting of drugs in which cumene sulfonic acid or cumene sulfonate is used in the process, and methanol or ethanol is used.
6. The method of claim 5, wherein the drug is acarbose or micafungin sodium.
7. The method according to claim 1, wherein the diluent in step 1 or step 2 is methanol, acetonitrile, water or a mixture thereof.
8. The method according to claim 7, wherein the diluent in step 1 or step 2 is aqueous methanol or acetonitrile.
9. The method of claim 7, wherein the diluent in step 1 or step 2 is aqueous methanol.
10. The method according to claim 7, wherein the diluent in step 1 or step 2 is 10% to 80% V/V aqueous methanol or acetonitrile.
11. The method according to claim 7, wherein the diluent in step 1 or step 2 is 10% to 60% V/V aqueous methanol or acetonitrile.
12. The method according to claim 7, wherein the diluent in step 1 or step 2 is 20% to 50% V/V aqueous methanol or acetonitrile.
13. The method according to claim 7, wherein the diluent in step 1 or step 2 is 25% to 50% V/V aqueous methanol or acetonitrile.
14. The method according to claim 7, wherein the diluent in step 1 or step 2 is 30% V/V aqueous methanol or acetonitrile.
15. The method according to claim 1, wherein the octadecylsilane chemically bonded silica column in step 3 has a column diameter of 2.1mm to 4.6mm, a column length of 50mm to 100mm and a particle diameter of 1.6 μm to 3.5 μm.
16. The method according to claim 15, wherein the octadecylsilane chemically bonded silica column in step 3 has a column diameter of 2.1 to 3.5mm, a column length of 50 to 100mm and a particle diameter of 1.6 to 2.5 μm.
17. The method according to claim 15, wherein the octadecylsilane chemically bonded silica column in step 3 has a column diameter of 2.1mm to 3.0mm, a column length of 50mm to 100mm and a particle diameter of 1.7 μm to 2.5 μm.
18. The method of claim 15, wherein the octadecylsilane chemically bonded silica column in step 3 is Agilent Eclipse Plus C 18 RRHD, column diameter of 2.1mm, column length of 50mm, and particle size of 1.8 μm.
19. The method according to claim 1, wherein the column temperature of the chromatographic column in step 3 is 25 ℃ to 45 ℃.
20. The method of claim 19, wherein the column temperature of the chromatographic column in step 3 is from 30 ℃ to 45 ℃.
21. The method of claim 19, wherein the column temperature of the chromatographic column in step 3 is 30 ℃ to 40 ℃.
22. The method of claim 19, wherein the column temperature of the chromatographic column in step 3 is 35 ℃.
23. The method according to claim 1, wherein the flow rate of the mobile phase in step 3 is 0.2mL/min to 0.6mL/min.
24. The method of claim 23, wherein the mobile phase in step 3 has a flow rate of 0.2mL/min to 0.5mL/min.
25. The method of claim 23, wherein the mobile phase in step 3 has a flow rate of 0.2mL/min to 0.4mL/min.
26. The method of claim 23, wherein the mobile phase in step 3 has a flow rate of 0.3mL/min.
27. The method according to claim 1, wherein the parameters of the mass spectrum in step 3 are:
scanning mode: monitoring mass spectrum multiple reactions;
ion source: an electrospray ion source;
ion source mode: a positive mode;
capillary voltage: 1.0 KV to 4.0KV;
drying gas temperature: 300-650 ℃;
source temperature: 150 ℃.
28. The method of claim 27, wherein the capillary voltage is 2.0 KV to 4.0KV.
29. The method of claim 27, wherein the capillary voltage is 3.5KV.
30. The method of claim 27, wherein the drying gas temperature is 350-600 ℃.
31. The method of claim 27, wherein the drying gas temperature is 400-550 ℃.
32. The method of claim 27, wherein the drying gas temperature is 500 ℃.
33. The method according to any one of claims 1 to 32, wherein the ultra performance liquid chromatography-mass spectrometer used in step 3 is Waters ACQUITY I UPLC Class-TQ-S micro or Agilent 1290-6470.
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