CN116718705A - Method for determining content of genotoxic impurities in ligustrazine phosphate - Google Patents
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- 239000012535 impurity Substances 0.000 title claims abstract description 51
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 50
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 35
- KWWLGXNRLABSMP-UHFFFAOYSA-N phosphoric acid;2,3,5,6-tetramethylpyrazine Chemical compound OP(O)(O)=O.CC1=NC(C)=C(C)N=C1C KWWLGXNRLABSMP-UHFFFAOYSA-N 0.000 title claims abstract description 31
- MRKOVWZAVHJNPG-UHFFFAOYSA-N 2,3,5,6-tetramethyl-1-oxidopyrazin-1-ium Chemical compound CC1=NC(C)=C(C)[N+]([O-])=C1C MRKOVWZAVHJNPG-UHFFFAOYSA-N 0.000 claims abstract description 22
- 150000002500 ions Chemical class 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 238000001819 mass spectrum Methods 0.000 claims abstract description 13
- 239000000243 solution Substances 0.000 claims description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000012071 phase Substances 0.000 claims description 15
- 239000012488 sample solution Substances 0.000 claims description 11
- 239000000523 sample Substances 0.000 claims description 10
- 238000012360 testing method Methods 0.000 claims description 9
- 238000005303 weighing Methods 0.000 claims description 8
- 238000007865 diluting Methods 0.000 claims description 7
- 239000011550 stock solution Substances 0.000 claims description 7
- 239000007791 liquid phase Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 4
- 239000013558 reference substance Substances 0.000 claims description 4
- 239000012086 standard solution Substances 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 3
- 238000000065 atmospheric pressure chemical ionisation Methods 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000005259 measurement Methods 0.000 claims 3
- 238000004458 analytical method Methods 0.000 abstract description 14
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- RQKFOGXUTRDQPB-UHFFFAOYSA-N hydron;2,3,5,6-tetramethylpyrazine;chloride Chemical compound Cl.CC1=NC(C)=C(C)N=C1C RQKFOGXUTRDQPB-UHFFFAOYSA-N 0.000 description 4
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000000691 measurement method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 206010008118 cerebral infarction Diseases 0.000 description 2
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- 238000010812 external standard method Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- -1 2,3,5, 6-tetramethyl pyrazine phosphate monohydrate Chemical compound 0.000 description 1
- 201000006474 Brain Ischemia Diseases 0.000 description 1
- 206010008120 Cerebral ischaemia Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 208000032109 Transient ischaemic attack Diseases 0.000 description 1
- 210000002565 arteriole Anatomy 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000916 dilatatory effect Effects 0.000 description 1
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- 238000011068 loading method Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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- 239000013557 residual solvent Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 238000013212 standard curve analysis Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 201000010875 transient cerebral ischemia Diseases 0.000 description 1
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention relates to the technical field of analysis and detection, in particular to a method for measuring the content of genotoxic impurities in ligustrazine phosphate. According to the analysis method, the contents of 2,3,5, 6-tetramethylpyrazine-1-oxide, 2,3,5, 6-tetramethylpyrazine-N, N-dioxide and (1-oxo-2, 5, 6-trimethylpyrazine-3-yl) methanol in ligustrazine phosphate are determined by using a triple quaternary-liquid chromatography-mass spectrometer, an ACE Excel C18-AR chromatographic column is adopted as a chromatographic column, an MRM positive ion mode is adopted as a mass spectrum, and the content of genotoxic impurities is analyzed by using a standard curve method. The analysis method has the advantages of good separation degree among three genotoxic impurities, strong specificity, good precision, low detection limit, high efficiency and accuracy, more comprehensive monitoring of the quality of ligustrazine phosphate and ensured effectiveness and consistency of the quality of products.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a method for measuring the content of genotoxic impurities in ligustrazine phosphate.
Background
Ligustrazine phosphate, chemical name of 2,3,5, 6-tetramethyl pyrazine phosphate monohydrate, molecular formula of C 8 H 12 N 2 ·H 3 PO 4 ·H 2 O. The traditional Chinese medicine composition has the effects of mainly improving clinical symptoms of ischemic cerebral apoplexy patients, and is mainly applied to patients with cerebral ischemia, cerebral infarction and transient ischemic attack, so that the traditional Chinese medicine composition is widely applied to neurology. Ligustrazine phosphate also has effects of resisting platelet aggregation, dilating arteriole, improving microcirculation, promoting blood circulation, and removing blood stasis, and depolymerizing aggregated platelet.
The quality standard of ligustrazine phosphate is received in the second part of the "Chinese pharmacopoeia" 2020 edition, wherein only detection items such as residual solvents and related substances are provided, no genotoxic impurity detection method exists at present, the quality condition of the product cannot be comprehensively reflected, and therefore, the genotoxic impurities in the medicine need to be analyzed and detected.
For example, patent CN111141841a discloses a method for determining the content of genotoxic impurities in ligustrazine hydrochloride, which adopts a liquid chromatography-mass spectrometry method, and the chromatographic conditions include: the chromatographic column is octadecylsilane chemically bonded silica chromatographic column, and the volume ratio of methanol to water is 20-40:60-80, the flow rate of the mixed solution is 0.2-0.6ml/min; the column temperature is 25-45 ℃, the four-stage rod mass spectrum detector is used for chemically ionizing an ion source at the atmospheric pressure, a positive ion mode detection of 153m/z of Selective Ions (SIM) is adopted, the flow rate of dry gas is 10L/min, the flow rate of atomized gas is 4.0L/min, the voltage of the ion source is 4.0-5.0KV, and the temperature of the ion source is 300400 ℃. The method can rapidly and accurately measure the content of the genotoxic impurities of the ligustrazine hydrochloride, and lays a foundation for guaranteeing and improving the quality of the ligustrazine hydrochloride.
However, the above-mentioned measurement method is mainly aimed at analyzing and detecting 1-oxo-2, 3,5, 6-tetramethylpyrazine, which is a genotoxic impurity contained in ligustrazine hydrochloride, whereas among ligustrazine phosphate, there are three types of 2,3,5, 6-tetramethylpyrazine-1-oxide, 2,3,5, 6-tetramethylpyrazine-N, N-dioxide and (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol, and therefore, it is necessary to design a measurement method for simultaneously measuring the above-mentioned three genotoxic impurities.
Disclosure of Invention
The purpose of the invention is that: the method for determining the content of the genotoxic impurities in the ligustrazine phosphate can rapidly and accurately determine the content of the genotoxic impurities in the ligustrazine phosphate, and lays a foundation for guaranteeing and improving the quality of the ligustrazine phosphate.
The method for determining the content of genotoxic impurities in ligustrazine phosphate comprises the steps of determining the content of three genotoxic impurities of 2,3,5, 6-tetramethylpyrazine-1-oxide, 2,3,5, 6-tetramethylpyrazine-N, N-dioxide and (1-oxo-2, 5, 6-trimethylpyrazine-3-yl) methanol in ligustrazine phosphate at one time by using a triple quaternary-liquid chromatography-mass spectrometry (LC-MS/MS), and determining the content of the genotoxic impurities by using a standard curve method;
the chromatographic conditions of the triple quaternary-liquid chromatography-mass spectrometer include: the chromatographic column is an ACE Excel C18-AR chromatographic column, the mobile phase is a mixed solution of methanol and water with the volume ratio of 40:60, the flow rate is 0.4-0.6 ml/min, and the column temperature is 25-40 ℃;
the mass spectrum conditions of the triple quaternary-liquid chromatograph include: atmospheric pressure chemical ionization source, adopting MRM positive ion mode detection, atomizing gas voltage 40-55 psi, temperature 400-550 ℃.
Specifically, the method for determining the content of genotoxic impurities in ligustrazine phosphate comprises the following steps:
(1) Setting the parameter conditions of liquid chromatography and mass spectrum of a triple quaternary-liquid chromatograph-mass spectrometer;
(2) Placing the prepared reference substance and the sample solution into a liquid phase small bottle, adopting direct sample injection, collecting data, and then carrying out analysis and test;
(3) After the analysis is finished, the concentration of genotoxic impurities in the solution is calculated according to the peak area measured in the sample solution by using a standard curve, and quantitative analysis is carried out.
The parameters of the liquid chromatograph of the triple quaternary-liquid chromatograph are as follows:
the chromatographic column is ACE Excel C18-AR chromatographic column, 4.6mm×150mm,3 μm;
the mobile phase is a mixed solution of methanol and water in a volume ratio of 40:60;
the flow rate is 0.5ml/min;
the column temperature is 35 ℃;
the sample injection amount is 5-10 mu l.
The parameters of the mass spectrum of the triple quaternary-liquid chromatograph-mass spectrometer are as follows:
in the mode of the MRM mode,
the ion pair of the 2,3,5, 6-tetramethylpyrazine-1-oxide is 153 to more than 135.9;
the ion pair of the 2,3,5, 6-tetramethylpyrazine-N, N-dioxide is 169 > 152.2;
the ion pair of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol is 169 > 151.9;
the scanning mode is a positive ion mode;
the atomizing gas voltage is 45psi;
the temperature is 500 ℃;
the declustering voltage of the 2,3,5, 6-tetramethylpyrazine-1-oxide is 110eV;
the declustering voltage of 2,3,5, 6-tetramethylpyrazine-N, N-dioxide was 170eV;
the declustering voltage of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol was 40eV;
the collision energy of the 2,3,5, 6-tetramethylpyrazine-1-oxide is 16eV;
the collision energy of 2,3,5, 6-tetramethylpyrazine-N, N-dioxide was 17eV;
the collision energy of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol was 15eV.
The standard curve is a regression curve of the concentration of each linear solution to the peak area by preparing the linear standard solution and sequentially injecting the linear standard solution into a liquid chromatography-mass spectrometer.
As a preferred embodiment, the standard curve can be prepared by the following method:
(1) Preparing mixed standard stock solution of genotoxic impurities with the concentration of 400ng/ml by taking a mobile phase as a solvent;
(2) Precisely weighing a proper amount of mixed standard stock solution of 400ng/ml respectively, and diluting with a mobile phase to prepare linear solutions of 4ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 60ng/ml and 80 ng/ml;
(3) Setting parameter conditions of liquid chromatography and mass spectrum, sequentially analyzing the linear solutions, and respectively making regression curves according to the concentration of each linear solution and the peak area.
Regression curve equation of genotoxic impurities is as follows:
2,3,5, 6-tetramethylpyrazine-1-oxide: y=2.99e 5 x+2.79e 5 ，r＝0.9996；
2,3,5, 6-tetramethylpyrazine-N, N-dioxide: y=6.85 e 4 x+5e 3 ，r＝1.0000；
(1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol: y=1.15e 4 x+1.9e 3 ，r＝0.9999。
The preparation method of the sample solution comprises the following steps: taking 40mg of a test sample, precisely weighing, placing into a 5ml measuring flask, adding a mobile phase for dissolution, diluting to a scale, and shaking uniformly to obtain a test sample solution.
Placing the prepared sample solution into a liquid phase small bottle, collecting data, calculating the concentration of genotoxic impurities in the solution according to peak area, and analyzing the content of genotoxic impurities by a standard curve method.
Compared with the prior art, the invention has the following advantages:
(1) The detection and determination method does not need pretreatment works such as extraction, concentration, purification, derivatization and the like, and the pretreatment work of the sample is simpler and can be carried out in one step;
(2) According to the invention, liquid chromatography-mass spectrometry (LC-MS/MS) is selected, and appropriate determination conditions are screened out, so that the determination of the toxic impurity content of the ligustrazine phosphate gene can be rapidly and accurately performed, the determination conditions are stable, 2,3,5, 6-tetramethylpyrazine-1-oxide, 2,3,5, 6-tetramethylpyrazine-N, N-dioxide and (1-oxo-2, 5, 6-trimethylpyrazine-3-yl) methanol are used as reference substances, and the determination is performed by an external standard method, so that the monitoring of the quality of ligustrazine phosphate is more comprehensive, and the effectiveness and consistency of the quality of products are ensured;
(3) The detection and measurement method of the invention reduces the pollution degree of the ion source of the mass spectrum detector and has relatively simple maintenance work.
Drawings
FIG. 1 is a graph of 2,3,5, 6-tetramethylpyrazine-1-oxide standards;
fig. 2 is a standard graph of 2,3,5, 6-tetramethylpyrazine-N, N-dioxide;
FIG. 3 is a graph of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol standard;
FIG. 4 is a diagram showing the specificity of 2,3,5, 6-tetramethylpyrazine-1-oxide;
fig. 5 is a diagram of the specificity of 2,3,5, 6-tetramethylpyrazine-N, N-dioxide;
FIG. 6 is a diagram showing the specificity of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol.
Detailed Description
The present invention will be described in detail with reference to specific examples.
The instrumentation used in the examples is as follows:
triple quaternary-liquid mass spectrometer, mass spectrum is SCIEX AB triple quaternary rod in America; the liquid phase is Shimadzu LC-20A.
The reagents, solutions used in the examples were as follows:
400ng/ml genotoxic impurity mixed standard stock solution prepared by taking mobile phase as solvent.
The analytical conditions parameters in the examples are as follows:
the parameters of the liquid chromatograph were: the chromatographic column is ACE Excel C18-AR chromatographic column, 4.6mm×150mm,3 μm; the mobile phase is a mixed solution of methanol and water in a volume ratio of 40:60; the flow rate is 0.5ml/min; the column temperature is 35 ℃; the sample loading was 10. Mu.l.
The parameters of the mass spectrum were as follows: adopting an MRM mode, wherein the ion pair of the 2,3,5, 6-tetramethylpyrazine-1-oxide is 153 to more than 135.9; the ion pair of the 2,3,5, 6-tetramethylpyrazine-N, N-dioxide is 169 > 152.2; the ion pair of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol is 169 > 151.9; the scanning mode is a positive ion mode; the atomization gas voltage is 45Psi; the temperature is 500 ℃;
the declustering voltage of the 2,3,5, 6-tetramethylpyrazine-1-oxide is 110eV; the declustering voltage of 2,3,5, 6-tetramethylpyrazine-N, N-dioxide was 170eV; the declustering voltage of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol was 40eV; the collision energy of the 2,3,5, 6-tetramethylpyrazine-1-oxide is 16eV; the collision energy of 2,3,5, 6-tetramethylpyrazine-N, N-dioxide was 17eV; the collision energy of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol was 15eV.
Example 1
Linear and range analysis:
the method comprises the following steps:
precisely weighing a proper amount of mixed standard stock solution of 400ng/ml respectively, and diluting with a mobile phase to prepare linear solutions of 4ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 60ng/ml and 80 ng/ml; and (3) sample injection detection, then sequentially analyzing the linear solutions, and respectively making regression curves according to the concentration of each linear solution and the peak area.
Discussion of results:
2,3,5, 6-tetramethylpyrazine-1-oxide standard curve as shown in fig. 1, y=2.99 e 5 x+2.79e 5 R= 0.9996, greater than 0.995.
The 2,3,5, 6-tetramethylpyrazine-N, N-dioxide standard curve is shown in fig. 2, y=6.85 e 4 x+5e 3 R=1.0000, greater than 0.995.
The (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol standard curve is shown in fig. 3: y=1.15e 4 x+1.9e 3 R=0.9999, greater than 0.995.
The genotoxic impurities are in good linear relation between 4 and 80 ng/m.
Example 2
Precision analysis:
the method comprises the following steps:
100% of standard test solution: taking 40mg of ligustrazine phosphate, precisely weighing, placing into a 5ml measuring flask, adding 40ng/ml of C 4 Dissolving the solution, fixing volume to scale, and shaking. 6 parts of the mixture were prepared in the same manner. .
Precisely measuring 10 μl of each solution, sequentially injecting into a liquid chromatograph-mass spectrometer, recording the retention time of the target peak, calculating RSD, and calculating the addition amount and RSD by standard curve method.
Discussion of results:
the results of the precision investigation of genotoxic impurities are shown in tables 1-3, respectively.
Table 1 results of examining precision of 2,3,5, 6-tetramethylpyrazine-1-oxide
| Name of the name | Retention time (min) | Peak area | Concentration (ng/ml) was measured |
| 1 | 6.64 | 11800000 | 38.35 |
| 2 | 6.63 | 11800000 | 38.65 |
| 3 | 6.64 | 11600000 | 37.98 |
| 4 | 6.63 | 11800000 | 38.64 |
| 5 | 6.64 | 12000000 | 39.13 |
| 6 | 6.62 | 12000000 | 39.12 |
| RSD(％) | 0.2 | — | 1.2 |
Table 2 results of examining the precision of 2,3,5, 6-tetramethylpyrazine-N, N-dioxide
| Name of the name | Retention time (min) | Peak area | Concentration (ng/ml) was measured |
| 1 | 4.34 | 2490000 | 36.25 |
| 2 | 4.33 | 2470000 | 36.04 |
| 3 | 4.33 | 2460000 | 35.93 |
| 4 | 4.33 | 2480000 | 36.10 |
| 5 | 4.33 | 2490000 | 36.25 |
| 6 | 4.32 | 2520000 | 36.74 |
| RSD(％) | 0.2 | — | 0.8 |
TABLE 3 precision investigation of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol results
Example 3
And (3) detection limit analysis:
the method comprises the following steps:
and respectively precisely measuring a proper amount of mixed standard stock solution of 400ng/ml, diluting with a mobile phase to prepare a detection limit solution of 2ng/ml, carrying out sample injection detection, and sequentially analyzing.
Discussion of results:
the results of the limit analysis of genotoxic impurities are shown in Table 4.
TABLE 4 detection limit analysis results
Example 4
Analysis of test article:
the method comprises the following steps:
respectively weighing 40mg of test samples of different batches, precisely weighing, placing into a 5ml measuring flask, adding mobile phase, dissolving, diluting to scale, and shaking. Placing the prepared sample solution into a liquid phase small bottle, collecting data, calculating the concentration of genotoxic impurities in the solution according to peak area, and analyzing the content of genotoxic impurities by a standard curve method.
Content (ppm) =measured concentration (ng/ml) ×diluted volume (ml)/weighed amount (mg) ×100%.
Discussion of results:
the results of analysis of the content of genotoxic impurities in the samples of different batches are shown in Table 5.
TABLE 5 analysis results of genotoxic impurity content in test sample
As can be seen from the above examples, the determination method of the invention can select liquid chromatography-mass spectrometry (LC-MS/MS) and screen out proper determination conditions, can rapidly and accurately determine the content of the genotoxic impurities of ligustrazine phosphate, has stable determination conditions, takes 2,3,5, 6-tetramethylpyrazine-1-oxide, 2,3,5, 6-tetramethylpyrazine-N, N-dioxide and (1-oxo-2, 5, 6-trimethylpyrazine-3-yl) methanol as reference substances, and performs determination by an external standard method, so that the quality of ligustrazine phosphate is monitored more comprehensively, the effectiveness and consistency of the product quality are ensured, the separation degree among genotoxic impurities is good, the specificity and precision are good, the detection limit is low, and the detection method is efficient and accurate.
Claims (9)
1. A method for determining the content of genotoxic impurities in ligustrazine phosphate is characterized by comprising the following steps of: determining the contents of three genotoxic impurities of 2,3,5, 6-tetramethylpyrazine-1-oxide, 2,3,5, 6-tetramethylpyrazine-N, N-dioxide and (1-oxo-2, 5, 6-trimethylpyrazine-3-yl) methanol in ligustrazine phosphate at one time by using a triple quaternary-liquid chromatography-mass spectrometer, and determining the contents of the genotoxic impurities by using a standard curve method;
the chromatographic conditions of the triple quaternary-liquid chromatography-mass spectrometer include: the chromatographic column is an ACE Excel C18-AR chromatographic column, the mobile phase is a mixed solution of methanol and water with the volume ratio of 40:60, the flow rate is 0.4-0.6 ml/min, and the column temperature is 25-40 ℃;
the mass spectrum conditions of the triple quaternary-liquid chromatograph include: atmospheric pressure chemical ionization source, adopting MRM positive ion mode detection, atomizing gas voltage 40-55 psi, temperature 400-550 ℃.
2. The method for determining the content of genotoxic impurities in ligustrazine phosphate according to claim 1, characterized in that: the method comprises the following steps:
(1) Setting the parameter conditions of liquid chromatography and mass spectrum of a triple quaternary-liquid chromatograph-mass spectrometer;
(2) Placing the prepared reference substance and the sample solution into a liquid phase small bottle, collecting data, and then performing measurement and test;
(3) After the measurement, the concentration of the genotoxic impurities in the solution is calculated according to the measured peak area in the sample solution by using a standard curve, and the quantitative measurement is carried out.
3. The method for determining the content of genotoxic impurities in ligustrazine phosphate according to claim 1 or 2, characterized in that: the parameters of the liquid chromatograph of the triple quaternary-liquid chromatograph are as follows:
the chromatographic column is ACE Excel C18-AR chromatographic column, 4.6mm×150mm,3 μm;
the mobile phase is a mixed solution of methanol and water in a volume ratio of 40:60;
the flow rate is 0.5ml/min;
the column temperature is 35 ℃;
the sample injection amount is 5-10 mu l.
4. The method for determining the content of genotoxic impurities in ligustrazine phosphate according to claim 1 or 2, characterized in that: the parameters of the mass spectrum of the triple quaternary-liquid mass spectrometer are as follows:
in the mode of the MRM mode,
the ion pair of the 2,3,5, 6-tetramethylpyrazine-1-oxide is 153 to more than 135.9;
the ion pair of the 2,3,5, 6-tetramethylpyrazine-N, N-dioxide is 169 > 152.2;
the ion pair of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol is 169 > 151.9;
the scanning mode is a positive ion mode;
the atomizing gas voltage is 45psi;
the temperature is 500 ℃;
the declustering voltage of the 2,3,5, 6-tetramethylpyrazine-1-oxide is 110eV;
the declustering voltage of 2,3,5, 6-tetramethylpyrazine-N, N-dioxide was 170eV;
the declustering voltage of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol was 40eV;
the collision energy of the 2,3,5, 6-tetramethylpyrazine-1-oxide is 16eV;
the collision energy of 2,3,5, 6-tetramethylpyrazine-N, N-dioxide was 17eV;
the collision energy of (1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol was 15eV.
5. The method for determining the content of genotoxic impurities in ligustrazine phosphate according to claim 2, characterized in that: the standard curve is a linear standard solution prepared by mixing three genotoxic impurities of 2,3,5, 6-tetramethylpyrazine-1-oxide, 2,3,5, 6-tetramethylpyrazine-N, N-dioxide and (1-oxo-2, 5, 6-trimethylpyrazine-3-yl) methanol, and the linear standard solution is sequentially injected into a liquid chromatograph, and a regression curve is formed by the concentration of each linear solution versus the peak area.
6. The method for determining the content of genotoxic impurities in ligustrazine phosphate according to claim 5, characterized in that: the standard curve is prepared by the following method:
(1) Preparing mixed standard stock solution of genotoxic impurities with the concentration of 400ng/ml by taking a mobile phase as a solvent;
(2) Precisely weighing a proper amount of mixed standard stock solution of 400ng/ml respectively, and diluting with a mobile phase to prepare linear solutions of 4ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 60ng/ml and 80 ng/ml;
(3) Setting parameter conditions of liquid chromatography and mass spectrum, sequentially measuring linear solutions, and respectively making regression curves according to the concentration of each linear solution and the peak area.
7. The method for determining the content of genotoxic impurities in ligustrazine phosphate according to claim 2 or 5 or 6, characterized in that: regression curve equation of genotoxic impurities is as follows:
2,3,5, 6-tetramethylpyrazine-1-oxide: y=2.99e 5 x+2.79e 5 ，r＝0.9996；
2,3,5, 6-tetramethylpyrazine-N, N-dioxide: y=6.85 e 4 x+5e 3 ，r＝1.0000；
(1-oxo-2, 5, 6-trimethylpyrazin-3-yl) methanol: y=1.15e 4 x+1.9e 3 ，r＝0.9999。
8. The method for determining the content of genotoxic impurities in ligustrazine phosphate according to claim 2, characterized in that: the preparation method of the sample solution comprises the following steps: taking 40mg of a test sample, precisely weighing, placing into a 5ml measuring flask, adding a mobile phase for dissolution, diluting to a scale, and shaking uniformly to obtain a test sample solution.
9. The method for determining the content of genotoxic impurities in ligustrazine phosphate according to claim 2 or 8, characterized in that: placing the prepared sample solution into a liquid phase small bottle, collecting data, calculating the concentration of genotoxic impurities in the solution according to peak area, and measuring the content of the genotoxic impurities by a standard curve method.
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