CN114487141A - Method for detecting genotoxic impurities in indobufen bulk drug - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
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- Life Sciences & Earth Sciences (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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Abstract
The invention discloses a method for detecting genotoxic impurities in indobufen bulk drugs. The method uses octadecylsilane chemically bonded silica or octylsilane chemically bonded silica as fixed relative genotoxic impurities for chromatographic separation, adopts a mass spectrometry detector for analysis and detection, can be used for detecting genotoxic impurities 2- (4-amino) phenylbutyric acid and/or 2- (4-nitro) phenylbutyric acid independently or simultaneously, can realize the independent or simultaneous effective separation of the genotoxic impurities 2- (4-amino) phenylbutyric acid and/or 2- (4-nitro) phenylbutyric acid, can also accurately determine the content of the genotoxic impurities, has strong specificity and high sensitivity, and has extremely important significance for realizing the quality control and safety guarantee of indobufen bulk drugs.
Description
Technical Field
The invention belongs to the field of pharmaceutical analytical chemistry, and particularly relates to a detection method for determining two genotoxic impurities, namely 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid, in an indobufen bulk drug, which is used for analyzing and determining the limit of the genotoxic impurities, namely 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid, in the indobufen bulk drug.
Background
Indobufen (Indobufen), an isoindolinylphenylbutyric acid derivative, an inhibitor of platelet aggregation, was first marketed in italy in 1984.
The chemical name is (+/-) 2- [4- (1-oxo-2-isoindolinyl) phenyl]Butyric acid of formula C18H17NO3Accurate molecular weight 295.12, formula shown below:
indobufen exerts an anti-platelet aggregation effect mainly through the following mechanisms: (1) reversibly inhibiting platelet cyclooxygenase to reduce thromboxane B2 (potent activator of platelet aggregation); (2) inhibiting platelet aggregation induced by Adenosine Diphosphate (ADP), epinephrine, and Platelet Activating Factor (PAF), collagen, and arachidonic acid; (3) reduce the levels of platelet adenosine triphosphate, serotonin, platelet factor 3, platelet factor 4 and beta-thromboglobulin, and reduce platelet adhesion. Indobufen is a reversible multi-target antithrombotic drug. For activator-induced platelet aggregation, the maximum inhibition effect can be achieved 2 hours after 200mg of indobufen is orally taken once, and the significant inhibition effect (90%) still remains after 12 hours, and the platelet aggregation is recovered within 24 hours.
For the most reported synthetic routes of indobufen involving 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid, patent CN106631974B discloses the following synthetic routes:
(1) hydrogenation reaction
(2) Cyclization reaction:
(3) zinc powder reduction reaction
The process route takes 2- (4-nitro) phenylbutyric acid as a starting material to obtain a key intermediate, and the 2- (4-aminophenyl) butyric acid and the 2- (4-nitrophenyl) butyric acid contain a genotoxic warning functional group, belong to genotoxic impurities and have the risk of residue in a finished product. The maximum daily intake of 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid should not exceed 1.5 μ g per the relevant guidelines of the ICHM7 guidelines combined with the maximum daily dose of the indobufen formulation (where indobufen is 400mg), so that the control limits of 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid in the indobufen drug substance should not exceed 3.75 ppm. However, the detection is difficult due to the lower limit, and no literature reports a method for detecting genotoxic impurities, namely 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid.
Therefore, a method for detecting genotoxic impurity 2- (4-amino) phenylbutyric acid and/or 2- (4-nitro) phenylbutyric acid with high sensitivity and good specificity is found, the control of the impurity in the indobufen is realized, and the method has very important significance for improving the quality of products and the safety of medication.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a method for measuring genotoxic impurities, namely 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid, which can effectively realize the measurement and limit control of the genotoxic impurities, namely 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid, in an indobufen raw material drug. The detection method has the advantages of high sensitivity, strong specificity and simple and rapid operation.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for detecting genotoxic impurities in indobufen bulk drug is characterized in that octadecylsilane chemically bonded silica or octylsilane chemically bonded silica is used as fixed relative genotoxic impurities for chromatographic separation, and a mass spectrometer is used for analysis and detection; the mobile phase is a mixture of a solvent A and a solvent B, the solvent A is selected from an aqueous solution system containing volatile acid, and the solvent B is selected from any one of methanol, acetonitrile or a mixture of the methanol and the acetonitrile.
Further, the genotoxic impurity is 2- (4-amino) phenylbutyric acid.
Further, the pH value of the mobile phase is controlled to be 2-7, preferably 2-3.
Further, preferably, the solvent A is selected from an aqueous solution containing any one of formic acid, acetic acid or a mixture of the two; still further, the volume concentration of the solvent A is preferably 0.01-1%; furthermore, the solvent A is preferably acetic acid water solution with the volume concentration of 0.01-1%.
Further, the mobile phase elution adopts gradient elution, and the gradient elution conditions are as follows: isocratic at 0.01-1 min, the volume ratio of the solvent A to the solvent B is 90: 10-70: 30, isocratic at 2-4 min, the volume ratio of the solvent A to the solvent B is 10: 90-5: 95, isocratic at 4.1-5 min, and the volume ratio of the solvent A to the solvent B is 90: 10-70: 30.
Further, the flow rate of the mobile phase is 0.3-0.5 mL/min.
Further, preferably, the specification of the column is 2.1X 50mm,1.7 μm or 4.6X 50mm, 1.8 μm or 4.6X 50mm, 2.7. mu.m.
Further, the method specifically comprises the following steps: dissolving and diluting a test sample to prepare a solution with the concentration of about 0.5-1 mg/mL as a test sample solution; dissolving and diluting genotoxic impurity 2- (4-amino) phenylbutyric acid to prepare a solution with the concentration of about 1.875-3.75 ng/mL, and taking the solution as a reference solution; respectively taking 5-20 mul of sample solutions of a test sample and a reference substance, respectively injecting samples, respectively recording ion flow diagrams under quantitative and qualitative ion pairs, and calculating the impurity content by the peak area of the quantitative ion pairs according to an external standard method; the diluent is the initial mobile phase.
The invention also discloses a detection method of genotoxic impurities in the indobufen bulk drug, which uses octadecylsilane chemically bonded silica or octylsilane chemically bonded silica as fixed relative genotoxic impurities to carry out chromatographic separation and adopts a mass spectrometer to carry out analysis and detection; the mobile phase is a mixture of a solvent C and a solvent D, the solvent C is selected from a volatile salt aqueous solution system, and the solvent D is selected from any one of methanol, acetonitrile or a mixture of the methanol and the acetonitrile.
Furthermore, the genotoxic impurity is 2- (4-amino) phenylbutyric acid and/or 2- (4-nitro) phenylbutyric acid.
Further, the pH value of the mobile phase is controlled to be between 3 and 7, and preferably between 6 and 7.
Further, the solvent C is one or a mixed aqueous solution of any several of ammonium formate and ammonium acetate; further, the concentration of the solvent C is preferably 5-50 mmol/L; still further, the solvent C is preferably 5-50 mmol/L ammonium acetate water solution.
Further, when the method is used for separately detecting the genotoxic impurity 2- (4-amino) phenylbutyric acid, gradient elution is adopted for the mobile phase elution, and the elution conditions are as follows: when 0.01min is reached, the volume ratio of the solvent C to the solvent D is 85: 15-70: 30; isocratic for 2-3 min, wherein the volume ratio of the solvent C to the solvent D is 30: 70-15: 85; isocratic at 3.1-5 min, and the volume ratio of the solvent C to the solvent D is 85: 15-70: 30.
Further, when the method is used for independently detecting the genotoxic impurity 2- (4-amino) phenylbutyric acid, the flow rate of the mobile phase is 0.9-1.1 mL/min.
Further, when the method is used for detecting the genotoxic impurity 2- (4-amino) phenylbutyric acid alone, the specification of the chromatographic column is 4.6 × 150mm,5 μm or 2.1 × 150mm,1.7 μm or 2.1 × 150mm,1.6 μm or 2.1 × 150mm,1.9 μm or 4.6 × 150mm,2.7 μm or 4.6 × 150mm,2.5 μm or 4.6 × 150mm,3.5 μm or 4.6 × 150mm and 3.0 μm.
Further, when the method is used for separately detecting the genotoxic impurity 2- (4-amino) phenylbutyric acid, the method specifically comprises the following steps: dissolving and diluting a test sample to prepare a solution with the concentration of about 5-10 mg/mL as a test sample solution; dissolving and diluting genotoxic impurity 2- (4-amino) phenylbutyric acid to prepare a solution with the concentration of about 18.75-37.5 ng/mL, and taking the solution as a reference solution; respectively taking 5-20 mul of sample solutions of a test sample and a reference substance, respectively injecting samples, respectively recording ion flow diagrams under quantitative and qualitative ion pairs, and calculating the impurity content by the peak area of the quantitative ion pairs according to an external standard method; the diluent is the initial mobile phase.
Further, when the method is used for separately detecting the genotoxic impurity 2- (4-nitro) phenylbutyric acid, gradient elution is adopted for the mobile phase elution, and the elution conditions are as follows: isocratic at 0.01-1 min, the volume ratio of the solvent C to the solvent D is 90: 10-70: 30, isocratic at 2-4 min, the volume ratio of the solvent C to the solvent D is 10: 90-5: 95, isocratic at 4.1-5 min, and the volume ratio of the solvent C to the solvent D is 90: 10-70: 30.
Further, when the method is used for independently detecting the genotoxic impurity 2- (4-nitro) phenylbutyric acid, the flow rate of the mobile phase is 0.3-0.5 mL/min.
Furthermore, when the method is used for separately detecting the genotoxic impurity 2- (4-nitro) phenylbutyric acid, the specification of the chromatographic column is 2.1 multiplied by 50mm,1.7 mu m or 4.6 multiplied by 50mm, 1.8 mu m or 4.6 multiplied by 50mm, and 2.7 mu m.
Further, when the method is used for separately detecting the genotoxic impurity 2- (4-nitro) phenylbutyric acid, the method specifically comprises the following steps: dissolving and diluting a test sample to prepare a solution with the concentration of about 5-10 mg/mL as a test sample solution; dissolving and diluting genotoxic impurity 2- (4-nitro) phenylbutyric acid to prepare a solution with the concentration of about 18.75-37.5 ng/mL, and taking the solution as a reference solution; respectively taking 2-5 mul of sample solutions of a test sample and a reference substance, respectively injecting samples, respectively recording ion flow diagrams under quantitative and qualitative ion pairs, and calculating the impurity content by the peak area of the quantitative ion pairs according to an external standard method; the diluent is any one of a mobile phase or an organic solvent in the mobile phase.
Further, when the method simultaneously detects genotoxic impurities as 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid, the mobile phase elution adopts gradient elution, and the elution conditions are as follows: when 0.01min is reached, the volume ratio of the solvent C to the solvent D is 90: 10-85: 15; isocratic within 5-6 min, wherein the volume ratio of the solvent C to the solvent D is 30: 70-25: 75; isocratic at 6.1-10 min, and the volume ratio of the solvent C to the solvent D is 90: 10-85: 15.
Further, when the method is used for simultaneously detecting genotoxic impurities 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid, the flow rate of the mobile phase is 0.8-1.2 mL/min.
Further, when the method simultaneously detects 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid, the size of the chromatographic column is 4.6 × 150mm,5 μm or 2.1 × 150mm,1.7 μm or 2.1 × 150mm,1.6 μm or 2.1 × 150mm,1.9 μm or 4.6 × 150mm,2.7 μm or 4.6 × 150mm,2.5 μm or 4.6 × 150mm,3.5 μm or 4.6 × 150mm,3.0 μm.
Further, when the method simultaneously detects genotoxic impurities 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid, the method specifically comprises the following steps: dissolving and diluting a test sample to prepare a solution with the concentration of about 5-10 mg/mL as a test sample solution; dissolving genotoxic impurities, namely- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid, and diluting to prepare a solution with the concentration of about 18.75-37.5 ng/mL, wherein the solution is used as a reference solution; respectively taking 10-20 mul of sample solutions of a test sample and a reference substance, respectively injecting samples, respectively recording ion flow diagrams under quantitative and qualitative ion pairs, and calculating the impurity content by the peak area of the quantitative ion pairs according to an external standard method; the diluent is any one of a mobile phase or an organic solvent in the mobile phase.
The detectors of the method are mass spectrometry detectors, an electrospray ionization source (ESI) and an MRM detection mode are adopted, a positive ion scanning mode is adopted for detecting genotoxic impurity 2- (4-aminophenyl) butyric acid, a negative ion scanning mode is adopted for detecting genotoxic impurity 2- (4-nitrophenyl) butyric acid, qualitative and quantitative analysis are carried out by adopting respective special ion pairs according to the chemical structures and the detection reality of daughter ions of genotoxic impurity 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid, and each specific quantitative ion pair and qualitative ion pair are as follows:
| genotoxic impurities | Quantitative ion pair | Qualitative ion pair |
| 2- (4-aminophenyl) butanoic acid | 180→106 | 180→134；180→93 |
| 2- (4-Nitrophenyl) butanoic acid | 164→149 | 164→119；164→93； |
Further, in the method, the temperature of the chromatographic column incubator is 20-50 ℃. Compared with the prior art, the invention has the beneficial effects that:
the invention provides three analysis methods for the separate or simultaneous determination of genotoxic impurities 2- (4-aminophenyl) butyric acid and 2- (4-nitro) phenylbutyric acid. The genotoxic impurities are nitrobenzene and aniline. The three methods can realize quantitative determination of genotoxic impurities, namely 2- (4-aminophenyl) butyric acid and 2- (4-nitro) phenylbutyric acid, in the indobufen bulk drug, and have the advantages of strong specificity, high accuracy and high sensitivity (the limit of quantitation is not more than 1/3 and the limit of detection is not more than 1/10); meanwhile, the method is simple, convenient and quick in experimental operation, and has important significance for quality control and medication safety guarantee of the indobufen.
Drawings
FIG. 1 is an HPLC-MS spectrum of a control solution of 2- (4-aminophenyl) butyric acid as a genotoxic impurity in example 1 (wherein A is an MRM total ion flux spectrum and B is an MRM 180- >106 extraction ion flux spectrum);
FIG. 2 is an HPLC-MS spectrum of the sample solution of Z191212 lot in example 1 (wherein A is MRM total ion current spectrum, and B is MRM 180- >106 extraction ion current spectrum);
FIG. 3 is an HPLC-MS spectrum of a control solution of 2- (4-nitrophenyl) butyric acid, a genotoxic impurity in example 3 (wherein A is an MRM total ion flux spectrum and B is an MRM 164- >149 extraction ion flux spectrum);
FIG. 4 is an HPLC-MS spectrum of the sample solution of Z191212 lot in example 3 (wherein A is MRM total ion current spectrum and B is MRM 164- >149 extract ion current spectrum);
FIG. 5 is an HPLC-MS spectrum of a control solution of 2- (4-aminophenyl) butyric acid as a genotoxic impurity in example 5 (wherein A is an MRM total ion flux spectrum and B is an MRM 180- >106 extraction ion flux spectrum);
FIG. 6 is an HPLC-MS spectrum of the sample solution of Z191212 lot in example 5 (wherein A is MRM total ion current spectrum and B is MRM 180- >106 extract ion current spectrum);
FIG. 7 is an HPLC-MS spectrum of detection limit test of 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid as genotoxic impurities in example 7 (wherein A is MRM total ion flux spectrum, B is MRM 180- >106 extraction ion flux spectrum, and C is MRM 164- >149 extraction ion flux spectrum);
FIG. 8 is an HPLC-MS spectrum of the quantitative limit test for the genotoxic impurity 2- (4-aminophenyl) butyric acid, 2- (4-nitrophenyl) butyric acid in example 7 (wherein A is an MRM total ion flux pattern, B is an MRM 180- >106 extraction ion flux pattern, and C is an MRM 164- >149 extraction ion flux pattern);
FIG. 9 is an HPLC-MS spectrum of a control solution of the genotoxic impurity 2- (4-nitrophenyl) butyric acid, 2- (4-nitrophenyl) butyric acid mixture of example 7 (wherein A is the MRM total ion flux spectrum, B is the MRM 180- >106 extraction ion flux spectrum, and C is the MRM 164- >149 extraction ion flux spectrum);
FIG. 10 is an HPLC-MS spectrum of the sample solution of Z191212 lot in example 7 (wherein A is MRM total ion current spectrum, B is MRM 180- >106 extract ion current spectrum, and C is MRM 164- >149 extract ion current spectrum).
Detailed Description
The invention will be further elucidated with reference to specific embodiments with reference to the drawing. The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
Sources of controls referred to in the examples:
genotoxic impurity 2- (4-aminophenyl) butyric acid, Shenzhen Dingli Biotech Limited, purity: 98.17%, batch number: DIF 18211-01;
genotoxic impurity 2- (4-nitrophenyl) butyric acid, Shenzhen Dingli Biotech Limited, purity: 99.72%, batch number: DIF 18210-0.
EXAMPLE 1 separation and determination of 2- (4-aminophenyl) butyric acid, a genotoxic impurity, in indobufen starting material by LC-MS
(1) Chromatographic conditions
The instrument comprises the following steps: agilent 6470 type high performance liquid chromatography triple quadrupole mass spectrometer
A chromatographic column: ACQUITY BEH C182.1mm X50 mm,1.7 μm
Mobile phase: solvent A: 0.01% aqueous acetic acid; solvent B: acetonitrile, elution with the following gradient:
diluent agent: acetonitrile-water (50:50)
Flow rate: 0.3mL/min
Column temperature: 35 deg.C
Sample introduction volume: 5 μ l
Mass spectrum conditions: using electrospray ion source, positive ion scan mode
The dry gas, the atomization gas and the sheath gas are all nitrogen; the collision gas is high-purity nitrogen;
the temperature of the drying gas is: 300 deg.C
The drying airflow rate is: 6L/min
Atomizing gas pressure 35psi
Temperature of sheath gas: 350 deg.C
Flow rate of sheath gas: 12L/min
Capillary voltage: +4000V
Nozzle voltage: +500V
MRM detection mode: quantitative ion pair 180 → 106, fragmentation voltage (Frag)115V, collision voltage (CE)28V, residence time (Dwell)170ms, acceleration voltage (Cell Acc) 3V;
qualitative ion pair 180 → 134, fragmentation voltage (Frag)115V, collision voltage (CE)18, residence time (Dwell)170ms, acceleration voltage (Cell Acc) 3V;
qualitative ion pair 180 → 93, fragmentation voltage (Frag)115V, collision voltage (CE) 28; dwell time (Dwell)170ms, acceleration voltage (Cell Acc) 3V;
(2) methodology survey
Checking the impurity limit according to a verification guiding principle of an analysis method of China pharmacopoeia 2020, year edition 9101, and investigating the specificity, detection limit and quantitative limit of the method;
(3) solution preparation
Control stock solution: accurately weighing 10.08mg of reference substance, placing in a 100mL volumetric flask, dissolving with acetonitrile-water (50:50), diluting to scale, and shaking to obtain the final product with concentration of 98.96 μ g/mL;
control solution: taking a proper amount of stock solution, diluting with diluent to a limit concentration (3.75ng/mL), and shaking up to obtain the final product;
quantitative limit and detection limit solution: taking a proper amount of reference solution, respectively placing in 100mL volumetric flasks, diluting to scale with diluent, shaking up, the concentration is respectively 30%, 25%, 10%, 5%, 3% of the limit concentration;
test solution: accurately weighing 100.2, 100.1 and 100.2mg of three batches of samples of indobufen raw materials, respectively placing in a 100mL volumetric flask, dissolving and diluting to scale with acetonitrile-water (50:50), filtering at 0.45 μm, and collecting the subsequent filtrate;
adding a standard test solution: accurately weighing 100.2mg of indobufen raw material sample (Z200203 batches), placing the indobufen raw material sample in a 100mL volumetric flask, dissolving and diluting the indobufen raw material sample to a scale with a reference substance solution, filtering the indobufen raw material sample by 0.45 mu m, and taking a subsequent filtrate to obtain the indobufen raw material sample;
(4) measurement method
The specificity determination method comprises the following steps: respectively injecting a diluent blank, a reference solution, a test solution and a standard test solution, wherein the blank solution is free of interference, and the sample and impurities in the sample are free of interference;
the method for measuring the quantitative limit and the detection limit comprises the following steps: respectively sampling a diluent blank and a test solution, recording a chromatogram, and calculating the signal-to-noise ratio of a main peak in the MRM 180 → 106 chromatogram, wherein the corresponding concentration is a detection limit when the signal-to-noise ratio is about 3, and the corresponding concentration is a quantification limit when the signal-to-noise ratio is about 10;
the method for measuring the residual amount of the 2- (4-aminophenyl) butyric acid in the sample comprises the following steps: respectively injecting 6 parts of diluent blank and reference substance solution, recording a chromatogram, extracting that RSD% of main peak area change in the chromatogram cannot exceed 20.0% by MRM 180 → 106, and calculating the residual amount of 2- (4-aminophenyl) butyric acid in the sample by peak area according to an external standard method;
(5) measurement results
The specificity is as follows: the peak of 2- (4-aminophenyl) butyric acid can achieve baseline separation under the condition, the blank solution has no interference, and the sample and impurities in the sample have no interference; the quantitative limit and detection limit test data are shown in table 1:
TABLE 1 quantitative Limit, detection Limit test data
The test data for 3 batches of raw material samples are shown in table 2:
table 23 raw material sample test data
The result of methodology investigation and sample determination shows that the method has good specificity and high sensitivity, and can be used for detecting the impurities in the sample. The typical control solution pattern is shown in figure 1, and the typical sample solution pattern is shown in figure 2 (batch Z191212).
Example 2
(1) The same conditions as in example 1 were used except for the column and elution gradient, ZORBAX SB-C83.0X 100mm,1.8 μm, and the elution gradient was:
| time (min) | 0.01 | 1 | 3 | 3.1 | 5 |
| Solvent A (%) | 90 | 90 | 10 | 90 | 90 |
| Solvent B (%) | 10 | 10 | 90 | 10 | 10 |
(2) Solution preparation and measurement methods were the same as in example 1
(3) Sample introduction and result analysis
Taking diluent blank, reference solution (with concentration of 30% of limit, 1.125ng/mL) and standard sample solution for sample injection, and recording chromatogram. The result shows that the peak of the 2- (4-aminophenyl) butyric acid can achieve baseline separation under the condition, the blank solution has no interference, and the sample and impurities in the sample have no interference; the S/N is 16.9 and is more than 10. The specificity and the sensitivity of the method can meet the requirements of the determination and limit formulation of the impurities in the sample, and the method is feasible.
Comparative example 1 influence of different mobile phase systems on the determination of sensitivity of 2- (4-aminophenyl) butyric acid, a genotoxic impurity in indobufen raw material, by means of liquid chromatography-mass spectrometry
(1) The chromatographic conditions were the same as in example 1 except for the mobile phase composition;
(2) the following two mobile phases were set to examine the effect on the sensitivity of the method:
(3) solution preparation the same as example 1;
(4) sample introduction and result analysis
Separately sampling diluent blank and reference solution (with concentration of 30% of limit, 1.125ng/mL), respectively performing gradient elution with the two mobile phases, and recording chromatogram. As a result, the peak was tailing and S/N was 7.9, 3.7 and less than 10, respectively. With both mobile phase assays, the sensitivity of the method does not meet the requirements for the determination of the impurities in the sample and for the limitation.
EXAMPLE 3 separation and determination of 2- (4-Nitrophenyl) butyric acid, a genotoxic impurity, in Indobufen raw Material by Liquid-Mass Spectrometry
(1) Chromatographic conditions
The instrument comprises the following steps: agilent 6470 type high performance liquid chromatography triple quadrupole mass spectrometer
A chromatographic column: ACQUITY BEH C182.1mm X50 mm,1.7 μm
Mobile phase: solvent A: 20mmol/L ammonium acetate solution; and (3) solvent B: acetonitrile, elution with the following gradient:
| time (min) | 0.01 | 1 | 2 | 4 | 4.1 | 5 |
| Solvent A (%) | 90 | 90 | 10 | 10 | 90 | 90 |
| Solvent B (%) | 10 | 10 | 90 | 90 | 10 | 10 |
Diluent agent: methanol
Flow rate: 0.3mL/min
Column temperature: 35 deg.C
Sample introduction volume: 2 μ l
Mass spectrum conditions: using electrospray ion source, negative ion scanning mode
The dry gas, the atomization gas and the sheath gas are all nitrogen; the collision gas is high-purity nitrogen;
the temperature of the drying gas is: 300 deg.C
The drying airflow rate is: 6L/min
Atomizing gas pressure 35psi
Temperature of sheath gas: 350 deg.C
Flow rate of sheath gas: 12L/min
Capillary voltage: -3500V
Nozzle voltage: -1000V
MRM detection mode: quantitative ion pair 164 → 149, fragmentation voltage (Frag)90V, collision voltage (CE)11V, residence time (Dwell)200ms, acceleration voltage (Cell Acc) 3V;
qualitative ion pair 164 → 119, fragmentation voltage (Frag)90V, collision voltage (CE)11, residence time (Dwell)200ms, acceleration voltage (Cell Acc) 3V;
qualitative ion pair 164 → 93, fragmentation voltage (Frag)90V, collision voltage (CE) 17; dwell time (Dwell)200ms, acceleration voltage (Cell Acc) 3V;
(2) methodology investigation
Checking the impurity limit according to a verification guiding principle of an analysis method of China pharmacopoeia 2020, year edition 9101, and investigating the specificity, detection limit and quantitative limit of the method;
(3) solution preparation
Control stock solution: accurately weighing 10.02mg of a reference substance, placing the reference substance in a 100mL volumetric flask, dissolving the reference substance with methanol, diluting the reference substance to a scale, and shaking up to obtain the product with the concentration of 99.92 mu g/mL;
control solution: taking a proper amount of stock solution, diluting with diluent to a limit concentration (18.75ng/mL), and shaking up to obtain the final product;
quantitative limit and detection limit solution: taking a proper amount of reference solution, respectively placing in 100mL volumetric flasks, diluting to scale with diluent, shaking up, the concentration is respectively 30%, 25%, 10%, 5%, 3% of the limit concentration;
test solution: precisely weighing three batches of samples (50.31 mg, 50.12 mg and 50.25 mg) of indobufen raw material, respectively placing in 10mL volumetric flasks, dissolving with methanol and diluting to scale, filtering with a filter of 0.45 μm, and collecting the subsequent filtrate to obtain a test solution;
adding a standard test solution: accurately weighing 50.22mg (Z200203 batches) of indobufen raw material samples, placing the indobufen raw material samples into a 10mL volumetric flask, dissolving the indobufen raw material samples by using a reference substance solution, diluting the indobufen raw material samples to a scale, and shaking up the indobufen raw material samples to obtain the indobufen liquid;
(4) measurement method
The specificity determination method comprises the following steps: respectively injecting a diluent blank, a reference solution, a test solution and a standard test solution, wherein the blank solution is free of interference, and the sample and impurities in the sample are free of interference;
the method for measuring the quantitative limit and the detection limit comprises the following steps: respectively sampling diluent blank and test solution, recording a chromatogram, calculating the signal-to-noise ratio of a main peak in the MRM 164 → 149 extraction chromatogram, wherein the corresponding concentration is a detection limit when the signal-to-noise ratio is about 3, and the corresponding concentration is a quantification limit when the signal-to-noise ratio is about 10;
the method for measuring the residual amount of the 2- (4-nitrophenyl) butyric acid in the sample comprises the following steps: and respectively injecting 6 parts of diluent blank and a reference substance solution, recording a chromatogram, wherein the RSD% of the peak area change of the main peak in the MRM 164 → 149 chromatogram is not more than 20.0%, and calculating the residual amount of the 2- (4-nitrophenyl) butyric acid in the sample by the peak area according to an external standard method.
(5) Measurement results
The specificity is as follows: the peak of 2- (4-nitrophenyl) butyric acid can achieve baseline separation under the condition, the blank solution is free from interference, and the sample and impurities in the sample are free from interference; the quantitative limit and detection limit test data are shown in a table 3;
TABLE 3 quantitative limit, detection limit test data
The test data of 3 batches of raw material samples are shown in Table 4
TABLE 4 quantitative limit, detection limit test data
The result of methodology investigation and sample determination shows that the method has good specificity and high sensitivity, and can be used for detecting the impurities in the sample. The typical control solution profile is shown in FIG. 3, and the typical sample solution profile is shown in FIG. 4 (batch Z191212).
Example 4
(1) The same conditions as in example 3 were used except for the column containing ZORBAX SB-C83.0X 100mm at 1.8 μm and the elution gradient:
| time (min) | 0.01 | 1 | 3.5 | 3.6 | 5 |
| Solvent A (%) | 90 | 90 | 10 | 90 | 90 |
| Solvent B (%) | 10 | 10 | 90 | 10 | 10 |
(2) Solution preparation and measurement methods were the same as in example 3
(3) Sample introduction and result analysis
Taking diluent blank, reference solution (with concentration of 30% of limit, 5.63ng/mL) and standard sample solution for sample injection, and recording chromatogram. The result shows that the peak of the 2- (4-nitrophenyl) butyric acid can achieve baseline separation under the condition, the blank solution is free from interference, and the sample and impurities in the sample are free from interference; the S/N is 15.2 and is more than 10. The specificity and the sensitivity of the method can meet the requirements of the determination and limit formulation of the impurities in the sample, and the method is feasible.
Comparative example 2
Influence of different mobile phase systems on sensitivity of separating and determining genotoxic impurity 2- (4-nitrophenyl) butyric acid in indobufen raw material by liquid chromatography-mass spectrometry
(1) The chromatographic conditions were the same as in example 3 except for the mobile phase composition
(2) The following two mobile phases were set to examine the influence on the sensitivity of the method
(3) solution preparation the same as in example 3
(4) Sample introduction and result analysis
Separately sampling diluent blank and reference solution (with concentration of 30% of limit, 5.63ng/mL), respectively performing gradient elution with the two mobile phases, and recording chromatogram. As a result, the peaks were smeared and S/N was 4.2, 6.8 and less than 10, respectively. With both mobile phase assays, the sensitivity of the method does not meet the requirements for the determination of the impurities in the sample and for the limitation.
EXAMPLE 5 separation and determination of the genotoxic impurity 2- (4-aminophenyl) butyric acid in Indobufen raw Material by Liquid chromatography-Mass Spectrometry
(1) Chromatographic conditions
The instrument comprises the following steps: agilent 6470 type high performance liquid chromatography triple quadrupole mass spectrometer
A chromatographic column: ACQUITY BEH C182.1mm X50 mm,1.7 μm
Mobile phase: solvent A: 20mmol/L ammonium acetate solution; solvent B: methanol, elution was performed with the following gradient:
diluent agent: 20mmol/L ammonium acetate-methanol (85:15) solution
Flow rate: 1.0mL/min
Column temperature: 35 deg.C
Sample introduction volume: 20 μ l
Mass spectrum conditions: using electrospray ion source, positive ion scan mode
The drying gas, the atomizing gas and the sheath gas are all nitrogen; the collision gas is high-purity nitrogen;
the temperature of the drying gas is: 300 deg.C
The drying airflow rate is: 6L/min
Atomizing gas pressure 35psi
Temperature of sheath gas: 350 deg.C
Flow rate of sheath gas: 12L/min
Capillary voltage: +4000V
Nozzle voltage: +500V
MRM detection mode:
quantitative ion pair 180 → 106, fragmentation voltage (Frag)119V, collision voltage (CE)30V, residence time (Dwell)180ms, acceleration voltage (Cell Acc) 3V;
qualitative ion pair 180 → 134, fragmentation voltage (Frag)115V, collision voltage (CE)20, residence time (Dwell)180ms, acceleration voltage (Cell Acc) 3V;
qualitative ion pair 180 → 93, fragmentation voltage (Frag)118V, collision voltage (CE) 32; residence time (Dwell)180ms, acceleration voltage (Cell Acc) 3V;
(2) methodology investigation
Checking the impurity limit according to a verification guiding principle of an analysis method of China pharmacopoeia 2020, year edition 9101, and investigating the specificity, detection limit and quantitative limit of the method;
(3) solution preparation
Control stock solution: accurately weighing 10.03mg of a reference substance, placing the reference substance in a 100mL volumetric flask, dissolving the reference substance in methanol, diluting the reference substance to a scale, and shaking up to obtain the product with the concentration of 98.46 mu g/mL;
control solution: taking a proper amount of stock solution, diluting with diluent to a limit concentration (37.5ng/mL), and shaking up to obtain the final product;
quantitative limit and detection limit solution: taking a proper amount of reference solution, respectively placing the reference solution in 100mL volumetric flasks, diluting the reference solution to the scale with a diluent, and shaking the reference solution uniformly, wherein the concentrations are respectively 30%, 25%, 10%, 5% and 3% of the limit concentration;
test solution: accurately weighing 100.8, 100.2 and 100.5mg of three batches of samples of indobufen raw materials, respectively placing the samples into 10mL volumetric flasks, dissolving the samples with 5mL of DMSO (dimethyl sulfoxide) and diluting the samples with a diluent to a scale, filtering the samples with the thickness of 0.45 mu m, and taking subsequent filtrate to obtain a sample solution;
adding a standard test solution: accurately weighing 100.2mg (Z200203 batches) of indobufen raw material samples, placing the indobufen raw material samples into a 10mL volumetric flask, dissolving the indobufen raw material samples in 5mL of DMSO (dimethyl sulfoxide), adding a proper amount of reference substance stock solution, diluting the indobufen raw material samples to a scale with a diluent, filtering the indobufen raw material samples by 0.45 mu m, and taking subsequent filtrate to obtain a test sample solution, wherein the concentration of 2- (4-aminophenyl) butyric acid is the limit concentration (37.5 ng/mL);
(4) measurement method
The specificity determination method comprises the following steps: respectively injecting a diluent blank, a reference solution, a test solution and a standard test solution, wherein the blank solution is free of interference, and the sample and impurities in the sample are free of interference;
the method for measuring the quantitative limit and the detection limit comprises the following steps: respectively sampling diluent blank and test solution, recording a chromatogram, calculating the signal-to-noise ratio of a main peak in the MRM 180 → 106 extraction chromatogram, wherein the corresponding concentration is a detection limit when the signal-to-noise ratio is about 3, and the corresponding concentration is a quantification limit when the signal-to-noise ratio is about 10;
the method for measuring the residual amount of the 2- (4-aminophenyl) butyric acid in the sample comprises the following steps: and respectively injecting 6 parts of diluent blank and a reference substance solution, recording a chromatogram, extracting RSD% of main peak area change in the chromatogram from MRM 180 → 106 to be not more than 20.0%, and calculating the residual amount of the 2- (4-aminophenyl) butyric acid in the sample by peak area according to an external standard method.
(5) Measurement results
The specificity is as follows: the peak of 2- (4-aminophenyl) butyric acid can achieve baseline separation under the condition, the blank solution has no interference, and the sample and impurities in the sample have no interference; the quantitative limit and detection limit test data are shown in a table 5;
TABLE 5 quantitative limit, detection limit test data
The test data of 3 batches of raw material samples are shown in Table 6
Table 63 raw material sample test data
The result of methodology investigation and sample determination shows that the method has good specificity and high sensitivity, and can be used for detecting the impurities in the sample. The pattern of a typical control solution is shown in FIG. 5, and the pattern of a typical sample solution is shown in FIG. 6 (lot Z191212).
Example 6(1) the chromatography conditions were the same as in example 5 except for the column of ZORBAX SB-C83.0X 100mm,1.8 μm, and the elution gradient:
| time (min) | 0.01 | 2 | 3.5 | 3.6 | 5 |
| Solvent A (%) | 70 | 15 | 15 | 70 | 70 |
| Solvent B (%) | 30 | 85 | 85 | 30 | 30 |
(3) Solution preparation and measurement methods were the same as in example 3
(3) Sample introduction and result analysis
Taking diluent blank, reference solution (with concentration of 30% of limit, 11.25ng/mL) and standard sample solution for sample injection, and recording chromatogram. The result shows that the peak of the 2- (4-aminophenyl) butyric acid can achieve baseline separation under the condition, the blank solution has no interference, and the sample and impurities in the sample have no interference; the S/N is 21.2 and is more than 10. The specificity and the sensitivity of the method can meet the requirements of the determination and limit formulation of the impurities in the sample, and the method is feasible.
Comparative example 3 Effect of different diluents and different sample injection volumes on chromatographic behavior of genotoxic impurity 2- (4-aminophenyl) butyric acid in indobufen raw material through separation and determination by LC-MS (liquid chromatography-Mass Spectrometry)
(1) The chromatographic conditions were the same as in example 5 except for the diluent composition and the additional 5. mu.l and 10. mu.l sample volumes;
(2) the following three diluents were set up to examine the effect on the chromatographic behavior of the main peak:
diluent 1: methanol;
diluent 2: ethanol;
diluent 3: DMSO (dimethylsulfoxide)
(3) Solution preparation the same as example 5 except that the diluents were changed to methanol, ethanol, DMSO, respectively;
(4) sample introduction and result analysis
Three diluent blanks and control solutions (with the concentration of 30% of the limit and 11.25ng/mL) of three different diluents methanol, ethanol and DMSO are respectively sampled by 5 mul, 10 mul and 20 mul, and chromatograms are recorded. As a result, in the chromatogram obtained by injecting 10. mu.l and 20. mu.l, the main peak was split into irregular multiple peaks. S/N in a chromatogram obtained by injecting 5 mu l of sample is respectively 6.4, 6.8 and 5.6 which are all less than 10, and the sensitivity of the method can not meet the requirements of the determination and limit formulation of the impurities in the sample.
Example 7 separation and determination of genotoxic impurities 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid in Indobufen raw Material by Liquid chromatography
(1) Chromatographic conditions
The instrument comprises the following steps: agilent 6470 type high performance liquid chromatography triple quadrupole mass spectrometer
A chromatographic column: agilent Eclipse XDB-C18 4.6×150mm,5μm
Mobile phase: solvent A: 20mmol/L ammonium acetate solution; solvent B: methanol, elution was performed with the following gradient:
| time (min) | 0.01 | 6 | 6.1 | 10 |
| Solvent A (%) | 90 | 30 | 90 | 90 |
| Solvent B (%) | 10 | 70 | 10 | 10 |
Diluent agent: 20mmol/L ammonium acetate-methanol (90:10) solution
Flow rate: 1.0mL/min
Column temperature: 35 deg.C
Sample introduction volume: 20 μ l
Mass spectrum conditions: adopting an electrospray ion source, wherein genotoxic impurity 2- (4-aminophenyl) butyric acid adopts a positive ion scanning mode, and genotoxic impurity 2- (4-aminophenyl) butyric acid adopts a negative ion scanning mode;
the dry gas, the atomization gas and the sheath gas are all nitrogen; the collision gas is high-purity nitrogen;
the temperature of the drying gas is: 300 deg.C
The drying airflow rate is: 6L/min
Atomizing gas pressure 35psi
Temperature of sheath gas: 350 deg.C
Flow rate of sheath gas: 12L/min
Capillary voltage: + 4000V; -3500V
Nozzle voltage: + 500V; 1000V
MRM detection mode:
quantitative ion pair 180 → 106 of genotoxic impurity 2- (4-aminophenyl) butyric acid, cleavage voltage (Frag)118V, collision voltage (CE)29V, residence time (Dwell)180ms, acceleration voltage (Cell Acc) 3V;
the genotoxic impurity 2- (4-aminophenyl) butyric acid qualitative ion pair 180 → 134, the cleavage voltage (Frag)114V, the collision voltage (CE)19V, the residence time (Dwell)180ms, the acceleration voltage (Cell Acc) 3V;
genotoxic impurity, 2- (4-aminophenyl) butyric acid, qualitative ion pair 180 → 93, cleavage voltage (Frag)116V, collision voltage (CE) 30; residence time (Dwell)180ms, acceleration voltage (Cell Acc) 3V;
the quantitative ion pair of the genotoxic impurity 2- (4-nitrophenyl) butyric acid is 164 → 149, the cleavage voltage (Frag) is 90V, the collision voltage (CE) is 11V, the residence time (Dwell) is 200ms, and the acceleration voltage (Cell Acc) is 3V;
the genotoxic impurity 2- (4-nitrophenyl) butyric acid qualitative ion pair 164 → 119, the cleavage voltage (Frag)90V, the collision voltage (CE)11V, the residence time (Dwell)200ms, the acceleration voltage (Cell Acc) 3V;
the genotoxic impurity, 2- (4-nitrophenyl) butyric acid, qualitative ion pair 164 → 93, cleavage voltage (Frag)90V, collision voltage (CE) 17; dwell time (Dwell)200ms, acceleration voltage (Cell Acc) 3V; (2) methodology investigation
Checking the impurity limit according to a verification guiding principle of an analysis method of China pharmacopoeia 2020, year edition 9101, and investigating the specificity, detection limit and quantitative limit of the method;
(3) solution preparation
2- (4-aminophenyl) butyric acid control stock solution: accurately weighing 10.02mg of 2- (4-aminophenyl) butyric acid reference substance, placing the reference substance in a 100mL volumetric flask, dissolving the reference substance in methanol, diluting the reference substance to a scale, and shaking up to obtain the product with the concentration of 98.37 mug/mL;
2- (4-nitrophenyl) butyric acid control stock solution: accurately weighing 10.08mg of 2- (4-nitrophenyl) butyric acid reference substance, placing the reference substance in a 100mL volumetric flask, dissolving the reference substance with methanol, diluting the reference substance to a scale, and shaking up to obtain the product with the concentration of 100.52 mu g/mL;
mixing the reference solution: taking appropriate amount of stock solutions of 2- (4-nitrophenyl) butyric acid and 2- (4-aminophenyl) butyric acid, placing in the same volumetric flask, diluting with diluent to make the concentrations of 2- (4-nitrophenyl) butyric acid and 2- (4-aminophenyl) butyric acid both reach limit concentration (37.5ng/mL), and shaking up to obtain the product.
Quantitative limit and detection limit solution: taking a proper amount of reference solution, respectively placing in 100mL volumetric flasks, diluting to scale with diluent, shaking up, the concentration is respectively 30%, 25%, 10%, 5%, 3% of the limit concentration;
test solution: accurately weighing 100.5, 100.2 and 100.1mg of three batches of samples of indobufen raw materials, respectively placing the samples in 10mL volumetric flasks, respectively adding 5mL of DMSO for dissolving, diluting to a scale with a diluent, filtering with a filter of 0.45 mu m, and taking a subsequent filtrate to obtain a test sample solution;
adding a standard test solution: accurately weighing 100.2mg (Z200203 batches) of indobufen raw material samples, placing the indobufen raw material samples into a 10mL volumetric flask, dissolving the indobufen raw material samples in 5mL of DMSO (dimethyl sulfoxide), adding appropriate amounts of reference stock solutions of 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid, diluting the reference stock solutions to a scale with a diluent, filtering the reference stock solutions by 0.45 mu m, and taking subsequent filtrate to obtain a test solution, wherein the concentrations of the 2- (4-aminophenyl) butyric acid and the 2- (4-nitrophenyl) butyric acid are both limited concentrations (37.5 ng/mL);
(4) measurement method
The specificity determination method comprises the following steps: respectively injecting a diluent blank, a reference solution, a test solution and a standard test solution, wherein the blank solution is free of interference, and the sample and impurities in the sample are free of interference;
the method for measuring the quantitative limit and the detection limit comprises the following steps: respectively sampling diluent blank and test solution, recording a chromatogram, calculating the signal-to-noise ratio of a main peak in the extraction chromatogram of MRM 180 → 106 and MRM 164 → 149, wherein the corresponding concentration is a detection limit when the signal-to-noise ratio is about 3, and the corresponding concentration is a quantification limit when the signal-to-noise ratio is about 10;
the method for measuring the residual quantity of 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid in the sample comprises the following steps: and respectively injecting 6 parts of diluent blank and a reference solution, recording a chromatogram, extracting RSD% of main peak area change in the chromatogram by using MRM 180 → 106 and MRM 164 → 149, wherein RSD% of the main peak area change is not more than 20.0%, and calculating the residual quantity of two impurities in the sample by using peak areas according to an external standard method.
(5) Measurement results
The specificity is as follows: 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid peaks can achieve baseline separation under the condition, a blank solution is free of interference, and a sample and impurities in the sample are free of interference;
the quantitative limit and the detection limit test data are shown in Table 7
TABLE 7 quantitative limit, detection limit test data
The test data of 3 batches of raw material samples are shown in Table 8
Table 8 raw material sample test data
The methodological investigation and sample determination results show that the method has good specificity and high sensitivity, and can be used for simultaneously detecting two impurities in a sample. Wherein the quantitative limit and detection limit test patterns are shown in figures 7 and 8 respectively; the typical control solution profile is shown in FIG. 9, and the typical sample solution profile is shown in FIG. 10 (batch Z191212).
Example 8
(1) The same conditions were used as in example 7 except for the column ZORBAX Eclipse XDB-C84.6X 150mm,5 μm and the elution gradient:
| time (min) | 0.01 | 6 | 6.1 | 10 |
| Solvent A (%) | 90 | 30 | 90 | 90 |
| Solvent B (%) | 10 | 70 | 10 | 10 |
(2) Solution preparation and measurement methods were the same as in example 7
(3) Sample introduction and result analysis
Taking diluent blank, reference solution (with concentration of 30% of limit, 11.25ng/mL) and standard sample solution for sample injection, and recording chromatogram. The result shows that the peak of 2- (4-aminophenyl) butyric acid and the peak of 2- (4-nitrophenyl) butyric acid can achieve baseline separation under the condition, the blank solution has no interference, and the sample and impurities in the sample have no interference; the S/N is respectively 19.6 and 21.2 which are more than 10. The specificity and the sensitivity of the method can meet the requirements of the determination and limit formulation of the impurities in the sample, and the method is feasible.
Comparative example 4 examination of the influence of mobile phases (mobile phase 1) of different systems or mobile phases (mobile phase 2 and mobile phase 3) of the same system and different pH values on the determination of the sensitivity of the genotoxic impurities 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid in indobufen raw materials by LC-MS (liquid chromatography-mass spectrometry) separation
(3) The chromatographic conditions were the same as in example 7 except for the mobile phase
(4) The following three mobile phases were set to examine the effect on the sensitivity of the method
diluent initial mobile phase
Elution was performed with the following gradient:
| time (min) | 0.01 | 6 | 6.1 | 10 |
| Solvent A (%) | 90 | 30 | 90 | 90 |
| Solvent B (%) | 10 | 70 | 10 | 10 |
(3) Solution preparation
The same as in example 7;
(4) method for measuring sensitivity
And (3) respectively sampling a diluent blank and a 11.25ng/mL reference substance solution (corresponding to 30% of the limit concentration), recording a chromatogram, calculating the signal-to-noise ratio of a main peak in the chromatogram, and determining the sensitivity of the method.
(5) Measurement results
The results of the sensitivity measurements are shown in Table 9, the effect of the flow of the different systems on the sensitivity of the method
TABLE 9 sensitivity test results
The results show that when the mobile phase 1 and the mobile phase 2 are used for elution according to the gradient, the S/N ratio of the 2- (4-nitrophenyl) butyric acid in the chromatogram of the reference solution (the concentration is 30 percent of the limit concentration) is less than 10, and the sensitivity can not meet the requirements of sample detection and quality control. And eluting with mobile phase 3 according to the gradient, wherein S/N of 2- (4-nitrophenyl) butyric acid and 2- (4-aminophenyl) butyric acid in a reference substance solution chromatogram (the concentration is 30% of the limit concentration) is more than 10, and the sensitivity can meet the detection and quality control of 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid in the product.
Comparative example 5 investigation of the Effect of different Mass Spectrometry detection modes (SIM) on the separation and determination of specificity of 2- (4-aminophenyl) butyric acid and 2- (4-nitrophenyl) butyric acid, genotoxic impurities in indobufen raw material by a liquid chromatography-mass spectrometry method
(1) Chromatographic conditions
The instrument comprises the following steps: agilent 6470 type high performance liquid chromatography triple quadrupole mass spectrometer
A chromatographic column: agilent Eclipse XDB-C18 4.6×150mm,5μm
Mobile phase: solvent A: 20mmol/L ammonium acetate solution; solvent B: methanol, elution was performed with the following gradient:
| time (min) | 0.01 | 6 | 6.1 | 10 |
| Solvent A (%) | 90 | 30 | 90 | 90 |
| Solvent B (%) | 10 | 70 | 10 | 10 |
Diluent agent: 20mmol/L ammonium acetate-methanol (90:10) solution
Flow rate: 1.0mL/min
Column temperature: 35 deg.C
Sample introduction volume: 20 μ l
Mass spectrum conditions: adopting an electrospray ion source, wherein genotoxic impurity 2- (4-aminophenyl) butyric acid adopts a positive ion scanning mode, and genotoxic impurity 2- (4-aminophenyl) butyric acid adopts a negative ion scanning mode;
the dry gas, the atomization gas and the sheath gas are all nitrogen; the collision gas is high-purity nitrogen;
the temperature of the drying gas is: 300 deg.C
The drying airflow rate is: 6L/min
Atomizing gas pressure 35psi
Temperature of sheath gas: 350 deg.C
Flow rate of sheath gas: 12L/min
Capillary voltage: + 4000V; -3500V
Nozzle voltage: + 500V; 1000V
SIM detection mode:
selecting a mass-to-charge ratio (m/z) of 2- (4-aminophenyl) butyric acid as a genotoxic impurity, wherein the mass-to-charge ratio (m/z) is 180, the cracking voltage (Frag) is 118V, the residence time (Dwell) is 180ms, and the accelerating voltage (Cell Acc) is 3V;
selecting a mass-to-charge ratio (m/z) of a genotoxic impurity 2- (4-nitrophenyl) butyric acid of 164, a cracking voltage (Frag) of 90V, a residence time (Dwell) of 200ms and an accelerating voltage (Cell Acc) of 3V;
(2) solution preparation
The same as in example 7;
(3) sample introduction and result analysis
And (4) sampling diluent blank and a mixed reference solution (limit concentration, 11.25ng/mL) respectively, and recording chromatograms. As a result, the peak shape was poor, and the S/N was 2.2, which was less than 10. The sensitivity of the method using this mode of measurement does not meet the requirements for the measurement and limit formulation of these two impurities in the sample.
Finally, the above embodiments are only used for illustrating the technical solutions of the present invention in detail and not for limiting, and those skilled in the art can modify or substitute the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and shall be covered by the claims of the present invention.
Claims (24)
1. A detection method of genotoxic impurities in indobufen bulk drugs is characterized in that: the method comprises the steps of carrying out chromatographic separation on relative genotoxic impurities by using octadecylsilane chemically bonded silica or octyl silane chemically bonded silica as fixed phase, and carrying out analysis and detection by using a mass spectrum detector; the mobile phase is a mixture of a solvent A and a solvent B, the solvent A is selected from an aqueous solution system containing volatile acid, and the solvent B is selected from any one of methanol, acetonitrile or a mixture of the methanol and the acetonitrile.
2. The detection method according to claim 1, characterized in that: the genotoxic impurity is 2- (4-amino) phenylbutyric acid.
3. The detection method according to claim 1, characterized in that: the pH value of the mobile phase is controlled to be 2-7, preferably 2-3.
4. The detection method according to claim 1, characterized in that: the solvent A is selected from aqueous solution containing any one of formic acid, acetic acid or a mixture of the two, and the volume concentration of the solvent A is preferably 0.01-1%; still further, the solvent A is preferably an acetic acid aqueous solution with a volume concentration of 0.01-1%.
5. The detection method according to claim 1, characterized in that: the mobile phase elution adopts gradient elution, and the gradient elution conditions are as follows: isocratic at 0.01-1 min, the volume ratio of the solvent A to the solvent B is 90: 10-70: 30, isocratic at 2-4 min, the volume ratio of the solvent A to the solvent B is 10: 90-5: 95, isocratic at 4.1-5 min, and the volume ratio of the solvent A to the solvent B is 90: 10-70: 30.
6. The detection method according to claim 1, characterized in that: the flow rate of the mobile phase is 0.3-0.5 mL/min.
7. The detection method according to claim 1, characterized in that: the specification of the chromatographic column is 2.1 × 50mm,1.7 μm or 4.6 × 50mm, 1.8 μm or 4.6 × 50mm,2.7 μm.
8. The detection method according to claim 1, characterized in that: the method specifically comprises the following steps: dissolving and diluting a test sample to prepare a solution with the concentration of about 0.5-1 mg/mL as a test sample solution; dissolving and diluting genotoxic impurity 2- (4-amino) phenylbutyric acid to prepare a solution with the concentration of about 1.875-3.75 ng/mL, and taking the solution as a reference solution; respectively taking 5-20 mu L of sample solution to be tested and reference solution to be injected, respectively recording ion flow diagrams under quantitative and qualitative ion pairs, and calculating the impurity content by the peak area of the quantitative ion pairs according to an external standard method; the diluent is the initial mobile phase.
9. A detection method of genotoxic impurities in indobufen bulk drugs is characterized in that: the method comprises the steps of carrying out chromatographic separation on relative genotoxic impurities by using octadecylsilane chemically bonded silica or octyl silane chemically bonded silica as fixed phase, and carrying out analysis and detection by using a mass spectrum detector; the mobile phase is a mixture of a solvent C and a solvent D, the solvent C is selected from a volatile salt aqueous solution system, and the solvent D is selected from any one of methanol, acetonitrile or a mixture of the methanol and the acetonitrile.
10. The detection method according to claim 9, characterized in that: the genotoxic impurity is 2- (4-amino) phenylbutyric acid and/or 2- (4-nitro) phenylbutyric acid.
11. The detection method according to claim 9, characterized in that: the pH value of the mobile phase is controlled to be 3-7, preferably 6-7.
12. The detection method according to claim 9, characterized in that: the solvent C is one or a mixed aqueous solution of any one of ammonium formate and ammonium acetate; further, the concentration of the solvent C is preferably 5-50 mmol/L; still further, the solvent C is preferably 5-50 mmol/L ammonium acetate water solution.
13. The detection method according to claim 10, characterized in that: when the genotoxic impurity 2- (4-amino) phenylbutyric acid is detected independently, gradient elution is adopted for the mobile phase elution, and the elution conditions are as follows: when 0.01min is reached, the volume ratio of the solvent C to the solvent D is 85: 15-70: 30; isocratic for 2-3 min, wherein the volume ratio of the solvent C to the solvent D is 30: 70-15: 85; isocratic at 3.1-5 min, and the volume ratio of the solvent C to the solvent D is 85: 15-70: 30.
14. The detection method according to claim 10, characterized in that: when the genotoxic impurity 2- (4-amino) phenylbutyric acid is detected independently, the flow rate of the mobile phase is 0.9-1.1 mL/min.
15. The detection method according to claim 10, characterized in that: when the genotoxic impurity 2- (4-amino) phenylbutyric acid is detected alone, the specification of the chromatographic column is 4.6 × 150mm,5 μm or 2.1 × 150mm,1.7 μm or 2.1 × 150mm,1.6 μm or 2.1 × 150mm,1.9 μm or 4.6 × 150mm,2.7 μm or 4.6 × 150mm,2.5 μm or 4.6 × 150mm,3.5 μm or 4.6 × 150mm,3.0 μm.
16. The detection method according to claim 10, characterized in that: when the genotoxic impurity 2- (4-amino) phenylbutyric acid is separately detected, the method specifically comprises the following steps: dissolving and diluting a test sample to prepare a solution with the concentration of about 5-10 mg/mL as a test sample solution; dissolving and diluting genotoxic impurity 2- (4-amino) phenylbutyric acid to prepare a solution with the concentration of about 18.75-37.5 ng/mL, and taking the solution as a reference solution; respectively taking 5-20 mu L of sample solution to be tested and reference solution to be injected, respectively recording ion flow diagrams under quantitative and qualitative ion pairs, and calculating the impurity content by the peak area of the quantitative ion pairs according to an external standard method; the diluent is the initial mobile phase.
17. The detection method according to claim 10, characterized in that: when the genotoxic impurity 2- (4-nitro) phenylbutyric acid is detected independently, gradient elution is adopted for the mobile phase elution, and the elution conditions are as follows: isocratic at 0.01-1 min, the volume ratio of the solvent C to the solvent D is 90: 10-70: 30, isocratic at 2-4 min, the volume ratio of the solvent C to the solvent D is 10: 90-5: 95, isocratic at 4.1-5 min, and the volume ratio of the solvent C to the solvent D is 90: 10-70: 30.
18. The detection method according to claim 10, characterized in that: when the genotoxic impurity 2- (4-nitro) phenylbutyric acid is detected independently, the flow rate of the mobile phase is 0.3-0.5 mL/min.
19. The detection method according to claim 10, characterized in that: when the genotoxic impurity 2- (4-nitro) phenylbutyric acid is detected separately, the specification of the chromatographic column is 2.1 multiplied by 50mm,1.7 mu m or 4.6 multiplied by 50mm, 1.8 mu m or 4.6 multiplied by 50mm, and 2.7 mu m.
20. The detection method according to claim 10, characterized in that: when the genotoxic impurity 2- (4-nitro) phenylbutyric acid is separately detected, the method specifically comprises the following steps: dissolving and diluting a test sample to prepare a solution with the concentration of about 5-10 mg/mL as a test sample solution; dissolving and diluting genotoxic impurity 2- (4-nitro) phenylbutyric acid to prepare a solution with the concentration of about 18.75-37.5 ng/mL, and taking the solution as a reference solution; respectively taking 2-5 mu L of sample solution of a test sample and 2-5 mu L of reference solution of a reference sample, respectively recording ion flow diagrams under quantitative and qualitative ion pairs, and calculating the impurity content by the peak area of the quantitative ion pairs according to an external standard method; the diluent is any one of a mobile phase or an organic solvent in the mobile phase.
21. The detection method according to claim 10, characterized in that: when genotoxic impurities are detected to be 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid at the same time, gradient elution is adopted for the mobile phase elution, and the elution conditions are as follows: when 0.01min is reached, the volume ratio of the solvent C to the solvent D is 90: 10-85: 15; isocratic within 5-6 min, wherein the volume ratio of the solvent C to the solvent D is 30: 70-25: 75; isocratic at 6.1-10 min, and the volume ratio of the solvent C to the solvent D is 90: 10-85: 15.
22. The detection method according to claim 10, characterized in that: and when genotoxic impurities, namely 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid, are detected at the same time, the flow rate is 0.8-1.2 mL/min.
23. The detection method according to claim 10, characterized in that: when the genotoxic impurities 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid are simultaneously detected, the specification of the chromatographic column is 4.6 × 150mm,5 μm or 2.1 × 150mm,1.7 μm or 2.1 × 150mm,1.6 μm or 2.1 × 150mm,1.9 μm or 4.6 × 150mm,2.7 μm or 4.6 × 150mm,2.5 μm or 4.6 × 150mm,3.5 μm or 4.6 × 150mm and 3.0 μm.
24. The detection method according to claim 10, characterized in that: when genotoxic impurities 2- (4-amino) phenylbutyric acid and 2- (4-nitro) phenylbutyric acid are detected simultaneously, the method specifically comprises the following steps: dissolving and diluting a test sample to prepare a solution with the concentration of about 5-10 mg/mL as a test sample solution; dissolving genotoxic impurities and diluting to prepare a solution with the concentration of about 18.75-37.5 ng/mL, wherein the solution is used as a reference solution; respectively taking 10-20 mu L of sample solution to be tested and reference solution to be injected, respectively recording ion flow diagrams under quantitative and qualitative ion pairs, and calculating the impurity content by the peak area of the quantitative ion pairs according to an external standard method; the diluent is the initial mobile phase.
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