CN113820426A - Method for detecting toxic impurities in ranolazine - Google Patents
Method for detecting toxic impurities in ranolazine Download PDFInfo
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- XKLMZUWKNUAPSZ-UHFFFAOYSA-N N-(2,6-dimethylphenyl)-2-{4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]piperazin-1-yl}acetamide Chemical compound COC1=CC=CC=C1OCC(O)CN1CCN(CC(=O)NC=2C(=CC=CC=2C)C)CC1 XKLMZUWKNUAPSZ-UHFFFAOYSA-N 0.000 title claims abstract description 95
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- G—PHYSICS
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- 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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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
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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
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Abstract
The invention provides a method for detecting toxic impurities in ranolazine. The method for detecting the toxic impurities in ranolazine comprises the following steps: (1) respectively preparing a test solution of ranolazine and a reference solution of 2, 6-xylidine; (2) and (2) respectively injecting the sample solution of ranolazine obtained in the step (1) and the reference solution of 2, 6-dimethylaniline into a high performance liquid chromatography-tandem mass spectrometer for detection, and calculating the content of the 2, 6-dimethylaniline in the ranolazine sample by adopting an external standard method. The method for detecting the toxic impurities in ranolazine has the advantages of strong specificity, quick analysis, strong anti-interference performance, high sensitivity and the like.
Description
Technical Field
The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a method for detecting toxic impurities in ranolazine.
Background
Ranolazine is an antianginal drug which is an inhibitor of calcium ion channel uptake and is used for treating chronic angina. The ranolazine raw material medicine/ranolazine sustained-release tablet is not recorded in pharmacopoeia, and reports of genotoxicity warning structure impurities in ranolazine raw material medicines are not seen at present.
According to the regulation of the international harmonization conference (ICH) M7(R1) of the technical requirement of drug registration of human, if the impurities with genotoxicity warning structures are not genotoxicity proved by bacterial mutagenesis experiments, namely potential toxic impurities, the daily intake of human cannot exceed 1.5 mu g when the human is taken for a long time. One of the starting materials of a key process intermediate of ranolazine bulk drug is 2, 6-xylidine, which belongs to class 2B carcinogens in the carcinogen list of the international cancer research institution of the world health organization. The maximum dose of ranolazine clinically administered is 2000mg, so the maximum limit of the impurities in the ranolazine raw material and the sustained release tablet is 0.75 ppm.
CN100581547C discloses a ranolazine sustained release tablet and a detection method, belonging to the field of drug analysis. Taking 20 tablets of the product, precisely weighing, grinding, precisely weighing a proper amount of fine powder, placing the fine powder in a 50mL measuring flask, adding mobile phase ultrasound to dissolve and dilute the fine powder to a scale, shaking up, filtering, precisely weighing 10mL of subsequent filtrate, placing the subsequent filtrate in a 50mL measuring flask, adding the mobile phase to dilute the subsequent filtrate to the scale, shaking up, precisely weighing 20 mu l of the subsequent filtrate, injecting the subsequent filtrate into a liquid chromatograph, recording a chromatogram, precisely weighing a proper amount of ranolazine reference substance, dissolving and quantitatively diluting the subsequent filtrate by using the mobile phase to prepare a solution containing about 80 mu g of the subsequent filtrate in each 1mL, measuring by the same method, and calculating by using a peak area according to an external standard method to obtain the ranolazine olanex. Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-acetonitrile-acetic acid solution 25:35:40 was used as the mobile phase, the pH was adjusted to 6.6 with ammonia, and the detection wavelength was 230 nm.
CN107144644A discloses a method for measuring a related substance of lidamycin, which comprises the following steps: (1) specificity test, (2) stability test, and (3) maximum related substance determination. The determination method comprises the steps of determining 2, 6-dimethylaniline by using HPLC, wherein a chromatographic column is a C18 column, a mobile phase is acetonitrile and phosphoric acid water, the ratio of acetonitrile to phosphoric acid water is 40:60, the detection wavelength comprises 225nm, the detection temperature is 30 ℃, the flow rate is 1.25mL/min, and the sample injection amount is 10 mu L.
However, the methods disclosed in the above patents cannot accurately and quantitatively detect the content of 2, 6-xylidine, a genotoxic impurity in ranolazine.
Therefore, the development of a detection method for ranolazine toxic impurities, which has the advantages of rapid analysis, strong interference resistance and high sensitivity, is the research focus in the field.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for detecting toxic impurities in ranolazine. The detection method is a high performance liquid chromatography-tandem mass spectrometry analysis method for determining specific genotoxic impurities in the ranolazine bulk drug and the ranolazine sustained-release tablets, and has the advantages of strong specificity, quick analysis, strong anti-interference performance and high sensitivity.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for detecting a toxic impurity in ranolazine, comprising the steps of:
(1) respectively preparing a test solution of ranolazine and a reference solution of 2, 6-xylidine;
(2) and (2) respectively injecting the sample solution of ranolazine obtained in the step (1) and the reference solution of 2, 6-dimethylaniline into a high performance liquid chromatography-tandem mass spectrometer for detection, and calculating the content of the 2, 6-dimethylaniline in the ranolazine sample by adopting an external standard method.
In the invention, high performance liquid chromatography-tandem mass spectrometry is adopted for detection, in a high performance liquid chromatograph, a sample solution and a mobile phase A, B solution are separated on a chromatographic column, and elution is carried out by adopting an isocratic elution mode; enabling the sample after isocratic elution to enter a tandem mass spectrum, and acquiring an ion spectrogram by using a mass spectrometer so as to obtain a sample spectrogram; the chromatographic peak of 2, 6-xylidine was determined.
In the invention, the chromatographic peak area of the 2, 6-xylidine is compared with the chromatographic peak area of the 2, 6-xylidine in the reference solution, and the content of the 2, 6-xylidine in the test solution is calculated by the following formula:
wherein Cs is the concentration of 2, 6-xylidine in the control solution, unit: mu g/mL; the area of the chromatographic peak of the 2, 6-xylidine in the test solution is equal to the area of the chromatographic peak; the chromatographic peak area of 2, 6-xylidine in the control solution; cu is the concentration of the test solution in units: g/mL.
The invention adopts high performance liquid chromatography-tandem mass spectrometry, can effectively improve the recognition capability of impurities, improve the specificity and sensitivity of analysis and accurately determine the content of the impurities; the problem of the determination of potential specific genotoxicity impurities in the ranolazine bulk drug and the ranolazine sustained release tablet is solved; therefore, the content of the potential specific genotoxic impurity 2, 6-xylidine in the ranolazine bulk drug and the ranolazine sustained release tablet can be simply, rapidly and accurately analyzed.
Preferably, in step (1), the concentration of the sample solution of ranolazine is 20-30mg/mL, and may be, for example, 20mg/mL, 21mg/mL, 22mg/mL, 23mg/mL, 24mg/mL, 25mg/mL, 26mg/mL, 27mg/mL, 28mg/mL, 29mg/mL, 30mg/mL, or the like.
Preferably, in step (1), the concentration of the control solution of 2, 6-xylidine is 7-50ng/mL, and may be, for example, 7ng/mL, 8ng/mL, 9ng/mL, 10ng/mL, 12ng/mL, 15ng/mL, 20ng/mL, 25ng/mL, 30ng/mL, 35ng/mL, 40ng/mL, 45ng/mL, 50ng/mL, etc.
Preferably, in step (1), the diluent used for the test solution and the reference solution is independently selected from methanol and/or water, preferably a mixture of methanol and water.
Preferably, the volume ratio of the methanol to the water is (70-90): (10-30), and can be, for example, 70:30, 75:25, 80:20, 85:15, 90:10, and the like.
Preferably, in the step (1), the ranolazine test sample comprises a ranolazine bulk drug and/or a ranolazine sustained-release tablet.
In the invention, when the ranolazine test sample is ranolazine raw material medicine, the ranolazine raw material medicine is precisely weighed, dissolved by methanol, ultrasonically treated, cooled, diluted by water to a scale constant volume, and shaken uniformly to prepare the test sample solution.
In the invention, when the ranolazine test sample is ranolazine sustained release tablets, the ranolazine sustained release tablets are firstly crushed into powder, an appropriate amount of ranolazine sustained release tablet powder is precisely weighed, dissolved by methanol, ultrasonically treated, cooled, diluted to a scale by water, shaken up, centrifuged at a high speed, and then the supernatant is taken as a test sample solution.
Preferably, in the step (2), the high performance liquid chromatography-tandem mass spectrometer is an Agilent 1260 high performance liquid chromatography and Agilent 6410 triple tandem quadrupole mass spectrometry-liquid chromatography mass spectrometer.
In the invention, an Agilent 1260HPLC-6410B Triple Quadrupole MSD system and a MassHunter workstation are used for analyzing a sample to be tested, and a chromatographic collection computer system is started to collect data; the content of the specific genotoxic impurity was calculated using an external standard method.
Preferably, in the step (2), the mobile phase a is 0.1 to 0.3 vol% (for example, 0.1 vol%, 0.12 vol%, 0.14 vol%, 0.16 vol%, 0.18 vol%, 0.2 vol%, 0.22 vol%, 0.24 vol%, 0.26 vol%, 0.28 vol%, 0.3 vol% and the like) of formic acid aqueous solution, and the mobile phase B is acetonitrile in the detection by high performance liquid chromatography.
Preferably, in the step (2), isocratic elution is adopted in the detection of the high performance liquid chromatography, and the volume ratio of the mobile phase A to the mobile phase B is (30-50): (50-70), and can be 30:70, 35:65, 40:60, 45:55, 50:50 and the like.
Preferably, in the step (2), the chromatographic column in the detection of the high performance liquid chromatography is YMC-Pack Pro-C18.
Preferably, in step (2), the specification of the chromatographic column is: the inner diameter is 4.6mm, the column length is 150-250mm (for example, 150mm, 250mm, etc.), and the particle diameter is 3.0-5.0 μm (for example, 3.0 μm, 5.0 μm, etc.).
Preferably, in step (2), the flow rate in the detection by high performance liquid chromatography is 0.3 to 0.7mL/min, and may be, for example, 0.3mL/min, 0.4mL/min, 0.5mL/min, 0.6mL/min, 0.7mL/min, or the like.
Preferably, in step (2), the sample injection volume in the detection by high performance liquid chromatography is 15-25 μ L, such as 15 μ L, 16 μ L, 17 μ L, 18 μ L, 19 μ L, 20 μ L, 21 μ L, 22 μ L, 23 μ L, 24 μ L, 25 μ L, etc.
Preferably, in the step (2), the column temperature in the detection by high performance liquid chromatography is 25 to 35 ℃, and may be, for example, 25 ℃, 27 ℃, 29 ℃, 30 ℃, 31 ℃, 33 ℃, 34 ℃, 35 ℃ or the like.
Preferably, in the step (2), the detection run time of the high performance liquid chromatography is 10-20min, such as 10min, 12min, 14min, 16min, 18min, 20min and the like.
Preferably, in the step (2), the ion source in the mass spectrometry detection is a pneumatically-assisted electrospray ion source (ESI), and the polarity is positive.
Preferably, in step (2), the mass spectrum is monitored in a multi-reaction monitoring mode, and the 122.1 → 105.1 channel is used for quantification.
Preferably, in step (2), the fragmentation voltage in the detection of the mass spectrum is 40-60V, such as 40V, 45V, 50V, 55V, 60V, etc., the collision energy is 10-20V, such as 10V, 12V, 14V, 16V, 18V, 20V, etc., the dry gas flow rate is 5-15L/min, such as 5L/min, 7L/min, 9L/min, 11L/min, 13L/min, 15L/min, etc., the dry gas temperature is 250-350 deg.C, such as 250 deg.C, 260 deg.C, 270 deg.C, 280 deg.C, 290 deg.C, 300 deg.C, 350 deg.C, the atomization chamber pressure is 45-55Psi, such as 45Psi, 47Psi, 49Psi, 51Psi, 53Psi, 55Psi, etc., the capillary voltage is 5500-6500volts, such as 5500volts, 5700volts, etc, 5900volts, 6100volts, 6300volts, 6500volts, and the like.
As a preferable technical scheme, the method for detecting the toxic impurities in ranolazine comprises the following steps:
(1) respectively preparing a 20-30mg/mL sample solution of ranolazine and a 7-50ng/mL reference solution of 2, 6-dimethylaniline;
(2) injecting the sample solution of ranolazine obtained in the step (1) and the reference solution of 2, 6-dimethylaniline into a high performance liquid chromatography-tandem mass spectrometer for detection respectively, and calculating the content of the 2, 6-dimethylaniline in the ranolazine sample by adopting an external standard method;
wherein, the testing conditions of the high performance liquid chromatography comprise: the mobile phase A is 0.1-0.3 vol% formic acid water solution, the mobile phase B is acetonitrile, isocratic elution is adopted, and the volume ratio of the mobile phase A to the mobile phase B is (30-50): (50-70); the chromatographic column is YMC-PackPro-C18, the inner diameter is 4.6mm, the column length is 150-250mm, the particle size is 3.0-5.0 mu m, the flow rate is 0.3-0.7mL/min, the sample injection volume is 15-25 mu L, the column temperature is 25-35 ℃, and the running time is 10-20 min;
wherein, the test conditions of the mass spectrum comprise: the ion source is a pneumatic auxiliary electrospray ion source, the polarity is positive, the monitoring mode is multi-reaction monitoring, a 122.1 → 105.1 channel is used for quantification, the fragmentation voltage is 40-60V, the collision energy is 10-20V, the flow rate of the dry gas is 5-15L/min, the temperature of the dry gas is 250-350 ℃, the pressure of the atomization chamber is 45-55Psi, and the voltage of a capillary tube is 5500-6500 volts.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention adopts high performance liquid chromatography-tandem mass spectrometry, can effectively improve the recognition capability of impurities, improve the specificity and sensitivity of analysis and accurately determine the content of the impurities;
(2) the invention solves the problem of the determination of potential specific genotoxicity impurities in the ranolazine bulk drug and the ranolazine sustained release tablet;
(3) the method can simply, conveniently, quickly and accurately analyze the content of the potential specific genotoxic impurity (2, 6-xylidine) in the ranolazine bulk drug and the ranolazine sustained release tablet.
Drawings
FIG. 1 is a solvent blank solution chromatogram.
FIG. 2 is a chromatogram of a control.
FIG. 3 is a chromatogram of a sample of example 1.
FIG. 4 is a chromatogram of a sample of example 2.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The following examples and comparative examples used the apparatus as follows:
high performance liquid chromatography-tandem mass spectrometer: agilent 1260HPLC-6410B Triple Quadrupole MSD system and MassHunter workstation.
A chromatographic column: YMC-Pack Pro-C18,150X 4.6mm,3 μm.
Example 1
The embodiment provides a method for detecting toxic impurities in ranolazine, which comprises the following steps:
(1) precisely weighing 1500mg of ranolazine bulk drug, placing the raw drug in a 50mL measuring flask, dissolving the raw drug in methanol, carrying out ultrasonic treatment, cooling the raw drug, diluting the raw drug with water to a scale, and shaking the raw drug uniformly to prepare a test solution with the concentration of 30 mg/mL; wherein, methanol is water (v/v) ═ 80: 20;
precisely weighing 2.3mg of 2, 6-xylidine impurity reference substance, placing in a 100mL measuring flask, dissolving with methanol, diluting with methanol to scale, shaking up, and making into reference substance stock solution-1 with concentration of 23 μ g/mL;
precisely measuring 4mL of reference stock solution-1, placing in a 200mL measuring flask, adding 40mL of water, diluting with methanol to scale, shaking up, and preparing into reference stock solution-2 with concentration of 0.46 μ g/mL;
precisely measuring 5mL of reference stock solution-2, placing in a 100mL measuring flask, diluting with diluent (methanol: water: 80:20, v/v) to scale, shaking up, and preparing into reference solution with concentration of 23 ng/mL;
(2) injecting the sample solution of ranolazine obtained in the step (1) and the reference solution of 2, 6-dimethylaniline into a high performance liquid chromatography-tandem mass spectrometer for detection respectively, and starting a chromatography acquisition computer system to acquire data; calculating the content of the specific genotoxic impurity by using an external standard method;
the chromatographic conditions include: mobile phase: 0.2% aqueous formic acid: acetonitrile 40:60 (v/v); a chromatographic column: YMC-Pack Pro-C18,150X 4.6mm,3 μm; flow rate: 0.5 mL/min; sample introduction volume: 20 mu L of the solution; column temperature: 30 ℃; operating time: and 15 min. The mass spectrometry conditions include: pneumatically assisted electrospray ion source (ESI), positive ion, multiple reactive ion monitoring (MRM, 122.1 → 105.1); fragmentation voltage: 50V, collision energy: 15V; flow rate of drying gas: 11L/min, drying gas temperature: 350 ℃, atomization chamber pressure: 50Psi, capillary voltage: 6000 volts.
The measurement results are shown in table 1 below:
TABLE 1
As can be seen from the test results in Table 1, the 2, 6-xylidine impurity levels in the 3 ranolazine test samples were 0.03ppm, 0.04ppm and 0.04ppm, respectively, which are below the quantitation limit (quantitation limit: 0.24ppm), and which are below the maximum 0.75ppm of the impurity in the sample.
Fig. 1 is a blank solution chromatogram, and as shown in fig. 1, the blank solution has no peak and the baseline of the chromatogram is stable. FIG. 2 is a control chromatogram of 23ng/mL, showing that 2, 6-xylidine peaked at 5.536min at an area of 25721, as shown in FIG. 2. FIG. 3 is a chromatogram of the sample solution of example 1, wherein 2, 6-xylidine has a peak at 5.614min and an area of 1765, as shown in FIG. 3.
Example 2
The embodiment provides a method for detecting toxic impurities in ranolazine, which comprises the following steps:
(1) accurately weighing a proper amount of ranolazine sustained release tablet powder, which is equivalent to 1500mg of ranolazine, placing the ranolazine sustained release tablet powder into a 50mL measuring flask, dissolving the powder by using methanol, carrying out ultrasonic treatment, cooling the solution, diluting the solution to a scale by using water, shaking the solution uniformly to prepare a test solution with the concentration of 30mg/mL, carrying out high-speed centrifugation, and taking supernatant as a test sample for analysis; wherein, methanol is water (v/v) ═ 80: 20;
precisely weighing 2.3mg of 2, 6-xylidine impurity reference substance, placing in a 100mL measuring flask, dissolving with methanol, diluting with methanol to scale, shaking up, and making into reference substance stock solution-1 with concentration of 23 μ g/mL;
precisely measuring 4mL of reference stock solution-1, placing in a 200mL measuring flask, adding 40mL of water, diluting with methanol to scale, shaking up, and preparing into reference stock solution-2 with concentration of 0.46 μ g/mL;
precisely measuring 5mL of reference stock solution-2, placing in a 100mL measuring flask, diluting with diluent (methanol: water: 80:20, v/v) to scale, shaking up, and preparing into reference solution with concentration of 23 ng/mL;
(2) injecting the sample solution of ranolazine obtained in the step (1) and the reference solution of 2, 6-dimethylaniline into a high performance liquid chromatography-tandem mass spectrometer for detection respectively, and starting a chromatography acquisition computer system to acquire data; calculating the content of the specific genotoxic impurity by using an external standard method;
the chromatographic conditions include: mobile phase: 0.2% aqueous formic acid: acetonitrile 40:60 (v/v); a chromatographic column: YMC-Pack Pro-C18,150X 4.6mm,3 μm; flow rate: 0.5 mL/min; sample introduction volume: 20 mu L of the solution; column temperature: 30 ℃; operating time: and 15 min. The mass spectrometry conditions include: pneumatically assisted electrospray ion source (ESI), positive ion, multiple reactive ion monitoring (MRM, 122.1 → 105.1); fragmentation voltage: 50V, collision energy: 15V; flow rate of drying gas: 11L/min, drying gas temperature: 350 ℃, atomization chamber pressure: 50Psi, capillary voltage: 6000 volts.
The measurement results are shown in table 2 below:
TABLE 2
As can be seen from the test results in Table 2, the 2, 6-xylidine impurity levels in the 3 batches of ranolazine sustained release tablets were 0.63ppm, 0.56ppm and 0.66ppm, respectively, but were below the maximum 0.75ppm of the impurity in the samples.
FIG. 4 is a chromatogram of the test solution of example 2, wherein 2, 6-xylidine peaked at 5.606min and had an area of 22676, as shown in FIG. 4.
Example 3
This example provides a method for detecting toxic impurities in ranolazine, which differs from example 1 only in that the mobile phase B is 0.1 vol% formic acid in water.
Example 4
This example provides a method for detecting toxic impurities in ranolazine, which is different from example 1 only in that the mobile phase a: 0.2 vol% acetic acid aqueous solution.
Example 5
This example provides a method for detecting toxic impurities in ranolazine, which is different from example 1 only in that the chromatographic column is
ODS-3(4.6×150mm,3μm)。
Example 6
This example provides a method for detecting toxic impurities in ranolazine, which differs from example 1 only in that the flow rate is 0.4 mL/min.
Example 7
This example provides a method for detecting toxic impurities in ranolazine, which differs from example 1 only in that the flow rate is 0.6 mL/min.
Example 8
This example provides a method for detecting toxic impurities in ranolazine, wherein the volume ratio of mobile phase a to mobile phase B is 35: 65.
Example 9
This example provides a method for detecting toxic impurities in ranolazine, wherein the volume ratio of mobile phase a to mobile phase B is 45: 55.
Comparative example 1
The comparative example provides a method for detecting toxic impurities in ranolazine, and the method is different from the method in example 1 only in that high performance liquid chromatography-tandem mass spectrometry is not adopted, and only high performance liquid chromatography is applied to detection.
The results show that: the sensitivity of the single high performance liquid chromatography is low, the reference solution cannot reach the lowest detection limit of the detector, and the peak of the 2, 6-xylidine chromatography cannot be detected.
Methodology investigation
Methodological studies of the methods for detecting toxic impurities in ranolazine provided in examples 1-9 above were conducted, and the specific test methods are as follows:
(1) reproducibility: taking a 23ng/mL reference substance solution and continuously feeding into 6 needles, and calculating the% RSD of the 2, 6-xylidine impurity peak area;
(2) the specificity is as follows: recording the retention time of liquid quality detection of 2, 6-dimethylaniline impurities;
(3) repeatability: preparing 6 samples (the concentration level of 2, 6-dimethylaniline is 0.75ppm) for recovery in parallel, and calculating the% RSD of the result of the recovery of the 2, 6-dimethylaniline;
(4) the system applicability is as follows: taking a 23ng/mL reference substance solution and continuously feeding into 6 needles, and calculating the% RSD of the 2, 6-xylidine impurity peak area;
the specific test results are shown in table 3 below:
TABLE 3
As can be seen from the test data in Table 3, the RSD of the peak area of the 2, 6-xylidine impurity was calculated to be less than 10% by taking the control solution and continuously feeding the solution into 6 needles, which indicates that the precision of the method is good. The retention time of the liquid quality detection of the 2, 6-xylidine impurity is about 5.5min, and the specificity is good. 6 parts of sample are prepared in parallel, RSD of the 2, 6-dimethylaniline impurity determination result is less than 10%, and the repeatability is good. And taking the reference substance solution to continuously feed into 6 needles, calculating the RSD of the 2, 6-xylidine impurity peak area to be less than 10 percent, meeting the requirement and having good system applicability.
In conclusion, the high performance liquid chromatography-tandem mass spectrometry can accurately determine the content of specific genotoxic impurities in the ranolazine bulk drug and the ranolazine sustained-release tablets; solves the problem of the determination method of trace genotoxicity impurities in the ranolazine bulk drug and the ranolazine sustained release tablets.
The applicant states that the present invention is illustrated by the above examples of the detection method of toxic impurities in ranolazine, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (10)
1. A method for detecting a toxic impurity in ranolazine, which is characterized by comprising the following steps:
(1) respectively preparing a test solution of ranolazine and a reference solution of 2, 6-xylidine;
(2) and (2) respectively injecting the sample solution of ranolazine obtained in the step (1) and the reference solution of 2, 6-dimethylaniline into a high performance liquid chromatography-tandem mass spectrometer for detection, and calculating the content of the 2, 6-dimethylaniline in the ranolazine sample by adopting an external standard method.
2. The method for detecting the toxic impurity of ranolazine as claimed in claim 1, wherein in step (1), the concentration of the test solution of ranolazine is 20-30 mg/mL;
preferably, in the step (1), the concentration of the 2, 6-xylidine control solution is 7-50 ng/mL;
preferably, in the step (1), the diluent used for the test solution and the reference solution is independently selected from methanol and/or water, preferably a mixed solution of methanol and water;
preferably, the volume ratio of the methanol to the water is (70-90): (10-30).
3. The method for detecting the toxic impurity in ranolazine as claimed in claim 1 or 2, wherein in step (1), the ranolazine test sample comprises a ranolazine bulk drug and/or a ranolazine sustained release tablet.
4. The method for detecting the toxic impurities in ranolazine, according to any one of claims 1 to 3, wherein in the step (2), the high performance liquid chromatography-tandem mass spectrometer is Agilent 1260 high performance liquid chromatography and Agilent 6410 triple tandem quadrupole mass spectrometry-liquid mass spectrometer.
5. The method for detecting the toxic impurity in ranolazine, as claimed in any one of claims 1 to 4, wherein in the step (2), in the detection of high performance liquid chromatography, the mobile phase A is 0.1-0.3 vol% formic acid water solution, and the mobile phase B is acetonitrile;
preferably, in the step (2), isocratic elution is adopted in the detection of the high performance liquid chromatography, and the volume ratio of the mobile phase A to the mobile phase B is (30-50): (50-70).
6. The method for detecting the toxic impurity in ranolazine, according to any one of claims 1 to 5, wherein in the step (2), the chromatographic column in the high performance liquid chromatography is YMC-Pack Pro-C18;
preferably, in step (2), the specification of the chromatographic column is: the inner diameter is 4.6mm, the column length is 150-250mm, and the particle size is 3.0-5.0 μm.
7. The method for detecting the toxic impurity in ranolazine, according to any one of claims 1 to 6, wherein in the step (2), the flow rate in the detection of the high performance liquid chromatography is 0.3-0.7 mL/min;
preferably, in the step (2), the injection volume in the detection of the high performance liquid chromatography is 15-25 μ L;
preferably, in the step (2), the column temperature in the detection of the high performance liquid chromatography is 25-35 ℃;
preferably, in the step (2), the running time in the detection of the high performance liquid chromatography is 10-20 min.
8. The method for detecting ranolazine toxic impurities as claimed in any one of claims 1-7, wherein in step (2), the ion source in the mass spectrometry detection is a pneumatically-assisted electrospray ion source, and the polarity is positive;
preferably, in step (2), the mass spectrum is monitored in a multi-reaction monitoring mode, and the 122.1 → 105.1 channel is used for quantification.
9. The method for detecting the toxic impurities in ranolazine, as claimed in any one of claims 1 to 8, wherein in step (2), the fragmentation voltage is 40-60V, the collision energy is 10-20V, the flow rate of the drying gas is 5-15L/min, the temperature of the drying gas is 250-350 ℃, the atomization pressure is 45-55Psi, and the voltage of the capillary is 5500-6500 volts.
10. The method for detecting ranolazine toxic impurities as claimed in any one of claims 1-9, wherein the method comprises the steps of:
(1) respectively preparing a 20-30mg/mL sample solution of ranolazine and a 7-50ng/mL reference solution of 2, 6-dimethylaniline;
(2) injecting the sample solution of ranolazine obtained in the step (1) and the reference solution of 2, 6-dimethylaniline into a high performance liquid chromatography-tandem mass spectrometer for detection respectively, and calculating the content of the 2, 6-dimethylaniline in the ranolazine sample by adopting an external standard method;
wherein, the testing conditions of the high performance liquid chromatography comprise: the mobile phase A is 0.1-0.3 vol% formic acid water solution, the mobile phase B is acetonitrile, isocratic elution is adopted, and the volume ratio of the mobile phase A to the mobile phase B is (30-50): (50-70); the chromatographic column is YMC-PackPro-C18, the inner diameter is 4.6mm, the column length is 150-250mm, the particle size is 3.0-5.0 mu m, the flow rate is 0.3-0.7mL/min, the sample injection volume is 15-25 mu L, the column temperature is 25-35 ℃, and the running time is 10-20 min;
wherein, the test conditions of the mass spectrum comprise: the ion source is a pneumatic auxiliary electrospray ion source, the polarity is positive, the monitoring mode is multi-reaction monitoring, a 122.1 → 105.1 channel is used for quantification, the fragmentation voltage is 40-60V, the collision energy is 10-20V, the flow rate of the dry gas is 5-15L/min, the temperature of the dry gas is 250-350 ℃, the pressure of the atomization chamber is 45-55Psi, and the voltage of a capillary tube is 5500-6500 volts.
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