CN110873765B - Detection method of pixantrone maleate related substance - Google Patents
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Abstract
The invention provides a high performance liquid chromatography analysis and detection method of a maleic acid pixantrone related substance, which comprises the following steps: a. taking a pixantrone maleate test sample, and preparing a test sample solution by using a mixed solvent or a mobile phase; b. taking a test solution, adding a mixed solvent or a mobile phase for dilution, and taking the diluted solution as a self control solution; c. taking a self-control solution and a test solution, washing by using a mobile phase, detecting and recording a chromatogram; d. c, calculating the impurity content according to the self-contrast solution chromatogram and the sample solution chromatogram obtained in the step c; e. taking a self-contrast solution and a test solution, washing by using a mobile phase, detecting and recording a chromatogram; f. and e, calculating the content of impurities according to the chromatogram obtained in the step e. The method adopts two different mobile phase systems to elute the maleic acid pixantrone sample solution, so that the separation degree between the main peak and the impurity peak of the pixantrone maleate and the separation degree between the impurity peak and the impurity peak reach more than 2.0, thereby accurately detecting the content of related substances in the maleic acid pixantrone sample, and providing a set of related substance detection method with simple operation, convenience and quickness for the quality control of the maleic acid pixantrone raw material medicine and the preparation thereof.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a high-performance liquid phase analysis and detection method for chemicals, and particularly relates to a high-performance liquid phase analysis method for pixantrone maleate and related substances of a preparation thereof.
Background
The related substances are closely related to the quality, safety and curative effect of the medicine, and the existence of the related substances may reduce the stability, curative effect and the like of the medicine and even may cause toxic and side effects, so the types and the contents of the related substances in the medicine must be controlled by a proper detection and analysis method to ensure that the quality of the medicine meets the related requirements of laws and regulations.
Pixantrone maleate (Pixantrone maleate) having the chemical name 6, 9-bis [ (2-aminoethyl) amino ] benzo [ G ] isoquinoline-5, 10-dione dimaleate and having the chemical structure shown below:
the pixantrone is a novel cytotoxic azaanthracenedione drug, can directly alkylate DNA to form a stable DNA adduct, leads to the cross-linking of the DNA or the double-strand break of the DNA, and simultaneously, the pixantrone is an inhibitor of topoisomerase II, can lead to the disintegration of genome DNA, thereby finally blocking the proliferation of B lymphocytes to lead to the death of the cells. Therefore, it was approved by the european union as the first monotherapy drug worldwide to date for the treatment of multiple relapsed and refractory aggressive non-hodgkin B-cell lymphomas.
Patent CN105699548 discloses a high performance liquid chromatography analysis detection method of pixantrone maleate, which is used for detecting the purity of pixantrone maleate. The process involves 5 impurities, namely 6-fluoro-9-aminoethylamine benzo [ g ] isoquinoline-5, 10-dione, 9-fluoro-6-aminoethylamine benzo [ g ] isoquinoline-5, 10-dione, 6-aminoethylamine-8, 9-piperazinebenzo [ g ] isoquinoline-5, 10-dione, 9-aminoethylamine-6, 7-piperazinebenzo [ g ] isoquinoline-5, 10-dione and 6, 9-difluorobenzo [ g ] isoquinoline-5, 10-dione. When the method is used for carrying out quantitative analysis on the related substances of the pixantrone, the separation degree of all impurities can not meet the requirements of the current Chinese pharmacopoeia. In order to ensure the safety of medication, the raw material medicine of the maleic acid pixantrone and the preparation thereof need to be subjected to more sufficient impurity research, and a high performance liquid chromatography analysis and detection method for the relevant substances of the raw material medicine of the maleic acid pixantrone and the preparation thereof, which has high sensitivity and good separation degree of each impurity peak, is established.
Disclosure of Invention
The invention relates to a pixantrone maleate raw material and a related substance high performance liquid chromatography analysis detection method of a preparation thereof, which have the advantages of high sensitivity, good specificity, excellent durability and impurity peak separation degree of more than 2.0. The theoretical plate number, the separation degree, the tailing factor and the like of the method can meet the requirements of the existing Chinese pharmacopoeia and drug registration laws and regulations on a high performance liquid analysis detection method of the drug.
The method synthesizes and obtains the pixantrone maleate to be detected according to the route provided by the patent CN104557704 (shown in figure 1).
Through impurity research, the pixantrone maleate prepared according to the route shown in figure 1 may contain known impurities including impurity A (6- [ (2-aminoethyl) amino ] -9-fluoro-benzo [ G ] isoquinoline-5, 10-dione and 9- [ (2-aminoethyl) amino ] -6-fluoro-benzo [ G ] isoquinoline-5, 10-dione mixture), impurity B (6, 7-bis [ (2-aminoethyl) amino ] -benzo [ G ] isoquinoline-5, 10-dione, 7, 8-bis [ (2-aminoethyl) amino ] -benzo [ G ] isoquinoline-5, 10-dione and 8, 9-bis [ (2-aminoethyl) amino ] -benzo [ G ] isoquinoline-5, 10-dione mixture), the impurity C ((Z) -4- ((2- ((9- ((2-aminoethyl) amino) -5, 10-dioxo-5, 10-dihydro-benzo [ G ] isoquinolin-6-yl) amino) ethyl) amino) -4-oxobut-2-enoic acid and (Z) -4- ((2- ((6- ((2-aminoethyl) amino) -5, 10-dioxo-5, 10-dihydro-benzo [ G ] isoquinolin-9-yl) amino) ethyl) amino) -4-oxobut-2-enoic acid mixture), the impurity D (6-amino-9- [ (2-aminoethyl) amino ] -benzo [ G ] isoquinoline-5, 10-dione and 9-amino-6- [ (2-aminoethyl) amino ] -benzo [ G ] isoquinoline-5, 10-dione mixtures) and impurities E (6- [ (2-aminoethyl) amino ] -1,2,3, 4-tetrahydro-isoquinolino [6,7-f ] quinoxaline-7, 12-dione and 6- [ (2-aminoethyl) amino ] -1,2,3, 4-tetrahydro-isoquinolino [6,7-h ] quinoxaline-7, 12-dione mixtures). The structural formula is shown in the following table:
as can be seen from the above table, impurities A, C, D and E are both mixtures of two isomers, while impurity B is a mixture of three isomers. The polarity of these impurities is very close to that of pixantrone maleate, and it becomes extremely difficult to simultaneously detect each impurity using a high performance liquid chromatography method. Through literature search, no document reports the high performance liquid phase detection method of the impurity content.
Aiming at the defects in the prior art, the technical problem to be solved by the invention is how to accurately and quickly detect the content of each impurity in the pixantrone maleate by adopting a high performance liquid chromatography method. In order to solve the technical problems, the method creatively adopts two different mobile phase systems to elute the pixantrone maleate sample solution, so that the main peak of the pixantrone maleate is effectively separated from the impurity peak, and the impurity peak is effectively separated from the impurity peak, thereby accurately detecting the content of each related substance in the pixantrone maleate sample. The method thoroughly solves the problems of difficult separation of the main peak and impurity peaks of the pixantrone and each impurity peak, and provides a feasible technical scheme for high performance liquid detection of the pixantrone maleate related substances. The method is simple to operate, high in sensitivity and good in specificity and accuracy.
The invention provides a mobile phase for detecting related substances of a maleic acid pixantrone test sample;
the mobile phase is selected from mobile phase (1) or mobile phase (2);
the mobile phase (1) is prepared from a saline solution, methanol and acetonitrile according to the weight ratio of (30-95): (2.5-35): (2.5-35) by volume ratio;
the mobile phase (2) is prepared from a saline solution and acetonitrile according to the weight ratio of (30-95): (5-70) by volume;
the saline solution is an aqueous solution containing sodium dihydrogen phosphate and sodium heptanesulfonate, and the pH is adjusted to 2-4 by using phosphoric acid;
the molar concentration of sodium dihydrogen phosphate in the saline solution is 5-60 mmol/L;
preferably, the molar concentration of the sodium dihydrogen phosphate is 10-40 mmol/L;
more preferably, the molar concentration of the sodium dihydrogen phosphate is 15-30 mmol/L;
the mass percentage of the sodium heptanesulfonate to the water in the saline solution is 0.01-2%;
preferably, the mass percentage of the sodium heptanesulfonate to the water is 0.05-1.5%;
more preferably, the mass percent of the sodium heptanesulfonate and the water is 0.1-1%.
The invention also provides a high performance liquid chromatography analysis and detection method of the pixantrone maleate related substance, which comprises the following steps:
a. weighing a proper amount of pixantrone maleate, and adding a mixed solvent or flowing phase to prepare a pixantrone maleate solution serving as a test sample solution;
the prepared solution contains 0.5-2 mg/mL of pixantrone maleate;
preferably, the prepared solution contains 0.5-1.5 mg/mL of pixantrone maleate;
more preferably, the prepared solution contains 0.8-1.2 mg/mL of pixantrone maleate;
the mixed solvent is formed by mixing acetonitrile and a sodium chloride aqueous solution according to a certain volume ratio;
the mass percentage of sodium chloride and water in the sodium chloride aqueous solution is 0.01-2%;
preferably, the mass percentage of the sodium chloride and the water is 0.5-1.5%;
more preferably, the mass percent of the sodium chloride and the water is 0.8-1.2%;
the volume ratio of the acetonitrile to the sodium chloride aqueous solution is (15-65): (35-85);
preferably, the volume ratio of the acetonitrile to the sodium chloride aqueous solution is (15-45): (55-85);
more preferably, the volume ratio of the acetonitrile to the sodium chloride aqueous solution is (20-30): (70-80);
the mobile phase is selected from mobile phase (1) or mobile phase (2);
the mobile phase (1) is prepared from a saline solution, methanol and acetonitrile according to the weight ratio of (30-95): (2.5-35): (2.5-35) by volume ratio;
the mobile phase (2) is prepared from a saline solution and acetonitrile according to the weight ratio of (30-95): (5-70) by volume ratio;
the saline solution is an aqueous solution containing sodium dihydrogen phosphate and sodium heptanesulfonate, and the pH value is adjusted to 2-4 by using phosphoric acid;
the molar concentration of sodium dihydrogen phosphate in the saline solution is 5-60 mmol/L;
preferably, the molar concentration of the sodium dihydrogen phosphate is 10-40 mmol/L;
more preferably, the molar concentration of the sodium dihydrogen phosphate is 15-30 mmol/L;
the mass percentage of the sodium heptanesulfonate to the water in the saline solution is 0.01-2%;
preferably, the mass percentage of the sodium heptanesulfonate to the water is 0.05-1.5%;
more preferably, the mass percent of the sodium heptanesulfonate and the water is 0.1-1%;
b. precisely measuring 1mL of a sample solution, putting the sample solution into a 100mL measuring flask, adding a mixed solvent or a mobile phase for diluting to a scale, shaking up, precisely measuring 1mL or 2mL of the diluted solution, putting the diluted solution into a 10mL measuring flask, adding the mixed solvent or the mobile phase for diluting to the scale, and shaking up to serve as a self-control solution;
c. precisely measuring proper amounts of self-contrast solution and sample solution, respectively injecting into a High Performance Liquid Chromatograph (HPLC) with phenyl column, washing with mobile phase 1, detecting with ultraviolet light, and recording chromatogram;
the phenyl column is selected from a pentafluorophenyl column, an ether phenyl column, a propyl phenyl column and a hexyl phenyl column;
the flow rate of the mobile phase is 0.5-1.5 mL/min;
the wavelength of the ultraviolet light is 220 nm;
d. calculating the content of unknown impurities and known impurities B, D, E by using a self-comparison method according to the self-comparison solution chromatogram and the sample solution chromatogram obtained in the step (c);
e. respectively and precisely measuring a proper amount of a self-contrast solution and a proper amount of a sample solution, respectively injecting into a high performance liquid chromatograph, wherein a chromatographic column is a phenyl column, washing with a mobile phase 2, detecting with ultraviolet light, and recording a chromatogram;
the phenyl column is selected from a pentafluorophenyl column, an ether phenyl column, a propyl phenyl column and a hexyl phenyl column;
the flow rate of the mobile phase is 0.5-1.5 mL/min;
the wavelength of the ultraviolet light is 220 nm;
f. and (e) calculating the contents of the impurity A and the impurity C by adopting a self-comparison method according to the self-comparison solution chromatogram and the sample solution chromatogram obtained in the step (e).
The invention also provides a high performance liquid chromatography analysis and detection method of the related substances of the pixantrone maleate freeze-dried powder injection for injection, which comprises the following steps:
a. weighing a proper amount of the pixantrone maleate freeze-dried powder injection preparation for injection, and adding a mixed solvent or a flowing phase to prepare a pixantrone maleate solution as a test solution;
the prepared solution contains 0.5-2 mg/mL of pixantrone maleate;
preferably, the prepared solution contains 0.5-1.5 mg/mL of pixantrone maleate;
more preferably, the prepared solution contains 0.8-1.2 mg/mL of pixantrone maleate;
the mixed solvent is formed by mixing acetonitrile and a sodium chloride aqueous solution according to a certain volume ratio;
the mass percentage of sodium chloride and water in the sodium chloride aqueous solution is 0.01-2%;
preferably, the mass percentage of the sodium chloride and the water is 0.5-1.5%;
more preferably, the mass percent of the sodium chloride and the water is 0.8-1.2%;
the volume ratio of the acetonitrile to the sodium chloride aqueous solution is (15-65): (35-85);
preferably, the volume ratio of the acetonitrile to the sodium chloride aqueous solution is (15-45): (55-85);
more preferably, the volume ratio of the acetonitrile to the sodium chloride aqueous solution is (20-30): (70-80);
the mobile phase is prepared from a saline solution, methanol and acetonitrile according to the weight ratio of (30-95): (2.5-35): (2.5-35) by volume ratio;
the saline solution is an aqueous solution containing sodium dihydrogen phosphate and sodium heptanesulfonate, and the pH is adjusted to 2-4 by using phosphoric acid;
the molar concentration of sodium dihydrogen phosphate in the saline solution is 5-60 mmol/L;
preferably, the molar concentration of the sodium dihydrogen phosphate is 10-40 mmol/L;
more preferably, the molar concentration of the sodium dihydrogen phosphate is 15-30 mmol/L;
the mass percentage of the sodium heptanesulfonate to the water in the saline solution is 0.01-2%;
preferably, the mass percentage of the sodium heptanesulfonate to the water is 0.05-1.5%;
more preferably, the mass percent of the sodium heptanesulfonate and the water is 0.1-1%;
b. precisely measuring 1mL of a sample solution, placing the sample solution in a 100mL measuring flask, adding a mixed solvent or a mobile phase for diluting to a scale, shaking up, precisely measuring 2mL of the diluted solution, placing the diluted solution in a 10mL measuring flask, adding the mixed solvent or the mobile phase for diluting to the scale, and shaking up to serve as a self-control solution;
c. precisely measuring proper amounts of self-contrast solution and sample solution, respectively injecting into High Performance Liquid Chromatograph (HPLC), wherein the chromatographic column is phenyl column, washing with mobile phase, detecting with ultraviolet light, and recording chromatogram;
the phenyl column is selected from a pentafluorophenyl column, an ether phenyl column, a propyl phenyl column and a hexyl phenyl column;
the flow rate of the mobile phase is 0.5-1.5 mL/min;
the wavelength of the ultraviolet light is 220 nm;
d. and (c) calculating the content of related substances of the test sample by adopting a self-comparison method according to the self-comparison solution and the chromatogram of the test sample solution obtained in the step (c).
The method provided by the invention can be used for detecting related substances such as a bulk drug of the maleic acid pixantrone, a freeze-dried powder injection preparation of the maleic acid pixantrone, degradation products of the pixantrone and the like.
The pixantrone degradation product comprises a pixantrone degradation product under the conditions of high temperature, high humidity and illumination and a degradation product under the action of acid, alkali, oxidant or reducer.
Compared with the prior art, the method for detecting the maleic acid pixantrone and related substances of the preparation thereof has the advantages and beneficial effects that:
the method creatively adopts two different mobile phase systems to elute the maleic acid pixantrone sample solution, so that the separation degree between the main peak and the impurity peak of the pixantrone and the impurity peak is more than 2.0, the content of related substances in the maleic acid pixantrone sample is accurately detected, and a set of related substance detection method which is simple in operation, convenient and fast is provided for the quality control of the maleic acid pixantrone raw material medicine and the preparation thereof.
Drawings
FIG. 1 is a synthetic route of pixantrone maleate provided in patent CN 104557704;
FIG. 2 is an HPLC chromatogram for detecting related substances in the bulk drug of pixantrone maleate in example 1;
FIG. 3 is an HPLC chromatogram of a system suitability solution eluted by mobile phase 1 in example 2;
FIG. 4 is an HPLC chromatogram of a system suitability solution eluted by mobile phase 2 in example 2;
FIG. 5 is an HPLC chromatogram of a system suitability solution eluted by mobile phase 1 in example 3;
FIG. 6 is an HPLC chromatogram of a system suitability solution eluted by mobile phase 2 in example 3;
FIG. 7 is an HPLC chromatogram of a system suitability solution in example 4;
FIG. 8 is an HPLC chromatogram of a self-control solution eluted with mobile phase 1 of example 5;
FIG. 9 is an HPLC chromatogram of a test sample of pixantrone maleate according to example 5 showing the detection of the unknown impurity, known impurity B, D, E;
FIG. 10 is an HPLC chromatogram of a self-control solution eluted by mobile phase 2 of example 5;
FIG. 11 is an HPLC chromatogram of a test sample of pixantrone maleate of example 5 showing detection of the known impurity A, C;
FIG. 12 is an HPLC chromatogram of the self-control solution of pixantrone maleate powder injection for example 6;
FIG. 13 is an HPLC chromatogram for detecting substances related to an pixantrone maleate powder injection for example 6.
Detailed Description
In order to facilitate a better understanding of the present disclosure by those skilled in the art, the applicant further describes the technical solutions of the present disclosure by the following specific examples, but the following should not limit the scope of the present disclosure claimed in the claims in any way.
The main instruments and raw materials used in the examples were as follows:
a high performance liquid chromatograph: shimadzu LC-20AT
One-tenth-ten-thousandth balance: danfo TB-2015D
Pixantrone maleate control: the applicant synthesizes and refines according to the route of patent CN104557704, and carries out structure confirmation by methods of infrared spectroscopy (IR), Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS), ultraviolet spectroscopy (UV), powder X-ray diffraction (PXRD), Differential Scanning Calorimetry (DSC), thermogravimetric analysis (TG), Circular Dichroism (CD) and the like, and then calibrates according to the conventional prior art.
Impurity reference substance: impurity A (6- [ (2-aminoethyl) amino ] -9-fluoro-benzo [ G ] isoquinoline-5, 10-dione and a mixture of 9- [ (2-aminoethyl) amino ] -6-fluoro-benzo [ G ] isoquinoline-5, 10-dione), impurity B (6, 7-bis [ (2-aminoethyl) amino ] -benzo [ G ] isoquinoline-5, 10-dione, 7, 8-bis [ (2-aminoethyl) amino ] -benzo [ G ] isoquinoline-5, 10-dione and a mixture of 8, 9-bis [ (2-aminoethyl) amino ] -benzo [ G ] isoquinoline-5, 10-dione), impurity C ((Z) -4- ((2- ((9- ((2-aminoethyl) amino) -5, 10-dioxo-5, 10-dihydro-benzo [ G ] isoquinolin-6-yl) amino) ethyl) amino) -4-oxobut-2-enoic acid and (Z) -4- ((2- ((6- ((2-aminoethyl) amino) -5, 10-dioxo-5, 10-dihydro-benzo [ G ] isoquinolin-9-yl) amino) ethyl) amino) -4-oxobut-2-enoic acid mixture), the impurity D (6-amino-9- [ (2-aminoethyl) amino ] -benzo [ G ] isoquinoline-5, 10-dione and 9-amino-6- [ (2-aminoethyl) amino ] -benzo [ G ] isoquinoline-5, 10-diketone mixture) and the impurities E (6- [ (2-aminoethyl) amino ] -1,2,3, 4-tetrahydro-isoquinolino [6,7-f ] quinoxaline-7, 12-dione and 6- [ (2-aminoethyl) amino ] -1,2,3, 4-tetrahydro-isoquinolino [6,7-h ] quinoxaline-7, 12-dione mixture) were purchased from a company with legal qualifications for the reference substance for the supply of chemical impurities and were subject to structural confirmation.
The crude drug of the maleopitron is synthesized by the applicant according to the route of the patent CN 104557704.
The maleopitron freeze-dried powder injection for injection is prepared by the applicant according to the conventional prior art.
Methanol, acetonitrile, sodium heptanesulfonate: and (4) carrying out chromatographic purification.
Other solvents and reagents are analytically pure.
Example 1 attempts of the literature method
A chromatographic column: alltima C18, 4.6mmx150mm, 5 μm.
Preparation of a mobile phase:
precisely weighing 3.0336g of sodium heptanesulfonate, adding 750mL of water, stirring to completely dissolve the sodium heptanesulfonate, then adding 200mL of acetonitrile and 50mL of dioxane, stirring to uniformly mix the solution, adding 3-4 drops of phosphoric acid to adjust the pH value to 3.0 (neglecting the influence of the added phosphoric acid on the volume of a mobile phase), and filtering through a 0.45-micron organic filter membrane to obtain a solution with the volume ratio of water to acetonitrile to dioxane of 75: 20: 5, the concentration of the sodium heptane sulfonate is 20 mmol/L.
Preparing a test solution:
9.98mg of pixantrone maleate raw material medicine is precisely weighed, placed in a 10mL volumetric flask, dissolved by adding a mobile phase and diluted to a scale, and shaken uniformly to obtain a test solution with the concentration of 998 mug/mL.
Injecting 20 μ l of the sample solution into a high performance liquid chromatograph, measuring the flow rate of mobile phase at 1.2mL/min and the column temperature at 40 deg.C, recording chromatogram (shown in figure 2), and calculating the contents of pixantrone maleate and various impurities by area normalization method.
In the attached figure 2, the retention time of the main peak of the maleic acid pixantrone is 37.184min, the theoretical plate number is only 4335, the peak height is only 191811, the theoretical plate number and the response value are both low, the peak shape symmetry is poor, and impurity peaks are contained in the main peak.
Example 2 System suitability experiment
A chromatographic column: welch Ultimate PFP, 4.6mmx250mm, 5 μm.
Preparation of a mobile phase:
3.9204g of sodium dihydrogen phosphate dihydrate and 5.0016g of sodium heptanesulfonate are weighed respectively, 1L of water is added for dissolution, the pH is adjusted to 2.5 by phosphoric acid, a saline solution is obtained, and the influence of the added sodium dihydrogen phosphate, sodium heptanesulfonate and phosphoric acid on the volume is ignored in the preparation process.
The brine solution, methanol and acetonitrile were mixed as 50: 25: 25 volume ratio, and filtering through a 0.45 mu m organic filter membrane to obtain a mobile phase 1.
The brine solution and acetonitrile were mixed as 50: 50 volume ratio, and filtering through a 0.45 mu m organic filter membrane to obtain a mobile phase 2.
Preparation of system applicability solution:
respectively and precisely weighing A, B, C, D, E known impurities and an appropriate amount of pixantrone maleate reference substance, adding the mobile phase 1 to dissolve and dilute the reference substance to prepare a mixed solution containing 0.8mg of pixantrone maleate and A, B, C, D, E known impurities of 1 mu g each in 1mL of the mixed solution as a system applicability solution.
Precisely measuring 20 μ l of the above system applicability solution, injecting into high performance liquid chromatograph, eluting with mobile phase 1 at flow rate of 1.0mL/min, column temperature of 45 deg.C, and recording chromatogram (shown in figure 3). The relative retention times, theoretical plate numbers, degrees of separation, and tailing factors for the unknown impurity, the known impurity B, D, E are shown in the following table:
injecting 20 μ l of the above system applicability solution into high performance liquid chromatograph, eluting with mobile phase 2 at flow rate of 1.0mL/min and column temperature of 45 deg.C, and recording chromatogram (shown in figure 4). The relative retention times, theoretical plate numbers, degrees of separation, and tailing factors for impurity A, C are known as shown in the following table:
example 3 System suitability experiment
A chromatographic column: welch Ultimate PFP, 4.6mmx250mm, 5 μm.
Preparation of a mobile phase:
3.1285g of sodium dihydrogen phosphate dihydrate and 2.0012g of sodium heptanesulfonate are weighed respectively, 1L of water is added for dissolution, the pH is adjusted to 2.5 by phosphoric acid, a saline solution is obtained, and the influence of the added sodium dihydrogen phosphate, sodium heptanesulfonate and phosphoric acid on the volume is ignored in the preparation process.
The brine solution, methanol and acetonitrile were mixed as a 90: 5: 5, and filtering the mixture through a 0.45 mu m organic filter membrane to obtain a mobile phase 1.
The brine solution and acetonitrile were mixed as 90: 10 volume ratio, and filtering through a 0.45 mu m organic filter membrane to obtain a mobile phase 2.
Preparation of system applicability solution:
an appropriate amount of known impurity A, B, C, D, E and an appropriate amount of pixantrone maleate control were precisely weighed, and dissolved and diluted with a mixed solvent (acetonitrile: 0.9% NaCl aqueous solution: 20: 80) to prepare a mixed solution containing about 1mg of pixantrone maleate and 1 μ g of known impurity A, B, C, D, E per 1mL, as a system suitability solution.
Precisely measuring 20 μ l of the above system applicability solution, injecting into high performance liquid chromatograph, eluting with mobile phase 1 at flow rate of 1.0mL/min, column temperature of 45 deg.C, and recording chromatogram (shown in figure 5). The relative retention times, theoretical plate numbers, degrees of separation, and tailing factors for the unknown impurity, the known impurity B, D, E are shown in the following table:
injecting 20 μ l of the above system applicability solution into high performance liquid chromatograph, eluting with mobile phase 2 at flow rate of 1.0mL/min and column temperature of 45 deg.C, and recording chromatogram (shown in figure 6). The relative retention times, theoretical plate numbers, degrees of separation, and tailing factors for impurity A, C are known as shown in the following table:
example 4 System suitability experiment
A chromatographic column: welch Ultimate PFP, 4.6mmx250mm, 5 μm.
Preparation of a mobile phase:
4.6801g of sodium dihydrogen phosphate dihydrate and 8.0003g of sodium heptanesulfonate are weighed respectively, 1L of water is added for dissolution, the pH is adjusted to 2.5 by phosphoric acid, a saline solution is obtained, and the influence of the added sodium dihydrogen phosphate, sodium heptanesulfonate and phosphoric acid on the volume is ignored in the preparation process.
The brine solution, methanol and acetonitrile were mixed as 30: 35: 35, and filtering the mixture through a 0.45 mu m organic filter membrane to obtain a mobile phase.
Preparation of system applicability solution:
respectively and precisely weighing B, C, D, E known impurities and an appropriate amount of pixantrone maleate reference substance, adding a mobile phase 1, dissolving and diluting to prepare a mixed solution containing 1.2mg of pixantrone maleate and B, C, D, E known impurities, each 2 mu g of the pixantrone maleate and the pixantrone maleate reference substance, and using the mixed solution as a system applicability solution.
Precisely measuring 20 μ l of the above system applicability solution, injecting into high performance liquid chromatograph, eluting with mobile phase at flow rate of 1.0mL/min, column temperature of 45 deg.C, and recording chromatogram (shown in figure 7). The relative retention times, theoretical plate numbers, degrees of separation, and tailing factors for the unknown impurity, the known impurity B, C, D, E are shown in the following table:
example 5 detection of pixantrone maleate related substances
A chromatographic column: welch Ultimate PFP, 4.6mmx250mm, 5 μm.
Preparing a test solution:
10.0014mg of pixantrone maleate was precisely weighed, and 10mL of a mixed solvent (acetonitrile: 0.9% aqueous NaCl solution: 20: 80) was added to dissolve and dilute the solution to prepare a solution containing about 1mg of pixantrone maleate per 1mL, as a test solution.
Preparation of a self-control solution:
precisely measuring 1mL of a sample solution, placing the sample solution into a 100mL measuring flask, adding a mixed solvent (acetonitrile: 0.9% NaCl aqueous solution ═ 20: 80) to dilute to a scale, shaking up, precisely measuring 1mL of the solution diluted by 100 times, placing the solution into a 10mL measuring flask, adding a mixed solvent (acetonitrile: 0.9% NaCl aqueous solution ═ 20: 80) to dilute to a scale, and shaking up to obtain a self-control solution;
preparation of a mobile phase:
6.2507g of sodium dihydrogen phosphate and 4.0018g of sodium heptanesulfonate are weighed respectively, 2L of water is added for dissolving, the pH is adjusted to 2.5 by phosphoric acid, a saline solution is obtained, and the influence of the added sodium dihydrogen phosphate, sodium heptanesulfonate and phosphoric acid on the volume is ignored in the preparation process.
The brine solution, methanol and acetonitrile were mixed as a 90: 5: 5, and filtering the mixture through a 0.45 mu m organic filter membrane to obtain a mobile phase 1.
The brine solution and acetonitrile were mixed as 90: 10 volume ratio, and filtering through a 0.45 mu m organic filter membrane to obtain a mobile phase 2.
Injecting 20 μ l of the self-control solution into high performance liquid chromatograph, eluting with mobile phase 1 at flow rate of 1.0mL/min and column temperature of 45 deg.C, and recording chromatogram (shown in figure 8) to obtain self-control peak area of 916619.
Injecting 20 μ l of the sample solution into high performance liquid chromatograph, eluting with mobile phase 1 at flow rate of 1.0mL/min and column temperature of 45 deg.C, and recording chromatogram (shown in figure 9).
In conjunction with fig. 5 of example 3, the assignment of each chromatographic peak in fig. 9 was identified and the content of each impurity was calculated according to the following formula:
unknown single impurity% ═ A unknown impurity/A vs. X0.1%
Known impurities% ═ a known impurities/a pair × 0.1% × f
Total impurities%
In the formula, impurity A: single maximum impurity peak area
Known impurities of A: known impurity peak area
f: correction factor, value 1.0
A pair: area of self-control peak
The contents of unknown impurity and known impurity B, D, E in the obtained sample are shown below:
injecting 20 μ l of the self-control solution into high performance liquid chromatograph, eluting with mobile phase 2 at flow rate of 1.0mL/min and column temperature of 45 deg.C, and recording chromatogram (shown in figure 10) to obtain self-control peak area 44968.
Injecting 20 μ l of the sample solution into high performance liquid chromatograph, eluting with mobile phase 2 at flow rate of 1.0mL/min and column temperature of 45 deg.C, and recording chromatogram (shown in figure 11).
With reference to fig. 6 of example 3, the assignment of each chromatographic peak in fig. 11 was identified, and the content of known impurity A, C was calculated according to the self-comparison calculation formula described above, with the following results:
in summary, the maximum unknown impurity content in the sample was 0.07%, the known impurity A, B, E was not detected, the known impurity C, D content was 0.07% and 0.01%, respectively, and the total impurity content was 0.19%.
Example 6 detection of related substances of pixantrone maleate freeze-dried powder injection for injection
As can be seen from example 5, the pixantrone maleate raw material drug prepared by the applicant does not contain the known impurity A, B, E, so that the pixantrone maleate freeze-dried powder injection prepared by the self-prepared raw material drug through a conventional technical means does not contain the known impurity A, B, E. Therefore, only one mobile phase is needed for eluting the relevant substances of the pixantrone maleate freeze-dried powder injection for injection.
A chromatographic column: welch Ultimate PFP, 4.6mmx250mm, 5 μm.
Preparation of a mobile phase:
4.6801g of sodium dihydrogen phosphate dihydrate and 8.0003g of sodium heptanesulfonate are weighed respectively, 1L of water is added for dissolution, the pH is adjusted to 2.5 by phosphoric acid, a saline solution is obtained, and the influence of the added sodium dihydrogen phosphate, sodium heptanesulfonate and phosphoric acid on the volume is ignored in the preparation process.
The brine solution, methanol and acetonitrile were mixed as 30: 35: 35, and filtering the mixture through a 0.45 mu m organic filter membrane to obtain a mobile phase.
Preparing a test solution:
9.9987mg of pixantrone maleate freeze-dried powder injection for injection is precisely weighed, 10mL of mobile phase is added for dissolution and dilution to prepare a solution containing 1mg of pixantrone maleate in each 1mL, and the solution is used as a test solution.
Preparation of a self-control solution:
precisely measuring 1mL of a sample solution, placing the sample solution in a 100mL measuring flask, adding a mobile phase to dilute the sample solution to a scale, shaking up, precisely measuring 2mL of the solution diluted by 100 times, placing the solution in a 10mL measuring flask, adding the mobile phase to dilute the solution to the scale, and shaking up to obtain a self-contrast solution;
injecting 20 μ l of the self-control solution into high performance liquid chromatograph, eluting with mobile phase 1 at flow rate of 1.0mL/min and column temperature of 45 deg.C, and recording chromatogram (shown in figure 12) to obtain self-control peak area of 1672563.
Injecting 20 μ l of the sample solution into high performance liquid chromatograph, eluting with mobile phase 1 at flow rate of 1.0mL/min and column temperature of 45 deg.C, and recording chromatogram (shown in figure 13).
In conjunction with fig. 5 of example 4, the assignment of each chromatographic peak in fig. 13 was identified and the content of each impurity was calculated according to the following formula:
unknown single impurity% ═ A unknown impurity/A vs. X0.2%
Known impurities% ═ a known impurities/a pair × 0.2% × f
Total impurities%
In the formula, impurity A: single maximum impurity peak area
Known impurities of A: known impurity peak area
f: correction factor, value 1.0
A pair: area of self-control peak
The contents of the unknown impurity and the known impurity D, C in the test sample are shown below:
as can be seen from the above table, the maximum unknown single impurity content in the test sample is 0.07%, the known impurity C, D content is 0.10% and 0.02%, respectively, and the total impurity content is 0.27%.
Claims (3)
1. A high performance liquid chromatography analysis and detection method for a maleic acid pixantrone related substance comprises the following steps:
a. taking a maleic acid pixantrone test sample, and preparing a maleic acid pixantrone-containing solution as a test sample solution by using a mixed solvent or a mobile phase; the volume ratio of the mixed solvent is (15-65): (35-85) mixing acetonitrile and a sodium chloride aqueous solution, wherein the mass percentage of sodium chloride and water in the sodium chloride aqueous solution is 0.01-2%; the mobile phase is selected from mobile phase (1) or mobile phase (2); the mobile phase (1) is prepared from (30-90) by volume: (5-35): (5-35) mixing a saline solution, methanol and acetonitrile; the mobile phase (2) is prepared from (50-90): (10-50) mixing a saline solution and acetonitrile; the saline solution is obtained by dissolving sodium dihydrogen phosphate and sodium heptanesulfonate with water and then adjusting the pH to 2-4 with phosphoric acid, wherein the molar concentration of the sodium dihydrogen phosphate is 5-60 mmol/L, and the mass percentage of the sodium heptanesulfonate to the water is 0.01-2%;
b. taking a test solution, adding a mixed solvent or a mobile phase for dilution, and taking the diluted solution as a self control solution;
c. injecting self-contrast solution and test solution into a high performance liquid chromatograph with a chromatographic column model of Welch Ultimate PFP, specification of 4.6mm x250mm and 5 μm, respectively, washing with a mobile phase (1), detecting with ultraviolet light, and recording chromatogram;
d. c, calculating the content of the unknown impurity and the known impurity B, D, E by adopting a self-comparison method according to the self-comparison solution chromatogram and the sample solution chromatogram obtained in the step c;
e. respectively injecting the self-contrast solution and the test solution into a high performance liquid chromatograph with a chromatographic column model of Welch Ultimate PFP, specification of 4.6mm x250mm and 5 mu m, washing with a mobile phase (2), detecting with ultraviolet light, and recording a chromatogram;
f. calculating the content of the known impurity A, C by using a self-comparison method according to the self-comparison solution chromatogram and the sample solution chromatogram obtained in the step e;
the structure of impurity A, B, C, D, E is shown in the following table:
。
2. the method of claim 1, wherein: in the step a, the pixantrone maleate test sample is a pixantrone maleate raw material medicine or a pixantrone maleate freeze-dried powder injection preparation for injection.
3. The method of claim 1, wherein: in the step a, the test solution contains 0.5-2 mg/mL of maleopitron.
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