CN113804793B - Impurity detection method for pimobendan soft chewing dosage form - Google Patents
Impurity detection method for pimobendan soft chewing dosage form Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 96
- 229960002164 pimobendan Drugs 0.000 title claims abstract description 66
- GLBJJMFZWDBELO-UHFFFAOYSA-N pimobendane Chemical compound C1=CC(OC)=CC=C1C1=NC2=CC=C(C=3C(CC(=O)NN=3)C)C=C2N1 GLBJJMFZWDBELO-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 238000001514 detection method Methods 0.000 title claims abstract description 48
- 239000002552 dosage form Substances 0.000 title claims abstract description 36
- 230000001055 chewing effect Effects 0.000 title description 25
- 239000000463 material Substances 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 33
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 8
- AUALQMFGWLZREY-UHFFFAOYSA-N acetonitrile;methanol Chemical compound OC.CC#N AUALQMFGWLZREY-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 5
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 5
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 5
- 230000001105 regulatory effect Effects 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 27
- 239000012488 sample solution Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 239000012085 test solution Substances 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000013558 reference substance Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 125000004172 4-methoxyphenyl group Chemical group [H]C1=C([H])C(OC([H])([H])[H])=C([H])C([H])=C1* 0.000 claims description 3
- WJMJXFIZZSXKST-UHFFFAOYSA-N n-[2-amino-4-(4-methyl-6-oxo-4,5-dihydro-1h-pyridazin-3-yl)phenyl]-4-methoxybenzamide Chemical compound C1=CC(OC)=CC=C1C(=O)NC1=CC=C(C=2C(CC(=O)NN=2)C)C=C1N WJMJXFIZZSXKST-UHFFFAOYSA-N 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 125000000336 imidazol-5-yl group Chemical group [H]N1C([H])=NC([H])=C1[*] 0.000 claims description 2
- 230000008859 change Effects 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 239000003755 preservative agent Substances 0.000 abstract description 4
- 230000002335 preservative effect Effects 0.000 abstract description 4
- 238000004811 liquid chromatography Methods 0.000 abstract description 3
- 230000004044 response Effects 0.000 abstract description 3
- 239000003814 drug Substances 0.000 description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 229940079593 drug Drugs 0.000 description 7
- 239000011159 matrix material Substances 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 4
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 4
- 241000282472 Canis lupus familiaris Species 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 description 2
- 206010019280 Heart failures Diseases 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- HDZOBAOPAVYPNF-UHFFFAOYSA-N 1h-benzimidazole;1h-pyridazin-6-one Chemical class O=C1C=CC=NN1.C1=CC=C2NC=NC2=C1 HDZOBAOPAVYPNF-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000000150 Sympathomimetic Substances 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- FGDQGIKMWOAFIK-UHFFFAOYSA-N acetonitrile;phosphoric acid Chemical group CC#N.OP(O)(O)=O FGDQGIKMWOAFIK-UHFFFAOYSA-N 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000006184 cosolvent Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- -1 imidazole-5-yl Chemical group 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000297 inotrophic effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000018984 mastication Effects 0.000 description 1
- 238000010077 mastication Methods 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001975 sympathomimetic effect Effects 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
-
- 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
- G01N30/74—Optical detectors
Abstract
The impurity detection method of the pimobendan soft chewable dosage form comprises the steps of measuring by using three-phase high performance liquid chromatography, wherein the chromatographic conditions are Bao Sepu columns: c18,4.6mm×250mm,4 μm; mobile phase a:2.6g/L sodium dihydrogen phosphate solution, and regulating the pH value to 3.0 by phosphoric acid; mobile phase B: acetonitrile-methanol, 80:20; the column temperature is 45 ℃; the detection wavelength is 290nm; the flow rate is 1.0ml/min; and (3) measuring the pimobendan soft chewable dosage form solution under the condition of liquid chromatography, and judging the impurity condition according to the main peak and the impurity peak. In addition, under the method, the preservative in the pimobendan soft chewable dosage form can be detected. According to the invention, the main peak of pimobendan is enabled to be out of a peak at the position of moderating gradient change by adjusting a mobile phase system and optimizing a mobile phase gradient, so that auxiliary materials are completely separated from the main peak and an impurity peak, the auxiliary materials do not interfere with the detection of impurities, and the pimobendan has the advantages of strong specificity, high sensitivity and high impurity response.
Description
Technical Field
The invention relates to a method for detecting impurities in a pimobendan soft chewable dosage form, and belongs to the technical field of medicine detection.
Background
Heart disease is one of the most common aging diseases of dogs and cats, pimobendan is a benzimidazole-pyridazinone derivative, is a non-sympathomimetic and non-glycoside positive inotropic medicament, and a large number of experiments prove that the pimobendan is superior to the conventional medicament in treating heart failure of dogs, has good safety and tolerance, can obviously improve the life quality and the life quality of dogs, and becomes a medicament for treating heart failure with wide clinical application. The soft chewing formulation has poor compliance for taking medicine of sick animals, and can greatly improve the problem, so the pimobendan soft chewing formulation is a research hot spot at present, but the matrix of the soft chewing formulation is complex, and contains more flavoring agent, so the stability of the main component is greatly affected.
The safety and effectiveness of a drug in clinical use are related to the pharmacological activity of the drug itself and also related to impurities in the drug. Impurity detection is an important link of medicine quality control, and impurity sources and contents can be ascertained through impurity inspection of medicines, so that medicine quality is ensured through optimizing prescriptions or improving medicine packaging forms and the like, and adverse reactions of medicines are reduced.
For soft chewing dosage forms containing the p Mo Ben pill, the impurity detection method has good specificity and high sensitivity, and because of the complexity of the soft chewing matrix and the fact that each auxiliary material basically appears a peak at the ultraviolet position, the auxiliary material is difficult to distinguish from the impurity, and the auxiliary material interferes with the detection of the impurity, the impurity detection method has high specificity and needs to detect the impurity as much as possible.
The European pharmacopoeia can not detect the impurity in the soft chewing dosage form at the same time due to the reasons, especially the auxiliary materials of the soft chewing matrix have obvious interference on the impurity, so that the detection result is inaccurate, and the accuracy of medicine quality judgment is further affected. Therefore, it is desirable to design an impurity detection method for pimobendan soft chewable dosage forms, which has good specificity for soft chewable dosage forms, can distinguish auxiliary materials from impurities, and can detect impurities as much as possible. In the prior art, the European pharmacopoeia analysis method comprises the following steps:
chromatographic column: c18 (125 mm 4.6mm,5 μm);
mobile phase a:3g/L potassium dihydrogen phosphate (pH 2.5);
mobile phase B: acetonitrile;
column temperature: 45 ℃;
wavelength: 290nm;
flow rate: 1.0ml/min;
gradient elution procedure
| Time (min) | Mobile phase a | Mobile phase B |
| 0-6 | 85→80 | 15→20 |
| 6-20 | 80→20 | 20→80 |
| 20-20.1 | 20→85 | 80→15 |
| 20.1-30 | 85 | 15 |
The impurity separation degree under the method is shown as a pimobendan impurity detection map in soft chewing under the EP method of the attached figure 1.
As can be seen from FIG. 1, in the EP method, the gradient change of the mobile phase at the position of 6-20min is more severe, the base line is uneven, the main peak appears at the position, and the auxiliary materials interfere the main peak and the 2 peaks of the known single impurities, so that the method is not suitable for detecting the pimobendan impurities in the soft chewing dosage form.
The prior art has the following defects:
1. in the prior art, the pH of the mobile phase is 2.5, the pimobendan impurity and the auxiliary material peak in the soft chewing dosage form cannot be completely separated under the pH of the mobile phase, and the pH tolerance range of a common chromatographic column is 2-8, so that the service life of the chromatographic column can be greatly influenced.
2. In the prior art, two-phase gradient elution is adopted, and for detecting impurities in pimobendan soft chewing, auxiliary materials, the impurities and main peaks cannot be separated, so that accurate detection of the impurities cannot be realized.
3. Under the prior art condition, the peak-out time of the main peak of pimobendan is equal to that of the peak-out position of the main peak of pimobendan at the position of the severe gradient change of the mobile phase, the severe gradient change of the mobile phase can cause obvious baseline fluctuation, the main peak is at the position of the peak-out position, the detection condition of impurities before and after the main peak can be influenced, the impurities before and after the main peak can not be distinguished from the baseline fluctuation, the detection sensitivity is influenced, and the impurity detection is influenced.
4. The mobile phase system in the prior art is phosphate-acetonitrile, wherein phosphate is mainly potassium salt, the tailing improvement effect of potassium salt on alkaline compounds is obvious, while pimobendan is alkaline compound, because the solubility of pimobendan is poor, proper cosolvent or surfactant is usually added in soft chewing to increase the solubility of pimobendan, and the compatibility of potassium salt and certain surfactants is poor, so that when the potassium salt is used as a mobile phase elution sample, the service life of the chromatographic column is greatly influenced.
5. For soft chewing of a dosage form with a complex matrix, the auxiliary material peak usually has ultraviolet absorption, and the auxiliary material peak cannot be completely separated from impurities and main peaks in the prior art, so that the detection of the impurities cannot be realized, which is the biggest defect in the prior art.
Disclosure of Invention
The invention aims to provide an impurity detection method of pimobendan in a soft chewing dosage form, so as to overcome the defects of the prior art.
The impurity detection method of the pimobendan soft chewable dosage form is characterized by measuring by using three-phase high performance liquid chromatography, wherein the chromatographic conditions are as follows:
chromatographic column: c18,4.6mm×250mm,4 μm;
mobile phase a:2.6g/L sodium dihydrogen phosphate solution, and regulating the pH value to 3.0 by phosphoric acid;
mobile phase B: acetonitrile-methanol, 80:20;
the column temperature is 45 ℃;
the detection wavelength is 290nm;
the flow rate is 1.0ml/min;
gradient elution:
| time (min) | Mobile phase a | Mobile phase B |
| 0-20 | 85-75 | 15-25 |
| 20-45 | 75-40 | 25-60 |
| 45-45.01 | 40-85 | 60-15 |
| 45.01-55 | 85 | 15 |
And (3) detecting the impurity of the pimobendan soft chewable dosage form, measuring the sample solution under the condition of liquid chromatography, and judging the impurity condition according to the main peak and the impurity peak.
The impurities of the pimobendan soft chewable dosage form comprise impurity A:4- (2- (4-methoxyphenyl) -1H-benzo [ d ] imidazol-5-yl) -3-methyl-4-oxobutanoic acid, or impurity B: n- (2-amino-4- (4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) -4-methoxybenzamide. Other impurities than the impurities a and B are also included.
Impurity A:4- (2- (4-methoxyphenyl) -1H-benzo [ d ] imidazole-5-yl) -3-methyl-4-oxobutanoic acid and pimobendan bulk drug process impurities have the following structural formula:
impurity B: n- (2-amino-4- (4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) -4-methoxybenzamide and pimobendan bulk drug process impurities have the following structural formula:
the method comprises the steps of preparing a sample solution, preparing a system applicability solution and preparing a blank auxiliary material solution before the step of measuring by using the three-phase high performance liquid chromatography;
the preparation of the test solution comprises the following steps: weighing and grinding pimobendan, soft chewing, adding methanol into a volumetric flask for ultrasonic dissolution, diluting to a scale, centrifuging for 10min at 10000r/min, taking supernatant, filtering, discarding primary filtrate, taking continuous filtrate as a test sample solution containing pimobendan, and diluting with methanol to ensure that the concentration of the pimobendan is 0.5mg/ml;
the system applicability solution preparation comprises the following steps: taking a proper amount of impurity A, B and pimobendan reference substance, precisely weighing, dissolving with methanol, and quantitatively diluting to prepare mixed reference substance solutions containing 1 mug of impurity A, B and 0.5mg of pimobendan in each 1ml, as a system applicability test solution;
the preparation of the blank auxiliary material solution comprises the following steps: obtaining a blank auxiliary material solution with the same volume as the sample solution according to the preparation mode of the sample solution;
the above solutions are measured under the condition of liquid chromatography, auxiliary material peaks are deducted from the solution of the test sample, 5 unknown impurities are detected besides the impurities A and B, the separation degree before and after the main peak is in accordance with the regulation (more than 2.0), the peak type of the main peak and the theoretical plate number can meet the detection requirement (theoretical plate number 32250), the auxiliary material does not interfere the detection of the main peak and each impurity peak, and the method can be used for detecting the impurities of the pimobendan soft chewing dosage form.
In addition, under the method, the preservative in the pimobendan soft chewable dosage form can be detected, namely the invention can detect impurities and the preservative in the preparation at the same time. Namely the application of the method in detecting the preservative of the pimobendan soft chewable dosage form.
In the prior art, the impurities of pimobendan in the soft chewable dosage form cannot be accurately measured, the auxiliary materials interfere a main peak or the impurities appear, and under the method, the auxiliary materials can be distinguished from the main peak and the impurities, the auxiliary materials do not interfere the detection of the impurities, and the method has the advantages of high sensitivity and high impurity response.
The invention optimizes the chromatographic column, optimizes the chromatographic column of the original method C18 (4.6 x 125mm,5 mu m) into the chromatographic column of C18 (4.6 x 250mm,4 mu m), and can better separate auxiliary materials in soft chewing from main peaks and impurity peaks by longer column length and smaller particle size.
The method can distinguish auxiliary materials from main peaks and impurities in the pimobendan soft chewing, the auxiliary materials do not interfere with impurity detection, the durability of the chromatographic column is increased, and the cost is saved to a certain extent.
The invention completely separates auxiliary materials from the main peak and impurity peak of the pimobendan under the soft chewing dosage form by adjusting the mobile phase system and optimizing the mobile phase gradient, the service life of the chromatographic column can be greatly prolonged by the optimized mobile phase system, the durability of the method is improved, the peak of the pimobendan is discharged from the position of the gradient change alleviation by the optimized mobile phase gradient, and the detection of impurities before and after the main peak is interfered by the baseline fluctuation caused by severe gradient change is avoided. Therefore, the invention realizes the impurity detection of pimobendan under soft chewing dosage forms.
Drawings
Fig. 1 is a graph of pimobendan impurity detection in soft mastication under EP method.
Figure 2 a map of pimobendan impurity detection under the method.
FIG. 3 shows an impurity detection spectrum under the method of example 1.
FIG. 4 impurity detection scheme under the method of example 2.
FIG. 5 impurity detection map under the method of example 3.
FIG. 6 impurity detection scheme under the method of example 5.
Detailed Description
The various reagents used in the present invention are as follows:
solvent: methanol
Preparing a system applicability solution: a mixed reference solution containing pimobendan 0.5mg/ml, impurity A1 μg/ml and impurity B1 μg/ml.
Preparing a test solution: weighing a proper amount of ground pimobendan soft chewable dosage form to obtain a weight equivalent to 5mg of pimobendan (the pimobendan in the soft chewable dosage form has a fixed proportion, so that the required weight of the pimobendan can be obtained by selecting a proper amount of the whole pimobendan soft chewable dosage form), placing into a 10ml volumetric flask, adding methanol for ultrasonic dissolution, diluting to a scale, centrifuging at 10000r/min for 10min, taking supernatant, filtering, discarding initial filtrate, and taking subsequent filtrate as a sample solution containing 0.5mg/ml of the pimobendan.
Preparing a blank auxiliary material solution: weighing a proper amount of ground pimobendan soft chewing blank auxiliary material (corresponding to the blank auxiliary material amount containing 5mg of pimobendan), placing into a 10ml volumetric flask, adding methanol for ultrasonic dissolution, diluting to a scale, centrifuging for 10min at 10000r/min, taking supernatant, filtering, removing primary filtrate, and taking subsequent filtrate as blank auxiliary material solution.
And (3) taking the solutions to measure according to the chromatographic conditions, wherein auxiliary materials do not interfere with detection of main peaks and impurity peaks.
The pimobendan impurity detection map in the method is shown in figure 2, and as can be seen from figure 2, the baseline at the peak outlet of the main peak is gentle, the separation degree of the main peak before and after is good, the auxiliary material peak is completely separated from the main peak and the 2 known impurity peaks, and the method has strong specificity. The invention is further illustrated by the following examples.
Example 1
Adjusting the ratio of chromatographic column and mobile phase based on EP method
Chromatographic conditions: agilent C18 column (4.6 x 250mm,5 μm); mobile phase a:3g/L potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid), mobile phase B: acetonitrile; detection wavelength: 290nm, flow rate 1.0ml/min; column temperature: 45 ℃;
| time (min) | Mobile phase a | Mobile phase B |
| 0.00 | 85 | 15 |
| 10.00 | 80 | 20 |
| 30.00 | 40 | 60 |
| 30.01 | 85 | 15 |
| 40.00 | 85 | 15 |
The detection results are shown in FIG. 3, and under the condition of example 1, the auxiliary materials interfere with main peak and impurity detection at 13min and 17min respectively.
Example 2
Chromatographic conditions: based on example 1, the pH of mobile phase a was adjusted to pH3.0 and the column was Agilent C18 (4.6 x 150mm,4 μm).
As shown in fig. 4, under the conditions of example 2, the main peak time was about 15min, but the auxiliary materials still interfered with the main peak and impurity detection.
Example 3
Observing the experimental result by adjusting the proportion of mobile phase
Chromatographic conditions: agilent C18 column (4.6 x 150mm,4 μm), mobile phase a:3g/L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid), mobile phase B: acetonitrile; detection wavelength: 290nm, flow rate 1.0ml/min, column temperature: 45 DEG C
| Time (min) | Mobile phase a | Mobile phase B |
| 0.00 | 85 | 15 |
| 10.00 | 80 | 20 |
| 20.00 | 70 | 30 |
| 40.00 | 40 | 60 |
| 40.01 | 85 | 15 |
| 50.00 | 85 | 15 |
As shown in FIG. 5, the gradient at 10-20min still needs to be improved.
Example 4
Chromatographic conditions: agilent C18 column (4.6 x 150mm,4 μm), mobile phase a:3g/L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid), mobile phase B: acetonitrile, detection wavelength: 290nm, flow rate 1.0ml/min, column temperature: 45 ℃;
| time (min) | Mobile phase a | Mobile phase B |
| 0.00 | 80 | 20 |
| 5.00 | 80 | 20 |
| 5.01 | 85 | 15 |
| 15.00 | 85 | 15 |
| 25.00 | 70 | 30 |
| 35.00 | 40 | 60 |
| 35.01 | 85 | 15 |
| 45.00 | 80 | 20 |
The method has low impurity response, poor main peak shape and poor auxiliary material interference impurity peak, and the effect is still not ideal.
Example 5
Chromatographic conditions: on the basis of example 3, the column was adjusted to Agilent C18 (4.6 x 250mm,4 μm), mobile phase a:2.6g/L sodium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid), mobile phase B: acetonitrile-methanol (80:20), detection wavelength: 290nm, flow rate 1.0ml/min, column temperature: 50 DEG C
As shown in FIG. 6, the separation degree is not improved obviously when the column temperature is increased to 50 ℃, and the chromatographic column is greatly lost due to the increase of the column temperature, and then the method is continuously carried out for adjusting the column temperature at 45 ℃.
Example 6
Chromatographic conditions (inventive protocol): agilent C18 column (4.6 x 250mm,4 μm), mobile phase a:2.6g/L sodium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid), mobile phase B: acetonitrile-methanol (80:20), detection wavelength: 290nm, flow rate 1.0ml/min, column temperature: 45 ℃;
| time (min) | Mobile phase a | Mobile phase B |
| 0.00 | 85 | 15 |
| 20.00 | 75 | 25 |
| 45.00 | 40 | 60 |
| 45.01 | 85 | 15 |
| 55.00 | 85 | 15 |
In summary, the invention mainly provides an impurity detection method of a pimobendan soft chewing dosage form, which has strong specificity and high sensitivity, can distinguish complex auxiliary materials from impurities, further well controls the impurities of pimobendan, and provides a basis for the detection method of the impurities of the pimobendan soft chewing dosage form. Meanwhile, the method can prolong the service life of the chromatographic column and save the cost.
Claims (2)
1. The impurity detection method of the pimobendan soft chewable dosage form is characterized by using three-phase high performance liquid chromatography to detect impurities in the pimobendan soft chewable dosage form, wherein the chromatographic conditions are as follows:
chromatographic column: c18,4.6mm×250mm,4 μm;
mobile phase a:2.6g/L sodium dihydrogen phosphate solution, and regulating the pH value to 3.0 by phosphoric acid;
mobile phase B: acetonitrile-methanol, 80:20;
the column temperature is 45 ℃;
the detection wavelength is 290nm;
the flow rate is 1.0ml/min;
gradient elution:
Measuring the sample solution of the pimobendan soft chewable dosage form under the chromatographic conditions, and judging the impurity condition according to the main peak and the impurity peak;
the impurities of the pimobendan soft chewable dosage form comprise impurity A:4- (2- (4-methoxyphenyl) -1H-benzo [ d ] imidazol-5-yl) -3-methyl-4-oxobutanoic acid;
the impurities of the pimobendan soft chewable dosage form comprise impurities B: n- (2-amino-4- (4-methyl-6-oxo-1, 4,5, 6-tetrahydropyridazin-3-yl) phenyl) -4-methoxybenzamide;
the method comprises the steps of preparing a sample solution, preparing a system applicability solution and preparing a blank auxiliary material solution before the step of measuring by using the three-phase high performance liquid chromatography;
the preparation of the test solution comprises the following steps: weighing the ground pimobendan soft chewable dosage form, placing the soft chewable dosage form into a volumetric flask, adding methanol for ultrasonic dissolution, diluting to a scale, centrifuging for 10min at 10000r/min, taking supernatant, filtering, discarding primary filtrate, taking subsequent filtrate as a test sample solution containing pimobendan, and diluting with methanol to ensure that the concentration of the pimobendan is 0.5mg/ml;
the system applicability solution preparation comprises the following steps: taking a proper amount of impurity A, B and pimobendan reference substance, precisely weighing, dissolving with methanol, and quantitatively diluting to prepare mixed reference substance solutions containing 1 mug of impurity A, B and 0.5mg of pimobendan in each 1ml, as a system applicability test solution;
the preparation of the blank auxiliary material solution comprises the following steps: obtaining a blank auxiliary material solution with the same volume as the sample solution according to the preparation mode of the sample solution;
and (3) measuring the solutions under the chromatographic conditions, wherein auxiliary material peaks are subtracted from the solution to be tested, the separation degree before and after the main peak accords with the regulation of more than 2.0, and the main peak type and the theoretical plate number meet the detection requirement of the plate number.
2. Use of the method according to claim 1, characterized in that the method is used for detecting impurities in a soft chewable dosage form of pimobendan.
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| EP2925305A1 (en) * | 2013-12-04 | 2015-10-07 | Boehringer Ingelheim Vetmedica GmbH | Improved pharmaceutical compositions of pimobendan |
Non-Patent Citations (2)
| Title |
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| EDQM.European Pharmacopoeia 9.0.2017,3342. * |
| N. Gayatri Devi.Low level determination of Genotoxic impurities in Pimobendan drug by RP-HPLC.American Journal of PharmTech Research.2016,第6卷(第6期),372-380. * |
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