CN114113346A - Detection method for ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in dabigatran etexilate bulk drug or preparation - Google Patents
Detection method for ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in dabigatran etexilate bulk drug or preparation Download PDFInfo
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- CN114113346A CN114113346A CN202010862582.XA CN202010862582A CN114113346A CN 114113346 A CN114113346 A CN 114113346A CN 202010862582 A CN202010862582 A CN 202010862582A CN 114113346 A CN114113346 A CN 114113346A
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- Prior art keywords
- toluenesulfonate
- solution
- ethyl
- isopropyl
- dabigatran etexilate
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- VRZVPALEJCLXPR-UHFFFAOYSA-N ethyl 4-methylbenzenesulfonate Chemical compound CCOS(=O)(=O)C1=CC=C(C)C=C1 VRZVPALEJCLXPR-UHFFFAOYSA-N 0.000 title claims abstract description 128
- SDQCGKJCBWXRMK-UHFFFAOYSA-N propan-2-yl 4-methylbenzenesulfonate Chemical compound CC(C)OS(=O)(=O)C1=CC=C(C)C=C1 SDQCGKJCBWXRMK-UHFFFAOYSA-N 0.000 title claims abstract description 109
- KSGXQBZTULBEEQ-UHFFFAOYSA-N dabigatran etexilate Chemical compound C1=CC(C(N)=NC(=O)OCCCCCC)=CC=C1NCC1=NC2=CC(C(=O)N(CCC(=O)OCC)C=3N=CC=CC=3)=CC=C2N1C KSGXQBZTULBEEQ-UHFFFAOYSA-N 0.000 title claims abstract description 71
- 229960000288 dabigatran etexilate Drugs 0.000 title claims abstract description 65
- 238000001514 detection method Methods 0.000 title claims abstract description 39
- 239000003814 drug Substances 0.000 title claims abstract description 33
- 229940079593 drug Drugs 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 40
- 239000012085 test solution Substances 0.000 claims abstract description 35
- 239000012088 reference solution Substances 0.000 claims abstract description 18
- 239000012535 impurity Substances 0.000 claims abstract description 14
- 231100000024 genotoxic Toxicity 0.000 claims abstract description 12
- 230000001738 genotoxic effect Effects 0.000 claims abstract description 12
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 89
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 84
- 238000012360 testing method Methods 0.000 claims description 41
- 239000000523 sample Substances 0.000 claims description 27
- 239000000126 substance Substances 0.000 claims description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000002775 capsule Substances 0.000 claims description 18
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- 150000002500 ions Chemical class 0.000 claims description 9
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- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 7
- 229960004951 dabigatran etexilate mesylate Drugs 0.000 claims description 6
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 4
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 4
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- 238000004587 chromatography analysis Methods 0.000 claims 1
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- 238000010812 external standard method Methods 0.000 abstract description 6
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- 238000011084 recovery Methods 0.000 description 29
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- 238000010998 test method Methods 0.000 description 13
- 230000000052 comparative effect Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 6
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- 238000007865 diluting Methods 0.000 description 5
- 206010051055 Deep vein thrombosis Diseases 0.000 description 4
- 208000010378 Pulmonary Embolism Diseases 0.000 description 4
- 206010047249 Venous thrombosis Diseases 0.000 description 4
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 2
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- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- LBLYYCQCTBFVLH-UHFFFAOYSA-N 2-Methylbenzenesulfonic acid Chemical compound CC1=CC=CC=C1S(O)(=O)=O LBLYYCQCTBFVLH-UHFFFAOYSA-N 0.000 description 1
- 206010003658 Atrial Fibrillation Diseases 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 206010013710 Drug interaction Diseases 0.000 description 1
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- 229920000084 Gum arabic Polymers 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000978776 Senegalia senegal Species 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940000635 beta-alanine Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
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- FPAFDBFIGPHWGO-UHFFFAOYSA-N dioxosilane;oxomagnesium;hydrate Chemical compound O.[Mg]=O.[Mg]=O.[Mg]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O FPAFDBFIGPHWGO-UHFFFAOYSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 125000004005 formimidoyl group Chemical group [H]\N=C(/[H])* 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 125000003707 hexyloxy group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])O* 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011540 hip replacement Methods 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
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- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
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- 231100000299 mutagenicity Toxicity 0.000 description 1
- 230000007886 mutagenicity Effects 0.000 description 1
- 229940127216 oral anticoagulant drug Drugs 0.000 description 1
- -1 p-toluenesulfonic acid ethyl ester ion Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000011003 system suitability test Methods 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
Images
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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
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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Abstract
The invention aims to provide a method for detecting genotoxic impurities of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in a dabigatran etexilate bulk drug or a preparation, which adopts a method combining Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry and comprises the steps of (1) preparing a test solution and a reference solution; respectively sampling a test solution and a reference solution, detecting by using an ultra-high performance liquid chromatography-mass spectrometer, recording a chromatogram, and calculating the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate according to an external standard method; the method is simple to operate, accurate in detection result, high in sensitivity and good in stability, and can be used for quality control of dabigatran etexilate bulk drugs or preparations.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting genotoxic impurities of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in a dabigatran etexilate bulk drug or a preparation.
Background
Dabigatran etexilate mesylate has the chemical name of beta-alanine, N- [ [2- [ [ [4- [ [ [ (hexyloxy) carbonyl ] amino ] iminomethyl ] phenyl ] amino ] methyl ] -1-methyl-1H-benzimidazol-5-yl ] carbonyl ] -N-2-pyrimidine-, ethyl ester and mesylate, and the chemical structure of the dabigatran etexilate mesylate is shown in a formula I, is an oral direct thrombin inhibitor developed by Boringer Yiger corporation, and is mainly used for: (1) prevention of stroke in patients with non-valvular atrial fibrillation; (2) deep Vein Thrombosis (DVT) and Pulmonary Embolism (PE) prophylaxis in patients who have been treated with anticoagulant injection for 5-10 days; (3) already treated patients reduce the risk of recurrence of DVT and PE; (4) prevention of DVT and PE following hip replacement.
The dabigatran etexilate is a new generation oral anticoagulant drug Direct Thrombin Inhibitors (DTIs), can provide effective, predictable and stable anticoagulant effects, simultaneously has less drug interaction, no drug-food interaction, does not need routine blood coagulation function monitoring or dose adjustment, and has very wide clinical application.
During the synthesis process of the dabigatran etexilate bulk drug, toluenesulfonic acid, ethanol, isopropanol and the like are used and react to generate byproducts of ethyl tosylate and isopropyl tosylate, the chemical structures of the byproducts are shown as formulas II and III, and the byproducts can be further transferred and exist in the dabigatran etexilate bulk drug and a final product of dabigatran etexilate preparation. According to the 'gene toxicity impurity limit guidance' issued by the European drug administration, sulfonate is a warning structure with gene toxicity, can directly or indirectly damage DNA, can cause the damage of human genetic substances at a very low concentration, has mutagenicity and carcinogenicity, and seriously threatens the health of human beings in the process of medication. Ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate belong to potential genotoxic impurities, sensitive, efficient and high-specificity detection methods are developed aiming at the two potential genotoxic impurities, and the strict control of the contents of the detection methods is a key ring for controlling the quality of the medicine.
However, no method for measuring the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in a dabigatran etexilate bulk drug or a preparation is reported at home and abroad at present. Therefore, a detection method should be established, and the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in the dabigatran etexilate bulk drug or preparation are strictly controlled, so as to ensure the safety of clinical medication.
Disclosure of Invention
The invention aims to provide a method for detecting ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in a dabigatran etexilate bulk drug or a preparation, which adopts a method of combining Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry to verify the dabigatran etexilate bulk drug or the preparation in the aspects of system applicability, specificity, precision, detection limit, quantification limit, accuracy, durability and the like, so that the dabigatran etexilate bulk drug or the preparation completely accords with the guide principle of Chinese pharmacopoeia method verification and can be used for quality control of the dabigatran etexilate bulk drug or the preparation.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for detecting genotoxic impurities of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in a dabigatran etexilate bulk drug or a preparation adopts Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry in a combined manner, and comprises the following steps:
preparing a test solution and a reference solution: dissolving a sample to be tested by taking an acetonitrile aqueous solution as a solvent to prepare a test solution; dissolving a standard substance of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in acetonitrile and/or acetonitrile aqueous solution as a solvent to prepare a reference substance solution;
respectively injecting sample into the test solution and the reference solution, detecting by using an ultra-high performance liquid chromatography-mass spectrometer, and recording a chromatogram;
wherein, the ultra-high performance liquid chromatography conditions are as follows: a chromatographic column: ultra high performance liquid chromatography column, 2.1mm × 50mm (diameter × length), 1.7 μm (particle size); flow rate: 0.4 plus or minus 0.05 ml/min; column temperature: 30 +/-5 ℃; mobile phase A: 0.05% -0.5% ammonium formate aqueous solution; mobile phase B: methanol; the gradient elution conditions were as follows:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 68-72 | 28-32 |
| 1.5 | 68-72 | 28-32 |
| 12.5 | 48-52 | 48-52 |
| 18.2 | 8-12 | 88-92 |
| 22.0 | 8-12 | 88-92 |
| 22.1 | 68-72 | 28-32 |
| 25.0 | 68-72 | 28-32 |
The gradient elution conditions are preferably:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 70 | 30 |
| 1.5 | 70 | 30 |
| 12.5 | 50 | 50 |
| 18.2 | 10 | 90 |
| 22.0 | 10 | 90 |
| 22.1 | 70 | 30 |
| 25.0 | 70 | 30 |
The mass spectrum conditions are as follows: mass spectrometry: a triple quadrupole mass spectrometer; mass spectrometry method mode: multiple reaction detection scan (MRM); ionization Mode (Ionization Mode): positive ion spectrum (ES +);
and calculating the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate according to an external standard method.
Further, in the above-mentioned case,
in the step (1), the acetonitrile water solution is 40-60% by volume; preferably 50% by volume of acetonitrile in water.
In the step (1), the mobile phase a is an aqueous ammonium formate solution with a concentration of 0.05% to 0.2%, preferably an aqueous ammonium formate solution with a concentration of 0.08% to 0.12%, and more preferably an aqueous ammonium formate solution with a concentration of 0.1%.
In the step (1), the sample to be detected is a dabigatran etexilate bulk drug or a dabigatran etexilate preparation, and the dabigatran etexilate preparation is preferably a dabigatran etexilate capsule.
In the step (1), the reference substance solutions of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate can be prepared separately, or the standard substances of the two impurities can be mixed and prepared in the same reference substance solution. The preparation method comprises the following specific steps: dissolving ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate standard substances in acetonitrile to obtain solutions with respective concentrations of 0.1-1mg/ml, and diluting with acetonitrile water solution to obtain reference substance solutions containing 0.1-100ng/ml of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate, respectively. The concentration of each of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in the control solution is preferably 1 to 20ng/ml, more preferably 2 to 10 ng/ml. In some embodiments, the control solution contains 4ng/ml ethyl p-toluenesulfonate and 4ng/ml isopropyl p-toluenesulfonate.
In the step (1), the test solution contains 0.25-100mg/ml of dabigatran etexilate, preferably 0.5-50mg/ml, and more preferably 10 mg/ml. The dabigatran etexilate provided by the invention comprises dabigatran etexilate and pharmaceutically acceptable salts thereof, and the concentration of the dabigatran etexilate in a test solution is calculated by the dabigatran etexilate.
In the step (1), when the sample to be tested is the dabigatran etexilate capsule, the preparation method of the test solution comprises the following steps: weighing a proper amount of the dabigatran etexilate capsule content, adding acetonitrile aqueous solution for dissolving, and filtering by a filter membrane to obtain a test sample solution.
In the step (2), the sample injection volume is 5-10 mu L.
In the step (2), the ultra-high performance liquid Phenyl chromatographic column is preferably an Acquity BEH Phenyl; the mass spectrometer is preferably Waters XEVO TQ-S.
In the step (2), the mass spectrometry conditions further include: the Capillary voltage (volts Capillary) was 3kV, the Desolvation Gas Flow rate (Gas Flow Desolvation) was 1000L/hr, and the Desolvation temperature (Desolvation temperature) was 500 ℃.
The peak-off time of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate is about 6.2min and 8.1min respectively, and the sample solution is changed into a master mode (without mass spectrum) before 6min and after 11 min.
The contents of the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate are not more than 5ppm, preferably not more than 2ppm calculated by an external standard method; more preferably not more than 1ppm, more preferably not more than 0.4 ppm.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a method for detecting genotoxic impurities of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in a dabigatran etexilate bulk drug or a preparation, belonging to a method developed for the first time in the field.
(2) According to the guideline of ICH M7 for the limit of genotoxic impurities, the limit value is set to 1.5 mug/d (day), which means that when the amount of genotoxic impurities ingested by each person per day is less than 1.5 mug, the risk of cancer in the life of the person (calculated by the life span of 70 years) is less than one ten-thousandth, the maximum daily dose of dabigatran etexilate is 300mg, and the limit values of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate are calculated to be 5 ppm. The liquid chromatography-mass spectrometry provided by the invention has the advantages of simple sample treatment process operation, high sensitivity (the detection limits of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate are respectively 0.08ppm and 0.02ppm), accurate detection result (the recovery rate reaches more than 85%), good stability and capability of detecting trace amounts of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate contained in a dabigatran etexilate bulk drug or a preparation, so that the method provided by the invention completely meets the requirements of the guide principle of the detection method verification related to Chinese pharmacopoeia and can be used for quality control of genotoxic impurities in the dabigatran etexilate bulk drug or the preparation.
Description of the drawings:
FIG. 1: chromatogram map of ethyl p-toluenesulfonate and ethyl p-toluenesulfonate in control solution of isopropyl p-toluenesulfonate in example 2
FIG. 2: exactly chromatogram for isopropyl p-toluenesulfonate in ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate control solution in example 2
FIG. 3: example 2 specificity chromatogram of an empty white solution at ethyl p-toluenesulfonate
FIG. 4: example 2 specificity chromatogram of an empty white solution at isopropyl p-toluenesulfonate
FIG. 5: example 2 specificity chromatogram of hollow white adjuvant at ethyl p-toluenesulfonate
FIG. 6: example 2 specificity chromatogram of hollow white adjuvant on isopropyl p-toluenesulfonate
FIG. 7: example 2 dabigatran etexilate control solution specificity chromatogram
FIG. 8: example 4 method precision 2 chromatogram of isopropyl p-toluenesulfonate in test sample solution
FIG. 9: limiting chromatogram for detecting ethyl p-toluenesulfonate in example 7
FIG. 10: limited chromatogram for detection of isopropyl p-toluenesulfonate in example 7
FIG. 11: quantitative limit chromatogram for ethyl p-toluenesulfonate in example 7
FIG. 12: quantitative limit chromatogram for isopropyl p-toluenesulfonate in example 7
FIG. 13: comparative example 1 chromatogram peculiar to ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate control solution
FIG. 14: comparative example 2 peak area chromatogram of ethyl p-toluenesulfonate under different quantitative ion pairs
FIG. 15: comparative example 3 Peak area chromatogram of Ethyl p-toluenesulfonate in different Mobile phases B
Detailed Description
The invention discloses a method for detecting ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in a dabigatran etexilate bulk drug or a preparation, which can be realized by combining the relevant principle of drug analysis and properly improving process parameters by taking the contents of the invention as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention.
For a better understanding of the invention, and not as a limitation on the scope thereof, all numbers expressing quantities, percentages, and other numerical values used in this application are to be understood as being modified in all instances by the term "about". At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
A detection instrument: waters XEVO TQ-S ultra-high performance liquid phase mass spectrometer
Ultra-high performance liquid chromatography conditions:
a chromatographic column: an ultra-high performance liquid chromatography column of Acquity BEH Phenyl, 2.1mm × 50mm,1.7 μm;
flow rate: 0.4 ml/min; column temperature: 30 ℃; sample introduction volume: 8 mu l of the solution;
mobile phase A: precisely measuring 0.5g of ammonium formate, dissolving in 500ml of ultrapure water, and performing ultrasonic degassing;
mobile phase B: methanol, ultrasonic degassing;
gradient elution table:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 70 | 30 |
| 1.5 | 70 | 30 |
| 12.5 | 50 | 50 |
| 18.2 | 10 | 90 |
| 22.0 | 10 | 90 |
| 22.1 | 70 | 30 |
| 25.0 | 70 | 30 |
The mass spectrometry conditions were as follows:
the mass spectrometry method comprises the following steps: MRM (multiple reaction detection scan); ionization Mode (Ionization Mode): ES + (positive ion spectrum); span: 0.2
The tuning method comprises the following steps:
voltages Capillary (Capillary voltage): 3.00 kV;
desolvation temperature (Desolvation temperature): 500 ℃;
gas Flow desorption (Desolvation Gas Flow): 1000L/Hr (hours)
Preparing a blank auxiliary material solution: taking the auxiliary materials of gum arabic, hydroxypropyl methylcellulose, hydroxypropyl cellulose, talcum powder and tartaric acid in the dabigatran etexilate capsule according to the dosage proportion of the formula of the dabigatran etexilate capsule, adding 50% acetonitrile aqueous solution for dissolving, shaking up, filtering by a 0.22 mu m filter membrane, and taking the filtrate as blank auxiliary material solution.
Preparing reference substance solutions of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate, namely precisely weighing about 10mg of each of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate standard substances, placing the weighed standard substances into a 25ml measuring flask with 10ml of acetonitrile, adding the acetonitrile to dilute to a scale, and shaking up; precisely measuring 1ml, putting the solution into a 100ml measuring flask, adding 50% acetonitrile aqueous solution to dilute the solution to a scale, and shaking up; and precisely measuring 25 mu l of the solution, putting the solution into a 25ml measuring flask, adding 50% acetonitrile aqueous solution to dilute the solution to a scale, and shaking the solution uniformly to obtain solutions respectively containing ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate, wherein the solutions are reference solutions of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate, and the concentrations of the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate are 4 ng/ml.
Preparing a dabigatran etexilate reference solution: precisely weighing about 10mg of the dabigatran etexilate standard substance into a 10ml measuring flask, adding 50% acetonitrile water solution to dissolve and dilute the dabigatran etexilate standard substance to a scale, and shaking up to obtain a solution with the concentration of 1mg/ml, wherein the solution is the standard solution of the dabigatran etexilate reference substance.
Preparation of a test sample (dabigatran etexilate bulk drug) solution 1: precisely weighing about 100mg of the dabigatran etexilate raw material medicine into a 10ml measuring flask, adding 50% acetonitrile water solution to dissolve and dilute the dabigatran etexilate raw material medicine to a scale, and shaking up to obtain a test solution 1 with the concentration of 10 mg/ml.
Preparation of test article (dabigatran etexilate capsule) solution 2: precisely weighing a proper amount of the content of the dabigatran etexilate capsules (about 100mg of dabigatran etexilate) and placing the dabigatran etexilate capsules into a 10ml measuring flask, adding 50% acetonitrile aqueous solution for ultrasonic dissolution and diluting to a scale, shaking up, and filtering through a 0.22 mu m filter membrane to obtain a sample solution 2 with the concentration of 10 mg/ml.
Example 1: system applicability
(1) The test method comprises the following steps:
and (3) continuously sampling 6 needles of the reference substance solution of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate, and recording the peak areas of the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate.
(2) And (3) test results:
as shown in Table 1, 6-pin RSD samples of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate control solution are 7.4% and 4.0% respectively in continuous sampling (standard regulation: RSD of 6-pin peak area is less than or equal to 10.0%), which indicates that the chromatographic system has good sampling precision and the applicability of the system meets the requirement.
Table 1: example 1 System suitability test results
Example 2: specificity
(1) The test method comprises the following steps:
blank solutions (50% acetonitrile water solution), blank auxiliary material solutions of (dabigatran etexilate capsules), ethyl p-toluenesulfonate, isopropyl p-toluenesulfonate reference substance solution and dabigatran etexilate reference substance solution are respectively injected.
The detection method of the dabigatran etexilate reference solution is high performance liquid chromatography, and the detection method comprises the steps of chromatographic column, flow rate, column temperature, sample injection volume, mobile phase A, mobile phase B and gradient elution; the detection wavelength is 226 nm.
(2) And (3) test results:
as shown in Table 2, the blank solution, the blank adjuvant solution and dabigatran etexilate do not interfere with the measurement of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate. See figures 1-7.
Table 2: example 2 results of the specificity test
Example 3: method precision 1 (dabigatran etexilate starting material)
(1) The test method comprises the following steps:
6 parts of test solution 1 is prepared in parallel for the same batch of dabigatran etexilate bulk drug, and the content of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate is measured.
6 test solutions (6 parts of 'sample + standard' solution) of 100% ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate with specified limits (0.4ppm) of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate are prepared in parallel, the concentration contained in the solutions is calculated by an external standard method by using the ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate reference solutions, the actual measured concentration is obtained by subtracting the content of the test sample from the concentration, and the average recovery rate and the relative standard deviation of the genotoxic impurities of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in 6 samples are calculated by comparing with the theoretical addition concentration.
(2) And (3) test results:
as shown in tables 3 and 4, the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in 6 parts of the test solution were both below the limit of quantitation, in which ethyl p-toluenesulfonate was not detected in test solution 1 and the content of isopropyl p-toluenesulfonate was 0.03 to 0.04ppm (below the limit of quantitation of 0.08ppm) (standard rule: RSD may not be calculated when the content is below the limit of quantitation); the average recovery rates of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in 6 portions of sample plus standard solution are respectively 99.9% and 89.6% (standard specification: average recovery rate 80.0% -120.0%), the RSD is respectively 8.8% and 3.8% (standard specification: RSD is less than or equal to 10.0%), which indicates that the precision of the method for determining the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in the dabigatran etexilate bulk drug meets the requirement.
Table 3: EXAMPLE 3 method precision 1 test results-results of measurement of 6 parts of test sample solution
Table 4: example 3 method precision 1 test results-6 parts sample + target solution recovery results
Example 4: method precision 2 (dabigatran etexilate capsules)
(1) The test method comprises the following steps:
6 parts of test solution 2 are prepared in parallel for the same batch of dabigatran etexilate capsules, and the content of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate are measured.
6 test solutions (6 parts of 'sample + standard' solution) of 100% ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate with specified limits (0.4ppm) of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate are prepared in parallel, the concentration contained in the solutions is calculated by an external standard method by using the ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate reference solutions, the actual measured concentration is obtained by subtracting the content of the test sample from the concentration, and the average recovery rate and the relative standard deviation of the genotoxic impurities of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in 6 samples are calculated by comparing with the theoretical addition concentration.
(2) And (3) test results:
as shown in tables 5 and 6, the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in 6 parts of test solution 2 were both below the limit of quantitation, in which ethyl p-toluenesulfonate was not detected in test solution 2 and the content of isopropyl p-toluenesulfonate was 0.04ppm (below the limit of quantitation of 0.08ppm) (standard stipulation: RSD may not be calculated when the content is below the limit of quantitation); the average recovery rates of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in 6 parts of sample plus standard solution are respectively 91.0% and 88.6% (standard specification: 80.0% -120.0%), the RSD is respectively 6.1% and 4.9% (standard specification: RSD is less than or equal to 10.0%), which indicates that the precision of the method for determining the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in the dabigatran etexilate capsule meets the requirement. The results of the content of isopropyl p-toluenesulfonate in the test solution 2 are shown in FIG. 8.
Table 5: example 4 method precision 2 test results-6 parts test solution 2 test results
Table 6: example 4 method precision 2 test results-6 parts sample + target solution recovery results
Example 5: intermediate precision 1 (dabigatran etexilate bulk drug)
(1) The test method comprises the following steps:
two laboratory technicians respectively detect the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in the same batch of dabigatran etexilate bulk drug by using two chromatographic columns with the same model on different dates, and parallelly prepare 6 samples of a sample solution 1 of a test sample (dabigatran etexilate bulk drug); and 6 test solutions containing 100% ethyl p-toluenesulfonate with ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate as specified limits, a control of isopropyl p-toluenesulfonate and a sample were prepared in parallel, and the relative average deviation of the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate and the average recovery rate measured by two persons was examined.
(2) And (3) test results:
as shown in Table 7, the average recovery rates RD of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate measured by two test personnel are respectively 1.9% and 1.7% (standard regulation: RD less than or equal to 10.0%), and the intermediate precision is in accordance with the requirement.
Table 7: example 5 comparison of results of intermediate precision 1 test
Example 6: intermediate precision 2 (dabigatran etexilate capsules)
(1) The test method comprises the following steps:
two assay workers respectively detect the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in the same batch of dabigatran etexilate capsules by using two chromatographic columns with the same model on different dates, and parallelly prepare 6 samples of a sample (dabigatran etexilate capsule) solution 2; and 6 test solutions containing 100% ethyl p-toluenesulfonate with ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate as specified limits, a control of isopropyl p-toluenesulfonate and a sample were prepared in parallel, and the relative average deviation of the contents of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate and the average recovery rate measured by two persons was examined.
(2) And (3) test results:
as shown in Table 8, the average recovery rates RD of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate measured by two test personnel are respectively 1.0% and 0.7% (standard regulation: RD less than or equal to 10.0%), and the intermediate precision is in accordance with the requirement.
Table 8: example 6 comparison of results of intermediate precision 2 test
Example 7: detection limit and quantification limit
(1) The detection limit test method comprises the following steps:
diluting ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate reference solution, measuring, recording a chromatogram, and determining by measuring S/N (signal-to-noise ratio) of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate; when the S/N is 3-10, the concentration is the minimum detection concentration of the substance to be detected, and the concentration/the concentration of the test solution obtains the detection limit (LOD, ppm);
(2) quantitative limit test method:
diluting ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate reference solution, measuring, recording a chromatogram, and determining by measuring S/N of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate; when the S/N is more than or equal to 10, the concentration is the minimum quantitative concentration of the substance to be detected, and the concentration/the concentration of the test solution obtains the quantitative limit (LOQ, ppm);
precision of quantitative limit: and continuously injecting 6 needles of LOQ solution, recording peak areas, and calculating the relative standard deviation of the peak areas.
Quantitative limit accuracy: 6 parts of LOQ solution were prepared in parallel and the average recovery and relative standard deviation of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate were determined. 6 parts of LOQ + auxiliary material solution are prepared in parallel, and the average recovery rate and relative standard deviation of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate are measured.
(3) Detection limit test results:
the detection limit test results are shown in Table 9, wherein the detection limits of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate are 0.08ppm and 0.02ppm respectively, and are shown in the attached figures 9-10.
Table 9: example 7 detection Limit results
| Name (R) | S/N | Corresponding concentration (ng/ml) | In ppm |
| P-toluenesulfonic |
5 | 0.79425 | 0.08 |
| P-toluene sulfonic |
3 | 0.20440 | 0.02 |
(4) Quantitative limit test results:
the results of the quantitative limit tests are shown in tables 10, 11, 12 and 13 below. The quantitative limits of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate were 0.2ppm and 0.08ppm, respectively. The quantitative limit precision of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate has RSD of 2.2% and 5.6% (RSD not more than 10.0% and standard specification), and the quantitative limit precision meets the requirement. The recovery rates of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in 6 parts of LOQ + auxiliary material solution are respectively 99.2% and 102.2% (standard specification: average recovery rate 80.0% -120.0%), the RSD is respectively 3.7% and 2.3% (standard specification: RSD is less than or equal to 10.0%), and the quantitative limit accuracy meets the requirement. The quantitative limit is shown in the attached figures 11-12.
Table 10: EXAMPLE 7 quantitative Limit results
| Name (R) | S/N | Corresponding concentration (ng/ml) | In ppm |
| P-toluenesulfonic |
18 | 1.9856 | 0.2 |
| P-toluene sulfonic |
14 | 0.81760 | 0.08 |
Table 11: EXAMPLE 7 Limited precision results
Table 12: example 7 quantitative Limited accuracy test-results on recovery of 6 parts of LOQ solution
| Recovery ratio of ethyl p-toluenesulfonate (%) | Recovery rate of isopropyl p-toluenesulfonate (%) | |
| 1 | 100.4 | 97.7 |
| 2 | 100.2 | 96.2 |
| 3 | 102.5 | 94.9 |
| 4 | 100.3 | 97.1 |
| 5 | 104.7 | 100.0 |
| 6 | 101.9 | 99.5 |
| Average | 101.7 | 97.6 |
| RSD(％) | 1.8 | 2.0 |
Table 13: example 7 quantitative Limited accuracy test-results on recovery of 6 parts of "LOQ + adjuvant" solution
| Recovery ratio of ethyl p-toluenesulfonate (%) | Recovery rate of isopropyl p-toluenesulfonate (%) | |
| 1 | 99.7 | 103 |
| 2 | 93.8 | 104.3 |
| 3 | 103.2 | 101.3 |
| 4 | 96.1 | 102.5 |
| 5 | 102.1 | 104.6 |
| 6 | 100.3 | 98.4 |
| Average | 99.2 | 102.4 |
| RSD(％) | 3.7 | 2.3 |
Example 8: stability of solution
(1) The test method comprises the following steps:
and after the ethyl p-toluenesulfonate, the isopropyl p-toluenesulfonate reference substance solution, the test solution 1 and the test solution 2 are placed in a refrigerator (at 2-8 ℃) in a closed manner for at least 12 hours, calculating the recovery rate of the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate reference substance and the content of the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate in the test solution according to an external standard method.
(2) And (3) test results:
the results of the solution stability are shown in tables 14, 15 and 16. The recovery rates of the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate reference substance solutions are respectively 101.8% and 104.5% after the solutions are placed in a closed manner at room temperature and a refrigerator (2-8 ℃) for 33 hours (the standard specifies that the solutions are stable when the recovery rates are 80.0% -120.0%), which indicates that the reference substance solutions are stable and meet the verification requirements. The sample solution 1 is placed in a refrigerator (2-8 ℃) in a closed manner for 23 hours at room temperature, ethyl p-toluenesulfonate is not detected before and after being placed, and the content ratio of isopropyl p-toluenesulfonate after and before being placed is 100 percent; the test solution 2 is placed in a refrigerator (2-8 ℃) in a closed manner at room temperature for 29 hours, the ethyl p-toluenesulfonate is not detected before and after being placed, the content ratio of the isopropyl p-toluenesulfonate after being placed to the content before being placed is 107.9 percent, which indicates that the test solution 1 and the test solution 2 are stable and meet the verification requirements (the standard specification is that the solution is stable if the ratio of the content after being placed to the content before being placed is 80.0-120.0 percent).
Table 14: example 8 solution stability test-results of recovery of control solution
Table 15: example 8 solution stability test-test solution 1 results
Table 16: example 8 solution stability test-test article solution 2 results
Example 9: durability
(1) Durability test method:
changing the column temperature to +/-5 ℃, continuously feeding samples of the ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate reference solution for 6 times, and calculating the RSD of the peak areas of the ethyl p-toluenesulfonate and the isopropyl p-toluenesulfonate.
(2) Durability test results:
the durability results are shown in tables 17 and 18, where the column temperature changes. + -. 5 ℃ and the RSD of the peak area of ethyl p-toluenesulfonate was 1.9% and 1.3%, respectively (standard regulation: RSD. ltoreq.10.0%); RSD of the peak area of the isopropyl p-toluenesulfonate is 1.1 percent and 1.1 percent respectively (the RSD is less than or equal to 10.0 percent according to the standard regulation); the system applicability meets the requirements.
Table 17: example 9 durability-column temperature 25 deg. -System suitability results
| Peak area of ethyl p-toluenesulfonate | Peak area of isopropyl p- |
|
| 1 st needle | 8645 | 29001 |
| 2 nd needle | 8356 | 29219 |
| No. 3 needle | 8497 | 28568 |
| The 4 th needle | 8385 | 28493 |
| The 5 th needle | 8739 | 28504 |
| The 6 th needle | 8384 | 28545 |
| Mean value of | 8501 | 28722 |
| RSD(％) | 1.9 | 1.1 |
Table 18: example 9 durability-column temperature 35 deg.C-System suitability results
| Peak area of ethyl p-toluenesulfonate | Peak area of isopropyl p- |
|
| 1 st needle | 8963 | 29471 |
| 2 nd needle | 8911 | 29695 |
| No. 3 needle | 8723 | 29489 |
| The 4 th needle | 8639 | 29029 |
| The 5 th needle | 8806 | 29233 |
| The 6 th needle | 8825 | 28851 |
| Mean value of | 8811 | 29295 |
| RSD(％) | 1.3 | 1.1 |
Comparative example 1: detection using liquid chromatography
1. Chromatographic conditions are as follows:
using liquid chromatography, column: acquisty BEH Phenyl, 2.1 mm. times.50 mm,1.7 μm; flow rate: 0.4 ml/min; column temperature: 30 ℃; sample introduction volume: 8 mu l of the solution; mobile phase A: precisely measuring 0.5g of ammonium formate, dissolving in 500ml of ultrapure water, and performing ultrasonic degassing; mobile phase B: methanol, ultrasonic degassing; detection wavelength: 226nm
Gradient elution table:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 70 | 30 |
| 1.5 | 70 | 30 |
| 12.5 | 50 | 50 |
| 18.2 | 10 | 90 |
| 22.0 | 10 | 90 |
| 22.1 | 70 | 30 |
| 25.0 | 70 | 30 |
2. Specificity test:
injecting ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate reference solution into liquid chromatograph, and obtaining a chromatogram shown in figure 13.
3. Quantitative limit test:
the test method was the same as in example 7, and the results showed that the quantitative limit of ethyl p-toluenesulfonate was 1.5ppm and the quantitative limit of isopropyl p-toluenesulfonate was 1.2 ppm.
4. And (4) conclusion:
the method has low sensitivity and cannot be used for detecting ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate.
Comparative example 2: test of influence of selection of p-toluenesulfonic acid ethyl ester ion pair on detection result
1. Chromatographic conditions are as follows:
the ion pair of ethyl p-toluenesulfonate is shown in the following table, and the other detection conditions are as described above.
2. Solution preparation:
preparing a reference solution 1 of ethyl p-toluenesulfonate, namely precisely weighing about 8mg of an ethyl p-toluenesulfonate standard substance, putting the ethyl p-toluenesulfonate standard substance into a 25ml measuring flask with 10ml of acetonitrile, adding the acetonitrile to dilute to a scale, and shaking up; precisely measuring 1ml, putting the solution into a 100ml measuring flask, adding 50% acetonitrile aqueous solution to dilute the solution to a scale, and shaking up; then, 100. mu.l of the solution was precisely measured and placed in a 25ml measuring flask, and 50% acetonitrile aqueous solution was added to dilute the solution to the scale, followed by shaking to obtain solutions each containing 20ng/ml of ethyl p-toluenesulfonate, which was referred to as ethyl p-toluenesulfonate control solution 1.
3. The test method comprises the following steps:
the ethyl p-toluenesulfonate control solution 1 was sampled and the peak areas of ethyl p-toluenesulfonate under different quantitative ion pairs were recorded, and the results are shown in FIG. 14.
4. And (4) quantitative limit:
diluting the ethyl p-toluenesulfonate reference solution 1, measuring, recording a chromatogram, and determining by measuring the S/N of the ethyl p-toluenesulfonate; when the concentration of S/N is more than or equal to 10, the concentration is the minimum quantitative concentration of the substance to be detected, and the concentration/the concentration of the test solution obtains the quantitative limit (LOQ, ppm).
5. And (4) conclusion:
under the same conditions, the peak area (38641) of the quantitative ion pair of ethyl p-toluenesulfonate 201/173 was 5.4 times that of the peak area (7093) of the quantitative ion pair of 218/173, and the limit of quantitation was 5 times different, so that the method was more sensitive when the quantitative ion pair of ethyl p-toluenesulfonate was 201/173.
Comparative example 3: test of Effect of selection of Mobile phase B on test results
1. Chromatographic conditions are as follows:
mobile phase B was acetonitrile and other assay conditions were as described previously.
2. Solution preparation:
preparing a p-toluenesulfonic acid ethyl ester reference solution 2, namely precisely weighing about 10mg of a p-toluenesulfonic acid ethyl ester standard substance, placing the p-toluenesulfonic acid ethyl ester standard substance into a 25ml measuring flask with 10ml of acetonitrile, adding the acetonitrile to dilute to a scale, and shaking up; precisely measuring 1ml, placing into a 100ml measuring flask, adding 50% acetonitrile water solution to dilute to scale, and shaking; then, 50. mu.l of the solution was precisely measured and placed in a 25ml measuring flask, and 50% acetonitrile aqueous solution was added to dilute the solution to the scale, followed by shaking to obtain solutions each containing ethyl p-toluenesulfonate at a concentration of 8ng/ml, which was ethyl p-toluenesulfonate control solution 2.
3. The test method comprises the following steps:
the ethyl p-toluenesulfonate control solution 2 was sampled and the peak area of ethyl p-toluenesulfonate under different mobile phases B was recorded, and the result is shown in FIG. 15.
| Mobile phase B | |
| Acetonitrile | |
| 5502 | |
| |
16198 |
4. And (4) conclusion:
under the same conditions, the peak area (16198) of the mobile phase B for methanol is 2.94 times the peak area (5502) of the mobile phase B for acetonitrile, so the method is more sensitive.
Comparative example 4: effect of gradient elution method on test results 2
1. Chromatographic conditions are as follows:
the gradient elution method is as follows, and the other detection conditions are as described previously.
The gradient elution method comprises the following steps:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 70 | 30 |
| 5 | 20 | 80 |
| 6 | 20 | 80 |
| 6.1 | 70 | 30 |
| 7 | 70 | 30 |
2. And (3) precision test:
precision was determined as in example 4.
The peak time of ethyl p-toluenesulfonate was 2.5min, and the peak time of isopropyl p-toluenesulfonate was 2.87 min.
The results of the precision test of the method are shown in Table 20: the average recovery rates of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in 6 portions of sample and standard solution are respectively 61.6 percent and 72.4 percent (standard specification: the average recovery rate is 80.0-120.0 percent), and the RSD is respectively 15.5 percent and 9.5 percent (standard specification: the RSD is less than or equal to 10.0 percent); not meeting the control requirement of precision experiment.
Table 20: comparative example 4 method precision 2 test results-6 parts sample + target solution recovery results
3. And (4) conclusion:
the method has poor precision and cannot be used for detecting ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate.
Claims (10)
1. A method for detecting genotoxic impurities of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in a dabigatran etexilate bulk drug or a preparation adopts ultra-high performance liquid chromatography and mass spectrometry, and comprises the following steps:
preparing a test solution and a reference solution: dissolving a sample to be tested by taking an acetonitrile aqueous solution as a solvent to prepare a test solution; dissolving a standard substance of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate in acetonitrile and/or acetonitrile aqueous solution as a solvent to prepare a reference substance solution;
respectively injecting sample into the test solution and the reference solution, detecting by using an ultra-high performance liquid chromatography-mass spectrometer, and recording a chromatogram;
wherein, the ultra-high performance liquid chromatography conditions are as follows: a chromatographic column: ultra-high performance liquid phenyl chromatographic column, 2.1mm × 50mm,1.7 μm; flow rate: 0.4 plus or minus 0.05 ml/min; column temperature: 30 +/-5 ℃; mobile phase A: an aqueous ammonium formate solution with a concentration of 0.05% to 0.2%, preferably 0.08% to 0.12%, more preferably 0.1%; mobile phase B: methanol; the gradient elution conditions were as follows:
The mass spectrum conditions are as follows: mass spectrometry: a triple quadrupole mass spectrometer; mass spectrometry method mode: multi-reaction detection scanning; ionization mode: (ii) a positive ion spectrum;
2. the detection method according to claim 1, wherein in the step (2), the gradient elution conditions are as follows:
3. the detection method of claim 1, wherein in step (2), the mass spectrometry conditions further comprise: the capillary voltage was 3kV, the desolvation gas flow rate was 1000L/hr, and the desolvation temperature was 500 ℃.
4. The detection method according to claim 1, wherein in the step (2), the ultra-high performance liquid Phenyl chromatography column is Acquity BEH Phenyl.
5. The detection method as claimed in claim 1, wherein in the step (2), the mass spectrometer is a Waters XEVO TQ-S.
6. The detection method according to claim 1, wherein in the step (1), the acetonitrile aqueous solution is 40-60% by volume; preferably 50% by volume of acetonitrile in water.
7. The detection method according to claim 1, wherein in the step (1), the concentration of the sample solution is 0.25-100mg/ml, preferably 0.5-50mg/ml, and more preferably 10 mg/ml.
8. The assay of claim 1, wherein in step (1), the control solution comprises 1 to 20ng/ml each of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate, more preferably 2 to 10ng/ml each of ethyl p-toluenesulfonate and isopropyl p-toluenesulfonate; more preferably, the composition contains 4ng/ml of ethyl p-toluenesulfonate and 4ng/ml of isopropyl p-toluenesulfonate.
9. The detection method according to claim 1, wherein in the step (1), the sample to be detected is a dabigatran etexilate mesylate raw material drug or a dabigatran etexilate mesylate preparation, and the dabigatran etexilate mesylate preparation is preferably a dabigatran etexilate mesylate capsule.
10. The detection method according to claim 8, wherein in the step (1), the sample to be detected is a dabigatran etexilate capsule, and the preparation method of the test solution comprises: weighing a proper amount of the dabigatran etexilate capsule content, adding acetonitrile aqueous solution for dissolving, and filtering by a filter membrane to obtain a test sample solution.
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