CN113933400A - Method for detecting genotoxic impurities in dabigatran etexilate bulk drug or preparation - Google Patents
Method for detecting genotoxic impurities in dabigatran etexilate bulk drug or preparation Download PDFInfo
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- CN113933400A CN113933400A CN202010604160.2A CN202010604160A CN113933400A CN 113933400 A CN113933400 A CN 113933400A CN 202010604160 A CN202010604160 A CN 202010604160A CN 113933400 A CN113933400 A CN 113933400A
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- Prior art keywords
- solution
- hexyl
- dabigatran etexilate
- toluenesulfonate
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- 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 77
- 229960000288 dabigatran etexilate Drugs 0.000 title claims abstract description 71
- 238000000034 method Methods 0.000 title claims abstract description 50
- 239000003814 drug Substances 0.000 title claims abstract description 35
- 229940079593 drug Drugs 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- 239000012535 impurity Substances 0.000 title claims description 16
- 230000001738 genotoxic effect Effects 0.000 title claims description 15
- 231100000024 genotoxic Toxicity 0.000 title claims description 14
- IVQOVYWBHRSGJI-UHFFFAOYSA-N hexyl 4-methylbenzenesulfonate Chemical compound CCCCCCOS(=O)(=O)C1=CC=C(C)C=C1 IVQOVYWBHRSGJI-UHFFFAOYSA-N 0.000 claims abstract description 98
- 238000001514 detection method Methods 0.000 claims abstract description 41
- 239000012085 test solution Substances 0.000 claims abstract description 29
- 239000012488 sample solution Substances 0.000 claims abstract description 27
- 239000012088 reference solution Substances 0.000 claims abstract description 21
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 12
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 81
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- 239000000523 sample Substances 0.000 claims description 33
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
- 239000002775 capsule Substances 0.000 claims description 17
- 239000000126 substance Substances 0.000 claims description 14
- 238000005303 weighing Methods 0.000 claims description 13
- 238000010828 elution Methods 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 10
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000013558 reference substance Substances 0.000 claims description 10
- 238000004807 desolvation Methods 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 229960004951 dabigatran etexilate mesylate Drugs 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims description 4
- 239000003643 water by type Substances 0.000 claims description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
- 239000005695 Ammonium acetate Substances 0.000 claims description 3
- 229940043376 ammonium acetate Drugs 0.000 claims description 3
- 235000019257 ammonium acetate Nutrition 0.000 claims description 3
- 238000001819 mass spectrum Methods 0.000 claims description 3
- 238000001228 spectrum Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 2
- 238000010812 external standard method Methods 0.000 abstract description 6
- 238000003908 quality control method Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 description 38
- 238000011084 recovery Methods 0.000 description 33
- 230000000052 comparative effect Effects 0.000 description 28
- 239000012071 phase Substances 0.000 description 13
- 238000010998 test method Methods 0.000 description 12
- 239000012490 blank solution Substances 0.000 description 10
- 239000012086 standard solution Substances 0.000 description 10
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 9
- 238000007865 diluting Methods 0.000 description 8
- 239000007789 gas Substances 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 239000000463 material Substances 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
- 239000002671 adjuvant Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 235000009518 sodium iodide Nutrition 0.000 description 3
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 3
- 235000019345 sodium thiosulphate Nutrition 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- ANOOTOPTCJRUPK-UHFFFAOYSA-N 1-iodohexane Chemical compound CCCCCCI ANOOTOPTCJRUPK-UHFFFAOYSA-N 0.000 description 2
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 2
- 239000012159 carrier gas Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000007872 degassing Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000011550 stock solution 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
- 229940123900 Direct thrombin inhibitor Drugs 0.000 description 1
- 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
- 239000002202 Polyethylene glycol Substances 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
- 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
- KMGBZBJJOKUPIA-UHFFFAOYSA-N butyl iodide Chemical compound CCCCI KMGBZBJJOKUPIA-UHFFFAOYSA-N 0.000 description 1
- LFLBHTZRLVHUQC-UHFFFAOYSA-N butyl methanesulfonate Chemical compound CCCCOS(C)(=O)=O LFLBHTZRLVHUQC-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 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
- VRZVPALEJCLXPR-UHFFFAOYSA-N ethyl 4-methylbenzenesulfonate Chemical compound CCOS(=O)(=O)C1=CC=C(C)C=C1 VRZVPALEJCLXPR-UHFFFAOYSA-N 0.000 description 1
- 125000004494 ethyl ester group Chemical group 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
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- KIWBRXCOTCXSSZ-UHFFFAOYSA-N hexyl carbonochloridate Chemical compound CCCCCCOC(Cl)=O KIWBRXCOTCXSSZ-UHFFFAOYSA-N 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
- 238000011540 hip replacement Methods 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- 229960003943 hypromellose Drugs 0.000 description 1
- 238000010813 internal standard method Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000299 mutagenicity Toxicity 0.000 description 1
- 230000007886 mutagenicity Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 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 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 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
- 239000003868 thrombin inhibitor Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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Abstract
The invention aims to provide a method for detecting hexyl 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 injecting a sample solution to be tested and a reference solution, detecting by using an ultra-high performance liquid chromatography-mass spectrometer, recording a chromatogram, and calculating the content of hexyl 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 a genotoxic impurity hexyl 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.
During the synthesis process of the dabigatran etexilate bulk drug, toluenesulfonic acid, hexyl chloroformate and the like are used and react to generate a byproduct of hexyl tosylate, the chemical structure of which is shown as formula II, and the byproduct can be further transferred and exists in the dabigatran etexilate bulk drug and a final product of dabigatran etexilate preparation. According to the 'genotoxic impurity limit guidance' issued by the European drug administration, sulfonate is a warning structure with genotoxicity, so that hexyl p-toluenesulfonate belongs to a potential genotoxic impurity, 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. The method is a sensitive, efficient and special detection method developed aiming at the potential genotoxic impurities, and the strict control of the content is a key ring for controlling the quality of the medicine.
However, no method for measuring the content of hexyl 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 content of the hexyl tosylate in the dabigatran etexilate bulk drug or the preparation is strictly controlled, so as to ensure the safety of clinical medication.
Disclosure of Invention
The invention aims to provide a method for detecting hexyl 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 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 guiding 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 impurity hexyl p-toluenesulfonate in a dabigatran etexilate bulk drug or a preparation adopts Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry in a combined way, and comprises the following steps:
preparing a test solution and a reference solution: dissolving a sample to be detected by using a methanol water solution as a solvent to prepare a test solution; dissolving a standard substance of hexyl p-toluenesulfonate by using an acetonitrile and/or methanol 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 C18 chromatography column, 2.1mm × 100mm (diameter × length), 1.8 μm (particle size); flow rate: 0.3 plus or minus 0.05 ml/min; column temperature: 30 +/-5 ℃; mobile phase A: 0.1% ammonium acetate aqueous solution; mobile phase B: methanol; the gradient elution conditions were as follows:
the gradient elution conditions are preferably:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 35 | 65 |
| 10 | 35 | 65 |
| 12 | 10 | 90 |
| 14 | 10 | 90 |
| 14.5 | 35 | 65 |
| 16.5 | 35 | 65 |
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 +); the quantitative ion pair for hexyl p-toluenesulfonate was 274.1/173.0, Cone (Cone voltage) 12V, and Collision (Collision energy) 16V.
The content of hexyl p-toluenesulfonate was calculated by the external standard method.
Further, in the above-mentioned case,
in the step (1), the methanol aqueous solution is 60-80% by volume; preferably 70% by volume of aqueous methanol.
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 hexyl p-toluenesulfonate standard substance is dissolved into a solution of 0.1-1mg/ml by adding acetonitrile, and then diluted into a control solution of 0.1-100ng/ml by using a methanol aqueous solution, preferably a control solution of 1-10ng/ml, and more preferably a control solution of 2-5 ng/ml. In some embodiments, the control solution has a concentration of 2-3 ng/ml.
In the step (1), the concentration of the test solution is 0.25-10mg/ml, preferably 0.5-5mg/ml, and more preferably 1mg/ml of dabigatran etexilate. 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 methanol water solution for dissolving, centrifuging at 8000rpm for 15min, and taking supernatant as a test solution.
In the step (2), the sample injection volume is 5-10 mu L.
In the step (2), the ultra-high performance liquid C18 chromatographic column is preferably Acquity UPLC HSS T3; 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 temperatures) was 500 ℃.
The peak time of hexyl p-toluenesulfonate was about 9.7min, and the sample solution was changed to the master mode before 8.5min and after 10.5min (without entering the mass spectrum).
The content of said hexyl p-toluenesulfonate should not exceed 5ppm, preferably not exceed 2.4ppm, calculated by external standard method.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a method for detecting genotoxic impurity hexyl 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 μ g/d (day), which means that when the amount of genotoxic impurities ingested per person per day is less than 1.5 μ g, the risk of cancer in the life of a human being (calculated according to the life of 70 years) is less than one ten-thousandth, the maximum daily dose of dabigatran etexilate is 300mg, and the calculated limit value is 5ppm, the liquid chromatography-mass spectrometry provided by the invention has the advantages of simple sample processing process operation, high sensitivity (the detection limit is 0.007ppm, the quantification limit is 0.5ppm), accurate detection result (the recovery rate is more than 90 percent), good stability, can detect that the dabigatran etexilate bulk drug or the preparation contains 0.007ppm of hexyl p-toluenesulfonate, therefore, the method provided by the invention completely meets the requirements of the guiding principles verified by the related detection method of Chinese pharmacopoeia, and can be used for quality control of genotoxic impurities in dabigatran etexilate bulk drugs or preparations.
Description of the drawings:
FIG. 1: example 2 chromatogram for specificity of hexyl p-toluenesulfonate control solution
FIG. 2 is a drawing: example 2 dabigatran etexilate control solution specificity chromatogram
FIG. 3: example 2 specificity chromatogram of blank solution
FIG. 4 is a drawing: example 2 chromatogram of specificity of hollow white excipients
FIG. 5: example 4 method precision 2 in the assay, "sample + target" chromatogram
FIG. 6: example 7 detection of limiting chromatograms
FIG. 7: example 7 quantitative limiting chromatogram
FIG. 8: comparative example 1 blank solution chromatogram
FIG. 9: comparative example 1 sample solution chromatogram
FIG. 10: comparative example 2 blank solution chromatogram
FIG. 11: comparative example 2 chromatogram of control solution
FIG. 12: comparative example 2 sample solution chromatogram
FIG. 13: chromatogram of control solution of hexyl p-toluenesulfonate
FIG. 14: solution chromatogram of dabigatran etexilate reference substance
Detailed Description
The invention discloses a method for detecting hexyl 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 the technical personnel in the field. 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: acquity UPLC HSS T3, 2.1mm × 100mm, 1.8 μm;
flow rate: 0.3 ml/min; column temperature: 30 ℃; sample introduction volume: 8 mu l of the solution;
mobile phase A: precisely measuring 0.5g of ammonium acetate, 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 | 35 | 65 |
| 10 | 35 | 65 |
| 12 | 10 | 90 |
| 14 | 10 | 90 |
| 14.5 | 35 | 65 |
| 16.5 | 35 | 65 |
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: mixing adjuvants in the dabigatran etexilate capsule, such as gum arabic, hypromellose, hydroxypropyl cellulose, pulvis Talci and tartaric acid, according to the dosage proportion of the dabigatran etexilate capsule prescription, adding 70% methanol water solution for dissolving, centrifuging at 8000rpm for 15min, and collecting supernatant as blank adjuvant solution.
Preparing a hexyl p-toluenesulfonate reference solution, namely precisely weighing about 10mg of a hexyl p-toluenesulfonate standard substance, putting the weighed hexyl 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, placing into a 100ml measuring flask, adding 70% methanol water solution, diluting to scale, and shaking; precisely measuring 30 μ l, placing in a 50ml measuring flask, adding 70% methanol water solution, diluting to scale, and shaking to obtain solution with concentration of 2.4ng/ml, which is hexyl p-toluenesulfonate control solution.
Preparing a dabigatran etexilate reference solution: precisely weighing about 10mg of the dabigatran etexilate standard substance, putting the dabigatran etexilate standard substance into a 10ml measuring flask, adding 70% methanol water solution for dissolving, diluting 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 10mg of the dabigatran etexilate raw material medicine, putting the dabigatran etexilate raw material medicine into a 10ml measuring flask, adding 70% methanol water solution to dissolve and dilute the dabigatran etexilate raw material medicine to a scale, and shaking up to obtain a test sample solution 1 with the concentration of 1 mg/ml.
Preparation of test article (dabigatran etexilate capsule) solution 2: precisely weighing a proper amount of the dabigatran etexilate capsule content (about 10mg of dabigatran etexilate) and placing the dabigatran etexilate capsule content into a 10ml measuring flask, adding 70% methanol water solution for ultrasonic dissolution and diluting to a scale, shaking up, centrifuging at 8000rpm for 15min, and taking supernatant to obtain a sample solution 2 with the concentration of 1 mg/ml.
Example 1: system applicability
(1) The test method comprises the following steps:
taking a hexyl p-toluenesulfonate reference solution and continuously injecting a sample for 6 needles, and recording the peak area of the hexyl p-toluenesulfonate.
(2) And (3) test results:
as shown in Table 1, the RSD of 6 needles of the hexyl tosylate reference solution is continuously injected with a sample of less than 0.8 percent (the RSD of the peak area of 6 needles is less than or equal to 10.0 percent), which indicates that the sample injection precision of the chromatographic system is good and the applicability of the system meets the requirement.
Table 1: example 1 System suitability test results
| Peak area of hexyl p- |
|
| 1 st needle | 23804 |
| 2 nd needle | 23429 |
| No. 3 needle | 23765 |
| The 4 th needle | 23725 |
| The 5 th needle | 23630 |
| The 6 th needle | 23394 |
| Mean value of | 23624 |
| RSD(％) | 0.8 |
Example 2: specificity
(1) The test method comprises the following steps:
blank solutions (70% methanol aqueous solution), blank auxiliary material solutions of (dabigatran etexilate capsules), hexyl p-toluenesulfonate reference substance solutions and dabigatran etexilate reference substance solutions are respectively injected.
Wherein: the detection method of the dabigatran etexilate reference solution comprises the following steps: the mass spectrometric parameters were as follows, and the remaining steps and parameters were as for the detection of hexyl p-toluenesulfonate.
(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 p-hexyl tosylate. See figures 1-4.
Table 2: example 2 results of the specificity test
Example 3: method precision 1 (dabigatran etexilate bulk drug)
(1) The test method comprises the following steps:
6 parts of test solution is prepared in parallel for the same batch of dabigatran etexilate raw material medicine, and the content of the hexyl p-toluenesulfonate is measured.
6 parts of a test solution (6 parts of a sample + standard solution) of 100% hexyl p-toluenesulfonate control and a sample, which contains hexyl p-toluenesulfonate to a specified limit (2.4ppm), were prepared in parallel, the concentration contained in the solution was calculated by an external standard method using the hexyl p-toluenesulfonate control solution, the amount of the sample was subtracted from the concentration to obtain an actual measured concentration, and the average recovery and the relative standard deviation of genotoxic impurities to hexyl p-toluenesulfonate in the 6 parts of the sample were calculated by comparing with the theoretical addition concentration.
(2) And (3) test results:
as shown in tables 3 and 4, the content RSD of hexyl p-toluenesulfonate in 6 parts of the test solution was 7.4% (RSD. ltoreq.10.0%) of the test solution; the average recovery rate of the hexyl p-toluenesulfonate in 6 parts of the sample and standard solution is 95.4 percent (standard specification: the average recovery rate is 80.0 to 120.0 percent), the RSD is 4.5 percent (standard specification: the RSD is less than or equal to 10.0 percent), and the method for determining the content of the hexyl p-toluenesulfonate in the dabigatran etexilate raw material medicine meets the requirement on precision.
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
| Recovery of hexyl p-toluenesulfonate (%) | |
| 1 | 95.1 |
| 2 | 96.9 |
| 3 | 91.5 |
| 4 | 98.5 |
| 5 | 87.9 |
| 6 | 102.4 |
| Average | 95.4 |
| RSD(％) | 4.5 |
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 the hexyl p-toluenesulfonate is determined.
6 parts of a test solution (6 parts of sample + standard solution) of 100% hexyl p-toluenesulfonate reference substance and a sample, which contain hexyl p-toluenesulfonate as a specified limit, are prepared in parallel, the concentration contained in the solution is calculated by using the hexyl p-toluenesulfonate reference substance solution according to an external standard method, the actually measured concentration is obtained after the content of the test substance is deducted by the concentration, and then the average recovery rate and the relative standard deviation of genotoxic impurities to hexyl p-toluenesulfonate in 6 parts of samples are calculated by comparing with the theoretical addition concentration.
(2) And (3) test results:
as shown in tables 5 and 6, the content RSD of hexyl p-toluenesulfonate in 6 parts of the test solution was 5.3% (RSD. ltoreq.10.0%); the average recovery rate of the hexyl p-toluenesulfonate in 6 parts of the sample and standard solution is 94.3 percent (standard specification: the average recovery rate is 80.0-120.0 percent), the RSD is 1.0 percent (standard specification: the RSD is less than or equal to 10.0 percent), and the method for determining the hexyl p-toluenesulfonate in the dabigatran etexilate capsule meets the requirement on precision. The detection of hexyl p-toluenesulfonate in the "sample + standard" solution is shown in FIG. 5.
Table 5: EXAMPLE 4 method precision 2 test results-results of 6 parts test sample solution
Table 6: example 4 method precision 2 test results-6 parts sample + target solution recovery results
| Recovery of hexyl p-toluenesulfonate (%) | |
| 1 | 93.4 |
| 2 | 94.7 |
| 3 | 93.7 |
| 4 | 93.8 |
| 5 | 95.7 |
| 6 | 94.3 |
| Average | 94.3 |
| RSD(％) | 1.0 |
Example 5: intermediate precision 1 (dabigatran etexilate bulk drug)
(1) The test method comprises the following steps:
two testers respectively detect the content of the hexyl p-toluenesulfonate in the same batch of dabigatran etexilate raw material medicaments by using two chromatographic columns with the same model on different dates, and parallelly preparing 6 samples; 6 parts of a 100% hexyl p-toluenesulfonate control plus sample test solution containing hexyl p-toluenesulfonate to the specified limit were prepared in parallel, and the relative average deviation of the measured hexyl p-toluenesulfonate content and the average recovery rate was examined by two persons.
(2) And (3) test results:
as shown in Table 7, the average recovery of hexyl p-toluenesulfonate by two test persons was 5.5% (standard: RD. ltoreq.10.0%) with intermediate precision.
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 testers respectively detect the content of the hexyl p-toluenesulfonate in the same batch of dabigatran etexilate capsules by using two chromatographic columns with the same model on different dates, and parallelly preparing 6 samples; 6 parts of a 100% hexyl p-toluenesulfonate control plus sample test solution containing hexyl p-toluenesulfonate to the specified limit were prepared in parallel, and the relative average deviation of the measured hexyl p-toluenesulfonate content and the average recovery rate was examined by two persons.
(2) And (3) test results:
as shown in Table 8, the average recovery of hexyl p-toluenesulfonate by two test persons was 0.8% (standard: RD. ltoreq.10.0%) with intermediate precision.
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 a hexyl p-toluenesulfonate reference solution, measuring, recording a chromatogram, and determining by measuring the S/N (signal-to-noise ratio) of hexyl 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 a hexyl p-toluenesulfonate reference solution, measuring, recording a chromatogram, and determining by measuring the S/N of hexyl 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: preparing 6 parts of LOQ solution in parallel, and measuring the average recovery rate and relative standard deviation of the hexyl p-toluenesulfonate; 6 parts of LOQ + auxiliary material solution are prepared in parallel, and the average recovery rate and relative standard deviation of the hexyl p-toluenesulfonate are measured.
(3) Detection limit test results:
the detection limit test results are shown in Table 9, wherein the detection limit is 0.007ppm, and the detection limit is shown in figure 6.
Table 9: example 7 detection Limit results
| Name (R) | S/N | Corresponding concentration (ng/ml) | In |
| Hexyltosylate | |||
| 9 | 0.0072981 | 0.007 |
(4) Quantitative limit test results:
the results of the quantitative limit tests are shown in tables 10, 11, 12 and 13 below, and the quantitative limit is 0.5 ppm; the RSD of the quantitative limit precision is 1.9 percent (the RSD is less than or equal to 10.0 percent in the standard specification), and the quantitative limit precision meets the requirement. The average recovery of 6 parts of LOQ solution was 103.7% (standard: 80.0% -120.0%) and RSD was 2.2% (standard: RSD. ltoreq.10.0%). The recovery rate of 6 parts of LOQ + auxiliary material solution is 97.4 percent (standard specification: average recovery rate is 80.0-120.0 percent), the RSD is 2.0 percent (standard specification: RSD is less than or equal to 10.0 percent), and the quantitative limit accuracy meets the requirement. The limit of quantitation is shown in figure 7.
Table 10: EXAMPLE 7 quantitative Limit results
| Name (R) | S/N | Corresponding concentration (ng/ml) | In ppm |
| Hexyltosylate | 48 | 0.48936 | 0.5 |
Table 11: EXAMPLE 7 Limited precision results
| Peak area of hexyl p- |
|
| 1 st needle | 5638 |
| 2 nd needle | 5594 |
| No. 3 needle | 5554 |
| The 4 th needle | 5529 |
| The 5 th needle | 5791 |
| The 6 th needle | 5731 |
| Mean value of | 5640 |
| RSD(％) | 1.9 |
Table 12: example 7 quantitative Limited accuracy test-results on recovery of 6 parts of LOQ solution
| Recovery of hexyl p-toluenesulfonate (%) | |
| 1 | 104.6 |
| 2 | 101.2 |
| 3 | 104.1 |
| 4 | 100.8 |
| 5 | 103.6 |
| 6 | 107.8 |
| Average | 103.7 |
| RSD(％) | 2.5 |
Table 13: example 7 quantitative Limited accuracy test-results on recovery of 6 parts of "LOQ + adjuvant" solution
| Recovery of hexyl p-toluenesulfonate (%) | |
| 1 | 98.2 |
| 2 | 94.7 |
| 3 | 99.7 |
| 4 | 95.7 |
| 5 | 97.6 |
| 6 | 98.7 |
| Average | 97.4 |
| RSD(％) | 2.0 |
Example 8: stability of solution
(1) The test method comprises the following steps:
and (3) after the hexyl p-toluenesulfonate reference solution, the test solution 1 and the test solution 2 are placed in a refrigerator (2-8 ℃) in a closed manner at room temperature for at least 12 hours, calculating the recovery rate of the hexyl p-toluenesulfonate reference solution and the content of hexyl 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 hexyl p-toluenesulfonate reference substance solution after being placed in a closed manner at room temperature and a refrigerator (2-8 ℃) for 37 hours are respectively 90.5 percent and 94.4 percent (the solution is stable if the recovery rate is 80.0-120.0 percent according to the standard specification), which indicates that the reference substance solution is stable and meets the verification requirements. The sample solution 1 is placed in a refrigerator (2-8 ℃) for 35 hours in a closed manner at room temperature, and the content ratio of the sample solution after placement to the sample solution before placement is 108.3 percent and 100 percent respectively; the sample solution 2 is placed in a refrigerator (2-8 ℃) closed at room temperature for 35 hours, the ratio of the content of the sample solution after placement to the content of the sample solution before placement is 111.8 percent and 100 percent respectively, which indicates that the sample solution 1 and the sample solution 2 are stable and meet the verification requirements (the standard specification is that the solution is stable if the ratio of the content of the sample solution after placement to the content of the sample solution before placement 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 the hexyl tosylate reference solution for 6 times, and calculating the RSD of the peak area of the hexyl tosylate.
(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 hexyl p-toluenesulfonate was 0.8% and 0.8%, respectively (standard regulation: RSD. ltoreq.10.0%); the system applicability meets the requirements.
Table 17: example 9 durability-column temperature 25 deg. -System suitability results
| Peak area of hexyl p- |
|
| 1 st needle | 24567 |
| 2 nd needle | 24592 |
| No. 3 needle | 24364 |
| The 4 th needle | 24229 |
| The 5 th needle | 24718 |
| The 6 th needle | 24358 |
| Mean value of | 24471 |
| RSD(％) | 0.8 |
Table 18: example 9 durability-column temperature 35 deg.C-System suitability results
| Peak area of hexyl p- |
|
| 1 st needle | 27454 |
| 2 nd needle | 24877 |
| No. 3 needle | 24545 |
| The 4 th needle | 24984 |
| The 5 th needle | 24909 |
| The 6 th needle | 25043 |
| Mean value of | 24852 |
| RSD(％) | 0.8 |
Comparative example 1 testing using the method in the European pharmacopoeia (2.5.38 in EP 9.7)
By using combination of derivative gas headspace chromatography and mass spectrometry
1, chromatographic conditions:
a GC chromatographic column: a capillary column (such as Thermo, TG-WAXMS, 30 m.times.0.25 mm, 0.25 μm or similar chromatographic column) with polyethylene glycol as stationary liquid;
carrier gas: high-purity nitrogen; flow rate: 0.5 ml/min;
a detector: a quadrupole mass spectrometer detector; sample inlet temperature: 110 ℃;
flow splitting rate: 10 ml/min; the split ratio is as follows: 20: 1;
pipetting line temperature: 250 ℃; EI ion source temperature: 250 ℃;
temperature rising procedure: the initial column temperature was 35 ℃ and held for 5 minutes; then the temperature is increased to 50 ℃ at the rate of 5 ℃ per minute and is kept for 3 minutes; then the temperature was raised to 220 ℃ at a rate of 90 ℃ per minute and held for 7 minutes.
The mass-to-charge ratio of the ion for quantitative analysis is as follows: 184 (iodobutane), 212 (iodohexane)
2 headspace conditions:
carrier gas: high purity nitrogen gas
Sample introduction mode: time; heating temperature: 60 ℃; mainfold Control: 80 ℃;
pipetting line temperature: 110 ℃; heating time: 30 minutes;
pressure balance time: 0.20 min; cycle time: 35 minutes;
3 preparation of the solution
3.1 diluent: 80% acetonitrile in water;
3.2 reaction solution, namely precisely weighing 60g of sodium iodide and 30mg of sodium thiosulfate, putting the sodium iodide and the sodium thiosulfate into a 50ml measuring flask, adding water to dissolve and dilute the sodium iodide and the sodium thiosulfate to a scale, and shaking up;
3.3 internal standard solution taking 60mg of butyl methanesulfonate, precisely weighing, placing in a 25ml measuring flask, diluting to scale with 80% acetonitrile water solution, and shaking up. Precisely measuring 50 mu l, placing the solution into a 250ml measuring flask, adding 80% acetonitrile aqueous solution to dilute the solution to a scale, and shaking up the solution to obtain the product.
3.4 blank solution of comparative example 10.5 ml of internal standard solution and 0.5ml of reaction solution were added precisely and placed in a 20ml headspace bottle as the blank solution of comparative example 1.
3.5 comparative example 1 sample solution taking 100mg of dabigatran etexilate raw material, precisely weighing, placing in a 20ml headspace bottle, and precisely adding 0.5ml of internal standard solution and 0.5ml of reaction solution to serve as the sample solution of comparative example 1.
3.6 comparative example 1 control solution: taking 25mg of each of ethyl p-toluenesulfonate, isopropyl p-toluenesulfonate and hexyl p-toluenesulfonate, precisely weighing, placing in the same 10ml measuring flask, diluting with toluene to the scale, shaking up, precisely weighing 0.3ml, placing in a 25ml measuring flask, adding an internal standard solution to the scale, shaking up, precisely weighing 0.25ml again, placing in a 25ml measuring flask, adding an internal standard solution to the scale, shaking up, using as a reference stock solution, precisely weighing 0.5ml of a reference stock solution and 0.5ml of a reaction solution, placing in a 20ml headspace flask, and using as a reference solution of comparative example 1.
4 test method: blank solution (80% acetonitrile water solution), comparative example 1 reference solution and comparative example 1 sample solution are respectively injected into a chromatograph for detection.
5, test results: see figures 8-9.
Under the experimental conditions, hexyl p-toluenesulfonate was iodinated to give iodohexane, which peaked at 13.745 min. The content of hexyl p-toluenesulfonate in the solution of the sample of comparative example 1 was calculated by the internal standard method and found to be 20ppm, which was significantly higher than the true content (0.24 ppm). It can be seen that the method of comparative example 1 is not suitable for detecting the content of hexyl p-toluenesulfonate in dabigatran etexilate.
Conclusion 6: this method cannot be used for the detection of hexyl p-toluenesulfonate.
Comparative example 2: detection using liquid chromatography
1, chromatographic conditions:
using liquid chromatography, ph5.0 phosphate buffer: acetonitrile (40:60) as a mobile phase, a chromatographic column of Waters symmetry C84.6X 250mm 5 μm, a detection wavelength of 226nm, a sample injection amount of 20 μ l, and a flow rate of 1.0 ml/min.
2, preparation of a solution:
comparative example 2 blank solution: 70% acetonitrile in water;
comparative example 2 control solution: hexyl p-toluenesulfonate was dissolved in 70% acetonitrile in water to a concentration of 0.1. mu.g/ml.
Comparative example 2 sample solution: the raw material drug of the dabigatran etexilate is dissolved by 70 percent acetonitrile water solution, and the concentration is 66.7 mg/ml.
3, special property test:
the blank solution of comparative example 2, the reference solution of comparative example 2 and the sample solution of comparative example 2 were injected into the chromatograph, as shown in FIGS. 10-12.
The peak appearance time of the hexyl p-toluenesulfonate in the control solution of the comparative example 2 is 15.985min, the impurity peaks of the sample solution of the comparative example 2 at the peak of 15-16min are more, and the measurement interference on the hexyl p-toluenesulfonate is large.
4, detection limit test:
the test procedure was as in example 7, and the results showed a detection limit of 0.025. mu.g/ml (25 ppm).
And 5, conclusion:
the method has low sensitivity, and the interference of other impurity peaks in the test solution is large, so that the method cannot be used for detecting the hexyl p-toluenesulfonate.
Comparative example 3: effect of gradient elution method on test results 1
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 | 50 | 50 |
| 3.0 | 50 | 50 |
| 6.0 | 10 | 90 |
| 8.0 | 10 | 90 |
| 8.5 | 50 | 50 |
| 10.0 | 50 | 50 |
Specificity test:
and respectively injecting a hexyl tosylate reference solution and a dabigatran etexilate reference solution.
The results show that: the peak time of the hexyl p-toluenesulfonate in the hexyl p-toluenesulfonate control solution was 6.85min, the peak time of the dabigatran etexilate in the dabigatran etexilate control solution was 6.57min, and the separation degree was poor. See fig. 13-14.
And (4) conclusion: the method has poor specificity and cannot be used for detecting the hexyl p-toluenesulfonate.
Comparative example 4: effect of gradient elution method on test results 2
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 | 50 | 50 |
| 1.5 | 50 | 50 |
| 2.8 | 35 | 65 |
| 7.98 | 35 | 65 |
| 10.2 | 10 | 90 |
| 12.5 | 10 | 90 |
| 12.95 | 50 | 50 |
| 15.0 | 50 | 50 |
And (3) precision test:
precision was determined as in example 4.
The results of the precision test of the method are shown in Table 20: the average recovery of hexyl p-toluenesulfonate in 6 "sample + standard" solutions was only 49.4% with an RSD of 5.3%. The recovery rate of the precision experiment is lower than 50 percent, and the control requirement of the precision experiment is not met (the standard specification is that the average recovery rate is 80.0 to 120.0 percent).
Table 20: comparative example 4 method precision 2 test results-6 parts sample + target solution recovery results
| Recovery of hexyl p-toluenesulfonate (%) | |
| 1 | 47.7％ |
| 2 | 50.6％ |
| 3 | 50.9％ |
| 4 | 45.2％ |
| 5 | 52.6％ |
| 6 | 49.5％ |
| Average | 49.4％ |
| RSD(％) | 5.3％ |
And (4) conclusion: the method has poor precision and cannot be used for detecting the hexyl p-toluenesulfonate.
Claims (11)
1. A method for detecting genotoxic impurity hexyl p-toluenesulfonate in a dabigatran etexilate bulk drug or a preparation adopts the combination of 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 detected by using a methanol water solution as a solvent to prepare a test solution; dissolving and fixing the volume of a standard substance of hexyl p-toluenesulfonate by taking acetonitrile and/or methanol 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 C18 chromatographic column, 2.1mm × 100mm, 1.8 μm; flow rate: 0.3 plus or minus 0.05 ml/min; column temperature: 30 +/-5 ℃; mobile phase A: 0.1% ammonium acetate aqueous solution; mobile phase B: methanol; the gradient elution conditions were as follows:
The mass spectrum conditions are as follows: the mass spectrometer is a triple quadrupole mass spectrometer; mass spectrometry method mode: multi-reaction detection scanning; ionization mode: (ii) a positive ion spectrum; the quantitative ion pair of the hexyl p-toluenesulfonate was 274.1/173.0, the cone voltage was 12V, and the collision energy was 16V.
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 C18 chromatographic column is Acquity UPLC HSS T3.
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 methanol aqueous solution is 60-80% by volume methanol aqueous solution; preferably 70% by volume of aqueous methanol.
7. The detection method according to claim 1, wherein in the step (1), the concentration of the sample solution is 0.25-10mg/ml, preferably 0.5-5mg/ml, and more preferably 1mg/ml of dabigatran etexilate.
8. The assay of claim 1, wherein in step (1), the concentration of the control solution is from 1 to 10ng/ml, preferably from 2 to 5ng/ml, more preferably from 2 to 3 ng/ml.
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 1, wherein in the step (1), the sample to be detected is a dabigatran etexilate capsule, and the preparation method of the test solution comprises the following steps: weighing a proper amount of the dabigatran etexilate capsule content, adding methanol water solution for dissolving, centrifuging at 8000rpm for 15min, and taking supernatant as a test solution.
11. The detection method according to claim 1, wherein in the step (2), the injection volume is 5 to 10 μ L.
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