Method for detecting genotoxic impurities in dabigatran etexilate mesylate

Abstract

The invention discloses a method for detecting genotoxic impurities in dabigatran etexilate mesylate, and belongs to the technical field of pharmaceutical analysis. The detection method comprises the following steps: pretreatment of a sample: dissolving a dabigatran etexilate mesylate sample in an acid solution, extracting, then centrifuging the extract liquor, and taking supernate to respectively obtain to-be-detected solutions; the method comprises the following steps: pre-treating a genotoxic impurity standard substance according to a method which is the same as a method for pre-treating a dabigatran etexilate mesylate sample to obtain a standard solution; and measuring the standard solution and the to-be-measured solution by adopting a gas chromatograph-tandem mass spectrometer, and calculating according to the standard curve to obtain the content of each genotoxic impurity in the to-be-measured solution. According to the method, various genotoxic impurities and dabigatran etexilate mesylate are effectively separated through a liquid-liquid extraction method, the influence of dabigatran etexilate on genotoxic impurity analysis is reduced, interfering substances are reduced, and the method is high in accuracy and good in repeatability.

Description

Technical Field

[0001] The invention relates to a method for detecting genotoxic impurities in dabigatran etexilate mesylate, and belongs to the technical field of drug analysis. Background Art

[0002] Dabigatran etexilate mesylate is a new antithrombin inhibitor developed by Boehringer Ingelheim in Germany. Compared with the traditional oral anticoagulant warfarin, it does not require routine monitoring of INR (International Normalized Ratio), has low adverse reactions and bleeding risks, and is the first new oral anticoagulant drug to be marketed in 50 years.

[0003] In the synthesis process of dabigatran etexilate mesylate raw material, toluenesulfonic acid, ethanol, isopropanol, n-hexyl chloroformate, etc. are used, and reactions will occur to produce by-products such as methyl methanesulfonate, ethyl methanesulfonate, n-hexyl methanesulfonate, diethyl sulfite, n-hexyl chloroformate, methyl p-toluenesulfonate, ethyl p-toluenesulfonate, n-hexyl p-toluenesulfonate, etc. These by-products can be further transferred and exist in the dabigatran etexilate raw material and the final product dabigatran etexilate preparation, all of which are genotoxic impurities. Sulfonate esters have a warning structure of genotoxicity, which can directly or indirectly damage DNA, and can cause damage to human genetic material at very low concentrations. They are mutagenic and carcinogenic, and seriously threaten human health during medication.

[0004] Developing sensitive, efficient and specific detection methods for these potential genotoxic impurities and strictly controlling their content is a key part of drug quality control.

[0005] A Chinese patent (publication number CN 113933400 A) discloses a method for detecting hexyl p-toluenesulfonate in dabigatran etexilate API or preparation, which adopts a method of ultra-high performance liquid chromatography (UPLC) coupled with mass spectrometry, including preparing a test solution and a reference solution; taking the test solution and the reference solution for injection respectively, detecting them with an ultra-high performance liquid chromatography-mass spectrometer, recording the chromatogram, and calculating the content of hexyl p-toluenesulfonate according to the external standard method. However, this method can only detect one genotoxic impurity, and some other impurities, such as diethyl sulfite, n-hexyl methanesulfonate, n-hexyl chloroformate, etc., have no response on LC-MS.

[0006] At present, there are many detection methods for mesylate and p-toluenesulfonate compounds, most of which use derivatization methods for detection. The European Pharmacopoeia 8.0 uses NaI and mesylate derivatization to generate iodomethane, iodoethane and other substances, which are detected by headspace injection. However, this method cannot be used to detect n-hexyl mesylate and n-hexyl p-toluenesulfonate in dabigatran mesylate, because dabigatran mesylate also contains a hexyl ester structure, which leads to inaccurate content testing of n-hexyl mesylate and n-hexyl p-toluenesulfonate.

[0007] Summary of the invention

[0008] For the problems existing in the above-mentioned prior art, the object of the invention is to provide a kind of detection method of genotoxic impurities in dabigatran mesylate, the method is by adopting acid solution to dissolve dabigatran mesylate, adopt the mixed solution of normal hexane and acetone to extract the medicine after dissolving, by centrifugation, take supernatant, then adopt gas chromatography-tandem mass spectrometry to measure, calculate the content of each genotoxic impurity in the liquid to be measured according to standard curve.The method can detect multiple genotoxic impurities simultaneously, and accuracy is high, repeatability is good, sensitivity is high, stability is good, can be used for the quality control of dabigatran mesylate bulk drug, simultaneously, the method is effectively separated multiple genotoxic impurities from dabigatran mesylate by the method for liquid-liquid extraction, reduce the impact of dabigatran etexilate on the analysis of genotoxic impurities, reduce interfering substances.

[0009] In order to achieve the above object, the present invention provides a method for detecting genotoxic impurities in dabigatran mesylate, wherein the genotoxic impurities include methyl methanesulfonate, ethyl methanesulfonate, n-hexyl methanesulfonate, diethyl sulfite, n-hexyl chloroformate, methyl p-toluenesulfonate, ethyl p-toluenesulfonate and n-hexyl p-toluenesulfonate; the detection method comprises the following steps: (1) Sample pretreatment: The dabigatran etexilate mesylate sample was dissolved in an acid solution and then extracted. The extract was then centrifuged and the supernatant was collected to obtain the test solution; The pH value of the acid solution is 0.5-1.0, and the extraction agent used in the extraction is composed of n-hexane and acetone in a volume ratio of 8.5-9.5:0.5-1.5; as well as, Pre-treating the genotoxic impurity standard substance in the same manner as the pre-treatment of the dabigatran etexilate mesylate sample to obtain a standard solution; (2) Use gas chromatography-tandem mass spectrometry to measure the standard solution and draw a standard curve; (3) The test solution is measured using a gas chromatography-tandem mass spectrometer, and the content of each genotoxic impurity in the test solution is calculated based on the standard curve.

[0010] The innovative point of the present invention is to first adopt acid solution that dabigatran mesylate sample is dissolved, then adopt the mixed solution of normal hexane and acetone to extract the medicine after dissolving, by centrifugation, take supernatant, dabigatran mesylate and various genotoxic impurities can be effectively separated like this, reduce the interference that dabigatran mesylate brings.Meanwhile, if only normal hexane is adopted to make extraction solvent, the recovery rate of methyl mesylate is very low, and extraction efficiency is poor, and the adding of acetone can improve the extraction efficiency of genotoxic impurities, but acetone is too high, and the content of dabigatran mesylate can be increased, produces matrix effect, and makes test result distorted.

[0011] In the preferred embodiment, the acid solution is a hydrochloric acid solution. The inventor of the present invention has found that dabigatran mesylate is soluble in organic solvents such as methanol, ethanol, but directly analyzes after adopting organic solvent dissolving, dabigatran mesylate can react at the injection port of temperament, can produce impurities such as methyl methanesulfonate, ethyl methanesulfonate, n-hexyl methanesulfonate and methyl p-toluenesulfonate, ethyl p-toluenesulfonate, n-hexyl p-toluenesulfonate, and concentration is very high, the chromatographic peaks of many impurities have reached saturation, causing the false positive of bulk drug, research has also found that dabigatran mesylate is insoluble in water, but, under the hydrochloric acid environment of 0.5≤pH≤1, solubility is better.

[0012] In the preferred embodiment, relative to the dabigatran mesylate sample of 100mg, the consumption of acid solution is 2~5mL.When the consumption of acid solution is less, the dissolving effect of dabigatran mesylate is not very good, and when more, the recovery rate of methyl mesylate is on the low side.

[0013] In the preferred embodiment, relative to 100mg of dabigatran mesylate sample, the consumption of the extractant is 4.5~5.5mL. Less extractant will make it difficult to take the upper organic phase after the organic phase and the aqueous phase are layered, and it is easy to take the aqueous phase, which damages the instrument. Too much extractant will reduce the concentration of impurities, thereby affecting the detection limit of the method and increasing the detection limit.

[0014] In a preferred embodiment, the centrifugal treatment conditions are: a rotation speed of 7000-9000 r / min and a time of 3-10 min.

[0015] In a preferred embodiment, the chromatographic column of the gas chromatography-tandem mass spectrometry is a Rtx-5MS column or a DB-1701MS chromatographic column.

[0016] In the preferred embodiment, the temperature control program of the gas chromatography-tandem mass spectrometry instrument is: initial temperature 30-50°C, maintained for 4-6 minutes, 10-20°C / min to 280-320°C and maintained for 4-6 minutes. These impurities have a small molecular weight and a low boiling point. If the initial temperature is too high, the peak time of the impurities will be too early, thereby affecting the separation degree of the impurities and the solvent. If the heating rate is too fast, the separation degree between the individual genotoxic impurities will be affected. Finally, using a higher temperature can bake out the high-boiling point substances entering the instrument, thereby extending the service life of the instrument.

[0017] In the preferred embodiment, the selected qualitative and quantitative ions of each genotoxic impurity are as follows: methyl methanesulfonate: 80, 95, 65; diethyl sulfite: 95, 110, 59; ethyl methanesulfonate: 79, 109, 97; n-hexyl chloroformate: 56, 69; n-hexyl methanesulfonate: 56, 84, 97; methyl p-toluenesulfonate: 91, 155, 186; ethyl p-toluenesulfonate: 91, 155, 200; n-hexyl p-toluenesulfonate: 91, 155, 173.

[0018] In the preferred embodiment, the gas chromatography setting parameters are: the carrier gas is helium, the purity is ≥99.999%, the constant pressure mode, the injection port temperature is 220-270°C, the split ratio is 8-12:1, and the injection volume is 1-2 μL.

[0019] In a preferred embodiment, the mass spectrometer parameters are: monitoring mode is single ion monitoring, ion source temperature: 220-280°C, interface temperature: 250-300°C, solvent delay: 4-6 min.

[0020] Compared with the prior art, the present invention has at least the following advantages: (1) The detection method provided by the present invention has a simple sample processing process, high sensitivity, can simultaneously detect multiple genotoxic impurities, and has a low detection limit (the detection limits of methyl methanesulfonate, ethyl methanesulfonate, n-hexyl methanesulfonate, diethyl sulfite, n-hexyl chloroformate, methyl p-toluenesulfonate, ethyl p-toluenesulfonate, and n-hexyl p-toluenesulfonate are all 10 ng / mL). The detection results are accurate (the recovery rate is between 80% and 105%) and the stability is good. Therefore, the method provided by the present invention fully meets the requirements of the guiding principles for the validation of detection methods in the Chinese Pharmacopoeia, and can be used for the quality control of genotoxic impurities in dabigatran etexilate raw materials or preparations.

[0021] (2) The detection method provided by the present invention effectively separates a variety of genotoxic impurities from dabigatran mesylate by liquid-liquid extraction, thereby reducing the impact of dabigatran on the analysis of genotoxic impurities and reducing interfering substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1This is the chromatogram of the first repeatability test of the spiked sample in Example 1; Figure 2 This is the chromatogram of the second repeatability test of the spiked sample in Example 1; Figure 3 This is the chromatogram of the third repeatability test of the spiked sample in Example 1; Figure 4 This is the chromatogram of the fourth repeatability test of the spiked sample in Example 1; Figure 5 This is the fifth repeatability test chromatogram of the spiked sample in Example 1; Figure 6 This is the chromatogram of the sixth repeatability test of the spiked sample in Example 1. DETAILED DESCRIPTION

[0023] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0024] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the following specific embodiments. Obviously, the embodiments described below are only a part of the embodiments, and all other embodiments obtained by professionals in this field without creative work still belong to the protection scope of the present invention.

[0025] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0026] Gas chromatography-tandem mass spectrometry: GC-MS (Shimadzu QP2020 NX).

[0027] Reagents and materials Reagents: methyl methanesulfonate, ethyl methanesulfonate, n-hexyl methanesulfonate, diethyl sulfite, n-hexyl chloroformate, methyl p-toluenesulfonate, ethyl p-toluenesulfonate, and n-hexyl p-toluenesulfonate are mixed standard solutions. The purity of the standard products is ≥99%. n-hexane and acetone are chromatographically pure, and water is ultrapure water.

[0028] Materials: 0.45μm organic phase syringe filter (without ester residue).

[0029] Example 1 1. Solution preparation and sample pretreatment The extracting liquid is a combination of n-hexane and acetone in a volume ratio of 9:1.

[0030] Acid solution: hydrochloric acid solution, pH 0.7.

[0031] Preparation of standard solution: Take a certain amount of 8 kinds of genotoxic mixed standards at 10μg / mL respectively, add hydrochloric acid solution and make up to 2mL, then add 5mL of extractant, shake vigorously, centrifuge at 8000r / min for 5min, and prepare standard solutions with concentrations of 20ng / mL, 50ng / mL, 100ng / mL, 150ng / mL and 200ng / mL, and take the supernatant for GC / MS analysis.

[0032] Dabigatran etexilate mesylate sample: weigh 100 mg of dabigatran etexilate mesylate sample into a 10 mL centrifuge tube, add 2 mL of hydrochloric acid solution, sonicate to dissolve it, add 5 mL of extractant, shake vigorously, centrifuge at 8000 r / min for 5 min, and take the supernatant for GC / MS analysis.

[0033] 2. Detection Gas chromatography parameters are as follows: chromatographic column: Rtx-5MS column 30m*0.25mm, 0.25μm; injection port temperature: 250℃; injection volume: 1μL; split ratio: 10:1; flow control mode: constant pressure mode; Heating program: initial temperature 40℃, hold for 5 min, increase to 300℃ at 15℃ / min and hold for 5 min.

[0034] The mass spectrometry parameters were: ion source temperature: 250°C, interface temperature: 280°C, solvent delay: 5 min; monitoring mode: single ion monitoring.

[0035]

[0036] 3. Instrument stability Determination method: Take the control solution (prepared with standard substances) with a concentration of 100% limit (corresponding to a concentration of 100 ng / ml) and inject it into GC-MS for 6 consecutive times.

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] From the data in the above table, it can be seen that the test precision of methyl methanesulfonate, ethyl methanesulfonate, n-hexyl methanesulfonate, diethyl sulfite, n-hexyl chloroformate, methyl p-toluenesulfonate, ethyl p-toluenesulfonate and n-hexyl p-toluenesulfonate is high and all meet the experimental requirements.

[0046] 4. Limit of detection and limit of quantification The stock solution was diluted and injected while diluting until the signal-to-noise ratio of each genotoxic impurity was 10:1. The concentration of the solution at this time was set as the minimum limit of quantification concentration (limit of quantification), and the sample was injected 6 times continuously to calculate the signal-to-noise ratio; the dilution was continued and the sample was injected while diluting until the signal-to-noise ratio of each genotoxic impurity was 3:1. The concentration at this time was set as the minimum detection limit concentration (limit of detection), and the sample was injected 3 times continuously to calculate the signal-to-noise ratio. The test results are shown in Table 9.

[0047]

[0048] 5. Regression equations and correlation coefficients of various genotoxic impurities A series of standard solutions were detected by gas chromatography-tandem mass spectrometry, and a standard curve was drawn using the external standard method to obtain a standard working curve for each genotoxic impurity.

[0049] Acceptance criteria (General Rules of Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition) 9101 Guiding Principles for Validation of Analytical Methods for Pharmaceutical Quality Standards): (1) Linear correlation coefficient R 2 It must not be less than 0.995, (2) the intercept / 100% limit must not be greater than 10%.

[0050]

[0051] It can be seen from the data in the above table that the preparation of the standard curve meets the standard requirements.

[0052] 6. Accuracy Each genotoxic impurity was added to the dabigatran etexilate mesylate sample solution at a limit concentration of 50%, 100% and 150%, and eight samples with different impurity concentrations were prepared. Each sample was measured 3 times, and the accuracy of the measurement results was calculated.

[0053] Dabigatran etexilate mesylate sample: weigh 100 mg of dabigatran etexilate mesylate sample into a 10 mL centrifuge tube, add 2 mL of hydrochloric acid solution, sonicate to dissolve it, add 5 mL of extractant, shake vigorously, centrifuge at 8000 r / min for 5 min, and take the supernatant for GC / MS analysis.

[0054] Preparation of 50% limit concentration solution: weigh 100 mg of dabigatran etexilate mesylate sample into a 10 mL centrifuge tube, add 25 μL of standard solution with a concentration of 10 μg / mL, then add 1.975 mL of hydrochloric acid solution, add 5 mL of extractant, shake vigorously, centrifuge at 8000 r / min for 5 minutes, and take the supernatant for GC / MS analysis.

[0055] Preparation of 100% limit concentration solution: weigh 100 mg of dabigatran etexilate mesylate sample into a 10 mL centrifuge tube, add 50 μL of standard solution with a concentration of 10 μg / mL, then add 1.95 mL of hydrochloric acid solution, add 5 mL of extractant, shake vigorously, centrifuge at 8000 r / min for 5 min, and take the supernatant for GC / MS analysis.

[0056] Preparation of 150% limit concentration solution: weigh 100 mg of dabigatran etexilate mesylate sample into a 10 mL centrifuge tube, add 75 μL of 10 μg / mL standard solution, then add 1.925 mL of hydrochloric acid solution, add 5 mL of extractant, shake vigorously, centrifuge at 8000 r / min for 5 min, and take the supernatant for GC / MS analysis.

[0057] Acceptance criteria (Guidelines for Validation of Analytical Methods for Pharmaceutical Quality Standards, Part IV, Pharmacopoeia of the People's Republic of China (2020 Edition) 9101): (1) The calculated recovery rate should be between 80% and 120%; (2) The RSD of the calculated 9 recovery rate data should be ≤10%.

[0058] The results are as follows:

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] From the above data, it can be seen that the accuracy of each genotoxic impurity meets the test requirements and is high in accuracy.

[0067] 7. Precision (repeatability test) Six spiked samples with a 100% limit concentration were prepared in parallel, tested, and the RSD of the six groups of content data was calculated. Acceptance criteria (General Rules of Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition) 9101 Guiding Principles for Validation of Analytical Methods for Pharmaceutical Quality Standards): The RSD of the reference substance content in the 6 test solution should not be greater than 10%. The results are as follows:

[0068]

[0069] It can be seen from the data in the above table that the repeatability of each genotoxic impurity meets the requirements and has good repeatability.

[0070] At the same time, the present invention also provides chromatograms of 6 repetitive tests, see Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 .

[0071] 8. Precision (intermediate precision test) At different times, another experimenter prepared 6 spiked samples with 100% limit concentration in parallel for determination. RSD was calculated together with the repeatability test results (6 groups) and this experiment (6 groups), a total of 12 groups of data.

[0072] Acceptance criteria (General Rules of Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition) 9101 Guiding Principles for Validation of Analytical Methods for Pharmaceutical Quality Standards): The RSD of the content of the reference substance in the test solution of 12 solutions in the repeatability test should not be greater than 20%.

[0073]

[0074]

[0075] From the above data, it can be seen that the precision of each genotoxic impurity meets the requirements and has high precision.

[0076] 9. Stability test Prepare a mixed control solution of eight genotoxic impurities at a concentration of 100 ng / mL, inject the sample for 12 hours, record the peak area, and the RSD value should be no more than 10%. The results are as follows:

[0077]

[0078] From the above data, it can be seen that the mixed control solution has good solution stability within 12 hours.

[0079] 10. Sample testing Three batches of different samples were taken for testing, and the test sample and 100% limit concentration (100ng / mL) reference sample solutions were prepared and tested. The test results are shown in the following table:

[0080]

[0081] Comparative Example 1 (Testing the accuracy of genotoxic impurity methyl methanesulfonate) 1. Solution preparation and sample pretreatment This comparative example was carried out with reference to the method of Example 1, except that the extractant used in this comparative example was a combination of n-hexane and acetone in a volume ratio of 9.8:0.2, and the amount of the extractant used was the same as that in Example 1.

[0082] 2. Detection The detection method is the same as in Example 1.

[0083] 3. Accuracy test The detection steps are the same as the 6 accuracy test steps in Example 1, and the test results are as follows:

[0084] It can be seen from the above data that the accuracy of each genotoxic impurity does not meet the test requirements and the accuracy is low.

Claims

1. A method for detecting genotoxic impurities in dabigatran mesylate, characterized in that: The genotoxic impurities include methyl methanesulfonate, ethyl methanesulfonate, n-hexyl methanesulfonate, diethyl sulfite, n-hexyl chloroformate, methyl p-toluenesulfonate, ethyl p-toluenesulfonate and n-hexyl p-toluenesulfonate; and the detection method includes the following steps: (1) Sample pretreatment: The dabigatran etexilate mesylate sample was dissolved in an acid solution and then extracted. The extract was then centrifuged and the supernatant was collected to obtain the test solution; The pH value of the acid solution is 0.5-1.0, and the extraction solvent used in the extraction is composed of n-hexane and acetone in a volume ratio of 8.5-9.5:0.5-1.5; as well as, Pre-treating the genotoxic impurity standard substance in the same manner as the pre-treatment of the dabigatran etexilate mesylate sample to obtain a standard solution; (2) Use gas chromatography-tandem mass spectrometry to measure the standard solution and draw a standard curve; (3) The test solution is measured using a gas chromatography-tandem mass spectrometer, and the content of each genotoxic impurity in the test solution is calculated based on the standard curve.

2. the detection method of genotoxic impurities in a kind of dabigatran mesylate according to claim 1, is characterized in that: Relative to 100 mg of dabigatran etexilate mesylate sample, the amount of acid solution used is 2-5 mL.

3. according to the detection method of genotoxic impurities in a kind of dabigatran mesylate according to claim 1 or 2, it is characterized in that: Relative to 100 mg of dabigatran etexilate mesylate sample, the amount of the extractant used is 4.5-5.5 mL.

4. according to the detection method of genotoxic impurities in a kind of dabigatran mesylate according to claim 1 or 2, it is characterized in that: The chromatographic column of the gas chromatography-tandem mass spectrometer is a Rtx-5MS column or a DB-1701MS chromatographic column.

5. according to the detection method of genotoxic impurities in a kind of dabigatran mesylate according to claim 1 or 2, it is characterized in that: The temperature control program of the gas chromatography-tandem mass spectrometry instrument is: initial temperature 30-50°C, maintained for 4-6 minutes, increased to 280-320°C at 10-20°C / min and maintained for 4-6 minutes.

6. according to the detection method of genotoxic impurities in a kind of dabigatran mesylate according to claim 1 or 2, it is characterized in that: The selected qualitative and quantitative ions for each genotoxic impurity are as follows: Methyl methanesulfonate: 80, 95, 65; diethyl sulfite: 95, 110, 59; ethyl methanesulfonate: 79, 109, 97; n-hexyl chloroformate: 56, 69; n-hexyl methanesulfonate: 56, 84, 97; methyl p-toluenesulfonate: 91, 155, 186; ethyl p-toluenesulfonate: 91, 155, 200; n-hexyl p-toluenesulfonate: 91, 155, 173.

7. according to the detection method of genotoxic impurities in a kind of dabigatran mesylate according to claim 1 or 2, it is characterized in that: The gas chromatography setting parameters are: carrier gas is helium, purity ≥99.999%, constant pressure mode, injection port temperature is 220-270°C, split ratio is 8-12:1, and injection volume is 1-2 μL.

8. The detection method of genotoxic impurities in a kind of dabigatran mesylate according to claim 1 or 2, characterized in that: The parameters of the mass spectrometer are: monitoring mode is single ion monitoring, ion source temperature: 220~280°C, interface temperature: 250~300°C, solvent delay: 4~6min.

9. The detection method of genotoxic impurities in a kind of dabigatran mesylate according to claim 1 or 2, characterized in that: The acid solution is a hydrochloric acid solution.

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