A method for detecting 5-chlorothiophene-2-carboxaldehyde

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to a method for detecting 5-chlorothiophene-2-formaldehyde. The present invention provides a new method for detecting 5-chlorothiophene-2-formaldehyde, which can simply, quickly and accurately determine the content of 5-chlorothiophene-2-formaldehyde impurities in raw materials, and the detection limit of 5-chlorothiophene-2-formaldehyde is extremely low and the sensitivity is high. The method can be used to effectively monitor the purity and quality of drugs during the production process.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and more specifically relates to a method for detecting 5-chlorothiophene-2-carboxaldehyde. Background Art

[0002] The chemical name of Rivaroxaban is 5-chloro-nitrogen-({(5S)-2-oxo-3-[-4-(3-oxo-4-morpholinyl)phenyl]-1,3-oxazolidin-5-yl}methyl)-2-thiophene-carboxamide, and its structural formula is The molecular formula is C 19 H 18 C1N 3 O 5 S, is a low molecular weight oral anticoagulant with high selectivity to directly inhibit factor Xa, used to prevent and / or treat various embolic diseases. However, rivaroxaban, during the production process, can introduce impurities such as starting materials and intermediates to affect the quality and efficacy of rivaroxaban. 5-Chlorothiophene-2-carboxaldehyde is one of the impurities, with genotoxicity, if rivaroxaban contains this impurity, it can not only affect the purity and efficacy of rivaroxaban, but also may cause great harm to human body, in addition, in other drugs, it is also easy to mix 5-chlorothiophene-2-carboxaldehyde impurities, therefore, the detection and monitoring of 5-chlorothiophene-2-carboxaldehyde impurities in drugs becomes a key problem that needs to be solved urgently. But the prior art lacks the detection method of 5-chlorothiophene-2-carboxaldehyde in rivaroxaban. For example, Wang Qianqian et al. disclosed a method for detecting 5-chlorothiophene-2-carbonyl chloride (Wang Qianqian, Gu Mingpan, Shen Zhichao. HPLC determination of the impurity 5-chlorothiophene-2-carbonyl chloride in the medium [J]. Guangdong Chemical Industry, 2022 (3)). However, the structure of 5-chlorothiophene-2-carbonyl chloride is different from that of 5-chlorothiophene-2-carboxaldehyde to be detected in the present invention. During the high performance liquid chromatography analysis, the different structures will cause significant differences in the analysis results. Therefore, their method is not suitable for detecting 5-chlorothiophene-2-carboxaldehyde. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the deficiency in the prior art that 5-chlorothiophene-2-carboxaldehyde impurities in rivaroxaban cannot be detected, and to provide a method for detecting 5-chlorothiophene-2-carboxaldehyde.

[0004] The above purpose of the present invention is achieved through the following technical solutions:

[0005] A method for detecting 5-chlorothiophene-2-carboxaldehyde, wherein a test sample is analyzed by a high performance liquid chromatography analysis method, wherein the chromatographic conditions of the high performance liquid chromatography analysis method are:

[0006] Chromatographic column: C8 chromatographic column;

[0007] Mobile phase: Phase A is 0.05-0.2% trifluoroacetic acid (TFA), phase B is acetonitrile (ACN);

[0008] Elution procedure: based on the total volume of mobile phase as 100%; from 0 to 6 min, the volume of mobile phase A decreases from 70% to 54%, and the volume of mobile phase B increases from 30% to 46%; from 6 to 15 min, the volume of mobile phase A is maintained at 54%, and the volume of mobile phase B is maintained at 46%; from 15 to 25 min, the volume of mobile phase A decreases from 54% to 20%, and the volume of mobile phase B increases from 46% to 80%; from 25 to 35 min, the volume of mobile phase A is maintained at 20%, and the volume of mobile phase B is maintained at 80%; from 35 to 36 min, the volume of mobile phase A increases from 20% to 70%, and the volume of mobile phase B decreases from 80% to 30%; from 36 to 43 min, the volume of mobile phase A is maintained at 70%, and the volume of mobile phase B is maintained at 30%.

[0009] We tried different mobile phases and elution procedures and found that only when the mobile phase A was 0.05-0.2% trifluoroacetic acid, the mobile phase B was acetonitrile, and the above elution procedure was used, could we obtain results with better peak shape and better separation.

[0010] Preferably, the phase A is 0.1-0.2% trifluoroacetic acid.

[0011] Preferably, the flow rate of the mobile phase is 0.65-1.2 mL / min.

[0012] Preferably, the detection wavelength of the HPLC is 250-260 nm.

[0013] Preferably, the C8 chromatographic column is Agilent ZORBAX SB-C8.

[0014] More preferably, the specifications of the C8 chromatographic column are 250×4.6 nm, 5 μm.

[0015] Preferably, the test article is rivaroxaban.

[0016] More preferably, the test sample is detected in the form of a solution, and the solvent of the test sample solution is a mixed solution of DMSO, acetonitrile and water.

[0017] Specifically, the volume ratio of DMSO, acetonitrile and water is 1:(2-3):(2-3).

[0018] Preferably, the limit of quantification of the 5-chlorothiophene-2-carboxaldehyde is 0.00003 mg / ml.

[0019] Specifically, the conditions of the high performance liquid chromatography are:

[0020] Chromatographic column: Agilent ZORBAX SB-C8, 250 × 4.6 nm, 5 μm;

[0021] Detection wavelength: 250~260nm;

[0022] Mobile phase: Phase A is 0.1-0.2% trifluoroacetic acid, phase B is acetonitrile;

[0023] Flow rate: 0.65~1.2ml / min;

[0024] Elution procedure: based on the total volume of mobile phase as 100%; from 0 to 6 min, the volume of mobile phase A decreases from 70% to 54%, and the volume of mobile phase B increases from 30% to 46%; from 6 to 15 min, the volume of mobile phase A is maintained at 54%, and the volume of mobile phase B is maintained at 46%; from 15 to 25 min, the volume of mobile phase A decreases from 54% to 20%, and the volume of mobile phase B increases from 46% to 80%; from 25 to 35 min, the volume of mobile phase A is maintained at 20%, and the volume of mobile phase B is maintained at 80%; from 35 to 36 min, the volume of mobile phase A increases from 20% to 70%, and the volume of mobile phase B decreases from 80% to 30%; from 36 to 43 min, the volume of mobile phase A is maintained at 70%, and the volume of mobile phase B is maintained at 30%;

[0025] Injection volume: 20-30 μL;

[0026] Column temperature: 25-35°C;

[0027] Running time: 40 to 45 minutes.

[0028] The present invention has the following beneficial effects:

[0029] The invention provides a new method for detecting 5-chlorothiophene-2-carboxaldehyde, which can simply, quickly and accurately determine the content of 5-chlorothiophene-2-carboxaldehyde impurities in raw materials, and the detection limit of 5-chlorothiophene-2-carboxaldehyde is extremely low and the sensitivity is high. The method can be used to effectively monitor the purity and quality of drugs in the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 HPLC spectrum of blank solution.

[0031] Figure 2 HPLC spectrum of the reference solution.

[0032] Figure 3 It is the HPLC spectrum of the test solution 1.

[0033] Figure 4 It is the HPLC spectrum of test solution 2.

[0034] Figure 5 This is the HPLC spectrum of the test solution 3.

[0035] Figure 6 HPLC chromatogram of the test sample spiked solution.

[0036] Figure 7 HPLC spectrum of the detection limit solution.

[0037] Figure 8 HPLC spectrum of the quantitative limit solution. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] The test solution was prepared with rivaroxaban, and the batch numbers of rivaroxaban used were CLF01-0002079-23-07, CLF01-0002079-23-08, and CLF01-0002079-23-09.

[0041] The preparation method of the reagent or sample to be tested used in the present invention is as follows:

[0042] Diluent: 500 mL of acetonitrile and 500 mL of purified water, mix well, and ultrasonicate to obtain;

[0043] Blank solution: Pipette 1.0 mL DMSO and 5.0 mL diluent, shake well, and you have it;

[0044] Control stock solution 1: weigh 25 mg of 5-chlorothiophene-2-carboxaldehyde into a 100 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain a concentration of 0.25 mg / ml of 5-chlorothiophene-2-carboxaldehyde;

[0045] Control stock solution 2: Pipette 5.0 mL of control stock solution 1 into a 50 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain a concentration of 0.025 mg / mL 5-chlorothiophene-2-carboxaldehyde.

[0046] Reference solution: Pipette 1.0 mL of reference stock solution 2 and 5.0 mL of DMSO into a 25 mL volumetric flask, dilute to scale with diluent, and shake well to obtain a concentration of 0.001 mg / mL 5-chlorothiophene-2-carboxaldehyde.

[0047] Test solution: weigh 50 mg of rivaroxaban, add 1.0 mL of DMSO to dissolve, then add 5.0 mL of diluent to mix, filter, and obtain;

[0048] Test sample spiked solution: weigh 50 mg of rivaroxaban, add 1.0 mL of DMSO to dissolve, then add 5.0 mL of reference solution, mix well, and filter to obtain;

[0049] Detection limit solution: Pipette 1.0 mL of the reference solution into a 10 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain a concentration of 5-chlorothiophene-2-carboxaldehyde of 0.0001 mg / ml.

[0050] Quantitation limit solution (sensitivity solution): Pipette 3.0 mL of the reference solution into a 10 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain a concentration of 5-chlorothiophene-2-carboxaldehyde of 0.00003 mg / ml.

[0051] Example 1 Detection of 5-chlorothiophene-2-carboxaldehyde in rivaroxaban

[0052] S1. Setting of chromatographic conditions: Set according to Table 1

[0053] Table 1 Chromatographic conditions

[0054]

[0055]

[0056] S2. After the system is balanced, take the corresponding solution and inject it according to the injection volume in Table 2, and record the corresponding chromatogram. Report the retention time, peak area and separation degree of each substance to be tested in the reference solution and the test solution, and record the chromatogram. The acceptable standard is: 5-chlorothiophene-2-carboxaldehyde ≤50ppm.

[0057] Table 2 Injection volume of corresponding solutions

[0058] Solution name Number of injection needles blank solution 1-2 needles Sensitivity Solution (SEN) 1 needle Reference solution (RS) 6-pin Test solution 1 2-pin Test solution 2 2-pin Test solution 3 3-pin

[0059] According to the measured peak response value, the residual content of 5-chlorothiophene-2-carboxaldehyde in the test sample was calculated according to the following formula, and the average of the two determination results was taken as the test result:

[0060]

[0061] Where: A i is the peak area of ​​5-chlorothiophene-2-carboxaldehyde measured in the test solution;

[0062] A S is the average value of the peak area of ​​5-chlorothiophene-2-carboxaldehyde measured in the reference solution;

[0063] W T is the sample weight of the test product, mg;

[0064] W S The weight of 5-chlorothiophene-2-carboxaldehyde weighed to prepare the reference solution, mg;

[0065] D T is the dilution multiple of the test product;

[0066] D S is the dilution multiple of the reference solution 5-chlorothiophene-2-carboxaldehyde;

[0067] P is the content of 5-chlorothiophene-2-carboxaldehyde in the reference solution.

[0068] Since the peaks in the chromatograms for the same sample tested with the same parameters and methods are similar, only one of the spectra obtained for the same sample injected with multiple needles is provided in this article. The peak areas are organized in the corresponding tables.

[0069] Test results such as Figure 1 to Figure 5 As shown, the test results are shown in Table 3: It can be seen from Table 3 that the blank solution has an obvious peak at about time 2.5, and no peak at about time 11.1; the reference solutions 1 to 6 all have new peaks at about time 11.1, the peak area of ​​reference solution 1 is 53.66, the peak area of ​​reference solution 2 is 53.81, the peak area of ​​reference solution 3 is 53.73, the peak area of ​​reference solution 4 is 53.80, the peak area of ​​reference solution 5 is 53.86, and the peak area of ​​reference solution 6 is 53.70. No new peaks appear at about time 11.1 for all test solutions, proving that the impurity 5-chlorothiophene-2-carboxaldehyde was not detected in the tested batches of test samples, and the safety is high.

[0070] Table 3 Detection results of 5-chlorothiophene-2-carboxaldehyde

[0071]

[0072] Example 2 Verification of the Applicability of the Detection Method for 5-Chlorothiophene-2-Carboxaldehyde in Rivaroxaban

[0073] Step S1 is the same as in Example 1;

[0074] S2. After the system is balanced, take 1 injection of blank solution and 6 injections of reference solution according to the above chromatographic conditions, record the chromatogram, and calculate according to the peak area.

[0075] Test results such as Figures 1-2As shown in Table 4: the blank solution had no peak at about time 11.1, and all reference solutions had new peaks at time 11.1, the RSD value of the peak area was 0.13%, and the signal-to-noise ratio of the peak was 101.9, which proved that the detection method of the present invention met the system suitability requirements and had good system suitability.

[0076] Table 4 Applicability results of the detection method for 5-chlorothiophene-2-carboxaldehyde in rivaroxaban

[0077]

[0078]

[0079] Example 3 Specificity Verification of the Detection Method for 5-Chlorothiophene-2-Carboxaldehyde in Rivaroxaban

[0080] Step S1 is the same as in Example 1;

[0081] S2. After the system suitability is qualified, take a needle each of the blank solution, reference solution, test solution 3, and test solution spiked with the test sample, and record the chromatogram.

[0082] Chromatograms see Figure 1 , Figure 2 , Figure 5 , Figure 6 The test results are shown in Table 5: the blank solution has no peak at about time 11.1, the reference solution has a new peak at about time 11.1 compared with the blank solution, and there is no adjacent peak near the peak, the test solution 3 has no detected peak at about time 11.1, and the test spiked solution 1 has a new peak at about time 11.1 and the separation degree with the nearest adjacent peak is 2.35. It can be seen from the above results that the detection method of the present invention meets the acceptable standard of specificity, has good specificity for the detection method of 5-chlorothiophene-2-carboxaldehyde.

[0083] Table 5 Specificity results of the detection method for 5-chlorothiophene-2-carboxaldehyde in rivaroxaban

[0084]

[0085]

[0086] Example 4 Accuracy Verification of the Detection Method for 5-Chlorothiophene-2-Carboxaldehyde in Rivaroxaban

[0087] Step S1 is the same as in Example 1;

[0088] S2. After the system is balanced, inject the above solutions into the chromatograph in the order of Table 6 and record the chromatogram.

[0089] Table 6 Number of injection needles for the corresponding solutions

[0090] Injection solution Number of injection needles blank solution 2-pin Sensitivity solution 1 needle Reference solution 6-pin Test solution 3 3-pin Test sample spike solution 3-pin

[0091] Chromatograms see Figure 1 , Figure 2 , Figure 5 , Figure 6 The test results are shown in Table 7: the blank solution had no peak at around time 11.1, the reference solutions 1 to 6 showed new peaks at around time 11.1 compared with the blank solution, the test solution 3 had no new peak at around time 11.1, the test spiked solution showed a new peak at around time 11.1, and the average recovery rate of the test spiked solution was 99.6%.

[0092] It can be seen from the corresponding spectra and Table 7 that the detection method of 5-chlorothiophene-2-carboxaldehyde in rivaroxaban of the present invention meets the acceptable standard of accuracy and has good accuracy.

[0093] Table 7 Accuracy results of 5-chlorothiophene-2-carboxaldehyde detection method in rivaroxaban

[0094]

[0095]

[0096] Example 5 LOQ (Limit of Quantification) and LOD (Limit of Detection) Verification of the Detection Method for 5-Chlorothiophene-2-Carboxaldehyde in Rivaroxaban

[0097] Step S1 is the same as in Example 1;

[0098] S2. After the system suitability is qualified, take the detection limit solution and the quantification limit solution respectively, inject 3 injections each continuously, and record the chromatogram.

[0099] The detection limit results are as follows Figure 7 As shown in Table 8: The detection limit solution showed a peak at around time 11.1, the RSD value of the peak area was 1.3%, and the signal-to-noise ratios were 47.8, 52.1, and 46.8, respectively, proving that the detection method of the present invention meets the acceptable standard for the detection limit, and the detection limit concentration is 0.0001 mg / ml.

[0100] Table 8 Detection limit results of 5-chlorothiophene-2-carboxaldehyde detection method in rivaroxaban

[0101]

[0102] The quantitative limit results are as follows Figure 8As shown in Table 9: The quantitative limit solutions (1, 2, 3) all showed a peak at about time 11.1, the RSD value of the peak area was 0.57%, and the signal-to-noise ratios were 117.0, 129.6, and 104.6, respectively, proving that the detection method of the present invention meets the acceptable standard of the detection limit, and the quantitative limit concentration is 0.00003 mg / ml.

[0103] Table 9 Quantitative limit results of 5-chlorothiophene-2-carboxaldehyde detection method in rivaroxaban

[0104]

[0105] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A method for detecting 5-chlorothiophene-2-carbaldehyde, characterized in that: The test sample was analyzed by high performance liquid chromatography analysis method, and the chromatographic conditions of the high performance liquid chromatography analysis method were: Chromatographic column: C8 chromatographic column; Mobile phase: Phase A is 0.1~0.2% trifluoroacetic acid, phase B is acetonitrile; Elution procedure: based on the total volume of mobile phase as 100%; from 0 to 6 min, the volume of mobile phase A decreased from 70% to 54%, and the volume of mobile phase B increased from 30% to 46%; from 6 to 15 min, the volume of mobile phase A was maintained at 54%, and the volume of mobile phase B was maintained at 46%; from 15 to 25 min, the volume of mobile phase A decreased from 54% to 20%, and the volume of mobile phase B increased from 46% to 80%; from 25 to 35 min, the volume of mobile phase A was maintained at 20%, and the volume of mobile phase B was maintained at 80%; from 35 to 36 min, the volume of mobile phase A increased from 20% to 70%, and the volume of mobile phase B decreased from 80% to 30%; from 36 to 43 min, the volume of mobile phase A was maintained at 70%, and the volume of mobile phase B was maintained at 30%; The test article is rivaroxaban; The flow rate of the mobile phase is 0.65~1.2 mL / min; the detection wavelength of the high performance liquid chromatography is 250~260 nm; the C8 chromatographic column is Agilent ZORBAX SB-C8; the specification of the C8 chromatographic column is 250×4.6 mm, 5 μm; the test sample is detected in the form of a solution, and the solvent of the test sample solution is a mixed solution of DMSO, acetonitrile and water.

2. The method according to claim 1, characterized in that: The volume ratio of DMSO, acetonitrile and water is 1:(2~3):(2~3).

3. The method according to claim 1 or 2, characterized in that: The limit of quantification of the 5-chlorothiophene-2-carboxaldehyde is 0.00003 mg / ml.

Images