Method for detecting potential mutagenic impurities in argatroban injection

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting potential mutagenic impurities in an argatroban injection, which comprises the following steps: preparing a test solution which is a mixture of an argatroban injection and a solvent, and the solvent is an acetonitrile aqueous solution; the test solution is subjected to high performance liquid chromatography detection under the detection conditions that mobile phases are a mobile phase A and a mobile phase B, the mobile phase A is a phosphoric acid aqueous solution with the mass concentration of 0.01-1%, the mobile phase B is a strong-polarity organic solvent, elution is performed by adopting a gradient elution procedure, the content of potential mutagenic impurities is detected after gradient elution, and the content of the potential mutagenic impurities is determined according to the content of the potential mutagenic impurities. The potential mutagenic impurities are 3-methyl-8-quinoline sulfonic acid ethyl ester and 3-methyl-8-quinoline sulfonic acid glyceride; according to the method, the potential mutagenic impurities can be separated from other specific impurities, and the content of the potential mutagenic impurities can be measured more accurately.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting potentially mutagenic impurities in argatroban injection. Background Technology

[0002] Argatroban is composed of R- and S-isomers in a 65% to 35% ratio. The S-isomer has twice the thrombin-inhibiting activity of the R-isomer. Argatroban is a small molecule with high selectivity, capable of reversibly and directly inhibiting thrombin activity. It rapidly binds to both free thrombin in circulation and thrombin in blood clots, producing an anticoagulant effect.

[0003] The main potential mutagenic impurities in argatroban injection are sulfonate esters. These sulfonate esters may originate from tetrahydroquinoline sulfonic acid produced by the photodegradation of argatroban, which, upon further reduction, produces quinoline sulfonic acid that reacts with excipients ethanol and glycerol in the formulation to form ethyl quinoline sulfonate and glyceryl quinoline sulfonate.

[0004] Argatroban injection degrades under alkaline, acidic, and light-exposed conditions, producing various types of degradation impurities. Six specific impurities are generated. Under alkaline conditions, the guanidinium group in its molecular structure undergoes an elimination reaction to produce the corresponding amino compound, i.e., impurity A. Under acidic conditions, the piperidine cyclamide bond in its molecular structure breaks to produce the corresponding acid degradation impurity, i.e., impurity D. Under ultraviolet light, the sulfonamide bond of its tetrahydroquinoline breaks to produce impurity C. Impurity C is further reduced to produce quinoline sulfonic acid, i.e., impurity I. Under prolonged light exposure, a hydroxyl compound, i.e., impurity J, is generated. Impurity B is an intermediate byproduct of the reduction reaction in the raw material synthesis process.

[0005] Ethyl quinoline sulfonate and glyceryl quinoline sulfonate are potential mutagenic impurities and require focused detection. However, these impurities are difficult to effectively separate from the six specific impurities mentioned above during detection, which reduces the accuracy of the detection.

[0006] Patent application CN116699015A discloses a method for detecting genotoxic impurities in argatroban injection. The genotoxic impurities are ethyl 3-methyl-8-quinoline sulfonate and glyceryl 3-methyl-8-quinoline sulfonate. The detection conditions are as follows: a C18 column is used, with a mixed solution of ammonium 3-methylbutyrate and ammonium acetate as mobile phase A and acetonitrile as mobile phase B, and gradient elution is performed. The detection wavelength is 259 nm. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for detecting potential mutagenic impurities in argatroban injection, which can separate potential mutagenic impurities from other specific impurities and make the determination of the content of potential mutagenic impurities more accurate.

[0008] This invention provides a method for detecting potential mutagenic impurities in argaban injection, comprising preparing a test solution, wherein the test solution is a mixture of argaban injection and a solvent, wherein the solvent is an aqueous acetonitrile solution; The test solution was analyzed by high-performance liquid chromatography (HPLC) under the following conditions: mobile phase A was a 0.01-1% (w / w) aqueous solution of phosphoric acid, and mobile phase B was a strongly polar organic solvent. A gradient elution program was used.

[0009] After gradient elution, the content of potential mutagenic impurities was detected, namely ethyl 3-methyl-8-quinoline sulfonate and glyceryl 3-methyl-8-quinoline sulfonate.

[0010] Preferably, the gradient elution procedure is as follows: .

[0011] Preferably, the highly polar organic solvent is methanol, ethanol, propanol, or acetonitrile.

[0012] Preferably, the highly polar organic solvent is acetonitrile.

[0013] Preferably, the mobile phase A is a 0.1% (w / w) aqueous solution of phosphoric acid.

[0014] Preferably, the volume ratio of acetonitrile to water in the solvent is 20:80.

[0015] Preferably, when using high performance liquid chromatography (HPLC) for detection, the column temperature is 20-35℃, the wavelength is 217nm, and the flow rate is 0.8-1.2ml / min.

[0016] Preferably, when using high performance liquid chromatography (HPLC) for detection, the column temperature is 35°C and the flow rate is 1.0 ml / min.

[0017] Preferably, the method further includes preparing a reference solution and performing high-performance liquid chromatography (HPLC) to detect the reference solution. The reference standard is ethyl 3-methyl-8-quinoline sulfonate or glyceryl 3-methyl-8-quinoline sulfonate, and the solvent in the reference solution is an aqueous solution of acetonitrile.

[0018] Preferably, in the acetonitrile aqueous solution, the volume ratio of acetonitrile to water is 20:80.

[0019] The beneficial effects of this invention are that it enables the effective separation of six specific impurities that may be present in argatroban injection, as well as the main component argatroban, from ethyl 3-methyl-8-quinoline sulfonate and glyceryl 3-methyl-8-quinoline sulfonate. Furthermore, it allows for the quantitative determination of ethyl 3-methyl-8-quinoline sulfonate and glyceryl 3-methyl-8-quinoline sulfonate. This method is simple to operate, has good specificity, high sensitivity, and is accurate and reliable. Using this method to determine the results of potential mutagenic impurities in sulfonate esters provides assurance for the quality control of argatroban injection. Attached Figure Description

[0020] Figure 1 This is the blank solvent chromatogram of the present invention.

[0021] Figure 2 This is a chromatogram of the spiked solution of the test sample of the present invention.

[0022] Figure 3 This is a chromatogram of a mixed solution of six specific impurities according to the present invention.

[0023] Figure 4 This is a chromatogram of the localization solution of ethyl 3-methyl-8-quinoline sulfonate (impurity G) of the present invention.

[0024] Figure 5 This is a chromatogram of the localization solution of 3-methyl-8-quinoline sulfonate glycerol (impurity H) of the present invention.

[0025] Figure 6 This is the chromatogram of Comparative Example 1.

[0026] Figure 7 This is the chromatogram of Comparative Example 2. Detailed Implementation

[0027] A method for detecting potential mutagenic impurities in argaban injection involves preparing a test solution and a reference solution. The test solution is a mixture of argaban injection and a solvent, wherein the solvent is an aqueous acetonitrile solution (acetonitrile to water volume ratio of 20:80). The reference standard is ethyl 3-methyl-8-quinoline sulfonate or glyceryl 3-methyl-8-quinoline sulfonate, and the solvent in the reference solution is an aqueous acetonitrile solution (acetonitrile to water volume ratio of 20:80).

[0028] The test solution and reference solution were analyzed by high-performance liquid chromatography (HPLC). The detection conditions were as follows: mobile phase A was 0.1% (w / w) aqueous phosphoric acid solution, and mobile phase B was acetonitrile. A gradient elution program was used. The gradient elution program was as follows:

[0029] For high performance liquid chromatography (HPLC) detection, the chromatographic column was Thermo Hypersil GOLD 4.6mm×250mm, 5μm, the column temperature was 35℃, the wavelength was 217nm, the flow rate was 1.0ml / min, and the injection volume was 20μl. After gradient elution, the content of potential mutagenic impurities was detected, namely ethyl 3-methyl-8-quinoline sulfonate and glyceryl 3-methyl-8-quinoline sulfonate.

[0030] Example 1 Specificity detection (1) Chromatographic conditions: Column: Thermo Hypersil GOLD 4.6mm × 250mm, 5μm Detection wavelength: 217nm Column temperature: 35℃ Flow rate: 1.0 ml / min Injection volume: 20 μl Mobile phase A: 0.1% aqueous phosphoric acid solution Mobile phase B: Acetonitrile Gradient elution procedure:

[0031] (2) Test methods Inject 20 µl each of the blank solution (solvent), impurity G positioning solution (ethyl 3-methyl-8-quinoline sulfonate), impurity H positioning solution (glyceryl 3-methyl-8-quinoline sulfonate), mixed solution of 6 specific impurities, test solution, and spiked solution of test solution into the liquid chromatograph and record the chromatogram.

[0032] (3) Test results As shown in Table 1, the blank solution, all impurities, and the chromatographic peaks of impurities G and H did not interfere with each other; the six specific impurities could be effectively separated from impurities G and H; the unknown impurities and main components in the test solution did not interfere with the chromatographic peaks of impurities G and H; indicating that the method has good specificity and meets the detection requirements. See Figure 1-5 .

[0033] Table 1: Specificity Test Results of Example 1

[0034] Example 2 System precision testing (1) Test methods The chromatographic conditions were the same as in Example 1. Six reference solutions were prepared in parallel, injected into the liquid chromatograph, and the chromatograms were recorded.

[0035] (2) Test results As shown in Table 2, the RSD of the peak area of ​​the six reference solutions was less than 2.0%, and the RSD of the retention time was less than 1.0%. The system has good precision.

[0036] Table 2: Results of System Precision Test in Example 2

[0037] Example 3 Intermediate precision testing (1) Test methods The chromatographic conditions were the same as in Example 1. Different analysts used different equipment to repeatedly determine the contents of ethyl 3-methyl-8-quinoline sulfonate and glyceryl 3-methyl-8-quinoline sulfonate in six spiked solutions of the test samples on different dates.

[0038] (2) Test results As shown in Table 3, different personnel used different instruments on different days to conduct parallel determinations of 6 spiked test solutions, and the RSD of each impurity content was less than 5.0%; together with the repeatability test of 12 spiked test solutions, the RSD of each impurity content was less than 10.0%, indicating that the method has good precision.

[0039] Table 3. Results of intermediate precision test in Example 3

[0040] Example 4 Accuracy testing (1) Test methods The chromatographic conditions were the same as in Example 1. Ethyl 3-methyl-8-quinoline sulfonate and glyceryl 3-methyl-8-quinoline sulfonate, along with the test sample, were taken and mixed solutions of the test sample at three concentration levels of 50%, 100%, and 150% were prepared according to the limit concentration. These solutions were injected into the chromatograph, the chromatograms were recorded, and the recovery rates of ethyl quinoline sulfonate and glyceryl quinoline sulfonate were calculated.

[0041] (2) Test results As shown in Tables 4 and 5, the impurity recovery rates were all between 80% and 120%, and the RSD values ​​were all less than 5.0%, indicating that the method had good accuracy.

[0042] Tables 4-5: Accuracy Results of Example 4 Table 4. Experimental results of ethyl quinoline sulfonate recovery rate

[0043] Table 5. Experimental results of the recovery rate of quinoline sulfonate glyceryl ester

[0044] Example 5: Linear (1) Experimental methods The chromatographic conditions were the same as in Example 1. Five series solutions of ethyl 3-methyl-8-quinoline sulfonate and glyceryl 3-methyl-8-quinoline sulfonate reference standards were prepared, and each solution was injected into the liquid chromatograph, with chromatograms recorded. Peak areas of each impurity at different concentrations were plotted as a function of their concentration, and the intercepts and correlation coefficients r were calculated.

[0045] (2) Test results As shown in Tables 6 and 7, within the concentration range from the limit of quantitation to 200% limit, the concentration and peak area of ​​each component showed a good linear relationship, with correlation coefficients r greater than 0.995. The ratio of the absolute value of the Y-axis intercept to the 100% response value was less than 20.0%, indicating a good linear relationship.

[0046] Table 6. Linearity and Range of Ethyl 3-Methyl-8-quinoline sulfonate

[0047] Table 7. Linearity and Range of 3-Methyl-8-quinoline sulfonate glycerol

[0048] Example 6: Limit of Quantification and Limit of Detection (1) Experimental methods The chromatographic conditions were the same as in Example 1. The reference solution was diluted and measured, and the chromatogram was recorded. When the signal-to-noise ratio (S / N) was 3-10, this concentration was the limit of detection; when the S / N was ≥10, this concentration was the limit of quantitation. The limit of quantitation solution was injected continuously for 6 injections, the peak area was recorded, and the relative standard deviation of the peak area was calculated.

[0049] (2) Test results As shown in Table 8, the quantitation limit concentrations of the analytes are all no more than 20% of the limit concentration, and the signal-to-noise ratios are all greater than 10; the signal-to-noise ratios of the detection limit concentration solutions are all greater than 3, indicating that the detection method meets the sensitivity requirements for impurity detection.

[0050] Table 8. Experimental results of limit of quantitation and limit of detection.

[0051] Comparative Example 1 Compared to Example 1, the difference lies in the mobile phase. In Comparative Example 1, mobile phase A was a 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. Everything else was the same as in Example 1.

[0052] Test methods Inject 20 µl each of the blank solution (solvent), impurity G positioning solution (ethyl 3-methyl-8-quinoline sulfonate), impurity H positioning solution (glyceryl 3-methyl-8-quinoline sulfonate), impurity E, impurity F, and a mixed solution of four specific impurities A, B, C, and D, and the test sample solution into the liquid chromatograph, and record the chromatogram according to the method in Example 1.

[0053] Test results As shown in Table 9, the blank solvent did not interfere with the detection of impurities G and H, but among the six specific impurities, impurity F and the main component argatroban interfered with the detection of impurity G. (See...) Figure 6 .

[0054] Table 9 Experimental Results of Comparative Example 1

[0055] Comparative Example 2 Compared to Example 1, the difference lies in the mobile phase. In Comparative Example 2, mobile phase A was a 0.1% ammonium acetate aqueous solution (with ammonia added to adjust the pH to 7.5), and mobile phase B was acetonitrile. Everything else was the same as in Example 1.

[0056] Test methods Take 20 µl each of blank solvent and spiked test solution, inject them into the liquid chromatograph, and record the chromatograms according to the method in Example 1.

[0057] Test results like Figure 7 As shown, the peak position of impurity G is on the upward baseline caused by the gradient, which will affect the accuracy of data processing.

[0058] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0059] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A method for detecting a potential mutagenic impurity in an argatroban injection, characterized by, A test sample solution is prepared, which is a mixture of agabamycin injection and a solvent, the solvent being an aqueous acetonitrile solution; The test sample solution is detected by high performance liquid chromatography, and the detection conditions are as follows: the mobile phase is mobile phase A and mobile phase B, the mobile phase A is a 0.01-1% phosphoric acid aqueous solution, the mobile phase B is a strong polar organic solvent, and a gradient elution program is used for elution, the gradient elution program being: After gradient elution, the content of potential mutagenic impurities, i.e., 3-methyl-8-quinoline sulfonic acid ethyl ester and 3-methyl-8-quinoline sulfonic acid glycerol ester, is detected.

2. The detection method as described in claim 1, characterized in that, The gradient elution program is: 。 3. The method of claim 1, wherein the step of detecting is characterized by, The strong polar organic solvent is methanol, ethanol, propanol or acetonitrile.

4. The detection method as described in claim 3, characterized in that, The strong polar organic solvent is acetonitrile.

5. The method of claim 1, wherein the step of detecting is characterized by, The mobile phase A is a 0.1% phosphoric acid aqueous solution.

6. The detection method as described in claim 1, characterized in that, In the solvent, the volume ratio of acetonitrile to water is 20:

80.

7. The detection method as described in claim 1, characterized in that, When detected by high performance liquid chromatography, the column temperature is 20-35℃, the wavelength is 217nm, and the flow rate is 0.8-1.2ml / min.

8. The method of claim 7, wherein the step of detecting is characterized by, When detected by high performance liquid chromatography, the column temperature is 35℃, and the flow rate is 1.0ml / min.

9. The detection method as described in claim 1, characterized in that, It also includes preparing a control sample solution, and detecting the control sample solution by high performance liquid chromatography, the control sample being 3-methyl-8-quinoline sulfonic acid ethyl ester and 3-methyl-8-quinoline sulfonic acid glycerol ester, and the solvent in the control sample solution being an aqueous acetonitrile solution.

10. The method of claim 9, wherein the step of detecting is characterized by, In the aqueous acetonitrile solution, the volume ratio of acetonitrile to water is 20:80.

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