Method for determining bedaquiline fumarate cleaning verification target residue

Abstract

The invention discloses a method for determining bedaquiline fumarate clean verification target residues, and relates to the technical field of instrument analysis of chemical pharmacy. The method comprises the following steps: preparing a sample wiping solution; determining the content of bedaquiline fumarate in the sample wiping solution by adopting a liquid chromatography; chromatographic conditions are as follows: a chromatographic column is a VP-ODS C18 column; mobile phases: a potassium hexafluorophosphate aqueous solution and acetonitrile, wherein the concentration of the potassium hexafluorophosphate aqueous solution is 0.01-0.03 mol / L; the elution mode is isocratic elution; the flow rate is 1 ml / min to 2 ml / min; the column temperature is 28-32 DEG C; the method has good specificity and sensitivity for determination of bedaquiline fumarate cleaning verification target residues, is simple and convenient to operate, rapid in determination, simple and easy to implement, and overcomes the defects that TOC, chemical titration methods, conductivity methods, pH methods and the like which are commonly used at present do not have specificity.

Description

Technical Field

[0001] This invention relates to the field of instrumental analysis technology in chemical pharmaceuticals, and more specifically, to a method for determining the target residue of bedaquiline fumarate for cleaning validation. Background Technology

[0002] After the production of bedaquiline fumarate is completed, equipment cleaning is a crucial step to avoid cross-contamination. Furthermore, the cleaning validation limit for the production equipment should be the lowest among all products produced using this equipment. Therefore, a specific and highly sensitive assay method is essential to ensure accurate and reliable results, while also considering ease and speed of operation. Currently, there is no clearly defined and easily implemented method for this purpose. There are no publicly reported methods, either domestically or internationally, for detecting the content of target residues in bedaquiline fumarate during cleaning validation.

[0003] In existing technologies, the content of residues on the surface of equipment after cleaning is mainly measured using TOC, chemical titration, conductivity, and pH determination methods. However, these methods lack specificity and have low sensitivity, and cannot accurately detect the target residues for cleaning verification.

[0004] Therefore, it is necessary to provide a new method specifically for determining the content of target residues in equipment after the production of bedaquiline fumarate. Summary of the Invention

[0005] This invention uses high performance liquid chromatography (HPLC) to quantitatively determine and analyze target residues. When using this method to determine the residue content, it is necessary to select chromatographic conditions based on the properties of the residue to be tested and the cleaning solvents used for cleaning the production equipment. The selection of chromatographic conditions has a critical impact on the accuracy of the determination results, and requires a significant investment of time, effort, and funds for exploration and verification.

[0006] In view of this, the present invention provides a method for determining the target residue of bedaquiline fumarate in cleaning verification; solving the problem of detecting the target residue in cotton swab samples after equipment cleaning, so as to determine the cleaning effect of production equipment and avoid cross-contamination.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: A method for determining bedaquiline fumarate cleaning validation target residues includes the following steps: (1) Prepare the sample wiping solution; (2) The content of bedaquiline fumarate in the sample wiping solution was determined by liquid chromatography; The chromatographic conditions are as follows: Chromatographic column: VP-ODS C18 column, 4.6 mm × 150 mm, 5 μm particle size; Mobile phase: aqueous solution of potassium hexafluorophosphate and acetonitrile, wherein the concentration of the aqueous solution of potassium hexafluorophosphate is 0.01~0.03 mol / L; Elution method: isocratic elution; Flow rate: 1.4 ml / min ~ 1.6 ml / min; Column temperature: 28℃~32℃.

[0008] Furthermore, the concentration of the potassium hexafluorophosphate aqueous solution in the mobile phase is 0.02 mol / L, and the pH value is adjusted to 3.4~3.6 with phosphoric acid.

[0009] Furthermore, the volume percentage of the potassium hexafluorophosphate aqueous solution in the mobile phase is 65%.

[0010] Furthermore, the detection wavelength is 226nm.

[0011] Furthermore, the flow rate is 1.5 ml / min; the column temperature is 30°C.

[0012] Furthermore, in the chromatographic conditions, the injection volume is 10 μl and the analysis time is 10 minutes.

[0013] Furthermore, the step of preparing the sample wiping solution includes: Wipe the equipment to be cleaned and verified with cotton swabs; Extract the swabs with acetonitrile, transfer the extract to a volumetric flask, and dilute with acetonitrile to the mark to obtain the sample wiping solution.

[0014] Furthermore, it also includes preparing multiple bedaquiline fumarate reference solutions with concentrations ranging from 0.015 mg / 100 ml to 30 mg / 100 ml, detecting the multiple bedaquiline fumarate reference solutions according to the chromatographic conditions in step (2), obtaining chromatograms of multiple bedaquiline fumarate reference solutions with known concentrations, and then calculating the content of bedaquiline fumarate in the sample wiping solution based on the peak area using the external standard method.

[0015] Optionally, in some embodiments, the content of bedaquiline fumarate in the sample wiping solution can also be calculated based on the peak height.

[0016] Optionally, the internal standard method can also be used to determine the content of bedaquiline fumarate in the sample wiping solution.

[0017] In this invention, a conventional C18 chromatographic column is used, the analysis time is short, the bedaquiline peak can be eluted within 5 to 7 minutes, and the operation is simple; the target residue bedaquiline is completely separated from the adjacent solvent peaks, and the resolution is >1.5.

[0018] Compared with the prior art, the present invention has the following advantages: This invention exhibits good specificity and sensitivity for the determination of target residues in bedaquiline fumarate cleaning validation. It is also simple to operate, rapid, and easy to perform, overcoming the shortcomings of commonly used methods such as TOC, chemical titration, conductivity, and pH, which lack specificity. Attached Figure Description

[0019] Figure 1 This is a typical spectrum of the specificity of the method of this invention.

[0020] Figure 2 This is a linear schematic diagram of the method of the present invention. Detailed Implementation

[0021] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0023] Unless otherwise specified, all materials and reagents used in this invention are available from commercially available products in the field.

[0024] The challenges of this invention lie in how to eliminate interference from impurities in the cleaning solvent after cleaning the bedaquiline fumarate production equipment, and how to accurately determine the content of the target residue bedaquiline and improve sensitivity when the equipment cleaning validation limit is low.

[0025] This invention employs the linear regression method in liquid chromatography to prepare a series of linear solutions and cotton swab sample solutions with bedaquiline fumarate concentrations ranging from approximately 0.015 mg / 100 ml to 30 mg / 100 ml. Parameters are selected based on the specific characteristics of the residues to be tested in the samples, and the content of the target residue bedaquiline in the equipment cleaning cotton swab samples is calculated using the linear regression method.

[0026] The following is a description using specific embodiments.

[0027] Example 1 I. The selection of liquid chromatography conditions is as follows: Chromatograph: Liquid Chromatograph; Chromatographic column: VP-ODS C18 column, 4.6 mm × 150 mm, 5 μm particle size; Mobile phase: Mobile phase A: 0.02 mol / L potassium hexafluorophosphate aqueous solution (weigh 3.68 g of potassium hexafluorophosphate, add 1000 ml of water to dissolve, and adjust the pH to 3.5 ± 0.1 with phosphoric acid). Mobile phase B: Acetonitrile; Elution method: Mobile phase A: Mobile phase B = 35:65 (V / V); Detection wavelength: 226nm; Flow rate: 1.5 ml / min; Injection volume: 10 μl; Column temperature: 30℃; Analysis time: 10 min; II. Reagent Selection: Acetonitrile (HPLC grade); isopropanol (HPLC grade); phosphoric acid (AR grade); purified water (HPLC grade); potassium hexafluorophosphate (AR grade).

[0028] III. Solution Preparation: 1. Sample preparation for cotton swab wiping: The sample from the swab was carefully extracted using acetonitrile and transferred to a 100ml volumetric flask, then diluted to the mark with acetonitrile.

[0029] 2. Preparation of linear solutions: Linear solution 1: Accurately weigh 30 mg of bedaquiline fumarate sample into a 100 ml volumetric flask, dissolve and dilute to the mark with acetonitrile.

[0030] Linear Solution 2: Accurately transfer 10.0 ml of Linear Solution 1 into a 20 ml volumetric flask and dilute to the mark with acetonitrile.

[0031] Linear solution 3: Accurately transfer 5.0 ml of linear solution 1 into a 50 ml volumetric flask and dilute to the mark with acetonitrile.

[0032] Linear Solution 4: Accurately pipette 5.0 ml of Linear Solution 1 into a 100 ml volumetric flask and dilute to the mark with acetonitrile.

[0033] Linear Solution 5: Accurately pipette 1.0 ml of linear solution 1 into a 100 ml volumetric flask and dilute to the mark with acetonitrile.

[0034] Linear Solution 6: Accurately pipette 5.0 ml of linear solution 5 into a 100 ml volumetric flask and dilute to the mark with acetonitrile.

[0035] IV. Calculation method: Linear regression method.

[0036] Based on the linear series spectral data, the linear regression equation of the peak area Y of bedaquiline versus the concentration X (mg / 100ml) was calculated.

[0037] The concentration of bedaquiline in the swab sample was calculated using a linear regression equation.

[0038] Example 2: Verification of Method Specificity 1. The chromatographic conditions and reagent selection remain unchanged, the same as in Example 1.

[0039] 2. Preparation of blank solution for the wiping method: Take a clean cotton swab, moisten it with isopropanol, and then extract the swab twice with acetonitrile (40 ml of acetonitrile each time). Transfer the extract to a 100 ml volumetric flask and dilute to the mark with acetonitrile.

[0040] 3. Sample solution preparation: Weigh 30 mg of bedaquiline fumarate sample into a 100 ml volumetric flask, dilute with acetonitrile, and shake until completely dissolved.

[0041] 4. Sample solution + blank swab: Take a clean cotton swab, moisten it with isopropanol, and then extract the swab twice with acetonitrile (40 ml of acetonitrile each time). Transfer the extract to a 100 ml volumetric flask, weigh 30 mg of bedaquiline fumarate sample and put it into this 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark.

[0042] 5. Solvent (acetonitrile), blank solution for wiping method, sample solution, and sample solution + blank swab are injected sequentially for determination.

[0043] The results of the determination of solvent, blank solution by wiping method, and sample solution are shown in the table below:

[0044] The results of the sample solution + blank swab test are shown in the table below:

[0045] Methodology Review: This method has good specificity for determination using solvents, blank solutions obtained by wiping, sample solutions, and sample solutions plus blank swabs. None of the above solvents interfere with the determination of the bedaquiline peak, and the resolution between bedaquiline and adjacent solvent peaks is >1.5.

[0046] Example 3: Validation of the method's limit of quantitation The chromatographic conditions and reagent selection remain unchanged, the same as in Example 1.

[0047] Preparation of limit-of-quantitation solutions: Prepare a solution with a bedaquiline fumarate concentration of approximately 0.002 mg / 100 ml at the limit of quantitation using a bedaquiline fumarate sample.

[0048] The results of the limit of quantitation determination are shown in the table below:

[0049] Methodology Review: This method can quantitatively determine bedaquiline fumarate at a concentration of approximately 0.002 mg / 100 ml, and the limit of quantitation is below the limit for equipment cleanliness of the product. It has high sensitivity and, from a method validation perspective, meets the requirements of the International Council for Validation (ICH) guidelines.

[0050] Example 4: Method Linearity Verification The chromatographic conditions and reagent selection remain unchanged, the same as in Example 1.

[0051] Preparation of linear solutions: Using a sample of bedaquiline fumarate, prepare a series of linear solutions with bedaquiline fumarate concentrations ranging from approximately 0.002 mg / 100 ml to 60.0 mg / 100 ml.

[0052] The linearity test results are shown in the table below:

[0053] Methodology Review: This method can accurately determine the concentration of bedaquiline fumarate in solutions ranging from approximately 0.002 mg / 100 ml to 60.0 mg / 100 ml, and the analyte concentration and response show a linear relationship. From a method validation perspective, it meets the requirements of the International Council for Validation (ICH) guidelines, with a linear correlation coefficient > 0.99. Figure 2 As shown.

[0054] Example 5: Method Precision Verification The chromatographic conditions and reagent selection remain unchanged, the same as in Example 1.

[0055] Preparation of precision solutions: Simulated cotton swab wiping of sample (stainless steel surface): Accurately weigh 30 mg of bedaquiline fumarate sample and distribute it evenly on a 10 cm × 10 cm stainless steel plate. Take 2 ml of isopropanol and add it dropwise to the bedaquiline fumarate sample until the bedaquiline fumarate sample is in a thin paste or dissolved (equivalent to distributing 30.0 mg of bedaquiline fumarate sample on a 10 cm × 10 cm stainless steel plate). Let it air dry naturally.

[0056] Take a clean cotton swab, moisten it with acetonitrile, and use the swab to wipe the bedaquiline fumarate sample distributed on a 10 cm × 10 cm stainless steel plate. Then extract the swab twice with acetonitrile (40 ml of acetonitrile each time). Transfer the extract to a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark.

[0057] The results of the precision measurement are shown in the table below:

[0058] Methodology Review: This method has good repeatability for the determination of bedaquiline fumarate residues in simulated cotton swab samples, and from the perspective of method validation, it meets the requirements of the International Council for Validation (ICH) guidelines.

[0059] Example 6: Verification of Method Sampling Efficiency The chromatographic conditions and reagent selection remain unchanged, the same as in Example 1.

[0060] Sampling efficiency solution preparation: Preparation of simulated cotton swab swab samples (bedaquiline fumarate concentration of 0.004 mg / 100 ml): Accurately pipette 1.0 ml of bedaquiline fumarate solution (0.4 mg / 100 ml) and evenly distribute it on a 10 cm × 10 cm stainless steel plate (equivalent to distributing approximately 0.004 mg of bedaquiline fumarate on a 10 cm × 10 cm stainless steel plate). After air drying, use a cotton swab to apply the solution to the 100 cm plate. 2 Wipe the area within the specified range, then carefully extract the contents of each swab into a 100ml volumetric flask twice with 40ml of acetonitrile. Dilute to the mark with acetonitrile. Prepare three parallel solutions, and test each solution three times.

[0061] Preparation of simulated cotton swab swab sample (bedaquiline fumarate concentration of 60 mg / 100 ml): Accurately weigh 60.0±0.5 mg of bedaquiline fumarate sample and evenly distribute it on a 10 cm × 10 cm stainless steel plate. Take 1 ml of isopropanol and add it dropwise to the bedaquiline fumarate sample until the bedaquiline fumarate sample is in a thin paste or dissolved (equivalent to distributing 60.0 mg of bedaquiline fumarate sample on a 10 cm × 10 cm stainless steel plate). Let it air dry naturally.

[0062] Take a clean cotton swab, moisten it with acetonitrile, and use the swab to wipe the bedaquiline fumarate sample distributed on a 10 cm × 10 cm stainless steel plate. Then extract the sample twice with acetonitrile (40 ml acetonitrile each time). Transfer the extract to a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark. Prepare three parallel aliquots of this solution.

[0063] The sampling efficiency test results are shown in the table below: Results of assay using simulated cotton swabs (bedaquiline fumarate concentration of 0.004 mg / 100 ml):

[0064] Results of assay using simulated cotton swabs (bedaquiline fumarate concentration of 60 mg / 100 ml):

[0065] Methodology Review: This method has high sampling efficiency, and the recovery rate of simulated cotton swab wiping samples has good repeatability. From the perspective of method validation, it meets the requirements of the International Council for Validation (ICH) and the Cleaning Validation Technical Guidelines issued by the National Medical Products Administration.

[0066] Comparative Example 1 The chromatographic conditions in Example 1 were followed, with the only difference being that a different specification VP-ODS C18 column (4.6 mm × 250 mm, 5 μm particle size) was used. The sample solution and blank cotton swab solution from Example 1 were taken and subjected to liquid chromatography detection according to the steps in Example 1.

[0067] In the chromatogram of the VP-ODS C18 column (4.6 mm × 250 mm, 5 μm particle size), the bedaquiline peak eluted late, had a small peak area, and low sensitivity, and could not be completely eluted within the 10-minute analysis time.

[0068] Comparative Example 2 The chromatographic conditions in Example 1 were followed, with the only difference being the ratio of potassium hexafluorophosphate aqueous solution in the mobile phase: potassium hexafluorophosphate aqueous solution: acetonitrile = 50:50 (V / V) and potassium hexafluorophosphate aqueous solution: acetonitrile = 20:80 (V / V). The sample solution and blank cotton swab solution from Example 1 were taken and liquid chromatography detection was performed according to the steps in Example 1.

[0069] In the chromatogram of the mobile phase detection using potassium hexafluorophosphate aqueous solution: acetonitrile = 50:50 (V / V), the bedaquinoline peak eluted later, had a smaller peak area, and low sensitivity, failing to fully elute within the 10-minute analysis time. In the chromatogram of the mobile phase detection using potassium hexafluorophosphate aqueous solution: acetonitrile = 20:80 (V / V), the bedaquinoline peak eluted earlier, and the bedaquinoline peak could not be effectively separated from the solvent peak (resolution < 1.5).

[0070] Comparative Example 3 The chromatographic conditions in Example 1 were followed, with the only difference being the column temperature setting, which was 35°C and 25°C respectively. The sample solution and blank cotton swab solution from Example 1 were taken and subjected to liquid chromatography detection according to the steps in Example 1.

[0071] In the chromatograms detected at a column temperature of 35℃, the bedaquiline peak eluted earlier, and the bedaquiline peak and solvent peak could not be effectively separated (resolution < 1.5). In the chromatograms detected at a column temperature of 25℃, the bedaquiline peak eluted later and could not be completely eluted within the 10-minute analysis time.

[0072] Comparative Example 4 The chromatographic conditions in Example 1 were followed, with the only difference being the flow rate setting, which was 1.8 ml / min and 1.0 ml / min respectively. The sample solution and blank cotton swab solution from Example 1 were taken and subjected to liquid chromatography detection according to the steps in Example 1.

[0073] In the chromatogram detected at a flow rate of 1.8 ml / min, the bedaquiline peak eluted earlier, and the bedaquiline peak could not be effectively separated from the solvent peak (resolution < 1.5), and the column pressure was high. In the chromatogram detected at a flow rate of 1.2 ml / min, the bedaquiline peak eluted later, had a small peak area, low sensitivity, and could not be completely eluted within the 10-minute analysis time.

[0074] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0075] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for determining bedaquiline fumarate cleaning validation target residues, characterized in that, Includes the following steps: (1) Prepare the sample wiping solution; (2) The content of bedaquiline fumarate in the sample wiping solution was determined by liquid chromatography; The chromatographic conditions are as follows: Chromatographic column: VP-ODS C18 column, 4.6 mm × 150 mm, 5 μm particle size; Mobile phase: aqueous solution of potassium hexafluorophosphate and acetonitrile, wherein the concentration of the aqueous solution of potassium hexafluorophosphate is 0.01~0.03 mol / L; Elution method: isocratic elution; Flow rate: 1.4 ml / min ~ 1.6 ml / min; Column temperature: 28℃~32℃.

2. The method according to claim 1, characterized in that, The concentration of potassium hexafluorophosphate aqueous solution in the mobile phase is 0.02 mol / L, and the pH value is adjusted to 3.4~3.6 with phosphoric acid.

3. The method according to claim 1, characterized in that, The volume percentage of potassium hexafluorophosphate aqueous solution in the mobile phase is 65%.

4. The method according to claim 1, characterized in that, The detection wavelength is 226nm.

5. The method according to claim 1, characterized in that, The flow rate is 1.5 ml / min; the column temperature is 30°C.

6. The method according to claim 1, characterized in that, The chromatographic conditions were as follows: injection volume was 10 μl, and analysis time was 10 minutes.

7. The method according to claim 1, characterized in that, The steps for preparing the sample wiping solution include: Wipe the equipment to be cleaned and verified with cotton swabs; Extract the swabs with acetonitrile, transfer the extract to a volumetric flask, and dilute with acetonitrile to the mark to obtain the sample wiping solution.

8. The method according to claim 1, characterized in that, It also includes preparing multiple bedaquiline fumarate reference solutions with concentrations ranging from 0.015 mg / 100 ml to 30 mg / 100 ml, detecting the multiple bedaquiline fumarate reference solutions according to the chromatographic conditions in step (2), obtaining chromatograms of multiple bedaquiline fumarate reference solutions with known concentrations, and then calculating the content of bedaquiline fumarate in the sample wiping solution based on the peak area using the external standard method.

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