Method for improving quality control of busulfan injection

Abstract

The invention provides a method for improving quality control of busulfan injection, which comprises the following steps: injecting a test solution of the busulfan injection into a high performance liquid chromatograph to separate and detect related substances in the busulfan injection by using an electric fog detector; a mobile phase A and a mobile phase B are adopted as eluents for gradient elution, the mobile phase A is a formic acid aqueous solution with the volume concentration of 0.02%-0.08%, the mobile phase B is acetonitrile, linear gradient elution is adopted when the elution time is controlled to range from 0 min to 10 min, the volume percent of the mobile phase A is reduced to 15% from 98%, and meanwhile the volume percent of the mobile phase B is increased to 85% from 2%. The method disclosed by the invention can be used for separating and detecting a main component busulfan and impurities methanesulfonic acid and RRT0.91, and has good separation degree, accuracy and sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analysis and relates to a method for improving the quality control of busulfan injection, comprising separating and detecting related substances of busulfan injection. Background Art

[0002] Busulfan, chemical name is 1,4-butanediol dimethanesulfonate, structural formula is .

[0003] Busulfan is a dimethylsulfonamide alkylating agent that has a strong inhibitory effect on bone marrow cell proliferation and can clear the bone marrow by killing hematopoietic progenitor cells and pluripotent stem cells. Compared with oral formulations, the advantages of intravenous formulations are that they avoid the influencing factors of unstable gastrointestinal absorption, are well tolerated, and are more pharmacokinetically stable. Therefore, intravenous formulations have gradually replaced oral busulfan in the pretreatment of hematopoietic stem cell transplantation and have been widely recognized in clinical practice.

[0004] As an intravenous dosage form, the detection method of related substances in busulfan injection is included in the "Registration Standards for Imported Drugs" (Standard No. JX20180237). This detection method uses a high-performance liquid chromatograph and sodium diethyldithiocarbamate (DEDC) as a derivatization reagent to detect a specific impurity, 1-methylsulfonyl-4-acetyl-butanediol (MABD). It can be seen that this method includes a derivatization step, so the operation is cumbersome and cannot meet the needs of high-throughput and automated detection; moreover, this method is limited to detecting a specific impurity.

[0005] However, busulfan injection also includes other impurities, such as methanesulfonic acid and RRT0.91, which are degradation products of busulfan injection and their content will gradually increase during the storage of busulfan injection. Therefore, the detection of impurities methanesulfonic acid and RRT0.91, as well as the determination of their content, are of great significance for the quality control of busulfan injection.

[0006] In particular, when the impurity methanesulfonic acid reaches a certain amount, it will cause gastrointestinal discomfort, inflammatory reaction, and even lead to adverse effects such as liver damage. However, the current detection methods for methanesulfonic acid, such as titration, are not suitable for busulfan injection because the titration method is easily interfered by other substances and has low accuracy.

[0007] In addition, impurities may also affect the content determination of active ingredients in drugs, such as the presence of unknown impurities that have not been detected, the content of impurities changes due to drug degradation, etc., which will cause the actual drug concentration to be inconsistent with the labeled concentration and fail to achieve the expected therapeutic effect. Therefore, it is important for drug quality and safety to be able to detect more impurities well and separate them for quantitative determination.

[0008] Therefore, in order to improve the quality control of busulfan injection, we need to develop a method for detecting related substances in busulfan injection, which can not only detect and separate the impurities methanesulfonic acid and RRT0.91 in busulfan injection, but also has good separation and accuracy. Summary of the invention

[0009] The object of the present invention is to provide a high performance liquid chromatography-charged aerosol detector (HPLC-CAD) method, which has good separation, sensitivity and accuracy, and can simultaneously separate and detect related substances in busulfan injection, including impurities methanesulfonic acid and RRT0.91, and accurately determine the contents of these impurities.

[0010] The object of the present invention is achieved in that: A method for improving the quality control of busulfan injection comprises: injecting a test solution of busulfan injection into a high performance liquid chromatograph to separate and detect related substances in the busulfan injection, wherein: Detection using a charged aerosol detector (CAD); Mobile phase A and mobile phase B were used as eluents for gradient elution, wherein mobile phase A was a formic acid aqueous solution with a volume concentration of 0.02% to 0.08%, and mobile phase B was acetonitrile, with a total flow rate of 0.8-1.2 mL / min. When the elution time was controlled from 0 to 10 minutes, a linear gradient elution was used, and the volume percentage of mobile phase A was linearly reduced from 98% to 15%; at the same time, the volume percentage of mobile phase B was linearly increased from 2% to 85%.

[0011] Compared with the prior art, the advantages and beneficial effects of the present invention are: 1. The present invention adopts a charged aerosol detector for detection, which can directly detect substances without UV absorption, such as the main component busulfan, and the test is stable and not easily interfered.

[0012] 2. The high performance liquid chromatography of the present invention, in particular, adopts specific elution conditions, especially when the elution time is controlled from 0 to 10 minutes, the volume percentage of mobile phase A is linearly reduced from 98% to 15%, and the volume percentage of mobile phase B is linearly increased from 2% to 85%; this is conducive to the simultaneous detection of related substances of busulfan injection, including impurities methanesulfonic acid and RRT0.91, in particular, the main component busulfan and the impurities methanesulfonic acid and RRT0.91 are completely separated, and the separation degrees are greater than 1.5, which shows that the method of the present invention has a good separation degree, thereby being able to accurately determine the content of impurities.

[0013] 3. By using the method of the present invention, within the concentration range of 0.53 μg / ml to 75.6 μg / ml of methanesulfonic acid, the logarithm of the concentration and the logarithm of the peak area have a good linear relationship, and also have good sensitivity, accuracy and injection precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The HPLC chart of the test solution of busulfan injection according to the chromatographic conditions of the embodiment is shown.

[0015] Figure 2 The HPLC chart of a control solution of busulfan injection according to the chromatographic conditions of the example is shown.

[0016] Figure 3 The standard curve of a series of methanesulfonic acid solutions measured by the external standard method according to the chromatographic conditions of the embodiment is shown.

[0017] Figure 4 The HPLC chart of the test solution of busulfan injection according to the chromatographic conditions of Comparative Example 1 is shown.

[0018] Figure 5 The HPLC chart of the test solution of busulfan injection according to the chromatographic conditions of Comparative Example 2 is shown. DETAILED DESCRIPTION

[0019] In the present invention, the "test sample" refers to busulfan injection containing the relevant substances to be determined.

[0020] In the present invention, "related substances" refer to impurities in busulfan injection, including methanesulfonic acid and RRT0.91.

[0021] In the present invention, "impurities" refer to substances in drugs that may have no therapeutic effect or affect the stability and efficacy of drugs, or even be harmful to human health. Usually, the purity of drugs can be evaluated as a whole with respect to the structure, appearance, physical and chemical constants, and impurity inspection and content determination of drugs. Impurities contained in drugs are the main factors affecting the purity of drugs. If the drugs contain impurities exceeding the limit (also called the limit), it is possible to change the physical and chemical constants, change the appearance, and affect the stability of drugs; the increase of impurities will inevitably reduce the content of drugs or reduce the activity, and significantly increase the toxic and side effects.

[0022] In the present invention, RRT0.91 is an unknown impurity. According to the experimental determination data of the present invention (as shown in Example Table 3), it is named by the ratio of the retention time of the impurity to the retention time of the main component busulfan, that is, it is named by relative retention time.

[0023] In the present invention, logarithm refers to the logarithm with the natural constant e (about 2.718) as the base, also called the natural logarithm; the logarithm of methanesulfonic acid concentration refers to the logarithm of the methanesulfonic acid concentration (in mg / ml) with the natural number e as the base, recorded as lnM; the logarithm of the methanesulfonic acid peak area refers to the logarithm of the methanesulfonic acid peak area with the natural number e as the base, recorded as lnA.

[0024] In the present invention, in order to more clearly show the impurity peaks, Figure 1 , 2 The HPLC graphs shown in 4 are enlarged graphs of the blue area (indicated by a blue box) in the upper right corner graph, wherein the upper right corner graph is the entire picture of the chromatogram obtained by the test.

[0025] The present invention provides a method for improving the quality control of busulfan injection, comprising: The test solution of busulfan injection was injected into a high performance liquid chromatograph to separate and detect the related substances in busulfan injection, among which, Detection is performed using a charged aerosol detector; Mobile phase A and mobile phase B are used as eluents for gradient elution, wherein mobile phase A is a formic acid aqueous solution with a volume concentration of 0.02% to 0.08%, and mobile phase B is acetonitrile, and the total flow rate is 0.8 to 1.2 mL / min; wherein when the elution time is controlled from 0 to 10 minutes, a linear gradient elution is used, and the volume percentage of mobile phase A is linearly reduced from 98% to 15%; and at the same time, the volume percentage of mobile phase B is linearly increased from 2% to 85%; The total volume percentage of mobile phase A and B is 100%.

[0026] Unexpectedly, the method of the present invention adopts the above-mentioned charged aerosol detector and a specific gradient elution program, especially when the elution time is from 0 to 10 minutes, so that the changes in the volume percentages of mobile phases A and B are linear, especially the mobile phase A is linearly reduced from 98% to 15%, and the mobile phase B is linearly increased from 2% to 85%. The impurities methanesulfonic acid and RRT0.91 in busulfan injection can be separated and detected simultaneously, and the main component busulfan, impurities methanesulfonic acid and RRT0.91 can be completely separated, so that the contents of these impurities can be accurately determined, and the quality of busulfan injection can be better controlled.

[0027] However, in contrast, according to the comparative examples of the present invention (as shown in Table 5 of the Examples section), if the elution time is from 0 to 10 minutes, the elution method is: first isocratic elution (from 0 to 3 minutes) and then linear gradient elution, and the initial volume percentage of mobile phase A is less than 93%, the impurities methanesulfonic acid, RRT0.91 and the main component busulfan cannot be completely separated (separation degree is less than 1.5), and even when the initial volume percentage is less than 85%, these impurities cannot be detected.

[0028] In a specific embodiment, the method of the present invention does not use isocratic elution when the elution time is from 0 to 10 minutes; in particular, only linear gradient elution is used.

[0029] Optionally, the method of the present invention adopts linear gradient elution when the elution time is from 0 to 10 minutes, and the initial mobile phase (at 0 minutes): the volume percentage of mobile phase A is any value from 98% to 95%, and the volume percentage of mobile phase B is any value from 2% to 5%; the final mobile phase (at 10 minutes): the volume percentage of mobile phase A is any value from 10% to 20%, and the volume percentage of mobile phase B is any value from 90% to 80%; the total volume percentage of mobile phases A and B is 100%.

[0030] Preferably, the elution procedure of the method of the present invention is shown in Table 1 below: .

[0031] The total volume percentage of mobile phase A and B is 100%.

[0032] In the present invention, the expression of the numerical range of the volume percentage of the mobile phase at a certain moment means that the volume percentage of the mobile phase can be selected as a numerical value at any end point of the range, or any numerical value within the range; for example, in the above Table 1, when the elution time is 0 minutes, the volume percentage of the mobile phase A is in the range of 98-95%, which means that at 0 minutes, the volume percentage of the mobile phase A is a certain specific numerical value, which can be 98% or 95%, or 95.5%, 96%, 96.5%, 97% or 97.5%.

[0033] More preferably, the gradient elution program is shown in Table 2 below: .

[0034] Table 2 can also be expressed as follows: when the elution time is from 0 to 10 minutes, mobile phase A decreases linearly from 98% to 15%, and mobile phase B increases linearly from 2% to 85%; when it is from 10 to 20 minutes, mobile phase A increases linearly from 15% to 98%, and mobile phase B decreases linearly from 85% to 2%; when it is from 20 to 30 minutes, mobile phase A is 98% and mobile phase B is 2%.

[0035] In a specific embodiment, when the elution time is from 0 to 10 minutes, the volume percentage of mobile phase A decreases by 7.8% to 8.5% per minute, preferably by 8.3% per minute, while the volume percentage of mobile phase B increases by 7.8% to 8.5% per minute, preferably by 8.3% per minute.

[0036] In the present invention, the related substances are methanesulfonic acid and RRT0.91, especially methanesulfonic acid.

[0037] In the method of the present invention, the injected busulfan injection test solution (referred to as the test solution) is a busulfan solution diluted with acetonitrile to a concentration of 0.3-0.8 mg / ml, preferably 0.5-0.6 mg / ml, more preferably 0.54 mg / ml.

[0038] In the method of the present invention, the atomizing gas used in the charged aerosol detector is nitrogen, and the evaporation temperature is 30-40°C, preferably 32-37°C, and more preferably 35°C.

[0039] Preferably, in the method of the present invention, the mobile phase A is preferably a formic acid aqueous solution with a volume concentration of 0.04% to 0.06%, more preferably a 0.05% formic acid aqueous solution; the total flow rate of the mobile phase is preferably 0.9 to 1.1 mL / min, more preferably 1.0 mL / min.

[0040] Preferably, in the method of the present invention, the chromatographic column used is a chromatographic column with octadecylsilane bonded silica as a filler, preferably Pheromone Titank C18 (4.6mm×250mm, 3 μm) or a chromatographic column with equivalent performance; the sample plate temperature is 5~10°C; and the injection volume is 15~25 μL.

[0041] In the method of the present invention, the column temperature of the chromatographic column is 25 to 35°C.

[0042] The method of the present invention also includes a method for determining the content of related substances in busulfan injection, such as an internal standard method, an external standard method, a main component self-control method, and a main component self-control method with a correction factor added.

[0043] The method of the present invention also includes a step of determining the content of RRT0.91 by the main component self-control method. Optionally, the step includes determining the chromatograms of the test solution and the control solution by the method of the present invention, obtaining the peak area of ​​the RRT0.91 impurity peak in the test solution and the peak area of ​​the main component busulfan in the control solution, substituting them into the following formula (1) to calculate the RRT0.91 content: ; in, Au is the peak area of ​​the RRT0.91 impurity peak in the test solution; As is the peak area of ​​busulfan, the main component in the control solution.

[0044] In a specific embodiment, the method of the present invention further comprises the step of determining the content of RRT0.91, the step comprising: (1) Solution preparation Preparation of test solution: Dissolve the busulfan injection to be tested in acetonitrile; Control solution preparation: Pipette a certain amount of the test solution and add acetonitrile to dilute it quantitatively so that the mass concentration of busulfan in the diluted solution is 1% of the mass concentration of busulfan in the test solution; (2) Determination method The same volume of the test solution and the control solution were measured and injected into the liquid chromatograph respectively, and the chromatogram was recorded according to the chromatographic conditions of the present invention; the peak area Au of the RRT0.91 impurity peak in the test solution and the peak area As of the main component busulfan in the control solution were substituted into the above formula (1) to obtain the content of the impurity RRT0.91 in the busulfan injection.

[0045] The method of the present invention also includes a step of determining the content of methanesulfonic acid by an external standard method, wherein the standard curve in the external standard method is obtained by plotting the logarithm (lnM) of the methanesulfonic acid concentration (in mg / ml) against the logarithm (lnA) of the methanesulfonic acid peak area; the methanesulfonic acid concentration is the mass concentration of methanesulfonic acid (in mg / ml).

[0046] In a specific embodiment, the method of the present invention further comprises the step of measuring the content of methanesulfonic acid, the step comprising: (1) Solution preparation Preparation of test solution: Dissolve the busulfan injection to be tested in acetonitrile; Preparation of methanesulfonic acid series solutions: The methanesulfonic acid reference substance was dissolved in acetonitrile to obtain a series of methanesulfonic acid solutions with different concentrations; (2) Determination method The test solution and the methanesulfonic acid series solution were measured and respectively injected into the liquid chromatograph, and the chromatogram was recorded according to the chromatographic conditions of the present invention; According to the recorded chromatograms of the methanesulfonic acid series solutions, the logarithm of the methanesulfonic acid peak area is used as the ordinate, and the logarithm of the mass concentration of methanesulfonic acid is used as the abscissa to obtain a standard curve, and a linear regression is performed to obtain a linear equation y=kx+b; The content of methanesulfonic acid in busulfan injection was calculated by the external standard method according to the following formula (2): ; in, Ai: peak area of ​​methanesulfonic acid in the test solution; k: slope of the linear equation; b: intercept of the linear equation; C: Concentration of the test solution.

[0047] In the method of the present invention, the test solution is prepared by adding acetonitrile to the test sample busulfan injection and fixing the volume. The busulfan concentration in the test solution is about 0.5-0.6 mg / ml, for example, 0.5 mg / ml, 0.54 mg / ml or 0.6 mg / ml; the busulfan concentration is the concentration of the test solution in formula (2).

[0048] In the present invention, if there are impurities in the chromatogram of the test solution, the methanesulfonic acid calculated by the peak area according to the external standard method shall not be greater than 7%, and the RRT0.91 shall not be greater than 4 times (4%) the peak area of ​​the main component busulfan of the control solution, and this limit is formulated based on the test results of the reference preparation at the end of the validity period; wherein, the reference preparation is busulfan injection, with a specification of 10ml:60mg, batch number 3H081A, Otsuka Pharmaceutical Co., Ltd, and a validity period of 24 months; on the last day of the validity period, the contents of methanesulfonic acid and RRT0.91 are determined according to the method of the present invention, and the measured methanesulfonic acid content is about 7%, and the RRT0.91 content is about 4%.

[0049] In the method of the present invention, the control solution is prepared by adding acetonitrile to the test solution, and the busulfan concentration in the control solution is 1% of the busulfan concentration in the test solution.

[0050] In the method of the present invention, the mass concentration of methanesulfonic acid in the methanesulfonic acid series solution is 0.5-80 μg / ml. In a specific embodiment, the mass concentration of methanesulfonic acid in the methanesulfonic acid series solution is 75.6 μg / ml, 56.7 μg / ml, 37.8 μg / ml, 18.9 μg / ml, 3.78 μg / ml, and 0.53 μg / ml, respectively; the logarithm of the methanesulfonic acid mass concentration (in mg / ml) and the logarithm of the methanesulfonic acid peak area are used to draw a standard curve, and the linear equation obtained is: y=0.946x+3.6555, and the correlation coefficient r = 0.9996.

[0051] The method of the present invention also includes using a blank solution, wherein acetonitrile is added to obtain a blank auxiliary material by mixing polyethylene glycol 400 with N,N-dimethylacetamide, so that each 100 ml of the blank solution contains 8 to 10 ml, preferably 9 ml, of the blank auxiliary material. In the blank auxiliary material, the volume ratio of polyethylene glycol 400 to N,N-dimethylacetamide is (1.8 to 2.2): 1, preferably a volume ratio of 2: 1. The blank solution does not interfere with the separation of the impurities methanesulfonic acid and RRT0.91 in the test solution determined by the method of the present invention.

[0052] In the method of the present invention, especially in the step of determining the methanesulfonic acid content of busulfan injection, it also includes recording the chromatogram of a blank solution according to the chromatographic conditions of the present invention, and the blank solution has no interfering peaks.

[0053] In the method of the present invention, busulfan and methanesulfonic acid positioning solutions are also used to determine the peak positions of each substance; wherein, in the busulfan positioning solution, the mass concentration of busulfan is 30 to 40 μg / ml, preferably 35 to 39 μg / ml, and especially 37.8 μg / ml; in the methanesulfonic acid positioning solution, the mass concentration of methanesulfonic acid is 30 to 40 μg / ml, preferably 35 to 39 μg / ml, and especially 37.8 μg / ml.

[0054] According to the detection method of the present invention, the linear range of the concentration of impurity methanesulfonic acid is 0.5-80 μg / ml, the limit of quantification is about 0.4 μg / ml, and the limit of detection is about 0.2 μg / ml; these concentrations are far less than 0.1% of the concentration of the test solution; this shows that the method of the present invention has a low limit of quantification and a detection limit, and is very sensitive to methanesulfonic acid. In other words, the method of the present invention allows the detection and quantitative determination of impurity methanesulfonic acid under low concentration conditions.

[0055] According to the detection performed by the method of the present invention, the retention time of the main component busulfan is 9.55 minutes, and the separation degree from the adjacent component (impurity RRT0.91) is 3.19, which can be completely separated (as shown in Table 3 of the embodiment), and according to the quantitative limit and detection limit determination method described in the embodiment, the quantitative limit of busulfan is 0.28 μg / ml, and the detection limit is 0.2 μg / ml. This shows that for the detection of busulfan, the method of the present invention is convenient and simple, and has good separation degree and sensitivity.

[0056] In the present invention, the separation degree calculation formula is as follows: ; in, is the retention time of the reference peak; is the retention time of the current peak; The peak width at 50% of the peak height of the reference peak; It is the peak width at 50% of the peak height of the current peak.

[0057] In the present invention, the current peak and the reference peak are two adjacent chromatographic peaks, wherein the current peak refers to the peak of the target component, and the reference peak refers to the next peak adjacent to the peak of the target component, or the previous peak. Thus, the separation calculated according to the above formula (3) is respectively referred to as "separation (with the next adjacent peak)" and "separation (with the previous adjacent peak)". In a specific embodiment, the target component is methanesulfonic acid, RRT0.91 or busulfan, as shown in Table 3 and Table 5 of the Examples.

[0058] The method of the present invention is applied to the control of related substances of busulfan injection, so as to perform quality control during the preparation and storage process and improve the safety of the medicine.

[0059] The method of the present invention will be described and illustrated in more detail below in conjunction with specific embodiments. It will be understood by those skilled in the art that the purpose of providing these embodiments is only for illustrative purposes and does not constitute any limitation to the scope of the present invention.

[0060] In the following examples, various processes and methods not described in detail are conventional methods known in the art, and the reagents used without indicating the source and specifications are all commercially available analytical grade or chromatographic grade.

[0061] I. General Description 1. Instruments High performance liquid chromatography (HPLC): Ultimate 3000, Thermo Fisher Scientific.

[0062] Charged aerosol detector (CAD): VF-D20-A (model), Thermo Fisher.

[0063] Electronic balance: AP135W (one hundred thousandth balance), Shimadzu.

[0064] Electronic balance: MSA6-6S-OCE-DM (one millionth balance), Sartorius.

[0065] Ultrapure water machine: WP-UP-YJ-10, Sichuan Water Treatment Equipment Co., Ltd.

[0066] 2. Reagents Formic acid (LC-MS): Lot No. 232524, Thermo Fisher Scientific.

[0067] Acetonitrile (LC-MS): Batch No. 231014, Tianjin Concord Technology Co., Ltd.

[0068] 3. Reference substances Busulfan: batch number BXAI230705A1, Guangzhou Aichi Pharmaceutical Technology Co., Ltd.

[0069] Methanesulfonic acid: batch number BXAR230522B1, Guangzhou Aici Pharmaceutical Technology Co., Ltd.

[0070] 4. Samples to be tested Busulfan injection: Specification 10ml: 60mg, batch number 3H081A, Otsuka Pharmaceutical Co., Ltd.

[0071] II. Examples 1. Chromatographic conditions Detector: The nebulizing gas is nitrogen, and the evaporation temperature is 35°C; Chromatographic column: Pheromones Titank C18 (4.6 mm × 250 mm, 3 μm); Sample pan temperature: 5°C; Column temperature: 30°C; Total flow rate: 1.0 mL / min; Injection volume: 20 μL; Mobile phase A: 0.05 vol% formic acid aqueous solution (measure 2.5 ml of formic acid, add 5000 ml of water, and mix well); Mobile phase B: acetonitrile; Mobile phase A and mobile phase B were used as eluents for gradient elution. The gradient elution program is shown in Table 2 above.

[0072] 2. Solution Preparation (1) Test solution: Take 0.9 ml of busulfan injection, accurately weigh it, place it in a 10 ml volumetric flask, add acetonitrile to dilute to the mark, and shake well to obtain the test solution of busulfan injection (busulfan concentration is 0.54 mg / ml).

[0073] (2) Control solution: Accurately measure 0.1 ml of the test solution and place it in a 10 ml volumetric flask. Add acetonitrile to dilute to the mark and shake well to obtain the control solution of busulfan injection.

[0074] (3) Blank solution: Mix polyethylene glycol 400 and N,N-dimethylacetamide in a volume ratio of 2:1 to prepare a blank auxiliary material. Take 0.9 ml of the blank auxiliary material and place it in a 10 ml volumetric flask. Add acetonitrile to dilute to the scale and shake well.

[0075] (4) Stock solution of busulfan reference substance: Weigh 3.80 mg busulfan (content 99.4%) of reference substance accurately into a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and the solution is ready (busulfan concentration is approximately 378 μg / ml).

[0076] (5) Methanesulfonic acid reference stock solution: Weigh 1.94 mg of methanesulfonic acid (content 97.4%) as the reference substance, accurately weigh it, place it in a 5 ml volumetric flask, add acetonitrile to dilute to the mark, shake well, and the solution is obtained (methanesulfonic acid concentration is approximately 378 μg / ml).

[0077] (6) Methanesulfonic acid series solutions: Accurately measure appropriate amounts of the above methanesulfonic acid reference stock solutions, add acetonitrile to dilute them quantitatively, and prepare 6 methanesulfonic acid solutions of different concentrations, which are 75.6 μg / ml, 56.7 μg / ml, 37.8 μg / ml, 18.9 μg / ml, 3.78 μg / ml, and 0.53 μg / ml, respectively.

[0078] (7) Busulfan reference solution: Accurately measure an appropriate amount of the above-mentioned busulfan reference substance stock solution and dilute it with acetonitrile to prepare a solution containing 37.8 μg of the reference substance busulfan per 1 ml.

[0079] (8) Methanesulfonic acid reference solution: Accurately measure an appropriate amount of the above methanesulfonic acid reference stock solution and dilute it with acetonitrile to prepare a solution containing 37.8 μg of reference methanesulfonic acid per 1 ml.

[0080] 3. Testing The method of the present invention was validated according to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 9101, Analytical Method Validation Guidelines.

[0081] 3.1 Determination of separation The blank solution, busulfan fixation solution, methanesulfonic acid fixation solution and the test solution were respectively injected into the liquid chromatograph, and the chromatogram was recorded according to the chromatographic conditions of the above embodiment (see item 1 of embodiment II); the chromatogram of the test solution is specifically shown in Figure 1 , the separation results (calculated by the above formula (3)) are shown in Table 3 below: .

[0082] Note: / indicates that the measurement result does not exist.

[0083] The blank solution was determined to have no interfering peaks; Figure 1 It is an enlarged view of the blue box area in the chromatogram of the test solution in the upper right corner, so as to more clearly present the impurity peaks and the main component peaks; wherein, number 1-represents the chromatographic peak of methanesulfonic acid, number 2-represents the chromatographic peak of RRT0.91, and number 3-represents the chromatographic peak of the main component busulfan; as can be seen from Table 3, in the test solution, the separation degrees between methanesulfonic acid, RRT0.91, the main component busulfan and their respective adjacent chromatographic peaks are all above 1.5, which meets the requirements; indicating that these peaks have reached a degree of complete separation, so the method for determining busulfan-related substances (impurity methanesulfonic acid and RRT0.91) provided by the present invention has good separation, can completely separate methanesulfonic acid, RRT0.91, and the main component busulfan, and can accurately determine the contents of impurities methanesulfonic acid and RRT0.91.

[0084] 3.2. Determination of related substances 3.2.1 Determination of peak areas of busulfan and related substances in test and control solutions Accurately measure 20 μl of the test solution and control solution prepared in Item 2 of the above embodiment, inject them into the liquid chromatograph respectively, and record the chromatogram according to the chromatographic conditions of the above embodiment (see Item 1 of Example II). The chromatogram of the test solution is as follows: Figure 1 As shown, in the figure, the chromatographic peaks of impurities methanesulfonic acid and RRT0.91 are numbered 1- and 2-, respectively, thereby obtaining the peak areas of impurities RRT0.91 and methanesulfonic acid in the test solution; Figure 2 This is an enlarged view of the blue box area in the chromatogram of the reference solution in the upper right corner. In this figure, the chromatographic peak of the main component busulfan is represented by the number 1-, thereby obtaining the peak area of ​​the main component busulfan in the reference solution.

[0085] 3.2.2 Content of impurity RRT0.91 The content of RRT0.91 in the test solution was calculated using the principal component self-reference method according to formula (1): ; in, Au: peak area of ​​RRT0.91 impurity peak in the test solution; As: Peak area of ​​the main component busulfan in the reference solution.

[0086] Au and As in the above formula (1) can be determined by the above step 3.2.1. It is calculated that the content of impurity RRT0.91 in busulfan injection is 2.69%.

[0087] 3.2.3 Content of impurity methanesulfonic acid The content of methanesulfonic acid in the test solution was measured by the external standard method.

[0088] (1) Drawing of standard curve Accurately measure 20 μl of the blank solution and methanesulfonic acid series solution prepared as in Item 2 of the above embodiment, and inject them into the liquid chromatograph in the order of blank solution and methanesulfonic acid series solution, and record the chromatogram according to the chromatographic conditions of the above embodiment (see Item 1 of Example II).

[0089] After determination, the blank solution had no interfering peak; according to the chromatogram of the methanesulfonic acid series solution recorded above, a standard curve was prepared with the logarithm of the methanesulfonic acid peak area (lnA) as the ordinate and the logarithm of the methanesulfonic acid concentration (lnM, concentration in mg / ml) as the abscissa. Figure 3 As shown. Through linear regression calculation, the linear equation is: y=0.946x+3.6555 (slope k=0.946, intercept b=3.6555), and the correlation coefficient r = 0.9996.

[0090] (2) Calculation of methanesulfonic acid content Calculate the methanesulfonic acid content according to the following formula: ; in, Ai: peak area of ​​methanesulfonic acid in the test solution; k: slope of the linear equation; b: intercept of the linear equation; C: concentration of the test solution; In the above formula (2), Ai can be determined by the above step 3.2.1, k and b are obtained from the linear equations obtained by plotting the above standard curve, and C is obtained from the test solution prepared in the above embodiment 2 (i.e., busulfan concentration 0.54 mg / ml). It is calculated that the content of impurity methanesulfonic acid in busulfan injection is 3.43%.

[0091] The results show that the logarithm of the methanesulfonic acid concentration (in mg / ml) and the logarithm of the methanesulfonic acid peak area have a good linear relationship when the methanesulfonic acid concentration is in the range of 0.53 μg / ml to 75.6 μg / ml as determined by the high performance liquid chromatography provided by the present invention. Moreover, the method of the present invention can determine the content of each of the impurities methanesulfonic acid and RRT0.91 in busulfan injection, which is conducive to the control of the content of a single impurity, so as to detect the quality of busulfan injection (especially during storage) and improve the safety of the product.

[0092] 3.3 Accuracy and sensitivity of methanesulfonic acid determination 3.3.1 Accuracy determination The proposed limit for the impurity methanesulfonic acid is 7%.

[0093] Preparation of accuracy solution 1: Take an appropriate amount of busulfan injection (specification, 10ml: 60mg) and 194μg of methanesulfonic acid reference substance (content 97.4%), place in a 10 ml volumetric flask, add acetonitrile to dilute to volume, and make a solution containing 0.54 mg busulfan and about 18.9 μg methanesulfonic acid per 1 ml. This solution is equivalent to an accuracy solution with a concentration level of 50% of the limit content of methanesulfonic acid. Prepare three replicates in parallel; the methanesulfonic acid concentration of the actual prepared solution is the "added impurity concentration" described in the following tables for accuracy 1-1, 1-2, and 1-3.

[0094] Preparation of accuracy solution 2: Take an appropriate amount of busulfan injection (specification, 10ml: 60mg) and 388μg of methanesulfonic acid reference substance (content 97.4%), place them in a 10 ml volumetric flask, add acetonitrile to dilute and fix the volume, and make a solution containing 0.54mg busulfan and about 37.8μg methanesulfonic acid per 1ml. This solution is equivalent to an accuracy solution with a horizontal concentration of 100% of the limit content of methanesulfonic acid. Prepare three replicates in parallel; the methanesulfonic acid concentration of the actual prepared solution is the "added impurity concentration" described in the following tables for accuracy 2-1, 2-2, and 2-3.

[0095] Preparation of accuracy solution 3: Take an appropriate amount of busulfan injection (specification, 10ml: 60mg) and 582μg of methanesulfonic acid reference substance (content 97.4%), place them in a 10 ml volumetric flask, add acetonitrile to dilute and fix the volume, and make a solution containing 0.54mg busulfan and about 56.7μg methanesulfonic acid per 1ml. This solution is equivalent to an accuracy solution with a concentration level of 150% of the limit content of methanesulfonic acid. Prepare three replicates in parallel; the methanesulfonic acid concentration of the actual prepared solution is the "added impurity concentration" described in the following tables for accuracy 3-1, 3-2, and 3-3.

[0096] The accuracy solution and two parallel test solutions (prepared as in Example 2 above) were respectively injected into a high performance liquid chromatograph, and the chromatogram was recorded according to the chromatographic conditions described in Example 1 above. The accuracy results of methanesulfonic acid are shown in Table 4 below: .

[0097] The linear equation of the methanesulfonic acid standard curve is: y=0.946x+3.6555, as described in item 3.2.3 of the above embodiment.

[0098] The results in the above table show that the average recovery rate of methanesulfonic acid at each concentration level is 84.26% to 94.73%, and the RSD (%) of the recovery rate is 5.21%, which is in compliance with the regulations. This indicates that the high performance liquid chromatography method provided by the present invention has good accuracy in determining the impurity methanesulfonic acid in busulfan injection.

[0099] 3.3.2 Determination of limit of quantification and limit of detection The reference substance methanesulfonic acid was accurately weighed and diluted with acetonitrile to prepare methanesulfonic acid solutions of different concentrations. These methanesulfonic acid solutions were respectively injected into a high performance liquid chromatograph, and the chromatograms were recorded according to the chromatographic conditions of the embodiment (see item 1 of embodiment II). The solution with a signal-to-noise ratio of about 10:1 was used as the quantitative limit solution, and the solution with a signal-to-noise ratio of about 3:1 was used as the detection limit solution.

[0100] The detection limit of methanesulfonic acid was determined to be 0.2004 μg / ml, which was 0.037% relative to the test sample concentration (0.54 mg / ml); the quantification limit was 0.4000 μg / ml, which was 0.074% relative to the test sample concentration (0.54 mg / ml), and the RSD of the peak area was 3.48%.

[0101] The above results show that for the impurity methanesulfonic acid in busulfan injection, the concentration that can be detected and the concentration that can be accurately quantified by the method of the present invention are both low, far less than the limit of 7%, and have good sensitivity; and the RSD is less than 5%, indicating that the method of the present invention has good injection precision.

[0102] III. Comparative Examples According to the above-mentioned "Chinese Pharmacopoeia" 2020 edition Part IV General Chapter 9101 analytical method, the verification results for separation stipulate that the separation of the target peaks is above 1.5.

[0103] 1. Comparative Example 1 The separation was determined according to the above-mentioned embodiment (see item 3.1 of embodiment II), except for the gradient elution procedure in the chromatographic conditions; the elution conditions and results are shown in Tables 5 and Figure 4 shown.

[0104] 2. Comparative Example 2 The separation was determined according to the above-mentioned embodiment (see item 3.1 of embodiment II), except for the gradient elution procedure in the chromatographic conditions; the elution conditions and results are shown in Tables 5 and Figure 5 shown.

[0105] .

[0106] Note: na means no measurement result was obtained, that is, no peak of the substance was detected.

[0107] The resolution indicates the resolution from the next adjacent peak.

[0108] *Indicates the separation degree from the adjacent previous peak.

[0109] From the above table 5 and Figure 1 It can be seen that the embodiments of the present invention can simultaneously detect the related substances of busulfan injection, including the impurities methanesulfonic acid and RRT0.91, and the separation between the main components busulfan, methanesulfonic acid, and RRT0.91 and their respective adjacent chromatographic peaks is greater than 1.5 (as shown in Table 3 above), which meets the requirements. The method of the present invention has good separation and can accurately determine the content of impurities methanesulfonic acid and RRT0.91.

[0110] On the contrary, the gradient elution conditions of Comparative Example 1 were used to analyze the test solution (the solution prepared in item 2 of the above embodiment). The chromatogram measured was as follows: Figure 4 As shown, Figure 4 It is an enlarged view of the blue box area in the chromatogram of the test solution in the upper right corner to more clearly present the impurity peaks and the main component peaks; wherein, numbers 1-, 3-, and 4-represent the chromatographic peaks of impurities methanesulfonic acid, RRT0.91, and busulfan, respectively, and their retention times are shown in Table 5, and number 2-represents the chromatographic peak adjacent to methanesulfonic acid, and its retention time is 3.350 minutes; according to the separation degree data of Comparative Example 1 in Table 5, it can be concluded that the separation degrees of impurities methanesulfonic acid, RRT0.91, and busulfan from their respective adjacent peaks are all less than 1.5, which does not meet the requirements; this indicates that they overlap with adjacent peaks and cannot reach the degree of complete separation, so the gradient elution procedure of Comparative Example 1, especially the elution conditions when the elution time is from 0 to 10 minutes, cannot completely separate the impurities methanesulfonic acid, RRT0.91, and busulfan, and cannot accurately determine the contents of impurities methanesulfonic acid and RRT0.91.

[0111] The test solution (the solution prepared in item 2 of the above embodiment) was analyzed using the gradient elution conditions of Comparative Example 2. The chromatogram measured is as follows: Figure 5 As shown, only the main component busulfan (as shown by number 1-), while the impurities methanesulfonic acid and RRT0.91 could not be detected. According to the separation data of Comparative Example 2 in Table 5, it can be concluded that the separation between busulfan and its adjacent peaks is less than 1.5, which does not meet the requirements. This indicates that busulfan overlaps with the adjacent peaks and has not reached the degree of complete separation. Therefore, the gradient elution procedure of Comparative Example 2, especially the elution condition when the elution time is from 0 to 10 minutes, cannot detect the impurities methanesulfonic acid, RRT0.91, and the main component busulfan at the same time, and it is even more impossible to completely separate these substances and accurately determine the contents of the impurities methanesulfonic acid and RRT0.91.

[0112] In summary, by comparing the above embodiment with Comparative Examples 1 and 2, it is shown that the method of the present invention, especially through a specific gradient elution program, especially when the elution time is controlled from 0 to 10 minutes, the volume percentage of mobile phase A is linearly reduced from 98% to 15%, and the volume percentage of mobile phase B is correspondingly linearly increased from 2% to 85%, which is conducive to the simultaneous separation and detection of impurities methanesulfonic acid and RRT0.91, and has good separation, and can achieve complete separation of the main component busulfan, impurity methanesulfonic acid and RRT0.91, thereby accurately determining the impurity content.

[0113] In summary, the advantages of the method of the present invention are that the impurities methanesulfonic acid and RRT0.91 in busulfan injection can be separated and detected simultaneously, and the contents of these impurities can be accurately determined, and the method has good separation. Moreover, using the method of the present invention, the linear range of the concentration of methanesulfonic acid is 0.53 μg / ml to 75.6 μg / ml, the detection limit concentration is 0.2004 μg / ml, and the quantitative limit concentration is 0.4000 μg / ml, which has good sensitivity, accuracy and injection precision.

Claims

1. A method for improving the quality control of busulfan injection, comprising: The test solution of busulfan injection was injected into a high performance liquid chromatograph to separate and detect the related substances in busulfan injection, among which, Detection is performed using a charged aerosol detector; Mobile phase A and mobile phase B were used as eluents for gradient elution, wherein mobile phase A was a formic acid aqueous solution with a volume concentration of 0.02% to 0.08%, and mobile phase B was acetonitrile, with a total flow rate of 0.8 to 1.2 mL / min; when the elution time was controlled from 0 to 10 minutes, a linear gradient elution was used, and the volume percentage of mobile phase A was linearly reduced from 98% to 15%, while the volume percentage of mobile phase B was linearly increased from 2% to 85%.

2. The method according to claim 1, wherein the elution gradient program is as follows: The total volume percentage of mobile phase A and B is 100%.

3. The method according to claim 1, wherein the related substance is methanesulfonic acid.

4. The method according to claim 1, wherein the injected test solution of busulfan injection is a solution of busulfan diluted with acetonitrile with a concentration of 0.3 to 0.8 mg / ml.

5. The method according to claim 1, wherein the nebulizing gas used in the charged aerosol detector is nitrogen gas, and the evaporation temperature is 30 to 40°C.

6. method according to claim 1, wherein also comprises the step of adopting external standard method to measure methanesulfonic acid content, and the typical curve in the described external standard method is that the logarithm of methanesulfonic acid concentration and the logarithm of methanesulfonic acid peak area are mapped to obtain.

7. The method according to claim 1, wherein the chromatographic column is a chromatographic column using octadecylsilane bonded silica gel as a filler.

8. The method according to claim 7, wherein the chromatographic column is a Pheromon Titank C18 chromatographic column.

9. The method according to claim 7, wherein the column temperature of the chromatographic column is 25 to 35°C.

10. The method of claim 1, wherein the injection volume is 15 to 25 μL.

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