Method for improving quality detection of busulfan injection

Abstract

The present invention provides a method for improving the quality control of busulfan injection, which includes: injecting a test solution of busulfan injection into a high performance liquid chromatograph to separate and detect related substances in the busulfan injection, wherein an evaporative light scattering detector is used for detection; gradient elution is carried out using mobile phases A and B as eluents, wherein mobile phase A is an aqueous formic acid solution with a volume concentration of 0.02% to 0.08%, and mobile phase B is acetonitrile. When controlling the elution time from 0 to 10 minutes, linear gradient elution is adopted, and the volume percentage of mobile phase A decreases from 98% to 15%, while the volume percentage of mobile phase B increases from 2% to 85%. The method of the present invention can separate and detect the main component busulfan and impurities methanesulfonic acid and RRT0.91, and has good resolution, accuracy and sensitivity.

Description

Technical Field

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

[0002] Busulfan, chemically named 1,4-butanediol dimethanesulfonate, has a structural formula of

[0003] 。

[0004] Busulfan is a dimethylsulfonate alkylating agent, which has a strong inhibitory effect on the proliferation of bone marrow cells and can play a myeloablative role by killing hematopoietic progenitor cells and pluripotent stem cells in the bone marrow. Since the advantages of the intravenous dosage form compared to the oral dosage form are that it avoids the influencing factors of unstable gastrointestinal absorption, has good tolerance, and is also more stable in pharmacokinetics, the intravenous dosage form has gradually replaced the oral busulfan in the pretreatment of hematopoietic stem cell transplantation and has been widely recognized clinically.

[0005] As for the detection method of the related substances in busulfan injection as an intravenous dosage form, it is included in the "Import Drug Registration Standard" (standard number JX20180237). This detection method uses a high-performance liquid chromatograph and uses sodium diethyldithiocarbamate (DEDC) as a derivatizing reagent to detect a specific impurity, 1-methylsulfonyl-4-acetyl-butanediol (MABD); it can be seen that since this method includes a derivatization step, the operation is cumbersome and it cannot meet the high-throughput and automated detection requirements; moreover, this method is only limited to detecting a specific impurity.

[0006] However, busulfan injection also includes other impurities, such as methanesulfonic acid and RRT0.91. These impurities are the degradation products of busulfan and their contents will gradually increase during the storage of busulfan injection. Therefore, detecting methanesulfonic acid and RRT0.91 and determining their contents are of great significance for the quality detection of busulfan injection.

[0007] In particular, when the content of methanesulfonic acid reaches a certain amount, it will cause adverse effects such as gastrointestinal discomfort, inflammatory reactions, and even liver function damage. However, the current detection methods for methanesulfonic acid, such as titration methods, are not applicable to busulfan injection because titration methods are easily interfered by other substances and have low accuracy.

[0008] In addition, impurities may also affect the determination of the content of the active ingredient in the drug. For example, there may be unknown impurities that are not detected, or the content of the impurities may change due to reasons such as drug degradation. All of these can lead to a discrepancy between the actual drug concentration and the labeled concentration, and thus the expected therapeutic effect cannot be achieved. Therefore, being able to detect more impurities well and separate them to achieve quantitative determination is important for the quality and safety of drugs.

[0009] Therefore, in order to improve the quality inspection of busulfan injection, we need to develop a method for detecting the related substances of busulfan injection. This method can not only detect and separate the impurity methanesulfonic acid and RRT0.91 in busulfan injection, but also has good resolution and accuracy. Summary of the Invention

[0010] The object of the present invention is to provide a high performance liquid chromatography - charged aerosol detector method (HPLC - CAD), which has good resolution, sensitivity and accuracy, can simultaneously separate and detect the related substances in busulfan injection, including the impurity methanesulfonic acid and RRT0.91, and accurately determine the content of these impurities.

[0011] The object of the present invention is achieved as follows:

[0012] A method for improving the quality inspection of busulfan injection, which includes: injecting the test solution of busulfan injection into a high performance liquid chromatograph to separate and detect the related substances in busulfan injection, wherein,

[0013] Detection is carried out using a charged aerosol detector (CAD);

[0014] Gradient elution is carried out using mobile phase A and mobile phase B as the eluent. Mobile phase A is an aqueous formic acid solution with a volume concentration of 0.02% to 0.08%, and mobile phase B is acetonitrile. The total flow rate is 0.8 - 1.2 mL / min. When controlling the elution time from 0 to 10 minutes, linear gradient elution is adopted, and the volume percentage of mobile phase A linearly decreases from 98% to 15%; at the same time, the volume percentage of mobile phase B linearly increases from 2% to 85%.

[0015] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0016] 1. The present invention uses a charged aerosol detector for detection, which can directly detect substances without ultraviolet absorption, such as the main component busulfan, and the test is stable and not easily interfered.

[0017] 2. The high performance liquid chromatography method of the present invention, especially with specific elution conditions, in particular when controlling the elution time from 0 to 10 minutes, the volume percentage of mobile phase A linearly decreases from 98% to 15%, while the volume percentage of mobile phase B linearly increases from 2% to 85%; this is beneficial to simultaneously detecting the related substances of busulfan injection, including impurity methanesulfonic acid and RRT0.91. In particular, the main component busulfan is completely separated from impurity methanesulfonic acid and RRT0.91, and the resolution is greater than 1.5. This indicates that the method of the present invention has good resolution, thus enabling accurate determination of the content of impurities.

[0018] 3. Using the method of the present invention, in the concentration range of 0.53 μg / ml to 75.6 μg / ml for methanesulfonic acid, there is a good linear relationship between the logarithm of the concentration and the logarithm of the peak area, and it also has good sensitivity, accuracy, and injection precision. Description of the Drawings

[0019] Figure 1 Shows the HPLC chromatogram of the test solution of busulfan injection under the chromatographic conditions according to the examples.

[0020] Figure 2 Shows the HPLC chromatogram of the control solution of busulfan injection under the chromatographic conditions according to the examples.

[0021] Figure 3 Shows the standard curve of the methanesulfonic acid series solution determined by the external standard method under the chromatographic conditions according to the examples.

[0022] Figure 4 Shows the HPLC chromatogram of the test solution of busulfan injection under the chromatographic conditions according to Comparative Example 1.

[0023] Figure 5 Shows the HPLC chromatogram of the test solution of busulfan injection under the chromatographic conditions according to Comparative Example 2. Detailed Description of the Invention

[0024] In the present invention, the "test sample" refers to the busulfan injection for which the related substances are to be determined.

[0025] In the present invention, the "related substances" refer to the impurities in busulfan injection, including methanesulfonic acid and RRT0.91.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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 chromatogram obtained by the test.

[0030] The present invention provides a method for improving the quality detection of busulfan injection, which comprises:

[0031] 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,

[0032] Detection is performed using a charged aerosol detector;

[0033] 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%;

[0034] The total volume percentage of mobile phase A and B is 100%.

[0035] Unexpectedly, by adopting the above-mentioned electrospray detector and a specific gradient elution program, especially when the elution time is from 0 to 10 minutes, the change in the volume percentages of mobile phases A and B both shows a linear change. In particular, mobile phase A linearly decreases from 98% to 15%, while mobile phase B linearly increases from 2% to 85%. The method of the present invention can simultaneously separate and detect impurity methanesulfonic acid and RRT0.91 in busulfan injection, and can completely separate the main component busulfan, impurity methanesulfonic acid and RRT0.91, thereby accurately determining the contents of these impurities and better detecting the quality of busulfan injection.

[0036] However, in contrast, as shown in the comparative example of the present invention (as shown in Table 5 in the Examples section), if the elution mode is: first isocratic elution (from 0 to 3 minutes) and then linear gradient elution when the elution time is from 0 to 10 minutes, and the initial volume percentage of mobile phase A is below 93%, impurity methanesulfonic acid, RRT0.91 and the main component busulfan cannot be completely separated (the resolution is less than 1.5). Even when the initial volume percentage is below 85%, these impurities cannot be detected.

[0037] In a specific embodiment, the method of the present invention does not adopt isocratic elution when the elution time is from 0 to 10 minutes; in particular, only linear gradient elution is adopted.

[0038] Optionally, the method of the present invention adopts linear gradient elution when the elution time is from 0 to 10 minutes. The starting mobile phase (at 0 minute): 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 sum of the volume percentages of mobile phases A and B is 100%.

[0039] Preferably, the elution program of the method of the present invention is as shown in Table 1 below:

[0040] 。

[0041] The sum of the volume percentages of mobile phases A and B is 100%.

[0042] 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 the value at either endpoint of the range or any value within the range; for example, in Table 1 above, when the elution time is 0 minutes, the volume percentage of mobile phase A is in the range of 98 - 95%, which means that at 0 minutes, the volume percentage of mobile phase A is a specific value, which can be 98% or 95%, or 95.5%, 96%, 96.5%, 97% or 97.5%.

[0043] More preferably, the gradient elution program is as shown in Table 2 below:

[0044] 。

[0045] Table 2 can also be expressed as: when the elution time is from 0 to 10 minutes, mobile phase A linearly decreases from 98% to 15%, and mobile phase B linearly increases from 2% to 85%; when from 10 to 20 minutes, mobile phase A linearly increases from 15% to 98%, and mobile phase B linearly decreases from 85% to 2%; when from 20 to 30 minutes, mobile phase A is 98% and mobile phase B is 2%.

[0046] 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 8.3% per minute, while the volume percentage of mobile phase B increases by 7.8% to 8.5% per minute, preferably 8.3% per minute.

[0047] In the present invention, the related substances are methanesulfonic acid and RRT 0.91, especially methanesulfonic acid.

[0048] In the method of the present invention, the test solution of busulfan injection (abbreviated as test solution) injected is a busulfan solution diluted with acetonitrile with a concentration of 0.3 - 0.8 mg / ml, preferably 0.5 - 0.6 mg / ml, more preferably 0.54 mg / ml.

[0049] In the method of the present invention, the nebulizing gas used in the charged aerosol detector is nitrogen, and the evaporation temperature is 30 - 40°C, preferably 32 - 37°C, more preferably 35°C.

[0050] Preferably, in the method of the present invention, mobile phase A is preferably an aqueous solution of formic acid with a volume concentration of 0.04% to 0.06%, more preferably 0.05% aqueous formic acid solution; the total flow rate of the mobile phase is preferably 0.9 - 1.1 mL / min, more preferably 1.0 mL / min.

[0051] Preferably, in the method of the present invention, the chromatographic column used is a chromatographic column filled with octadecylsilyl-bonded silica gel, preferably Phenomenex Titank C18 (4.6 mm × 250 mm, 3 μm) or a chromatographic column with equivalent efficiency; the sample tray temperature is 5 to 10 °C; the injection volume is 15 to 25 μL.

[0052] In the method of the present invention, the column temperature of the chromatographic column is 25 to 35 °C.

[0053] The method of the present invention also includes a method for determining the content of related substances in busulfan injection, such as the internal standard method, the external standard method, the self-control method of the main component, and the self-control method of the main component with a correction factor, etc.

[0054] In the method of the present invention, it also includes the step of determining the content of RRT0.91 by the self-control method of the main component. Optionally, this step includes measuring 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 into the following formula (1), and calculating to obtain the content of RRT0.91:

[0055] ;

[0056] Wherein,

[0057] Au is the peak area of the RRT0.91 impurity peak in the test solution;

[0058] As is the peak area of the main component busulfan in the control solution.

[0059] In a specific embodiment, the method of the present invention also includes the step of determining the content of RRT0.91, and this step includes:

[0060] (1) Solution preparation

[0061] Preparation of the test solution:

[0062] Dissolve the busulfan injection to be determined in acetonitrile;

[0063] Preparation of the control solution:

[0064] Pipette a certain amount of the test solution, and quantitatively dilute it with acetonitrile so that the mass concentration of busulfan in the diluted solution is 1% of the busulfan mass concentration of the test solution;

[0065] (2) Determination method

[0066] Measure the same volume of the test solution and the reference solution, and inject them into the liquid chromatograph respectively. Record the chromatogram according to the chromatographic conditions of the present invention; substitute the peak area Au of the impurity peak with RRT 0.91 in the test solution and the peak area As of the main component busulfan in the reference solution into the above formula (1) to obtain the content of the impurity with RRT 0.91 in the busulfan injection.

[0067] In the method of the present invention, it further includes the step of determining the content of methanesulfonic acid by the 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 units of mg / ml).

[0068] In a specific embodiment, the method of the present invention further includes the step of determining the content of methanesulfonic acid, and this step includes:

[0069] (1) Solution preparation

[0070] Preparation of the test solution:

[0071] Dissolve the busulfan injection to be determined in acetonitrile;

[0072] Preparation of the methanesulfonic acid series solutions:

[0073] Dissolve the methanesulfonic acid reference substance in acetonitrile to obtain a series of methanesulfonic acid solutions with different concentrations;

[0074] (2) Determination method

[0075] Measure the test solution and the methanesulfonic acid series solutions respectively and inject them into the liquid chromatograph. Record the chromatogram according to the chromatographic conditions of the present invention;

[0076] According to the recorded chromatogram of the methanesulfonic acid series solutions, with the logarithm of the methanesulfonic acid peak area as the ordinate and the logarithm of the mass concentration of methanesulfonic acid as the abscissa, obtain the standard curve, and perform linear regression to obtain the linear equation y = kx + b;

[0077] Calculate the content of methanesulfonic acid in the busulfan injection by the external standard method according to the following formula (2):

[0078] ;

[0079] Wherein,

[0080] Ai: the peak area of methanesulfonic acid in the test solution;

[0081] k: the slope of the linear equation;

[0082] b: the intercept of the linear equation;

[0083] C: the concentration of the test solution.

[0084] In the method of the present invention, the test solution is prepared by adding acetonitrile to the test busulfan injection and making up the volume. The concentration of busulfan in the test solution is about 0.5 - 0.6 mg / ml, such as 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).

[0085] In the present invention, if there are impurities in the chromatogram of the test solution, the content of methanesulfonic acid calculated by the external standard method based on the peak area shall not be greater than 7%, and the RRT0.91 shall not be greater than 4 times (4%) of the peak area of the main component busulfan in the control solution. This limit is formulated according to the test results at the end of the validity period of the reference preparation; among them, the reference preparation is busulfan injection, with a specification of 10 ml: 60 mg, batch number 3H081A, Otsuka Pharmaceutical Co., Ltd, and the validity period is 24 months; on the last day of the validity period, the contents of methanesulfonic acid and RRT0.91 are measured according to the method of the present invention, and the measured content of methanesulfonic acid is about 7%, and the content of RRT0.91 is about 4%.

[0086] In the method of the present invention, the control solution is prepared by adding acetonitrile to the test solution, and the concentration of busulfan in the control solution is 1% of the concentration of busulfan in the test solution.

[0087] 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 concentrations of methanesulfonic acid in the methanesulfonic acid series solution are 75.6 μg / ml, 56.7 μg / ml, 37.8 μg / ml, 18.9 μg / ml, 3.78 μg / ml, 0.53 μg / ml respectively; a standard curve is plotted with the logarithm of the methanesulfonic acid mass concentration (in mg / ml) and the logarithm of the methanesulfonic acid peak area, and the linear equation obtained is: y = 0.946x + 3.6555, and the correlation coefficient r = 0.9996.

[0088] In the method of the present invention, it also includes using a blank solution. After mixing polyethylene glycol 400 and N,N-dimethylacetamide to obtain blank excipients, acetonitrile is added so that each 100 ml of the blank solution contains 8 - 10 ml, preferably 9 ml of blank excipients. In the blank excipients, the volume ratio of polyethylene glycol 400 to N,N-dimethylacetamide is (1.8 - 2.2):1, preferably the volume ratio is 2:1. The blank solution does not interfere with the resolution of the impurity methanesulfonic acid and RRT0.91 in the test solution determined by the method of the present invention.

[0089] In the method of the present invention, especially in the step of determining the content of methanesulfonic acid in busulfan injection, it further includes recording the chromatogram of the blank solution according to the chromatographic conditions of the present invention, and there are no interfering peaks in this blank solution.

[0090] In the method of the present invention, localization solutions of busulfan and methanesulfonic acid are also used to determine the peak positions of each substance; among them, in the busulfan localization solution, the mass concentration of busulfan is 30 to 40 μg / ml, preferably 35 to 39 μg / ml, especially 37.8 μg / ml; in the methanesulfonic acid localization solution, the mass concentration of methanesulfonic acid is 30 to 40 μg / ml, preferably 35 to 39 μg / ml, especially 37.8 μg / ml.

[0091] For the detection by the method of the present invention, the linear range of the concentration of the impurity methanesulfonic acid is 0.5 - 80 μg / ml, the quantitation limit is about 0.4 μg / ml, and the detection limit is about 0.2 μg / ml; these concentrations are all far less than 0.1% of the concentration of the test solution; this indicates that the method of the present invention has a lower quantitation limit and detection limit and is very sensitive to the response of methanesulfonic acid. In other words, the method of the present invention allows the detection and quantitative determination of the impurity methanesulfonic acid under lower concentration conditions.

[0092] For the detection by the method of the present invention, the retention time of the main component busulfan is 9.55 minutes, and the resolution from the adjacent component (impurity RRT 0.91) is 3.19, enabling complete separation (as shown in Table 3 of the examples), and according to the quantitation limit and detection limit determination methods described in the examples, the quantitation 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 resolution and sensitivity.

[0093] In the present invention, the resolution calculation formula is as follows:

[0094] ;

[0095] Among them,

[0096] t RefPeak is the retention time of the reference peak;

[0097] t R is the retention time of the current peak;

[0098] W 50%,RefPeak is the peak width at 50% of the peak height of the reference peak;

[0099] W 50%,R is the peak width at 50% of the peak height of the current peak.

[0100] 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.

[0101] The method of the present invention is applied to the detection 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.

[0102] 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.

[0103] 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.

[0104] I. General Description

[0105] 1. Instruments

[0106] High performance liquid chromatography (HPLC): Ultimate 3000, Thermo Fisher Scientific.

[0107] Charged aerosol detector (CAD): VF-D20-A (model), Thermo Fisher.

[0108] Electronic balance: AP135W (one hundred thousandth balance), Shimadzu.

[0109] Electronic balance: MSA6-6S-OCE-DM (one millionth balance), Sartorius.

[0110] Ultrapure water machine: WP-UP-YJ-10, Sichuan Water Treatment Equipment Co., Ltd.

[0111] 2. Reagents

[0112] Formic acid (LC-MS): Lot No. 232524, Thermo Fisher Scientific.

[0113] Acetonitrile (LC-MS): Batch No. 231014, Tianjin Concord Technology Co., Ltd.

[0114] 3. Reference substances

[0115] Busulfan: Batch number BXAI230705A1, Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.

[0116] Methanesulfonic acid: Batch number BXAR230522B1, Guangzhou Aiqixi Pharmaceutical Technology Co., Ltd.

[0117] 4. Samples to be tested

[0118] Busulfan injection: Specification 10 ml: 60 mg, Batch number 3H081A, Otsuka Pharmaceutical Co., Ltd.

[0119] II. Examples

[0120] 1. Chromatographic conditions

[0121] Detector: The nebulizing gas is nitrogen and the evaporation temperature is 35 °C;

[0122] Chromatographic column: Phenomenex Titank C18 (4.6 mm × 250 mm, 3 μm);

[0123] Sample tray temperature: 5 °C;

[0124] Column temperature: 30 °C;

[0125] Total flow rate: 1.0 mL / min;

[0126] Injection volume: 20 μL;

[0127] Mobile phase A: 0.05% (v / v) formic acid aqueous solution (measure 2.5 ml of formic acid and add 5000 ml of water, mix well);

[0128] Mobile phase B: Acetonitrile;

[0129] Mobile phases A and B are used as eluents for gradient elution, and the gradient elution program is shown in Table 2 above.

[0130] 2. Solution preparation

[0131] (1) Test solution: Take 0.9 ml of busulfan injection, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the mark with acetonitrile, and shake well to obtain the test solution of busulfan injection (the concentration of busulfan is 0.54 mg / ml).

[0132] (2) Control solution: Accurately measure 0.1 ml of the above test solution, place it in a 10 ml volumetric flask, dilute it to the mark with acetonitrile, and shake well to obtain the control solution of busulfan injection.

[0133] (3)Blank solution: Mix polyethylene glycol 400 and N,N-dimethylacetamide evenly at a volume ratio of 2:1 to prepare a blank excipient. Take 0.9 ml of the above blank excipient, place it in a 10-ml volumetric flask, dilute it to the mark with acetonitrile, and shake well to obtain the blank solution.

[0134] (4)Busulfan reference stock solution: Weigh 3.80 mg of busulfan reference substance (content 99.4%) accurately, place it in a 10-ml volumetric flask, dissolve it with acetonitrile and dilute it to the mark, and shake well to obtain the solution (the concentration of busulfan is about 378 μg / ml).

[0135] (5)Methanesulfonic acid reference stock solution: Weigh 1.94 mg of methanesulfonic acid reference substance (content 97.4%) accurately, place it in a 5-ml volumetric flask, dilute it to the mark with acetonitrile, and shake well to obtain the solution (the concentration of methanesulfonic acid is about 378 μg / ml).

[0136] (6)Methanesulfonic acid series solutions: Accurately measure appropriate amounts of the above methanesulfonic acid reference stock solution, and quantitatively dilute them with acetonitrile respectively to prepare 6 methanesulfonic acid solutions with different concentrations, and the concentrations 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.

[0137] (7)Busulfan positioning solution: Accurately measure an appropriate amount of the above busulfan reference stock solution, dilute it with acetonitrile, and prepare a solution containing 37.8 μg of busulfan reference substance per 1 ml.

[0138] (8)Methanesulfonic acid positioning solution: Accurately measure an appropriate amount of the above methanesulfonic acid reference stock solution, dilute it with acetonitrile, and prepare a solution containing 37.8 μg of methanesulfonic acid reference substance per 1 ml.

[0139] 3. Testing

[0140] Verify the method of the present invention according to the General Principles for Verification of Analytical Methods 9101 in Part IV of the Chinese Pharmacopoeia (2020 Edition).

[0141] 3.1 Resolution determination

[0142] Inject the blank solution, busulfan positioning solution, methanesulfonic acid positioning solution and test solution into the liquid chromatograph respectively, and record the chromatogram according to the chromatographic conditions in the above example (see item 1 of Example II); the chromatogram of the test solution is specifically shown in Figure 1 , and the resolution results (calculated from the above formula (3)) are shown in Table 3 below:

[0143] .

[0144] Note: / indicates that the measurement result does not exist.

[0145] Upon measurement, there are no interfering peaks in the blank solution; Figure 1 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; among them, No. 1- represents the chromatographic peak of methanesulfonic acid, No. 2- represents the chromatographic peak with RRT 0.91, and No. 3- represents the chromatographic peak of the main component busulfan; as can be seen from Table 3, in the test solution, the resolution between methanesulfonic acid, RRT 0.91, the main component busulfan and their respective adjacent chromatographic peaks is all above 1.5, meeting the requirements; indicating that complete separation has been achieved between these peaks. Therefore, the method for determining the related substances (impurity methanesulfonic acid and RRT 0.91) of busulfan provided by the present invention has good resolution, can completely separate methanesulfonic acid, RRT 0.91, and the main component busulfan, and can accurately determine the contents of impurity methanesulfonic acid and RRT 0.91.

[0146] 3.2. Determination of the content of related substances

[0147] 3.2.1 Determination of the peak areas of busulfan and related substances in the test solution and the control solution

[0148] Precisely measure 20 μl of the test solution and the control solution prepared in Item 2 of the above-mentioned example, and inject them into the liquid chromatograph respectively, and record the chromatogram according to the chromatographic conditions of the above-mentioned example (see Item 1 of Example II). The chromatogram of the test solution is as Figure 1 shown. In this figure, the chromatographic peaks of impurity methanesulfonic acid and RRT 0.91 are represented by No. 1- and No. 2- respectively, and thus the peak areas of impurity RRT 0.91 and methanesulfonic acid in the test solution are obtained; Figure 2 It is an enlarged view of the blue box area in the chromatogram of the control solution in the upper right corner. In this figure, the chromatographic peak of the main component busulfan is represented by No. 1-, and thus the peak area of the main component busulfan in the control solution is obtained.

[0149] 3.2.2 Content of impurity RRT 0.91

[0150] The content of RRT 0.91 in the test solution is determined by the self-control method of the main component; calculated according to formula (1):

[0151] ;

[0152] Wherein,

[0153] Au: Peak area of the RRT 0.91 impurity peak in the test solution;

[0154] As: Peak area of the main component busulfan in the control solution.

[0155] In the above formula (1), both Au and As can be determined by the above step 3.2.1. Through calculation, in the busulfan injection, the content of impurity RRT 0.91 is 2.69%.

[0156] 3.2.3 Content of Impurity Methanesulfonic Acid

[0157] The content of methanesulfonic acid in the test solution is determined by the external standard method.

[0158] (1) Preparation of Standard Curve

[0159] Precisely measure 20 μl of the blank solution and the methanesulfonic acid series solutions prepared in Item 2 of the above example, and inject them into the liquid chromatograph in the order of the blank solution and the methanesulfonic acid series solutions respectively. Record the chromatogram according to the chromatographic conditions of the above example (see Item 1 of Example II).

[0160] After determination, there is no interference peak in the blank solution; according to the chromatograms of the methanesulfonic acid series solutions recorded above, with the logarithm of the methanesulfonic acid peak area (lnA) as the ordinate and the logarithm of the methanesulfonic acid concentration (lnM, the concentration is in mg / ml) as the abscissa, draw a standard curve as Figure 3 shown. Through linear regression calculation, the linear equation is obtained as: y = 0.946x + 3.6555 (slope k = 0.946, intercept b = 3.6555), and the correlation coefficient r = 0.9996.

[0161] (2) Calculation of Methanesulfonic Acid Content

[0162] Calculate the methanesulfonic acid content according to the following formula:

[0163] ;

[0164] Wherein,

[0165] Ai: Peak area of methanesulfonic acid in the test solution;

[0166] k: Slope of the linear equation;

[0167] b: Intercept of the linear equation;

[0168] C: Concentration of the test solution;

[0169] In the above formula (2), Ai can be determined by the above step 3.2.1, k and b are obtained from the linear equation obtained by the above preparation of the standard curve, and C is obtained from the test solution prepared in Item 2 of the above example (i.e., the busulfan concentration is 0.54 mg / ml); through calculation, in the busulfan injection, the content of impurity methanesulfonic acid is 3.43%.

[0170] The results show that by the determination using the high performance liquid chromatography method provided by the present invention, the concentration of mesylate is in the range of 0.53 μg / ml to 75.6 μg / ml, and there is a good linear relationship between the logarithm of the mesylate concentration (in mg / ml) and the logarithm of the mesylate peak area. Moreover, the method of the present invention can determine the respective contents of the impurity mesylate and RRT0.91 in busulfan injection,

[0171] which is conducive to the detection of the content of individual impurities, so as to detect the quality of busulfan injection (especially during storage), and improve the safety of the product.

[0172] 3.3 Accuracy and sensitivity of mesylate determination

[0173] 3.3.1 Accuracy determination

[0174] The limit of the impurity mesylate is proposed to be 7%.

[0175] Preparation of accuracy solution 1: Take an appropriate amount of busulfan injection (specification, 10 ml: 60 mg) and 194 μg of mesylate reference substance (content 97.4%), place them in a 10 ml volumetric flask, dilute and make up the volume with acetonitrile to prepare a solution containing 0.54 mg of busulfan and about 18.9 μg of mesylate per 1 ml. This solution is equivalent to the accuracy solution at 50% of the mesylate limit content, and prepare three parallel portions; the mesylate concentration of the actually prepared solution is the "added impurity concentration" as described in Accuracy 1-1, 1-2, 1-3 in the following table.

[0176] Preparation of accuracy solution 2: Take an appropriate amount of busulfan injection (specification, 10 ml: 60 mg) and 388 μg of mesylate reference substance (content 97.4%), place them in a 10 ml volumetric flask, dilute and make up the volume with acetonitrile to prepare a solution containing 0.54 mg of busulfan and about 37.8 μg of mesylate per 1 ml. This solution is equivalent to the accuracy solution at 100% of the mesylate limit content, and prepare three parallel portions; the mesylate concentration of the actually prepared solution is the "added impurity concentration" as described in Accuracy 2-1, 2-2, 2-3 in the following table.

[0177] Preparation of accuracy solution 3: Take an appropriate amount of busulfan injection (specification, 10 ml: 60 mg) and 582 μg of mesylate reference substance (content 97.4%), place them in a 10 ml volumetric flask, dilute and make up the volume with acetonitrile to prepare a solution containing 0.54 mg of busulfan and about 56.7 μg of mesylate per 1 ml. This solution is equivalent to the accuracy solution at 150% of the mesylate limit content, and prepare three parallel portions; the mesylate concentration of the actually prepared solution is the "added impurity concentration" as described in Accuracy 3-1, 3-2, 3-3 in the following table.

[0178] Inject the above accuracy solution and two parallel test solutions (prepared as described in Item 2 of the above Example) into a high-performance liquid chromatograph respectively, and record the chromatogram according to the chromatographic conditions described in Item 1 of the above Example. The accuracy results of methanesulfonic acid are shown in Table 4 below:

[0179] 。

[0180] The linear equation of the standard curve of methanesulfonic acid is: y = 0.946x + 3.6555, as described in Item 3.2.3 of the above Example.

[0181] The results in the above table show that the average recovery rate of methanesulfonic acid at each level concentration is 84.26% to 94.73%, and the RSD (%) of the recovery rate is 5.21%. The results meet the requirements. It shows that the high-performance liquid chromatography method provided by the present invention for the determination of methanesulfonic acid in busulfan injection has good accuracy.

[0182] 3.3.2 Determination of quantification limit and detection limit

[0183] Precisely weigh the reference substance methanesulfonic acid, dilute it with acetonitrile to prepare methanesulfonic acid solutions with different concentrations, inject these methanesulfonic acid solutions into a high-performance liquid chromatograph respectively, and record the chromatogram according to the chromatographic conditions of the Example (refer to Item 1 of Example II). Among them, the solution with a signal-to-noise ratio of about 10:1 is used as the quantification limit solution, and the solution with a signal-to-noise ratio of about 3:1 is used as the detection limit solution.

[0184] After determination, the detection limit concentration of methanesulfonic acid is 0.2004 μg / ml, and this concentration is 0.037% relative to the test sample concentration (0.54 mg / ml); the quantification limit concentration is 0.4000 μg / ml, and this concentration is 0.074% relative to the test sample concentration (0.54 mg / ml), and the RSD of the peak area is 3.48%.

[0185] The above results show that for methanesulfonic acid, an impurity 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; moreover, the RSD is less than 5%, indicating that the method of the present invention has good injection precision.

[0186] III. Comparative example

[0187] According to the general rules 9101 analysis method of the fourth part of the Chinese Pharmacopoeia 2020 edition above, among which for the verification result of the resolution, the resolution of the target peak is above 1.5.

[0188] 1. Comparative example 1

[0189] It was carried out according to the process of resolution determination in the above-mentioned embodiment (see item 3.1 of Embodiment II), with the difference being the gradient elution program in the chromatographic conditions; the elution conditions and results are as shown in Table 5 below and Figure 4 as shown.

[0190] 2. Comparative Example 2

[0191] It was carried out according to the process of resolution determination in the above-mentioned embodiment (see item 3.1 of Embodiment II), with the difference being the gradient elution program in the chromatographic conditions; the elution conditions and results are as shown in Table 5 below and Figure 5 as shown.

[0192] .

[0193] Note: n.a. indicates that no measurement result was obtained, that is, no peak of this substance was detected.

[0194] The resolution indicates the resolution from the next adjacent peak.

[0195] * indicates the resolution from the previous adjacent peak.

[0196] From Table 5 above and Figure 1 it can be seen that the examples of the present invention can simultaneously detect the related substances of busulfan injection, including impurity methanesulfonic acid and RRT0.91, and the resolutions between the main components busulfan, methanesulfonic acid, RRT0.91 and their respective adjacent chromatographic peaks are all greater than 1.5 (as shown in Table 3 above), meeting the requirements. The method of the present invention has good resolution and can accurately determine the contents of impurity methanesulfonic acid and RRT0.91.

[0197] On the contrary, when analyzing the test solution using the gradient elution conditions of Comparative Example 1 (the solution preparation in item 2 of the above-mentioned embodiment), the measured chromatogram is as Figure 4 shown, Figure 4 which 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; among them, numbers 1-, 3-, and 4- respectively represent the chromatographic peaks of impurity methanesulfonic acid, RRT0.91, and the main component busulfan, and their retention times are as shown in Table 5. Number 2- represents the chromatographic peak adjacent to methanesulfonic acid, and its retention time is 3.350 minutes; according to the resolution data of Comparative Example 1 in Table 5, it can be obtained that the resolutions of impurity methanesulfonic acid, RRT0.91, and the main component busulfan from their respective adjacent peaks are all less than 1.5, not meeting the requirements; this indicates that they overlap with the adjacent peaks and cannot be completely separated. Therefore, using the gradient elution program of Comparative Example 1, especially the elution conditions from 0 to 10 minutes of the elution time, cannot completely separate impurity methanesulfonic acid, RRT0.91, and the main component busulfan, and cannot accurately determine the contents of impurity methanesulfonic acid and RRT0.91.

[0198] Analyze the test solution (the solution preparation in item 2 of the above-mentioned examples) under the gradient elution conditions of Comparative Example 2. The measured chromatogram is as Figure 5 shown. Only the main component busulfan (as shown by No. 1 -) is detected, while neither impurity methanesulfonic acid nor RRT0.91 can be detected. Moreover, according to the resolution data of Comparative Example 2 in Table 5, the resolution between busulfan and its adjacent peak is less than 1.5, which does not meet the requirements; this indicates that busulfan overlaps with the adjacent peak and does not achieve complete separation. Therefore, using the gradient elution program of Comparative Example 2, especially the elution conditions from 0 to 10 minutes of the elution time, it is impossible to simultaneously detect impurity methanesulfonic acid, RRT0.91, and the main component busulfan, let alone completely separate these substances and accurately determine the contents of impurity methanesulfonic acid and RRT0.91.

[0199] In summary, comparing the above-mentioned examples with Comparative Examples 1 and 2 shows that the method of the present invention, especially through a specific gradient elution program, especially when controlling the elution time from 0 to 10 minutes, the volume percentage of mobile phase A linearly decreases from 98% to 15%, while the volume percentage of mobile phase B linearly increases from 2% to 85% correspondingly. This is beneficial to simultaneously separate and detect impurity methanesulfonic acid and RRT0.91, and has good resolution, enabling complete separation of these substances such as the main component busulfan, impurity methanesulfonic acid, and RRT0.91, thereby accurately determining the impurity content.

[0200] In conclusion, the advantages of the method of the present invention are that it can simultaneously separate and detect impurity methanesulfonic acid and RRT0.91 in busulfan injection, and can accurately determine the contents of these impurities. The method has good resolution. 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 quantitation limit concentration is 0.4000 μg / ml. It has good sensitivity, as well as good accuracy and injection precision.

Claims

1. A method for improving the quality detection of busulfan injection, comprising: Injecting the test solution of busulfan injection into a high performance liquid chromatograph to separate and detect related substances in the busulfan injection, wherein the related substance is methanesulfonic acid; in, The detection was performed using a charged aerosol detector, wherein the atomizing gas used in the charged aerosol detector was nitrogen gas with an evaporation temperature of 30 to 40°C; 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%, 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%, while the volume percentage of mobile phase B is linearly increased from 2% to 85%; The Pheromone Titank C18 chromatographic column was used.

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 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.

4. 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. The method according to claim 1 , wherein the column temperature of the chromatographic column is 25 to 35° C.

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

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