Detection method of chiral isomer of bedaquiline fumarate

Abstract

The invention discloses a method for detecting chiral isomers of bedaquiline fumarate, and belongs to the technical field of chiral substance separation and detection. The method comprises the following steps: dissolving a sample with a diluent to obtain a sample solution; a mobile phase composed of normal hexane, isopropanol and amines is prepared, and a CHIRALPAK AD-H chromatographic column is adopted for separation and detection. The volume ratio of normal hexane to isopropanol in the mobile phase is 98: 2, and 0.1%-0.5% of amine is added to improve the peak shape and the separation degree. By optimizing the composition of the mobile phase and chromatographic conditions, efficient separation of the bedaquiline fumarate and the chiral isomer thereof is realized, the separation degree is greater than 1.5, and the detection result is accurate and reliable. The method is simple and convenient to operate, is suitable for detection of chiral impurities in drug quality control, and meets strict requirements of ICH on chiral impurity control.

Description

Technical Field

[0001] This invention relates to the field of chiral substance separation and detection technology, and more specifically, to a method for detecting chiral isomers of bedaquiline fumarate. Background Technology

[0002] Bedaquiline fumarate is an anti-tuberculosis drug primarily used to treat multidrug-resistant tuberculosis in adults. It works by inhibiting the energy metabolism of Mycobacterium tuberculosis. However, the synthesis of bedaquiline fumarate inevitably produces its chiral isomers. These chiral isomers may possess different pharmacological or toxicological properties, potentially leading to reduced efficacy or even side effects. Chiral impurities may also interfere with the anti-tuberculosis activity of bedaquiline fumarate or increase cardiotoxicity (such as the risk of QT interval prolongation). The presence of chiral impurities may affect the dose-response relationship, resulting in unpredictable clinical efficacy. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) requires strict control of chiral impurities (typically below 0.1%-0.5%). Unseparated chiral impurities may be considered process impurities and require toxicological evaluation. Regulatory agencies require clear identification of the structure, content, and potential risks of chiral impurities; separation is a critical step in quality control. Therefore, how to detect the chiral isomers of bedaquiline fumarate has become a problem that needs to be addressed in quality control.

[0003] In the prior art, patent application number 201710670318.4 discloses "a method for separating and analyzing bedaquiline optical isomers". This method can effectively separate (1R,2S)-bedaquiline and / or (1S,2R)-bedaquiline, and / or detect the purity and / or content of (1R,2S)-bedaquiline and the purity of (1S,2R)-bedaquiline. However, its detection time is too long, with the elution time of the main peak (1S,2R)-bedaquiline exceeding 30 minutes, and mostly exceeding 35 minutes, i.e., the detection time is long. In addition, the width of its main peak is relatively wide, which can easily affect the quantitative accuracy.

[0004] Therefore, it is necessary to provide a detection method that can effectively separate the chiral isomers of bedaquiline fumarate, with short detection time and high accuracy. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a method for detecting chiral isomers of bedaquiline fumarate. The detection method provided by this invention can complete the peak elution of bedaquiline fumarate and its chiral isomers in a short time, and the main peak width is small, resulting in high detection accuracy.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A method for detecting the chiral isomer of bedaquiline fumarate includes the following steps:

[0008] Step 1: Dissolve the sample in a diluent to obtain a sample solution;

[0009] Step 2: Prepare the mobile phase and use a liquid chromatograph to detect the sample solution;

[0010] The liquid chromatograph is a forward high-performance liquid chromatograph; the diluent and the mobile phase are both a compound solution of hexane, isopropanol and organic amine.

[0011] Furthermore, the compound solution is prepared by adding an organic amine with a concentration of 0.1% to 0.5% at a volume ratio of n-hexane to isopropanol of 98:2.

[0012] Furthermore, the organic amine is triethylamine.

[0013] Furthermore, the liquid chromatograph uses a CHIRALPAK AD-H column with dimensions of 4.6 × 250 mm 5 μm.

[0014] Furthermore, during liquid chromatography detection, the flow rate was set to 0.5 mL / min, the column temperature to 35℃, and the detection wavelength was set to 210 nm to 240 nm.

[0015] Furthermore, during liquid chromatography detection, isocratic elution is used.

[0016] Furthermore, the injection volume for liquid chromatography when detecting sample solutions is 5-10 μL.

[0017] Furthermore, step 2 specifically involves:

[0018] Step 2.1: Select the chromatographic column;

[0019] Step 2.2: Prepare the mobile phase;

[0020] The mobile phase comprises n-hexane and isopropanol in a volume ratio of 98:2, with the addition of 0.1% to 0.5% organic amine;

[0021] Step 2.3, Liquid Chromatography System Setup;

[0022] Based on the characteristics of the chromatographic column in step 2.1 and the sample solution in step 1, set the parameters of the liquid chromatograph;

[0023] Step 2.4, Sample injection;

[0024] Inject the sample solution from step 1 into the liquid chromatograph;

[0025] Step 2.5: Separation and Detection;

[0026] Turn on the liquid chromatograph and use a mobile phase to separate and detect the sample solution from step 1, thereby obtaining the liquid chromatogram of the sample solution;

[0027] Step 2.6: Detect bedaquiline fumarate and its chiral isomers at multiple fixed concentrations using liquid chromatography to obtain the corresponding liquid chromatograms.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] (1) This invention provides a method for detecting chiral isomers of bedaquiline fumarate. The method involves adding a certain proportion of amine to the mobile phase and diluent to achieve the separation and detection of bedaquiline fumarate and its chiral isomers in the test system. Compared to traditional detection methods, this invention can detect chiral isomers of bedaquiline fumarate more quickly, with a resolution greater than 1.5, resulting in more accurate detection results.

[0030] (2) The present invention provides a method for detecting chiral isomers of bedaquiline fumarate. The mobile phase, diluent, and chromatographic column used are simple and readily available, and the operation process is simple and fast, which greatly reduces the detection time compared with the existing liquid chromatography method.

[0031] (3) The present invention provides a method for detecting the chiral isomer of bedaquiline fumarate. The mobile phase used contains a small proportion of isopropanol. Compared with the case without the addition of isopropanol, this method allows bedaquiline fumarate and its chiral isomer to be better separated and eluted. The amine added to the mobile phase is preferably triethylamine. Compared with the diethylamine added in the existing method, triethylamine has weaker basicity and lower nucleophilicity while improving the peak shape, and is less likely to react with impurities that may be contained in the analyte itself.

[0032] (4) The present invention provides a method for detecting chiral isomers of bedaquiline fumarate. The preferred chromatographic column is the CHIRALPAK AD-H column, which is filled with a linear starch derivative. It has a better separation effect on enantiomers and is more suitable for the detection of bedaquiline fumarate than chromatographic columns filled with cellulose derivatives.

[0033] (5) The present invention provides a method for detecting chiral isomers of bedaquiline fumarate. The diluent used is the same as the mobile phase as the existing method, which minimizes the possibility of solvent peaks affecting the main peak, isomer peaks and other impurity peaks in the chromatogram. Attached Figure Description

[0034] Figure 1 This is the liquid phase spectrum of bedaquiline fumarate in Example 1 of the present invention;

[0035] Figure 2 This is the liquid chromatogram of the chiral isomer of bedaquiline fumarate in Example 1 of the present invention;

[0036] Figure 3 This is the liquid chromatogram of bedaquiline fumarate with a certain amount of chiral isomer added in Example 1 of the present invention.

[0037] Figure 4 This is a diagram showing the detection results in Comparative Example 1 of the present invention.

[0038] Figure 5 This is a graph showing the detection results of the blank system when diethylamine was added as the amine in Comparative Example 2 of this invention.

[0039] Figure 6 This is a graph showing the sample separation results when diethylamine was added as the amine in Comparative Example 2 of this invention.

[0040] Figure 7 This is a diagram showing the detection results in Comparative Example 3 of the present invention. Detailed Implementation

[0041] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0043] Unless otherwise specified, all materials and reagents used in this invention are available from commercially available products in the field.

[0044] Example 1

[0045] A method for detecting the chiral isomer of bedaquiline fumarate includes the following steps:

[0046] Step 1: Weigh 5 mg of bedaquiline fumarate, dissolve it in a diluent (composed of n-hexane and isopropanol in a volume ratio of 98:2, with the addition of 0.3% triethylamine), and bring the volume to the preset mark of 50 mL to obtain the sample solution.

[0047] Step 2: Weigh 5 mg of bedaquiline fumarate chiral isomer, dissolve it in diluent (composed of n-hexane and isopropanol in a volume ratio of 98:2, with the addition of 0.3% triethylamine), and bring the volume to the preset mark of 50 mL to obtain impurity stock solution A; accurately transfer 1.5 mL of impurity stock solution A, dilute it with diluent and bring the volume to 100 mL, shake well, and obtain a 1.5% chiral impurity solution.

[0048] Step 3: Detect the sample solution and the chiral impurity solution using liquid chromatography to obtain the separation degree data between bedaquiline fumarate and the chiral isomer impurity.

[0049] Step 3 is as follows:

[0050] Step 3.1: Select the chromatographic column;

[0051] The chromatographic column used was a CHIRALPAK AD-H 4.6×250mm 5μm column, which has good separation effect and reproducibility for chiral substances;

[0052] Step 3.2: Prepare the mobile phase;

[0053] The mobile phase consisted of n-hexane and isopropanol in a volume ratio of 98:2, with 0.3% triethylamine added; the triethylamine was used to improve the peak elution.

[0054] Step 3.3, Chromatograph settings;

[0055] Based on the characteristics of the chromatographic column in step 3.1 and the sample solution in step 1, the parameters of the chromatogram were set as follows: the flow rate was set to 0.5 mL / min, the column temperature to 35℃, the liquid chromatograph was set to an Agilent 1260 liquid chromatograph, and the detection wavelength of the liquid chromatograph was set to 227 nm. This wavelength has good response for both bedaquiline fumarate and its chiral isomer.

[0056] Step 3.4, Sample injection;

[0057] The sample solution obtained in step 1 and the impurity stock solution A obtained in step 2 were injected into the liquid chromatograph, with an injection volume of 10 μL respectively.

[0058] Step 3.5: Separation and Detection;

[0059] Turn on the liquid chromatograph and use a mobile phase to separate and detect the sample solution obtained in step 1 and the impurity stock solution A obtained in step 2, thereby obtaining the liquid chromatograms of the sample solution and the impurity stock solution A, as shown below. Figure 1 , Figure 2 As shown, peaks can be clearly observed at the bedaquiline site of fumarate and at the chiral isomer site.

[0060] The time is 30 minutes, which is three times the elution time of the main peak, ensuring that the substance is completely eluted.

[0061] Step 3.6: Perform data analysis on the liquid chromatography spectrum of the sample solution from Step 3.5;

[0062] Step 3.6.1: Prepare comparison samples;

[0063] Bedaquiline fumarate sample: Weigh 5 mg of bedaquiline fumarate, dissolve it in a diluent (composed of n-hexane and isopropanol in a volume ratio of 98:2, with the addition of 0.3% triethylamine), and bring the volume to the preset mark of 50 mL to obtain the sample solution. The peak elution time was 8.48 min.

[0064] Chiral isomer sample: Weigh 5 mg of bedaquiline fumarate chiral isomer, dissolve and dilute to the preset mark of 50 mL with the same diluent to obtain impurity solution A, with a peak elution time of 10.53 min;

[0065] Step 3.6.2: Detect the test system, the sample solutions from Step 1 and Step 2, and the comparison sample from Step 3.6.1 using a liquid chromatograph to obtain the corresponding liquid chromatograms, and compare them with the liquid chromatogram of the sample solution from Step 3.5 to determine the peak sites of the chiral isomers.

[0066] In the liquid phase spectrum of the blank system, no peaks were observed at the elution sites of bedaquiline fumarate and its chiral isomer, indicating that there was no significant interference between the two elution sites in the blank system.

[0067] In the liquid chromatography spectrum of the sample solution in step 1, a peak can be clearly observed at the bedaquiline fumarate site, while no obvious peak is observed at the chiral isomer site, indicating that the sample solution contains bedaquiline fumarate but does not contain the chiral isomer.

[0068] Mixed localization: (Mixed localization involves adding a known amount of the chiral isomer to the sample solution and using an established method to detect its separation from bedaquiline fumarate):

[0069] Reference Figure 3 A quantitative amount of 1.5% chiral isomer impurity was added to the sample solution, and the above method was used for testing to obtain the liquid phase spectrum of the mixed solution, as shown below. Figure 3 As shown, peaks were observed at both the bedaquiline site and the chiral isomer site of fumarate; and the separation was excellent.

[0070] Depend on Figure 3 The test data shows that after adding 1.5% chiral isomer impurities, the proportion of chiral isomers obtained was 1.6%, and the recovery rate was 106.7%, which is within the recovery range of 90% to 110%.

[0071] Comparative Example 1

[0072] This comparative example also uses the bedaquiline fumarate raw material from Example 1, with the addition of a known amount of chiral isomer, and is tested according to the steps in Example 1. The only difference between this comparative example and Example 1 is that:

[0073] Isopropanol was not added to the mobile phase or diluent used.

[0074] The results are as follows Figure 4 As shown, by Figure 4 The test data shows that the chiral isomer impurity (the elution time under this condition is 10.53 min) was clearly tailed in the sample without the addition of isopropanol, and the separation from the adjacent peaks failed to reach 1.5.

[0075] Comparative Example 2

[0076] This comparative example also uses the bedaquiline fumarate raw material from Example 1, with the addition of a known amount of chiral isomer, and is tested according to the steps in Example 1. The only difference between this comparative example and Example 1 is that:

[0077] Replace triethylamine in the mobile phase and diluent with diethylamine in equal amounts.

[0078] The results are as follows Figure 5 As shown, Figure 5 This is a blank sample containing diethylamine as the amine added to the mobile phase and diluent. Figure 6 This describes the sample separation. It is known that adding diethylamine as an amine results in a large solvent peak, which poses a risk to sample detection as it may mask other unspecified impurities emitting peaks at this location.

[0079] Comparative Example 3

[0080] This comparative example also uses the bedaquiline fumarate raw material from Example 1, with the addition of a known amount of chiral isomer, and is tested according to the steps in Example 1. The only difference between this comparative example and Example 1 is that:

[0081] The chromatographic column was a YMC Pack Pro C18, 150 mm × 3.0 mm, 5 μm.

[0082] Figure 7 The figure shows the detection of chiral isomers in bedaquiline fumarate samples after changing the column and chromatographic conditions. It indicates that bedaquiline fumarate and its chiral isomers cannot be separated after the column change. (Other impurities in the figure are identified achiral isomers).

[0083] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0084] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for detecting a chiral isomer of bedaquiline fumarate, characterized by, The method comprises the following steps: Step 1, dissolving the sample with a diluent to obtain a sample solution; Step 2, configuring a mobile phase, and detecting the sample solution by using a liquid chromatograph; The liquid chromatograph is a forward high-performance liquid chromatograph; the diluent and the mobile phase are both complex solutions of n-hexane, isopropyl alcohol and an organic amine.

2. The detection method according to claim 1, characterized in that, In the complex solution, n-hexane and isopropyl alcohol are configured in a volume ratio of 98:2, and the organic amine is configured in a concentration of 0.1% to 0.5%.

3. The detection method according to claim 1 or 2, characterized in that, The organic amine is triethylamine.

4. The method of claim 1, wherein, The chromatographic column used by the liquid chromatograph is CHIRALPAK AD-H, with a specification of 4.6*250mm 5μm.

5. The method of claim 1, wherein, When the liquid chromatograph is detected, the flow rate is set to 0.5mL / min, the column temperature is set to 35℃, and the detection wavelength of the liquid chromatograph is set to 210nm to 240nm.

6. The method of claim 1, wherein, When the liquid chromatograph is detected, the elution program used is isocratic elution.

7. The method of claim 1, wherein, When the liquid chromatograph detects the sample solution, the injection amount is 5-10μL.

8. The method of claim 1, wherein, Step 2 specifically comprises: Step 2.1, selecting a chromatographic column; Step 2.2, preparing a mobile phase; The mobile phase comprises n-hexane and isopropyl alcohol in a volume ratio of 98:2, and 0.1% to 0.5% of an organic amine is added; Step 2.3, setting the liquid chromatograph; According to the characteristics of the chromatographic column in step 2.1 and the sample solution in step 1, the parameters of the liquid chromatograph are set; Step 2.4, sample injection; The sample solution in step 1 is injected into the liquid chromatograph; Step 2.5, separation and detection; The liquid chromatograph is turned on, the mobile phase is used to separate and detect the sample solution in step 1, and then the liquid chromatogram of the sample solution is obtained; Step 2.6, detecting the bedaquiline fumarate and its chiral isomers at multiple fixed concentrations by using the liquid chromatograph to obtain corresponding liquid chromatograms.

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