Method for detecting maleic acid residue in bedaquiline fumarate

Abstract

The invention discloses a pH-regulated liquid-liquid extraction-HPLC (High Performance Liquid Chromatography) coupling technique, which solves the problem of research on maleic acid residues in a medicine bedaquiline fumarate. The method is characterized in that maleic acid / fumaric acid is ionized into salt to retain a water phase under the condition that the pH value is 7.5-8.5, bedaquiline enters an organic phase to realize physical separation, and the separation degree of maleic acid and fumaric acid reaches 2.5 or above in combination with optimized chromatographic conditions. The limit LOD of the method is as low as 6 ppb, the recovery rate is 90-105%, the method is successfully applied to quality control of maleic acid residues in bedaquiline fumarate, and the method has the advantages of being high in selectivity, low in cost and environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analytical chemistry, and more specifically, to a method for detecting maleic acid residues in bedaquiline fumarate. Background Technology

[0002] Maleic acid (maleic acid) and fumaric acid (trans-maleic acid) are cis-trans isomers with similar polarity and strong water solubility. The target drug, bedaquiline fumarate, is a diarylquinoline compound with strong hydrophobicity; its solubility in the organic phase is more than 1000 times that in the aqueous phase. Traditional liquid-liquid extraction (such as an ethyl acetate / water system) leads to co-extraction of the three components, resulting in insufficient separation (R<1.5) and inaccurate quantification of maleic acid residues. Existing methods have certain drawbacks: ion chromatography is highly susceptible to matrix interference and requires complex pretreatment; derivatization GC / MS is cumbersome and has fluctuating recovery rates; conventional HPLC suffers from overlapping retention times between maleic acid and fumaric acid. Summary of the Invention

[0003] In view of this, the present invention addresses the shortcomings of the existing technology in the lack of a rigorous and reliable testing method for detecting maleic acid residues in bedaquiline fumarate, and provides a testing method for detecting maleic acid residues in bedaquiline fumarate.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows:

[0005] A method for detecting maleic acid residues in bedaquiline fumarate includes the following steps:

[0006] (1) Mix bedaquiline fumarate with an organic solvent, dissolve and stir until homogeneous;

[0007] (2) Add alkaline solution to adjust the pH of the system to 7.5-8.5;

[0008] (3) After heating and stirring, cool to room temperature and separate to obtain the water layer, which is the sample solution;

[0009] (4) The content of maleic acid in bedaquiline fumarate was determined by detecting the content of maleic acid in the aqueous layer using liquid chromatography.

[0010] In step (3), during separation, a separatory funnel can be used to separate the water layer and the organic layer. The organic layer is discarded and the water layer is retained. The water layer contains maleic acid and fumaric acid, and the organic layer contains bedaquiline.

[0011] Further, in step (1), the organic solvent is toluene, dichloromethane, or ethyl acetate.

[0012] Further, in step (2), the alkaline solution is any one of potassium carbonate solution, potassium hydroxide solution, ammonia water, and sodium bicarbonate, and the concentration of the alkaline solution is 8-12%.

[0013] Further, in step (3), the heating temperature is 70-90°C, preferably 80°C; the heating time is 30-40 min, preferably 35 min.

[0014] In step (4) of this invention, specifically:

[0015] Step 4.1: Select the chromatographic column;

[0016] Step 4.2: Select the mobile phase;

[0017] The mobile phase is prepared from purified water and phosphoric acid, i.e., an aqueous phosphoric acid solution;

[0018] Step 4.3, Liquid Chromatography System Setup;

[0019] Based on the selection of the chromatographic column in step 4.1 and the characteristics of the sample solution (aqueous layer) in step (3), set the parameters of the liquid chromatograph;

[0020] Step 4.4, Sample injection;

[0021] Inject the sample from step (3) into the liquid chromatograph;

[0022] Step 4.5: Separation and Detection;

[0023] Turn on the liquid chromatograph and use a mobile phase to separate and detect the sample solution from step 3, thereby obtaining the liquid chromatogram of the sample solution; and prepare control samples of different concentrations for detection and analysis, thereby obtaining the liquid chromatogram of the control sample solution, and then perform data analysis and processing on the liquid chromatogram of the sample solution.

[0024] Furthermore, in step (4), when using liquid chromatography for detection, the chromatographic column is: BDSHYPERSIL-C18, with specifications of 250×4.6mm and 5μm.

[0025] Furthermore, in step (4), when using liquid chromatography for detection, the mobile phase is an aqueous solution, and the pH is adjusted to 2-3.5 with phosphoric acid.

[0026] Furthermore, in step (4), during liquid chromatography detection, the flow rate is 1.2 mL / min, the column temperature is room temperature, and the detection wavelength of the liquid chromatograph is set to 210 nm.

[0027] Furthermore, in step (4), the detection time for the liquid chromatograph is 10-20 min.

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

[0029] (1) This invention combines a triple selective separation mechanism; chemical selection layer: pH control of carboxyl ionization state; physical partitioning layer: water / organic phase solubility difference; chromatographic separation layer: HPLC ion suppression separation;

[0030] (2) The detection limit of maleic acid is reduced to 6 ppb (far lower than 100 ppm in the prior art), and the recovery rate is 90-105%; the cost of a single detection is reduced by 70% (compared to LC-MS / MS); the detection specificity of maleic acid in bedaquiline fumarate is 100%; it has been successfully applied to the quality control of maleic acid residues in bedaquiline fumarate. This invention has the advantages of high selectivity, low cost and green environmental protection. Attached Figure Description

[0031] Figure 1 This is the liquid chromatogram of sample A with a concentration of 0.2 mg / mL in Example 1 of the present invention;

[0032] Figure 2 The liquid chromatogram of a maleic acid sample with a concentration of 0.02 μg / mL in Example 1 of this invention;

[0033] Figure 3 The liquid chromatogram of a maleic acid sample with a concentration of 0.006 μg / mL in Example 1 of this invention;

[0034] Figure 4 This is the liquid chromatogram of sample A spiked with maleic acid recovered at 0.02 μg / mL in Example 1 of this invention;

[0035] Figure 5 This is the liquid chromatogram of sample A spiked with maleic acid recovered at 0.02 μg / mL in Example 2 of the present invention;

[0036] Figure 6 The image shows the liquid chromatogram of sample A spiked with maleic acid recovered at 0.02 μg / mL in Example 3 of this invention.

[0037] Figure 7 This is the liquid chromatogram of sample B with a concentration of 1 mg / mL in Example 4 of the present invention.

[0038] Figure 8 This is the liquid chromatogram of sample C in Example 5 of the present invention. Detailed Implementation

[0039] 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.

[0040] 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.

[0041] Unless otherwise specified, all materials and reagents used in this invention are available from commercially available products in the field.

[0042] It should be noted that in the embodiments of the present invention, the raw material bedaquiline fumarate used has almost no maleic acid residue. In order to prove that the detection method provided by the present invention can detect maleic acid, spike recovery test and other operations were performed.

[0043] Example 1

[0044] Step 1: Accurately weigh 0.5g ± 0.05g bedaquiline fumarate into a 100mL reaction flask, add 5.0g ± 0.1g toluene solution and stir until homogeneous.

[0045] Step 2: Weigh 5 mL of 10% potassium carbonate solution and add it to the reaction flask. The pH value is measured to be 8.

[0046] Step 3: Heat the water bath to 80℃, stir and maintain the temperature for 35 minutes, then stop heating. After allowing it to cool to room temperature, separate the aqueous layer and the organic layer using a separatory funnel. At this point, the aqueous layer contains maleic acid and fumaric acid, with a concentration of 100 mg / mL, which is sample solution A.

[0047] Step 4: The limit of maleic acid is detected by liquid chromatography, which can be used as a method for detecting maleic acid residues in bedaquiline fumarate.

[0048] Step 4 is as follows:

[0049] Step 4.1: Select the chromatographic column;

[0050] The chromatographic column used was a BDSHYPERSIL C18250×4.6mm, 5μm column. Step 4.2: Prepare the mobile phase;

[0051] The mobile phase was an aqueous solution, and the pH was adjusted to 2.5 with phosphoric acid; phosphoric acid was used to improve peak elution.

[0052] Step 4.3, Chromatograph settings;

[0053] Based on the characteristics of the chromatographic column in step 4.1 and sample solution A in step 3, the parameters of the chromatogram were set as follows: flow rate 1.2 mL / min, column temperature room temperature, Shimadzu 2050 liquid chromatograph, and detection wavelength of 210 nm. Maleic acid and fumaric acid both have good responses at this wavelength.

[0054] Step 4.4, Sample injection;

[0055] Inject 10 μL of sample A from step 3 into the liquid chromatograph.

[0056] Step 4.5: Separation and Detection;

[0057] Turn on the liquid chromatograph and use the mobile phase to detect sample A in step 3, thereby obtaining the liquid chromatogram of sample A, where the peak of fumaric acid can be clearly observed.

[0058] The elution time is 20 minutes, which is three times the elution time of the last peak, to ensure that the substance is completely eluted; isocratic elution is used during the process.

[0059] Step 4.6: Perform data analysis on the liquid chromatogram of sample A from step 4.5;

[0060] Step 4.6.1, Preparation of Sample A Solution:

[0061] Accurately pipette 1.0 ml of the obtained sample solution A into a 100 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain sample A with a concentration of 1 mg / mL; dilute sample A to obtain sample A with a concentration of 0.2 mg / mL.

[0062] Prepare control samples:

[0063] Maleic acid control sample: Accurately weigh 20 mg of maleic acid solution, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well to obtain a maleic acid control sample with a concentration of 0.2 mg / mL; dilute the maleic acid sample to obtain maleic acid control samples with concentrations of 0.02 μg / mL and 0.006 μg / mL, respectively.

[0064] In step 4.6.1, the solvent is an aqueous solution, and the pH is adjusted to 2.5 with phosphoric acid.

[0065] Step 4.6.2: Analyze sample solution A with a concentration of 0.2 mg / mL from Step 4.6.1 using a liquid chromatograph to obtain the corresponding liquid chromatogram, as shown below. Figure 1 As shown; and compared with the liquid chromatograms of maleic acid at concentrations of 0.02 μg / mL and 0.006 μg / mL in step 4.6.1 ( Figure 2 and Figure 3The comparison showed that no maleic acid (limit of detection) residue was found in sample A solution;

[0066] In the liquid chromatogram of the test system, no obvious interfering peaks appeared at the elution sites of maleic acid and fumaric acid, indicating that there was no maleic acid (limit of detection) residue in sample A solution;

[0067] The peak area of ​​maleic acid at a known concentration of 0.02 μg / mL is set as S. In this example, S is 1359 (the same in subsequent examples 2 and 3). Maleic acid is added to sample solution A to a concentration of 0.02 μg / mL, and the peak area of ​​maleic acid is M. The recovery rate of maleic acid is calculated to be (M / S)%.

[0068] Spiked recovery (spiked recovery involves adding a known amount of maleic acid to a blank sample or a background with a known concentration, and then using an established method to determine the ratio of the measured concentration to the spiked concentration):

[0069] A known amount of maleic acid was quantitatively added to sample A with a concentration of 0.2 mg / mL, and the above method was used for detection. The resulting liquid chromatogram of sample solution A was obtained, as shown below. Figure 4 As shown, peaks of maleic acid and fumaric acid can be observed at specific sites; at the part per million concentration level, the recovery rate of maleic acid (M / S)% = (1374 / 1359)% = 101.1% > 95%.

[0070] Example 2

[0071] Step 1: Accurately weigh 0.5g ± 0.05g of bedaquiline fumarate into a 100mL reaction flask, add 5.0g ± 0.1g of dichloromethane solution and stir until homogeneous.

[0072] Step 2: Weigh 5 mL of 8% potassium hydroxide solution and add it to the reaction flask. Measure the pH to be 7.5–8.5.

[0073] Step 3: Heat the water bath to 80℃, stir and maintain the temperature for 35 minutes, then stop heating. After allowing it to cool to room temperature, separate the aqueous layer and the organic layer using a separatory funnel. At this point, the aqueous layer contains maleic acid and fumaric acid, with a concentration of 100 mg / mL, which is sample solution A.

[0074] Step 4: The limit of maleic acid is detected by liquid chromatography, which can be used as a method for detecting maleic acid residues in bedaquiline fumarate.

[0075] Step 4 is as follows:

[0076] Step 4.1: Select the chromatographic column;

[0077] The chromatographic column used was a BDSHYPERSIL C18250 × 4.6 mm, 5 μm column.

[0078] Step 4.2: Prepare the mobile phase;

[0079] The mobile phase was an aqueous solution, and the pH was adjusted to 2.5 with phosphoric acid; phosphoric acid was used to improve peak elution.

[0080] Step 4.3, Chromatograph settings;

[0081] Based on the characteristics of the chromatographic column in step 4.1 and sample A in step 3, the parameters of the chromatogram were set as follows: flow rate 1.2 mL / min, column temperature room temperature, Shimadzu 2050 liquid chromatograph, and detection wavelength of 210 nm. Maleic acid and fumaric acid both showed good response at this wavelength.

[0082] Step 4.4, Sample injection;

[0083] Inject 10 μL of sample A from step 3 into the liquid chromatograph.

[0084] Step 4.5: Separation and Detection;

[0085] Turn on the liquid chromatograph and use the mobile phase to detect sample A in step 3, thereby obtaining the liquid chromatogram of sample A, where the peak of fumaric acid can be clearly observed.

[0086] The elution time is 20 minutes, which is three times the elution time of the last peak, to ensure that the substance is completely eluted; isocratic elution is used during the process.

[0087] Step 4.6: Perform data analysis on the liquid chromatogram of sample A from step 4.5;

[0088] Step 4.6.1, Preparation of Sample A Solution:

[0089] Accurately pipette 1.0 ml of the obtained sample solution A into a 100 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain sample A with a concentration of 1 mg / mL; dilute sample A to obtain sample A with a concentration of 0.2 mg / mL.

[0090] Prepare control samples:

[0091] Maleic acid control sample: Accurately weigh 20 mg of maleic acid solution, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well to obtain a maleic acid control sample with a concentration of 0.2 mg / mL; dilute the maleic acid sample to obtain maleic acid control samples with concentrations of 0.02 μg / mL and 0.006 μg / mL, respectively.

[0092] In step 4.6.1, the solvent is an aqueous solution, and the pH is adjusted to 2.5 with phosphoric acid.

[0093] Step 4.6.2: The sample solution A with a concentration of 0.2 mg / mL from Step 4.6.1 was detected by liquid chromatography to obtain the corresponding liquid chromatogram. The chromatogram was then compared with the liquid chromatograms of maleic acid with concentrations of 0.02 μg / mL and 0.006 μg / mL from Step 4.6.1. It was found that there was no maleic acid residue in the sample solution A (limit of detection).

[0094] In the liquid chromatogram of the test system, no obvious interfering peaks appeared at the elution sites of maleic acid and fumaric acid, indicating that there was no maleic acid (limit of detection) residue in sample A solution;

[0095] Let S be the peak area of ​​maleic acid at a known concentration of 0.02 μg / mL; let M be the peak area of ​​maleic acid in sample solution A after maleic acid is added to a concentration of 0.02 μg / mL. Calculate the recovery rate of maleic acid as (M / S)%.

[0096] Spiked recovery (spiked recovery involves adding a known amount of maleic acid to a blank sample or a background with a known concentration, and then using an established method to determine the ratio of the measured concentration to the spiked concentration):

[0097] Similarly, a known amount of maleic acid was quantitatively added to sample A with a concentration of 0.2 mg / mL, and the above method was used for detection to obtain the liquid chromatogram of sample solution A, as shown below. Figure 5 As shown, peaks of maleic acid and fumaric acid can be observed at specific sites; at the part per million concentration level, the recovery rate of maleic acid (M / S)% = (1294 / 1359)% = 95.22% > 95%.

[0098] Example 3

[0099] Step 1: Accurately weigh 0.5g ± 0.05g bedaquiline fumarate into a 100mL reaction flask, add 5.0g ± 0.1g ethyl acetate solution and stir until homogeneous.

[0100] Step 2: Weigh 6 mL of 12% sodium bicarbonate solution and add it to the reaction flask. Measure the pH to be 7.5–8.5.

[0101] Step 3: Heat the water bath to 80℃, stir and maintain the temperature for 35 minutes, then stop heating. After allowing it to cool to room temperature, separate the aqueous layer and the organic layer using a separatory funnel. At this point, the aqueous layer contains maleic acid and fumaric acid, with a concentration of 100 mg / mL, which is sample solution A.

[0102] Step 4: The limit of maleic acid is detected by liquid chromatography, which can be used as a method for detecting maleic acid residues in bedaquiline fumarate.

[0103] Step 4 is as follows:

[0104] Step 4.1: Select the chromatographic column;

[0105] The chromatographic column used was a BDSHYPERSIL C18250 × 4.6 mm, 5 μm column.

[0106] Step 4.2: Prepare the mobile phase;

[0107] The mobile phase was an aqueous solution, and the pH was adjusted to 2.5 with phosphoric acid; phosphoric acid was used to improve peak elution.

[0108] Step 4.3, Chromatograph settings;

[0109] Based on the characteristics of the chromatographic column in step 4.1 and sample A in step 3, the parameters of the chromatogram were set as follows: flow rate 1.2 mL / min, column temperature room temperature, Shimadzu 2050 liquid chromatograph, and detection wavelength of 210 nm. Maleic acid and fumaric acid both showed good response at this wavelength.

[0110] Step 4.4, Sample injection;

[0111] Inject 10 μL of sample A from step 3 into the liquid chromatograph.

[0112] Step 4.5: Separation and Detection;

[0113] Turn on the liquid chromatograph and use the mobile phase to detect sample A in step 3, thereby obtaining the liquid chromatogram of sample A, where the peak of fumaric acid can be clearly observed.

[0114] The elution time is 20 minutes, which is three times the elution time of the last peak, to ensure that the substance is completely eluted; isocratic elution is used during the process.

[0115] Step 4.6: Perform data analysis on the liquid chromatogram of sample A from step 4.5;

[0116] Step 4.6.1, Preparation of Sample A Solution:

[0117] Accurately pipette 1.0 ml of the obtained sample solution A into a 100 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain sample A with a concentration of 1 mg / mL; dilute sample A to obtain sample A with a concentration of 0.2 mg / mL.

[0118] Prepare control samples:

[0119] Maleic acid control sample: Accurately weigh 20 mg of maleic acid solution, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with solvent, shake well to obtain a maleic acid control sample with a concentration of 0.2 mg / mL; dilute the maleic acid sample to obtain maleic acid control samples with concentrations of 0.02 μg / mL and 0.006 μg / mL, respectively.

[0120] In step 4.6.1, the solvent is an aqueous solution, and the pH is adjusted to 2.5 with phosphoric acid.

[0121] Step 4.6.2: The sample solution A with a concentration of 0.2 mg / mL from Step 4.6.1 was detected by liquid chromatography to obtain the corresponding liquid chromatogram. The chromatogram was then compared with the liquid chromatograms of maleic acid with concentrations of 0.02 μg / mL and 0.006 μg / mL from Step 4.6.1. It was found that there was no maleic acid residue in the sample solution A (limit of detection).

[0122] In the liquid chromatogram of the test system, no obvious interfering peaks appeared at the elution sites of maleic acid and fumaric acid, indicating that there was no maleic acid (limit of detection) residue in sample A solution;

[0123] Let S be the peak area of ​​maleic acid at a known concentration of 0.02 μg / mL; let M be the peak area of ​​maleic acid in sample solution A after maleic acid is added to a concentration of 0.02 μg / mL. Calculate the recovery rate of maleic acid as (M / S)%.

[0124] Spiked recovery (spiked recovery involves adding a known amount of maleic acid to a blank sample or a background with a known concentration, and then using an established method to determine the ratio of the measured concentration to the spiked concentration):

[0125] Similarly, a known amount of maleic acid was quantitatively added to sample A with a concentration of 0.2 mg / mL, and the above method was used for detection to obtain the liquid chromatogram of sample solution A, as shown below. Figure 6 As shown, peaks of maleic acid and fumaric acid can be observed at specific sites; at the part per million concentration level, the recovery rate of maleic acid (M / S)% = (1393 / 1359)% = 102.50% > 95%.

[0126] Example 4

[0127] Step 1: Accurately weigh 0.5g ± 0.05g bedaquiline fumarate and 2.5mL maleic acid with a concentration of 0.002mg / mL into a 100mL reaction flask, add 5.0g ± 0.1g toluene solution and stir until homogeneous.

[0128] Step 2: Weigh 2.5 mL of 20% potassium carbonate solution and add it to the reaction flask. The pH value is measured to be 8.

[0129] Step 3: After heating in a water bath to 80℃ and stirring for 35 minutes, stop heating and allow to stand at room temperature. Separate the aqueous layer and organic layer using a separatory funnel. The aqueous layer contains maleic acid and fumaric acid, with a concentration of approximately 100 mg / mL, which is sample solution B. Accurately pipette 1.0 mL of the obtained sample solution B into a 100 mL volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain sample B with a concentration of 1 mg / mL.

[0130] Step 4: The limit of maleic acid is detected by liquid chromatography, which can be used as a method for detecting maleic acid residues in bedaquiline fumarate.

[0131] Step 4 is as follows:

[0132] Step 4.1: Select the chromatographic column;

[0133] The chromatographic column used was a BDSHYPERSIL C18250×4.6mm, 5μm column. Step 4.2: Prepare the mobile phase;

[0134] The mobile phase was an aqueous solution, and the pH was adjusted to 2.5 with phosphoric acid; phosphoric acid was used to improve peak elution.

[0135] Step 4.3, Chromatograph settings;

[0136] Based on the characteristics of the chromatographic column in step 4.1 and sample solution B in step 3, the parameters of the chromatogram were set as follows: flow rate 1.2 mL / min, column temperature room temperature, Shimadzu 2050 liquid chromatograph, and detection wavelength of 210 nm. Maleic acid and fumaric acid both showed good response at this wavelength.

[0137] Step 4.4, Sample injection;

[0138] Sample B with a concentration of 1 mg / mL from step 3 was injected into the liquid chromatograph in a volume of 10 μL.

[0139] Step 4.5: Separation and Detection;

[0140] Turn on the liquid chromatograph and use the mobile phase to detect sample B from step 3, thereby obtaining the liquid chromatogram of sample B with a concentration of 1 mg / mL. The peaks of maleic acid and fumaric acid can be clearly observed.

[0141] The elution time is 20 minutes, which is three times the elution time of the last peak, to ensure that the substance is completely eluted; isocratic elution is used during the process.

[0142] Step 4.6: Perform data analysis on the liquid chromatogram of sample B from step 4.5;

[0143] Step 4.6.1, Preparation of Sample B Solution:

[0144] Accurately pipette 1.0 ml of the obtained sample solution B into a 100 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain sample B with a concentration of 1 mg / mL;

[0145] In step 4.6.1, the solvent is an aqueous solution, and the pH is adjusted to 2.5 with phosphoric acid.

[0146] Step 4.6.2: Analyze sample solution B with a concentration of 1 mg / mL from Step 4.6.1 using a liquid chromatograph to obtain the corresponding liquid chromatogram, as shown below. Figure 7 As shown.

[0147] In the liquid chromatogram of the test system, no obvious interfering peaks appeared at the elution sites of maleic acid and fumaric acid, indicating that maleic acid residue and fumaric acid in sample B solution can be detected simultaneously by this method.

[0148] Comparative Example 1: Conventional HPLC Detection

[0149] Step 1: Accurately weigh 20 mg of bedaquiline fumarate into a 100 ml volumetric flask, dissolve and dilute to the mark with the mobile phase, and shake well to obtain stock solution A.

[0150] Step 2: Accurately weigh 20 mg of maleic acid into a 100 ml volumetric flask, dissolve and dilute it to the mark with the mobile phase, and shake well to obtain stock solution B.

[0151] Step 3: Accurately pipette 1.0 ml of stock solution A and stock solution B into the same 100 ml volumetric flask, dissolve and dilute to the mark with the mobile phase, shake well, and you will get sample C.

[0152] Step 4: The limit of maleic acid is detected by liquid chromatography, which can be used as a method for detecting maleic acid residues in bedaquiline fumarate.

[0153] Step 4 is as follows:

[0154] Step 4.1: Select the chromatographic column;

[0155] The chromatographic column used was a BDSHYPERSIL C18250 × 4.6 mm, 5 μm column.

[0156] Step 4.2: Prepare the mobile phase;

[0157] The mobile phase was an aqueous solution, and the pH was adjusted to 2.5 with phosphoric acid; phosphoric acid was used to improve peak elution.

[0158] Step 4.3, Chromatograph settings;

[0159] Based on the characteristics of the chromatographic column in step 4.1 and sample C in step 3, the parameters of the chromatogram were set as follows: flow rate 1.2 mL / min, column temperature room temperature, Shimadzu 2050 liquid chromatograph, and detection wavelength of 210 nm. Maleic acid and fumaric acid both showed good response at this wavelength.

[0160] Step 4.4, Sample injection;

[0161] Inject 10 μL of sample C from step 3 into the liquid chromatograph.

[0162] Step 4.5: Separation and Detection;

[0163] Turn on the liquid chromatograph and use a mobile phase to detect sample C from step 3, thereby obtaining the liquid chromatogram of sample C, as shown below. Figure 8 As shown, it can be clearly observed that the peak position of maleic acid overlaps with that of bedaquiline fumarate, making effective separation impossible and thus the residual amount of maleic acid in bedaquiline fumarate cannot be detected.

[0164] Comparative Example 2

[0165] This comparative example also uses the bedaquiline fumarate raw material from Example 1, adds a known amount of maleic acid, and performs the test according to the steps in Example 1. The only difference is:

[0166] In step 2, 5 mL of purified water (10% potassium carbonate solution in Example 1) was weighed and added to the reaction flask, and the pH was measured to be 4.5 (fumaric acid itself is acidic).

[0167] In this comparative example, during liquid-liquid extraction and separation, the volume of the aqueous phase decreased significantly, while the volume of the organic phase increased and became turbid. The three phases were mixed—maleic acid and fumaric acid failed to remain completely in the aqueous phase; bedaquiline failed to completely enter the organic phase. This resulted in an emulsified or poorly partitioned system, leading to complete separation failure. The separated aqueous layer was analyzed using mobile phase A.

[0168] Chromatographic results:

[0169] Significantly reduced resolution: The chromatographic peaks of maleic acid and fumaric acid severely overlapped, resulting in a low resolution (R<1.0), making accurate quantitative analysis impossible; extremely low recovery: The recovery rate of maleic acid in the aqueous phase was <10%; severe matrix interference: Numerous extraneous peaks and elevated baselines appeared near the peak positions of maleic acid / fumaric acid, indicating that a large number of hydrophobic bedaquiline bases were not effectively extracted into the organic phase, but instead partially remained or emulsified in the aqueous phase, severely interfering with the detection of the target analyte.

[0170] Comparative Example 3

[0171] This comparative example also uses the bedaquiline fumarate raw material from Example 1, adds a known amount of maleic acid, and performs testing according to the steps in Example 1. The only difference between this comparative example and Example 1 is:

[0172] In step 2, the pH of the system is adjusted to 6 or 9.

[0173] In this comparative example, the liquid chromatograph was turned on, and mobile phase A was used to detect sample A from step 3, thereby obtaining the liquid chromatogram of sample A for data analysis. Following the same sample preparation method, it was found that when the pH was adjusted to 9, fumaric acid underwent partial hydrolysis, resulting in impurity peaks in the chromatogram and decreased resolution. When the pH was adjusted to 6.0, maleic acid was not fully ionized, with an aqueous phase residue of only 60% and a recovery rate (M / S)% < 75%.

[0174] Comparative Example 4

[0175] This comparative example also uses the bedaquiline fumarate raw material from Example 1, adds a known amount of maleic acid, and performs testing according to the steps in Example 1. The only difference between this comparative example and Example 1 is:

[0176] In step 4, the chromatographic column is a SHIMADZU VP-ODS C18 250×4.6mm, 5μm column.

[0177] In this comparative example, the liquid chromatograph was turned on, and mobile phase A was used to detect sample A in step 3, thereby obtaining the liquid chromatogram of sample A and performing data analysis; it can be clearly observed that the fumaric acid peak has severe tailing, and the reduced column efficiency leads to the detection time being extended to 30 minutes.

[0178] In summary, this invention solves the problem of maleic acid residue research in the drug bedaquiline fumarate. The core of the method lies in utilizing the ionization of maleic acid / fumaric acid into salts retained in the aqueous phase under pH 7.5–8.5 conditions, while bedaquiline enters the organic phase for physical separation. Combined with optimized chromatographic conditions, the separation degree of maleic acid and fumarate reaches above 2.5. The method has a limit of elimination (LOD) as low as 30 ppm and a recovery rate of 90–105%. It has been successfully applied to the quality control of maleic acid residue in bedaquiline fumarate, demonstrating that the crystallization process can completely remove 0.12% maleic acid. This technology combines the advantages of high selectivity, low cost, and environmental friendliness.

[0179] 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.

[0180] 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 maleic acid residues in bedaquiline fumarate, characterized in that, Includes the following steps: (1) Mix bedaquiline fumarate with an organic solvent, dissolve and stir until homogeneous; (2) Add alkaline solution to adjust the pH of the system to 7.5-8.5; (3) After heating and stirring, cool to room temperature and separate to obtain the water layer; (4) The content of maleic acid in bedaquiline fumarate was determined by detecting the content of maleic acid in the aqueous layer using liquid chromatography.

2. The detection method according to claim 1, characterized in that, In step (1), the organic solvent is toluene, dichloromethane or ethyl acetate.

3. The detection method according to claim 1, characterized in that, In step (2), the alkaline solution is any one of potassium carbonate solution, potassium hydroxide solution, ammonia water, and sodium bicarbonate, and the concentration of the alkaline solution is 8-12%.

4. The detection method according to claim 1, characterized in that, In step (3), the heating temperature is 70-90℃ and the heating time is 30-40min.

5. The detection method according to claim 1, characterized in that, In step (4), when using liquid chromatography for detection, the chromatographic column is BDSHYPERSIL-C18, with dimensions of 250×4.6mm and 5μm.

6. The detection method according to claim 1, characterized in that, In step (4), when using liquid chromatography for detection, the mobile phase is an aqueous solution, and the pH is adjusted to 2-3.5 with phosphoric acid.

7. The detection method according to claim 1, characterized in that, In step (4), the flow rate of the liquid chromatograph is 1.2 mL / min, the column temperature is room temperature, and the detection wavelength of the liquid chromatograph is set to 210 nm.

8. The detection method according to claim 1, characterized in that, In step (4), the detection time for the liquid chromatograph is 10-20 min.

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