Method for detecting related substances of amphotericin B lipidosome

Abstract

The invention relates to the technical field of drug impurity analysis, and discloses a method for detecting related substances of amphotericin B lipidosome. The amphotericin B lipidosome related substances comprise amphotericin B, an amphotericin impurity A, an amphotericin impurity B, an amphotericin impurity C, an amphotericin impurity D, an amphotericin impurity E, an amphotericin B glycosidic ligand and amphotericin B methyl ester, a mobile phase A of the liquid chromatography comprises a citric acid solution with the pH value of 4.7, acetonitrile and methanol, and a mobile phase B of the liquid chromatography comprises a mobile phase B of the liquid chromatography and a mobile phase B of the liquid chromatography; a mobile phase B of the liquid chromatography comprises a citric acid solution with the pH value of 3.9, acetonitrile and methanol. According to the method, the degree of separation of various amphotericin B liposome related substances is good, baseline separation is achieved, each impurity is attributed, a basis is provided for process research, tracking of the destination and trend of each impurity is facilitated, and stability research is further facilitated.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical impurity analysis technology, and in particular to a method for detecting related substances in injectable amphotericin B liposomes. Background Technology

[0002] Amphotericin B is a polyene antifungal drug, considered the most effective treatment for deep fungal infections and the first-line clinical drug for treating invasive fungal infections. Amphotericin B liposomes for injection are sterile lyophilized liposomes prepared from amphotericin B, distearate phosphatidylglycerol sodium, hydrogenated soybean phosphatidylcholine, and cholesterol. It is a complex formulation with a complex impurity profile, containing numerous process and degradation impurities; therefore, the determination of related substances is particularly important.

[0003] Currently, the relevant substance detection methods for injectable amphotericin B liposomes mainly refer to the Chinese Pharmacopoeia and imported registration standards. Using the Chinese Pharmacopoeia method, impurities B and C, along with the main peak, all elute at the same position, making effective separation impossible. Using the imported registration standard method (standard number: JX20230008), impurities B and C also elute at the same position, again failing to separate them effectively. Using the European Pharmacopoeia method, the main peak fails to achieve baseline separation from adjacent impurities. Furthermore, the methods provided by the Chinese Pharmacopoeia, imported registration standards, and the European Pharmacopoeia are all rather general, calculating impurities as a sum without assigning specific peaks to each impurity. Therefore, it is impossible to track the growth or decrease trends of each impurity at each step of the process, increasing the difficulty of process research and hindering subsequent stability studies.

[0004] CN114315932A discloses a method for separating and purifying impurity A or impurity B of amphotericin B, which can only separate one of impurity A and impurity B at a time. Specifically, it involves pretreatment of amphotericin B with NMP, acetonitrile, and concentrated hydrochloric acid to finally separate impurity A; and pretreatment of amphotericin B with NMP, methanol, and concentrated hydrochloric acid to finally separate impurity B. Similarly, CN114315921A discloses a method for separating and purifying impurity C or impurity E of amphotericin B, which also achieves the separation of one impurity through a specific solvent. The purified impurity is then used as a calibrator, etc. However, it cannot separate multiple impurities simultaneously, therefore, this separation and purification method cannot be directly used for liquid chromatography analysis.

[0005] Therefore, it is necessary to develop analytical detection methods for related substances (process impurities and degradation impurities) in injectable amphotericin B liposomes in order to achieve effective separation of each impurity in liquid chromatography and achieve baseline separation, so as to meet the needs of process development such as impurity tracking. Summary of the Invention

[0006] To address the problems in existing technologies where baseline separation and accurate tracking of related substances in amphotericin B liposomes are not achieved in liquid chromatography, this invention, through extensive theoretical analysis and experimental verification, proposes a method for detecting related substances in injectable amphotericin B liposomes. This method effectively separates amphotericin B, amphotericin B impurities A, B, C, D, and E as recorded in the Chinese Pharmacopoeia, amphotericin B glycosides and methyl amphotericin B as recorded in the imported registered standard method (standard number: JX20230008), as well as process substances, achieving baseline separation and accurately tracking various related substances in amphotericin B liposomes.

[0007] The first aspect of this invention provides a method for detecting amphotericin B liposome-related substances. The method includes performing liquid chromatography (LC) on a test sample containing amphotericin B liposome-related substances, wherein the amphotericin B liposome-related substances include amphotericin B, amphotericin B impurity A, amphotericin B impurity C, amphotericin D, amphotericin E, amphotericin B glycoside, and amphotericin B methyl ester. The mobile phase A for LC detection comprises a citric acid solution at pH 4.7, acetonitrile, and methanol, and the mobile phase B for LC detection comprises a citric acid solution at pH 3.9, acetonitrile, and methanol.

[0008] In some embodiments, the volume ratio of citric acid solution at pH 4.7, acetonitrile, and methanol in mobile phase A is 60:29:13; and / or, the volume ratio of citric acid solution at pH 3.9, acetonitrile, and methanol in mobile phase B is 20:68:12.

[0009] Using the two mobile phases described above, along with conventional chromatographic techniques, baseline separation can be achieved. The following are preferred chromatographic parameters, which, in addition to baseline separation, can further improve detection sensitivity and accuracy.

[0010] In some methods, the preparation of the citric acid solution with pH 4.7 includes adjusting the pH of the citric acid solution to 4.7 with ammonia; and / or, the preparation of the citric acid solution with pH 3.9 includes adjusting the pH of the citric acid solution to 3.9 with ammonia.

[0011] In some embodiments, the gradient elution procedure of the liquid chromatography includes:

[0012] When executing the gradient elution program, time is plotted on the x-axis, and the amount of mobile phase A or B used is plotted on the y-axis according to the proportions set in the table above (e.g., starting at 23 minutes, the liquid chromatography mobile phase includes mobile phases A and B, with mobile phase A accounting for 70% of the volume and mobile phase B accounting for 30% of the volume. At 3 minutes, only mobile phase A is used).

[0013] In some embodiments, the chromatographic column of the liquid chromatography includes a C18 column.

[0014] In some methods, the column temperature of the liquid chromatography column is 10-60°C.

[0015] In some methods, the sample chamber temperature of the liquid chromatograph is 2-40°C.

[0016] In some embodiments, the liquid chromatography includes ultraviolet detection, wherein the wavelength of the ultraviolet detection includes 303 nm and / or 383 nm.

[0017] In some methods, the flow rate of the liquid chromatography is 0.1-1.5 mL / min.

[0018] In some methods, the injection volume of the test sample containing amphotericin B liposome-related substances in liquid chromatography is 5-50 μL.

[0019] In summary, the present invention has the following beneficial technical effects: The present invention proposes a method for determining related substances of amphotericin B liposomes for injection that can simultaneously achieve baseline separation of amphotericin B, amphotericin B impurities A, B, C, D, and E as recorded in the Chinese Pharmacopoeia, amphotericin B glycosides and methyl amphotericin B as recorded in the imported registration standard method (standard number: JX20230008), and process substances. Each impurity is assigned, providing a basis for process research, facilitating the tracking of the destination and trend of each impurity, and thus benefiting stability studies. Attached Figure Description

[0020] Figure 1 : System suitability chromatogram (303 nm) for the detection of amphotericin B liposome-related substances by liquid chromatography. Figure 2 : System suitability chromatogram (383 nm) for the detection of amphotericin B liposome-related substances by liquid chromatography; Figure 3 : Spectrum of control solution b for liquid chromatography detection of related substances in amphotericin B liposomes; Figure 4 : chromatogram of control solution d for detection of related substances of amphotericin B liposomes by liquid chromatography; Figure 5 Chromatogram (303 nm) for the system suitability of the Chinese Pharmacopoeia method for detecting related substances in amphotericin B liposomes. Figure 6 Chromatogram (383 nm) for the system suitability of the Chinese Pharmacopoeia method for detecting related substances in amphotericin B liposomes. Figure 7 Chromatogram (303 nm) of system suitability solution for the detection of amphotericin B liposome related substances using the imported registered standard method. Figure 8 Chromatogram (383 nm) of system suitability solution for the detection of related substances in amphotericin B liposomes using the imported registered standard method. Figure 9 Chromatogram (303 nm) of a system suitability solution for the detection of amphotericin B liposome related substances by the European Pharmacopoeia method. Figure 10 Chromatogram (383 nm) of a system suitability solution for the detection of amphotericin B liposome related substances using the European Pharmacopoeia method. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0022] Example 1 1. Instruments, reagents, consumables, and reference standards High-performance liquid chromatograph (including UV detector), chromatographic column, electronic analytical balance, pH meter, citric acid, ammonia, hydrochloric acid, ammonium acetate, methanol, acetonitrile, ethanol, dimethyl sulfoxide (DMSO), amphotericin B reference standard, and amphotericin B liposome reference standard.

[0023] 2. Solution preparation a) Citric acid solution: Weigh 4.2g of citric acid, add 1000mL of water to dissolve, mix well, and filter (can be prepared in equal proportions).

[0024] b) Citric acid solution (pH 4.7): Take citric acid solution and adjust the pH to 4.7 with concentrated ammonia.

[0025] c) Citric acid solution (pH 3.9): Take citric acid solution and adjust the pH to 3.9 with concentrated ammonia.

[0026] d) Mobile phase A: Measure 600 mL of citric acid solution (pH 4.7), 290 mL of acetonitrile, and 130 mL of methanol, mix well, and sonicate to obtain the mobile phase (can be prepared in equal proportions).

[0027] e) Mobile phase B: Measure 200 mL of citric acid solution (pH 3.9), 680 mL of acetonitrile, and 120 mL of methanol, mix well, and sonicate to obtain the mobile phase (can be prepared in equal proportions).

[0028] f) Blank solvent: namely methanol.

[0029] g) Dilute hydrochloric acid: Take 23.4 mL of hydrochloric acid and place it in a 100 mL volumetric flask. Dilute with water to the mark and shake well (can be prepared in equal proportions).

[0030] h) Ammonium acetate solution: Dissolve 1g of ammonium acetate in 100mL of water and mix well (it can be prepared in equal proportions).

[0031] i) Methanol-ethanol mixed solution: Measure 10 mL of anhydrous methanol and 40 mL of anhydrous ethanol and mix well (can be prepared in equal proportions).

[0032] j) Control solution a: Accurately weigh approximately 20 mg of amphotericin B reference standard into a 50 mL volumetric flask, dissolve in 15 mL of DMSO, and dilute to the mark with blank solvent within 2 hours, then shake well. Then accurately measure 5 mL of the above solution into a 25 mL volumetric flask, dilute to the mark with blank solvent, and shake well (amphotericin B concentration is 80 μg / mL).

[0033] k) Control solution b: Accurately measure 1 mL of control solution a into a 100 mL volumetric flask and dilute to the mark with blank solvent (use within 24 hours of preparation; amphotericin B concentration is 0.8 μg / mL).

[0034] l) Control solution d: Accurately weigh about 10 mg of amphotericin B reference standard into a 50 mL volumetric flask, dissolve it in 5 mL of DMSO, then add 35 mL of a methanol-ethanol mixture over 2 hours, add 0.1 mL of dilute hydrochloric acid, shake well and let stand for 2.5 hours, then add 10 mL of ammonium acetate solution and shake well (for system suitability).

[0035] m) System suitability solution: Accurately weigh about 106 mg of amphotericin B liposome reference powder, place it in a 50 ml volumetric flask, add methanol to about 80% of the volume, shake for about 20 min, then dilute to the mark with methanol and shake well.

[0036] n) Test solution: Take 32 mg of this product, place it in a 50 mL volumetric flask, add about 80% methanol, shake for 20 min, dilute to the mark with methanol, and shake well.

[0037] 3. Chromatographic conditions The chromatographic conditions are shown in Table 1.

[0038] Table 1

[0039] 4. Chromatographic results Chromatogram as shown Figure 1 , Figure 2 As shown, based on the structures of various impurities in amphotericin B liposomes (i.e., the amphotericin B impurity AE, amphotericin B glycoside ligand, and amphotericin B methyl ester involved in this invention) as recorded in the Chinese Pharmacopoeia and the imported registration standard method (standard number: JX20230008), the impurities corresponding to each peak were identified by mass spectrometry. All impurities achieved baseline separation, providing a good foundation for process research and facilitating the tracking of impurity destinations. Specific results are shown in Table 2.

[0040] Table 2

[0041] 5. Methodological Validation 5.1 System Applicability Inject one sample each of the blank solvent, system suitability solution, and control solution d, and five samples of control solution b. Record the chromatograms. The system suitability chromatogram is shown below. Figure 1 , Figure 2 As shown, the spectrum of control solution b is as follows: Figure 3 As shown, the spectrum of the control solution d is as follows. Figure 4 As shown.

[0042] Blank solvent: The solvent peak does not interfere with the main peak of related substances and amphotericin B.

[0043] Reference solution d: In the chromatogram (383 nm) of reference solution d, the resolution between the two peaks (impurity B and impurity C) with a relative retention time of about 0.85 is 2.06.

[0044] Control solution b: The peak area RSD of 5 control solutions b was 1.30%.

[0045] System suitability solution: In the chromatogram (383 nm) of the system suitability solution, the resolution between the impurity peak with a relative retention time of about 0.96 (which has significant absorption at 303 nm) and the main peak is 4.87.

[0046] 5.2 Limit of Quantitation and Limit of Detection Each impurity localization solution is diluted to an appropriate concentration and injected. The limits of detection and quantitation are the signal-to-noise ratios of the impurity peaks, which are approximately 3 and 10, respectively. Some examples are given below.

[0047] Detection limits: The detection limit concentration (LOC) for impurity A (303 nm) is 0.025 μg / mL, with a relative limit concentration (RLC) of 1.50%; the signal-to-noise ratio (SNR) is 4. The LOC concentration for nystatin (303 nm) is 0.021 μg / mL, with a RLC of 1.50%; the SNR is 3. The LOC concentration for impurity B (383 nm) is 0.022 μg / mL, with a RLC of 3.00%; the SNR is 11. The LOC concentration for impurity C (383 nm) is 0.022 μg / mL, with a RLC of 1.50%; the SNR is 10. The LOC concentration for impurity D (383 nm) is 0.074 μg / mL, with a RLC of 5.00%; the SNR is 6. The detection limit for glycoside aglycone (383 nm) was 0.020 μg / mL, with a relative limit of 0.50% and a signal-to-noise ratio of 10. The detection limit for amphotericin B (383 nm) was 0.022 μg / mL, with a relative limit of 1.50% and a signal-to-noise ratio of 25.

[0048] Limits of quantitation (LOQ): The LOQ concentration of impurity B (383 nm) was 0.07 μg / mL, the relative limit concentration was 10%, and the RSD of the peak area of ​​6 LOQ solutions was 0.79%; the LOQ concentration of glycoside agglutinin (383 nm) was 0.07 μg / mL, the relative limit concentration was 2%, and the RSD of the peak area of ​​6 LOQ solutions was 1.57%; the LOQ concentration of amphotericin B (383 nm) was 0.07 μg / mL, the relative limit concentration was 5%, and the RSD of the peak area of ​​6 LOQ solutions was 2.33%.

[0049] 5.3 Linearity and Range Prepare solutions with limits of quantitation, 25% linearity, 50% linearity, 100% linearity, and 200% linearity respectively, inject the samples, and record the chromatograms. The following examples use impurity B, amphotericin B, and amphotericin B glycosides as examples.

[0050] The linear equation for impurity B in the validation concentration range of 0.0001 mg / mL to 0.0014 mg / mL is y = 219285X + 0.2206, with a correlation coefficient of 1.000. The ratio of the absolute value of the Y-axis intercept of this curve to the peak area of ​​the 100% linear solution is 0.14%.

[0051] The linear equation for amphotericin B in the validation concentration range of 0.0001 mg / mL to 0.0089 mg / mL is y = 234891X + 9.5196, with a correlation coefficient of 0.9999. The ratio of the absolute value of the Y-axis intercept of this curve to the peak area of ​​the 100% linear solution is 0.91%.

[0052] The linear equation for the glycoside aglycone in the validation concentration range of 0.0001 mg / mL to 0.0081 mg / mL is y = 188400X + 2.3868, with a correlation coefficient of 0.9999. The ratio of the absolute value of the Y-axis intercept of this curve to the peak area of ​​the 100% linear solution is 0.31%.

[0053] 5.4 Precision Each of the two individuals prepared 6 solutions for injection and recorded the chromatograms.

[0054] For 2 servings (12 servings), the RSD of each impurity content is ≤10.0%. 5.5 Accuracy Prepare solutions with quantitation limit accuracy, 20% accuracy, 100% accuracy, and 120% accuracy respectively, inject them, and record the chromatograms.

[0055] The recovery rates of all impurities were between 80% and 120%, with RSD ≤ 10.0%. 5.6 Stability Prepare reference solution and test solution separately, inject them, and record the chromatograms.

[0056] Control solution b is stable after being stored at refrigeration (2℃-8℃) for 75 hours.

[0057] The sample solution is stable when stored under refrigeration (2℃-8℃) for 25 hours.

[0058] Comparative Example 1 Analysis was performed according to the methods described in the Chinese Pharmacopoeia, and the liquid chromatogram is as follows: Figure 5 , Figure 6 As shown in Table 3, the retention times of each impurity are as follows.

[0059] Table 3

[0060] It is evident that, using the Chinese Pharmacopoeia method, impurity B, impurity C, and the main peak all elute at the same position, indicating that the impurities cannot be effectively separated.

[0061] Comparative Example 2 Analysis was performed according to the import registration standard method (standard number: JX20230008), and the liquid chromatogram is shown below. Figure 7 , Figure 8 As shown in Table 4, the retention times of each impurity are as follows.

[0062] Table 4

[0063] As can be seen, using the imported registration standard method, impurities B and C precipitate at the same position, and the impurities cannot be effectively separated. Furthermore, impurities after the main peak also fail to achieve baseline separation.

[0064] Comparative Example 3 Analysis was performed according to the European Pharmacopoeia method, and the liquid chromatography chromatogram is as follows: Figure 9 , Figure 10 As shown in Table 5, the retention times of each impurity are as follows.

[0065] Table 5

[0066] It is evident that, using the European Pharmacopoeia method, the main peak and amphotericin B methyl ester did not achieve baseline separation, and impurity E and amphotericin B glycoside ligand merged into peaks with poor peak shape.

[0067] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent modifications or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting amphotericin B liposome-related substances, the method comprising performing liquid chromatography on a test sample containing amphotericin B liposome-related substances, wherein the amphotericin B liposome-related substances include amphotericin B, amphotericin B impurity A, amphotericin B impurity C, amphotericin D, amphotericin E, amphotericin B glycoside, and amphotericin B methyl ester, characterized in that, The mobile phase A for liquid chromatography detection includes a citric acid solution at pH 4.7, acetonitrile, and methanol, while the mobile phase B for liquid chromatography detection includes a citric acid solution at pH 3.9, acetonitrile, and methanol.

2. The method according to claim 1, wherein, In mobile phase A, the volume ratio of citric acid solution at pH 4.7, acetonitrile, and methanol is 60:29:13; and / or, in mobile phase B, the volume ratio of citric acid solution at pH 3.9, acetonitrile, and methanol is 20:68:

12.

3. The method according to claim 1 or 2, wherein, The method for preparing the citric acid solution with pH 4.7 includes: adjusting the pH of the citric acid solution to 4.7 with ammonia; and / or, the method for preparing the citric acid solution with pH 3.9 includes: adjusting the pH of the citric acid solution to 3.9 with ammonia.

4. The method according to claim 1 or 2, wherein, The gradient elution program for liquid chromatography includes: 。 5. The method according to claim 1 or 2, wherein, The chromatographic column used in the liquid chromatography includes a C18 column.

6. The method according to claim 1 or 2, wherein, The column temperature of the liquid chromatography column is 10-60℃.

7. The method according to claim 1 or 2, wherein, The sample chamber temperature of the liquid chromatograph is 2-40℃.

8. The method according to claim 1 or 2, wherein, The liquid chromatography detection includes ultraviolet detection; the wavelength of the ultraviolet detection includes 303 nm and / or 383 nm.

9. The method according to claim 1 or 2, wherein, The flow rate of the liquid chromatography is 0.1-1.5 mL / min.

10. The method according to claim 1 or 2, wherein, The injection volume of the test sample containing amphotericin B liposome-related substances in liquid chromatography is 5-50 μL.

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