HPLC-UV (High Performance Liquid Chromatography-Ultraviolet) detection method for components of amphotericin B liposome
Through the HPLC-UV detection method combined with equivalent elution technology, the problem of complexity and stability of bisexilin B liposomal composition in the existing technologies has been solved, and a simple, sensitive and simple analysis effect is achieved.
Patent Information
- Application Number
- CN202411832012.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-01-21
AI Technical Summary
Existing detection methods such as ELSD and RI detection have problems such as complex components of amphotericin B liposomes, such as complex operation, poor stability and long equilibrium time, making it difficult to achieve fast, sensitive and simple operation analysis.
Adopt the HPLC-UV detection method, composed of YMC Triart C18 columns and optimized flow phases, combined with equivalent elution technology, to realize the lipid-glycolic glyceride sodium and hydrogenated soybean phospholipidal gallbladder in bisexin B liposomes The qualitative and quantitative analysis of alkali, cholesterol, and narnium.
The effective separation and quantitative analysis of the four components of the bisexin B liposomes are achieved, and the separation is greater than 2.0, which improves the sensitivity and accuracy of the analysis, and simplifies the operation process.
Smart Images
Figure CN119335101A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of drug analysis and detection, and in particular to an HPLC-UV detection method for components of amphotericin B liposomes. Background Art
[0002] AmBisome® amphotericin B (AmB) liposomes are currently the most effective antifungal drugs in clinical practice. The development of generic amphotericin B liposomes must first solve the problem of analyzing drug ingredients and excipient ingredients. However, due to the complex ingredients, the analysis of amphotericin B excipients is also quite complicated, and generally requires multiple analytical methods to analyze each component separately. It is known that the excipients contained in amphotericin B liposomes include sucrose, sodium succinate, sodium distearoylphosphatidylglycerol (DSPG), hydrogenated soy phosphatidylcholine (HSPC), cholesterol, and tocopherol. Hydrogenated soy phosphatidylcholine (HSPC) contains two structures, PSPC and DSPC. Developing a series of methods to analyze each component separately requires a lot of time and reagents, and the actual operation is relatively complicated. A simpler solution is to develop a method to analyze as many components as possible at the same time, reducing the number of methods required to analyze each component.
[0003] Since sodium distearoylphosphatidylglycerol (DSPG), hydrogenated soy phosphatidylcholine (HSPC), and cholesterol do not have strong ultraviolet absorption, the evaporative light scattering (ELSD) method is often used in the literature to separate and analyze the mixture of phospholipids and cholesterol. ELSD can be gradient eluted, which is relatively easy to achieve the purpose of separating multiple auxiliary material components. However, the operation of the ELSD instrument is relatively complicated and the instrument stability is relatively poor, so it is not the preferred detection method for the quality inspection of the final product.
[0004] In addition, differential refractive index detection (RI) is also commonly used in phospholipid analysis, but this method requires a long equilibrium time, and the unstable background baseline caused by system equilibrium problems is common in the actual application of this method. Ultraviolet (UV) detection is the most stable detection method, especially UV detection has the simplicity of operation and stability that neither ELSD nor RI detection can match. For substances with weak UV absorption, you can try UV absorption at a shorter wavelength.
[0005] Amphotericin B liposomes are antibiotics commonly used to treat severe fungal infections. In clinical applications, they are usually in the form of liposomes to reduce their toxicity and improve the therapeutic effect. In order to ensure the quality and efficacy of the drug, it is very necessary to accurately analyze the components of amphotericin B liposomes. At present, among the existing detection methods, the ELSD instrument operation is relatively complicated and the instrument stability is relatively poor, and the differential refractive index detection requires a long equilibrium time. For the complex components of amphotericin B liposomes, especially the separation and quantification of its lipid components, it is necessary to seek a detection method with rapid analysis, high sensitivity and simple operation. Summary of the invention
[0006] In the prior art, the operation of ELSD instrument is relatively complicated and the instrument stability is relatively poor, and differential refractive index detection requires a long equilibrium time. For the complex components of amphotericin B liposomes, especially the separation and quantification of its lipid components, it is necessary to seek a detection method with rapid analysis, high sensitivity and simple operation. Therefore, the present invention provides a HPLC-UV detection method for the components of amphotericin B liposomes.
[0007] To achieve the above object, in a first aspect, the present invention provides a HPLC-UV detection method for the components of amphotericin B liposomes, which comprises the following specific steps: S1, the stationary phase provided is YMC Triart C 18 The column is used for chromatography, wherein the pH of the mobile phase in the column is adjusted to 9-11 with n-butylamine or triethylamine, and the amphotericin B liposomes are isocratically eluted at a temperature of 28-32°C; S2. Performing qualitative and quantitative analysis on sodium stearoylphosphatidylglycerol, hydrogenated soybean phosphatidylcholine, cholesterol and tocopherol in the amphotericin B liposomes by HPLC-UV based on the principle of reverse phase partition chromatography in the ultraviolet region of 200-240 nm; The mobile phase gradient phase A is a mixture of methanol and acetonitrile, and phase B is water containing ammonium acetate, and the volume ratio of phase A to phase B is 96:4.
[0008] In one implementation, in S1, it further includes: dissolving the amphotericin B liposomes by mixing a reagent to dissolve its components, wherein the mixed reagent includes methanol, chloroform and water in a volume ratio of 3:1:1.
[0009] In one implementation, in S1, the mobile phase is adjusted to pH 10 using n-butylamine.
[0010] In one implementation, the volume ratio of methanol to acetonitrile in phase A of the mobile phase is 86:14, and the concentration of ammonium acetate is 77 mg / mL.
[0011] In one implementation, in S2, HPLC-UV includes a chromatograph and an ultraviolet detector, the chromatograph is a Waters 2695 series high performance liquid chromatograph, and the ultraviolet detector is a Waters 2487 ultraviolet detector.
[0012] In one implementation, the chromatograph uses a 4.6 x 150 mm, 3.0 µm YMC Triart C 18 column; the injection volume of the chromatograph is 20 µL; the flow rate of the mobile phase is 1.2 mL / min; the column temperature is 30°C; and the detector wavelength is 214 nm.
[0013] Beneficial effects: The HPLC-UV detection method for the components of amphotericin B liposomes provided by the present invention combines the stationary phase and the mobile phase, and realizes the effective separation of the four components in the amphotericin B liposomes at the same time by isocratic elution. The final separation degree between each component is greater than 2.0. By optimizing the composition and pH value of the mobile phase and selecting a suitable detection wavelength, the sensitivity and accuracy of the analysis are improved. In addition, the use of a specific mixed reagent to dissolve the sample ensures the full dissolution and separation of each component in the sample, improves the reproducibility and reliability, and provides a rapid analysis, high sensitivity and simple operation detection method. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 It is a flow chart of the steps of the HPLC-UV detection method of the components of the amphotericin B liposome provided by the present invention; Figure 2 This is the HPLC spectrum obtained by the detection method provided in Example 1 using triethylamine to adjust the pH of the mobile phase; Figure 3 This is the HPLC spectrum obtained by the detection method provided in Example 2 using n-butylamine to adjust the pH of the mobile phase.
[0015] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION
[0016] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the description of the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" described below means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms is not necessarily for the same embodiment or example. Moreover, the technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.
[0017] Specific reference Figure 1 , Figure 1 The present invention provides a HPLC-UV detection method for the components of amphotericin B liposomes, which comprises the following specific steps: S1, the stationary phase provided is YMC Triart C 18 The column is used for chromatography, wherein the pH of the mobile phase in the column is adjusted to 9-11 with n-butylamine or triethylamine, and the amphotericin B liposomes are isocratically eluted at a temperature of 28-32°C; S2. Performing qualitative and quantitative analysis of sodium stearoylphosphatidylglycerol (DSPG), hydrogenated soybean phosphatidylcholine (HSPC), cholesterol and tocopherol in the amphotericin B liposomes by HPLC-UV based on the principle of reverse phase partition chromatography in the ultraviolet region of 200-240 nm; The mobile phase gradient phase A is a mixture of methanol and acetonitrile, and phase B is water containing ammonium acetate, and the volume ratio of phase A to phase B is 96:4.
[0018] Specifically, in S1, it also includes: dissolving the amphotericin B liposomes by a mixed reagent to dissolve its components, wherein the mixed reagent includes methanol, chloroform and water, and the volume ratio thereof is 3:1:1. Furthermore, the volume ratio of methanol to acetonitrile in the mobile phase A is 86:14, and the concentration of ammonium acetate is 77 mg / mL. Preferably, the mobile phase is adjusted to a pH of 10 using n-butylamine. Adjusting the mobile phase pH with n-butylamine can optimize the peak shape. At the same time, selecting an injection volume of 20 µL not only improves the detection sensitivity, but also makes the peak shape of the chromatographic peaks of each component have good symmetry.
[0019] Among them, YMC Triart C 18The column packing is a material based on organic / inorganic hybrid silica, which provides excellent chemical stability and mechanical strength and can be used in a wide pH range; octadecyl (C18) groups are bonded to the surface of the hybrid silica, making it suitable for reversed-phase high performance liquid chromatography (RP-HPLC) separation. Due to its unique packing structure and surface properties, YMC Triart C 18 Columns usually have good separation efficiency and selectivity and are suitable for the analysis of various complex samples.
[0020] Specifically, in S2, the HPLC-UV includes a chromatograph and a UV detector, the chromatograph is a Waters 2695 series high performance liquid chromatograph, and the UV detector is a Waters 2487 UV detector. Further, the chromatograph uses a 4.6 x 150 mm, 3.0 µm YMC Triart C 18 column; the injection volume of the chromatograph is 20 µL; the flow rate of the mobile phase is 1.2 mL / min; the column temperature is 30°C; and the detector wavelength is 214 nm.
[0021] In the present invention, ammonium acetate buffer, acetonitrile, methanol and a small amount of water are used as the organic phase, and sodium distearoylphosphatidylglycerol (DSPG), hydrogenated soybean phosphatidylcholine (HSPC), cholesterol and tocopherol can be effectively detected at a shorter ultraviolet absorption wavelength, such as around 214 nm.
[0022] Among them, the mixed solvent (methanol: chloroform: water, volume ratio of 3:1:1) can effectively dissolve the amphotericin B liposome sample and standard; the isocratic elution method can effectively separate the four components in the amphotericin B liposome at the same time, so that the separation degree between each component is greater than 2.0. This method can also be used to analyze other liposome drugs.
[0023] Compared with the differential detection method, the ultraviolet detection method of the present invention does not require a long system equilibration time, and the system is more stable. Compared with the evaporative light scattering detection method, ultraviolet detection is also simpler, easier to operate, and has a lower probability of system error. In general, the use of the ultraviolet (UV) detection method greatly shortens the system equilibration time compared with the differential detection method, is simpler and easier to operate than the evaporative light scattering detection method, has a lower probability of instrument error, and is more suitable for final product production inspection.
[0024] Furthermore, the specific steps before isocratic elution include: Prepare the mobile phase: First, select and prepare a suitable mobile phase, set the ratio of the mobile phase according to the experimental requirements, and adjust the pH value; set the chromatograph parameters; select a suitable chromatographic column; set the volume ratio of the mobile phase; set the flow rate, usually between 1.0-2.0 mL / min.
[0025] The specific steps of isocratic elution include: Regulation system: A preset proportion of mobile phase is pumped into the chromatographic column by a pump to ensure that the mobile phase remains constant during the entire analysis process and avoid gradient changes; Sample injection: Use an injector to inject the sample to be analyzed into the chromatographic system; Detection: After the sample is separated in the chromatographic column, it enters the UV detector for detection; under isocratic elution, the sample components are separated according to the interaction between their chemical properties and the chromatographic column, and quantitative and qualitative analysis is performed by detecting their UV absorption peaks; Maintain constant conditions: During the entire analysis process, the composition, flow rate and other conditions of the mobile phase do not change, ensuring that the various components of the sample are separated under constant conditions on the chromatographic column until all components are completely eluted from the chromatographic column.
[0026] Example 1 Instruments and conditions Chromatograph: Waters 2695 series high performance liquid chromatograph; Detector: Waters 2487 ultraviolet (UV) detector; Chromatographic column: YMC Triart C 18 Column (4.6 x 150 mm, 3.0 µm); Mobile phase: NH4Ac (308 mg): H2O (4 mL): MeOH (860 mL): ACN (140 mL), adjust the pH of the mobile phase to 10.0 with triethylamine; Detection wavelength: 214 nm; Flow rate: 1.2 mL / min; Column temperature: 30°C; Injection volume: 20 µL; Running time: 17 minutes.
[0027] Example 2 Instruments and conditions Chromatograph: Waters 2695 series high performance liquid chromatograph; Detector: Waters 2487 ultraviolet (UV) detector; Chromatographic column: YMC Triart C 18Column (4.6 x 150 mm, 3.0 µm); Mobile phase: NH4Ac (308 mg): H2O (4 mL): MeOH (860 mL): ACN (140 mL), adjust the pH of the mobile phase to 10.0 with n-butylamine; Detection wavelength: 214nm; Flow rate: 1.2 mL / min; Column temperature: 30°C; Injection volume: 20 µL; Running time: 17 minutes.
[0028] Combined with reference Figure 2 and Figure 3 , Figure 2 This is the HPLC spectrum obtained by the detection method provided in Example 1 using triethylamine to adjust the pH of the mobile phase, Figure 3 This is the HPLC spectrum obtained by the detection method provided in Example 2 using n-butylamine to adjust the pH of the mobile phase. It can be seen that the detection methods of Example 1 and Example 2 are both effective in qualitatively and quantitatively analyzing sodium stearoylphosphatidylglycerol (DSPG), hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (Cholesterol) and tocopherol (α-Tocopherol) in amphotericin B liposomes, with clear peak shape, good separation effect, and high accuracy of quantitative analysis. Among them, when n-butylamine is used to adjust the pH of the mobile phase, the separation effect of each component in the detection result is better, therefore, n-butylamine is preferably used to adjust the pH of the mobile phase.
[0029] In summary, the HPLC-UV detection method for the components of amphotericin B liposomes provided by the present invention combines the stationary phase and the mobile phase, and realizes the effective separation of the four components in the amphotericin B liposomes by isocratic elution. The final separation degree between each component is greater than 2.0. By optimizing the composition and pH value of the mobile phase and selecting a suitable detection wavelength, the sensitivity and accuracy of the analysis are improved. In addition, a specific mixed reagent is used to dissolve the sample, which ensures the full dissolution and separation of each component in the sample, and improves the reproducibility and reliability.
[0030] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. A HPLC-UV detection method for the components of amphotericin B liposomes, characterized in that: The specific steps include: S1, the stationary phase provided is YMC Triart C 18 The column is used for chromatography, wherein the pH of the mobile phase in the column is adjusted to 9-11 with n-butylamine or triethylamine, and the amphotericin B liposomes are isocratically eluted at a temperature of 28-32°C; S2. Performing qualitative and quantitative analysis on sodium stearoylphosphatidylglycerol, hydrogenated soybean phosphatidylcholine, cholesterol and tocopherol in the amphotericin B liposomes by HPLC-UV based on the principle of reverse phase partition chromatography in the ultraviolet region of 200-240 nm; The mobile phase gradient phase A is a mixture of methanol and acetonitrile, and phase B is water containing ammonium acetate, and the volume ratio of phase A to phase B is 96:
4.
2. The HPLC-UV detection method for the components of amphotericin B liposomes according to claim 1, characterized in that: In S1, it also includes: dissolving the amphotericin B liposomes by mixing reagents to dissolve its components, wherein the mixed reagents include methanol, chloroform and water in a volume ratio of 3:1:
1.
3. The HPLC-UV detection method for the components of amphotericin B liposomes according to claim 1, characterized in that: In S1, the mobile phase was adjusted to pH 10 using n-butylamine.
4. The HPLC-UV detection method for the components of amphotericin B liposomes according to claim 1, characterized in that: The volume ratio of methanol to acetonitrile in phase A of the mobile phase is 86:14, and the concentration of ammonium acetate is 77 mg / mL.
5. The HPLC-UV detection method for the components of amphotericin B liposomes according to claim 1, characterized in that: In S2, HPLC-UV includes a chromatograph and an ultraviolet detector, the chromatograph is a Waters 2695 series high performance liquid chromatograph, and the ultraviolet detector is a Waters 2487 ultraviolet detector.
6. The HPLC-UV detection method for the components of amphotericin B liposomes according to claim 1, characterized in that: The chromatograph used was a 4.6 x 150 mm, 3.0 µm YMC Triart C 18 column; the injection volume of the chromatograph is 20 µL; the flow rate of the mobile phase is 1.2 mL / min; the column temperature is 30°C; and the detector wavelength is 214 nm.