Detailed Description
In order to make the purpose, technical solution and effect of the present application clearer and clearer, the present application is further described in detail below with reference to the accompanying drawings and examples.
The application provides a high performance liquid detection method for impurities of abacavir, wherein a plurality of impurities can be generated in the synthesis process of abacavir, and the details are shown in table 1. Specifically, abacavir is generally prepared by:
the present inventors found that the compound of formula II and cyclopropylamine have similar primary amine reaction sites during the synthesis when the residual (1S-cis) -4-amino-2-cyclopentenyl-1-methanol (II) is carried into the intermediate (1S-4R) -4- (2-amino-6-chloro-9H-purin-9-yl) -2-cyclopentene-1-methanol hydrochloride (V), so that the compound of formula II and the compound of formula V react to generate impurities during the synthesis of abacavir, and the impurities are ((1S,4R) -4- (2-amino-6- ((1R,4S) -4- (hydroxymethyl) cyclopentyl-2-alkenylamino) -9H-purine- 9-yl) cyclopentyl-2-enyl) methanol (I), with reference to figures 1 and 2 for a detailed analysis of the results, figure 1 is a mass spectrum of an abacavir compound impurity of the present application, wherein the abscissa on the graph represents the mass to charge ratio (m/z) value of the ion; the ordinate on the graph represents the intensity of the ion flow; in the present application, compound I has a characteristic peak at 343.5m/z in the mass spectrometric assay; FIG. 2 is a nuclear magnetic hydrogen spectrum of an impurity of an abacavir compound of the present application, wherein the ordinate on the graph is the nuclear magnetic resonance peak signal intensity; the abscissa on the graph is the resonant magnetic field strength (resonant frequency); in the present application, the NMR spectrum of the above-mentioned Compound I: (1H NMR spectrum) of1H NMR(400MHzMeOD), δ 1.45 to 1.73(m, 2H), 2.60 to 2.80(m, 2H), 2.81 to 2.88(m, 1H), 2.90 to 3.09(m, 1H), 3.57 to 3.61(m, 2H), 3.63 to 3.66(m, 2H), 5.529 to 5.531(m, 1H), 5.571 to 5.591(m, 1H), 5.87 to 5.90(m, 1H), 5.91 to 5.93(m, 1H), 5.95 to 5.97(m, 1H), 6.18 to 6.20(m, 1H), 7.74(s, 1H). The compound (formula I) is not mentioned in the past literature and lacks a corresponding analysis method, but is generated in the actual production of abacavir, so that the application provides a detection method for detecting such impurities.
Table 1: abacavir impurity name and structure
Referring to fig. 3, fig. 3 is a schematic flow chart of a first embodiment of a method for high performance liquid detection of impurities in abacavir according to the present application. In this embodiment, the detection method comprises the steps of:
s301: a sample analysis solution is provided.
Wherein, when preparing the sample analysis solution, the content of impurities in the sample analysis solution should be controlled within a proper range in consideration of the detection limit, for example, the content of impurities may be 0.1-10 μ g.
Wherein the structural formula of the impurity compound is
Wherein R is alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen, hydroxyalkyl, carboxyl or ester group; in particular, the compound may be
S302: injecting the sample analysis solution into a high performance liquid chromatograph for chromatographic analysis, and recording a chromatogram; wherein the chromatographic column is a reversed phase chromatographic column, and the mobile phase is a mixed solution of an acid solution and a solvent phase.
According to the specification requirement of a chromatograph, firstly setting detection conditions, injecting sample after the instrument runs stably, eluting by using a mobile phase, recording a chromatogram, and analyzing the chromatogram to obtain the content of impurities.
In this embodiment, by using a reverse phase chromatography column and performing elution using a mixed solvent of an acid solution and a solvent phase, the separation degree and accuracy of detection of impurities can be improved.
The abacavir contains various impurities, the impurities may influence each other, and researches show that due to large polarity difference among the impurities, the other impurities do not influence the determination of the compound I (impurity M) in the abacavir, and the impurities J and L influence the determination of the compound (I) in the abacavir, so that the influence of the impurities J and L on the determination of the compound (I) in the abacavir is researched while the detection method of the compound I is developed.
In one embodiment, the detection is performed by reverse phase High Performance Liquid chromatography (RT-HPLC or RP-HPLC), which is a Liquid chromatography system consisting of a non-polar stationary phase and a polar mobile phase, and is suitable for separating non-polar, polar or ionic compounds. Therefore, the used chromatographic column is a reverse phase chromatographic column, and the reverse phase chromatographic column is selected from one or more of a phenylsilane chemically bonded silica chromatographic column, a cyano-bonded silica chromatographic column, an amino-bonded silica chromatographic column, an octadecylsilane chemically bonded silica chromatographic column or an octaalkylsilane chemically bonded silica chromatographic column.
The specific specifications of the reversed phase chromatographic column are as follows: the column length is 100mm to 300mm, such as 150mm, 200mm, 250mm and the like; an inner diameter of 1mm to 10mm, for example, 2.5mm, 5.0mm, 8.0mm, etc.; the particle size is 1 to 10 μm, for example, 2, 3, 5, 7 μm. For example, it may be a Waters symmetry C18(3.9 × 150mm,5um) octadecylsilane bonded silica chromatography column.
The mobile phase is a mixed solution of an acid solution and a solvent phase, and the solvent phase is a mixed solution of water and an organic phase; the organic phase is methanol or acetonitrile; the acid solution is formic acid solution, acetic acid solution or trifluoroacetic acid solution; the acid solution is an aqueous solution, and the volume concentration of the acid solution is 0.01% -0.1%, such as 0.03%, 0.05%, 0.08%, 0.10% and the like. For example, the acid solution is a trifluoroacetic acid-water solution of 0.05% concentration by volume. The pH value of the mobile phase is 1.0-9.0, such as pH values of 1.5, 3.5, 5.5, 6.0, 7.5, 8.5, etc.
Wherein the volume ratio of the acid solution to the solvent phase in the mobile phase is 98: 2-2: 98; such as 90:10, 70:30, 50:50, 30:70, 15:85, etc. The volume ratio of water to the organic phase in the solvent phase is 5: 95-25: 75; e.g., 10:90, 15:85, 20:80, 25:75, etc. For example, the solvent phase may be water: methanol is a 15:85 mixture.
Specifically, chromatographic conditions are set, wherein the flow rate of the mobile phase is 0.8-1.3 ml/min, such as 0.9ml/min, 1.0ml/min, 1.1ml/min, 1.2ml/min and the like; the temperature of the chromatographic column is 25-50 ℃, such as 30 ℃, 35 ℃, 40 ℃, 45 ℃ and the like; the detection wavelength of the detector is 205-300 nm, such as 225nm and 256 nm; the running time is 30-70 min.
In one embodiment, a gradient elution method is adopted for elution, specifically, during the gradient elution, the volume ratio of the acid solution in the mobile phase is 95-70% within 0-20 minutes of the operation time, the volume ratio of the acid solution in the mobile phase is 70-10% within 20-35 minutes of the operation time, the volume ratio of the acid solution in the mobile phase is 10-95% within 35-35.1 minutes of the operation time, and the volume ratio of the acid solution in the mobile phase is 95-95% within 35.1-50 minutes of the operation time.
In one embodiment, a sample is dissolved using water, methanol, ethanol, acetonitrile, a methanol-water mixture, an ethanol-water mixture, or an acetonitrile-water mixture as a solvent to prepare a sample analysis solution. Specifically, the method can also be applied to detecting impurities in the abacavir bulk drug, the abacavir intermediate or the abacavir preparation, namely in a sample analysis solution, the sample can be the abacavir bulk drug, the intermediate or the preparation, and when the sample is different, the solubility difference of the sample is utilized, and different solvents are selected for dissolving. Wherein the abacavir preparation can be tablet, capsule, granule, eye preparation, nasal preparation, suppository, pill, ointment cream, cataplasm, inhalation preparation, spray, aerosol, gel, powder, syrup, liniment, plastics, tincture, patch, oral solution, implant, membrane, lotion, rinse, decocted extract, plaster, distillate and liniment.
In the following, the scheme of the present application will be illustrated and explained by several groups of specific experimental examples, but these experimental examples are only some exemplary schemes and should not be used to limit the scope of the present application. In the experimental examples, the experimental method not specified under the specific conditions is generally performed under the conventional conditions or the conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Experimental example 1
(1) Instrumentation and chromatographic conditions
High performance liquid chromatograph: e2965 high-performance liquid chromatography system and workstation.
A chromatographic column: thermo Syncronis C18(4.6 mm. times.250 mm,5 μm) octadecylsilane bonded silica chromatography column.
Mobile phase: preparing a trifluoroacetic acid-water solution with the volume concentration of 0.05%, preparing a water/methanol (15/85) mixed solution, mixing the trifluoroacetic acid-water solution with the water-methanol mixed solution to prepare a mobile phase, wherein the trifluoroacetic acid solution- (water/methanol ═ 15/85) in the mobile phase is subjected to gradient elution at the time points of 0-20 min, 20-35 min, 35-35.1 min and 35.1-50 min, and the volume ratio of the acid solution is 95-70%, 70-10%, 10-95% and 95-95%.
Detection conditions are as follows: the flow rate was set at 1.0ml/min, the detection wavelength at 254nm, and the column temperature at 30 ℃.
(2) Experimental procedure
Preparation of sample analysis solutions:
predetermined amounts of a standard sample of impurity J, impurity L, compound I and abacavir, respectively, were dissolved and diluted with methanol-water (20:80) to obtain sample analysis solutions containing about 2.5. mu.g of impurity J, impurity L, compound I and 250. mu.g of abacavir per 1ml of sample analysis solution.
Testing of sample analysis solutions:
precisely measuring 20 μ l of the above analysis solution, injecting into high performance liquid chromatograph, performing gradient elution with the mobile phase, and recording chromatogram, wherein the chromatogram is shown in figure 4. Fig. 4 is a chromatogram for detection in experimental example 1 of the present application, in which the Retention Time (RT) ═ 26.541min peak is impurity J, the RT ═ 27.921min peak is compound I, the RT ═ 28.898min peak is abacavir, and the RT ═ 31.103min peak is impurity L.
According to the chromatogram, the impurity J, the impurity L, the compound I and the abacavir can be completely separated under the condition, and the content of each impurity can be accurately measured.
Experimental example 2
(1) Instrumentation and chromatographic conditions
High performance liquid chromatograph: e2965 high-performance liquid chromatography system and workstation.
A chromatographic column: waters symmetry C18 (3.9X 150mm,5um) octadecylsilane bonded silica chromatography column.
Mobile phase: preparing a trifluoroacetic acid-water solution with the volume concentration of 0.02%, preparing a water/methanol (15/85) mixed solution, mixing the trifluoroacetic acid-water solution with the water-methanol mixed solution to prepare a mobile phase, wherein the trifluoroacetic acid solution- (water/methanol ═ 15/85) in the mobile phase is subjected to gradient elution at the time points of 0-20 min, 20-35 min, 35-35.1 min and 35.1-50 min, and the acid solution is subjected to gradient elution at the volume ratios of 95-70%, 70-10%, 10-95% and 95-95%.
Detection conditions are as follows: the flow rate was set at 1.0ml/min, the detection wavelength at 254nm, and the column temperature at 30 ℃.
(2) Experimental procedure
Preparation of sample analysis solutions:
predetermined amounts of a standard sample of impurity J, impurity L, compound I and abacavir, respectively, were dissolved and diluted with methanol-water (20:80) to obtain sample analysis solutions containing about 2.5. mu.g of impurity J, impurity L, compound I and 250. mu.g of abacavir per 1ml of sample analysis solution.
Testing of sample analysis solutions:
precisely measuring 20 μ l of the above analysis solution, injecting into high performance liquid chromatograph, performing gradient elution with the mobile phase, and recording chromatogram, wherein the chromatogram is shown in figure 5. Fig. 5 is a chromatogram for detection of experimental example 2 of the present application, in which the Retention Time (RT) ═ 25.729min peak is impurity J, the RT ═ 27.036min peak is compound I, the RT ═ 27.767min peak is abacavir, and the RT ═ 29.739min peak is impurity L.
According to the chromatogram, the impurity J, the impurity L, the compound I and the abacavir can be completely separated under the condition, and the content of each impurity can be accurately measured.
Experimental example 3
(1) Instrumentation and chromatographic conditions
High performance liquid chromatograph: e2965 high-performance liquid chromatography system and workstation.
A chromatographic column: waters symmetry C18 (3.9X 150mm,5um) octadecylsilane bonded silica chromatography column.
Mobile phase: preparing a trifluoroacetic acid-water solution with the volume concentration of 0.05%, preparing a water/methanol (15/85) mixed solution, mixing the trifluoroacetic acid-water solution with the water-methanol mixed solution to prepare a mobile phase, wherein the trifluoroacetic acid solution- (water/methanol ═ 15/85) in the mobile phase is subjected to gradient elution at the time points of 0-20 min, 20-35 min, 35-35.1 min and 35.1-50 min, and the volume ratio of the acid solution is 95-70%, 70-10%, 10-95% and 95-95%.
Detection conditions are as follows: the flow rate was set at 1.0ml/min, the detection wavelength at 254nm, and the column temperature at 30 ℃.
(2) Experimental procedure
Preparation of sample analysis solutions:
predetermined amounts of a standard sample of impurity J, impurity L, compound I and abacavir, respectively, were dissolved and diluted with methanol-water (20:80) to obtain sample analysis solutions containing about 2.5. mu.g of impurity J, impurity L, compound I and 250. mu.g of abacavir per 1ml of sample analysis solution.
Testing of sample analysis solutions:
precisely measuring 20 μ l of the above analysis solution, injecting into high performance liquid chromatograph, performing gradient elution with the mobile phase, and recording chromatogram, wherein the chromatogram is shown in figure 6. Fig. 6 is a detection chromatogram of experimental example 3 of the present application, in which the Retention Time (RT) ═ 26.717min peak is impurity J, the RT ═ 28.013min peak is compound I, the RT ═ 29.022min peak is abacavir, and the RT ═ 31.033min peak is impurity L.
According to the chromatogram, the impurity J, the impurity L, the compound I and the abacavir can be completely separated under the condition, and the content of each impurity can be accurately measured.
Experimental example 4
(1) Instrumentation and chromatographic conditions
High performance liquid chromatograph: e2965 high-performance liquid chromatography system and workstation.
A chromatographic column: waters symmetry C18 (3.9X 150mm,5um) octadecylsilane bonded silica chromatography column.
Mobile phase: preparing a trifluoroacetic acid-water solution with the volume concentration of 0.05%, preparing a water/acetonitrile (15/85) mixed solution, mixing the trifluoroacetic acid-water solution with the water-acetonitrile mixed solution to prepare a mobile phase, wherein the trifluoroacetic acid solution- (water/acetonitrile ═ 15/85) in the mobile phase is subjected to gradient elution at the time points of 0-20 min, 20-35 min, 35-35.1 min and 35.1-50 min, and the acid solution is subjected to gradient elution at the volume ratios of 95-70%, 70-10%, 10-95% and 95-95%.
Detection conditions are as follows: the flow rate was set at 1.0ml/min, the detection wavelength at 254nm, and the column temperature at 30 ℃.
(2) Experimental procedure
Preparation of sample analysis solutions:
predetermined amounts of a standard sample of impurity J, impurity L, compound I and abacavir, respectively, were dissolved and diluted with methanol-water (20:80) to obtain sample analysis solutions containing about 2.5. mu.g of impurity J, impurity L, compound I and 250. mu.g of abacavir per 1ml of sample analysis solution.
Testing of sample analysis solutions:
precisely measuring 20 μ l of the above analysis solution, injecting into high performance liquid chromatograph, performing gradient elution with the mobile phase, and recording chromatogram, wherein the chromatogram is shown in figure 7. Fig. 7 is a chromatogram for detection of experimental example 4 of the present application, in which the Retention Time (RT) ═ 27.946min peak is impurity J, the RT ═ 29.372min peak is compound I, the RT ═ 30.422min peak is abacavir, and the RT ═ 32.645min peak is impurity L.
According to the chromatogram, the impurity J, the impurity L, the compound I and the abacavir can be completely separated under the condition, and the content of each impurity can be accurately measured.
Experimental example 5
(1) Instrumentation and chromatographic conditions
High performance liquid chromatograph: e2965 high-performance liquid chromatography system and workstation.
A chromatographic column: waters symmetry C18 (3.9X 150mm,5um) octadecylsilane bonded silica chromatography column.
Mobile phase: preparing a trifluoroacetic acid-water solution with the volume concentration of 0.05%, preparing a water/methanol (15/85) mixed solution, mixing the trifluoroacetic acid-water solution with the water-methanol mixed solution to prepare a mobile phase, wherein the trifluoroacetic acid solution- (water/methanol ═ 15/85) in the mobile phase is subjected to gradient elution at the time points of 0-20 min, 20-35 min, 35-35.1 min and 35.1-50 min, and the volume ratio of the acid solution is 95-70%, 70-10%, 10-95% and 95-95%.
Detection conditions are as follows: the flow rate was set at 1.0ml/min, the detection wavelength at 254nm, and the column temperature at 30 ℃.
(2) Experimental procedure
Preparation of sample analysis solutions:
predetermined amounts of a standard sample of impurity J, impurity L, compound I and abacavir, respectively, were dissolved and diluted with methanol-water (20:80) to obtain sample analysis solutions containing about 2.5. mu.g of impurity J, impurity L, compound I and 250. mu.g of abacavir per 1ml of sample analysis solution.
Testing of sample analysis solutions:
precisely measuring 20 μ l of the above analysis solution, injecting into high performance liquid chromatograph, performing gradient elution with the mobile phase, and recording chromatogram, wherein the chromatogram is shown in figure 8. Fig. 8 is a detection chromatogram of experimental example 5 of the present application, in which the Retention Time (RT) ═ 28.462min peak is impurity J, the RT ═ 29.965min peak is compound I, the RT ═ 31.030min peak is abacavir, and the RT ═ 33.422min peak is impurity L.
According to the chromatogram, the impurity J, the impurity L, the compound I and the abacavir can be completely separated under the condition, and the content of each impurity can be accurately measured.
According to the scheme, a new impurity in the preparation process of abacavir is discovered, a high performance liquid detection method of the impurity is provided, and the detection separation degree and the detection limit of the impurity can be improved by utilizing a reversed phase chromatographic column and eluting with a mixed solvent of an acid solution and a solvent phase.
The above description is only for the purpose of illustrating embodiments of the present application and is not intended to limit the scope of the present application, and all modifications of equivalent structures and equivalent processes, which are made by the contents of the specification and the drawings of the present application or are directly or indirectly applied to other related technical fields, are also included in the scope of the present application.