CN109239253A - A kind of efficient liquid phase detection method of the impurity of Abacavir - Google Patents
A kind of efficient liquid phase detection method of the impurity of Abacavir Download PDFInfo
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Abstract
This application discloses a kind of efficient liquid phase detection method of the impurity of Abacavir, the structural formula of impurity isWherein, R is alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen, hydroxyalkyl, carboxyl or ester group；The described method includes: providing sample analysis solution；Sample analysis solution injection high performance liquid chromatograph is subjected to chromatography, and records chromatogram；Chromatographic column used in it is reverse-phase chromatographic column, and mobile phase used is the mixed liquor of acid solution and solvent phase.By the above-mentioned means, the application can be improved the separating degree and accuracy of detection.
Description
Technical field
This application involves Pharmaceutical Analysis technical fields, detect more particularly to a kind of efficient liquid phase of the impurity of Abacavir
Method.
Background technique
Abacavir (abacavir, ABC) is a kind of anti-AIDS new drug, is new carbocyclic ring 2'- deoxyguanosine ucleosides
Drug, oral administration biaavailability is high, easily infiltration central nervous system.As other efabirenzs, it
It is an inactive prodrug, is metabolized into active triguaiacyl phosphate through 4 steps in vivo, and pass through following 2 approach
It plays the effect of HIV inhibiting (HIV) reverse transcriptase: 1. competitively inhibiting 2'- deoxyguanosine triphosphate ester
(dGTP) (one of DNA synthesis segment) is combined into nucleic acid chains.2. by preventing the addition of new base from effectively terminating DNA chain
Synthesis.The chemical structural formula of Abacavir is as follows:
Abacavir (Formula IV) the preparation method is as follows: anti-under alkaline condition by Formula II compound and formula III compound
Formula IV should be obtained, triethyl orthoformate and acidic alcohol reaction is added, (1S, 4R) -4- is obtained after filtering, and (2- amino -6- is chloro-
9H- purine -9- base) -2- cyclopentene -1- methoxide hydrochlorate (Formula V), then react to obtain Abacavir (Formula IV) with cyclopropylamine
Present inventor is in long-term R&D process, during discovery prepares Abacavir (VI), due to chemistry
The relationship for reacting balance always has a small amount of raw material to remain, is brought into subsequent reaction step, and can be with the original in subsequent reactions
Material or product react, therefore generate many impurity during synthesizing Abacavir, these impurity will affect Abacavir
The purity of product, and there are partial impurities to be disclosed at present, and have coherent detection control means, and there is no quilts for some impurity
It is open, detection control more is carried out to these impurity without good method, therefore need a kind of scientific and effective analysis method pair
Impurity carries out detection control.
Summary of the invention
The application is mainly solving the technical problems that provide a kind of efficient liquid phase detection method of the impurity of Abacavir, energy
Enough separating degrees and accuracy for improving detection.
In order to solve the above technical problems, the technical solution that the application uses is: providing a kind of impurity of Abacavir
Efficient liquid phase detection method, the structural formula of impurity isWherein, R is alkyl, alkenyl, alkynyl,
Aryl, hydroxyl, halogen, hydroxyalkyl, carboxyl or ester group；The described method includes: providing sample analysis solution；By sample analysis solution
It injects high performance liquid chromatograph and carries out chromatography, and record chromatogram；Chromatographic column used in it is reverse-phase chromatographic column, used
Mobile phase be acid solution and solvent phase mixed liquor.
Wherein, solvent is mutually water-organic phase mixed liquor；Organic phase is methanol or acetonitrile.
Wherein, the volume ratio of acid solution and solvent phase is 98:2~2:98 in mobile phase；Water and organic phase in solvent phase
Volume ratio is 5:95~25:75；The pH value of mobile phase is 1.0~9.0.
Wherein, eluted by the way of gradient elution, wherein when gradient elution, at runtime between 0~20 minute
In range, in mobile phase the volume ratio of acid solution be 95~70%, at runtime between 20~35 minutes within the scope of, in mobile phase
The volume ratio of acid solution be 70~10%, at runtime between 35~35.1 minutes within the scope of, the volume of acid solution in mobile phase
Than being 10~95%, at runtime between 35.1~50 minutes within the scope of, in mobile phase the volume ratio of acid solution be 95~95%.
Wherein, acid solution is aqueous solution, and the volumetric concentration of acid solution is 0.01%-0.1%, acid solution be formic acid solution,
Acetic acid solution or trifluoroacetic acid solution.
Wherein, reverse-phase chromatographic column is phenyl silane bonded silica gel chromatographic column, cyano bonded silica gel chromatographic column, amino bonded silicon
Glue chromatographic column, octadecylsilane chemically bonded silica chromatographic column or eight alkyl silane bonded silica gel chromatographic columns.
Wherein, the column length of reverse-phase chromatographic column is 100mm~300mm, and internal diameter is 1mm~10mm, and partial size is 1 μm~10 μm.
Wherein, the flow velocity of mobile phase is 0.8~1.3ml/min；The column temperature of chromatographic column is 25~50 DEG C, and Detection wavelength is
205~300nm；Runing time is 30~70min.
Wherein, water, methanol, ethyl alcohol, acetonitrile, methanol-water mixture, ethanol-water mixture, acetonitrile-water mixed liquor are utilized
Sample is dissolved, sample analysis solution is made.
Wherein, sample is Abacavir bulk pharmaceutical chemicals, Abacavir intermediate or Abacavir preparation.
The beneficial effect of the application is: being in contrast to the prior art, the application has found Abacavir preparation process
One of new impurity, and the efficient liquid phase detection method of the impurity is provided, by utilizing reverse-phase chromatographic column, and molten using acid
Liquid and the mixed solvent of solvent phase are eluted, and can be improved the detection separating degree and detection limit of impurity.
Detailed description of the invention
Fig. 1 is the mass spectrogram of the application Abacavir impurity compound；
Fig. 2 is the nucleus magnetic hydrogen spectrum figure of the application Abacavir impurity compound；
Fig. 3 is the flow diagram of the efficient liquid phase detection method first embodiment of the impurity of the application Abacavir；
Fig. 4 is the detection chromatogram of the application experimental example 1；
Fig. 5 is the detection chromatogram of the application experimental example 2；
Fig. 6 is the detection chromatogram of the application experimental example 3；
Fig. 7 is the detection chromatogram of the application experimental example 4；
Fig. 8 is the detection chromatogram of the application experimental example 5.
Specific embodiment
It is right as follows in conjunction with drawings and embodiments to keep the purpose, technical solution and effect of the application clearer, clear
The application is further described.
The application provides a kind of efficient liquid phase detection method of the impurity of Abacavir, wherein Abacavir synthesis process
In can generate plurality of impurities, it is specific that see Table 1 for details.Specifically, Abacavir is made usually in the following way:
Present inventor has found in the synthesis process, when remaining (1S-cis) -4- amino -2- cyclopentenyl -1- first
Alcohol (II) is brought into intermediate (1S -4R) -4- (the chloro- 9H- purine -9- base of 2- amino -6-) -2- cyclopentene -1- methanolic hydrochloric acid
When in salt (V), compound Formula II has similar primary amine reaction site with cyclopropylamine, therefore during synthesizing Abacavir
Close object Formula II and Formula V and can react and generates impurity, confirm after nuclear-magnetism and mass spectral analysis the impurity be ((1S,
4R) -4- (2- amino -6- ((1R, 4S) -4- (methylol) cyclopenta -2- alkenyl amino) -9H- purine -9- base) cyclopenta -2-
Alkenyl) methanol (I), it makes a concrete analysis of result figure and please refers to Fig. 1 and Fig. 2, Fig. 1 is the mass spectrum of the application Abacavir compound impurities
Figure, wherein abscissa indicates mass-to-charge ratio (m/z) value of ion on figure；Ordinate indicates the intensity of ion stream on figure；In the application
In, compound I is in mass spectroscopy, with the characteristic peak at 343.5m/z；Fig. 2 is the application Abacavir compound impurities
Nucleus magnetic hydrogen spectrum figure, wherein ordinate is nuclear magnetic resonance peak signal strength on figure；Abscissa is the (resonance of resonant field intensity on figure
Frequency)；In this application, above compound I nuclear magnetic resonance spectroscopy (1H H NMR spectroscopy) be1H NMR (400MHz, MeOD), δ
1.45~1.73 (m, 2H), 2.60~2.80 (m, 2H), 2.81~2.88 (m, 1H), 2.90~3.09 (m, 1H), 3.57~
3.61 (m, 2H), 3.63~3.66 (m, 2H), 5.529~5.531 (m, 1H), 5.571~5.591 (m, 1H), 5.87~5.90
(m, 1H), 5.91~5.93 (m, 1H), 5.95~5.97 (m, 1H), 6.18~6.20 (m, 1H), 7.74 (s, 1H).And the substance
Compound (Formulas I) is not mentioned in passing document, and lacks corresponding analysis method, but in Abacavir actual production
In again can generate, therefore, the application provides a kind of detection method for detecting this kind of impurity thing.
Table 1: the impurity title and structure of Abacavir
Referring to Fig. 3, Fig. 3 is the stream of the efficient liquid phase detection method first embodiment of the impurity of the application Abacavir
Journey schematic diagram.In this embodiment, detection method includes the following steps:
S301: sample analysis solution is provided.
Wherein, when preparing sample analysis solution, it is contemplated that detection limit problem should control impurity in sample analysis solution
Content in the appropriate range, as impurity content can be 0.1~10 μ g.
Wherein, the structural formula of this kind of impurity compound isWherein, R is alkyl, alkenyl,
Alkynyl, aryl, hydroxyl, halogen, hydroxyalkyl, carboxyl or ester group；Specifically, compound can be
S302: sample analysis solution injection high performance liquid chromatograph is subjected to chromatography, and records chromatogram；Wherein color
Spectrum column is reverse-phase chromatographic column, and mobile phase is the mixed liquor of acid solution and solvent phase.
Wherein, according to chromatographic specification requirement, testing conditions are first set, after instrument is stable, sample introduction utilizes stream
Dynamic phase is eluted, and records chromatogram, is analyzed chromatogram, obtains impurity content.
In this embodiment, it by utilizing reverse-phase chromatographic column, and is carried out using acid solution and the mixed solvent of solvent phase
Elution, can be improved the detection separating degree and accuracy of impurity.
Wherein, it due to containing plurality of impurities in Abacavir, may influence each other between impurity, it has been investigated that due to
Impurity is intermediate polarity to differ greatly, remaining impurity does not influence the measurement of compound I (impurity M) in Abacavir, and impurity J, miscellaneous
The influence that matter L measures compound in Abacavir (I), therefore while developing the detection method of compound I, study impurity
J, the influence that impurity L measures compound in Abacavir (I).
Wherein, in one embodiment, reversed-phased high performace liquid chromatographic (Reversed-phase High is utilized
Performance Liquid Chromatogra, RT-HPLC or RP-HPLC) it is detected, reversed-phased high performace liquid chromatographic is
The liquid chromatographic system as composed by non-polar stationary phase and polarity mobile phase, this method be suitable for separation nonpolarity, polarity or from
Subtype compound.Therefore chromatographic column used is reverse-phase chromatographic column, and reverse-phase chromatographic column is selected from phenyl silane bonded silica gel chromatographic column, cyanogen
Base bonded silica gel chromatographic column, amino bonded silica gel chromatographic column, octadecylsilane chemically bonded silica chromatographic column or eight alkyl silane keys
Close one of silica gel chromatographic column or a variety of.
The concrete specification of reverse-phase chromatographic column are as follows: column length 100mm~300mm, such as 150mm, 200mm, 250mm etc.；Internal diameter
1mm~10mm, such as 2.5mm, 5.0mm, 8.0mm etc.；1 μm~10 μm of partial size, such as 2 μm, 3 μm, 5 μm, 7 μm.Such as it can be with
It is Waters symmetry C18 (3.9*150mm, 5um) octadecylsilane chemically bonded silica chromatographic column.
Mobile phase is the mixed liquor of acid solution and solvent phase, and solvent is mutually water-organic phase mixed liquor；Organic phase is methanol
Or acetonitrile；Acid solution is formic acid solution, acetic acid solution or trifluoroacetic acid solution；Acid solution is aqueous solution, and the volume of acid solution is dense
Spending is 0.01%-0.1%, such as 0.03%, 0.05%, 0.08%, 0.10% etc..For example, acid solution is that 0.05% volume is dense
Trifluoroacetic acid-aqueous solution of degree.The pH value of mobile phase be 1.0~9.0, such as pH value be 1.5,3.5,5.5,6.0,7.5,
8.5 waiting.
Wherein, the volume ratio of acid solution and solvent phase is 98:2~2:98 in mobile phase；As 90:10,70:30,50:50,
30:70,15:85 etc..The volume ratio of water and organic phase is 5:95~25:75 in solvent phase；Such as 10:90,15:85,20:80,
25:75 etc..Such as solvent mutually can be water: methanol=15:85 mixed liquor.
Specifically, be arranged chromatographic condition, wherein the flow velocity of mobile phase be 0.8~1.3ml/min, such as 0.9ml/min,
1.0ml/min, 1.1ml/min, 1.2ml/min etc.；The column temperature of chromatographic column is 25~50 DEG C, such as 30 DEG C, 35 DEG C, 40 DEG C, 45
DEG C etc.；The Detection wavelength of detector is 205~300nm, such as 225nm, 256nm etc.；30~70min of runing time.
Wherein, in one embodiment, it is eluted using the method for gradient elution, specifically, when gradient elution, is being transported
Within the scope of 0~20 minute of row time, in mobile phase the volume ratio of acid solution be 95~70%, at runtime between 20~35 points
Within the scope of clock, in mobile phase the volume ratio of acid solution be 70~10%, at runtime between 35~35.1 minutes within the scope of, flowing
In phase the volume ratio of acid solution be 10~95%, at runtime between 35.1~50 minutes within the scope of, acid solution in mobile phase
Volume ratio is 95~95%.
Wherein, in one embodiment, it is mixed using water, methanol, ethyl alcohol, acetonitrile, methanol-water mixture, alcohol-water
Sample analysis solution is made as solvent sample dissolution in liquid, acetonitrile-water mixed liquor.Specifically, this method can also be applied
Impurity in detection Abacavir bulk pharmaceutical chemicals, Abacavir intermediate or Abacavir preparation, i.e., in sample analysis solution, sample
Product can be Abacavir bulk pharmaceutical chemicals, intermediate or preparation, when sample difference, utilizes the dissolution sex differernce of sample, selects different
Solvent go to dissolve.Wherein Abacavir preparation can be tablet, capsule, granule, eye-drops preparations, nasal formulations, suppository,
Pill, ointment cream, paste, sucking preparation, spray, aerosol, gelling agent, powder, syrup, liniment, paint, painting
Film, tincture, patch, oral solution, implant, film, lotion, irrigation, soft extract, plaster, distillate medicinal water, liniment.
In the following, being illustrated, being explained to the scheme of the application by several groups of specific experiment examples, but these experimental examples are only
Some exemplary arrangements, should not be taken to limit scope of the present application.Wherein, the experiment side of actual conditions is not specified in experimental example
Method, usually according to conventional conditions or according to the manufacturer's recommendations.Unless otherwise defined, as used herein all special
Industry and scientific words are identical as meaning well-known to those skilled in the art.
Experimental example 1
(1) instrument and chromatographic condition
High performance liquid chromatograph: e2965 highly effective liquid phase chromatographic system and work station.
Chromatographic column: Thermo Syncronis C18The octadecylsilane chemically bonded silica color of (4.6mm × 250mm, 5 μm)
Compose column.
Mobile phase: preparing trifluoroacetic acid-aqueous solution of 0.05% volumetric concentration, and the mixing for preparing water/methanol (15/85) is molten
Liquid carries out trifluoroacetic acid-aqueous solution and water-methanol mixture to be mixed to prepare mobile phase, trifluoroacetic acid solution-in mobile phase
The ratio of (water/methanol=15/85) presses 0~20min, 20~35min, 35~35.1min, 35.1~50min time point, and acid is molten
Liquid volume ratio is 95~70%, 70~10%, 10~95%, 95~95% progress gradient elutions.
Testing conditions: setting flow velocity be 1.0ml/min, Detection wavelength 254nm, 30 DEG C of column temperature.
(2) experimental procedure
Prepare sample analysis solution:
The standard sample for taking the impurity J of predetermined amount, impurity L, compound I and Abacavir respectively, with methanol-water (20:
80) dissolve and dilute to obtain sample analysis solution, wherein in every 1ml sample analysis solution containing about the impurity J of 2.5 μ g, impurity L,
The Abacavir of compound I and 250 μ g.
The test of sample analysis solution:
Precision measures above-mentioned 20 μ l of analytical solution, injects high performance liquid chromatograph, carries out gradient using above-mentioned mobile phase and washes
It is de-, and chromatogram is recorded, specific chromatogram is shown in attached drawing 4.Fig. 4 is the detection chromatogram of the application experimental example 1, is wherein retained in figure
Time (RT)=peak 26.541min is impurity J, and the peak RT=27.921min is compound I, and the peak RT=28.898min is A Baka
Wei, the peak RT=31.103min are impurity L.
Impurity J, impurity L, compound I and Abacavir can be kept completely separate under this condition it can be seen from chromatogram, can
Accurately measure each impurity content.
Experimental example 2
(1) instrument and chromatographic condition
High performance liquid chromatograph: e2965 highly effective liquid phase chromatographic system and work station.
Chromatographic column: the octadecylsilane chemically bonded silica chromatography of Waters symmetry C18 (3.9*150mm, 5um)
Column.
Mobile phase: preparing trifluoroacetic acid-aqueous solution of 0.02% volumetric concentration, and the mixing for preparing water/methanol (15/85) is molten
Liquid carries out trifluoroacetic acid-aqueous solution and water-methanol mixture to be mixed to prepare mobile phase, trifluoroacetic acid solution-in mobile phase
The ratio of (water/methanol=15/85) presses 0~20min, 20~35min, 35~35.1min, 35.1~50min time point, and acid is molten
Liquid volume ratio is 95~70%, 70~10%, 10~95%, 95~95% progress gradient elutions.
Testing conditions: setting flow velocity be 1.0ml/min, Detection wavelength 254nm, 30 DEG C of column temperature.
(2) experimental procedure
Prepare sample analysis solution:
The standard sample for taking the impurity J of predetermined amount, impurity L, compound I and Abacavir respectively, with methanol-water (20:
80) dissolve and dilute to obtain sample analysis solution, wherein in every 1ml sample analysis solution containing about the impurity J of 2.5 μ g, impurity L,
The Abacavir of compound I and 250 μ g.
The test of sample analysis solution:
Precision measures above-mentioned 20 μ l of analytical solution, injects high performance liquid chromatograph, carries out gradient using above-mentioned mobile phase and washes
It is de-, and chromatogram is recorded, specific chromatogram is shown in attached drawing 5.Fig. 5 is the detection chromatogram of the application experimental example 2, is wherein retained in figure
Time (RT)=peak 25.729min is impurity J, and the peak RT=27.036min is compound I, and the peak RT=27.767min is A Baka
Wei, the peak RT=29.739min are impurity L.
Impurity J, impurity L, compound I and Abacavir can be kept completely separate under this condition it can be seen from chromatogram, can
Accurately measure each impurity content.
Experimental example 3
(1) instrument and chromatographic condition
High performance liquid chromatograph: e2965 highly effective liquid phase chromatographic system and work station.
Chromatographic column: the octadecylsilane chemically bonded silica chromatography of Waters symmetry C18 (3.9*150mm, 5um)
Column.
Mobile phase: preparing trifluoroacetic acid-aqueous solution of 0.05% volumetric concentration, and the mixing for preparing water/methanol (15/85) is molten
Liquid carries out trifluoroacetic acid-aqueous solution and water-methanol mixture to be mixed to prepare mobile phase, trifluoroacetic acid solution-in mobile phase
The ratio of (water/methanol=15/85) presses 0~20min, 20~35min, 35~35.1min, 35.1~50min time point, and acid is molten
Liquid volume ratio is 95~70%, 70~10%, 10~95%, 95~95% progress gradient elutions.
Testing conditions: setting flow velocity be 1.0ml/min, Detection wavelength 254nm, 30 DEG C of column temperature.
(2) experimental procedure
Prepare sample analysis solution:
The standard sample for taking the impurity J of predetermined amount, impurity L, compound I and Abacavir respectively, with methanol-water (20:
80) dissolve and dilute to obtain sample analysis solution, wherein in every 1ml sample analysis solution containing about the impurity J of 2.5 μ g, impurity L,
The Abacavir of compound I and 250 μ g.
The test of sample analysis solution:
Precision measures above-mentioned 20 μ l of analytical solution, injects high performance liquid chromatograph, carries out gradient using above-mentioned mobile phase and washes
It is de-, and chromatogram is recorded, specific chromatogram is shown in attached drawing 6.Fig. 6 is the detection chromatogram of the application experimental example 3, is wherein retained in figure
Time (RT)=peak 26.717min is impurity J, and the peak RT=28.013min is compound I, and the peak RT=29.022min is A Baka
Wei, the peak RT=31.033min are impurity L.
Impurity J, impurity L, compound I and Abacavir can be kept completely separate under this condition it can be seen from chromatogram, can
Accurately measure each impurity content.
Experimental example 4
(1) instrument and chromatographic condition
High performance liquid chromatograph: e2965 highly effective liquid phase chromatographic system and work station.
Chromatographic column: the octadecylsilane chemically bonded silica chromatography of Waters symmetry C18 (3.9*150mm, 5um)
Column.
Mobile phase: preparing trifluoroacetic acid-aqueous solution of 0.05% volumetric concentration, and the mixing for preparing water/acetonitrile (15/85) is molten
Liquid carries out trifluoroacetic acid-aqueous solution and water-acetonitrile mixture to be mixed to prepare mobile phase, trifluoroacetic acid solution-in mobile phase
The ratio of (water/acetonitrile=15/85) presses 0~20min, 20~35min, 35~35.1min, 35.1~50min time point, and acid is molten
Liquid volume ratio is 95~70%, 70~10%, 10~95%, 95~95% progress gradient elutions.
Testing conditions: setting flow velocity be 1.0ml/min, Detection wavelength 254nm, 30 DEG C of column temperature.
(2) experimental procedure
Prepare sample analysis solution:
The standard sample for taking the impurity J of predetermined amount, impurity L, compound I and Abacavir respectively, with methanol-water (20:
80) dissolve and dilute to obtain sample analysis solution, wherein in every 1ml sample analysis solution containing about the impurity J of 2.5 μ g, impurity L,
The Abacavir of compound I and 250 μ g.
The test of sample analysis solution:
Precision measures above-mentioned 20 μ l of analytical solution, injects high performance liquid chromatograph, carries out gradient using above-mentioned mobile phase and washes
It is de-, and chromatogram is recorded, specific chromatogram is shown in attached drawing 7.Fig. 7 is the detection chromatogram of the application experimental example 4, is wherein retained in figure
Time (RT)=peak 27.946min is impurity J, and the peak RT=29.372min is compound I, and the peak RT=30.422min is A Baka
Wei, the peak RT=32.645min are impurity L.
Impurity J, impurity L, compound I and Abacavir can be kept completely separate under this condition it can be seen from chromatogram, can
Accurately measure each impurity content.
Experimental example 5
(1) instrument and chromatographic condition
High performance liquid chromatograph: e2965 highly effective liquid phase chromatographic system and work station.
Chromatographic column: the octadecylsilane chemically bonded silica chromatography of Waters symmetry C18 (3.9*150mm, 5um)
Column.
Mobile phase: preparing trifluoroacetic acid-aqueous solution of 0.05% volumetric concentration, and the mixing for preparing water/methanol (15/85) is molten
Liquid carries out trifluoroacetic acid-aqueous solution and water-methanol mixture to be mixed to prepare mobile phase, trifluoroacetic acid solution-in mobile phase
The ratio of (water/methanol=15/85) presses 0~20min, 20~35min, 35~35.1min, 35.1~50min time point, and acid is molten
Liquid volume ratio is 95~70%, 70~10%, 10~95%, 95~95% progress gradient elutions.
Testing conditions: setting flow velocity be 1.0ml/min, Detection wavelength 254nm, 30 DEG C of column temperature.
(2) experimental procedure
Prepare sample analysis solution:
The standard sample for taking the impurity J of predetermined amount, impurity L, compound I and Abacavir respectively, with methanol-water (20:
80) dissolve and dilute to obtain sample analysis solution, wherein in every 1ml sample analysis solution containing about the impurity J of 2.5 μ g, impurity L,
The Abacavir of compound I and 250 μ g.
The test of sample analysis solution:
Precision measures above-mentioned 20 μ l of analytical solution, injects high performance liquid chromatograph, carries out gradient using above-mentioned mobile phase and washes
It is de-, and chromatogram is recorded, specific chromatogram is shown in attached drawing 8.Fig. 8 is the detection chromatogram of the application experimental example 5, is wherein retained in figure
Time (RT)=peak 28.462min is impurity J, and the peak RT=29.965min is compound I, and the peak RT=31.030min is A Baka
Wei, the peak RT=33.422min are impurity L.
Impurity J, impurity L, compound I and Abacavir can be kept completely separate under this condition it can be seen from chromatogram, can
Accurately measure each impurity content.
Above scheme, it was found that the new impurity of one of Abacavir preparation process, and provide the efficient liquid of the impurity
Phase detection method by utilizing reverse-phase chromatographic column, and is eluted using acid solution and the mixed solvent of solvent phase, be can be improved
The detection separating degree and detection limit of impurity.
The foregoing is merely presently filed embodiments, are not intended to limit the scope of the patents of the application, all to utilize this
Equivalent structure or equivalent flow shift made by application specification and accompanying drawing content, it is relevant to be applied directly or indirectly in other
Technical field similarly includes in the scope of patent protection of the application.
Claims (10)
1. a kind of efficient liquid phase detection method of the impurity of Abacavir, which is characterized in that the structural formula of the impurity isWherein, R is alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen, hydroxyalkyl, carboxyl or ester group；
The described method includes:
Sample analysis solution is provided；
Sample analysis solution injection high performance liquid chromatograph is subjected to chromatography, and records chromatogram；Used in it
Chromatographic column is reverse-phase chromatographic column, and mobile phase used is the mixed liquor of acid solution and solvent phase.
2. the efficient liquid phase detection method of the impurity of Abacavir according to claim 1, which is characterized in that the solvent
Xiang Weishui-organic phase mixed liquor；The organic phase is methanol or acetonitrile.
3. the efficient liquid phase detection method of the impurity of Abacavir according to claim 2, which is characterized in that the flowing
The volume ratio of acid solution and solvent phase is 98:2~2:98 in phase；In the solvent phase volume ratio of water and organic phase be 5:95~
25:75；The pH value of the mobile phase is 1.0~9.0.
4. the efficient liquid phase detection method of the impurity of Abacavir according to claim 1, which is characterized in that use gradient
The mode of elution is eluted, wherein when gradient elution, at runtime between 0~20 minute within the scope of, it is sour in the mobile phase
The volume ratio of solution be 95~70%, at runtime between 20~35 minutes within the scope of, the volume of acid solution in the mobile phase
Than being 70~10%, at runtime between 35~35.1 minutes within the scope of, in the mobile phase volume ratio of acid solution be 10~
95%, at runtime between 35.1~50 minutes within the scope of, in the mobile phase volume ratio of acid solution be 95~95%.
5. the efficient liquid phase detection method of the impurity of Abacavir according to claim 1, which is characterized in that the acid is molten
Liquid is aqueous solution, and the volumetric concentration of the acid solution is 0.01%-0.1%, the acid solution be formic acid solution, acetic acid solution or
Trifluoroacetic acid solution.
6. the efficient liquid phase detection method of the impurity of Abacavir according to claim 1, which is characterized in that reverse-phase chromatography
Column is phenyl silane bonded silica gel chromatographic column, cyano bonded silica gel chromatographic column, amino bonded silica gel chromatographic column, octadecylsilane
Bonded silica gel chromatographic column or eight alkyl silane bonded silica gel chromatographic columns.
7. the efficient liquid phase detection method of the impurity of Abacavir according to claim 1, which is characterized in that the reverse phase
The column length of chromatographic column is 100mm~300mm, and internal diameter is 1mm~10mm, and partial size is 1 μm~10 μm.
8. the efficient liquid phase detection method of the impurity of Abacavir according to claim 1, which is characterized in that mobile phase
Flow velocity is 0.8~1.3ml/min；The column temperature of chromatographic column is 25~50 DEG C, and Detection wavelength is 205~300nm；Runing time is 30
~70min.
9. the efficient liquid phase detection method of the impurity of Abacavir according to claim 1, which is characterized in that the offer
Sample analysis solution includes: mixed using water, methanol, ethyl alcohol, acetonitrile, methanol-water mixture, ethanol-water mixture, acetonitrile-water
It closes liquid to dissolve sample, the sample analysis solution is made.
10. the efficient liquid phase detection method of the impurity of Abacavir according to claim 9, which is characterized in that the sample
Product are Abacavir bulk pharmaceutical chemicals, Abacavir intermediate or Abacavir preparation.
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