CN106918672A - Using the method in HPLC measure Chinese mugwort Fluconazole bulk drug and its preparation about material - Google Patents
Using the method in HPLC measure Chinese mugwort Fluconazole bulk drug and its preparation about material Download PDFInfo
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- CN106918672A CN106918672A CN201511004811.XA CN201511004811A CN106918672A CN 106918672 A CN106918672 A CN 106918672A CN 201511004811 A CN201511004811 A CN 201511004811A CN 106918672 A CN106918672 A CN 106918672A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses the method in a kind of utilization HPLC measure Chinese mugwort Fluconazole bulk drug and its preparation about material, the analysis condition of methods described is：Chromatographic column uses octadecylsilane chemically bonded silica post；Eluted as eluent gradient using the mixed solvent of formate buffer and the first organic solvent.The separation that will can be carried out under same chromatographic condition about material rapidly and efficiently in end Fluconazole bulk drug and its preparation using the method, effectively controls the quality of bulk drug and preparation.The detection method specificity is strong, precision is high, accuracy is strong, easy to operate, can effective control for product quality.
Description
Technical field
The invention belongs to Pharmaceutical Analysis technical field, specifically, HPLC (high performance liquid chromatography is utilized the present invention relates to one kind
Method) determine Chinese mugwort Fluconazole bulk drug and its preparation in about material method.
Background technology
Onychomycosis (onychomycosis) is that a kind of generation refers to being commonly called as the communicable disease in (toe) first in people, is big by one
The microorganism infection that class is referred to as disease fungus causes, and pathogenic bacteria first invade nail matrix under the first of distal end, hyperkeratosis under nail matrix, thickens,
Deck can be changed into white, brown to taupe, and surface irregularity tarnishes, deck missing, crisp.The disease is controlled at present
Therapeutic effect is not good, is largely because existing treatment method and has some limitations.At present, OTC or prescription external application
The curative effect that medicine is provided is extremely limited, frequent debridement is generally required while medication, or scrape, cut or remove nail.Place's square opening
Medication is then applied extremely limited due to drug interaction and serious safety issue.
In the prior art, the optimal therapy for the treatment of onychomycosis is Topical therapy, wherein the easiest especially in the way of local application.
The curative effect of topical antifungal agents is most direct, and wherein paint has splendid penetration to deck, can be with after the outside coating on sick deck
Deck inner side is penetrated into, antibacterial or bactericidal activity can be directly played.
Chinese mugwort Fluconazole (Efinaconazole) is a kind of new antifungal drug in triazole class, is by Canadian DOW drugmakers
Research and development, were approved listing by U.S. FDA on June 6th, 2014, and (containing ending, Fluconazole mass fraction is trade name Jublia
10% externally used solution).Jublia is first external application triazole antifungal agent, can locally apply use, by a kind of unique built-in use
Amount control type nail brush is coated, and the medicine can be dried quickly, without removing overdose of medicine thing, is mainly used in treatment by red hair
Hand, sufficient moss (i.e. onychomycosis) that tinea bacterium, trichophyton gypseum cause, while without the systemic side effect of misgivings, such as medicine is mutual
Effect or acute liver damage, side effect are minimum.
However, the method in present analysis Chinese mugwort Fluconazole bulk drug and its preparation about material still has much room for improvement.
The content of the invention
It is contemplated that at least solving one of technical problem in correlation technique to a certain extent.Therefore, a mesh of the invention
Be the high performance liquid chromatography that a kind of quick Analyze ＆ separate Chinese mugwort Fluconazole catabolite, technique side reaction impurity and intermediate are provided
Method, it is achieved thereby that the separation determination in Chinese mugwort Fluconazole bulk drug and its preparation about material under same chromatographic condition.
The chemical name of Chinese mugwort Fluconazole is (2R, 3R) -2- (2,4 difluorobenzene base) -3- (4- methylenepiperidines -1- bases) -1- (1H-1,2,4- triazoles
- 1- bases) -2- butanol, its structural formula is：
Chinese mugwort Fluconazole synthesis uses route：
Chinese mugwort Fluconazole bulk drug and its relevant material of preparation have three below to originate：One be this product building-up process in bring into raw material,
Intermediate, degradation product；Two is the accessory substance of generation in synthetic reaction；Three drops produced for the influence in storage process due to environment
Solution product, mainly has：
Impurity 1 is raw material：1- (((2R, 3S) -2- (2,4 difluorobenzene base) -3- methyl epoxy ethyl -2- bases) methyl) -1H-1,2,4- triazoles；
Impurity 2 is side reaction product, diastereoisomer impurity：(2S, 3R) -2- (2,4 difluorobenzene base) -3- (4- methylenepiperidines -1-
Base) -1- (1H-1,2,4- triazol-1-yls) -2- butanol；
Impurity 3 is side reaction product：E- (3R) -2- (2,4 difluorobenzene base) -3- (4- methylenepiperidines -1- bases) -1- (1H-1,2,4- triazoles -1-
Base) -1- butylene；
Impurity 4 is side reaction product：(2R, 3R) -2- (2,4 difluorobenzene base) -3- (4- methylenepiperidines -1- bases) -1- (4H-1,2,4- triazoles -4-
Base) -2- butanol；
Impurity 5 is side reaction product：(R, E) -3- (2,4- difluorophenyl) -4- (1H-1,2,4- triazol-1-yls)) -2- hydroxyl -3- butylene；
Impurity 6 is side reaction product：1- ((6- fluoro-2-methylbenzenes and furans -3- bases) methyl) -1H-1,2,4- triazoles；
Impurity 7 is side reaction product, degradation product：(2R, 3R) -2- (2,4 difluorobenzene base) -3 (4- methyl -3,6- dihydropyridines -1 (2H) -
Base) -1- (1H-1,2,4- triazol-1-yls) -2- butanol.
For synthesis, production, storage Chinese mugwort Fluconazole bulk drug and preparation, storage Chinese mugwort fluconazole formulation during all there may be
Above impurity, be both needed to strictly be controlled, to ensure product quality.Therefore, relevant thing in Chinese mugwort Fluconazole and its preparation is realized
The quick separating of matter and analysis all have important practical significance in terms of synthesis and the quality control of production process.
There is not been reported for the relevant substance detecting method of Chinese mugwort Fluconazole, and the difficulty of its detection method is that may produce in its building-up process
The various process byproducts impurity of life, diastereoisomer impurity, degradation product etc., the impurity that cannot be still removed after refining and main ingredient
Chromatographic behavior is more similar, is difficult to be kept completely separate.
In one aspect of the invention, have in determining Chinese mugwort Fluconazole bulk drug and its preparation using HPLC the invention provides one kind
The method for closing material.The chromatographic condition of methods described is：Chromatographic column is octadecylsilane chemically bonded silica post；Mobile phase is formic acid
The mixed solvent of salt buffer and the first organic solvent.
Thus, it is possible to by relevant material in end Fluconazole bulk drug and its preparation carried out under same chromatographic condition rapidly and efficiently point
From can effectively control the quality of bulk drug and preparation.The detection method sensitivity is high, specificity is strong, precision is high, accuracy
By force, it is easy to operate, can effectively control the quality of product.
About the side of material in the utilization HPLC measure Chinese mugwort Fluconazole bulk drug and its preparation of some embodiments of the invention
Method, can also have following additional technical feature：
Some embodiments of the invention, the condition of gradient elution is：At 0-3 minutes, the formate buffer with it is described
The volume ratio of the first organic solvent is (65-55) %：(35-45) %；At 6-25 minutes, the formate buffer with it is described
The volume ratio of the first organic solvent is 45%:55%；At 30-35 minutes, the formate buffer and first organic solvent
Volume ratio be 20%:80%；The formate buffer is (20-65) % with the volume ratio of first organic solvent：(80-35)
%.Thus obtained separating effect is optimal, and peak shape is best.
According to one embodiment of present invention, the condition of the gradient elution is：
| Time/min | Formate buffer/% | First organic solvent/% |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 6 | 45 | 55 |
| 25 | 45 | 55 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Some embodiments of the invention, the particle diameter of the chromatographic column is 3 μm~5 μm, preferably 3 μm.Thus, it is possible to
Further improve separating degree.
Some embodiments of the invention, the formate buffer is Ammonium formate buffer.Thus, it is possible to further improve
Peak shape.
Some embodiments of the invention, the pH value of the formate buffer is adjusted to 5.6-6.0 using formic acid or ammoniacal liquor,
Preferably 5.8.Thus, it is possible to further improve the separating degree between adjacent chromatographic peak.
Embodiments in accordance with the present invention, the concentration of formates is 0.001mol/L~0.10mol/L in the formate buffer, preferably
0.005mol/L~0.05mol/L, most preferably 0.01mol/L.Thus, it is possible to will be to instrument in the case where separating effect is ensured
It is decreased to the infringement of chromatographic column minimum.
Embodiments in accordance with the present invention, first organic solvent is methyl alcohol and/or acetonitrile, preferably acetonitrile.Thus, it is possible to enter
One step improves separative efficiency.
Embodiments in accordance with the present invention, analysis method of the invention is further included：Using the second organic solvent and the mixed liquor of water
Detected sample is configured to sample solution, sample solution is injected into high performance liquid chromatograph, complete Chinese mugwort Fluconazole bulk drug and its
About the measure of material in preparation.
Embodiments in accordance with the present invention, second organic solvent is methyl alcohol and/or acetonitrile, preferably acetonitrile.
Embodiments in accordance with the present invention, the volume ratio of the mixed liquor reclaimed water and the second organic solvent of second organic solvent and water is
(0%:100%)~(60%:40%), preferably 50%:50%.
Some embodiments of the invention, the volume for injecting the sample solution of the high performance liquid chromatograph is 2 μ L~20 μ L,
Preferably 10 μ L.
Some embodiments of the invention, the Detection wavelength of high performance liquid chromatograph is 254nm~262nm, preferably 260nm.
Thus, it is possible to significantly improve detection sensitivity.
Embodiments in accordance with the present invention, the flow velocity of the mobile phase is 0.8mL/min~1.2mL/min, preferably 1.0mL/min.
Thus, it is possible to further improve separating degree.
Some embodiments of the invention, the column temperature of the chromatographic column is 25 DEG C~40 DEG C, preferably 35 DEG C.Thus, it is possible to
Significantly improve the separating degree between adjacent chromatographic peak.
According to a particular embodiment of the invention, determine Chinese mugwort Fluconazole and its preparation in about material method, can be according to following step
It is rapid to realize：
(1) take detected sample appropriate, use acetonitrile：(volume ratio is 50 to water:50) mixed liquor dissolving, be configured to every 1ml containing about
The sample solution of 10mg/ml.
(2) 25 DEG C~40 DEG C, preferably 35 DEG C of chromatographic column column temperature is set.The flow velocity of mobile phase is 0.8~1.2mL/min, preferably
1.0ml/min, Detection wavelength is 254nm~262nm, preferably 260nm.
(3) sample solution sample size 2 μ L~20 μ L, the preferably 10 μ L of (1) are taken, high performance liquid chromatograph is injected, Chinese mugwort fluorine is completed
About the measure of material in health azoles and its preparation.
Relevant each composition of material in Chinese mugwort Fluconazole bulk drug and its preparation can be separated thorough, impurity peaks and master before and after main peak by the present invention
Peak separating degree is good, and workable, analysis method specificity is strong, precision is high, accuracy is strong, easy to operate, can be effective
The quality of control bulk drug and its preparation.
Additional aspect of the invention and advantage will be set forth in part in the description, and partly will become apparent from the description below,
Or recognized by practice of the invention.
Brief description of the drawings
Fig. 1：According to the condition of embodiment 1 gained Chinese mugwort Fluconazole and its seven high-efficient liquid phase chromatograms of impurity；
Fig. 2：According to the condition of embodiment 2 gained Chinese mugwort Fluconazole and its seven high-efficient liquid phase chromatograms of impurity；
Fig. 3：According to the condition of embodiment 3 gained Chinese mugwort Fluconazole and its seven high-efficient liquid phase chromatograms of impurity；
Fig. 4：According to the condition of embodiment 4 gained Chinese mugwort Fluconazole and its seven high-efficient liquid phase chromatograms of impurity；
Fig. 5：According to the high-efficient liquid phase chromatogram of the condition of embodiment 4 gained Chinese mugwort Fluconazole bulk drug；
Fig. 6：According to the condition of embodiment 5 gained Chinese mugwort Fluconazole and its seven high-efficient liquid phase chromatograms of impurity；
Fig. 7：According to the high-efficient liquid phase chromatogram of the condition of embodiment 5 gained blank auxiliary；
Fig. 8：According to the high-efficient liquid phase chromatogram of the condition of embodiment 5 gained Chinese mugwort fluconazole formulation；
Fig. 9：According to the high-efficient liquid phase chromatogram of the condition of comparative example 1 gained Chinese mugwort Fluconazole impurity 3；
Figure 10：According to the condition of comparative example 1 gained Chinese mugwort Fluconazole and its seven high-efficient liquid phase chromatograms of impurity；
Figure 11：According to the high-efficient liquid phase chromatogram of the condition of comparative example 2 gained Chinese mugwort Fluconazole impurity 3；
Figure 12：According to the condition of comparative example 2 gained Chinese mugwort Fluconazole and its seven high-efficient liquid phase chromatograms of impurity.
Specific embodiment
Following examples are provided the present invention and embodiments thereof is further illustrated.What is be given in embodiment is specific interior in detail
Appearance is only in order at the purpose of illustration, of the invention rather than that should be construed as limitation.In embodiment unreceipted particular technique or
Condition, carried out according to the technology or condition described by document in the art or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, be can by city available from conventional products.
Chinese mugwort Fluconazole bulk drug used in the embodiment of the present invention is that applicant is made by oneself by said synthesis route.Chinese mugwort Fluconazole used
Preparation is made by oneself for applicant unit one belongs to.
Embodiment 1
Instrument：The high performance liquid chromatographs of Agilent 1260,1260Infinity PDADs
Chromatographic column：The C18 of CAPCELL PAK MG II (150*4.6mm, 3 μm)
Mobile phase A：0.005mol/L Ammonium formate buffers (adjust pH value to 5.6) with formic acid or ammoniacal liquor
Mobile phase B：Acetonitrile
Gradient elution see the table below：
| Time/min | Formate buffer/% | First organic solvent/% |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 6 | 45 | 55 |
| 25 | 45 | 55 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Flow velocity：1.0mL/min
Detection wavelength：260nm
Column temperature：30℃
Sampling volume：10μL
Diluent：Acetonitrile：Water=50:50(v/v)
Run time：42 minutes
Experimental procedure：
Impurity positioning solution is prepared：Precision weighs impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, miscellaneous respectively
Each about 20mg, 20mg, 10mg, 20mg, 20mg, 20mg, the 20mg of matter 7 is placed in 20mL volumetric flasks, plus diluent
Scale is diluted to, is shaken up, as solution a.Precision weighs Chinese mugwort Fluconazole 100mg, is placed in 10ml volumetric flasks, pipettes 0.2mL
Solution a is in the measuring bottle, plus diluent is diluted to scale, shakes up, and obtains final product.
Impurity positioning solution is taken, efficient liquid phase chromatographic analysis are carried out under above-mentioned chromatographic condition, record chromatogram, as a result see accompanying drawing
1。
In Fig. 1 retention time be 4.250min, 6.380min, 7.922min, 11.384min, 17.446min, 18.170min,
The chromatographic peak of 18.637min, 19.724min is followed successively by impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7, miscellaneous
The chromatographic peak of matter 3 and Chinese mugwort Fluconazole, wherein impurity 2 are 2.09 with the separating degree of impurity 7, the separating degree of impurity 7 and impurity 3
It is 1.25, impurity 3 is 2.45 with the separating degree of Chinese mugwort Fluconazole.
Relevant material can be issued to good separation in same chromatographic condition during result shows Chinese mugwort Fluconazole and its preparation.
Embodiment 2
Instrument：The high performance liquid chromatographs of Agilent 1260,1260Infinity PDADs
Chromatographic column：CAPCELL PAK MGⅡ C18(150*4.6mm,3μm)
Mobile phase A：0.01mol/L Ammonium formate buffers (adjust pH value to 5.8) with formic acid or ammoniacal liquor
Mobile phase B：Acetonitrile
Gradient elution see the table below：
| Time/min | Formate buffer/% | First organic solvent/% |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 6 | 45 | 55 |
| 25 | 45 | 55 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Flow velocity：1.0mL/min
Detection wavelength：260nm
Column temperature：35℃
Sampling volume：10μL
Diluent：Acetonitrile：Water=50:50(v/v)
Run time：42 minutes
Experimental procedure：
Impurity positioning solution is prepared：Precision weighs impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, miscellaneous respectively
Each about 20mg, 20mg, 10mg, 20mg, 20mg, 20mg, the 20mg of matter 7 is placed in 20mL volumetric flasks, plus diluent
Scale is diluted to, is shaken up, as solution a.Precision weighs Chinese mugwort Fluconazole 100mg, is placed in 10ml volumetric flasks, pipettes 0.2mL
Solution a is in the measuring bottle, plus diluent is diluted to scale, shakes up, and obtains final product.
Impurity positioning solution is taken, efficient liquid phase chromatographic analysis are carried out under above-mentioned chromatographic condition, record chromatogram, as a result see accompanying drawing
2。
In Fig. 2 retention time be 4.247min, 6.343min, 7.882min, 11.355min, 17.240min, 17.935min,
The chromatographic peak of 18.451min, 19.412min is respectively impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7, miscellaneous
The chromatographic peak of matter 3, Chinese mugwort Fluconazole, impurity 2 is 2.08 with the separating degree of impurity 7, and impurity 7 is 1.45 with the separating degree of impurity 3,
Impurity 3 is 2.27 with the separating degree of Chinese mugwort Fluconazole.
Relevant material can be issued to good separation in same chromatographic condition during chromatogram shows Chinese mugwort Fluconazole and its preparation.
Embodiment 3
Instrument：The high performance liquid chromatographs of Agilent 1260,1260Infinity PDADs
Chromatographic column：CAPCELL PAK MGⅡ C18(150*4.6mm,3μm)
Mobile phase A：0.05mol/L Ammonium formate buffers (adjust pH value to 6.0) with formic acid or ammoniacal liquor
Mobile phase B：Acetonitrile
Gradient elution see the table below：
| Time/min | Formate buffer/% | First organic solvent/% |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 6 | 45 | 55 |
| 25 | 45 | 55 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Flow velocity：1.0mL/min
Detection wavelength：260nm
Column temperature：40℃
Sampling volume：10μL
Diluent：Methyl alcohol：Water=80:20(v/v)
Run time：42 minutes
Experimental procedure：
Impurity positioning solution is prepared：Precision weighs impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, miscellaneous respectively
Each about 20mg, 20mg, 10mg, 20mg, 20mg, 20mg, the 20mg of matter 7 is placed in 20mL volumetric flasks, plus diluent
Scale is diluted to, is shaken up, as solution a.Precision weighs Chinese mugwort Fluconazole 100mg, is placed in 10ml volumetric flasks, pipettes 0.2mL
Solution a is in the measuring bottle, plus diluent is diluted to scale, shakes up, and obtains final product.
Impurity positioning solution is taken, efficient liquid phase chromatographic analysis are carried out under above-mentioned chromatographic condition, record chromatogram, as a result see accompanying drawing
3。
In Fig. 3 retention time be 4.171min, 6.197min, 7.761min, 11.206min, 16.832min, 17.559min,
The chromatographic peak of 18.144min, 18.835min is respectively impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7, miscellaneous
The chromatographic peak of matter 3, Chinese mugwort Fluconazole, impurity 2 is 2.21 with the separating degree of impurity 7, and impurity 7 is 1.67 with the separating degree of impurity 3,
Impurity 3 is 1.68 with the separating degree of Chinese mugwort Fluconazole.
Relevant material can be issued to good separation in same chromatographic condition during chromatogram shows Chinese mugwort Fluconazole and its preparation.
Embodiment 4
Instrument：The high performance liquid chromatographs of Agilent 1260,1260Infinity PDADs
Chromatographic column：CAPCELL PAK MGⅡ C18(150*4.6mm,3μm)
Mobile phase A：0.01mol/L Ammonium formate buffers (adjust pH value to 5.8) with formic acid or ammoniacal liquor
Mobile phase B：Acetonitrile
Gradient elution see the table below：
| Time/min | Formate buffer/% | First organic solvent/% |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 6 | 45 | 55 |
| 25 | 45 | 55 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Flow velocity：1.0mL/min
Detection wavelength：260nm
Column temperature：35℃
Sampling volume：10μL
Diluent：Acetonitrile：Water=50:50(v/v)
Run time：42 minutes
Experimental procedure：
Impurity positioning solution is prepared：Precision weighs impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, miscellaneous respectively
Each about 20mg, 20mg, 10mg, 20mg, 20mg, 20mg, the 20mg of matter 7 is placed in 20mL volumetric flasks, plus diluent
Scale is diluted to, is shaken up, as solution a.Precision weighs Chinese mugwort Fluconazole 100mg, is placed in 10ml volumetric flasks, pipettes 0.2mL
Solution a is in the measuring bottle, plus diluent is diluted to scale, shakes up, and obtains final product.
Need testing solution is prepared：Precision weighs self-control Chinese mugwort Fluconazole bulk drug about 100mg and is placed in 10mL volumetric flasks, plus dilution
Dilution agent shakes up to scale, obtains final product.
Impurity positioning solution, need testing solution are taken, efficient liquid phase chromatographic analysis are carried out respectively under above-mentioned chromatographic condition, color is recorded
Spectrogram, is as a result shown in accompanying drawing 4~5.
Impurity positioning solution collection of illustrative plates in retention time be 4.247min, 6.343min, 7.882min, 11.355min, 17.240min,
The chromatographic peak of 17.935min, 18.451min, 19.412min be respectively impurity 5, impurity 1, impurity 6, impurity 4, impurity 2,
Impurity 7, impurity 3, the chromatographic peak of Chinese mugwort Fluconazole, impurity 2 is 2.08 with the separating degree of impurity 7, and impurity 7 divides with impurity 3
It is 1.45 from degree, impurity 3 is 2.27 with the separating degree of Chinese mugwort Fluconazole, is specifically shown in Fig. 4.
Retention time is 6.255min in need testing solution collection of illustrative plates, the chromatographic peak of 17.934min, 19.260min be respectively impurity 1,
The chromatographic peak of impurity 7, Chinese mugwort Fluconazole, its area normalization content is respectively 0.01%, 0.02%, 99.97%, and remaining is known miscellaneous
Matter is not detected, is specifically shown in Fig. 5.The relevant material list of Chinese mugwort Fluconazole bulk drug is miscellaneous to be less than 0.1%, always miscellaneous to be less than 0.5%, as a result
Show that Chinese mugwort Fluconazole bulk drug conforms to quality requirements, this method can be used for the quality testing of Chinese mugwort Fluconazole bulk drug.
Chromatogram shows that the relevant material of Chinese mugwort Fluconazole bulk drug can be issued to good separation in same chromatographic condition.
Embodiment 5
Instrument：The high performance liquid chromatographs of Agilent 1260,1260Infinity PDADs
Chromatographic column：CAPCELL PAK MGⅡ C18(150*4.6mm,3μm)
Mobile phase A：0.01mol/L Ammonium formate buffers (adjust pH value to 5.8) with formic acid or ammoniacal liquor
Mobile phase B：Acetonitrile
Gradient elution see the table below：
Flow velocity：1.0mL/min
Detection wavelength：260nm
Column temperature：35℃
Sampling volume：10μL
Diluent：Acetonitrile：Water=50:50(v/v)
Run time：42 minutes
Experimental procedure：
Impurity positioning solution is prepared：Precision weighs impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, miscellaneous respectively
Each about 20mg, 20mg, 10mg, 20mg, 20mg, 20mg, the 20mg of matter 7 is placed in 20mL volumetric flasks, plus diluent
Scale is diluted to, is shaken up, as solution a.Precision weighs Chinese mugwort Fluconazole 100mg, is placed in 10ml volumetric flasks, pipettes 0.2mL
Solution a is in the measuring bottle, plus diluent is diluted to scale, shakes up, and obtains final product.
Blank auxiliary solution：Precision weighs the auxiliary material (absolute ethyl alcohol of Chinese mugwort Fluconazole：Water：Weight ratio=38 of diisopropyl adipate：
2：5) about 0.9g (auxiliary material of the Fluconazole that ended equivalent to 100mg), in 10ml measuring bottles, dissolves and is settled to scale with acetonitrile,
Shake up, filter.
Need testing solution：Precision weighs Chinese mugwort fluconazole formulation (Chinese mugwort Fluconazole bulk drug：Absolute ethyl alcohol：Water：Adipic acid diisopropyl
Weight ratio=5 of ester：38：2：5) in about 1g to 10ml measuring bottles, scale is dissolved and be settled to acetonitrile, shake up, filter.
Impurity positioning solution, blank auxiliary solution, need testing solution are taken, high-efficient liquid phase color is carried out respectively under above-mentioned chromatographic condition
Analysis of spectrum, records chromatogram, as a result sees accompanying drawing 6~8.
Impurity positioning solution collection of illustrative plates in retention time be 4.247min, 6.343min, 7.882min, 11.355min, 17.240min,
The chromatographic peak of 17.935min, 18.451min, 19.412min be respectively impurity 5, impurity 1, impurity 6, impurity 4, impurity 2,
Impurity 7, impurity 3, the chromatographic peak of Chinese mugwort Fluconazole, impurity 2 is 2.08 with the separating degree of impurity 7, and impurity 7 divides with impurity 3
It is 1.45 from degree, impurity 3 is 2.27 with the separating degree of Chinese mugwort Fluconazole, is specifically shown in Fig. 6.
Go out the auxiliary material peak that a retention time is 35.199min in blank auxiliary solution collection of illustrative plates, be specifically shown in Fig. 7.
Retention time is 6.282min in need testing solution, and the chromatographic peak of 17.933min, 19.248min is respectively impurity 1, impurity
7th, the chromatographic peak of Chinese mugwort Fluconazole, its area normalization content is respectively 0.01%, 0.02%, 99.96%, when separately having one to retain
Between for 10.131min unknown impuritie peak, its area normalization content be 0.01%, remaining known impurities is not detected, is specifically shown in
Fig. 8.The relevant material list of Chinese mugwort fluconazole formulation is miscellaneous to be less than 0.1%, always miscellaneous to be less than 0.5%, as a result shows that Chinese mugwort fluconazole formulation meets
Quality requirement, this method can be used for the quality testing of Chinese mugwort fluconazole formulation.
Chromatogram shows that the relevant material of Chinese mugwort fluconazole formulation can be issued to good separation in same chromatographic condition.
Comparative example 1
Instrument：The high performance liquid chromatographs of Agilent 1260,1260Infinity PDADs
Chromatographic column：CAPCELL PAK MGⅡ C18(150*4.6mm,3μm)
Mobile phase A：0.01mol/L Ammonium formate buffers (adjust pH value to 5.8) with formic acid or ammoniacal liquor
Mobile phase B：Acetonitrile
Gradient elution see the table below：
| Time/min | Formate buffer/% | First organic solvent/% |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 8 | 42 | 58 |
| 25 | 42 | 58 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Flow velocity：1.0mL/min
Detection wavelength：260nm
Column temperature：35℃
Sampling volume：10μL
Diluent：Acetonitrile：Water=50:50(v/v)
Run time：42 minutes
Experimental procedure：
Impurity positioning solution is prepared：Precision weighs impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, miscellaneous respectively
Each about 20mg, 20mg, 10mg, 20mg, 20mg, 20mg, the 20mg of matter 7 is placed in 20mL volumetric flasks, plus diluent
Scale is diluted to, is shaken up, as solution a.Precision weighs Chinese mugwort Fluconazole 100mg, is placed in 10ml volumetric flasks, pipettes 0.2mL
Solution a is in the measuring bottle, plus diluent is diluted to scale, shakes up, and obtains final product.
The positioning solution of impurity 3 is prepared：Precision weighs the about 20mg of impurity 3 and is placed in 20mL volumetric flasks, plus diluent is diluted to quarter
Degree, shakes up.Another 1.0mL this solution of taking is in 100mL measuring bottles, plus diluent is diluted to scale, shakes up, and obtains final product.
Impurity positioning solution, the positioning solution of impurity 3 are taken, efficient liquid phase chromatographic analysis are carried out respectively under above-mentioned chromatographic condition, remembered
Record chromatogram, is as a result shown in accompanying drawing 9 and 10.
Retention time is that the chromatographic peak of 16.526min is impurity 3 in the positioning solution collection of illustrative plates of impurity 3, is specifically shown in Fig. 9.
Impurity positioning solution collection of illustrative plates in retention time be 4.002min, 6.078min, 8.048min, 11.045min, 15.084min,
The chromatographic peak of 15.997min, 16.520min is respectively impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7, Chinese mugwort
(chromatographic peak of impure 3), impurity 2 is 3.85 with the separating degree of impurity 7 to Fluconazole, and impurity 7 is separated with Chinese mugwort Fluconazole
It is 1.82 to spend, and impurity 3 is not separated with Chinese mugwort Fluconazole, is specifically shown in Figure 10.
Chromatogram show on the basis of embodiment 2 change gradient elution be 8min-25min when, keep formate buffer with
The ratio of acetonitrile is 42%:58% is constant, and relevant material can not be issued to well in same chromatographic condition in Chinese mugwort Fluconazole and its preparation
Separation.
Comparative example 2
Instrument：The high performance liquid chromatographs of Agilent 1260,1260Infinity PDADs
Chromatographic column：CAPCELL PAK MGⅡ C18(150*4.6mm,3μm)
Mobile phase A：0.01mol/L ammonium acetate buffers (adjust pH value to 5.8) with acetic acid or ammoniacal liquor
Mobile phase B：Acetonitrile
Gradient elution see the table below：
Flow velocity：1.0mL/min
Detection wavelength：260nm
Column temperature：35℃
Sampling volume：10μL
Diluent：Acetonitrile：Water=50:50(v/v)
Run time：42 minutes
Experimental procedure：
Impurity positioning solution is prepared：Precision weighs impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, miscellaneous respectively
Matter 7 about 20mg, 20mg, 10mg, 20mg, 20mg, 20mg, 20mg are placed in 20mL volumetric flasks, plus diluent is dilute
Release to scale, shake up, as solution a.Precision weighs Chinese mugwort Fluconazole 100mg, is placed in 10ml volumetric flasks, pipettes 0.2mL
Solution a is in the measuring bottle, plus diluent is diluted to scale, shakes up, and obtains final product.
The positioning solution of impurity 3 is prepared：Precision weighs the about 20mg of impurity 3 and is placed in 20mL volumetric flasks, plus diluent is diluted to quarter
Degree, shakes up.Another 1.0mL this solution of taking is in 100mL measuring bottles, plus diluent is diluted to scale, shakes up, and obtains final product.
Impurity positioning solution, the positioning solution of impurity 3 are taken, efficient liquid phase chromatographic analysis are carried out respectively under above-mentioned chromatographic condition, remembered
Record chromatogram, is as a result shown in accompanying drawing 11 and 12.
Retention time is that the chromatographic peak of 19.474min is impurity 3 in the positioning solution collection of illustrative plates of impurity 3, is specifically shown in Figure 11.
Impurity positioning solution collection of illustrative plates in retention time be 4.206min, 6.282min, 7.842min, 11.362min, 17.408min,
The chromatographic peak of 18.739min, 19.479min is respectively impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7, Chinese mugwort
(chromatographic peak of impure 3), impurity 2 is 3.92 with the separating degree of impurity 7 to Fluconazole, and impurity 7 is separated with Chinese mugwort Fluconazole
It is 1.71 to spend, and impurity 3 is not separated with Chinese mugwort Fluconazole, is specifically shown in Figure 12.
Chromatogram shows when it is ammonium acetate to change buffer salt species on the basis of embodiment 2, relevant in Chinese mugwort Fluconazole and its preparation
Material can not be issued to good separation in same chromatographic condition.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specific example " or
The description of " some examples " etc. means that the specific features, structure, material or the feature that are described with reference to the embodiment or example are contained in
In at least one embodiment of the invention or example.In this manual, the schematic representation of above-mentioned term is necessarily directed to
It is identical embodiment or example.And, the specific features of description, structure, material or feature can be at any one or more
Combined in an appropriate manner in embodiment or example.Additionally, in the case of not conflicting, those skilled in the art can be with
The feature of the different embodiments or example described in this specification and different embodiments or example is combined and combined.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment be it is exemplary,
It is not considered as limiting the invention, one of ordinary skill in the art can enter to above-described embodiment within the scope of the invention
Row change, modification, replacement and modification.
Claims (10)
1. a kind of utilization HPLC determines the method about material in Chinese mugwort Fluconazole bulk drug and its preparation, it is characterised in that chromatographic condition
For：
Chromatographic column is octadecylsilane chemically bonded silica post；
Mobile phase is the mixed solvent of formate buffer and the first organic solvent.
2. method according to claim 1, it is characterised in that the condition of gradient elution is：At 0-3 minutes, the formates
Buffer solution is (65-55) % with the volume ratio of first organic solvent：(35-45) %；At 6-25 minutes, the formates
Buffer solution is 45% with the volume ratio of first organic solvent:55%；At 30-35 minutes, the formate buffer with it is described
The volume ratio of the first organic solvent is 20%:80%；At 36-42 minutes, the formate buffer and first organic solvent
Volume ratio be (20-65) %：(80-35) %；
Optionally, the condition of the gradient elution is：
3. method according to claim 1, it is characterised in that the particle diameter of the chromatographic column is 3 μm~5 μm, preferably 3 μm.
4. method according to claim 1, it is characterised in that the formate buffer is Ammonium formate buffer.
5. method according to claim 4, it is characterised in that the pH value of the formate buffer is adjusted using formic acid or ammoniacal liquor
To 5.6-6.0, preferably 5.8.
6. method according to claim 1, it is characterised in that the concentration of the formate buffer is
0.001mol/L~0.10mol/L, preferably 0.005mol/L~0.05mol/L, most preferably 0.01mol/L.
7. method according to claim 1, it is characterised in that first organic solvent is methyl alcohol and/or acetonitrile, preferably second
Nitrile.
8. method according to claim 1, it is characterised in that further include：
Detected sample is configured to by sample solution using the mixed liquor of the second organic solvent and water；
Sample solution is injected into high performance liquid chromatograph, about the measure of material in completion Chinese mugwort Fluconazole bulk drug and its preparation.
9. method according to claim 8, it is characterised in that second organic solvent is methyl alcohol and/or acetonitrile, preferably second
Nitrile；
Optional, the volume ratio of the mixed liquor reclaimed water and the second organic solvent of second organic solvent and water is (0%：100%)~
(60%:40%), preferably 50%:50%；
Optionally, the volume for injecting the sample solution of the high performance liquid chromatograph is 2 μ L~20 μ L, preferably 10 μ L；
Optionally, the Detection wavelength of the high performance liquid chromatograph is 254nm~262nm, preferably 260nm.
10. method according to claim 1, it is characterised in that the flow velocity of the mobile phase is 0.8mL/min~1.2mL/min,
Preferably 1.0mL/min；
Optionally, the column temperature of the chromatographic column is 25 DEG C~40 DEG C, preferably 35 DEG C.
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| CN115327000A (en) * | 2022-10-12 | 2022-11-11 | 深圳市海滨制药有限公司 | Method for detecting 4-methylenepiperidine in efinaconazole |
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