CN107132297A - A kind of analyzing detecting method of pramiconazole optical isomer - Google Patents
A kind of analyzing detecting method of pramiconazole optical isomer Download PDFInfo
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- CN107132297A CN107132297A CN201710520143.9A CN201710520143A CN107132297A CN 107132297 A CN107132297 A CN 107132297A CN 201710520143 A CN201710520143 A CN 201710520143A CN 107132297 A CN107132297 A CN 107132297A
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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
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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 a kind of analyzing detecting method of pramiconazole optical isomer, using liquid chromatography, the stationary phase of liquid chromatography is octadecylsilane chemically bonded silica, mobile phase is acetonitrile solution, acetonitrile concentration expressed in percentage by volume is 30 50%, 4 tert. butyl cyclohexanols containing valid density in the acetonitrile solution, and 4 tert. butyl cyclohexanols are cis-trans-isomer mixture.Efficiently separating for pramiconazole optical isomer is can be achieved in the method provided using the present invention in common C18 chromatographic columns, and application cost is significantly reduced, and can save a large amount of chromatographic column consumptive material expenses.
Description
Technical field
The invention belongs to Pharmaceutical Analysis field, and in particular to a kind of analyzing detecting method of pramiconazole optical isomer.
Background technology
Pramiconazole (Pramiconazole, SR type), chemical name 1- (4- { 4- [4- ({ (2S, 4R) -4- (2,4- difluorobenzenes
Base) -4- [(1H-1,2,4- triazol-1-yls) methyl] -1,3- dioxolanes -2- bases } methoxyl group) phenyl] piperazine -1- bases } benzene
Base) -3- (propane -2- bases) imidazolidinone -2- alkane is one developed by Belgian BarrierTherapeutics companies
Novel triazole antifungal is planted, was still in for II a clinical trial phase stages in European Union at present, not yet lists, with determined curative effect,
The characteristics of better tolerance, adverse reaction lesser extent, be a kind of more promising broad-spectrum antifungal medicine.
There are 2 chiral carbons in pramiconazole structure, 4 kinds of optical isomers may be produced in building-up process, except mesh
Mark outside compound pramiconazole SR types, also RS types isomers, RR types isomers and SS type isomers.According to research and development alloisomerism
The associated specifications of body new drug, it is necessary to set up a set of perfect analysis method and enter to the optical isomer impurity of pramiconazole
Row quality control and Product Quality Evaluation.Isomers chemical structural formula is as follows：
Zheng Xizi provide a kind of pramiconazole in four kinds of optical isomers HPLC assay methods (Chinese Medicine Leader,
In April, 2016), but this method uses chiral chromatographic column (DaicelChiralpakIC chiral columns).It is well known that hand
Property chromatographic column costly, and service life is short, and the liquid phase analysis method developed using chiral chromatographic column has higher application
Cost.
The content of the invention
Pula is determined based on common C18 chromatographic columns it is an object of the invention to overcome the deficiencies of the prior art and provide one kind
The liquid phase analysis method of four kinds of optical isomers in health azoles, to reduce chromatogram consumptive material expenditure, saves development costs.
The present invention is achieved by following technical scheme：
A kind of analyzing detecting method of pramiconazole optical isomer, using liquid chromatography, the fixation of liquid chromatography
It is mutually octadecylsilane chemically bonded silica, mobile phase is acetonitrile solution, and acetonitrile concentration expressed in percentage by volume is 30-50%, the acetonitrile
4- tert. butyl cyclohexanols containing valid density in the aqueous solution, and 4- tert. butyl cyclohexanols are cis-trans-isomer mixture.
Preferably, in the mobile phase, the valid density of 4- tert. butyl cyclohexanols refers to mass-volume concentration for 1-2g/L.
Preferably, described analyzing detecting method includes following chromatographic parameter：
Chromatographic column：Octadecylsilane chemically bonded silica (C18) chromatographic column；
Mobile phase A phase：30% acetonitrile solution, the mass-volume concentration of 4- tert. butyl cyclohexanols is 1.5g/L；
Mobile phase B phase：50% acetonitrile solution, the mass-volume concentration of 4- tert. butyl cyclohexanols is 1.5g/L；
Elution program：0-3min, 0%B phase；3-5min, 0% → 50%B phase；5-10min, 50%B phase；
Flow velocity：0.8-1.2mL/min；
Column temperature：28-32℃；
Detection wavelength：208-212nm.
Preferably, flow velocity is 1.0mL/min.
Preferably, column temperature is 30 DEG C.
Preferably, Detection wavelength is 210nm.
Preferably, the specification of octadecylsilane chemically bonded silica chromatographic column is long 250mm, internal diameter 4.6mm, the μ of packing material size 5
m。
Preferably, the preferred Thermo of octadecylsilane chemically bonded silica chromatographic column
ScientificTMAcclaimTM120C18。
Advantages of the present invention：
The effective of pramiconazole optical isomer can be achieved in the method provided using the present invention in common C18 chromatographic columns
Separation, application cost is significantly reduced, and can save a large amount of chromatographic column consumptive material expenses.
Brief description of the drawings
Fig. 1 compares for the chemical structural formula of four kinds of optical isomers of pramiconazole；
Fig. 2 is the HPLC chromatogram of four kinds of optical isomer mixed reference substance solutions of pramiconazole；
Fig. 3 is chromatogram of the need testing solution under chromatographic condition of the present invention；
Fig. 4 is chromatogram of the mixed reference substance solution under contrast chromatographic condition.
Embodiment
Technical scheme is further described with reference to specific embodiment.
The separation of the pramiconazole optical isomer of embodiment 1 and measure
First, experiment material
Shimadzu LC-20A high performance liquid chromatographs；
CPA225D electronic balances (Beijing Sai Duolisi instrument systems Co., Ltd)；
Chromatographic column：Thermo ScientificTMAcclaimTM120C18；
Pramiconazole bulk drug is made by oneself, and pramiconazole, RS types isomers, RR types isomers and SS type isomer controls product are certainly
System (purity is more than 98.5%), the chemical structural formula of pramiconazole and its isomers is as shown in Figure 1；
4- tert. butyl cyclohexanols (CAS 98-52-2, cis-trans-isomer mixture, lark prestige science and technology, production code member
107223)；
Acetonitrile is chromatographically pure, and water is deionized water, and remaining reagent is that analysis is pure.
2nd, experimental method and result
1st, solution is prepared
Reference substance and test sample dilution：30% acetonitrile solution, the mass-volume concentration of 4- tert. butyl cyclohexanols is
1.5g/L。
Reference substance solution：Precision weighs each about 10mg of 4 kinds of isomers standard items of pramiconazole respectively, is respectively placed in 100mL amounts
In bottle, add and state dilution and dissolve and be settled to scale, shake up, be used as the reference substance solution that concentration is 100 μ g/mL.
Mixed reference substance solution：4 kinds of isomer control product solution are mixed in equal volume, 4 kinds of isomer concentrations point are configured to
Not Yue Wei 25 μ g/mL mixed reference substance solution.
Need testing solution：Precision weighs pramiconazole sample 100mg, is placed in 100mL measuring bottles, adds and states dilution dissolving
And scale is settled to, shake up, be used as the need testing solution that concentration is 1mg/mL.
2nd, chromatographic condition
Chromatographic column：Thermo ScientificTMAcclaimTM120C18；
Mobile phase A phase：30% acetonitrile solution, the mass-volume concentration of 4- tert. butyl cyclohexanols is 1.5g/L；
Mobile phase B phase：50% acetonitrile solution, the mass-volume concentration of 4- tert. butyl cyclohexanols is 1.5g/L；
Elution program：0-3min, 0%B phase；3-5min, 0% → 50%B phase；5-10min, 50%B phase；
Flow velocity：1.0mL/min；
Column temperature：30℃；
Detection wavelength：210nm.
3rd, separating degree is investigated
Pramiconazole 4 kinds of isomer control product solution, mixed reference substance solutions are taken, sample introduction is determined respectively, is as a result shown, 4
The peak sequence for planting isomers is followed successively by RR types isomers, SR types isomers, RS types isomers, SS isomer forms, pramiconazole
And its optical isomer baseline reaches and is kept completely separate, separating degree is all higher than 1.5, and number of theoretical plate is all higher than 10000, and tailing factor is equal
Less than 1.15, solvent peak not interference measurement.Chromatogram is shown in Fig. 2.
4th, linear test
To weigh 4 kinds of isomer control product appropriate for precision respectively, plus dilution dissolves and shakes up dilution, be configured to about 0.5,5,
10th, 20,50,80,100,120 μ g/mL series concentration solution, sample introduction is determined, and concentration is mapped with peak area, linearly returned
Return, respectively obtain the regression equation of 4 kinds of isomers, linear relationship is good, R2＞ 0.9998.
5th, sensitivity is investigated
It is each appropriate that precision weighs 4 kinds of isomer control product, plus dilution dissolves and is diluted to 4 kinds of isomers peak heights about base
3 times of line noise.As a result the minimum detectability of Isomers is each about 1.2 × 10-3μ g, sensitivity is high.
6th, average recovery is tested
Determination of recovery rates solution is prepared according to a conventional method, the rate of recovery of Isomers is calculated, and as a result proves 4 kinds of pramiconazole
The rate of recovery of isomers (RSD ＜ 1.5%, n=3) between 98-102%, meets the requirements.
7th, precision and stability test
Above-mentioned mixed reference substance solution and need testing solution are taken, respectively continuous sample introduction 6 times, record chromatogram.As a result RSD is equal
Less than 2.0% (n=6), show that this method precision is good.
Above-mentioned mixed reference substance solution and need testing solution are taken respectively, are placed, are entered respectively at 0,4,8,12,24h in room temperature
Sample is determined, and records chromatogram.As a result RSD of the peak area of 4 kinds of isomers of pramiconazole in 24h is respectively less than 2.0% (n=5),
Show that mixed reference substance solution room temperature places 24h stable.
8th, content of isomer is determined in raw material
Take sample appropriate, mixed reference substance solution and need testing solution are prepared as stated above, sample introduction is determined respectively, record
Chromatogram.If any miscellaneous with Isomers retention time identical in mixed reference substance solution chromatogram in need testing solution chromatogram
Mass peak, by external standard method with the content of calculated by peak area Isomers.Need testing solution chromatogram is as shown in Figure 3.
Content of the RR types isomers in three batches of samples is respectively 0.02%, 0.03%, 0.02%；
Content of the RS types isomers in three batches of samples is respectively 0.03%, 0.02%, 0.02%；
Content of the SS types isomers in three batches of samples is respectively 0.03%, 0.02%, 0.03%.
The comparative example of embodiment 2, uses common acetonitrile water elution
First, experiment material
Shimadzu LC-20A high performance liquid chromatographs；
CPA225D electronic balances (Beijing Sai Duolisi instrument systems Co., Ltd)；
Chromatographic column：Thermo ScientificTMAcclaimTM120C18；
Pramiconazole bulk drug is made by oneself, and pramiconazole, RS types isomers, RR types isomers and SS type isomer controls product are certainly
System (purity is more than 98.5%), the chemical structural formula of pramiconazole and its isomers is as shown in Figure 1；
Acetonitrile is chromatographically pure, and water is deionized water, and remaining reagent is that analysis is pure.
2nd, experimental method and result
1st, solution is prepared
Reference substance and test sample dilution：30% acetonitrile solution.
Reference substance solution：Precision weighs each about 10mg of 4 kinds of isomers standard items of pramiconazole respectively, is respectively placed in 100mL amounts
In bottle, add and state dilution and dissolve and be settled to scale, shake up, be used as the reference substance solution that concentration is 100 μ g/mL.
Mixed reference substance solution：4 kinds of isomer control product solution are mixed in equal volume, 4 kinds of isomer concentrations point are configured to
Not Yue Wei 25 μ g/mL mixed reference substance solution.
2nd, chromatographic condition
Chromatographic column：Thermo ScientificTMAcclaimTM120C18；
Mobile phase A phase：30% acetonitrile solution；
Mobile phase B phase：50% acetonitrile solution；
Elution program：0-3min, 0%B phase；3-5min, 0% → 50%B phase；5-10min, 50%B phase；
Flow velocity：1.0mL/min；
Column temperature：30℃；
Detection wavelength：210nm.
3rd, separating degree is investigated
Pramiconazole mixed reference substance solution sample introduction is taken to determine, as a result 4 kinds of isomers can not be realized under the chromatographic condition
Separate two-by-two.Chromatogram of the mixed reference substance solution under the chromatographic condition is shown in Fig. 4.
The effective of pramiconazole optical isomer can be achieved in the method provided using the present invention in common C18 chromatographic columns
Separation, application cost is significantly reduced, and can save a large amount of chromatographic column consumptive material expenses.
Claims (8)
1. a kind of analyzing detecting method of pramiconazole optical isomer, using liquid chromatography, it is characterised in that：Liquid chromatogram
The stationary phase of method is octadecylsilane chemically bonded silica, and mobile phase is acetonitrile solution, and acetonitrile concentration expressed in percentage by volume is 30-
50%, the 4- tert. butyl cyclohexanols containing valid density in the acetonitrile solution, and 4- tert. butyl cyclohexanols are cis-trans-isomer
Mixture.
2. analyzing detecting method according to claim 1, it is characterised in that：In the mobile phase, 4- tert. butyl cyclohexanols
Valid density refer to mass-volume concentration for 1-2g/L.
3. analyzing detecting method according to claim 1 or 2, it is characterised in that including following chromatographic parameter：
Chromatographic column：Octadecylsilane chemically bonded silica (C18) chromatographic column；
Mobile phase A phase：30% acetonitrile solution, the mass-volume concentration of 4- tert. butyl cyclohexanols is 1.5g/L；
Mobile phase B phase：50% acetonitrile solution, the mass-volume concentration of 4- tert. butyl cyclohexanols is 1.5g/L；
Elution program：0-3min, 0%B phase；3-5min, 0% → 50%B phase；5-10min, 50%B phase；
Flow velocity：0.8-1.2mL/min；
Column temperature：28-32℃；
Detection wavelength：208-212nm.
4. analyzing detecting method according to claim 3, it is characterised in that：Flow velocity is 1.0mL/min.
5. analyzing detecting method according to claim 3, it is characterised in that：Column temperature is 30 DEG C.
6. analyzing detecting method according to claim 3, it is characterised in that：Detection wavelength is 210nm.
7. analyzing detecting method according to claim 3, it is characterised in that：Octadecylsilane chemically bonded silica (C18) color
The specification for composing post is long 250mm, internal diameter 4.6mm, 5 μm of packing material size.
8. analyzing detecting method according to claim 7, it is characterised in that：The octadecylsilane chemically bonded silica chromatogram
The preferred Thermo Scientific of postTMAcclaimTM120C18 chromatographic columns.
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Cited By (2)
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| CN110672753A (en) * | 2019-11-04 | 2020-01-10 | 青海省农林科学院 | Method for splitting and detecting fluorochloridone isomer |
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| CN107764922A (en) * | 2017-10-05 | 2018-03-06 | 南京普氟生物检测技术有限公司 | A kind of method for separating and detecting of Doxazosin optical isomer |
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| CN110672753A (en) * | 2019-11-04 | 2020-01-10 | 青海省农林科学院 | Method for splitting and detecting fluorochloridone isomer |
| CN110672753B (en) * | 2019-11-04 | 2022-05-20 | 青海省农林科学院 | Method for splitting and detecting fluorochloridone isomer |
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