CN107014909A - The separation of posaconazole intermediate Z and its genotoxicity impurity and assay method - Google Patents
The separation of posaconazole intermediate Z and its genotoxicity impurity and assay method Download PDFInfo
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- CN107014909A CN107014909A CN201610056478.5A CN201610056478A CN107014909A CN 107014909 A CN107014909 A CN 107014909A CN 201610056478 A CN201610056478 A CN 201610056478A CN 107014909 A CN107014909 A CN 107014909A
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- 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 belongs to analytical chemistry field, and in particular to a kind of posaconazole intermediate Z and its separation and the assay method of related gene toxic impurities.Described method uses octadecylsilane chemically bonded silica for stationary phase, is separated using solvent orange 2 A with solvent B mixture as mobile phase, and this method can realize efficiently separating for intermediate Z and related gene toxic impurities.Described method of separating and assaying is to use high performance liquid chromatography, intermediate Z and related gene toxic impurities reference substance is taken to be prepared into reference substance solution and need testing solution respectively, carry out efficient liquid phase chromatographic analysis, record chromatogram, the content of contained posaconazole intermediate Z and related gene toxic impurities in test sample is calculated according to area normalization method, realize efficiently separating and detecting for intermediate Z and related gene toxic impurities, it is simple to operate, specificity is strong, ensures that the quality safety of posaconazole raw material and its preparation is controllable.
Description
Technical field
The invention belongs to analytical chemistry field, and in particular to a kind of posaconazole intermediate Z and its correlation
The separation of genotoxicity impurity and assay method.
Background technology
Posaconazole is a kind of wide spectrum triazole antifungal agent, available for the aggressive Aspergillus of prevention and thought
Pearl bacterium infect, while available for treatment oropharynx candidiasis, including Itraconazole and/or Fluconazole it is refractory
Property oropharynx candidiasis.Posaconazole intermediate Z (posaconazole crude product) chemistry is entitled
4-[4-[4-[4-[[(3R,5R)-5-(2,4-difluorophenyl)tetrahydro-5-(1H-1,2,4-triazol-1-ylm
ethyl)-3-furanyl]methoxy]phenyl]-1-piperazinyl]phenyl]-2-[(1S,2S)-1-ethyl-2-h
Ydroxypropyl] -2,4-dihydro-3H-1,2,4-triazol-3-one, molecular formula C37H42F2N8O4, point
Son amount 700.78, its structural formula is as follows：
Genotoxicity impurity refers to compound directly or indirectly damaging cells DNA in itself, causes biology
Body produces gene mutation or occurs mutagenesis in vivo, with carcinogenicity or undertone.With gene
The group of toxic action typically has the property of electrophilic reagent, and these groups are in physiological conditions with vivo
Substitution reaction occurs for the nucleophilic center in nucleic acid, protein or other important components, makes these into distribution
Raw irreversible damage, shows as toxicity, mutagenesis or carcinogenic etc. acts on.Show as toxicity, cause to dash forward
Become or the group of the effect such as carcinogenic is exemplified below：
In process of production, it is possible to create the link of genotoxicity impurity is pure including new drug synthesis, raw material
Change, storage transports (being contacted with packing material) etc., therefore should be noted that what is be related in new drug synthesis phase
All chemical substances that there is genotoxicity or have carcinogenicity, such as agents useful for same, intermediate, byproduct
Deng.Further, there is no the genotoxicity reactant occurred in pharmaceutically active substance and have gene poison
The material of property structure, should all be considered.In the building-up process of posaconazole, in its intermediate Z
It there may be the genotoxicity impurity brought into by key starting material：Methyl tosylate and to toluene
Sulfonic acid.Genotoxicity impurity in posaconazole intermediate Z derives from key starting material SM1,
Synthesize SM1During the paratoluensulfonyl chloride used can hydrolyze generation methyl tosylate and to toluene
Sulfonic acid, SM1Synthetic route is as follows：
According to EMA《Genotoxicity limit of impurities guide》Provide within (2006), tackle genotoxicity
Content of the impurity in posaconazole is strictly controlled, by the maximum day metering of posaconazole and most long use
The limit of such impurity of medicine Time Calculation should control to be no more than 0.015%.Therefore, come for posaconazole
Say, the content of genotoxicity impurity in its key intermediate Z should be controlled, so as to effectively ensure that pool is husky
The quality safety of health azoles raw material and preparation is controllable.
The content of the invention
In view of this, an object of the present invention be to provide a kind of separation posaconazole intermediate Z and
The method of its related gene toxic impurities, this method can realize posaconazole intermediate Z and dependency basis
Because of efficiently separating for toxic impurities, so that genotoxicity impurity contains in effectively control key intermediate Z
Amount；The second object of the present invention is to provide in a kind of high efficiency liquid chromatography for separating and determining posaconazole
Mesosome Z and related gene toxic impurities method, using high performance liquid chromatography, can efficiently, essence
True separation and the measure of realizing intermediate Z and related gene toxic impurities, to posaconazole raw material and
The quality safety of its preparation is controllable significant.
To achieve the above object, the technical scheme is that：
The method for separating posaconazole intermediate Z and related gene toxic impurities, described method uses ten
Eight alkyl silane bonded silica gels are stationary phase, are divided using solvent orange 2 A and solvent B mixture as mobile phase
From the solvent orange 2 A is water, and the solvent B is the one or more in methanol, acetonitrile, isopropanol.
It is miscellaneous that method of the present invention is applied to above-mentioned posaconazole intermediate Z and related gene toxicity
The separation of matter, available for a certain material is separately separated, can also separate simultaneously posaconazole intermediate Z and
Related gene toxic impurities.
Separation posaconazole intermediate Z of the present invention and related gene toxic impurities method, preferably
, methods described is particularly suitable for genotoxicity impurity for methyl tosylate and/or p-methyl benzenesulfonic acid second
The separation of ester.
As preferred scheme, the solvent B is methanol or acetonitrile.
Present invention also offers a kind of high efficiency liquid chromatography for separating and determining posaconazole intermediate Z and phase
The method of correlation gene toxic impurities, is comprised the following steps that：Take test sample plus diluent to dissolve and be prepared into confession
Test sample solution, takes need testing solution sample introduction, and mobile phase elution carries out efficient liquid phase chromatographic analysis, note
Chromatogram is recorded, contained posaconazole intermediate Z and dependency basis in test sample are calculated according to area normalization method
Because of the content of toxic impurities；The efficient liquid phase chromatographic analysis use octadecylsilane chemically bonded silica for
The chromatographic column of filler, the mobile phase is the mixture of solvent orange 2 A and solvent B, and the solvent orange 2 A is water,
The solvent B is the one or more in methanol, acetonitrile, isopropanol.
As preferred scheme, in the middle of a kind of described high efficiency liquid chromatography for separating and determining posaconazole
Body Z and related gene toxic impurities method, the solvent B are methanol or acetonitrile.
High efficiency liquid chromatography for separating and determining posaconazole intermediate Z and related gene of the present invention
The method of toxic impurities is applied to the separation and survey of posaconazole intermediate Z and related gene toxic impurities
It is fixed, available for a certain material of independent detection, also can separate and detect simultaneously posaconazole intermediate Z and
Related gene toxic impurities.
As preferred scheme, in the middle of a kind of described high efficiency liquid chromatography for separating and determining posaconazole
Body Z and related gene toxic impurities method, also comprise the following steps：Posaconazole intermediate is taken respectively
Z and related gene toxic impurities reference substance, dissolving are prepared into reference substance solution, take respectively in posaconazole
Mesosome Z and related gene toxic impurities reference substance solution sample introduction, mobile phase elution, carry out high-efficient liquid phase color
Analysis of spectrum, records chromatogram, when determining the reservation of posaconazole intermediate Z and related gene toxic impurities
Between.
High efficiency liquid chromatography for separating and determining posaconazole intermediate Z and related gene of the present invention
The method of toxic impurities, it is preferred that it is to toluene that methods described, which is particularly suitable for related gene toxic impurities,
The separation of methylmesylate and/or ethyl p-toluenesulfonate.
As preferred scheme, in the middle of a kind of described high efficiency liquid chromatography for separating and determining posaconazole
Body Z and related gene toxic impurities method, the diluent are the mixture of solvent orange 2 A and solvent B,
The solvent orange 2 A is water, and the solvent B is the one or more in methanol, acetonitrile, isopropanol；It is preferred that
, solvent orange 2 A and solvent B volume ratio are 25-75：75-25.
As preferred scheme, in the middle of a kind of described high efficiency liquid chromatography for separating and determining posaconazole
Body Z and related gene toxic impurities method, the mobile phase elution use gradient elution, the gradient
Elution requirement is：During 0.01min, solvent orange 2 A and solvent B volume ratio are 58：42；It is molten during 20min
Agent A and solvent B volume ratio is 58：42；During 35min, solvent orange 2 A and solvent B volume ratio are 5：
95；During 40min, solvent orange 2 A and solvent B volume ratio are 5：95；During 40.01min, solvent orange 2 A with it is molten
Agent B volume ratio is 58：42；During 48min, solvent orange 2 A and solvent B volume ratio are 58：42.
As preferred scheme, the flow rate of mobile phase is 0.5-2.0ml/min.
As preferred scheme, in the middle of a kind of described high efficiency liquid chromatography for separating and determining posaconazole
Body Z and related gene toxic impurities method, the chromatographic column column temperature are 25-40 DEG C, preferably described color
The specification for composing post is 4.6 × 250mm, 5 μm.
As preferred scheme, in the middle of a kind of described high efficiency liquid chromatography for separating and determining posaconazole
Body Z and related gene toxic impurities method, the efficient liquid phase chromatographic analysis are entered using UV-detector
Row detection, Detection wavelength is 205-280nm.
As preferred scheme, in the middle of a kind of described high efficiency liquid chromatography for separating and determining posaconazole
In body Z and related gene toxic impurities method, the need testing solution, posaconazole intermediate Z's
Concentration is preferably in the range of 0.5mg/ml-3.0mg/ml.
The high efficiency liquid chromatography for separating and determining posaconazole intermediate Z and related gene toxicity of the present invention
During the method for impurity, sample detection, solvent peak can not disturb posaconazole intermediate Z and its gene
Between the measure of toxic impurities, and genotoxicity impurity peaks and posaconazole intermediate Z main peaks, genotoxicity
Separating degree between impurity peaks is required to be more than 1.5.
In one particular embodiment of the present invention, one kind high effective liquid chromatography for measuring is also disclosed
Posaconazole intermediate Z and its genotoxicity impurity method, specifically include following steps：
(1) blank solution is prepared：Take solvent orange 2 A and solvent B, using solvent orange 2 A and solvent B volume ratios as
40-60% is prepared, and obtains blank solution；
(2) need testing solution is prepared：Posaconazole intermediate Z samples, plus diluent is taken to dissolve and dilute
Release, obtain need testing solution；
(3) genotoxicity dirt solution is prepared：Take each posaconazole intermediate Z genotoxicity impurity pair
According to product, dissolved with organic solvent, then add dilution dilution agent, obtain genotoxicity dirt solution；It is described to have
Machine solvent is the one or more in methanol, acetonitrile, isopropanol.
(4) blank solution, the need testing solution of step (2) and step (3) of step (1) are taken respectively
Genotoxicity dirt solution sample introduction, carry out efficient liquid phase chromatographic analysis, record chromatogram, it is determined that pool
Saperconazole intermediate Z and its genotoxicity impurity retention time, calculate pool husky according to area normalization method
The content of each related gene toxic impurities in health azoles intermediate Z.
The beneficial effects of the present invention are：(1) a kind of separation posaconazole intermediate Z of the invention and
The method of related gene toxic impurities, this method can realize posaconazole intermediate Z and related gene
Efficiently separating for toxic impurities, realizes impurity and effectively controls, product quality is fundamentally determined,
With simplicity, quickly, the advantages of degree of accuracy is high；(2) a kind of high performance liquid chromatography of the invention point
From the method for determining posaconazole intermediate Z and related gene toxic impurities, using common C18Chromatogram
Post, can efficiently separate and detect posaconazole intermediate Z and its genotoxicity impurity, operation letter
Single, the degree of accuracy is high；Solvent peak does not disturb posaconazole intermediate Z and its genotoxicity in detection process
The measure of impurity, between genotoxicity impurity peaks and posaconazole intermediate Z main peaks, genotoxicity impurity
Separating degree between peak is all higher than 1.5；(3) method simple possible of the present invention, specificity is strong,
Sensitivity is high, solves the problems, such as posaconazole intermediate Z and its genotoxicity impurity separation determination,
Ensure that the quality safety of posaconazole raw material and its preparation is controllable, compared with prior art, this
The method of invention is faster, improves efficiency, with significant progressive.
Brief description of the drawings
The high-efficient liquid phase chromatogram of Fig. 1 blank solvents.
Fig. 2 posaconazole intermediates Z high-efficient liquid phase chromatogram.
The high-efficient liquid phase chromatogram of Fig. 3 methyl tosylates.
The high-efficient liquid phase chromatogram of Fig. 4 ethyl p-toluenesulfonates.
Fig. 5 posaconazole intermediates Z and its genotoxicity impurity mixed solution high-efficient liquid phase chromatogram.
The posaconazole intermediate Z of Fig. 6 embodiments 3 and its genotoxicity impurity mixed solution efficient liquid phase
Chromatogram.
The posaconazole intermediate Z of Fig. 7 embodiments 4 and its genotoxicity impurity mixed solution efficient liquid phase
Chromatogram.
Genotoxicity defects inspecting limits the high performance liquid chromatography of solution in Fig. 8 posaconazole intermediates Z
Figure.
Genotoxicity impurity adds the high-efficient liquid phase chromatogram of solution in Fig. 9 posaconazole intermediates Z.
The batch posaconazole intermediate Z of Figure 10 150101 high-efficient liquid phase chromatogram.
The batch posaconazole intermediate Z of Figure 11 150102 high-efficient liquid phase chromatogram.
The batch posaconazole intermediate Z of Figure 12 150103 high-efficient liquid phase chromatogram.
The batch posaconazole intermediate Z of Figure 13 150501 high-efficient liquid phase chromatogram.
The batch posaconazole intermediate Z of Figure 14 150502 high-efficient liquid phase chromatogram.
The batch posaconazole intermediate Z of Figure 15 150601 high-efficient liquid phase chromatogram.
The batch posaconazole intermediate Z of Figure 16 150602 high-efficient liquid phase chromatogram.
Embodiment
The regulations that are preferable to carry out of the present invention are described in detail hereinafter with reference to accompanying drawing.It is preferable to carry out bar
The test method of unreceipted actual conditions in example, with reference to normal condition, illustrated embodiment is in order to more preferable
Ground is illustrated to present disclosure, but is not that present disclosure is only limitted to illustrated embodiment.
So those skilled in the art carry out nonessential change according to foregoing invention content to embodiment
Enter and adjust, still fall within protection scope of the present invention.
In following examples, in method of the invention, the instrument and chromatographic condition of use are as follows：
High performance liquid chromatograph：Shimadzu：LC-20ATvp, SPD-M20Avp；
Chromatographic condition is with following preferably example：
Flow velocity：1.0ml/min；
Detection wavelength：210nm；
Column temperature：30℃；
Sampling volume：20μl.
Genotoxicity impurity in the high efficiency liquid chromatography for separating and determining posaconazole intermediate Z of embodiment 1
Method
Chromatographic column：C18(VP-ODS or Agilent ZORBAX SB, 250mm × 4.6mm, 5 μm)；
Mobile phase：Solvent orange 2 A：Water；Solvent B：Acetonitrile or methanol, are eluted by with Gradient：
| Time (min) | 0.01 | 20 | 35 | 40 | 40.01 | 48 |
| A (%) | 58 | 58 | 5 | 5 | 58 | 58 |
| B (%) | 42 | 42 | 95 | 95 | 42 | 42 |
Diluent：(volume ratio of water and acetonitrile is 40 to acetonitrile solution：60).
Test procedure：
(1) blank solution is prepared：Fetch water 40ml, puts in 100ml measuring bottles, plus dilution in acetonitrile is to scale,
Shake up, produce；
(2) need testing solution is prepared：Posaconazole intermediate Z about 50mg are taken, it is accurately weighed, put
In 25ml measuring bottles, plus diluent dissolves and is diluted to scale, shakes up, is used as need testing solution；
(3) genotoxicity dirt solution is prepared：
Methyl tosylate solution (1.0mg/ml)：Methyl tosylate about 25mg is taken, precision claims
It is fixed, put in 25ml measuring bottles, plus acetonitrile dissolves and is diluted to scale with diluent, shakes up, produces；
Ethyl p-toluenesulfonate solution (0.5mg/ml)：Ethyl p-toluenesulfonate about 12.5mg is taken, it is accurate
It is weighed, put in 25ml measuring bottles, plus acetonitrile dissolves and is diluted to scale with diluent, shakes up, produces；
(4) blank solution, the need testing solution of step (2) and step (3) of step (1) are taken respectively
Methyl tosylate solution and ethyl p-toluenesulfonate solution sample introduction, carry out high performance liquid chromatography point
Analyse and record chromatogram, determine posaconazole intermediate Z and methyl tosylate and p-methyl benzenesulfonic acid
The retention time of ethyl ester, calculates genotoxicity in posaconazole intermediate Z miscellaneous according to area normalization method
The content of confrontation methyl tosylate and ethyl p-toluenesulfonate.
The chromatographic system of the present invention of embodiment 2 is to posaconazole intermediate Z, methyl tosylate and right
The separating degree experiment 1 of toluenesulfonic acid ethyl ester
Chromatographic column：C18(VP-ODS, 250mm × 4.6mm, 5 μm)；
Mobile phase：Solvent orange 2 A：Water；Solvent B：Acetonitrile, is eluted by with Gradient：
| Time (min) | 0.01 | 20 | 35 | 40 | 40.01 | 48 |
| A (%) | 58 | 58 | 5 | 5 | 58 | 58 |
| B (%) | 42 | 42 | 95 | 95 | 42 | 42 |
Diluent：(volume ratio of water and acetonitrile is 40 to acetonitrile solution：60).
Test procedure：
(1) blank solution is prepared：Fetch water 40ml, puts in 100ml measuring bottles, plus dilution in acetonitrile is to scale,
Shake up, produce；
(2) need testing solution is prepared：Precision weighs intermediate Z 50.55mg, puts in 25ml measuring bottles,
Plus diluent makes dissolving and is diluted to scale, shakes up, produces, being computed need testing solution concentration is
2.022mg/ml。
(3) genotoxicity impurity positioning solution is prepared：
Methyl tosylate stock solution：Precision weighs methyl tosylate 23.79mg, puts 25ml
In measuring bottle, plus acetonitrile makes dissolving and is diluted to scale with diluent, shakes up, produces, is computed concentration
For 951.6 μ g/ml.
Ethyl p-toluenesulfonate stock solution：Precision weighs ethyl p-toluenesulfonate 12.88mg, puts 25ml
In measuring bottle, plus acetonitrile makes dissolving and is diluted to scale with diluent, shakes up, produces, is computed concentration
For 515.2 μ g/ml.
Methyl tosylate positions solution：Precision pipettes methyl tosylate stock solution 0.5ml, puts
In 25ml measuring bottles, plus diluent is diluted to scale, shakes up, and produces, and is computed concentration for 19.032 μ g/ml.
Ethyl p-toluenesulfonate positions solution：Precision pipettes ethyl p-toluenesulfonate stock solution 1.0ml, puts
In 25ml measuring bottles, plus diluent dissolves and is diluted to scale, shakes up, produces, and being computed concentration is
20.608μg/ml。
(4) mixed solution is prepared：Precision weighs intermediate Z 51.26mg, puts in 25ml measuring bottles, essence
Close methyl tosylate stock solution 0.5ml, the ethyl p-toluenesulfonate stock solution 1.0ml of pipetting is to the amount
In bottle, plus diluent makes dissolving and is diluted to scale, shakes up, produces；
(5) take respectively above-mentioned blank solution, need testing solution, methyl tosylate positioning solution,
Ethyl p-toluenesulfonate positioning solution sample introduction, mixed solution sample introduction, record chromatogram, as a result such as Fig. 1-5
Shown, separating degree test data is shown in Table 1.
The separating degree test data of table 1
Illustrate that the present invention can be effectively by posaconazole intermediate Z by Fig. 1-Fig. 5 and the data of table 1
Separated with its genotoxicity impurity (methyl tosylate and ethyl p-toluenesulfonate), separating degree is equal
More than 1.5, specificity is strong.
The chromatographic system of the present invention of embodiment 3 is to posaconazole intermediate Z, methyl tosylate and right
The separating degree experiment 2 of toluenesulfonic acid ethyl ester
Chromatographic column：C18(VP-ODS, 250mm × 4.6mm, 5 μm)；
Mobile phase：Solvent orange 2 A：Water；Solvent B：Methanol, is eluted by with Gradient：
| Time (min) | 0.01 | 20 | 35 | 40 | 40.01 | 48 |
| A (%) | 58 | 58 | 5 | 5 | 58 | 58 |
| B (%) | 42 | 42 | 95 | 95 | 42 | 42 |
Diluent：(volume ratio of water and acetonitrile is 40 to acetonitrile solution：60).
Test procedure：
(1) blank solution is prepared：Fetch water 40ml, puts in 100ml measuring bottles, plus dilution in acetonitrile is to scale,
Shake up, produce；
(2) need testing solution is prepared：Posaconazole intermediate Z 50.20mg are taken, it is accurately weighed, put
In 25ml measuring bottles, plus diluent dissolves and is diluted to scale, shakes up, is used as need testing solution；Through meter
It is 2.008mg/ml to calculate need testing solution concentration.
(3) genotoxicity dirt solution is prepared：
Methyl tosylate solution：Methyl tosylate 26.56mg is taken, it is accurately weighed, put 25ml
In measuring bottle, plus acetonitrile dissolves and is diluted to scale with diluent, shakes up, produces；It is computed test sample
Solution concentration is 1062.4 μ g/ml.
Ethyl p-toluenesulfonate solution：Ethyl p-toluenesulfonate 12.51mg is taken, it is accurately weighed, put 25ml
In measuring bottle, plus acetonitrile dissolves and is diluted to scale with diluent, shakes up, produces；It is computed test sample
Solution concentration is 500.4 μ g/ml.
(4) mixed solution is prepared：Precision weighs intermediate Z 50.26mg, puts in 25ml measuring bottles, essence
Close methyl tosylate stock solution 0.5ml, the ethyl p-toluenesulfonate stock solution 1.0ml of pipetting is to the amount
In bottle, plus diluent makes dissolving and is diluted to scale, shakes up, produces；
(5) mixed solution sample introduction is taken, chromatogram is recorded, as a result as shown in Figure 6.
It will be appreciated from fig. 6 that retention time is 20.669min, 28.723min, 37.208min chromatographic peak point
Wei not methyl tosylate, ethyl p-toluenesulfonate and posaconazole intermediate Z chromatographic peaks, separation
Degree is followed successively by 14.320,27.619；Separating degree is all higher than 1.5, and specificity is strong.
The chromatographic system of the present invention of embodiment 4 is to posaconazole intermediate Z, methyl tosylate and right
The separating degree experiment 3 of toluenesulfonic acid ethyl ester
Chromatographic column：C18(Agilent ZORBAX SB, 250mm × 4.6mm, 5 μm)；
Mobile phase：Solvent orange 2 A：Water；Solvent B：Acetonitrile, is eluted by with Gradient：
| Time (min) | 0.01 | 20 | 35 | 40 | 40.01 | 48 |
| A (%) | 58 | 58 | 5 | 5 | 58 | 58 |
| B (%) | 42 | 42 | 95 | 95 | 42 | 42 |
Diluent：(volume ratio of water and acetonitrile is 40 to acetonitrile solution：60).
Test procedure：
(1) blank solution is prepared：Fetch water 40ml, puts in 100ml measuring bottles, plus dilution in acetonitrile is to scale,
Shake up, produce；
(2) need testing solution is prepared：Posaconazole intermediate Z 50.20mg are taken, it is accurately weighed, put
In 25ml measuring bottles, plus diluent dissolves and is diluted to scale, shakes up, is used as need testing solution；Through meter
It is 2.008mg/ml to calculate need testing solution concentration.
(3) genotoxicity dirt solution is prepared：
Methyl tosylate solution：Methyl tosylate 26.56mg is taken, it is accurately weighed, put 25ml
In measuring bottle, plus acetonitrile dissolves and is diluted to scale with diluent, shakes up, produces；It is computed test sample
Solution concentration is 1062.4 μ g/ml.
Ethyl p-toluenesulfonate solution：Ethyl p-toluenesulfonate 12.51mg is taken, it is accurately weighed, put 25ml
In measuring bottle, plus acetonitrile dissolves and is diluted to scale with diluent, shakes up, produces；It is computed test sample
Solution concentration is 500.4 μ g/ml.
(4) mixed solution is prepared：Precision weighs intermediate Z 50.26mg, puts in 25ml measuring bottles, essence
Close methyl tosylate stock solution 0.5ml, the ethyl p-toluenesulfonate stock solution 1.0ml of pipetting is to the amount
In bottle, plus diluent makes dissolving and is diluted to scale, shakes up, produces；
(5) mixed solution sample introduction is taken, chromatogram is recorded, as a result as shown in Figure 7.
As shown in Figure 7, retention time is 11.696min, 16.694min, 28.904min chromatographic peak point
Wei not methyl tosylate, ethyl p-toluenesulfonate and posaconazole intermediate Z chromatographic peaks, separation
Degree is followed successively by 8.211,16.647；Separating degree is all higher than 1.5, and specificity is strong.
In above-mentioned method, chromatographic column uses C18Post, solvent B can use methanol, acetonitrile, isopropanol
In one or more, can reach identical effect, following examples using solvent B as acetonitrile,
Chromatographic column is C18(VP-ODS, 250mm × 4.6mm, 5 μm), is illustrated.Wherein, mobile phase：
Solvent orange 2 A：Water；Solvent B：Acetonitrile, is eluted by with Gradient：
| Time (min) | 0.01 | 20 | 35 | 40 | 40.01 | 48 |
| A (%) | 58 | 58 | 5 | 5 | 58 | 58 |
| B (%) | 42 | 42 | 95 | 95 | 42 | 42 |
Diluent：(volume ratio of water and acetonitrile is 40 to acetonitrile solution：60).
The detection hydraulic test of the methyl tosylate of embodiment 5 and ethyl p-toluenesulfonate
Test procedure：
(1) blank solution is prepared：Fetch water 40ml, put in 100ml measuring bottles, plus dilution in acetonitrile is extremely carved
Degree, shakes up, produces.
(2) need testing solution is prepared：Precision weighs intermediate Z about 50mg, puts in 25ml measuring bottles,
Plus diluent makes dissolving and is diluted to scale, shakes up, produces, concentration is computed for 2.0mg/ml.
(3) test limit solution is prepared：
Methyl tosylate stock solution：Precision weighs methyl tosylate 23.79mg, puts 25ml
In measuring bottle, plus acetonitrile makes dissolving and is diluted to scale with diluent, shakes up, produces, is computed concentration
For 951.6 μ g/ml.
Ethyl p-toluenesulfonate stock solution：Precision weighs ethyl p-toluenesulfonate 12.88mg, puts 25ml
In measuring bottle, plus acetonitrile makes dissolving and is diluted to scale with diluent, shakes up, produces, is computed concentration
For 515.2 μ g/ml.
Methyl tosylate positions solution：Precision pipettes methyl tosylate stock solution 0.5ml, puts
In 25ml measuring bottles, plus diluent is diluted to scale, shakes up, and produces, and is computed concentration for 19.032 μ g/ml.
Ethyl p-toluenesulfonate positions solution：Precision pipettes ethyl p-toluenesulfonate stock solution 1.0ml, puts
In 25ml measuring bottles, plus diluent dissolves and is diluted to scale, shakes up, produces, and being computed concentration is
20.608μg/ml。
Quantitative limit solution：Precision pipettes methyl tosylate solution positioning solution 1.0ml, to first respectively
Ethyl benzenesulfonat positions solution 1.0ml, puts in same 100ml measuring bottles, plus diluent is diluted to scale,
Shake up, produce.
Test limit solution：Precision pipettes above-mentioned quantitative limit solution 3ml, puts in 10ml measuring bottles, plus dilution
Dilution agent shakes up to scale, produces, be computed methyl tosylate concentration for 0.05710 μ g/ml,
Ethyl p-toluenesulfonate concentration is 0.06182 μ g/ml.
(4) test limit solution sample introduction is taken, efficient liquid phase chromatographic analysis is carried out, chromatogram is recorded, sees figure
8, calculate the test limit concentration (S/N=3 of methyl tosylate and ethyl p-toluenesulfonate：1), try
Test data and be shown in Table 2.
The test limit test data of table 2
As known to Fig. 8 and table 2, methyl tosylate test limit concentration is 0.05710 μ g/ml, in
There is concentration in mesosome Z and be expressed as 0.0029%；Ethyl p-toluenesulfonate test limit concentration is
0.06182 μ g/ml, 0.0031% is expressed as there is concentration in intermediate Z；Test limit S/N=3:1,
Meet the requirements.
The Detection capability experiment of the methyl tosylate of embodiment 6 and ethyl p-toluenesulfonate
Test procedure：
(1) blank solution is prepared：Fetch water 40ml, puts in 100ml measuring bottles, plus dilution in acetonitrile is to scale,
Shake up, produce.
(2) need testing solution is prepared：Precision weighs posaconazole intermediate Z about 50mg, puts 25ml
In measuring bottle, plus diluent makes dissolving and is diluted to scale, shakes up, produces, and being computed concentration is
2.0mg/ml。
(3) prepare and add solution (two parts of configured in parallel)：Precision weighs posaconazole intermediate Z about
50mg, puts in 25ml measuring bottles, and precision pipettes quantitative limit solution 7.5ml, puts in same volumetric flask, plus
Diluent makes dissolving and is diluted to scale, shakes up, produces.
(4) addition solution sample introduction is taken, efficient liquid phase chromatographic analysis is carried out, chromatogram is recorded, sees Fig. 9,
The test limit S/N of methyl tosylate and ethyl p-toluenesulfonate is calculated, experimental data is shown in Table 3.
Table 3 adds test data
In Fig. 9 and the data of table 3, posaconazole intermediate Z add test limit concentration to first
Methyl benzene sulfonate, ethyl p-toluenesulfonate can be detected effectively.
The detection of the posaconazole intermediate Z samples of embodiment 7
Test procedure：
(1) blank solution is prepared：Fetch water 40ml, puts in 100ml measuring bottles, plus dilution in acetonitrile is to scale,
Shake up, produce.
(2) need testing solution is prepared：Take posaconazole intermediate Z (totally seven batches of posaconazole intermediates
Z, lot number is respectively：1510101、150102、150103、150501、150502、150601、
150602) about 50mg, accurately weighed, puts in 25ml measuring bottles, plus diluent dissolves and is diluted to quarter
Degree, shakes up, obtains need testing solution.
(3) genotoxicity dirt solution is prepared：
Methyl tosylate solution：Methyl tosylate about 25mg is taken, it is accurately weighed, put 25ml
In measuring bottle, plus acetonitrile dissolves and is diluted to scale with diluent, shakes up, produces, being computed concentration is
1.0mg/ml。
Ethyl p-toluenesulfonate solution：Ethyl p-toluenesulfonate about 12.5mg is taken, it is accurately weighed, put 25ml
In measuring bottle, plus acetonitrile dissolves and is diluted to scale with diluent, shakes up, produces, being computed concentration is
0.5mg/ml。
(4) need testing solution is taken, liquid-phase chromatographic analysis is carried out by above-mentioned condition, chromatogram is recorded, sees
Figure 10-Figure 16, detection data are shown in Table 4.
Genotoxicity impurity detects data in the multiple batches of intermediate Z of table 4
| Lot number | Sample weighting amount (mg) | Methyl tosylate | Ethyl p-toluenesulfonate |
| POS-Z-150101 | 50.58 | Do not detect | Do not detect |
| POS-Z-150102 | 50.21 | Do not detect | Do not detect |
| POS-Z-150103 | 51.26 | Do not detect | Do not detect |
| POS-Z-150501 | 50.13 | Do not detect | Do not detect |
| POS-Z-150502 | 50.05 | Do not detect | Do not detect |
| POS-Z-150601 | 50.25 | Do not detect | Do not detect |
| POS-Z-150602 | 50.19 | Do not detect | Do not detect |
Illustrate that method of the invention can be effectively by posaconazole by Fig. 1-Figure 16, table 1-4
Mesosome Z is separated with its genotoxicity impurity (methyl tosylate and ethyl p-toluenesulfonate), and
Can accurately it be measured, to control the content of genotoxicity impurity described in posaconazole intermediate Z,
So as to ensure that the quality safety of final posaconazole raw material and preparation is controllable.
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted,
Although the present invention is described in detail with reference to preferred embodiment, one of ordinary skill in the art should
Work as understanding, technical scheme can be modified or equivalent substitution, without departing from this hair
The objective and scope of bright technical scheme, it all should cover among scope of the presently claimed invention.
Claims (10)
1. separate the method for posaconazole intermediate Z and related gene toxic impurities, it is characterised in that described
Method uses octadecylsilane chemically bonded silica for stationary phase, and the mixture using solvent orange 2 A and solvent B is mobile phase
Separated, the solvent orange 2 A is water, the solvent B is the one or more in methanol, acetonitrile, isopropanol.
2. separation posaconazole intermediate Z according to claim 1 and related gene toxic impurities side
Method, it is characterised in that the related gene toxic impurities are methyl tosylate and/or p-methyl benzenesulfonic acid second
Ester.
3. separation posaconazole intermediate Z according to claim 1 and related gene toxic impurities side
Method, it is characterised in that the solvent B is methanol or acetonitrile.
4. a kind of high efficiency liquid chromatography for separating and determining posaconazole intermediate Z and related gene toxic impurities
Method, it is characterised in that comprise the following steps that：Take test sample plus diluent to dissolve and be prepared into need testing solution,
Need testing solution sample introduction is taken, mobile phase elution carries out efficient liquid phase chromatographic analysis, records chromatogram, according to
Area normalization method calculates containing for contained posaconazole intermediate Z and related gene toxic impurities in test sample
Amount；The efficient liquid phase chromatographic analysis uses octadecylsilane chemically bonded silica for the chromatographic column of filler, institute
Mixture of the mobile phase for solvent orange 2 A and solvent B is stated, the solvent orange 2 A is water, and the solvent B is methanol, second
One or more in nitrile, isopropanol.
5. a kind of high efficiency liquid chromatography for separating and determining posaconazole intermediate Z according to claim 4 and
The method of related gene toxic impurities, it is characterised in that methods described also comprises the following steps：Pool is taken respectively
Saperconazole intermediate Z and related gene toxic impurities reference substance, dissolving are prepared into reference substance solution, take respectively
Posaconazole intermediate Z and related gene toxic impurities reference substance solution sample introduction, mobile phase elution are carried out high
Effect liquid phase chromatogram is analyzed, and records chromatogram, determines posaconazole intermediate Z and related gene toxic impurities
Retention time.
6. a kind of high efficiency liquid chromatography for separating and determining posaconazole intermediate according to claim 4 or 5
Z and related gene toxic impurities method, it is characterised in that the related gene toxic impurities are to toluene
Methylmesylate and/or ethyl p-toluenesulfonate.
7. a kind of high efficiency liquid chromatography for separating and determining posaconazole intermediate according to claim 4 or 5
Z and related gene toxic impurities method, it is characterised in that the diluent is the mixed of solvent orange 2 A and solvent B
Compound, solvent orange 2 A and solvent B volume ratio are 25-75：75-25, the solvent orange 2 A is water, the solvent B
For the one or more in methanol, acetonitrile, isopropanol.
8. a kind of high efficiency liquid chromatography for separating and determining posaconazole intermediate Z according to claim 6 and
The method of related gene toxic impurities, it is characterised in that the mobile phase elution uses gradient elution, described
Condition of gradient elution is：During 0.01min, solvent orange 2 A and solvent B volume ratio are 58：42；It is molten during 20min
Agent A and solvent B volume ratio is 58：42；During 35min, solvent orange 2 A and solvent B volume ratio are 5：95；
During 40min, solvent orange 2 A and solvent B volume ratio are 5：95；During 40.01min, the body of solvent orange 2 A and solvent B
Product is than being 58：42；During 48min, solvent orange 2 A and solvent B volume ratio are 58：42.
9. a kind of high efficiency liquid chromatography for separating and determining posaconazole intermediate according to claim 4 or 5
Z and related gene toxic impurities method, it is characterised in that the chromatographic column column temperature is 25-40 DEG C.
10. in the middle of a kind of high efficiency liquid chromatography for separating and determining posaconazole according to claim 4 or 5
Body Z and related gene toxic impurities method, it is characterised in that the efficient liquid phase chromatographic analysis is using purple
External detector is detected that Detection wavelength is 205-280nm.
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| CN115128177A (en) * | 2021-06-18 | 2022-09-30 | 上药康丽（常州）药业有限公司 | Analysis and determination of genotoxic impurities in ganciclovir condensate by HPLC |
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| CN108828105A (en) * | 2018-08-29 | 2018-11-16 | 四川百特芳华医药科技有限公司 | A kind of posaconazole method for detecting impurities |
| CN113671048A (en) * | 2020-05-13 | 2021-11-19 | 昆药集团股份有限公司 | Method for detecting methyl p-toluenesulfonate and ethyl p-toluenesulfonate in high piperazine residue |
| CN111638281A (en) * | 2020-05-28 | 2020-09-08 | 上海宣泰海门药业有限公司 | Analysis method of related substances of posaconazole enteric-coated tablets |
| CN115128177A (en) * | 2021-06-18 | 2022-09-30 | 上药康丽（常州）药业有限公司 | Analysis and determination of genotoxic impurities in ganciclovir condensate by HPLC |
| CN113533574A (en) * | 2021-07-20 | 2021-10-22 | 成都倍特药业股份有限公司 | Composition for drug synthesis and detection method of p-toluenesulfonyl chloride in composition |
| CN115448911A (en) * | 2022-10-12 | 2022-12-09 | 成都海博为药业有限公司 | Preparation method of posaconazole intermediate |
| CN116297978A (en) * | 2023-03-30 | 2023-06-23 | 重庆华邦制药有限公司 | Method for separation and determination of posaconazole Z3 and its impurities and solvents by HPLC |
| CN117405792A (en) * | 2023-10-24 | 2024-01-16 | 北京阳光诺和药物研究股份有限公司 | Method for detecting genotoxic impurities in posaconazole |
| CN117405792B (en) * | 2023-10-24 | 2024-06-21 | 北京阳光诺和药物研究股份有限公司 | Method for detecting genotoxic impurities in posaconazole |
| CN118688370A (en) * | 2024-08-27 | 2024-09-24 | 天津辰欣药物研究有限公司 | A quality control method for new antiviral drug intermediates |
| CN118688370B (en) * | 2024-08-27 | 2024-12-13 | 天津辰欣药物研究有限公司 | A quality control method for new antiviral drug intermediates |
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