CN105784867B - Make the purposes of reference standard for analyzing HPLC method and these impurity of the Fimasartan in relation to substance - Google Patents
Make the purposes of reference standard for analyzing HPLC method and these impurity of the Fimasartan in relation to substance Download PDFInfo
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- CN105784867B CN105784867B CN201610181939.1A CN201610181939A CN105784867B CN 105784867 B CN105784867 B CN 105784867B CN 201610181939 A CN201610181939 A CN 201610181939A CN 105784867 B CN105784867 B CN 105784867B
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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
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The present invention relates to the methods analyzed the impurity occurred in Fimasartan potassium (the thio formyl methyl -6- methyl -3- of 2- butyl -5- dimethylamino [[2 '-(1H-TETRAZOLE -5- base) biphenyl -4- base] methyl] (3H) -one of pyrimidine -4 sylvite) or its hydrate preparation process, wherein impurity is selected from compound A-L, which comprises (1) will be containing Fimasartan or the dissolution of its hydrate sample in a solvent to prepare sample solution；(, 2) sample any one or more of in compound A~L is dissolved in a solvent to prepare reference standard solution or reference substance solution；(3) chromatographic technique is implemented to sample solution and reference standard solution；And (4) measure the presence of any one or more of compound A~L in sample by referring to the one or more of known compound A-L present in the reference standard solution.
Description
Technical field
The invention belongs to pharmaceutical synthesis fields.In particular it relates to Fimasartan potassium (2- butyl -5- dimethylamino
Thio formyl methyl -6- methyl-the 3- of base [[2 '-(1H-TETRAZOLE -5- base) biphenyl -4- base] methyl] (3H) -one of pyrimidine -4 sylvite)
Or the method that the impurity occurred in its hydrate preparation process is analyzed.
Background technique
Fimasartan (Fimasartan) is a kind of novel selectivity AT1 receptor blocker, in a variety of Type of Hypertension
In show quickly and efficiently anti-hypertension effect, very safety and have preferable tolerance, be better than other same type drugs.
The Fimasartan (sylvite) of 2011 Boryung Pharmaceutical Co., Ltd. of Nian9Yue South Korea (BorYung) is in Korean market.Successively exist within 2013
The listing of the country such as Russia, Mexico, Brazil, Cuba.Phase III clinical trial is carried out in the U.S. at present, it is contemplated that will to 2016
It is declared in FDA.
It there is no the Chinese translation of standard at present, therefore its transliteration is herein " Fimasartan " by the applicant.Fimasartan
Chemical name are as follows: the thio formyl methyl -6- methyl -3- of 2- butyl -5- dimethylamino [[2 '-(1H-TETRAZOLE -5- bases) connection
Benzene -4- base] methyl] pyrimidine -4 (3H) -one sylvite, structural formula are as follows:
The preparation process route of Fimasartan potassium has been reported that in patent KR20040032639, the strategy of the route are as follows: with
2- (2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-n,N-dimethylacetamide (SM1) and 5- (4 '-(bromine first
Base)-[1,1 '-diphenyl] -2- base) -1- triphenyl -1H- tetrazole (SM2) be starting material, under the action of lithium hydride, into
Row nucleophilic substitution obtains intermediate 2- (2- butyl -4- methyl -6- oxygen -1- ((2 '-(1- triphenyl -1H- tetrazole -5-
Base)-[1,1 '-diphenyl] -4- base) methyl) 1,6- dihydro-pyrimidin -5- base)-n,N-dimethylacetamide (FMST-1), in this
Mesosome acts on obtaining intermediate 2- (1- ((2 '-(1H- tetrazole -5- bases)-[1,1 '-two with Lawesson reagent in toluene
Phenyl] -4- base) methyl -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide
(FMST-2).Using salt-forming reaction, intermediate Fimasartan sylvite (FMST) is obtained, in aqueous tetrahydrofuran solution transfer crystalline substance system
Obtain Fimasartan potassium finished product.Reaction equation are as follows:
In disclosed patent or document not for the method for substance related in Fimasartan and its hydrate analyze
It appears in the newspapers.Report in open source literature (9 (2014) 123-128 of ASIAN JOURNAL OF PHARMACEUTICAL SCIENCES)
The road method of Fimasartan and amlodipine besylate compound preparation principal component assay, but this method is not suitable for analyzing
In relation to the measurement of substance in Fimasartan and its hydrate.
Summary of the invention
The present invention provides new, interchangeable methods, are used for Fimasartan and its hydrate (such as trihydrate or a water
Close object) in the qualitative and quantitative analysis in relation to substance, and use these impurity as reference standard or reference substance to carry out determining for impurity
Amount and qualitative analysis.It is a further object to provide reference standard or reference substances, are preparing Fimasartan for detecting
And its impurity for being known as A~L formed during hydrate (such as trihydrate or monohydrate).
Provided by the present invention in analysis Fimasartan or Fimasartan hydrate (such as trihydrate or monohydrate)
HPLC method in relation to substance, wherein mobile phase includes two or more liquid, and the relative concentration of liquid is with scheduled ladder
It spends and changes.Inventor utilizes the impurity (claiming compound A~L) of 12 kinds of preparation and authentication structures for Fimasartan or Fei Masha
The reference standard or reference substance of Related substance method in smooth hydrate (such as trihydrate or monohydrate).In the prior art
Not yet disclose quantitative, the qualitative detection method for detecting these impurity in Fimasartan.
In the present invention, statement " Fimasartan hydrate " includes, but are not limited to three hydrations regardless of whether specifically noting
Object or monohydrate.
Therefore, the first aspect of the invention provides compound A, with chemical name: (E) -2- (- 6 methyl of 2- butyl -
4- carbonyl pyrimidine base -5- (4H)-subunit)-n,N-dimethylacetamide, and have a structure that
The second aspect provides compound B, with chemical name: triphenyl methane, and has a structure that
Compound B reference substance is purchased from Sigma-aldrich, and HPLC purity is greater than 98.0%.
Compound C is provided in terms of third, with chemical name: triphenyl alcohol, and have a structure that
Compound C reference substance is purchased from Sigma-aldrich, and HPLC purity is greater than 98.0%.
4th aspect provides compound D, with chemical name: triphenyl methyl ether, and has a structure that
Compound D reference substance is purchased from Sigma-aldrich, and HPLC purity is greater than 98.0%.
5th aspect provides compound E, with chemical name: 2- (1- ((2 '-(1H- tetrazole -5- bases)-[1,1 ' -
Diphenyl] -4- base) methyl) -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-n,N-dimethylacetamide, and
It has a structure that
6th aspect provides compound F, with chemical name: 2- (2- butyl -4- methyl -6- oxygen -1- ((2 '-(1- tri-
Benzyl -1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) 1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl
Thioacetamide, and have a structure that
Compound E, F are purchased from TRC company, and purity is all larger than 98.0%.
7th aspect provides compound G, with chemical name: 2- (1- ((2 '-(1H- tetrazole -5- bases)-[1,1 ' -
Diphenyl] -4- base) methyl) -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetic acid, and have a structure that
8th aspect provides compound H, with chemical name: 2- (2- butyl-4- methyl-1-((2 '-(1- methyl-
1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl disulfide
For acetamide, and have a structure that
J&K is purchased from outside compound H, HPLC purity is greater than 98%.
9th aspect provides compound I, with chemical name: 2- (2- butyl -1- ((2 '-itrile groups-[1,1 '-hexichol
Base] -4- base) methyl) -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide, and have following
Structure:
Tenth aspect provides compound J, with chemical name: 2- (1- ([1,1 '-diphenyl] -4- base-methyl) -2-
Butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide, and have a structure that
Tenth provides compound K on one side, with chemical name: 2- (1- ((2 '-(1H- tetrazole -5- bases)-[1,
1 '-diphenyl] -4- base) methyl) -2- butyl -6- thiocarbonyl -1,6- dihydroxy-pyrimidine -5- base)-N, N '-dimethyl is thio
Acetamide, and have a structure that
Compound K is bought in Toronto Research Chemicals, (abbreviation TRC), purity 97.5%.
12nd aspect provides compound L, with chemical name: 2- (1- ((2 '-(1H- tetrazole -5- bases)-[1,
1 '-diphenyl] -4- base) methyl) -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetoxymethyl ester, and have with
Flowering structure:
The way of production of compound A (impurity A) is as follows:
Containing the structure of lactams in starting material SM1, it is alkylated and reacts with SM2 under the action of lithium hydride, it is main
Wanting product is N- alkylate: intermediate FMST-1, but due to the influence of steric hindrance, it is miscellaneous to generate a small amount of O- alkylation
Matter (structure sees below formula), the impurity have thermodynamic phase, further decompose under alkaline condition, generate impurity A, generate
Mechanism is shown below.After the impurity generates, does not change in subsequent reactions, be transferred in finished product with prototype.
The way of production of compound L (impurity B) is as follows:
Starting material (SM1) uses recrystallizing methanol during the preparation process, therefore can generate esterification impurity, impurity ginseng
It is reacted with the subsequent preparation of Fimasartan potassium, generates impurity B precursor.Compared with amide, methyl esters is reacted with Lawesson reagent
Activity wants much lower.Therefore, under this process conditions, the methyl esters position of impurity B precursor is not reacted with Lawesson reagent, cannot
It is enough to be replaced by sulphur, only under the acid condition of Lawesson reagent, sloughs protection and generate impurity B.
Compound I (impurity C), the way of production of compound J (impurity D) are as follows:
Starting material (SM2) can generate impurity SM2- impurity -1 and SM2- impurity -2, two impurity during preparation
The subsequent synthetic procedures of Fimasartan can be participated in, alkylated reaction and thio reaction occurs, generates corresponding impurity C and D.
Compound A~L is the centre in Fimasartan or its hydrate (such as trihydrate or monohydrate) synthesis process
Body, by-product or degradation impurity are suitable as reference standard or reference substance, and the quality for the product controls.
In a particularly preferred embodiment, compound A~L according to the present invention is monomeric compound, most preferably
Be pure form, preferably purity is greater than about 95%, preferably purity is greater than about 98%, most preferably purity and is greater than about 99%, preferably
It is measured by HPLC.
12nd aspect of the invention provides a kind of hydrate (such as trihydrate for detecting Fimasartan or Fimasartan
Or monohydrate) sample purity method, this method includes measurement Fimasartan or its hydrate (such as trihydrate or a water
Close object) in sample containing one of the compound of the present invention A~L or a variety of.In the method for the invention, the compound is used
Make the reference standard or reference substance of impurity.
According to the twelfth aspect of the present invention, a kind of method for characterization of compound A~L is provided, this method utilizes
Such as HPLC method analyzes the impurity A~L in Fimasartan or its hydrate.
It thus provides compound A~L (one or more) according to the present invention is in detection Fimasartan or Fimasartan
Purposes in the sample purity of hydrate (such as trihydrate or monohydrate) as reference standard or reference substance.
On the other hand, the present invention also provides one kind for detecting Fimasartan or Fimasartan hydrate (such as trihydrate
Or monohydrate) sample purity method, which comprises pass through utilize reference standard according to the present invention or control
Product measure one of compound A~L or a variety of presence in sample by chromatography.
Another aspect, provide it is a kind of by measurement containing Fimasartan or Fimasartan hydrate (such as trihydrate or
Monohydrate) sample in presence any one or more of in compound A~L detect Fimasartan or its hydrate sample
Purity method, which comprises
(1) by Fimasartan or the dissolution of its hydrate sample in a solvent to prepare sample solution；
(2) by sample dissolution any one or more of in compound A~L in a solvent with prepare reference standard solution or
Reference substance solution；
(3) chromatographic technique is implemented to sample solution and reference standard solution；And
(4) by referring to the presence of known compound A-L (one or more) present in the reference standard solution, measurement
The presence of any one or more of compound A~L in sample.
In one embodiment, which is liquid chromatography, such as HPLC, UPLC, LC-MS；It is preferred that the chromatography
Method is HPLC method, preferred gradient HPLC method.
Present invention preferably uses stationary phase be reverse phase.Suitable stationary phase includes octadecylsilane chemically bonded silica or pungent
Base silane bonded silica gel.
In a preferred embodiment of the invention, a kind of analysis Fimasartan or Fimasartan hydrate (Ru Sanshui are provided
Close object or monohydrate) gradient HPLC method, wherein mobile phase includes containing the group of buffer solution (A) and organic solvent (B)
It closes.It is preferred that buffer solution (A) is water-containing buffering liquid, preferably acetate, formates, phosphate, phosphoric acid, trifluoroacetic acid, formic acid
Or the aqueous solution of their mixture.Preferred buffer solution (A) is phosphoric acid or phosphate, most preferably phosphoric acid and phosphate
Or phosphatic aqueous solution adjusted the aqueous solution of pH with phosphoric acid.Most preferably.Phosphoric acid exists in particularly preferred embodiments
Concentration be about 0.03 volume % to 1.5 volume %, preferably 0.05 volume %-1.3 volume %, more preferable 0.08 volume %-1.2
Volume %, most preferably 0.1 volume of volume %~1.0 %.Preferable organic solvent (B) be polar aprotic solvent, as methanol, ethyl alcohol or
Isopropanol；Or dipolar aprotic solvent, such as acetonitrile.Preferable organic solvent (B) be selected from including methanol, acetonitrile, ethyl alcohol, isopropanol or
In the group of their mixture, the most preferably mixture of acetonitrile and methanol.
Mobile phase specifically preferred according to the invention includes the group of phosphoric acid and phosphatic aqueous solution (A) and acetonitrile and methanol (B)
It closes.
Gradient HPLC method according to the present invention is further provided for, wherein mobile phase includes following gradient design:
In further preferred embodiment, the HPLC analysis method about 25~60 DEG C at a temperature of carry out, preferably
25~50 DEG C, 25~45 DEG C, 25~40 DEG C, most preferably 25~35 DEG C.
In other embodiments, which carries out under the flow velocity of about 0.6~1.5ml/min, preferably
0.7~1.4ml/min, 0.7~1.3ml/min, most preferably 0.8~1.2ml/min.
In other embodiments, in the HPLC analysis method, organic phase B be methanol and acetonitrile mixture, methanol and
The mixed proportion of acetonitrile is about 0: 100, and preferably 5: 95,10: 90,20: 80,25: 75, most preferably 30: 70.
HPLC method according to the present invention is effectively detected with single operation and quantitatively includes that those are selected from following compound
In all impurity:
Compound A:(E) -2- (- 6 methyl -4- carbonyl pyrimidine base -5- (4H) of 2- butyl-subunit)-N, N- dimethylacetamide
Amine.
Compound B: triphenyl methane.
Compound C: triphenyl alcohol.
Compound D: triphenyl methyl ether.
Compound E:2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -4-
Methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-n,N-dimethylacetamide.
Compound F:2- (2- butyl -4- methyl -6- oxygen -1- ((2 '-(1- trityl -1H- tetrazole -5- bases)-[1,
1 '-diphenyl] -4- base) methyl) 1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide.
Compound G:2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -4-
Methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetic acid.
Compound H:2- (2- butyl-4- methyl-1-((2 '-(1- methyl-1 H- tetrazole-5- base)-[1,1 '-hexichol
Base] -4- base) methyl) -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide.
Compound I:2- (2- butyl-1- ((2 '-itrile groups-[1,1 '-diphenyl]-4- base) methyl) oxygen-1-4- methyl-6-,
6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide.
(1- ([1,1 '-diphenyl] -4- base-methyl) -2- butyl -4- methyl -6- oxygen -1,6- dihydro is phonetic by compound J:2-
Pyridine -5- base)-N, N- dimethyl thio acetamide.
Compound K: 2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -6-
Thiocarbonyl -1,6- dihydroxy-pyrimidine -5- base)-N, N '-dimethyl thioacetamide.
Compound L: 2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -4-
Methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetoxymethyl ester.
The present invention can be used for analyzing as API Fimasartan and its hydrate (such as trihydrate or monohydrate) and/
Or pharmaceutical composition.The pharmaceutical composition that the present invention can analyze includes solid or liquid composition, and optionally includes one kind
Or a variety of pharmaceutically acceptable excipient.The composition of solid form includes pulvis, tablet, capsule, pill and can divide
Scattered granule etc..Liquid composition includes solution or suspension, can be administered by oral, injection or instillation approach.Explanation
Book and claims in the whole text used in term " Fimasartan " refer to the solvent of Fimasartan potassium and/or Fimasartan sylvite
Compound (such as hydrate, such as trihydrate or monohydrate).Specification in the whole text used in term " impurity " or " related substance "
It can refer to the impurity that is formed and/or API in manufacture API or pharmaceutical composition to degrade the impurity to be formed or in storage
Pharmaceutical composition or preparation in the impurity that is formed.
As described above, the HPLC method reported in the prior art, is not suitable for analyzing Fimasartan and contains non-horse
The preparation or pharmaceutical composition of Sha Tan.
However, method of the invention solves the problems, such as this, and it is effectively detected and is quantified with single operation, qualitative point
Analyse all impurity and intermediate formed in this specific synthesis process or preparation preparation.Present invention has an advantage that for the first time
Disclose the impurity in Fimasartan or Fimasartan hydrate and composition comprising Fimasartan or Fimasartan hydrate
Specific structure, and polar impurity and non polar impurities be eluted out simultaneously with gradient HPLC method, and carries out qualitative, quantitative
Analysis.
The invention is particularly suited to measure and quantify one of compound A~L or a variety of presence in sample.Unless another
It is described, otherwise under range relevant to compound A~L, can be interchanged makes for term herein " impurity " and " compound "
With.
It is also an advantage of the invention that this method for or Fimasartan hydrate and include Fimasartan or Fei Masha
The spies such as the analysis of the related substance in the composition of smooth hydrate has specificity strong, and accuracy, precision height, durability are good
Point.In addition, the present invention has height sensitivity, and allow detection and quantitative Fimasartan or Fimasartan hydrate API or medicine
Horizontal amount is substantially less than the related substance of acceptable limits specified in drug administration department and ICH guideline in compositions.
In addition, method of the invention can be used for detecting and quantifying the sample or medicine of Fimasartan or Fimasartan hydrate
All degradation impurities formed during compositions storage.By carrying out forced degradation research according to ICH Q1A guide come really
Determine this method, and verified according to ICH Q2A guide, which covers following items: specificity, linearity and range, precision
Degree, accuracy, detection limit, quantitative limit, durability and system suitability.
The novel method that the present inventor develops such as eight kinds of impurity A~L of gradient HPLC qualitative determination.The method can
Disposable analysis is produced according in Fimasartan preparation process manufactured in the present embodiment and during commercially available Fimasartan and storage
The impurity that raw by-product, degradation impurity isopolarity differ greatly, therefore, inventors believe that gradient design is the most suitable.
In the embodiment of this invention, the inventors found that containing octadecylsilane chemically bonded silica or octyl silane key
The stationary phase for closing silica gel is the most advantageous.Particularly preferred stationary phase contains Aglea Venusil XBP C8 5μm 4.6
×250mm、Aglea Venusil XBP C18 5μm 4.6×150mm、Phemomenex Luna C8 5μm 4.6×
250mm、Phemomenex Luna C85 μm of 4.6 × 150mm and Phemomenex Luna C185 μm of 4.6 × 150mm's
Chromatographic column.The method of the present invention preferably includes gradient design, so that mobile phase A and the relative concentration of B are in 10~60 minutes allusion quotations
Change to type the gradient for 100%A: 0%B to 0%A: 100%B.Preferably, 15 to 55 minutes of process, gradient 95%
A: 5%B to 5%A: 95%B, it is highly preferred that by 20 to 50 minutes, gradient 90%A: 10%B to 10%A: 90%
B, most preferably, by about 30 minutes, gradient 65%A: 35%B to 0%A: 100%B or 60%A: 40%B to 5%A:
95%B or 55%A: 45%B to 10%A: 90%B or 50%A: 50%B to 15%A: 85%B.The advantage of this gradient method exists
In, can by Fimasartan or Fimasartan hydrate (such as trihydrate, monohydrate) API or Fimasartan (or hydrate,
Such as trihydrate, monohydrate) various opposed polarities or the very similar impurity of polarity are totally separated out in pharmaceutical composition, just
In accurately qualitative and quantitative.
The mobile phase used is preferably selected from the group of one or more buffer solutions (A) and one or more organic solvents (B)
It closes.
Buffer solution (A) is preferably selected from including phosphate, phosphoric acid, acetate, formates, trifluoroacetic acid, formic acid or acetic acid
The aqueous solution of their mixture combines.
The concentration of buffer solution (A) can be the 0.03 volume % of volume %~1.5, preferred concentration be 0.05 volume %~
1.3 volume %, more preferable concentration are the 0.08 volume % of volume %~1.2, most preferably 0.1 volume of volume %~1.0 %.It is especially excellent
The mobile phase of choosing includes (A): phosphoric acid and phosphatic aqueous solution or phosphoric acid solution ammonium hydroxide, sodium hydroxide, potassium hydroxide tune
Save the aqueous solution and (B) of pH: the combination of the mixture of acetonitrile or acetonitrile and methanol.
In particularly preferred embodiment according to the present invention, it is further provided a kind of method of gradient HPLC, wherein
Mobile phase includes following gradient design:
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 65 | 35 |
| 25 | 0 | 100 |
| 25.01 | 65 | 35 |
| 30 | 65 | 35 |
The present invention also provides particularly preferred HPLC gradient method, wherein mobile phase includes that phosphoric acid and/or phosphate are made
For buffer solution (A).In other particularly preferred embodiment, mobile phase includes acetonitrile as organic solvent (B), and/or
Acetonitrile-methanol mixed solvent is as organic solvent (B).Inventor's discovery includes phosphoric acid and/or phosphate (A) and second when mobile phase
Nitrile and gradient design is particularly effective when carbinol mixture (B).
Buffer solution (A) can be methanol, ethyl alcohol, isopropyl containing one or more of added solvents, these added solvents
Alcohol or their mixture are as organic solvent.Added solvent in buffer solution (A) may or may not be and organic solvent (B)
Identical solvent.Added solvent in buffer solution (A) is preferably the mixture of acetonitrile or acetonitrile and methanol.
The pH of buffer solution (A) is about 2.0~4.0, preferably from about 2.2~3.8,2.5~3.5, most preferably 2.5~
3.0。
HPLC method of the invention about 25~60 DEG C at a temperature of carry out, preferably 25~50 DEG C, 25~45 DEG C, 25~
40 DEG C, most preferably 25~35 DEG C.
The analysis method carries out under the flow velocity of about 0.6~1.5ml/min, preferably 0.7~1.4ml/min, most preferably
For 0.8~1.2ml/min.
Organic solvent (B) can be a kind of organic solvent, and this organic solvent can be methanol, ethyl alcohol, acetonitrile, isopropanol.
Organic solvent (B) is also possible to the mixed solvent of methanol and acetonitrile or the mixed solution of acetonitrile and ethyl alcohol, and inventor has found methanol
Mixed solvent with acetonitrile is as organic phase (B) optimal effect.In the HPLC analysis method, organic phase B is the mixed of methanol and acetonitrile
The mixed proportion of conjunction object, methanol and acetonitrile is about 0: 100, and preferably 5: 95,10: 90,20: 80,25: 75, most preferably 30:
70。
Another aspect of the present invention provides a kind of reference standard solution.The solution includes to be dissolved in suitable solvent (acetonitrile)
One or more compound A~L.The reference standard solution can be used for measure using chromatographic technique according to the present invention into
Presence in the sample of row analysis as any compound A~L of impurity.
According to another aspect of the present invention, a kind of reference standard solution is provided, wherein one or more chemical combination of known quantity
Object A~L is dissolved in suitable solvent (acetonitrile).The reference standard solution, which can be used for measuring, is utilizing color according to the present invention
As the qualitative of any compound A~L of impurity and quantitatively in the sample that spectral technology is analyzed.The method of the analysis for
Technical staff important and be conveniently obvious.
The present inventor verifies method of the invention extensively, and verification result shows that the specificity of this method is strong, accuracy, essence
Density and high sensitivity, durability are good.
Detailed description of the invention
Fig. 1: compound A (impurity A): (E) -2- (- 6 methyl -4- carbonyl pyrimidine base -5- (4H) of 2- butyl-subunit)-N, N-
Dimethyl acetamide1H-NMR；
Fig. 2: compound A (impurity A): (E) -2- (- 6 methyl -4- carbonyl pyrimidine base -5- (4H) of 2- butyl-subunit)-N, N-
The high resolution mass spectrum of dimethyl acetamide；
Fig. 3: compound L (impurity B): 2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) first
Base) -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetoxymethyl ester1H-NMR；
Fig. 4: compound L (impurity B): 2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) first
Base) -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetoxymethyl ester high resolution mass spectrum；
Fig. 5: compound I (impurity C): 2- (2- butyl -1- ((2 '-itrile groups-[1,1 '-diphenyl] -4- base) methyl) -4-
Methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide1H-NMR；
Fig. 6: compound I (impurity C): 2- (2- butyl -1- ((2 '-itrile groups-[1,1 '-diphenyl] -4- base) methyl) -4-
Methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, the high resolution mass spectrum of N- dimethyl thio acetamide；
Fig. 7: compound J (impurity D): 2- (1- ([1,1 '-diphenyl] -4- base-methyl) -2- butyl -4- methyl -6- oxygen -
1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide1H-NMR；
Fig. 8: compound J (impurity D): 2- (1- ([1,1 '-diphenyl] -4- base-methyl) -2- butyl -4- methyl -6- oxygen -
1,6- dihydro-pyrimidin -5- base)-N, the high resolution mass spectrum of N- dimethyl thio acetamide.
Fig. 9: compound G:2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- fourth
Base -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetic acid1H-NMR；
Figure 10: compound G:2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- fourth
Base -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetic acid high resolution mass spectrum；
Figure 11: Fimasartan potassium crude product HPLC analysis map.
Figure 12: the HPLC spectrogram of Fimasartan mark-on mixed solution.
Figure 13: the HPLC spectrogram of Fimasartan test solution.
Specific embodiment
Although the present invention is described its specific embodiment, certain modifications and equivalent are for this field skill
Art personnel are it will be apparent that and it is intended to be included within the scope of the present invention.
The present invention is illustrated by following embodiment, following embodiment is not limit the invention in any way.
Embodiment 1: compound A:(E) -2- (- 6 methyl -4- carbonyl pyrimidine base -5- (4H) of 2- butyl-subunit)-N, N- bis-
The synthesis of methylacetamide.
The mixing of 20ml n,N-Dimethylformamide and ethyl acetate (volume ratio 1: 1) is added into the there-necked flask of 100mL
Solvent is added with stirring 1.00g SM1 (2- (2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl
Acetamide), nitrogen protection is cooled to 0~10 DEG C, and 0.48 gram of LiH is added, continues stirring 20~30 minutes, and 2.08 grams of SM2 are added
(5- (4 '-(bromomethyls)-[1,1 '-diphenyl] -2- base) -1- triphenyl -1H- tetrazole) is warming up to 60 DEG C, and stirring 48 is small
When；Sampling TLC detection, (methanol: methylene chloride=1: 10) raw material disappears.Add water 20ml, there are a large amount of solids to be precipitated, continue to stir
30 minutes.Filtering, drain, 40 DEG C forced air drying 30 minutes, obtain solid crude product, will obtain crude product carry out column chromatographic purifying, wash
De- ratio are as follows: methanol: methylene chloride=1: 50, collect the total 160mL of eluent, 30~40 DEG C of temperature control, vacuum degree: -0.08MPa,
Vacuum distillation, solvent to no fraction is evaporated off and steams, obtains faint yellow solid 105mg, yield: 5.05% to get arrive impurity A: (E)-
2- (- 6 methyl -4- carbonyl pyrimidine base -5- (4H) of 2- butyl-subunit)-n,N-dimethylacetamide.1H-NMR (400Mz, CDCl3)
δ: 5.765 (s, 1H), 3.164 (s, 1H), 3.117 (m, 2H), 2.866 (s, 3H), 1.852~1.813 (m, 2H), 1.454~
1.398 (m, 2H), 0.967~0.930 (m, 3H), are specifically detailed in attached drawing 1.High resolution mass spectrum (ESI-HRMS) spectrogram shows molecule
Quasi-molecular ions m/z=250.15378 [M+H]+, the structural formula calculated value (250.15500) of corresponding molecular weight and offer
It is consistent.Absolute error is 4.87ppm, within high resolution mass spectrum error range, referring specifically to attached drawing 2.
Embodiment 2: compound L: 2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -
2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetoxymethyl ester preparation
The mixing of 20ml n,N-Dimethylformamide and ethyl acetate (volume ratio 1: 1) is added into the there-necked flask of 100mL
Solvent, is added with stirring the impurity (SM1 impurity shown in impurity B the way of production as described above) of 1.00g SM1, and nitrogen is protected
Shield, is cooled to 0~10 DEG C, 0.48 gram of LiH is added, continues stirring 20~30 minutes, 2.08 grams of SM2 are added, is warming up to 60 DEG C, stirs
It mixes 48 hours；Sampling TLC detection, (methanol: methylene chloride=1: 10) raw material disappears.Add water 20ml, there are a large amount of solids to be precipitated, after
Continuous stirring 30 minutes.Filtering, drain, 40 DEG C forced air drying 30 minutes, obtain 2.18 grams of solid crude product, which be dissolved in
In 100mL toluene, it is added 1.57 grams of Lawesson reagents, under nitrogen protection, reaction solution is heated to 70 DEG C, is stirred 4 hours, it will
Reaction solution is evaporated under vacuum conditions.It will obtain crude product and carry out column chromatographic purifying, and elute ratio are as follows: methanol: methylene chloride=1:
30, the total 50mL of eluent is collected, 30~40 DEG C of temperature control, vacuum degree: -0.08MPa, vacuum distillation are evaporated off solvent to no fraction and steam
Out, obtain faint yellow solid 0.99g, yield: 50.20% to get to impurity B: 2- (1- ((2 '-(1H- tetrazole -5- bases)-[1,
1 '-diphenyl] -4- base) methyl) -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetoxymethyl ester.1H-NMR
(400Mz, CDCl3) δ: 7.951 (d, J=1.20Hz, 1H), 7.934~7.556 (m, 1H)；7.524~7.486 (m, 1H),
7.400 (d, J=1.2,1H), 7.095~7.090 (m, 2H)；7.060~7.039 (m, 2H)；5.210 (s, 1H)；3.640 (s,
3H)；3.525 (s, 2H)；2.631 (t, J=7.60Hz, 2H)；2.260 (s, 3H)；1.684~1.645 (m, 2H)；1.391~
1.335 (m, 2H)；0.890 (t, J=7.20Hz, 3H), is specifically detailed in attached drawing 3.ESI-HRMS spectrogram shows molecular ion peak m/z
=473.23083 [M+H]+, corresponding molecular weight is consistent with the structural formula calculated value (473.22957) of offer.Absolutely
Error is 2.66ppm, within high resolution mass spectrum error range, is specifically detailed in attached drawing 4.
Embodiment 3: compound I:2- (2- butyl -1- ((2 '-itrile groups-[1,1 '-diphenyl] -4- base) methyl) -4- first
Base -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, the preparation of N- dimethyl thio acetamide
The mixing of 20ml n,N-Dimethylformamide and ethyl acetate (volume ratio 1: 1) is added into the there-necked flask of 100mL
Solvent is added with stirring 1.00g SM1, and nitrogen protection is cooled to 0~10 DEG C, and 0.30 gram of LiH is added, continues stirring 20~30
Minute, 1.30 gram of 4 '-(bromomethyl)-[1,1 '-diphenyl] -2- nitrile (SM2- impurity -1) is added, is warming up to 60 DEG C, stirring 48 is small
When；Sampling TLC detection, (methanol: methylene chloride=1: 20) raw material disappears.Add water 15ml, there are a large amount of solids to be precipitated, continue to stir
30 minutes.Filtering, drain, 40 DEG C forced air drying 30 minutes, obtain 1.41 grams of solid crude product, which be dissolved in 50mL toluene
In, it is added 1.28 grams of Lawesson reagents, under nitrogen protection, reaction solution is heated to 70 DEG C, stirs 4 hours, reaction solution is existed
It is evaporated under vacuum condition.It will obtain crude product and carry out column chromatographic purifying, and elute ratio are as follows: ethyl acetate: petroleum ether=50: 50, it receives
Collect the total 73mL of eluent, 30~40 DEG C of temperature control, vacuum degree: -0.08MPa, vacuum distillation are evaporated off solvent to no fraction and steam, obtain
Faint yellow solid 0.78g, yield: 43.20% to get arrive impurity C:2- (2- butyl -1- ((2 '-itrile groups-[1,1 '-diphenyl] -
4- yl) methyl) -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide.1H-NMR (400Mz,
CDCl3) δ: 7.753~7.733 (m, 1H), 7.623~7.603 (m, 1H)；7.529~7.509 (m, 2H), 7.466~7.428
(m, 2H), 7.277~7.256 (m, 2H)；5.363 (br, 1H)；3.859 (s, 3H)；3.524 (s, 3H)；3.485 (s, 3H)；
2.738 (t, J=7.20Hz, 2H)；2.378 (s, 3H)；1.699~1.660 (m, 2H)；1.409~1.353 (m, 2H)；0.889
(t, J=7.20Hz, 3H) is specifically detailed in attached drawing 5.ESI-HRMS spectrogram shows molecular ion peak m/z=459.22289 [M+H
]+, corresponding molecular weight is consistent with the structural formula calculated value (459.62569) of offer.Absolute error is 3.38ppm,
Within high resolution mass spectrum error range, it is specifically detailed in attached drawing 6.
Embodiment 4: compound J:2- (oxygen -1 1- ([1,1 '-diphenyl] -4- base-methyl) -2- butyl -4- methyl -6-,
6- dihydro-pyrimidin -5- base)-N, the preparation of N- dimethyl thio acetamide
The mixed of 20ml n,N-Dimethylformamide and ethyl acetate (volume ratio 1: 1) is added into the there-necked flask of 100mL
Bonding solvent is added with stirring 1.00g SM1, and nitrogen protection is cooled to 0~10 DEG C, and 0.30 gram of LiH is added, continue stirring 20~
30 minutes, 1.18 grams of 4- (bromomethyl) -1 are added, 1 '-biphenyl 4- (bromomethyl) -1, (SM2- is miscellaneous for 1 '-biphenyl
Matter -2), 60 DEG C are warming up to, is stirred 48 hours；Sampling TLC detection, (methanol: methylene chloride=1: 25) raw material disappears.Add water
20ml has a large amount of solids to be precipitated, and continues stirring 30 minutes.Filtering, drain, 40 DEG C forced air drying 30 minutes, obtain solid crude product
1.27 grams, which is dissolved in 50mL toluene, is added 1.58 grams of Lawesson reagents, under nitrogen protection, reaction solution is heated
To 70 DEG C, stirs 4 hours, reaction solution is evaporated under vacuum conditions.It will obtain crude product and carry out column chromatographic purifying, and elute ratio
Are as follows: ethyl acetate: petroleum ether=40: 60, the total 66mL of eluent is collected, 30~40 DEG C of temperature control, vacuum degree: -0.08MPa is depressurized
Distillation, solvent to no fraction is evaporated off and steams, obtains faint yellow solid 0.90g, yield: 51.30% to get arrive impurity D:2- (1-
([1,1 '-diphenyl] -4- base-methyl) -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl disulfide
For acetamide.1H-NMR (400Mz, CDCl3) δ: 7.545~7.525 (m, 4H), 7.434~7.415 (m, 2H)；7.345~
7.327 (m, 1H), 7.222~7.202 (m, 2H)；5.337 (br, 1H)；3.872 (s, 3H)；3.533 (s, 3H)；3.490 (s,
3H)；2.677 (t, J=7.60Hz, 2H)；2.365 (s, 3H)；1.687~1.629 (m, 2H)；1.393~1.336 (m, 2H)；
0.880 (t, J=7.20Hz, 3H), is specifically detailed in attached drawing 7.ESI-HRMS spectrogram shows molecular ion peak m/z=434.22725
[M+H]+, corresponding molecular weight is consistent with the structural formula calculated value (434.22606) of offer.Absolute error is
2.74ppm is specifically detailed in attached drawing 8 within high resolution mass spectrum error range.
Embodiment 5: compound G:2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -
2- butyl -4- methyl -6- oxygen -1,6- dihydroxy-pyrimidine -5- base) acetic acid preparation
The mixed solvent of 20ml tetrahydrofuran and water (volume ratio 5: 1) is added into the there-necked flask of 100mL, is added with stirring
1.00g tri- is hydrated Fimasartan potassium, and 0.1 gram of sodium hydroxide is added in nitrogen protection.Reaction solution is warming up to 50 DEG C, stirs 12 hours
After be cooled to room temperature, it is 2.0 or so that the hydrochloric acid solution of 1N to pH value, which is added dropwise, there is white precipitate precipitation, be collected by filtration solid, 40 DEG C
Lower forced air drying obtains 0.41 gram of white solid for 12 hours, yield: 53.25%, obtain impurity E: 2- (1- ((2 '-(tetra- nitrogen of 1H-
Azoles -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -4- methyl -6- oxygen -1,6- dihydroxy-pyrimidine -5- base) second
Acid.1H-NMR (400Mz, CDCl3) δ: 7.939~7.936 (d, J=1.20Hz, 1H), 7.920~7.551 (m, 1H)；7.523
~7.485 (m, 1H), 7.408~7.386 (m, 1H)；7.104~7.047 (m, 4H)；5.200 (br, 2H)；3.538 (s,
3H)；2.694 (t, J=7.60Hz, 2H)；2.275 (s, 3H)；1.696~1.658 (m, 2H)；1.404~1.437 (m, 2H)；
0.885 (t, J=7.20Hz, 3H), (being detailed in attached drawing 9).ESI-HRMS spectrogram shows molecular ion peak m/z=459.21581 [M+
H]+, corresponding molecular weight is consistent with the structural formula calculated value (459.21392) of offer.Absolute error is 4.12ppm,
Within high resolution mass spectrum error range, the high resolution mass spectrum of compound G is shown in attached drawing 10.
Embodiment 6:2- (2- butyl -4- methyl -6- oxygen -1- ((2 '-(1- trityl -1H- tetrazole -5- bases)-[1,
1 '-diphenyl] -4- base) methyl) -1,6- dihydro-pyrimidin -5- base)-n,N-dimethylacetamide (FMST-1) preparation
600mL ethyl acetate is added into 1L there-necked flask, opens stirring, sequentially adds 100g 2- (2- butyl -4- methyl -
6- oxygen -1,6- dihydro-pyrimidin -5- base)-n,N-dimethylacetamide (FMST-SM1), 8g lithium hydride, reaction 5 is small under room temperature
When, 210g 5- (4 '-(bromomethyls)-[1,1 '-diphenyl] -2- base) -1- trityl -1H- tetrazole (FMST- is added
SM2), stir 72 hours under room temperature, TLC monitoring reaction terminates；Reaction solution is added in 1L pure water under stirring, is stirred
It mixes 1 hour, filters, it is pure to be washed to filtrate and be in neutrality, filter cake forced air drying 12 hours (45 DEG C -50 DEG C of drying temperature), obtain 2-
(2- butyl -4- methyl -6- oxygen -1- ((2 '-(1- trityl -1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) first
Base) -1,6- dihydro-pyrimidin -5- base)-n,N-dimethylacetamide 178g is off-white powder, yield: 61.6%；
Embodiment 7:2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -4-
Methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, the preparation of N- dimethyl thio acetamide (FMST-2)
750mL toluene is added into the reaction flask of 1L, opens stirring, sequentially adds 150g 2- (2- butyl -4- methyl -6-
Oxygen -1- ((2 '-(1- trityl -1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -1,6- dihydro-pyrimidin -
5- yl)-n,N-dimethylacetamide (FMST-1), 105g Lawesson ' s reagent starts to warm up, reacts under the conditions of 105 DEG C
5h, HPLC monitor reaction process.Reaction terminates, and is down to room temperature.2L purified water, filtering, filter cake 1.05L is added to reaction system
Purified water elution.45 DEG C forced air drying 4 hours, obtain 2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base)
Methyl) -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide 85g, for white powder
End, yield: 82.5%.
Embodiment 8: the preparation of Fimasartan potassium (FMST)
350mL ethyl alcohol is added into the there-necked flask of 500mL, opens stirring, sequentially adds 80g 2- (1- ((2 '-(1H- tetra-
Nitrogen azoles -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N,
N- dimethyl thio acetamide (FMST-2), 10g potassium hydroxide, system are warming up to reflux, react 12 hours under reflux state, instead
It should terminate, be further continued for stirring and crystallizing 12 hours after being cooled to room temperature.Filtering, 4 hours (45 DEG C of drying temperatures-of filter cake forced air drying
50 DEG C), Fimasartan potassium crude product 82g is obtained, is white powder, yield: 95.4%.
Embodiment 9: Fimasartan potassium trihydrate preparation
The mixed solvent of 250mL isopropanol/water (10: 1) is added into the there-necked flask of 500mL, opens stirring, 60g is added
Fimasartan potassium, system are warming up to reflux dissolved clarification, start to cool down, and keep the temperature crystallization 4 hours after being cooled to room temperature.Filtering, filter cake drum
It air-dries dry 4 hours (40 DEG C~50 DEG C of drying temperature), obtains Fimasartan potassium trihydrate 65g, be white crystalline powder, receive
Rate: 98.5%.
Embodiment 10: the preparation of Fimasartan potassium monohydrate
180mL Isosorbide-5-Nitrae-dioxane/water (5: 1) mixed solvent is added into the there-necked flask of 500mL, opens stirring, adds
Enter 60g Fimasartan potassium, system is warming up to 60 DEG C of dissolved clarifications, starts to cool down, and keeps the temperature crystallization 12 hours after being cooled to room temperature.Filtering,
Filter cake forced air drying 12 hours (45 DEG C -50 DEG C of drying temperature), Fimasartan potassium monohydrate 58g is obtained, is white crystalline powder
End, yield: 93.5%.
Embodiment 11: Fimasartan is analyzed in relation to the HPLC of substance
Using impurity external standard method (version Chinese Pharmacopoeia in 2015), analyze the API Fimasartan prepared according to the method described above or
Related substance in hydrate, and each impurity is quantified with external standard method, referring specifically to 2010 editions two annex V of Chinese Pharmacopoeia
D.Method for carrying out the analysis is gradient HPLC method.The result is shown in Figure 11.
The preparation of Fimasartan or hydrate and sample preparation are the same as embodiment 6~10.
The gradient design used is as follows:
Chromatographic condition:
Chromatographic column: 5 μm of C8 of Aglea Venusil XBP4.6×250mm
The concentration of test solution: 0.5mg/ml
The concentration of each impurity of reference standard solution: 1000ppm
Mobile phase: A:0.05% phosphoric acid (ammonium hydroxide or sodium hydroxide tune pH to 2.5), B: acetonitrile
Detection wavelength: 210nm
Sampling volume: 20 μ l
Diluent: 50% acetonitrile-water
Column temperature: 25 DEG C
Flow velocity: 1.2ml/min
Sample preparation:
Prepare impurity positioning solution
(1) compound A~L standard setting solution: taking compound A~L about 10mg respectively, accurately weighed, sets 100ml respectively
In volumetric flask, acetonitrile ultrasound is added to make to dissolve, and with dilution in acetonitrile to scale, shakes up, the solution that concentration is about 100 μ g/ml is made,
As compound A~L stock solution.
(2) mark-on mixed solution: take above-mentioned preparation Fimasartan bulk pharmaceutical chemicals about 50mg (in terms of anhydride, Fimasartan
The conversion coefficient of potassium and Fimasartan potassium trihydrate and monohydrate is respectively as follows: 0.90 and 0.96), accurately weighed, sets 100mg
In measuring bottle, then accurate addition each 0.5ml of compound A~L stock solution, add 50% acetonitrile-water appropriate, ultrasound makes to dissolve, then uses
50% acetonitrile-water is diluted to scale, shakes up, and every 1ml is made and contains Fimasartan about 0.5mg, A containing compound~L about 0.5 μ g respectively
Mixed solution, as mark-on mixed solution.
(3) prepare compound A~L reference standard solution or reference substance solution: precision measures compound A~L stock solution
0.5ml is set in 100ml volumetric flask, is diluted to scale with 50% acetonitrile-water, is shaken up, and every 1ml is made containing about compound A~L points
Not Wei 0.5 μ g solution, reference standard solution or reference substance solution as compound A~L.
(4) it prepares Fimasartan test solution: taking the Fimasartan bulk pharmaceutical chemicals about 10mg of above-mentioned preparation (with anhydride
The conversion coefficient of meter, Fimasartan potassium and Fimasartan potassium trihydrate and monohydrate is respectively as follows: 0.90 and 0.96), accurate
It is weighed, it sets in 20ml volumetric flask, adds 50% acetonitrile-water appropriate, ultrasound makes to dissolve, and is diluted to scale with 50% acetonitrile-water, shakes
It is even to get.The concentration of Fimasartan bulk pharmaceutical chemicals test solution is about 0.5mg/ml.
Embodiment 12:
The preparation of Fimasartan and sample preparation are with embodiment 6~10, and gradient design is the same as embodiment 11.
Chromatographic condition:
Chromatographic column: Aglea Venusil XBP C8 5μm4.6×250mm
The concentration of test solution: 0.5mg/ml
The concentration of each impurity of reference standard solution: 1000ppm
Mobile phase: A:0.1% phosphoric acid (ammonium hydroxide or sodium hydroxide tune pH to 3.0), B: acetonitrile
Detection wavelength: 210nm
Sampling volume: 20 μ l
Diluent: 50% acetonitrile-water
Column temperature: 25 DEG C
Flow velocity: 1.0ml/min
Embodiment 13:
The preparation of Fimasartan and sample preparation are with embodiment 6~10, and gradient design is the same as embodiment 11.
Chromatographic condition:
Chromatographic column: Aglea Venusil XBP C8 5μm 4.6×250mm
The concentration of test solution: 0.5mg/ml
The concentration of each impurity of reference standard solution: 1000ppm
Mobile phase: A:1.0% phosphoric acid (ammonium hydroxide or sodium hydroxide tune pH to 2.5), B: acetonitrile
Detection wavelength: 210nm
Sampling volume: 20 μ l
Diluent: 50% acetonitrile-water
Column temperature: 35 DEG C
Flow velocity: 0.8ml/min
Embodiment 14:
The preparation of Fimasartan and sample preparation are with embodiment 6~10, and gradient design is the same as embodiment 11.
Chromatographic condition:
Chromatographic column: Aglea Venusil XBP C8 5μm 4.6×250mm
The concentration of test solution: 0.5mg/ml
The concentration of each impurity of reference standard solution: 1000ppm
Mobile phase: A:0.1% phosphoric acid (ammonium hydroxide or sodium hydroxide tune pH to 2.5), B: acetonitrile
Detection wavelength: 210nm
Sampling volume: 20 μ l
Diluent: 50% acetonitrile-water
Column temperature: 30 DEG C
Flow velocity: 1.0ml/min
Embodiment 15:
The preparation of Fimasartan and sample preparation are with embodiment 6~10, and gradient design is the same as embodiment 11.
Chromatographic column: Aglea Venusil XBP C8 5μm 4.6×250mm
The concentration of test solution: 0.5mg/ml
The concentration of each impurity of reference standard solution: 1000ppm
Mobile phase: A:1.0% phosphoric acid (ammonium hydroxide or sodium hydroxide tune pH to 3.0), B: acetonitrile
Detection wavelength: 210nm
Sampling volume: 20 μ l
Diluent: 50% acetonitrile-water
Column temperature: 35 DEG C
Flow velocity: 0.8ml/min
Embodiment 16:
The preparation of Fimasartan and sample preparation are with embodiment 6~10, and gradient design is the same as embodiment 11.
Chromatographic column: Aglea Venusil XBP C8 5μm 4.6×250mm
The concentration of test solution: 0.5mg/ml
The concentration of each impurity of reference standard solution: 1000ppm
Mobile phase: A:0.1% phosphoric acid (ammonium hydroxide or sodium hydroxide tune pH to 3.0), B: acetonitrile
Detection wavelength: 210nm
Sampling volume: 20 μ l
Diluent: 50% acetonitrile-water
Column temperature: 30 DEG C
Flow velocity: 1.2ml/min
Embodiment 17:
The preparation of Fimasartan and sample preparation are with embodiment 6~10, and gradient design is the same as embodiment 11.
Chromatographic column: Aglea Venusil XBP C8 5μm 4.6×250mm
The concentration of test solution: 0.5mg/ml
The concentration of each impurity of reference standard solution: 1000ppm
Mobile phase: A:0.1% phosphoric acid (ammonium hydroxide or sodium hydroxide tune pH to 3.0), B: acetonitrile
Detection wavelength: 210nm
Sampling volume: 20 μ l
Diluent: 50% acetonitrile-water
Column temperature: 30 DEG C
Flow velocity: 0.8ml/min
Embodiment 18:
The preparation of Fimasartan and sample preparation are with embodiment 6~10, and gradient design is the same as embodiment 11.
Chromatographic column: Aglea Venusil XBP C8 5μm 4.6×250mm
The concentration of test solution: 0.5mg/ml
The concentration of each impurity of reference standard solution: 1000ppm
Mobile phase: A:1.0% phosphoric acid (ammonium hydroxide or sodium hydroxide tune pH to 3.0), B: acetonitrile
Detection wavelength: 210nm
Sampling volume: 20 μ l
Diluent: 50% acetonitrile-water
Column temperature: 25 DEG C
Flow velocity: 1.2ml/min
Embodiment 19:
Chromatographic column: Aglea Venusil XBP C8 5μm 4.6×250mm
The concentration of test solution: 0.5mg/ml
The concentration of each impurity of reference standard solution: 1000ppm
Mobile phase: A:0.05% phosphoric acid (ammonium hydroxide or sodium hydroxide tune pH to 2.5), B: acetonitrile
Detection wavelength: 210nm
Sampling volume: 20 μ l
Diluent: 50% acetonitrile-water
Column temperature: 35 DEG C
Flow velocity: 0.8ml/min
Test procedure:
(1) under gradient HPLC method of the present invention, in Agilent 1260HPLC instrument (Agilent company of the U.S.),
According to the chromatographic condition of embodiment 14, take compound A~L positioning solution, mark-on mixed solution, compound A~L reference solution,
Each 20 μ l of Fimasartan test solution injects liquid chromatograph, records chromatogram.
(2) under gradient HPLC method of the present invention, according to the chromatographic condition of embodiment 11~19, mark-on mixed solution 20 is taken
μ l injects liquid chromatograph, records chromatogram.
Test result:
The positioning result of compound A~L and principal component Fimasartan in the present embodiment 14 under gradient HPLC method is shown in Table
1；Under the chromatographic condition of the embodiment of the present invention 11~19, separating degree, each impurity in mark-on mixed solution between each impurity with
The relative retention time at Fimasartan peak is shown in Table 2.
Using the chromatographic condition of embodiment 14, containing for the compound A~L contained in test solution is calculated according to external standard method
Amount and total impurities amount the results are shown in Table 3, and the HPLC spectrogram of mark-on mixed solution, the HPLC spectrogram of Fimasartan test solution divide
Figure 12~Figure 13 is not seen.
Analysis according to compound A~L and principal component positioning result and Fimasartan in relation to substance is as a result, according to embodiment
The related substance for the Fimasartan raw material that the preparation method of middle Fimasartan is prepared is as follows: the color of Fimasartan test solution
In spectrogram, the retention time at Fimasartan peak (top) is about 11.91min, and opposite Fimasartan peak retention time is about
0.63 impurity is compound A；The impurity that opposite Fimasartan peak retention time is about 0.70 is compound L；When opposite reservation
Between 0.77 impurity be compound E；The impurity that relative retention time is about 0.85 is compound G；Relative retention time is about
1.12 impurity is compound H；The impurity that relative retention time is about 1.34 is compound I；Relative retention time is about 1.41
Impurity be compound K；The impurity that relative retention time is about 1.54 is compound J；Relative retention time be about 1.66 it is miscellaneous
Matter is compound C；The impurity that relative retention time is about 2.00 is compound B；The impurity that relative retention time is about 2.04 is
Compound D；The impurity that relative retention time is about 2.10 is compound F；Compound is not detected in Fimasartan test solution
E、G、K、F。
1 compound A~L of table and Fimasartan prepared by the present invention in 14 method of embodiment positioning and each peak between
Separating degree
The relative retention time of each impurity under the conditions of 2 embodiment 11~19 of table
In relation to substance according to the testing result of the chromatographic condition of embodiment 14 in the Fimasartan bulk pharmaceutical chemicals of the above-mentioned preparation of table 3
-- representative is not detected.
Claims (19)
1. the sample purity of the Fimasartan or its hydrate or the drug containing Fimasartan or its hydrate of detection Formula II
Method, including passing through the impurity in chromatography determination sample, the impurity is selected from following compound A, B, C, D, E, F, G, H, I,
One of J, K and L or a variety of,
Wherein Fimasartan is the thio formyl methyl -6- methyl -3- of 2- butyl -5- dimethylamino [[2'- (1H-TETRAZOLE -5-
Base) biphenyl -4- base] methyl] pyrimidine -4 (3H) -one sylvite, structure is as shown in Formula II:
Compound A is (E) -2- (- 6 methyl -4- carbonyl pyrimidine base -5- (4H) of 2- butyl-subunit)-n,N-dimethylacetamide,
And it has a structure that
Compound B is triphenyl methane, and is had a structure that
Compound C is triphenyl alcohol, and is had a structure that
Compound D is triphenyl methyl ether, and is had a structure that
Compound E is 2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -4- first
Base -6- oxygen -1,6- dihydro-pyrimidin -5- base)-n,N-dimethylacetamide, and have a structure that
Compound F is 2- (2- butyl -4- methyl -6- oxygen -1- ((2 '-(1- trityl -1H- tetrazole -5- bases)-[1,1 ' -
Diphenyl] -4- base) methyl) 1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide, and have a structure that
Compound G is 2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -4- first
Base -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetic acid, and have a structure that
Compound H is 2- (2- butyl-4- methyl-1-((2 '-(1- methyl-1 H- tetrazole-5- base)-[1,1 '-diphenyl]-4-
Base) methyl) -6- oxygen -1,6- dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide, and have a structure that
Compound I is 2- (2- butyl -1- ((2 '-itrile groups-[1,1 '-diphenyl] -4- base) methyl) -4- methyl -6- oxygen -1,6-
Dihydro-pyrimidin -5- base)-N, N- dimethyl thio acetamide, and have a structure that
Compound J is 2- (1- ([1,1 '-diphenyl] -4- base-methyl) -2- butyl -4- methyl -6- oxygen -1,6- dihydro-pyrimidin -
5- yl)-N, N- dimethyl thio acetamide, and have a structure that
Compound K is 2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -6- sulphur
For carbonyl -1,6- dihydroxy-pyrimidine -5- base)-N, N '-dimethyl thioacetamide, and have a structure that
Compound L is 2- (1- ((2 '-(1H- tetrazole -5- base)-[1,1 '-diphenyl] -4- base) methyl) -2- butyl -4- first
Base -6- oxygen -1,6- dihydro-pyrimidin -5- base) acetoxymethyl ester, and have a structure that
The described method includes:
It (1) will be containing Fimasartan or the dissolution of its hydrate sample in a solvent to prepare sample solution；
(2) by sample dissolution any one or more of in compound A, B, C, D, E, F, G, H, I, J, K and L in a solvent with system
Standby reference standard solution or reference substance solution；
(3) chromatographic technique is implemented to sample solution and reference standard solution；And
(4) by referring to known compound A present in the reference standard solution, B, C, D, E, F, G, H, I, one kind of J, K and L
Or it is a variety of, the presence of any one or more of compound A, B, C, D, E, F, G, H, I, J, K and L in sample are measured,
Wherein the chromatography is gradient HPLC method, wherein the solid phase that the chromatography uses is octadecylsilane bonded silica
Glue,
In the gradient HPLC method, Detection wavelength 210nm, mobile phase includes the group of buffer solution A and organic solvent B
It closes, the buffer solution A is the aqueous solution that phosphoric acid and phosphate or phosphatic aqueous solution adjusted pH with phosphoric acid, described to have
Solvent B is acetonitrile, wherein concentration existing for the phosphoric acid is 0.03 volume % to 1.5 volume %, wherein the buffer solution A
PH be 2.0~4.0,
Wherein in the gradient HPLC method, the mobile phase includes following gradient design:
2. the method for claim 1 wherein concentration existing for the phosphoric acid is 0.05 volume %-1.3 volume %.
3. the method for claim 1 wherein concentration existing for the phosphoric acid is 0.08 volume %-1.2 volume %.
4. the method for claim 1 wherein concentration existing for the phosphoric acid is 0.1 volume of volume %~1.0 %.
5. the method for any one of claim 1-4, wherein the gradient HPLC method 25~60 DEG C at a temperature of carry out.
6. the method for any one of claim 1-4, wherein the gradient HPLC method 25~50 DEG C at a temperature of carry out.
7. the method for any one of claim 1-4, wherein the gradient HPLC method 25~45 DEG C at a temperature of carry out.
8. the method for any one of claim 1-4, wherein the gradient HPLC method 25~40 DEG C at a temperature of carry out.
9. the method for any one of claim 1-4, wherein the gradient HPLC method 25~35 DEG C at a temperature of carry out.
10. the method for any one of claim 1-4, wherein the gradient HPLC method under the flow velocity of 0.6~1.5ml/min into
Row.
11. the method for any one of claim 1-4, wherein the gradient HPLC method under the flow velocity of 0.7~1.4ml/min into
Row.
12. the method for any one of claim 1-4, wherein the gradient HPLC method under the flow velocity of 0.7~1.3ml/min into
Row.
13. the method for any one of claim 1-4, wherein the gradient HPLC method under the flow velocity of 0.8~1.2ml/min into
Row.
14. the method for any one of claim 1-4, wherein the pH of the buffer solution A is 2.2~3.8.
15. the method for any one of claim 1-4, wherein the pH of the buffer solution A is 2.5~3.5.
16. the method for any one of claim 1-4, wherein the pH of the buffer solution A is 2.5~3.0.
17. the described in any item methods of claim 1-4, wherein the drug containing Fimasartan or its hydrate is solid
Or liquid composition.
18. the method for claim 17, wherein the solid or liquid composition include one or more pharmaceutically acceptable
Excipient.
19. the method for claim 17, wherein the composition of solid form includes pulvis, tablet, capsule, pill and can divide
Scattered granule, liquid composition include solution or suspension, can be administered by oral, injection or instillation approach.
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