CN105388244B - A kind of ACT-064992 is about the HPLC (high performance liquid chromatography) of material - Google Patents
A kind of ACT-064992 is about the HPLC (high performance liquid chromatography) of material Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/89—Inverse chromatography
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- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities
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Abstract
The invention discloses a kind of ACT-064992 is about the HPLC (high performance liquid chromatography) of material, the method adopts reversed phase chromatographic column and UV-detector, with the acid of acetonitrile water beetle as mobile phase, carries out gradient elution.This method can analyze all known impurities in ACT-064992 raw material and its preparation simultaneously, and each known impurity level of main composition Self-control method effective control of the correction up factor can be passed through, separating degree between each impurity peaks and between main peak and other impurities peak is all higher than 1.5, and main peak and each impurity peaks peak purity are 1.0.The present invention provides simple, reliable analysis method for the Quality Control Analysis of ACT-064992 raw material and its preparation.
Description
First, technical field
The present invention relates to a kind of analysis method of chemicalses purity, the relevant material of specifically a kind of ACT-064992
HPLC (high performance liquid chromatography).
2nd, background technology
ACT-064992 (Macitentan), chemical entitled N- [5- (4- bromophenyl) -6- [2- [(5- bromo- 2- pyrimidine radicals) oxygen]
Ethyoxyl] -4- pyrimidine radicals]-N'- sulfonyl propyl amine, chemical structural formula such as following formula (I)：
ACT-064992 is a kind of two-way endothelin-receptor antagonists, can drugmaker of ACE Semi (Actelion by Switzerland's love
Pharmaceuticals Inc.) exploitation.On October 18th, 2013 obtains U.S. FDA approval, for treating pulmonary hypertension
(PAH), to delay progression of disease.
ACT-064992 impurity is more, and from structure, 5- (4- bromophenyl) -4,6- disubstituted pyrimidines parent nucleus and diethoxy are tied
The reaction site (chlorine atom, hydroxyl) all having two activity to be equal in structure, can produce in building-up process double condensation impurity (impurity A,
Impurity B, impurity C, impurity D) and hydrolysis impurity (impurity E, impurity F, impurity G)；Unstable sulfonamide in ACT-064992 molecule
Structure hydrolysis produces impurity H, impurity I and impurity J, and the hydrolysis of aryl oxide structure produces impurity E and impurity K, and oxidative degradation produces impurity I
And impurity J.In terms of source, the impurity of ACT-064992 raw material be mainly derived from the reaction raw materials being introduced in building-up process, synthesis in
Mesosome, byproduct of reaction and degradation impurity, the impurity of ACT-064992 preparation derives from raw material and introduces impurity, process contaminants and degraded
Impurity.
The analysis method of ACT-064992 raw material and its preparation does not also have the document report of correlation at present.
3rd, content of the invention
Present invention aim at providing a kind of ACT-064992 about the HPLC (high performance liquid chromatography) of material.
For the disappearance to ACT-064992 raw material and its preparation impurity identification research for the existing document, inventor is tried by technique
System, forced degradation test carry out to ACT-064992 impurity being enriched with, separating-purifying, identify 18 main known impurities, and right
Impurity has carried out ownership of tracing to the source, and closes including ACT-064992 synthetic intermediate (impurity O, impurity P, impurity Q, impurity R), ACT-064992
Become by-product (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G) and degradation impurity (impurity H, impurity I, miscellaneous
Matter J, impurity K, impurity L, impurity M, impurity N).
Inventor attempts to separate each known impurities by isocratic elution, but reaches the feelings of baseline separation in each impurity of guarantee
Under condition, chromatographic retention is more than 2h, takes, impracticable.Therefore, use linear gradient elution method instead, through to flowing phase composition and ratio
Optimal screening, determines the analysis method of the present invention.Press Chinese Pharmacopoeia two annex V D (high performance liquid chromatography of version in 2010 simultaneously
Method) and the definition of annex XIX A (drug standard analysis method verification guide principle) and verification method, carry out specificity and test
Card, result shows that the method can analyze all known impurities in ACT-064992 raw material and its preparation simultaneously, and can be by adding
The each known impurity level of main composition Self-control method effective control of correction factor, between each impurity peaks and main peak and other impurities
Separating degree between peak is all higher than 1.5, and main peak and each impurity peaks peak purity are 1.0.
Described " peak purity " refers to using the photodiode array detector being furnished with corresponding analysis software, gathers, records, divides
The spectroscopic data through chromatographic column separation component for the analysis, the corresponding spectral signature of sign particular separation component automatically generating is conforming
Weighted value.
ACT-064992 of the present invention about the HPLC (high performance liquid chromatography) of material, using reversed phase chromatographic column and ultraviolet detection
Device, is with the mixed solution of acetonitrile-water-formic acid as mobile phase, carries out gradient elution, comprise the steps：
(1) sample preparation：Take ACT-064992 raw material, (take ammonium hydrogen carbonate with the ammonium bicarbonate buffers that volume ratio is 1: 4
1.58g, the 1000ml that adds water makes dissolving, with ammonia regulation pH value to 9.0)-acetonitrile solution ultrasonic dissolution, filter or be centrifuged, prepare
Become the ACT-064992 solution that concentration is 0.1-1.5mg/ml；
(2) chromatographic condition setting：Using anti-phase C18 post, column temperature is arranged on 20-50 DEG C；With volume ratio for 49: 49-54:
The acetonitrile-water of 0.05-0.15-formic acid mixed liquor is mobile phase A, with volume ratio for 85: 14-17: 0.05-0.15 acetonitrile-water-
Formic acid mixed liquor is Mobile phase B, carries out gradient elution；Flow velocity is 0.5-2.0ml/min；Detection wavelength is 250-270nm；Eluting
Liquid is made up of mobile phase A and Mobile phase B；
(3) detect：Take the ACT-064992 solution that step (1) is prepared, sample introduction 10-50 μ l, record chromatogram.
In step (2), anti-phase C18 post is selected from Grace Smart C18 (250 × 4.6mm, 5 μm), Agilent C18 (250
× 4.6mm, 5 μm) or Apollo C18 (250 × 4.6mm, 5 μm), preferably Agilent C18 (250 × 4.6mm, 5 μm).
In step (2), mobile phase A is that 49: 50-52: 0.08-0.12 mixing is constituted by volume for acetonitrile, water and formic acid, excellent
Volume ratio is selected to be 49: 51: 0.1；It is acetonitrile, water and formic acid 85: 14-16: 0.08-0.12 mixing structure by volume in Mobile phase B
Become, preferred volume ratio is 85: 15: 0.1.
In step (2), gradient elution program is：The percent by volume that 0-10min mobile phase A accounts for eluent is 100%,
The percent by volume that 10min-35min mobile phase A accounts for eluent is progressively decremented to 0%, 35min-50min mobile phase A by 100%
The percent by volume that the percent by volume accounting for eluent accounts for eluent for 0%, 50min-52min mobile phase A is progressively incremented by by 0%
The percent by volume accounting for eluent to 100%, 52min-60min mobile phase A is 100%.
More excellent elution program is：
The percent by volume that 0-10min mobile phase A accounts for eluent accounts for eluent for 100%, 10min-35min mobile phase A
Percent by volume by 100% be progressively decremented to 0%, 35min-40min mobile phase A account for eluent percent by volume be 0%,
The percent by volume that 40min-42min mobile phase A accounts for eluent is progressively incremented to 100%, 42min-50min mobile phase A by 0%
The percent by volume accounting for eluent is 100%.
In step (2), flow velocity is 0.5-2.0ml/min, preferably 0.5-1.5ml/min；Detection wavelength is 250-270nm, excellent
Select 255-265nm.
In step (3), sample size is 10 μ l-20 μ l.
The relevant material in ACT-064992 raw material and preparation, 18 known impurities can be efficiently controlled using the inventive method
All can analyze out on a collection of illustrative plates, the separating degree between each impurity peaks and between main peak and other impurities peak is all higher than 1.5,
Main peak and each impurity peaks peak purity are 1.0.Analysis process is shown in embodiment 1, and typical chromatogram is shown in Fig. 1, and result of calculation is shown in Table 1.
Table 1 ACT-064992 and each known impurities chromatographic isolation parametric results
| Title | Relative retention time | Separating degree | Peak purity |
| Impurity G | 0.11 | / | 1.0 |
| Impurity K | 0.14 | 5.97 | 1.0 |
| Impurity I | 0.15 | 1.56 | 1.0 |
| Impurity E | 0.23 | 11.48 | 1.0 |
| Impurity F | 0.27 | 4.90 | 1.0 |
| Impurity P | 0.28 | 1.53 | 1.0 |
| Impurity N | 0.32 | 2.88 | 1.0 |
| Impurity H | 0.33 | 2.65 | 1.0 |
| Impurity R | 0.35 | 1.89 | 1.0 |
| Impurity J | 0.49 | 9.02 | 1.0 |
| Impurity C | 0.51 | 2.20 | 1.0 |
| Impurity Q | 0.76 | 18.64 | 1.0 |
| Impurity L | 0.81 | 1.66 | 1.0 |
| Impurity D | 0.84 | 2.71 | 1.0 |
| Impurity M | 0.86 | 1.57 | 1.0 |
| Impurity A | 0.91 | 4.60 | 1.0 |
| Impurity O | 0.95 | 3.00 | 1.0 |
| ACT-064992 | 1.00 | 3.75 | 1.0 |
| Impurity B | 1.20 | 17.51 | 1.0 |
Degradation impurity under various complex environments for the ACT-064992, method specificity can be analyzed using the inventive method
By force.By Chinese Pharmacopoeia two annex V D (high performance liquid chromatography) of version in 2010 and annex XIX A, (drug standard is analyzed
Method validation guideline) definition and verification method, by ACT-064992 respectively with acid, alkali, high temperature, oxidation, illumination destroy, make
Sample must be destroyed, gather each destruction sample chromatogram figure respectively by the inventive method, analysis process is shown in embodiment 2, typical chromatogram
See accompanying drawing 2～7.Result shows that the method can analyze the sample destroying through acid, alkali, high temperature, oxidation, illumination, and main peak is miscellaneous with each
Mass peak all can reach baseline separation, and main peak purity is 1.0.
Quantitative analyses can be carried out to each known impurities using the inventive method.By Chinese Pharmacopoeia two annex V of version in 2010
The definition of D (high performance liquid chromatography) and annex XIX A (drug standard analysis method verification guide principle) and authentication
Method, detects, result shows the method using impurity counter point to the test limit of 18 known impurities of ACT-064992, quantitative limit
High to the response value of each impurity, the energy each known impurities of effective control, the results are shown in Table 2.
Table 2 ACT-064992 known impurities quantitative analyses certificate parameter result
| Title | Test limit (μ g/ml) | Quantitative limit (μ g/ml) | Correction factor |
| ACT-064992 | 0.018 | 0.060 | / |
| Impurity O | 0.014 | 0.048 | 0.89 |
| Impurity F | 0.019 | 0.063 | 0.93 |
| Impurity P | 0.038 | 0.128 | 0.78 |
| Impurity G | 0.059 | 0.196 | 0.98 |
| Impurity Q | 0.018 | 0.060 | 0.88 |
| Impurity E | 0.022 | 0.073 | 0.91 |
| Impurity H | 0.019 | 0.062 | 1.07 |
| Impurity R | 0.017 | 0.058 | 0.75 |
| Impurity D | 0.014 | 0.048 | 1.12 |
| Impurity B | 0.011 | 0.037 | 0.77 |
| Impurity I | 0.006 | 0.020 | 0.92 |
| Impurity A | 0.013 | 0.038 | 0.96 |
| Impurity K | 0.044 | 0.147 | 0.89 |
| Impurity C | 0.092 | 0.306 | 0.95 |
| Impurity J | 0.019 | 0.062 | 1.06 |
| Impurity N | 0.068 | 0.228 | 1.04 |
| Impurity L | 0.034 | 0.098 | 1.06 |
| Impurity M | 0.029 | 0.087 | 0.91 |
The present invention carries out, to ACT-064992 raw material and its preparation impurity, ownership of tracing to the source first, identifies 18 known impurities,
It is ACT-064992 raw material and its research of preparation relevant material provides the spectrum reference of reliable impurity, there is larger actively progressive effect
Fruit and actual application value.
4th, brief description
Fig. 1 is the mixed chromatogram (in figure A is artwork, and B is enlarged drawing) of embodiment 1 ACT-064992 and known impurities.
Fig. 2 is that embodiment 2 acid destroys chromatogram.
Fig. 3 is that embodiment 2 alkali destroys chromatogram.
Fig. 4 is embodiment 2 high temperature chromatogram.
Fig. 5 is embodiment 2 Oxidative demage chromatogram.
Fig. 6 is that embodiment 2 illumination destroys chromatogram.
Fig. 7 is that embodiment 2 does not destroy sample chromatogram figure.
Fig. 8 is the relevant material chromatogram of embodiment 3 ACT-064992.
Fig. 9 is the relevant material chromatogram of embodiment 4 ACT-064992 piece (10mg).
5th, specific embodiment
Embodiment 1：
Detecting instrument and chromatographic condition：
High performance liquid chromatograph：LC-10AD pump, SPD-M10A detector
Chromatographic column：Agilent C18 (250 × 4.6mm, 5 μm)；Mobile phase A：Volume ratio be 49: 51: 0.1 acetonitrile-
Water-formic acid solution；Mobile phase B：Volume ratio is 85: 15: 0.1 acetonitrile-water-formic acid solution；Detection wavelength：260nm；Flow velocity：
1.0ml/min；Sample size：20μl.
Experimental procedure：
(1) sample preparation：
Take ACT-064992, known impurities A-R each appropriate, (take ammonium hydrogen carbonate with the ammonium bicarbonate buffers of volume ratio 1: 4
1.58g, the 1000ml that adds water makes dissolving, adjusts pH value to 9.0 with ammonia)-acetonitrile solution ultrasonic dissolution diluting makes containing Ma Xi
It is about the solution that 1mg/ml, impurity A-R are each about 1 μ g/ml for smooth, as sample solution；
(2) gradient elution program setting：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 10 | 100 | 0 |
| 35 | 0 | 100 |
| 50 | 0 | 100 |
| 52 | 100 | 0 |
| 60 | 100 | 0 |
(3) detect：Take above-mentioned sample solution, respectively sample introduction 20 μ l, record chromatogram respectively.
Typical chromatogram is shown in Fig. 1.
Embodiment 2：
Detecting instrument and chromatographic condition：
High performance liquid chromatograph：LC-10AD pump, SPD-M10A detector
Chromatographic column：Agilent C18 (250 × 4.6mm, 5 μm)；Mobile phase A：With embodiment 1, Mobile phase B：Same embodiment
1；Flow velocity：1.0ml/min；Detection wavelength：260nm；Sampling volume：20μl.
(1) sample preparation：
Acid destroys：Take ACT-064992 piece, finely ground, take fine powder appropriate (being approximately equivalent to 50mg containing ACT-064992), take Ma Xi to replace
Smooth raw material 50mg, puts in 50ml measuring bottle, plus 1mol/L hydrochloric acid solution 5ml, puts 80 DEG C of water-baths 1 hour, rapid cooling, plus 1mol/L
Sodium hydroxide solution 5ml neutralizes, and (takes ammonium hydrogen carbonate 1.58g, the 1000ml that adds water makes with the ammonium bicarbonate buffers of volume ratio 1: 4
Dissolving, adjusts pH value to 9.0 with ammonia)-acetonitrile solution dissolves and is diluted to scale, shake up, filter, and destroys sample as acid；
Alkali destroys：Take ACT-064992 piece, finely ground, take fine powder appropriate (being approximately equivalent to 50mg containing ACT-064992), put 50ml amount
In bottle, plus 1mol/L sodium hydroxide solution 5ml, put 40 DEG C of water-baths 30 minutes, rapid cooling, plus in 1mol/L hydrochloric acid solution 5ml
With (take ammonium hydrogen carbonate 1.58g, the 1000ml that adds water makes dissolving, adjusts pH with ammonia with the ammonium bicarbonate buffers of volume ratio 1: 4
It is worth to 9.0)-acetonitrile solution ultrasonic dissolution be diluted to scale, shake up, filter, destroy sample as alkali；
High temperature：Take ACT-064992 piece, finely ground, take fine powder appropriate (being approximately equivalent to 50mg containing ACT-064992), put 50ml
In measuring bottle, heat 4 hours in 105 DEG C, (taken ammonium hydrogen carbonate 1.58g, add water with the ammonium bicarbonate buffers of volume ratio 1: 4
1000ml makes dissolving, adjusts pH value to 9.0 with ammonia)-acetonitrile solution ultrasonic dissolution be diluted to scale, shake up, filter, as
High temperature sample；
Oxidative demage：Take ACT-064992 piece, finely ground, take fine powder appropriate (being approximately equivalent to 50mg containing ACT-064992), put 50ml
In measuring bottle, plus 30% hydrogenperoxide steam generator 5ml, put 60 DEG C of water-baths 1 hour, rapid cooling, delayed with the ammonium hydrogen carbonate of volume ratio 1: 4
- acetonitrile solution ultrasonic dissolution is simultaneously to rush liquid (taking ammonium hydrogen carbonate 1.58g, the 1000ml that adds water makes dissolving, with ammonia regulation pH value to 9.0)
It is diluted to scale, shakes up, filter, as Oxidative demage sample；
Photo damage：Take ACT-064992 piece, finely ground, take fine powder appropriate (being approximately equivalent to 50mg containing ACT-064992), put 50ml amount
In bottle, (take ammonium hydrogen carbonate 1.58g, the 1000ml that adds water makes dissolving, is adjusted with ammonia with the ammonium bicarbonate buffers of volume ratio 1: 4
PH value is to 9.0)-acetonitrile solution ultrasonic dissolution be diluted to scale, put and irradiate 72 hours under high light (5000Lx), centrifugation, as
Photo damage sample.
Do not destroy：Take ACT-064992 piece, finely ground, take fine powder appropriate (being approximately equivalent to 50mg containing ACT-064992), put 50ml amount
In bottle, (take ammonium hydrogen carbonate 1.58g, the 1000ml that adds water makes dissolving, is adjusted with ammonia with the ammonium bicarbonate buffers of volume ratio 1: 4
PH value is to 9.0)-acetonitrile solution ultrasonic dissolution be diluted to scale, shake up, filter, as not bad sample.
(2) gradient elution program setting：With embodiment 1.
(3) detect：Take above-mentioned each sample solution, respectively sample introduction 20 μ l, record chromatogram respectively.
Typical chromatogram is shown in Fig. 2-7.
Embodiment 3：
Detecting instrument and chromatographic condition：
High performance liquid chromatograph：SSI Series 1500 pump, Series 1500PDA detector
Chromatographic column：Agilent C18 (250 × 4.6mm, 5 μm)；Mobile phase A：With embodiment 1, Mobile phase B：Same embodiment
1；Flow velocity：1.0ml/min；Detection wavelength：260nm；Sampling volume：20μl.
Experimental procedure：
(1) sample preparation：Take ACT-064992 appropriate, (take ammonium hydrogen carbonate with the ammonium bicarbonate buffers of volume ratio 1: 4
1.58g, the 1000ml that adds water makes dissolving, adjusts pH value to 9.0 with ammonia)-acetonitrile solution ultrasonic dissolution diluting makes containing Ma Xi
For the solution of smooth 1mg/ml, as sample solution.
(2) gradient elution program setting：
| Time (minute) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 10 | 100 | 0 |
| 35 | 0 | 100 |
| 40 | 0 | 100 |
| 42 | 100 | 0 |
| 50 | 100 | 0 |
(3) detect：Take above-mentioned each sample solution, respectively sample introduction 20 μ l, record chromatogram respectively.
Typical chromatogram is shown in Fig. 8.
Embodiment 4：
Detecting instrument and chromatographic condition：
High performance liquid chromatograph：SSI Series 1500 pump, Series 1500PDA detector
Chromatographic column：Agilent C18 (250 × 4.6mm, 5 μm)；Mobile phase A：With embodiment 1, Mobile phase B：Same embodiment
1；Flow velocity：1.0ml/min；Detection wavelength：260nm；Sampling volume：20μl.
Experimental procedure：
(1) sample preparation：Take ACT-064992 piece, finely ground, take fine powder appropriate (being approximately equivalent to 50mg containing ACT-064992), put
In 50ml measuring bottle, (take ammonium hydrogen carbonate 1.58g, the 1000ml that adds water makes dissolving, uses ammonia with the ammonium bicarbonate buffers of volume ratio 1: 4
Water adjusts pH value to 9.0)-acetonitrile solution ultrasonic dissolution be diluted to scale, shake up, filter, as sample solution.
(2) gradient elution program setting：With embodiment 3.
(3) detect：Take above-mentioned each sample solution, respectively sample introduction 20 μ l, record chromatogram respectively.
Typical chromatogram is shown in Fig. 9.
Claims (8)
1. a kind of ACT-064992 about the HPLC (high performance liquid chromatography) of material it is characterised in that comprising the steps：
(1) sample preparation：Take ACT-064992 raw material, the ammonium bicarbonate buffers-acetonitrile solution being 1: 4 with volume ratio is ultrasonic molten
Solution, filters or is centrifuged, and is configured to the ACT-064992 solution that concentration is 0.1-1.5mg/ml；
(2) chromatographic condition setting：Using anti-phase C18 post, column temperature is arranged on 20-50 DEG C；With volume ratio for 49: 49-54: 0.05-
0.15 acetonitrile-water-formic acid mixed liquor is mobile phase A, with volume ratio for 85: 14-17: 0.05-0.15 acetonitrile-water-formic acid
Mixed liquor is Mobile phase B, carries out gradient elution；Flow velocity is 0.5-2.0ml/min；Detection wavelength is 250-270nm；Eluent by
Mobile phase A and Mobile phase B are constituted；
In step (2), gradient elution program is：The percent by volume that 0-10min mobile phase A accounts for eluent is 100%, 10min-
The percent by volume that 35min mobile phase A accounts for eluent is progressively decremented to 0%, 35min-50min mobile phase A by 100% and accounts for eluting
The percent by volume that the percent by volume of liquid accounts for eluent for 0%, 50min-52min mobile phase A is progressively incremented to by 0%
The percent by volume that 100%, 52min-60min mobile phase A accounts for eluent is 100%；
(3) detect：Take the ACT-064992 solution that step (1) is prepared, sample introduction 10-50 μ l, record chromatogram；
The relevant material of described ACT-064992 is 18 main known impurities, respectively：
2. HPLC (high performance liquid chromatography) according to claim 1 it is characterised in that：
In step (2), anti-phase C18 post is selected from 250 × 4.6mm, the Grace Smart C18 of 5 μm of particle diameter, 250 × 4.6mm, particle diameter
5 μm of Agilent C18 or 250 × 4.6mm, the Apollo C18 of 5 μm of particle diameter.
3. HPLC (high performance liquid chromatography) according to claim 1 and 2 it is characterised in that：
In step (2), anti-phase C18 post is 250 × 4.6mm, the Agilent C18 of 5 μm of particle diameter.
4. HPLC (high performance liquid chromatography) according to claim 1 it is characterised in that：
In step (2), mobile phase A is that 49: 50-52: 0.08-0.12 mixing is constituted by volume for acetonitrile, water and formic acid；Mobile phase B
In for acetonitrile, water and formic acid, 85: 14-16: 0.08-0.12 mixing is constituted by volume.
5. the HPLC (high performance liquid chromatography) according to claim 1 or 4 it is characterised in that：
In step (2), mobile phase A is that 49: 51: 0.1 mixing are constituted by volume for acetonitrile, water and formic acid；In Mobile phase B be acetonitrile,
85: 15: 0.1 mixing are constituted by volume for water and formic acid.
6. HPLC (high performance liquid chromatography) according to claim 1 it is characterised in that：
In step (2), gradient elution program is：The percent by volume that 0-10min mobile phase A accounts for eluent is 100%, 10min-
The percent by volume that 35min mobile phase A accounts for eluent is progressively decremented to 0%, 35min-40min mobile phase A by 100% and accounts for eluting
The percent by volume that the percent by volume of liquid accounts for eluent for 0%, 40min-42min mobile phase A is progressively incremented to by 0%
The percent by volume that 100%, 42min-50min mobile phase A accounts for eluent is 100%.
7. the HPLC (high performance liquid chromatography) according to claim 1 or 6 it is characterised in that：
In step (2), flow velocity is 0.5-1.5ml/min；Detection wavelength is 255-265nm.
8. HPLC (high performance liquid chromatography) according to claim 1 it is characterised in that：
In step (3), sample size is 10 μ l-20 μ l.
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|---|---|---|---|---|
| CN106478520B (en) * | 2016-10-11 | 2018-09-11 | 合肥久诺医药科技有限公司 | A kind of synthetic method of macitentan contamination levels product |
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