Impurity in the compound of a kind of angiotensin receptor antagonist and nep inhibitorSeparation methodTechnical field
The present invention relates to the composite technology fields more particularly to one of angiotensin receptor antagonist and nep inhibitorThe separation method of impurity in the compound of kind angiotensin receptor antagonist and nep inhibitor.
Background technology
Angiotensin receptor blocker (ARBs, angiotensin-ii antagonist) can be by preventing Angiotensin IIIt is combined with its receptor on vascular wall, is reduced so as to cause blood pressure.Since AT1 receptors can be inhibited, so such antagonistIt can be used for anti-hypertension, or for treating congestive heart failure and other indications.Neutral endopeptidase (EC3.4.24.11；Enkephalinase；atriopeptidase；NEP it is) metalloproteinases containing zinc, it can crack various hydrophobic residualPeptide substrates on the aminoterminal of base, the blood plasma level that nep inhibitor (neutral endopeptidase inhibitor, NEPi) can reduce ANP, becauseAnd natruresis can be promoted and there is diuresis.Angiotensin receptor antagonist and nep inhibitor compound ([3- ((1S,3R) -1- biphenyl -4- ylmethyls -3- ethoxy carbonyl -1- butylcarbamoyls) -3 '-methyl -2 ' of propionic acid-(S)-(valeryl{ 2 "-(tetrazolium -5- bases) biphenyl -4 '-ylmethyl } amino) butyric acid] half pentahydrate of trisodium) it is a kind of answering with double actionObject is closed, there is structure shown in Formulas I：
Angiotensin receptor antagonist and nep inhibitor compound have extensive clinical generalization value.
Since the compound drug products of angiotensin receptor antagonist and nep inhibitor are from synthesis starting material to endProduct, during which all there may be impurity for each process, as that may bring starting material, reagent, intermediate and pair in production process intoThe impurity such as product, these Impurities Upon Product Qualities produce a very large impact.Therefore, in order to ensure the safe and effective of drug, to drugIn the separation of each impurity be of great significance.
Currently, yet there are no about angiotensin receptor antagonist in nep inhibitor compound impurity detach it is relatedReport.
Invention content
In view of this, the purpose of the present invention is to provide a kind of angiotensin receptor antagonist and nep inhibitor are compoundThe separation method of impurity in object, method provided by the invention can efficiently separate angiotensin receptor antagonist and NEP suppressionsPlurality of impurities in inhibitor complex.
The present invention provides the separation sides of impurity in the compound of a kind of angiotensin receptor antagonist and nep inhibitorMethod, including：
The pharmaceutical composition of angiotensin receptor antagonist and nep inhibitor is carried out using high performance liquid chromatography miscellaneousMatter detaches；
Mobile phase in the high performance liquid chromatography includes water phase and organic phase；
The water phase includes phosphate buffer, trifluoroacetic acid solution or formic acid solution；
The organic phase includes organic mixture or acetonitrile, and the organic mixture includes acetonitrile and methanol；
The volume ratio of the water phase and organic phase is (45%~65%):(55%~35%).
Preferably, the molar concentration of the phosphate buffer is 25mmol/L~45mmol/L.
Preferably, the volumetric concentration of the trifluoroacetic acid solution is 0.05%~0.15%.
Preferably, the volumetric concentration of the formic acid solution is 0.05%~0.15%.
Preferably, the volume ratio of the acetonitrile in the organic mixture and methanol is (50~60)：(50~40).
Preferably, the volume ratio of the water phase in the high performance liquid chromatography and organic phase is (50%~60%)：(50%~40%).
Preferably, the flow rate of mobile phase in the high performance liquid chromatography is 0.5mL/min~1.5mL/min.
Preferably, the column temperature in the high performance liquid chromatography is 25 DEG C~45 DEG C.
Preferably, the chromatography column packing in the high performance liquid chromatography is octadecylsilane chemically bonded silica or eight alkaneBase silane bonded silica gel.
Preferably, the Detection wavelength in the high performance liquid chromatography is 240nm~260nm.
The separation method of impurity is adopted in the compound of angiotensin receptor antagonist and nep inhibitor provided by the inventionImpurity separation is carried out with high performance liquid chromatography, the mobile phase selected during high performance liquid chromatography includes the water of special componentMutually and organic phase, answering for angiotensin receptor antagonist and nep inhibitor can be effectively separated using this mobile phaseClose the plurality of impurities in object.The experimental results showed that the angiotensins obtained using impurity separation method provided by the invention byThe mass content ＜ 0.1% of single impurity in body antagonist and nep inhibitor compound sterling.
In addition, in the compound of angiotensin receptor antagonist provided by the invention and nep inhibitor impurity separationMethod and process is simple, favorable reproducibility, and this separation method is accurate and reliable.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show belowThere is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only thisThe embodiment of invention for those of ordinary skill in the art without creative efforts, can also basisThe attached drawing of offer obtains other attached drawings.
Fig. 1 is the preparation of the compound of angiotensin receptor antagonist and nep inhibitor that the embodiment of the present invention 1 providesThe flow chart of technique；
Fig. 2 is the compound of angiotensin receptor antagonist and nep inhibitor that the embodiment of the present invention 1 is preparedNmr spectrum；
Fig. 3 is compound of the method that provides of the embodiment of the present invention 2 to angiotensin receptor antagonist and nep inhibitorThe chromatogram that middle impurity obtains after being detached；
Fig. 4 is compound of the method that provides of the embodiment of the present invention 3 to angiotensin receptor antagonist and nep inhibitorThe chromatogram that middle impurity obtains after being detached；
Fig. 5 is compound of the method that provides of the embodiment of the present invention 4 to angiotensin receptor antagonist and nep inhibitorThe chromatogram that middle impurity obtains after being detached；
Fig. 6 is compound of the method that provides of comparative example 1 of the present invention to angiotensin receptor antagonist and nep inhibitorThe chromatogram that middle impurity obtains after being detached.
Specific implementation mode
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementationExample is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is commonThe every other embodiment that technical staff is obtained without making creative work belongs to the model that the present invention protectsIt encloses.
The present invention provides the separation sides of impurity in the compound of a kind of angiotensin receptor antagonist and nep inhibitorMethod, including：
The pharmaceutical composition of angiotensin receptor antagonist and nep inhibitor is carried out using high performance liquid chromatography miscellaneousMatter detaches；
Mobile phase in the high performance liquid chromatography includes water phase and organic phase；
The water phase includes phosphate buffer, trifluoroacetic acid solution or formic acid solution；
The organic phase includes organic mixture or acetonitrile, and the organic mixture includes acetonitrile and methanol；
The volume ratio of the water phase and organic phase is (45%~65%):(55%~35%).
The present invention is carried out the compound of angiotensin receptor antagonist and nep inhibitor using high performance liquid chromatographyImpurity detaches.In the present invention, the compound of the angiotensin receptor antagonist and nep inhibitor is [3- ((1S, 3R)-1- biphenyl -4- ylmethyl -3- ethoxy carbonyl -1- butylcarbamoyls) -3 '-methyl -2 ' of propionic acid-(S)-(valeryl { 2 " -(tetrazolium -5- bases) biphenyl -4 '-ylmethyl } amino) butyric acid] half pentahydrate of trisodium, there is structure shown in Formulas I：
The present invention does not have the source of the compound of the angiotensin receptor antagonist and nep inhibitor special limitSystem, can by market buy obtain, can be also prepared by method well known to those skilled in the art, such as can according to application No. is200680001733.0 Chinese patent " pharmaceutical combination product of the inhibitor of angiotensin receptor antagonist and NEP " institute is publicThe method opened is prepared.In an embodiment of the present invention, the angiotensin receptor antagonist and nep inhibitor compoundPreparation method be：
It will be reacted with the compound of structure, Valsartan and sodium hydroxide shown in formula 1, obtain angiotensin receptorThe compound of antagonist and nep inhibitor：
In an embodiment of the present invention, the temperature of the compound of structure, Valsartan shown in the formula 1 and sodium hydroxide reactionIt is 20 DEG C~30 DEG C.In an embodiment of the present invention, the compound of structure, Valsartan shown in the formula 1 and sodium hydroxide reactionTime be 10h.In an embodiment of the present invention, mole of the compound of structure, Valsartan and sodium hydroxide shown in the formula 1Than being 1:1:3.In an embodiment of the present invention, the preparation method of the compound of structure shown in the formula 1 is：
It will be reacted with the compound of structure and hydrochloric acid shown in formula 2, obtain the compound with structure shown in formula 1：
In an embodiment of the present invention, the compound of structure shown in the formula 2 and the temperature of hydrochloric acid reaction are 20 DEG C~30℃.In an embodiment of the present invention, the time of the compound of structure shown in the formula 2 and hydrochloric acid reaction is 2h.The present invention'sIn embodiment, the compound of structure shown in the formula 2 and the molar ratio of hydrochloric acid ratio are 1:3.In an embodiment of the present invention, describedThe preparation method of the compound of structure shown in formula 2 is：
It will be reacted with the compound of structure and calcium chloride shown in formula 3, obtain the chemical combination with structure shown in formula 2Object：
In an embodiment of the present invention, the compound of structure shown in the formula 3 and calcium chloride reaction temperature be 60 DEG C~70℃.In an embodiment of the present invention, the time of the compound of structure shown in the formula 3 and calcium chloride reaction is 2h.In this hairIn bright embodiment, the compound of structure and the molar ratio of calcium chloride shown in the formula 3 are 1:1.In an embodiment of the present invention,The preparation method of the compound of structure shown in the formula 3 is：
It will in water be reacted with the compound of structure shown in formula 4 and sodium hydroxide, obtain the change of structure shown in formula 4Close object：
In an embodiment of the present invention, the compound of structure shown in the formula 4 and the temperature of sodium hydroxide reaction are 20 DEG C~30 DEG C.In an embodiment of the present invention, the time of the compound of structure shown in the formula 4 and sodium hydroxide reaction is 1h.In the embodiment of the present invention, the compound of structure and the molar ratio of sodium hydroxide shown in the formula 4 are 1:2.The present invention is to 4 institute of formulaShow that the source of the compound of structure does not have special limitation, can be bought and be obtained by market, it also can be ripe according to those skilled in the artThe method known is prepared.
In an embodiment of the present invention, miscellaneous in the compound of the angiotensin receptor antagonist and nep inhibitorMatter is one or more of formula a~formula o：
In the present invention, the high performance liquid chromatography, using high pressure transfusion system, will have not using liquid as mobile phaseThe mobile phases such as the single solvent of same polarity or the mixed solvent of different proportion, buffer solution are pumped into the chromatographic column equipped with stationary phase,It after each ingredient is detached in column, is detected into detector, to realize that the separation to sample is analyzed.
In the present invention, the mobile phase in the high performance liquid chromatography includes water phase and organic phase.In the reality of the present inventionIt applies in example, the water phase includes phosphate buffer, trifluoroacetic acid solution or formic acid solution.In an embodiment of the present invention, instituteThe molar concentration for stating phosphate buffer is 25mmol/L~45mmol/L；In other examples, the phosphate-bufferedThe molar concentration of liquid is 30mmol/L~40mmol/L；In a further embodiment, the molar concentration of the phosphate bufferFor 32mmol/L~38mmol/L；In a further embodiment, the molar concentration of the phosphate buffer be 34mmol/L~36mmol/L；In a further embodiment, the molar concentration of the phosphate buffer is 35mmol/L.
In an embodiment of the present invention, the volumetric concentration of the trifluoroacetic acid solution is 0.05%~0.15%；At otherEmbodiment in, the volumetric concentration of the trifluoroacetic acid solution is 0.08%~0.12%；In a further embodiment, described threeThe volumetric concentration of fluoroacetic acid solution is 0.1%.In an embodiment of the present invention, the trifluoroacetic acid solution is the water of trifluoroacetic acidSolution.
In an embodiment of the present invention, the volumetric concentration of the formic acid solution is 0.05%~0.15%；Other realIt applies in example, the volumetric concentration of the formic acid solution is 0.08%~0.12%；In a further embodiment, the formic acid solutionVolumetric concentration is 0.1%.In an embodiment of the present invention, the formic acid solution is the aqueous solution of formic acid.
In the present invention, the organic phase includes organic mixture or acetonitrile, and the organic mixture includes acetonitrile and firstAlcohol.In an embodiment of the present invention, the volume ratio of the acetonitrile in the organic mixture and methanol is (50~60)：(50~40)；In other examples, the volume ratio of the acetonitrile in the organic mixture and methanol is (52~58)：(48~42)；In a further embodiment, the volume ratio of the acetonitrile in the organic mixture and methanol is (54~56)：(46~44)；AnotherIn outer embodiment, the volume ratio of acetonitrile and methanol in the organic mixture is 55：45.
In an embodiment of the present invention, the initial volume ratio of the water phase in the high performance liquid chromatography and organic phase is(45%~65%)：(55%~35%)；In other examples, the water phase and organic phase in the high performance liquid chromatographyInitial volume ratio be (50%~60%)：(50%~40%)；In a further embodiment, in the high performance liquid chromatographyWater phase and organic phase initial volume ratio be (52%~58%)：(48%~42%)；In a further embodiment, the heightThe initial volume ratio of water phase and organic phase in effect liquid phase chromatogram method is (54%~56%)：(46%~44%)；OtherIn embodiment, the initial volume ratio of water phase and organic phase in the high performance liquid chromatography is 50：50.
In an embodiment of the present invention, the flow rate of mobile phase in the high performance liquid chromatography be 0.5mL/min~1.5mL/min；In other examples, the flow rate of mobile phase in the high performance liquid chromatography be 0.8mL/min~1.2mL/min；In a further embodiment, the flow rate of mobile phase in the high performance liquid chromatography is 1mL/min.
In an embodiment of the present invention, the method that gradient elution may be used makes mobile phase pass through chromatographic column, the gradientThe method of elution is specially：
In 0~35min, volume content of the water phase in mobile phase is reduced to 5%~15% by 45%~55%, organic phaseVolume content in mobile phase has 45%~55% to increase to 85%~95%；
In 35min~45min, volume content of the water phase in mobile phase keeps 5%~15%, and organic phase is in mobile phaseVolume content keep 85%~95%.
In other examples, the method for the gradient elution is specially：
In 0~35min, volume content of the water phase in mobile phase is reduced to 8%~12% by 48%~52%, organic phaseVolume content in mobile phase has 48%~52% to increase to 88%~92%；
In 35min~45min, volume content of the water phase in mobile phase keeps 8%~12%, and organic phase is in mobile phaseVolume content keep 88%~92%.
In a further embodiment, the method for the gradient elution is specially：
In 0~35min, volume content of the water phase in mobile phase is reduced to 10% by 50%, and organic phase is in mobile phaseVolume content there is 50% to increase to 90%；
In 35min~45min, volume content of the water phase in mobile phase keeps 10%, body of the organic phase in mobile phaseProduct content keeps 90%.
In an embodiment of the present invention, the column temperature in the high performance liquid chromatography is 25 DEG C~45 DEG C；Other realIt applies in example, the column temperature in the high performance liquid chromatography is 30 DEG C~40 DEG C；In a further embodiment, the high-efficient liquid phase colorColumn temperature in spectrometry is 35 DEG C.
In an embodiment of the present invention, the chromatography column packing in the high performance liquid chromatography is octadecylsilane keyClose silica gel or eight alkyl silane bonded silica gels；In other examples, the chromatographic column filling in the high performance liquid chromatographyAgent is octadecylsilane chemically bonded silica.
In an embodiment of the present invention, the Detection wavelength in the high performance liquid chromatography is 240nm~260nm；At itIn his embodiment, the Detection wavelength in the high performance liquid chromatography is 245nm~255nm；In a further embodiment, instituteIt is 250nm to state the Detection wavelength in high performance liquid chromatography.
In an embodiment of the present invention, impurity in the compound of the angiotensin receptor antagonist and nep inhibitorSeparation method be：
The pharmaceutical composition of angiotensin receptor antagonist and nep inhibitor is carried out using high performance liquid chromatography miscellaneousMatter detaches；
Mobile phase in the high performance liquid chromatography includes the phosphate buffer and body that molar concentration is 35mmol/LProduct is than being 55：45 acetonitrile and the mixed liquor of methanol；
The initial volume ratio of the mixed liquor of phosphate buffer and acetonitrile and methanol is 50 in the high performance liquid chromatography：50；
Flow rate of mobile phase in the high performance liquid chromatography is 1mL/min；
Column temperature in the high performance liquid chromatography is 35 DEG C；
Chromatography column packing is octadecylsilane chemically bonded silica in the high performance liquid chromatography；
Detection wavelength in the high performance liquid chromatography is 250nm；
Mobile phase is set to pass through chromatographic column using the method for gradient elution, the method for the gradient elution is specially：
In 0~35min, volume content of the water phase in mobile phase is reduced to 10% by 50%, and organic phase is in mobile phaseVolume content there is 50% to increase to 90%；
In 35min~45min, volume content of the water phase in mobile phase keeps 10%, body of the organic phase in mobile phaseProduct content keeps 90%.
Impurity is carried out to the compound of angiotensin receptor antagonist and nep inhibitor using method provided by the inventionSeparation, the angiotensin receptor antagonist obtained after detaching using efficient liquid-phase chromatography method, test and nep inhibitor are answeredClose the impurity content in object sterling；Testing result is the angiotensin receptor that impurity separation method provided by the invention obtainsThe mass content ＜ 0.1% of single impurity, vasotonia provided by the invention in the compound sterling of antagonist and nep inhibitorThe method that impurity detaches in the compound of hormone receptor antagonists and nep inhibitor has preferable effect.
According to《Chinese Pharmacopoeia》The 4th 0512 efficient liquid-phase chromatography method of general rule of version in 2015, test are provided by the inventionThe reproducibility for the method that impurity detaches in the compound of angiotensin receptor antagonist and nep inhibitor, by counting impurityRetention time reproducibility and peak area reproducibility result are it is found that angiotensin receptor antagonist provided by the invention and NEP suppressionsThe method that impurity detaches in the compound of preparation has preferable reproducibility, method accurate and reliable.
Embodiment 1
The compound of angiotensin receptor antagonist and nep inhibitor is prepared according to flow chart shown in FIG. 1, Fig. 1 isThe flow chart of the preparation process of the compound of angiotensin receptor antagonist and nep inhibitor that the embodiment of the present invention 1 provides：
Compound with structure shown in formula 4 is commercial product.
There is the compound of structure and the sodium hydroxide of 1.9g shown in formula 4 to carry out 1 at 20~30 DEG C in water 10gThe reaction of hour, obtains the compound with structure shown in formula 3；
10g is subjected to 2 hours anti-with the compound of structure and the calcium chloride of 2.6g shown in formula 3 at 60~70 DEG CIt answers, obtains the compound with structure shown in formula 2；
15g is subjected to 2 hours anti-with the compound of structure and the 1N hydrochloric acid of 52g shown in formula 2 at 20~30 DEG CIt answers, obtains the compound with structure shown in formula 1；
By the sodium hydroxide with the compound of structure, the Valsartan of 10.6g and 2.9g shown in formula 1 of 10g at 20-30 DEG CThe lower reaction for carrying out 10 hours, obtains the compound of angiotensin receptor antagonist and nep inhibitor.
According to《Chinese Pharmacopoeia》The 4th 0441 NMR spectrum method of general rule of version in 2015 makes the embodiment of the present invention 1The compound of standby obtained angiotensin receptor antagonist and nep inhibitor carries out structure detection, testing result such as Fig. 2 institutesShow, Fig. 2 is the nuclear-magnetism of the compound of angiotensin receptor antagonist and nep inhibitor that the embodiment of the present invention 1 is preparedResonate spectrogram, and as shown in Figure 2, angiotensin receptor antagonist that the embodiment of the present invention 1 is prepared and nep inhibitor are answeredClosing object has structure shown in Formulas I.
Embodiment 2
The angiotensin receptor antagonist and NEP that the embodiment of the present invention 1 is prepared using high performance liquid chromatographyThe compound of inhibitor carries out impurity separation, and the chromatography column packing in the high performance liquid chromatography is octadecylsilane keySilica gel is closed, mobile phase includes the phosphate buffer (water phase) that molar concentration is 35mmol/L and volume ratio is 55：45 acetonitrileWith the mixed liquor (organic phase) of methanol, the initial volume ratio of phosphate buffer and the mixed liquor of acetonitrile and methanol is 50：50, streamThe flow velocity of dynamic phase is 1mL/min, and column temperature is 35 DEG C, Detection wavelength 250nm；Mobile phase is set to pass through using the method for gradient elutionThe method of chromatographic column, the gradient elution is specially：In 0~35min, volume content of the water phase in mobile phase is reduced by 50%To 10%, volume content of the organic phase in mobile phase has 50% to increase to 90%；In 35min~45min, water phase is in mobile phaseIn volume content keep 10%, volume content of the organic phase in mobile phase keeps 90%.
After the embodiment of the present invention 2 carries out impurity separation to the compound of angiotensin receptor antagonist and nep inhibitorObtained chromatogram as shown in figure 3, the method that Fig. 3, which is the embodiment of the present invention 2, to be provided to angiotensin receptor antagonist and NEPThe chromatogram that impurity obtains after being detached in the compound of inhibitor, from the figure 3, it may be seen that the method that the embodiment of the present invention 2 providesLC-Z09 impurity (the structure chemical combination shown in formula h in the compound of angiotensin receptor antagonist and nep inhibitor is detachedObject), LC-01 impurity (compound of structure shown in formula o), LC-Z01 impurity (compound of structure shown in formula a), LC-Z17 impurity(compound of structure shown in formula m), LC-Z02 (compound of structure shown in formula b), the LC-Z03 (chemical combination of structure shown in formula cObject), LC-Z18 (compound of structure shown in formula n), LC-Z15 impurity (compound of structure shown in formula k), LC-Z04 impurity (formulasThe compound of structure shown in d), LC-Z12 impurity (compound of structure shown in formula i), (change of structure shown in formula f of LC-Z06 impurityClose object), LC-Z14 (compound of structure shown in formula j), LC-Z08 impurity (compound of structure shown in formula g), LC-Z16 impurity(compound of structure shown in formula l), LC-Z05 impurity (compound of structure shown in formula e).
According to the method described in above-mentioned technical proposal, the angiotensin receptor antagonist and NEP that are obtained after test separationImpurity content and yield in the compound sterling of inhibitor；Testing result is the impurity separation side that the embodiment of the present invention 2 providesThe mass content of single impurity is respectively less than in the compound sterling for the angiotensin receptor antagonist and nep inhibitor that method obtains0.1%.
According to the method described in above-mentioned technical proposal, angiotensin receptor antagonist and NEP provided by the invention are testedThe reproducibility for the method that impurity detaches in the compound of inhibitor, test result is as shown in Table 1 and Table 2, and table 1 is that the present invention is implementedThe method impurity retention time test result that example 2 provides, table 2 are the survey for the method impurity peak area that the embodiment of the present invention 2 providesTest result.
The method impurity retention time test result that 1 embodiment of the present invention 2 of table provides
The test result for the method impurity peak area that 2 embodiment of the present invention 2 of table provides
Note：In Tables 1 and 2, a~o is related impurities (impurity of structure shown in formula a~formula o)；Based on LC-03 and LC-2Peak；SS is system suitability solution
By Tables 1 and 2 it is found that angiotensin receptor antagonist that the embodiment of the present invention 2 provides and nep inhibitor are answeredClosing the method that impurity detaches in object has preferable reproducibility, method accurate and reliable.
Embodiment 3
The angiotensin receptor antagonist and NEP that the embodiment of the present invention 1 is prepared using high performance liquid chromatographyThe compound of inhibitor carries out impurity separation, and the chromatography column packing in the high performance liquid chromatography is octadecylsilane keySilica gel is closed, mobile phase includes the trifluoroacetic acid aqueous solution (water phase) that mass concentration is 0.1% and mass ratio is 50：50 acetonitrile andThe initial volume ratio of the mixed liquor (organic phase) of methanol, trifluoroacetic acid aqueous solution and the mixed liquor of acetonitrile and methanol is 55：45, streamThe flow velocity of dynamic phase is 0.8mL/min, and column temperature is 25 DEG C, Detection wavelength 250nm；Flowing is set to communicate using the method for gradient elutionChromatographic column is crossed, the method for the gradient elution is specially：In 0~35min, volume content of the water phase in mobile phase is by 55% dropAs low as 5%, volume content of the organic phase in mobile phase has 45% to increase to 95%；In 35min~45min, water phase is flowingVolume content in phase keeps 5%, and volume content of the organic phase in mobile phase keeps 95%.
After the embodiment of the present invention 3 carries out impurity separation to the compound of angiotensin receptor antagonist and nep inhibitorObtained chromatogram as shown in figure 4, the method that Fig. 4, which is the embodiment of the present invention 3, to be provided to angiotensin receptor antagonist and NEPThe chromatogram that impurity obtains after being detached in the compound of inhibitor, as shown in Figure 4, the method that the embodiment of the present invention 3 providesLC-Z09 impurity (the structure chemical combination shown in formula h in the compound of angiotensin receptor antagonist and nep inhibitor is detachedObject), LC-01 impurity (compound of structure shown in formula o), LC-Z01 impurity (compound of structure shown in formula a), LC-Z17 impurity(compound of structure shown in formula m), LC-Z02 (compound of structure shown in formula b), the LC-Z03 (chemical combination of structure shown in formula cObject), LC-Z18 (compound of structure shown in formula n), LC-Z15 impurity (compound of structure shown in formula k), LC-Z04 impurity (formulasThe compound of structure shown in d), LC-Z12 impurity (compound of structure shown in formula i), (change of structure shown in formula f of LC-Z06 impurityClose object), LC-Z14 (compound of structure shown in formula j), LC-Z08 impurity (compound of structure shown in formula g), LC-Z16 impurity(compound of structure shown in formula l), LC-Z05 impurity (compound of structure shown in formula e).
According to the method described in above-mentioned technical proposal, the angiotensin receptor antagonist and NEP that are obtained after test separationImpurity content and yield in the compound sterling of inhibitor；Testing result is the impurity separation side that the embodiment of the present invention 3 providesThe mass content of single impurity is respectively less than in the compound sterling for the angiotensin receptor antagonist and nep inhibitor that method obtains0.1%.
According to the method described in above-mentioned technical proposal, angiotensin receptor antagonist and NEP provided by the invention are testedThe reproducibility for the method that impurity detaches in the compound of inhibitor, test result is as shown in Table 3 and Table 4, and table 3 is that the present invention is implementedThe method impurity retention time test result that example 3 provides, table 4 are the survey for the method impurity peak area that the embodiment of the present invention 3 providesTest result.
The method impurity retention time test result that 3 embodiment of the present invention 3 of table provides
The test result for the method impurity peak area that 4 embodiment of the present invention 3 of table provides
Note：A-o is related impurities；LC-03 and LC-2 is main peak；SS is system suitability solution
By table 3 and table 4 it is found that angiotensin receptor antagonist that the embodiment of the present invention 3 provides and nep inhibitor are answeredClosing the method that impurity detaches in object has preferable reproducibility, method accurate and reliable.
Embodiment 4
The angiotensin receptor antagonist and NEP that the embodiment of the present invention 1 is prepared using high performance liquid chromatographyThe compound of inhibitor carries out impurity separation, and the chromatography column packing in the high performance liquid chromatography is octadecylsilane keyClose silica gel, mobile phase include mass concentration be 0.1% aqueous formic acid (water phase) and acetonitrile (mobile phase), aqueous formic acid withThe initial volume ratio of acetonitrile is 45：55, the flow velocity of mobile phase is 1.2mL/min, and column temperature is 30 DEG C, Detection wavelength 250nm；It adoptsMobile phase is set to pass through chromatographic column with the method for gradient elution, the method for the gradient elution is specially：In 0~35min, water phase existsVolume content in mobile phase is reduced to 15% by 45%, and volume content of the organic phase in mobile phase has 55% to increase to85%；In 35min~45min, volume content of the water phase in mobile phase keeps 15%, and volume of the organic phase in mobile phase containsAmount keeps 85%.
After the embodiment of the present invention 4 carries out impurity separation to the compound of angiotensin receptor antagonist and nep inhibitorObtained chromatogram as shown in figure 5, the method that Fig. 5, which is the embodiment of the present invention 4, to be provided to angiotensin receptor antagonist and NEPThe chromatogram that impurity obtains after being detached in the compound of inhibitor, as shown in Figure 5, the method that the embodiment of the present invention 4 providesLC-Z09 impurity (the structure chemical combination shown in formula h in the compound of angiotensin receptor antagonist and nep inhibitor is detachedObject), LC-01 impurity (compound of structure shown in formula o), LC-Z01 impurity (compound of structure shown in formula a), LC-Z17 impurity(compound of structure shown in formula m), LC-Z02 (compound of structure shown in formula b), the LC-Z03 (chemical combination of structure shown in formula cObject), LC-Z18 (compound of structure shown in formula n), LC-Z15 impurity (compound of structure shown in formula k), LC-Z04 impurity (formulasThe compound of structure shown in d), LC-Z12 impurity (compound of structure shown in formula i), (change of structure shown in formula f of LC-Z06 impurityClose object), LC-Z14 (compound of structure shown in formula j), LC-Z08 impurity (compound of structure shown in formula g), LC-Z16 impurity(compound of structure shown in formula l), LC-Z05 impurity (compound of structure shown in formula e).
According to the method described in above-mentioned technical proposal, the angiotensin receptor antagonist and NEP that are obtained after test separationImpurity content and yield in the compound sterling of inhibitor；Testing result is the impurity separation side that the embodiment of the present invention 4 providesThe mass content of single impurity is respectively less than in the compound sterling for the angiotensin receptor antagonist and nep inhibitor that method obtains0.1%.
According to the method described in above-mentioned technical proposal, angiotensin receptor antagonist and NEP provided by the invention are testedThe reproducibility for the method that impurity detaches in the compound of inhibitor, as shown in table 5 and table 6, table 5 is that the present invention is implemented to test resultThe method impurity retention time test result that example 4 provides, table 6 are the survey for the method impurity peak area that the embodiment of the present invention 4 providesTest result.
The method impurity retention time test result that 5 embodiment of the present invention 4 of table provides
The test result for the method impurity peak area that 6 embodiment of the present invention 4 of table provides
Note：A-o is related impurities；LC-03 and LC-2 is main peak；SS is system suitability solution
By table 5 and table 6 it is found that angiotensin receptor antagonist that the embodiment of the present invention 5 provides and nep inhibitor are answeredClosing the method that impurity detaches in object has preferable reproducibility, method accurate and reliable.
Comparative example 1
The angiotensin receptor antagonist and NEP that the embodiment of the present invention 1 is prepared using high performance liquid chromatographyThe compound of inhibitor carries out impurity separation, and the chromatography column packing in the high performance liquid chromatography is octadecylsilane keySilica gel is closed, mobile phase includes the glacial acetic acid aqueous solution (water phase) and acetonitrile (organic phase) that mass concentration is 0.1%, 0.1% iceThe initial volume of aqueous acetic acid and acetonitrile ratio is 45：55, the flow velocity of mobile phase is 1.0mL/min, and column temperature is 35 DEG C, detects waveA length of 250nm；Mobile phase is set to pass through chromatographic column using the method for gradient elution, the method for the gradient elution is specially：0~35min, volume content of the water phase in mobile phase are reduced to 10% by 45%, and volume content of the organic phase in mobile phase has55% increases to 90%；In 35min~45min, volume content of the water phase in mobile phase keeps 10%, and organic phase is in mobile phaseIn volume content keep 90%.
After comparative example 1 of the present invention carries out impurity separation to the compound of angiotensin receptor antagonist and nep inhibitorObtained chromatogram as shown in fig. 6, the method that Fig. 6, which is comparative example 1 of the present invention, to be provided to angiotensin receptor antagonist and NEPThe chromatogram that impurity obtains after being detached in the compound of inhibitor, it will be appreciated from fig. 6 that the method that comparative example of the present invention 1 providesImpurity in the compound of separating blood vessel angiotensin receptor antagonist and nep inhibitor, impurity cannot reach baseline point with main peakFrom separating effect is poor.
As seen from the above embodiment, answering the present invention provides a kind of angiotensin receptor antagonist and nep inhibitorThe separation method of impurity in object is closed, including：Using high performance liquid chromatography to angiotensin receptor antagonist and nep inhibitorPharmaceutical composition carry out impurity separation；Mobile phase in the high performance liquid chromatography includes water phase and organic phase；The waterInclude mutually phosphate buffer, trifluoroacetic acid solution or formic acid solution；The organic phase includes organic mixture or acetonitrile, describedOrganic mixture includes acetonitrile and methanol；The volume ratio of the water phase and organic phase is (45%~65%)：(55%~35%).The present invention carries out impurity separation using high performance liquid chromatography, and the mobile phase selected during high performance liquid chromatography includes specificThe water phase and organic phase of ingredient can be effectively separated angiotensin receptor antagonist using this mobile phase and NEP press downPlurality of impurities in the compound of preparation.