CN109929011A - A kind of refining methd of pitressin [5-Asp] impurity - Google Patents
A kind of refining methd of pitressin [5-Asp] impurity Download PDFInfo
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Abstract
The invention discloses a kind of refining methds of pitressin [5-Asp] impurity.The refining methd of pitressin [5-Asp] impurity includes the following steps: pitressin [5-Asp] impurity crude product solution successively carried out reverse phase enrichment using efficient liquid phase RP chromatography, reverse phase turns salt, reverse phase purifying；The filler of efficient liquid phase RP chromatography is super resistance to water packing；Reverse phase enrichment, reverse phase turn salt, reverse phase purifying is completed in the reverse phase elution process of a step.The waste liquid that the refining methd of pitressin [5-Asp] impurity of the present invention generates during purifying is largely waste water, can be reused through sewage plant simple process, economic and environment-friendly.
Description
Technical field
The present invention relates to a kind of refining methds of pitressin [5-Asp] impurity.
Background technique
The synthesis polypeptide that pitressin is made of nine amino acid residues, chemical structure areThe theoretical molecular weight 1084.24 of pitressin.Belong to neurohypophysis to swash
Element is called vasopressin or vasopressins, and there are two types of receptor V1 and V2 for it.V1 is mainly distributed on vascular smooth muscle cells film
On, it is played a role by receptor G protein-second messenger's approach, makes vessel retraction, blood pressure increases.V2 in kidney distal tubule and
Concetrated pipe epithelial cell, physiological dose can promote the reabsorption of kidney distal tubule and concetrated pipe to water, play antidiuresis and make
With.
For a drug, a small amount of impurity contained therein is the initiation most important reason of drug side effect, therefore right
The inspection of its purity is one of the important foundation of guarantee drug safety validity, and the content of purity test, according to each drug
Property and feature it is somewhat different, but be substantially intended to be related to the inspection research of respective " in relation to substance ".Polypeptide drug
" in relation to substance " process impurity in synthesis polypeptide class pharmaceutical procedures and the degradation generated since polypeptide is unstable
The impurity such as product, polymer, although the purifying process of synthesis polypeptide has had great progress at present, process impurity is still
The important sources of " in relation to substance ", this is mainly due to some process impurities of synthesis polypeptide (such as peptide disappearance, fracture peptide, oxidation
Peptide, product of disulfide bond exchange etc.) may be very approximate with the property of drug itself, to cause certain difficulty to purifying
Degree.Research shows that the most common catabolite is deamidation product, oxidation product, hydrolysate in synthesis polypeptide.It is more forming
In the various amino acid of peptide, C sections of asparagine, glutamine and peptide chain amides are prone to deamidation reaction (especially in pH
Under value raising and hot conditions).
Wherein, pitressin [5-Asp] impurity is pitressin common impurity in the synthesis process, which can pressurize
Impurity reference substance is used as in plain quality testing, therefore, pitressin [5-Asp] impurity for preparing purity is high controls pitressin quality
It is of great significance.
The common purification process of peptide material mostly uses preparative high performance liquid chromatography at present, which is to obtain height
The most efficient method of purity polypeptide target molecule.General peptide material purifying preparation process design is first mesolow chromatograph enrichment
Target polypeptides, then high pressure chromatographic refining, it is contemplated that the molecular weight about 1kDa of target polypeptides pitressin [5-Asp] impurity,
Without suitable molecular sieve gel column, (its applied sample amount is small, and flow velocity is low, and treating capacity is small, relatively molecular weight is suitble to be greater than 10kDa albumen
Desalination) or ultrafiltration membrane selection.And common separation method has molecular sieve chromatography, ion-exchange chromatography in mesolow chromatography
And hydrophobic interaction chromatography, the partial size for the filler used in these chromatographic processes usually from tens microns to several hundred microns not
Deng void size is mostly several hundred nanometers etc., is unable to get the target polypeptides of high-purity.It is cyclized using synthesis in solid state+dilution
Pitressin [5-Asp] the impurity crude product solution concentration arrived is diluter, is purified using general reverse-phase chromatographic column, only loading
With turn salt during just generate a large amount of organic liquid waste, cannot be in line or can be reused through sewage plant simple process,
The especially Sample Purification on Single processing of low concentration, waste liquid amount is bigger, and the processing cost of dangerous waste is very high.Therefore, there is an urgent need to develop new
Suitable purifying low concentration polypeptide and salt cost-effective technique.
Summary of the invention
Technical problem to be solved by the present invention lies in the purifications in order to overcome pitressin [5-Asp] impurity in the prior art
Generate a large amount of organic liquid wastes in the process, and dangerous waste liquid measure is big, caused by treatment cost of waste liquor it is high, uneconomic defect, and providing
A kind of refining methd of pitressin [5-Asp] impurity.The present invention refines the mistake of the method in purifying of pitressin [5-Asp] impurity
The waste liquid generated in journey is largely waste water, can be reused through sewage plant simple process, economic and environment-friendly.
The present invention is to solve above-mentioned technical problem by the following technical programs:
The present invention provides a kind of refining methds of pitressin [5-Asp] impurity comprising following step:
Pitressin [5-Asp] impurity crude product solution is successively carried out by reverse phase enrichment, anti-using efficient liquid phase RP chromatography
Phase turns salt, reverse phase purifying；
The filler of the efficient liquid phase RP chromatography is super resistance to water packing；
The reverse phase is enriched with, reverse phase turns salt, reverse phase purifying is completed in the reverse phase elution process of a step；Described
It is as follows that reverse phase enrichment, reverse phase turn salt, the condition of reverse phase purifying:
The mobile phase A is the acetic acid/water solution that percent by volume is 0.005~0.1%, and the Mobile phase B is
Acetic acid/acetonitrile that percent by volume is 0.005~0.1%, the sample C1 are described pitressin [5-Asp] the impurity crude product
Solution, the mobile phase C2 are 5~50mM NH4Ac-NH4OH aqueous solution, the pH of the mobile phase C2 are 7.0~9.0, institute
The flow velocity for the eluent stated is 80~100mL/min.It collects the eluent that retention time is 96~105min and obtains pitressin
[5-Asp] dirt solution.
In the present invention, during 45~46min, the eluent is changed to the flowing by the sample C1
Phase C2.During 65~66min, the eluent is changed to the mobile phase A by the mobile phase C2.According to this
Field is conventional, and above-mentioned time interval should not be construed as the restriction to elution requirement, and length of time can be according to high performance liquid chromatograph factory
The difference of family's model adjusts accordingly.
In the present invention, the conversion rate of the eluent in the elution step (4) and (5) is one and at the uniform velocity becomes
The process of change.The process at the uniform velocity changed in the elution step (4) is increases by 2% institute on the basis of former eluent per minute
The Mobile phase B stated, while mobile phase A described in corresponding reduction 2%.The mistake at the uniform velocity changed described in the elution step (5)
Journey is the Mobile phase B described in increase by 0.333% on the basis of former eluent per minute, while described in corresponding reduction 0.333%
Mobile phase A.
The preparation method of described pitressin [5-Asp] the impurity crude product solution includes the following steps:
(1) reduced form pitressin [5-Asp] impurity crude product is made using solid-phase synthesis；
(2) reduced form pitressin [5-Asp] the impurity crude product dissolved, dilute reduced form pitressin [5-Asp] is miscellaneous
Matter crude product solution；
(3) reduced form pitressin [5-Asp] the impurity crude product solution obtains the pitressin [5-Asp] through oxidation process
Impurity crude product solution.
The solid-phase synthesis includes the following steps: to protect using Rink Amide MBHA resin as starting material with Fmoc
The amino acid of shield successively connects amino acid using HOBt/DIC as condensing agent for monomer one by one；Addition cuts peptide reagent and carries out cutting peptide,
Methyl tertiary butyl ether(MTBE) is added to be precipitated, reduced form pitressin [5-Asp] impurity crude product is obtained.
Wherein, the described peptide reagent of cutting can be conventional for this field, preferably the volume ratio TFA/ that is 90:7.5:2.5
TIS/H2O。
The dissolution can be conventional for this field, is preferably carried out with the acetic acid/water solution that percent by volume is 50% molten
Solution.
The dilution can be conventional for this field, is preferably diluted with water.
The oxidation process can be conventional for this field, preferably with alkaline matter by the reduced form pitressin [5-
Asp] pH of impurity crude product solution is adjusted to 7.0-9.0, and the hydrogen peroxide that percent by volume is 30% is added and carries out oxidation process.Institute
The dosage for stating hydrogen peroxide is 0.5mL/1g reduced form pitressin [5-Asp] impurity crude product.
Wherein, the alkaline matter can be conventional for this field, preferably NaOH.
In the present invention, reduced form pitressin [5-Asp] is miscellaneous in described reduced form pitressin [5-Asp] the impurity crude product solution
The concentration of matter crude product is 0.1~4mg/mL, preferably 0.5~2mg/mL, for example, 0.8mg/mL, 1mg/mL and 1.5mg/mL.
In the present invention, the mobile phase A is preferably the acetic acid/water solution that percent by volume is 0.02~0.05%.
The Mobile phase B is preferably acetic acid/acetonitrile that percent by volume is 0.02~0.05%.
The mobile phase C2 is preferably 10~20mM NH4Ac-NH4OH aqueous solution.
The pH of the mobile phase C2 is preferably 7.5~8.5.
The present invention is preferably, the eluent that the collection retention time is 98~103min obtains pitressin [5-
Asp] dirt solution.
In the present invention, pitressin [5-Asp] impurity described in the pitressin [5-Asp] impurity crude product solution
HPLC purity is 60~85%, preferably 70%~80%.
The structure of pitressin [5-Asp] impurity described in described pitressin [5-Asp] the impurity crude product solution is
In the present invention, the solvent in the pitressin [5-Asp] impurity crude product solution is to contain trifluoroacetic acid and acetic acid
Aqueous solution.
In the present invention, the super resistance to water packing isThe super resistance to water packing of ODS-AQ, preferably Suzhou is received micro-
Science and Technology Co., Ltd.The super resistance to water packing of ODS-AQ.The aperture of the super resistance to water packing is preferably 7~
The partial size of 10nm, the super resistance to water packing are preferably 10 μm.
In the present invention, the Detection wavelength of the efficient liquid phase RP chromatography is 220nm.
Wherein, 110~111min after the elution step (5), the eluent are stream described in 80%
Mobile phase B described in dynamic phase A+20% is replaced to Mobile phase B described in mobile phase A+50% described in 50%.111~120min,
The eluent is Mobile phase B described in mobile phase A+50% described in 50%.By quickly improving the Mobile phase B
Ratio achievees the purpose that clean chromatographic column.
The described reverse phase enrichment is the elution step (1), the reverse phase turn salt be the elution step (2)~
(3).Specifically, the elution step (2) is with the NH4Ac-NH4The OH aqueous solution removal pitressin [5-Asp]
The process of trifluoroacetic acid root in impurity crude product solution, the elution step (3) are in the removal elution step (2)
The process of ammonium ion, the reverse phase purifying is the elution step (4) and (5), wherein the elution step (4)
It is the process for removing weaker adsorbing contaminant.
Described pitressin [5-Asp] impurity is a kind of peptide material, unstable under high ph conditions, degradable, especially
It is that the integrated survey of the present invention reverse phase turns eluent pH and the time of salt, to guarantee to reduce the reverse phase under alkali environment
Turn the destruction and loss of sample during salt.
In a certain better embodiment, with Load&Lock dynamic axial compression and static locking technology, filler is institute
It statesThe super resistance to water packing of ODS-AQ, aperture 10nm, 10 μm of partial size, being filled to column bed pressure is 1000psi, is used
Varian chromatography loading system, 300g dry powder-shaped it is describedThe super resistance to water packing of ODS-AQ, the stirring of 600mL isopropanol
After homogenate, the Load&Lock4002 for pouring into internal diameter 50mm prepares column, compression ratio 1.5:1, carrier gas N2, adjust the carrier gas
Pressure makes oil pressure meter pressure be 1500psi, and dynamic axial compression to column bed height 25cm turns salt as reverse phase enrichment, reverse phase
Column is prepared with used in reverse phase purification schemes.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
(1) method that the present invention uses on-line preconcentration first will using the super water resistance and absorption property of super resistance to water packing
Pitressin [5-Asp] impurity crude product, which is adsorbed onto stationary phase, in pitressin [5-Asp] impurity crude product solution is enriched with, pitressin
The hydrophobic binding of [5-Asp] impurity crude product and reverse phase filler.
(2) present invention uses on-line preconcentration, can directly convert and carry out gradient elution purifying after mobile phase, obtains final pure
Product are suitble to continuous production.
(3) present invention innovatively turns salt with reverse phase enrichment, reverse phase and reverse phase purifies and pitressin is made through one-step method
The sterling of [5-Asp] impurity, optimizes production technology, is suitble to industrialization continuous production.
(4) more recent application of the water-fast filler of excess of export is designed, column equilibration stage, reverse phase concentration stage and reverse phase turn salt phase
Eluent is aqueous solution, environment friendly and pollution-free, and efflux is immediately discharged to Sewage Disposal, the recoverable after simple process,
The yield of dangerous waste, economical environment-protective greatly reduces in relatively traditional preparation process.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
In following embodiments, HPLC method detects pitressin [5-Asp] impurity crude product and after purification pitressin in product solution
The appointed condition of [5-Asp] impurity purity is as follows:
Instrument: Agilent1260 high performance liquid chromatograph
Chromatographic column: WatersXBridgeC184.6 × 250mm, 5 μm
Mobile phase: A is that percent by volume is 0.1%TFA aqueous solution, and B is the second that percent by volume is 0.1%TFA-50%
Nitrile aqueous solution (TFA is trifluoroacetic acid)
Flow velocity is 1.0mL/min, and Detection wavelength 210nm, column temperature: 25 DEG C, gradient see the table below, and percentage is volume
Percentage.
| Elution step | Elution time | Eluent |
| 1 | 0~2min | 95%A+5%B |
| 2 | 2~12min | 95%A+5%B → 85%A+15%B |
| 3 | 12~22min | 85%A+15%B |
| 4 | 22~30min | 85%A+15%B → 77%A+23%B |
| 5 | 30~30.1min | 77%A+23%B → 50%A+50%B |
| 6 | 30.1~35min | 50%A+50%B |
In following embodiments, the preparation method of pitressin [5-Asp] impurity crude product solution includes: (1) using synthesis in solid state
Legal system obtains reduced form pitressin [5-Asp] impurity crude product；(2) reduced form pitressin [5-Asp] the impurity crude product is dissolved, is dilute
Release to obtain reduced form pitressin [5-Asp] impurity crude product solution；(3) reduced form pitressin [5-Asp] the impurity crude product solution warp
Oxidation process obtains described pitressin [5-Asp] the impurity crude product solution.
The solid-phase synthesis includes the following steps: to protect using Rink Amide MBHA resin as starting material with Fmoc
The amino acid of shield successively connects amino acid using HOBt/DIC as condensing agent for monomer one by one；Addition cuts peptide reagent and carries out cutting peptide,
Methyl tertiary butyl ether(MTBE) is added to be precipitated, reduced form pitressin [5-Asp] impurity crude product is obtained.The peptide reagent of cutting is volume ratio
For the TFA/TIS/H of 90:7.5:2.52O.It is described be dissolved as being carried out with the acetic acid/water solution that percent by volume is 50% it is molten
Solution.Described is diluted to be diluted with water.It is described to be oxidized to reduced form the pitressin [- NH with alkaline matter2] impurity
The pH of crude product solution is adjusted to 7.0~9.0, and the hydrogen peroxide that percent by volume is 30% is added and carries out oxidation process.The dioxygen
The dosage of water is 0.5mL/1g reduced form pitressin [- NH2] impurity crude product.The alkaline matter is NaOH.
1 internal diameter 50mm Load&Lock4002 of embodiment prepares column filling
With Load&Lock dynamic axial compression and static locking technology, filler is describedODS-AQ, hole
Diameter 10nm, 10 μm of partial size, being filled to column bed pressure is 1000psi, using Varian chromatography loading system, the institute of 300g dry powder-shaped
It statesODS-AQ super resistance to water packing pours into the Load& that internal diameter is 50mm after the stirring homogenate of 600mL isopropanol
Lock4002 prepares column, compression ratio 1.5:1, carrier gas N2, adjust the nebulizer gas pressure and make the oil pressure meter pressure to be
1500psi, dynamic axial compression to column bed height 25cm turn used in salt and reverse phase purification schemes as reverse phase enrichment, reverse phase
Prepare column.
The reverse phase enrichment of 2 pitressin of embodiment [5-Asp] impurity crude product solution, reverse phase turn salt and reverse phase purifying
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: embodiment 1 prepares column 50 × 250mm of Load&Lock4002, and filler isODS-AQ
Diameter is 10 μm, aperture 10nm.
The structural formula of pitressin [5-Asp] impurity isReduced form pitressin
The concentration of reduced form pitressin [5-Asp] impurity crude product is 1mg/mL, pitressin [5-Asp] in [5-Asp] impurity crude product solution
Solvent in impurity crude product solution is the aqueous solution containing trifluoroacetic acid and acetic acid.
Mobile phase A is the acetic acid/water solution that percent by volume is 0.02%, and Mobile phase B is that percent by volume is 0.02%
Acetic acid/acetonitrile, sample C1 be pitressin [5-Asp] impurity crude product solution, according to HPLC method measure HPLC purity be
75.33%, mobile phase C2 are the NH of 10mM4Ac-NH4OH aqueous solution, the pH of mobile phase C2 are 7.5.
The reverse phase enrichment of the present embodiment, reverse phase turns salt and reverse phase purification condition is as follows: flow velocity 100mL/min, detects wave
A length of 220nm, purifying gradient see the table below shown, and percentage is percent by volume；
It collects the eluent that retention time is 96~105min and obtains pitressin [5-Asp] dirt solution.According to HPLC
The HPLC purity of pitressin [5-Asp] impurity that the collection retention time of method measurement is 96~105min is 99.34%, collects and protects
The HPLC purity for staying pitressin [5-Asp] impurity that the time is 98~103min is 99.76%.
The reverse phase enrichment of 3 pitressin of embodiment [5-Asp] impurity crude product solution, reverse phase turn salt and reverse phase purifying
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: embodiment 1 prepares column 50 × 250mm of Load&Lock4002, and filler isODS-AQ
Diameter is 10 μm, aperture 10nm.
The structural formula of pitressin [5-Asp] impurity isReduced form pitressin
The concentration of reduced form pitressin [5-Asp] impurity crude product is 1.5mg/mL, pitressin [5- in [5-Asp] impurity crude product solution
Asp] solvent in impurity crude product solution is the aqueous solution containing trifluoroacetic acid and acetic acid.
Mobile phase A is the acetic acid/water solution that percent by volume is 0.05%, and Mobile phase B is that percent by volume is 0.05%
Acetic acid/acetonitrile, sample C1 be pitressin [5-Asp] impurity crude product solution, according to HPLC method measure HPLC purity be
75.33%, mobile phase C2 are the NH of 20mM4Ac-NH4OH aqueous solution, the pH of mobile phase C2 are 8.5.
The reverse phase enrichment of the present embodiment, reverse phase turns salt and reverse phase purification condition is as follows: flow velocity 100mL/min, detects wave
A length of 220nm, purifying gradient see the table below shown, and percentage is percent by volume；
It collects the eluent that retention time is 96~105min and obtains pitressin [5-Asp] dirt solution.According to HPLC
The HPLC purity of pitressin [5-Asp] impurity that the collection retention time of method measurement is 96~105min is 99.31%, collects and protects
The HPLC purity for staying pitressin [5-Asp] impurity that the time is 98~103min is 99.79%.
The reverse phase enrichment of 4 pitressin of embodiment [5-Asp] impurity crude product solution, reverse phase turn salt and reverse phase purifying
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: embodiment 1 prepares column 50 × 250mm of Load&Lock4002, and filler isODS-AQ
Diameter is 10 μm, aperture 10nm
The structural formula of pitressin [5-Asp] impurity isReduced form pitressin
The concentration of reduced form pitressin [5-Asp] impurity crude product is 0.8mg/mL, pitressin [5- in [5-Asp] impurity crude product solution
Asp] solvent in impurity crude product solution is the aqueous solution containing trifluoroacetic acid and acetic acid.
Mobile phase A is the acetic acid/water solution that percent by volume is 0.05%, and Mobile phase B is that percent by volume is 0.05%
Acetic acid/acetonitrile, sample C1 be pitressin [5-Asp] impurity crude product solution, according to HPLC method measure HPLC purity be
74.26%, mobile phase C2 are the NH of 20mM4Ac-NH4OH aqueous solution, the pH of mobile phase C2 are 7.5.
The reverse phase enrichment of the present embodiment, reverse phase turns salt and reverse phase purification condition is as follows: flow velocity 100mL/min, detects wave
A length of 220nm, purifying gradient see the table below shown, and percentage is percent by volume；
It collects the eluent that retention time is 96~105min and obtains pitressin [5-Asp] dirt solution.According to HPLC
The HPLC purity of pitressin [5-Asp] impurity that the collection retention time of method measurement is 96~105min is 99.44%, collects and protects
The HPLC purity for staying pitressin [5-Asp] impurity that the time is 98~103min is 99.81%.
The Mass Spectrometer Method of 5 pitressin of embodiment [5-Asp] impurity
It is obtained using Waters micromass ZQ substance level four bars electrospray ionization mass spectrum (ESI-MS) measurement embodiment 2,3 and 4
Pitressin [5-Asp] impurity arrived, test condition are as follows: use the source electrospray ionisation (ESI), carried out in positive ion electrospray under mode
Mass spectral analysis, capillary ionization voltage 3.0kV sample orifice potential 35kV；115 DEG C of ion source temperature, desolventizing temperature 350
DEG C, desolventizing nitrogen flow rate 700L/h, taper hole blowback nitrogen 50L/h, 50.0~1500m/z of level four bars surface sweeping range.
The result of detection are as follows: molecular ion peak [M+H]+Mass-to-charge ratio (m/z) is 1085.41, leading ion fragment peak [M+
2H]2+Mass-to-charge ratio (m/z) is 543.21, and all meeting theoretical value, (relative molecular mass of pitressin [5-Asp] impurity is
1085.25)。
Claims (10)
1. a kind of refining methd of pitressin [5-Asp] impurity, which is characterized in that it includes the following steps: using efficient liquid phase
Pitressin [5-Asp] impurity crude product solution is successively carried out reverse phase enrichment by RP chromatography, reverse phase turns salt, reverse phase purifying；
The filler of the efficient liquid phase RP chromatography is super resistance to water packing；
The reverse phase is enriched with, reverse phase turns salt, reverse phase purifying is completed in the reverse phase elution process of a step；The reverse phase
It is as follows that enrichment, reverse phase turn salt, the condition of reverse phase purifying:
The mobile phase A is the acetic acid/water solution that percent by volume is 0.005~0.1%, and the Mobile phase B is volume
Acetic acid/acetonitrile that percentage is 0.005~0.1%, the sample C1 are that described pitressin [5-Asp] the impurity crude product is molten
Liquid, the mobile phase C2 are 5~50mM NH4Ac-NH4OH aqueous solution, the pH of the mobile phase C2 is 7.0~9.0, described
Eluent flow velocity be 80~100mL/min；It collects the eluent that retention time is 96~105min and obtains pitressin [5-
Asp] dirt solution.
2. the refining methd of pitressin [5-Asp] impurity as described in claim 1, it is characterised in that: in 45~46min phase
Between, the eluent is changed to the mobile phase C2 by the sample C1；It, will be described during 65~66min
Eluent is changed to the mobile phase A by the mobile phase C2.
3. the refining methd of pitressin [5-Asp] impurity as described in claim 1, it is characterised in that: the elution step
(4) conversion rate of the eluent and in (5) is the process at the uniform velocity changed.
4. the refining methd of pitressin [5-Asp] impurity as described in claim 1, it is characterised in that: the pitressin [5-
Asp] preparation method of impurity crude product solution includes the following steps:
(1) reduced form pitressin [5-Asp] impurity crude product is made using solid-phase synthesis；
(2) reduced form pitressin [5-Asp] the impurity crude product is dissolved, dilutes to obtain reduced form pitressin [5-Asp] impurity crude
Product solution；
(3) reduced form pitressin [5-Asp] the impurity crude product solution obtains described pitressin [5-Asp] impurity through oxidation process
Crude product solution.
5. the refining methd of pitressin [5-Asp] impurity as claimed in claim 4, it is characterised in that: the reduced form adds
The concentration for pressing reduced form pitressin [5-Asp] impurity crude product in plain [5-Asp] impurity crude product solution is 0.1~4mg/mL, preferably
Ground is 0.5~2mg/mL.
6. the refining methd of pitressin [5-Asp] impurity as described in claim 1, it is characterised in that: the mobile phase A is
The acetic acid/water solution that percent by volume is 0.02~0.05%；
And/or the Mobile phase B is acetic acid/acetonitrile that percent by volume is 0.02~0.05%；
And/or the mobile phase C2 is 10~20mM NH4Ac-NH4OH aqueous solution；
And/or the pH of the mobile phase C2 is 7.5~8.5；
And/or the eluent that the collection retention time is 98~103min obtains pitressin [5-Asp] dirt solution；
And/or the HPLC purity of pitressin [5-Asp] impurity described in described pitressin [5-Asp] the impurity crude product solution
It is 60~85%, preferably 70%~80%.
7. the refining methd of pitressin [5-Asp] impurity as described in claim 1, it is characterised in that: the pitressin [5-
Asp] structure of pitressin [5-Asp] impurity described in impurity crude product solution isSolvent is the aqueous solution containing trifluoroacetic acid and acetic acid.
8. the refining methd of pitressin [5-Asp] impurity as described in claim 1, it is characterised in that: described super water-fast to fill out
The aperture of material is 7~10nm, and the partial size of the super resistance to water packing is 10 μm.
9. the refining methd of pitressin [5-Asp] impurity as described in claim 1, it is characterised in that: described super water-fast to fill out
Material isThe super resistance to water packing of ODS-AQ.
10. the refining methd of pitressin [5-Asp] impurity as described in claim 1, it is characterised in that: the efficient liquid phase
The Detection wavelength of RP chromatography is 220nm.
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