CN109942686A - A kind of refining methd of pitressin acetylation impurity - Google Patents
A kind of refining methd of pitressin acetylation impurity Download PDFInfo
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Abstract
The invention discloses a kind of refining methds of pitressin acetylation impurity.The refining methd of pitressin acetylation impurity includes the following steps: pitressin acetylation 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 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 waste liquid that the refining methd of pitressin acetylation 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 acetylation 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 acetylation impurity is pitressin common impurity in the synthesis process, which can pressurize
Impurity reference substance is used as in plain quality testing, therefore, the pitressin acetylation 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 polypeptide drugs 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 acetylation impurity, nothing
(its applied sample amount is small, and flow velocity is low, and treating capacity is small, and molecular weight is relatively suitble to be greater than the de- of 10kDa albumen for suitable molecular sieve gel column
Salt) or ultrafiltration membrane selection.And in mesolow chromatography common separation method have molecular sieve chromatography, ion-exchange 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
The pitressin acetylation impurity crude product solution concentration arrived is diluter, is purified using general reverse-phase chromatographic column, only loading
During just generate a large amount of organic liquid waste, cannot be in line or can be reused through sewage plant simple process, especially
The Sample Purification on Single of low concentration is handled, and waste liquid amount is bigger, and the processing cost of dangerous waste is very high.Therefore, there is an urgent need to develop new to be suitble to
Purify the cost-effective technique of low concentration polypeptide and salt.
Summary of the invention
Technical problem to be solved by the present invention lies in the purifications in order to overcome pitressin acetylation 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 acetylation impurity.The method of purification pitressin acetylation impurity of the invention is in purification of target
The waste liquid generated during product is largely waste water, through sewage treatment can direct reuse, it is 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 acetylation impurity comprising following step: using efficient liquid
Pitressin acetylation impurity crude product solution is successively carried out reverse phase enrichment by phase RP chromatography, reverse phase turns salt, reverse phase purifying, i.e.,
It can；
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:
It collects the eluent that retention time is 84~92min and obtains pitressin acetylation dirt solution；
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 the pitressin acetylation 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.
In the present invention, during 25~26min, the eluent is changed to the flowing by the sample C1
Phase C2；During 35~36min, 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 process at the uniform velocity changed in the elution step (4) is per minute on the basis of former eluent
Mobile phase B described in increasing by 2%, while mobile phase A described in corresponding reduction 2%；Described in the elution step (5) at the uniform velocity
The process of variation is the Mobile phase B described in increase by 0.333% on the basis of former eluent, while corresponding reduction per minute
Mobile phase A described in 0.333%.
The pitressin acetylation impurity crude product solution is using reduced form pitressin acetyl made from solid-phase synthesis
Change impurity crude product to first pass through dissolution, be diluted to reduced form pitressin acetylation impurity crude product solution, then the reduced form is added
Plain acetylation impurity crude product solution is pressed to obtain through oxidation process.
Specific preparation process is as follows for the pitressin acetylation impurity crude product solution: with Rink Amide MBHA tree
Rouge is starting material, using fmoc-protected amino acid as monomer, using HOBt/DIC as condensing agent, successively connects amino acid one by one；
Addition cuts peptide reagent and carries out cutting peptide, and methyl tertiary butyl ether(MTBE) is added and is precipitated, reduced form pitressin acetylation impurity crude product is obtained；It will
The reduced form pitressin acetylation impurity crude product is dissolved, is diluted, and reduced form pitressin acetylation impurity crude product solution is obtained；
The pH of the reduced form pitressin acetylation impurity crude product solution is adjusted to 7.0-9.0 with alkaline matter, concentration, which is added, is
30% hydrogen peroxide carries out the oxidation process, and every gram of reduced form pitressin acetylation impurity crude product adds the 30% of 0.5ml
Hydrogen peroxide obtains oxidized form pitressin acetylation impurity crude product solution, as the pitressin acetylation impurity crude product solution.
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 acetyl
The pH for changing 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.It is described
The dosage of hydrogen peroxide is 0.5mL/1g reduced form pitressin acetylation impurity crude product.
Wherein, the alkaline matter can be conventional for this field, preferably NaOH.
In the present invention, the reduced form pitressin acetylation impurity crude product solution concentration is 0.1~4mg/mL, preferably
For 0.5~2mg/mL, for example, 0.8mg/mL, 1mg/mL and 1.5mg/mL.
In the present invention, the HPLC of pitressin acetylation impurity described in the pitressin acetylation impurity crude product solution
Purity is 60%~85%, preferably 70%~80%.
The structural formula of pitressin acetylation impurity described in the pitressin acetylation impurity crude product solution is
In the present invention, the solvent in the pitressin acetylation impurity crude product solution is the water containing trifluoroacetic acid and acetic acid
Solution.
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 Detection wavelength of the efficient liquid phase RP chromatography is 220nm.
In the present invention, the aperture of the super resistance to water packing is preferably 7~10nm, the partial size of the super resistance to water packing
Preferably 10 μm；
The super resistance to water packing isThe super resistance to water packing of ODS-AQ, preferably Suzhou receive micro- scientific and technological share
Co., LtdThe super resistance to water packing of ODS-AQ.
In addition, (6) 93~94min of step ,+20% Mobile phase B of 80% mobile phase A →+50% mobile phase of 50% mobile phase A
(7) 94~109min of B and step ,+50% Mobile phase B of 50% mobile phase A.By quickly improving the ratio of organic phase, reach clear
Wash the purpose of chromatographic column.
Wherein, reverse phase enrichment is the elution step (1), and it is the elution step that the reverse phase, which turns salt,
(2)~(3), specifically, the elution step (2) are with the NH4Ac-NH4The OH aqueous solution removal pitressin second
The process of trifluoroacetic acid root in acylated impurity crude product solution, the elution step (3) are the removal elution steps (2)
In ammonium ion process, the described reverse phase purifying is elution step (4)~(5), wherein the elution step
It (4) is the process for removing weaker adsorbing contaminant.
The pitressin acetylation impurity is a kind of peptide material, unstable under high ph conditions, degradable, especially
Under alkali environment, the integrated survey of the present invention reverse phase turns eluent pH and the time of salt, to guarantee to reduce the reverse phase
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) more recent application of the water-fast filler of excess of export, column equilibration stage, loading concentration stage and the flowing for turning salt phase are designed
Xiang Kewei aqueous solution, environment friendly and pollution-free, efflux is immediately discharged to Sewage Disposal, the recoverable after simple process, phase
To traditional preparation process, the yield of dangerous waste, economical environment-protective greatly reduces.
(2) method that the present invention uses on-line preconcentration, it is first that polypeptide is thick using the super water resistance and absorption property of filler
Thick peptide, which is adsorbed onto stationary phase, in product solution is enriched with, polypeptide and reverse phase filler hydrophobic binding.
(3) 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.
(4) present invention has innovatively used reverse phase absorption enrichment, has turned the obtained polypeptide sterling of salt, desalination one-step method, optimization
Production technology is suitble to industrialization continuous production.
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 acetylation impurity crude product and after purification pitressin second in product solution
The appointed condition of acylated 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 acetylation impurity crude product solution includes: (1) using solid-phase synthesis
Reduced form pitressin acetylation impurity crude product is made；(2) the reduced form pitressin acetylation impurity crude product is dissolved, is diluted
Reduced form pitressin acetylation impurity crude product solution；(3) the reduced form pitressin acetylation impurity crude product solution was through aoxidizing
Journey obtains the pitressin acetylation 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 acetylation impurity crude product is obtained.Wherein, the peptide reagent of cutting is body
Product is than the TFA/TIS/H for 90:7.5:2.52O.The dissolution is carried out molten with the acetic acid/water solution that percent by volume is 50%
Solution.Described is diluted to be diluted with water.The oxidation process is with alkaline matter by the reduced form pitressin acetylation
The 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.It is described double
The dosage of oxygen water is 0.5mL/1g reduced form pitressin acetylation impurity crude product.Wherein, 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, described in 300g dry powder-shaped
'sODS-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 acetylation impurity crude product solution of embodiment, reverse phase turn salt and reverse phase purifying
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: embodiment 1 prepares column Load&Lock400250 × 250mm, and filler isODS-AQ, grain
Diameter is 10 μm, aperture 10nm.
The structural formula of pitressin acetylation impurity isReduced form pressurization
Plain acetylation impurity crude product solution concentration is 1mg/mL, the solvent of pitressin acetylation impurity crude product solution be containing trifluoroacetic acid and
The aqueous solution of 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 acetylation impurity crude product solution, according to HPLC method measure the pitressin acetyl
The HPLC purity for changing impurity is 78.36%, and mobile phase C2 is 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 84~92min and obtains pitressin acetylation dirt solution.According to HPLC method
The HPLC purity of the pitressin acetylation impurity of measurement is 99.68%.
The reverse phase enrichment of 3 pitressin acetylation impurity crude product solution of embodiment, reverse phase turn salt and reverse phase purifying
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: embodiment 1 prepares column Load&Lock400250 × 250mm, and filler isODS-AQ, grain
Diameter is 10 μm, aperture 10nm.
The structural formula of pitressin acetylation impurity isReduced form pressurization
Plain acetylation impurity crude product solution concentration is 1.5mg/mL, and the solvent of pitressin acetylation impurity crude product solution is containing trifluoroacetic acid
With the aqueous solution of 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 acetylation impurity crude product solution, according to HPLC method measure the pitressin acetyl
The HPLC purity for changing impurity is 72.13%, and mobile phase C2 is 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 84~92min and obtains pitressin acetylation dirt solution.According to HPLC method
The HPLC purity of the pitressin acetylation impurity of measurement is 99.25%.
The reverse phase enrichment of 4 pitressin acetylation impurity crude product solution of embodiment, reverse phase turn salt and reverse phase purifying
Instrument: Varian SD-1 high pressure liquid phase preparation system
Chromatographic column: embodiment 1 prepares column Load&Lock400250 × 250mm, and filler isODS-AQ, grain
Diameter is 10 μm, aperture 10nm.
The structural formula of pitressin acetylation impurity isReduced form pressurization
Plain acetylation impurity crude product solution concentration is 0.8mg/mL, and the solvent of pitressin acetylation impurity crude product solution is containing trifluoroacetic acid
With the aqueous solution of 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 acetylation impurity crude product solution, according to HPLC method measure the pitressin acetyl
The HPLC purity for changing impurity is 75.46%, and mobile phase C2 is 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 84~92min and obtains pitressin acetylation dirt solution.According to HPLC method
The HPLC purity of the pitressin acetylation impurity of measurement is 99.30%.
The Mass Spectrometer Method of 5 pitressin acetylation impurity of embodiment
It is obtained using Waters micromass ZQ substance level four bars electrospray ionization mass spectrum (ESI-MS) measurement embodiment 2,3 and 4
The pitressin acetylation 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 1128.43, leading ion fragment peak [M+
2H]2+Mass-to-charge ratio (m/z) is 564.73, and all meeting theoretical value, (relative molecular mass of pitressin acetylation impurity is
1128.34)。
Claims (10)
1. a kind of refining methd of pitressin acetylation impurity, which is characterized in that it includes the following steps: anti-using efficient liquid phase
Pitressin acetylation impurity crude product solution is successively carried out reverse phase enrichment by phase 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:
It collects the eluent that retention time is 84~92min and obtains pitressin acetylation dirt solution；
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 the pitressin acetylation impurity crude product solution,
The mobile phase C2 is 5~50mM NH4Ac-NH4OH aqueous solution, the pH of the mobile phase C2 is 7.0~9.0, described
The flow velocity of eluent is 80~100mL/min.
2. the refining methd of pitressin acetylation impurity as described in claim 1, it is characterised in that: during 25~26min,
The eluent is changed to the mobile phase C2 by the sample C1；During 35~36min, by the elution
Liquid is changed to the mobile phase A by the mobile phase C2.
3. the refining methd of pitressin acetylation impurity as described in claim 1, it is characterised in that: elution step (4) and (5)
In, the conversion rate of the eluent is the process at the uniform velocity changed.
4. the refining methd of pitressin acetylation impurity as described in claim 1, it is characterised in that: the pitressin acetyl
Changing impurity crude product solution is that dissolution, dilution are first passed through using reduced form pitressin acetylation impurity crude product made from solid-phase synthesis
For reduced form pitressin acetylation impurity crude product solution, then by the reduced form pitressin acetylation impurity crude product solution through oxygen
Change process and obtain.
5. the refining methd of pitressin acetylation impurity as described in claim 1, it is characterised in that: the reduced form pressurization
Plain acetylation impurity crude product solution concentration is 0.1~4mg/mL, preferably 0.5~2mg/mL.
6. the refining methd of pitressin acetylation impurity as described in claim 1, it is characterised in that: the pitressin acetyl
Change impurity crude product solution described in pitressin acetylation impurity HPLC purity be 60%~85%, preferably 70%~
80%.
7. the refining methd of pitressin acetylation impurity as described in claim 1, it is characterised in that: the pitressin acetylation
The structural formula of pitressin acetylation impurity described in impurity crude product solution is
Solvent is the aqueous solution containing trifluoroacetic acid and acetic acid.
8. the refining methd of pitressin acetylation 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 Detection wavelength of the efficient liquid phase RP chromatography is 220nm.
9. the refining methd of pitressin acetylation impurity as described in claim 1, it is characterised in that: the super resistance to water packing
Aperture be 7~10nm, the partial size of the super resistance to water packing is 10 μm.
10. the refining methd of pitressin acetylation 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.
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| CN113698456A (en) * | 2021-05-31 | 2021-11-26 | 海南双成药业股份有限公司 | Method for purifying argirelin |
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