CN110078796B - Method for refining oxytocin [4-Glu,5-Asp ] impurity - Google Patents
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Abstract
The invention discloses a refining method of oxytocin [4-Glu,5-Asp ] impurities, which comprises the following steps: adopting a high performance liquid phase reverse phase chromatography to sequentially carry out reverse phase enrichment, reverse phase salt conversion and reverse phase purification on the oxytocin [4-Glu,5-Asp ] impurity crude product solution; the oxytocin [4-Glu,5-Asp ] impurity crude product solution is prepared by oxidizing a solid-phase synthesized reduction type oxytocin [4-Glu,5-Asp ] impurity crude product solution; the filler of the high performance liquid phase reverse phase chromatography is super water-resistant filler. The invention adopts one-step method of on-line reversed-phase enrichment, reversed-phase salt conversion and reversed-phase purification to prepare the pure polypeptide product, the removal rate of impurities in the crude product solution is higher, the production process is optimized, the mobile phase in the stages of column balance, sample loading enrichment and salt conversion is aqueous solution, the invention is environment-friendly and pollution-free, and the effluent waste liquid can be directly treated and recycled by sewage.
Description
Technical Field
The invention relates to a refining method of oxytocin [4-Glu,5-Asp ] impurities.
Background
Oxytocin, also known as Oxytocin, is known as oxyytocin and has the structural formula:
the molecular formula is: c43H66N12O12S2Molecular weight of 1007.2
The oxytocin is used for induced labor, postpartum and postpartum metrorrhagia caused by uterine weakness or poor abdomen contraction; understanding placental reserve function (oxytocin rage test); it can promote milk excretion by dripping into nose. Oxytocin can indirectly stimulate uterine smooth muscle to shrink, simulate uterine contraction effect of normal delivery, and cause cervix dilatation, and uterine response to oxytocin gradually increases in the pregnancy process, and reaches peak at term. Oxytocin may also stimulate contraction of the smooth muscle of the breast, facilitating the drainage of milk from the breast, but does not increase the milk production of the breast.
In the case of a drug, the small amount of impurities contained therein is the most important cause for the side effects of the drug, so that the purity inspection is one of the important bases for ensuring the safety and effectiveness of the drug, and the content of the purity inspection is somewhat different according to the properties and characteristics of each drug, but basically involves respective inspection research on "related substances". Although the purification process of the synthesized polypeptide has been greatly improved at present, the process impurities are still important sources of the synthesized polypeptide-related substances, mainly because some process impurities of the synthesized polypeptide, such as deletion peptides, broken peptides, oxidized peptides, products of disulfide bond exchange, and the like, may be very similar to the properties of the drug per se, thereby causing certain difficulty in purification. Studies have shown that the most common degradation products in the synthesis of polypeptides are deamidates, oxygenates, and hydrolysates. Among the various amino acids that make up a polypeptide, asparagine, glutamine and peptide chain C-segment amides are susceptible to deamidation reactions, particularly at elevated pH and elevated temperatures.
At present, most of common purification methods for polypeptide drugs on the market adopt preparative high performance liquid chromatography, which is the most effective method for obtaining high-purity polypeptide target molecules. The general polypeptide medicine purification preparation process design is that target polypeptide is enriched by medium-low pressure chromatography and then refined by high pressure chromatography, but considering that the molecular weight of the target polypeptide oxytocin is about 1kDa, no proper molecular sieve gel column (the sample size is small, the flow rate is low, the treatment capacity is small, and the method is more suitable for desalting protein with the molecular weight of more than 10 kDa) or ultrafiltration membrane selection. And common separation methods in medium and low pressure chromatography include molecular sieve chromatography, ion exchange chromatography and hydrophobic interaction chromatography, the particle size of the filler used in the chromatography methods is usually different from dozens of micrometers to hundreds of micrometers, the size of the gap is mostly different from hundreds of nanometers, and the target polypeptide with high purity cannot be obtained. The concentration of the oxytocin crude product solution obtained by adopting solid phase synthesis and dilution cyclization is relatively dilute, and when a general reverse phase chromatographic column is adopted for purification, a large amount of organic waste liquid is generated only in the sample loading process, so that the treatment cost of hazardous waste is very high. There is still a lack of an efficient method for preparing polypeptide salt drug substances, and there is still a great need to develop new economical and efficient processes suitable for purifying low concentrations of polypeptides and salts.
In the conventional polypeptide purification process, in the sample loading and salt transferring process, the effluent liquid of a chromatographic column contains an organic phase, so that the organic phase cannot be directly discharged or can be recycled by simple treatment of a sewage station, and particularly, the purification treatment of a low-concentration sample has larger waste liquid amount and higher treatment cost. Chinese patent document CN106518976A discloses a preparation method of oxytocin [4-Glu,5-Asp ]. The preparation method comprises the following steps: carrying out reversed-phase cyclization, reversed-phase purification and reversed-phase desalting on the oxytocin [4-Glu,5-Asp ] precursor crude product solution in sequence by adopting a high performance liquid reversed-phase chromatography; the filler of the high performance liquid reverse phase chromatography is C18; the oxytocin [4-Glu,5-Asp ] precursor crude product is an oxytocin [4-Glu,5-Asp ] precursor crude product containing two free sulfydryl groups. In the preparation process of the patent, a reduced oxytocin [4-Glu,5-Asp ] precursor crude product is used as a starting material, NaOH is used as an alkaline substance in a mobile phase, pH is not controlled favorably, and the stability of a product is possibly influenced. In addition, the method needs a large amount of mobile phase containing organic solvent when the cyclization is carried out on a chromatographic column, a large amount of organic hazardous waste liquid is generated in the steps of purification and salt conversion, and the treatment cost of the subsequent waste liquid is high and the subsequent waste liquid is difficult to recycle.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects that in the prior art, the amount of hazardous waste liquid generated in the process of refining oxytocin [4-Glu,5-Asp ] impurities is large, and the hazardous waste liquid is organic hazardous waste liquid, so that the waste liquid treatment cost is high and the waste liquid is not economical, and provides a method for refining oxytocin [4-Glu,5-Asp ] impurities. Most of waste liquid generated in the process of purifying a target product by the method for refining the oxytocin [4-Glu,5-Asp ] impurity is waste water, can be directly recycled by sewage treatment, and is economic and environment-friendly.
The invention solves the technical problems through the following technical scheme:
the invention provides a refining method of oxytocin [4-Glu,5-Asp ] impurities, which comprises the following steps:
adopting a high performance liquid phase reverse phase chromatography to sequentially carry out reverse phase enrichment, reverse phase salt conversion and reverse phase purification on the oxytocin [4-Glu,5-Asp ] impurity crude product solution;
the filler of the high performance liquid reverse phase chromatography is super water-resistant filler;
the reverse phase enrichment, reverse phase salt conversion and reverse phase purification are all completed in one-step reverse phase elution process, and the conditions of the reverse phase enrichment, the reverse phase salt conversion and the reverse phase purification are as follows:
wherein the mobile phase A is acetic acid/water with the volume percentage of 0.005-0.1%, the mobile phase B is acetic acid/acetonitrile with the volume percentage of 0.005-0.1%, and the sample C1 is oxytocin [4-Glu, 5-Asp%]Crude impurity solution with a mobile phase C2 of 5-50 mM NH4Ac-NH4The pH value of the mobile phase C2 is 7.0-9.0, and the flow rate of the eluent is 80-100 ml/min;
and collecting the eluent with the retention time of 65-73 min to obtain the oxytocin [4-Glu,5-Asp ] impurity solution.
In the invention, the crude oxytocin [4-Glu,5-Asp ] impurity solution is prepared by dissolving, diluting and oxidizing a solid-phase synthesized crude reduced oxytocin [4-Glu,5-Asp ] impurity.
In the invention, the concrete preparation steps of the oxytocin [4-Glu,5-Asp ] impurity crude product solution are as follows: taking Rink Amide MBHA resin as an initial raw material, taking amino acid protected by Fmoc as a monomer, taking HOBt/DIC as a condensing agent, and sequentially connecting the amino acid one by one; adding a peptide cutting reagent for peptide cutting, adding methyl tert-butyl ether for precipitation to obtain a crude product of reduced oxytocin [4-Glu,5-Asp ] impurities; dissolving the crude reduced oxytocin [4-Glu,5-Asp ] impurity product in 50% acetic acid/water, and diluting with water to obtain crude reduced oxytocin [4-Glu,5-Asp ] impurity solution; and (2) adjusting the pH value of the reduced oxytocin [4-Glu,5-Asp ] impurity crude product solution to 7.0-9.0 by using an alkaline substance, adding hydrogen peroxide with the concentration of 30% for oxidation, wherein 0.5ml of 30% hydrogen peroxide is added into each gram of the reduced oxytocin [4-Glu,5-Asp ] impurity crude product to obtain an oxidized oxytocin [4-Glu,5-Asp ] impurity crude product solution, namely the oxytocin [4-Glu,5-Asp ] impurity crude product solution.
Wherein 50% acetic acid/water can fully dissolve the crude reduced oxytocin [4-Glu,5-Asp ] impurity.
Wherein, the concentration of the crude solution of the reduced oxytocin [4-Glu,5-Asp ] impurity is 0.1-4 mg/ml, preferably 0.5-2 mg/ml.
Wherein the peptide cutting reagent is conventional in the field, and preferably TFA/TIS/H with the volume ratio of 90:7.5:2.52O。
Wherein, the alkaline substance is conventional in the field, and is preferably NaOH.
In the invention, the oxytocin [4-Glu,5-Asp ]]Oxytocin [4-Glu,5-Asp in impurity crude product solution]The structural formula of the impurity is
The oxytocin [4-Glu,5-Asp ]]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
In the invention, the super waterproof filler is
ODS-AQ super water-resistant filler.
In the invention, the aperture of the super-waterproof filler is 7-10 nm, and the particle size is 10 μm.
In a preferred embodiment, Load is used&The Lock dynamic axial compression and static locking technology adopts the packing material of
ODS-AQ super water-resistant filler with pore diameter of 10nm and particle diameter of 10 μm, loading to column bed pressure of 1000psi, adopting Varian chromatography loading system, 300g dry powder
ODS-AQ super water-resistant filler is stirred and homogenized by 600mL of isopropanol, and then Load with the inner diameter of 50mm is poured into the mixture&Lock4002 column preparation, compression ratio of 1.5:1, carrier gas N2The carrier gas pressure is adjusted so that the oil pressure gauge pressure is1500psi, dynamic axial compression to a bed height of 25cm, as a preparative column for reverse phase enrichment, reverse phase salt conversion and reverse phase purification schemes.
In the invention, the detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
In the invention, the mobile phase A is an acetic acid/water solution with the volume percentage of 0.02-0.05%;
and/or the mobile phase B is acetic acid/acetonitrile with the volume percentage of 0.02-0.05%;
and/or the mobile phase C2 is 10-20 mM NH4Ac-NH4An aqueous OH solution;
and/or the pH of the mobile phase C2 is 7.5-8.5;
and/or the HPLC purity of the oxytocin [4-Glu,5-Asp ] impurity crude product solution is 60-85%, preferably 70-85%, more preferably 70-80%;
and/or collecting the eluent with the retention time of 68-71 min to obtain the oxytocin [4-Glu,5-Asp ] impurity solution.
In the invention, the eluent is completely changed from a sample C1 to a mobile phase C2 within 30-31 min; and completely replacing the eluent from the mobile phase C2 to the mobile phase A within 40-41 min. According to the routine in the field, the time interval is not understood to be the limit of the elution condition, and the time can be adjusted according to the different types of the manufacturers of the high performance liquid chromatograph.
In the invention, within 86-87 min after the step (5) is finished, the eluent is reduced from 80% of the mobile phase A to 50% of the mobile phase A at a constant speed, the mobile phase B is correspondingly increased to 50% at a constant speed, and the elution of 50% of the mobile phase A and 50% of the mobile phase B is kept within 87-100 min, so that the aim of cleaning the chromatographic column is fulfilled.
In the invention, the reversed-phase enrichment is an elution step (1), the reversed-phase salt conversion is steps (2) to (3), and specifically, the step (2) is performed by using weak base NH4Ac-NH4OH removal of oxytocin [4-Glu,5-Asp ]]The process of trifluoroacetic acid radical in the crude product of impurity, the step (3) is the process of removing ammonium radical ion in the step (2), the reverse phase purificationSteps (4) and (5), wherein step (4) is a process for removing weakly adsorbed impurities.
Wherein, the mobile phase in post equilibrium stage, the enrichment stage of going up a sample and commentaries on classics salt stage is the aqueous solution that contains salt, and the environmental protection is pollution-free, and the effluent waste liquid can directly carry out simple sewage treatment and recycle, very big reduction the cost that the danger waste liquid was handled.
In the invention, the conversion rate of the eluent in the elution steps (4) and (5) is a process of uniform speed change, the uniform speed change rate in the elution step (4) is 2% B/min, namely, 2% of the mobile phase B is increased on the basis of the original eluent every minute, and 2% of the mobile phase A is correspondingly reduced at the same time; the uniform speed change rate in the elution step (5) is 0.333% B/min, namely, 0.333% of the mobile phase B is increased on the basis of the original eluent every minute, and 0.333% of the mobile phase A is correspondingly reduced at the same time.
The oxytocin [4-Glu,5-Asp ] impurity is a polypeptide substance, is unstable and easy to degrade under the condition of high pH, and particularly under the alkaline environment, the pH and the time of salt transfer elution are comprehensively considered, so that the damage and the loss of a sample in the salt transfer process are reduced.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
(1) the invention adopts an on-line enrichment method, utilizes the super-water-resistant performance and the adsorption performance of the filler to adsorb the crude polypeptide product onto the stationary phase for enrichment, and the polypeptide and the reversed-phase filler are combined in a hydrophobic manner. After on-line enrichment, the mobile phase can be directly transformed and then gradient elution purification is carried out to obtain the final pure product, and the method is suitable for continuous production.
(2) The invention innovatively applies a one-step method of reversed-phase adsorption enrichment, salt conversion and desalting to prepare a pure polypeptide product, optimizes the production process, is suitable for industrial continuous production, and has higher removal rate of impurities in a crude oxytocin [4-Glu,5-Asp ] impurity solution.
(3) The latest application of the super-waterproof filler is designed, the eluents in the column balance stage, the reversed-phase enrichment stage and the reversed-phase salt conversion stage are aqueous solutions, the environment is protected, no pollution is caused, the effluent liquid is directly discharged to a sewage treatment station, the effluent liquid can be recycled after simple treatment, the production amount of dangerous waste liquid is greatly reduced compared with the traditional preparation process, and the environment is saved.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
The water-resistant fillers used in the examples were obtained from Suzhou Nami microbial science and technology Ltd
ODS-AQ super water-resistant filler with a pore diameter of 10nm and a particle size of 10 μm.
Detecting the purity of the oxytocin [4-Glu,5-Asp ] impurity crude product and the purified product solution by an HPLC method:
the instrument comprises the following steps: agilent 1200 high performance liquid chromatograph
Separating the column: waters Xbridge-C18, 4.6X 150mm, 5 μm
Mobile phase: a is acetonitrile water solution with the volume percentage of 50 percent, B is 0.02M KH2PO4pH3.0 water solution, flow rate of 1.0ml/min, detection wavelength of 220nm, room temperature detection, and elution gradient shown in the following table, the percentage is volume percentage.
| Step of elution | Elution time | Eluent |
| 1 | 0～15min | 35％A+65％B |
| 2 | 15～25min | 35％A+65％B→100％A |
| 3 | 25～28min | 100％A |
| 4 | 28～28.1min | 100％A→35％A+65％B |
| 5 | 28.1～32min | 35％A+65％B |
In the following examples, oxytocin [4-Glu,5-Asp ] is described]The crude impurity solution is reduced oxytocin [4-Glu,5-Asp ] synthesized by solid phase]The crude product of the impurity is obtained by dissolution, dilution and oxidation. The solid phase synthesis method comprises the following steps: taking Rink Amide MBHA resin as an initial raw material, taking amino acid protected by Fmoc as a monomer, taking HOBt/DIC as a condensing agent, and sequentially connecting the amino acid one by one; adding peptide cutting reagent to cut peptide, adding methyl tert-butyl ether to precipitate to obtain reduced oxytocin [4-Glu,5-Asp]Crude product of impurities. The peptide cutting reagent is TFA/TIS/H with the volume ratio of 90:7.5:2.52And O. The dissolution is carried out by using 50 percent by volume of acetic acid/water solution. The dilution is water dilution. The oxidation is carried out by using alkaline substancesThe reduction type oxytocin [4-Glu,5-Asp ]]Adjusting the pH value of the impurity crude product solution to 7.0-9.0, and adding 30% hydrogen peroxide by volume percentage for oxidation. The dosage of the hydrogen peroxide is 0.5mL/1g of reduced oxytocin [4-Glu,5-Asp ]]Crude product of impurities. The alkaline substance is NaOH.
Example 1 preparation of column packing with 50mm inner diameter L & L4002:
application of Load&The Lock dynamic axial compression and static locking technology adopts the packing material of
ODS-AQ with aperture of 10nm and particle diameter of 10 μm is packed to column bed pressure of 1000psi, and stirred and homogenized by Varian chromatography packing system, 300g dry powder packing and 600ml isopropanol, and poured into L with inner diameter of 50mm&L4002 column preparation, compression ratio 1.5:1, carrier gas N2The carrier gas pressure was adjusted to 1500psi oil gauge pressure and dynamic axial compression to 25cm column bed height was used as a preparative column for reverse phase enrichment, reverse phase salt conversion and reverse phase purification protocols.
EXAMPLE 2 reverse phase enrichment, reverse phase salt conversion and reverse phase purification of crude oxytocin [4-Glu,5-Asp ] impurity solution
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: EXAMPLE 1 self-contained preparative column Load&Lock400250×250mm，
ODS-AQ 10μm 10nm
Oxytocin [4-Glu,5-Asp]The structural formula of the impurity is
Reduced oxytocin [4-Glu,5-Asp ]]Reduced oxytocin [4-Glu,5-Asp ] in impurity crude product solution]The concentration of the crude product of the impurity is 1.0mg/ml, and the oxytocin [4-Glu,5-Asp ]]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
The mobile phase A is 0.02% by volume acetic acid/water, the mobile phase B is 0.02% by volume acetic acid/acetonitrile, sample C1 is oxytocin [ alpha ], [ beta ], [4-Glu,5-Asp]Crude solution of impurities, oxytocin [4-Glu,5-Asp ] determined by HPLC]The HPLC purity of the crude solution of the impurity was 73.44%, 10mM NH in mobile phase C24Ac-NH4OH pH7.5 aqueous solution.
The reverse phase enrichment, reverse phase salt conversion and reverse phase purification conditions of this example are as follows: the flow rate was 100ml/min, the detection was at 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
And collecting the eluent with the retention time of 65-73 min to obtain the oxytocin [4-Glu,5-Asp ] impurity solution. The HPLC purity of the oxytocin [4-Glu,5-Asp ] impurity determined by an HPLC method is 99.60 percent, and the removal rate of the impurity in the oxytocin [4-Glu,5-Asp ] impurity crude product solution is 26.16 percent; and collecting the eluent with the retention time of 68-71 min to obtain the oxytocin [4-Glu,5-Asp ] impurity with the purity of 99.79%.
The eluent in the steps (1) - (3) is aqueous solution, is environment-friendly and pollution-free, the effluent liquid of the eluent is directly discharged to a sewage treatment station, and can be recycled after simple treatment, so that the generation amount of hazardous waste liquid is greatly reduced, and the environment is saved compared with the traditional preparation process.
EXAMPLE 3 reverse phase enrichment, reverse phase salt conversion and reverse phase purification of crude oxytocin [4-Glu,5-Asp ] impurity solution
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: EXAMPLE 1 self-contained preparative column Load&Lock4002 50×250mm，
ODS-AQ 10μm 10nm
Oxytocin [4-Glu,5-Asp]The structural formula of the impurity is
Reduced oxytocin [4-Glu,5-Asp ]]Reduced oxytocin [4-Glu,5-Asp ] in impurity crude product solution]The concentration of the crude product of the impurity is 1.5mg/ml, and the concentration of oxytocin [4-Glu,5-Asp ] is]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
Mobile phase a was 0.05% acetic acid/water by volume, mobile phase B was 0.05% acetic acid/acetonitrile by volume, sample C1 was oxytocin [4-Glu,5-Asp ]]Crude solution of impurities, oxytocin [4-Glu,5-Asp ] determined by HPLC]The crude solution of the impurity had an HPLC purity of 71.22% and a mobile phase C2 of 20mM NH4Ac-NH4OH pH8.5 aqueous solution.
The reverse phase enrichment, reverse phase salt conversion and reverse phase purification conditions of this example are as follows: the flow rate was 100ml/min, the detection was at 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
And collecting the eluent with the retention time of 65-73 min to obtain the oxytocin [4-Glu,5-Asp ] impurity solution. The HPLC purity of the oxytocin [4-Glu,5-Asp ] impurity determined by an HPLC method is 99.33 percent, and the impurity removal rate in the crude product solution is 28.11 percent; and collecting the eluent with the retention time of 68-71 min to obtain the oxytocin [4-Glu,5-Asp ] impurity with the purity of 99.76%.
EXAMPLE 4 reverse phase enrichment, reverse phase salt conversion and reverse phase purification of crude oxytocin [4-Glu,5-Asp ] impurity solution
The instrument comprises the following steps: varian SD-1 high-pressure liquid phase preparation system
A chromatographic column: EXAMPLE 1 self-contained preparative column Load&Lock4002 50×250mm，
ODS-AQ 10μm 10nm
Oxytocin [4-Glu,5-Asp]The structural formula of the impurity is
Reduced oxytocin [4-Glu,5-Asp ]]Reduced oxytocin [4-Glu,5-Asp ] in impurity crude product solution]The concentration of the crude product of the impurity is 0.8mg/ml, and the oxytocin [4-Glu,5-Asp ]]The solvent in the crude impurity solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
Mobile phase a was 0.05% acetic acid/water by volume, mobile phase B was 0.05% acetic acid/acetonitrile by volume, sample C1 was oxytocin [4-Glu,5-Asp]Crude solution of impurities, oxytocin [4-Glu,5-Asp ] determined by HPLC]The crude solution of the impurity had an HPLC purity of 76.31% and a mobile phase C2 of 20mM NH4Ac-NH4OH pH7.5 aqueous solution.
The reverse phase enrichment, reverse phase salt conversion and reverse phase purification conditions of this example are as follows: the flow rate was 100ml/min, the detection was at 220nm, and the purification elution gradient is shown in the following table, with percentages being by volume.
And collecting the eluent with the retention time of 65-73 min to obtain the oxytocin [4-Glu,5-Asp ] impurity solution. The purity of oxytocin [4-Glu,5-Asp ] impurity HPLC determined by HPLC method is 99.31%, and the impurity removal rate in the crude product solution is 23%; and collecting the eluent with the retention time of 68-71 min to obtain the oxytocin [4-Glu,5-Asp ] impurity with the purity of 99.80%.
Example 5 Mass Spectrometry detection of oxytocin [4-Glu,5-Asp ] impurity
Oxytocin [4-Glu,5-Asp ] impurities obtained in examples 2, 3 and 4 were determined by Waters micromass ZQ single quadrupole electrospray mass spectrometry (ESI-MS) under the following test conditions: performing mass spectrometry by adopting an electrospray ionization (ESI) source in a positive ionization mode, wherein the ionization voltage of a capillary tube is 3.0kV, and the sampling taper hole voltage is 35 kV; the ion source temperature is 115 ℃, the desolventizing temperature is 350 ℃, the desolventizing nitrogen flow rate is 700L/h, the cone hole back flushing nitrogen flow rate is 50L/h, and the sweep range of the four-level rod is 50.0-1500 m/z.
The detection result is as follows: molecular ion Peak [ M + H]+Mass to charge ratio (M/z) of 1009.41, main ion fragment peak [ M +2H]2+The mass-to-charge ratio (m/z) is 505.20, and the mass-to-charge ratios all accord with the theoretical value that is oxytocin [4-Glu,5-Asp]The molecular weight of the impurity is 1009.19.
Claims (11)
1. A refining method of oxytocin [4-Glu,5-Asp ] impurities is characterized by comprising the following steps: adopting a high performance liquid phase reverse phase chromatography to sequentially carry out reverse phase enrichment, reverse phase salt conversion and reverse phase purification on the oxytocin [4-Glu,5-Asp ] impurity crude product solution;
the oxytocin [4-Glu,5-Asp ]]Oxytocin [4-Glu,5-Asp ] in impurity crude product solution]The structural formula of the impurity is
；
The filler of the high performance liquid reverse phase chromatography is super water-resistant filler; the super water-resistant filler is UniSil
ODS-AQ super water-resistant filler;
the reverse phase enrichment, reverse phase salt conversion and reverse phase purification are all completed in one-step reverse phase elution process, and the conditions of the reverse phase enrichment, the reverse phase salt conversion and the reverse phase purification are as follows:
wherein the mobile phase A is acetic acid/water solution with volume percentage of 0.005-0.1%, the mobile phase B is acetic acid/acetonitrile solution with volume percentage of 0.005-0.1%, and the sample C1 is oxytocin [4-Glu, 5-Asp%]Crude product of impuritiesThe mobile phase C2 is 5-50 mM NH4Ac-NH4The pH value of the mobile phase C2 is 7.0-9.0, and the flow rate of the eluent is 80-100 ml/min;
and collecting the eluent with the retention time of 68-71 min to obtain the oxytocin [4-Glu,5-Asp ] impurity solution.
2. The method for refining oxytocin [4-Glu,5-Asp ] impurity according to claim 1, characterized in that: the crude oxytocin [4-Glu,5-Asp ] impurity solution is prepared by dissolving, diluting and oxidizing a solid-phase synthesized crude reduced oxytocin [4-Glu,5-Asp ] impurity.
3. The method for refining oxytocin [4-Glu,5-Asp ] impurity according to claim 2, characterized in that: dissolving and diluting the crude reduced oxytocin [4-Glu,5-Asp ] impurity product to obtain a crude reduced oxytocin [4-Glu,5-Asp ] impurity product solution, wherein the concentration of the crude reduced oxytocin [4-Glu,5-Asp ] impurity product in the crude reduced oxytocin [4-Glu,5-Asp ] impurity product solution is 0.1-4 mg/ml; the solvent for dissolving the crude product of the reduced oxytocin [4-Glu,5-Asp ] impurity is an acetic acid/water solution with the volume percentage of 50%.
4. The method for purifying oxytocin [4-Glu,5-Asp ] impurities according to claim 3, characterized in that: the concentration of the crude reduced oxytocin [4-Glu,5-Asp ] impurity in the crude reduced oxytocin [4-Glu,5-Asp ] impurity solution is 0.5-2 mg/ml.
5. The method for refining oxytocin [4-Glu,5-Asp ] impurity according to claim 1, characterized in that: the solvent in the oxytocin [4-Glu,5-Asp ] impurity crude product solution is an aqueous solution containing trifluoroacetic acid and acetic acid.
6. The method for refining oxytocin [4-Glu,5-Asp ] impurity according to claim 1, characterized in that: the aperture of the super water-resistant filler is 7-10 nm, and the particle size is 10 microns.
7. The method for refining oxytocin [4-Glu,5-Asp ] impurity according to claim 1, characterized in that: the detection wavelength of the high performance liquid reverse phase chromatography is 220 nm.
8. The method for refining oxytocin [4-Glu,5-Asp ] impurity according to claim 1, characterized in that: the mobile phase A is an acetic acid/water solution with the volume percentage of 0.02-0.05%;
and/or the mobile phase B is an acetic acid/acetonitrile solution with the volume percentage of 0.02-0.05%;
and/or the mobile phase C2 is 10-20 mM NH4Ac-NH4An aqueous OH solution;
and/or the pH of the mobile phase C2 is 7.5-8.5;
and/or the HPLC purity of the oxytocin [4-Glu,5-Asp ] impurity crude product solution is 60-85%.
9. The method for purifying oxytocin [4-Glu,5-Asp ] impurity according to claim 8, characterized in that: the HPLC purity of the oxytocin [4-Glu,5-Asp ] impurity crude product solution is 70-85%.
10. The method for purifying oxytocin [4-Glu,5-Asp ] impurities according to claim 9, characterized in that: the HPLC purity of the oxytocin [4-Glu,5-Asp ] impurity crude product solution is 70-80%.
11. The method for refining oxytocin [4-Glu,5-Asp ] impurity according to claim 1, characterized in that: completely replacing the eluent with a mobile phase C2 from a sample C1 in a period of 30-31 min; and completely replacing the eluent from the mobile phase C2 to the mobile phase A within 40-41 min.
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| CN106518976A (en) * | 2017-01-03 | 2017-03-22 | 上海上药第生化药业有限公司 | Preparation method of oxytocin [4-Glu, 5-Asp] |
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