CN117659127A - Method for purifying octreotide - Google Patents
Method for purifying octreotide Download PDFInfo
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- CN117659127A CN117659127A CN202311853091.9A CN202311853091A CN117659127A CN 117659127 A CN117659127 A CN 117659127A CN 202311853091 A CN202311853091 A CN 202311853091A CN 117659127 A CN117659127 A CN 117659127A
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- DEQANNDTNATYII-OULOTJBUSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-16-benzyl-n-[(2r,3r)-1,3-dihydroxybutan-2-yl]-7-[(1r)-1-hydroxyethyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carboxa Chemical compound C([C@@H](N)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CC=2C=CC=CC=2)NC1=O)C(=O)N[C@H](CO)[C@H](O)C)C1=CC=CC=C1 DEQANNDTNATYII-OULOTJBUSA-N 0.000 title claims abstract description 134
- 108010016076 Octreotide Proteins 0.000 title claims abstract description 134
- 229960002700 octreotide Drugs 0.000 title claims abstract description 134
- 238000000034 method Methods 0.000 title claims abstract description 24
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 165
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 93
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 50
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 44
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims abstract description 44
- 238000000746 purification Methods 0.000 claims abstract description 38
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- 239000000945 filler Substances 0.000 claims abstract description 30
- 150000003839 salts Chemical class 0.000 claims abstract description 21
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 20
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 18
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 43
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 21
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- 238000001514 detection method Methods 0.000 claims description 21
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- 238000002360 preparation method Methods 0.000 claims description 15
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
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- 239000012528 membrane Substances 0.000 claims description 8
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
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- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 2
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- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
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- 230000003248 secreting effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
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Abstract
The invention relates to a method for purifying octreotide, belonging to the fields of drug analysis and peptide purification and separation. Weighing octreotide crude product, dissolving in mobile phase solution, dissolving fully, and filtering for use; performing a first HPLC purification by taking octadecylsilane chemically bonded silica filler as a stationary phase, perchloric acid as a mobile phase A and acetonitrile as a mobile phase B to remove oxidized impurities in octreotide; octadecylsilane chemically bonded silica filler is used as a stationary phase, and a phosphate solution is used as a mobile phase A; acetonitrile is used as a mobile phase B, and a second HPLC purification is carried out to remove impurities with physical and chemical properties similar to octreotide; octaalkylsilane bonded silica gel filler is used as a stationary phase, acetic acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, salt conversion is carried out, and solvent is removed, so that octreotide is obtained. According to the invention, impurities with similar physical and chemical properties to octreotide in the sample can be removed through twice HPLC purification, and the sample with the purity of more than 99% can be obtained; the yield is about 67.4%, and the purity and yield of the octreotide finished product are improved.
Description
Technical Field
The invention relates to the fields of pharmaceutical analysis and peptide purification and separation, in particular to a method for purifying octreotide.
Background
Octreotide (Octreotide) is an octapeptide derivative of natural somatostatin which is synthesized artificially, the molecular sequence is D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol, and the pharmacological action is similar to that of somatostatin but the action duration is longer.
The medicine is marketed in the United states in 1988 for the treatment of gastrointestinal secretory tumors and pituitary tumors. The product has various physiological activities such as growth hormone inhibition and thyrotropin inhibition; has inhibitory effect on secretion of gastric acid, pancreatin, glucagon and insulin; can reduce gastric motility and gallbladder emptying, inhibit secretion of cholecystokinin-pancreatin, reduce pancreatic secretion, and directly protect pancreatic parenchymal cell membrane; reducing visceral blood flow, reducing portal pressure, reducing excessive secretion in the intestine, and increasing water and sodium absorption in the intestine. The structural formula is as follows:
。
therefore, it is of great importance to study the purification process of octreotide with high purity and yield.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a novel method for purifying octreotide, which improves the purity and yield of octreotide through two HPLC purifications.
The object of the invention is achieved by the following technical scheme. A method for purifying octreotide, characterized by comprising the following steps:
(1) Pretreatment: weighing octreotide crude product, dissolving in mobile phase solution, dissolving fully, and filtering for use;
(2) Primary purification: performing a first HPLC purification by taking octadecylsilane chemically bonded silica filler as a stationary phase, perchloric acid as a mobile phase A and acetonitrile as a mobile phase B to remove oxidized impurities in octreotide;
(3) Removing the solvent to obtain octreotide first-step sample solution;
(4) And (3) secondary purification: octadecylsilane chemically bonded silica filler is used as a stationary phase, and a phosphate solution is used as a mobile phase A; acetonitrile is used as a mobile phase B, and a second HPLC purification is carried out to remove impurities with physical and chemical properties similar to octreotide;
(5) Removing the solvent to obtain octreotide sample solution;
(6) Salt conversion: performing salt transfer by taking octaalkylsilane bonded silica gel filler as a stationary phase, acetic acid as a mobile phase A and acetonitrile as a mobile phase B to obtain octreotide solution only containing acetate;
(7) And removing the solvent to obtain octreotide finished product solution.
The invention relates to a method for purifying octreotide, which further adopts the preferable technical scheme that: the pretreatment step of the step (1) comprises the following steps:
1) Dissolving the crude octreotide in acetonitrile aqueous solution and dilute ammonia water solution to obtain crude octreotide aqueous solution;
2) Standing at room temperature for 10 hr, filtering octreotide water solution with filter membrane to remove insoluble particles, and collecting filtrate.
The invention relates to a method for purifying octreotide, which further adopts the preferable technical scheme that: in the step (2) of primary purification: octadecylsilane chemically bonded silica filler is used as a stationary phase, 0.1% perchloric acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, and the first HPLC linear gradient elution and the gradient are carried out: 0 to 40min, 22 to 25 percent of mobile phase B; collecting a fraction containing octreotide sample; the particle size of the stationary phase is 10 μm; the preparation method of the 0.1% perchloric acid comprises the following steps: 1000ml of water was taken, 1ml of perchloric acid was added thereto, and the mixture was uniformly mixed to obtain 0.1% perchloric acid.
The invention relates to a method for purifying octreotide, which further adopts the preferable technical scheme that: in the step (3), removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 25-30 ℃ and the vacuum degree of below-0.09 MPa to obtain an octreotide sample solution in the first step, wherein the sample solution is acidic; and (3) adjusting the pH to 7.5-8 by using dilute ammonia water.
The invention relates to a method for purifying octreotide, which further adopts the preferable technical scheme that: and (4) in the secondary purification process: taking octadecylsilane chemically bonded silica filler as a stationary phase, taking phosphate solution as a mobile phase A, acetonitrile as a mobile phase B, performing a second HPLC gradient elution with the detection wavelength of 220nm, and collecting fractions containing octreotide samples; the preparation method of the phosphate solution comprises the following steps: 1000ml of water was added with 0.0.5% ammonia water and phosphoric acid was adjusted to ph=2.5 to give a phosphate solution.
The invention relates to a method for purifying octreotide, which further adopts the preferable technical scheme that: in the step (5), partial acetonitrile is removed by using a rotary evaporator at a water bath temperature of 35 ℃ and a vacuum degree of below-0.09 MPa, so as to obtain octreotide sample solution, wherein the sample solution is acidic.
The invention relates to a method for purifying octreotide, which further adopts the preferable technical scheme that: and (3) salt transferring in the step (6): octaalkylsilane bonded silica gel filler is used as a stationary phase, 0.5% acetic acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, salt transfer linear gradient elution is carried out, and fractions containing octreotide samples are collected; then, dilute ammonia water is used for adjusting the pH value to 7.5-8; the particle size of the stationary phase is 10 mu m, and the preparation method of 0.5% acetic acid comprises the following steps: 1000ml of water was taken, 5ml of acetic acid was added thereto, and the mixture was uniformly mixed to obtain 0.5% acetic acid.
The invention relates to a method for purifying octreotide, which further adopts the preferable technical scheme that: in the step (7), partial acetonitrile is removed by using a rotary evaporator at a water bath temperature of 35 ℃ and a vacuum degree of below-0.09 MPa, so as to obtain octreotide sample solution, wherein the sample solution is acidic.
Compared with the prior art, the invention has the following advantages:
the method can remove impurities with the physical and chemical properties similar to those of octreotide in the sample through twice purification, and can obtain the sample with the purity of more than 99% at one time; improves the purity and yield of octreotide finished products.
Detailed Description
The present invention will be specifically described with reference to the following specific examples.
Example 1, a method of purifying octreotide, comprising the steps of:
pretreatment: weighing octreotide crude product, dissolving in mobile phase solution, dissolving fully, and filtering for use;
the pretreatment steps comprise:
1) Dissolving the crude octreotide in acetonitrile aqueous solution and dilute ammonia water solution to obtain crude octreotide aqueous solution;
2) Standing at room temperature for 10 hr, filtering octreotide water solution with filter membrane to remove insoluble particles, and collecting filtrate.
(2) Primary purification: octadecylsilane chemically bonded silica filler is used as a stationary phase, 0.1% perchloric acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, and the first HPLC linear gradient elution and the gradient are carried out: 0 to 40min, 22 to 25 percent of mobile phase B; collecting a fraction containing octreotide sample; the particle size of the stationary phase is 10 μm; the preparation method of the 0.1% perchloric acid comprises the following steps: 1000ml of water was taken, 1ml of perchloric acid was added thereto, and the mixture was uniformly mixed to obtain 0.1% perchloric acid. Removing oxidized impurities in octreotide by first HPLC purification;
(3) Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 25 ℃ and the vacuum degree of below-0.09 MPa to obtain octreotide sample solution in the first step, wherein the sample solution is acidic; it was adjusted to pH 7.5 with dilute ammonia.
(4) And (3) secondary purification: taking octadecylsilane chemically bonded silica filler as a stationary phase, taking phosphate solution as a mobile phase A, acetonitrile as a mobile phase B, performing a second HPLC gradient elution with the detection wavelength of 220nm, and collecting fractions containing octreotide samples; the preparation method of the phosphate solution comprises the following steps: 1000ml of water was added with 0.0.5% ammonia water and phosphoric acid was adjusted to ph=2.5 to give a phosphate solution. The second HPLC purification to remove impurities with similar physical and chemical properties to octreotide;
(5) Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 35 ℃ and the vacuum degree of below-0.09 MPa to obtain octreotide sample solution, wherein the sample solution is acidic.
(6) Salt conversion: octaalkylsilane bonded silica gel filler is used as a stationary phase, 0.5% acetic acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, salt transfer linear gradient elution is carried out, and fractions containing octreotide samples are collected; then, dilute ammonia water is used for adjusting the pH value to 7.5; the particle size of the stationary phase is 10 mu m, and the preparation method of 0.5% acetic acid comprises the following steps: 1000ml of water was taken, 5ml of acetic acid was added thereto, and the mixture was uniformly mixed to obtain 0.5% acetic acid. Obtaining octreotide solution only containing acetate after salt conversion;
(7) Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 35 ℃ and the vacuum degree of below-0.09 MPa to obtain octreotide finished product solution, wherein the solution is acidic.
Example 2, a method of purifying octreotide, comprising the steps of:
(1) Pretreatment: weighing octreotide crude product, dissolving in mobile phase solution, dissolving fully, and filtering for use;
the pretreatment steps comprise:
1) Dissolving the crude octreotide in acetonitrile aqueous solution and dilute ammonia water solution to obtain crude octreotide aqueous solution;
2) Standing at room temperature for 10 hr, filtering octreotide water solution with filter membrane to remove insoluble particles, and collecting filtrate.
(2) Primary purification: octadecylsilane chemically bonded silica filler is used as a stationary phase, 0.1% perchloric acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, and the first HPLC linear gradient elution and the gradient are carried out: 0 to 40min, 22 to 25 percent of mobile phase B; collecting a fraction containing octreotide sample; the particle size of the stationary phase is 10 μm; the preparation method of the 0.1% perchloric acid comprises the following steps: 1000ml of water was taken, 1ml of perchloric acid was added thereto, and the mixture was uniformly mixed to obtain 0.1% perchloric acid. Removing oxidized impurities in octreotide by first HPLC purification;
(3) Removing part of acetonitrile by using a rotary evaporator at a water bath temperature of 30 ℃ and a vacuum degree of below-0.09 MPa to obtain octreotide sample solution in the first step, wherein the sample solution is acidic; and (3) adjusting the pH to be between 8 and pH by using dilute ammonia water.
(4) And (3) secondary purification: taking octadecylsilane chemically bonded silica filler as a stationary phase, taking phosphate solution as a mobile phase A, acetonitrile as a mobile phase B, performing a second HPLC gradient elution with the detection wavelength of 220nm, and collecting fractions containing octreotide samples; the preparation method of the phosphate solution comprises the following steps: 1000ml of water was added with 0.0.5% ammonia water and phosphoric acid was adjusted to ph=2.5 to give a phosphate solution. The second HPLC purification to remove impurities with similar physical and chemical properties to octreotide;
(5) Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 35 ℃ and the vacuum degree of below-0.09 MPa to obtain octreotide sample solution, wherein the sample solution is acidic.
(6) Salt conversion: octaalkylsilane bonded silica gel filler is used as a stationary phase, 0.5% acetic acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, salt transfer linear gradient elution is carried out, and fractions containing octreotide samples are collected; then, dilute ammonia water is used for adjusting the pH value to 8; the particle size of the stationary phase is 10 mu m, and the preparation method of 0.5% acetic acid comprises the following steps: 1000ml of water was taken, 5ml of acetic acid was added thereto, and the mixture was uniformly mixed to obtain 0.5% acetic acid. Obtaining octreotide solution only containing acetate after salt conversion;
(7) Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 35 ℃ and the vacuum degree of below-0.09 MPa to obtain octreotide finished product solution, wherein the solution is acidic.
Example 3 octreotide purification experiment one:
crude octreotide solution
Sample treatment: a sample containing 10g of octreotide was dissolved in acetonitrile water and a diluted ammonia solution, and after complete dissolution, the sample was left at room temperature for 10 hours and filtered through a 0.45 μm filter membrane. And collecting the filtered octreotide water solution for later use.
First HPLC purification
Octadecylsilane chemically bonded silica filler is used as a stationary phase, 0.1% perchloric acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, and the first HPLC linear gradient elution and the gradient are carried out: 0-40 min, 22-25% of mobile phase B, and flow rate: 80ml.min-1. Collecting a fraction containing octreotide sample; the particle size of the stationary phase was 10. Mu.m.
Fractions of octreotide samples with purity greater than 90% were collected and single impurities did not exceed 1.0% before and after. Removing part of acetonitrile by rotary evaporator at 30deg.C and vacuum degree below-0.09 Mpa. Obtaining octreotide first-step sample solution.
Second HPLC purification
Octadecylsilane chemically bonded silica filler is used as a stationary phase, a phosphate solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, the second HPLC gradient elution is carried out, the gradient is 0-50 min, the mobile phase B comprises 35% -55%, and the flow rate is as follows: 80ml.min-1. The fraction containing octreotide sample was collected.
Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 35 ℃ and the vacuum degree of below-0.09 MPa to obtain octreotide sample solution, wherein the sample solution is acidic.
Salt conversion
Octaalkylsilane bonded silica gel filler is used as a stationary phase, 0.5% acetic acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, salt transfer linear gradient elution is carried out, and fractions containing octreotide samples are collected; then, dilute ammonia water is used for adjusting the pH value to 7.5; the particle size of the stationary phase is 10 mu m, and the preparation method of 0.5% acetic acid comprises the following steps: 1000ml of water was taken, 5ml of acetic acid was added thereto, and the mixture was uniformly mixed to obtain 0.5% acetic acid.
Collecting fraction of octreotide sample with purity greater than 99%, removing part of acetonitrile with rotary evaporator at water bath temperature of 35deg.C and vacuum degree below-0.09 MPa to obtain octreotide sample solution, and quantitatively containing octreotide 6.65g with yield up to 66.5% by reference substance.
Example 4 octreotide purification experiment two:
crude octreotide solution
Sample treatment: a sample containing 50g of octreotide was dissolved in acetonitrile water and a diluted ammonia solution, and after complete dissolution, the sample was left at room temperature for 10 hours and filtered through a 0.45 μm filter membrane. And collecting the filtered octreotide water solution for later use.
First HPLC purification
Octadecylsilane chemically bonded silica filler is used as a stationary phase, 0.1% perchloric acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, and the first HPLC linear gradient elution and the gradient are carried out: 0-40 min, 22-25% of mobile phase B, and flow rate: 150ml.min-1. Collecting a fraction containing octreotide sample; the particle size of the stationary phase was 10. Mu.m.
Fractions of octreotide samples with purity greater than 90% were collected and single impurities did not exceed 1.0% before and after. The water bath temperature of the rotary evaporator is 25 ℃, and the vacuum degree is below-0.09 Mpa to remove part of acetonitrile. Obtaining octreotide first-step sample solution.
Second HPLC purification
Octadecylsilane chemically bonded silica filler is used as a stationary phase, a phosphate solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, the second HPLC gradient elution is carried out, the gradient is 0-50 min, the mobile phase B comprises 35% -55%, and the flow rate is as follows: 150ml.min-1. The fraction containing octreotide sample was collected.
Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 35 ℃ and the vacuum degree of below-0.09 MPa to obtain octreotide sample solution, wherein the sample solution is acidic.
Salt conversion
Octaalkylsilane bonded silica gel filler is used as a stationary phase, 0.5% acetic acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, salt transfer linear gradient elution is carried out, and fractions containing octreotide samples are collected; then, dilute ammonia water is used for adjusting the pH value to 7.7; the particle size of the stationary phase is 10 mu m, and the preparation method of 0.5% acetic acid comprises the following steps: 1000ml of water was taken, 5ml of acetic acid was added thereto, and the mixture was uniformly mixed to obtain 0.5% acetic acid.
Collecting fraction of octreotide sample with purity greater than 99%, removing part of acetonitrile with rotary evaporator at water bath temperature of 35deg.C and vacuum degree below-0.09 MPa to obtain octreotide sample solution, and quantitatively containing octreotide 33.6g with yield up to 67.2% by reference substance.
Example 5 octreotide purification experiment three:
crude octreotide solution
Sample treatment: a sample containing 100g of octreotide was dissolved in acetonitrile water and a diluted ammonia solution, and after complete dissolution, the sample was left at room temperature for 10 hours and filtered through a 0.45 μm filter membrane. And collecting the filtered octreotide water solution for later use.
First HPLC purification
Octadecylsilane chemically bonded silica filler is used as a stationary phase, 0.1% perchloric acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, and the first HPLC linear gradient elution and the gradient are carried out: 0-40 min, 22-25% of mobile phase B, and flow rate: 250ml.min-1. Collecting a fraction containing octreotide sample; the particle size of the stationary phase was 10. Mu.m.
Fractions of octreotide samples with purity greater than 90% were collected and single impurities did not exceed 1.0% before and after. Removing part of acetonitrile by rotary evaporator at water bath temperature of 28deg.C and vacuum degree below-0.09 Mpa. Obtaining octreotide first-step sample solution.
Second HPLC purification
Octadecylsilane chemically bonded silica filler is used as a stationary phase, a phosphate solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, the second HPLC gradient elution is carried out, the gradient is 0-50 min, the mobile phase B comprises 35% -55%, and the flow rate is as follows: 250ml.min-1. The fraction containing octreotide sample was collected.
Removing part of acetonitrile by using a rotary evaporator at the water bath temperature of 35 ℃ and the vacuum degree of below-0.09 MPa to obtain octreotide sample solution, wherein the sample solution is acidic.
Salt conversion
Octaalkylsilane bonded silica gel filler is used as a stationary phase, 0.5% acetic acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, salt transfer linear gradient elution is carried out, and fractions containing octreotide samples are collected; then, dilute ammonia water is used for adjusting the pH value to 8; the particle size of the stationary phase is 10 mu m, and the preparation method of 0.5% acetic acid comprises the following steps: 1000ml of water was taken, 5ml of acetic acid was added thereto, and the mixture was uniformly mixed to obtain 0.5% acetic acid.
Collecting fraction of octreotide sample with purity greater than 99%, removing part of acetonitrile with rotary evaporator at water bath temperature of 35deg.C and vacuum degree below-0.09 MPa to obtain octreotide sample solution, and quantitatively containing octreotide 68.4g with yield up to 68.4% by reference substance.
Three experiments show that impurities with the physical and chemical properties similar to those of octreotide in the sample can be removed through two times of HPLC purification, and the sample with the purity of more than 99% can be obtained; the yield is about 67.4%, and the purity and yield of the octreotide finished product are improved.
Claims (8)
1. A method for purifying octreotide, comprising the steps of:
(1) Pretreatment: weighing octreotide crude product, dissolving in mobile phase solution, dissolving fully, and filtering for use;
(2) Primary purification: performing a first HPLC purification by taking octadecylsilane chemically bonded silica filler as a stationary phase, perchloric acid as a mobile phase A and acetonitrile as a mobile phase B to remove oxidized impurities in octreotide;
(3) Removing the solvent to obtain octreotide first-step sample solution;
(4) And (3) secondary purification: octadecylsilane chemically bonded silica filler is used as a stationary phase, and a phosphate solution is used as a mobile phase A; acetonitrile is used as a mobile phase B, and a second HPLC purification is carried out to remove impurities with physical and chemical properties similar to octreotide;
(5) Removing the solvent to obtain octreotide sample solution;
(6) Salt conversion: performing salt transfer by taking octaalkylsilane bonded silica gel filler as a stationary phase, acetic acid as a mobile phase A and acetonitrile as a mobile phase B to obtain octreotide solution only containing acetate;
(7) And removing the solvent to obtain octreotide finished product solution.
2. The method of purifying octreotide according to claim 1, wherein the step (1) of pretreating step comprises:
1) Dissolving the crude octreotide in acetonitrile aqueous solution and dilute ammonia water solution to obtain crude octreotide aqueous solution;
2) Standing at room temperature for 10 hr, filtering octreotide water solution with filter membrane to remove insoluble particles, and collecting filtrate.
3. The method of purifying octreotide according to claim 1, wherein in the step (2) of one purification: octadecylsilane chemically bonded silica filler is used as a stationary phase, 0.1% perchloric acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, and the first HPLC linear gradient elution and the gradient are carried out: 0 to 40min, 22 to 25 percent of mobile phase B; collecting a fraction containing octreotide sample; the particle size of the stationary phase is 10 μm; the preparation method of the 0.1% perchloric acid comprises the following steps: 1000ml of water was taken, 1ml of perchloric acid was added thereto, and the mixture was uniformly mixed to obtain 0.1% perchloric acid.
4. The method for purifying octreotide according to claim 1, wherein in the step (3), partial acetonitrile is removed by a rotary evaporator at a water bath temperature of 25-30 ℃ and a vacuum degree of-0.09 MPa to obtain a first sample solution of octreotide, wherein the sample solution is acidic; and (3) adjusting the pH to 7.5-8 by using dilute ammonia water.
5. The method of purifying octreotide according to claim 1, wherein in the step (4) of secondary purification: taking octadecylsilane chemically bonded silica filler as a stationary phase, taking phosphate solution as a mobile phase A, acetonitrile as a mobile phase B, performing a second HPLC gradient elution with the detection wavelength of 220nm, and collecting fractions containing octreotide samples; the preparation method of the phosphate solution comprises the following steps: 1000ml of water was added with 0.0.5% ammonia water and phosphoric acid was adjusted to ph=2.5 to give a phosphate solution.
6. The method for purifying octreotide according to claim 1, wherein in the step (5), a portion of acetonitrile is removed at a water bath temperature of 35 ℃ and a vacuum degree of-0.09 MPa or less by a rotary evaporator to obtain an octreotide sample solution, and the sample solution is acidic.
7. The method of purifying octreotide according to claim 1, wherein in the salt transfer step (6): octaalkylsilane bonded silica gel filler is used as a stationary phase, 0.5% acetic acid is used as a mobile phase A, acetonitrile is used as a mobile phase B, the detection wavelength is 220nm, salt transfer linear gradient elution is carried out, and fractions containing octreotide samples are collected; then, dilute ammonia water is used for adjusting the pH value to 7.5-8; the particle size of the stationary phase is 10 mu m, and the preparation method of 0.5% acetic acid comprises the following steps: 1000ml of water was taken, 5ml of acetic acid was added thereto, and the mixture was uniformly mixed to obtain 0.5% acetic acid.
8. The method for purifying octreotide according to claim 1, wherein in the step (7), a portion of acetonitrile is removed at a water bath temperature of 35 ℃ and a vacuum degree of-0.09 MPa or less by a rotary evaporator to obtain an octreotide sample solution, and the sample solution is acidic.
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