CN116818935B - Method for detecting encapsulation rate of alprostadil injection - Google Patents
Method for detecting encapsulation rate of alprostadil injection Download PDFInfo
- Publication number
- CN116818935B CN116818935B CN202310651697.8A CN202310651697A CN116818935B CN 116818935 B CN116818935 B CN 116818935B CN 202310651697 A CN202310651697 A CN 202310651697A CN 116818935 B CN116818935 B CN 116818935B
- Authority
- CN
- China
- Prior art keywords
- alprostadil
- solution
- detecting
- encapsulated drug
- injection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229940070940 alprostadil injection Drugs 0.000 title claims abstract description 54
- 238000005538 encapsulation Methods 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 31
- GMVPRGQOIOIIMI-UHFFFAOYSA-N (8R,11R,12R,13E,15S)-11,15-Dihydroxy-9-oxo-13-prostenoic acid Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CCCCCCC(O)=O GMVPRGQOIOIIMI-UHFFFAOYSA-N 0.000 claims abstract description 81
- 229960000711 alprostadil Drugs 0.000 claims abstract description 81
- GMVPRGQOIOIIMI-DWKJAMRDSA-N prostaglandin E1 Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1CCCCCCC(O)=O GMVPRGQOIOIIMI-DWKJAMRDSA-N 0.000 claims abstract description 81
- 239000000243 solution Substances 0.000 claims abstract description 60
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 45
- 229920005654 Sephadex Polymers 0.000 claims abstract description 32
- 239000003814 drug Substances 0.000 claims abstract description 32
- 239000012488 sample solution Substances 0.000 claims abstract description 31
- 229940079593 drug Drugs 0.000 claims abstract description 28
- 239000012507 Sephadex™ Substances 0.000 claims abstract description 27
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 23
- 239000002904 solvent Substances 0.000 claims abstract description 23
- 239000003480 eluent Substances 0.000 claims abstract description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000012856 packing Methods 0.000 claims abstract description 11
- 239000007864 aqueous solution Substances 0.000 claims abstract description 10
- 238000003255 drug test Methods 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 238000001121 post-column derivatisation Methods 0.000 claims abstract description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 239000000523 sample Substances 0.000 claims description 9
- 239000000945 filler Substances 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 239000003643 water by type Substances 0.000 claims description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 229940111688 monobasic potassium phosphate Drugs 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims 1
- 239000000741 silica gel Substances 0.000 claims 1
- 229910002027 silica gel Inorganic materials 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 14
- 238000005406 washing Methods 0.000 description 14
- 235000011187 glycerol Nutrition 0.000 description 13
- 238000012360 testing method Methods 0.000 description 10
- 238000005119 centrifugation Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 238000007865 diluting Methods 0.000 description 7
- 239000000839 emulsion Substances 0.000 description 7
- 238000010828 elution Methods 0.000 description 6
- 239000013558 reference substance Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- CMDGQTVYVAKDNA-UHFFFAOYSA-N propane-1,2,3-triol;hydrate Chemical compound O.OCC(O)CO CMDGQTVYVAKDNA-UHFFFAOYSA-N 0.000 description 5
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 239000002960 lipid emulsion Substances 0.000 description 4
- -1 polytetrafluoroethylene Polymers 0.000 description 4
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 4
- 239000004810 polytetrafluoroethylene Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- 239000003957 anion exchange resin Substances 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 229940090044 injection Drugs 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 208000002249 Diabetes Complications Diseases 0.000 description 1
- 208000010228 Erectile Dysfunction Diseases 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 206010033647 Pancreatitis acute Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 201000003229 acute pancreatitis Diseases 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 208000023652 chronic gastritis Diseases 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000000916 dilatatory effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000003020 exocrine pancreas Anatomy 0.000 description 1
- 230000027119 gastric acid secretion Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000002464 muscle smooth vascular Anatomy 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 208000002815 pulmonary hypertension Diseases 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the technical field of medicine analysis, and particularly discloses a detection method of the encapsulation efficiency of alprostadil injection. The detection method provided by the invention comprises the steps of adding an alcohol solvent into the alprostadil injection to obtain a sample solution with the total alprostadil content; adding the alprostadil injection into a chromatographic column filled with sephadex, centrifuging the chromatographic column, and collecting centrifugate to obtain a primary alprostadil encapsulated drug solution; adding glycerol aqueous solution into a chromatographic column for eluting, collecting eluent, mixing the primary alprostadil encapsulated drug solution with the eluent, and adding an alcohol solvent to obtain alprostadil encapsulated drug test sample solution; and detecting the total content of the alprostadil sample solution and the alprostadil encapsulated drug sample solution by adopting a high performance liquid chromatography combined post-column derivatization device. The invention can minimize the influence of each operation step on the encapsulation rate of the alprostadil injection in the treatment process by utilizing the specific column packing and the specific eluting solvent.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a detection method of the encapsulation efficiency of alprostadil injection.
Background
The alprostadil has physiological effects of dilating vascular smooth muscle, inhibiting platelet aggregation, protecting vascular endothelial cells, inhibiting gastric acid secretion, stimulating intestinal canal and uterine smooth muscle, inhibiting exocrine pancreas, inhibiting lipid plaque, and stimulating insulin secretion. Is widely used for treating a plurality of diseases such as cerebrovascular diseases, cardiovascular diseases, respiratory diseases, diabetes complications, liver diseases, kidney diseases, acute pancreatitis, chronic gastritis, pulmonary hypertension, male erectile dysfunction, infertility and the like clinically.
The alprostadil injection prepared in the prior art is fat emulsion injection. The encapsulation efficiency is an important index for controlling the quality of the fat emulsion preparation, and reflects the degree of the medicine being coated by the carrier. Therefore, the method for accurately detecting the encapsulation efficiency has important significance for quality control of the fat emulsion injection.
At present, most of the commonly used encapsulation efficiency detection methods for the alprostadil injection are ultrafiltration centrifugation or microcolumn centrifugation for preparing microcolumns by taking weak-alkaline anion exchange resin as a filling material. The inventor adopts ultrafiltration centrifugation to measure the encapsulation efficiency of the alprostadil injection, but in practice, different filter membranes have different degrees of adsorption on the alprostadil, which can cause larger measurement results; when the entrapment rate of the alprostadil injection is measured by adopting a microcolumn centrifugation method by adopting weak-alkaline anion exchange resin as a filling material, fat emulsion is adsorbed in the resin due to negative charge, so that the measurement result is smaller. Therefore, the development of a novel detection method for the encapsulation efficiency of the alprostadil injection, which is convenient, quick and high in accuracy, has important significance.
Disclosure of Invention
In view of the above, the invention provides a method for detecting the encapsulation efficiency of the alprostadil injection, which can completely separate the encapsulated alprostadil from the free alprostadil by limiting a specific column packing and a specific eluting solvent and adopting mild conditions, so that the influence of each operation step in the treatment process on the encapsulation efficiency of the alprostadil injection can be reduced to the greatest extent.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
the invention provides a detection method of the encapsulation efficiency of alprostadil injection, which comprises the following steps:
Step a, adding an alcohol solvent into the alprostadil injection to obtain a sample solution with the total alprostadil content;
Step b, adding the alprostadil injection into a chromatographic column with a packing of sephadex, centrifuging the chromatographic column, and collecting centrifugate to obtain a primary alprostadil encapsulated drug solution;
c, adding 11.05mg/mL-44.20mg/mL of glycerol aqueous solution into the centrifuged chromatographic column for eluting, collecting eluent, mixing the primary alprostadil encapsulated drug solution with the eluent, and adding an alcohol solvent to obtain a alprostadil encapsulated drug test sample solution;
and d, detecting the total content of the test sample solution and the test sample solution of the alprostadil encapsulated drug by adopting a high performance liquid chromatography combined post-column derivatization device.
Compared with the prior art, the detection method of the encapsulation efficiency of the alprostadil injection provided by the invention has the advantages that the sephadex is selected as the column filler, the specific column filler can adsorb free alprostadil, and the encapsulated alprostadil is blocked outside the column filler, so that the rapid separation of the encapsulated alprostadil and the free alprostadil is realized; further, the solvent introduced by the injection in the sephadex can be removed to the greatest extent through centrifugal treatment, the dosage of the subsequent eluent is reduced, the encapsulation alprostadil and the free alprostadil can be completely separated through centrifugal treatment, and the accuracy of the encapsulation rate is improved; the special column packing is cooperated with centrifugal treatment, so that the encapsulated alprostadil can be blocked from entering the column packing, further, the solvent in the sephadex is thrown out of the column packing by centrifugal treatment, and meanwhile, a small amount of encapsulated alprostadil remained outside the sephadex is thrown out of the column packing along with the solvent, thereby maximally realizing the separation of the encapsulated alprostadil and the free alprostadil and greatly improving the accuracy of detecting the encapsulation rate; according to the invention, the dextran gel after centrifugation is eluted by using the glycerol aqueous solution with specific concentration, wherein the glycerol aqueous solution can increase the stability of the encapsulated alprostadil, and the encapsulated alprostadil is prevented from diffusing to the outer water phase in the elution process; the specific eluting solvent and the concentration of the eluting solvent can collect and encapsulate the alprostadil to the greatest extent, thereby improving the accuracy of detecting the encapsulation rate.
Preferably, in the step b, the type of the Sephadex is Sephadex G-50.
The preferred dextran gel is advantageous for improving the accuracy of the encapsulation efficiency of the detection.
Preferably, in step b, the sephadex is immersed in water to be fully expanded, and then dehydrated for use.
Preferably, in step a and step c, the alcohol solvent is at least one of methanol, ethanol or isopropanol.
Preferably, in the step b, the rotation speed of the centrifugation is 500-2000 r/min, and the centrifugation time is 1-3 min.
Preferably, in the step b, the volume ratio of the alprostadil injection to the sephadex is 0.05-0.2:1.
Preferably, in step c, the elution is centrifugation.
Further preferably, in the step c, the centrifugal speed is 500r/min-2000r/min during centrifugal elution, and the centrifugal time is 1min-3min.
The centrifugal speed provided by the steps b and c is low, the centrifugal time is short, and the problem that the encapsulation efficiency is inaccurate due to diffusion of the encapsulated alprostadil into the water phase can be avoided.
Preferably, in the step c, the volume ratio of the glycerol aqueous solution to the sephadex is 2.8-3.2:10.
The preferable proportion can elute and encapsulate the alprostadil, so that the accuracy of the encapsulation efficiency detection is improved.
Further preferably, in step c, the number of times of elution may be 3.
The preferred number of elution will maximize elution of the non-eluted encapsulated alprostadil, but will not elute the free alprostadil in the dextran gel.
It should be further noted that in step b and step c, the centrifuge tube collecting the centrifugate and the eluent is further washed with water or methanol, and the collected washing liquid is mixed with the collected primary alprostadil encapsulated drug solution and the eluent.
Preferably, in the step c, the content of the alcohol solvent in the alprostadil test sample solution is more than or equal to 50%.
Preferably, in step d, the high performance liquid chromatography column is a Waters Xbridge C18,4.6mm x 250mm,5 μm; the detection wavelength of the high performance liquid chromatography is 278nm; the mobile phase of the high performance liquid chromatography is a mixed solution of 0.0067mol/L phosphate solution and acetonitrile according to the volume ratio of 3:1; the post-column reaction solution is 1mol/L potassium hydroxide solution, the post-column reaction tube is a polytetrafluoroethylene tube, the specification is 0.5mm multiplied by 10mm, and the column temperature is 60 ℃.
Preferably, the phosphate solution is a monobasic potassium phosphate solution.
According to the invention, the encapsulation of the alprostadil and the free alprostadil are completely separated by using a specific column packing and a specific eluting solvent under mild conditions, so that the influence of each operation step in the treatment process on the encapsulation rate of the alprostadil injection can be reduced to the greatest extent; the invention provides a method for rapidly and accurately measuring the encapsulation efficiency of the alprostadil injection, which is simple and convenient to operate and can be used for researching the prescription process and controlling the quality of the alprostadil injection.
Drawings
FIG. 1 is a chromatogram of an interference experiment in an application example;
wherein A is alprostadil reference substance solution, B is blank solvent, C is blank emulsion solution, and D is alprostadil encapsulated drug test substance solution.
Detailed Description
The present invention will be described in further detail with reference to the following examples and drawings in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
The embodiment provides a method for detecting the encapsulation efficiency of alprostadil injection, which comprises the following steps:
Step a, adding methanol into the alprostadil injection to obtain a sample solution with the total alprostadil content;
Step b, adding the alprostadil injection into a chromatographic column with a filler of dehydrated Sephadex G-50, centrifuging the chromatographic column for 3min at a rotating speed of 500r/min, and collecting centrifugate to obtain a primary alprostadil encapsulated drug solution; adding 11.05mg/mL glycerol water solution into the chromatographic column, centrifuging and eluting for 3 times, centrifuging for 3min at 500r/min each time, and collecting eluent; washing a centrifuge tube for collecting centrifugate and eluent by using methanol, and collecting washing liquid; mixing the primary alprostadil encapsulated drug solution, the washing solution and the eluent to obtain a primary alprostadil encapsulated drug test sample solution; wherein, the volume ratio of the alprostadil injection to the sephadex is 0.05:1, and the volume ratio of the glycerol aqueous solution to the sephadex is 2.8:10;
Step c, adding methanol into the primary alprostadil encapsulated drug sample solution to obtain the alprostadil encapsulated drug sample solution; the content of methanol in the solution of the test sample of the alprostadil encapsulated drug is more than or equal to 50 percent;
step d, detecting the total content of the sample solution and the sample solution of the alprostadil encapsulated drug by adopting a high performance liquid chromatography combined post-column derivatization device; wherein the chromatographic column of the high performance liquid chromatography is Waters Xbridge C18,4.6mm×250mm,5 μm; the detection wavelength of the high performance liquid chromatography is 278nm; the mobile phase of the high performance liquid chromatography is a mixed solution of 0.0067mol/L potassium dihydrogen phosphate solution and acetonitrile according to the volume ratio of 3:1; the post-column reaction solution is 1mol/L potassium hydroxide solution, the post-column reaction tube is a polytetrafluoroethylene tube, the specification is 0.5mm multiplied by 10mm, and the column temperature is 60 ℃.
The results of the three replicates were as follows:
Example 2
The embodiment provides a method for detecting the encapsulation efficiency of alprostadil injection, which comprises the following steps:
Step a, adding methanol into the alprostadil injection to obtain a sample solution with the total alprostadil content;
step b, adding the alprostadil injection into a chromatographic column with a filler of dehydrated Sephadex G-50, centrifuging the chromatographic column at a rotating speed of 1000r/min for 2min, and collecting centrifugate to obtain a primary alprostadil encapsulated drug solution; adding 22.1mg/mL glycerol water solution into the chromatographic column, centrifuging for eluting for 3 times, centrifuging for 2min at 1000r/min each time, and collecting eluent; washing a centrifuge tube for collecting centrifugate and eluent by using methanol, and collecting washing liquid; mixing the primary alprostadil encapsulated drug solution, the washing solution and the eluent to obtain a primary alprostadil encapsulated drug test sample solution; wherein, the volume ratio of the alprostadil injection to the sephadex is 0.1:1, and the volume ratio of the glycerol aqueous solution to the sephadex is 3:10;
Step c, adding methanol into the primary alprostadil encapsulated drug sample solution to obtain the alprostadil encapsulated drug sample solution; the content of methanol in the solution of the test sample of the alprostadil encapsulated drug is more than or equal to 50 percent;
step d, detecting the total content of the sample solution and the sample solution of the alprostadil encapsulated drug by adopting a high performance liquid chromatography combined post-column derivatization device; wherein the chromatographic column of the high performance liquid chromatography is Waters Xbridge C18,4.6mm×250mm,5 μm; the detection wavelength of the high performance liquid chromatography is 278nm; the mobile phase of the high performance liquid chromatography is a mixed solution of 0.0067mol/L potassium dihydrogen phosphate solution and acetonitrile according to the volume ratio of 3:1; the post-column reaction solution is 1mol/L potassium hydroxide solution, the post-column reaction tube is a polytetrafluoroethylene tube, the specification is 0.5mm multiplied by 10mm, and the column temperature is 60 ℃.
The results of the three replicates were as follows:
Example 3
The embodiment provides a method for detecting the encapsulation efficiency of alprostadil injection, which comprises the following steps:
Step a, adding methanol into the alprostadil injection to obtain a sample solution with the total alprostadil content;
Step b, adding the alprostadil injection into a chromatographic column with a filler of dehydrated Sephadex G-50, centrifuging the chromatographic column for 1min at a rotating speed of 2000r/min, and collecting centrifugate to obtain a primary alprostadil encapsulated drug solution; adding 44.20mg/mL glycerol water solution into the chromatographic column, centrifuging for eluting for 3 times, centrifuging for 1min at 2000r/min each time, and collecting eluent; washing a centrifuge tube for collecting centrifugate and eluent by using methanol, and collecting washing liquid; mixing the primary alprostadil encapsulated drug solution, the washing solution and the eluent to obtain a primary alprostadil encapsulated drug test sample solution; wherein, the volume ratio of the alprostadil injection to the sephadex is 0.2:1, and the volume ratio of the glycerol aqueous solution to the sephadex is 3.2:10;
Step c, adding methanol into the primary alprostadil encapsulated drug sample solution to obtain the alprostadil encapsulated drug sample solution; the content of methanol in the solution of the test sample of the alprostadil encapsulated drug is more than or equal to 50 percent;
step d, detecting the total content of the sample solution and the sample solution of the alprostadil encapsulated drug by adopting a high performance liquid chromatography combined post-column derivatization device; wherein the chromatographic column of the high performance liquid chromatography is Waters Xbridge C18,4.6mm×250mm,5 μm; the detection wavelength of the high performance liquid chromatography is 278nm; the mobile phase of the high performance liquid chromatography is a mixed solution of 0.0067mol/L potassium dihydrogen phosphate solution and acetonitrile according to the volume ratio of 3:1; the post-column reaction solution is 1mol/L potassium hydroxide solution, the post-column reaction tube is a polytetrafluoroethylene tube, the specification is 0.5mm multiplied by 10mm, and the column temperature is 60 ℃.
The results of the three replicates were as follows:
Comparative example 1
Compared with example 2, this comparative example differs from example 2 in that: the procedure of example 2 was repeated except that the aqueous glycerol solution in step c was replaced with an equal amount of water.
The results of the three replicates were as follows:
Comparative example 2
Compared with example 2, this comparative example differs from example 2 in that: the procedure of example 2 was repeated except that the aqueous glycerol solution in step c was replaced with an equal amount of 0.9wt% aqueous sodium chloride solution.
The results of the three replicates were as follows:
Comparative example 3
Compared with example 2, this comparative example differs from example 2 in that: the procedure of example 2 was repeated except that the aqueous glycerol solution in step c was replaced with an equal amount of 5wt% aqueous glucose solution.
The results of the three replicates were as follows:
Comparative example 4
Compared with example 2, this comparative example differs from example 2 in that: the concentration of the aqueous glycerin solution in step c was adjusted to 8.84mg/mL, and the other steps were the same as in example 2.
The results of the three replicates were as follows:
Comparative example 5
Compared with example 2, this comparative example differs from example 2 in that: the concentration of the aqueous glycerin solution in step c was adjusted to 53.04mg/mL, and the other steps were the same as in example 2.
The results of the three replicates were as follows:
Comparative example 6
Compared with example 2, this comparative example differs from example 2 in that: the procedure of example 2 was repeated except that Sephadex G-50 was replaced with an equal amount of Sephadex Sepharose 6B.
The results of the three replicates were as follows:
Comparative example 7
The embodiment provides a method for detecting the encapsulation efficiency of alprostadil injection, which comprises the following steps:
adding the alprostadil injection into a 100KD ultrafiltration centrifuge tube, centrifuging at 5000r/min and 2-8 ℃ for 45min, and directly measuring the water solution filtered by the lower layer of the ultrafiltration centrifuge tube.
The results of the three replicates were as follows:
Comparative example 8
The embodiment provides a method for detecting the encapsulation efficiency of alprostadil injection, which comprises the following steps:
Filling DEAE weak-base anion exchange resin into a 5mL column tube, and centrifugally dehydrating to form a 5mL dry column; 1mL of the alprostadil injection is loaded and centrifuged for 5 minutes at a centrifugation speed of 1500 rpm; centrifugally eluting with 1mL of water for 5 times; transferring the collected and eluted emulsion drops into a 10mL measuring flask, and determining the volume to obtain the content of the encapsulated medicine. And diluting the alprostadil injection to the theoretical concentration after passing through the column, measuring by the same method, and then calculating the encapsulation efficiency of the alprostadil injection.
The results of the three replicates were as follows:
Application example
Based on the detection method of the invention, the detection method is verified, and the detection method is concretely as follows:
1. Specialization of
(1) Interference experiment of solvent and auxiliary materials
The blank solvent is a mixed solution of methanol and water in a volume ratio of 1:1;
alprostadil control solution: taking a proper amount of alprostadil reference substance, dissolving the alprostadil reference substance with absolute ethanol and diluting the alprostadil reference substance with a mobile phase to prepare a solution with the alprostadil content of about 0.25 mug in each 1 ml;
Blank emulsion solution: precisely measuring the upper end of Yu Weizhu (dehydrated Sephadex G-50 as column packing, volume of 2.5 mL) added with 0.25mL of blank emulsion, centrifuging, and collecting centrifugate; then centrifugally eluting the microcolumn by 0.25mL of glycerin water solution (with the concentration of 22.1 mg/mL) each time, washing for 3 times, transferring the centrifugate and the eluent into a 5mL measuring flask, rinsing the centrifuge tube by methanol, transferring the centrifuge tube into the 5mL measuring flask, diluting to a scale by methanol, and shaking uniformly to obtain an empty white emulsion solution;
Respectively taking 100 μl of each of blank solvent, blank emulsion solution, alprostadil reference substance solution and alprostadil encapsulated drug test substance solution prepared in example 2, injecting into a liquid chromatograph, and recording the chromatograms, see fig. 1.
Results: neither the blank solvent nor the blank emulsion solution interfered with the detection of alprostadil.
2. Quantitative limit
Taking a alprostadil reference substance solution, gradually diluting with a mobile phase until the S/N of the alprostadil peak is between 10 and 20, detecting by a high performance liquid chromatography, preparing 6 parts in parallel, and calculating the relative standard deviation of the peak area.
Quantitative limit test results
Conclusion: the limit concentration of the alprostadil is 0.019 mug/mL, which is equivalent to 7.6% of the concentration of the main component, and the sensitivity of the method meets the detection requirement; the quantitative limit is repeatedly prepared for 6 times, the S/N is more than 10, the RSD of the peak area is 6.1%, and the quantitative limit repeatability is good.
3. Detection limit
And taking a quantitative limiting solution, and gradually diluting until the S/N of the alprostadil peak is 3-5.
Detection limit test results
Conclusion: the detection limit concentration of the alprostadil is 0.0063 mug/ml, which is equivalent to 2.5% of the concentration of the main component, and the S/N is more than 3, and the method sensitivity meets the detection requirement.
4. Sample loading volume investigation
Taking alprostadil injection, adding Yu Weizhu (dehydrated Sephadex G-50 as column packing with volume of 2.5 mL) into three volumes of high (0.5 mL), medium (0.25 mL) and low (0.125 mL), centrifuging, and collecting centrifugate; and then centrifugally eluting the microcolumn by 0.25mL of glycerol water (with the concentration of 22.1 mg/mL) each time, washing for 3 times, transferring the centrifugate and the eluent into a 5mL measuring flask, washing a centrifuge tube by methanol, transferring the centrifuge tube into the 5mL measuring flask, diluting to a scale by methanol, shaking uniformly, measuring the encapsulation efficiency of the test sample, and measuring the average volume of each sample for 3 times. As a result, there was no significant difference in the encapsulation efficiency measured for the different loading volumes.
Encapsulation efficiency measurement results of different loading volumes
5. Sample recovery test
Taking alprostadil injection, loading samples according to three volumes of 0.225mL, 0.250mL and 0.275mL, respectively sequentially loading 25 mu L of solutions with mass concentration of 20 mu g/mL, 10 mu g/mL and 5 mu g/mL respectively as low, medium and high concentration standard adding samples, centrifuging, and collecting centrifugate; and then centrifugally eluting the microcolumn by 0.25mL of glycerol water (with the concentration of 22.1 mg/mL) each time, washing for 3 times, transferring the centrifugate and the eluent into a 5mL measuring flask, rinsing the centrifuge tube by methanol, transferring the centrifuge tube into the measuring flask, diluting to a scale by methanol, and shaking uniformly. For each concentration of 3 parts, the sample recovery rate was calculated, and as a result, the average recovery rate was 98.5%, RSD was 1.1% (n=9), and the accuracy of the method was good.
Sample-adding recovery rate of encapsulation rate of alprostadil injection
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.
Claims (8)
1. The method for detecting the encapsulation efficiency of the alprostadil injection is characterized by comprising the following steps:
Step a, adding an alcohol solvent into the alprostadil injection to obtain a sample solution with the total alprostadil content;
Step b, adding the alprostadil injection into a chromatographic column with a packing of sephadex, centrifuging the chromatographic column, and collecting centrifugate to obtain a primary alprostadil encapsulated drug solution;
c, adding 11.05mg/mL-44.20mg/mL of glycerol aqueous solution into the centrifuged chromatographic column for eluting, collecting eluent, mixing the primary alprostadil encapsulated drug solution with the eluent, and adding an alcohol solvent to obtain a alprostadil encapsulated drug test sample solution;
Step d, detecting the total content of the sample solution and the sample solution of the alprostadil encapsulated drug by adopting a high performance liquid chromatography combined post-column derivatization device; the detection wavelength of the high performance liquid chromatography is 278nm; the mobile phase of the high performance liquid chromatography is a mixed solution of 0.0067mol/L phosphate solution and acetonitrile according to the volume ratio of 3:1; the chromatographic column filling agent of the high performance liquid chromatography is octadecylsilane chemically bonded silica gel.
2. The method for detecting the encapsulation efficiency of alprostadil injection according to claim 1, wherein in the step b, the type of the Sephadex is Sephadex G-50; and/or
In the step a and the step c, the alcohol solvent is at least one of methanol, ethanol or isopropanol.
3. The method for detecting the encapsulation efficiency of alprostadil injection according to claim 1, wherein in the step b, the centrifugal rotating speed is 500r/min-2000r/min, and the centrifugal time is 1min-3min.
4. The method for detecting the encapsulation efficiency of a alprostadil injection according to claim 1, wherein in the step b, the volume ratio of the alprostadil injection to the dextran gel is 0.05-0.2:1.
5. The method for detecting the encapsulation efficiency of alprostadil injection according to claim 1, wherein in the step c, the volume ratio of the glycerol aqueous solution to the dextran gel is 2.8-3.2:10.
6. The method for detecting the entrapment rate of a alprostadil injection according to claim 1, wherein in the step c, the content of an alcohol solvent in a solution of the alprostadil encapsulated drug test sample is more than or equal to 50%.
7. The method for detecting the encapsulation efficiency of alprostadil injection according to claim 1, wherein in the step d, the chromatographic column of the high performance liquid chromatography is Waters Xbridge C18,4.6mm x 250mm,5 μm.
8. The method for detecting the encapsulation efficiency of alprostadil injection according to claim 1, wherein the phosphate solution is a monobasic potassium phosphate solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310651697.8A CN116818935B (en) | 2023-06-02 | 2023-06-02 | Method for detecting encapsulation rate of alprostadil injection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310651697.8A CN116818935B (en) | 2023-06-02 | 2023-06-02 | Method for detecting encapsulation rate of alprostadil injection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116818935A CN116818935A (en) | 2023-09-29 |
| CN116818935B true CN116818935B (en) | 2024-04-26 |
Family
ID=88125002
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310651697.8A Active CN116818935B (en) | 2023-06-02 | 2023-06-02 | Method for detecting encapsulation rate of alprostadil injection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116818935B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106474066A (en) * | 2016-10-26 | 2017-03-08 | 沈阳医学院附属中心医院 | Lornoxicam flexible lipidosome is prepared in Box Behnken effect surface method optimization |
-
2023
- 2023-06-02 CN CN202310651697.8A patent/CN116818935B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106474066A (en) * | 2016-10-26 | 2017-03-08 | 沈阳医学院附属中心医院 | Lornoxicam flexible lipidosome is prepared in Box Behnken effect surface method optimization |
Non-Patent Citations (4)
| Title |
|---|
| 前列地尔长循环前体脂质体的研究;时念秋;中国优秀博硕士学位论文全文数据库 (硕士)医药卫生科技辑(第01期);E079-104 * |
| 国家食品药品监督管理局 发布.国家药品标准 新药转正标准第29册.2002,139-141. * |
| 微柱离心 - 药脂比测定脂质体药物包封率;王汀 等;沈阳药科大学学报;第25卷(第1期);10-14 * |
| 葡聚糖凝胶微柱-HPLC测定奥沙利铂脂质体包封率;刘阳 等;中国现代应用药学;第31卷(第1期);74-77 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116818935A (en) | 2023-09-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Blanchard et al. | Improved liquid-chromatographic determination of caffeine in plasma. | |
| Enquist et al. | Separation and quantitation of (R)‐and (S)‐atenolol in human plasma and urine using an α1‐AGP column | |
| CN116818935B (en) | Method for detecting encapsulation rate of alprostadil injection | |
| CN103822975B (en) | A kind of detection method of formulation of ' Sheng Mai ' of Radix Codonopsis side | |
| Ren-Dan et al. | Enantioseparation and determination of propranolol in human plasma on a new derivatized β-cyclodextrin-bonded phase by HPLC | |
| CN113237973B (en) | Method for detecting content of impurities in eldecalcitol soft capsules | |
| Lensmeyer et al. | Stabilized analysis of antidepressant drugs by solvent-recycled liquid chromatography: procedure and proposed resolution mechanisms for chromatography. | |
| CN109541158A (en) | A kind of measuring method of the encapsulation rate of liposome medicament | |
| CN113504325A (en) | Detection method of changyanning preparation | |
| CN110208396B (en) | High performance liquid chromatography for simultaneously determining contents of two main drugs in ivermectin cloxolone injection | |
| CN114577937A (en) | Method for detecting taurine content in formula milk powder for special medical purposes | |
| CN107300599A (en) | A kind of method of acidic drug in SPE liquid chromatogram combination examination liquid medicine | |
| CN110412164B (en) | Method for detecting related substances of mexiletine hydrochloride | |
| CN112649516A (en) | Derivatization-based milk powder containing 4 human milk oligosaccharides and qualitative and quantitative method thereof | |
| US4477346A (en) | Method for the removal of interfering substances in theophylline assays | |
| Gianesello et al. | Determination by coupled high-performance liquid chromatography—gas chromatography of the β-blocker levomoprolol in plasma following ophthalmic administration | |
| JP2021128005A (en) | Blood pretreatment method and examination method | |
| US4460695A (en) | Method for the removal of interfering substances, including caffeine, in theophylline assays | |
| CN112730680B (en) | Method for determining dissolution curve of mycophenolate mofetil | |
| CN113398900B (en) | Molecular imprinting material for aflatoxin and preparation method and application thereof | |
| CN110988182B (en) | Quality detection method of Zhuang medicine ethnic medicine Huatuo Fengtongbao capsule | |
| CN109307725B (en) | Analysis method of trimetazidine hydrochloride | |
| CN111323517B (en) | Content determination method | |
| CN117665139A (en) | Rapid determination method for encapsulation efficiency of complex injection | |
| CN109781905B (en) | Method for synchronously detecting coniferyl alcohol and pinoresinol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |