CN109541158A - A kind of measuring method of the encapsulation rate of liposome medicament - Google Patents
A kind of measuring method of the encapsulation rate of liposome medicament Download PDFInfo
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- CN109541158A CN109541158A CN201811522748.2A CN201811522748A CN109541158A CN 109541158 A CN109541158 A CN 109541158A CN 201811522748 A CN201811522748 A CN 201811522748A CN 109541158 A CN109541158 A CN 109541158A
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- 239000003814 drug Substances 0.000 title claims abstract description 128
- 238000005538 encapsulation Methods 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 35
- 239000002502 liposome Substances 0.000 title claims abstract description 32
- 229940079593 drug Drugs 0.000 claims abstract description 96
- 239000011347 resin Substances 0.000 claims abstract description 67
- 229920005989 resin Polymers 0.000 claims abstract description 67
- 239000002250 absorbent Substances 0.000 claims abstract description 48
- 230000002745 absorbent Effects 0.000 claims abstract description 48
- 238000005259 measurement Methods 0.000 claims abstract description 25
- 239000003480 eluent Substances 0.000 claims abstract description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 26
- 239000000872 buffer Substances 0.000 claims description 26
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 claims description 23
- 229940115080 doxil Drugs 0.000 claims description 19
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 18
- 239000003960 organic solvent Substances 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 13
- 150000003839 salts Chemical class 0.000 claims description 13
- 239000007975 buffered saline Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 7
- 235000019441 ethanol Nutrition 0.000 claims description 7
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 6
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 3
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 claims description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 3
- 229910002651 NO3 Inorganic materials 0.000 claims description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 3
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 125000003118 aryl group Chemical group 0.000 claims description 3
- ZCDOYSPFYFSLEW-UHFFFAOYSA-N chromate(2-) Chemical compound [O-][Cr]([O-])(=O)=O ZCDOYSPFYFSLEW-UHFFFAOYSA-N 0.000 claims description 3
- 150000001860 citric acid derivatives Chemical class 0.000 claims description 3
- 229910052736 halogen Inorganic materials 0.000 claims description 3
- 150000002367 halogens Chemical class 0.000 claims description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-M methacrylate group Chemical group C(C(=C)C)(=O)[O-] CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 claims description 3
- 150000003891 oxalate salts Chemical class 0.000 claims description 3
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical compound [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 2
- 238000005406 washing Methods 0.000 abstract description 8
- 238000001514 detection method Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 239000003463 adsorbent Substances 0.000 description 8
- 230000002209 hydrophobic effect Effects 0.000 description 7
- MWWSFMDVAYGXBV-RUELKSSGSA-N Doxorubicin hydrochloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 MWWSFMDVAYGXBV-RUELKSSGSA-N 0.000 description 6
- 229960002918 doxorubicin hydrochloride Drugs 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000002105 nanoparticle Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000001212 derivatisation Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 229960004679 doxorubicin Drugs 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000790917 Dioxys <bee> Species 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- KMSKQZKKOZQFFG-HSUXVGOQSA-N Pirarubicin Chemical compound O([C@H]1[C@@H](N)C[C@@H](O[C@H]1C)O[C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1CCCCO1 KMSKQZKKOZQFFG-HSUXVGOQSA-N 0.000 description 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Substances CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229960001221 pirarubicin Drugs 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- RYVMUASDIZQXAA-UHFFFAOYSA-N pyranoside Natural products O1C2(OCC(C)C(OC3C(C(O)C(O)C(CO)O3)O)C2)C(C)C(C2(CCC3C4(C)CC5O)C)C1CC2C3CC=C4CC5OC(C(C1O)O)OC(CO)C1OC(C1OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OCC(O)C(O)C1O RYVMUASDIZQXAA-UHFFFAOYSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention provides a kind of measuring method of the encapsulation rate of liposome medicament, belongs to chemicals detection technique field.The measuring method of the encapsulation rate of liposome medicament, comprising: measure the amount of entrapped drug: liposome medicament is mixed with macroporous absorbent resin, is obtained the first efflux using no machine eluate washing macroporous absorbent resin, is measured the amount of the entrapped drug in the first efflux.Measure the amount of free drug: after no machine eluate washing macroporous absorbent resin, macroporous absorbent resin also continues to wash proper second efflux using organic eluent, measures the amount of the free drug in the second efflux.Measure encapsulation rate: the amount of entrapped drug/(amount+free drug amount of entrapped drug) × 100%.The measuring method of this encapsulation rate is convenient for operation, and measurement result is more accurate.
Description
Technical field
The present invention relates to chemicals detection technique field, in particular to a kind of encapsulation rate of liposome medicament
Measuring method.
Background technique
Encapsulation rate is a kind of important index of liposome and the control of nanoparticle quality, and it is coated by carrier to reflect drug
Degree.The concentration of drug and carrier, the condition in preparation process are to influence the principal element of its encapsulation rate.Encapsulation rate includes percentage
The measurement of encapsulation rate and package volume, it is general main to investigate percentage encapsulation rate, expression formula be encapsulation rate=(1-Cf/Ct) ×
100%.Cf is the amount of free drug in formula；Ct is the total amount of drug in nanoparticle or Liposomal suspensions.
It is crucial step by nanoparticle or the separation of liposome and free drug for Accurate Determining encapsulation rate.Due to lipid
Body or nanoparticle bigger than the drug particle wrapped up can use the of different sizes of them and do not wrap up to be separated off more
Drug, or collect not wrapped drug.
Inventors have found that in the prior art, measurement encapsulation rate either uses liposome to separate with entrapped drug still not
The technical issues of isolated method can all cause entrapped drug to leak, to keep measurement result inaccurate.
Summary of the invention
The purpose of the present invention is to provide a kind of measuring methods of the encapsulation rate of liposome medicament, and measuring method is simple, and
Measurement result is more accurate.
The embodiment of the present invention provides a kind of measuring method of the encapsulation rate of liposome medicament, comprising:
It measures the amount of entrapped drug: liposome medicament is mixed with macroporous absorbent resin, washed using no machine eluate big
Macroporous adsorbent resin obtains the first efflux, measures the amount of the entrapped drug in the first efflux.
Measure the amount of free drug: after no machine eluate washing macroporous absorbent resin, macroporous absorbent resin is also using having
Machine eluate continues to wash proper second efflux, measures the amount of the free drug in the second efflux；
Measure encapsulation rate: the amount of entrapped drug/(amount+free drug amount of entrapped drug) × 100%.
The amount of entrapped drug and the amount of free drug are measured, needs first to separate entrapped drug with free drug, due to big
The duplicature of macroporous adsorbent resin have buffer action, when liposome medicament mix with macroporous absorbent resin, free drug with greatly
Macroporous adsorbent resin occur hydrophobic adsorbent and combine, entrapped drug due to film isolation and cannot be contacted with macroporous absorbent resin, from
And hydrophobic effect can not be played, it is flowed out by inorganic elution, that is, entrapped drug outflow, determines entrapped drug
Amount.
Then, the hydrophobic adsorbent effect of free drug and macroporous absorbent resin is destroyed using organic eluent and make free medicine
Object is dissociated from macroporous absorbent resin, to calculate the amount of free drug, finally obtains encapsulation rate, measurement result is more quasi-
Really.
With reference to first aspect, in another embodiment, no machine eluate includes water, physiological saline and buffered saline buffer
It is one or more；Optionally, buffered saline buffer includes the salt with buffer function and/or the salt without buffer function；Optionally,
Salt with buffer function includes the one or more of phosphate, citrate salt and acetate；Optionally, the salt of no buffer function
Including sulfate, nitrate, one kind containing halogen, carbonate, silicate, persulfate, chromate, lactate and oxalates or
It is a variety of；Optionally, the concentration of buffered saline buffer is no more than 0.9%.
So as to elute entrapped drug from macroporous absorbent resin, finally to measure encapsulation rate.Further
The concentration on ground, buffered saline buffer is no more than 0.8%, and further, the concentration of buffered saline buffer is no more than 0.5%, into one
The concentration on step ground, buffered saline buffer is no more than 0.1%.
With reference to first aspect, in another embodiment, organic eluent includes organic solvent, organic solvent include methanol,
Ethyl alcohol, propyl alcohol, butanol, acetone and butanone it is one or more；Optionally, organic solvent includes the one of methanol, ethyl alcohol and acetone
Kind is a variety of.To dissociate free drug and macroporous absorbent resin.
With reference to first aspect, in another embodiment, organic eluent includes organic solvent and water, and organic solvent occupies machine
The mass percent of eluent are as follows: 50%-100%；Optionally, organic solvent occupies the mass percent of machine eluate are as follows:
80%-100%, optionally, organic solvent occupy the mass percent of machine eluate are as follows: 90%-100%.
With reference to first aspect, in another embodiment, the amount of the entrapped drug in the first efflux is measured, comprising: first will
First outflow of first efflux using isopropanol demulsification constant volume, after reusing high-performance liquid chromatography measurement demulsification constant volume
The amount of entrapped drug in liquid.Keep the measurement of the amount of entrapped drug more accurate.
With reference to first aspect, in another embodiment, the amount of the free drug in the second efflux is measured, comprising: first will
Second efflux reuses the free medicine in high-performance liquid chromatography the second efflux of measurement using isopropanol demulsification constant volume
The amount of object.Keep the measurement of the amount of free drug more accurate.
With reference to first aspect, in another embodiment, liposome medicament is Doxil drug.
With reference to first aspect, in another embodiment, the weight of Doxil drug and macroporous absorbent resin
Amount volume ratio is 1-15mg/ml.Further, the bulking value of Doxil drug and macroporous absorbent resin
Than for 5-15mg/ml, further, the w/v of Doxil drug and macroporous absorbent resin is 5-
10mg/ml。
With reference to first aspect, in another embodiment, macroporous absorbent resin include polymerize by styrene with divinylbenzene and
At nonpolar aromatic series absorption resin and moderately polar methacrylic acid with methacrylate unit structure inhale
One or both of attached resin.Macroreticular resin is more preferable to the adsorption effect of free drug.
With reference to first aspect, in another embodiment, by liposome medicament loading to macroporous absorbent resin.Macroreticular resin pair
The adsorption effect of free drug is more preferable.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
The measuring method of the encapsulation rate of the liposome medicament of the embodiment of the present invention is specifically described below.
The measuring method of the encapsulation rate of liposome medicament, includes the following steps:
(1), it measures the amount of entrapped drug: liposome medicament being mixed with macroporous absorbent resin, is washed using no machine eluate
It washs macroporous absorbent resin and obtains the first efflux, measure the amount A of the entrapped drug in the first effluxPacket。
Since the duplicature of macroporous absorbent resin has buffer action, when liposome medicament is mixed with macroporous absorbent resin
When, free drug and macroporous absorbent resin occur hydrophobic adsorbent and combine, entrapped drug due to film isolation and cannot be with macropore
Resin contact is adsorbed, so that hydrophobic effect can not be played, is flowed out by inorganic elution, that is, entrapped drug stream
Out, the amount of entrapped drug is determined.
Optionally, by liposome medicament loading to macroporous absorbent resin.Liposome medicament, which flows through, is mounted with macroporous absorption tree
The chromatographic column of rouge makes free drug be adsorbed to macroporous absorbent resin, and entrapped drug is encapsulated in liposome, can not be inhaled with macropore
Attached resin contact, to flow directly out, obtains the first efflux.
Optionally, macroporous adsorption resin chromatography column can be UniHRPhenyl-60s, and no machine eluate flows through chromatographic column
Speed be 0.01-50 bed volume per minute, optionally, on condition that an external force is not applied naturally outflow.
Wherein, the aperture of macroporous absorbent resin is between 100-1000nm.Optionally, macroporous absorbent resin includes by benzene
The nonpolar aromatic series that ethylene and divinylbenzene are polymerized adsorbs resin and in methacrylate unit structure
One or both of methacrylic acid absorption resin of isopolarity.
No machine eluate includes the one or more of water, physiological saline and buffered saline buffer.Optionally, salt buffer is buffered
Liquid includes the salt with buffer function and/or the salt without buffer function；Optionally, with buffer function salt include phosphate,
Citrate salt and acetate it is one or more；Optionally, the salt of no buffer function includes sulfate, nitrate, containing halogen, carbon
Hydrochlorate, silicate, persulfate, chromate, lactate and oxalates it is one or more；Optionally, buffered saline buffer
Concentration is no more than 0.9%.
The first efflux is first reused into high-performance liquid chromatography measurement demulsification constant volume using isopropanol demulsification constant volume
The amount of entrapped drug in the first efflux afterwards.
(2), measure the amount of free drug: after no machine eluate washing macroporous absorbent resin, macroporous absorbent resin also makes
Continued to wash proper second efflux with organic eluent, measures the amount A of the free drug in the second effluxTrip。
Using organic eluent destroy the hydrophobic adsorbent effect of free drug and macroporous absorbent resin and make free drug from
It is dissociated on macroporous absorbent resin, to calculate the amount of free drug.
Optionally, macroporous absorbent resin is rinsed using organic eluent, the speed that organic eluent flows through chromatographic column is every
0.01-50 bed volume of minute optionally flows out naturally on condition that an external force is not applied.
Organic eluent includes organic solvent, and organic solvent includes the one of methanol, ethyl alcohol, propyl alcohol, butanol, acetone and butanone
Kind is a variety of；Optionally, organic solvent includes the one or more of methanol, ethyl alcohol and acetone.Organic eluent includes organic molten
Agent and water, organic solvent occupy the mass percent of machine eluate are as follows: 50%-100%；Optionally, organic solvent occupies machine washing
The mass percent of de- liquid are as follows: 80%-100%.
Measure the amount of the free drug in the second efflux, comprising: the second efflux is first used into isopropanol demulsification constant volume,
Reuse the amount for the free drug that high-performance liquid chromatography measures in the second efflux.
In the present embodiment, the condition of high-performance liquid chromatography is: octadecylsilane chemically bonded silica is filler
Chromatographic column；Mobile phase is 0.07mol/L ammonium acetate solution-acetonitrile (80:20), and flow velocity is 1.0ml per minute, uses ultraviolet detection
Device, Detection wavelength 254nm.
(3), encapsulation rate: the amount of entrapped drug/(amount+free drug amount of entrapped drug) × 100%, encapsulation rate is measured
=APacket/(APacket+ATrip) × 100%.
Liposome medicament is Doxil drug.Doxil is a kind of injection, is
Antitumor antibiotics class drug.Its chemical name is: (1S, 3S) -3- glycollyl -1,2,3,4,6,11- six oxygen -3,5,
12- trihydroxy -10- methoxyl group -6,13- dioxy aphthacene -1- base -3- amino -2,3, tri- deoxidation-α of 6- -
L- lysol pyranoside.Molecular formula: C27H29NO11HCl, structural formula are as follows:
When Doxil drug flows through macroporous absorbent resin, free Doxorubicin (HCl, that is,
Free drug) hydrophobic adsorbent occurs with macroporous absorbent resin and combines, the Doxorubicin (C of encapsulating27H29NO11, that is, encapsulate
Drug) due to film isolation and cannot be contacted with macroporous absorbent resin, so, when use without machine eluate wash macroporous absorption tree
When rouge, entrapped drug goes out with no machine eluate together as the first outflow liquid stream, and the macropore for having adsorbed free drug is inhaled
After organic eluent washing, free drug and organic eluent go out attached resin together as the second outflow liquid stream.
The w/v of Doxil drug and macroporous absorbent resin is 1-15mg/ml.Keep hydrochloric acid more
The soft encapsulation rate than star liposome medicament can be accurately out.
It should be noted that liposome medicament may also is that NSC 654509 liposome medicament, hydrochloric acid table pirarubicin
Liposome medicament.
Embodiment 1
Doxil lot number 20181102, encapsulation rate about 95%.
The measuring method of the encapsulation rate of Doxil, includes the following steps:
(1), the amount of entrapped drug is measured:
Take the Doxil 0.1ml loading of 2mg/ml to being equipped with 0.3mlUniHRPhenyl-60s resin
Column in, with 2ml water washing macroporous absorbent resin；The first efflux is collected into 20ml volumetric flask, with 75% isopropanol constant volume,
It shakes up, the amount A of doxorubicin hydrochloride in the first efflux is measured using aforementioned high-performance liquid chromatographyPacket=0.1905mg.
(2), the amount of free drug is measured:
Continued to rinse macroporous absorbent resin with 3ml90% methanol；The second efflux is collected into 20ml volumetric flask, with 75%
Isopropanol constant volume shakes up, and the amount A of doxorubicin hydrochloride in the second efflux is measured using aforementioned high-performance liquid chromatographyTrip=
0.0102mg。
(3), the calculating of encapsulation rate:
Encapsulation rate=APacket/(APacket+ATrip) × 100%
Result such as table 1 is obtained by the above method,
The analysis of 1 measurement result of table
Above-mentioned derivatization method measuring method specifically:
(1) it the measurement of total drug: takes the Doxil 2ml of 2mg/ml in the volumetric flask of 50ml, adds first
Alcohol to scale shakes up.Take wherein 1ml add methanol to scale in the volumetric flask of 10ml, shake up, be blank in 259nm using methanol
Place's measurement trap is A1=0.461.
(2) free drug measures: take 2mg/ml is with a batch of Doxil 2ml with step (1)
In the volumetric flask of 50ml, adds water to scale and shake up.It takes wherein that 1ml is in the volumetric flask of 10ml, at room temperature, adds 1mol/L's
Potassium hydroxide shakes up, measures immediately to scale；
The Doxil for taking same concentration (same batch), takes 2ml in the volumetric flask of 50ml, adds water to quarter
Degree shakes up, then takes wherein that 1ml is in the volumetric flask of 10ml, at room temperature, adds water to scale, shakes up, and is blank.
Using ultraviolet-visible photometer, it is A that trap is measured at 600nm2=0.016.
(3) pass through correction factor computational envelope rate, encapsulation rate=(1-A2×f/A1) × 100%, correction factor f are 1.5.
It is above-mentioned from fast centrifugal determination, carried out referring to State Food and Drug Administration's standard (YBH02522012).
As it can be seen from table 1 the encapsulation rate result of measurement of the embodiment of the present invention is more accurate.
Embodiment 2
Doxil lot number 20181102, encapsulation rate about 85%.
(1), the amount of entrapped drug is measured:
Take the Doxil 0.1ml loading of 2mg/ml to being equipped with 0.3mlUniHRPhenyl-60s resin
Column in, with 2ml water washing macroporous absorbent resin；The first efflux is collected into 20ml volumetric flask, with 75% isopropanol constant volume,
It shakes up, uses the amount A of doxorubicin hydrochloride in aforementioned high-performance liquid chromatography measurement effluxPacket=0.3396mg.
(2), the amount of free drug is measured:
Continued to rinse macroporous absorbent resin with 3ml methanol；The second efflux is collected into 20ml volumetric flask, with 75% isopropyl
Alcohol constant volume, shakes up, and the amount A of doxorubicin hydrochloride in the second efflux is measured using aforementioned high-performance liquid chromatographyTrip=
0.0603mg。
(3), the calculating of encapsulation rate:
Encapsulation rate=APacket/(APacket+ATrip) × 100%
Result such as table 2 is obtained by the above method,
The analysis of 2 measurement result of table
Above-mentioned derivatization method measuring method is identical as the method for embodiment 1.
It is above-mentioned from fast centrifugal determination, carried out referring to State Food and Drug Administration's standard (YBH02522012).
From table 2 it can be seen that the encapsulation rate result of measurement of the embodiment of the present invention is more accurate.
Embodiment 3
Doxil lot number 20181102, encapsulation rate about 85%.
(1), the amount of entrapped drug is measured:
Take the Doxil 0.1ml loading of 2mg/ml to being equipped with 0.3mlUniHRPhenyl-60s resin
Column in, wash macroporous absorbent resin with the sulfate liquor of 2ml0.8%；The first efflux is collected into 20ml volumetric flask, is used
75% isopropanol constant volume, shakes up, and uses the amount A of doxorubicin hydrochloride in aforementioned high-performance liquid chromatography measurement effluxPacket
=0.3409mg.
(2), the amount of free drug is measured:
Continued to rinse macroporous absorbent resin with 3ml methanol；The second efflux is collected into 20ml volumetric flask, with 75% isopropyl
Alcohol constant volume, shakes up, and the amount A of doxorubicin hydrochloride in the second efflux is measured using aforementioned high-performance liquid chromatographyTrip=
0.0591mg。
(3), the calculating of encapsulation rate:
Encapsulation rate=APacket/(APacket+ATrip) × 100%
Result such as table 3 is obtained by the above method,
The analysis of 3 measurement result of table
Above-mentioned derivatization method measuring method is identical as the method for embodiment 1.
It is above-mentioned from fast centrifugal determination, carried out referring to State Food and Drug Administration's standard (YBH02522012).
From table 3 it can be seen that the encapsulation rate result of measurement of the embodiment of the present invention is more accurate.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
1. a kind of measuring method of the encapsulation rate of liposome medicament characterized by comprising
It measures the amount of entrapped drug: the liposome medicament is mixed with macroporous absorbent resin, wash institute using no machine eluate
It states macroporous absorbent resin and obtains the first efflux, measure the amount of the entrapped drug in first efflux；
Measure the amount of free drug: after the no machine eluate washs the macroporous absorbent resin, the macroporous absorbent resin
Also continue to wash proper second efflux using organic eluent, measures the amount of the free drug in second efflux；
Measure encapsulation rate: the amount of the entrapped drug/(amount of the entrapped drug+free drug amount) × 100%.
2. measuring method according to claim 1, which is characterized in that the no machine eluate include water, physiological saline and
Buffered saline buffer it is one or more；
Optionally, the buffered saline buffer includes the salt with buffer function and/or the salt without buffer function；
Optionally, the salt with buffer function includes the one or more of phosphate, citrate salt and acetate；
Optionally, the salt of the no buffer function include sulfate, nitrate, containing halogen, carbonate, silicate, persulfate,
Chromate, lactate and oxalates it is one or more；
Optionally, the concentration of the buffered saline buffer is no more than 0.9%.
3. measuring method according to claim 1, which is characterized in that the organic eluent includes organic solvent, described
Organic solvent includes the one or more of methanol, ethyl alcohol, propyl alcohol, butanol, acetone and butanone；Optionally, the organic solvent packet
Include the one or more of methanol, ethyl alcohol and acetone.
4. measuring method according to claim 3, which is characterized in that the organic eluent includes organic solvent and water,
The organic solvent accounts for the mass percent of the organic eluent are as follows: 50%-100%；
Optionally, the organic solvent accounts for the mass percent of the organic eluent are as follows: 80%-100%.
5. measuring method according to claim 1, which is characterized in that the encapsulating medicine in measurement first efflux
The amount of object, comprising:
First efflux is first reused into high-performance liquid chromatography measurement demulsification constant volume using isopropanol demulsification constant volume
The amount of entrapped drug in first efflux afterwards.
6. measuring method according to claim 1, which is characterized in that the free medicine in measurement second efflux
The amount of object, comprising: second efflux is first reused into high-performance liquid chromatography measurement using isopropanol demulsification constant volume
The amount of free drug in second efflux.
7. measuring method according to claim 1-6, which is characterized in that the liposome medicament is that hydrochloric acid is more
It is soft than star liposome medicament.
8. measuring method according to claim 7, which is characterized in that the Doxil drug and described
The w/v of macroporous absorbent resin is 1-15mg/ml.
9. measuring method according to claim 7, which is characterized in that the macroporous absorbent resin includes by styrene and two
The nonpolar aromatic series that Ethenylbenzene is polymerized adsorbs resin and with the moderately polar of methacrylate unit structure
Methacrylic acid adsorbs one or both of resin.
10. measuring method according to claim 9, which is characterized in that by the liposome medicament loading to the macropore
Adsorb resin.
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Application publication date: 20190329 |