CN113049727A - Method for detecting polysorbate 80 content - Google Patents
Method for detecting polysorbate 80 content Download PDFInfo
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- CN113049727A CN113049727A CN202110250370.0A CN202110250370A CN113049727A CN 113049727 A CN113049727 A CN 113049727A CN 202110250370 A CN202110250370 A CN 202110250370A CN 113049727 A CN113049727 A CN 113049727A
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- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 title claims abstract description 86
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 title claims abstract description 86
- 229920000053 polysorbate 80 Polymers 0.000 title claims abstract description 86
- 229940068968 polysorbate 80 Drugs 0.000 title claims abstract description 83
- 238000000034 method Methods 0.000 title claims abstract description 48
- 239000000243 solution Substances 0.000 claims abstract description 84
- 238000001514 detection method Methods 0.000 claims abstract description 44
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 39
- 238000012360 testing method Methods 0.000 claims abstract description 31
- 239000000126 substance Substances 0.000 claims abstract description 25
- 238000005886 esterification reaction Methods 0.000 claims abstract description 14
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 12
- 239000012085 test solution Substances 0.000 claims abstract description 12
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 10
- 238000007069 methylation reaction Methods 0.000 claims abstract description 9
- 238000010790 dilution Methods 0.000 claims abstract description 8
- 239000012895 dilution Substances 0.000 claims abstract description 8
- 239000012086 standard solution Substances 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims description 35
- 239000011259 mixed solution Substances 0.000 claims description 33
- 238000010438 heat treatment Methods 0.000 claims description 32
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 31
- 239000003513 alkali Substances 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 21
- WCYAALZQFZMMOM-UHFFFAOYSA-N methanol;sulfuric acid Chemical compound OC.OS(O)(=O)=O WCYAALZQFZMMOM-UHFFFAOYSA-N 0.000 claims description 21
- 239000012047 saturated solution Substances 0.000 claims description 16
- LOHGGLZYTJNUAL-UHFFFAOYSA-M sodium;ethanol;chloride Chemical compound [Na+].[Cl-].CCO LOHGGLZYTJNUAL-UHFFFAOYSA-M 0.000 claims description 16
- 102000008946 Fibrinogen Human genes 0.000 claims description 15
- 108010049003 Fibrinogen Proteins 0.000 claims description 15
- 229940012952 fibrinogen Drugs 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 13
- 238000000926 separation method Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 claims description 8
- QGPGUZIKJKOKRF-UHFFFAOYSA-M potassium;acetonitrile;dihydrogen phosphate Chemical compound [K+].CC#N.OP(O)([O-])=O QGPGUZIKJKOKRF-UHFFFAOYSA-M 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 6
- 239000002585 base Substances 0.000 claims description 6
- 238000006386 neutralization reaction Methods 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 abstract description 2
- 229950008882 polysorbate Drugs 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 25
- 238000002474 experimental method Methods 0.000 description 23
- 239000013558 reference substance Substances 0.000 description 20
- 239000011550 stock solution Substances 0.000 description 13
- 239000011159 matrix material Substances 0.000 description 12
- 238000007865 diluting Methods 0.000 description 9
- 238000005119 centrifugation Methods 0.000 description 6
- 239000012488 sample solution Substances 0.000 description 6
- 239000007791 liquid phase Substances 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- -1 polyoxyethylene Polymers 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 4
- 229920001214 Polysorbate 60 Polymers 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 238000001212 derivatisation Methods 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000010926 purge Methods 0.000 description 3
- 229950004959 sorbitan oleate Drugs 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 2
- 229920006192 POE isosorbide Polymers 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 230000000379 polymerizing effect Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000012088 reference solution Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000011272 standard treatment Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 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
- KLDXJTOLSGUMSJ-JGWLITMVSA-N Isosorbide Chemical compound O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 KLDXJTOLSGUMSJ-JGWLITMVSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- 239000004147 Sorbitan trioleate Substances 0.000 description 1
- PRXRUNOAOLTIEF-ADSICKODSA-N Sorbitan trioleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCC\C=C/CCCCCCCC)[C@H]1OC[C@H](O)[C@H]1OC(=O)CCCCCCC\C=C/CCCCCCCC PRXRUNOAOLTIEF-ADSICKODSA-N 0.000 description 1
- TTZKGYULRVDFJJ-GIVMLJSASA-N [(2r)-2-[(2s,3r,4s)-3,4-dihydroxyoxolan-2-yl]-2-[(z)-octadec-9-enoyl]oxyethyl] (z)-octadec-9-enoate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](OC(=O)CCCCCCC\C=C/CCCCCCCC)[C@H]1OC[C@H](O)[C@H]1O TTZKGYULRVDFJJ-GIVMLJSASA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 229960002479 isosorbide Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- JZMJDSHXVKJFKW-UHFFFAOYSA-N methyl sulfate Chemical compound COS(O)(=O)=O JZMJDSHXVKJFKW-UHFFFAOYSA-N 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 125000000963 oxybis(methylene) group Chemical group [H]C([H])(*)OC([H])([H])* 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000019337 sorbitan trioleate Nutrition 0.000 description 1
- 229960000391 sorbitan trioleate Drugs 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to the technical field of polysorbate content detection, and particularly relates to a method for detecting polysorbate 80 content. The method for detecting the polysorbate 80 content comprises the following steps: respectively carrying out high performance liquid chromatography detection on the pretreated dilution of the polysorbate 80 standard substance and the pretreated dilution of the test sample, and calculating the content of polysorbate 80 in the test sample according to the detection peak area of the standard substance and the detection peak area of the test sample; the pretreatment of the polysorbate 80 standard product comprises the following steps: performing a first hydrolysis reaction on the polysorbate 80 standard solution, and then performing a first methyl esterification reaction; the pretreatment of the test article comprises the following steps: and carrying out a second hydrolysis reaction on the test solution, and then carrying out a second methylation reaction. The detection method disclosed by the invention is stable and has good specificity, strong specificity and high accuracy, and the content of polysorbate 80 in the solution can be simply, accurately and efficiently evaluated.
Description
Technical Field
The invention relates to the technical field of polysorbate content detection, and particularly relates to a method for detecting polysorbate 80 content.
Background
Polysorbate 80, polyoxyethylene sorbitan monooleate, the main component has a chemical formula of C24H44O6(C2H4O) n. Is easily soluble in water, soluble in ethanol, vegetable oil, ethyl acetate, methanol, and toluene, and insoluble in mineral oil. It is gelatinized at low temperature and heated to recover. It is very smelly and slightly bitter. Is a commonly used preparation auxiliary material in traditional Chinese medicine, chemical medicine and biological pharmacy, and mainly plays the roles of an emulsifier, a solubilizer, a wetting agent, a dispersant, a stabilizer and the like in the preparation process of various medicines.
Polysorbate 80 is prepared by esterifying and polymerizing oleic acid, sorbitan and ethylene oxide, and is polyoxyethylene 20 sorbitan oleate synthesized by polymerizing sorbitan oleate and ethylene oxide according to Chinese pharmacopoeia. In practice, however, the synthesis of 1mol of sorbitan oleate and 20mol of polyoxyethylene is only the total dosage ratio, and the number of polyoxyethylene polymerized on each molecule is different, so that the polysorbate 80 is a mixture with different polyoxyethylene polymerized numbers. Due to the difference of raw material sources and production processes, the chemical components and the proportion of products of different manufacturers may have larger difference. Through the research of multiple students, it is confirmed that polysorbate 80 mainly comprises 8 chemical substances such as polyoxyethylene sorbitan PS, polyoxyethylene sorbitan monooleate PSM, polyoxyethylene sorbitan dioleate PSD, polyoxyethylene sorbitan trioleate PSTri, polyoxyethylene sorbitan tetraoleate PSTetra, polyoxyethylene isosorbide PI, polyoxyethylene isosorbide monooleate PIM, ethylene isosorbide dioleate PID and the like.
Polysorbate 80 is not a single chemical component, and the difference between the main component and impurities of samples from different sources is large, so that the actual situation provides a challenge for the quality control of the auxiliary material. The determination of polysorbate 80 content and residual levels in formulations is a hot spot and challenge of current concern.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for detecting the polysorbate 80 content, which can simply, accurately and efficiently evaluate the polysorbate 80 content in a medicament, meets the requirements on specificity, accuracy, precision and the like in the process of method verification, and has important significance on the dosage, quality control and even clinical application of the polysorbate 80.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for detecting the polysorbate 80 content comprises the following steps:
respectively carrying out high performance liquid chromatography detection on the pretreated dilution of the polysorbate 80 standard substance and the pretreated dilution of the test sample, and calculating the content of polysorbate 80 in the test sample according to the detection peak area of the standard substance and the detection peak area of the test sample;
the pretreatment of the polysorbate 80 standard product comprises the following steps: performing a first hydrolysis reaction on the polysorbate 80 standard solution, and then performing a first methyl esterification reaction;
the pretreatment of the test article comprises the following steps: and carrying out a second hydrolysis reaction on the test solution, and then carrying out a second methylation reaction.
Preferably, the first hydrolysis reaction comprises: heating the mixed solution of the polysorbate 80 standard substance solution and the alkali liquor;
preferably, the temperature of the mixed solution of the polysorbate 80 standard substance solution and the alkali liquor for heating treatment is 65-75 ℃, and the time is 2.5-3.5 h;
preferably, the base comprises potassium hydroxide;
preferably, the first hydrolysis reaction is followed by a neutralization treatment with an acid.
Preferably, the first methyl esterification reaction comprises: heating the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution;
preferably, the temperature for heating the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution is 65-75 ℃, and the time is 0.8-1.2 h;
preferably, after the first methyl esterification reaction, adjusting the pH to be 5-9 by using alkali;
preferably, the mixture adjusted to pH 5-9 is filtered with a 0.45 μm filter membrane.
Preferably, the second hydrolysis reaction comprises: heating the mixed solution of the test solution and the alkali liquor;
preferably, the temperature of the mixed solution of the test solution and the alkali liquor for heating treatment is 65-75 ℃, and the time is 2.5-3.5 h;
preferably, the base comprises potassium hydroxide;
preferably, the second hydrolysis reaction is followed by a neutralization treatment with an acid.
Preferably, the second methylation reaction comprises: heating the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution;
preferably, the temperature for heating the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution is 65-75 ℃, and the time is 0.8-1.2 h;
preferably, after the second methylation reaction, adjusting the pH value to 5-9 by using alkali;
preferably, the mixture adjusted to pH 5-9 is filtered with a 0.45 μm filter membrane.
Preferably, the test article comprises fibrinogen;
preferably, the fibrinogen is concentrated prior to the pretreatment as follows:
carrying out first centrifugal treatment on a mixed solution of a fibrinogen solution and an ethanol-sodium chloride saturated solution in a centrifugal tube, carrying out first solid-liquid separation, and cleaning the centrifugal tube by using the ethanol-sodium chloride saturated solution; performing second centrifugal treatment, performing second solid-liquid separation, and cleaning the centrifugal tube by adopting an ethanol-sodium chloride saturated solution; combining the supernatant obtained after the first solid-liquid separation, the supernatant obtained after the second solid-liquid separation and an ethanol-sodium chloride saturated solution to wash the centrifuge tube to obtain a washing solution, and carrying out third centrifugal treatment; and concentrating the supernatant after the third centrifugal treatment at 50-60 ℃.
Preferably, the rotating speed of the first centrifugal treatment is 2900-3100 r/min, and the time is 8-12 min;
preferably, the rotation speed of the second centrifugal treatment is 2900-3100 r/min, and the time is 3-5 min;
preferably, the rotation speed of the third centrifugal treatment is 2900-3100 r/min, and the time is 20-30 min.
Preferably, the detection conditions of the high performance liquid chromatography comprise: a C8 chromatography column; the mobile phase is 18-22 mM potassium dihydrogen phosphate-acetonitrile solution; the detection wavelength is 235-242 nm; the flow rate is 0.8-1.2 mL/min.
Preferably, the mobile phase is a 20mM potassium dihydrogen phosphate-acetonitrile solution; the detection wavelength is 240 nm; the flow rate was 1.0 mL/min.
Preferably, the instrument for detecting high performance liquid chromatography comprises an Agilent 1260II high performance liquid chromatograph.
Compared with the prior art, the invention has the beneficial effects that:
the method for detecting the polysorbate 80 content has the advantages of good stability and specificity, high accuracy and 0.5ppm of lowest detectable solution concentration; the precision is high, the repeatability and the accuracy error RSD are both less than 8%, and R2 in the linear range of the reference product is more than 0.998. Therefore, the method provided by the invention can simply, conveniently, accurately and efficiently evaluate the polysorbate 80 content in the solution, and has important significance for the polysorbate 80 dosage, product quality control and even clinical application in the development of pharmaceutical preparations.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
A method for detecting the polysorbate 80 content comprises the following steps:
respectively carrying out high performance liquid chromatography detection on the pretreated dilution of the polysorbate 80 standard substance and the pretreated dilution of the test sample, and calculating the content of polysorbate 80 in the test sample according to the detection peak area of the standard substance and the detection peak area number of the test sample;
the pretreatment of the polysorbate 80 standard product comprises the following steps: performing a first hydrolysis reaction on the polysorbate 80 standard solution, and then performing a first methyl esterification reaction;
the pretreatment of the test article comprises the following steps: and carrying out a second hydrolysis reaction on the test solution, and then carrying out a second methylation reaction.
The method has good stability and specificity, strong specificity and high accuracy, and the lowest detectable solution concentration is 0.5 ppm; the precision is high, the repeatability and the accuracy error RSD are both less than 8%, and R2 in the linear range of the reference product is more than 0.998. Therefore, the method provided by the invention can simply, conveniently, accurately and efficiently evaluate the polysorbate 80 content in the solution, and has important significance for the polysorbate 80 dosage, product quality control and even clinical application in the development of pharmaceutical preparations.
Polysorbate 80 standards of the present invention were purchased from Sigma.
Preferably, the first hydrolysis reaction comprises: and heating the mixed solution of the polysorbate 80 standard substance solution and the alkali liquor.
Preferably, the temperature of the mixed solution of the polysorbate 80 standard substance solution and the alkali liquor for heating treatment is 65-75 ℃, and the time is 2.5-3.5 h.
In one embodiment, the temperature of the mixed solution of the polysorbate 80 standard solution and the alkali solution for the water-bath heating treatment is 65-75 ℃, and may be 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃ or 75 ℃.
In one embodiment, the mixed solution of the polysorbate 80 standard solution and the alkali liquor is subjected to water-bath heating for 2.5-3.5 hours, and optionally 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours or 3.5 hours.
Preferably, the base comprises potassium hydroxide.
Preferably, the volume ratio of the polysorbate 80 standard substance solution to the alkali liquor is (1.1-1.5): (1.8-2.2), wherein the concentration of the alkali is 0.8-1.5 mol/L.
Preferably, the concentration of the polysorbate 80 standard solution is 250-350 mug/mL.
The main component of polysorbate 80, polyoxyethylene sorbitan monooleate and other derivatives are not easy to be detected by a common detector, so that the macromolecular organic matter is firstly digested into fatty acid salt with smaller molecular weight by alkali, and detection by the detector is convenient after methyl esterification.
Preferably, the first hydrolysis reaction is followed by a neutralization treatment with an acid.
Preferably, the first methyl esterification reaction comprises: and carrying out water bath heating treatment on the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution.
Preferably, the temperature of the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution for water bath heating treatment is 65-75 ℃, and the time is 0.8-1.2 h.
In one embodiment, the temperature of the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution subjected to the water bath heating treatment is 65-75 ℃.
The methyl esterification reaction can enhance the stability of the sample and is convenient to detect. The specific temperature and time setting is more beneficial to enhancing the stability of the sample.
In one embodiment, the temperature of the water bath heating treatment of the mixture of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution is 65 to 75 ℃, and may be 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃ or 75 ℃.
In one embodiment, the time for performing the water-bath heating treatment on the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution is 0.8 to 1.2 hours, and may be selected from 0.8 hour, 0.9 hour, 1 hour, 1.1 hour, or 1.2 hours.
Preferably, the volume ratio of the supernatant to the sulfuric acid-methanol solution is 1: (1.8-2.5), wherein the mass content of sulfuric acid in the sulfuric acid-methanol is 9-11%.
Preferably, the pH value is adjusted to 5-9 by using alkali after the first methyl esterification reaction.
Adjusting the pH value to 5-9 by using 1M potassium hydroxide, and optionally adjusting the pH value to 6, 7 or 8.
Preferably, the mixture adjusted to pH 5-9 is filtered with a 0.45 μm filter membrane.
Preferably, the second hydrolysis reaction comprises: and carrying out water bath heating treatment on the mixed solution of the test solution and the alkali.
Preferably, the temperature of the mixed solution of the test solution and the alkali for water bath heating treatment is 65-75 ℃, and the time is 2.5-3.5 h.
In one embodiment, the temperature of the mixed solution of the sample solution and the alkali in the water bath heating treatment is 65 to 75 ℃, and may be selected from 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃ or 75 ℃.
In one embodiment, the time of the water-bath heating treatment of the mixed solution of the sample solution and the alkali is 2.5 to 3.5 hours, and may be 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9 hours, 3 hours, 3.1 hours, 3.2 hours, 3.3 hours, 3.4 hours or 3.5 hours.
Preferably, the base comprises potassium hydroxide.
Preferably, the volume ratio of the test solution to the alkali liquor is (1.1-1.5): (1.8-2.2), wherein the concentration of the alkali is 0.8-1.5 mol/L.
Preferably, the second hydrolysis reaction is followed by a neutralization treatment with an acid.
Preferably, the second methylation reaction comprises: and carrying out water bath heating treatment on the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution.
Preferably, the temperature of the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution for water bath heating treatment is 65-75 ℃, and the time is 0.8-1.2 h.
In one embodiment, the temperature of the water bath heating treatment of the mixture of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution is 65 to 75 ℃, and may be 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃ or 75 ℃.
Preferably, the volume ratio of the supernatant to the sulfuric acid-methanol solution is 1: (1.8-2.5), wherein the mass content of sulfuric acid in the sulfuric acid-methanol is 9-11%.
Preferably, the pH is adjusted to 5-9 by using alkali after the second methylation reaction.
Adjusting the pH value to 5-9 by using 1M potassium hydroxide, and optionally adjusting the pH value to 6, 7 or 8.
Preferably, the mixture adjusted to pH 5-9 is filtered with a 0.45 μm filter membrane.
Preferably, the test article comprises fibrinogen.
Preferably, the fibrinogen is concentrated prior to the pretreatment as follows:
carrying out first centrifugal treatment on a mixed solution of a fibrinogen solution and an ethanol-sodium chloride saturated solution in a centrifugal tube, carrying out first solid-liquid separation, and cleaning the centrifugal tube by using the ethanol-sodium chloride saturated solution; performing second centrifugal treatment, performing second solid-liquid separation, and cleaning the centrifugal tube by adopting an ethanol-sodium chloride saturated solution; combining the supernatant obtained after the first solid-liquid separation, the supernatant obtained after the second solid-liquid separation and an ethanol-sodium chloride saturated solution to wash the centrifuge tube to obtain a washing solution, and carrying out third centrifugal treatment; and concentrating the supernatant after the third centrifugal treatment at 50-60 ℃.
Preferably, the rotation speed of the first centrifugal treatment is 2900-3100 r/min, and the time is 8-12 min.
In one embodiment, the rotation speed of the first centrifugal treatment is 2900-3100 r/min, and may be 2900r/min, 2950r/min, 3000r/min, 3050r/min or 3100 r/min.
In one embodiment, the time of the first centrifugation treatment is 8-12 min, and can be selected from 8min, 9min, 10min, 11min or 12 min.
Preferably, the rotation speed of the second centrifugal treatment is 2900-3100 r/min, and the time is 3-5 min.
In one embodiment, the rotation speed of the second centrifugal treatment is 2900-3100 r/min, and may be 2900r/min, 2950r/min, 3000r/min, 3050r/min or 3100 r/min.
In one embodiment, the time of the second centrifugation treatment is 3-5 min, and can be 3min, 3.5min, 4min, 4.5min or 5 min.
Preferably, the rotation speed of the third centrifugal treatment is 2900-3100 r/min, and the time is 20-30 min.
In one embodiment, the rotation speed of the third centrifugal treatment is 2900-3100 r/min, and may be 2900r/min, 2950r/min, 3000r/min, 3050r/min or 3100 r/min.
In one embodiment, the time of the third centrifugation treatment is 20-30 min, and can be selected from 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min or 30 min.
In one embodiment, 1 freeze-dried fibrinogen finished product with the specification of 2.0mL is taken, 2.0mL of water is added for dissolving, 1.0mL of the sample is measured and put into a centrifuge tube, 5.0mL of ethanol-sodium chloride saturated solution is added, shaking is carried out uniformly, centrifugation is carried out for 8-12 minutes at 2900-3100 rpm, supernatant liquid is taken, the tube wall is carefully washed by 1.0mL of ethanol-sodium chloride saturated solution, centrifugation is carried out for 3-5 minutes at 2900-3100 rpm for 2-3 times, washing liquid and supernatant liquid are combined, centrifugation is carried out for 20-30 minutes at 2900-3100 rpm, the supernatant liquid is placed at 50-60 ℃, concentration is carried out to about 0.1-0.5mL by air purging, and 1mL of water is added for dissolving.
Preferably, the detection conditions of the high performance liquid chromatography comprise: a C8 chromatography column; the mobile phase is 18-22 mM potassium dihydrogen phosphate-acetonitrile solution; the detection wavelength is 235-242 nm; the flow rate is 0.8-1.2 mL/min.
The parameters of the C8 column were: 4.6 × 150mm, 4 μm.
In one embodiment, the mobile phase is 18-22 mM potassium dihydrogen phosphate-acetonitrile solution, and optionally 18mM, 19mM, 20mM, 21mM or 22 mM.
In one embodiment, the detection wavelength is 235-242 nm, and can be 235nm, 236nm, 237nm, 238nm, 239nm, 240nm, 241nm or 242 nm.
Preferably, the mobile phase is a 20mM potassium dihydrogen phosphate-acetonitrile solution; the detection wavelength is 240 nm; the flow rate was 1.0 mL/min.
Preferably, the instrument for detecting high performance liquid chromatography comprises an Agilent 1260II high performance liquid chromatograph.
The invention will be further explained with reference to specific examples.
Example 1
A method for detecting the polysorbate 80 content comprises the following steps:
1. preparation of test solution
(a) Taking 1 finished freeze-dried fibrinogen product with the specification of 2.0mL, adding 2.0mL of water for dissolving, measuring 1.0mL of a sample into a centrifuge tube, adding 5.0mL of ethanol-sodium chloride saturated solution, shaking up, centrifuging for 10 minutes at 3000r/min, taking supernatant, carefully washing the tube wall by using 1.0mL of ethanol-sodium chloride saturated solution, repeatedly washing and centrifuging for 2-3 times at 3000r/min for 4 minutes, combining the washing solution with the supernatant, centrifuging for 25 minutes at 3000r/min, placing the supernatant at 55 ℃, concentrating to about 0.1-0.5mL by using an air purging method, and adding 1mL of water for dissolving;
(b) adding 2mL of 1mol/l KOH solution into the concentrated and dissolved sample obtained in the step (a), uniformly mixing by vortex, placing in a 70 ℃ water bath for 3h, and neutralizing with hydrochloric acid for later use;
(c) taking 1mL of the upper layer solution in the step (b), adding 2mL of 10% sulfuric acid-methanol solution, carrying out water bath at 70 ℃ for 1 hour, neutralizing with 1M potassium hydroxide until the pH value is 7, and filtering with a 0.45-micrometer filter membrane to obtain a sample solution;
2. preparation of control solution
Accurately weighing a proper amount of polysorbate 80 reference solution (30mg accurately weighed), placing in a 100mL volumetric flask, adding water to a constant volume to reach a scale, shaking uniformly to prepare a polysorbate 80 aqueous solution with the concentration of 300 mug/mL, and treating the reference solution according to a test sample hydrolysis and esterification method;
3. sample testing
Detecting by using an Agilent 1260II high performance liquid chromatograph and a C8(4.6x150mM, 4 mu m) chromatographic column, wherein a chromatographic buffer solution is a 20mM potassium dihydrogen phosphate-acetonitrile solution, the detection wavelength is 240nm, and the flow rate is 1 mL/min;
4. content calculation
The content was calculated from the peak area of the control.
Examples of the experiments
Polysorbate 80 standard substance linear relation and sample inspection repeatability
The freeze-dried fibrinogen reference substance and the test substance are used as materials, and the detection method is adopted to detect and examine the biological activity of the test substance relative to the reference substance. The detection results of the polysorbate 80 content in the fibrinogen freeze-dried product are shown in table 1, the curve fitting conditions of the detection results of standard samples with different concentrations prepared by using the standard are good, the curve fitting constant R2 is more than 0.998, and the repeatability RSD of the sample content detection is 0.185%.
TABLE 1 polysorbate 80 Standard Linear relationship and sample test repeatability
| Serial number | Peak area 1 | Peak area 2 | Peak area 3 | Sample (μ g/mL) |
| 1 | 10.18 | 42.44 | 41.62 | 720.12 |
| 2 | 20.56 | 61.41 | 61.23 | 721.11 |
| 3 | 41.12 | 119.54 | 119.22 | 719.94 |
| 4 | 82.24 | 235.47 | 235.07 | 719.88 |
| 5 | 102.80 | 291.08 | 290.54 | 722.01 |
| R2 | 0.9984 | 0.9986 | 0.9986 | RSD：0.185％ |
Method verification of polysorbate 80 content detection method
1. And (3) derivatization treatment:
1) standard processing
Adding 1mL of 3M potassium hydroxide solution into 1mL of standard substance, hydrolyzing at 70 ℃ for 1 hour, cooling to room temperature, adjusting the pH to be below 9 with hydrochloric acid, centrifuging at 5000rpm for 5min, adding 2mL of 10% methanol sulfate solution into 1mL of upper layer liquid, carrying out water bath at 70 ℃ for 2 hours, filtering at 0.45 mu M, and detecting;
2) sample processing
Taking 5mL of sample, precipitating with 25mL of ethanol-saturated sodium chloride solution, centrifuging at 3000rpm for 10 minutes, separating the supernatant from the precipitate, carefully cleaning the centrifuge tube wall and the precipitate with 5mL of ethanol-saturated sodium chloride solution, centrifuging at 3000rpm for 5 minutes, repeating the operation for 2 times, combining the cleaning solution and the supernatant, purging at 55 ℃, evaporating to about 0.5-2.5mL, transferring to a 5mL volumetric flask, fixing the volume with water, and taking 1mL of the solution to carry out pre-column derivatization detection by a standard derivatization method.
2. The system applicability is as follows:
1) experimental methods
Processing 1000 mug/mL polysorbate 80 reference substance solution according to specified experimental conditions, detecting according to specified chromatographic conditions, carrying out parallel sample injection for 6 times, and calculating the retention time and RSD of peak area;
2) determination of results
The RSD of the retention time is less than 1%, the RSD of the peak area is less than 4%, the resolution is more than 1.5, and the tailing factor and the number of theoretical plates are proper;
3) results of the experiment
The RSD of the retention time was 0.03%, the RSD of the peak area was 0.08%, the degree of separation was 2.6, the tailing factor was 0.96, and the number of theoretical plates was 19071, and it was judged that the system applicability was acceptable.
TABLE 2 System suitability results
3. Limit of quantification
3.1 limit of quantitation
1) Experimental methods
Weighing a proper amount of polysorbate 80 reference substance, preparing a reference substance stock solution with the concentration of 1000 mug/mL, diluting with water respectively to obtain polysorbate 80 reference substance solutions with the concentrations of 10 mug/mL, 8 mug/mL and 5 mug/mL, treating according to a specified experimental method to obtain polysorbate 80 solutions with the concentrations of 1 mug/mL, 0.8 mug/mL and 0.5 mug/mL, and detecting according to specified chromatographic conditions;
2) determination of results
Selecting the concentration with the signal-to-noise ratio closest to 10 as the quantitative limit of the experiment;
3) results of the experiment
The signal-to-noise ratio was 19.13 at a concentration of 1.027. mu.g/mL, 25.32 at a concentration of 0.8216. mu.g/mL, and 13.46 at a concentration of 0.5315. mu.g/mL, so the concentration of 0.5. mu.g/mL was used as the limit of quantitation in the experiment;
3.2 quantitative limit repeatability:
1) experimental methods
Taking a proper amount of polysorbate 80 reference substance, preparing a polysorbate 80 reference substance stock solution with the concentration of 1000 mug/mL, diluting the polysorbate 80 reference substance stock solution with water into a reference substance solution with the concentration of 5 mug/mL, processing the reference substance solution according to a specified experimental method to obtain a polysorbate 80 solution with the concentration of 0.5 mug/mL, preparing 6 samples in parallel, and detecting under a specified chromatographic condition;
2) determination of results
RSD of the peak area of 6 samples is less than 8%;
3) results of the experiment
The RSD of the peak area of 6 samples is 7.86 percent and is less than 8 percent, so that the repeatability of the determination limit is qualified.
4. Standard curve
1) Experimental methods
Preparing a polysorbate 80 reference substance into a solution with the concentration of about 1mg/mL, diluting the solution with water to solutions of 100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL, 800. mu.g/mL and 1000. mu.g/mL respectively, treating 1mL of the solution in a standard treatment mode, repeatedly treating 3 parallel samples, and detecting according to a determined method;
preparing a polysorbate 80 reference substance into a solution with the concentration of about 1mg/mL, diluting the solution with water to solutions with the concentrations of 20 mu g/mL, 50 mu g/mL, 100 mu g/mL, 150 mu g/mL and 200 mu g/mL respectively, treating 1mL of the solution in a standard treatment manner, repeatedly treating 3 parallel samples, and detecting according to a determined method;
2) determination of results
Each experiment is subjected to linear fitting according to peak area and content, and R2 is more than 0.998;
3) results of the experiment
TABLE 3 Linear relationship at Low concentration
Linear relationship under concentration conditions in Table 4
As shown in tables 3 and 4, the method has a good linear relationship between 2-20 μ g/mL or 10-100 μ g/mL and the corresponding standard substance content is about 20.1 μ g/mL-0.201 mg/mL or 51.4 μ g/mL-1.028 mg/mL.
5. Repeatability of
1) Experimental methods
Accurately weighing a proper amount of polysorbate 80 reference substance solution (30mg accurately weighed), placing the weighed solution into a 100mL volumetric flask, adding water to a constant volume to scale, shaking up, preparing a polysorbate 80 aqueous solution with the concentration of 300 mug/mL, treating the reference substance solution according to a test sample hydrolysis method, preparing two reference substance solutions in parallel for detection, preparing 6 test sample solutions with the same concentration, carrying out sample injection detection by an analyst under the same condition as far as possible, and calculating the content of the test sample according to the peak area of a standard sample;
2) determination of results
The concentration RSD of the test sample is less than 4.0 percent;
3) the results of the experiment are shown in table 5;
TABLE 5 repeatability results
As can be seen from Table 5, the repetitive RSD of the sample content detection is less than 4%, which meets the requirements of the method.
6. Intermediate precision
1) Experimental methods
Preparing 6 parts of sample solution repeatedly and parallelly at different time by 2 testers (A, B), totaling 12 parts of sample solution, respectively injecting samples for 6 times by the 2 testers (A, B), and calculating the content of the test sample according to a content calculation formula in a repeated experiment;
2) determination of results
RSD of 12 detection content results is less than 8.0%;
3) the results of the experiment are shown in Table 6
TABLE 6 intermediate precision results
As can be seen from Table 6, the intermediate precision RSD of the sample content detection is less than 8%, which meets the requirements of the method.
7. Accuracy of
1) Experimental methods
And precisely weighing a proper amount of polysorbate 80 reference substances, putting the reference substances into a 100mL volumetric flask, diluting the reference substances to a scale by using a blank matrix, shaking the reference substances uniformly, and preparing a polysorbate 80 standard-added blank matrix stock solution.
Precisely measuring an appropriate amount of stock solution with the added standard blank matrix, placing the stock solution in a 10mL volumetric flask, and diluting the stock solution with the blank matrix to a scale mark, namely (1: 0.8) solution with the added standard blank matrix E1;
precisely measuring a proper amount of stock solution with the added standard blank matrix, placing the stock solution in a 10mL volumetric flask, and diluting the stock solution with the blank matrix to a scale mark, namely (1: 1) the solution with the added standard blank matrix E2;
precisely measuring an appropriate amount of stock solution with the added standard blank matrix, placing the stock solution in a 10mL volumetric flask, and diluting the stock solution with the blank matrix to a scale, namely (1: 1.2) solution with the added standard blank matrix E3;
taking the blank matrix solution E1, E2 and E3, treating the control solution according to the test product hydrolysis method, detecting according to the chromatographic conditions determined above, repeating the experiment three times, and calculating the recovery rate of the results of 9 experiments.
2) Determination of results
The recovery rate of 9 detections is 85-110%.
3) The results of the experiments are shown in Table 7;
TABLE 7 recovery results
As can be seen from Table 7, the recovery rates of the samples were 85% to 110%, and the method requirements were met. Thirdly, the comparison research of the detection method and the ultraviolet method of the invention
1. Comparative study of sample detection
Taking different batches of freeze-dried fibrinogen samples as test samples, and respectively carrying out comparative study by adopting a liquid phase method and an ultraviolet method.
The liquid phase method operation is the same as the sample detection method in example 2, and the ultraviolet method is the same as the measurement method of the residual quantity of the polybergine pear vinegar 80 in the third part 3023 of pharmacopeia, and the measurement results are shown in table 8:
TABLE 8 test results of different test methods
| Batch number | Theoretical addition amount | Liquid phase method detection result | Ultraviolet method detection result |
| BC201909003 | 800μg/mL | 788.97μg/mL | 467.16μg/mL |
| BC201910005 | 800μg/mL | 789.98μg/mL | 482.78μg/mL |
| BC201910006 | 800μg/mL | 783.64μg/mL | 507.51μg/mL |
| BC201912007 | 800μg/mL | 788.29μg/mL | 477.92μg/mL |
| BC201912008 | 800μg/mL | 794.31μg/mL | 523.77μg/mL |
| BC201912009 | 800μg/mL | 788.73μg/mL | 548.76μg/mL |
2. Comparative study of sample stability
Taking freeze-dried fibrinogen samples in a uniform batch, diluting and temporarily storing in a refrigerator. Appropriate amounts of samples were taken at 1, 4, 8, 12 and 24 hours and examined by the liquid phase method and the ultraviolet method, respectively, and the results are shown in table 9:
TABLE 9 comparative results of sample stability
The result shows that the liquid phase method for detecting the polysorbate 80 can truly and accurately reflect the content of the polysorbate 80 in the solution, the esterified sample is stable and not easy to degrade, and the content can still be accurately detected after the sample is prepared and stored for 24 hours. The result is stable and has good repeatability, and the accuracy, the repeatability, the stability and the like are all superior to those of a pharmacopoeia method.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for detecting the polysorbate 80 content is characterized by comprising the following steps:
respectively carrying out high performance liquid chromatography detection on the pretreated dilution of the polysorbate 80 standard substance and the pretreated dilution of the test sample, and calculating the content of polysorbate 80 in the test sample according to the detection peak area of the standard substance and the detection peak area of the test sample;
the pretreatment of the polysorbate 80 standard product comprises the following steps: performing a first hydrolysis reaction on the polysorbate 80 standard solution, and then performing a first methyl esterification reaction;
the pretreatment of the test article comprises the following steps: and carrying out a second hydrolysis reaction on the test solution, and then carrying out a second methylation reaction.
2. The method for detecting the polysorbate 80 content according to claim 1, wherein the first hydrolysis reaction comprises: heating the mixed solution of the polysorbate 80 standard substance solution and the alkali liquor;
preferably, the temperature of the mixed solution of the polysorbate 80 standard substance solution and the alkali liquor for heating treatment is 65-75 ℃, and the time is 2.5-3.5 h;
preferably, the base comprises potassium hydroxide;
preferably, the first hydrolysis reaction is followed by a neutralization treatment with an acid.
3. The method for detecting the polysorbate 80 content according to claim 1, wherein the first methyl esterification reaction comprises: heating the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution;
preferably, the temperature for heating the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution is 65-75 ℃, and the time is 0.8-1.2 h;
preferably, after the first methyl esterification reaction, adjusting the pH to be 5-9 by using alkali;
preferably, the mixture adjusted to pH 5-9 is filtered with a 0.45 μm filter membrane.
4. The method for detecting the polysorbate 80 content according to claim 1, wherein the second hydrolysis reaction comprises: heating the mixed solution of the test solution and the alkali liquor;
preferably, the temperature of the mixed solution of the test solution and the alkali liquor for heating treatment is 65-75 ℃, and the time is 2.5-3.5 h;
preferably, the base comprises potassium hydroxide;
preferably, the second hydrolysis reaction is followed by a neutralization treatment with an acid.
5. The method for detecting the polysorbate 80 content according to claim 1, wherein the second esterification reaction comprises: heating the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution;
preferably, the temperature for heating the mixed solution of the supernatant after the hydrolysis reaction and the sulfuric acid-methanol solution is 65-75 ℃, and the time is 0.8-1.2 h;
preferably, after the second methylation reaction, adjusting the pH value to 5-9 by using alkali;
preferably, the mixture adjusted to pH 5-9 is filtered with a 0.45 μm filter membrane.
6. The method for detecting the polysorbate 80 content of claim 1, wherein the sample comprises fibrinogen;
preferably, the fibrinogen is concentrated prior to the pretreatment as follows:
carrying out first centrifugal treatment on a mixed solution of a fibrinogen solution and an ethanol-sodium chloride saturated solution in a centrifugal tube, carrying out first solid-liquid separation, and cleaning the centrifugal tube by using the ethanol-sodium chloride saturated solution; performing second centrifugal treatment, performing second solid-liquid separation, and cleaning the centrifugal tube by adopting an ethanol-sodium chloride saturated solution; combining the supernatant obtained after the first solid-liquid separation, the supernatant obtained after the second solid-liquid separation and an ethanol-sodium chloride saturated solution to wash the centrifuge tube to obtain a washing solution, and carrying out third centrifugal treatment; and concentrating the supernatant after the third centrifugal treatment at 50-60 ℃.
7. The method for detecting the polysorbate 80 content according to claim 6, wherein the rotation speed of the first centrifugal treatment is 2900-3100 r/min, and the time is 8-12 min;
preferably, the rotation speed of the second centrifugal treatment is 2900-3100 r/min, and the time is 3-5 min;
preferably, the rotation speed of the third centrifugal treatment is 2900-3100 r/min, and the time is 20-30 min.
8. The method for detecting the polysorbate 80 content according to claim 1, wherein the detection conditions of the high performance liquid chromatography comprise: a C8 chromatography column; the mobile phase is 18-22 mM potassium dihydrogen phosphate-acetonitrile solution; the detection wavelength is 235-242 nm; the flow rate is 0.8-1.2 mL/min.
9. The method for detecting the polysorbate 80 content of claim 8, wherein the mobile phase is a 20mM potassium dihydrogen phosphate-acetonitrile solution; the detection wavelength is 240 nm; the flow rate was 1.0 mL/min.
10. The method for detecting the polysorbate 80 content of claim 1, wherein the instrument for detecting the high performance liquid chromatography comprises an Agilent 1260II high performance liquid chromatograph.
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| CN117470792B (en) * | 2023-12-22 | 2024-05-24 | 华通福源生物技术(北京)股份有限公司 | Analysis method for detecting polysorbate 80 content in protein freeze-dried preparation |
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