CN113049727B - Method for detecting content of polysorbate 80 - Google Patents
Method for detecting content of polysorbate 80 Download PDFInfo
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- CN113049727B CN113049727B CN202110250370.0A CN202110250370A CN113049727B CN 113049727 B CN113049727 B CN 113049727B CN 202110250370 A CN202110250370 A CN 202110250370A CN 113049727 B CN113049727 B CN 113049727B
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- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 title claims abstract description 84
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 title claims abstract description 84
- 229920000053 polysorbate 80 Polymers 0.000 title claims abstract description 84
- 229940068968 polysorbate 80 Drugs 0.000 title claims abstract description 81
- 238000000034 method Methods 0.000 title claims abstract description 47
- 239000000243 solution Substances 0.000 claims abstract description 70
- 238000001514 detection method Methods 0.000 claims abstract description 51
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 39
- 239000000126 substance Substances 0.000 claims abstract description 29
- 238000012360 testing method Methods 0.000 claims abstract description 27
- 238000005886 esterification reaction Methods 0.000 claims abstract description 25
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 24
- 239000012488 sample solution Substances 0.000 claims abstract description 19
- 239000012086 standard solution Substances 0.000 claims abstract description 14
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 11
- 238000010790 dilution Methods 0.000 claims abstract description 8
- 239000012895 dilution Substances 0.000 claims abstract description 8
- 239000000523 sample Substances 0.000 claims description 37
- 239000006228 supernatant Substances 0.000 claims description 34
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 31
- 239000003513 alkali Substances 0.000 claims description 29
- 239000011259 mixed solution Substances 0.000 claims description 28
- 238000010438 heat treatment Methods 0.000 claims description 26
- 239000007788 liquid Substances 0.000 claims description 23
- WCYAALZQFZMMOM-UHFFFAOYSA-N methanol;sulfuric acid Chemical compound OC.OS(O)(=O)=O WCYAALZQFZMMOM-UHFFFAOYSA-N 0.000 claims description 20
- 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
- 238000000926 separation method Methods 0.000 claims description 14
- 238000005119 centrifugation Methods 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 12
- 239000012530 fluid Substances 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 claims description 9
- QGPGUZIKJKOKRF-UHFFFAOYSA-M potassium;acetonitrile;dihydrogen phosphate Chemical compound [K+].CC#N.OP(O)([O-])=O QGPGUZIKJKOKRF-UHFFFAOYSA-M 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 5
- 230000003472 neutralizing effect Effects 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims 1
- 229920000136 polysorbate Polymers 0.000 abstract description 2
- 229950008882 polysorbate Drugs 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 26
- 239000013558 reference substance Substances 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 15
- 239000011159 matrix material Substances 0.000 description 15
- 239000011550 stock solution Substances 0.000 description 13
- 238000007865 diluting Methods 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- -1 polyoxyethylene Polymers 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 5
- 239000007791 liquid phase Substances 0.000 description 5
- 239000012071 phase Substances 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
- 239000002585 base Substances 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006386 neutralization reaction 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
- 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
- 230000000052 comparative effect Effects 0.000 description 3
- 238000001212 derivatisation Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000825 pharmaceutical preparation Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000010926 purge Methods 0.000 description 3
- 229950004959 sorbitan oleate Drugs 0.000 description 3
- 238000005303 weighing Methods 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
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 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
- 230000000379 polymerizing effect Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000011160 research 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
- 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
- 239000012670 alkaline solution Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 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
- 239000002270 dispersing agent Substances 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
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012535 impurity Substances 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
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 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
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000005070 sampling Methods 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
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- 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
Abstract
The invention relates to the technical field of polysorbate content detection, in particular to a method for detecting polysorbate 80 content. The method for detecting the content of polysorbate 80 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 substance, and calculating the content of polysorbate 80 in the test substance according to the detection peak area of the standard substance and the detection peak area of the test substance; the pretreatment of the polysorbate 80 standard comprises: performing a first hydrolysis reaction on the polysorbate 80 standard solution, and performing a first methyl esterification reaction; the pretreatment of the test article comprises the following steps: and (3) carrying out a second hydrolysis reaction on the sample solution, and then carrying out a second methyl esterification reaction. The detection method disclosed by the invention has the advantages of good stability and specificity, strong specificity and high accuracy, and can be used for simply, conveniently, accurately and efficiently evaluating the content of polysorbate 80 in the solution.
Description
Technical Field
The invention relates to the technical field of polysorbate content detection, in particular to a method for detecting polysorbate 80 content.
Background
Polysorbate 80 is polyoxyethylene sorbitan monooleate, and the main component has a chemical formula of C 24H44O6(C2H4 O) n. Is easily soluble in water, ethanol, vegetable oil, ethyl acetate, methanol, toluene, and insoluble in mineral oil. Cheng Jiaozhuang at low temperature, recovering after heating. Has special odor and slightly bitter taste. Is a common preparation auxiliary material in traditional Chinese medicine, chemical medicine and biological pharmacy, and mainly plays roles of emulsifying agent, solubilizer, wetting agent, dispersing agent, stabilizing agent and the like in the process of various pharmaceutical preparations.
Polysorbate 80 is produced by esterifying and polymerizing oleic acid, sorbitan and ethylene oxide, and is polyoxyethylene 20 sorbitan oleate synthesized by polymerizing sorbitan oleate and ethylene oxide in Chinese pharmacopoeia. However, in practice, the synthesis of 1mol of sorbitan oleate and 20mol of polyoxyethylene is only a total feed ratio, and the number of polyoxyethylene polymerized per molecule is not the same, so that polysorbate 80 is a mixture of different polyoxyethylene polymerization numbers. Because of different raw material sources and production processes, the chemical components and proportions of products of different factories can have larger differences. Through researches of multiple scholars, it is confirmed that polysorbate 80 is mainly composed of 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 other 8 chemical substances.
Polysorbate 80 is not a single chemical component, and the main component and impurities of samples from different sources are greatly different, so that the actual situation presents challenges for quality control of the auxiliary materials. The amount of polysorbate 80 and residual levels in the formulation are hot spots and difficulties of current concern.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for detecting the content of polysorbate 80, which can simply, conveniently, accurately and efficiently evaluate the content of polysorbate 80 in a medicament, meets the requirements on specificity, accuracy, precision and the like in the verification process of the method, and has important significance on the dosage, quality control and even clinical application of polysorbate 80.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
the method for detecting the content of polysorbate 80 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 substance, and calculating the content of polysorbate 80 in the test substance according to the detection peak area of the standard substance and the detection peak area of the test substance;
The pretreatment of the polysorbate 80 standard comprises: performing a first hydrolysis reaction on the polysorbate 80 standard solution, and performing a first methyl esterification reaction;
the pretreatment of the test article comprises the following steps: and (3) carrying out a second hydrolysis reaction on the sample solution, and then carrying out a second methyl esterification reaction.
Preferably, the first hydrolysis reaction comprises: heating the mixed solution of the polysorbate 80 standard solution and the alkali liquor;
Preferably, the temperature of the mixed solution of the polysorbate 80 standard solution and the alkali liquor for heating treatment is 65-75 ℃ for 2.5-3.5 hours;
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 includes: heating the mixed solution of the supernatant fluid after the hydrolysis reaction and sulfuric acid-methanol solution;
preferably, the temperature of the mixed solution of the supernatant fluid after the hydrolysis reaction and the sulfuric acid-methanol solution is 65-75 ℃ and the time is 0.8-1.2 h;
preferably, alkali is adopted to adjust the pH value to 5-9 after the first methyl esterification reaction;
Preferably, the mixture adjusted to pH 5-9 is filtered using a 0.45 μm filter.
Preferably, the second hydrolysis reaction comprises: heating the mixed solution of the sample solution and the alkali liquor;
preferably, the temperature of the mixed solution of the sample 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 methyl esterification reaction includes: heating the mixed solution of the supernatant fluid after the hydrolysis reaction and sulfuric acid-methanol solution;
preferably, the temperature of the mixed solution of the supernatant fluid after the hydrolysis reaction and the sulfuric acid-methanol solution is 65-75 ℃ and the time is 0.8-1.2 h;
Preferably, the pH value is adjusted to 5-9 by alkali after the second methyl esterification reaction;
Preferably, the mixture adjusted to pH 5-9 is filtered using a 0.45 μm filter.
Preferably, the test sample comprises fibrinogen;
Preferably, the fibrinogen is subjected to the following concentration treatment prior to the pretreatment:
Performing first centrifugal treatment on mixed solution of fibrinogen solution and ethanol-sodium chloride saturated solution in a centrifugal tube, performing first solid-liquid separation, and cleaning the centrifugal tube by adopting the ethanol-sodium chloride saturated solution; performing second centrifugal treatment, performing second solid-liquid separation, and cleaning the centrifuge tube by adopting an ethanol-sodium chloride saturated solution; combining the supernatant after the first solid-liquid separation, the supernatant after the second solid-liquid separation and the washing liquid obtained after the centrifuge tube is washed by the ethanol-sodium chloride saturated solution, and performing third centrifugation; concentrating the supernatant after the third centrifugation 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 rotating 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 include: a C8 chromatographic 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 240nm; the flow rate was 1.0mL/min.
Preferably, the apparatus 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 detection method of the polysorbate 80 content has the advantages of good stability and specificity, strong specificity and high accuracy, and the concentration of the lowest detectable solution is 0.5ppm; the precision is high, the repeatability and the accuracy error RSD are all less than 8%, and R2 in the linear range of the reference is more than 0.998. Therefore, the method provided by the invention can simply, conveniently, accurately and efficiently evaluate the content of the polysorbate 80 in the solution, and has important significance for the use amount of the polysorbate 80 in the development of the pharmaceutical preparation, the quality control of the product and even the clinical application.
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 for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The method for detecting the content of polysorbate 80 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 substance, and calculating the content of polysorbate 80 in the test substance according to the detection peak area of the standard substance and the detection peak area number of the test substance;
The pretreatment of the polysorbate 80 standard comprises: performing a first hydrolysis reaction on the polysorbate 80 standard solution, and performing a first methyl esterification reaction;
the pretreatment of the test article comprises the following steps: and (3) carrying out a second hydrolysis reaction on the sample solution, and then carrying out a second methyl esterification reaction.
The method has the advantages of good stability and specificity, strong specificity and high accuracy, and the concentration of the lowest detectable solution is 0.5ppm; the precision is high, the repeatability and the accuracy error RSD are all less than 8%, and R2 in the linear range of the reference is more than 0.998. Therefore, the method provided by the invention can simply, conveniently, accurately and efficiently evaluate the content of the polysorbate 80 in the solution, and has important significance for the use amount of the polysorbate 80 in the development of the pharmaceutical preparation, the quality control of the product and even the clinical application.
Polysorbate 80 standard of the present invention was purchased from Sigma.
Preferably, the first hydrolysis reaction comprises: and heating the mixed solution of the polysorbate 80 standard solution and the alkali liquor.
Preferably, the temperature of the mixed solution of the polysorbate 80 standard 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 is 65-75deg.C, and optionally 65 deg.C, 66 deg.C, 67 deg.C, 68 deg.C, 69 deg.C, 70 deg.C, 71 deg.C, 72 deg.C, 73 deg.C, 74 deg.C or 75 deg.C.
In one embodiment, the mixed solution of the polysorbate 80 standard solution and the alkali solution is subjected to water bath heating treatment for 2.5-3.5 h, and 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, 3.1h, 3.2h, 3.3h, 3.4h or 3.5h can be selected.
Preferably, the base comprises potassium hydroxide.
Preferably, the volume ratio of the polysorbate 80 standard solution to the alkali solution 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, is not easily detected by a common detector, so that macromolecular organic matters are firstly digested into fatty acid salts with smaller molecular weight by alkali, and the fatty acid salts are conveniently detected by the detector after methyl esterification.
Preferably, the first hydrolysis reaction is followed by a neutralization treatment with an acid.
Preferably, the first methyl esterification reaction includes: and carrying out water bath heating treatment on the mixed solution of the supernatant fluid after the hydrolysis reaction and the sulfuric acid-methanol solution.
Preferably, the temperature of the mixed solution of the supernatant fluid after the hydrolysis reaction and the sulfuric acid-methanol solution 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 mixed solution of the supernatant fluid after the hydrolysis reaction and the sulfuric acid-methanol solution is 65-75 ℃.
The methyl esterification reaction of the invention can enhance the stability of the sample and is convenient for detection. The specific temperature and time settings are 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 and the sulfuric acid-methanol solution after the hydrolysis reaction is 65 to 75 ℃, and 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃ or 75 ℃ can be selected.
In one embodiment, the time for heating the mixture of the supernatant liquid after the hydrolysis reaction and the sulfuric acid-methanol solution in water bath is 0.8-1.2 h, and 0.8h, 0.9h, 1h, 1.1h, or 1.2h may be selected.
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 alkali after the first methyl esterification reaction.
The pH is adjusted to 5-9 by 1M potassium hydroxide, and the pH can be adjusted to 6, 7 or 8.
Preferably, the mixture adjusted to pH 5-9 is filtered using a 0.45 μm filter.
Preferably, the second hydrolysis reaction comprises: and carrying out water bath heating treatment on the mixed solution of the sample solution and the alkali.
Preferably, the temperature of the mixed solution of the sample 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 mixture of the sample solution and the alkali is 65 to 75℃in the water bath heating treatment, and optionally 65℃66℃67℃68℃69℃70℃71℃72℃73℃74℃75 ℃.
In one embodiment, the water bath heating treatment is performed for 2.5-3.5 h, and 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, 3.1h, 3.2h, 3.3h, 3.4h or 3.5h may be selected.
Preferably, the base comprises potassium hydroxide.
Preferably, the volume ratio of the sample solution to the alkaline solution 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 methyl esterification reaction includes: and carrying out water bath heating treatment on the mixed solution of the supernatant fluid after the hydrolysis reaction and the sulfuric acid-methanol solution.
Preferably, the temperature of the mixed solution of the supernatant fluid after the hydrolysis reaction and the sulfuric acid-methanol solution 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 and the sulfuric acid-methanol solution after the hydrolysis reaction is 65 to 75 ℃, and 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃ or 75 ℃ can be selected.
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 alkali after the second methyl esterification reaction.
The pH is adjusted to 5-9 by 1M potassium hydroxide, and the pH can be adjusted to 6, 7 or 8.
Preferably, the mixture adjusted to pH 5-9 is filtered using a 0.45 μm filter.
Preferably, the test sample comprises fibrinogen.
Preferably, the fibrinogen is subjected to the following concentration treatment prior to the pretreatment:
Performing first centrifugal treatment on mixed solution of fibrinogen solution and ethanol-sodium chloride saturated solution in a centrifugal tube, performing first solid-liquid separation, and cleaning the centrifugal tube by adopting the ethanol-sodium chloride saturated solution; performing second centrifugal treatment, performing second solid-liquid separation, and cleaning the centrifuge tube by adopting an ethanol-sodium chloride saturated solution; combining the supernatant after the first solid-liquid separation, the supernatant after the second solid-liquid separation and the washing liquid obtained after the centrifuge tube is washed by the ethanol-sodium chloride saturated solution, and performing third centrifugation; concentrating the supernatant after the third centrifugation 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 2900r/min, 2950r/min, 3000r/min, 3050r/min or 3100r/min can be selected.
In one embodiment, the time of the first centrifugation is 8-12 min, and 8min, 9min, 10min, 11min or 12min may be selected.
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 2900r/min, 2950r/min, 3000r/min, 3050r/min or 3100r/min can be selected.
In one embodiment, the second centrifugation is performed for 3 to 5 minutes, and may be performed for 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, or 5 minutes.
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 2900r/min, 2950r/min, 3000r/min, 3050r/min or 3100r/min can be selected.
In one embodiment, the time of the third centrifugation is 20-30 min, and 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min or 30min may be selected.
In one embodiment, 1 branch of 2.0mL of freeze-dried fibrinogen finished product is taken, 2.0mL of water is added for dissolution, 1.0mL of the sample is measured into a centrifuge tube, 5.0mL of ethanol-sodium chloride saturated solution is added for shaking, centrifugation is carried out for 8-12 minutes at 2900-3100 rpm, the wall of the tube is taken out, the supernatant 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, the washing liquid is combined with the supernatant, centrifugation is carried out for 20-30 minutes at 2900-3100 rpm, the supernatant is concentrated to about 0.1-0.5 mL by an air purging method at 50-60 ℃, and 1mL of water is added for dissolution.
Preferably, the detection conditions of the high performance liquid chromatography include: a C8 chromatographic 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 chromatographic column were: 4.6X150mm,4 μm.
In one embodiment, the mobile phase is 18 to 22mM potassium dihydrogen phosphate-acetonitrile solution, and optionally 18mM, 19mM, 20mM, 21mM, or 22mM.
In one embodiment, the detection wavelength is 235-242 nm, and 235nm, 236nm, 237nm, 238nm, 239nm, 240nm, 241nm, or 242nm may also be selected.
Preferably, the mobile phase is a 20mM potassium dihydrogen phosphate-acetonitrile solution; the detection wavelength is 240nm; the flow rate was 1.0mL/min.
Preferably, the apparatus for detecting high performance liquid chromatography comprises an Agilent 1260II high performance liquid chromatograph.
The invention will be further illustrated with reference to specific examples.
Example 1
The method for detecting the content of polysorbate 80 comprises the following steps:
1. preparation of sample solution
(A) Taking 1 branch of freeze-dried fibrinogen finished product with the specification of 2.0mL, adding 2.0mL of water for dissolution, measuring 1.0mL of a test sample into a centrifuge tube, adding 5.0mL of ethanol-sodium chloride saturated solution, shaking uniformly, centrifuging for 10 minutes at 3000 rpm, taking supernatant, carefully flushing the tube wall with 1.0mL of ethanol-sodium chloride saturated solution, centrifuging for 4 minutes at 3000 rpm, repeatedly cleaning and centrifuging for 2-3 times, combining the washing liquid and the supernatant, centrifuging for 25 minutes at 3000 rpm, concentrating the supernatant to about 0.1-0.5mL by an air purging method under the condition of 55 ℃, and adding 1mL of water for dissolution;
(b) Adding 2mL of 1mol/l KOH solution into the sample after the concentration step (a) and dissolution, uniformly mixing by vortex, placing in a water bath at 70 ℃ 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 7,0.45 mu M, and filtering to obtain a sample solution;
2. Preparation of control solution
Precisely weighing a proper amount of polysorbate 80 reference substance solution (30 mg precisely weighed), placing into a 100mL volumetric flask, adding water to a certain volume to scale, shaking uniformly to prepare a polysorbate 80 aqueous solution with the concentration of 300 mug/mL, and treating the reference substance solution according to the hydrolysis and esterification method of the sample;
3. Sample testing
Agilent 1260II high performance liquid chromatograph is selected to be matched with a C8 (4.6x150mm, 4 mu m) chromatographic column for detection, chromatographic buffer solution is 20mM potassium dihydrogen phosphate-acetonitrile solution, the detection wavelength is 240nm, and the flow rate is 1mL/min for sample loading detection;
4. Content calculation
And calculating the content according to the peak area of the reference substance.
Experimental example
1. Polysorbate 80 standard linear relationship and sample test repeatability
The freeze-dried fibrinogen reference and the test sample are used as materials, and the biological activity of the test sample relative to the reference sample is detected and inspected by adopting the detection method. The detection results of the polysorbate 80 content in the fibrinogen freeze-dried product are shown in table 1, the curve fitting condition of the detection results of standard samples with different concentrations prepared by the standard products is good, the curve fitting constant R2 meets the requirement of > 0.998, and the sample content detection repeatability RSD is 0.185%.
TABLE 1 Polysorbate 80 Standard linear relationship and sample test repeatability
| Sequence number | Peak area 1 | Peak area 2 | Peak area 3 | Sample (μg/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％ |
2. Method verification of polysorbate 80 content detection method
1. And (3) derivatization treatment:
1) Standard substance treatment
1ML of a standard substance is taken, 1mL of 3M potassium hydroxide solution is added, hydrolysis is carried out for 1 hour at 70 ℃, the pH is regulated to be below 9 by hydrochloric acid after cooling to room temperature, centrifugation is carried out for 5min at 5000rpm, 2mL of 10% sulfuric acid methanol solution is added into 1mL of an upper layer liquid, water bath is carried out for 2 hours at 70 ℃, and detection is carried out after filtration at 0.45 μm;
2) Sample processing
Taking 5mL of sample, precipitating with 25mL of ethanol-saturated sodium chloride solution, centrifuging at 3000rpm for 10 minutes, separating supernatant and precipitate, carefully cleaning the wall and the precipitate of the centrifuge tube with 5mL of ethanol-saturated sodium chloride solution, centrifuging at 3000rpm for 5 minutes, repeating the operation for 2 times, combining the cleaning liquid and the supernatant, purging and evaporating to about 0.5-2.5mL at 55 ℃, transferring to a 5mL volumetric flask for constant volume with water, and taking 1mL of the supernatant and performing pre-column derivatization detection by a standard derivatization method.
2. System applicability:
1) Experimental method
Taking 1000 mug/mL of polysorbate 80 reference solution, processing according to specified experimental conditions, detecting according to specified chromatographic conditions, sampling for 6 times in parallel, and calculating the retention time and RSD of peak area;
2) Result determination
The retention time RSD is less than 1%, the peak area RSD is less than 4%, the separation degree is greater than 1.5, and the tailing factor and the theoretical plate number are proper;
3) Experimental results
The retention time RSD was 0.03%, the peak area RSD was 0.08%, the separation degree was 2.6, the tailing factor was 0.96, and the theoretical plate number was 19071, which can be judged as acceptable system applicability.
Table 2 system applicability results
3. Quantitative limit
3.1 Quantitative limit
1) Experimental method
Measuring a proper amount of polysorbate 80 reference substance, preparing reference substance stock solution with the concentration of 1000 mug/mL, respectively diluting with water to obtain polysorbate 80 reference substance solutions with the concentration of 10 mug/mL, 8 mug/mL and 5 mug/mL, processing according to a specified experimental method to obtain polysorbate 80 solution with the concentration of 1 mug/mL, 0.8 mug/mL and 0.5 mug/mL, and detecting according to specified chromatographic conditions;
2) Result determination
Selecting the concentration with the signal-to-noise ratio closest to 10 as the quantitative limit of the experiment;
3) Experimental results
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 that the concentration of 0.5. Mu.g/mL was used as a quantitative limit for the experiment;
3.2 quantitative limit reproducibility:
1) Experimental method
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 to obtain 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 specified chromatographic conditions;
2) Result determination
RSD for the 6 sample peak areas should be less than 8%;
3) Experimental results
Since the RSD of the peak area of 6 samples was 7.86% and less than 8%, the reproducibility of the quantitative limit was judged to be acceptable.
4. Standard curve
1) Experimental method
Taking a polysorbate 80 reference substance, preparing a solution with the concentration of about 1mg/mL, diluting the solution with water to obtain solutions with the concentration of 100 mug/mL, 200 mug/mL, 400 mug/mL, 800 mug/mL and 1000 mug/mL respectively, taking 1mL of the solution, treating the solution in a standard substance treatment mode, repeatedly treating 3 parallel samples, and detecting according to a determined method;
taking a polysorbate 80 reference substance, preparing a solution with the concentration of about 1mg/mL, diluting the solution with water to obtain solutions with the concentration of 20 mug/mL, 50 mug/mL, 100 mug/mL, 150 mug/mL and 200 mug/mL respectively, taking 1mL of the solution, treating the solution in a standard substance treatment mode, repeatedly treating 3 parallel samples, and detecting according to a determined method;
2) Result determination
Each experiment is linearly fitted with peak area and content, R2 should be greater than 0.998;
3) Experimental results
TABLE 3 Linear relationship under Low concentration conditions
The linear relationship under the concentration conditions in Table 4
As is clear from tables 3 and 4, the method can achieve a good linear relationship between the corresponding standard contents of about 20.1. Mu.g/mL-0.201 mg/mL or 51.4. Mu.g/mL-1.028 mg/mL in the range of 2 to 20. Mu.g/mL or 10 to 100. Mu.g/mL.
5. Repeatability of
1) Experimental method
Precisely weighing a proper amount of polysorbate 80 reference substance solution (30 mg precisely weighed), placing the solution into a 100mL volumetric flask, adding water to a fixed volume to scale, shaking uniformly, preparing a polysorbate 80 aqueous solution with the concentration of 300 mug/mL, treating the reference substance solution according to a sample hydrolysis method, preparing two reference substance solutions in parallel for detection, preparing 6 sample solutions with the same concentration, carrying out sample injection detection by an analyst under the condition that the sample injection is the same as possible, and calculating the content of the sample according to the peak area of the standard sample;
2) Result determination
The concentration RSD of the test sample is less than 4.0%;
3) The experimental results are shown in table 5;
TABLE 5 repeatability results
As can be seen from Table 5, the sample content detection repeatability RSD is less than 4%, which meets the method requirements.
6. Intermediate precision
1) Experimental method
Repeatedly preparing 6 parts of sample solutions in parallel by 2 testers (A, B) at different times, adding the total of 12 parts of sample solutions, respectively injecting the sample solution into the sample solution for 6 times by the 2 testers (A, B), and calculating the content of the sample according to a content calculation formula in a repeatability experiment;
2) Result determination
RSD of the content detection result of 12 times should be less than 8.0%;
3) The experimental results are shown in Table 6
TABLE 6 intermediate precision results
As shown in Table 6, the sample content detection intermediate precision RSD is less than 8%, which meets the method requirements.
7. Accuracy of
1) Experimental method
Precisely weighing a proper amount of polysorbate 80 reference substance, placing into a 100mL volumetric flask, diluting to scale with blank matrix, shaking, and preparing into polysorbate 80 stock solution with blank matrix.
Precisely measuring a proper amount of blank matrix stock solution with a standard, placing the blank matrix stock solution into a 10mL volumetric flask, and diluting the blank matrix stock solution to a scale to obtain (1:0.8) blank matrix solution E1 with the standard;
Precisely measuring a proper amount of blank matrix stock solution with a standard, placing the blank matrix stock solution into a 10mL volumetric flask, and diluting the blank matrix stock solution to a scale to obtain (1:1) blank matrix solution E2 with the standard;
precisely measuring a proper amount of blank matrix stock solution with a standard, placing the blank matrix stock solution into a 10mL volumetric flask, and diluting the blank matrix stock solution to a scale to obtain (1:1.2) blank matrix solution E3 with the standard;
And (3) adding the standard blank matrix solutions E1, E2 and E3, treating the reference substance solution according to a sample hydrolysis method, detecting according to the determined chromatographic conditions, repeating the experiment three times, and calculating the recovery rate of the results of 9 experiments.
2) Result determination
The recovery rate of the 9 times of detection is between 85% and 110%.
3) The experimental results are shown in table 7;
TABLE 7 recovery results
As shown in Table 7, the sample detection recovery rates are all between 85% and 110%, which meets the method requirements. 3. Comparative study of the detection method and the ultraviolet method of the invention
1. Comparative study of sample detection
Different batches of freeze-dried fibrinogen samples are taken as test samples, and a liquid phase method and an ultraviolet method are respectively adopted for comparison research.
The liquid phase method is operated by the same sample detection method in example 2, the ultraviolet method is the same as the method for measuring the residual quantity of polysorbate 80 in three parts 3023 of pharmacopoeia, and the measurement results are shown in table 8:
TABLE 8 detection results for different detection methods
| Lot 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
And taking a unified batch of freeze-dried fibrinogen samples, diluting and temporarily storing in a refrigerator. Appropriate amounts of samples were taken at 1, 4, 8, 12 and 24 hours and tested by liquid phase method and ultraviolet method, respectively, and the results are shown in Table 9:
table 9 comparison of sample stability results
The result shows that the detection of the polysorbate 80 by the liquid phase method can truly and accurately reflect the content of the polysorbate 80 in the solution, the sample is stable and not easy to degrade after being esterified, and the content can still be accurately detected after the sample is prepared and stored for 24 hours. The result is stable and good in repeatability, and the accuracy, the repeatability, the stability and the like are superior to those of the pharmacopoeia method.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (7)
1. The method for detecting the content of polysorbate 80 is characterized by comprising the following steps of:
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 substance, and calculating the content of polysorbate 80 in the test substance according to the detection peak area of the standard substance and the detection peak area of the test substance;
The pretreatment of the polysorbate 80 standard comprises: performing a first hydrolysis reaction on the polysorbate 80 standard solution, and performing a first methyl esterification reaction;
The pretreatment of the test article comprises the following steps: carrying out a second hydrolysis reaction on the sample solution, and then carrying out a second methyl esterification reaction;
the first hydrolysis reaction includes: heating the mixed solution of the polysorbate 80 standard solution and the alkali liquor; the temperature of the mixed solution of the polysorbate 80 standard solution and the alkali liquor for heating treatment is 65-75 ℃ and the time is 2.5-3.5 h; the alkali in the alkali liquor comprises potassium hydroxide;
neutralizing with acid after the first hydrolysis reaction; the first methyl esterification reaction includes: heating the supernatant fluid after the first hydrolysis reaction and the mixed solution of sulfuric acid-methanol solution at 65-75 ℃ for 0.8-1.2 h; after the first methyl esterification reaction, adjusting the pH to be 5-9 by adopting alkali;
The second hydrolysis reaction comprises: heating the mixed solution of the sample solution and the alkali liquor; the temperature of the heating treatment of the mixed solution of the sample solution and the alkali liquor is 65-75 ℃ and the time is 2.5-3.5 h; in the second hydrolysis reaction, the alkali in the alkali liquor comprises potassium hydroxide;
neutralizing with acid after the second hydrolysis reaction; the second methyl esterification reaction includes: heating the supernatant after the second hydrolysis reaction with a mixed solution of sulfuric acid-methanol solution at 65-75 ℃ for 0.8-1.2 h; after the second methyl esterification reaction, adjusting the pH to be 5-9 by adopting alkali;
The test sample is fibrinogen;
the fibrinogen is subjected to the following concentration treatment before the pretreatment:
Performing first centrifugal treatment on mixed solution of fibrinogen solution and ethanol-sodium chloride saturated solution in a centrifugal tube, performing first solid-liquid separation, and cleaning the centrifugal tube by adopting the ethanol-sodium chloride saturated solution; performing second centrifugal treatment, performing second solid-liquid separation, and cleaning the centrifuge tube by adopting an ethanol-sodium chloride saturated solution; combining the supernatant after the first solid-liquid separation, the supernatant after the second solid-liquid separation and the washing liquid obtained after the centrifuge tube is washed by the ethanol-sodium chloride saturated solution, and performing third centrifugation; concentrating the supernatant after the third centrifugal treatment at 50-60 ℃;
the detection conditions of the high performance liquid chromatography comprise: a C8 chromatographic column; the mobile phase is 20mM potassium dihydrogen phosphate-acetonitrile solution; the detection wavelength is 240nm; the flow rate was 1.0mL/min.
2. The method for detecting the content of polysorbate 80 according to claim 1, wherein the mixture adjusted to pH 5 to 9 after the first methyl esterification reaction is filtered with a 0.45 μm filter.
3. The method for detecting the content of polysorbate 80 according to claim 1, wherein the mixture adjusted to pH 5 to 9 after the second methyl esterification reaction is filtered with a filter membrane of 0.45 μm.
4. The method for detecting the content of polysorbate 80 according to claim 1, wherein the rotation speed of the first centrifugal treatment is 2900-3100 r/min, and the time is 8-12 min.
5. The method for detecting the content of polysorbate 80 according to claim 1, wherein the rotation speed of the second centrifugation is 2900-3100 r/min for 3-5 min.
6. The method for detecting the content of polysorbate 80 according to claim 1, wherein the rotation speed of the third centrifugal treatment is 2900-3100 r/min for 20-30 min.
7. The method for detecting the content of polysorbate 80 according to claim 1, wherein the apparatus for detecting the high performance liquid chromatography comprises an agilent 1260II high performance liquid chromatography apparatus.
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| 玉米胚芽油脂肪酸衍生化工艺;王大为等;《食品科学》;第32卷(第22期);143-147 * |
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