CN115420822A - Method for detecting content of glycerol in vaccine - Google Patents
Method for detecting content of glycerol in vaccine Download PDFInfo
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- CN115420822A CN115420822A CN202211023567.1A CN202211023567A CN115420822A CN 115420822 A CN115420822 A CN 115420822A CN 202211023567 A CN202211023567 A CN 202211023567A CN 115420822 A CN115420822 A CN 115420822A
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- 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
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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- 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
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- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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Abstract
The invention provides a method for detecting the content of glycerol in a vaccine. The vaccine is selected from an adsorption acellular diphtheria-pertussis series combined vaccine and/or a pertussis intermediate product thereof, and the detection method comprises the following steps: pretreating a vaccine sample to obtain a loading solution; performing on-machine detection on the sample solution by using a high-efficiency anion exchange chromatograph equipped with an ampere detector, and calculating by using an external standard method to obtain the content of glycerol; wherein the chromatographic conditions comprise: the ion chromatographic column is a Dionex CarboPac MA-1 ion chromatographic column, and the column temperature is 30-32 ℃; the mobile phase is 450-580mM sodium hydroxide solution, the flow rate is 0.3-0.4mL/min, and the elution mode is isocratic elution; the amperometric detector temperature was 30 ℃. The detection method has the advantages of simple sample pretreatment steps, high test speed, strong specificity, good durability, high precision, high accuracy and the like.
Description
Technical Field
The invention relates to the technical field of vaccine detection, in particular to a method for detecting the content of glycerol in a vaccine.
Background
Vaccines are an effective means of preventing and controlling infectious diseases. The adsorption cell-free Baikukui (three-component) Baikui (DTacP) series combined vaccine is prepared by combining a plurality of antigen components, and when the quality control research is carried out on the combined vaccine, besides the consideration of the interaction among the components in the combined vaccine, the influence of auxiliary material components such as excipient, preservative, adjuvant and the like on the active components and the detection of the combined vaccine also needs to be considered. Pertussis Toxin (PT) in DTacp series combined vaccines is an important vaccine antigen and has a plurality of biological activities, such as reactions of peripheral blood leukocyte increase, histamine sensitization, vasoactive substance anaphylaxis enhancement and the like after nanogram non-detoxified PT is injected into experimental animals, so that PT can be used for vaccine preparation after being detoxified. Other antigens such as Filamentous Haemagglutinin (FHA) and pertussis adhesin (PRN) also need to be detoxified to ensure vaccine safety. The common detoxification method is to treat antigens by using detoxifiers such as formaldehyde, glutaraldehyde and the like, but because the detoxifiers belong to protein cross-linking agents, the structure of target proteins is greatly influenced, and even epitope of the antigens can be changed, glycerol is required to be added in the detoxification process as an antigen protective agent, so that the detoxification process of the antigens is milder, the antigen degradation and epitope loss are prevented, and the immunogenicity of the detoxified antigens is ensured.
Glycerol is a saturated polyhydroxy compound, has the effect of improving protein stability, is widely used in pharmaceutical, food and daily chemical industries, and is commonly used as a lubricant, a humectant, a preservative, a solvent, a protective agent and the like in pharmaceutical preparations. Ion chromatography is a mature instrument analysis method, is widely applied in the fields of environment, medicines, foods and the like, and is used for qualitative and quantitative analysis of inorganic anions, inorganic cations, organic acids, sugar alcohols, amino sugars, amino acids, proteins, glycoproteins and the like. Although the toxicity of the glycerol is low, the high-concentration glycerol can cause side reactions of different degrees after entering a human body through intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, vertebral cavity injection and other routes.
The existing methods for detecting glycerol comprise sodium periodate oxidation titration, density method, refractive index method, electrochemical analysis method, enzyme method, ultraviolet spectroscopy, high performance liquid chromatography, gas chromatography and the like. Because the detection wavelength of the glycerol is 200nm, the absorption is very weak, the glycerol peak form formed during the detection by using an infrared spectrophotometry is poor, and the glycerol peak form is not suitable for measuring the residual content and is often used as an identification test. The glycerol content is measured by adopting a sodium periodate titration method in 'Chinese pharmacopoeia' 2020 edition, but the specificity and the accuracy are poor, and if a sample to be measured contains an o-hydroxyl component, a component capable of being oxidized by sodium periodate and a component capable of reacting with a sodium hydroxide solution (an aluminum hydroxide adjuvant), the accuracy of a measurement result is influenced; the density method and the refractive index method must ensure that the sample does not contain other impurities; the electrode is easy to be polluted in the electrochemical method; although the enzyme method has good specificity, the needed reagent is expensive; high performance liquid chromatography and gas chromatography have good accuracy, but the sample pretreatment step is complicated.
CN107421975A discloses that the content of glycerin in injection is measured based on quantitative nuclear magnetic resonance technology (1H-NMR and 13 CNMR), although the method has the characteristics of good stability, high precision and the like, the equipment price is high, and the method is difficult to popularize in recent years for most biological product enterprises; the glycerol detection kit (Sigma company) utilizes a coupling enzyme determination method of glycerol kinase and glycerol phosphate oxidase, has strong specificity, is rarely influenced by impurities contained in a sample, has simple operation, high sensitivity, good reproducibility, good precision and high determination speed, is suitable for the detection of glycerol in a sample with complex components, but has expensive required reagent price, and is not beneficial to industrial application and the detection of a large number of samples. The combined vaccine contains various antigens, and the buffer solution has complex components and is easy to interfere the detection of the content of the glycerol.
In conclusion, it is urgently needed to develop a detection method with simple operation and low cost for detecting the glycerol residual quantity in the combined vaccine.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a simple, accurate and low-cost method for detecting the content of glycerol in the combined vaccine and the intermediate product.
In order to achieve the above object, the present invention provides a method for detecting the content of glycerol in a vaccine selected from an adsorbed acellular pertussis-intermediate and/or pertussis-diphtheria-pertussis-associated vaccine, comprising the steps of:
pretreating a vaccine sample to obtain a loading solution; performing on-machine detection on the sample solution by using a high-efficiency anion exchange chromatograph equipped with an ampere detector, and calculating by using an external standard method to obtain the content of glycerol;
wherein the chromatographic conditions comprise: the ion chromatographic column is Dionex CarboPac MA-1 ion chromatographic column (specification 4mm × 250 mm), and the column temperature is 30-32 deg.C; the mobile phase is 450-580mM sodium hydroxide solution, the flow rate is 0.3-0.4mL/min, and the elution mode is isocratic elution; the ampere detector temperature is 28-32 ℃.
In the invention, the Dionex CarboPac MA-1 ion chromatographic column is an ion chromatographic column with a vinyl benzyl chloride/divinylbenzene microporous matrix as a filler.
In the method for detecting the content of glycerol in the vaccine, preferably, the adsorption acellular hundred (three-component) diphtheria-pertussis-tetanus (DTacP) series combined vaccine is selected from adsorption acellular hundred (three-component) diphtheria-pertussis-tetanus combined vaccine (DTacP combined vaccine, triple vaccine), adsorption acellular hundred (three-component) diphtheria-inactivated poliomyelitis combined vaccine (DTacP-sIPV combined vaccine, quadruple vaccine), and adsorption acellular hundred (three-component) diphtheria-inactivated poliomyelitis b-type influenza haemophilus combined vaccine (DTacP-sIPV-Hib combined vaccine, quintuplet vaccine).
In the method for detecting the content of glycerol in the vaccine, the pertussis intermediate is preferably selected from detoxified Pertussis Toxin (PT), detoxified Filamentous Hemagglutinin (FHA) or detoxified pertussis adhesin (PRN).
In the method for detecting the content of glycerol in vaccine, preferably, the chromatographic conditions further include: the sample injection volume is 20-25 μ L, and the signal collection rate is 1Hz.
In the method for detecting the content of glycerol in the vaccine, preferably, the external standard method comprises the steps of feeding glycerol standard solutions with different concentrations, fitting a standard curve equation between the concentration of the standard substance and the peak area of the standard substance according to the detection result of the glycerol standard solution, and calculating the concentration of glycerol according to the standard curve equation and the detection result of the peak area of the glycerol substance in the sample feeding solution.
In the method for detecting the content of glycerol in the vaccine, the glycerol standard solution is preferably prepared by quantifying the glycerol standard substance with water and then diluting the glycerol standard solution.
In the method for detecting the content of glycerol in the vaccine, preferably, the concentration of the working solution of each glycerol standard curve is distributed between 0.90 and 28.66 mu g/mL.
In the method for detecting the content of glycerol in the vaccine, the pretreatment of the vaccine preferably comprises: the vaccine samples were centrifuged and the centrifuged supernatant was subjected to membrane filtration.
In the method for detecting the content of the glycerol in the vaccine, preferably, the centrifugation temperature of the vaccine is 2-8 ℃, the centrifugation rotating speed is 6000-8000rpm, and the centrifugation time is 4-7min.
In the method for detecting the content of glycerol in the vaccine, the pore size of membrane filtration is preferably less than or equal to 0.22 μm.
In the method for detecting the content of glycerol in the vaccine, the high-efficiency anion exchange chromatograph is preferably further equipped with a protective column and an online degassing machine.
In the method for detecting the content of glycerol in the vaccine, preferably, the method specifically comprises the following steps:
1. preparation of a loading solution: placing the sample in a centrifuge, centrifuging, sucking a supernatant sample, and filtering the supernatant sample to an upper sample bottle through a 0.22 mu m membrane to obtain an upper sample solution;
2. high performance anion exchange chromatography-amperometric detection:
(1) Preparing an external standard solution: the glycerol reference substance is precisely weighed, placed in a 100mL volumetric flask, dissolved and diluted by deionized water to scale and shaken up to be used as a glycerol reference substance stock solution.
(2) Preparing a special solution: precisely weighing appropriate amount of ribitol, glycerol, and ethylene glycol reference substance, dissolving with ultrapure water, and making into mixed solution containing ribitol, glycerol, and ethylene glycol as special solution for use.
(3) The detection parameters were as follows:
the column temperature is 30-32 ℃, the sample injection volume is 25 mu L, and the signal collection rate is 1Hz; the mobile phase is 450-580mM sodium hydroxide solution, and the flow rate is 0.3-0.4mL/min; and (3) calculating the glycerol content according to a formula y = ax + b by using an external standard method according to the glycerol peak area, wherein x is the area of a glycerol quantitative peak, y is the mass concentration of a glycerol standard substance, and a and b are coefficients of a standard curve equation respectively.
The technical scheme provided by the invention has the following beneficial effects:
the detection method adopts a high-efficiency anion exchange chromatography-pulse amperometric detection method (HPAEC-PAD) to detect the content of the glycerol in the DTacP series combined vaccine and the pertussis intermediate product, and through systematic verification, the detection method has the advantages of simple sample pretreatment step, high test speed, strong specificity, good durability, high precision and accuracy and the like, can effectively solve the problem of quantitative analysis of the glycerol components in the combined vaccine and the intermediate product, and can accurately detect the glycerol components in the combined vaccine and the intermediate product.
The invention provides a new means for measuring the content of the glycerol component in the combined vaccine, and has wide application prospect in the aspect of quality detection of the glycerol component of the combined vaccine.
Drawings
FIG. 1A is an ion chromatogram of a blank control solution in the specificity test of proof example 1;
FIG. 1B is an ion chromatogram of a proprietary solution in the proprietary study of proof example 1;
fig. 2 is an ion chromatogram of each standard curve working solution in the linear and range investigation of the validation example 2, wherein each symbol in the figure is:
1:28.66 μ g/mL glycerol standard; 2:14.33 μ g/mL glycerol standard; 3:7.17 μ g/mL glycerol standard; 4:3.58 μ g/mL glycerol standard; 5:1.79 μ g/mL glycerol standard; 6:0.90 mug/mL glycerol standard;
FIG. 3 is a standard curve of glycerol peak area and standard concentration obtained by fitting the results of FIG. 2 in validation example 2.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention should not be construed as limiting the implementable scope of the present invention.
Example 1
The embodiment provides a method for detecting the content of glycerol in a vaccine, which specifically comprises the following steps:
1. combination vaccine pretreatment
1.1 samples and instruments
And (3) testing the sample: DTacP combined vaccine pertussis intermediates, DTacP combined vaccine, DTacP-sIPV combined vaccine and DTacP-sIPV-Hib combined vaccine, wherein the DTacP combined vaccine pertussis intermediates comprise: detoxified PT, detoxified FHA and detoxified PRN. All the samples were provided by the company Limited liability of the institute of biological products, beijing.
The instrument comprises: sorvall ST4R Plus centrifuge from Thermo scientific.
1.2 preparation of the loading solution: 1.5mL of each sample is put into a centrifuge tube, centrifuged at 7500rpm for 5min at 4 ℃ and then the supernatant sample is absorbed, and filtered to an upper sample bottle through a 0.22 mu m membrane to obtain a loading solution.
2. Standard substance solution
2.1 preparation of standard solution: precisely weighing 20.0651g of a glycerol standard, putting the glycerol standard into a 100mL volumetric flask, dissolving the glycerol standard with deionized water, diluting the glycerol standard to scale, and shaking up the glycerol standard to obtain a glycerol standard stock solution; taking a glycerol standard stock solution, diluting the glycerol standard stock solution to 28.66 mu g/mL by using ultrapure water in three steps, and then diluting the glycerol standard stock solution to 14.33 mu g/mL, 7.17 mu g/mL, 3.58 mu g/mL, 1.79 mu g/mL and 0.90 mu g/mL in stages.
3. Establishment of Glycerol detection method
3.1 reagents and instruments
Reagent: sodium hydroxide solution (chromatographically pure) was purchased from Thermo corporation; the experimental water was deionized water.
The instrument comprises the following steps: dionex ion chromatograph (ICS-5000) available from Thermo corporation, USA, including autosampler, column oven, online degasser, amperometric detector, ion chromatography column (Dionex CarboPac MA-1), guard column (Dionex CarboPac MA-1), and chameleon 6.8 chromatographic workstation;
10KD ultrafiltration centrifuge tube (15 mL); precision analytical balance (BS 224S) and ultra pure Water Instrument (aril)
pro) from Sartorius, usa.
3.2 chromatographic Condition establishment
Chromatographic conditions are as follows: the ion chromatographic column is a Dionex CarboPac MA-1 ion chromatographic column, and the column temperature is 30 ℃; the mobile phase is 45mM sodium hydroxide solution, the flow rate is 0.4mL/min, and the elution mode is isocratic elution; the ampere detector temperature is 30 ℃; the sample injection volume is 25 muL, and the sample injection times are 2 times.
3.3 operating machine detection
And (3) using a mobile phase equilibrium chromatographic system, respectively injecting the standard curve working solution and the sample loading solution into an ion chromatographic column after the base line is stable, collecting the chromatogram, and calculating by using an external standard method to obtain the glycerol content.
Verification example 1
This verification example was conducted to examine the method for detecting the content of glycerol in the vaccine of example 1 specifically.
The purpose of specificity inspection is to confirm that the tested target substance is glycerol but not other substances in the vaccine under the specified conditions of the DTacP series combined vaccine and the method for testing the content of the glycerol in the intermediate product, and verify that the method has specificity on the detection of the glycerol. In the verification example, ribitol (a component of Hib in the DTacp-sIPV-Hib combined vaccine) and ethylene glycol (the property of the ribitol and the ethylene glycol is similar to that of glycerol) are taken as reference substances, and the specific method is as follows:
preparing a special solution: weighing appropriate amount of ribitol, glycerol, and ethylene glycol as reference substances, dissolving with ultrapure water to obtain a mixed solution containing ribitol about 1.5 μ g/mL, glycerol about 4 μ g/mL, and ethylene glycol about 100 μ g/mL per 1mL, and preparing as a special solution.
Deionized water was used as a blank solution.
Performing on-machine detection on the blank control solution (deionized water) and the special solution according to the chromatographic conditions of example 1 to obtain chromatograms shown in fig. 1A and 1B respectively, wherein the results show that the retention time of the ethylene glycol, glycerol and ribitol peaks is 2.885min, 9.350min and 18.667min respectively; by comparing spectrograms, the detection method can effectively separate substance peaks of ethylene glycol, glycerol and ribitol peaks, and has good specificity for the detection of the glycerol.
Verification example 2
This verification example performs a linear and range investigation of the method for detecting the glycerol content of the vaccine of example 1.
The stock solution of the glycerol standard of example 1 was diluted stepwise with ultrapure water to give standard curve working solutions of 28.66. Mu.g/mL, 14.33. Mu.g/mL, 7.17. Mu.g/mL, 3.58. Mu.g/mL, 1.79. Mu.g/mL, and 0.90. Mu.g/mL. The assay was repeated 6 times for each standard curve working solution using the chromatographic conditions of example 1 and the ion chromatogram is shown in figure 2. The peak area (x) is taken as the abscissa and the concentration (y) of the working solution of the glycerol standard curve is taken as the ordinate, the standard curve shown in figure 3 is drawn, and the correlation coefficient is counted. The result shows that the glycerol standard curve solution has good linear relation with the peak area in the concentration range of 0.9-28.66 mu g/mL, and R is 2 ＞0.99。
Verification example 3
This verification example examined the detection limit and the quantification limit of the method for detecting the content of glycerol in the vaccine of example 1.
Gradually diluting 28.66. Mu.g/mL of glycerol standard solution to 60ng/mL, and performing machine detection on each diluted standard solution according to the chromatographic conditions of example 1, wherein the detection Limit (LOD) is determined by the signal-to-noise ratio (S/N) of glycerol being not less than 3 and the quantification Limit (LOQ) is determined by the signal-to-noise ratio (S/N) being not less than 10 and being 500ng/mL.
Verification example 4
This verification example was conducted to examine the accuracy of the method for detecting the content of glycerin in the vaccine of example 1.
Taking DTacP-sIPV-Hib combined vaccine (batch No. 202111S 04) as a test sample (namely a sample to be tested), pretreating the test sample according to the method of example 1, diluting the test sample step by step to 3.341 mu g/mL, respectively taking 1mL of diluted solution, putting the diluted solution into a 4mL centrifuge tube, respectively adding 1mL of glycerol standard substance with high (50.0 mu g/mL), medium (25.0 mu g/mL) and low (3.0 mu g/mL) mass concentration into each tube, mechanically detecting each added standard solution according to the chromatographic conditions of example 1, and calculating the added standard recovery rate according to the following formula:
the results are shown in Table 1. The results show that the glycerol recovery rates after the DTacP-sIPV-Hib combined vaccine is added with the standard substances with high, medium and low concentrations are 99.56%, 100.75% and 103.15%, respectively, and are all between 95 and 105%, and the Relative Standard Deviation (RSD) is 1.81%.
TABLE 1 accuracy examination results (I)
In this verification example, detoxified PT was also used as a test sample, which was pretreated according to the method of example 1, and was stepwise diluted to 1.673 μ g/mL, 1mL of the diluted solution was put into a 4mL centrifuge tube, and 1mL of glycerol standard with high (50.0 μ g/mL) and medium (25.0 μ g/mL) and low (3.0 μ g/mL) mass concentrations was added to each tube, and each spiked solution was mechanically tested according to the chromatographic conditions of example 1, and the spiked recovery rate was calculated, and the results are shown in table 2. As can be seen from the table, the average recovery was 99.31% and the RSD was 2.45%.
TABLE 2 accuracy examination results (II)
The examination results show that the detection method has higher accuracy.
Verification example 5
This verification example was conducted to examine the precision of the method for detecting the content of glycerin in the vaccine of example 1.
1. Repeatability of
The DTacP-sIPV-Hib combination vaccine (batch No. 202111S 04) was tested in duplicate 6 times according to the chromatographic conditions of example 1, and the results are shown in tables 3 and 4.
TABLE 3 precision review-repeatability (peak area)
TABLE 4 precision investigation-repeatability (Glycerol content)
As can be seen from tables 3 and 4, RSD (n = 6) of the glycerol peak area and content were 0.63% and 0.64%, respectively, indicating good reproducibility.
2. Intermediate precision
The detection results of the same operator at different times and different operators at the same time for the glycerol standard substance are shown in table 5, wherein the detection values of the glycerol contents of the 1 st group and the 2 nd group in the table are obtained by the same operator at different times, and the detection value of the glycerol content of the 3 rd group is obtained by the other operator and the 1 st group by detection at the same time.
TABLE 5 results of intermediate precision investigation
The result shows that the relative standard deviation RSD is respectively 0.67%, 0.69% and 0.38%, and the detection method of the invention has good intermediate precision.
Verification example 6
This verification example was a durability examination of the method for detecting the content of glycerin in the vaccine of example 1.
This proof case tests the degree of tolerance of the detection method at different mobile phase concentrations, column temperatures and flow rates. Each test was carried out on a machine testing the separation degree (R) of the glycerol and ethylene glycol peaks, the tailing factor (T) of the glycerol peak, the content of glycerol in the DTacP-sIPV-Hib combination vaccine (batch No. 202010S 004) under different conditions, respectively, recorded and examined the durability of the testing method, the results of which are shown in Table 6, with the single variable being determined on the basis of the specific solution of example 2 (the mobile phase concentration changed from 450mM to 580mM, the column temperature changed from 30 ℃ to 32 ℃ and the flow rate changed from 0.4mL/min to 0.3 mL/min).
TABLE 6 durability examination results
As can be seen from the above table, without changing the parameters (same chromatographic conditions as in example 1), the glycerol peak retention time was 9.350min, the tailing factor (T) was 1.38, and the degree of separation (R) of ethylene glycol and glycerol was 1.78. When the flow rate was changed to 0.3mL/min, the glycerol peak retention time was 12.467min, the tailing factor (T) was 1.34, and the ethylene glycol and glycerol separation (R) was 1.86; when the column temperature was changed to 32 ℃, the glycerol peak retention time was 9.317min, the tailing factor (T) was 1.39, and the ethylene glycol and glycerol separation degree (R) was 1.78. The above conditions are in accordance with the validation regulations, i.e. the separation degree (R) of the glycol and the glycerol should be more than 1.5, and the tailing factor (T) of the glycerol peak should be between 0.95 and 1.50. When the mobile phase concentration was changed to 580mM, the glycerol peak retention time was 9.100min, the tailing factor (T) was 1.40, and the ethylene glycol and glycerol separation degree (R) was 1.47, a criterion of slightly < 1.5. Furthermore, the RSD of the glycerol content of the quintuplet (batch No. 202010S 004) measured under varying parameters was only 0.09%.
In addition, in this verification example, the durability of the detection method of the present invention was also examined by using a DTacP combination vaccine pertussis intermediate as a test sample, wherein the DTacP combination vaccine pertussis intermediate comprises: detoxified PT (batch ACP20211022S 07), detoxified FHA (batch ACP20211012S 07), and detoxified PRN (batch ACP20211032S 07). The test results are shown in Table 7.
TABLE 7 Glycerol assay results for various pertussis intermediates under different chromatographic conditions
The result shows that the detection method of the invention has good durability for each test sample.
Application example 1
In the application example, the detection method for detecting the glycerol content in the vaccine in example 1 is adopted to detect the glycerol content in the DTacP series combined vaccines of different batches and pertussis intermediates (detoxified PT, FHA and PRN), and the detection results are shown in table 8, wherein the volume percentage is the volume percentage of the glycerol in the vaccine after the glycerol is added in the vaccine detoxification process.
TABLE 8 statistics of test results
The result shows that the method established by the embodiment can be used for measuring the content of glycerol in the pertussis intermediate products and finished products of DTacP series combined vaccines, and has good consistency in batches and among batches.
Claims (10)
1. A method for detecting the content of glycerol in a vaccine selected from an adsorbed acellular diphtheria-pertussis-series combined vaccine and/or a pertussis intermediate thereof, comprising the following steps:
pretreating a vaccine sample to obtain a loading solution; performing on-machine detection on the sample loading solution by using a high-efficiency anion exchange chromatograph equipped with an ampere detector, and calculating by using an external standard method to obtain the content of glycerol;
wherein the chromatographic conditions comprise: the ion chromatographic column is a Dionex CarboPac MA-1 ion chromatographic column, and the column temperature is 30-32 ℃; the mobile phase is 450-580mM sodium hydroxide solution, the flow rate is 0.3-0.4mL/min, and the elution mode is isocratic elution; the amperometric detector temperature was 28-32 ℃.
2. The method for detecting the content of glycerol in vaccine according to claim 1, wherein said adsorption acellular hundred (three-component) diphtheria-pertussis-series combined vaccine is selected from the group consisting of adsorption acellular hundred (three-component) diphtheria-pertussis-series combined vaccine, adsorption acellular hundred (three-component) diphtheria-inactivated polio-polio combined vaccine, and adsorption acellular hundred (three-component) diphtheria-inactivated Haemophilus influenzae type b combined vaccine.
3. The method for detecting glycerol content in a vaccine according to claim 1, wherein said pertussis intermediate is selected from the group consisting of detoxified pertussis toxin, detoxified filamentous hemagglutinin and detoxified pertussis adhesin.
4. The method for detecting the content of glycerol in vaccine according to any one of claims 1-3, wherein said chromatographic conditions further comprise: the injection volume is 20-25 mu L; the signal collection rate was 1Hz.
5. The method for detecting the content of glycerol in the vaccine according to any one of claims 1 to 3, wherein the external standard method comprises the steps of feeding glycerol standard solutions with different concentrations, fitting a standard curve equation between the concentration of the standard solution and the peak area of the standard solution according to the detection result of the glycerol standard solutions, and calculating the concentration of glycerol according to the standard curve equation and the detection result of the peak area of the glycerol substance in the sample feeding solution.
6. The method for detecting the content of glycerol in vaccine according to claim 5, wherein the concentration of the working solution of each glycerol standard curve is distributed between 0.90-28.66 μ g/mL.
7. The method for detecting the content of glycerol in vaccine according to any one of claims 1-3, wherein the pretreatment of the vaccine comprises: the vaccine samples were centrifuged and the centrifuged supernatant was subjected to membrane filtration.
8. The method for detecting the content of glycerol in vaccine according to claim 7, wherein the centrifugation temperature of the vaccine is 2-8 ℃, the centrifugation speed is 6000-8000rpm, and the centrifugation time is 4-7min.
9. The method for detecting the content of glycerol in vaccine according to claim 7, wherein the pore size of membrane filtration is less than or equal to 0.22 μm.
10. The method for detecting the content of glycerol in vaccine according to any one of claims 1-3, wherein said HPLC is further equipped with a guard column and an on-line degassing machine.
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