Atm detection method for migration of simethicone on the surface of medicinal halogenated butyl rubber stopper in human rabies vaccine

Abstract

The application relates to the technical field of dimethicone content detection, and specifically discloses an ATR detection method for the migration amount of dimethicone on the surface of a medicinal halogenated butyl rubber plug in a human rabies vaccine. The method comprises the following steps: S1, taking a human rabies vaccine aqueous solution, adding ethanol and an extractant, extracting, leaving an oleophilic phase, and discarding an aqueous phase; wherein the volume ratio of the human rabies vaccine aqueous solution to the ethanol is 1:1-2.5, and the volume ratio of the human rabies vaccine aqueous solution to the extractant is 1:0.5-0.8; the extractant is selected from any one of ethyl ether, toluene, n-hexane and chloroform; S2, after removing the extractant in the oleophilic phase, a crude product is obtained, chloroform is added to the crude product for redissolution, a sample injection sample is obtained, and the sample injection sample is detected by using the ATR method. The detection method can improve the accuracy of detecting the migration amount of dimethicone on the surface of the halogenated butyl rubber plug in the human rabies vaccine.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of dimethicone content detection, and more particularly to an ATR detection method for the migration amount of dimethicone on the surface of a medicinal halogenated butyl rubber plug in a human rabies vaccine. BACKGROUND

[0002] Dimethicone is a colorless, odorless, nontoxic, and nonvolatile oily liquid. In the production process of the medicinal halogenated butyl rubber plug, dimethicone is used for the siliconization treatment of the medicinal halogenated butyl rubber plug.

[0003] The human rabies vaccine is generally divided into a freeze-dried human rabies vaccine and a water injection human rabies vaccine. For the freeze-dried human rabies vaccine, dimethicone is an inert material, but when the drug powder on the freeze-dried human rabies cake falls off, the drug powder can contact the medicinal halogenated butyl rubber plug, thereby contacting the dimethicone on the surface of the medicinal halogenated butyl rubber plug. After the drug powder falls off is adsorbed by the dimethicone, the dimethicone migrates into the drug and forms a colloid or a drug powder mass with the drug powder. When the contact area of the drug powder with the medicinal halogenated butyl rubber plug is larger, the colloid or the drug powder mass formed is more. When the vaccine is used, after the vaccine is diluted by sterile water for injection, because dimethicone is not soluble in water, the drug solution appears turbid and the drug powder is difficult to dissolve, thereby affecting the clarity of the drug solution.

[0004] For the water injection human rabies vaccine, after the drug solution contacts the medicinal halogenated butyl rubber plug for a long time, the dimethicone on the surface of the medicinal halogenated butyl rubber plug migrates into the drug solution, and the migration amount of the dimethicone increases with the increase of the contact time. In addition, the dimethicone on the medicinal halogenated butyl rubber plug also increases the amount of insoluble particles in the solution, thereby affecting the clarity of the drug solution.

[0005] Therefore, it is necessary to establish a detection method for determining the migration amount of dimethicone on the medicinal halogenated butyl rubber plug in the freeze-dried human rabies vaccine (Vero cell) and the human rabies vaccine (Vero cell). At present, the method for detecting the content of dimethicone in the related research is as follows: dimethicone is extracted by using an organic solvent such as dichloromethane, chloroform, n-hexane, toluene, diethyl ether and the like, and then the content of dimethicone is detected by infrared spectrophotometry or Fourier transform attenuated total reflection infrared spectroscopy (ATR-FTIR). However, there is no legal standard for the detection of the residual amount of dimethicone on the surface of the medicinal halogenated butyl rubber plug or the dimethicone in the drug solution in China, and the domestic production enterprises also do not control the quality of the residual amount of dimethicone on the surface of the plug, and there is no detection method and quality control method for the migration amount of dimethicone in the drug solution. When the above method is directly used to detect the content of dimethicone, the detection result is not accurate. SUMMARY

[0006] In order to improve the accuracy of detecting the migration amount of dimethyl silicone oil on the surface of a halogenated butyl rubber stopper in a human rabies vaccine, the present application provides an ATR detection method for the migration amount of dimethyl silicone oil on the surface of a pharmaceutical halogenated butyl rubber stopper in a human rabies vaccine.

[0007] The ATR detection method for the migration amount of dimethyl silicone oil on the surface of a pharmaceutical halogenated butyl rubber stopper in a human rabies vaccine provided by the present application adopts the following technical scheme:

[0008] The ATR detection method for the migration amount of dimethyl silicone oil on the surface of a pharmaceutical halogenated butyl rubber stopper in a human rabies vaccine comprises the following steps:

[0009] S1, take a human rabies vaccine aqueous solution, add ethanol and an extractant, shake and extract thoroughly to form a two-phase system, which is an oleophilic phase and an aqueous phase respectively, keep the oleophilic phase and discard the aqueous phase; wherein the volume ratio of the human rabies vaccine aqueous solution to ethanol is 1:1-2.5, and the volume ratio of the human rabies vaccine aqueous solution to the extractant is 1:0.5-0.8, and the extractant is selected from any one of ethyl ether, toluene, n-hexane and chloroform;

[0010] S2, after removing the extractant in the oleophilic phase, a crude product is obtained, chloroform is added to the crude product to dissolve, a sample injection sample is obtained, and the sample injection sample is detected by ATR method.

[0011] The present application is to detect the content of dimethyl silicone oil in human rabies vaccine. The dimethyl silicone oil is from halogenated butyl rubber stopper, so the content of dimethyl silicone oil in human rabies vaccine is relatively low. In addition, the main components in human rabies vaccine are rabies virus related proteins, including glycoprotein, nucleoprotein, phosphoprotein, matrix protein and transcriptase large protein, etc. Through organic solvent extraction, dimethyl silicone oil and most of the impurity proteins (glycoprotein, nucleoprotein, etc.) can be separated, but part of the impurity proteins are difficult to separate from dimethyl silicone oil by this method. The applicant found that when extracting dimethyl silicone oil, if some proteins in human rabies vaccine are more inclined to dissolve in water than in lipophilic, they will remain in the aqueous solution of human rabies vaccine and thus separate from dimethyl silicone oil; if some proteins in human rabies vaccine are more inclined to dissolve in the extractant, they will remain in the extractant together with dimethyl silicone oil and thus are difficult to separate from dimethyl silicone oil. In addition, the distribution of proteins in the aqueous solution of human rabies vaccine and the extractant is related to the polarity of the aqueous solution of human rabies vaccine and the extractant; the same type of proteins only tend to remain in the aqueous solution of human rabies vaccine or the extractant, but not completely; especially some types of proteins (such as part of the matrix protein and phosphoprotein) contain both relatively hydrophilic groups and hydrophobic groups, and these types of proteins may also carry part of the dimethyl silicone oil to distribute in the aqueous solution of human rabies vaccine. The content of dimethyl silicone oil in human rabies vaccine is relatively low, so the extraction is incomplete from the beginning of the extraction stage, resulting in inaccurate final test results.

[0012] By adopting the above technical solution, the applicant firstly combines ethanol, water and an extractant to fully separate dimethicone and impurity proteins at the very beginning: among them, ethanol is miscible with water in any proportion. Therefore, compared with the lipophilic phase where the extractant is located, ethanol is more inclined to be distributed in the human rabies vaccine aqueous solution; therefore, in fact, in this process, there are a total of two phases, namely the hydrophilic phase formed by ethanol and the human rabies vaccine aqueous solution, and the lipophilic phase where the extractant is located. Dimethicone is insoluble in water and slightly soluble in ethanol, but is easily soluble in the extractant. Therefore, compared with the hydrophilic phase, dimethicone tends to be completely distributed in the extractant and is repelled by the hydrophilic phase; while water-soluble proteins are dissolved in the hydrophilic phase; in addition, since the ethanol in the hydrophilic phase is also lipophilic, some proteins containing both hydrophilic and hydrophobic groups are more easily distributed in the hydrophilic phase, thereby achieving the separation of dimethicone and such proteins containing both hydrophilic and hydrophobic groups, ensuring that dimethicone is more fully extracted, thereby ensuring the accuracy of the detection method. In this scheme, the volume ratio of the human rabies vaccine aqueous solution to ethanol is 1:1-2.5, and the volume ratio of the human rabies vaccine aqueous solution to the extractant is 1:0.5-0.8. This can further promote the distribution of dimethicone in the lipophilic phase, while other impurity proteins (especially proteins containing both hydrophilic and hydrophobic groups) are distributed in the hydrophilic phase, further and more fully extracting dimethicone, thereby ensuring the accuracy and sensitivity of the detection method. In addition, the first thing to do in this scheme is to separate out a class of proteins containing both hydrophilic and hydrophobic groups that are difficult to separate from dimethicone, so as to avoid the influence of such impurity proteins on the test results at the beginning.

[0013] Optionally, in step S2, before removing the extractant, the step further includes treating the lipophilic phase with hot water at 70-80° C. and then cooling it to room temperature.

[0014] By adopting the above technical solution, hot water treatment denatures the protein, further fully removing hydrophilic proteins and ensuring the accuracy of test results. These impurity proteins may carry some dimethicone, so hot water treatment directly removes these impurity proteins, avoiding the loss of dimethicone caused by these impurity proteins. At the same time, incomplete treatment of these impurity proteins may also affect subsequent test results as impurities.

[0015] Optionally, in step S2, when the lipophilic phase is treated with hot water, the following steps are included:

[0016] Add water to the lipophilic phase in an amount of 1-1.8 times the volume of the extractant, then keep warm in a water bath and cool, leaving the lipophilic phase.

[0017] By adopting the above technical solution and the above volume ratio of water to extractant, the detection results can be made more accurate.

[0018] Optionally, the water bath temperature is 90-100℃, and the time is 5-10min.

[0019] Optionally, in step S2, before the hot water treatment, the step of first treating the lipophilic phase with a 0.11-0.18mol / L sodium chloride solution is further included.

[0020] By using the above technical solution, before the hot water treatment, the nucleoprotein is first removed by the low-concentration sodium chloride, and then the hot water treatment is performed to remove all the proteins, which can more completely remove the impurity proteins and reduce the influence of the impurity proteins on the detection result.

[0021] Optionally, in step S2, when the sodium chloride solution is used to treat the lipophilic phase, the following steps are included:

[0022] The 0.11-0.18mol / L sodium chloride solution is added to the lipophilic phase, the addition amount of the sodium chloride solution is 40%-80% of the volume of the extractant, after sufficient shaking, the lipophilic phase is left after standing for 5-10min.

[0023] By using the above technical solution, on the one hand, the sodium chloride solution can precipitate the nucleoprotein, and on the other hand, the sodium chloride solution can further promote the distribution of the simethicone in the lipophilic phase.

[0024] In this scheme, the "volume of the extractant" refers to the addition volume of the extractant in step S1.

[0025] Optionally, the initial sample of the human rabies vaccine aqueous solution is selected from any one of the freeze-dried human rabies vaccine and the water-injection human rabies vaccine.

[0026] Optionally, when the initial sample of the human rabies vaccine aqueous solution is the freeze-dried human rabies vaccine, before step S1, the freeze-dried human rabies vaccine is first dissolved with water to obtain the human rabies vaccine aqueous solution, and then the detection is performed; when the initial sample of the human rabies vaccine is the water-injection human rabies vaccine, the detection is directly performed.

[0027] Optionally, in step S2, the ATR method is used to directly sample on the ZnSe surface of the Fourier transform infrared spectrometer, and the infrared spectrum is immediately scanned to obtain the detection result.

[0028] In summary, the present application has the following beneficial effects:

[0029] 1、The application forms a new extraction system with extractant, water and ethanol to reduce the influence of part of the hydrophilic and lipophilic impurity protein in human rabies vaccine on the extraction of simethicone, so as to more fully extract simethicone from human rabies vaccine, and then realize the purpose of improving the accuracy of detecting the migration amount of simethicone on the surface of halogenated butyl rubber plug in human rabies vaccine.

[0030] 2、In the application, further treatment with hot water can more fully remove other impurity proteins, so as to avoid that the impurity proteins carry part of simethicone, resulting in inaccurate detection results.

[0031] 3、The method of the application further effectively removes nucleoprotein by treatment with sodium chloride solution, so that the detection result is more accurate. DETAILED DESCRIPTION

[0032] The application will be further described in detail below in combination with examples.

[0033] Examples

[0034] The related equipment for implementing the scheme of the application is IS10 Fourier transform infrared spectrometer with attenuated total reflection accessory (Thermo).

[0035] The reagents for implementing the scheme of the application are simethicone, diethyl ether, toluene, n-hexane, chloroform and ethanol, which are all ordinary commercial products; the halogenated butyl rubber plug is commercially available and is of pharmaceutical grade.

[0036] The freeze-dried human rabies vaccine (Vero cell) and water needle human rabies vaccine (Vero cell) are both products of Liaoning Chengda Biological Co., Ltd., and are stored at 2-8℃. All vaccine samples are near expiration samples; among them, the specification of the water needle human rabies vaccine (Vero cell) is 0.5 mL per branch, and the effective period is 18 months. The water needle human rabies vaccine (Vero cell) selected in the application is 16 months away from the production date; the freeze-dried human rabies vaccine (Vero cell) is used by adding diluent to 0.5 mL per branch before use, and the effective period is 36 months. The freeze-dried human rabies vaccine (Vero cell) selected in the application is 33 months away from the production date.

[0037] When detecting, the instrument test parameters are: sample scanning times: 16 times; resolution: 4.00 cm -1 ; wave number range: 4000-650 cm -1 ; OMNIC Specta operation software is used to determine the sample; TQ Analyst EZ Edition operation software is used for quantitative analysis.

[0038] Example 1

[0039] An ATR detection method for the migration amount of dimethyl silicone oil on the surface of a medicinal halogenated butyl rubber plug in a human rabies vaccine, comprising the following steps:

[0040] S1, take 400 water needles of human rabies vaccine (Vero cell) (i.e. 200 mL) into the same liquid separation funnel, add 200 mL of ethanol and 100 mL of toluene, shake thoroughly, and then stand still to form double liquid phases, i.e. the toluene-containing oleophilic phase and the ethanol and water needle human rabies vaccine-containing aqueous phase, wherein the dimethyl silicone oil is distributed in the oleophilic phase, then the oleophilic phase is reserved and the aqueous phase is discarded.

[0041] S2, place the oleophilic phase in a 40℃ water bath to heat and remove the toluene to obtain a crude product, add 250 μL of chloroform to the crude product to redissolve, obtain a sample for injection, and then use the ATR method to detect the sample for injection.

[0042] Example 2

[0043] An ATR detection method for the migration amount of dimethyl silicone oil on the surface of a medicinal halogenated butyl rubber plug in a human rabies vaccine, comprising the following steps:

[0044] S1, take 400 water needles of human rabies vaccine (Vero cell) (i.e. 200 mL) into the same liquid separation funnel, add 400 mL of ethanol and 140 mL of toluene, shake thoroughly, and then stand still to form double liquid phases, i.e. the toluene-containing oleophilic phase and the ethanol and water needle human rabies vaccine-containing aqueous phase, wherein the dimethyl silicone oil is distributed in the oleophilic phase, then the oleophilic phase is reserved and the aqueous phase is discarded.

[0045] S2, place the oleophilic phase in a 40℃ water bath to heat and remove the toluene to obtain a crude product, add 250 μL of chloroform to the crude product to redissolve, obtain a sample for injection, and then use the ATR method to detect the sample for injection.

[0046] Example 3

[0047] An ATR detection method for the migration amount of dimethyl silicone oil on the surface of a medicinal halogenated butyl rubber plug in a human rabies vaccine, comprising the following steps:

[0048] S1, take 400 water needles of human rabies vaccine (Vero cell) (i.e. 200 mL) into the same liquid separation funnel, add 500 mL of ethanol and 160 mL of toluene, shake thoroughly, and then stand still to form double liquid phases, i.e. the toluene-containing oleophilic phase and the ethanol and water needle human rabies vaccine-containing aqueous phase, wherein the dimethyl silicone oil is distributed in the oleophilic phase, then the oleophilic phase is reserved and the aqueous phase is discarded.

[0049] S2, place the oleophilic phase in a 40℃ water bath to heat and remove the toluene to obtain a crude product, add 250 μL of chloroform to the crude product to redissolve, obtain a sample for injection, and then use the ATR method to detect the sample for injection.

[0050] Example 4

[0051] The difference between this example and Example 2 is that, in step S2, before removing the extraction agent, the lipophilic phase is treated with hot water at 75°C and then cooled to room temperature, and the other steps are the same as in Example 2.

[0052] Step S2 is specifically:

[0053] S2, 210 mL of water is added to the lipophilic phase obtained in step S1, and shaken thoroughly and mixed; then treated in a water bath, the water bath temperature is 75°C, and the time is 7 min; then cooled to room temperature, and the lipophilic phase in which the extraction agent is located is taken, and the lipophilic phase is rotary evaporated under vacuum at 40°C to remove the toluene to obtain a crude product, 250 μL of chloroform is added to the crude product to redissolve, and an injection sample is obtained, which can be detected by ATR method.

[0054] Example 5

[0055] The difference between this example and Example 4 is that, in step S2, before hot water treatment, the lipophilic phase is first treated with a 0.14 mol / L sodium chloride solution, and the other steps are the same as in Example 4.

[0056] Step S2 is specifically:

[0057] S2, 80 mL of a 0.14 mol / L sodium chloride solution is added to the lipophilic phase obtained in step S1, shaken thoroughly and then left to stand for 8 min, and then the lipophilic phase is reserved.

[0058] 210 mL of water is added to the above treated lipophilic phase, shaken thoroughly and mixed; then treated in a water bath, the water bath temperature is 75°C, and the time is 7 min; then cooled to room temperature, and the lipophilic phase in which the extraction agent is located is taken, and the lipophilic phase is rotary evaporated under vacuum at 40°C to remove the toluene to obtain a crude product, 250 μL of chloroform is added to the crude product to redissolve, and an injection sample is obtained, which can be detected by ATR method.

[0059] Example 6

[0060] The difference between this example and Example 5 is that, in step S1, the extraction agent is diethyl ether, and the amount of diethyl ether added is 140 mL, and the others are the same as in Example 5.

[0061] Example 7

[0062] The difference between this example and Example 5 is that, in step S1, the extraction agent is n-hexane, and the amount of n-hexane added is 140 mL, and the others are the same as in Example 5.

[0063] Example 8

[0064] The difference between this example and Example 5 is that the initial sample for preparing the aqueous solution of human rabies vaccine is a lyophilized human rabies vaccine, which is prepared by taking one lyophilized human rabies vaccine and adding sterile water for injection (i.e. diluent) to 0.5 mL, and then mixing 400 diluted vaccines, thereby obtaining the required 200 mL aqueous solution of human rabies vaccine of this example. The rest is the same as Example 5.

[0065] Comparative Example

[0066] Comparative Example 1

[0067] An ATR detection method for the migration amount of dimethyl silicone oil on the surface of a medicinal halogenated butyl rubber stopper in human rabies vaccine, comprising the following steps:

[0068] Take 400 vials (i.e. 200 mL) of water needle human rabies vaccine (Vero cell) into the same separation funnel, add 50 mL of ether in three times, shake well and extract, place the ether layer on a water bath to evaporate, and then add 250 μL of chloroform to dilute to obtain a sample for injection.

[0069] Comparative Example 2

[0070] An ATR detection method for the migration amount of dimethyl silicone oil on the surface of a medicinal halogenated butyl rubber stopper in human rabies vaccine, comprising the following steps:

[0071] Take 400 vials (i.e. 200 mL) of water needle human rabies vaccine (Vero cell) into the same separation funnel, add 50 mL of toluene in three times, shake well and extract, place the toluene layer on a water bath to evaporate, and then add 250 μL of chloroform to dilute to obtain a sample for injection.

[0072] Comparative Example 3

[0073] An ATR detection method for the migration amount of dimethyl silicone oil on the surface of a medicinal halogenated butyl rubber stopper in human rabies vaccine, comprising the following steps:

[0074] Take 400 vials (i.e. 200 mL) of water needle human rabies vaccine (Vero cell) into the same separation funnel, add 50 mL of n-hexane in three times, shake well and extract, place the n-hexane layer on a water bath to evaporate, and then add 250 μL of chloroform to dilute to obtain a sample for injection.

[0075] Comparative Example 4

[0076] The difference between this comparative example and Example 5 is that the amount of toluene added in step S1 is different, and the rest is the same as Example 5.

[0077] Step S1 is specifically:

[0078] Take 400 of water injection human rabies vaccine (Vero cell) (i.e. 200 mL) into the same separation funnel, add 400 mL of ethanol, 60 mL of toluene, shake thoroughly and stand, forming two liquid phases, the toluene-containing lipophilic phase and the ethanol and water injection human rabies vaccine-containing aqueous phase, with dimethyl silicone oil distributed in the lipophilic phase, then retain the lipophilic phase and discard the aqueous phase.

[0079] Comparative Example 5

[0080] The difference between this comparative example and Example 5 is that the amount of ethanol added in step S1 is different, and the others are the same as in Example 5.

[0081] Step S1 is specifically:

[0082] Take 400 of water injection human rabies vaccine (Vero cell) (i.e. 200 mL) into the same separation funnel, add 600 mL of ethanol, 140 mL of toluene, shake thoroughly and stand, forming two liquid phases, the toluene-containing lipophilic phase and the ethanol and water injection human rabies vaccine-containing aqueous phase, with dimethyl silicone oil distributed in the lipophilic phase, then retain the lipophilic phase and discard the aqueous phase.

[0083] Comparative Example 6

[0084] The difference between this comparative example and Example 5 is that the amount of ethanol added in step S1 is different, and the others are the same as in Example 5.

[0085] Step S1 is specifically:

[0086] Take 400 of water injection human rabies vaccine (Vero cell) (i.e. 200 mL) into the same separation funnel, add 600 mL of ethanol, 140 mL of toluene, shake thoroughly and stand, forming two liquid phases, the toluene-containing lipophilic phase and the ethanol and water injection human rabies vaccine-containing aqueous phase, with dimethyl silicone oil distributed in the lipophilic phase, then retain the lipophilic phase and discard the aqueous phase.

[0087] Place in a 40°C water bath to heat to remove the toluene, obtain a crude product, add chloroform 250 μL to the crude product in three portions to redissolve, obtain a sample for injection, and use ATR method to detect the sample for injection.

[0088] Comparative Example 7

[0089] The difference between this comparative example and Comparative Example 1 is that the initial sample for preparing the water solution of human rabies vaccine is a lyophilized human rabies vaccine, and the preparation method is: take one lyophilized human rabies vaccine and add sterile water for injection (i.e. diluent) to 0.5 mL, then mix 400 of the diluted vaccine, to obtain the required 200 mL of water solution of human rabies vaccine. The others are the same as in Comparative Example 1.

[0090] Comparative Example 8

[0091] An ATR detection method for the migration amount of dimethicone on the surface of a medicinal halogenated butyl rubber stopper in a human rabies vaccine, comprising the following steps:

[0092] S1, take 400 water needles human rabies vaccine (Vero cell) into the same liquid funnel, add 50 mL of toluene, shake well and stand, form double liquid phase, toluene is in the oil phase, water needle human rabies vaccine is in the water phase, dimethicone is distributed in the oil phase, then leave the oil phase, discard the water phase.

[0093] S2, add 80 mL of 0.14 mol / L sodium chloride solution to the oil phase obtained in step S1, shake well and stand for 8 min, then leave the oil phase. Add 210 mL of water to the above treated oil phase, shake well and mix; then treat in water bath, the water bath temperature is 75℃, the time is 7 min; then cool to room temperature.

[0094] Then add 400 mL of ethanol and 140 mL of toluene, shake well and stand, form double liquid phase, toluene is in the oil phase, ethanol and water needle human rabies vaccine are in the water phase, dimethicone is distributed in the oil phase, then leave the oil phase, discard the water phase. The oil phase is rotary evaporated under vacuum at 40℃ to remove toluene, and the crude product is obtained. Add 250 μL of chloroform to the crude product to dissolve, and obtain the sample for injection. The sample for injection can be detected by ATR method.

[0095] Detection method

[0096] 1. Preparation of standard solution and drawing of standard curve

[0097] Precisely weigh about 2.5 g of dimethicone and place it in a 25 mL volumetric flask, add chloroform to constant volume, shake well, and obtain 100 mg / mL standard curve stock solution; precisely take 1.0, 2.0, 3.0, 4.0 and 5.0 mL of standard curve stock solution, and place them in 10 mL volumetric flasks, dilute to the mark with chloroform, shake well, and obtain standard curve solutions with concentrations of 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL and 50 mg / mL, respectively, which are named as standard solution 1, standard solution 2, standard solution 3, standard solution 5 and standard solution 5.

[0098] Respectively, standard solutions 1-5 are detected by attenuated total reflection (ATR method), directly added to the surface of ZnSe of fourier transform infrared spectrometer, and the infrared spectrum is immediately scanned. The characteristic peak of dimethicone is 1260±10 cm -1Quantitative peak height, establish regression equation; with the standard curve solution peak height quantitative calculation, to make linear equation of mass concentration (mg / mL) to peak height: Y = 0.000572X + 0.000201, R 2 = 0.991.

[0099] The results show that dimethicone in the concentration in the range of 10 mg / mL ~ 50 mg / mL shows a good linear relationship (R 2 = 0.991).

[0100] 2. Precision test

[0101] Take 30 mg / mL standard solution, continuously determine 6 times, the RSD of dimethicone concentration is 1.8%, the precision of the method is good.

[0102] 3. Quantitative limit With the standard solution of 10 mg / mL, the quantitative peak is 10:1 to the baseline noise signal, the lower limit of dimethicone quantification is 3.03 mg / mL.

[0103] 4. Sample determination The sample injection sample prepared by each example and comparative example is taken 50 μL to the surface of ZnSe crystal, and the infrared spectrum is immediately scanned, the quantitative peak height at (1260 ± 10) cm -1 is measured, the concentration (mg / mL) of dimethicone is calculated by substituting the standard curve equation, and then the residual amount of dimethicone on the surface of each rubber plug (μg / each) is calculated. The results are shown in Table 1.

[0104] Table 1 The residual amount of dimethicone in human rabies vaccine (μg / branch) detected by the method of different examples and comparative examples

[0105]

[0106] From the data results in Table 1, it can be seen that the results of Example 2 (or Example 4 or Example 5) are compared with the results of Comparative Example 2 (or the results of Example 8 and Comparative Example 7 are compared, the results of Example 6 and Comparative Example 1 are compared, the results of Example 7 and Comparative Example 3 are compared), it can be seen that the detection of the amount of dimethicone on the surface of the pharmaceutical halogenated butyl rubber plug in the human rabies vaccine is more sufficient. The method of the application effectively reduces the loss of dimethicone when removing impurity proteins in the vaccine, thereby realizing more accurate detection of the amount of dimethicone on the surface of the pharmaceutical halogenated butyl rubber plug in the human rabies vaccine.

[0107] In the above embodiments, the detection results of Example 2 and Example 4 (or Example 5) are compared, and the sample injection sample in Example 2 contains more impurities, that is, the impurity content in the spectrum is obviously more.

[0108] In addition, in the scheme of the present application, it is shown by Examples 6-7 that when the extractant is selected as toluene, n-hexane and diethyl ether, the purpose of effectively detecting the migration amount of simethicone on the surface of the medicinal halogenated butyl rubber stopper in the human rabies vaccine can be achieved, but different extractants will also have an impact on the detection results; it is possible that the different extractant and water, ethanol form different extraction systems, which further affects the phase distribution of simethicone and impurity protein, thereby leading to different final detection results.

[0109] Examples 4-6 further illustrate that in the extraction system of the extractant, water and ethanol, the volume ratio of the extractant, water and ethanol should be properly selected, otherwise it will also be difficult to achieve the purpose of more fully extracting simethicone in the human rabies vaccine, which will further affect the detection results.

[0110] 5. Accuracy: Take the injection sample prepared in Example 5, 4 parts, 1 part is dissolved with 250 μL of chloroform, and the content of simethicone in the sample is directly determined according to the determination method in "4, sample determination". The other 3 parts are precisely added with 250 μL of simethicone standard solution with a concentration of 10 mg / mL, 30 mg / mL and 50 mg / mL respectively, and directly determined according to the determination method, and the average recovery rate is 99.3% (n=9) and RSD=2.1% by standard curve method.

[0111] Take the injection sample prepared in Example 5, 4 parts, 1 part is dissolved with 250 μL of chloroform, and the content of simethicone in the sample is directly determined according to the determination method in "4, sample determination". The other 3 parts are precisely added with 250 μL of simethicone standard solution with a concentration of 10 mg / mL, 30 mg / mL and 50 mg / mL respectively, and directly determined according to the determination method, and the average recovery rate is 99.3% (n=9) and RSD=2.1% by standard curve method.

[0112] The specific embodiments are only an explanation of the present application, which is not a limitation of the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. An ATR detection method for the migration of dimethicone on the surface of a medicinal halogenated butyl rubber stopper in a human rabies vaccine, characterized in that: The following steps are involved: S1. Take an aqueous solution of human rabies vaccine, add ethanol and an extractant, and fully shake and extract to form a two-phase solution, namely a lipophilic phase and an aqueous phase. Keep the lipophilic phase and discard the aqueous phase; wherein the volume ratio of the aqueous solution of human rabies vaccine to ethanol is 1:1-2.5, and the volume ratio of the aqueous solution of human rabies vaccine to the extractant is 1:0.5-0.8, and the extractant is selected from any one of ether, toluene, n-hexane and chloroform; S2. After removing the extractant from the lipophilic phase, a crude product is obtained, chloroform is added to the crude product for redissolution to obtain an injection sample, and the injection sample is detected by ATR method. Before removing the extractant, the lipophilic phase is treated with hot water at 70-80° C. and then cooled to room temperature. When the lipophilic phase is treated with hot water, the following steps are included: Add water to the lipophilic phase in an amount of 1-1.8 times the volume of the extractant, then keep warm in a water bath and cool, leaving the lipophilic phase.

2. The detection method according to claim 1, wherein The water bath temperature is 90-100℃ and the time is 5-10 minutes.

3. The detection method according to any one of claims 1 to 2, characterized in that In step S2, before the hot water treatment, the lipophilic phase is treated with a 0.11-0.18 mol / L sodium chloride solution.

4. The detection method according to claim 3, characterized in that In step S2, when the sodium chloride solution is used to treat the lipophilic phase, the following steps are included: Add 0.11-0.18 mol / L sodium chloride solution to the lipophilic phase. The amount of sodium chloride solution added is 40%-80% of the volume of the extractant. After sufficient shaking, let it stand for 5-10 minutes to retain the lipophilic phase.

5. The detection method according to claim 1, wherein The initial sample of the human rabies vaccine aqueous solution is selected from any one of freeze-dried human rabies vaccine and water-injected human rabies vaccine.

6. The detection method according to claim 1, characterized in that When the initial sample of the human rabies vaccine aqueous solution is a freeze-dried human rabies vaccine, before performing step S1 during testing, the freeze-dried human rabies vaccine is first dissolved in water to obtain a human rabies vaccine aqueous solution, and then the test is performed; when the initial sample of the human rabies vaccine is a water-injectable human rabies vaccine, the test is performed directly.

7. The detection method according to claim 1, characterized in that In step S2, the sample is directly added to the ZnSe surface of the Fourier transform infrared spectrometer using the ATR method during detection, and the infrared spectrum is immediately scanned to obtain the detection result.

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