Method for detecting content of cracking agent Triton N-101

Abstract

The invention belongs to the technical field of detection, and provides a Triton N-101 content determination method, which comprises the operation of detecting a to-be-detected sample by adopting an RP-HPLC-ELSD (Reverse P-High Performance Liquid Chromatography-Evaporative Light Scattering Detector) method, mobile phases adopted in the RP-HPLC-ELSD method comprise a mobile phase A, a mobile phase B and a mobile phase C; wherein the mobile phase A is water, the mobile phase B is methanol, and the mobile phase C is isopropanol; according to the method, the mobile phase is adopted for gradient elution. The method provided by the invention can be used for accurately determining the residual quantity of Triton N-101 in the subunit influenza vaccine containing the milk adjuvant, is rapid, accurate and good in linearity and specificity, and is not interfered by all components of the milk adjuvant and other components in components of a sample preparation.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and particularly relates to a method for detecting the content of a lysing agent Triton N-101. Background Art

[0002] Triton N-101, also known as polyethylene glycol monooctylphenyl ether, is a non-ionic surfactant with a certain degree of irritation. In the production process of influenza virus split vaccine and subunit vaccine, a splitting agent (such as Triton N-101) needs to be added to split the virus, and then purified to prepare influenza virus split vaccine and subunit vaccine. The current edition of the "Chinese Pharmacopoeia" Part III "Influenza Virus Split Vaccine" stipulates that the residual amount of Triton N-101 in the semi-finished product should be less than 300μg / mL, so the detection of Triton N-101 residue is more important for monitoring the quality of vaccine products.

[0003] At present, there are few studies on the detection of Triton N-101 residues. Zhang Pengyan reported the detection of Triton N-101 residues by thiocyanocobalamin colorimetric method (Zhang Pengyan, Yang Xin. Detection of Triton N-101 residues in influenza split vaccines by thiocyanocobalamin colorimetric method [C] / / Collection of papers from the Fifth National Immunodiagnosis and Vaccine Academic Symposium. 2011.). The detection was carried out according to the "3203 Polysorbate 80 Residue Determination Method" of the "Chinese Pharmacopoeia". The principle is that Triton N-101 contains the same polyethoxy group as polysorbate 80 (Tween 80). This group can react with ammonium cobalt thiocyanate to form a blue complex that is soluble in dichloromethane. Therefore, the Triton N-101 residues in influenza split vaccines and subunit vaccines can be determined by colorimetry.

[0004] However, this detection method is only applicable to vaccines that do not contain polysorbate 80 and / or polyethoxylated interfering substances, and the experimental steps are too complicated. Zhang Yan reported another colorimetric method for determining the residual amount of Triton N-101 (Zhang Yan, Pan Xiaonan, Ren Jing. Rapid detection method for residual amount of lysing agent Triton N-101 in influenza vaccine [J]. Chinese Journal of Biological Products, 2007, 20 (9): 3.), which calculates the content of lysing agent in the sample by deducting the difference in protein absorbance values ​​in the sample, but the experimental steps are complicated and the specificity is poor. Therefore, in practice, a method for rapid, accurate and stable detection of Triton N-101 residual amount in vaccines is urgently needed. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides a method for determining the content of Triton N-101, the method comprising the operation of detecting a sample to be tested by using an RP-HPLC-ELSD method; the mobile phase used in the RP-HPLC-ELSD method comprises a mobile phase A, a mobile phase B and a mobile phase C; wherein the mobile phase A is water, the mobile phase B is methanol, and the mobile phase C is isopropanol; and the method uses the mobile phase for gradient elution.

[0006] Preferably, the process and parameters of the gradient elution include:

[0007] At 0.0-1.0min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is maintained at (40-60):(40-60):0; preferably maintained at 50:50:0;

[0008] At 1.0-3.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C changes linearly from (40-60):(40-60):0 to (10-30):(70-90):0; preferably, it changes linearly from 50:50:0 to 20:80:0;

[0009] At 3.0-6.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C changes linearly from (10-30):(70-90):0 to (0-10):(90-100):0; preferably, it changes linearly from 20:80:0 to 5:95:0;

[0010] At 6.0-7.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is (0-10):(90-100):0 linearly changed to (0-5):(0-10):(85-100); preferably, it is linearly changed from 5:95:0 to 0:0:100;

[0011] At 7.0-9.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is maintained at (0-5): (0-10): (85-100); preferably maintained at 0:0:100;

[0012] At 9.0-9.01 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C changes linearly from (0-5):(0-10):(85-100) to (40-60):(40-60):0; preferably, it changes linearly from 0:0:100 to 50:50:0;

[0013] At 9.01-11.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is maintained at (40-60):(40-60):0, preferably maintained at 50:50:0.

[0014] The method for detecting the content of the lytic agent Triton N-101 provided by the present invention is particularly suitable for accurately determining the residual amount of Triton N-101 in a subunit influenza vaccine containing an emulsion adjuvant. The detection method is rapid, accurate, linear, and has good specificity, is not interfered by other components in the components of the emulsion adjuvant and the sample preparation components, and has guiding significance for detecting the residual amount of Triton N-101 in a subunit influenza vaccine containing an emulsion adjuvant. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a chromatogram of the sample solution to be tested, the emulsified adjuvant diluent, the blank solution, the Triton N-101 standard solution, and the spiked sample solution detected by RP-HPLC-ELSD in Example 1 of the present invention.

[0016] Figure 2 This is a standard curve drawn after five standard solutions of Triton N-101 were detected by RP-HPLC-ELSD in Example 1 of the present invention.

[0017] Figure 3 This is a chromatogram of the sample solution to be tested, the emulsified adjuvant dilution, the blank solution, and the Triton N-101 standard solution detected by RP-HPLC-ELSD in Example 2 of the present invention.

[0018] Figure 4 This is a chromatogram of the sample solution to be tested, the emulsified adjuvant dilution, the blank solution, and the Triton N-101 standard solution detected by RP-HPLC-ELSD in Example 3 of the present invention.

[0019] Figure 5 This is a standard curve drawn after five standard solutions of Triton N-101 were detected by RP-HPLC-ELSD in Example 3 of the present invention.

[0020] Figure 6 This is a chromatogram of the sample solution to be tested, the emulsified adjuvant dilution, the blank solution, and the Triton N-101 standard solution detected by RP-HPLC-ELSD in Example 4 of the present invention.

[0021] Figure 7 This is a standard curve drawn after five standard solutions of Triton N-101 were detected by RP-HPLC-ELSD in Example 4 of the present invention.

[0022] Figure 8 This is a chromatogram of the sample solution to be tested, the emulsified adjuvant dilution, the blank solution, and the Triton N-101 standard solution detected by RP-HPLC-ELSD in Example 5 of the present invention.

[0023] Fig. 9 This is a standard curve drawn after five standard solutions of Triton N-101 were detected by RP-HPLC-ELSD in Example 5 of the present invention.

[0024] Fig.10 This is a chromatogram of the sample solution to be tested, the emulsified adjuvant diluent, the blank solution, and the Triton N-101 standard solution detected by RP-HPLC-ELSD in the comparative example of the present invention. DETAILED DESCRIPTION

[0025] To make the technical solution, purpose and advantages of the present invention clearer, the present invention is further described in detail below through specific implementation examples. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0026] The invention provides a method for detecting the content of a lytic agent Triton N-101, and the method is particularly suitable for detecting the residual amount of Triton N-101 in a subunit influenza vaccine containing an emulsion adjuvant.

[0027] A method for determining the content of Triton N-101 comprises the following steps: using RP-HPLC-ELSD (reverse phase high performance liquid chromatography-evaporative light scattering) to detect a sample to be tested.

[0028] According to the method for determining the content of Triton N-101 of the present invention, the sample to be tested may be a vaccine using Triton N-101 in the preparation process, such as a subunit influenza vaccine containing an emulsion adjuvant.

[0029] According to the method for determining the content of Triton N-101 of the present invention, the mobile phases used in the RP-HPLC-ELSD method include mobile phase A, mobile phase B and mobile phase C; wherein mobile phase A is water, mobile phase B is methanol, and mobile phase C is isopropanol. The method uses the mobile phases for gradient elution.

[0030] The process and parameters of the gradient elution may include:

[0031] At 0.0-1.0min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is maintained at (40-60):(40-60):0; preferably maintained at 50:50:0;

[0032] At 1.0-3.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C changes linearly from (40-60):(40-60):0 to (10-30):(70-90):0; preferably, it changes linearly from 50:50:0 to 20:80:0;

[0033] At 3.0-6.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C changes linearly from (10-30):(70-90):0 to (0-10):(90-100):0; preferably, it changes linearly from 20:80:0 to 5:95:0;

[0034] At 6.0-7.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is (0-10):(90-100):0 linearly changed to (0-5):(0-10):(85-100); preferably, it is linearly changed from 5:95:0 to 0:0:100;

[0035] At 7.0-9.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is maintained at (0-5): (0-10): (85-100); preferably maintained at 0:0:100;

[0036] At 9.0-9.01 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C changes linearly from (0-5):(0-10):(85-100) to (40-60):(40-60):0; preferably, it changes linearly from 0:0:100 to 50:50:0;

[0037] At 9.01-11.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is maintained at (40-60):(40-60):0, preferably maintained at 50:50:0.

[0038] According to the method for determining the content of Triton N-101 described in the present invention, the instruments and parameters used in the RP-HPLC-ELSD method include: the detector is an evaporative light scattering detector, and the detector parameters are: atomizing gas pressure of 25psi, nebulizer mode heating (power 60%), drift tube temperature of 60°C, and gain value of 200; the chromatographic column can be a C8 chromatographic column; the column temperature can be 35°C-50°C, preferably 40°C; the sample plate temperature can be 12-18°C, preferably 15°C; the flow rate is 0.4-0.8mL / min, preferably 0.6mL / min.

[0039] According to the method for determining the content of Triton N-101 of the present invention, the method further comprises setting the operation of Triton N-101 standard solution. The concentration of the Triton N-101 standard solution may include: at least 5 of 0.2 mg / mL, 0.19 mg / mL, 0.18 mg / mL, 0.17 mg / mL, 0.16 mg / mL, 0.15 mg / mL, 0.14 mg / mL, 0.13 mg / mL, 0.12 mg / mL, 0.11 mg / mL, 0.1 mg / mL, 0.09 mg / mL, 0.08 mg / mL, 0.07 mg / mL, 0.06 mg / mL, 0.05 mg / mL, 0.04 mg / mL, and 0.03 mg / mL, and appropriate multiple standard concentrations may be selected according to actual needs; for example, the following set of standard concentrations may be selected: 0.2 mg / mL, 0.1 mg / mL, 0.06 mg / mL, 0.04 mg / mL, and 0.03 mg / mL. The Triton N-101 standard solution may be prepared using methanol.

[0040] According to the method for determining the content of Triton N-101 of the present invention, the method further comprises the operation of setting a blank control. The blank solution used in the blank control is methanol and a demulsifier; the demulsifier is isopropanol-trichloroform in a volume ratio of 9:1.

[0041] According to the method for determining the content of Triton N-101 of the present invention, the method further comprises the operation of setting an emulsion adjuvant control. The emulsion adjuvant may be MF59 emulsion adjuvant.

[0042] According to the method for determining the content of Triton N-101 of the present invention, the method further includes an operation of demulsifying the original solution of the sample to be tested. The demulsifier of the demulsification operation can be isopropanol-chloroform with a volume ratio of 9:1. In the demulsification operation, the volume ratio between the original solution of the sample to be tested and the demulsifier is 1:(3-6), preferably 1:4. Through the demulsification operation, the original solution of the sample to be tested can be used as a solution of the sample to be tested for detection. After demulsification, the Triton N-101 and the components of the emulsion adjuvant (such as squalene, Tween 80 and Span 85, etc.) in the original solution of the sample to be tested can be fully released, and the components of Triton N-101 and the emulsion adjuvant are dissolved in the demulsification solvent; the sample after demulsification and dilution is separated by a C8 chromatographic column, and the other components in the sample can be effectively separated from Triton N-101 after gradient elution.

[0043] According to the method for determining the content of Triton N-101 of the present invention, taking into account the characteristics of the emulsion of the emulsion adjuvant-related vaccine, the sample to be tested will be slightly stratified after being placed for a long time, so after demulsification and before detection, the sample solution to be tested can also be mixed. The mixing operation can be, for example, vortex mixing, and the vortex mixing time can be 15 seconds to 1 minute, preferably 30 seconds. Further preferably, it can also include standing the mixed sample solution to be tested (for example, standing for 5 minutes), and then centrifuging (for example, in a desktop high-speed centrifuge, 13000rpm centrifugation for 10 minutes, and transferring the upper clear liquid of the centrifuged sample for detection).

[0044] According to the method for determining the content of Triton N-101 of the present invention, the method further comprises setting an operation of adding a spiked sample solution. The spiked sample is a standard solution of Triton N-101 with a known amount added to the sample solution to be tested.

[0045] According to the determination method of Triton N-101 content of the present invention, the method includes detecting the Triton N-101 standard solution using RP-HPLC-ELSD, and obtaining the linear equation of the standard curve according to the detection result; then the sample solution to be tested is detected, and the detection result is substituted into the linear equation, so as to calculate the content of Triton N-101. Specifically, the above-mentioned Triton N-101 standard solution, the sample solution to be tested, the blank control solution, the emulsified adjuvant control solution, and the spiked sample solution can be measured by RP-HPLC-ELSD respectively to obtain the corresponding chromatogram and peak area; with the logarithm of the standard concentration of Triton N-101 as the abscissa X, the logarithm of the peak area as the ordinate Y, the standard curve is drawn, and the linear equation of the standard curve is calculated; the peak area obtained by measuring the sample solution to be tested and the spiked sample solution by RP-HPLC-ELSD is substituted into the linear equation of the standard curve, and the content of Triton N-101 in the sample solution to be tested and the spiked sample solution is calculated.

[0046] In order to improve the peak shape of Triton N-101 and effectively separate Triton N-101 in the vaccine and the components in the emulsifier (squalene, Tween 80 and Span 85), the inventors of the present invention investigated the Waters XBridge BEH C8 DirectConnect HP ( 10μm, 2.1mm×30mm) and Waters XBridge BEH C8 ( 2.5μm, 3mm×75mm) two C8 columns. The former has a slightly poorer separation effect on Triton N-101 and emulsifier, so WatersXBridge BEH C8 ( 2.5μm, 3mm×75mm) chromatographic column. In order to reduce the contamination of the detector, the switching valve was adjusted to set each component to enter the detector from 3.0min to 5.5min, and the rest of the time was switched to waste liquid.

[0047] Triton N-101 is one of the lysing agents often introduced in the production process of influenza virus split vaccines and subunit vaccines. Its residual amount must be strictly controlled within a certain limit standard. At present, there is no rapid, stable, uninterfered by other substances, and good specificity detection method for the detection of Triton N-101 residual amount in subunit influenza vaccines. Based on the problems existing in the prior art, the present invention provides a method for detecting the content of the lysing agent Triton N-101, which is particularly suitable for the detection of Triton N-101 residual amount in subunit influenza vaccines containing emulsion adjuvants.

[0048] The invention comprises the following steps: respectively obtaining a sample solution to be tested, a blank solution and a Triton N-101 standard solution of different concentrations, injecting an ultra-high performance liquid chromatography, and detecting by an evaporative light scattering detector; determining a characteristic peak of Triton N-101 by a chromatogram of the Triton N-101 standard solution and a chromatogram of the blank solution, that is, selecting a chromatographic peak in the standard solution that is different from the chromatogram of the blank solution as the characteristic peak of Triton N-101, establishing a linear equation by using the logarithmic values ​​of the Triton N-101 standard solutions of different concentrations and the logarithmic value of the peak area of ​​the Triton N-101 characteristic peak, substituting the logarithmic value of the peak area of ​​the Triton N-101 characteristic peak in the sample solution to be tested after demulsification treatment into the linear equation, and calculating the residual amount of Triton N-101 in the sample solution to be tested. The method provided by the present invention can realize the accurate determination of the Triton N-101 residue in the subunit influenza vaccine containing emulsion adjuvant. The detection method is rapid, accurate, linear, and has good specificity. It is not interfered by other components in the components of the emulsion adjuvant and the sample preparation components. It has guiding significance for the detection of Triton N-101 residue in the subunit influenza vaccine containing emulsion adjuvant.

[0049] The various reagents, materials, etc. used in the following examples, unless otherwise specified, are all products that can be obtained from commercial channels; the various tests and detection methods used in the following examples, unless otherwise specified, are all conventional tests and detection methods in the art and can be obtained from textbooks, reference books or academic journals.

[0050] In the following examples, the ultra-high performance liquid chromatograph with an evaporative light scattering detector was purchased from Waters, model Acquity Premier; the chromatographic column used was XBridge BEH C8 ( 2.5μm, 3mm×75mm), purchased from Waters, catalog number 186006046; Triton N-101 (reduced) standard was purchased from Sigma-Aldrich, catalog number 303135; emulsifier was SEPIVAC SWE purchased from SEPPIC, catalog number 80748J TM , containing squalene, Tween 80 and Span 85.

[0051] Example 1

[0052] This example is used to illustrate the specificity and accuracy of the method for detecting the content of the lysing agent Triton N-101 provided by the present invention when detecting the residual amount of Triton N-101 in a subunit influenza vaccine containing an emulsion adjuvant.

[0053] In this embodiment, the sample solution to be tested is the subunit influenza vaccine sample 1 containing an emulsion adjuvant, which is known to contain the following ingredients: 2.4 mg / mL Span 85, 2.5 mg / mL Tween 80, 21.5 mg / mL squalene, and 300 μg / mL influenza antigen protein.

[0054] In this embodiment, the emulsifier is a product SEPIVAC SWE purchased from SEPPIC with a product number of 80748J. TM The ingredients contained are: 4.8mg / mL Span 85, 5.0mg / mL Tween 80, and 43.0mg / mL squalene.

[0055] Preparation of demulsifier:

[0056] Pipette 9 mL of isopropanol into a 15 mL centrifuge tube, add 1 mL of chloroform, vortex mix at medium speed for 1 min, and prepare an isopropanol-chloroform volume ratio of 9:1, which is the demulsifier.

[0057] Preparation of Triton N-101 standard solution:

[0058] Accurately weigh an appropriate amount of Triton N-101 and place it in a volumetric flask. Dissolve it in methanol and dilute to the mark. Shake well to prepare a 1 mg / mL Triton N-101 standard solution stock solution. Then use methanol as a diluent to dilute in steps to 0.2 mg / mL, 0.1 mg / mL, 0.06 mg / mL, 0.04 mg / mL, and 0.03 mg / mL as 5 standard curve concentration points for use.

[0059] Preparation of sample solution to be tested:

[0060] After vortexing the test sample solution (i.e., the above-mentioned subunit influenza vaccine test sample 1) for 30 seconds, take 200 μL of the test sample solution, add 800 μL of demulsifier, vortex for 30 seconds, let stand for 5 minutes, and then centrifuge at 13000 rpm for 10 minutes. Take the upper clear liquid of the test sample solution after centrifugation for sampling (equivalent to diluting the subunit influenza vaccine test sample 1 5 times for detection).

[0061] Preparation of emulsion adjuvant dilution:

[0062] After vortexing the emulsion adjuvant for 30 seconds, take 200 μL of the emulsion adjuvant, add 800 μL of demulsifier, vortex for 30 seconds, let stand for 5 minutes, and then centrifuge at 13000 rpm for 10 minutes. Take the upper clear liquid of the emulsion adjuvant solution after centrifugation, which is the emulsion adjuvant dilution.

[0063] Preparation of spiked sample solution:

[0064] After vortexing the sample solution for 30 seconds, take three portions of 200 μL of the sample solution, add 100 μL of Triton N-101 standard solution (concentrations are 0.4 mg / mL, 0.6 mg / mL, 1.0 mg / mL), and finally add 700 μL of demulsifier to prepare low, medium, and high concentration spiked samples with spiked amounts (i.e., the final added concentration of Triton N-101) of 0.04 mg / mL, 0.06 mg / mL, and 0.1 mg / mL, respectively. Vortex the spiked sample for 30 seconds, let it stand for 5 minutes, and then centrifuge it at 13000 rpm for 10 minutes. Take the upper clear liquid of the centrifuged spiked sample solution for injection.

[0065] The prepared Triton N-101 standard solution, blank solution (methanol), blank solution (demulsifier), sample solution, emulsion adjuvant dilution, and spiked sample solution were injected into the instrument for detection (chromatographic conditions are shown in Table 1), and the corresponding chromatograms were recorded (see Figure 1 ) and the Triton N-101 peak areas of the sample solution, spiked sample solution and standard solution.

[0066] Table 1 RP-HPLC-ELSD chromatographic parameters

[0067]

[0068] like Figure 1As shown, the blank solution (methanol), blank solution (demulsifier), and emulsion adjuvant diluent have no interference with the characteristic peak position of the Triton N-101 standard solution (retention time is 4.641min), indicating that the method of the present invention has good specificity. Comparing the sample solution to be tested and the standard solution, the peak retention time of Triton N-101 in the sample solution to be tested is 4.638min, which is consistent with the standard solution.

[0069] Drawing of standard curve: With the logarithm of the five standard concentrations of Triton N-101 prepared above as the abscissa X and the logarithm of the peak area as the ordinate Y, draw the standard curve and obtain the linear equation: Y = 1.829136X + 6.218259, the linear correlation coefficient R 2 =0.999220 (e.g. Figure 2 As shown), this shows that the method has good linear correlation in the range of Triton N-101 concentration of 0.03mg / mL-0.2mg / mL. Among them, the signal-to-noise ratio S / N of the standard curve concentration of 0.03mg / mL is 12, which meets the requirement of the quantitative limit signal-to-noise ratio S / N greater than 10 in the "9101 Validation Guidelines for Analytical Methods" of the Chinese Pharmacopoeia 2020 edition, so 0.03mg / mL is set as the quantitative lower limit of the method provided by the present invention.

[0070] Result calculation: Substitute the peak area values ​​obtained by RP-HPLC-ELSD detection of the above-mentioned sample solution and spiked sample solution into Figure 2 From the linear equation shown, the content of Triton N-101 in the above-mentioned test samples and spiked samples was obtained, and the specific results are shown in Table 2 (RSD in Table 2 represents relative standard deviation).

[0071] Table 2 Detection results of Triton N-101 content in the sample solution and the spiked sample solution

[0072]

[0073] It can be seen from the data in Table 2 that the recovery rate of the spiked sample solution is between 82% and 97%, which meets the accuracy requirements of conventional trace detection (recovery rate 80%-115%), indicating that the method of the present invention can accurately and quantitatively detect Triton N-101 in the range of 0.03 mg / mL-0.2 mg / mL, and the method has good accuracy.

[0074] The concentration of Triton N-101 in the test sample solution (unspiked solution) was detected and calculated to be less than 0.03 mg / mL, which is lower than the above-mentioned limit of quantification. The concentration of Triton N-101 in the corresponding test sample stock solution (i.e., the above-mentioned subunit influenza vaccine test sample 1) was less than 0.15 mg / mL (since the above-mentioned test sample solution was obtained by diluting the test sample stock solution 5 times after the above-mentioned demulsifier treatment, the concentration of Triton N-101 in the test sample stock solution was less than 5*0.03 mg / mL). Based on the Chinese Pharmacopoeia 2020 edition "Influenza Virus Split Vaccine", the residual amount of the splitting agent Triton N-101 is less than 0.3 mg / mL, which meets the requirements of the marketed influenza vaccine products. Therefore, the sensitivity of the current detection method can be used for the evaluation of the residual amount of the splitting agent Triton N-101 in the vaccine. At the same time, combined with the relevant research on the toxicology of this vaccine, the content of Triton N-101 in the above-mentioned test sample concentrate (i.e. the above-mentioned subunit influenza vaccine test sample 1) is within the safe content range and has been approved for use in human clinical trials.

[0075] Example 2

[0076] This example is used to illustrate the detection method of the lysing agent Triton N-101 content provided by the present invention to detect the residual amount of Triton N-101 in the subunit influenza vaccine containing emulsion adjuvant.

[0077] In this embodiment, except that the sample stock solution to be tested is the subunit influenza vaccine sample 2 containing an emulsion adjuvant, the other reagents used are the same as those in Example 1, including the blank solution (methanol), the blank solution (demulsifier), the Triton N-101 standard solution, the emulsion adjuvant diluent, etc., which are the same as those in Example 1; wherein the sample solution to be tested is prepared using the above-mentioned subunit influenza vaccine sample 2 according to the method of Example 1 (equivalent to diluting the subunit influenza vaccine sample 2 5 times for detection).

[0078] The RP-HPLC-ELSD chromatographic parameters used are the same as those in Example 1, as shown in Table 1.

[0079] The chromatogram obtained by RP-HPLC-ELSD detection is as follows Figure 3 As shown, the peak retention time of Triton N-101 in the sample solution is 4.640 min, which is consistent with that of the standard solution.

[0080] Result calculation: Substitute the peak area value obtained by RP-HPLC-ELSD test of the sample solution into Figure 2 The equation of the straight line shown (Y = 1.829136X + 6.218259, R 2=0.999220), the content of Triton N-101 in the above-mentioned sample solution was obtained, and then the content of Triton N-101 in the subunit influenza vaccine test sample 2 was calculated. The specific results are shown in Table 3.

[0081] Table 3 Detection results of Triton N-101 content in the sample solution and vaccine sample 2

[0082]

[0083] As shown in Table 3, the method of the present invention can accurately detect the content of Triton N-101 in the subunit influenza vaccine.

[0084] Example 3

[0085] This example is used to illustrate the detection method of the lysing agent Triton N-101 content provided by the present invention to detect the residual amount of Triton N-101 in the subunit influenza vaccine containing emulsion adjuvant.

[0086] In this example, the reagents used are the same as those in Example 2, including the blank solution (methanol), blank solution (demulsifier), Triton N-101 standard solution, test sample solution, emulsion adjuvant diluent, etc.

[0087] Except for the gradient elution program of the RP-HPLC-ELSD chromatography in Table 4, everything else is the same as Example 2.

[0088] Table 4 Gradient elution program of RP-HPLC-ELSD chromatography

[0089]

[0090]

[0091] like Figure 4 As shown, the blank solution (methanol), blank solution (demulsifier), and emulsion adjuvant diluent have no interference with the characteristic peak position of the Triton N-101 standard solution (retention time is 4.922min), indicating that the method of the present invention has good specificity. Comparing the sample solution to be tested and the standard solution, the peak retention time of Triton N-101 in the sample solution to be tested is 4.917min, which is consistent with the standard solution.

[0092] Drawing of standard curve: With the logarithm of the five standard concentrations of Triton N-101 prepared above as the abscissa X and the logarithm of the peak area as the ordinate Y, draw the standard curve and obtain the linear equation: Y = 1.965433X + 6.601272, the linear correlation coefficient R 2=0.999358 (e.g. Figure 5 shown).

[0093] Result calculation: The specific results of the Triton N-101 content in the test sample solution and the subunit influenza vaccine test sample 2 are shown in Table 5.

[0094] Table 5 Detection results of Triton N-101 content in the sample solution and vaccine sample 2

[0095]

[0096] As can be seen from Table 5, the method of the present invention can accurately detect the content of Triton N-101 in the subunit influenza vaccine.

[0097] Example 4

[0098] This example is used to illustrate the detection method of the lysing agent Triton N-101 content provided by the present invention to detect the residual amount of Triton N-101 in the subunit influenza vaccine containing emulsion adjuvant.

[0099] In this example, the reagents used are the same as those in Example 2, including the blank solution (methanol), blank solution (demulsifier), Triton N-101 standard solution, test sample solution, emulsion adjuvant diluent, etc.

[0100] Except for the gradient elution program of the RP-HPLC-ELSD chromatography in Table 6, everything else is the same as Example 2.

[0101] Table 6 Gradient elution program of RP-HPLC-ELSD chromatography

[0102]

[0103] like Figure 6 As shown, the blank solution (methanol), blank solution (demulsifier), and emulsion adjuvant diluent have no interference with the characteristic peak position of the Triton N-101 standard solution (retention time is 4.464min), indicating that the method of the present invention has good specificity. Comparing the sample solution to be tested and the standard solution, the peak retention time of Triton N-101 in the sample solution to be tested is 4.453min, which is consistent with the standard solution.

[0104] Drawing of standard curve: With the logarithm of the five standard concentrations of Triton N-101 prepared above as the abscissa X and the logarithm of the peak area as the ordinate Y, draw a standard curve and obtain the linear equation: Y = 1.915493X + 6.602546, the linear correlation coefficient R 2 =0.999805 (e.g. Figure 7shown).

[0105] Result calculation: The specific results of the Triton N-101 content in the test sample solution and the subunit influenza vaccine test sample 2 are shown in Table 7.

[0106] Table 7 Detection results of Triton N-101 content in the sample solution and vaccine sample 2

[0107]

[0108] As can be seen from Table 7, the method of the present invention can accurately detect the content of Triton N-101 in the subunit influenza vaccine.

[0109] Example 5

[0110] This example is used to illustrate the detection method of the lysing agent Triton N-101 content provided by the present invention to detect the residual amount of Triton N-101 in the subunit influenza vaccine containing emulsion adjuvant.

[0111] In this example, the reagents used are the same as those in Example 2, including the blank solution (methanol), blank solution (demulsifier), Triton N-101 standard solution, test sample solution, emulsion adjuvant diluent, etc.

[0112] Except for the gradient elution program of the RP-HPLC-ELSD chromatography in Table 8, everything else is the same as Example 2.

[0113] Table 8 Gradient elution program of RP-HPLC-ELSD chromatography

[0114]

[0115] like Figure 8 As shown, the blank solution (methanol), blank solution (demulsifier), and emulsion adjuvant diluent have no interference with the characteristic peak position of the Triton N-101 standard solution (retention time is 3.980min), indicating that the method of the present invention has good specificity. Comparing the sample solution to be tested and the standard solution, the peak retention time of Triton N-101 in the sample solution to be tested is 3.975min, which is consistent with the standard solution.

[0116] Drawing of standard curve: With the logarithm of the five standard concentrations of Triton N-101 prepared above as the abscissa X and the logarithm of the peak area as the ordinate Y, draw the standard curve and obtain the linear equation: Y = 1.920237X + 6.651587, the linear correlation coefficient R 2 =0.999213 (e.g. Fig. 9 shown).

[0117] Result calculation: The specific results of the Triton N-101 content in the test sample solution and the subunit influenza vaccine test sample 2 are shown in Table 9.

[0118] Table 9 Test results of Triton N-101 content in the sample solution and vaccine sample 2

[0119]

[0120] As can be seen from Table 9, the method of the present invention can accurately detect the Triton N-101 content in the subunit influenza vaccine.

[0121] Comparative Example

[0122] In this example, the reagents used are the same as those in Example 2, including the blank solution (methanol), blank solution (demulsifier), Triton N-101 standard solution, test sample solution, emulsion adjuvant diluent, etc.

[0123] Except for the gradient elution program of the RP-HPLC-ELSD chromatography in Table 10, everything else is the same as Example 2.

[0124] Table 10 Gradient elution program of RP-HPLC-ELSD chromatography

[0125]

[0126]

[0127] like Fig.10 As shown in the figure, the blank solution (methanol) and the blank solution (demulsifier) ​​have no interference on the characteristic peak position of the Triton N-101 standard solution, but the chromatographic peak of the emulsion adjuvant dilution has obvious overlap with the characteristic peak of Triton N-101, and the Triton N-101 and emulsion adjuvant components in the sample solution to be tested are not baseline separated. It can be seen that the gradient elution procedure cannot effectively separate the Triton N-101 and emulsion adjuvant components in the vaccine test sample 2, has poor specificity, and is not suitable for detecting the content of Triton N-101 in influenza vaccines containing emulsion adjuvants.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for determining the content of Triton N-101, the method comprising the operation of detecting a sample to be tested by using the RP-HPLC-ELSD method; the mobile phase used in the RP-HPLC-ELSD method comprises mobile phase A, mobile phase B and mobile phase C; wherein, Mobile phase A is water, mobile phase B is methanol, and mobile phase C is isopropanol; the method uses the mobile phases for gradient elution.

2. The method according to claim 1, characterized in that: The process and parameters of the gradient elution include: At 0.0-1.0min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is maintained at (40-60):(40-60):0; preferably maintained at 50:50:0; At 1.0-3.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C changes linearly from (40-60):(40-60):0 to (10-30):(70-90):0; preferably, it changes linearly from 50:50:0 to 20:80:0; At 3.0-6.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C changes linearly from (10-30):(70-90):0 to (0-10):(90-100):0; preferably, it changes linearly from 20:80:0 to 5:95:0; At 6.0-7.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is (0-10):(90-100):0 linearly changed to (0-5):(0-10):(85-100); preferably, it is linearly changed from 5:95:0 to 0:0:100; At 7.0-9.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is maintained at (0-5): (0-10): (85-100); preferably maintained at 0:0:100; At 9.0-9.01 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C changes linearly from (0-5):(0-10):(85-100) to (40-60):(40-60):0; preferably, it changes linearly from 0:0:100 to 50:50:0; At 9.01-11.0 min, the volume ratio of mobile phase A, mobile phase B and mobile phase C is maintained at (40-60):(40-60):0, preferably maintained at 50:50:

0.

3. The method according to claim 1 or 2, characterized in that: The sample to be tested is a vaccine using Triton N-101 during the preparation process; Preferably, the sample to be tested is a subunit influenza vaccine containing an emulsion adjuvant.

4. The method according to any one of claims 1 to 3, characterized in that: The method also includes setting an operation of Triton N-101 standard solution; Preferably, the concentration of the Triton N-101 standard solution includes: at least 5 of 0.2 mg / mL, 0.19 mg / mL, 0.18 mg / mL, 0.17 mg / mL, 0.16 mg / mL, 0.15 mg / mL, 0.14 mg / mL, 0.13 mg / mL, 0.12 mg / mL, 0.11 mg / mL, 0.1 mg / mL, 0.09 mg / mL, 0.08 mg / mL, 0.07 mg / mL, 0.06 mg / mL, 0.05 mg / mL, 0.04 mg / mL, and 0.03 mg / mL; Preferably, the concentration of the Triton N-101 standard solution includes: 0.2 mg / mL, 0.1 mg / mL, 0.06 mg / mL, 0.04 mg / mL, and 0.03 mg / mL.

5. The method according to any one of claims 1 to 4, characterized in that: The method further comprises the operation of setting a blank control and / or an emulsion adjuvant control; Preferably, the blank solution used in the blank control is methanol and a demulsifier; Preferably, the demulsifier is isopropanol-chloroform in a volume ratio of 9:1; Preferably, the emulsion adjuvant is MF59 emulsion adjuvant.

6. The method according to any one of claims 1 to 5, characterized in that: The method further comprises the operation of demulsifying the original solution of the sample to be tested; Preferably, the demulsifier used in the demulsification operation is isopropanol-chloroform in a volume ratio of 9:1; Preferably, in the demulsification operation, the volume ratio between the sample solution to be tested and the demulsifier is 1:(3-6), preferably 1:

4.

7. The method according to any one of claims 1 to 6, characterized in that: The method also includes setting up an operation of spiked sample solution; The spiked sample is a standard solution of Triton N-101 with a known amount added to the sample solution to be tested.

8. The method according to any one of claims 1 to 7, characterized in that: The method comprises the steps of detecting the Triton N-101 standard solution by using RP-HPLC-ELSD, and obtaining a linear equation of a standard curve according to the detection result; then detecting the sample solution to be tested, and substituting the detection result into the linear equation, so as to calculate the content of Triton N-101.

9. The method according to claim 8, characterized in that: The method comprises: measuring a Triton N-101 standard solution, a sample solution to be tested, a blank control solution, an emulsified adjuvant control solution, and a spiked sample solution by RP-HPLC-ELSD, respectively, to obtain corresponding chromatograms and peak areas; drawing a standard curve with the logarithm of the standard concentration of Triton N-101 as the abscissa X and the logarithm of the peak area as the ordinate Y, and calculating the linear equation of the standard curve; substituting the peak areas obtained by measuring the sample solution to be tested and the spiked sample solution by RP-HPLC-ELSD into the linear equation of the standard curve, and calculating the content of Triton N-101 in the sample solution to be tested and the spiked sample solution.

10. The method according to any one of claims 1 to 9, characterized in that: The RP-HPLC-ELSD method uses Waters XBridge BEH C8 ( 2.5μm, 3mm×75mm) chromatographic column.

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