Method for detecting content of Span 85

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting the content of Span 85. The method comprises the following steps: pretreating a sample to be detected; establishing a standard curve equation; and detecting a to-be-detected sample. According to the present invention, the Span 85 is subjected to the esterification reaction under the alkaline condition to derive, the high boiling point Span 85 is converted into the low boiling point Span 85 derivative, the gas chromatography analysis can be performed on the Span 85 derivative, the mass spectrum positioning is performed on the esterified Span 85 derivative, and the technical support is provided for the quality monitoring of the vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting the content of Span 85. Background Art

[0002] Currently, most subunit vaccines (including natural products, recombinant products, and synthetic peptides) are weak antigens, and subunit antigens often fail when administered alone. The possible reasons are misprocessing by the immune system, rapid clearance, stimulation of inappropriate immune responses, and lack of key B cell or T cell epitopes.

[0003] In order to enhance the immunogenicity, speed and durability of immune response of antigens, aluminum adjuvants, MF59 and other adjuvants are often added in vaccine production. Among them, Span 85 is used in vaccine products.

[0004] Sorbitan Trioleate is a pale yellow to yellow oily liquid, insoluble in water, with a molecular formula of C 60 H 108 O8, with a molecular weight of 957.495 and a boiling point of 887.4±65.0°C, is a non-volatile substance and cannot be directly analyzed by gas chromatography. Summary of the invention

[0005] In order to solve at least one of the above technical problems, the present invention provides a method for detecting the content of Span 85.

[0006] As one aspect, the present invention provides a method for detecting the content of Span 85, which comprises the following steps: pretreatment of the sample to be tested: mixing the sample to be tested with a demulsifier and an organic solvent, performing demulsification treatment to obtain a supernatant, performing esterification reaction on the supernatant to obtain a Span 85 derivative solution, and treating the Span 85 derivative solution with a saturated NaCl solution to obtain an injection sample to be tested; establishing a standard curve equation: preparing at least three standard solutions of Span 85 with different concentrations, treating the standard solutions according to the same steps as the pretreatment of the sample to be tested, detecting by gas chromatography, and establishing a standard curve equation of Span 85 derivative chromatographic peak area-Span 85 concentration according to the detection results; detecting the sample to be tested: detecting the injection sample to be tested by gas chromatography, and calculating the content of Span 85 in the sample to be tested according to the detection results and the standard curve equation.

[0007] In some embodiments of the present invention, the demulsification treatment step includes: ultrasound and centrifugation to obtain a supernatant.

[0008] In some embodiments of the present invention, the ultrasound time is 0.1~20 min, for example, it can be 5 min, 8.9 min, 11.3 min, 14.6 min, 16.8 min, 17.3 min, 19.6 min, etc.; and / or the centrifugal acceleration is 1000~15000 g, for example, it can be 1509 g, 3896 g, 8623 g, 9836 g, 10000 g, 11239 g, 15000 g, 14239 g, etc., and the time is 0.1~20 min, for example, it can be 6.8 min, 3 min, 11.4 min, 14.9 min, 19.6 min, etc.

[0009] In some embodiments of the present invention, the step of esterification reaction comprises: mixing the supernatant with a base and an alcohol, and performing an esterification reaction to obtain a Span 85 derivative.

[0010] In some embodiments of the present invention, the base is an alkali metal hydroxide; and / or the alcohol is methanol.

[0011] In some embodiments of the present invention, the temperature of the esterification reaction is 10~100°C, for example, it can be 13.6°C, 24.9°C, 46.7°C, 63.2°C, 78.4°C, 96.9°C, etc.; the time is 0.1~30min, for example, it can be 8.3min, 13.8min, 16.4min, 19.6min, 23.6min, 25.6min, 29.6min, etc.

[0012] In some embodiments of the present invention, the conditions of gas chromatography include: carrier gas: nitrogen; and / or, flow rate: 1-3 mL / min, for example, 1.2 mL / min, 1.6 mL / min, 2 mL / min, 2.1 mL / min, 2.3 mL / min, 2.6 mL / min, 2.9 mL / min, etc., constant flow mode; and / or, chromatographic column: capillary column or equivalent chromatographic column; and / or, detector: FID detector; and / or, injection port temperature: 200-280°C, for example, 212°C, 236°C, 240°C, 243°C, 259°C, 271°C, 278°C, etc.; And / or, detector temperature: 240~320℃, for example, it can be 259℃, 271℃, 280℃, 283℃, 297℃, 309℃, 318℃, etc.; and / or, injection volume: 0.01~20.0μL, for example, it can be 1.0μL, 1.2μL, 3.4μL, 5.9μL, 7.8μL, 9.1μL, 12.3μL, 14.8μL, 16.1μL, 19.3μL, etc.; and / or, split ratio: (0.1~100):1, for example, it can be 0.8:1, 1:1, 13:1, 29:1, 42:1, 68:1, 73:1, 82:1, 96:1, etc.

[0013] In some embodiments of the present invention, the temperature program of the gas chromatography includes: column temperature: initial temperature 40-280°C, for example, 57°C, 79°C, 92°C, 118°C, 139°C, 160°C, 178°C, 216°C, 238°C, 279°C, etc., increased at 0-100°C / min (for example, 12°C / min, 27°C / min, 43°C / min, 62°C / min, 89°C / min, 96°C / min, etc.) to 150-300°C (for example, 169°C, 183°C, 220°C, 228°C, 249°C, 286°C, 298°C, etc.), maintained for 0-20min (for example, 1.9min, 3.1min, 5.5min, 6.9min). The temperature may be preferably 200°C / min, 300°C, 400°C, 800°C, etc.), then increased at a rate of 0-50°C / min (for example, 3°C / min, 7°C / min, 13°C / min, 20°C / min, 23°C / min, 37°C / min, 48°C / min, etc.) to 220-330°C (for example, 231°C, 249°C, 258°C, 280°C, 283°C, 307°C, 329°C, etc.), and maintained for 0-10 min (for example, 0.9 min, 3.1 min, 5 min, 5.9 min, 6.4 min, 7.6 min, 8.9 min, 9.7 min, etc.).

[0014] In some embodiments of the present invention, the organic solvent is n-hexane; and / or the demulsifier is acetonitrile.

[0015] In some embodiments of the present invention, the sample to be tested is selected from any one of a finished vaccine, an adjuvant, and a placebo.

[0016] The technical solution provided by the embodiment of the present invention has the following advantages: The method for detecting the content of Span 85 of the present invention is to derive Span 85 by esterifying Span 85 under alkaline conditions, converting the high-boiling-point Span 85 into a Span 85 derivative with a lower boiling point. The Span 85 derivative can be analyzed by gas chromatography, and the esterified Span 85 derivative is positioned by mass spectrometry, providing technical support for the quality monitoring of vaccines. At the same time, the content of Span 85 in the sample to be tested (for example, any one of the finished vaccine, adjuvant, and placebo) can be accurately detected, the stability of the vaccine product and the consistency between different batches can be ensured, and the problem of interference from other components in the sample is overcome, thereby enhancing the specificity of the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0019] Figure 1 is a gas chromatogram of the blank matrix of Example 1 of the present invention; Figure 2 is a gas chromatogram of the Span 85 derivative of the Span 85 standard solution of Example 1 of the present invention; Figure 3 This is a gas chromatogram of the supernatant obtained after the esterification reaction of the finished recombinant protein vaccine of Example 1 of the present invention. DETAILED DESCRIPTION

[0020] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0021] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0022] According to an embodiment of one aspect of the present invention, a method for detecting the content of Span 85 is provided, and the method comprises the following steps: pretreatment of the sample to be tested: mixing the sample to be tested with a demulsifier and an organic solvent, performing demulsification treatment to obtain a supernatant, performing esterification reaction on the supernatant to obtain a Span 85 derivative solution, and treating the Span 85 derivative solution with a saturated NaCl solution to obtain an injection sample to be tested; establishing a standard curve equation: preparing at least three standard solutions of Span 85 with different concentrations, treating the standard solutions according to the same steps as the pretreatment of the sample to be tested, detecting by gas chromatography, and establishing a standard curve equation of Span 85 derivative chromatographic peak area-Span 85 concentration according to the detection results; detecting the sample to be tested: detecting the injection sample to be tested by gas chromatography, and calculating the content of Span 85 in the sample to be tested according to the detection results and the standard curve equation.

[0023] In some embodiments of the present invention, a method for detecting the content of Span 85 comprises the following steps: (1) Pretreatment of samples to be tested: The sample to be tested is dissolved in an organic solvent n-hexane to prepare a sample solution to be tested; an emulsifier acetonitrile is added to the sample solution to be tested, and an ultrasonic demulsification treatment is performed for 5 minutes. After centrifugation at 10000g for 3 minutes, a supernatant is obtained; the supernatant is evenly mixed with a KOH-methanol solution, and an ultrasonic treatment is performed to perform an esterification reaction to obtain a Span 85 derivative. The Span 85 derivative is treated with saturated NaCl to obtain a sample to be tested.

[0024] (2) Establishment of standard curve equation: At least three standard solutions of Span 85 with different concentrations were prepared using n-hexane as an organic solvent, and the standard solutions with different concentrations were respectively demulsified using acetonitrile as a demulsifier to obtain supernatants. The supernatants were mixed evenly with a KOH-methanol solution, and subjected to ultrasound to perform an esterification reaction to obtain Span 85 derivatives. The derivatives were detected by gas chromatography, and a standard curve equation of the chromatographic peak area-concentration of the derivatives was established according to the detection results.

[0025] (3) Testing of samples to be tested: The sample to be tested obtained in step (1) is tested by gas chromatography, and the content of Span 85 in the sample to be tested is calculated based on the test result and the standard curve equation obtained in step (2).

[0026] In some embodiments of the present invention, representative peaks of Span 85 derivatives are determined by chromatograms of Span 85 standard solution and blank matrix chromatograms, and mass spectrometry positioning is performed on Span 85 derivatives; and a standard curve equation is established based on the concentration and peak area of ​​the Span 85 standard curve solution.

[0027] In some embodiments of the present invention, the content of Span 85 in the test sample (selected from any one of the finished vaccine, adjuvant, and placebo) is calculated based on the standard curve equation and the peak area of ​​the representative peak of the Span 85 derivative in the chromatogram of the test sample (selected from any one of the finished vaccine, adjuvant, and placebo).

[0028] In order to further illustrate the effect of the present invention, specific embodiments are used for illustration below.

[0029] Example 1 Detection of Span 85 in a finished recombinant protein vaccine (1) Preparation of the finished vaccine solution to be tested: Take 100μL of the sample vaccine finished product-1 to be tested in a 2mL EP tube, add 100μL of n-hexane and 100μL of acetonitrile respectively, mix well and sonicate for 5min, add 800μL of n-hexane, continue to mix well and sonicate for 5min, centrifuge at 10000g for 3min. Accurately measure 500μL of the supernatant in a 2mL EP tube, add 50μL of KOH-methanol solution and mix well, sonicate for 5min to cause esterification reaction, finally add 1mL of saturated NaCl and mix well and sonicate for 2min, let stand to clarify, accurately measure 1μL of the supernatant and inject it into GC analysis.

[0030] (2) Preparation of standard solution: Standard mother solution: accurately weigh 100 mg of Span 85 standard and dissolve it in n-hexane. Mix thoroughly with a vortex mixer for more than 30 seconds to prepare a mother solution with a mass concentration of 10.0 mg / mL; Standard working solution: use n-hexane solution to prepare Span 85 working solutions with mass concentrations of 1.0, 1.6, 2.0, 3.0, 4.0, 5.0, 6.0, and 10.0 mg / mL, take 100 μL of each into a 2 mL EP tube, add 100 μL ultrapure water and 100 μL acetonitrile, mix well and sonicate for 5 minutes, add 800 μL n-hexane, continue mixing well and sonicate for 5 minutes, and centrifuge at 10000 g for 3 minutes. The remaining steps are the same as (1) Preparation of the finished vaccine to be tested for gas chromatography determination. The 1.6 and 5.0 mg / mL working solutions are used as quality control solutions QC-L and QC-H, respectively.

[0031] (3) Gas chromatography analysis: Accurately measure 1.0 μL of blank matrix solution, standard curve solution and finished vaccine solution to be tested, inject into gas chromatograph, and perform detection under the following chromatographic conditions: Carrier gas: nitrogen; Flow rate: 2mL / min, constant flow mode; Chromatographic column: Agilent HP-5 capillary column or equivalent, 30m×0.32mm×0.25μm; Temperature program: Column temperature: initial temperature 160℃, increase to 220℃ at 12℃ / min, hold for 5min, then increase to 280℃ at 20℃ / min, hold for 5min; Detector: FID detector; Inlet temperature: 240°C, FID detector temperature: 280°C; Injection volume: 1.0 μL, split ratio: 10:1.

[0032] (4) Test result analysis: The blank matrix chromatogram of the embodiment and the reference chromatogram of the standard curve solution with a concentration of 2.0 mg / mL were measured as follows: Figure 1 , Figure 2 As shown, the chromatogram of the finished vaccine to be tested is as follows Figure 3 shown.

[0033] The blank matrix chromatogram of Example 1 was used as a blank reference, and the chromatographic peak in the standard curve chromatogram that was different from the blank matrix chromatogram was selected as the representative peak. The interference of the blank matrix was eliminated, and the standard curve equation was established with the concentration of the standard curve solution and the representative peak area of ​​the Span 85 derivative, wherein the standard curve equation was: y=0.0078x-0.0810; the chromatographic peak area corresponding to the representative peak in the chromatogram of the finished vaccine to be tested was substituted into the linear regression equation to calculate the accurate content of Span 85 in the finished vaccine to be tested. The test results are listed in Table 1.

[0034] Example 2 Except that the "sample to be tested" in "(1) Preparation of the finished vaccine solution to be tested" is "finished vaccine product-2", the rest is the same as Example 1.

[0035] Example 3 Except that the "sample to be tested" in "(1) Preparation of the finished vaccine solution to be tested" is "finished vaccine product-3", the rest is the same as Example 1.

[0036] Example 4 Except that the "sample to be tested" in "(1) Preparation of the finished vaccine solution to be tested" is "finished vaccine product-4", the rest is the same as Example 1.

[0037] Example 5 Except that the "sample to be tested" in "(1) preparation of the finished vaccine solution to be tested" is "finished vaccine product-5", the rest is the same as Example 1.

[0038] Table 1

[0039] Example 6 Except that the "test sample" in "(1) Preparation of the test vaccine finished solution" is "adjuvant MF59 (2×)", the rest is the same as Example 1.

[0040] The test results are listed in Table 2.

[0041] Table 2

[0042] Example 7 Except that the "test sample" in "(1) Preparation of test vaccine finished solution" is "placebo", the rest is the same as Example 1.

[0043] The test results are listed in Table 3.

[0044] Table 3

[0045] In summary, the method for detecting the content of Span 85 in the embodiment of the present invention, under alkaline conditions, derives Span 85 by esterification reaction, converts the high-boiling point Span 85 into a derivative with a lower boiling point, and performs gas chromatography analysis, so as to accurately detect the content of Span 85 in the sample to be tested (any one of the finished vaccine, adjuvant, and placebo).

[0046] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0047] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting the content of Span 85, characterized in that: The method comprises the following steps: Pretreatment of samples to be tested: The sample to be tested is mixed with a demulsifier and an organic solvent, and subjected to demulsification treatment to obtain a supernatant, and the supernatant is subjected to an esterification reaction to obtain a Span 85 derivative solution, and the Span 85 derivative solution is treated with a saturated NaCl solution to obtain a sample to be tested; Establishment of standard curve equation: Prepare at least three standard solutions of Span 85 with different concentrations, treat the standard solutions according to the same steps as the pretreatment of the sample to be tested, detect them by gas chromatography, and establish a standard curve equation of Span 85 derivative chromatographic peak area-Span 85 concentration according to the test results; Sample testing: The sample to be tested is tested by gas chromatography, and the content of Span 85 in the sample to be tested is calculated according to the test result and the standard curve equation.

2. The method according to claim 1, characterized in that The steps of the demulsification treatment include: Ultrasonication and centrifugation were performed to obtain the supernatant.

3. The method according to claim 2, characterized in that The ultrasonic time is 0.1~20min; And / or, the centrifugal acceleration is 1000~15000g, and the time is 0.1~20min.

4. The method according to claim 1, characterized in that The steps of the esterification reaction include: The supernatant is mixed with a base and an alcohol to undergo an esterification reaction to obtain a Span 85 derivative.

5. The method according to claim 4, characterized in that The base is an alkali metal hydroxide; And / or, the alcohol is methanol.

6. The method according to claim 4, characterized in that The temperature of the esterification reaction is 10-100° C., and the time is 0.1-30 min.

7. The method according to claim 1, characterized in that The gas chromatography conditions include: Carrier gas: nitrogen; and / or, flow rate: 1~3mL / min, constant flow mode; and / or, Chromatographic column: capillary column or equivalent; and / or, detector: FID detector; and / or, injection port temperature: 200~280℃; and / or, detector temperature: 240~320°C; and / or, injection volume: 0.01~20.0μL; And / or, split ratio: (0.1~100):

1.

8. The method according to claim 1, characterized in that The temperature raising program of the gas chromatography comprises: Column temperature: initial temperature 40~280℃, increase to 150~300℃ at 0~100℃ / min, maintain for 0~20min, then increase to 220~330℃ at 0~50℃ / min, maintain for 0~10min.

9. The method according to claim 1, characterized in that: The organic solvent is n-hexane; And / or, the demulsifier is acetonitrile.

10. The method according to claim 1, characterized in that The sample to be tested is selected from any one of a finished vaccine product, an adjuvant, and a placebo.

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