Method for detecting content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane

Abstract

The invention provides a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane, which comprises the following steps: pretreating a sample to be detected based on a double-solvent oscillation extraction method so as to precipitate an interference matrix in the medicine; extracting to obtain a to-be-detected sample solution enriched with octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5); and carrying out gas mass spectrometry detection on the extracted sample solution to be detected so as to carry out high-sensitivity targeted quantitative test on D4 and D5 in the sample solution to be detected. The method comprises the following steps: rapidly precipitating an interference matrix in a to-be-detected sample through a double-solvent oscillation extraction method, enriching residual D4 and D5 components, fully shaking through an oscillator without concentration, and directly taking the to-be-detected sample for gas chromatography mass spectrometry (GCMS) detection; then, a gas phase mass spectrum is adopted to select an ion mode, and selective targeted quantitative testing with the sensitivity as high as 0.01 mu g / mL is carried out on D4 and D5 in the sample.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane. Background Art

[0002] During the production and preparation of inactivated vaccines and other drugs, cyclosiloxanes may be produced when process fluids come into contact with silicone materials (such as silicone tubes and seals used in production). These substances may cause harm to human health after migrating into the liquid medicine. Cyclosiloxanes are mainly harmful to the endocrine system. They interfere with the normal secretion of hormones and cause damage to the liver and respiratory system. In particular, some small molecule cyclosiloxanes, such as octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5), have been shown to be carcinogenic and bioaccumulative.

[0003] In the existing technology, D4 and D5 are mainly determined by gas chromatography and gas chromatography-mass spectrometry, which have low detection efficiency and insufficient sensitivity to meet the detection needs of high-concern compounds. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane, aiming to solve the technical problems in the prior art that the detection efficiency is low and the sensitivity is not enough to meet the detection needs of high-concern compounds.

[0005] In order to achieve the above object, the present invention is implemented by the following technical scheme: a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane, comprising the following steps:

[0006] Step 1) pre-treating the sample to be tested based on a dual solvent oscillation extraction method to precipitate the interfering matrix in the drug to extract and obtain a sample solution to be tested enriched in octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane, wherein the step of pre-treating the sample to be tested based on the dual solvent oscillation extraction method specifically includes:

[0007] Add the sample to be tested into a centrifuge tube, add a double solvent consisting of 2 to 3 parts of the first solvent and 10 to 20 parts of the second solvent and 1 to 3 parts of the internal standard solution, seal and shake thoroughly with an oscillator, transfer the sample extraction solution to a separatory funnel after shaking, add anhydrous sodium sulfate until the bottom of the bottle is in the form of quicksand, shake for 1 to 2 minutes, let stand for 4 to 6 minutes, take 2 parts of the organic phase into a volumetric flask, add dichloromethane to dilute to the target scale, and shake well;

[0008] Step 2), performing gas phase mass spectrometry detection on the extracted sample solution to perform high-sensitivity targeted quantitative testing on octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane in the sample solution.

[0009] According to one aspect of the above technical solution, the gas phase mass spectrometry detection parameters in step 2) include:

[0010] Chromatographic column: DB-5MS chromatographic column; chromatographic column specifications: 30m×0.25mm×0.25μm; column flow rate is 0.9-1.1mL / min; injection volume is 1μL; injection mode is split, and the split ratio is 5:1; carrier gas is He gas; gas phase inlet temperature is 250℃; heating program is initial temperature 40℃, hold for 5 minutes, heat to 150℃ at 20℃ / min, hold for 3min, then heat to 280℃ at 15℃ / min, hold for 15min.

[0011] According to one aspect of the above technical solution, the gas phase mass spectrometry detection parameters in step 2) also include:

[0012] The scanning ion mode was SCAN, the scanning range was 30-800 m / z; the ion source temperature was 230°C; the quadrupole temperature was 150°C; and the solvent delay was 3 min.

[0013] According to one aspect of the above technical solution, the gas phase mass spectrometry detection parameters in step 2) include:

[0014] Chromatographic column: DB-624UI chromatographic column; chromatographic column specifications: 30m×0.25mm×1.4μm; column flow rate is 0.9-1.1mL / min; injection volume is 1μL; injection mode is split, and the split ratio is 20:1; carrier gas is He gas; gas phase inlet temperature is 200℃; heating program is initial temperature 60℃, hold for 2min, increase to 220℃ at 20℃ / min, and hold for 2min.

[0015] According to one aspect of the above technical solution, the gas phase mass spectrometry detection parameters in step 2) also include:

[0016] The scanning ion mode was SIM, the ion source temperature was 230°C; the quadrupole temperature was 150°C; the solvent delay was 3 min; the octamethylcyclotetrasiloxane quantitative ion: 281 m / z, the octamethylcyclotetrasiloxane qualitative ion: 133 m / z; the decamethylcyclopentasiloxane quantitative ion: 355 m / z, the decamethylcyclopentasiloxane qualitative ion: 267 m / z.

[0017] According to one aspect of the above technical solution, the gas phase mass spectrometry detection parameters in step 2) also include: ion bombardment energy is 50 eV.

[0018] According to one aspect of the above technical solution, in step 1), the step of pre-treating the sample to be tested based on the dual solvent oscillation extraction method to precipitate the interfering matrix in the drug to extract and obtain the sample solution to be tested enriched with octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane specifically includes:

[0019] The sample to be tested is added into a sealed PTFE centrifuge tube, and a double solvent consisting of 2-3 mL of the first solvent and 10-20 mL of the second solvent and 1-3 mL of the internal standard solution are added. After sealing, the sample is fully shaken with an oscillator. After shaking, the sample extraction solution to be tested is transferred to a separatory funnel, and anhydrous sodium sulfate is added until the bottom of the bottle becomes a quicksand state. After shaking for 1 minute, the tube is allowed to stand for 5 minutes, 2 mL of the organic phase is taken into a 10 mL volumetric flask, and dichloromethane is added to dilute to the target scale, and the tube is shaken well to obtain a sample solution to be tested enriched with octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane.

[0020] According to one aspect of the above technical solution, the first solvent is one of acetone, methanol, acetonitrile and ethanol, and the second solvent is one of dichloromethane, chloroform, ethyl acetate and n-hexane.

[0021] According to one aspect of the above technical solution, the frequency of the oscillator is 50 rpm-70 rpm, and the shaking time of the oscillator is 2-5 minutes.

[0022] According to one aspect of the above technical solution, the sample to be tested is a new coronavirus inactivated vaccine liquid.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: the novel coronavirus inactivated vaccine stock solution can be quickly pretreated by the dual-solvent oscillation extraction method, and the interfering matrix in the novel coronavirus inactivated vaccine stock solution can be precipitated while the residual octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5) components can be enriched. After sufficient shaking with an oscillator, the test sample can be directly taken for gas chromatography-mass spectrometry (GCMS) detection without concentration; then, the gas chromatography-mass spectrometry (GCMS) selected ion mode is used to perform selective targeted quantitative testing of D4 and D5 in the extracted sample with a sensitivity of up to 0.01 μg / mL. This method has the advantages of short detection time, simple operation, and high sensitivity, and can effectively eliminate interference and achieve accurate quantification. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a flow chart of a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane in the first embodiment of the present invention;

[0025] Figure 2 The specificity chromatogram of the reagent blank solution in the first embodiment of the present invention;

[0026] Figure 3It is a specific chromatogram of the standard curve solution (L-3) in the first embodiment of the present invention;

[0027] Figure 4 The specific chromatogram of the sample solution to be tested in the first embodiment of the present invention;

[0028] Figure 5 The specific chromatogram of the quantitative limit spiked solution 1 in the first embodiment of the present invention;

[0029] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0031] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be a central element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be a central element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0033] Figure 1 Flow chart of a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane according to the first embodiment of the present invention, as shown in FIG. Figure 1 As shown, the method comprises the following steps:

[0034] Step 1) pre-treating the sample to be tested based on a dual solvent oscillation extraction method to precipitate the interfering matrix in the drug to extract and obtain a sample solution to be tested enriched with octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane. In this embodiment, the sample to be tested is preferably a novel coronavirus inactivated vaccine solution.

[0035] Furthermore, the steps of pre-treating the sample to be tested based on the dual solvent oscillation extraction method specifically include:

[0036] Add the sample to be tested into a centrifuge tube, and add a double solvent consisting of 2 to 3 parts of the first solvent and 10 to 20 parts of the second solvent and 1 to 3 parts of the internal standard solution. After sealing, shake thoroughly with an oscillator. After shaking, transfer the sample extraction solution to a separatory funnel, and then add anhydrous sodium sulfate until the bottom of the bottle becomes quicksand. After shaking for 1 to 2 minutes, let it stand for 4 to 6 minutes, take 2 parts of the organic phase into a volumetric flask, add dichloromethane to dilute to the target scale, and shake well.

[0037] Preferably, in this embodiment, the above step 1) specifically includes:

[0038] The sample to be tested is added into a sealed PTFE centrifuge tube, and a double solvent consisting of 2-3 mL of the first solvent and 10-20 mL of the second solvent and 1-3 mL of the internal standard solution are added. After sealing, the sample is fully shaken with an oscillator. After shaking, the sample extraction solution to be tested is transferred to a separatory funnel, and anhydrous sodium sulfate is added until the bottom of the bottle becomes a quicksand state. After shaking for 1 minute, the tube is allowed to stand for 5 minutes, 2 mL of the organic phase is taken into a 10 mL volumetric flask, and dichloromethane is added to dilute to the target scale, and the tube is shaken well to obtain a sample solution to be tested enriched with octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane.

[0039] Specifically, in this embodiment, the frequency of the oscillator in the above-mentioned dual solvent oscillation extraction method is 50 rpm-70 rpm, and the shaking time of the oscillator is 2-5 minutes.

[0040] In addition, in this embodiment, the first solvent is one of acetone, methanol, acetonitrile and ethanol, and the second solvent is one of dichloromethane, chloroform, ethyl acetate and n-hexane. That is, the two solvents are one of acetone / dichloromethane, acetone / chloroform, acetone / ethyl acetate, acetone / n-hexane, methanol / dichloromethane, methanol / chloroform, methanol / ethyl acetate, methanol / n-hexane, acetonitrile / dichloromethane, acetonitrile / chloroform, acetonitrile / ethyl acetate, acetonitrile / n-hexane, ethanol / dichloromethane, ethanol / chloroform, ethanol / ethyl acetate, ethanol / n-hexane. In this solution, the first solvent is preferably acetone, and the second solvent is preferably dichloromethane.

[0041] Step 2), performing gas phase mass spectrometry detection on the extracted sample solution to perform high-sensitivity targeted quantitative testing on octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane in the sample solution.

[0042] Specifically, in this embodiment, the gas phase mass spectrometry detection parameters in the above step 2) specifically include:

[0043] Gas chromatography conditions:

[0044] Chromatographic column: DB-624UI chromatographic column; chromatographic column specifications: 30m×0.25mm×1.4μm; column flow rate: 0.9-1.1mL / min; injection volume: 1μL; injection mode: split flow, split ratio: 20:1; carrier gas: He gas; gas phase injection port temperature: 200℃; heating program: initial temperature: 60℃, hold for 2min, increase to 220℃ at 20℃ / min, hold for 2min. In this embodiment, the column flow rate is preferably 1mL / min.

[0045] Mass spectrometry conditions: scanning ion mode is SIM, ion source temperature is 230°C; quadrupole temperature is 150°C; solvent delay is 3 min; octamethylcyclotetrasiloxane quantitative ion: 281 m / z, octamethylcyclotetrasiloxane qualitative ion: 133 m / z; decamethylcyclopentasiloxane quantitative ion: 355 m / z, decamethylcyclopentasiloxane qualitative ion: 267 m / z. Preferably, in this embodiment, under the conditions of ion source temperature of 230°C and quadrupole temperature of 150°C, the ion bombardment energy is preferably 50 eV, which can achieve higher detection sensitivity.

[0046] In some application scenarios of this embodiment, the sample configuration is as follows:

[0047] Diluent: dichloromethane;

[0048] Preparation of standard curve solution: Take appropriate amount of octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5), accurately weigh them, put them into a volumetric flask, add diluent to dissolve them, and quantitatively dilute them to make standard curve solutions containing D4: 0.948μg, 1.58μg, 3.16μg, 4.74μg, 6.34μg per 1mL; and D5: 7.25μg, 12.1μg, 24.2μg, 36.2μg, 48.4μg per 1mL.

[0049] Test solution: Measure 10.0 μL of the original solution of the inactivated new coronavirus vaccine into a 20mL sample bottle, add 2-3mL of acetone, 10-20mL of dichloromethane and 1-3mL of internal standard solution, seal and shake thoroughly with an oscillator, let stand for 5 minutes, then add anhydrous sodium sulfate until the bottom of the bottle becomes quicksand, shake for 1 minute, let stand for 5 minutes, take 2.000mL of the lower layer solution into a 10mL volumetric flask, add dichloromethane to dilute to the scale, shake well, and take about 1mL of the solution into the injection bottle.

[0050] The standard curve solution and the test solution were accurately measured and injected into a gas chromatography-mass spectrometer (GC-MS) for detection and analysis, and the chromatogram was recorded to perform a highly sensitive targeted quantitative test on octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane in the sample solution.

[0051] The content detection method in the first embodiment of the present invention was methodologically validated, including specificity, linear range, limit of quantitation, accuracy, precision (repeatability), precision (intermediate precision), and solution stability.

[0052] The preparation method of the standard substance solution A used for the specificity, linearity, limit of quantitation, accuracy, precision (repeatability) and solution stability tests is as follows:

[0053] Extraction solvent: dichloromethane

[0054] Preparation of standard substance D4 and D5 solution A:

[0055] Weigh 0.9040 mg of D4 standard substance into a 10 mL volumetric flask, add n-hexane to dilute to the scale, shake well for later use, and mark it as D4 stock solution.

[0056] Weigh 0.9580 mg of D5 standard substance into a 10 mL volumetric flask, add n-hexane to dissolve and dilute to the scale, shake well for later use, and mark it as D5 stock solution.

[0057] Separately measure 1105 μL of the above-mentioned D4 standard stock solution and 3985 μL of the D5 stock solution into a 10 mL volumetric flask, add ethanol to dilute to the scale, shake well, and obtain a standard substance solution A with a D4 concentration of 9.98 μg / mL and a D5 concentration of 38.17 μg / mL.

[0058] The preparation method of standard substance solution B used for precision (intermediate precision) test and durability test is as follows:

[0059] Weigh 0.9030 mg of D4 standard substance into a 10 mL volumetric flask, add n-hexane to dilute to the scale, shake well for later use, and mark it as D4 stock solution.

[0060] Weigh 0.9965 mg of D5 standard substance into a 10 mL volumetric flask, add n-hexane to dissolve and dilute to the scale, shake well for later use, and mark it as D5 stock solution.

[0061] Separately measure 1105 μL of the above-mentioned D4 standard stock solution and 3830 μL of the D5 stock solution into a 10 mL volumetric flask, add ethanol to dilute to the scale, shake well, and obtain a standard substance solution B with a D4 concentration of 9.970 μg / mL and a D5 concentration of 38.17 μg / mL.

[0062] (1) Preparation of standard curve solution:

[0063] Separately measure D4 and D5 standard substance solution A into a 10 mL volumetric flask, add dichloromethane to dilute to the scale, shake well, and take about 1 mL of the solution into a sampling bottle to prepare a standard curve solution.

[0064] The preparation parameters of the standard curve solution are shown in Table 1 below:

[0065]

[0066] Table 1;

[0067] The concentrations of the standard substances in the standard curve solution are shown in Table 2 below:

[0068]

[0069] Table 2;

[0070] (2) Preparation of system suitability (STT) solution: Take 1 mL of L-3 solution into an injection vial.

[0071] (3) Preparation of quality control (QC) solution: Take 1 mL of L-3 solution into an injection vial.

[0072] (4) Preparation of limit of quantitation (LOQ) solution: Take 1 mL of L-1 solution into an injection vial.

[0073] (5) Preparation of sample stock solution: Take one bottle of inactivated novel coronavirus vaccine stock solution and mix well.

[0074] (6) Preparation of reagent blank solution: Measure 10 μL of purified water into a 20 mL sample bottle, add 2-3 mL of acetone, 10-20 mL of dichloromethane and 1-3 mL of internal standard solution, seal and shake thoroughly with an oscillator, let stand for 5 minutes, then add anhydrous sodium sulfate until the bottom of the bottle becomes a quicksand, shake for 1 minute, let stand for 5 minutes, take 2 mL of the lower layer solution into a 10 mL volumetric flask, add dichloromethane to dilute to the mark, shake well, and take about 1 mL of the solution into a sampling bottle.

[0075] (7) Preparation of accuracy solution:

[0076] Low concentration horizontal accuracy solution: Measure 10.0 μL of the sample mother solution into a 20 mL sample bottle, add 7.5 μL of D4 and D5 standard substance solution A4, then add 10 mL of dichloromethane as the extractant, shake for 1 minute, let stand for 5 minutes, then add anhydrous sodium sulfate until the bottom of the bottle is in the form of quicksand, shake for 1 minute, let stand for 5 minutes, take 2 mL of the lower layer solution into a 10 mL volumetric bottle, then add dichloromethane to dilute to the scale, shake well, and take about 1 mL of the solution into the injection bottle. Prepare a total of 3 solutions in parallel using the same method, and obtain low concentration horizontal accuracy solution 1, low concentration horizontal accuracy solution 2, and low concentration horizontal accuracy solution 3 in turn.

[0077] Medium concentration level accuracy solution: Measure 10μL of sample mother solution into a 20mL sample bottle, add 158.5μL of D4 and D5 standard substance solution A, then add 10mL of dichloromethane as extractant, shake for 1 minute, let stand for 5 minutes, then add anhydrous sodium sulfate until the bottom of the bottle is in the form of quicksand, shake for 1 minute, let stand for 5 minutes, take 2mL of the lower layer solution into a 10mL volumetric bottle, then add dichloromethane to dilute to the scale, shake well, and take about 1mL of the solution into the injection bottle. Prepare a total of 3 solutions in parallel by the same method, and obtain medium concentration level accuracy solution 1, medium concentration level accuracy solution 2, and medium concentration level accuracy solution 3 in turn.

[0078] High-concentration horizontal accuracy solution: Measure 10.0 μL of the sample mother solution into a 20 mL sample bottle, add 237.5 μL of D4 and D5 standard substance solution A, then add 10 mL of dichloromethane as the extractant, shake for 1 minute, let stand for 5 minutes, then add anhydrous sodium sulfate until the bottom of the bottle is in the form of quicksand, shake for 1 minute, let stand for 5 minutes, take 2 mL of the lower layer solution into a 10 mL volumetric bottle, then add dichloromethane to dilute to the scale, shake well, and take about 1 mL of the solution into the injection bottle. Prepare a total of 3 solutions in parallel using the same method, and obtain high-concentration horizontal accuracy solution 1, high-concentration horizontal accuracy solution 2, and high-concentration horizontal accuracy solution 3 in turn.

[0079] The spike concentrations of the target substances in the accuracy concentrations are shown in Table 3 below.

[0080]

[0081] Table 3;

[0082] (8) Preparation of quantitation limit spiked solutions: Low concentration level accuracy solution 1, low concentration level accuracy solution 2, and low concentration level accuracy solution 3 were used as quantitation limit spiked solution 1, quantitation limit spiked solution 2, and quantitation limit spiked solution 3, respectively.

[0083] (9) Preparation of specific solutions: The reagent blank solution, the standard curve solution (L-3), the sample solution to be tested and the quantitative limit spike solution 1 are used as specific solutions.

[0084] Specifically, Figure 2 Shown is a specific chromatogram of the reagent blank solution in the embodiment of the present invention, Figure 3 Shown is a specific chromatogram of the standard curve solution (L-3) in the embodiment of the present invention, Figure 4 is the specific chromatogram of the sample solution to be tested, Figure 5 The specific chromatogram of the quantitation limit spike solution 1 is shown.

[0085] (10) Preparation of reproducible solutions: 10 μL of the sample mother solution was measured and placed in a 20 mL sample bottle, 158.5 μL of D4 and D5 standard substance solution A was added, and then 10 mL of dichloromethane was added as the extractant. The solution was shaken for 1 minute, and allowed to stand for 5 minutes. Anhydrous sodium sulfate was added until the bottom of the bottle was in a quicksand state. The solution was shaken for 1 minute, and allowed to stand for 5 minutes. 2 mL of the lower layer solution was placed in a 10 mL volumetric bottle, and then dichloromethane was added to dilute to the mark. The solution was shaken well, and about 1 mL of the solution was placed in a sample injection bottle. A total of 6 solutions were prepared in parallel by the same method, and reproducible solution 1, reproducible solution 2, reproducible solution 3, reproducible solution 4, reproducible solution 5, and reproducible solution 6 were obtained in turn.

[0086] (11) Preparation of durability solution: Take about 1 mL of L-3 solution into an injection vial.

[0087] (12) Preparation of solution stability solution: Take the above-mentioned repetitive solution 1, repetitive solution 2, repetitive solution 3, repetitive solution 4, repetitive solution 5 and repetitive solution 6, and leave them at room temperature for 18 hours to obtain solution stability solution 1, solution stability solution 2, solution stability solution 3, solution stability solution 4, solution stability solution 5 and solution stability solution 6, respectively.

[0088] (13) Preparation of intermediate precision solution: prepared by different analysts on different days, and analyzed using different instruments:

[0089] Measure 10 μL of the sample mother solution into a 20 mL sample bottle, add 158.5 μL of D4 and D5 standard substance solution B, then add 10 mL of dichloromethane as the extractant, shake for 1 minute, let stand for 5 minutes, then add anhydrous sodium sulfate until the bottom of the bottle is in a quicksand state, shake for 1 minute, let stand for 5 minutes, take 2 mL of the lower layer solution into a 10 mL volumetric bottle, then add dichloromethane to dilute to the scale, shake well, and take about 1 mL of the solution into the injection bottle. Prepare a total of 6 solutions in parallel using the same method, and obtain intermediate precision solution 1, intermediate precision solution 2, intermediate precision solution 3, intermediate precision solution 4, intermediate precision solution 5, and intermediate precision solution 6 in turn.

[0090] The verification results of octamethylcyclotetrasiloxane (D4) are summarized in Tables 4 and 5 below:

[0091] Table 4;

[0092]

[0093] Table 5: The verification results of decamethylcyclopentasiloxane (D5) are summarized in Tables 6 and 7 below:

[0094]

[0095] Table 6;

[0096]

[0097] Table 7;

[0098] In summary, the method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane in the above embodiments of the present invention can quickly pretreat the new coronavirus inactivated vaccine stock solution through a dual solvent oscillation extraction method, precipitate the interfering matrix in the new coronavirus inactivated vaccine stock solution, and at the same time achieve the enrichment of the residual octamethylcyclotetrasiloxane (D4) and decamethylcyclopentasiloxane (D5) components. After sufficient shaking with an oscillator, there is no need to concentrate, and the sample to be tested is directly taken for gas chromatography-mass spectrometry (GCMS) detection; then, the gas chromatography-mass spectrometry (GCMS) selected ion mode is used to perform selective targeted quantitative testing of D4 and D5 in the extracted sample with a sensitivity of up to 0.01 μg / mL. This method has the advantages of short detection time, simple operation, and high sensitivity, and can effectively eliminate interference and achieve accurate quantification.

[0099] The second embodiment of the present invention provides a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane. The detection method in this embodiment is basically the same as that in the first embodiment, except that:

[0100] In this embodiment, the gas phase mass spectrometry detection parameters in the above step 2) specifically include:

[0101] Gas chromatography conditions:

[0102] Chromatographic column: DB-5MS chromatographic column; chromatographic column specifications: 30m×0.25mm×0.25μm; column flow rate is 0.9-1.1mL / min; injection volume is 1μL; injection mode is split, and the split ratio is 5:1; carrier gas is He gas; gas phase inlet temperature is 250℃; heating program is initial temperature 40℃, hold for 5 minutes, heat to 150℃ at 20℃ / min, hold for 3min, then heat to 280℃ at 15℃ / min, hold for 15min.

[0103] Mass spectrometry conditions:

[0104] The scanning ion mode was SCAN, the scanning range was 30-800 m / z; the ion source temperature was 230°C; the quadrupole temperature was 150°C; and the solvent delay was 3 min.

[0105] The third embodiment of the present invention provides a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane. The detection method in this embodiment is basically the same as that in the first embodiment, except that:

[0106] The gas phase mass spectrometry detection parameters in step 2) include: a column flow rate of 0.9 mL / min.

[0107] The fourth embodiment of the present invention provides a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane. The detection method in this embodiment is basically the same as that in the first embodiment, except that:

[0108] The column flow rate was 1.1 mL / min.

[0109] The detection results of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane under different column flow rate conditions of the first embodiment, the third embodiment and the fourth embodiment were compared and observed, and the area change RSD was ≤1%, and the method had good durability and met the requirements.

[0110] Comparative Example 1

[0111] This comparative example 1 provides a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane. The detection method in this example is basically the same as that in the first example, except that:

[0112] The shaking time of the oscillator in the above-mentioned dual solvent shaking extraction method is 1.5 minutes.

[0113] Comparative Example 2

[0114] This comparative example 1 provides a method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane. The detection method in this example is basically the same as that in the first example, except that:

[0115] The shaking time of the oscillator in the above-mentioned dual solvent shaking extraction method is 5.5 minutes.

[0116] The test solution samples prepared in the first embodiment, comparative example 1 and comparative example 2 were tested. The test solution sample obtained in comparative example 1 could not be fully extracted, while the solvent of the test solution sample obtained in comparative example 2 volatilized, both of which affected the accuracy of the analysis results.

[0117] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0118] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane, characterized in that: The following steps are involved: Step 1) pre-treating the sample to be tested based on a dual solvent oscillation extraction method to precipitate the interfering matrix in the drug to extract and obtain a sample solution to be tested enriched in octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane, wherein the step of pre-treating the sample to be tested based on the dual solvent oscillation extraction method specifically includes: Add the sample to be tested into a centrifuge tube, add a double solvent consisting of 2 to 3 parts of the first solvent and 10 to 20 parts of the second solvent and 1 to 3 parts of the internal standard solution, seal and shake thoroughly with an oscillator, transfer the sample extraction solution to a separatory funnel after shaking, add anhydrous sodium sulfate until the bottom of the bottle is in the form of quicksand, shake for 1 to 2 minutes, let stand for 4 to 6 minutes, take 2 parts of the organic phase into a volumetric flask, add dichloromethane to dilute to the target scale, and shake well; Step 2), performing gas phase mass spectrometry detection on the extracted sample solution to perform high-sensitivity targeted quantitative testing on octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane in the sample solution.

2. The method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane according to claim 1, characterized in that: The gas phase mass spectrometry detection parameters in step 2) include: Chromatographic column: DB-5MS chromatographic column; chromatographic column specifications: 30m×0.25mm×0.25μm; column flow rate is 0.9-1.1mL / min; injection volume is 1μL; injection mode is split, and the split ratio is 5:1; carrier gas is He gas; gas phase inlet temperature is 250℃; heating program is initial temperature 40℃, hold for 5 minutes, heat to 150℃ at 20℃ / min, hold for 3min, then heat to 280℃ at 15℃ / min, hold for 15min.

3. The method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane according to claim 2, characterized in that: The gas phase mass spectrometry detection parameters in step 2) also include: The scanning ion mode was SCAN, the scanning range was 30-800 m / z; the ion source temperature was 230°C; the quadrupole temperature was 150°C; and the solvent delay was 3 min.

4. The method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane according to claim 1, characterized in that: The gas phase mass spectrometry detection parameters in step 2) include: Chromatographic column: DB-624UI chromatographic column; chromatographic column specifications: 30m×0.25mm×1.4μm; column flow rate is 0.9-1.1mL / min; injection volume is 1μL; injection mode is split, and the split ratio is 20:1; carrier gas is He gas; gas phase inlet temperature is 200℃; heating program is initial temperature 60℃, hold for 2min, increase to 220℃ at 20℃ / min, and hold for 2min.

5. The method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane according to claim 4, characterized in that: The gas phase mass spectrometry detection parameters in step 2) also include: The scanning ion mode was SIM, the ion source temperature was 230°C; the quadrupole temperature was 150°C; the solvent delay was 3 min; the octamethylcyclotetrasiloxane quantitative ion: 281 m / z, the octamethylcyclotetrasiloxane qualitative ion: 133 m / z; the decamethylcyclopentasiloxane quantitative ion: 355 m / z, the decamethylcyclopentasiloxane qualitative ion: 267 m / z.

6. The method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane according to claim 2 or 4, characterized in that: The gas phase mass spectrometry detection parameters in step 2) also include: ion bombardment energy is 50 eV.

7. The method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane according to claim 1, characterized in that: In step 1), the step of pretreating the sample to be tested based on the dual solvent oscillation extraction method to precipitate the interfering matrix in the drug to extract and obtain the sample solution to be tested enriched with octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane specifically includes: The sample to be tested is added into a sealed PTFE centrifuge tube, and a double solvent consisting of 2-3 mL of the first solvent and 10-20 mL of the second solvent and 1-3 mL of the internal standard solution are added. After sealing, the sample is fully shaken with an oscillator. After shaking, the sample extraction solution to be tested is transferred to a separatory funnel, and anhydrous sodium sulfate is added until the bottom of the bottle becomes a quicksand state. After shaking for 1 minute, the tube is allowed to stand for 5 minutes, 2 mL of the organic phase is taken into a 10 mL volumetric flask, and dichloromethane is added to dilute to the target scale, and the tube is shaken well to obtain a sample solution to be tested enriched with octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane.

8. The method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane according to claim 7, characterized in that: The first solvent is one of acetone, methanol, acetonitrile and ethanol, and the second solvent is one of dichloromethane, chloroform, ethyl acetate and n-hexane.

9. The method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane according to claim 7, characterized in that: The frequency of the shaker is 50 rpm-70 rpm, and the shaking time of the shaker is 2-5 minutes.

10. The method for detecting the content of octamethylcyclotetrasiloxane and decamethylcyclopentasiloxane according to claim 1, characterized in that: The sample to be tested is a new coronavirus inactivated vaccine liquid.

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