Application of capillary electrophoresis-mass spectrometry on-line, and detection method of tissue plasminogen activator drug heterogeneity

Abstract

The application provides application of capillary electrophoresis-mass spectrometry online connection, a detection method of tissue plasminogen activator drug heteromer, and relates to the technical field of analysis and detection.The application utilizes online capillary electrophoresis and mass spectrometry connection, separates t-PA through a capillary zone electrophoresis (CZE) mode, uses a neutral coating capillary, uses an acid-water mixed solution or an acid-acetonitrile-water mixed solution as an electrophoresis background buffer, adopts air pressure sampling, applies a separation voltage to perform capillary zone electrophoresis, effectively separates the heteromer in the t-PA drug through CZE, then introduces a mass spectrometer through an electric osmosis flow driven coaxial sheath flow liquid electrospray ion source for detection and analysis, realizes high-efficiency, accurate and high-sensitivity detection of the heteromer in the t-PA drug.Further, the capillary electrophoresis-mass spectrometry online connection detection adopted by the application is short in time consumption, small in required sample amount, and provides a reliable means for evaluating the quality and stability of the t-PA drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analytical detection, and particularly relates to application of capillary electrophoresis-mass spectrometry online combination and detection method of t-PA drug heterogeneity. BACKGROUND

[0002] t-PA (Tissue plasminogen activator) is an important recombinant protein drug, which is often used for the regulation of fibrinolytic system and the treatment of thrombotic diseases. The main function of t-PA is to activate plasminogen and promote the dissolution of thrombus, and the heterogeneity (i.e. variation in chemical structure) of t-PA has an important influence on its drug efficacy and stability.

[0003] The heterogeneity of t-PA can include active isomers, degradation products and modifiers, etc. The existence of these heterogeneity can cause differences in the activity, stability, immunogenicity and other aspects of the drug, thereby affecting its therapeutic effect and safety. Therefore, it is of great significance to accurately evaluate the heterogeneity of t-PA drug.

[0004] At present, the commonly used analysis methods for t-PA drug heterogeneity include high performance liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography (HPLC) and gel electrophoresis. However, the above analysis methods have some limitations, such as inability to qualify, unsatisfactory separation effect, long analysis time, large sample amount requirement, etc. Therefore, it is necessary to develop an efficient and accurate analysis method for t-PA drug heterogeneity. SUMMARY

[0005] Therefore, the purpose of the present application is to provide the application of capillary electrophoresis-mass spectrometry online combination and the detection method of t-PA drug heterogeneity. The detection method provided by the present application can realize efficient and accurate detection of heterogeneity in t-PA.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] The present application provides the application of capillary electrophoresis-mass spectrometry online combination in biological medicine detection or food detection.

[0008] The present application provides a detection method of t-PA drug heterogeneity, comprising the following steps:

[0009] Dissolve the t-PA drug to be tested in an aqueous ammonium salt solution to obtain a sample solution to be tested;

[0010] Perform capillary electrophoresis-mass spectrometry online combination detection on the sample solution to be tested to obtain a qualitative detection result of t-PA drug heterogeneity;

[0011] The online capillary electrophoresis-mass spectrometry (CES-MS) detection includes capillary electrophoresis separation and mass spectrometry detection. The online coupling of capillary electrophoresis separation and mass spectrometry detection is achieved by an electroosmotic flow-driven coaxial sheath electrospray ion source.

[0012] The conditions for capillary electrophoresis separation include: using capillary zone electrophoresis (CZE) mode; using neutral coated capillaries; using an electrophoresis background buffer solution including an acid-water mixture or an acid-acetonitrile-water mixture; and using pneumatic injection.

[0013] Preferably, the ammonium salt in the ammonium salt aqueous solution includes ammonium bicarbonate and / or ammonium acetate; the concentration of the ammonium salt aqueous solution is 1–50 mmol / L.

[0014] Preferably, the concentration of the tissue plasminogen activator drug to be tested in the sample solution is 0.5–2 mg / mL.

[0015] Preferably, the electrospray voltage of the electroosmotic flow driven coaxial sheath fluid electrospray ion source is 1.5-3kV, and the sheath fluid is a formic acid-isopropanol-water mixed solution; the volume fraction of formic acid in the formic acid-isopropanol-water mixed solution is 0.5-5%, and the volume fraction of isopropanol is 10-20%; the flow rate of the sheath fluid is <50nL / min.

[0016] Preferably, the capillary is made of elastic quartz, and the inner diameter of the neutral coated capillary is 50 μm, and the length is 75 to 100 cm.

[0017] Preferably, the volume fraction of acid in the electrophoresis background buffer is 1-35%, and the acid includes formic acid and / or acetic acid;

[0018] The volume fraction of acetonitrile in the acid-acetonitrile-water mixed solution is 1-35%.

[0019] Preferably, the conditions for capillary electrophoresis separation include: the sample injection method is pneumatic injection, the pneumatic injection pressure is 100 mbar, the sample injection time is 20 s, the electrophoresis background buffer injection time is 10 s; the separation voltage is 1-30000 V; and the separation time is 20 min.

[0020] Preferably, the conditions for the mass spectrometry detection include: the ion source is a CE-MS ion source; a first-level mass spectrometry full scan mode with selective ion monitoring is used; the scan range is 1000–4000 m / z; the ion mode is positive ion mode; the number of microscans is 3; the resolution is 17500; the automatic gain control is 3E6; the maximum injection time is 150 ms; and the mass spectrometry data type is Profile.

[0021] Preferably, the heterogeneous material includes one or more of the following: active isomers, degradation products, and modifiers.

[0022] Capillary electrophoresis possesses excellent separation capabilities, efficiently separating charged substances, including ions, polar compounds, and macromolecules. Based on the difference in migration speed under an electric field, capillary electrophoresis achieves high-resolution separation, enabling it to separate complex mixtures and provide accurate analytical results. Mass spectrometry, a highly sensitive detection technique, can detect trace amounts of analytes. This invention, by coupling capillary electrophoresis with mass spectrometry, allows substances separated by capillary electrophoresis to be directly introduced into the mass spectrometer for detection, achieving highly sensitive detection of the separated substances. Simultaneously, mass spectrometry provides precise mass and structural information of molecules, further enhancing the accuracy and reliability of the analysis. The coupling of capillary electrophoresis and mass spectrometry also provides complementary analytical information. Capillary electrophoresis provides separation and quantification information, while mass spectrometry provides mass and structural information. Combining these two techniques yields more comprehensive analytical results, aiding in the separation, identification, and characterization of components in complex samples. Furthermore, capillary electrophoresis coupled with mass spectrometry typically requires a small sample volume for analysis, which is highly advantageous when samples are scarce or limited in quantity. Capillary electrophoresis has a small inner diameter, requiring less sample, while mass spectrometry provides high sensitivity for even trace amounts of sample. Online capillary electrophoresis-mass spectrometry combines the advantages of online capillary electrophoresis and mass spectrometry, showing great promise for applications in biopharmaceutical and food testing.

[0023] This invention utilizes online capillary electrophoresis and mass spectrometry, separating t-PA drugs through capillary zone electrophoresis (CZE) mode to effectively control drug migration rate, separation efficiency, and separation selectivity, thus improving separation performance. Using a neutral-coated capillary avoids interaction between the drug and the capillary material, reducing sample loss and bias. Using an acid-water mixture or an acid-acetonitrile-water mixture as the electrophoresis background buffer (BGE) facilitates drug separation and analysis. Gas pressure injection ensures accurate entry of the sample solution and BGE into the neutral-coated capillary. Applying a separation voltage allows for effective CZE separation of heterogeneous components in t-PA drugs through capillary zone electrophoresis. The separated t-PA drugs are then introduced into the mass spectrometer via an electroosmotic flow-driven coaxial sheath electrospray ionization source for detection and analysis, obtaining more detailed drug information and heterogeneity analysis results, thereby achieving efficient, accurate, and highly sensitive detection of heterogeneous components in t-PA drugs. Moreover, the online capillary electrophoresis-mass spectrometry detection method used in this invention is time-efficient and requires a small amount of sample, providing a reliable means to evaluate the quality and stability of t-PA drugs. Attached Figure Description

[0024] Figure 1 This is a schematic diagram showing the positional relationship between the capillary electrophoresis apparatus and the mass spectrometer.

[0025] Figure 2 The TIC plot (top) and Base Peak plot (bottom) of the test sample solution in Comparative Example 1 are shown.

[0026] Figure 3 The average mass spectrum of the t-PA peak in the sample solution of Comparative Example 1;

[0027] Figure 4 The images show the TIC (top) and Base Peak (bottom) plots of the sample solution in Example 1.

[0028] Figure 5 The average mass spectrum of the t-PA peak in the sample solution to be tested in Example 1;

[0029] Figure 6 The images show the TIC (top) and Base Peak (bottom) plots of the sample solution in Example 2.

[0030] Figure 7 The average mass spectrum of the t-PA peak in the sample solution to be tested in Example 2;

[0031] Figure 8 The TIC plot (top) and Base Peak plot (bottom) of the test sample solution in Comparative Example 2 are shown.

[0032] Figure 9 The average mass spectrum of the t-PA peak in the sample solution of Comparative Example 2;

[0033] Figure 10 The images show the TIC (top) and Base Peak (bottom) plots of the sample solution in Example 3.

[0034] Figure 11 The average mass spectrum of the t-PA peak in the sample solution to be tested in Example 3;

[0035] Figure 12 The images show the TIC (top) and Base Peak (bottom) plots of the sample solution in Example 41.

[0036] Figure 13 The average mass spectrum of the t-PA peak in the sample solution to be tested in Example 4 is shown. Detailed Implementation

[0037] This invention provides the application of online capillary electrophoresis-mass spectrometry in biomedical or food detection. In this invention, the biomedical detection preferably includes the detection of one or more of cytokines, antibodies, antibody-drug conjugates, fusion proteins, hormones, enzymes, gene therapy drugs, cell therapy drugs, and therapeutic vaccines. In this invention, the food detection is preferably the detection of proteins in food.

[0038] This invention provides a method for detecting tissue-type plasminogen activator drug isoforms, comprising the following steps:

[0039] The tissue plasminogen activator drug to be tested was dissolved in an ammonium salt aqueous solution to obtain the test sample solution;

[0040] The sample solution to be tested was subjected to online capillary electrophoresis-mass spectrometry to obtain the qualitative detection results of tissue-type plasminogen activator drug isoforms.

[0041] The online capillary electrophoresis-mass spectrometry (CES-MS) detection includes capillary electrophoresis separation and mass spectrometry detection. The online coupling of capillary electrophoresis separation and mass spectrometry detection is achieved by an electroosmotic flow-driven coaxial sheath electrospray ion source.

[0042] The conditions for capillary electrophoresis separation include: using capillary zone electrophoresis (CZE) mode; using neutral coated capillaries; using an electrophoresis background buffer solution including an acid-water mixture or an acid-acetonitrile-water mixture; and using pneumatic injection.

[0043] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0044] This invention dissolves the tissue plasminogen activator drug to be tested in an ammonium salt aqueous solution to obtain the test sample solution.

[0045] In this invention, the tissue plasminogen activator drug to be tested is preferably t-PA stock solution.

[0046] In this invention, the ammonium salt in the ammonium salt aqueous solution preferably includes ammonium bicarbonate and / or ammonium acetate; the concentration of the ammonium salt aqueous solution is preferably 1 to 50 mmol / L (mM), more preferably 15 to 30 mM.

[0047] In this invention, the concentration of the tissue plasminogen activator drug to be tested in the sample solution is preferably 0.5 to 2 mg / mL, more preferably 1 to 1.5 mg / mL.

[0048] After obtaining the sample solution to be tested, the present invention performs capillary electrophoresis-mass spectrometry online detection on the sample solution to obtain the qualitative detection results of tissue plasminogen activator drug isoforms.

[0049] In this invention, the online capillary electrophoresis-mass spectrometry (CES-MS) coupling detection includes capillary electrophoresis separation and mass spectrometry detection. The online coupling of capillary electrophoresis separation and mass spectrometry detection is achieved through an electroosmotic flow-driven coaxial sheath electrospray ion source, more preferably the Yongdao Zhiyuan EMASS-II type CE-MS online coupling ion source. Based on an ultra-low flow rate coaxial sheath nano-current electrospray interface using an electroosmotic pump, online electrospraying is achieved through an external high voltage. In this invention, Figure 1 This diagram illustrates the positional relationship between the capillary electrophoresis apparatus and the mass spectrometer. The distance between the tip of the capillary nozzle and the mass spectrometer is preferably 1–18 mm, more preferably 2 mm; the distance between the tip of the capillary nozzle and the tip of the nozzle is preferably 0.1–2 mm, more preferably 0.65 mm. In this invention, the ion source electrospray voltage (external high voltage) of the electroosmotic flow-driven coaxial sheath flow electrospray ion source is preferably +1.5–+3.0 kV, more preferably 2.4 kV. In this invention, the sheath flow solution is preferably a formic acid-isopropanol-water mixed solution. The volume fraction of formic acid (FA) in the formic acid-isopropanol-water mixed solution is preferably 0.5–5%, more preferably 0.5–2%, and further preferably 0.5–1%; the volume fraction of isopropanol (IPA) in the formic acid-isopropanol-water mixed solution is preferably 10–20%, more preferably 10–15%, and further preferably 10–12%. In this invention, the flow rate of the sheath fluid is preferably <50 nL / min.

[0050] In this invention, the conditions for capillary electrophoresis separation include: using capillary zone electrophoresis (CZE) mode; the capillary is a neutral-coated capillary, preferably made of elastic quartz, with an inner diameter of 50 μm and a length of 75–100 cm; the electrophoresis background buffer includes an acid-water mixture or an acid-acetonitrile-water mixture; the volume fraction of acid in the electrophoresis background buffer is preferably 1–35%, more preferably 2–30%, and even more preferably 15–30%; the acid preferably includes formic acid and / or acetic acid; the volume fraction of acetonitrile in the acid-acetonitrile-water mixture is preferably 1–35%, more preferably 5–30%, and even more preferably 15–20%. In this invention, the electrophoresis background buffer is preferably a 15wt% acetic acid aqueous solution, a 30wt% acetic acid aqueous solution, or a 2wt% formic acid-20wt% acetonitrile aqueous solution; the injection method is pneumatic injection, the pneumatic injection pressure is preferably 100 mbar, the injection time of the sample solution is preferably 20 s, and the injection time of the electrophoresis background buffer is preferably 10 s; the separation voltage is preferably 1-30000 V, more preferably 10000-30000 V, and even more preferably 20000-30000 V; the separation time is preferably 20 min.

[0051] Using capillaries without a neutral coating may result in the following effects and interactions: (1) Non-specific adsorption: The inner wall of a capillary usually has a certain affinity, and proteins may interact with the inner wall under non-specific adsorption conditions. This may lead to protein adsorption, degradation, or aggregation, thereby affecting the accuracy and reproducibility of the analytical results; (2) Chromatographic peak deformation: Proteins may undergo non-specific adsorption or interaction under non-ideal surface conditions, leading to chromatographic peak deformation. This may manifest as peak diffusion, splitting, drastic forward or backward shift, making the analytical results difficult to interpret and resolve; (3) Decreased separation efficiency: The affinity of the inner wall of the capillary may lead to poor protein separation. In the absence of a neutral coating, interactions between proteins may affect their migration speed and separation performance, leading to fuzzy or inaccurate analytical results; (4) Reduced sensitivity: Non-specific adsorption and interactions may cause protein loss or degradation in the capillary, thereby reducing the detection sensitivity of the target protein, which may require higher sample concentrations or more sensitive detection methods to obtain reliable results; (5) Reproducibility issues: The presence of non-specific adsorption and interactions may lead to reproducibility issues in analytical results, resulting in large variability in repeated analysis results of the same sample. This is a serious problem for quantitative and comparative analysis. The present invention uses a neutral-coated capillary, which avoids the interaction between the drug and the capillary material, reduces sample loss and bias, significantly improves the separation effect of capillary electrophoresis on the interaction between t-PA drug and capillary material, reduces sample loss and bias, and improves the separation effect of heteroplasms in t-PA drug.

[0052] In capillary zone electrophoresis (CZE), the background buffer plays several important roles, including: (1) providing conductivity: BGE usually contains ionic compounds (such as salts), which can increase the conductivity of the solution, ensuring the formation and conduction of the electric field. High conductivity BGE helps maintain a stable electric field strength and separation effect; (2) adjusting sample pH: BGE can be used to adjust the sample pH in the capillary. In CZE, the sample pH is very important for the charge and migration behavior of substances. By selecting appropriate buffer components and pH, the charge of the sample can be adjusted, thereby affecting the substances' electrostatic properties. (3) Controlling current and potential: The ion concentration and type of BGE can affect the distribution of current and potential. Appropriate selection of the ion concentration and type of BGE can control the uniformity and stability of the electric field, ensuring accurate separation and quantitative analysis; (4) Affecting separation selectivity: The composition of BGE can affect the selectivity of separated substances. By adjusting the ion type and concentration in BGE, the interaction between ions and charged compounds in the capillary can be changed, thereby achieving selective separation of substances; (5) Controlling electroosmotic flow and solvent accumulation: The composition of BGE can affect the intensity and direction of electroosmotic flow (EOF). EOF is the flow of solution in the charged capillary under the action of an electric field, which is crucial for sample transport and separation. By selecting appropriate BGE composition, the intensity and direction of EOF can be controlled to optimize the separation effect. This invention achieves control of its pH value and ionic strength by controlling the composition and concentration of the electrophoresis background buffer, further improving the separation effect of capillary electrophoresis on heteroplasms in t-PA drugs.

[0053] This invention uses pneumatic injection and precise control of injection time, which can further ensure that the sample solution to be tested and the electrophoresis background buffer solution accurately enter the capillary.

[0054] This invention, by controlling the separation voltage, can provide sufficient electric field strength to further achieve the separation and zone electrophoresis of heteroplasms in effective t-PA drugs.

[0055] In this invention, the preferred conditions for mass spectrometry detection include: the ion source is a CE-MS ion source; a first-level mass spectrometry full scan mode with selective ion monitoring is used; the scan range is 1000–4000 m / z; the ion mode is positive ion mode; the number of microscopic scans is 3; the resolution is 17500; the automatic gain control is 3E6; the maximum injection time is 150 ms; and the mass spectrometry data type is Profile.

[0056] In capillary zone electrophoresis, the migration velocity depends on factors such as the charge, size, and shape of the charged substance. Under the influence of an electric field, charged substances pass through the capillary at different speeds. Substances with faster speeds reach the detector earlier, while substances with slower speeds reach the detector later. This invention further controls the migration speed, separation efficiency, and separation selectivity of capillary zone electrophoresis by controlling various conditions of online capillary electrophoresis-mass spectrometry, thereby further improving the separation and detection effect.

[0057] In this invention, the heteroform preferably includes one or more of the following: active isomers, degradation products, and modifiers; the active isomer preferably includes one or more of the following: splicing isomers, mutant isomers, post-translational modification isomers, protein folding isomers, and protein charge isomers; the degradation product preferably includes one or more of the following: amino acid degradation products, peptide degradation products, oxidation products, and aggregation products; the modifier preferably includes one or more of the following: glycosylation products, sialylation products, phosphorylation modification products, acetylation modification products, methylation modification products, and sulfation modification products.

[0058] To further illustrate the present invention, the detection method of tissue plasminogen activator drug isoforms is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0059] The following examples and comparative examples illustrate the conditions for online capillary electrophoresis-mass spectrometry detection:

[0060] A neutral-coated capillary was connected to a capillary electrophoresis system, and the capillary electrophoresis instrument was connected to the mass spectrometer online using the Yongdao Zhiyuan EMASS-II CE-MS online coupling ion source.

[0061] Capillary electrophoresis apparatus model: ECE-001;

[0062] Mass spectrometer (MS) model: QE PLUS;

[0063] Distance from the nozzle tip to the mass spectrometer: 2.500 mm;

[0064] Distance between capillary tip and nozzle tip: 0.65mm

[0065] Ion source electrospray voltage: +2.4kV;

[0066] Sheath fluid: 0.5 v / v % FA - 10 v / v % IPA - water;

[0067] Flow rate of sheath fluid: <50 nL / min;

[0068] CE voltage: +30kV;

[0069] Sample injection: pneumatic injection, pressure is 100 mbar, sample injection time is 20 s, BGE injection time is 10 s;

[0070] Level 1 mass spectrometry full scan mode - selective ion monitoring;

[0071] Ion mode: Positive ion mode;

[0072] Scan speed: 3 spectrum / s;

[0073] Resolution: 17500;

[0074] Automatic gain control: 3E6;

[0075] Maximum injection time: 150ms;

[0076] Scan range: 1000~4000m / z

[0077] Mass spectrometry data type: Profile.

[0078] The water used in the examples and comparative examples was ultrapure water.

[0079] Comparative Example 1

[0080] The t-PA stock solution was diluted with 30 mM ammonium bicarbonate aqueous solution to obtain a test sample solution with a t-PA concentration of 1 mg / mL. The test sample solution was then subjected to online capillary electrophoresis-mass spectrometry to obtain qualitative detection results of the t-PA isoform. The capillary was a quartz capillary (50 μm * 75 cm, BFS) without a neutral coating, and BGE was 30 v / v% HAc aqueous solution.

[0081] Figure 2 The images show the TIC plot (top) and Base Peak plot (bottom) of the sample solution to be tested. Figure 3 The average mass spectrum of the t-PA peak is obtained from... Figures 2-3 It can be seen that using an uncoated capillary to separate t-PA in CZE mode has virtually no separation effect.

[0082] Example 1

[0083] The t-PA stock solution was diluted with 15 mM ammonium bicarbonate aqueous solution to obtain a test sample solution with a t-PA concentration of 1 mg / mL. The test sample solution was then subjected to online capillary electrophoresis-mass spectrometry (CES-MS) to obtain qualitative detection results of the t-PA isoform. The capillary was a neutral-coated quartz capillary (50 μm * 100 cm, purchased from CMS Scientific, model PS2; no detectable electroosmotic flow (EOF) was observed using this capillary); BGE was 30 v / v% HAc aqueous solution.

[0084] Figure 4 The images show the TIC plot (top) and Base Peak plot (bottom) of the sample solution to be tested. Figure 5 The average mass spectrum of the t-PA peak is obtained from... Figures 4-5 It can be seen that using a 30 v / v% HAc aqueous solution as BGE, with the sample dissolved in a 15 mM ammonium bicarbonate aqueous solution, and performing capillary zone electrophoresis with a neutral-coated capillary, the separation effect of t-PA is good.

[0085] Example 2

[0086] The heteroplasm in t-PA was detected according to the method of Example 1. The difference from Example 1 is that the diluent for the t-PA stock solution was a 15 mM ammonium bicarbonate aqueous solution; BGE: 15 v / v% HAc aqueous solution.

[0087] Figure 6 The images show the TIC plot (top) and Base Peak plot (bottom) of the sample solution to be tested. Figure 7 The average mass spectrum of the t-PA peak is obtained from... Figures 6-7 It can be seen that using 15 v / v% HAc aqueous solution as BGE, and dissolving the sample in 15 mM ammonium bicarbonate aqueous solution, capillary zone electrophoresis with neutral coated capillary tubes has a good separation effect on t-PA.

[0088] Comparative Example 2

[0089] The heteroplasm in t-PA was detected according to the method of Example 1. The difference from Example 1 is that the diluent for the t-PA stock solution was 15 mM ammonium bicarbonate aqueous solution; BGE: 15 v / v% FA aqueous solution.

[0090] Figure 8 The images show the TIC plot (top) and Base Peak plot (bottom) of the sample solution to be tested. Figure 9 The average mass spectrum of the t-PA peak is obtained from... Figures 8-9 It can be seen that the separation effect of using formic acid aqueous solution as BGE is worse than that of using formic acid-acetonitrile-aqueous solution as BGE.

[0091] Example 3

[0092] The heteroplasm in t-PA was detected according to the method of Example 1. The difference from Example 1 is that the diluent for the t-PA stock solution was 15 mM ammonium acetate aqueous solution; BGE: 30 v / v% HAc aqueous solution.

[0093] Figure 10 The images show the TIC plot (top) and Base Peak plot (bottom) of the sample solution to be tested. Figure 11 The average mass spectrum of the t-PA peak is obtained from... Figures 10-11It can be seen that using a 30 v / v% HAc aqueous solution as BGE, and dissolving the sample in a 15 mM ammonium acetate aqueous solution, capillary zone electrophoresis with a neutral-coated capillary showed good separation effect for t-PA.

[0094] Example 4

[0095] The heteroplasm in t-PA was detected according to the method of Example 1. The difference from Example 1 is that the diluent for the t-PA stock solution was 15mM ammonium bicarbonate aqueous solution; BGE: 2v / v% FA-20v / v% acetonitrile-aqueous solution.

[0096] Figure 12 The images show the TIC plot (top) and Base Peak plot (bottom) of the sample solution to be tested. Figure 13 The average mass spectrum of the t-PA peak is obtained from... Figures 12-13 It can be seen that using 2v / v% FA-20v / v% acetonitrile-water solution as BGE, and dissolving the sample in 15mM ammonium bicarbonate aqueous solution, capillary zone electrophoresis with neutral coated capillary showed good separation effect for t-PA.

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting tissue-type plasminogen activator drug isoforms, comprising the following steps: The tissue plasminogen activator drug to be tested is dissolved in an ammonium salt aqueous solution to obtain the test sample solution; the ammonium salt in the ammonium salt aqueous solution includes ammonium bicarbonate and / or ammonium acetate; the concentration of the ammonium salt aqueous solution is 15~30 mmol / L; The sample solution to be tested was subjected to online capillary electrophoresis-mass spectrometry to obtain the qualitative detection results of tissue-type plasminogen activator drug isoforms. The online capillary electrophoresis-mass spectrometry (CES-MS) detection includes capillary electrophoresis separation and mass spectrometry detection. The online coupling of capillary electrophoresis separation and mass spectrometry detection is achieved by an electroosmotic flow-driven coaxial sheath electrospray ion source. The conditions for capillary electrophoresis separation include: using capillary zone electrophoresis (CZE) mode; using neutral-coated capillaries; using an acid-acetonitrile-water mixture as the electrophoresis background buffer, wherein the volume fraction of acid in the background buffer is 1-35% and the volume fraction of acetonitrile is 15-20%, and the acid includes formic acid and / or acetic acid; and using pneumatic injection.

2. The detection method according to claim 1, characterized in that, The concentration of the tissue plasminogen activator drug to be tested in the sample solution is 0.5~2 mg / mL.

3. The detection method according to claim 1, characterized in that, The electrospray voltage of the electroosmotic flow-driven coaxial sheath fluid electrospray ion source is 1.5~3kV, and the sheath fluid is a formic acid-isopropanol-water mixed solution; the volume fraction of formic acid in the formic acid-isopropanol-water mixed solution is 0.5~5%, and the volume fraction of isopropanol is 10~20%; the flow rate of the sheath fluid is <50nL / min.

4. The detection method according to claim 1, characterized in that, The capillary is made of elastic quartz, and the inner diameter of the neutral-coated capillary is 50μm, with a length of 75~100cm.

5. The detection method according to claim 1, characterized in that, The conditions for capillary electrophoresis separation include: a gas pressure of 100 mbar for gas injection, an injection time of 20 s for the sample solution to be tested, an injection time of 10 s for the electrophoresis background buffer, a separation voltage of 1~30000V, and a separation time of 20 min.

6. The detection method according to claim 1, characterized in that, The conditions for mass spectrometry detection include: the ion source is a CE-MS ion source; a first-level mass spectrometry full scan mode with selective ion monitoring is used; the scan range is 1000~4000 m / z; the ion mode is positive ion mode; the number of microscans is 3; the resolution is 17500; the maximum injection time is 150 ms; and the mass spectrometry data type is Profile.

7. The detection method according to claim 1, characterized in that, The heterogenes include one or more of the following: active isomers, degradation products, and modifiers.

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