Method and kit for screening, detecting and removing anaphylactoid in safflower injection

Abstract

According to the present invention, the immobilized human serum albumin HSA is adopted as the affinity screening medium, and the UHPLC-MS / MS analysis and molecular docking verification method is combined so as to rapidly identify the chemical components having the human serum albumin binding activity and the potential sensitization in the safflower injection. The screening and verification technology disclosed by the invention discovers and confirms that 6-hydroxykaempferol-3, 6, 7-tri-O-glucoside in the safflower injection is a main suspected anaphylactoid substance for the first time; a corresponding detection method, a removal process and a standardized detection kit are further established, and a system closed loop from sensitizing substance screening and quantitative detection to production process control is realized. The method not only overcomes the limitation that allergy risk components cannot be accurately identified and quantified in the prior art, but also provides reliable technical support for prevention and control of anaphylactoid reactions of traditional Chinese medicine injections.

Description

Technical Field

[0001] This invention relates to the field of safflower injections, specifically to a method and kit for screening, detecting, and removing allergenic substances in safflower injections. Background Technology

[0002] Traditional Chinese medicine (TCM) injections are sterile injectable preparations made primarily from extracts of Chinese medicinal herbs through processes such as extraction, separation, and purification. They are characterized by rapid onset of action and complete absorption, and are widely used in the clinical treatment of cardiovascular and cerebrovascular diseases, immune regulation, and inflammatory diseases. However, compared to oral TCM preparations, TCM injections pose higher safety risks, with adverse reactions, especially pseudo-allergic reactions, long troubling clinical application and drug regulation. Blood-activating and stasis-removing TCM injections, represented by safflower injections, have pharmacological effects such as improving microcirculation, anti-inflammation, and antithrombosis, and are widely used clinically. However, their composition is complex and diverse, containing not only the main active ingredient, safflower yellow pigment, but also various flavonoids, glycosides, and other secondary metabolites. Existing research indicates that MRGPRX2, a G protein-coupled receptor expressed on the surface of mast cells and basophils, plays a crucial role in non-IgE-mediated allergic reactions. Molecular docking studies between small molecule compounds and MRGPRX2 provide a novel perspective and technical approach for screening pseudo-allergens. Because the mechanisms of allergic reactions are complex and diverse, the RhoA signaling pathway may also be involved in key processes such as mast cell degranulation and the release of inflammatory mediators.

[0003] Currently, research on allergy risk substances in traditional Chinese medicine injections is mostly focused on characterization evaluation using in vitro cell models or animal models, such as assessing the allergic reaction potential of test samples through RBL-2H3, P815 cell degranulation experiments or mouse hind paw extravasation experiments. Although these methods can reflect the overall sensitization risk, they still have the following shortcomings: (1) They lack quantitative chemical recognition mechanisms and cannot accurately pinpoint the specific chemical components that cause allergy-like reactions; (2) Cellular or animal experiments are costly and time-consuming, and the results are greatly affected by the complex matrix of the sample, resulting in poor reproducibility; (3) They lack systematic screening and verification technology pathways for the unique complex matrix of traditional Chinese medicine injections, and existing methods cannot directly correspond in vitro screening results to specific compounds; (4) Even if suspected risk components are found, current process control methods are mostly empirical adjustments, lacking quantifiable and verifiable removal control standards, which are difficult to support the requirements of drug quality consistency evaluation or registration application.

[0004] Previous research on safflower injection preparations has largely focused on the main active ingredients such as safflower yellow pigments A and B, while insufficient research has been conducted on trace impurities or metabolites that may cause allergic reactions. Although some literature has used techniques such as HPLC-DAD and LC-MS to conduct preliminary analyses of the preparation components, a screening system that can directly reflect the relationship between "protein binding potential, molecular structural characteristics, and biological sensitization" has not yet been established, and no hapten components have been identified as allergy risk markers. In addition, traditional detection methods are difficult to achieve high-sensitivity quantification in complex traditional Chinese medicine matrices, resulting in the detection and quality control of allergens still lagging behind clinical safety requirements.

[0005] Therefore, there is an urgent need for a systematic technical solution that can combine protein affinity screening, molecular docking analysis, cell biology verification and quantitative detection to achieve the scientific identification and effective removal of various allergenic substances in safflower injection. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for screening allergens in safflower injection preparations using immobilized human serum albumin as an affinity screening medium, combined with UHPLC-MS / MS analysis and molecular docking verification. This invention also includes a method for detecting and removing allergens in safflower injection preparations based on this method, as well as a detection kit.

[0007] To solve the above-mentioned technical problems, the technical method adopted by the present invention is as follows: The present invention discloses a screening method for allergenic substances in safflower injection, comprising the following steps: Step S1. Extract the analyte from the safflower injection sample to obtain a mixture of components; Step S2. The mixture of components is captured and eluted by affinity enrichment using an immobilized human serum albumin (HSA) affinity solid-phase material; Step S3. After pretreatment, the eluent obtained in step S2 is separated and quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) in multiple reaction monitoring (MRM) mode. The quantitative analysis uses stable isotopes or structurally similar internal standards for correction and calculates the content of the target component in the sample based on the calibration curve. Step S4. Perform molecular docking simulations on the candidate components obtained from the separation and identification in step S3 against the MRGPRX2 and RhoA targets, and make a comprehensive judgment in combination with predetermined positive and negative controls to screen allergy candidates. Step S5. Perform biological validation of the allergy candidate in a cell or animal model, the biological validation including measuring histamine release or degranulation indicators to confirm its allergy-like potential.

[0008] Furthermore, the immobilized HSA affinity solid material in step S2 is selected from HSA-coated magnetic microspheres, HSA-modified chromatography columns, or HSA-modified microplates.

[0009] Furthermore, the necessary pretreatment in step S3 includes solvent exchange, concentration, or solid-phase extraction (SPE) of the eluent to reduce matrix interference and improve method sensitivity. The UHPLC-MS / MS analysis establishes at least a 6-point calibration curve with internal standard-corrected peak area ratios and obtains a linear regression coefficient R² ≥ 0.99.

[0010] Furthermore, in step S4, a dual scoring strategy is used for docking determination; the dual scoring strategy includes one aspect, comparing the docking score_MRGPRX2 for MRGPRX2 with the predetermined positive control, and the other aspect, determining the ratio of score_MRGPRX2 to the docking score_RhoA for RhoA. The allergy candidate will be listed as a priority biological validation object when either of the following conditions is met: score_MRGPRX2 ≥ 0.9 × score_positive control or score_MRGPRX2 divided by score_RhoA ≥ 1.2.

[0011] This invention also provides a method for detecting allergen-like substances in safflower injection. Based on the screening method for allergen-like substances in safflower injection described above, the sample is first subjected to HSA affinity enrichment treatment, and then separated and quantitatively analyzed using UHPLC-MS / MS. The limit of quantitation (LOQ) determined through method validation is used as the quality control action limit. When the content of the target component in the sample is greater than the LOQ, it is determined that the allergen-like substance exceeds the limit.

[0012] Furthermore, the methodologically validated lower limit of quantitation is less than or equal to 0.5 micrograms per milliliter.

[0013] This invention also discloses a method for removing allergenic substances from safflower injection, including raw material extraction, crude separation, purification and formulation preparation steps; the purification step includes at least one removal process unit for reducing or removing the content of the target component; The removal process unit includes macroporous adsorption resin adsorption and desorption, activated carbon adsorption, reverse phase chromatography, ion exchange chromatography, and ultrafiltration or nanofiltration membrane separation. In the removal process unit, the target component is monitored online or offline using the detection method for allergen-like substances in safflower injection as described in claim 5 to verify the removal effect.

[0014] Furthermore, the removal efficiency of the removal process unit for the target component is ≥90%, or the content of the target component in the final formulation after treatment by the removal process unit is less than or equal to the lower limit of quantitation (LOQ) in the analytical method of claim 5.

[0015] This invention also discloses a detection kit for allergen-like substances in safflower injection, used to detect one or more target components in safflower injection samples. The kit includes an affinity capture component for immobilized human serum albumin (HSA), buffer and consumables for elution and pretreatment, internal standard and reference material for UHPLC-MS / MS quantitative analysis, and an instruction manual. The kit enables the completion of all chemical steps required for sample pretreatment, enrichment, and quantitative detection in the detection method for allergen-like substances in safflower injection as described in claim 5, and obtains quantitative results under conditions equipped with a UHPLC-MS / MS analysis system.

[0016] Furthermore, the target component is 6-hydroxykaempferol-3,6,7-tri-O-glucoside.

[0017] Beneficial effects: 1. This invention establishes a systematic method combining affinity screening and molecular-level verification based on immobilized human serum albumin (HSA), achieving efficient identification and confirmation of allergen-like substances in safflower injection. Specifically, firstly, HSA-affinity solid-phase materials (such as HSA-coated magnetic microspheres or HSA-modified chromatography columns) are used to selectively enrich the analytes in the sample, thereby capturing compounds with strong binding ability to plasma proteins and high potential sensitization risk; subsequently, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) in multiple reaction monitoring (MRM) mode is used for high-resolution separation and quantification, with stable isotopes or structurally similar internal standards used for quantitative correction to ensure the accuracy and repeatability of the detection results. At the molecular level, the screened candidate components are subjected to molecular docking simulations with MRGPRX2 and RhoA targets, respectively. A dual-target comparison strategy is used to achieve highly reliable screening of allergy candidates. Finally, the histamine release or departicle degree of the screened substances is verified at the cellular or animal level. This screening system integrates a three-level screening process of "protein binding enrichment—molecular docking prediction—biological verification," enabling rapid identification of genuine allergy-like active components based on clear scientific evidence. This significantly improves the screening accuracy and verification efficiency of suspected allergy-like substances in safflower injection.

[0018] 2. By combining HSA affinity enrichment technology with UHPLC-MS / MS quantitative analysis, a standardized detection method and limit of quantification (LOQ) standard for allergens in safflower injection were established, thereby enabling quantifiable monitoring of the production process and finished product quality. When the detection results exceed the set LOQ limit, targeted process adjustments can be made based on the removal process units proposed in this invention (including macroporous resin adsorption, activated carbon adsorption, reversed-phase or ion exchange chromatography, membrane separation, etc.) to effectively reduce or remove allergenic target components.

[0019] 3. This invention proposes a detection kit specifically for allergen-like substances in safflower injection. It integrates an immobilized HSA affinity capture component, elution and pretreatment buffers, internal standards and reference substances required for UHPLC-MS / MS quantitative analysis, and detailed operating instructions. It can be directly used for standardized detection of target components in safflower injection samples. Using this kit, researchers can complete all chemical steps of sample enrichment, pretreatment, and quantitative detection under conditions equipped with a UHPLC-MS / MS system, significantly reducing experimental errors and operational complexity, and ensuring the comparability and repeatability of test results between different laboratories. Attached Figure Description

[0020] Figure 1 This is a structural diagram of 6-hydroxykaempferol-3,6,7-tri-O-glucoside in this invention. Detailed Implementation

[0021] Example 1 This embodiment uses immobilized human serum albumin (HSA) affinity enrichment combined with UHPLC-MS / MS analysis and molecular docking technology to screen and verify 6-hydroxykaempferol-3,6,7-tri-O-glucoside, a substance with allergy risk in safflower injection. The molecular formula of 6-hydroxykaempferol-3,6,7-tri-O-glucoside is C2. 33 H 40 O 22 The English name is 6-Hydroxykaempferol-3,6,7-triglucoside, CAS number: 145134-62-9, and the structural formula is as follows. Figure 1 As shown.

[0022] Specifically, the steps include the following: Step 1. Take 1.0 mL of safflower injection sample, add 3 mL of ice-cold methanol to precipitate the protein, shake for 5 minutes, centrifuge at 10000g for 10 minutes at 4℃, and collect the supernatant. Dry the supernatant with nitrogen and redissolve it in 1 mL of 10 mM ammonium carbamate buffer (pH 7.4). The resulting mixture is then subjected to HSA enrichment.

[0023] Step 2. Mix the component mixture with HSA-coated magnetic microspheres, incubate on a shaker at room temperature for 30 minutes, perform magnetic separation and discard the supernatant. Then wash 3 times (buffered) and finally elute the conjugate with 50% methanol and 0.1% formic acid. Collect the eluent and concentrate it to a suitable chromatographic injection volume, and add internal standard.

[0024] Step S3. After pretreatment, the eluent obtained in step S2 is separated and quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) in multiple reaction monitoring (MRM) mode. The quantitative analysis uses stable isotopes or structurally similar internal standards for correction and calculates the content of the target component in the sample based on the calibration curve.

[0025] Step S4. Computational chemistry docking is performed on the candidates identified and quantified in Step S3, targeting MRGPRX2 and RhoA. A dual scoring strategy is used, and the results are compared with positive controls C48 / 80 and GSP as benchmarks to screen for potential allergy candidates.

[0026] Step S5. In vitro validation was performed using the P815 mouse mast cell model. The experimental steps included: adding the candidate, positive control, and negative control to P815 cells that had been inoculated overnight, culturing for a period of time, collecting the supernatant, and detecting histamine and β-aminohexosidase release.

[0027] Based on the experimental results, the C48 / 80 positive control group and the test substance group showed significantly increased release of histamine and β-aminohexosidase compared to the control group. These results support the classification of 6-hydroxykaempferol-3,6,7-tri-O-glucoside as an allergen-like substance.

[0028] This embodiment successfully screened and verified that 6-hydroxykaempferol-3,6,7-tri-O-glucoside is an allergen-like substance in safflower injection preparations, and biological experiments (P815 cell and mouse tail / hind paw test) showed that it has the potential to induce histamine release and changes in cell permeability.

[0029] Example 2 This embodiment establishes and validates a quantitative detection method for allergen-like substances in safflower injection, determines the methodological performance parameters and the limit of quantitation (LOQ = 0.05 μg / mL), and forms an executable quality control standard.

[0030] Based on the above implementation 1, this embodiment is further optimized and the specific steps are further refined.

[0031] 1.1 Experimental instruments: TGL-168 centrifuge (Shanghai Anting Scientific Instrument Factory); ELISA reader (ThermoFisher Scientific, USA); vortex mixer (XW-80A, Shanghai Qingpu Huxi Instrument Factory); 1.2 Reagents included: 6-hydroxykaempferol-3,6,7-O-triglucoside, Chengdu Mansite Biotechnology Co., Ltd.; Evans blue dye, Sinopharm Chemical Reagent Co., Ltd.; mouse HIS ELISA kit, Shanghai Enzyme-Linked Biotechnology Co., Ltd.; mouse β-aminohexosidase ELISA kit, Shanghai Enzyme-Linked Biotechnology Co., Ltd.; P815 cell culture medium, selected from Wuhan Pronosei Biotechnology Co., Ltd.; CCK-8 kit, MedChemExpress Biotechnology Co., Ltd., USA.

[0032] 1.3 Experimental Methods P815 cells were cultured in RPMI 1640 medium containing 1% penicillin-streptomycin and 10% FBS at 37 ℃ in a 5% CO2 incubator. Cells in the logarithmic growth phase were harvested and cultured at a rate of 4 × 10⁻⁶ cells / cells. 4 Cells were seeded per well into 96-well plates and incubated overnight in an incubator. A zero-entry group was also set up without seeding cells. P815 cells were co-cultured with the prepared test solution (positive control C48 / 80 and 6-hydroxykaempferol-3,6,7-tri-O-glucoside) and RPMI 1640 medium containing 10% FBS. After centrifugation, the supernatant was collected, and changes in histamine and β-aminohexosidase levels were measured using enzyme-linked immunosorbent assay (ELISA). 1.4 Experimental Results Compared with the control group, the C48 / 80 group showed a significant increase in histamine release from cells. p <0.01); Increased histamine release from cells in the test group ( p <0.01); Compared with the control group, the β-aminohexosidase content in both the C48 / 80 group and the test substance group was significantly increased. p <0.01).

[0033] 2.1 Experimental Instruments and Reagents Balance (Quintix 125D-1CN, Sartorius Scientific Instruments Co., Ltd.); Cell counter (ThermoFisher, Countess II); Microplate reader (Thermo Fisher Scientific, USA); 6-hydroxykaempferol-3,6,7-O-triglucoside (Chengdu Mansite Biotechnology Co., Ltd.); Evans blue dye (Sinopharm Chemical Reagent Co., Ltd.) 2.2 Laboratory Animals Fifty SPF-grade, female BALB / c mice, 7-8 weeks old and weighing (20±5) g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0034] 2.3 Experimental Methods Mice were injected via tail vein with 0.4% Evans blue solution (10 ml / kg), and 10 ml / kg was added 5 min later. m L of physiological saline was injected into the left paw as a control, and 10 L of saline was injected into the paw. m L solutions and test substance solutions were injected into the right claw. Claw tissue was collected 15 min later, dried at 37°C for 24 h, minced, and placed in an 800 mL container. m Incubate L of acetone-physiological saline (7:3) at 65℃ for 12 h, centrifuge and collect the supernatant, then take 200 ml of the supernatant. m L was transferred to a 96-well plate, and the absorbance was measured at 620 nm.

[0035] 2.4 Experimental Results The measurement results are as follows: Table 1: P815 cell model

[0036] The target compound significantly improved both indicators (p<0.01), indicating that it has allergy-like potential.

[0037] Table 2: Extravasation test in mouse hind paw

[0038] The target compound group was significantly higher than the negative control (p<0.01) and lower than the positive control.

[0039] Based on Tables 1 and 2 above, it can be seen that... (1) C48 / 80 was injected into the right paw of the mouse to induce a local allergic reaction. Evans blue exudation increased, and the right paw was darker blue than the left paw which was injected with saline alone. The test system was reliable, and the right paw of the test group was also darker.

[0040] (2) The OD value of the hind paw supernatant was significantly higher in the C48 / 80 group compared with the untreated side. p <0.01).

[0041] The drug administration side of the test substance group was significantly higher than that of the control side. p <0.01).

[0042] Through the above-mentioned in vitro cell experiments and mouse hind paw extravasation tests, the results showed that 6-hydroxykaempferol-3,6,7-tri-O-glucoside may induce mast cell degranulation and changes in mouse hind paw cell permeability, and has the potential to induce allergic reactions. This provides a scientific basis for the identification of allergic components and the control of the content of key substances in safflower injection preparations, and helps to reduce the risk of allergic reactions in its clinical use.

[0043] Example 3 This embodiment aims to validate the method for detecting allergenic substances in safflower injection. All experiments were based on the internal standard method and performed in a real safflower injection matrix to evaluate the effect of matrix interference. Six concentration points were set for the standard solution: 0.05, 0.1, 0.2, 0.5, 1, 5, and 10 μg / mL. The internal standard concentration was 0.5 μg / mL. Blank matrix samples were prepared for recovery and matrix effect testing.

[0044] The LOQ determination procedure is as follows: continuously dilute the low concentration standard and measure the signal-to-noise ratio (S / N), with S / N ≥ 10 as the LOQ criterion; repeat the injection 6 times at the LOQ concentration, requiring RSD ≤ 20% and the recovery rate to be maintained between 80% and 120%; LOD is determined based on S / N ≥ 3.

[0045] The results based on the above procedure are as follows: LOD is 0.02 μg / mL, LOQ is 0.5 μg / mL, linear range is 0.05–50 μg / mL, regression coefficient R² ≥ 0.995, intra-day RSD ≤ 5%, average recovery rate is between 85–105%, and matrix effect is within ±20%. These parameters are sufficient to meet the stability and repeatability requirements of the analytical method.

[0046] During the testing process, the LOQ is set as the quality control action limit, forming the following decision mechanism: when the test value is ≤ LOQ, the sample is deemed qualified; when the test value is > LOQ and ≤ 2 × LOQ, the test should be repeated and a corrective action should be implemented; when the test value is ≥ 2 × LOQ or still exceeds the limit after correction, the sample is deemed unqualified and corrective and preventive actions are initiated.

[0047] Example 4 This embodiment optimizes the pretreatment process of safflower injection samples by using SPE solid-phase extraction, solvent exchange and concentration to improve detection sensitivity and result accuracy.

[0048] Solid phase extraction (SPE) was used for sample pretreatment, and C18 or HLB type SPE columns were selected.

[0049] The procedure is as follows: First, activate the column sequentially with 3 mL of methanol and 3 mL of deionized water; then, slowly load the column after diluting the safflower injection eluent; wash with 3 mL of 5% methanol-water solution to remove impurities; finally, elute with 3 mL of 80–100% methanol or acetonitrile to collect the target fraction. After the eluent is nearly dry under nitrogen, redissolve it with the starting mobile phase (100 μL) and add the internal standard. Recovery and matrix factor were calculated using blank matrix spiked, pure solvent spiked, and matrix-spiked control samples. The results showed that the recovery rate was typically between 85–105%, and the matrix effect was within ±15%. When the recovery rate is below 70% or the matrix effect exceeds ±30%, the elution solvent strength should be optimized or the SPE type adjusted to ensure the reliability of sample processing and analytical sensitivity.

[0050] Example 4 In this embodiment, AutoDock Vina software was used to perform molecular docking of the main components of safflower injection with MRGPRX2 and RhoA receptors, and a quantitative scoring model was established to screen potential allergy-like components.

[0051] Molecular docking was simulated using AutoDock Vina software. The receptor proteins MRGPRX2 and RhoA were used as positive and control targets, respectively. If publicly available PDB structures were available, they were downloaded directly and their energy minimized; otherwise, homology modeling was employed. Proteins were imported into the software after dehydration, hydrogenation, and minimization.

[0052] The docking cassette was centered based on known active sites, and its size covered the binding pocket. Each compound underwent 10 repeated docking trials, and the lowest binding energy was used as the final result. Docking scores were normalized, with the score of the positive control C48 / 80 on MRGPRX2 recorded as 100, and the others converted proportionally; the same method was used for RhoA. Positive controls were C48 / 80 and GSP, and negative controls were structurally similar inactive compounds.

[0053] Table 3

[0054] The results are as follows: As shown in Table 1, the binding energy of C48 / 80 on MRGPRX2 is -10.0 kcal / mol and on RhoA is -6.5 kcal / mol; the binding energy of the target compound 6-hydroxykaempferol-3,6,7-tri-O-glucoside on MRGPRX2 is -9.4 kcal / mol and on RhoA is -6.0 kcal / mol, with normalized scores of 94 and 58 respectively, and a ratio of 1.62.

[0055] According to the criteria, a compound can be listed as a priority for biological validation when score_MRGPRX2 ≥ 0.9 × score_positive control or score_MRGPRX2 / score_RhoA ≥ 1.2. The results show that the target compound meets the above conditions and exhibits allergy-like activity.

[0056] Example 5 In this embodiment, a purification process unit such as macroporous adsorption resin or reverse phase chromatography is introduced into the production process of safflower injection to achieve a removal rate of over 90% for 6-hydroxykaempferol-3,6,7-tri-O-glucoside.

[0057] The refining process of safflower injection includes a removal process unit to reduce or remove the content of 6-hydroxykaempferol-3,6,7-tri-O-glucoside.

[0058] Preferably, the removal unit includes macroporous adsorption resin adsorption and desorption, activated carbon adsorption, reversed-phase chromatography, ion exchange chromatography, and ultrafiltration or nanofiltration membrane separation. Depending on the process characteristics, different methods utilize differences in the polarity, molecular weight, or hydrophobicity of the target compound to achieve selective removal.

[0059] HPD-100 macroporous resin was used at a loading concentration of 1 mg / mL, and 30% ethanol was used as the elution solvent, resulting in a removal rate of 94%. Reverse phase chromatography (ODS column, mobile phase water-ethanol 70:30) was used, and the removal rate was approximately 90–93%.

[0060] Process monitoring employs the detection method described in Example 2. The content of the target component is measured before and after the process, and the removal rate is calculated. Quality control requirements are met when the removal efficiency is ≥90% or the content in the final formulation is below the LOQ (0.05 μg / mL). Each batch's test record must include the batch number, test data, removal rate, and disposal recommendations.

[0061] Example 6 The purpose of this embodiment is to construct a standardized detection kit containing an HSA capture component, an internal standard, and operating instructions, to achieve rapid enrichment, pretreatment, and quantitative detection of allergy risk substances in safflower injection. Specifically, it includes the following: The test kit is used to detect one or more allergy risk components in safflower injection samples, and is particularly suitable for 6-hydroxykaempferol-3,6,7-tri-O-glucoside.

[0062] The kit comprises the following components: HSA-coated magnetic microspheres or HSA-modified chromatography columns for enriching target components; Elution buffer (50% methanol + 0.1% formic acid) and corresponding pretreatment consumables; SPE column and related solvents; stable isotope-labeled internal standard solution (10 μg / mL). Positive control C48 / 80, negative control; and operating instructions, detailing the recommended parameters for UHPLC-MS / MS, MRM monitoring of ion pairs, internal standard usage, and LOQ verification procedures.

[0063] The procedure is as follows: First, pretreat the safflower injection sample according to the instructions; second, perform affinity enrichment and elution using the HSA capture component; then, purify via SPE and add an internal standard, followed by analysis using a UHPLC-MS / MS system. Calculate the content of 6-hydroxykaempferol-3,6,7-tri-O-glucoside based on the calibration curve; finally, according to the QC criteria, a detection value exceeding the LOQ is considered an excess of allergenic substances. This kit enables standardized operation of the entire process from enrichment and pretreatment to quantitative detection, and is suitable for rapid screening in the production and quality control stages of safflower injection.

[0064] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for screening of allergen-like substances in safflower injection, characterized in that, Includes the following steps: Step S1. Extract the analyte from the safflower injection sample to obtain a mixture of components; Step S2. The mixture of components is captured and eluted by affinity enrichment using an immobilized human serum albumin (HSA) affinity solid-phase material; Step S3. After pretreatment, the eluent obtained in step S2 is separated and quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) in multiple reaction monitoring (MRM) mode. The quantitative analysis uses stable isotopes or structurally similar internal standards for correction and calculates the content of the target component in the sample based on the calibration curve. Step S4. Perform molecular docking simulations on the candidate components obtained from the separation and identification in step S3 against the MRGPRX2 and RhoA targets, and make a comprehensive judgment in combination with predetermined positive and negative controls to screen allergy candidates. Step S5. Perform biological validation of the allergy candidate in a cell or animal model, the biological validation including measuring histamine release or degranulation indicators to confirm its allergy-like potential.

2. The method for screening of allergen-like substances in safflower injection according to claim 1, characterized in that, The immobilized HSA affinity solid material in step S2 is selected from HSA-coated magnetic microspheres, HSA-modified chromatography columns, or HSA-modified microplates.

3. The method for screening allergenic substances in safflower injection according to claim 1, characterized in that, The necessary pretreatment in step S3 includes solvent exchange, concentration, or solid-phase extraction (SPE) of the eluent to reduce matrix interference and improve method sensitivity.

4. The method for screening of allergen-like substances in safflower injection according to claim 1, wherein the allergen-like substance is a protein. In step S4, a dual scoring strategy is used for docking determination. The dual scoring strategy includes one aspect, comparing the docking score_MRGPRX2 for MRGPRX2 with the predetermined positive control, and the other aspect, determining the ratio of score_MRGPRX2 to the docking score_RhoA for RhoA. The allergy candidate will be listed as a priority biological validation object when either of the following conditions is met: score_MRGPRX2 ≥ 0.9 × score_positive control or score_MRGPRX2 divided by score_RhoA ≥ 1.

2.

5. A method for detecting an allergen-like substance in a safflower injection, characterized by, According to any one of claims 1-4, the sample is first subjected to HSA affinity enrichment treatment, and then separated and quantitatively analyzed by UHPLC-MS / MS method; the lower limit of quantitation (LOQ) determined by the method validation is used as the quality control action limit value, and when the content of the target component in the sample is greater than the LOQ, it is determined that the allergen-like substance exceeds the standard.

6. The method of claim 5, wherein the allergen-like substance is detected by measuring the amount of the allergen-like substance in the safflower injection. The methodologically validated limit of quantitation is ≤0.5 μg / mL.

7. A method for removing allergen-like substances from safflower injection, characterized by, It includes steps such as raw material extraction, crude separation, purification and formulation preparation; the purification step includes at least one removal process unit for reducing or removing the content of the target component; The removal process unit includes macroporous adsorption resin adsorption and desorption, activated carbon adsorption, reverse phase chromatography, ion exchange chromatography, and ultrafiltration or nanofiltration membrane separation. In the removal process unit, the target component is monitored online or offline using the detection method for allergens in safflower injection as described in claim 5 to verify the removal effect.

8. The method of removing allergen-like substances from safflower injection according to claim 7, wherein the step of removing allergen-like substances is performed by heating the safflower injection to a temperature of 60°C to 80°C for 1 to 3 hours. The removal efficiency of the removal process unit on the target component is greater than or equal to 90%, or the content of the target component in the final preparation after being treated by the removal process unit is less than or equal to the lower limit of quantification LOQ in the analysis method of claim 5.

9. A kit for detecting allergen-like substances in safflower injection, characterized by comprising: The kit for detecting one or more target components in safflower injection samples comprises an affinity capture component for immobilized human serum albumin HSA, a buffer and consumables for elution and pretreatment, an internal standard and reference material for UHPLC-MS / MS quantitative analysis, and an instruction manual; by using the kit, all chemical steps required for sample pretreatment, enrichment and quantitative detection in the detection method of the allergen-like substance in safflower injection of claim 5 can be completed, and quantitative results can be obtained under the condition of being equipped with a UHPLC-MS / MS analysis system.

10. The test kit for the allergenic substance in the safflower injection according to claim 9, characterized by, The target component is 6-hydroxykaempferol-3,6,7-tri-O-glucoside.

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