Reagent combination, blank biological liquid matrix, preparation method and application of blank biological liquid matrix, and kit

Abstract

The invention discloses a reagent combination for preparing a blank biological liquid matrix, the blank biological liquid matrix, a preparation method and application of the blank biological liquid matrix and a kit. The reagent combination comprises a stripping solvent and an additive, the stripping solvent comprises alcohol and ether, and the additive comprises at least one of a dealcoholization solvent, an excipient, a pH regulator and a protective agent; the dealcoholization solvent is suitable for removing alcohol in the stripped biological liquid; the excipient is suitable for forming the dry powder in the process of freeze-drying a blank biological liquid matrix; the pH regulator is suitable for regulating the pH value of the blank biological liquid matrix reconstitution fluid; the protective agent is suitable for corrosion prevention of a blank biological liquid matrix reconstitution fluid.

Description

Technical Field

[0001] The present invention relates to the technical field of laboratory testing, and more specifically, to a reagent combination for preparing a blank biological liquid matrix, a blank biological liquid matrix, a preparation method thereof, an application thereof, and a kit. Background Art

[0002] In the related art, in the reagent combination used to detect the concentration of target substances, such as fat-soluble vitamins, ceramides, steroid hormones, etc., calibrants and quality control products are usually prepared using alternative matrices. These alternative matrices include organic solvents, bovine serum albumin solutions or human serum albumin solutions. However, these alternative matrices have large differences in components such as protein composition and concentration from the sample to be tested (e.g., blood), resulting in inaccurate detection results of the target substance.

[0003] Therefore, it is necessary to provide a new technical solution to solve the above technical problems. Summary of the invention

[0004] An object of the present invention is to provide a new technical solution for the reagent combination for preparing a blank biological fluid matrix.

[0005] According to a first aspect of the present invention, a reagent combination for preparing a blank biological liquid matrix is ​​provided. The reagent combination comprises a stripping solvent and an additive, wherein the stripping solvent comprises alcohol and ether, and the additive comprises at least one of a dealcoholization solvent, an excipient, a pH regulator, and a protective agent;

[0006] The de-alcoholization solvent is suitable for removing alcohol from the biological liquid matrix after stripping;

[0007] The excipient is suitable for forming a dry powder during freeze-drying of a blank biological liquid matrix;

[0008] The pH regulator is suitable for adjusting the pH of the blank biological liquid matrix reconstitution solution;

[0009] The protective agent is suitable for the preservation of blank biological liquid matrix re-solution.

[0010] Optionally, in the stripping solvent, calculated by mass percentage, the content of the alcohol is 1% to 35%, and the content of the ether is 65% to 99%; preferably, the content of the alcohol is 5% to 35%, and the content of the ether is 65% to 95%.

[0011] Optionally, the alcohol includes at least one of a monohydric alcohol and a polyhydric alcohol; preferably, the ether includes at least one of a monoether and a mixed ether.

[0012] Optionally, the ether includes at least one of dimethyl ether, diethyl ether, dipropyl ether, methyl ethyl ether, ethyl propyl ether, methyl propyl ether, and methyl tert-butyl ether, and the alcohol includes at least one of methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, and sorbitol; preferably, the ratio of the alcohol content to the methyl tert-butyl ether content is 5:95 to 25:75.

[0013] Optionally, the alcohol includes isopropanol, the ether includes methyl tert-butyl ether, and the ratio of the content of the isopropanol to the content of the methyl tert-butyl ether is 25:75 to 10:90;

[0014] Optionally, the alcohol includes isopropanol and ethanol, the ether includes methyl tert-butyl ether, and the ratio of the content of the isopropanol, the content of the ethanol and the content of the methyl tert-butyl ether is (5-15): (1-10): (75-94); or

[0015] Optionally, the alcohol includes n-butanol and ethanol, the ether includes methyl tert-butyl ether, and the ratio of the content of n-butanol, the content of ethanol and the content of methyl tert-butyl ether is (2-10): (6-15): (75-92);

[0016] Optionally, the alcohol includes isopropanol and glycerol, the ether includes methyl tert-butyl ether, and the ratio of the isopropanol content, the glycerol content and the methyl tert-butyl ether content is (10-20): (2-8): (88-72).

[0017] Optionally, the dealcoholization solvent includes ether, preferably, the ether includes at least one of monoether and mixed ether, more preferably includes methyl tert-butyl ether;

[0018] Optionally, the excipient includes at least one of mannitol, trehalose, and sucrose;

[0019] Optionally, the pH adjuster includes at least one of sodium dihydrogen phosphate and acetic acid;

[0020] Optionally, the protective agent includes at least one of ProClean 300, Buffpro 300, ProClin 300, ProClin150, ProClin200, and ProClin950.

[0021] According to a second aspect of the present application, a method for preparing a blank biological liquid matrix is ​​provided. The preparation method comprises: mixing a biological liquid with the stripping solvent described in the present application, separating the layers to obtain a first organic phase layer and a first main phase layer, removing the first organic phase layer to obtain the first main phase layer, wherein the first main phase layer is a blank biological liquid matrix.

[0022] Optionally, the biological fluid includes at least one of whole blood, plasma, serum, saliva, urine, cerebrospinal fluid, and milk.

[0023] Optionally, the volume ratio of the stripping solvent to the biological fluid is greater than or equal to 1:2.

[0024] Optionally, the blank biological liquid matrix is ​​dried to prepare a dry powder; or

[0025] The method also includes purifying the blank biological matrix liquid, including: adding a dealcoholizing agent to the obtained blank biological liquid matrix, mixing, layering to obtain a second organic phase layer and a second main phase layer, and removing the second organic phase layer.

[0026] Optionally, the second main phase layer is subjected to rotary evaporation to remove the dealcoholization agent in the second main phase layer; or

[0027] The second main phase layer is dried to prepare a dry powder; preferably, the second main phase layer is dried by freeze-drying.

[0028] Optionally, the method further comprises adding an excipient before the blank biological liquid matrix is ​​prepared into dry powder. Preferably, the mass ratio of the excipient to the blank biological liquid matrix is ​​1% to 10%.

[0029] Optionally, the method further comprises re-dissolving the dry powder prepared from the blank biological liquid matrix to obtain a blank biological liquid matrix re-solution, and preferably, adding a protective agent and / or a pH regulator to the blank biological liquid matrix re-solution;

[0030] Preferably, the mass ratio of the protective agent to the blank biological liquid matrix reconstituted solution is 0.01% to 0.2%;

[0031] Preferably, the mass ratio of the pH regulator to the blank biological fluid matrix reconstituted solution is 0.1% to 0.5%.

[0032] According to a third aspect of the present application, a blank biological liquid matrix is ​​provided. The blank biological liquid matrix is ​​prepared according to the preparation method described in the present application.

[0033] According to a fourth aspect of the present application, there is provided a use of a blank biological liquid matrix in detecting fat-soluble vitamins, ceramides or steroid hormones.

[0034] According to a fifth aspect of the present application, a kit is provided, which comprises the reagent combination described in the present application and / or the blank biological liquid matrix described.

[0035] In an embodiment of the present application, the stripping solvent in the reagent combination can effectively remove target substances in the biological liquid, such as fat-soluble vitamins, ceramides, steroid hormones and other substances, thereby forming a blank biological liquid matrix. The blank biological liquid matrix has a similar protein concentration to the unstripped biological liquid, so it can be used as a preparation of calibration products and quality control products for detecting target substances, such as fat-soluble vitamins, ceramides, steroid hormones, etc. The dealcoholization solvent can remove alcohol from the stripped biological liquid, thereby effectively purifying the blank biological liquid matrix. The excipient can form dry powder during the freeze-drying process of the blank biological liquid matrix; the pH regulator can adjust the pH of the blank biological liquid matrix reconstitution solution; the protective agent is suitable for the preservation of the blank biological liquid matrix reconstitution solution.

[0036] Further features and advantages of the present invention will become apparent from the following detailed description of exemplary embodiments of the present invention with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the present invention and, together with the description, serve to explain the effects of the present invention.

[0038] Figure 1 This is a chromatogram of 7 types of ceramides in the mixed plasma of Example 2 of the present application.

[0039] Figure 2 This is the chromatogram of 7 ceramides in the optimized blank plasma matrix of Example 2 of the present application.

[0040] Figure 3 This is a picture of the dry powder of the blank serum matrix prepared in Example 1 of the present application.

[0041] Figure 4 This is a picture of the dry powder of the blank serum matrix prepared in Comparative Example 5.

[0042] Figure 5 Example 1 Optimization of blank serum matrix reconstitution solution after storage at 2-8°C for 20 days.

[0043] Figure 6 Comparative Example 6: Photo of the blank serum matrix reconstituted solution after being stored at 2-8°C for 20 days.

[0044] Reference numerals: Figure 1 and Figure 21. Cer(d18:1 / 14:0); 2. Cer(d18:1-d7 / 14:0); 3. Cer(d18:1 / 16:0); 4. Cer(d18:1 -d7 / 16:0); 5. Cer(d18:1 / 18:0); 6. Cer(d18:1-d7 / 18:0); 7. Cer(d18:1 / 20:0); 8. C er(d18:1-d7 / 20:0); 9. Cer(d18:1 / 22:0); 10. Cer(d18:1-d7 / 22:0); 11. Cer(d18:1 / 24:0); 12. Cer(d18:1-d7 / 24:0); 13. Cer(d18:1 / 24:1); 14. Cer(d18:1-d7 / 24:1). DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0046] The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses.

[0047] Technologies, methods, and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and equipment should be considered as part of the specification.

[0048] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0049] It should be noted that like reference numerals and letters refer to similar items in the following figures, and therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0050] According to the first embodiment of the present application, a reagent combination for preparing a blank biological liquid matrix is ​​provided. The reagent combination includes a stripping solvent and an additive, wherein the stripping solvent includes alcohol and ether, and the additive includes at least one of a dealcoholization solvent, an excipient, a pH regulator, and a protective agent;

[0051] The de-alcoholization solvent is suitable for removing alcohol from the biological liquid after stripping;

[0052] The excipient is suitable for forming a dry powder during freeze-drying of a blank biological liquid matrix;

[0053] The pH regulator is suitable for adjusting the pH of the blank biological liquid matrix reconstitution solution;

[0054] The protective agent is suitable for the preservation of blank biological liquid matrix re-solution.

[0055] In the embodiment of the present application, the stripping solvent can effectively strip target substances in biological fluids, such as fat-soluble vitamins, ceramides, steroid hormones, and the like, thereby forming a blank biological fluid matrix. The blank biological fluid matrix has a similar protein concentration to the unstripped biological fluid, and the retention time of the target substances in the matrix before and after stripping is similar, and can be used for the preparation of calibration products and quality control products for detecting target substances, such as fat-soluble vitamins, ceramides, steroid hormones, and the like.

[0056] The de-alcoholization solvent can remove alcohol in the biological liquid after stripping, thereby effectively purifying the blank biological liquid matrix. Optionally, the de-alcoholization solvent includes ether. Preferably, the ether includes at least one of a monoether and a mixed ether. For example, the ether includes at least one of dimethyl ether, diethyl ether, dipropyl ether, methyl ethyl ether, ethyl propyl ether, methyl propyl ether, and methyl tert-butyl ether. More preferably, it includes methyl tert-butyl ether. All of the above substances can effectively remove alcohol in the biological liquid after stripping.

[0057] The blank biological liquid matrix can be further purified by rotary evaporation and drying to remove residual alcohol and ether in the blank biological liquid matrix. The blank liquid matrix from which the organic solvent is removed by rotary evaporation can be directly used for the preparation of calibration products and quality control products.

[0058] Alternatively, the blank biological liquid matrix can be prepared into a dry powder. For example, the blank biological liquid matrix can be prepared into a dry powder using a freeze-drying process for easy storage. When in use, the dry powder is reconstituted to obtain a blank biological liquid matrix reconstituted solution. The reconstituted solution can be used for preparation with calibration products and quality control products. The dry powder can effectively extend the shelf life of the blank biological liquid matrix, for example, the dry powder can be kept for more than 1 year.

[0059] The excipient can form the dry powder during the freeze-drying process of the blank biological liquid matrix. For example, before freeze-drying the blank biological liquid matrix, the excipient is added to the blank biological liquid matrix and mixed; during the freeze-drying process, the excipient can promote the crystallization of the blank biological liquid matrix, and can resist the damage of the protein by stress such as low temperature and drying, thereby improving the speed and quality of dry powder formation. Finally, a dry powder with a cake-like structure is formed. Optionally, the excipient includes at least one of mannitol, trehalose, and sucrose, and the above substances can promote the formation of dry powder. The mass ratio of the excipient to the blank biological liquid matrix is ​​1% to 10%. Optionally, the mass ratio is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. Within this range, the dry powder is formed quickly, the quality is high, and the size of the cake structure is uniform.

[0060] The pH regulator can adjust the pH of the blank biological liquid matrix reconstitution solution. The dry powder needs to be reconstituted when used to form a blank biological liquid matrix reconstitution solution. Preferably, adding a pH regulator to the reconstitution solution can make the pH of the reconstitution solution meet the use requirements. Optionally, the pH regulator includes at least one of sodium dihydrogen phosphate and acetic acid. The above two substances are acidic and can effectively adjust the pH of the reconstitution solution. Optionally, the mass ratio of the pH regulator to the blank biological liquid matrix reconstitution solution is 0.1% to 0.5%. For example, the mass ratio is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, etc. Within the above range, the pH regulator can effectively adjust the pH of the blank biological liquid matrix reconstitution solution.

[0061] The protective agent can effectively prevent the corruption of the blank biological liquid matrix reconstitution solution and extend the shelf life of the blank biological liquid matrix reconstitution solution. For example, adding a protective agent to the reconstitution solution can prevent the reconstitution solution from being corrupted during long-term storage. Optionally, the protective agent includes at least one of ProClean 300, Buffpro 300, ProClin 300, ProClin150, ProClin200, and ProClin950. The mass ratio of the protective agent to the blank biological liquid matrix reconstitution solution is 0.01% to 0.2%. For example, the mass ratio is 0.01%, 0.05%, 0.08%, 0.1%, 0.13%, 0.15%, 0.17%, 0.2%, etc. The above substances and mass ratios can effectively prevent the reconstitution solution from being corrupted.

[0062] In one example, in the stripping solvent, calculated by mass percentage, the content of the alcohol is 1% to 35%, and the content of the ether is 65% to 99%.

[0063] Optionally, the content of alcohol is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, etc. Correspondingly, the content of ether is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, etc.

[0064] Within the above range, the contents of fat-soluble vitamins, ceramides and steroid hormones in the blank biological liquid matrix obtained by stripping with a stripping solvent are extremely low. Therefore, it can be used for the preparation of calibrants and quality control products in the detection of fat-soluble vitamin concentrations, ceramide concentrations and steroid hormone concentrations in biological liquids.

[0065] By LC-MS / MS detection, the content of target substances (e.g., fat-soluble vitamins, ceramides, steroid hormones) in the blank biological liquid matrix can be obtained, and the removal rate and content of the target substances after removal in the blank biological liquid matrix can be determined.

[0066] In one example, the content of the ether is 65% to 95%, and the content of the alcohol is 5% to 35%.

[0067] If the alcohol content is too high, protein precipitation will be formed during the stripping of target substances (e.g., fat-soluble vitamins, ceramides, steroid hormones) in the biological fluid, resulting in a large difference in the matrix between the blank biological fluid and the biological fluid; conversely, if the alcohol content is too low, the removal rate of the target substance is low. Optionally, the alcohol content is 5%, 10%, 15%, 20%, 25%, 30%, 35%, etc. Correspondingly, the ether content is 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc. Within the above range, the stripping solvent can effectively remove the target substance in the biological fluid, and no protein precipitation will be formed during the stripping process.

[0068] In one example, in the stripping solvent, the alcohol includes at least one of a monohydric alcohol and a polyhydric alcohol.

[0069] Optionally, the monohydric alcohol includes methanol, ethanol, propanol, butanol, isopropanol, etc. The polyhydric alcohol includes ethylene glycol, propylene glycol, glycerol, sorbitol, etc.

[0070] Preferably, the alcohol does not include structurally unstable enols, which have poor stability and whose hydroxyl groups may become carbonyl groups during use, resulting in failure to effectively remove the target substance.

[0071] In one example, in the stripping solvent, the ether includes at least one of a monoether and a mixed ether.

[0072] Optionally, the monoether includes dimethyl ether, diethyl ether (ie, diethyl ether), dipropyl ether, etc. The mixed ether includes methyl ethyl ether, ethyl propyl ether, methyl propyl ether, methyl tert-butyl ether, etc.

[0073] Preferably, the ether does not include water-miscible ethers, active cyclic ethers, and crown ethers that can complex metal ions in biological matrices. Water-miscible ethers cannot be separated after mixing with biological fluids, resulting in the inability to strip the target substance. Active cyclic ethers easily react with substances in biological fluids, thereby affecting the composition of blank biological fluid matrix. Crown ethers can complex metal ions in biological fluids, thereby causing a difference in ionic strength between the prepared blank biological fluid matrix and the biological fluid.

[0074] In one example, in the stripping solvent, the ether includes at least one of dimethyl ether, diethyl ether, dipropyl ether, methyl ethyl ether, ethyl propyl ether, methyl propyl ether, and methyl tert-butyl ether, and the alcohol includes at least one of methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, and sorbitol.

[0075] In this example, the ether can effectively remove the target substance in the biological fluid. The ether is miscible with methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, sorbitol, etc., thereby further improving the stripping efficiency of the stripping solvent.

[0076] In one example, in the stripping solvent, the ratio of the alcohol content to the ether content is 5:95 to 25:75.

[0077] In this example, the stripping solvent includes ether and alcohol mixed together. Optionally, the ratio of the content of the alcohol to the content of the ether is 5:95, 10:90, 12:88, 15:85, 18:82, 20:80, 22:78, 25:75, etc. Within the above range, the stripping solvent can effectively remove fat-soluble vitamins, ceramides, steroid hormones and other substances in the biological fluid, and no protein precipitation will be formed during the stripping process.

[0078] In one example, in the stripping solvent, the alcohol includes isopropanol, the ether includes methyl tert-butyl ether, and the ratio of the content of the isopropanol to the content of the methyl tert-butyl ether is 25:75 to 10:90; in this example, the stripping solvent includes methyl tert-butyl ether and isopropanol mixed together. Optionally, the above content ratio is 10:90, 12:88, 15:85, 18:82, 20:80, 22:78, 25:75, etc. Within the above range, the stripping solvent has a good stripping effect on substances such as fat-soluble vitamins, ceramides, and steroid hormones.

[0079] In one example, in the stripping solvent, the alcohol includes isopropanol and ethanol, the ether includes methyl tert-butyl ether, and the ratio of the isopropanol content, the ethanol content and the methyl tert-butyl ether content is (5-15):(1-10):(75-94).

[0080] In this example, the stripping solvent includes methyl tert-butyl ether, ethanol and isopropanol mixed together. Optionally, the above content ratio is 5:1:94, 6:4:90, 7:4:89, 8:5:87, 10:6:84, 12:8:80, 14:8:78, 15:10:75, etc. Within the above range, the stripping solvent has a good stripping effect on substances such as fat-soluble vitamins, ceramides, steroid hormones, etc.

[0081] In one example, in the stripping solvent, the alcohol includes n-butanol and ethanol, the ether includes methyl tert-butyl ether, and the ratio of the content of n-butanol, the content of ethanol and the content of methyl tert-butyl ether is (2-10):(6-15):(75-92).

[0082] In this example, the stripping solvent includes n-butanol, ethanol and methyl tert-butyl ether mixed together. Optionally, the above content ratio is: 2:6:92, 4:7:89, 5:10:85, 6:12:82, 7:13:80, 9:11:80, 10:15:75, etc. Within the above range, the stripping solvent has a good stripping effect on substances such as fat-soluble vitamins, ceramides, steroid hormones, etc.

[0083] In one example, in the stripping solvent, the alcohol includes isopropanol and glycerol, the ether includes methyl tert-butyl ether, and the ratio of the isopropanol content, the glycerol content and the methyl tert-butyl ether content is (10-20): (2-8): (88-72).

[0084] In this example, the stripping solvent includes isopropyl alcohol, glycerol and methyl tert-butyl ether mixed together. Optionally, the above content ratio is: 10:6:84, 12:8:80, 15:5:80, 16:5:79, 17:5:78, 18:4:78, 19:7:74, 20:8:72, etc. Within the above range, the stripping solvent has a good stripping effect on substances such as fat-soluble vitamins, ceramides, steroid hormones, etc.

[0085] In one example, as mentioned above, the dealcoholization solvent includes an ether, preferably, the ether includes at least one of a monoether and a mixed ether, and more preferably includes methyl tert-butyl ether;

[0086] Optionally, the excipient includes at least one of mannitol, trehalose, and sucrose;

[0087] Optionally, the pH adjuster includes at least one of sodium dihydrogen phosphate and acetic acid;

[0088] Optionally, the protective agent includes at least one of ProClean 300, Buffpro 300, ProClin 300, ProClin150, ProClin200, and ProClin950.

[0089] According to a second embodiment of the present application, a method for preparing a blank biological liquid matrix is ​​provided. The preparation method comprises mixing a biological liquid with a stripping solvent provided in an embodiment of the present application, layering to obtain a first organic phase layer and a first main phase layer, removing the first organic phase layer to obtain the first main phase layer, wherein the first main phase layer is a blank biological liquid matrix.

[0090] In the specific implementation, first, according to the lipophilicity of the target analyte, the types and proportions of alcohol and ether are adjusted. Preferably, the content of the alcohol is 1 to 35%, and the content of the ether is 65% to 99% by mass. Within this range, the ratio of alcohol and ether is adjusted to avoid protein precipitation in the biological fluid. After the stripping solvent is prepared, it can be placed in a brown reagent bottle and stored away from light.

[0091] Then, according to a fixed value that the volume ratio of the stripping solvent to the biological liquid is greater than or equal to 1:2, the stripping solvent is added to the container containing the biological liquid, and mixed evenly, for example, centrifuged by a centrifuge to perform stratification. For example, the first main phase layer is the aqueous phase layer. Since the density of the first organic phase layer is less than the density of the aqueous phase layer, the first organic phase layer is located on the first main phase layer. The target substance is dissolved in the first organic phase layer. The first organic phase layer is removed, and the remaining first main phase layer is a blank biological liquid matrix.

[0092] After the above preparation method, the protein concentration of the blank biological fluid matrix is ​​similar to the protein concentration of the biological fluid before stripping.

[0093] The preparation method can effectively remove target substances in biological fluids, such as fat-soluble vitamins, steroid hormones, ceramides, etc., to obtain a blank biological fluid matrix.

[0094] Preferably, in order to better preserve the blank biological liquid matrix, the obtained blank biological liquid matrix is ​​dried to prepare a dry powder. For example, the blank biological liquid matrix is ​​prepared into a dry powder by freeze drying. This method can remove the organic solvent in the blank biological liquid matrix and preserve the blank biological liquid matrix. When in use, the dry powder can be reconstituted with high-purity water.

[0095] Optionally, the biological fluid includes at least one of whole blood, plasma, serum, saliva, urine, cerebrospinal fluid, and milk.

[0096] In one example, the volume ratio of the stripping solvent to the biological fluid is greater than or equal to 1:2.

[0097] In the above range, the removal efficiency of the target substance in the biological fluid is high.

[0098] Optionally, the volume ratio of the stripping solvent to the biological liquid is 1:2, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, etc. During preparation, setting the amount of biological liquid and stripping solvent according to the above volume ratio can effectively remove the target substance in the biological liquid.

[0099] In one example, the preparation method further includes purifying the blank biological liquid matrix, including: adding a dealcoholizing agent to the obtained blank biological liquid matrix, mixing, layering to obtain a second organic phase layer and a second main phase layer, and removing the second organic phase layer.

[0100] Since the stripping solvent contains alcohol and ether, the two substances will be partially miscible in the blank biological liquid matrix during the preparation of the blank biological liquid matrix. In this example, by adding a dealcoholizing agent to the blank biological liquid matrix and fully mixing it, after layering, the alcohol in the blank biological liquid matrix can be effectively removed. The dealcoholizing agent is as described above. For example, layering is performed by centrifugation to obtain a second organic phase layer and a second main phase layer, and the second organic phase layer is removed. The second main phase layer is an aqueous phase layer. Since the density of the aqueous phase layer is greater than the density of the second organic phase layer, the second organic phase layer is located above the second main phase layer. After removing the second organic phase layer, the alcohol content in the blank biological liquid matrix is ​​greatly reduced, thereby effectively purifying the blank biological liquid matrix.

[0101] To further purify the blank biological fluid matrix, the addition of the dealcoholizing agent and the removal of the second organic phase layer can be performed multiple times.

[0102] Preferably, the volume ratio of the alcohol removal agent to the first main phase layer is greater than or equal to 1:1. The types of alcohol removal agents are as described above. Optionally, the volume ratio of the alcohol removal agent to the first main phase layer is 1:1, 1.5:1, 2:1, 2.5:1, 3:1, etc. Within this range, the alcohol in the first main phase layer can be effectively removed.

[0103] In one example, the preparation method further comprises rotary evaporating the second main phase layer to remove the ether in the second main phase layer.

[0104] Rotary evaporation is a method of rapidly evaporating the solvent of a sample by rotating and heating the sample container under reduced pressure, thereby concentrating or separating the solutes in the sample. This method can effectively remove the ether in the second main phase layer.

[0105] The method for removing the ether in the second main phase layer may also be to dry the second main phase layer to prepare a dry powder; preferably, the second main phase layer is dried by freeze-drying.

[0106] During the drying process, ether gradually evaporates. By drying the blank biological liquid matrix and preparing it into a dry powder, the ether and other organic solvents in the blank biological liquid matrix can be effectively removed. In addition, the dry powder is conducive to the preservation of the blank biological liquid matrix. When used, the dry powder can be re-dissolved with a solvent, such as ultrapure water. The volume of the solvent can be determined according to the ratio of volatile matter to dry powder after the corresponding biological liquid is dried without stripping treatment and the mass of the dry powder.

[0107] In one example, the preparation method further comprises adding an excipient before the blank biological liquid matrix is ​​prepared into a dry powder. Preferably, the mass ratio of the excipient to the blank biological liquid matrix is ​​1% to 10%.

[0108] Excipients are as described above. Excipients can effectively improve the speed and quality of dry powder formation, and ultimately form dry powder with a cake-like structure.

[0109] In one example, the preparation method further comprises re-dissolving the dry powder prepared from the blank biological liquid matrix to obtain a blank biological liquid matrix re-solution, preferably, adding a protective agent and / or a pH regulator to the blank biological liquid matrix re-solution;

[0110] Preferably, the mass ratio of the protective agent to the blank biological liquid matrix reconstituted solution is 0.01% to 0.2%;

[0111] Preferably, the mass ratio of the pH regulator to the blank biological fluid matrix reconstituted solution is 0.1% to 0.5%.

[0112] According to a third embodiment of the present application, a blank biological liquid matrix is ​​provided, which is prepared according to the preparation method described in the present application.

[0113] The content of the target substance in the blank biological liquid matrix is ​​low, and can be used to prepare quality control products and calibration products for target substance detection, so that the accuracy of the target substance in the biological liquid is high.

[0114] According to a fourth embodiment of the present application, there is provided a use of a blank biological liquid matrix in detecting fat-soluble vitamins, ceramides or steroid hormones.

[0115] The blank biological fluid matrix has a similar protein concentration to that of the unstripped biological fluid and can be used to detect target substances in the biological fluid, such as fat-soluble vitamins, ceramides, steroid hormones, etc. For example, it can be used for the preparation of calibrators and quality control products.

[0116] According to a fifth embodiment of the present application, a kit is provided, which includes the reagent combination provided in the present application and / or a blank biological liquid matrix.

[0117] The reagent combination can be used to prepare a blank biological liquid matrix. The blank biological liquid matrix can be prepared into a dry powder. When used, ultrapure water is used to dissolve the dry powder to obtain a liquid blank biological liquid matrix. Since the kit uses the blank biological liquid matrix of the embodiment of the present application to prepare calibration products and quality control products, the detection of the target substance is more accurate.

[0118] <Example 1>

[0119] Preparation of blank serum matrix for fat-soluble vitamins

[0120] The biological fluid is human serum. Human sera from multiple sources are mixed together, for example, sera from 30 people are mixed together, i.e., mixed serum. Part of the mixed human serum is used to prepare a blank biological fluid matrix, and part is used for comparison.

[0121] In the stripping solvent, the mass percentages of isopropanol, ethanol and methyl tert-butyl ether are 10%, 5% and 85% respectively, and all three reagents are chromatographically pure reagents. The stripping solvent is mixed evenly by vortex mixing. The stripping solvent is added to human serum at a volume ratio of 1:1.5 between the first main phase layer and the stripping solvent, mixed at 1000rpm for 40 minutes, and the mixed solution is centrifuged at a speed of 4000rpm for 10min to obtain a stratified liquid, wherein the first organic phase layer is on the upper layer and the first main phase layer is on the lower layer. The first organic phase layer is removed. The first main phase layer is mixed with methyl tert-butyl ether at a volume ratio of 1:1.5 between the first main phase layer and methyl tert-butyl ether, and mixed at 1000rpm for 40 minutes. Centrifuge at 4000rpm for 10min to obtain a stratified liquid. The second organic phase layer is on the upper layer. The second main phase layer is on the lower layer. The second organic phase layer was removed, and 5% mannitol was added to the second main phase layer according to a mass ratio, and then freeze-dried to obtain a dry powder of a blank serum matrix.

[0122] test:

[0123] The dry powder of the blank serum matrix is ​​reconstituted with ultrapure water to obtain a blank serum matrix in a solution state. The ratio of ultrapure water to dry powder in this embodiment is 7.06, that is, when the mass of the dry powder of the blank serum matrix is ​​1g, 7.06g of ultrapure water is added for reconstitution and vortex mixing. Then, the pH value of the blank serum matrix reconstituted solution is adjusted to 7.4 with acetic acid; next, 0.05% ProClin 300 is added to the blank serum matrix reconstituted solution according to the mass ratio, and vortex mixing is continued to obtain an optimized blank serum matrix. The optimized blank serum matrix is ​​sealed and stored in a dark environment at -20°C.

[0124] The results were analyzed by liquid chromatography-tandem mass spectrometry (SCIEX Triple Quad TM 4500MD LC-MS / MS system) detected the contents of the five fat-soluble vitamins in the optimized blank serum matrix of this example and the unstripped human serum of this example, and calculated the removal rates. The detection results are shown in Table 1.

[0125] Table 1 - Comparison of the contents of five fat-soluble vitamins in the blank serum matrix optimized in Example 1 and in unstripped human serum

[0126]

[0127] Note: If the target substance peak signal-to-noise ratio in the test results is less than 3, the content is marked as 0.

[0128] As shown in Table 1, the contents of fat-soluble vitamin A1, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E and vitamin K1 in the optimized blank serum matrix are all reduced by more than 99% compared with the unstripped human serum. Among them, the detected contents of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in the optimized blank serum matrix are 0, and the detected contents of vitamin A1, vitamin E and vitamin K1 are less than 1% of the detected contents in the unstripped human serum. The stripping solvent can effectively strip the fat-soluble vitamins in human serum.

[0129] <Example 2>

[0130] Ceramide blank plasma preparation

[0131] The biological liquid is human plasma. Human plasma from multiple sources is mixed together, for example, plasma from 30 people is mixed together, i.e., mixed plasma. A portion of the mixed plasma is used to prepare a blank plasma matrix, and a portion is used for comparison. In the stripping solvent, the mass percentages of isopropanol, ethanol, and methyl tert-butyl ether are 20%, 10%, and 70%, respectively. The stripping solvent is mixed evenly by vortex mixing. The stripping solvent is added to the mixed plasma in a volume ratio of 1:2 of the mixed plasma: the stripping solvent, and the mixture is mixed head to head for 40 minutes. The mixed solution is centrifuged at a speed of 4000 rpm for 15 minutes to obtain a stratified liquid. The first organic phase layer is on the upper layer. The first main phase layer is on the lower layer. The first organic phase layer is removed. The first main phase layer is mixed with methyl tert-butyl ether in a volume ratio of 1:2 of the mixed first main phase layer: methyl tert-butyl ether, and the mixture is mixed head to head for 10 minutes. Next, the mixture is centrifuged at a speed of 4000 rpm for 10 minutes to obtain a stratified liquid. The second organic phase layer is on the upper layer. The second main phase layer is on the lower layer. The second organic phase layer was removed, and 3% mannitol was added to the second main phase layer according to the mass ratio, and then freeze-dried to obtain blank plasma matrix dry powder.

[0132] test:

[0133] The dry powder of the blank plasma matrix is ​​reconstituted with ultrapure water to obtain a blank plasma matrix in a solution state. The ratio of ultrapure water to dry powder in this embodiment is 6.86, that is, when the mass of the dry powder of the blank plasma matrix is ​​1g, 6.86g of ultrapure water is added for reconstitution and vortex mixing. Then, the pH value of the blank plasma matrix reconstituted solution is adjusted to 7.4 with acetic acid; next, 0.04% ProClin 300 is added according to the mass ratio, and vortex mixing is continued to obtain an optimized blank plasma matrix. The optimized blank plasma matrix is ​​sealed and stored in a dark environment at -20°C.

[0134] The results were analyzed by liquid chromatography-tandem mass spectrometry (SCIEX Triple Quad TM The contents of seven ceramides in the optimized blank plasma matrix and unstripped mixed plasma were detected by 4500MD LC-MS / MS system. The measurement results are shown in Figure 1 and Figure 2 .

[0135] Figure 1These are the peaks of 7 ceramides and the corresponding internal standards in the unstripped mixed plasma, where the 7 ceramides include: 1, Cer (d18:1 / 14:0); 3, Cer (d18:1 / 16:0); 5, Cer (d18:1 / 18:0); 7, Cer (d18:1 / 20:0); 9, Cer (d18:1 / 22:0); 11, Cer (d18:1 / 24:0); 13, Cer (d18:1 / 24:1). The 7 internal standards corresponding to the 7 ceramides include: 2, Cer (d18:1-d7 / 14:0); 4, Cer (d18:1-d7 / 16:0); 6, Cer (d18:1-d7 / 18:0); 8, Cer (d18:1-d7 / 20:0); 10, Cer (d18:1-d7 / 22:0); 12, Cer (d18:1-d7 / 24:0); 14, Cer (d18:1-d7 / 24:1).

[0136] Figure 2 are the peaks of the internal standards corresponding to the 7 ceramides in the optimized blank plasma matrix. Figure 2 It can be seen that the peak heights of the seven ceramides in the blank plasma matrix are close to the baseline level. Only the peaks of the seven internal standards can be detected.

[0137] It can be seen that the stripping solvent can effectively strip the seven ceramides in the mixed plasma. Figure 1 It can be seen that the retention times of the internal standards of the seven ceramides in the unstripped mixed plasma chromatogram are the same as those of the internal standards of the seven ceramides in the blank plasma matrix.

[0138] Table 2 - Comparison of the content and removal rate of 7 ceramides in the optimized blank plasma matrix and unstripped mixed plasma of Example 2

[0139] Ceramide (ng / mL) Unstripped plasma pool Optimized blank plasma matrix Removal rate Cer(d18:1 / 14:0) 1.74 0 100.00％ Cer(d18:1 / 16:0) 166.50 0 100.00％ Cer(d18:1 / 18:0) 53.98 0 100.00％ Cer(d18:1 / 20:0) 57.87 0 100.00％ Cer(d18:1 / 22:0) 355.17 0 100.00％ Cer(d18:1 / 24:0) 1183.33 0 100.00％ Cer(d18:1 / 24:1) 555.17 0 100.00％

[0140] Note: If the target substance peak signal-to-noise ratio in the test results is less than 3, the content is marked as 0.

[0141] As shown in Table 2, the detected contents of the seven ceramides in the optimized blank plasma matrix were all 0. The stripping solvent can effectively strip the above seven ceramides in the mixed plasma.

[0142] <Example 3>

[0143] Preparation of steroid hormone blank serum matrix

[0144] The biological fluid is human serum. Human sera from multiple sources are mixed together, for example, sera from 30 people are mixed together. Part of the mixed human serum is used to prepare a blank biological fluid matrix, and part is used for comparison.

[0145] In the stripping solvent, the mass percentages of isopropanol, glycerol and methyl tert-butyl ether are 15%, 5% and 80% respectively, and all three reagents are chromatographically pure reagents. The stripping solvent is mixed evenly by vortex mixing. The stripping solvent is added to the human serum in a ratio of 1:1 by volume of human serum to stripping solvent, mixed for 60 minutes at a speed of 1200 rpm, and then the mixed solution is centrifuged for 10 minutes at a speed of 4000 rpm to obtain a stratified liquid. The first organic phase layer is on the upper layer. The first main phase layer is on the lower layer. The first organic phase layer is removed. The first main phase layer is mixed with methyl tert-butyl ether in a ratio of 1:1 by volume of the first main phase layer: methyl tert-butyl ether, and mixed for 20 minutes at a speed of 1200 rpm. Then centrifuged at a speed of 4000 rpm for 10 minutes to obtain a stratified liquid. The second organic phase layer is on the upper layer. The second main phase layer is on the lower layer. The second organic phase layer was removed, and 6% mannitol was added to the second main phase layer according to the mass ratio, and then freeze-dried to obtain a dry powder of a blank serum matrix.

[0146] The dry powder of the blank serum matrix is ​​reconstituted with ultrapure water to obtain a blank serum matrix in a solution state. The ratio of ultrapure water to dry powder in this embodiment is 6.87, that is, when the mass of the dry powder of the blank serum matrix is ​​1g, 6.87g of ultrapure water is added for reconstitution and vortex mixing. Then, the pH value of the blank serum matrix reconstituted solution is adjusted to 7.4 with acetic acid; next, 0.05% ProClin 300 is added according to the mass ratio, and vortex mixing is continued to obtain an optimized blank serum matrix. The optimized blank serum matrix is ​​sealed and stored in a dark environment at -20°C.

[0147] The results were analyzed by liquid chromatography-tandem mass spectrometry (SCIEX Triple Quad TM 6500 LC-MS / MS system) was used to detect the steroid hormone content in the blank serum matrix optimized in this example and the unstripped human serum, and the removal rate was calculated. The test results are shown in Table 3.

[0148] Table 3 - Comparison of steroid hormone content in blank serum matrix optimized in Example 3 and unstripped human serum

[0149]

[0150]

[0151] Note: If the target substance peak signal-to-noise ratio in the test results is less than 3, the content is marked as 0.

[0152] As shown in Table 3, the contents of 13 steroid hormones in the optimized blank serum matrix were reduced by more than 95% compared with those in unstripped human serum. TM The detection was carried out on a 6500 mass spectrometer, and the detected contents of testosterone, 11-deoxycorticosterone, 17-hydroxyprogesterone, corticosterone, aldosterone, estrone, estriol, 17-hydroxypregnenolone, and estrone sulfate in the optimized blank serum matrix were 0. The detected contents of androstenedione, progesterone, cortisone, and cortisol were less than 5% of the detected contents in unstripped human serum. The stripping solvent can effectively strip steroid hormones in human serum.

[0153] <Example 4>

[0154] The biological fluid is mixed serum. The mixed serum is the same as that in Example 1, and the stripping solvent composition and mixing process are the same as those in Example 1. The mixed serum stripping process is the same as that in Example 1. The difference from Example 1 is that after one stripping process is completed, the stripping process is repeated again using the same stripping solvent. The subsequent freeze-drying process, re-dissolution process, pH adjustment and addition of ProClin 300, sample pre-treatment process and detection process are the same as those in Example 1.

[0155] The contents and removal rates of the five fat-soluble vitamins in the optimized blank serum matrix after stripping and in the unstripped human serum of this example are shown in Table 4.

[0156] Table 4 - Comparison of the content of fat-soluble vitamins in the blank serum matrix optimized in Example 4 and in the unstripped human serum

[0157]

[0158] Note: When the target substance peak signal-to-noise ratio in the test results is <3, the content is marked as 0.

[0159] As shown in Table 4, the contents of the five fat-soluble vitamins in the optimized blank serum matrix are lower, and the detected contents are zero or close to zero. Among them, the removal rates of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E and vitamin K1 all reach 100%. The removal rate of vitamin A1 is close to 100%.

[0160] <Example 5>

[0161] The biological fluid is mixed serum. The mixed serum is the same as that in Example 3, the composition of the stripping solvent is isopropanol: methyl tert-butyl ether = 20:80, and the stripping solvent mixing process is the same as that in Example 3. The stripping treatment process of the mixed serum is the same as that in Example 3. The freeze-drying process, the reconstitution process, the operation mode of adjusting the pH value and adding ProClin 300, the sample pre-treatment process and the detection process are the same as those in Example 3.

[0162] The steroid hormone content and removal rate in the optimized blank serum matrix after stripping and the unstripped human serum of this example are shown in Table 5.

[0163] Table 5 - Comparison of steroid hormone content in blank serum matrix optimized in Example 5 and unstripped human serum

[0164] Steroid hormones (ng / mL) Unstripped human serum Optimized blank serum matrix Removal rate Androstenedione 1.32 0.05 96.35％ Testosterone 0.75 0.05 93.43％ Progesterone 25.71 0.10 99.60％ 11-Deoxycorticosterone 0.11 0.00 100.00％ 17-Hydroxyprogesterone 1.53 0.01 99.28％ Corticosterone 7.33 0.00 100.00％ Aldosterone 0.68 0.09 86.81％ Cortisone 19.25 0.90 95.32％ Cortisol 178.95 4.94 97.24％ Estrone 0.97 0.00 100.00％ Estriol 0.80 0.07 91.51％ 17-Hydroxypregnenolone 15.03 0.00 100.00％ Estrone sulfate 1.10 0.10 90.74％

[0165] Note: When the target substance peak signal-to-noise ratio in the test results is <3, the content is marked as 0.

[0166] As shown in Table 5, the contents of 13 steroid hormones in the optimized blank serum matrix were reduced by more than 85% compared with those in unstripped human serum. TM The detection was performed on a 6500 mass spectrometer, and the detected contents of 11-deoxycorticosterone, corticosterone, estrone, and 17-hydroxypregnenolone in the blank serum matrix were 0. The detected contents of androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisone, cortisol, estriol, and estrone sulfate in the optimized blank serum matrix were less than 10% of the detected contents in the unstripped human serum. The stripping solvent can effectively strip the steroid hormones in the mixed serum.

[0167] <Example 6>

[0168] The biological fluid is mixed serum. The mixed serum is the same as that in Example 1, and the stripping solvent, stripping treatment process, freeze-drying process, re-dissolution process, pH adjustment and addition of ProClin 300 are the same as those in Example 1. The unstripped human serum of this example and the blank serum matrix of this example are transferred. Three portions of unstripped human serum and optimized blank serum matrix are transferred, each portion is 400 μL, and added to 6 centrifuge tubes with a capacity of 2.0 mL. Then, 1000 μL of acetonitrile is added to each centrifuge tube and vortexed for 5 minutes. Next, centrifuge for 15 minutes at a temperature of 4°C and a speed of 15000 rpm / min to stratify the solution. Next, remove the supernatant to obtain a protein precipitate. The mass of the protein precipitate is weighed after freeze-drying. The mass of the protein precipitate in the six centrifuge tubes is shown in Table 6.

[0169] Table 6 - Comparison of protein content of unstripped human serum and the optimized blank serum matrix of this example

[0170]

[0171] As shown in Table 6, the difference between the mass of protein precipitate of the optimized blank serum matrix and the mass of protein precipitate of the unstripped mixed serum is only 3.45%, which indicates that the stripping solvent does not substantially change the protein content of the blank serum matrix.

[0172] <Example 7>

[0173] The biological fluid is human urine. Human urine from multiple sources is mixed together, for example, urine from 30 people is mixed together. Part of the mixed human urine is used to prepare a blank biological fluid matrix, and part is used for comparison.

[0174] The human urine stripping solution and stripping process are the same as those in Example 3. The difference from Example 3 is that 10% mannitol is added to the blank urine matrix according to the mass ratio before lyophilization, and the lyophilization process is the same as that in Example 3. The dry powder of the blank urine matrix is ​​reconstituted with ultrapure water to obtain a blank urine matrix reconstituted solution. In this embodiment, the ratio of ultrapure water to dry powder is 6.24, that is, when the mass of the blank urine matrix dry powder is 1g, 6.24g of ultrapure water is added and vortexed to mix. Then, the pH value of the blank urine matrix reconstituted solution is adjusted to 6.5 with acetic acid; next, 0.05% ProClin 300 is added according to the mass ratio, vortexed to mix, and an optimized blank urine matrix is ​​obtained. The optimized blank urine matrix is ​​sealed and stored in a dark place at -20°C.

[0175] The results were analyzed by liquid chromatography-tandem mass spectrometry (SCIEX Triple Quad TM 4500MD LC-MS / MS system) detected the free cortisol and cortisone in the blank urine matrix optimized in this example and the unstripped urine in this example, and calculated the removal rate. The test results are shown in Table 7.

[0176] Table 7 - Comparison of free cortisol and cortisone content in the optimized blank urine matrix and unstripped urine of Example 7

[0177] Free cortisol (ng / mL) Cortisol Cortisone Unstripped urine 3.270 20.34 Optimized blank urine matrix 0.000 0.68 Removal rate 100.00％ 96.66％

[0178] Note: If the target substance peak signal-to-noise ratio in the test results is less than 3, the content is marked as 0.

[0179] As shown in Table 7, the free cortisol and cortisone content in the optimized blank urine matrix were reduced by more than 99% compared with the unstripped urine. Among them, the detected content of free cortisol in the optimized blank urine matrix was 0. The stripping solvent can effectively strip free cortisol and cortisone in human urine.

[0180] <Comparative Example 1>

[0181] The biological fluid is mixed serum. The mixed serum is the same as that in Example 1, and the sum of the mass percentages of isopropanol and methanol and the mass percentage of methyl tert-butyl ether in the stripping solvent are 40% and 60%, respectively. The stripping solvent mixing process is the same as that in Example 1. The preparation process of the stripping serum matrix, the freeze-drying process, the reconstitution process, the operation mode of adjusting the pH value and adding ProClin 300, the fat-soluble vitamin sample pretreatment and the detection process are the same as those in Example 1.

[0182] The content and removal rate of fat-soluble vitamins in the stripped serum matrix prepared in Comparative Example 1 and the unstripped human serum are shown in Table 8.

[0183] Table 8 - Comparison of the content of fat-soluble vitamins in the stripped serum matrix of Comparative Example 1 and the unstripped human serum

[0184]

[0185]

[0186] Note: If the target substance peak signal-to-noise ratio in the test results is less than 3, the content is marked as 0.

[0187] As can be seen from Table 8, the stripped serum matrix obtained by stripping human serum with the stripping solvent of Comparative Example 1 has a 25-hydroxyvitamin D2 detection content of zero, and the removal rates of the other four are all below 95%, among which the removal rate of vitamin E is close to 85%. The residual vitamin A1, 25-hydroxyvitamin D3, vitamin E and vitamin K1 of the stripped serum matrix are high. Using the stripped serum matrix prepared in Comparative Example 1 as a diluent may result in a lower accuracy of the detection result of the target substance.

[0188] <Comparative Example 2>

[0189] The biological fluid is mixed serum. The mixed serum is the same as that in Example 1, and the sum of the mass percentages of isopropanol and methanol and the mass percentage of methyl tert-butyl ether in the stripping solvent are 60% and 40%, respectively. The stripping solvent mixing process is the same as that in Example 1. After adding the stripping solvent and mixing, protein precipitation is found in the mixed serum.

[0190] This indicates that the protein concentration of the stripped serum matrix obtained by stripping with the stripping solvent of Comparative Example 2 is significantly different from that of the unstripped mixed serum, and does not meet the requirement that the blank matrix and the unstripped human serum have similar compositions.

[0191] <Comparative Example 3>

[0192] The biological fluid is mixed serum. The mixed serum is the same as that in Example 1, and the stripping solvent is 100% methyl tert-butyl ether. The stripping serum matrix preparation process, freeze-drying process, reconstitution process, pH adjustment and ProClin 300 addition operation mode, sample pretreatment and detection process are the same as those in Example 1.

[0193] The contents and removal rates of the five fat-soluble vitamins in the stripped serum matrix and unstripped human serum prepared in Comparative Example 3 are shown in Table 9.

[0194] Table 9 - Comparison of the contents of five fat-soluble vitamins in the stripped serum matrix of Comparative Example 3 and unstripped human serum

[0195]

[0196]

[0197] Note: If the target substance peak signal-to-noise ratio in the test results is less than 3, the content is marked as 0.

[0198] As can be seen from Table 9, in the stripped serum matrix after stripping with the stripping solvent of Comparative Example 3, the removal rates of vitamin A1, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin K1 are about 50%, and the removal rate of vitamin E is about 40%. Therefore, the residual contents of vitamin A1, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin K1, and vitamin E in the stripped serum matrix after stripping are all high. Using the stripped serum matrix prepared in Comparative Example 3 as a diluent may result in a lower accuracy of the detection result of the target substance.

[0199] <Comparative Example 4>

[0200] The biological fluid is mixed plasma. The mixed plasma is the same as that in Example 2. Activated carbon is used to strip ceramide from the mixed plasma.

[0201] Step 1: Weigh activated carbon and mixed plasma in the same container at a ratio of 1 mL mixed plasma plus 30 mg activated carbon, mix well, sonicate for 40 min, then centrifuge at 10,000 rpm for 15 min, and take the supernatant;

[0202] Step 2: Repeat step 1 twice;

[0203] Step 3: Centrifuge the supernatant after three strippings at 10,000 rpm for 15 min, and then take the supernatant;

[0204] Step 4, repeating step 3 6 times to obtain a stripped matrix;

[0205] Step 5: dilute according to the ratio of stripping matrix to ultrapure water = 1:1 (v / v). Then,

[0206] Vortex to mix. Next, adjust the pH of the stripped plasma matrix to 7.4 with acetic acid, add 0.05% ProClin 300 according to the mass ratio, and continue to vortex to mix to obtain the optimized stripped plasma matrix plasma. The optimized stripped plasma matrix is ​​sealed and stored in a dark environment at -20°C.

[0207] The sample pretreatment and sample detection were performed in the same manner as in Example 2.

[0208] The contents and removal rates of seven ceramides in the optimized stripping matrix prepared in Comparative Example 4 and in unstripped human plasma are shown in Table 10.

[0209] Table 10 - Comparison of the contents of seven ceramides in the stripped plasma matrix of Comparative Example 4 and in unstripped human plasma

[0210]

[0211]

[0212] As can be seen from Table 10, activated carbon as a stripping agent has a good stripping effect on some ceramides in mixed plasma, such as Cer (d18:1 / 24:0), but has a poor stripping effect on some ceramides, such as Cer (d18:1 / 14:0), Cer (d18:1 / 16:0), and Cer (d18:1 / 18:0). Therefore, the residual contents of Cer (d18:1 / 14:0), Cer (d18:1 / 16:0), and Cer (d18:1 / 18:0) in the stripping matrix after stripping treatment are all high. Using the stripping matrix prepared in Comparative Example 4 as a calibrant and quality control diluent may result in a lower accuracy of the detection results of the target substance.

[0213] <Comparative Example 5>

[0214] The mixed serum was the same as that in Example 1, and the stripping solvent composition, the preparation process of the blank serum matrix, and the freeze-drying curve were the same as those in Example 1. Unlike Example 1, mannitol was not added to the blank serum matrix before freeze-drying in this comparative example. The dry powder of the blank serum matrix prepared in Example 1 was compared with the dry powder of the blank serum matrix prepared in Comparative Example 5. Figure 3 and Figure 4 .

[0215] Depend on Figure 3 and Figure 4It can be seen that in the blank serum matrix of Comparative Example 5 without adding mannitol, ice crystals are formed unevenly during the freeze-drying process, the dry powder has a poor structure and low mechanical strength, and is easily broken during transportation, storage or use, affecting the stability of the dry powder; and the dry powder prepared in Comparative Example 5 is more susceptible to environmental factors, such as humidity, temperature, etc., resulting in a shortened shelf life and an increased risk of dry powder deterioration. However, in Example 1, the blank serum matrix with mannitol added has uniform ice crystal formation during the freeze-drying process, the dry powder has a strong structure and high mechanical strength, and is not easily broken during transportation, storage or use. The dry powder is not easily affected by the environment and has a longer shelf life.

[0216] <Comparative Example 6>

[0217] The mixed serum is the same as that in Example 1, and the stripping solvent composition, the preparation process of the blank serum matrix, the freeze-drying process, the reconstitution process, and the pH adjustment process are the same as those in Example 1. Unlike Example 1, in this comparative example, ProClin 300 is not added to the blank serum matrix reconstitution solution. The state of the blank serum matrix reconstitution solution prepared in Comparative Example 6 after being placed in an environment of 2°C-8°C for 20 days is compared with the state of the blank serum matrix reconstitution solution prepared in Example 1 after being placed in an environment of 2°C-8°C for 20 days, see Figure 5 and Figure 6 .

[0218] The optimized blank serum matrix reconstituted solution of Example 1 is clear and transparent. Figure 5 As shown, the blank serum matrix reconstituted solution of Comparative Example 6 was turbid and had floccules, such as Figure 6 As shown, ProClin 300 can effectively antibacterial blank serum matrix reconstitution solution, and blank serum matrix reconstitution solution after adding ProClin 300 can be stored longer at 2℃-8℃. Calibrators and quality control products prepared with blank serum matrix reconstitution solution containing ProClin 300 have a longer storage time, thereby reducing the impact of repeated freezing and thawing on detection.

[0219] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will be appreciated by those skilled in the art that the above embodiments may be modified without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A reagent combination for preparing a blank biological liquid matrix, It is characterized in that The method comprises a stripping solvent and an additive, wherein the stripping solvent comprises alcohol and ether, and the additive comprises at least one of a dealcoholization solvent, an excipient, a pH regulator, and a protective agent; The de-alcoholization solvent is suitable for removing alcohol from the biological liquid after stripping; The excipient is suitable for forming a dry powder during freeze-drying of a blank biological liquid matrix; The pH regulator is suitable for adjusting the pH of the blank biological liquid matrix reconstitution solution; The protective agent is suitable for the preservation of blank biological liquid matrix re-solution.

2. The reagent combination according to claim 1, It is characterized in that In the stripping solvent, calculated by mass percentage, the content of the alcohol is 1% to 35%, and the content of the ether is 65% to 99%; preferably, the content of the alcohol is 5% to 35%, and the content of the ether is 65% to 95%.

3. The reagent combination according to claim 1, It is characterized in that The alcohol includes at least one of a monohydric alcohol and a polyhydric alcohol; preferably, the ether includes at least one of a monoether and a mixed ether.

4. The reagent combination according to claim 2, It is characterized in that The ether includes at least one of dimethyl ether, diethyl ether, dipropyl ether, methyl ethyl ether, ethyl propyl ether, methyl propyl ether, and methyl tert-butyl ether; the alcohol includes at least one of methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, and sorbitol; preferably, the ratio of the alcohol content to the methyl tert-butyl ether content is 5:95 to 25:

75.

5. The reagent combination according to claim 2, It is characterized in that The alcohol includes isopropanol, the ether includes methyl tert-butyl ether, and the ratio of the isopropanol content to the methyl tert-butyl ether content is 25:75 to 10:90; The alcohol includes isopropanol and ethanol, the ether includes methyl tert-butyl ether, and the ratio of the content of the isopropanol, the content of the ethanol and the content of the methyl tert-butyl ether is (5-15): (1-10): (75-94); or The alcohol includes n-butanol and ethanol, the ether includes methyl tert-butyl ether, and the ratio of the content of n-butanol, the content of ethanol and the content of methyl tert-butyl ether is (2-10): (6-15): (75-92); The alcohol includes isopropanol and glycerol, the ether includes methyl tert-butyl ether, and the ratio of the content of the isopropanol, the content of the glycerol and the content of the methyl tert-butyl ether is (10-20): (2-8): (88-72).

6. The reagent combination according to claim 1, It is characterized in that The dealcoholization solvent includes ether, preferably, the ether includes at least one of monoether and mixed ether, more preferably includes methyl tert-butyl ether; Optionally, the excipient includes at least one of mannitol, trehalose, and sucrose; Optionally, the pH adjuster includes at least one of sodium dihydrogen phosphate and acetic acid; Optionally, the protective agent includes at least one of ProClean 300, Buffpro 300, ProClin 300, ProClin150, ProClin200, and ProClin950.

7. A method for preparing a blank biological liquid matrix, It is characterized in that include: The biological liquid is mixed with the stripping solvent as claimed in any one of claims 1 to 6, and the layers are separated to obtain a first organic phase layer and a first main phase layer, and the first organic phase layer is removed to obtain the first main phase layer, wherein the first main phase layer is a blank biological liquid matrix.

8. The preparation method according to claim 7, It is characterized in that The biological fluid includes at least one of whole blood, plasma, serum, saliva, urine, cerebrospinal fluid, and milk.

9. The preparation method according to claim 7, It is characterized in that The volume ratio of the stripping solvent to the biological fluid is greater than or equal to 1:

2.

10. The preparation method according to claim 7, It is characterized in that drying the blank biological liquid matrix to prepare a dry powder; or The method also includes purifying the blank biological matrix liquid, including: adding a dealcoholizing agent to the obtained blank biological liquid matrix, mixing, layering to obtain a second organic phase layer and a second main phase layer, and removing the second organic phase layer.

11. The preparation method according to claim 10, It is characterized in that performing rotary evaporation on the second main phase layer to remove the dealcoholization agent in the second main phase layer; or The second main phase layer is dried to prepare a dry powder; preferably, the second main phase layer is dried by freeze-drying.

12. The preparation method according to claim 11, It is characterized in that The method further comprises adding an excipient before the blank biological liquid matrix is ​​prepared into dry powder. Preferably, the mass ratio of the excipient to the blank biological liquid matrix is ​​1% to 10%.

13. The preparation method according to claim 11, It is characterized in that The method further comprises re-dissolving the dry powder prepared from the blank biological liquid matrix to obtain a blank biological liquid matrix re-solution, and preferably, adding a protective agent and / or a pH regulator to the blank biological liquid matrix re-solution; Preferably, the mass ratio of the protective agent to the blank biological liquid matrix reconstituted solution is 0.01% to 0.2%; Preferably, the mass ratio of the pH regulator to the blank biological fluid matrix reconstituted solution is 0.1% to 0.5%.

14. A blank biological liquid matrix, It is characterized in that Prepared according to the preparation method according to any one of claims 7 to 13.

15. Application of blank biological liquid matrix in the detection of fat-soluble vitamins, ceramides or steroid hormones.

16. A kit, It is characterized in that Comprising the reagent combination according to any one of claims 1 to 6 and / or the blank biological liquid matrix according to any one of claims 7 to 13.

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