Reagent combinations, blank biological liquid matrix and its preparation methods, applications, and kits

Abstract

The application discloses a reagent combination for preparing a blank biological liquid matrix, a blank biological liquid matrix and a preparation method, application and kit thereof. 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 biological liquid after stripping. The excipient is suitable for forming a dry powder in the process of freeze-drying the blank biological liquid matrix. The pH regulator is suitable for adjusting the pH value of a reconstituted solution of the blank biological liquid matrix. The protective agent is suitable for preserving the reconstituted solution of the blank biological liquid matrix.

Description

Technical Field

[0001] This invention relates to the field of laboratory testing technology, and more specifically, to a reagent combination for preparing a blank biological liquid matrix, the blank biological liquid matrix and its preparation method, application, and kit. Background Technology

[0002] In related technologies, calibrators and quality control samples used in reagent combinations for detecting target substances, such as fat-soluble vitamins, ceramides, and steroid hormones, are typically prepared using alternative matrices. These alternative matrices include organic solvents, bovine serum albumin solutions, or human serum albumin solutions. However, these alternative matrices differ significantly from the composition of the sample being tested (e.g., blood), such as protein composition and concentration, leading to inaccurate detection results for the target substances.

[0003] Therefore, a new technical solution is needed to solve the above-mentioned technical problems. Summary of the Invention

[0004] One object of the present invention is to provide a new technical solution for reagent combinations for preparing blank biological liquid matrices.

[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 includes a stripping solvent and an additive, the stripping solvent comprising an alcohol and an ether, and the additive comprising at least one selected from a dealcoholizing solvent, an excipient, a pH adjuster, and a protective agent;

[0006] The alcohol removal solvent is suitable for removing alcohols from the stripped biological liquid matrix;

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

[0008] The pH adjuster is suitable for adjusting the acidity or alkalinity of blank biological liquid matrix reconstituted solutions;

[0009] The protective agent is suitable for the preservation of blank biological liquid matrix reconstituted solutions.

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

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

[0012] Optionally, the ether includes at least one selected from 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 selected from 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 from 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 isopropanol to the content of 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 isopropanol, the content of ethanol, and the content of 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 content of isopropanol, glycerol and methyl tert-butyl ether is (10-20):(2-8):(88-72).

[0017] Optionally, the dealcoholizing solvent includes an ether; preferably, the ether includes at least one of a monoether and a mixture of ethers; more preferably, it 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, ProClin 150, ProClin 200, and ProClin 950.

[0021] According to a second aspect of this application, a method for preparing a blank biological liquid matrix is ​​provided. The method includes: mixing a biological liquid with the stripping solvent described in this application, separating the layers to obtain a first organic phase layer and a first main phase layer, and 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 breast 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] It also includes purifying the blank biological matrix liquid, including: adding a dealcoholizing agent to the obtained blank biological liquid matrix, mixing, separating into layers 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 alcohol-removing agent from 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 includes adding an excipient before preparing the blank biological liquid matrix into a dry powder, preferably, the mass ratio of the excipient to the blank biological liquid matrix is ​​1% to 10%.

[0029] Optionally, it also includes reconstituted the dry powder prepared from the blank biological liquid matrix to obtain a blank biological liquid matrix reconstituted solution. Preferably, a protective agent and / or pH adjuster are added to the blank biological liquid matrix reconstituted solution.

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

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

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

[0033] According to a fourth aspect of this application, a blank biological liquid matrix is ​​provided for the detection of fat-soluble vitamins, ceramides, or steroid hormones.

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

[0035] In this embodiment, the stripping solvent in the reagent combination effectively removes target substances, such as fat-soluble vitamins, ceramides, and steroid hormones, from the biological liquid, thereby forming a blank biological liquid matrix. This blank biological liquid matrix has a similar protein concentration to the unstripped biological liquid, and can therefore be used as a calibrator or quality control sample for detecting target substances, such as fat-soluble vitamins, ceramides, and steroid hormones. The alcohol removal solvent removes alcohols from the stripped biological liquid, effectively purifying the blank biological liquid matrix. The excipient enables the dry powder to solidify during the freeze-drying process of the blank biological liquid matrix; the pH adjuster adjusts the pH of the blank biological liquid matrix reconstituted solution; and the preservative is suitable for preserving the blank biological liquid matrix reconstituted solution.

[0036] Other features and advantages of the invention will become clear from the following detailed description of exemplary embodiments of the invention with reference to the accompanying drawings. Attached Figure Description

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

[0038] Figure 1 This is a chromatogram of seven ceramides in the mixed plasma of Example 2 of this application.

[0039] Figure 2 This is a chromatogram of seven ceramides in the optimized blank plasma matrix of Example 2 of this application.

[0040] Figure 3 This is an image of the dry powder of the blank serum matrix prepared in Example 1 of this application.

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

[0042] Figure 5 Example 1: Photograph of the optimized blank serum matrix reconstituted solution after storage at 2-8℃ for 20 days.

[0043] Figure 6 Photograph of the blank serum matrix reconstituted solution in Comparative Example 6 after being stored at 2-8℃ for 20 days.

[0044] Figure label: 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 Implementation

[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, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

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

[0047] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and equipment should be considered part of the specification.

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

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

[0050] According to a first embodiment of this 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 alcohols and ethers, and the additive includes at least one selected from a dealcoholizing solvent, an excipient, a pH adjuster, and a protective agent.

[0051] The alcohol removal solvent is suitable for removing alcohols from the biofluid after stripping.

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

[0053] The pH adjuster is suitable for adjusting the acidity or alkalinity of blank biological liquid matrix reconstituted solutions;

[0054] The protective agent is suitable for the preservation of blank biological liquid matrix reconstituted solutions.

[0055] In this embodiment, the stripping solvent can effectively strip target substances, such as fat-soluble vitamins, ceramides, and steroid hormones, from the biological liquid, thereby forming a blank biological liquid matrix. This blank biological liquid matrix has a similar protein concentration to the unstripped biological liquid, and the retention time of the target substances in the matrix before and after stripping is similar. It can be used as a calibrator and quality control sample for detecting target substances, such as fat-soluble vitamins, ceramides, and steroid hormones.

[0056] The alcohol removal solvent can remove alcohol from the stripped biofluid, thereby effectively purifying the blank biofluid matrix. Optionally, the alcohol removal solvent includes ethers. Preferably, the ether includes at least one of monoethers and mixed ethers. For example, the ether includes at least one of dimethyl ether, diethyl ether, dipropyl ether, methyl ethyl ether, ethylpropyl 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 from the stripped biofluid.

[0057] The blank biological liquid matrix can be further purified by rotary evaporation and drying to remove residual alcohols and ethers. The blank liquid matrix after removing organic solvents by rotary evaporation can be directly used for the preparation of calibrators and quality control samples.

[0058] Alternatively, the blank biological liquid matrix can be prepared as a dry powder. For example, a freeze-drying process can be used to prepare the blank biological liquid matrix into a dry powder for easy storage. When needed, the dry powder is reconstituted to obtain a blank biological liquid matrix reconstituted solution. This reconstituted solution can be used to prepare calibrators and quality control samples. The dry powder effectively extends the shelf life of the blank biological liquid matrix; for example, it can be stored for more than one year.

[0059] The excipient enables the formation of dry powder during the freeze-drying of a blank biological liquid matrix. For example, the excipient is added to and mixed with the blank biological liquid matrix before freeze-drying; during freeze-drying, the excipient promotes the crystallization of the blank biological liquid matrix and resists damage to proteins caused by stresses such as low temperature and drying, thus improving the speed and quality of dry powder formation. The final product is a cake-like dry powder. Optionally, the excipient includes at least one of mannitol, trehalose, and sucrose, all of which promote dry powder formation. 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 forms quickly and with high quality, exhibiting a uniform cake-like structure.

[0060] The pH adjuster can adjust the pH of the blank biological liquid matrix reconstituted solution. The dry powder needs to be reconstituted before use to form the blank biological liquid matrix reconstituted solution. Preferably, adding a pH adjuster to the reconstituted solution ensures that the pH of the reconstituted solution meets the usage requirements. Optionally, the pH adjuster includes at least one of sodium dihydrogen phosphate and acetic acid. Both of these substances are acidic and can effectively adjust the pH of the reconstituted solution. Optionally, the mass ratio of the pH adjuster to the blank biological liquid matrix reconstituted solution is 0.1% to 0.5%. For example, mass ratios of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, etc. Within the above range, the pH adjuster can effectively adjust the pH of the blank biological liquid matrix reconstituted solution.

[0061] The preservative effectively prevents the reconstituted blank biological liquid matrix solution from spoiling, thus extending its shelf life. For example, adding a preservative to the reconstituted solution can prevent spoilage during long-term storage. Optionally, the preservative includes at least one of ProClean 300, Buffpro 300, ProClin 300, ProClin 150, ProClin 200, and ProClin 950. The mass ratio of the preservative to the blank biological liquid matrix reconstituted solution is 0.01% to 0.2%. For example, mass ratios of 0.01%, 0.05%, 0.08%, 0.1%, 0.13%, 0.15%, 0.17%, and 0.2% are possible. The above substances and mass ratios effectively prevent spoilage of the reconstituted solution.

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

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

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

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

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

[0067] Excessive alcohol content can lead to protein precipitation during the stripping of target substances (e.g., fat-soluble vitamins, ceramides, steroid hormones) from the biological fluid, resulting in a significant difference in matrix between the blank biological fluid and the biological fluid. Conversely, insufficient alcohol content leads to low removal rates of target substances. Optionally, the alcohol content can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, etc. Correspondingly, the ether content can be 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc. Within the above ranges, the stripping solvent can effectively remove target substances from the biological fluid without forming protein precipitation during the stripping process.

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

[0069] Optionally, monohydric alcohols include methanol, ethanol, propanol, butanol, isopropanol, etc. Polyhydric alcohols include ethylene glycol, propylene glycol, glycerol, sorbitol, etc.

[0070] Preferably, the alcohol does not include structurally unstable enols. Enols have poor stability, and during use, the hydroxyl group can convert to a carbonyl group, making it impossible to effectively remove the target substance.

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

[0072] Optionally, monoethers include dimethyl ether, diethyl ether (i.e., diethyl ether), dipropyl ether, etc. Mixed ethers include methyl ethyl ether, ethylpropyl ether, methyl propyl ether, methyl tert-butyl ether, etc.

[0073] Preferably, the ether does not include water-miscible ethers, highly reactive cyclic ethers, or crown ethers capable of complexing metal ions in the biological matrix. Water-miscible ethers cannot be separated after mixing with the biological liquid, resulting in the inability to strip the target substance. Highly reactive cyclic ethers readily react with substances in the biological liquid, thereby affecting the composition of the blank biological liquid matrix. Crown ethers can undergo complexation reactions with metal ions in the biological liquid, causing a difference in ionic strength between the prepared blank biological liquid matrix and the biological liquid.

[0074] In one example, in the stripping solvent, the ether comprises 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 comprises at least one of methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, and sorbitol.

[0075] In this example, the ether effectively removes target substances from biological fluids. 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, the ratio of the alcohol content to the ether content in the stripping solvent is from 5:95 to 25:75.

[0077] In this example, the stripping solvent comprises a mixture of ethers and alcohols. Optionally, the ratio of the alcohol to the ether content is 5:95, 10:90, 12:88, 15:85, 18:82, 20:80, 22:78, 25:75, etc. Within the above ranges, the stripping solvent can effectively remove fat-soluble vitamins, ceramides, steroid hormones, and other substances from biological fluids without forming protein precipitation during the stripping process.

[0078] In one example, the alcohol in the stripping solvent 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; in this example, the stripping solvent comprises a mixture of methyl tert-butyl ether and isopropanol. Optionally, the above content ratios are 10:90, 12:88, 15:85, 18:82, 20:80, 22:78, 25:75, etc. Within the above ranges, the stripping solvent has a good stripping effect on fat-soluble vitamins, ceramides, steroid hormones, and other substances.

[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 content of isopropanol, the content of ethanol and the content of methyl tert-butyl ether is (5-15):(1-10):(75-94).

[0080] In this example, the stripping solvent comprises a mixture of methyl tert-butyl ether, ethanol, and isopropanol. Optionally, the ratio of these components 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 these ranges, the stripping solvent exhibits good stripping effects on fat-soluble vitamins, ceramides, steroid hormones, and other substances.

[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 comprises a mixture of n-butanol, ethanol, and methyl tert-butyl ether. Optionally, the ratios of these components are: 2:6:92, 4:7:89, 5:10:85, 6:12:82, 7:13:80, 9:11:80, 10:15:75, etc. Within these ranges, the stripping solvent exhibits good stripping effects on fat-soluble vitamins, ceramides, steroid hormones, and other substances.

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

[0084] In this example, the stripping solvent comprises a mixture of isopropanol, glycerol, and methyl tert-butyl ether. Optionally, the ratios of these components are: 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 these ranges, the stripping solvent exhibits good stripping effects on fat-soluble vitamins, ceramides, steroid hormones, and other substances.

[0085] In one example, as previously stated, the dealcoholizing solvent comprises an ether, preferably, the ether comprises at least one of a monoether and a mixture of ethers, more preferably, 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, ProClin 150, ProClin 200, and ProClin 950.

[0089] According to a second embodiment of this application, a method for preparing a blank biological liquid matrix is ​​provided. The method includes mixing a biological liquid with a stripping solvent provided in the embodiments of this application, separating the layers to obtain a first organic phase layer and a first main phase layer, and 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 practice, firstly, based on the lipophilicity of the target analyte, the types and ratios of alcohol and ether are adjusted. Preferably, the alcohol content is 1% to 35% and the ether content is 65% to 99% by mass percentage. Within this range, the ratio of alcohol to 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 volume ratio of stripping solvent to biological liquid of 1:2 or greater, the stripping solvent is added to the container containing the biological liquid and mixed thoroughly, for example, by centrifugation to separate the layers. For example, the first main phase layer is an aqueous phase layer. Since the density of the first organic phase layer is less than that of the aqueous phase layer, the first organic phase layer is located on top of the first main phase layer. The target substance is dissolved in the first organic phase layer. After removing the first organic phase layer, the remaining first main phase layer is the blank biological liquid matrix.

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

[0093] The above preparation method can effectively remove target substances, such as fat-soluble vitamins, steroid hormones, and ceramides, from biological liquids to obtain blank biological liquid matrix.

[0094] Preferably, to better preserve the blank biological liquid matrix, the obtained blank biological liquid matrix is ​​dried to prepare a dry powder. For example, freeze-drying can be used to prepare the blank biological liquid matrix into a dry powder. This method can both remove the organic solvents in the blank biological liquid matrix and preserve it. When using, 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 breast 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] Within the aforementioned range, the stripping efficiency of target substances in biological fluids is relatively high.

[0098] Optionally, the volume ratio of the stripping solvent to the bio-liquid is 1:2, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, etc. During preparation, setting the amounts of bio-liquid and stripping solvent according to the above volume ratios can effectively remove the target substances from the bio-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, separating into layers to obtain a second organic phase layer and a second main phase layer, and removing the second organic phase layer.

[0100] Because the stripping solvent contains alcohols and ethers, these two substances are partially miscible into the blank biological liquid matrix during preparation. In this example, by adding a dealcoholizing agent to the blank biological liquid matrix and mixing thoroughly, followed by 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 that 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 liquid matrix, the addition of dealcoholizing agent and removal of the second organic phase layer can be performed multiple times.

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

[0103] In one example, the preparation method further includes rotary evaporation of the second main phase layer to remove the ether from the second main phase layer.

[0104] Rotary evaporation is a method of concentrating or separating solutes from a sample by rapidly evaporating the solvent in the sample container under reduced pressure through rotation and heating. This method can effectively remove ethers from the second master phase.

[0105] Another method for removing ether from the second main phase layer is 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, ethers gradually evaporate. By drying the blank biological liquid matrix and preparing it into a dry powder, organic solvents such as ethers in the blank biological liquid matrix can be effectively removed. Furthermore, the dry powder facilitates the preservation of the blank biological liquid matrix. When using it, the dry powder can be reconstituted with a solvent, such as ultrapure water. The volume of solvent added can be determined based on the ratio of volatiles to dry powder after the corresponding biological liquid drying process (without stripping treatment) and the mass of the dry powder.

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

[0108] The excipients are as described above. The excipients effectively improve the speed and quality of dry powder formation, ultimately resulting in a cake-like dry powder structure.

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

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

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

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

[0113] The target substance in this blank biological liquid matrix is ​​present in low concentrations, making it suitable for preparing quality control and calibrators for target substance detection, thus ensuring high accuracy of target substances in biological liquids.

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

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

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

[0117] This reagent combination can be used to prepare a blank biological liquid matrix. This blank biological liquid matrix can be prepared as a dry powder. During use, the dry powder is dissolved in ultrapure water to obtain a liquid blank biological liquid matrix. Because this kit uses the blank biological liquid matrix from the embodiments of this application to prepare calibrators and quality control samples, the detection of target substances is more accurate.

[0118] <Example 1>

[0119] Preparation of fat-soluble vitamin blank serum matrix

[0120] The biological liquid is human serum. Human serum from multiple sources is mixed together, for example, serum from 30 individuals, resulting in a mixed serum. A portion of the mixed human serum is used to prepare a blank biological liquid matrix, and another portion is used for comparison.

[0121] The stripping solvent contained isopropanol, ethanol, and methyl tert-butyl ether at mass percentages of 10%, 5%, and 85%, respectively, all of which were chromatographically pure. The stripping solvent was vortexed to ensure homogeneity. The stripping solvent was added to human serum at a volume ratio of 1:1.5 (first main phase to stripping solvent), and the mixture was stirred at 1000 rpm for 40 minutes. The resulting solution was then centrifuged at 4000 rpm for 10 minutes, yielding a layered liquid with the first organic phase on top and the first main phase on the bottom. The first organic phase was removed. The first main phase and methyl tert-butyl ether were then mixed together at a volume ratio of 1:1.5 (first main phase to methyl tert-butyl ether), and stirred at 1000 rpm for 40 minutes. The mixture was then centrifuged at 4000 rpm for 10 minutes, yielding a layered liquid with the second organic phase on top and the second main phase on the bottom. Remove the second organic phase layer, add 5% mannitol to the second main phase layer according to the mass ratio, and then freeze-dry to obtain the dry powder of blank serum matrix.

[0122] test:

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

[0124] Using liquid chromatography-tandem mass spectrometry (SCIEX Triple Quad) TM The content of five fat-soluble vitamins in the optimized blank serum matrix and the unpeeled human serum of this embodiment was detected by a 4500MD LC-MS / MS system, and the removal rate was calculated. The detection results are shown in Table 1.

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

[0126]

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

[0128] As shown in Table 1, the optimized blank serum matrix exhibited a reduction of over 99% in the levels of fat-soluble vitamins A1, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, and vitamin K1 compared to unstripped human serum. Specifically, the optimized blank serum matrix showed zero detection levels of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, and the detection levels of vitamins A1, E, and K1 were less than 1% of those found in unstripped human serum. This stripping solvent effectively removes fat-soluble vitamins from human serum.

[0129] <Example 2>

[0130] Preparation of ceramide blank plasma

[0131] The biological liquid is human plasma. Human plasma from multiple sources is mixed together, for example, plasma from 30 individuals, to form a mixed plasma. A portion of the mixed plasma is used to prepare a blank plasma matrix, and another portion is used for control. 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 vortexed to ensure homogeneity. The stripping solvent is added to the mixed plasma at a volume ratio of 1:2 (mixed plasma:stripping solvent), and the mixture is mixed head-to-head for 40 min. The resulting solution is then centrifuged at 4000 rpm for 15 min to obtain a layered liquid. The first organic phase layer is on top. The first main phase layer is on the bottom. The first organic phase layer is removed. The first main phase layer is then mixed with methyl tert-butyl ether at a volume ratio of 1:2 (mixed main phase layer:stripping ether), and the mixture is mixed head-to-head for 10 min. Next, the mixture is centrifuged at 4000 rpm for 10 min to obtain a layered liquid. The second organic phase layer is on top. The second main phase layer is on the bottom. Remove the second organic phase layer, add 3% mannitol to the second main phase layer by mass ratio, and then freeze-dry to obtain blank plasma matrix dry powder.

[0132] test:

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

[0134] Using liquid chromatography-tandem mass spectrometry (SCIEX Triple Quad) TM The levels of seven ceramides in the optimized blank plasma matrix and unpeeled mixed plasma were detected using a 4500MD LC-MS / MS system. The results are shown in [Figure number missing]. Figure 1 and Figure 2 .

[0135] Figure 1The peaks of seven ceramides and their corresponding internal standards in unseparated mixed plasma are shown. The seven ceramides are: 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 seven internal standards corresponding to the seven ceramides are: 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 The peaks of the seven ceramides corresponding to the internal standards in the optimized blank plasma matrix are shown. Figure 2 As can be seen, the peak heights of the seven ceramides in the blank plasma matrix were close to the baseline level. Only the peaks of the seven internal standards could be detected.

[0137] It is evident that the stripping solvent can effectively strip seven types of ceramides from mixed plasma. (From...) Figure 1 It is evident that the retention times of the internal standards for the seven ceramides in the unpeeled mixed plasma chromatogram were the same as those in the blank plasma matrix.

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

[0139] Ceramide (ng / mL) Unstripped mixed plasma 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 signal-to-noise ratio of the target substance peak in the test results is <3, the content is marked as 0.

[0141] As shown in Table 2, the detection levels of all seven ceramides in the optimized blank plasma matrix were 0. This stripping solvent effectively stripped the aforementioned seven ceramides from the mixed plasma.

[0142] <Example 3>

[0143] Preparation of blank serum matrix for steroid hormones

[0144] The biological liquid is human serum. Human serum from multiple sources is mixed together, for example, serum from 30 individuals. A portion of the mixed human serum is used to prepare a blank biological liquid matrix, and another portion is used for comparison.

[0145] The stripping solvent contained isopropanol, glycerol, and methyl tert-butyl ether at mass percentages of 15%, 5%, and 80%, respectively, all of which were chromatographically pure. The stripping solvent was thoroughly mixed using a vortex mixer. The stripping solvent was added to the human serum at a volume ratio of 1:1, and the mixture was stirred at 1200 rpm for 60 min. The resulting solution was then centrifuged at 4000 rpm for 10 min to obtain a layered liquid. The first organic phase was on top, and the first main phase was on the bottom. The first organic phase was removed. The first main phase and methyl tert-butyl ether were then mixed together at a volume ratio of 1:1, and stirred at 1200 rpm for 20 min. The mixture was then centrifuged at 4000 rpm for 10 min to obtain a layered liquid. The second organic phase was on top, and the second main phase was on the bottom. Remove the second organic phase layer, add 6% mannitol to the second main phase layer by mass ratio, and then freeze-dry to obtain a dry powder of blank serum matrix.

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

[0147] Using liquid chromatography-tandem mass spectrometry (SCIEX Triple Quad) TM The steroid hormone content in the optimized blank serum matrix and unpeeled human serum in this embodiment was detected by a 6500LC-MS / MS system, and the removal rate was calculated. The detection results are shown in Table 3.

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

[0149]

[0150]

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

[0152] As shown in Table 3, the levels of all 13 steroid hormones in the optimized blank serum matrix were reduced by more than 95% compared to those in unstripped human serum. This was achieved using the highly sensitive SCIEX Triple Quad... TM Detection was performed using a 6500 mass spectrometer. The optimized blank serum matrix showed zero levels of testosterone, 11-deoxycorticosterone, 17-hydroxyprogesterone, corticosterone, aldosterone, estrone, estriol, 17-hydroxypregnenolone, and estrone sulfate. The levels of androstenedione, progesterone, cortisone, and cortisol were less than 5% of those found in unstripped human serum. This stripping solvent effectively strips steroid hormones from human serum.

[0153] <Example 4>

[0154] The biological liquid was a mixed serum. This mixed serum was the same as in Example 1, with the same stripping solvent composition and mixing process. The stripping process for the mixed serum was also the same as in Example 1. The difference from Example 1 was that after one stripping treatment, the same stripping solvent was used to repeat the stripping process once more. Subsequent lyophilization, reconstitution, pH adjustment, and the addition of ProClin 300, as well as the sample pretreatment and detection processes, were the same as in Example 1.

[0155] The content and removal rate of five fat-soluble vitamins in the optimized blank serum matrix and the unpeeled human serum of this embodiment are shown in Table 4.

[0156] Table 4 - Comparison of fat-soluble vitamin content in optimized blank serum matrix and unpeeled human serum in Example 4

[0157]

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

[0159] As shown in Table 4, the optimized blank serum matrix contained even lower levels of the five fat-soluble vitamins, with detection levels at or near zero. Specifically, the removal rates of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, and vitamin K1 all reached 100%. The removal rate of vitamin A1 was close to 100%.

[0160] <Example 5>

[0161] The biological liquid was a mixed serum. This mixed serum was the same as in Example 3, with the stripping solvent consisting of isopropanol:methyl tert-butyl ether = 20:80. The mixing process of the stripping solvent was the same as in Example 3. The stripping process of the mixed serum was the same as in Example 3. The lyophilization process, reconstitution process, pH adjustment, and addition of ProClin 300, as well as the sample pretreatment and detection processes, were the same as in Example 3.

[0162] Table 5 shows the steroid hormone content and removal rate in the optimized blank serum matrix and the un-peeled human serum in this embodiment.

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

[0164] Steroid hormones (ng / mL) Unpeeled 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 signal-to-noise ratio of the target substance peak in the detection results is <3, the content is marked as 0.

[0166] As shown in Table 5, the levels of all 13 steroid hormones in the optimized blank serum matrix were reduced by more than 85% compared to those in unstripped human serum. This was achieved using the highly sensitive SCIEX Triple Quad... TM Detection was performed using a 6500 mass spectrometer. The blank serum matrix showed zero levels of 11-deoxycorticosterone, corticosterone, estrone, and 17-hydroxypregnenolone. The optimized blank serum matrix showed levels of androstenedione, testosterone, progesterone, 17-hydroxyprogesterone, cortisone, cortisol, estriol, and estrone sulfate less than 10% of those found in unstripped human serum. This stripping solvent effectively strips steroid hormones from mixed serum.

[0167] <Example 6>

[0168] The biological liquid was a mixed serum. This mixed serum was identical to that in Example 1, with the same procedures for solvent stripping, stripping treatment, lyophilization, reconstitution, pH adjustment, and addition of ProClin 300. Unstripped human serum and the optimized blank serum matrix from this example were transferred. Three 400 μL portions of both the unstripped human serum and the optimized blank serum matrix were transferred into six 2.0 mL centrifuge tubes. Then, 1000 μL of acetonitrile was added to each tube, and the mixture was vortexed for 5 minutes. Next, the mixture was centrifuged at 15000 rpm for 15 minutes at 4°C to separate the solutions. The supernatant was then removed to obtain protein precipitate. The protein precipitate was lyophilized and weighed. The masses of the protein precipitate in the six centrifuge tubes are shown in Table 6.

[0169] Table 6 - Comparison of protein content between unpeeled human serum and the optimized blank serum matrix of this embodiment.

[0170]

[0171] As shown in Table 6, the difference in protein precipitation quality between the optimized blank serum matrix and the unexfoliated mixed serum matrix was only 3.45%. This indicates that the exfoliation solvent essentially did not change the protein content of the blank serum matrix.

[0172] <Example 7>

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

[0174] The human urine stripping solution and stripping process were the same as in Example 3. The difference from Example 3 was that 10% mannitol was added to the blank urine matrix at a specific mass ratio before freeze-drying, and the freeze-drying process was the same as in Example 3. The dry powder of the blank urine matrix was reconstituted with ultrapure water to obtain a blank urine matrix reconstituted solution. In this example, the ratio of ultrapure water to dry powder was 6.24, that is, when the mass of the blank urine matrix dry powder was 1g, 6.24g of ultrapure water was added, and the mixture was vortexed. Then, the pH of the blank urine matrix reconstituted solution was adjusted to 6.5 with acetic acid; next, 0.05% ProClin 300 was added at a specific mass ratio, and the mixture was vortexed to obtain the optimized blank urine matrix. The optimized blank urine matrix was sealed and stored in the dark at -20°C.

[0175] Using liquid chromatography-tandem mass spectrometry (SCIEX Triple Quad) TM The optimized blank urine matrix and the unremoved urine in this embodiment were detected by a 4500MD LC-MS / MS system, and the removal rate was calculated. The results are shown in Table 7.

[0176] Table 7 - Comparison of free cortisol and cortisol content in optimized blank urine matrix and unstripped urine in 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 signal-to-noise ratio of the target substance peak in the test results is <3, the content is marked as 0.

[0179] As shown in Table 7, the levels of free cortisol and cortisol in the optimized blank urine matrix were reduced by more than 99% compared to the unstripped urine. Specifically, the detection rate of free cortisol in the optimized blank urine matrix was 0. This stripping solvent effectively strips free cortisol and cortisol from human urine.

[0180] <Comparative Example 1>

[0181] The biological liquid was a mixed serum. This mixed serum was identical to that in Example 1, with the sum of the mass percentages of isopropanol and methanol in the stripping solvent and the mass percentages of methyl tert-butyl ether being 40% and 60%, respectively. The mixing process of the stripping solvent was the same as in Example 1. The preparation process of the stripped serum matrix, the lyophilization process, the reconstitution process, the pH adjustment and addition of ProClin 300, and the pretreatment and detection process of the fat-soluble vitamin samples were the same as in Example 1.

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

[0183] Table 8 - Comparison of fat-soluble vitamin content in exfoliated serum matrix and unexfoliated human serum in Comparative Example 1

[0184]

[0185]

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

[0187] As shown in Table 8, the stripped serum matrix obtained after stripping human serum using the stripping solvent of Comparative Example 1 showed zero detection of 25-hydroxyvitamin D2, and the removal rates of the other four vitamins were all below 95%, with vitamin E removal approaching 85%. The stripped serum matrix contained high levels of vitamin A1, 25-hydroxyvitamin D3, vitamin E, and vitamin K1. Using the stripped serum matrix prepared in Comparative Example 1 as a diluent may lead to lower accuracy in the detection results of the target substances.

[0188] <Comparative Example 2>

[0189] The biological fluid was a mixed serum. This mixed serum was the same as in Example 1, with the sum of the mass percentages of isopropanol and methanol in the stripping solvent being 60% and 40% by mass of methyl tert-butyl ether, respectively. The stripping solvent mixing process was the same as in Example 1. After adding the stripping solvent and mixing, protein precipitation was observed in the mixed serum.

[0190] This indicates that the protein concentration of the stripped serum matrix obtained by the stripping solvent treatment in Comparative Example 2 differs significantly from the protein concentration of the unstripped mixed serum, failing to meet the requirement that the blank matrix and the unstripped human serum have similar composition.

[0191] <Comparative Example 3>

[0192] The biological liquid was a mixed serum. This mixed serum was the same as in Example 1, with 100% methyl tert-butyl ether as the stripping solvent. The preparation process of the stripped serum matrix, the lyophilization process, the reconstitution process, the pH adjustment and addition of ProClin 300, the sample pretreatment, and the detection process were all the same as in Example 1.

[0193] The content and removal rate of five fat-soluble vitamins in the stripped serum matrix and the 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 exfoliated serum matrix and the unexfoliated human serum of Comparative Example 3

[0195]

[0196]

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

[0198] As shown in Table 9, the removal rates of vitamin A1, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin K1 in the stripped serum matrix treated with the stripping solvent of Comparative Example 3 were approximately 50%, and the removal rate of vitamin E was approximately 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 treatment were all relatively high. Using the stripped serum matrix prepared in Comparative Example 3 as a diluent may result in lower accuracy of the detection results for the target substances.

[0199] <Comparative Example 4>

[0200] The biological fluid was a mixed plasma. This mixed plasma was the same as in Example 2. Activated charcoal was used to remove ceramides from the mixed plasma.

[0201] Step 1: Weigh out activated charcoal and measure out mixed plasma into the same container according to the ratio of 1mL of mixed plasma to 30mg of activated charcoal, mix them evenly, sonicate for 40min, and then centrifuge at 10000rpm for 15min and take the supernatant.

[0202] Step 2: Repeat step 1 twice;

[0203] Step 3: Centrifuge the supernatant after three separations at 10000 rpm for 15 minutes, and then take the supernatant.

[0204] Fourth step: Repeat step three six times to obtain the exfoliation matrix;

[0205] Fifth, dilute the stripping matrix with ultrapure water at a ratio of 1:1 (v / v). Then,

[0206] Vortex mix. Next, adjust the pH of the stripped plasma matrix to 7.4 with acetic acid, add 0.05% ProClin 300 by mass, and continue vortex mixing to obtain optimized stripped plasma matrix. The optimized stripped plasma matrix is ​​stored sealed and protected from light at -20°C.

[0207] Sample pretreatment and sample detection were performed using the same method as in Example 2.

[0208] Table 10 shows the content and removal rate of seven ceramides in the optimized stripping matrix and unstripped human plasma prepared in Comparative Example 4.

[0209] Table 10 - Comparison of the contents of seven ceramides in human plasma stroma and unstrapped plasma in Comparative Example 4

[0210]

[0211]

[0212] As shown in Table 10, activated carbon as a stripping agent exhibits good stripping effect on some ceramides in mixed plasma, such as Cer(d18:1 / 24:0), but 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 content of Cer(d18:1 / 14:0), Cer(d18:1 / 16:0), and Cer(d18:1 / 18:0) in the stripping matrix after stripping treatment is relatively high. Using the stripping matrix prepared in Comparative Example 4 as a diluent for calibrators and quality control samples may lead to lower accuracy in the detection results of target substances.

[0213] <Comparative Example 5>

[0214] The mixed serum was prepared in the same manner as in Example 1, including the solvent composition, preparation process of the blank serum matrix, and lyophilization curve. The difference from Example 1 is that mannitol was not added to the blank serum matrix before lyophilization in this comparative example. A comparison of the dry powder of the blank serum matrix prepared in Example 1 and the dry powder of the blank serum matrix prepared in Comparative Example 5 is shown below. Figure 3 and Figure 4 .

[0215] Depend on Figure 3 and Figure 4As can be seen, the blank serum matrix of Comparative Example 5 without mannitol exhibited uneven ice crystal formation during freeze-drying, resulting in a poorer dry powder structure and lower mechanical strength. This made it prone to breakage during transportation, storage, or use, affecting the stability of the dry powder. Furthermore, the dry powder prepared in Comparative Example 5 was more susceptible to environmental factors such as humidity and temperature, leading to a shorter shelf life and increasing the risk of deterioration. In contrast, the blank serum matrix of Example 1 with mannitol exhibited uniform ice crystal formation during freeze-drying, resulting in a stronger dry powder structure and higher mechanical strength, making it less prone to breakage during transportation, storage, or use. This dry powder was less affected by environmental factors and had a longer shelf life.

[0216] <Comparative Example 6>

[0217] The mixed serum was prepared in the same manner as in Example 1, including the solvent composition, preparation process of the blank serum matrix, lyophilization process, reconstitution process, and pH adjustment process. The difference from Example 1 is that ProClin 300 was not added to the blank serum matrix reconstitution solution in this comparative example. The state of the blank serum matrix reconstitution solution prepared in Comparative Example 6 after being stored at 2℃-8℃ for 20 days was compared with the state of the blank serum matrix reconstitution solution prepared in Example 1 after being stored at 2℃-8℃ for 20 days. See [link to relevant documentation]. Figure 5 and Figure 6 .

[0218] The optimized blank serum matrix reconstituted solution from Example 1 was clear and transparent, as shown in the image. Figure 5 As shown, the blank serum matrix reconstituted solution in Comparative Example 6 was turbid and contained flocculent matter, such as... Figure 6 As shown, ProClin 300 effectively inhibits the bacterial count of blank serum matrix reconstituted solutions. Blank serum matrix reconstituted solutions containing ProClin 300 have a longer shelf life at 2℃-8℃. Calibrators and quality control samples prepared using blank serum matrix reconstituted solutions containing ProClin 300 have a longer shelf life, thus reducing the impact of repeated freeze-thaw cycles on the assay.

[0219] While specific embodiments of the invention have been described in detail by way of examples, those skilled in the art should understand that the examples are for illustrative purposes only and not intended to limit the scope of the invention. Those skilled in the art should understand that modifications can be made to the above embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.

Claims

1. A reagent combination for preparing a blank biological liquid matrix, characterized in that, The stripping solvent comprises alcohol and ether, and the additive comprises dealcoholizing solvent, excipient, pH regulator and protective agent; The dealcoholizing solvent is suitable for removing alcohol in the stripped biological liquid, and the dealcoholizing solvent comprises ether, and the ether comprises at least one of dimethyl ether, diethyl ether, dipropyl ether, methyl ethyl ether, ethyl propyl ether and methyl tert-butyl ether; The excipient is suitable for forming dry powder during lyophilization of the blank biological liquid matrix, and the excipient comprises at least one of mannitol, trehalose and sucrose; The pH regulator is suitable for adjusting the pH of the reconstituted blank biological liquid matrix solution, and the pH regulator comprises at least one of sodium dihydrogen phosphate and acetic acid; The protective agent is suitable for the preservation of the reconstituted blank biological liquid matrix solution, and the protective agent comprises at least one of ProClean 300, Buffpro 300, ProClin 300, ProClin 150, ProClin 200 and ProClin 950; The alcohol comprises isopropyl alcohol, and the ether comprises methyl tert-butyl ether, and the ratio of the content of the isopropyl alcohol to the content of the methyl tert-butyl ether is 10:90 to 25:75; or The alcohol comprises isopropyl alcohol and ethanol, and the ether comprises methyl tert-butyl ether, and the ratio of the content of the isopropyl alcohol, the content of the ethanol and the content of the methyl tert-butyl ether is (5-15):(1-10):(75-94); or The alcohol comprises isopropyl alcohol and glycerol, and the ether comprises methyl tert-butyl ether, and the ratio of the content of the isopropyl alcohol, the content of the glycerol and the content of the methyl tert-butyl ether is (10-20):(2-8):(72-88).

2. A method for the preparation of a blank biological liquid matrix, characterized in that, Comprising: Mixing the biological liquid with the stripping solvent in the reagent combination of claim 1, and separating into a first organic phase layer and a first main phase layer, removing the first organic phase layer to obtain the first main phase layer, and the first main phase layer is a blank biological liquid matrix.

3. The preparation method according to claim 2, characterized in that, The biological liquid comprises at least one of whole blood, plasma, serum, saliva, urine, cerebrospinal fluid and milk.

4. The preparation method according to claim 2, characterized in that, The volume ratio of the stripping solvent to the biological liquid is greater than or equal to 1:

2.

5. The preparation method according to claim 2, characterized in that, Drying the blank biological liquid matrix to prepare dry powder; or Further comprising purifying the blank biological liquid matrix, comprising: adding a dealcoholizing agent to the obtained blank biological liquid matrix, mixing, and separating into a second organic phase layer and a second main phase layer, and removing the second organic phase layer.

6. The preparation method according to claim 5, characterized in that, Rotary evaporation of the second main phase layer to remove the dealcoholizing agent in the second main phase layer; or Drying the second main phase layer to prepare dry powder.

7. The preparation method according to claim 6, characterized in that, Drying the second main phase layer by lyophilization.

8. The preparation method according to claim 6, characterized in that, Further comprising adding an excipient before the blank biological liquid matrix is prepared into dry powder.

9. The production method according to claim 8, characterized by, The mass ratio of the excipient to the blank biological liquid matrix is 1% to 10%.

10. The method of claim 6, wherein, Further comprising reconstituting the dry powder prepared from the blank biological liquid matrix to obtain a reconstituted blank biological liquid matrix solution.

11. The method of claim 10, wherein, Adding a protective agent and / or a pH regulator to the reconstituted blank biological liquid matrix solution.

12. The method of claim 11, wherein, The mass ratio of the protective agent to the blank biological liquid matrix reconstitution solution is 0.01% to 0.2%.

13. The method of claim 11, wherein, The mass ratio of the pH regulator to the blank biological liquid matrix reconstitution solution is 0.1% to 0.5%.

14. A blank biological liquid substrate, characterized in that, Prepared according to the preparation method as claimed in any one of claims 2 to 13.

15. Use of the blank biological liquid matrix prepared by the preparation method as claimed in any one of claims 2 to 13 in detecting fat-soluble vitamins, ceramides or steroid hormones.

16. A kit comprising, The reagent combination as claimed in claim 1 and / or the blank biological liquid matrix as claimed in claim 14 are included.

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