Detection kit for ceramide and method for detecting ceramide by using detection kit

Abstract

The invention discloses a ceramide detection kit and a method for detecting ceramide by using the detection kit. The detection kit comprises a calibration product, a quality control product, a protein precipitant and a mobile phase additive, substrates adopted by the calibration product and the quality control product are alternative substrates, and the alternative substrates are blank biological liquid substrates prepared by the following method: mixing biological liquid with a stripping solvent, and stirring to obtain a mixed solution; layering 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 which is a blank biological liquid matrix; the stripping solvent comprises alcohol and ether, the content of the alcohol is 1%-35% by mass, and the content of the ether is 65%-99% by mass.

Description

Technical Field

[0001] The present invention relates to the technical field of laboratory testing, and more specifically, to a detection kit for ceramide and a method for detecting ceramide using the detection kit. Background Art

[0002] In the related art, the detection kit of ceramide usually uses organic solvents (such as isopropanol, methanol) or bovine serum albumin solution as a substitute matrix. However, the composition of organic solvents and bovine serum albumin solution is quite different from that of the sample to be tested, such as blood, resulting in inaccurate detection of ceramide. Summary of the invention

[0003] One object of the present invention is to provide a new technical solution for a ceramide detection kit.

[0004] According to the first aspect of the present application, a ceramide detection kit is provided. The detection kit comprises a calibrator, a quality control product, a protein precipitant and a mobile phase additive, wherein the matrix used by the calibrator and the quality control product is a substitute matrix,

[0005] Wherein, the surrogate matrix is ​​a blank biological liquid matrix prepared by the following method:

[0006] Mixing the biological liquid with a stripping solvent, 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;

[0007] The stripping solvent includes alcohol and ether. Calculated by mass percentage, the content of the alcohol is 1% to 35%, and the content of the ether is 65% to 99%.

[0008] Optionally, the content of the ether is 65% to 95%, and the content of the alcohol is 5% to 35%.

[0009] Optionally, the alcohol includes at least one of a monohydric alcohol and a polyhydric alcohol.

[0010] Optionally, the ether includes at least one of a monoether and a mixed ether.

[0011] Optionally, the ether includes dimethyl ether, diethyl ether, dipropyl ether, methyl ethyl ether, ethyl propyl ether, methyl propyl ether, methyl tert-butyl ether, and the alcohol includes at least one of methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, and sorbitol;

[0012] Optionally, the ratio of the alcohol content to the 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 isopropanol content to the methyl tert-butyl ether content 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 isopropanol content, the ethanol content and the methyl tert-butyl ether content is (5-15):(1-10):(75-94).

[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); or

[0016] 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): (72-88).

[0017] 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 a stripping solvent for preparing a blank biological liquid matrix as described in 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.

[0018] Optionally, the biological fluid includes at least one of whole blood, plasma, and serum.

[0019] Optionally, the volume ratio of the stripping solvent to the biological fluid is greater than or equal to 1:2.

[0020] Optionally, the blank biological liquid matrix is ​​dried to prepare a dry powder; or

[0021] The method also includes purifying the blank biological liquid matrix, including: adding ether 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.

[0022] Optionally, the second main phase layer is subjected to rotary evaporation to remove the ether in the second main phase layer.

[0023] Optionally, the second main phase layer is dried to prepare a dry powder; preferably, the second main phase layer is dried by freeze-drying.

[0024] Optionally, the ceramide includes at least one of Cer (d18:1 / 14:0), Cer (d18:1 / 16:0), Cer (d18:1 / 18:0), Cer (d18:1 / 20:0), Cer (d18:1 / 22:0), Cer (d18:1 / 24:0) and Cer (d18:1 / 24:1).

[0025] According to the third aspect of the present application, a method for detecting ceramide using the above detection kit is provided, wherein the method comprises detecting ceramide in a sample by liquid chromatography tandem mass spectrometry.

[0026] In the embodiments of the present application, a blank biological liquid matrix is ​​used as a diluent to prepare a calibrant and a quality control product. The blank biological liquid matrix is ​​prepared by stripping a biological liquid with a stripping solvent. The stripping solvent can effectively remove the target substance in the biological liquid, such as ceramide, thereby forming a blank biological liquid matrix. The blank biological liquid matrix has a similar composition to the unstripped biological liquid and can be used for the preparation of calibrants and quality control products for detecting ceramide.

[0027] 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

[0028] 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.

[0029] Figure 1 This is a chromatogram of seven ceramides in the mixed plasma of Example 1 of the present application.

[0030] Figure 2 This is a chromatogram of 7 ceramides in the blank plasma matrix of Example 1 of the present application.

[0031] Figure numerals: 1. 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 , Cer(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

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0036] 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.

[0037] According to the first embodiment of the present application, a ceramide detection kit is provided. The detection kit includes a calibrator, a quality control product, a protein precipitant and a mobile phase additive, wherein the matrix used by the calibrator and the quality control product is a blank biological liquid matrix.

[0038] Wherein, the blank biological liquid matrix is ​​prepared according to the following method:

[0039] Mixing the biological liquid with a stripping solvent, 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;

[0040] The stripping solvent includes alcohol and ether. Calculated by mass percentage, the content of the alcohol is 1% to 35%, and the content of the ether is 65% to 99%.

[0041] Specifically, the calibrator is used to form a standard curve. The quality control product is used to perform quality control on the standard curve. The protein precipitant is used to precipitate proteins in the calibrator, the quality control product and the sample to be tested. The mobile phase additive is added to the mobile phase during detection to increase the target ceramide response, improve the signal-to-noise ratio and optimize the peak shape. The kit may also contain a diluent, which is used to dissolve the internal standard, and the diluent may be isopropanol, methanol or a mixed solution of the two. Optionally, the biological fluid includes whole blood, plasma, and serum. Depending on the type of sample to be tested, the biological fluid is whole blood, plasma, and serum from a human or animal body. After the above-mentioned biological fluids are stripped, a blank biological fluid matrix can be formed. The biological fluid matrix is ​​similar in composition to the sample to be tested, but does not contain the target substance, i.e., ceramide.

[0042] In the specific implementation, first, according to the lipophilicity of the target substance, i.e., ceramide, 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% calculated by mass percentage. Within this range, the ratio of alcohol and ether is adjusted to avoid protein precipitation in the biological fluid. After the stripping solvent is configured, it can be placed in a brown reagent bottle and stored away from light.

[0043] 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 using a centrifuge to perform stratification. For example, the first main phase layer is an 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. The blank biological liquid matrix is ​​purified to remove the organic solvent.

[0044] 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.

[0045] The above preparation method can effectively remove target substances, such as ceramide, in biological fluids to obtain blank biological fluid matrix. The blank biological fluid matrix has a low content of target substances and can be used for the preparation of calibrators and quality control products in in vitro diagnostic reagents or scientific research reagents to detect the content of target substances in biological fluids.

[0046] In the embodiments of the present application, a blank biological liquid matrix is ​​used as a diluent to prepare a calibrant and a quality control product. The blank biological liquid matrix is ​​prepared by stripping a biological liquid with a stripping solvent. The stripping solvent can effectively strip the target substance, such as ceramide, in the biological liquid, thereby forming a blank biological liquid matrix. The blank biological liquid matrix has a similar protein concentration to the unstripped biological liquid matrix, and the retention time of the target substance in the matrix before and after stripping is similar, and can be used for the preparation of a calibrant and a quality control product for detecting a target substance, such as ceramide.

[0047] 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.

[0048] 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.

[0049] The content of ceramide in the blank biological liquid matrix is ​​extremely low, and therefore, the blank biological liquid matrix can be used for the preparation of calibration products and quality control products in the detection of ceramide concentration in biological liquids.

[0050] By LC-MS / MS detection, the content of the target substance (eg, ceramide) in the blank biological liquid matrix is ​​obtained, and the removal rate and the content of the target substance after removal in the blank biological liquid matrix can be determined.

[0051] In one example, the content of the ether is 65% to 95%, and the content of the alcohol is 5% to 35%.

[0052] If the alcohol content is too high, protein precipitation will be formed during the stripping process of the target substance (such as ceramide) 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.

[0053] In one example, the alcohol includes at least one of a monohydric alcohol and a polyhydric alcohol.

[0054] Optionally, the monohydric alcohol includes methanol, ethanol, propanol, butanol, isopropanol, etc. The polyhydric alcohol includes ethylene glycol, propylene glycol, glycerol, sorbitol, etc.

[0055] 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.

[0056] In one example, the ether includes at least one of a monoether and a mixed ether.

[0057] 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.

[0058] 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 are prone to react with certain substances in biological fluids, thereby affecting the composition of blank biological fluid matrix. Crown ethers can complex with metal ions in biological fluids, thereby causing a difference in ionic strength between the prepared blank biological fluid matrix and the biological fluid.

[0059] In one example, the ether includes 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.

[0060] 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.

[0061] In one example, the ratio of the alcohol content to the ether content is 5:95 to 25:75.

[0062] 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 ceramide in the biological fluid, and no protein precipitation will be formed during the stripping process.

[0063] In one example, 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 ceramide.

[0064] In one example, 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).

[0065] 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 ceramide.

[0066] In one example, 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).

[0067] 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 ceramide.

[0068] In one example, 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).

[0069] 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 ceramide.

[0070] In one example, the volume ratio of the stripping solvent to the biological fluid is greater than or equal to 1:2.

[0071] In the above range, the removal efficiency of the target substance in the biological fluid is high.

[0072] 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.

[0073] In one example, the preparation method further includes purifying the blank biological liquid matrix, including: adding ether 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.

[0074] 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 ether to the blank biological liquid matrix and mixing it thoroughly, after stratification, the alcohol in the blank biological liquid matrix can be effectively removed. For example, stratification 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.

[0075] To further purify the blank biological fluid matrix, the addition of ether and removal of the second organic phase layer can be performed multiple times.

[0076] Preferably, the volume ratio of the ether to the first main phase layer is greater than or equal to 1:1. The types of ether are as described above. Optionally, the volume ratio of the ether 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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. And the dry powder is conducive to the preservation of the blank biological liquid matrix. When in use, the dry powder can be re-dissolved with a solvent, such as ultrapure water. The volume of the re-dissolved 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 blank biological liquid matrix dry powder.

[0081] According to the second embodiment of the present application, a method for detecting ceramide using the above detection kit is provided, wherein the method comprises detecting ceramide in a sample by liquid chromatography-tandem mass spectrometry.

[0082] Example 1

[0083] (I) Preparation of ceramide blank plasma

[0084] 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 is removed to obtain a blank plasma matrix. Finally, the blank plasma matrix is ​​freeze-dried to obtain a dry powder of the blank plasma matrix.

[0085] test:

[0086] The dry powder of the blank plasma matrix is ​​reconstituted with ultrapure water to obtain a blank plasma matrix in a solution state. In this embodiment, the ratio of ultrapure water to dry powder is 10.0295, that is, when the mass of the dry powder of the blank plasma matrix is ​​1g, 10.0295g of ultrapure water is added for reconstitution.

[0087] (II) Detection of ceramide

[0088] (1) The main components of the ceramide detection kit are shown in Table 1

[0089] Table 1-Main components of ceramide detection kit

[0090]

[0091]

[0092] (2) Items required for testing

[0093] Table 2 Instruments required for testing

[0094] Device Name Specifications Liquid chromatography-mass spectrometry <![CDATA[Shimadzu Jasper HPLC - SCIEX Triple Quad TM 4500MD]]> Pipette 0.5-10μL Pipette 2-20μL Pipette 100-1000μL Vortex mixer Vrtex-6 Microplate thermostat oscillator MB100-4A High-speed refrigerated centrifuge D3024R

[0095] Table 3- Consumables required for testing

[0096] name Specifications Pipette Tips 10μL Pipette Tips 200μL Pipette Tips 1000μL 1.5mL microtube 1.5mL Microtube rack 8×12 array Chromatographic columns Phenomenex XB-C18(50*4.6mm,2.6μm) 96-well V-plate and plate mat /

[0097] Table 4 - Reagents required for testing

[0098]

[0099] (3) Detection method:

[0100] 1) Before testing, restore the test kit and the sample to room temperature (20℃~30℃);

[0101] 2) Reconstitution of calibrators and quality control products: At room temperature, accurately pipette 1 mL of ultrapure water into each calibrator and quality control product bottle and mix thoroughly for 3 to 5 minutes to obtain calibrators and quality control product solutions at various concentration levels;

[0102] 3) Redissolving the internal standard: At room temperature, accurately pipette 3 mL of diluent into the internal standard bottle and mix thoroughly for 3 to 5 minutes to obtain the internal standard solution.

[0103] 4) Sample processing and testing process: Vortex the sample before testing, accurately pipette 100μL of calibrators, quality control products, and plasma samples into a 1.5mL centrifuge tube, add 20μL of internal standard solution and 880μL of protein precipitant, and vortex for 2-3 minutes. Centrifuge at 4°C and 15000rpm for 10 minutes to separate the supernatant. Transfer 200μL of the supernatant to a new 96-well V-plate for LC-MS / MS testing.

[0104] (4) Data analysis and results:

[0105] Calibration curve drawing: take the analyte concentration of the calibrator as the independent variable X, take the peak area ratio of the analyte of the calibrator and the internal standard at the corresponding concentration as the dependent variable Y, calculate the linear regression equation Y=a+bX and the correlation coefficient r;

[0106] Quality control product data analysis: When the correlation coefficient of the calibration curve is r≥0.990, the signal intensity of the quality control product is substituted into the regression equation to obtain the concentration of the quality control product;

[0107] Sample data analysis: When the test results of the quality control products are within the expected range, the ratio of the analyte detection signal in the plasma sample to the internal standard is substituted into the regression equation to obtain the concentration of the target substance in the plasma sample.

[0108] Instruments and equipment: Mass spectrometer uses Sciex Triple Quad TM 4500MD; the liquid chromatograph used was Shimadzu Sciex Jasper HPLC.

[0109] Instrumental methods

[0110] Ion source parameters

[0111] The ion source used was ESI source, positive ion scanning, MRM mode, the ion source parameters are shown in Table 5, and the mass spectrometry parameters of the compounds to be tested are shown in Table 6.

[0112] Table 5 - Ion source parameters

[0113]

[0114] Table 6 - Mass spectrometry parameters of the tested compounds

[0115] Q1 Q3 DWELL TIME ID DP CE CXP 538.6 264.5 30 Cer(d18:1 / 16:0) 40 35 15 545.4 271.4 30 Cer(d18:1-d7 / 16:0) 40 35 15 566.4 264.5 30 Cer(d18:1 / 18:0) 40 35 15 573.4 271.4 30 Cer(d18:1-d7 / 18:0) 40 35 15 650.4 264.5 15 Cer(d18:1 / 24:0) 40 35 15 657.4 271.4 15 Cer(d18:1-d7 / 24:0) 40 35 15 648.4 264.5 15 Cer(d18:1 / 24:1) 40 35 15 655.4 271.4 15 Cer(d18:1-d7 / 24:1) 40 35 15 594.4 264.5 30 Cer(d18:1 / 20:0) 40 35 15 622.4 264.5 20 Cer(d18:1 / 22:0) 40 35 15 510.4 264.3 40 Cer(d18:1 / 14:0) 40 35 12 517.6 271.4 40 Cer(d18:1-d7 / 14:0) 40 35 12 601.4 271.4 30 Cer(d18:1-d7 / 20:0) 40 35 15 629.4 271.4 20 Cer(d18:1-d7 / 22:0) 40 35 15

[0116] Liquid Phase Method

[0117] Reverse phase chromatography was used to establish the separation conditions of the analytes. The chromatographic column was Phenomenex Kinetex XB-C18 (50*4.6mm, 2.6μm), the flow rate was 0.3mL / min, and the column temperature was 40°C. Gradient elution was used. The mobile phase A was an aqueous solution containing 0.1% formic acid and 2mmol / L ammonium acetate, and the mobile phase B was a mixture of 0.1% formic acid and 2mmol / L ammonium acetate and isopropanol and acetonitrile (isopropanol: acetonitrile = 3:1 (v / v). The gradient elution program is shown in the following table:

[0118] Table 7 - Elution gradient

[0119] Time (min) Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) 0 0.3 15 85 1.5 0.3 4 96 4.4 0.3 4 96 4.6 0.3 15 85 5 0.3 15 85

[0120] Evaluation of background content of blank plasma matrix reconstitution solution

[0121] Figure 1 It is the total ion current graph of 7 ceramides and corresponding internal standards in unstripped mixed plasma. Among them, 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).

[0122] Figure 2 The total ion chromatograms of the seven ceramides and the corresponding internal standards in the blank plasma reconstitution solution are shown in Figure 1 It can be seen that the interfering Cer (d18:1 / 24:0) and Cer (d18:1 / 24:1) achieved baseline separation, and the seven ceramides achieved good separation from the interfering peaks, with sharp and symmetrical peaks. Figure 2 ) showed that the peak heights of the seven ceramides in the blank plasma matrix reconstitution solution were close to the baseline level, the ceramide removal effect was very good, and the retention times of the internal standards of the seven ceramides in the unstripped mixed plasma chromatogram were the same as those of the seven ceramides in the blank plasma matrix, and the internal standard peaks were sharp and symmetrical.

[0123] Table 8 - Comparison of the contents and removal rates of seven ceramides in blank plasma matrix and unstripped mixed plasma in Example 1

[0124]

[0125]

[0126] Note: If the target substance peak signal-to-noise ratio in the test results is less than 3, the content is marked as 0.

[0127] As shown in Table 8, the detected contents of the 7 ceramides in the blank plasma matrix were all 0. The stripping solvent can effectively strip the above 7 ceramides in human plasma.

[0128] Linear evaluation

[0129] A kit of this embodiment within the validity period is randomly selected, and the calibrator of the standard curve of the kit is reconstituted with ultrapure water. The concentration of the target substance is used as the horizontal coordinate, and the ratio of the peak area of ​​the target substance to the peak area of ​​the internal standard is used as the vertical coordinate. The weighted (W=1 / X2) least squares method is used for linear regression to obtain the standard curve regression equation (y=a+bx).

[0130] Table 9 - Evaluation of regression equation parameters of 7 ceramide standard curves

[0131] Compound Fitting equation r Linear range Cer(d18:1 / 14:0) y=0.411x-0.000249 0.9991 1-50 ng / mL Cer(d18:1 / 16:0) y=0.0379x+0.0182 0.9989 10-500ng / mL Cer(d18:1 / 18:0) y=0.0281x+0.0405 0.9986 10-500ng / mL Cer(d18:1 / 20:0) y=0.0284x+0.0192 0.9989 10-500ng / mL Cer(d18:1 / 22:0) y=0.0199x-0.0198 0.9981 30-1500ng / mL Cer(d18:1 / 24:0) y=0.0052x+0.00259 0.9995 100-5000ng / mL Cer(d18:1 / 24:1) y=0.000683x-0.00173 0.9984 100-5000ng / mL

[0132] Therefore, when blank plasma matrix reconstitution solution was used as the surrogate matrix, the ceramide detection method was within the linear range of the seven ceramides set in the debugging, and the correlation coefficient of the standard curve was r≥0.990, and the standard curve evaluation met the methodological requirements.

[0133] Precision and Accuracy Assessment

[0134] Six test kits of this example within the validity period were randomly selected, and quality control products with low, medium and high concentrations were prepared by reconstitution with ultrapure water. One portion of each concentration was prepared for each kit, and each portion was tested once. The calibrator of one of the kits was used as the standard curve to evaluate the precision (RSD) and accuracy (RE) of the intra-batch quality control products.

[0135] Table 10-7 Ceramide Kit Precision and Accuracy

[0136]

[0137]

[0138] As shown in Table 10, the imprecision RSD of the seven ceramide kits was ≤10%, and the accuracy deviation RE was ≤15%. The precision and accuracy evaluation met the methodological requirements.

[0139] Matrix effect evaluation

[0140] Experimental process: The matrix effect was evaluated by evaluating the signal value of the target substance in the plasma matrix and the surrogate matrix. Six clinical individual plasma samples from different sources were selected for matrix effect evaluation. After sample pretreatment, seven ceramides at three concentration levels were added to the six clinical individual plasma samples from different sources and the blank plasma matrix reconstitution solution. The matrix effect was evaluated by comparing the signal intensity of the target substance in the plasma matrix (ratio to the internal standard) with the signal intensity of the target substance in the blank plasma matrix reconstitution solution (ratio to the internal standard), and finally the matrix effect was evaluated by the internal standard normalized matrix factor. The calculation formula is as follows: Internal standard normalized matrix factor = (analyte peak area in plasma matrix / internal standard peak area) / (analyte peak area in blank plasma matrix / internal standard peak area).

[0141] Table 11-Matrix effect of Cer (d18:1 / 14:0)

[0142]

[0143]

[0144] Table 12-Matrix effect of Cer (d18:1 / 16:0)

[0145]

[0146] Table 13-Matrix effect of Cer (d18:1 / 18:0)

[0147]

[0148]

[0149] Table 14-Matrix effect of Cer (d18:1 / 20:0)

[0150]

[0151] Table 15-Matrix effect of Cer (d18:1 / 22:0)

[0152]

[0153] Table 16-Matrix effect of Cer (d18:1 / 24:0)

[0154]

[0155]

[0156] Table 17-Matrix effect of Cer (d18:1 / 24:1)

[0157]

[0158] The experimental results show that the normalized matrix effect factors of 7 ceramide internal standards at three spiked concentrations for 6 clinical samples from different sources are between 0.96 and 1.06, indicating that the matrix effects of the internal standard and the target substance are very close, which can compensate for the matrix effects of the target substance in the sample. The matrix effect does not affect the final accurate quantitative analysis, and the matrix effect meets the methodological evaluation requirements.

[0159] Example 2

[0160] The blank plasma matrix was prepared according to the method described in Example 1, except that the mass percentages of isopropanol, ethanol and methyl tert-butyl ether in the stripping solvent were 10%, 5% and 85%, respectively.

[0161] The sample pretreatment and detection were performed in the same manner as in Example 1. The ceramide results are shown in Table 18.

[0162] Table 18 Comparison of ceramide content in blank plasma matrix and unstripped human plasma in Example 2

[0163] Ceramide (ng / mL) Unstripped human plasma Blank plasma matrix Removal rate Cer(d18:1 / 14:0) 1.74 0.13 92.51％ Cer(d18:1 / 16:0) 166.50 1 99.40％ 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％

[0164] As shown in Table 18, the removal rates of the seven ceramides in the blank plasma matrix all reached more than 90%, and the detected contents of the five ceramides were all 0. The stripping solvent can effectively strip the above seven ceramides in human plasma.

[0165] Example 3

[0166] The blank plasma matrix was prepared according to the method described in Example 1, except that the mass percentages of isopropanol, glycerol and methyl tert-butyl ether in the stripping solvent were 5%, 5% and 90%, respectively.

[0167] The sample pretreatment and detection were performed in the same manner as in Example 1. The ceramide results are shown in Table 19.

[0168] Table 19 Comparison of ceramide content in blank plasma matrix and unstripped human plasma in Example 3

[0169] Ceramide (ng / mL) Unstripped human plasma Blank plasma matrix Removal rate Cer(d18:1 / 14:0) 1.74 0.08 95.39％ 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％

[0170] As shown in Table 19, the removal rates of the seven ceramides in the blank plasma matrix all reached more than 95%, and the detected contents of the six ceramides were all 0. The stripping solvent can effectively strip the above seven ceramides in human plasma.

[0171] Example 4

[0172] The blank plasma matrix was prepared according to the method described in Example 1, except that the composition of the stripping solvent was isopropanol:methyl tert-butyl ether=20:80.

[0173] The sample pretreatment and detection were performed in the same manner as in Example 1. The ceramide results are shown in Table 20.

[0174] Table 20 Comparison of ceramide content in blank plasma matrix and unstripped human plasma in Example 4

[0175] Ceramide (ng / mL) Unstripped human plasma Blank plasma matrix Removal rate Cer(d18:1 / 14:0) 1.74 0.06 96.54％ 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％

[0176] As shown in Table 20, the removal rates of the seven ceramides in the blank plasma matrix all reached more than 95%, and the detected contents of the six ceramides were all 0. The stripping solvent can effectively strip the above seven ceramides in human plasma.

[0177] Comparative Example 1

[0178] The target substance is stripped according to the method described in Example 1 to prepare a stripping matrix, except that the sum of the mass percentages of isopropanol and ethanol in the stripping solvent and the mass percentage of methyl tert-butyl ether are 40% and 60%, respectively.

[0179] The sample pretreatment and detection were performed in the same manner as in Example 1. The ceramide results are shown in Table 21.

[0180] Table 21 - Comparative Example 1 Comparison of ceramide content in human plasma between stripped matrix and unstripped matrix

[0181] Ceramide (ng / mL) Unstripped human plasma Stripping matrix Removal rate Cer(d18:1 / 14:0) 1.74 0.32 81.56％ Cer(d18:1 / 16:0) 166.50 11.5 93.09％ Cer(d18:1 / 18:0) 53.98 2.1 96.11％ 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％

[0182] As can be seen from Table 21, the removal rates of the seven ceramides in the stripping matrix are different, the removal rate of Cer (d18:1 / 14:0) is about 80%, the removal rate of Cer (d18:1 / 16:0) is between 90% and 95%, and the detection content of the four ceramides is 0. The stripping matrix with a high content of Cer (d18:1 / 14:0) will lead to inaccurate detection of ceramides.

[0183] Comparative Example 2

[0184] The stripping matrix was prepared according to the method described in Example 1, except that the sum of the mass percentages of isopropanol and ethanol and the mass percentage of methyl tert-butyl ether in the stripping solvent were 60% and 40%, respectively. The stripping solvent mixing process was the same as in Example 1. After adding the stripping solvent and mixing, protein precipitation was found in the mixed plasma.

[0185] This indicates that the protein concentration of the stripped matrix obtained by stripping with the stripping solvent of Comparative Example 2 is significantly different from that of the unstripped mixed plasma, and does not meet the requirement that the composition of the surrogate matrix is ​​similar to that of unstripped human plasma.

[0186] Comparative Example 3

[0187] A release matrix was prepared as described in Example 1, except that the release solvent was 100% methyl tert-butyl ether.

[0188] Table 22 Comparison of ceramide content in human plasma between stripped matrix and unstripped matrix in comparative example 3

[0189] Ceramide (ng / mL) Unstripped human plasma Blank plasma matrix Removal rate Cer(d18:1 / 14:0) 1.74 0.58 66.79％ Cer(d18:1 / 16:0) 166.50 28.15 83.09％ Cer(d18:1 / 18:0) 53.98 12.48 76.89％ Cer(d18:1 / 20:0) 57.87 18.07 68.78％ Cer(d18:1 / 22:0) 355.17 157.00 55.80％ Cer(d18:1 / 24:0) 1183.33 647.00 45.32％ Cer(d18:1 / 24:1) 555.17 157.00 71.72％

[0190] As can be seen from Table 22, the removal rate of 100% methyl tert-butyl ether for 7 ceramides is between 45% and 83%, which is relatively low. High residual ceramide in the stripping matrix will lead to inaccurate detection of ceramide.

[0191] Comparative Example 4

[0192] The biological fluid is mixed plasma. The mixed plasma is the same as that in Example 1. Activated carbon is used to strip ceramide from the mixed plasma.

[0193] 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;

[0194] Step 2: Repeat step 1 twice;

[0195] Step 3: Centrifuge the supernatant after three strippings at 10,000 rpm for 15 min, and then take the supernatant;

[0196] Step 4, repeat step 3 6 times to obtain a stripping matrix;

[0197] Step 5: dilute the sample according to the ratio of stripping matrix to ultrapure water = 1:1 (v / v). Then, perform sample pretreatment and sample detection in the same manner as in Example 2.

[0198] The contents and removal rates of seven ceramides in the stripped matrix prepared in Comparative Example 4 and in unstripped human plasma are shown in Table 23.

[0199] Table 23 - Comparison of the contents of seven ceramides in the stripped matrix of Comparative Example 4 and in the unstripped human plasma

[0200] Ceramide (ng / mL) Unstripped human plasma Stripping matrix Removal rate Cer(d18:1 / 14:0) 1.74 0.51 70.61％ Cer(d18:1 / 16:0) 166.50 42.5 74.47％ Cer(d18:1 / 18:0) 53.98 5.06 90.63％ Cer(d18:1 / 20:0) 57.87 3.42 94.09％ Cer(d18:1 / 22:0) 355.17 20.83 94.14％ Cer(d18:1 / 24:0) 1183.33 51.19 95.67％ Cer(d18:1 / 24:1) 555.17 40.28 92.74％

[0201] As can be seen from the above table, 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 diluent for the calibration and quality control products will result in some ceramides, such as

[0202] The detection results of Cer(d18:1 / 14:0), Cer(d18:1 / 16:0), and Cer(d18:1 / 18:0) have low accuracy.

[0203] Comparative Example 5

[0204] Ceramide was detected according to the method of Example 1, except that the matrix was replaced by isopropanol. The results of the matrix effect detection were as follows:

[0205] Table 24-Matrix effect of Cer (d18:1 / 14:0)

[0206]

[0207] Table 25-Matrix effect table of Cer (d18:1 / 16:0)

[0208]

[0209]

[0210] Table 26-Matrix effect of Cer (d18:1 / 18:0)

[0211]

[0212] Table 27-Matrix effect of Cer (d18:1 / 20:0)

[0213]

[0214]

[0215] Table 28-Matrix effect table of Cer (d18:1 / 22:0)

[0216]

[0217] Table 29-Matrix effect table of Cer (d18:1 / 24:0)

[0218]

[0219]

[0220] Table 30-Matrix effect table of Cer (d18:1 / 24:1)

[0221]

[0222] The above results show that when isopropanol is used as an alternative matrix, the average matrix effect factors of the seven ceramides after internal standard correction are between 0.83 and 1.12. Among them, some target substances have larger matrix effects in some samples. For example, the average matrix effect of the third sample L spike level in Cer (d18:1 / 22:0) is 0.69, and the average matrix effect factor of the fifth sample L spike level in Cer (d18:1 / 16:0) reaches 1.18. These strong matrix effects will lead to inaccurate ceramide detection results in the corresponding samples.

[0223] 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 ceramide detection kit, characterized in that: It includes a calibrator, a quality control product, a protein precipitant and a mobile phase additive, wherein the matrix used by the calibrator and the quality control product is a substitute matrix, Wherein, the surrogate matrix is ​​a blank biological liquid matrix prepared by the following method: Mixing the biological liquid with a stripping solvent, 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; The stripping solvent includes alcohol and ether. Calculated by mass percentage, the content of the alcohol is 1% to 35%, and the content of the ether is 65% to 99%.

2. The detection kit according to claim 1, characterized in that The content of the ether is 65% to 95%, and the content of the alcohol is 5% to 35%.

3. The detection kit according to claim 1, 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 detection kit according to claim 1, characterized in that The ether includes 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 ether content is 5:95 to 25:

75.

5. The detection kit according to claim 1, 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; or 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); or 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): (72-88).

6. The detection kit according to claim 1, characterized in that The biological fluids include whole blood, plasma, and serum.

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

2.

8. The detection kit according to claim 1, characterized in that drying the blank biological liquid matrix to prepare a dry powder; or The method also includes purifying the blank biological liquid matrix, including: adding ether 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.

9. The detection kit according to claim 8, characterized in that performing rotary evaporation on the second main phase layer to remove the ether 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.

10. The detection kit according to claim 1, characterized in that The ceramide includes at least one of Cer (d18:1 / 14:0), Cer (d18:1 / 16:0), Cer (d18:1 / 18:0), Cer (d18:1 / 20:0), Cer (d18:1 / 22:0), Cer (d18:1 / 24:0) and Cer (d18:1 / 24:1).

11. A method for detecting ceramide using the detection kit according to claims 1-10, characterized in that: It involves detecting ceramide in samples using liquid chromatography tandem mass spectrometry.

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