Ceramide detection kit, method for detecting ceramide using the detection kit

Abstract

The present invention discloses a detection kit for ceramide and a method for detecting ceramide by using the detection kit. The detection kit includes a calibrator, a quality control product, a protein precipitant and a mobile phase additive. The matrix used for the calibrator and the quality control product is a surrogate matrix, wherein the surrogate matrix is a blank biological fluid matrix prepared by the following method: mixing a biological fluid with a stripping solvent, 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, and the first main phase layer is the blank biological fluid matrix; the stripping solvent includes an alcohol and an ether. Calculated by mass percentage, the content of the alcohol is 1% to 35%, and the content of the ether is 65% to 99%.

Description

Technical Field

[0001] The present invention relates to the technical field of laboratory tests, 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, organic solvents (such as isopropanol, methanol) or bovine serum albumin solution are usually used as the replacement matrix for the detection kit of ceramide. However, the component compositions of organic solvents and bovine serum albumin solution are quite different from those of the sample to be tested, such as blood, resulting in inaccurate detection of ceramide. Summary of the Invention

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

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

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

[0006] Mix the biological liquid with a stripping solvent, and layer to obtain a first organic phase layer and a first main phase layer. Remove the first organic phase layer to obtain the first main phase layer, and the first main phase layer is the 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 monohydric alcohol and polyhydric alcohol.

[0010] Optionally, the ether includes at least one of monoether and 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 content of the alcohol to the content of the ether is 5:95 to 25:75.

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

[0014] Optionally, the alcohol includes isopropyl alcohol and ethanol, the ether includes methyl tert-butyl ether, and the ratio of the content of isopropyl alcohol, the content of ethanol, and the content of methyl tert-butyl ether 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 isopropyl alcohol and glycerol, the ether includes methyl tert-butyl ether, and the ratio of the content of isopropyl alcohol, the content of glycerol, and the content of methyl tert-butyl ether is (10-20):(2-8):(72-88).

[0017] According to the second aspect of the present application, a method for preparing a blank biological liquid matrix is provided. The preparation method includes: mixing a biological liquid with the stripping solvent for preparing the blank biological liquid matrix according to the present application, 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 the blank biological liquid matrix.

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

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

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

[0021] It further includes purifying the blank biological liquid matrix, including: adding an 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 rotary evaporated 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. The method includes detecting ceramide in a sample by liquid chromatography-tandem mass spectrometry.

[0026] In the embodiments of the present application, a blank biological fluid matrix is used as a diluent to prepare calibration products and quality control products. The blank biological fluid matrix is prepared by stripping a biological fluid with a stripping solvent. The stripping solvent can effectively remove the target substance in the biological fluid, such as ceramide, thereby forming a blank biological fluid matrix. The blank biological fluid matrix is similar in composition to the non-stripped biological fluid and can be used for the preparation of calibration products and quality control products for detecting ceramide.

[0027] Other features and advantages of the present invention will become clear from the following detailed description of the exemplary embodiments of the present invention with reference to the accompanying drawings. Description of the Drawings

[0028] The drawings incorporated in the specification and constituting a part of the specification illustrate embodiments of the present invention and, together with the description thereof, are used to explain the effects of the present invention.

[0029] Figure 1 is the chromatogram of 7 ceramides in the mixed plasma of Example 1 of the present application.

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

[0031] Reference 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 Embodiments

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

[0033] The following description of at least one exemplary embodiment is merely illustrative in nature and in no way limits the present invention, its application, or its use.

[0034] Technologies, methods, and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, such technologies, methods, and devices should be regarded as part of the specification.

[0035] In all the examples shown and discussed herein, any specific values should be construed as merely exemplary and not as a limitation. Thus, other examples of the exemplary embodiments may have different values.

[0036] It should be noted that: like reference numerals and letters denote like items in the following drawings, and thus, once an item is defined in one drawing, further discussion thereof is not required in subsequent drawings.

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

[0038] Wherein, the blank biological fluid matrix is prepared by the following method:

[0039] Mix a biological fluid with a stripping solvent, and layer to obtain a first organic phase layer and a first main phase layer. Remove the first organic phase layer to obtain the first main phase layer, and the first main phase layer is the blank biological fluid 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 sample is used for quality control of the standard curve. The protein precipitant is used to precipitate the proteins in the calibrator, the quality control sample, and the sample to be tested. The mobile phase additive is added to the mobile phase during detection to enhance the response of the target ceramide, 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. The diluent can 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 the sample to be tested, the biological fluid is whole blood, plasma, or serum of a human or an animal. After stripping the above biological fluids, blank biological fluid matrices can be formed. The components of the biological fluid matrix are similar to those of the sample to be tested, but it does not contain the target substance, i.e., ceramide.

[0042] In 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, calculated by mass percentage, the content of the alcohol is 1% to 35%, and the content of the ether is 65% to 99%. 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 in the dark.

[0043] Then, according to a fixed value that the volume ratio of the stripping solvent to the biological fluid is greater than or equal to 1:2, the stripping solvent is added to the container containing the biological fluid and mixed evenly. For example, centrifugation is used to perform layering. For example, the first main phase layer is the aqueous layer. Since the density of the first organic phase layer is less than that of the aqueous layer, the first organic phase layer is located above the first main phase layer. Among them, 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 the blank biological fluid matrix. The blank biological fluid matrix is purified to remove the organic solvent.

[0044] Through the above preparation method, the protein concentration of the blank biological fluid matrix is similar to that of the biological fluid before stripping.

[0045] The above preparation method can effectively remove the target substance in the biological fluid, such as ceramide, etc., to obtain a blank biological fluid matrix. The content of the target substance in the blank biological fluid matrix is low, and it can be used for the preparation of calibrators and quality control samples in in vitro diagnostic reagents or research reagents for detecting the content of the target substance in biological fluids.

[0046] In the embodiments of the present application, a blank biological liquid matrix is used as a diluent to prepare calibration products and quality control products. The blank biological liquid matrix is prepared by stripping a biological liquid with a stripping solvent. The stripping solvent can effectively strip the target substances in the biological liquid, such as ceramides, thereby forming a blank biological liquid matrix. The blank biological liquid matrix has a similar protein concentration to the un-stripped biological liquid, and the retention time of the target substances in the matrix before and after stripping is similar, and it can be used for the preparation of calibration products and quality control products for detecting target substances, such as ceramides.

[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 not only remove the organic solvent in the blank biological liquid matrix but also 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 ceramides in the blank biological liquid matrix is extremely low. Therefore, it 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 (such as ceramide) in the blank biological liquid matrix can be obtained, and the removal rate and the content after removal of the target substance 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 content of alcohol is too high, protein precipitation will form during the process of stripping the target substance (such as ceramide) in the biological liquid, resulting in a large difference in the matrix between the blank biological liquid and the biological liquid; on the contrary, if the content of alcohol is too low, the removal rate of the target substance will be low. Optionally, the content of alcohol is 5%, 10%, 15%, 20%, 25%, 30%, 35%, etc. Correspondingly, the content of ether is 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc. Within the above range, the stripping solvent can effectively remove the target substances in the biological liquid and will not form protein precipitation during the stripping process.

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

[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 enols with unstable structures. Enols have poor stability, and the hydroxyl group will turn into a carbonyl group during use, resulting in the inability to effectively remove the target substance.

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

[0057] Optionally, the monoether includes dimethyl ether, diethyl ether (i.e., ethyl 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 ethers miscible with water, active cyclic ethers, and crown ethers that can complex metal ions in the biological matrix. Ethers miscible with water cannot be separated after mixing with biological liquids, resulting in the inability to strip the target substance. Active cyclic ethers are prone to react with certain substances in biological liquids, thus affecting the composition of the blank biological liquid matrix. Crown ethers will undergo complexation reactions with metal ions in biological liquids, thus causing differences in the ionic strength between the prepared blank biological liquid matrix and biological liquids.

[0059] In one example, 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, sorbitol.

[0060] In this example, the ether can effectively remove the target substance in the biological liquid. The ether can be 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 content of the alcohol to the content of the ether is 5:95 to 25:75.

[0062] In this example, the stripping solvent includes the ether and the 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 ceramides in biological liquids and will not form protein precipitates during the stripping process.

[0063] In one example, the alcohol includes isopropyl alcohol, the ether includes methyl tert-butyl ether, and the ratio of the content of isopropyl alcohol to the content of methyl tert-butyl ether is 25:75 to 10:90; in this example, the stripping solvent includes methyl tert-butyl ether and isopropyl alcohol mixed together. Optionally, the above 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 isopropyl alcohol and ethanol, the ether includes methyl tert-butyl ether, and the ratio of the content of isopropyl alcohol, the content of ethanol, and the content of methyl tert-butyl ether is (5 - 15):(1 - 10):(75 - 94).

[0065] In this example, the stripping solvent includes methyl tert-butyl ether, ethanol, and isopropyl alcohol mixed together. Optionally, the above 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 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 isopropyl alcohol and glycerol, the ether includes methyl tert-butyl ether, and the ratio of the content of isopropyl alcohol, the content of glycerol, and the content of methyl tert-butyl ether 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 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 liquid is greater than or equal to 1:2.

[0071] Within the above range, the stripping efficiency of the target substance in the biological liquid is relatively 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 amounts of the biological liquid and the 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, and 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, during the preparation of the blank biological liquid matrix, the two substances will partially dissolve into the blank biological liquid matrix. In this example, by adding ether to the blank biological liquid matrix and mixing well, after layering, the alcohol in the blank biological liquid matrix can be effectively removed. For example, centrifugation is used for layering to obtain the second organic phase layer and the 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.

[0075] To further purify the blank biological liquid matrix, adding ether and removing 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 includes rotary evaporation of the second main phase layer to remove the ether in the second main phase layer.

[0078] Rotary evaporation refers to 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 solute in the sample. Through this method, the ether in the second main phase layer can be effectively removed.

[0079] Another method for removing the ether in 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.

[0080] During the drying process, the ether gradually volatilizes. By drying the blank biological liquid matrix and preparing it into a dry powder, organic solvents such as ether 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, it can be reconstituted with a solvent, such as ultrapure water. The volume of the reconstituting solvent can be determined according to the ratio of the volatile matter to the dry powder after drying the corresponding biological liquid without stripping treatment and the mass of the dry powder of the blank biological liquid matrix.

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

[0082] Example 1

[0083] (1) Preparation of ceramide blank plasma

[0084] The biological liquid is human plasma. Human plasma from multiple sources is mixed together. For example, the plasma of 30 people is mixed together, that is, the mixed plasma. Part of the mixed plasma is used to prepare the blank plasma matrix, and part is used for comparison. In the stripping solvent, the mass percentage contents of isopropanol, ethanol, and methyl tert-butyl are 20%, 10%, and 70% respectively. The stripping solvent is mixed evenly by vortex mixing. According to the volume ratio of mixed plasma:stripping solvent of 1:2, the stripping solvent is added to the mixed plasma, and they are mixed head-to-head for 40 min. The mixed solution is centrifuged at 4000 rpm for 15 min to obtain a layered 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. Then, according to the volume ratio of the first main phase layer after mixing:methyl tert-butyl ether of 1:2, the first main phase layer is mixed with methyl tert-butyl ether, and they are mixed head-to-head for 10 minutes. Next, it is centrifuged at 4000 rpm for 10 min to obtain a layered 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 the blank plasma matrix. Finally, the blank plasma matrix is freeze-dried to obtain the 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 the blank plasma matrix in solution state. The ratio of ultrapure water to dry powder in this example is 10.0295, that is, when the mass of the dry powder of the blank plasma matrix is 1 g, 10.0295 g of ultrapure water is added for reconstitution.

[0087] (2) Detection of ceramide

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

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

[0090]

[0091]

[0092] (2) Items required for detection

[0093] Table 2 - Instruments required for detection

[0094] Equipment Name Specification Model Liquid Chromatography-Mass Spectrometer <![CDATA[Shimadzu Jasper HPLC - SCIEX Triple Quad TM 4500MD]]> Pipette 0.5 - 10 μL Pipette 2 - 20 μL Pipette 100 - 1000 μL Vortex Oscillator Vrtex-6 Microplate Thermostatic Oscillator MB100-4A High-Speed Refrigerated Centrifuge D3024R

[0095] Table 3 - Consumables required for detection

[0096] Name Specification Model Pipette Tips 10 μL Pipette Tips 200 μL Pipette Tips 1000 μL 1.5 mL Microtube 1.5 mL Microtube Rack 8×12 Array Chromatographic Column Phenomenex XB-C18(50*4.6mm,2.6μm) 96-Well V-Plate and Plate Mat /

[0097] Table 4 - Reagents required for detection

[0098]

[0099] (3) Detection method:

[0100] 1) Before testing, restore the kit and the sample to be tested to room temperature (20°C - 30°C);

[0101] 2) Reconstitution of calibrators and quality control materials: At room temperature, accurately pipette 1 mL of ultrapure water into the vials of each calibrator and quality control material, and mix well for 3 - 5 min to obtain calibrator and quality control material solutions at each concentration level;

[0102] 3) Reconstitution of internal standard: At room temperature, accurately pipette 3 mL of diluent into the vial of the internal standard, and mix well for 3 - 5 min to obtain the internal standard solution;

[0103] 4) Sample processing and detection procedure: Before detection, vortex the sample to mix well. Accurately pipette 100 μL of calibrator, quality control material, and plasma sample into a 1.5 mL centrifuge tube, add 20 μL of the internal standard solution and 880 μL of protein precipitant, and vortex for 2 - 3 min. Centrifuge at 4°C and 15000 rpm for 10 min to separate the supernatant. Transfer 200 μL of the supernatant to a new 96-well V-bottom plate for LC-MS / MS detection.

[0104] (4) Data analysis and results:

[0105] Calibration curve plotting: Taking the analyte concentration of the calibration standard as the independent variable X, and the ratio of the peak areas of the analyte and internal standard in the calibration standard corresponding to the concentration as the dependent variable Y, calculate the linear regression equation Y = a + bX and the correlation coefficient r;

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

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

[0108] Instrumentation: The mass spectrometer uses Sciex Triple Quad TM 4500MD; The liquid chromatograph uses Shimadzu SciexJasper HPLC.

[0109] Instrument method

[0110] Ion source parameters

[0111] The ion source uses an 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 measured are shown in Table 6.

[0112] Table 5 - Ion source parameters

[0113]

[0114] Table 6 - Mass spectrometry parameters of the compounds to be measured

[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] The separation conditions of the compounds to be measured are established using reverse-phase chromatography. The chromatographic column is Phenomenex Kinetex XB-C18 (50 * 4.6 mm, 2.6 μm), the flow rate is 0.3 mL / min, and the column temperature is 40 °C. Gradient elution is used. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2 mmol / L ammonium acetate, and mobile phase B is a mixed solution containing 0.1% formic acid, 2 mmol / L ammonium acetate, 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 the background content of the blank plasma matrix reconstituted solution

[0121] Figure 1 Total ion current chromatograms of 7 ceramides and their corresponding internal standards in unpeeled mixed plasma. Among them, the 7 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 7 internal standards corresponding to the 7 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).

[0122] Figure 2 Total ion current chromatograms of 7 ceramides and their corresponding internal standards in blank plasma reconstitution solution are shown by Figure 1 It can be seen that Cer(d18:1 / 24:0) and Cer(d18:1 / 24:1) with interference relationship achieve baseline separation, and the 7 ceramides achieve good separation from the interference peaks, with sharp and symmetrical peak shapes. The total ion current chromatogram of blank plasma matrix reconstitution solution ( Figure 2 ) shows that the peak heights of the 7 ceramides in the blank plasma matrix reconstitution solution are close to the baseline level, the ceramide removal effect is very good, and the retention times of the internal standards of the 7 ceramides in the chromatogram of unpeeled mixed plasma are the same as those of the internal standards of the 7 ceramides in the blank plasma matrix, and the internal standard peak shapes are sharp and symmetrical.

[0123] Table 8 - Comparison of the contents and removal rates of 7 ceramides in blank plasma matrix and unpeeled mixed plasma in Example 1

[0124]

[0125]

[0126] Note: For the target substance peaks with signal-to-noise ratio < 3 in the detection results, the content is marked as 0.

[0127] As can be seen from Table 8, the detected contents of the 7 ceramides in the blank plasma matrix are all 0. This peeling solvent can effectively peel the above 7 ceramides from human plasma.

[0128] Linear evaluation

[0129] Randomly select a kit of this example within the validity period. Reconstitute the calibrator of the kit standard curve with ultrapure water. Use the concentration of the target substance as the abscissa and the ratio of the peak area of the target substance to the peak area of the internal standard as the ordinate. Perform linear regression using weighted (W = 1 / X2) least squares method to obtain the standard curve regression equation (y = a + bx).

[0130] Table 9 - Evaluation of parameters of the standard curve regression equation for 7 kinds of ceramides

[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 - 500 ng / mL Cer(d18:1 / 18:0) y = 0.0281x + 0.0405 0.9986 10 - 500 ng / mL Cer(d18:1 / 20:0) y = 0.0284x + 0.0192 0.9989 10 - 500 ng / Ml Cer(d18:1 / 22:0) y = 0.0199x - 0.0198 0.9981 30 - 1500 ng / mL Cer(d18:1 / 24:0) y = 0.0052x + 0.00259 0.9995 100 - 5000 ng / mL Cer(d18:1 / 24:1) y = 0.000683x - 0.00173 0.9984 100 - 5000 ng / mL

[0132] Therefore, when using the blank plasma matrix reconstitution solution as the alternative matrix, within the linear range of the 7 kinds of ceramides set during debugging for the detection method of ceramides, the correlation coefficient r of the standard curve is ≥ 0.990, and the evaluation of the standard curve meets the methodological requirements.

[0133] Evaluation of precision and accuracy

[0134] Randomly select six kits of this example within the validity period. Reconstitute and prepare quality control products with low, medium, and high concentration values using ultrapure water. Prepare 1 portion for each concentration of each kit and detect each portion once. Use the calibrator of one of the kits to make the standard curve to evaluate the precision (RSD) and accuracy (RE) of the within-batch quality control products.

[0135] Table 10 - Precision and accuracy of 7 kinds of ceramide kits

[0136]

[0137]

[0138] As can be seen from Table 10, for the 7 kinds of ceramide kits, the imprecision RSD ≤ 10%, the accuracy deviation RE ≤ 15%, and the evaluation of precision and accuracy meet the methodological requirements.

[0139] Evaluation of matrix effect

[0140] Experimental procedure: Evaluate the matrix effect by evaluating the signal values of the target substance in the plasma matrix and the alternative matrix. Select 6 plasma samples from clinical individuals with different sources for matrix effect evaluation. After sample pretreatment of the 6 plasma samples from clinical individuals with different sources and the blank plasma matrix reconstitution solution, add 7 kinds of ceramides at three concentration levels respectively. Evaluate the matrix effect by comparing the signal intensity of the target substance (ratio to the internal standard) in the plasma matrix with the signal intensity of the target substance (ratio to the internal standard) in the blank plasma matrix reconstitution solution. Finally, evaluate the matrix effect through the matrix factor normalized by the internal standard. The calculation formula is as follows: Matrix factor normalized by the internal standard = (peak area of the analyte in the plasma matrix / peak area of the internal standard) / (peak area of the analyte in the blank plasma matrix / peak area of the internal standard).

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

[0142]

[0143]

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

[0145]

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

[0147]

[0148]

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

[0150]

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

[0152]

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

[0154]

[0155]

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

[0157]

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

[0159] Example 2

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

[0161] The detection sample pretreatment and detection were carried out according to the same method as in Example 1, and the ceramide results are shown in Table 18.

[0162] Table 18 Comparison of ceramide contents in the blank plasma matrix of Example 2 and unpeeled human plasma

[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 can be seen from Table 18, the removal rates of the 7 ceramides in the blank plasma matrix all reached over 90%, and the detected contents of the 5 ceramides were all 0. This stripping solvent can effectively strip the above 7 ceramides from human plasma.

[0165] Example 3

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

[0167] The sample pretreatment and detection were carried out according to the same method as in Example 1, and the ceramide results are shown in Table 19.

[0168] Table 19 Comparison of ceramide contents in the blank plasma matrix of Example 3 and unpeeled human plasma

[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 can be seen from Table 19, the removal rates of the 7 ceramides in the blank plasma matrix all reached over 95%, and the detected contents of the 6 ceramides were all 0. This stripping solvent can effectively strip the above 7 ceramides from 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 carried out according to the same method as in Example 1, and the ceramide results are shown in Table 20.

[0174] Table 20 Comparison of ceramide contents in the blank plasma matrix of Example 4 and unpeeled human plasma

[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 can be seen from Table 20, the removal rates of the 7 ceramides in the blank plasma matrix all reached over 95%, and the detected contents of the 6 ceramides were all 0. This stripping solvent can effectively strip the above 7 ceramides from human plasma.

[0177] Comparative Example 1

[0178] The target substance was stripped according to the method described in Example 1 to prepare a stripped 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 were 40% and 60% respectively.

[0179] Sample pretreatment and detection were carried out according to the same method as in Example 1, and the ceramide results are shown in Table 21.

[0180] Table 21 - Comparison of ceramide contents in the stripped matrix of Comparative Example 1 and non-stripped human plasma

[0181] Ceramide (ng / mL) Unstripped human plasma Stripped 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 7 ceramides in the stripped matrix were different. The removal rate of Cer(d18:1 / 14:0) was approximately 80%, the removal rate of Cer(d18:1 / 16:0) was between 90% and 95%, and the detected contents of 4 ceramides were 0. The stripped matrix with a high content of Cer(d18:1 / 14:0) would lead to inaccurate detection of ceramides.

[0183] Comparative Example 2

[0184] A stripped matrix was prepared according to the method described in Example 1, 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 were 60% and 40% respectively. The mixing process of the stripping solvent was the same as that in Example 1. Protein precipitation was found in the mixed plasma after adding the stripping solvent and mixing evenly.

[0185] This indicates that the difference in protein concentration between the stripped matrix obtained by stripping treatment with the stripping solvent of Comparative Example 2 and the protein concentration of the non-stripped mixed plasma is relatively large, not meeting the requirement that the composition of the substitute matrix is similar to that of the non-stripped human plasma.

[0186] Comparative Example 3

[0187] A stripped matrix was prepared according to the method described in Example 1, except that the stripping solvent was 100% methyl tert-butyl ether.

[0188] Table 22 - Comparison of ceramide contents in the stripped matrix of Comparative Example 3 and non-stripped human plasma

[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 rates of 100% methyl tert-butyl ether for 7 ceramides range from 45% to 83%, with relatively low removal rates. High residual ceramides in the stripping matrix will lead to inaccurate detection of ceramides.

[0191] Comparative Example 4

[0192] The biological liquid is mixed plasma. This mixed plasma is the same as that in Example 1. Activated carbon was used to strip ceramides from the mixed plasma.

[0193] First step: Weigh activated carbon and measure the mixed plasma according to the ratio of 1 mL of mixed plasma to 30 mg of activated carbon and place them in the same container, mix evenly, sonicate for 40 min, then centrifuge at 10000 rmp for 15 min, and take the supernatant.

[0194] Second step: Repeat the first step 2 times;

[0195] Third step: Centrifuge the supernatant after three stripping processes at 10000 rmp for 15 min, and then take the supernatant again;

[0196] Fourth step: Repeat the third step 6 times to obtain the stripping matrix;

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

[0198] The contents and removal rates of 7 ceramides in the stripping matrix prepared in Comparative Example 4 and non-stripped human plasma are shown in Table 23.

[0199] Table 23 - Comparison of the contents of 7 ceramides in the stripping matrix and non-stripped human plasma of Comparative Example 4

[0200] Ceramide (ng / mL) Unstripped human plasma Stripped 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 reagent has a good stripping effect on some ceramides in the 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 relatively high. Using the stripping matrix prepared in Comparative Example 4 as the dilution diluent for calibration products and quality control products will lead to some ceramides, such as

[0202] The accuracy of the test results for Cer(d18:1 / 14:0), Cer(d18:1 / 16:0), and Cer(d18:1 / 18:0) is low.

[0203] Comparative Example 5

[0204] Detect the ceramide according to the method of Example 1, except that the alternative matrix is isopropanol, and the matrix effect test results are as follows:

[0205] Table 24 - Matrix effect table 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 table of Cer(d18:1 / 18:0)

[0211]

[0212] Table 27 - Matrix effect table 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 indicate that when isopropanol is used as a surrogate matrix, the average matrix effect factors of the 7 ceramides after internal standard correction range from 0.83 to 1.12. Among them, the matrix effects of some target substances are relatively large in some samples. For example, the average matrix effect of the third sample at the L spiking level in Cer(d18:1 / 22:0) is 0.69, and the average matrix effect factor of the fifth sample at the L spiking level in Cer(d18:1 / 16:0) reaches 1.18. These strong matrix effects will lead to inaccurate detection results of ceramides in the corresponding samples.

[0223] Although some specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and not for limiting the scope of the present invention. Those skilled in the art should understand that the above embodiments can 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: The method 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. 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. The ether includes dimethyl ether, diethyl ether, dipropyl ether, methyl ethyl ether, ethyl propyl ether, methyl propyl ether, and methyl tert-butyl ether in terms of mass percentage. The alcohol includes at least one of methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol, propylene glycol, glycerol, and sorbitol. The ratio of the alcohol content to the ether content is 5:95 to 25:

75. The volume ratio of the stripping solvent to the biological fluid is greater than or equal to 1:

2.

2. 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 content of the isopropanol to the content of the methyl tert-butyl ether 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).

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

4. 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.

5. The detection kit according to claim 4, 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.

6. The detection kit according to claim 5, characterized in that The second main phase layer is dried by freeze drying.

7. 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).

8. A method for detecting ceramide using the detection kit according to any one of claims 1 to 7, characterized in that: It involves detecting ceramide in samples using liquid chromatography tandem mass spectrometry.

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