In vitro quantitative detection of four ceramide compounds in human serum

Abstract

The application provides a detection kit for quantitatively detecting four ceramide compounds in human serum in vitro, relates to the technical field of ceramide compound detection, and comprises a calibrant, a quality control product, an internal standard, a mobile phase additive, an extraction solution and a reconstitution solution; the calibrant and the quality control product adopt a nano matrix as a matrix. The amino-functionalized mesoporous silica nanoparticles are introduced, amino functional groups on the surface of the amino-functionalized mesoporous silica nanoparticles can specifically chelate metal ions in a capture solution, the path of metal ion catalytic oxidative degradation is fundamentally blocked, and the stability of ceramide in the calibrant is improved; especially, L-ascorbic acid, Tween 80 and the amino-functionalized mesoporous silica nanoparticles are mixed with sample serum in a sample collection stage, ceramide quality change in the sample serum during a sample serum delivery process is hindered, the stability of ceramide in the sample is significantly improved, and the accuracy and sensitivity of ceramide quantification are also significantly improved.

Description

Technical Field

[0001] This invention relates to the field of ceramide compound detection technology, specifically a detection kit for the in vitro quantitative detection of four ceramide compounds in human serum. Background Technology

[0002] In clinical practice, six indicators of ceramides—Cer(16:0), Cer(18:0), Cer(24:1), Cer(16:0) / Cer(24:0), Cer(18:0) / Cer(24:0), and Cer(24:1) / Cer(24:0)—are used to classify patients with coronary artery disease into low, medium, high, and high-risk groups to predict the risk of cardiovascular death in patients with acute coronary syndrome and stable coronary artery disease.

[0003] Currently, the detection methods for ceramides are mainly divided into mass spectrometry and non-mass spectrometry. In the past, non-mass spectrometry methods were often used to determine ceramides in tissues or cells, including immunochemistry, thin-layer chromatography, gas chromatography, high-performance liquid chromatography, and enzymatic methods (the most commonly used). With the continuous development of mass spectrometry, it can distinguish different types of ceramides based on the mass-to-charge ratio of molecules. Moreover, chromatographic tandem mass spectrometry is very suitable for detecting low levels of ceramides due to its high selectivity and high sensitivity, and it has become a common method for detecting ceramides in plasma or serum.

[0004] In the clinical determination of the concentrations of four key ceramides, Cer(d18∶1 / 16∶0), Cer(d18∶1 / 18∶0), Cer(d18∶1 / 24∶0), and Cer(d18∶1 / 24∶1), trace metal ions in serum samples can catalyze the oxidative degradation of ceramides, leading to low and unstable detection results. Furthermore, they can inhibit the ionization efficiency of the target analytes in mass spectrometry analysis, introducing a significant matrix effect that affects the accuracy and sensitivity of quantification. Therefore, this invention provides a detection kit for the in vitro quantitative detection of four ceramide compounds in human serum with stable and reliable detection results. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a detection kit for the in vitro quantitative detection of four ceramide compounds in human serum. This kit solves the problems that trace metal ions in serum samples can catalyze the oxidative degradation of ceramides, leading to low and unstable detection results; and that they can suppress the ionization efficiency of target compounds in mass spectrometry analysis, introducing a significant matrix effect that affects the accuracy and sensitivity of quantification.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A kit for the in vitro quantitative detection of four ceramide compounds in human serum includes: calibrators, quality control samples, internal standards, mobile phase additives, extraction solutions, and reconstitution solutions. The calibrators and quality control samples use nano-matrixes.

[0008] The nanomatrix includes: bovine serum albumin, phosphate buffer, L-ascorbic acid, Tween 80, lecithin, and amino-functionalized mesoporous silica nanoparticles.

[0009] The amino density of the amino-functionalized mesoporous silica nanoparticles is 0.20-0.55 mmol / g.

[0010] Preferably, the nanomatrix comprises: 3% bovine serum albumin, 0.3% Tween 80, 1.25% lecithin, 0.05% L-ascorbic acid, and amino-functionalized mesoporous silica nanoparticles, wherein the concentration of the amino-functionalized mesoporous silica nanoparticles in the matrix is ​​0.01 mg / mL to 1.22 mg / mL.

[0011] Preferably, the extract is an ethyl acetate or isopropanol solution.

[0012] Preferably, the internal standard is an isopropanol solution containing ceramides Cer(16:0)-D7, Cer(18:0)-D7, Cer(24:0)-D7, and Cer(24:1)-D7.

[0013] Preferably, the mobile phase additive is one or a mixture of formic acid solution, acetonitrile solution, and ammonium acetate solution.

[0014] Preferably, the reconstitution solution is an acetonitrile solution.

[0015] Preferably, the method for preparing the amino-functionalized mesoporous silica nanoparticles includes:

[0016] (1) Synthesis of mesoporous silica nanoparticles:

[0017] a. Dissolve hexadecyltrimethylammonium bromide in a mixed solvent of deionized water and ethanol, and stir at a constant temperature in a 35°C water bath. After the hexadecyltrimethylammonium bromide is completely dissolved, add NaOH solution and continue stirring for 30 minutes.

[0018] b. Stir vigorously at a speed of 600 rpm or higher, and add tetraethyl orthosilicate dropwise at a rate of 1-2 mL / min using a constant pressure dropping funnel;

[0019] c. After the addition is complete, maintain the reaction temperature at 35°C for 2 hours, then transfer the reaction system to an oven at 80°C for static aging for 24 hours to form a regular mesoporous structure.

[0020] d. Allow to cool naturally to room temperature, centrifuge at 10,000 rpm for 15 min using a high-speed centrifuge to collect the white precipitate, and wash it several times alternately with anhydrous ethanol and deionized water to remove residual reactants and obtain the preproduct;

[0021] e. The preproduct was redispersed in an acidic ethanol solution, centrifuged, washed, and dried to obtain pure mesoporous silica nanoparticles;

[0022] (2) Amin functionalization:

[0023] f. Vacuum activation of pure mesoporous silica nanoparticles at 100℃ for 2 hours;

[0024] g. Disperse the activated pure mesoporous silica nanoparticles in anhydrous toluene and sonicate for 30 minutes to form a dispersion.

[0025] h. Under nitrogen protection and stirring, the catalyst and 3-aminopropyltriethoxysilane were added to the dispersion, and then the mixture was heated to 90°C and refluxed for 12 h.

[0026] i. Solid products are collected and dried to obtain amino-functionalized mesoporous silica nanoparticles.

[0027] Preferably, the pure mesoporous silica nanoparticles have a BET specific surface area (referring to the total surface area per unit mass of material) of not less than 800 m² / g, and the pore size distribution is concentrated in the range of 2.5-3.5 nm.

[0028] This invention provides a detection kit for the in vitro quantitative detection of four ceramide compounds in human serum. It has the following beneficial effects:

[0029] This invention introduces amino-functionalized mesoporous silica nanoparticles, whose surface amino functional groups can specifically chelate and capture metal ions in solution, fundamentally blocking the pathway of metal ion catalytic oxidation degradation and improving the stability of ceramide in standards. In particular, mixing L-ascorbic acid, Tween 80, and amino-functionalized mesoporous silica nanoparticles with sample serum during the sample collection stage prevents the degradation of ceramide during the sample serum delivery process, significantly improving the stability of ceramide in the sample and also significantly improving the accuracy and sensitivity of ceramide quantification.

[0030] In the nanomatrix system of this invention, bovine serum albumin, phosphate buffer, and lecithin simulate the components of human serum. L-ascorbic acid, Tween 80, and amino-functionalized mesoporous silica nanoparticles are added to chelate metal ions. Tween 80, as a surfactant, effectively inhibits ion formation. During the detection stage, L-ascorbic acid, Tween 80, and amino-functionalized mesoporous silica nanoparticles are mixed with the sample serum during sample collection, ensuring that the resulting treated sample serum matrix is ​​consistent with the nanomatrix of this invention, thus avoiding matrix effects that could affect the accuracy and sensitivity of quantification. Compared to traditional, simple simulated human serum matrices, this application uses L-ascorbic acid, Tween 80, and amino-functionalized mesoporous silica nanoparticles to adjust the components of both simulated and sample serum, resulting in higher reliability during high-performance liquid chromatography-tandem mass spectrometry detection. Attached Figure Description

[0031] Figure 1 This is a standard curve diagram of the detection kit for the in vitro quantitative detection of four ceramide compounds in human serum according to Embodiment 1 of the present invention.

[0032] Figure 2 This is a standard curve diagram of the detection kit for the in vitro quantitative detection of four ceramide compounds in human serum according to Embodiment 2 of the invention.

[0033] Figure 3 This is a chromatogram of the detection kit for the in vitro quantitative detection of four ceramide compounds in human serum proposed in this invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] The preparation of amino-functionalized mesoporous silica nanoparticles specifically includes the following steps:

[0036] (1) Synthesis of mesoporous silica nanoparticles:

[0037] a. Weigh 1.0 g of cetyltrimethylammonium bromide and dissolve it in a mixed solvent consisting of 480 mL of deionized water and 160 mL of anhydrous ethanol; stir at a constant temperature in a 35 °C water bath until it is completely dissolved, then add 0.28 g of sodium hydroxide and continue stirring for 30 minutes to form a homogeneous template micelle solution.

[0038] b. Stir vigorously at a speed of 600 rpm or higher (using a magnetic stirrer), and add tetraethyl orthosilicate dropwise at a rate of 1-2 mL / min using a constant pressure dropping funnel.

[0039] c. After the addition is complete, maintain the temperature at 35°C and continue the reaction for 2 hours. Then, transfer the reaction system to an oven at 80°C for static aging for 24 hours to form a regular mesoporous structure.

[0040] d. Allow to cool naturally to room temperature, centrifuge at 10,000 rpm for 15 min using a high-speed centrifuge to collect the white precipitate, and wash it several times alternately with anhydrous ethanol and deionized water to remove residual reactants and obtain the preproduct.

[0041] e. The preproduct is redispersed in an acidic ethanol solution (nitric acid ethanol solution), centrifuged, washed, and dried to obtain pure mesoporous silica nanoparticles. Generally, the BET specific surface area of ​​pure mesoporous silica nanoparticles is not less than 800 m² / g, and the pore size distribution is concentrated in the range of 2.5-3.5 nm.

[0042] (2) Amin functionalization:

[0043] f. Pure mesoporous silica nanoparticles were vacuum activated at 100°C for 2 hours.

[0044] g. Disperse the activated pure mesoporous silica nanoparticles in anhydrous toluene in a volume more than 5 times that of the pure mesoporous silica nanoparticles, and sonicate for 30 minutes to form a dispersion.

[0045] h. Under nitrogen protection and stirring, add an appropriate amount of catalyst and 3-aminopropyltriethoxysilane to the dispersion, and then heat the mixture to 90°C and reflux for 12 hours; specifically, under nitrogen protection and mechanical stirring at 300-500 rpm, add triethylamine (volume ratio of triethylamine to 3-aminopropyltriethoxysilane is (0.05-0.2):1) and 3-aminopropyltriethoxysilane (mass ratio of 3-aminopropyltriethoxysilane to pure mesoporous silica nanoparticles is (0.5-2):1) as catalysts to the dispersion obtained in step g, and then heat the mixture to 90°C and reflux for 12-24 hours.

[0046] i. Wash with toluene and anhydrous ethanol 3-5 times each, then collect the solid product (by centrifugation) and dry it (in a vacuum drying oven at 60-80℃ for 6-12 hours) to obtain amino-functionalized mesoporous silica nanoparticles.

[0047] A kit for the in vitro quantitative detection of four ceramide compounds in human serum includes: calibrators, quality control samples, internal standards, mobile phase additives, extraction solutions, and reconstitution solutions. The calibrators and quality control samples use nano-matrixes.

[0048] The nanomatrix comprises bovine serum albumin, phosphate buffer, L-ascorbic acid, Tween 80, lecithin, and amino-functionalized mesoporous silica nanoparticles, wherein the amino density of the amino-functionalized mesoporous silica nanoparticles is 0.20-0.55 mmol / g.

[0049] The amino density of the amino-functionalized mesoporous silica nanoparticles can be adjusted by controlling the ratio of 3-aminopropyltriethoxysilane to pure mesoporous silica nanoparticles in the preparation process. For example, the initial mass ratio of 3-aminopropyltriethoxysilane to pure mesoporous silica nanoparticles can be controlled within the range of 0.2:1 to 0.6:1.

[0050] In a preferred embodiment, the nanomatrix comprises: bovine serum albumin at a mass-volume concentration of 3%, Tween 80 at a mass-volume concentration of 0.3%, lecithin at a mass-volume concentration of 1.25%, L-ascorbic acid at a mass-volume concentration of 0.05%, and amino-functionalized mesoporous silica nanoparticles, wherein the concentration of the amino-functionalized mesoporous silica nanoparticles in the matrix is ​​0.01 mg / mL to 1.22 mg / mL.

[0051] In a preferred embodiment, the extract is an ethyl acetate or isopropanol solution.

[0052] In a preferred embodiment, the mobile phase additive is one or a mixture of formic acid solution, acetonitrile solution, and ammonium acetate solution.

[0053] In a preferred embodiment, the reconstitution solution is an acetonitrile solution.

[0054] The internal standard is an isopropanol solution containing ceramides Cer(16:0)-D7, Cer(18:0)-D7, Cer(24:0)-D7, and Cer(24:1)-D7. It is used to accurately correct the quantitative analysis of the four corresponding ceramide targets during sample pretreatment to eliminate errors caused by pretreatment loss and mass spectrometry ionization fluctuations.

[0055] Example 1:

[0056] Configure calibrators:

[0057] Table 1 shows the composition of calibrators S1-S6 in Example 1:

[0058]

[0059] In Table 1, nanomatrix 1 consists of: 3% bovine serum albumin (BSA), 0.3% Tween 80 (TWA), 1.25% lecithin (LSA), 0.05% L-ascorbic acid (LSA), and amino-functionalized mesoporous silica nanoparticles. The concentration of amino-functionalized mesoporous silica nanoparticles in the matrix is ​​0.52 mg / mL, and the amino density of the amino-functionalized mesoporous silica nanoparticles is 0.25 mmol / g.

[0060] Table 2 shows the concentrations of ceramide in calibrators S1-S6 in Example 1:

[0061]

[0062] Table 3 shows the liquid phase conditions in Example 1:

[0063]

[0064] Table 4 shows the gradient elution conditions in Example 1:

[0065]

[0066] Pre-processing steps:

[0067] ① Accurately transfer 50 μL of serum sample (Note: during the serum sample collection stage, add L-ascorbic acid, Tween 80, and amino-functionalized mesoporous silica nanoparticles with the same content as those in nanomatrix 1 above to chelate metal ions) into a 2 mL 96-well plate;

[0068] ② Accurately transfer 10 μL of internal standard solution into each of the above 2 mL 96-well plates and vortex mix for 1 min (1200 RPM).

[0069] ③ Accurately transfer 600 μL of protein precipitant (isopropanol) into each of the above 2 mL 96-well plates, cap, shake for 10 min, and centrifuge for 10 min (4℃, 4000 RPM).

[0070] ④ After centrifugation, accurately transfer 50 μL of supernatant to another 2 mL 96-well plate, add 150 μL of reconstitution solution (acetonitrile), cover with a silica gel cap, and shake for 5 min; place the 2 mL 96-well plate in LC-MS / MS, edit the sequence, balance the instrument, and start the detection.

[0071] Example 2:

[0072] Table 5 shows the composition of calibrators S1-S6 in Example 2:

[0073]

[0074] Table 5 shows that nanomatrix 2 consists of bovine serum albumin (3% by volume), Tween 80 (0.3% by volume), lecithin (1.25% by volume), L-ascorbic acid (0.05% by volume), and amino-functionalized mesoporous silica nanoparticles. The concentration of amino-functionalized mesoporous silica nanoparticles in the matrix is ​​0.11 mg / mL, and the amino density of the amino-functionalized mesoporous silica nanoparticles is 0.25 mmol / g.

[0075] Pre-processing steps:

[0076] ① Accurately transfer 50 μL of serum sample (Note: during the serum sample collection stage, L-ascorbic acid, Tween 80, and amino-functionalized mesoporous silica nanoparticles with the same content as those in nanomatrix 2 above are added to chelate metal ions) into a 2 mL 96-well plate; the remaining steps are the same as in Example 1.

[0077] Comparative Example 1:

[0078] Table 6 shows the composition of calibrators S1-S6 in Comparative Example 1:

[0079]

[0080] In Table 6, nanomatrix 3 consists of: 3% bovine serum albumin, 0.3% Tween 80, 1.25% lecithin, 0.05% L-ascorbic acid, and amino-functionalized mesoporous silica nanoparticles. The concentration of amino-functionalized mesoporous silica nanoparticles in the matrix is ​​0.98 mg / mL, and the amino density of the amino-functionalized mesoporous silica nanoparticles is 0.25 mmol / g.

[0081] Pre-processing steps:

[0082] ① Accurately transfer 50 μL of serum sample (Note: during the serum sample collection stage, L-ascorbic acid, Tween 80, and amino-functionalized mesoporous silica nanoparticles with the same content as those in nanomatrix 3 above are added to chelate metal ions) into a 2 mL 96-well plate; the remaining steps are the same as in Example 1.

[0083] Comparative Example 2:

[0084] Table 7 shows the composition of calibrators S1-S6 in Comparative Example 2:

[0085]

[0086] The table above shows: 3% bovine serum albumin and 1.25% lecithin.

[0087] Pre-processing steps:

[0088] ① Accurately transfer 50 μL of serum sample into a 2 mL 96-well plate; the remaining steps are the same as in Example 1.

[0089] Comparative Example 3:

[0090] Table 8 shows the composition of calibrators S1-S6 in Comparative Example 3:

[0091]

[0092] Table 8 lists nanomatrix 4 as follows: 3% bovine serum albumin, 0.3% Tween 80, 1.25% lecithin, 0.05% L-ascorbic acid, and amino-functionalized mesoporous silica nanoparticles. The concentration of amino-functionalized mesoporous silica nanoparticles in the matrix is ​​0.11 mg / mL, and the amino density of the amino-functionalized mesoporous silica nanoparticles is 0.65 mmol / g.

[0093] Pre-processing steps:

[0094] ① Accurately transfer 50 μL of serum sample (Note: during the serum sample collection stage, L-ascorbic acid, Tween 80, and amino-functionalized mesoporous silica nanoparticles with the same content as those in nanomatrix 4 above are added to chelate metal ions) into a 2 mL 96-well plate; the remaining steps are the same as in Example 1.

[0095] Comparative Example 4:

[0096] Table 9 shows the composition of calibrators S1-S6 in Comparative Example 4:

[0097]

[0098] In Table 9, nanomatrix 5 consists of 3% bovine serum albumin, 1.25% lecithin, 0.05% L-ascorbic acid, and amino-functionalized mesoporous silica nanoparticles. The concentration of amino-functionalized mesoporous silica nanoparticles in the matrix is ​​0.52 mg / mL, and the amino density of the amino-functionalized mesoporous silica nanoparticles is 0.25 mmol / g.

[0099] Pre-processing steps:

[0100] ① Accurately transfer 50 μL of serum sample (Note: during the serum sample collection stage, L-ascorbic acid and amino-functionalized mesoporous silica nanoparticles with the same content as those in nanomatrix 5 above are added to chelate metal ions) into a 2 mL 96-well plate; the remaining steps are the same as in Example 1.

[0101] The steps of Examples 1 to 2 and Comparative Examples 1 to 4 were repeated 5 times respectively, and the data in Table 10 below were obtained. The recovery yield was obtained by testing the calibrator S2 under the corresponding examples or comparative examples.

[0102] Table 10 shows the recovery yield and in-batch accuracy data for Examples 1 to 2 and Comparative Examples 1 to 4:

[0103]

[0104] Under the test conditions of Examples 1 and 2, using the following concentrations (w / v): 3% bovine serum albumin, 0.3% Tween 80, 1.25% lecithin, 0.05% L-ascorbic acid, and amino-functionalized mesoporous silica nanoparticles, with an amino density of 0.25 mmol / g and concentrations of 0.52 mg / mL and 0.11 mg / mL (between 0.01 mg / mL and 1.22 mg / mL), respectively, the average recovery rate was greater than 85%, demonstrating excellent quantification accuracy. Furthermore, the intra-batch precision was less than 8%, indicating excellent detection reliability. Compared with Comparative Example 2, both the average recovery rate and intra-batch precision were significantly improved.

[0105] Comparing Comparative Example 1 with Examples 1 and 2, when the concentration of amino-functionalized mesoporous silica nanoparticles in the matrix is ​​0.98 mg / mL, the concentration is relatively high, which will affect the average recovery rate. The possible mechanism is that the high concentration of amino-functionalized mesoporous silica nanoparticles affects the operation of the experimental steps, and the ceramide compound is lost during the intermediate processing, increasing the difficulty of the experiment.

[0106] In Comparative Example 3, the amino density of the amino-functionalized mesoporous silica nanoparticles was 0.65 mmol / g, which caused ceramide to be adsorbed by the amino-functionalized mesoporous silica nanoparticles themselves, making it impossible to fully extract and detect. Its average recovery rate was lower than that of Examples 1 and 2.

[0107] In Comparative Example 4, the matrix aggregation of amino-functionalized mesoporous silica nanoparticles was controlled by the surfactant Tween 80, and the average recovery rate was lower than that of Examples 1 and 2.

[0108] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A test kit for the in vitro quantitative determination of four ceramide compounds in human serum, characterized in that, The application relates to a calibration sample, a quality control sample, an internal standard sample, a mobile phase additive, an extraction solution and a reconstitution solution, wherein the matrix of the calibration sample and the quality control sample is a nano-matrix. The nano-matrix comprises bovine serum albumin, phosphate buffered saline, L-ascorbic acid, Tween 80, lecithin and amino-functionalized mesoporous silica nanoparticles. The amino-functionalized mesoporous silica nanoparticles have an amino density of 0.20-0.55 mmol / g. The nano-matrix comprises 3% bovine serum albumin, 0.3% Tween 80, 1.25% lecithin, 0.05% L-ascorbic acid and amino-functionalized mesoporous silica nanoparticles, and the concentration of the amino-functionalized mesoporous silica nanoparticles in the matrix is 0.01 mg / mL-1.22 mg / mL.

2. The in vitro quantitative test kit for detecting four ceramide compounds in human serum according to claim 1, characterized by, The extraction solution is an ethyl acetate solution or an isopropanol solution.

3. The kit for in vitro quantitative determination of four ceramide compounds in human serum according to claim 1, characterized by, The internal standard sample is an isopropanol solution containing ceramide Cer (16:0)-D7, Cer (18:0)-D7, Cer (24:0)-D7 and Cer (24:1)-D7.

4. The kit for in vitro quantitative determination of four ceramide compounds in human serum according to claim 1, characterized by, The preparation method of the amino-functionalized mesoporous silica nanoparticles comprises the following steps:

5. The in vitro test kit for quantitatively measuring four ceramide compounds in human serum according to any one of claims 1 to 4, characterized by, (1) synthesis of mesoporous silica nanoparticles: a. dissolve cetyltrimethylammonium bromide in a mixed solvent of deionized water and ethanol, and stir in a 35 DEG C water bath; after the cetyltrimethylammonium bromide is completely dissolved, add NaOH solution, and continuously stir for 30 minutes; b. perform strong stirring at a rotating speed of more than 600 rpm, and drop add tetraethyl orthosilicate at a rate of 1-2 mL / min by using a constant-pressure dropping funnel; c. after the dropping is completed, continue to react for 2 hours at 35 DEG C, and then transfer the reaction system to an oven at 80 DEG C for static aging for 24 hours, so as to form a regular mesoporous structure; d. naturally cool to room temperature, collect the white precipitate by using a high-speed centrifuge at a rotating speed of 10000 rpm for 15 min, and wash the white precipitate with anhydrous ethanol and deionized water alternately for multiple times to remove residual reactants, so as to obtain a pre-product; e. disperse the pre-product in an acidic ethanol solution, and obtain pure mesoporous silica nanoparticles after centrifugal washing and drying; (2) amino functionalization: f. activate the pure mesoporous silica nanoparticles at 100 DEG C for 2 hours under vacuum; g. disperse the activated pure mesoporous silica nanoparticles in anhydrous toluene, and ultrasonically treat for 30 minutes to form a dispersion liquid; h. under the protection of nitrogen and stirring, add a catalyst and 3-aminopropyltriethoxysilane into the dispersion liquid, then heat the mixture to 90 DEG C, and heat to reflux for 12 hours; i. collect and dry the solid product to obtain amino-functionalized mesoporous silica nanoparticles. The pure mesoporous silica nanoparticles have a BET specific surface area of not less than 800 m² / g, and the pore size distribution is concentrated in 2.5-3.5 nm.

6. The kit for in-vitro quantitative detection of four ceramide compounds in human serum according to claim 5, characterized in that, The mobile phase additive is a mixture of one or more of formic acid solution, acetonitrile solution and ammonium acetate solution.

7. The kit for in vitro quantitative determination of four ceramide compounds in human serum according to claim 1, characterized by, The reconstitution solution is an acetonitrile solution.

8. The kit for in vitro quantitative determination of four ceramide compounds in human serum according to claim 1, characterized by, ​

Images