A method for determining ceramide in a sample, and its product and application

Abstract

The present invention discloses a method for determining ceramide in a sample, a product and an application thereof, and relates to the technical field of biological detection. The present invention provides a method suitable for simultaneously detecting multiple ceramides, wherein the method optimizes the mobile phase and the gradient elution program used in chromatographic elution, so that multiple ceramides can be separated and detected within 4.5 minutes, the analysis time is short, the throughput is high, and the influence of interference peaks is avoided or reduced, so that the detection signal of the ceramide to be detected can be effectively separated from the interference peak, thereby obtaining a detection result with high stability and good accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for determining ceramide in a sample, and a product and application thereof. Background Art

[0002] Given the high morbidity and mortality of coronary artery disease, preventing fatal and non-fatal myocardial infarction in patients with coronary artery disease remains a clinical challenge. The annual mortality rate of patients with stable coronary artery disease is between 1-3%, and the non-fatal morbidity is between 1-2%. Among patients with acute coronary syndrome who survive the acute illness, the mortality rate of myocardial infarction is significantly increased (especially within the first year). However, the risk of disease varies greatly among patients, which requires effective diagnostic, risk stratification, disease monitoring and efficacy evaluation tools to improve patient identification and management.

[0003] Coronary artery disease markers are bioactive substances that increase or decrease during the development of atherosclerosis and can be used for the diagnosis, risk stratification, disease monitoring and efficacy evaluation of coronary artery disease. They will have a huge impact on the clinical treatment of coronary artery disease. Therefore, in order to meet the needs of clinical diagnosis and treatment of coronary artery disease, the development of efficient and accurate markers needs to be accelerated.

[0004] Most of the markers currently used for diagnosis, risk stratification, disease monitoring and efficacy evaluation of coronary artery disease cannot provide more reference value for clinicians due to lack of sensitivity and specificity. For example, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in serum are associated with atherosclerosis and have long been used for diagnosis, risk stratification and prognosis of cardiovascular disease. However, the determination of these traditional risk molecules cannot identify a large proportion of patients with high risk of cardiovascular disease. Therefore, in addition to traditional risk molecules such as low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), there is an urgent need to find a more accurate and predictive risk factor.

[0005] Free lipidomics analysis shows that some lipids may serve as effective markers for diagnosis, risk stratification and prognosis of coronary artery disease. Lipidomics studies have found that specific ceramide species are significantly associated with cardiovascular death in patients with coronary artery disease (Meikle, PJ et al. Plasma lipidomic analysis of stable and unstable coronary artery disease. Arterioscler Thromb Vasc Biol 31, 2723-2732, doi: 10.1161 / ATVBAHA.111.234096 (2011).). Ceramide is composed of sphingosine and fatty acids. Ceramide is mainly produced through the sphingomyelinase pathway, which decomposes phospholipids on the membrane of sphingomyelinase and releases ceramide. At the same time, ceramide can be synthesized by six fatty acyl-selective ceramide synthases using simple molecules through a new synthesis pathway. Different ceramide molecules have specific physiological functions. Several key cytokines (such as tumor necrosis factor and interleukin) can induce inflammatory responses (such as atherosclerosis and ischemia-reperfusion injury), and ceramide levels are increased in these inflammatory responses. Some cardiovascular risk factors and markers (such as oxidized low-density lipoprotein and homocysteine) can increase the production of ceramide. In addition, ceramide, as a signaling molecule, participates in the regulation of many cellular responses and functions, including differentiation, proliferation, apoptosis, reactive oxygen production, and gene expression, which are directly involved in the molecular mechanisms of cardiovascular disease.

[0006] Ceramide is also an important target for the development of drugs for the treatment of coronary artery disease. Studies have shown that the metabolic process of sphingomyelin is a dynamic process, and the metabolites of sphingomyelin (including ceramide and sphingosine) play an important role in coronary artery disease as second messengers (Kolesnick, R. The therapeutic potential of modulating the ceramide / sphingomyelin pathway. J Clin Invest 110, 3-8, doi: 10.1172 / JCI16127 (2002).). When ceramide analogs are directly applied to damaged arteries, they can show a strong effect of resisting the proliferation and spread of malignant cells (Bourbon, NA, Yun, J., Berkey, D., Wang, Y. & Kester, M. Inhibitory actions of ceramide upon PKC-epsilon / ERK interactions. Am J Physiol Cell Physiol 280, C1403-1411 (2001).). Neointimal hyperplasia of vascular smooth muscle cells and secondary obstruction of coronary arteries are the causes of restenosis after balloon angioplasty and arterial stent implantation, affecting 3 million patients undergoing coronary angioplasty worldwide. Ceramide can inhibit the hyperplasia of vascular smooth muscle induced by ERK kinase and AKT kinase cascade. C6 ceramide-coated balloon catheters can prevent stretch-induced neointimal hyperplasia of vascular smooth muscle cells (Charles, R. et al. Ceramide-coated balloon catheters limit neointimal hyperplasia after stretch injury in carotid arteries. Circ Res 87, 282-288 (2000).).

[0007] There are a large number of analytical methods for measuring ceramides, including thin layer chromatography, conventional liquid chromatography, immunochemical methods, gas chromatography and tandem mass spectrometry. However, most of the methods are designed for research rather than clinical use. The common disadvantages of these methods are that they cannot analyze ceramides at the molecular species level, lack selectivity, low throughput, low precision and accuracy, and therefore cannot meet the requirements of clinical laboratories. We also cannot use traditional immunoassay methods to distinguish these four ceramides because the antigenic epitopes of each ceramide are too similar. Anti-ceramide antibodies recognize a class of ceramide molecules that differ only in the length of the fatty acid chain. Therefore, the low selectivity of existing antibodies has hindered the development of clinically effective immunoassay methods (Krishnamurthy, K., Dasgupta, S. & Bieberich, E. Development and characterization of a novel anti-ceramide antibody. J Lipid Res 48, 968-975, doi: 10.1194 / jlr.D600043-JLR200 (2007).). The main advantage of LC-MS / MS is that it combines chromatographic separation with precise monitoring of mass spectrometry, which can clearly detect each ceramide molecule (Kauhanen, D. et al. Development and validation of a high-throughput LC-MS / MS assay for routine measurement of molecular ceramides. Anal Bioanal Chem 408, 3475-3483, doi: 10.1007 / s00216-016-9425-z (2016).).

[0008] In view of this, the present invention is proposed. Summary of the invention

[0009] The purpose of the present invention is to provide a method for determining ceramide in a sample and a product and application thereof.

[0010] The present invention is achieved in that:

[0011] In a first aspect, an embodiment of the present invention provides a method for determining ceramide in a sample, which comprises detecting ceramide in the sample using a liquid chromatography tandem mass spectrometry method; the chromatography comprises gradient elution using mobile phase A and mobile phase B, the mobile phase A comprises an aqueous solution of ammonium acetate containing formic acid, and the mobile phase B comprises a mixed solution of acetonitrile and methyl tert-butyl ether containing formic acid. The elution conditions of the chromatography are as follows: from 0 to 1 minute, the volume percentage of the mobile phase A is maintained at 30% to 40%, and the volume percentage of the mobile phase B is maintained at 60% to 70%; from 1 to 1.5 minutes, the volume percentage of the mobile phase A is reduced from 30% to 40% to 20% to 30%, and the volume percentage of the mobile phase B is increased from 60% to 70% to 70%; from 1.5 to 3.5 minutes, the volume percentage of the mobile phase A is reduced from 20% to 30% to 1% to 10%, and the volume percentage of the mobile phase B is increased from 70% to 80% to 90% to 99%; from 3.5 to 3.6 minutes, the volume percentage of the mobile phase A is increased from 1% to 10% to 30% to 40%, and the volume percentage of the mobile phase B is reduced from 90% to 99% to 60% to 70%; from 3.6 to 4.5 minutes, the volume percentage of the mobile phase A is maintained at 30% to 40%, and the volume percentage of the mobile phase B is maintained at 60% to 70%. 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), and the method is not directly intended for the diagnosis or treatment of a disease.

[0012] In a second aspect, an embodiment of the present invention provides an application of a reagent combination in preparing a product for measuring ceramide in a sample, the reagent combination comprising reagents for implementing the method as described in the aforementioned embodiment, the ceramide comprising 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).

[0013] In a third aspect, an embodiment of the present invention provides a product for determining ceramide in a sample, which includes reagents for implementing the method for determining ceramide in a sample described in the above embodiment.

[0014] The present invention has the following beneficial effects: (1) There is an interference peak in the Cer (d18:1 / 20:0) plasma sample. By adjusting the liquid phase gradient, the interference peak is separated from Cer (d18:1 / 20:0) to avoid interference with the quantification of Cer (d18:1 / 20:0).Figure 1 , the retention time 3.43min is the C20:0 chromatographic peak in plasma, and the retention time 3.58min is the interference peak. The two are completely separated. (2) Ceramide itself is a lipid and is difficult to elute. The addition of methyl tert-butyl ether in the mobile phase enhances the elution ability, which can achieve a faster separation of the seven substances without causing residue. (3) Phospholipids can easily affect the detection of ceramides. By monitoring the phospholipid channel and adjusting the liquid phase gradient, the separation of phospholipids and seven ceramides is achieved, avoiding interference caused by phospholipids. (4) The method of the present invention can complete the separation and detection of multiple ceramides within 4.5 minutes, with short analysis time and high throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 It is a schematic diagram of the detection process;

[0017] Figure 2 is the total ion current diagram;

[0018] Figure 3 is the chromatogram of C20:0;

[0019] Figure 4 is a standard curve graph of C16:0;

[0020] Figure 5 This is the chromatogram of spiked plasma with C16:0 and C16:0 internal standard;

[0021] Figure 6 is a standard curve graph of C18:0;

[0022] Figure 7 Chromatograms of spiked plasma with C18:0 and C18:0 internal standard;

[0023] Figure 8 is a standard curve graph of C24:0;

[0024] Fig. 9 This is the chromatogram of spiked plasma with C24:0 and C24:0 internal standard;

[0025] Fig.10 This is the C24:1 standard curve diagram;

[0026] Fig.11 This is the chromatogram of spiked plasma with C24:1 and C24:1 internal standard;

[0027] Fig.12 is a standard curve graph of C14:0;

[0028] Fig.13 This is the chromatogram of spiked plasma with C14:0 and C14:0 internal standard;

[0029] Fig.14 is the C20:0 standard curve;

[0030] Fig.15 This is the chromatogram of spiked plasma with C20:0 and C20:0 internal standard;

[0031] Fig.16 is a standard curve graph of C22:0;

[0032] Fig.17 This is the chromatogram of spiked plasma with C22:0 and C22:0 internal standard;

[0033] Fig.18 This is the separation chromatogram without adding methyl tert-butyl ether;

[0034] Fig.19 This is the separation chromatogram of gradient 1

[0035] Fig. 20 is the separation chromatogram of gradient 2;

[0036] Fig.21 It is a phenyl column, phase A is 2mM ammonium acetate + 0.1% formic acid + water; phase B is 0.1% formic acid methanol chromatogram;

[0037] Fig. 22 This is the separation chromatogram of the Fenome Kinetex C8 (50×3mm, 2.6μm) column;

[0038] Fig.23 This is the separation chromatogram of the Fenome Kinetex Phenyl-Hexyl (50×4.6mm, 2.6μm) chromatographic column. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0040] An embodiment of the present invention provides a method for determining ceramide in a sample, comprising: detecting ceramide in the sample by liquid chromatography tandem mass spectrometry; the chromatography comprises gradient elution using mobile phase A and mobile phase B, the mobile phase A comprises an aqueous solution of ammonium acetate containing formic acid, and the mobile phase B comprises a mixed solution of acetonitrile and methyl tert-butyl ether containing formic acid.

[0041] The elution conditions of the chromatography are as follows: from 0 to 1 minute, the volume percentage of the mobile phase A is maintained at 30% to 40%, and the volume percentage of the mobile phase B is maintained at 60% to 70%; from 1 to 1.5 minutes, the volume percentage of the mobile phase A is reduced from 30% to 40% to 20% to 30%, and the volume percentage of the mobile phase B is increased from 60% to 70% to 70%; from 1.5 to 3.5 minutes, the volume percentage of the mobile phase A is reduced from 20% to 30% to 1% to 10%, and the volume percentage of the mobile phase B is increased from 70% to 80% to 90% to 99%; from 3.5 to 3.6 minutes, the volume percentage of the mobile phase A is increased from 1% to 10% to 30% to 40%, and the volume percentage of the mobile phase B is reduced from 90% to 99% to 60% to 70%; from 3.6 to 4.5 minutes, the volume percentage of the mobile phase A is maintained at 30% to 40%, and the volume percentage of the mobile phase B is maintained at 60% to 70%.

[0042] The ceramides include 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). The "determining ceramides in a sample" herein specifically refers to the qualitative and / or quantitative detection of ceramides.

[0043] The method is not aimed at diagnosing or treating a disease directly, but is aimed at detecting ceramide or its content in a sample directly.

[0044] Preferably, the elution conditions of the chromatography are as follows: from 0 to 1 min, the volume percentage of mobile phase A is maintained at 35%, and the volume percentage of mobile phase B is maintained at 65%; from 1 to 1.5 min, the volume percentage of mobile phase A is reduced from 35% to 25%, and the volume percentage of mobile phase B is increased from 65% to 75%; from 1.5 to 3.5 min, the volume percentage of mobile phase A is reduced from 25% to 5%, and the volume percentage of mobile phase B is increased from 75% to 95%; from 3.5 to 3.6 min, the volume percentage of mobile phase A is increased from 5% to 35%, and the volume percentage of mobile phase B is reduced from 95% to 65%; from 3.6 to 4.5 min, the volume percentage of mobile phase A is maintained at 35%, and the volume percentage of mobile phase B is maintained at 65%.

[0045] In some embodiments, the flow rate of the gradient elution is 0.1-1 mL / min, specifically any one of 0.01 mL / min, 0.2 mL / min, 0.4 mL / min, 0.6 mL / min, 0.8 mL / min, 1 mL / min, or a range between any two of them.

[0046] In some embodiments, the chromatographic column used in the chromatography includes any one of a C8 chromatographic column and a phenyl chromatographic column; optionally, the C8 chromatographic column is selected from any one of Fenom Kinetex C8, Shimadzu shim-pack-GIST-HP C8, and a chromatographic column with a similar type of stationary phase bonded phase or model as Fenom Kinetex C8 or Shimadzu shim-pack-GIST-HP C8. Preferably, the model of Fenom Kinetex C8 is 50×3mm, 2.6μm; preferably, the model of Shimadzu shim-pack-GIST-HP C8 is 50×2.1, 3μm. Preferably, the phenyl chromatographic column comprises Fenom Kinetex Phenyl-Hexyl or a chromatographic column with a similar type of stationary phase bonded phase or model; preferably, the model of Fenom Kinetex Phenyl-Hexyl is 50×4.6, 2.6μm.

[0047] The traditional C18 column has strong retention for the seven ceramides and is not easy to elute, while the C8 column has weaker retention for the seven ceramides than C18 and is not easy to cause residues. Preferably, the chromatographic column is Shimadzu Shim-pack-GIST-HP C8.

[0048] In some embodiments, the chromatographic conditions include: a column temperature of 35 to 45° C., an injector temperature of 5 to 15° C., and an injection volume of 2 to 5 μL.

[0049] In some embodiments, in the mobile phase B, the volume ratio of acetonitrile to methyl tert-butyl ether is (70-80): (20:30), specifically any one of 70:30, 72:28, 74:26, 76:24, 78:22, 80:20 or a range between any two thereof, preferably 75:25. In the mobile phase B, the volume concentration fraction of formic acid is 0.01% to 0.5%, specifically any one of 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5% or a range between any two thereof. The volume concentration fraction of formic acid in the mobile phase A is 0.01% to 0.5%, specifically any one of 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, or any two thereof. In the mobile phase A, the concentration of ammonium acetate is 1 to 10 mM, specifically any one of 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, or 10 mM, or any two thereof, preferably 2 mM. In some embodiments, the mass spectrometer uses an electrospray ion source, the detection mode is a positive ion mode, the scanning mode is a multiple reaction monitoring, the desolvation temperature is 550°C, the nebulizing gas flow rate (GAS1) is 55, the desolvation flow rate (GAS2) is 55, and the ion source voltage (IS) is 5500V.

[0050] In some embodiments, the sample comprises a blood sample, or an environmental sample containing a blood sample. The blood sample may be any one of a whole blood sample, a serum sample, and a plasma sample.

[0051] In some embodiments, the liquid chromatography tandem mass spectrometry detection includes a sample pretreatment step, and the sample pretreatment includes: mixing the sample to be tested, the internal standard and the precipitant to perform protein precipitation, removing the precipitate and using it for liquid chromatography tandem mass spectrometry detection. The pretreatment is performed by protein precipitation method, which is simple to operate, has a short processing cycle, and greatly shortens the pretreatment time. Preferably, the precipitant includes methanol.

[0052] In some embodiments, the internal standard solution includes: at least one of an internal standard of Cer (d18:1 / 16:0), an internal standard of Cer (d18:1 / 18:0), an internal standard of Cer (d18:1 / 24:0), and an internal standard of Cer (d18:1 / 24:1). By optimizing pretreatment, liquid phase method, etc., using a shared isotope internal standard, 4 isotope internal standards are used to complete the detection of 7 ceramides, solving the matrix effect problem of the remaining 3 non-isotope internal standard ceramides. Preferably, the internal standard solution includes: at least one of Cer (d18:1 / 16:0) deuterated, Cer (d18:1 / 18:0) deuterated, Cer (d18:1 / 24:0) deuterated, and Cer (d18:1 / 24:1) deuterated.

[0053] The embodiment of the present invention also provides the use of a reagent combination in preparing a product for measuring ceramide in a sample, wherein the reagent combination includes a reagent for implementing the method described in any of the foregoing embodiments, and 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). The sample is the same as that described in any of the foregoing embodiments and will not be described in detail.

[0054] Specifically, the reagent combination includes at least one of the mobile phase A, mobile phase B, protein precipitant and internal standard as described in any of the above embodiments.

[0055] In some embodiments, the reagent combination further comprises at least one of the calibrators and quality control products corresponding to the seven ceramides, and the diluent used for each calibrator and quality control product is isopropanol.

[0056] In some embodiments, the concentration of the calibrator and the quality control product is 20× the target concentration, and they are packaged in different carrying containers, air-dried and stored in the form of dry powder, which prolongs the shelf life and facilitates storage and transportation. When used, the diluent is used to reconstitute (the volume is expanded by 20 times) to obtain the reagent of the target concentration.

[0057] In some embodiments, the reagent combination further includes: at least one of purified water, formic acid, acetonitrile, methyl tert-butyl ether and ammonium acetate.

[0058] In addition, an embodiment of the present invention further provides a product for measuring ceramide in a sample, which includes reagents for implementing the method for measuring ceramide in a sample as described in any of the above embodiments.

[0059] In some embodiments, the product is a kit, which may include the reagent combination described in any of the preceding embodiments.

[0060] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0061] Example 1

[0062] [Main components] The main components of the kit are shown in the following table.

[0063]

[0064] The items required for testing are shown in the following table.

[0065] name Require Purified water Deionized water Formic acid Chromatographic purity or above Acetonitrile Chromatographic purity or above Methyl tert-butyl ether Chromatographic purity or above Ammonium acetate Chromatographic purity or above 96-well V-shaped plate 450μL 96-well V-plate and 96-well plate membrane Polypropylene centrifuge tube 1.5-2mL Pipette 10-100μL, 20-200μL, 100-1000μL Vortex mixer /

[0066] Sample type: This kit is suitable for human plasma samples, and the sample volume is required to be ≥0.5mL.

[0067] [Test method] Before testing, restore the test kit and samples to room temperature (10℃~30℃); 2. Preparation of calibrators and quality control products - reconstitution: At room temperature, accurately pipette 1mL of diluent into each level of calibrators and quality control bottles, mix thoroughly for 3~5min, and obtain each level of calibrators and quality control solutions; 3. Preparation of internal standards - reconstitution: At room temperature, accurately pipette 3mL of diluent into the internal standard bottle, mix thoroughly for 3~5min, and obtain the internal standard solution; 4. Sample processing and detection process: Add sample: Accurately pipette 100μL of calibrators / quality control products / plasma samples, add 20μL of internal standard solution and 880μL of diluent, and vortex for 2~3min; Centrifugation: Centrifuge at 4℃, 15000rpm for 10min to separate the supernatant; Detection: Transfer 100-200μL of supernatant to a clean 96-well V-plate for LC-MS / MS detection. See the detection process diagram for details. Figure 1 .

[0068] Data Analysis and Results:

[0069] Calibration curve drawing: the concentration of the calibrator is the independent variable x i , the mean of the peak area ratio of the corresponding concentration calibrator and internal standard is taken as the dependent variable y i , calculate the linear regression equation y=ax+b and the correlation coefficient r; quality control product data analysis: when the r of the calibration curve is ≥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; sample data analysis: when the test result of the quality control product is within the expected range, the test signal of the sample is substituted into the regression equation to obtain the concentration of the target substance in the sample.

[0070] Instruments: The mass spectrometer used was AB Sciex 4500 (S / N EB252831905); the liquid chromatograph used was Jasper Sciex.

[0071]

Instrumental method

[0072] Table 1. Ion source parameters

[0073] CUR CAD IS(v) TEM(℃) GAS1 GAS1 35 5 5500 550 55 55

[0074] Table 2 Mass spectrometry methods

[0075] MRM channels Compound Q1 Q3 DWELL(msec) DP(V) Collision(V) 1 C16:0 520.5 264.5 15 58 35 2 C16-d7 527.4 271.4 15 58 35 3 C18:0 548.5 264.5 15 58 35 4 C18-d7 555.5 271.4 15 58 35 5 C24:0 632.5 264.5 15 70 22 6 C24:0-d7 639.5 271.4 15 70 40 7 C24:1 630.5 264.5 15 70 22 8 C24:1-d7 637.5 271.4 15 70 40 9 C14:0 492.5 264.5 15 58 35 10 C20:0 576.5 264.5 15 50 25 11 C22:0 604.5 264.5 15 58 25

[0076] Table 3. Liquid phase methods

[0077]

[0078] The total ion current corresponding to the kit and method is shown in Figure 2 , C20:0 chromatogram is shown in Figure 3 . From the above total ion current, it can be seen that the seven ceramides have achieved good retention and separation (c22 and c24:1 have the same retention time, but different quantitative ion pairs, and the mass spectrometer can distinguish them). The peaks of all compounds are sharp and symmetrical, and are completely separated from the phospholipid peaks that may affect the test results. Figure 3 It can be seen that the retention time of 3.43min is the C20:0 chromatographic peak in plasma, and the retention time of 3.58min is the interference peak. The two are completely separated.

[0079]

Method evaluation content

[0080] 1. Standard curve linearity evaluation

[0081] Experimental procedure: According to the series of working solutions provided by the kit, a standard curve was prepared by adding isopropanol, with the analyte concentration as the horizontal axis and the peak area ratio of the analyte to the internal standard as the vertical axis. The weighted (W=1 / X2) least squares method was used for linear regression to obtain the linear regression equation (Y=bX+a), and the correlation coefficient r was calculated, which was required to be r≥0.99.

[0082] Table 4. Experimental results

[0083]

[0084] Table 5. Evaluation results of the standard curve regression equation parameters for three consecutive analysis batches of C16:0

[0085]

[0086] C16:0 standard curve Figure 4, C16:0 and C16:0 internal standard plasma spiked chromatograms are shown in Figure 5 .

[0087] Table 6. Evaluation results of the standard curve regression equation parameters for three consecutive analysis batches of C18:0

[0088]

[0089] C18:0 standard curve is shown in Figure 6 , C18:0 and C18:0 internal standard plasma spiked chromatograms are shown in Figure 7 .

[0090] Table 7. Evaluation results of the standard curve regression equation parameters for three consecutive analysis batches of C24:0

[0091]

[0092] C24:0 standard curve is shown in Figure 8 , C24:0 and C24:0 internal standard plasma spiked chromatograms are shown in Fig. 9 .

[0093] Table 7. Evaluation results of the standard curve regression equation parameters for three consecutive analysis batches of C24:1

[0094]

[0095] C24:1 standard curve diagram Fig.10 , C24:1 and C24:1 internal standard plasma spiked chromatograms are shown in Fig.11 .

[0096] Table 8. Evaluation results of the standard curve regression equation parameters for three consecutive analysis batches of C14:0

[0097]

[0098] C14:0 standard curve Fig.12 , C14:0 and C14:0 internal standard plasma spiked chromatograms are shown in Fig.13 .

[0099] Table 9. Evaluation results of the standard curve regression equation parameters for three consecutive analysis batches of C20:0

[0100]

[0101] C20:0 standard curve is shown in Fig.14 , C20:0 and C20:0 internal standard plasma spiked chromatograms are shown in Fig.15 .

[0102] Table 10. Evaluation results of the standard curve regression equation parameters for three consecutive analysis batches of C22:0

[0103]

[0104] C22:0 standard curve is shown in Fig.16 , C22:0 and C22:0 internal standard plasma spiked chromatograms are shown in Fig.17 .

[0105] Based on the above experimental results, it can be seen that according to the linear range of C16:0, C18:0, C24:0, C24:1, C14:0, C20:0, and C22:0 set by the method debugging, three batches of standard curves, and the correlation coefficient R of each batch of standard curves is ≥ 0.990, and the standard curve evaluation meets the methodological requirements. Using the chromatographic method of the present invention, the compounds can be completely separated, the peak shape is sharp and symmetrical, and the signal-to-noise ratio is high. The standard curve has a high linear fit and can achieve accurate quantification.

[0106] Test Example 1

[0107] Precision and Accuracy Evaluation

[0108] Experimental process: According to the three levels of quality control solutions provided by the kit of the embodiment, low, medium and high value quality control products were prepared by spiking with isopropanol. Six copies of each concentration were prepared in parallel, and each copy was tested once to evaluate the intra-batch precision (RSD) and accuracy (RE). Three batches were tested continuously to evaluate the inter-batch precision and accuracy.

[0109] Experimental Results

[0110] Table 11. Results of intra-batch and inter-batch precision and accuracy evaluation of C16:0

[0111]

[0112] Table 12. Results of intra-batch and inter-batch precision and accuracy evaluation of C18:0

[0113]

[0114] Table 13. Results of intra-batch and inter-batch precision and accuracy evaluation of C24:0

[0115]

[0116]

[0117] Table 14. Results of intra-batch and inter-batch precision and accuracy evaluation of C24:1

[0118]

[0119]

[0120] Table 15. Results of intra-batch and inter-batch precision and accuracy evaluation of C14:0

[0121]

[0122]

[0123] Table 16. Results of intra-batch and inter-batch precision and accuracy evaluation of C20:0

[0124]

[0125]

[0126] Table 17. Results of intra-batch and inter-batch precision and accuracy evaluation of C22:0

[0127]

[0128]

[0129] Experimental Summary

[0130] The data in Table 11 show that the precision and accuracy experimental data of three consecutive batches, C16:0 intra-batch and inter-batch precision RSD≤3.96%, C16:0 intra-batch and inter-batch accuracy RE≤11.56%, and the precision and accuracy evaluation meets the methodological requirements. The data in Table 12 show that the precision and accuracy experimental data of three consecutive batches, C18:0 intra-batch and inter-batch precision RSD≤5.78%, C18:0 intra-batch and inter-batch accuracy RE≤11.83%, and the precision and accuracy evaluation meets the methodological requirements. The data in Table 13 show that the precision and accuracy experimental data of three consecutive batches, C24:0 intra-batch and inter-batch precision RSD≤5.37%, C24:0 intra-batch and inter-batch accuracy RE≤11.44%, and the precision and accuracy evaluation meets the methodological requirements. The data in Table 14 show that the precision and accuracy experimental data of three consecutive batches, C24:1 intra-batch and inter-batch precision RSD≤3.24%, C24:1 intra-batch and inter-batch accuracy RE≤12.22%, and the precision and accuracy evaluation meets the methodological requirements. The data in Table 15 show that the precision and accuracy experimental data of three consecutive batches, C14:0 intra-batch and inter-batch precision RSD≤8.44%, C14:0 intra-batch and inter-batch accuracy RE≤10.78%, and the precision and accuracy evaluation meets the methodological requirements. The data in Table 16 show that the precision and accuracy experimental data of three consecutive batches, C20:0 intra-batch and inter-batch precision RSD≤7.64%, C20:0 intra-batch and inter-batch accuracy RE≤11.94%, and the precision and accuracy evaluation meets the methodological requirements. The data in Table 17 show that for three consecutive batches of precision and accuracy experimental data, the intra-batch and inter-batch precision RSD of C22:0 was ≤3.64%, and the intra-batch and inter-batch accuracy RE of C20:0 was ≤11.37%. The precision and accuracy evaluation met the methodological requirements.

[0131] Test Example 2: Evaluation of spike recovery

[0132] Experimental procedure: The ratio of the concentration difference between the sample with medium and high concentration of quality control sample working solution added to the plasma matrix and the sample without spiked solution to the theoretical value was used for evaluation. Six parallel samples were prepared for each concentration.

[0133] Experimental Results

[0134] Table 18. Evaluation results of C16:0 spike recovery

[0135]

[0136]

[0137] Table 19. Evaluation results of C18:0 spike recovery

[0138]

[0139] Table 20. Evaluation results of C24:0 spike recovery

[0140]

[0141] Table 21. Evaluation results of C24:1 spike recovery

[0142]

[0143] Table 22. Evaluation results of C14:0 spike recovery

[0144]

[0145]

[0146] Table 23. C20:0 spike recovery evaluation results

[0147]

[0148] Table 24. Evaluation results of C22:0 spike recovery

[0149]

[0150] The experimental results show that the spiked recovery rates of C16:0 at different spiked concentrations in plasma samples are between 94.50%-100.47%, the spiked recovery rates of C18:0 are between 100.50%-103.13%, the spiked recovery rates of C24:0 are between 94.67%-103.13%, the spiked recovery rates of C24:1 are between 90.55%-94.42%, the spiked recovery rates of C14:0 are between 104.13%-117.45%, the spiked recovery rates of C20:0 are between 77.65%-79.99%, and the spiked recovery rates of C22:0 are between 88.44%-92.64%, which meet the requirements for spiked recovery evaluation.

[0151] Experimental Example 3: Evaluation of Matrix Effects

[0152] Experimental procedure: The matrix effect was evaluated by evaluating the signal values ​​of the target compound in plasma matrix and in the absence of matrix. Two concentration levels of standard solutions were added to 6 different clinical individual plasma matrix samples and pure solvents, and the matrix effect was evaluated by comparing the signal intensity of the target compound (internal standard) in the presence of plasma matrix with the signal intensity of the target compound (internal standard) in pure solvent. Finally, the matrix effect was evaluated by the matrix factor normalized by the internal standard. The calculation formula is as follows: Matrix factor normalized by internal standard = (Analyte peak area in the presence of matrix / internal standard peak area) / (Analyte peak area in the absence of matrix / internal standard peak area).

[0153] Experimental Results

[0154] Table 25. Evaluation results of C16:0 matrix effect

[0155]

[0156]

[0157] Table 26. Evaluation results of C18:0 matrix effect

[0158]

[0159] Table 27. Evaluation results of C24:0 matrix effect

[0160]

[0161] Table 28. Evaluation results of C24:1 matrix effect

[0162]

[0163] Table 29. Evaluation results of C14:0 matrix effect

[0164]

[0165] Table 30. Evaluation results of C20:0 matrix effect

[0166]

[0167] Table 31. Evaluation results of C22:0 matrix effect

[0168]

[0169] The experimental results showed that for 6 clinical samples from different sources, the normalized matrix factors of C16:0 internal standard with different spiked concentrations were between 1.05-1.08; the normalized matrix factors of C18:0 internal standard were between 1.01-1.07; the normalized matrix factors of C24:0 internal standard were between 1.03-1.10; the normalized matrix factors of C24:1 internal standard were between 0.90-0.96, the normalized matrix factors of C14:0 internal standard were between 1.02-1.12, the normalized matrix factors of C20:0 internal standard were between 0.85-0.90, and the normalized matrix factors of C22:0 internal standard were between 0.88-0.93, indicating that the matrix effects of the internal standard and the analyte were close, and the matrix effects of the analyte in the sample could be compensated, so the matrix effect would not affect the final accurate quantitative analysis, and the matrix effect investigation met the requirements of methodological evaluation.

[0170] Test Example 4: Residue Evaluation

[0171] Experimental procedure: Test the blank matrix sample after high quality control for 5 consecutive times in the order of HQC-DB-HQC-DB-HQC-DB-HQC-DB-HQC-DB. Determine the peak area of ​​the analyte and internal standard in the blank matrix sample and compare it with the calibration standard at the lowest concentration point to confirm the effect of the residue on the accurate quantification of the analyte.

[0172] Experimental Results

[0173] Table 32. Evaluation results of C16:0 residue experiment

[0174]

[0175] Note: HQC stands for high concentration quality control sample, DB stands for blank matrix sample

[0176] Table 33. C18:0 residue test evaluation results

[0177]

[0178] Table 34. C24:0 residue test evaluation results

[0179]

[0180] Table 35. C24:1 residue test evaluation results

[0181]

[0182] Table 36. Evaluation results of C14:0 residue experiment

[0183]

[0184]

[0185] Table 37. C20:0 residue test evaluation results

[0186]

[0187] Table 38. C22:0 residue test evaluation results

[0188]

[0189] The experimental results showed that the C16:0 residue was 1.27-7.41%, and the C16:0 internal standard residue was 0.00%; the C18:0 residue was 3.23-6.21%, and the C18:0 internal standard residue was 0.00%; the C24:0 residue was 6.90-9.62%, and the C24:0 internal standard residue was 0.00%; the C24:1 residue was 3.82-6.01%, and the C24:1 internal standard residue was 0.00%, and the C14:0 residue was 0.0 0%, C14:0 internal standard residue is 0.00%; C20:0 residue is 0.00%; C22:0 residue is 5.09-8.91%, C22:0 internal standard residue is 0.00%; it meets the requirements of the Chinese Pharmacopoeia that the response of the interference peak at the retention time of the analyte does not exceed 20% of the response of the analyte in the lower limit of quantification, and the response of the interference peak at the retention time of the internal standard does not exceed 5% of the response of the internal standard in the low concentration sample. The residue experiment evaluation meets the methodological evaluation requirements.

[0190] Test Example 5: Evaluation of Quantitative Detection Limit (LOQ)

[0191] Experimental process: The quantitative detection limit of the method was investigated by detecting the linear minimum point of the standard curve, and the sample was injected and tested 6 times continuously, and the CV and accuracy deviation (RE) were used to investigate.

[0192] Experimental Results

[0193] Table 39. Evaluation results of quantitative detection limit of C16:0

[0194]

[0195] Table 40. Evaluation results of quantitative detection limit of C18:0

[0196]

[0197] Table 41. Evaluation results of quantitative detection limit of C24:0

[0198]

[0199] Table 42. Evaluation results of quantitative detection limit of C24:1

[0200]

[0201] Table 43. Evaluation results of quantitative detection limit of C14:0

[0202]

[0203] Table 44. Evaluation results of quantitative detection limit of C20:0

[0204]

[0205] Table 45. Evaluation results of quantitative detection limits of C22:0

[0206]

[0207] The experimental results showed that the CV and RE of the lowest linear point (LOQ) of C16:0, C18:0, C24:0, and C24:1 were all less than 20%, indicating that the quantitative detection limit evaluation met the methodological evaluation.

[0208] Test Example 6: Sample stability evaluation

[0209] Experimental Procedure

[0210] (1) Stability at room temperature for 8 hours: Ceramide is an endogenous substance and contains background in clinical samples. Therefore, when evaluating the stability experiment, the plasma sample stability does not use the spike method. The content of the analyte contained in it is directly tested at each experimental node. Take a portion of mixed plasma, mix it evenly, and place it at room temperature for 8 hours for testing according to the operating instructions. Process 6 samples in parallel at each concentration level. Based on the test data at hour 0, calculate the accuracy and uniformity of the sample indicator components to evaluate the stability of the plasma sample at room temperature.

[0211] (2) Freeze-thaw stability: Take a portion of mixed plasma, mix well, and place in a -20°C refrigerator. After freezing and thawing three times, test according to the operating instructions. Based on the test data at hour 0, calculate the accuracy and uniformity of the sample index components to evaluate the freeze-thaw stability of the plasma sample.

[0212] (3) Stability of samples frozen at -20°C for 7 days: Take a portion of mixed plasma, mix well and place in a -20°C refrigerator, freeze for 7 days and then test according to the operating instructions. Based on the test data of the unfrozen sample on day 0, calculate the accuracy and uniformity of the sample index components to evaluate the stability of the plasma sample frozen for 7 days.

[0213] (4) Sample stability test plan after preparation: Take a portion of mixed plasma and process the sample according to the operating instructions. The processed solution is placed in an automatic sampler at 8°C for 24 hours before testing. Based on the test data at hour 0, calculate the accuracy and uniformity of the sample index components to evaluate the stability of the plasma sample after preparation.

[0214] (5) Sample stability at 2-8°C for 24 hours: Take a portion of mixed plasma, mix well, store at 2-8°C for 24 hours, and then test according to the operating instructions. Process 6 samples in parallel at each concentration level. Based on the test data at hour 0, calculate the accuracy and uniformity of the sample index components to evaluate the room temperature stability of the plasma sample.

[0215] Experimental Results

[0216] Table 46. Room temperature stability of various ceramides

[0217]

[0218] Table 47. Freeze-thaw stability of various ceramides

[0219]

[0220] Table 48. Stability of various ceramides stored frozen at -20°C for 7 days

[0221]

[0222] Table 49. Stability of various ceramide samples after preparation

[0223]

[0224] Table 50. Stability of various ceramides stored at 2-8°C for 24 hours

[0225]

[0226] This experiment evaluated the standard curve, linearity, precision accuracy, spike recovery, matrix effect, residue, quantitative detection limit, and sample stability of the C14:0, C16:0, C18:0, C20:0, C22:0, C24:0, and C24:1 mass spectrometry detection methods. The evaluation results showed that the method can meet the needs of clinical blood sample testing.

[0227] Test Example 7: Comparison of the effect of adding methyl tert-butyl ether to phase B

[0228] Based on the method and product provided in Example 1, two test groups were set up, one of which was the technical solution of Example 1, and the other was different from Example 1 only in that methyl tert-butyl ether was not added. Under the same liquid phase gradient: phase A: 2mM ammonium acetate + 0.1% formic acid + water, phase B: 0.1% acetonitrile. The results are shown in Fig.18 , it can be seen that without adding methyl tert-butyl ether to phase B, the elution ability of pure acetonitrile is weak, and only three compounds can elute within 4.5 min.

[0229] Experimental Example 8: Chromatographic separation under different liquid phase gradients under fixed mobile phase composition

[0230] Based on the technical solution of Example 1, the separation conditions under different liquid phase gradients were verified.

[0231] Gradient 1:

[0232] TIme Flow rate (mL / min) A(％) B(％) 0.00 0.7 25 75 0.30 0.7 25 75 1.20 0.7 5 95 1.80 0.7 5 95 1.90 0.7 25 75 3.00 0.7 25 75

[0233] The results of gradient 1 are shown in Fig.19 As can be seen from the figure above: the green is the phospholipid channel, which is not completely separated from the other ceramides, and the interference peak of C20 in the figure below is not separated from C20, but merged into one peak. The interference peak will interfere with the detection of C20.

[0234] Gradient 2:

[0235] TIme Flow rate (mL / min) A(％) B(％) 0.00 0.6 35 65 1.00 0.6 35 65 1.50 0.6 15 85 3.00 0.6 5 95 3.10 0.6 35 65 4.00 0.6 35 65

[0236] The results of gradient 2 are shown in Fig. 20 The results show that the interference peak in C20 is not completely separated from C20, which will interfere with the detection of C20.

[0237] Experimental Example 9: Effects of different chromatographic columns and mobile phases on chromatographic separation

[0238] Based on the technical solution of Example 1, a control group was set up. The difference between the control group and Example 1 is: phenyl chromatographic column, phase A: 2mM ammonium acetate + 0.1% formic acid + water; phase B: 0.1% formic acid methanol. The results are shown in Fig.21 The results show that when using a phenyl column and methanol as the mobile phase, the compounds in plasma interfere more. Based on the technical solution of Example 1, the same mobile phase and liquid phase gradient were used to separate the chromatograms on a Fenome Kinetex (50x3mm, 2.6μm) C8 column. Fig. 22 As shown in the figure, phospholipids and 7 analytes can be well separated, and the interference peak of C20 can be completely separated from C20, which will not interfere with the detection of C20. Based on the technical solution of Example 1, using the same mobile phase and liquid phase gradient, the separation chromatogram of the Fenome Kinetex Phenyl-Hexyl (50x4.6mm, 2.6μm) chromatographic column is as follows Fig.23 As can be seen from the figure, phospholipids and the seven analytes can be well separated, and the interference peak of C20 can be completely separated from C20, and will not interfere with the detection of C20.

[0239] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for determining ceramide in a sample, characterized in that: It includes: The ceramide in the sample is detected by liquid chromatography-tandem mass spectrometry; the chromatography comprises gradient elution using mobile phase A and mobile phase B, the mobile phase A comprises an aqueous solution of ammonium acetate containing formic acid, and the mobile phase B comprises a mixed solution of acetonitrile and methyl tert-butyl ether containing formic acid; in the mobile phase B, the volume ratio of acetonitrile to methyl tert-butyl ether is (70-80): (20:30); The elution conditions of the chromatography were as follows: From 0 to 1 min, the volume percentage of mobile phase A was maintained at 35%, and the volume percentage of mobile phase B was maintained at 65%; From 1 to 1.5 min, the volume percentage of mobile phase A decreased from 35% to 25%, and the volume percentage of mobile phase B increased from 65% to 75%; From 1.5 to 3.5 min, the volume percentage of mobile phase A decreased from 25% to 5%, and the volume percentage of mobile phase B increased from 75% to 95%; From 3.5 to 3.6 min, the volume percentage of mobile phase A increased from 5% to 35%, and the volume percentage of mobile phase B decreased from 95% to 65%; 3.6-4.5min, the volume percentage of mobile phase A is maintained at 35%, and the volume percentage of mobile phase B is maintained at 65%; the ceramides include: 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); The chromatographic column used in the chromatography is any one of a C8 chromatographic column and a phenyl chromatographic column; The C8 chromatographic column is selected from Kinetex C8 and shim-pack-GIST-HP C8; the model of the Kinetex C8 is 50×3mm, 2.6μm, and the model of the shim-pack-GIST-HP C8 is 50×2.1, 3μm; The phenyl chromatographic column comprises Kinetex Phenyl-Hexyl, and the model of the Kinetex Phenyl-Hexyl is 50×4.6, 2.6 μm; The mass spectrometer used an electrospray ion source, the detection mode was positive ion mode, the scanning mode was multiple reaction monitoring, the desolvation temperature was 550°C, the nebulizer gas flow rate was 55, the desolvation flow rate was 55, and the ion source voltage was 5500V; The method is not directed toward the diagnosis or treatment of disease.

2. The method for measuring ceramide in a sample according to claim 1, characterized in that: The flow rate of the gradient elution is 0.1-1 mL / min.

3. The method for measuring ceramide in a sample according to claim 1, characterized in that The chromatographic conditions include: the column temperature of the chromatographic column is 35-45°C, the injector temperature is 5-15°C, and the injection volume is 2-5 μL.

4. The method for measuring ceramide in a sample according to claim 1, characterized in that: In the mobile phase B, the volume ratio of acetonitrile to methyl tert-butyl ether is 75:

25.

5. The method for measuring ceramide in a sample according to claim 1, characterized in that: In the mobile phase B, the volume concentration fraction of formic acid is 0.01%~0.5%.

6. The method for measuring ceramide in a sample according to claim 1, characterized in that: The volume concentration fraction of formic acid in the mobile phase A is 0.01% to 0.5%.

7. The method for measuring ceramide in a sample according to any one of claims 1 to 6, characterized in that: The sample comprises a blood sample.

8. The method for measuring ceramide in a sample according to claim 7, characterized in that: The liquid chromatography tandem mass spectrometry detection includes a sample pretreatment step, and the sample pretreatment includes: mixing the sample to be tested, the internal standard and the precipitant to perform protein precipitation, and removing the precipitate before using for liquid chromatography tandem mass spectrometry detection.

9. The method for measuring ceramide in a sample according to claim 8, characterized in that: The precipitating agent includes methanol.

10. The method for measuring ceramide in a sample according to claim 8, characterized in that: The internal standard solution includes at least one of an internal standard of Cer (d18:1 / 16:0), an internal standard of Cer (d18:1 / 18:0), an internal standard of Cer (d18:1 / 24:0), and an internal standard of Cer (d18:1 / 24:1).

11. The method for measuring ceramide in a sample according to claim 8, characterized in that: The internal standard solution includes at least one of Cer (d18:1 / 16:0) deuterated substance, Cer (d18:1 / 18:0) deuterated substance, Cer (d18:1 / 24:0) deuterated substance and Cer (d18:1 / 24:1) deuterated substance.

12. Use of a reagent combination in preparing a product for measuring ceramide in a sample, characterized in that: The reagent combination includes reagents for implementing the method according to any one of claims 1 to 11, and the ceramides include: 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).

13. The use according to claim 12, characterized in that: The sample comprises a blood sample.

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