CN115508466A

CN115508466A - Method for determining ceramide in sample, product and application thereof

Info

Publication number: CN115508466A
Application number: CN202210986777.4A
Authority: CN
Inventors: 肖冰心; 王春静; 栗琳; 张红; 雒琴; 贾子强; 周立; 丁亮
Original assignee: Hunan Haosi Biotechnology Co ltd; Jiangsu Haosi Muke Biotechnology Co ltd; Beijing Haosi Biotechnology Co ltd
Current assignee: Hunan Haosi Biotechnology Co ltd; Jiangsu Haosi Muke Biotechnology Co ltd; Beijing Haosi Biotechnology Co ltd
Priority date: 2022-08-17
Filing date: 2022-08-17
Publication date: 2022-12-23

Abstract

The invention discloses a method for measuring ceramide in a sample, a product and application thereof, relates to the technical field of biological detection, and provides a method suitable for simultaneously detecting multiple ceramides, wherein the method optimizes a mobile phase and a gradient elution procedure adopted by chromatographic elution, so that the multiple ceramides can be separated and detected within 4.5min, the analysis time is short, the flux is high, the influence of an interference peak is avoided or reduced, and a 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 high accuracy.

Description

Method for determining ceramide in sample, product and application thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a method for determining ceramide in a sample, a product and application thereof.

Background

In view of the high morbidity and mortality of coronary artery disease, prevention of fatal and non-fatal myocardial infarction in coronary artery patients remains a clinical challenge. Annual mortality rates in stable coronary patients range from 1 to 3% and non-fatal morbidity rates range from 1 to 2%. Mortality from myocardial infarction is significantly elevated (especially within the first year) in patients with acute coronary syndrome where acute conditions survive. However, the risk of morbidity varies greatly among individual patients, and effective tools for diagnosis, risk stratification, disease monitoring, and efficacy assessment are needed to improve patient identification and management.

The coronary artery disease marker is a bioactive substance which is increased or reduced in the occurrence and development process of atherosclerosis, and can be used for diagnosis, risk stratification, disease monitoring and curative effect evaluation of coronary artery disease. It will bring great influence to the clinical treatment of coronary artery disease, so in order to meet the clinical diagnosis and treatment needs of coronary artery disease, the research and development of efficient, accurate marker is urgently needed to be accelerated.

The markers for coronary artery disease diagnosis, risk stratification, disease monitoring and efficacy evaluation at present cannot provide more reference values for clinicians mostly 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, which has long been used for diagnosis, risk stratification and prognosis of cardiovascular disease. However, these traditional risk molecule assays do not identify a significant proportion of patients with high risk cardiovascular disease. Therefore, in addition to the traditional risk molecules of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C), there is a need to find a more accurate and predictive risk factor.

Free lipidomics analysis shows that some lipids may be useful markers for the diagnosis, risk stratification and prognosis of coronary artery disease. Lipidomic studies found that specific ceramide species were significantly associated with cardiovascular death in patients with coronary artery disease (Meikle, p.j.et al. Plasma lipid analysis of stable and unstable coronary heart disease. Ariioscope Thromb Vasc Biol 31,2723-2732, doi. Ceramides are composed of sphingosine and fatty acids. Ceramides are mainly produced by the sphingomyelinase pathway, which breaks down phospholipids on the membrane of the sphingomyelin and releases ceramides. Meanwhile, ceramide can be synthesized by six kinds of fatty acyl selective ceramide synthetases through a new synthesis path by using simple molecules. Different ceramide-like molecules have specific physiological functions. Several key cytokines (e.g., tumor necrosis factor and interleukins) can induce inflammatory responses (e.g., atherosclerosis and ischemia reperfusion injury) in which ceramide levels are elevated. Some cardiovascular risk factors and markers (such as oxidized low density lipoproteins and homocysteine) can increase ceramide production. In addition, ceramides are involved as signaling molecules in the regulation of many cellular responses and functions, including differentiation, proliferation, apoptosis, reactive oxygen species production and gene expression, which are directly involved in the molecular mechanisms of cardiovascular disease.

Ceramide is also an important target for drug development for treating coronary artery diseases at present. Studies have shown that the metabolic process of sphingomyelin is a dynamic process, and that sphingomyelin metabolites (including ceramides and sphingosines) play an important role as second messengers in coronary artery disease (Kolesnik, 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 directed against damaged arteries, they may exhibit a strong effect against the spread of malignant Cell proliferation (Bourbon, N.A., yun, J., berkey, D., wang, Y. & Kester, M.inhibit actions of ceramide up on PKC-epsilon/ERK interactions, am J Physiol 280, C1403-1411 (2001)). Neointimal hyperplasia of vascular smooth muscle cells and secondary occlusion of coronary arteries are the cause of restenosis following balloon angioplasty and arterial stent implantation, affecting 300 million patients undergoing coronary angioplasty worldwide. Ceramides inhibit vascular smooth muscle proliferation induced by the ERK kinase and AKT kinase cascades. 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 nerve tissue in vascular hypertension animals in vascular organs. Circuit Res 87,282-288 (2000)).

A number of analytical methods have been used to measure 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 work rather than clinical use, and they have a common disadvantage in that they cannot analyze ceramide at the molecular species level and lack selectivity, low throughput, low accuracy and precision, and thus cannot meet the requirements of clinical laboratories. We were also unable to distinguish the four ceramides using traditional immunoassay methods because the epitopes of each ceramide were too similar. Anti-ceramide antibodies recognize a class of ceramide molecules that differ only in fatty acid chain length. Thus, the low selectivity of existing antibodies has hampered the development of clinically effective immunoassays (Krishnmurgy, K., dasguppt, S. & Bieberich, E.development and characterization of a novel anti-ceramide antibody. J. Lipid Res 48,968-975, doi. The main advantage of LC-MS/MS is that in combination with chromatographic separation and accurate monitoring of mass spectra, each ceramide molecule can be unambiguously detected (Kauhanen, D.et al. Development and validation of a high-throughput LC-MS/MS assay for a routine measurement of molecular ceramides. Anal Bioanal chemical Chem 408,3475-3483, doi 10.1007/s 00216-9425-z (2016)).

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for measuring ceramide in a sample, and a product and application thereof.

The invention is realized by the following steps:

in a first aspect, embodiments of the present invention provide a method for determining ceramide in a sample, which includes detecting ceramide in a sample by using a liquid chromatography tandem mass spectrometry method; the chromatography comprises gradient elution by adopting a mobile phase A and a mobile phase B, wherein the mobile phase A comprises ammonium acetate aqueous solution containing formic acid, and the mobile phase B comprises mixed solution of acetonitrile containing formic acid and methyl tert-butyl ether. Elution conditions for chromatography were as follows: 0-1 min, the volume percentage of the mobile phase A is maintained at 30-40%, and the volume percentage of the mobile phase B is maintained at 60-70%; 1-1.5 min, the volume percentage of the mobile phase A is reduced from 30-40% to 20-30%, and the volume percentage of the mobile phase B is increased from 60-70% to 70-80%; 1.5-3.5 min, the volume percentage of the mobile phase A is reduced from 20-30% to 1-10%, and the volume percentage of the mobile phase B is increased from 70-80% to 90-99%; 3.5-3.6 min, the volume percentage of the mobile phase A is increased from 1-10% to 30-40%, and the volume percentage of the mobile phase B is reduced from 90-99% to 60-70%; 3.6-4.5 min, the volume percentage of the mobile phase A is maintained at 30-40%, and the volume percentage of the mobile phase B is maintained at 60-70%. The ceramide comprises: cer (d 18: 1/14), cer (d 18: 1/16.

In a second aspect, embodiments of the present invention provide the use of a reagent combination comprising reagents for carrying out a method according to the preceding embodiments in the manufacture of a product for the determination of ceramide in a sample, the ceramide comprising: cer (d 18: 1/14), cer (d 18: 1/16.

In a third aspect, embodiments of the present invention provide a product for determining ceramide in a sample, which includes reagents for performing the method for determining ceramide in a sample described in the previous embodiments.

The invention has the following beneficial effects: (1) The presence of interfering peaks in the Cer (d 18: 1/20) plasma sample, which are separated from the Cer (d 18: 1/20. Complete separation of the two is achieved. (2) Ceramide belongs to lipid and is difficult to elute, methyl tert-butyl ether is added into a mobile phase, the elution capability is enhanced, 7 substances can be separated more quickly, and no residue is caused. (3) The phospholipid is easy to influence the detection of ceramide, and the phospholipid channel is monitored, and the liquid phase gradient is adjusted, so that the separation of the phospholipid and 7 kinds of ceramide is realized, and the interference caused by the phospholipid is avoided. (4) The method can complete the separation and detection of various ceramides within 4.5min, and has short analysis time and high flux.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic view of a detection process;

FIG. 2 is a total ion flow diagram;

FIG. 3 is a chromatogram of C20: 0;

FIG. 4 is a C16:0 standard graph;

FIG. 5 is a C16:0 and C16:0 internal standard plasma spiked chromatogram;

FIG. 6 is a C18:0 standard graph;

FIG. 7 is a C18:0 and C18:0 internal standard plasma labeling chromatogram;

FIG. 8 is a C24:0 standard graph;

FIG. 9 is a C24:0 and C24:0 internal standard plasma spiking chromatogram;

FIG. 10 is a C24:1 standard graph;

FIG. 11 is a C24:1 and C24:1 internal standard plasma spiking chromatogram;

FIG. 12 is a C14:0 standard graph;

FIG. 13 is a C14:0 and C14:0 internal standard plasma spiked chromatogram;

FIG. 14 is a C20:0 standard graph;

FIG. 15 is a C20:0 and C20:0 internal standard plasma labeling chromatogram;

FIG. 16 is a C22:0 standard graph;

FIG. 17 is a C22:0 and C22:0 internal standard plasma labeling chromatogram;

FIG. 18 is a separation chromatogram without addition of methyl t-butyl ether;

FIG. 19 is a separation chromatogram of gradient 1

FIG. 20 is a separation chromatogram of gradient 2;

FIG. 21 is a phenyl chromatography column with phase A being 2mM ammonium acetate +0.1% formic acid + water; chromatogram of 0.1% formic acid methanol for phase B;

FIG. 22 is a Heinomei Kinetex C8 (50X 3mm,2.6 μm) column separation chromatogram;

FIG. 23 is a separation chromatogram of a Fenomei Kinetex Phenyl-Hexyl (50X 4.6mm,2.6 μm) column.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiment of the invention provides a method for measuring ceramide in a sample, which comprises the following steps: detecting ceramide in the sample by adopting a liquid chromatography-tandem mass spectrometry method; the chromatography comprises gradient elution by adopting a mobile phase A and a mobile phase B, wherein the mobile phase A comprises an ammonium acetate aqueous solution containing formic acid, and the mobile phase B comprises a mixed solution of acetonitrile containing formic acid and methyl tert-butyl ether.

Elution conditions for chromatography were as follows: 0-1 min, the volume percentage of the mobile phase A is maintained at 30-40%, and the volume percentage of the mobile phase B is maintained at 60-70%; 1-1.5 min, the volume percentage of the mobile phase A is reduced from 30-40% to 20-30%, and the volume percentage of the mobile phase B is increased from 60-70% to 70-80%; 1.5-3.5 min, the volume percentage of the mobile phase A is reduced from 20-30% to 1-10%, and the volume percentage of the mobile phase B is increased from 70-80% to 90-99%; 3.5-3.6 min, the volume percentage of the mobile phase A is increased from 1-10% to 30-40%, and the volume percentage of the mobile phase B is decreased from 90-99% to 60-70%; 3.6-4.5 min, the volume percentage of the mobile phase A is maintained at 30-40%, and the volume percentage of the mobile phase B is maintained at 60-70%.

The ceramide includes: the term "assaying ceramide in a sample" as used herein specifically refers to qualitative and/or quantitative detection of ceramide, and includes at least one of Cer (d 18: 1/14), cer (d 18: 1/16.

The method does not have the direct objective of diagnosis or treatment of a disease, which is to detect ceramide or its content in a sample.

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

In some embodiments, the flow rate of the gradient elution is 0.1 to 1mL/min, and specifically can be any one or a range between any two of 0.01mL/min, 0.2mL/min, 0.4mL/min, 0.6mL/min, 0.8mL/min, and 1mL/min.

In some embodiments, the chromatography employs a chromatography column comprising any one of a C8 chromatography column and a phenyl chromatography column; optionally, the C8 column is selected from any one of femomelette Kinetex C8, shimadzum-pack-GIST-HP C8 and a column having a stationary phase bound phase or type of similar type to femomelette Kinetex C8 or shimadzum-pack-GIST-HP C8. Preferably, said femomei Kinetex C8 is of type 50x3mm,2.6 μm; preferably, the Shimadzu-pack-GIST-HP C8 model is 50X 2.1,3 μm. Preferably, the Phenyl chromatography column comprises a femomeniex Kinetex Phenyl-Hexyl or chromatography column having a similar type of stationary phase bound phase or model; preferably, the model number of the Feinuomei Kinetex Phenyl-Hexyl is 50 × 4.6,2.6 μm.

The traditional C18 column is adopted, the retention of 7 kinds of ceramide is strong, elution is not easy, and the C8 column has weaker retention to 7 kinds of ceramide than C18 and is not easy to cause residue. Preferably, the chromatographic column is Shijin-pack-GIST-HP C8.

In some embodiments, the chromatographic conditions comprise: the temperature of the chromatographic column is 35-45 ℃, the temperature of the sample injector is 5-15 ℃, and the sample injection amount is 2-5 mu L.

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

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.

In some embodiments, the liquid chromatography tandem mass spectrometry detection comprises a sample pre-treatment step comprising: and mixing the sample to be detected, the internal standard and the precipitant to precipitate the protein, and removing the precipitate to be used for the detection of the liquid chromatography tandem mass spectrometry. The pretreatment is carried out by adopting a protein precipitation method, the operation is simple, the treatment period is short, and the pretreatment time is greatly shortened. Preferably, the precipitating agent comprises methanol.

In some embodiments, the internal standard solution comprises: at least one of an internal standard of Cer (d 18: 1/16) and an internal standard of Cer (d 18: 1/18. By means of optimization pretreatment, a liquid phase method and the like, a mode of sharing isotope internal standards is adopted, 4 isotope internal standards are used for completing the detection of 7 ceramides, and the problem of matrix effect of the remaining 3 non-isotope internal standards ceramides is solved. Preferably, the internal standard solution comprises: at least one of Cer (d 18: 1/16) deuteron, cer (d 18: 1/18.

Embodiments of the present invention also provide the use of a combination of reagents in the preparation of a product for the determination of ceramide in a sample, the combination of reagents comprising reagents for carrying out a method as described in any preceding embodiment, the ceramide comprising: cer (d 18: 1/14), cer (d 18: 1/16. The sample is the same as any of the foregoing embodiments, and is not repeated.

Specifically, the reagent combination comprises the following components in any of the embodiments: at least one of mobile phase a, mobile phase B, a protein precipitant, and an internal standard.

In some embodiments, the reagent combination further comprises at least one of a calibrator and a quality control for each of the 7 ceramides. The diluent adopted by each calibrator and quality control material is isopropanol.

In some embodiments, the concentration of the calibrator and the quality control product is 20 x of the target concentration, and the calibrator and the quality control product are respectively loaded in different carrying containers, air-dried and stored in a dry powder form, so that the validity period is prolonged, and the calibrator and the quality control product are convenient to store and transport. When the reagent is used, the reagent is redissolved by using a diluent (the volume is enlarged by 20 times) to obtain a reagent with a target concentration.

In some embodiments, the reagent combination further comprises: purifying at least one of water, formic acid, acetonitrile, methyl tert-butyl ether and ammonium acetate.

In addition, the embodiment of the invention also provides a product for measuring ceramide in a sample, which comprises a reagent for implementing the method for measuring ceramide in the sample, as described in any of the preceding embodiments.

In some embodiments, the product is a kit that may include a combination of reagents as described in any of the preceding embodiments.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

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

The items required for testing are shown in the table below.

Name(s)	Require to make a request for
		Purified water	Deionized water
Formic acid	The color spectrum is pure
		Acetonitrile	The color spectrum is pure
Methyl tert-butyl ether	The color spectrum is pure
		Ammonium acetate	The color spectrum is pure
96-hole V-shaped plate	450 mu L96-hole V-shaped plate and 96-hole plate film
		Polypropylene centrifuge tube	1.5-2mL
Liquid transfer device	10-100μL，20-200μL，100-1000μL
		Vortex mixer	/

Sample type: the kit is suitable for human plasma samples, and the sample size is required to be more than or equal to 0.5mL.

[ test methods ] before testing, the kit and sample were returned to room temperature (10-30 ℃); 2. preparing a calibrator and a quality control product, and redissolving: precisely transferring 1mL of diluent into each horizontal calibrator and quality control material bottle at room temperature, and fully mixing for 3-5 min to obtain each horizontal calibrator and quality control material solution; 3. preparation of internal standard substance-redissolution: precisely transferring 3mL of diluent into an internal standard bottle at room temperature, and fully mixing for 3-5 min to obtain an internal standard solution; 4. sample treatment and detection flow: adding a sample: precisely sucking 100 mu L of calibrator/quality control/plasma sample, adding 20 mu L of internal standard solution and 880 mu L of diluent, and performing vortex oscillation for 2-3 min; centrifuging: centrifuging at 15000rpm at 4 deg.C for 10min, and separating supernatant; and (3) detection: transfer 100-200. Mu.L of the supernatant to a clean 96-well V-plate for LC-MS/MS detection. The detection process is schematically shown in FIG. 1.

Data analysis and results:

and (3) drawing a calibration curve: with the concentration of the calibrator as the independent variable x _i Taking the peak area ratio mean value of the corresponding concentration calibrator and internal standard as dependent variable y _i Calculating a linear regression equation y = ax + b and a correlation coefficient r; analyzing quality control material data: when r of the calibration curve is more than or equal to 0.990, substituting the signal intensity of the quality control product into a regression equation to obtain the concentration of the quality control product; sample data analysis: and when the detection result of the quality control product is in an expected range, substituting the detection signal of the sample into the regression equation to obtain the concentration of the target object in the sample.

The instrument equipment comprises: the mass spectrometer adopts AB Sciex 4500 (S/N EB 252831905); the liquid chromatograph adopts Jasper Sciex.

[ MEANS FOR MEASURING AND METHOD ]

TABLE 1 ion Source parameters

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

TABLE 2 Mass Spectrometry method

MRM channel	Compound (I)	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

TABLE 3 liquid phase Process

The total ion flow chart corresponding to the kit and the method is shown in figure 2, and the C20:0 chromatogram chart is shown in figure 3. As can be seen from the above total ion current, better retention and separation was achieved for the 7 ceramides (c 22 and c24:1 retention times are identical, but the quantitative ion pair is different and the mass spectra can be distinguished). All the compound peaks are sharp and symmetrical in shape, and are completely separated from phospholipid peaks which may influence the detection result. As can be seen from FIG. 3, the retention time of 3.43min is the C20:0 peak in the plasma, and the interference peak is at 3.58 min. Complete separation of the two is achieved.

[ methods evaluation contents ]

1. Linear evaluation of standard curve

The experimental process comprises the following steps: according to a series of working solutions with concentration provided by the kit, an isopropanol standard adding mode is adopted to prepare a standard curve, the concentration of an analyte is used as a horizontal coordinate, the peak area ratio of the analyte to an internal standard is used as a vertical coordinate, linear regression is carried out by a weighted (W = 1/X2) least square method to obtain a linear regression equation (Y = bX + a), a correlation coefficient r is calculated, and the r is required to be more than or equal to 0.99.

TABLE 4 Experimental results

Table 5.C16 standard curve regression equation parameter evaluation results for 3 consecutive analytical batches of 0

The C16:0 standard curve is shown in FIG. 4, and the C16:0 and C16:0 internal standard plasma spiked chromatograms are shown in FIG. 5.

Table 6.C18 standard curve regression equation parameter evaluation results for 3 consecutive analytical batches

The standard curve of C18:0 is shown in FIG. 6, and the standard curve of C18:0 and C18:0 plasma-labeled chromatogram is shown in FIG. 7.

Table 7.C24 standard curve regression equation parameter evaluation results for 3 consecutive analytical batches

The C24:0 standard curve is shown in FIG. 8, and the C24:0 and C24:0 internal standard plasma spiked chromatograms are shown in FIG. 9.

Table 7.C24 standard curve regression equation parameter evaluation results for 1 consecutive 3 analysis batches

The standard curve of C24:1 is shown in FIG. 10, and the standard curve of C24:1 and C24:1 plasma standard chromatogram is shown in FIG. 11.

Table 8.C14 standard curve regression equation parameter evaluation results for 3 consecutive analytical batches of 0

The standard curve of C14:0 is shown in FIG. 12, and the standard curve of plasma with C14:0 and C14:0 is shown in FIG. 13.

Table 9.C20 standard curve regression equation parameter evaluation results for 3 consecutive analytical batches of 0

The standard curve of C20:0 is shown in FIG. 14, and the standard curve of C20:0 and C20:0 plasma-labeled chromatogram is shown in FIG. 15.

Table 10.C22 standard curve regression equation parameter evaluation results for 3 consecutive analytical batches of 0

The standard curve of C22:0 is shown in FIG. 16, and the standard curve of C22:0 and C22:0 plasma-labeled chromatogram is shown in FIG. 17.

Based on the experimental results, 3 batches of standard curves are continuously set according to the linear ranges of C16:0, C18:0, C24:1, C14:0, C20:0 and C22:0 set by the method, the correlation coefficient R of each batch of standard curves is more than or equal to 0.990, and the standard curve evaluation meets the methodological requirements. By adopting the chromatographic method, the compounds can be completely separated, the peak shapes are sharp and symmetrical, and the signal-to-noise ratio is high. The standard curve has high linear fitting degree and can realize accurate quantification.

Test example 1

Precision and accuracy evaluation

The experimental process comprises the following steps: according to three levels of quality control solutions provided by the kit of the embodiment, low, medium and high value quality control products are prepared by adopting an isopropanol standard adding mode, 6 parts of each concentration is prepared in parallel, each part is detected for 1 time, the precision (RSD) and the accuracy (RE) in a batch are evaluated, 3 batches are continuously detected, and the precision and the accuracy between batches are evaluated.

Results of the experiment

Table 11.C16 precision accuracy evaluation results within and between batches of 0

Table 12.C18

Table 13.C24

Table 14.C24 precision accuracy evaluation results within and between batches of 1

Table 15.C14 precision accuracy evaluation results within and between batches of 0

Table 16.C20 precision accuracy evaluation results within and between batches of 0

Table 17.C22 precision accuracy evaluation results within and between batches of 0

Experiment summary

The data in Table 11 show that for the precision and accuracy experimental data of 3 consecutive batches, the precision RSD between the C16:0 batches and the batch-to-batch precision RSD is less than or equal to 3.96 percent, the precision RE between the C16:0 batches is less than or equal to 11.56 percent, and the precision and accuracy evaluation meets the methodology requirements. The data in Table 12 show that for the precision and accuracy experimental data of 3 consecutive batches, the precision RSD between the C18:0 batches and the batch-to-batch precision RSD is less than or equal to 5.78 percent, the precision RE between the C18:0 batches is less than or equal to 11.83 percent, and the precision and accuracy evaluation meets the methodology requirements. The data in Table 13 show that the precision and accuracy experimental data of 3 consecutive batches, the precision RSD of the C24:0 batch and the inter-batch precision RSD are less than or equal to 5.37 percent, the precision RE of the C24:0 batch and the inter-batch accuracy RE are less than or equal to 11.44 percent, and the precision and accuracy evaluation meets the methodological requirements. The data in Table 14 show that for the precision and accuracy experimental data of 3 consecutive batches, the precision RSD between the C24:1 batches and the batch-to-batch precision is less than or equal to 3.24 percent, the precision RE between the C24:1 batches is less than or equal to 12.22 percent, and the precision and accuracy evaluation meets the methodology requirements. The data in Table 15 show that the precision and accuracy experimental data of 3 continuous batches have the precision RSD of less than or equal to 8.44% in the C14:0 batch and between batches, the accuracy RE of less than or equal to 10.78% in the C14:0 batch and meet the methodological requirements for precision and accuracy evaluation. The data in Table 16 show that for the precision and accuracy experimental data of 3 consecutive batches, the precision RSD between the C20:0 batches and the batch-to-batch precision RSD is less than or equal to 7.64 percent, the precision RE between the C20:0 batches is less than or equal to 11.94 percent, and the precision and accuracy evaluation meets the methodology requirements. The data in Table 17 show that the precision and accuracy experimental data of 3 continuous batches, the precision RSD of the C22:0 batch and the batch-to-batch precision RSD are less than or equal to 3.64 percent, the precision RE of the C20:0 batch and the batch-to-batch precision RE are less than or equal to 11.37 percent, and the precision and accuracy evaluation meets the methodological requirements.

Test example 2: evaluation of recovery by adding standard

The experimental process comprises the following steps: the ratio of the concentration difference value of the sample of the medium and high concentration quality control sample working solution added with the plasma matrix and the sample obtained without the added standard to the theoretical value is adopted for evaluation, and 6 parallel samples are prepared for each concentration sample.

Results of the experiment

Table 18.C16

Table 19.C18

Table 20.C24

Table 21.C24

Table 22.C14

Table 23.C20 addition standard recovery evaluation results of

Table 24.C22

The experimental results show that: the recovery rate of different standard addition concentrations C16:0 in the plasma sample is between 94.50% and 100.47%, the recovery rate of C18:0 is between 100.50% and 103.13%, the recovery rate of C24:0 is between 94.67% and 103.13%, the recovery rate of C24:1 is between 90.55% and 94.42%, the recovery rate of C14:0 is between 104.13% and 117.45%, the recovery rate of C20:0 is between 77.65% and 79.99%, and the recovery rate of C22:0 is between 88.44% and 92.64%, thus meeting the evaluation requirements of the recovery rates.

Test example 3: evaluation of matrix Effect

The experimental process comprises the following steps: the matrix effect was evaluated by evaluating the signal value of the target compound in plasma matrix and in the absence of matrix. Adding 2 concentration levels of standard solution to 6 clinical individual plasma matrix samples of different sources and pure solvent respectively, evaluating matrix effect by comparing signal intensity of target compound (internal standard) with signal intensity of pure solvent target compound (internal standard) in the presence of plasma matrix, and finally evaluating matrix effect by internal standard normalized matrix factor, wherein the calculation formula is as follows: internal standard normalized matrix factor = (analyte peak area in the presence of matrix/internal standard peak area)/(analyte peak area in the absence of matrix/internal standard peak area).

Results of the experiment

Table 25.C16

Table 26.C18

Table 27.C24

Table 28.C24

Table 29.C14

Table 30.C20

Table 31.C22

The experimental result shows that 6 clinical samples from different sources have different standard adding concentrations of C16:0 internal standard normalized matrix factor of 1.05-1.08; the normalized matrix factor of the C18:0 internal standard is between 1.01 and 1.07; the normalized matrix factor of the C24:0 internal standard is between 1.03 and 1.10; the normalized matrix factor of the C24:1 internal standard is between 0.90 and 0.96, the normalized matrix factor of the C14:0 internal standard is between 1.02 and 1.12, the normalized matrix factor of the C20:0 internal standard is between 0.85 and 0.90, and the normalized matrix factor of the C22:0 internal standard is between 0.88 and 0.93, which indicates that the matrix effect of the internal standard and the analyte is close, and can compensate the matrix effect which may appear in the analyte in the sample, so that the matrix effect does not influence the final accurate quantitative analysis, and the matrix effect investigation meets the requirements of the methodological evaluation.

Test example 4: residue evaluation

The experimental process comprises the following steps: and (3) continuously detecting a high-quality-controlled blank matrix sample for 5 times by using an HQC-DB-HQC-DB-HQC-DB-HQC-DB sequence, and determining the peak areas of an analyte and an internal standard in the blank matrix sample and comparing the peak areas with a minimum concentration point calibration standard sample to confirm the influence of residues on the accurate quantification of the analyte.

Results of the experiment

Table 32.C16

Note: HQC represents a high-concentration quality control sample, DB represents a blank matrix sample

Table 33.C18

Table 34.C24

Table 35.C24

Table 36.C14

Table 37.C20

Table 38.C22

The experimental result shows that the residue of C16:0 is 1.27-7.41%, and the residue of the internal standard C16:0 is 0.00%; 3.23-6.21% of C18:0 residue and 0.00% of C18:0 internal standard residue; a C24:0 residue of 6.90-9.62%, a C24:0 internal standard residue of 0.00%; 3.82-6.01% of C24:1 residue, 0.00% of C24:1 internal standard residue, 0.00% of C14:0 residue and 0.00% of C14:0 internal standard residue; 0.00% of C20:0 residue; 5.09-8.91% of C22:0 residues and 0.00% of C22:0 internal standard residues; 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 the quantification, 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, and the residual experimental evaluation meets the methodological evaluation requirement.

Test example 5: quantitative detection limit evaluation (LOQ)

The experimental process comprises the following steps: the quantitative detection limit of the method is inspected by detecting the linear lowest point of the standard curve, the sample is continuously injected and detected for 6 times, and the inspection is performed by CV and accuracy deviation (RE).

Results of the experiment

Table 39.C16

Table 40.C18

Table 41.C24

Table 42.C24

Table 43.C14

Table 44.C20

Table 45.C22

The experimental results show that both CV and RE for the linear minima (LOQ) of C16:0, C18:0, C24:1 are less than 20%, indicating that the quantitative detection limit evaluation satisfies the methodological evaluation.

Test example 6: evaluation of sample stability

Experimental procedure

(1) Standing at room temperature for 8h for stability: ceramide is an endogenous substance and contains background in clinical samples, so that a labeling method is not adopted for evaluating the stability of a plasma sample in a stability evaluation experiment. And directly testing the content of the substance to be tested in each experimental node. Taking a part of mixed plasma, uniformly mixing, standing at room temperature for 8 hours, detecting according to an operation instruction, and processing 6 samples in parallel at each concentration level. And (3) calculating the accuracy and the uniformity of the index components of the sample according to the detection data at the 0 th hour so as to evaluate the stability of the plasma sample at room temperature.

(2) Freeze-thaw stability: taking a part of mixed plasma, uniformly mixing, respectively placing in a refrigerator at the temperature of-20 ℃, performing freeze thawing for 3 times, and detecting according to an operation instruction. And (3) calculating the accuracy and the uniformity of the index components of the sample according to the detection data at the 0 th hour so as to evaluate the freeze-thaw stability of the plasma sample.

(3) Stability of the samples (-20 ℃) frozen for 7 days: taking a part of the mixed plasma, uniformly mixing, respectively placing in a refrigerator at the temperature of-20 ℃, freezing for 7 days, and detecting according to an operation instruction. Calculating the accuracy and uniformity of index components of the sample based on the detection data of the unfrozen sample on day 0 to evaluate the stability of the plasma sample in freezing for 7 days

(4) Sample post-preparation stability protocol: a portion of the mixed plasma was sampled and processed according to the instructions, and the processed solution was placed in an autosampler at 8 ℃ for 24 hours and then examined. And calculating the accuracy and uniformity of the index components of the sample based on the detection data at the 0 th hour so as to evaluate the stability of the plasma sample after preparation.

(5) The sample is stored for 24 hours at the temperature of 2-8 ℃ and has stability: taking a part of mixed plasma, uniformly mixing, placing at 2-8 ℃, storing for 24 hours, detecting according to an operation instruction, and parallelly processing 6 samples in each concentration level. And (3) calculating the accuracy and the uniformity of the index components of the sample according to the detection data at the 0 th hour so as to evaluate the stability of the plasma sample at room temperature.

Results of the experiment

TABLE 46 stability at room temperature of various ceramides

TABLE 47 repeated freeze thaw stability of various ceramides

TABLE 48 stability of various ceramides by cryopreservation at-20 ℃ for 7 days

TABLE 49 stability after preparation of various ceramide samples

TABLE 50 stability of various ceramides stored at 2-8 deg.C for 24h

According to the experiment, the standard curve, linearity, precision accuracy, standard recovery rate, matrix effect, residue, quantitative detection limit and sample stability of the mass spectrum detection methods of C14:0, C16:0, C18:0, C20:0, C22:0, C24:0 and C24:1 are evaluated, and the evaluation result display method can meet the clinical blood sample detection requirements.

Test example 7: comparison of the effects of whether to add MTBE to phase B

Based on the method and products provided in example 1,2 sets of test groups were set, and 1 set was the technical solution of example 1, and the other set was different from example 1 only in that no methyl t-butyl ether was 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, and it can be seen that phase B has no methyl tert-butyl ether added, pure acetonitrile has a weak elution capacity, and only three compounds peak within 4.5 min.

Test example 8: chromatographic separation under different liquid phase gradients with fixed mobile phase composition

The separation conditions under different liquid phase gradients were verified based on the technical scheme of example 1.

Gradient 1:

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

The results of gradient 1 are shown in FIG. 19. From the above figure, it can be seen that: the green color is a phospholipid channel, which is not completely separated from other ceramides, and the interference peak of C20 in the lower graph is not separated from C20 and is combined into one peak, and the interference peak can interfere the detection of C20.

Gradient 2:

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

The results of gradient 2 are shown in FIG. 20. From the results, it is understood that the interference peak in C20 is not completely separated from C20, and thus interferes with the detection of C20.

Test example 9: effect of different chromatography columns and flow on chromatographic separation

Taking the technical scheme of the embodiment 1 as a reference, a comparison group is set, and the difference between the comparison group and the embodiment 1 is as follows: phenyl chromatographic column, phase A is 2mM ammonium acetate, 0.1% formic acid and water; phase B0.1% methanoic acid. The results are shown in FIG. 21. From the results, it was found that the interference of each compound in plasma was large by using a phenyl column and methanol as a mobile phase. FIG. 22 shows a chromatogram obtained by separating a Heanomei Kinetex (50x3mm, 2.6 μm) C8 column using the same mobile phase and liquid phase gradient based on the protocol of example 1. As can be seen from the figure, the phospholipid can be well separated from the 7 analytes, the interference peak of C20 can be completely separated from C20, and the detection of C20 cannot be interfered. FIG. 23 shows a separation chromatogram of a Fenomei Kinetex Phenyl-Hexyl (50x4.6mm, 2.6 μm) column using the same mobile phase and liquid phase gradient based on the technical scheme of example 1. As can be seen from the figure, the phospholipid and 7 analytes can be well separated, the interference peak of C20 can be completely separated from C20, and the detection of C20 cannot be interfered.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining ceramide in a sample, comprising: detecting ceramide in the sample by adopting a liquid chromatography-tandem mass spectrometry method; the chromatography comprises gradient elution by adopting a mobile phase A and a mobile phase B, wherein the mobile phase A comprises an ammonium acetate aqueous solution containing formic acid, and the mobile phase B comprises a mixed solution of acetonitrile containing formic acid and methyl tert-butyl ether;

the elution conditions for the chromatography were as follows:

0-1 min, the volume percentage of the mobile phase A is maintained at 30-40%, and the volume percentage of the mobile phase B is maintained at 60-70%;

1-1.5 min, the volume percentage of the mobile phase A is reduced from 30-40% to 20-30%, and the volume percentage of the mobile phase B is increased from 60-70% to 70-80%;

1.5-3.5 min, the volume percentage of the mobile phase A is reduced from 20-30% to 1-10%, and the volume percentage of the mobile phase B is increased from 70-80% to 90-99%;

3.5-3.6 min, the volume percentage of the mobile phase A is increased from 1-10% to 30-40%, and the volume percentage of the mobile phase B is decreased from 90-99% to 60-70%;

3.6-4.5 min, the volume percentage of the mobile phase A is maintained at 30-40%, and the volume percentage of the mobile phase B is maintained at 60-70%;

the ceramide comprises: cer (d 18: 1/14), cer (d 18: 1/16; the methods are not directed towards the diagnosis or treatment of disease.

2. The method for detecting ceramide in a sample according to claim 1, wherein the elution conditions of the chromatogram are as follows:

0-1 min, the volume percentage of the mobile phase A is maintained at 35%, and the volume percentage of the mobile phase B is maintained at 65%;

1-1.5 min, the volume percentage of the mobile phase A is reduced from 35% to 25%, and the volume percentage of the mobile phase B is increased from 65% to 75%;

1.5-3.5 min, the volume percentage of the mobile phase A is reduced from 25% to 5%, and the volume percentage of the mobile phase B is increased from 75% to 95%;

3.5-3.6 min, the volume percentage of the mobile phase A is increased from 5% to 35%, and the volume percentage of the mobile phase B is decreased from 95% to 65%;

3.6-4.5 min, the volume percentage of the mobile phase A is maintained at 35%, and the volume percentage of the mobile phase B is maintained at 65%.

3. The method according to claim 2, wherein the flow rate of the gradient elution is 0.1 to 1mL/min.

4. The method for detecting ceramide in a sample according to claim 1, wherein the chromatography uses a column including any one of a C8 column and a phenyl column;

preferably, the C8 column is selected from any one of the femomei Kinetex C8, shimadzum-pack-GIST-HP C8 and a column having a stationary phase bonding phase or type similar to the femomei Kinetex C8 or shimadzum-pack-GIST-HP C8;

preferably, said femomei Kinetex C8 is of type 50x3mm,2.6 μm;

preferably, the model of the Shimadzu-pack-GIST-HP C8 is 50X 2.1,3 μm;

preferably, the Phenyl chromatographic column comprises a penomes Kinetex Phenyl-Hexyl or a chromatographic column with a similar type of stationary phase bonded phase or model;

preferably, said fenomei Kinetex Phenyl-Hexyl is of type 50 × 4.6,2.6 μm;

preferably, the chromatographic column is Shijin-pack-GIST-HP C8;

preferably, the chromatographic conditions comprise: the temperature of the chromatographic column is 35-45 ℃, the temperature of the sample injector is 5-15 ℃, and the sample injection amount is 2-5 mu L.

5. The method for detecting ceramide in a sample according to claim 1, wherein the volume ratio of acetonitrile to methyl tert-butyl ether in the mobile phase B is (70-80): (20;

preferably, in the mobile phase B, the volume ratio of acetonitrile to methyl tert-butyl ether is 75:25;

preferably, in the mobile phase B, the volume concentration fraction of formic acid is 0.01-0.5%;

preferably, the volume concentration fraction of formic acid in the mobile phase A is 0.01-0.5%.

6. The method of claim 1, wherein the mass spectrometry IS performed using an electrospray ion source, the detection mode IS positive ion mode, the scanning mode IS multiplex reaction monitoring, the desolvation temperature IS 550 ℃, the flow rate of the atomizing GAS (GAS 1) IS 55, the flow rate of the desolvation GAS (GAS 2) IS 55, and the ion source voltage (IS) IS 5500V.

7. The method for detecting ceramide in a sample according to any one of claims 1 to 6, wherein the sample comprises a blood sample.

8. The method of claim 7, wherein the LC-MS detection comprises a sample pre-treatment step, the sample pre-treatment step comprising: mixing a sample to be detected, an internal standard and a precipitator to carry out protein precipitation, and removing the precipitate for liquid chromatography tandem mass spectrometry detection;

preferably, the precipitating agent comprises methanol;

preferably, the internal standard solution comprises: at least one of an internal standard for Cer (d 18: 1/16) and an internal standard for Cer (d 18: 1/18;

preferably, the internal standard solution comprises: cer (d 18: 1/16) deuteron, cer (d 18: 1/18) deuteron, cer (d 18: 1/24) deuteron and Cer (d 18: 1/24).

9. Use of a combination of reagents for the preparation of a product for the determination of a ceramide in a sample, said combination of reagents comprising reagents for carrying out the method according to any one of claims 1 to 8, said ceramide comprising: cer (d 18: 1/14), cer (d 18: 1/16;

preferably, the sample comprises a blood sample.

10. A product for measuring ceramide in a specimen, characterized by comprising a reagent for carrying out the method for measuring ceramide in a specimen according to any one of claims 1 to 8.