CN114563505A - Alpha-hydroxyceramide detection method based on ultra-high performance liquid chromatography-mass spectrometry combined technology - Google Patents
Alpha-hydroxyceramide detection method based on ultra-high performance liquid chromatography-mass spectrometry combined technology Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention relates to the technical field of alpha-hydroxyceramide detection, in particular to an alpha-hydroxyceramide detection method based on an ultra-high performance liquid chromatography-mass spectrometry combined technology. The method specifically comprises the following steps: (1) mixing the serum and the extracting solution, carrying out first standing, centrifuging to obtain the lowest layer to obtain a material 1, mixing the material 1 with an internal standard solution, volatilizing to obtain a material 2, mixing the material 2 with a double solvent, carrying out second standing, and obtaining a supernatant to obtain a material 3; (2) and (3) carrying out ultra performance liquid chromatography tandem mass spectrometry on the material 3 obtained in the step (1). The invention establishes an analytical method for simultaneously measuring 6 types of alpha-hydroxyceramide in serum for the first time based on LC-MS/MS, and the method has good precision, accuracy and wider linear range.
Description
Technical Field
The invention relates to the technical field of alpha-hydroxyceramide detection, in particular to an alpha-hydroxyceramide detection method based on an ultra-high performance liquid chromatography-mass spectrometry combined technology.
Background
Ceramide is generated by connecting one molecule of sphingosine long-chain base and one molecule of fatty acid chain through amido bond, and is a precursor substance for synthesizing complex sphingolipid. Ceramides, which are central to sphingolipid metabolism, are not only important components of cell membranes, but also play important roles in biological processes such as induction of apoptosis, regulation of cell growth, proliferation and differentiation, regulation of immune function, and inflammatory response. In metabolic diseases such as dyslipidemia and inflammation, ceramide accumulates in tissues and blood circulation. Research has shown that ceramide levels in blood are associated with the pathophysiological processes of sepsis, cardiovascular diseases, cancer, type 2 diabetes, etc. Elevated levels of 4 ceramides Cer (d18:1/16:0), Cer (d18:1/18:0), Cer (d18:1/20:0) and Cer (d18:1/24:0) in plasma are considered to be predictive of Alzheimer's disease.
Ceramides have a high degree of structural diversity due to differences in the number of carbon atoms, the number of unsaturated bonds in the fatty acid chain, and the presence of hydroxyl groups in the fatty acid chain. Of all ceramides, mammals are most abundantly characterized by non-hydroxylic ceramides, followed by α -hydroxylic ceramides found in the skin, kidneys and brain. Ceramides containing alpha-hydroxy fatty acid chains account for about one third of the ceramides that can be extracted from the skin. The hydroxyl groups of Alpha-hydroxyceramide interact with other lipid molecules through hydrogen bonds to enhance lipid interaction, thereby influencing the size, fluidity and stability of micro-domains and providing a barrier for the stratum corneum, which indicates that Alpha-hydroxyceramide may mediate pro-apoptotic signaling pathways. Current evidence suggests that α -hydroxyceramides have specific functions in membrane homeostasis and cell signaling. Alpha-hydroxyceramides are precursors to many complex sphingolipids, such as Alpha-hydroxygalactosylceramide, which play a key role in maintaining the normal structure and stability of myelin sheaths and the differentiation of oligodendrocytes. However, there are limited reports of studies on the physiological function of α -hydroxyceramide in vivo, probably due to the lack of sensitive detection methods, and the fact that compound standards have not been marketed until recently. Therefore, an accurate and reliable alpha-hydroxyceramide detection method is urgently needed to be established, and the possibility is provided for further discussing the pathophysiological action of the alpha-hydroxyceramide in vivo.
The detection method of ceramide includes benzoylation-ultraviolet detection method, thin layer chromatography, liquid chromatography-tandem mass spectrometry, etc. Among them, the liquid chromatography-tandem mass spectrometry is the most common detection method due to its high selectivity and sensitivity. As a gold standard for mass spectrometry quantification, the multiple reaction monitoring mode (MRM) shows its powerful advantages in selecting specific ions (parent and fragment ions). LC-MRM based detection methods have been widely used to detect non-hydroxylceramide or complex lipids containing non-hydroxylceramide. Kasumov et al developed and validated a method of reverse phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS) for the determination of 7 ceramide Cer (d18:1/14:0), Cer (d18:1/16:0), Cer (d18:1/18:0), Cer (d18:1/20:0), Cer (d18:1/24:0), Cer (d18:1/18:1) and Cer (d18:1/24:1) (Kasumov T, Huang H, Chung YM, Zhang RL, Mcuoulgh AJ, Kirwan JP quantification of nuclear reactions in biological samples by liquid chromatography amplification analysis (2010) 154), biochemical analysis (401). Olga et al used the RPLC-MS/MS method to quantify Cer (d18:1/16:0), Cer (d18:1/18:0), Cer (d18:1/24:0) and Cer (d18:1/24:1) in human serum (Begou OA, Deda O, Karagianidis E, Sinanos G, Theodidis G, Gika HG. development and evaluation of a RPLC-MS/MS method for the identification of ceramides in human serum. J chromosome B1175 (2021)). However, studies on α -hydroxyceramides have focused mainly on their structural identification, and their quantitative determination in vivo has not been carried out.
Therefore, how to solve the problem that the prior art lacks a detection method related to alpha-hydroxyceramide and establish an LC-MS-based alpha-hydroxyceramide quantification method is urgently needed to be solved by the technical personnel in the field.
Disclosure of Invention
The invention aims to provide a method for detecting alpha-hydroxyceramide based on an ultra-high performance liquid chromatography-mass spectrometry combined technology, which can simultaneously carry out quantitative detection on 6 kinds of alpha-hydroxyceramides, has high detection accuracy and has a wider linear range.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an alpha-hydroxyceramide detection method based on an ultra-high performance liquid chromatography-mass spectrometry combined technology, which comprises the following steps:
(1) mixing the serum and the extracting solution, performing first standing, centrifuging to obtain the lowest layer to obtain a material 1, mixing the material 1 with an internal standard solution, volatilizing to obtain a material 2, mixing the material 2 with a double solvent, performing second standing, and taking the supernatant to obtain a material 3;
(2) carrying out ultra-high performance liquid chromatography tandem mass spectrometry on the material 3 obtained in the step (1);
the alpha-hydroxyceramides are Cer (d18:1/16:0(2OH)), Cer (d18:1/18:0(2OH)), Cer (d18:1/20:0(2OH)), Cer (d18:1/22:0(2OH)), Cer (d18:1/18:1(2OH)) and Cer (d18:1/24:1(2 OH)).
Preferably, the volume ratio of the serum to the extracting solution in the step (1) is 3-5: 14-18; the extracting solution is chloroform and methanol; the volume ratio of the chloroform to the methanol is 2-6: 1-3.
Preferably, the first standing time and the second standing time in the step (1) are independent for 8-12 min; the centrifugation time is 13-17 min; the centrifugation temperature is 3-5 ℃; the centrifugal rotating speed is 12000-14000 rpm.
Preferably, the volume ratio of the material 1 to the internal standard liquid in the step (1) is 80-100: 8-12; the internal standard solution is a Cer (d18:1-d7/16:0) solution with the concentration of 90-110 ng/mL.
Preferably, the volume ratio of the material 1 to the double solvent in the step (1) is 8-10: 4-6; the double solvent is isopropanol, acetonitrile and water; the volume ratio of the isopropanol to the acetonitrile to the water is 2-6: 1-3.
Preferably, the mass spectrum in the ultra performance liquid chromatography tandem mass spectrum in the step (2) is analyzed by adopting a multiple reaction monitoring mode and a positive ion mode: the ion source is an electrospray ion source, the temperature of the dry gas is 250-350 ℃, the flow rate of the dry gas is 9-13L/min, the pressure of the atomization gas is 18-22 psi, the voltage of a capillary tube is 3800-4200V, the temperature of the sheath gas is 380-420 ℃, the flow rate of the sheath gas is 9-13L/min, and the voltage of a nozzle is 800-1200V.
Preferably, the chromatographic column of the ultra performance liquid chromatography in the ultra performance liquid chromatography tandem mass spectrum in the step (2) is a BEHC18 chromatographic column; the mobile phase A is acetonitrile and water, and the mobile phase B is isopropanol and acetonitrile.
Preferably, the volume ratio of acetonitrile to water in the phase A is 5-7: 3-5; the volume ratio of the isopropanol to the acetonitrile in the phase B is 8-10: 1-2.
Preferably, the phase A and the phase B independently contain 8-12 mM ammonium acetate.
Preferably, the mobile phase elution gradient of the ultra performance liquid chromatography is as follows:
TABLE 1 ultra performance liquid chromatography mobile phase elution gradient
Compared with the prior art, the invention has the following beneficial effects:
the analytical method for simultaneously determining 6 types of alpha-hydroxyceramides in serum is established for the first time based on LC-MS/MS, has good precision, accuracy and wider linear range, and can provide technical support for determining the content of more types of alpha-hydroxyceramides.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 shows the MRM chromatogram of α -hydroxyceramide standard (wherein A: Cer (d18:1-d7/16:0), B: Cer (d18:1/16:0(2 OH)));
FIG. 2 is an MRM chromatogram of an α -hydroxyceramide standard (where C: Cer (D18:1/18:0(2OH)), D: Cer (D18:1/20:0(2 OH)));
FIG. 3 is an MRM chromatogram of an α -hydroxyceramide standard (where E: Cer (d18:1/22:0(2OH)), F: Cer (d18:1/18:1(2 OH)));
FIG. 4 is an MRM chromatogram of an α -hydroxyceramide standard (G: Cer (d18:1/24:1(2 OH)));
FIG. 5 shows the results of detection of inflammatory factors in sera of different test groups (where A is IL-1. beta. and B is TNF-. alpha.);
FIG. 6 shows the results of measurement of α -hydroxyceramide in the serum of mice in the normal control group (CON group) and LPS-induced sepsis group (LPS group) (where A is Cer (D18:1/16:0(2OH)), B is Cer (D18:1/18:0(2OH)), C is Cer (D18:1/18:1(2OH)), D is Cer (D18:1/20:0(2OH)), E is Cer (D18:1/22:0(2OH)), and F is Cer (D18:1/24:1(2 OH)).
Detailed Description
The invention provides an alpha-hydroxyceramide detection method based on an ultra-high performance liquid chromatography-mass spectrometry combined technology, which comprises the following steps:
(1) mixing the serum and the extracting solution, carrying out first standing, centrifuging to obtain the lowest layer to obtain a material 1, mixing the material 1 with an internal standard solution, volatilizing to obtain a material 2, mixing the material 2 with a double solvent, carrying out first standing for 8-12 min, and taking the supernatant to obtain a material 3;
(2) carrying out ultra-high performance liquid chromatography tandem mass spectrometry on the material 3 obtained in the step (1);
the alpha-hydroxyceramides are Cer (d18:1/16:0(2OH)), Cer (d18:1/18:0(2OH)), Cer (d18:1/20:0(2OH)), Cer (d18:1/22:0(2OH)), Cer (d18:1/18:1(2OH)) and Cer (d18:1/24:1(2 OH)).
In the present invention, the serum is derived from an animal or human.
In the present invention, the mixing in step (1) is followed by vortexing.
In the invention, the volume ratio of the serum and the extracting solution in the step (1) is 3-5: 14-18; preferably 4: 15-17; further preferably 4: 16.
In the invention, the extracting solution in the step (1) is chloroform and methanol.
In the invention, the volume ratio of chloroform to methanol in the step (1) is 2-6: 1-3; preferably 3-5: 2; further preferred is 4: 2.
In the invention, the first standing time and the second standing time in the step (1) are independent for 8-12 min; preferably 9-11 min; more preferably 10 min.
In the invention, the centrifugation time in the step (1) is 13-17 min; preferably 14-16 min; further preferably 15 min; the centrifugation temperature is 3-5 ℃; preferably 4 ℃; the centrifugal rotating speed is 12000-14000 rpm; preferably 12500-13500 rpm; further preferably 12700-13200 rpm; more preferably 13000 rpm.
In the invention, the volume ratio of the material 1 in the step (1) to the internal standard liquid is 80-100: 8-12; preferably 84-96: 9-11; further preferably 88-92: 10; more preferably 90: 10.
In the invention, the internal standard solution in the step (1) is a Cer (d18:1-d7/16:0) solution with the concentration of 90-110 ng/mL; preferably 94-106 ng/mL; further preferably 98-102 ng/mL; more preferably 100 ng/mL.
In the invention, the volume ratio of the material 1 and the double solvent in the step (1) is 8-10: 4-6; preferably 9: 4-6; further preferably 9: 5.
In the invention, the double solvent in the step (1) is isopropanol, acetonitrile and water; the volume ratio of the isopropanol to the acetonitrile to the water is 2-6: 1-3; preferably 3-5: 2; further preferred is 4: 2.
In the invention, the mass spectrum in the ultra performance liquid chromatography tandem mass spectrum in the step (2) adopts a multi-reaction monitoring mode and a positive ion mode for analysis: the ion source is an electrospray ion source, the temperature of dry gas is 250-350 ℃, the flow rate of the dry gas is 9-13L/min, the pressure of the atomization gas is 18-22 psi, the voltage of a capillary tube is 3800-4200V, the temperature of sheath gas is 380-420 ℃, the flow rate of the sheath gas is 9-13L/min, and the voltage of a nozzle is 800-1200V;
preferably, the temperature of the drying gas is 270-330 ℃, the flow of the drying gas is 10-12L/min, the pressure of the atomization gas is 19-21 psi, the voltage of a capillary tube is 3900-4100V, the temperature of the sheath gas is 390-410 ℃, the flow of the sheath gas is 10-12L/min, and the voltage of a nozzle is 900-1100V;
further preferably, the temperature of the drying gas is 280-320 ℃, the flow of the drying gas is 11L/min, the pressure of the atomization gas is 20psi, the voltage of a capillary tube is 4000V, the temperature of the sheath gas is 400 ℃, the flow of the sheath gas is 11L/min, and the voltage of a nozzle is 1000V;
more preferably, the temperature of the drying gas is 300 ℃, the flow rate of the drying gas is 11L/min, the pressure of the atomization gas is 20psi, the voltage of the capillary tube is 4000V, the temperature of the sheath gas is 400 ℃, the flow rate of the sheath gas is 11L/min, and the voltage of the nozzle is 1000V.
In the invention, the chromatographic column of the ultra performance liquid chromatography in the ultra performance liquid chromatography tandem mass spectrum in the step (2) is a BEHC18 chromatographic column; the mobile phase A is acetonitrile and water, and the mobile phase B is isopropanol and acetonitrile.
In the invention, the volume ratio of acetonitrile to water in the phase A in the step (2) is 5-7: 3-5; preferably 6: 3-5; further preferred is 6: 4.
In the invention, the volume ratio of isopropanol to acetonitrile in the phase B in the step (2) is 8-10: 1-2; preferably 9: 1-2; further preferred is 9: 1.
In the invention, the phase A and the phase B in the step (2) independently contain 8-12 mM ammonium acetate; preferably 9 to 11 mM; more preferably 10 mM.
In the invention, the mobile phase elution gradient of the ultra-high performance liquid chromatography is as follows:
TABLE 2 ultra performance liquid chromatography mobile phase elution gradient
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The alpha-hydroxyceramide detection method based on the ultra performance liquid chromatography-mass spectrometry combined technology comprises the following steps:
1. animal experiments and sample Collection
Selecting C57B/L6 mice with male non-specific pathogenic bacteria, weighing 22-25 g, purchasing from Shanghai Ricius experimental animal center, (Shanghai, China), placing under constant temperature condition, breeding for a week in an adaptive manner, circulating for 12h day and night, and feeding food and water.
Mice were injected intraperitoneally with LPS (5mg/kg) to induce the development of sepsis. After LPS injection for 3h, blood samples were collected from both CON group and LPS group mice by orbital puncture, and after the blood was allowed to stand in a refrigerator at 4 ℃ for 2h, it was centrifuged at 4 ℃ and 3500rpm for 15min, and the supernatant serum was taken and stored in a refrigerator at-80 ℃ for further experiments. All animal experiments were performed exactly according to the animal experimental principles.
2. Evaluation of mouse animal models for sepsis
Evaluation of a septic mouse animal model was performed from mouse serum biochemical indicators. Serum cytokines (IL-1. beta., TNF-. alpha.) are important indicators for the degree of sepsis in mice. Mice were sacrificed after abdominal anesthesia. The chest of the mouse was cut along the manubrium to expose the heart. Blood was drawn using a 1mL syringe, stored in an EP tube, centrifuged at (4000rpm, 4 ℃) for 15min, and the supernatant was removed and stored at-80 ℃. IL-1. beta. and TNF-. alpha.were detected using enzyme-linked immunosorbent assay (ELASA).
3. The specific detection steps are as follows:
(1) preparation of alpha-hydroxy ceramide standard substance stock solution and working solution
Preparation of stock solution: accurately weighing appropriate amount of ceramide standard, respectively, adding appropriate amount of methanol solution for dissolving, preparing into 1.0mg/mL standard stock solution for subsequent experiment, and storing in a refrigerator at-20 deg.C.
Preparation of working solution: precisely sucking the stock solution of the standard substance of 1.0mg/mL, diluting with methanol step by step to obtain a series of required working solutions, and storing in a refrigerator at-20 ℃.
(2) Preparation of surrogate matrix (5% BSA):
500mg of bovine serum albumin (BSA, > 95%) was weighed accurately, and 10mL of Phosphate Buffered Saline (PBS) was added to prepare a 5% BSA substitute matrix.
(3) Pretreatment of serum samples
Preparing an Internal Standard (IS) solution: an appropriate amount of internal standard Cer (d18:1-d7/16:0) standard is precisely weighed, and methanol is added for dissolution to prepare 100ng/mL methanol solution of Cer (d18:1-d7/16: 0).
(4) Unfreezing the serum obtained in the step 2 in a refrigerator at 4 ℃, mixing the serum and an extracting solution according to the volume ratio of 3: 14, carrying out vortex oscillation for 30s, standing for 8min at room temperature, centrifuging to obtain the lowest layer (a liquid-liquid extraction method is used in the invention, three layers are formed after extraction, the uppermost layer is a polar molecular layer, an intermediate protein layer and the lowest layer is a lipid layer, namely a required part) to obtain a material 1, mixing the material 1 with a Cer (d18:1-d7/16:0) solution with the concentration of 90ng/mL according to the volume ratio of 80: 8, volatilizing to obtain a material 2 by using a vacuum centrifugal concentrator, mixing the material 1 with a double solvent according to the volume ratio of 8: 4, standing for 8min, and taking the supernatant to obtain a material 3;
(5) performing ultra-high performance liquid chromatography tandem mass spectrometry on the material 3 obtained in the step (4);
the alpha-hydroxyceramide is Cer (d18:1/16:0(2OH)), Cer (d18:1/18:0(2OH)), Cer (d18:1/20:0(2OH)), Cer (d18:1/22:0(2OH)), Cer (d18:1/18:1(2OH)) and Cer (d18:1/24:1(2 OH));
the centrifugation time is 13 min; the temperature of the centrifugation is 3 ℃; the rotation speed of the centrifugation is 12000 rpm; the extracting solution is chloroform and methanol; the volume ratio of the chloroform to the methanol is 2: 1;
the double solvent is isopropanol, acetonitrile and water; the volume ratio of the isopropanol to the acetonitrile to the water is 2: 1;
the mass spectrum in the ultra-high performance liquid chromatography tandem mass spectrum adopts a multi-reaction monitoring mode and a positive ion mode for analysis: the ion source is an electrospray ion source, the temperature of dry gas is 250 ℃, the flow of the dry gas is 9L/min, the pressure of the atomization gas is 18psi, the voltage of a capillary tube is 3800V, the temperature of sheath gas is 380 ℃, the flow of the sheath gas is 9L/min, and the voltage of a nozzle is 800V;
the chromatographic column of the ultra-high performance liquid chromatography in the ultra-high performance liquid chromatography tandem mass spectrum is a BEH C18 chromatographic column; mobile phase a was (acetonitrile and water 5: 3, containing 8mM ammonium acetate) and phase B was (isopropanol and acetonitrile 8:1, containing 8mM ammonium acetate);
the mobile phase elution gradient of the ultra-high performance liquid chromatography is as follows:
TABLE 3 ultra performance liquid chromatography mobile phase elution gradient
Example 2
The alpha-hydroxyceramide detection method based on the ultra performance liquid chromatography-mass spectrometry combined technology comprises the following steps:
1. animal experiments and sample Collection
Selecting a C57B/L6 mouse with male non-specific pathogenic bacteria, weighing 22-25 g, purchasing from Shanghai Rice laboratory animal center, (Shanghai, China), placing under a constant temperature condition, adaptively feeding for a week, circulating for 12h day and night, and feeding food and water.
Mice were injected intraperitoneally with LPS (5mg/kg) to induce the development of sepsis. After LPS injection for 3h, blood samples were collected from both CON group and LPS group mice by orbital puncture, and after the blood was allowed to stand in a refrigerator at 4 ℃ for 2h, it was centrifuged at 4 ℃ and 3500rpm for 15min, and the supernatant serum was taken and stored in a refrigerator at-80 ℃ for further experiments. All animal experiments were performed exactly according to the animal experimental principles.
2. Evaluation of mouse animal models of sepsis
Evaluation of a septic mouse animal model was performed from mouse serum biochemical indicators. Serum cytokines (IL-1. beta., TNF-. alpha.) are important indicators for the degree of sepsis in mice. Mice were sacrificed after abdominal anesthesia. The chest of the mouse was cut along the manubrium to expose the heart. Blood was drawn using a 1mL syringe, stored in an EP tube (4000rpm, 4 ℃) and centrifuged for 15min, and the supernatant was collected and stored at-80 ℃. IL-1. beta. and TNF-. alpha.were detected using enzyme-linked immunosorbent assay (ELASA).
3. The specific detection steps are as follows:
(1) preparation of alpha-hydroxy ceramide standard substance stock solution and working solution
Preparation of stock solution: accurately weighing a proper amount of ceramide standard substances respectively, adding a proper amount of methanol solution for dissolving, preparing a standard substance stock solution of 1.0mg/mL for subsequent experiments, and storing in a refrigerator at the temperature of-20 ℃.
Preparation of working solution: precisely sucking the stock solution of the standard substance of 1.0mg/mL, diluting with methanol step by step to obtain a series of required working solutions, and storing in a refrigerator at-20 ℃.
(2) Preparation of surrogate matrix (5% BSA):
500mg of bovine serum albumin (BSA, > 95%) was weighed accurately, and 10mL of Phosphate Buffered Saline (PBS) was added to prepare a 5% BSA substitute matrix.
(3) Pretreatment of serum samples
Preparing an Internal Standard (IS) solution: an appropriate amount of internal standard Cer (d18:1-d7/16:0) standard is precisely weighed, and methanol is added for dissolution to prepare 100ng/mL methanol solution of Cer (d18:1-d7/16: 0).
(4) Unfreezing the serum obtained in the step 2 in a refrigerator at 4 ℃, mixing the serum and an extracting solution according to the volume ratio of 5: 18, carrying out vortex oscillation for 30s, standing at room temperature for 12min, centrifuging to obtain the lowest layer to obtain a material 1, mixing the material 1 and a Cer (d18:1-d7/16:0) solution with the concentration of 110ng/mL according to the volume ratio of 100: 12, volatilizing by using a vacuum centrifugal concentrator to obtain a material 2, mixing the material 1 and a double solvent (isopropanol, acetonitrile and water are 6: 3) according to the volume ratio of 10: 6, standing for 12min, and taking the supernatant to obtain a material 3;
(5) performing ultra-high performance liquid chromatography tandem mass spectrometry on the material 3 obtained in the step (4);
the alpha-hydroxyceramide is Cer (d18:1/16:0(2OH)), Cer (d18:1/18:0(2OH)), Cer (d18:1/20:0(2OH)), Cer (d18:1/22:0(2OH)), Cer (d18:1/18:1(2OH)) and Cer (d18:1/24:1(2 OH));
the centrifugation time is 17 min; the temperature of the centrifugation is 5 ℃; the rotation speed of the centrifugation is 14000 rpm; the extracting solution is chloroform and methanol; the volume ratio of the chloroform to the methanol is 6: 3;
the double solvent is isopropanol, acetonitrile and water; the volume ratio of the isopropanol to the acetonitrile to the water is 6: 3;
the mass spectrum in the ultra-high performance liquid chromatography tandem mass spectrum adopts a multi-reaction monitoring mode and a positive ion mode for analysis: the ion source is an electrospray ion source, the temperature of dry gas is 350 ℃, the flow rate of the dry gas is 13L/min, the pressure of the atomization gas is 22psi, the voltage of a capillary tube is 4200V, the temperature of sheath gas is 420 ℃, the flow rate of the sheath gas is 13L/min, and the voltage of a nozzle is 1200V;
the chromatographic column of the ultra-high performance liquid chromatography in the ultra-high performance liquid chromatography tandem mass spectrum is a BEH C18 chromatographic column; mobile phase a was (acetonitrile and water 7: 5, containing 12mM ammonium acetate) and phase B was (isopropanol and acetonitrile 10: 2, containing 12mM ammonium acetate);
the mobile phase elution gradient of the ultra-high performance liquid chromatography is as follows:
TABLE 4 ultra performance liquid chromatography mobile phase elution gradient
Example 3
The alpha-hydroxyceramide detection method based on the ultra performance liquid chromatography-mass spectrometry combined technology comprises the following steps:
1. animal experiments and sample Collection
Selecting C57B/L6 mice with male non-specific pathogenic bacteria, weighing 22-25 g, purchasing from Shanghai Ricius experimental animal center, (Shanghai, China), placing under constant temperature condition, breeding for a week in an adaptive manner, circulating for 12h day and night, and feeding food and water.
Mice were injected intraperitoneally with LPS (5mg/kg) to induce the development of sepsis. After LPS injection for 3h, blood samples were collected from both CON group and LPS group mice by orbital puncture, and after the blood was allowed to stand in a refrigerator at 4 ℃ for 2h, it was centrifuged at 4 ℃ and 3500rpm for 15min, and the supernatant serum was taken and stored in a refrigerator at-80 ℃ for further experiments. All animal experiments were performed exactly according to the animal experimental principles.
2. Evaluation of mouse animal models of sepsis
Evaluation of a septic mouse animal model was performed from mouse serum biochemical indicators. Serum cytokines (IL-1. beta., TNF-. alpha.) are important indicators for the degree of sepsis in mice. Mice were sacrificed after abdominal anesthesia. The chest of the mouse was cut along the manubrium to expose the heart. Blood was drawn using a 1mL syringe, stored in an EP tube, centrifuged at (4000rpm, 4 ℃) for 15min, and the supernatant was removed and stored at-80 ℃. IL-1. beta. and TNF-. alpha.were detected using enzyme-linked immunosorbent assay (ELASA).
3. The specific detection steps are as follows:
(1) preparation of alpha-hydroxy ceramide standard substance stock solution and working solution
Preparation of stock solution: accurately weighing appropriate amount of ceramide standard, respectively, adding appropriate amount of methanol solution for dissolving, preparing into 1.0mg/mL standard stock solution for subsequent experiment, and storing in a refrigerator at-20 deg.C.
Preparation of working solution: precisely sucking the stock solution of the standard substance of 1.0mg/mL, diluting with methanol step by step to obtain a series of required working solutions, and storing in a refrigerator at-20 ℃.
(2) Preparation of surrogate matrix (5% BSA):
500mg of bovine serum albumin (BSA, > 95%) was weighed accurately, and 10mL of Phosphate Buffered Saline (PBS) was added to prepare a 5% BSA substitute matrix.
(3) Pretreatment of serum samples
Preparing an Internal Standard (IS) solution: an appropriate amount of internal standard Cer (d18:1-d7/16:0) standard is precisely weighed, and methanol is added for dissolution to prepare 100ng/mL methanol solution of Cer (d18:1-d7/16: 0).
(4) Unfreezing the serum obtained in the step 2 in a refrigerator at 4 ℃, mixing the unfrozen serum with an extracting solution according to the volume ratio of 4: 16, carrying out vortex oscillation for 30s, standing for 10min at room temperature, centrifuging to obtain the lowest layer to obtain a material 1, mixing the material 1 with a Cer (d18:1-d7/16:0) solution with the concentration of 100ng/mL according to the volume ratio of 90: 10, volatilizing by using a vacuum centrifugal concentrator to obtain a material 2, mixing the material 1 with a double solvent according to the volume ratio of 9: 5, standing for 10min, and taking the supernatant to obtain a material 3;
(5) performing ultra-high performance liquid chromatography tandem mass spectrometry on the material 3 obtained in the step (4);
the alpha-hydroxyceramide is Cer (d18:1/16:0(2OH)), Cer (d18:1/18:0(2OH)), Cer (d18:1/20:0(2OH)), Cer (d18:1/22:0(2OH)), Cer (d18:1/18:1(2OH)) and Cer (d18:1/24:1(2 OH));
the centrifugation time is 15 min; the temperature of the centrifugation is 4 ℃; the rotating speed of the centrifugation is 13000 rpm; the extracting solution is chloroform and methanol; the volume ratio of the chloroform to the methanol is 4: 2;
the double solvent is isopropanol, acetonitrile and water; the volume ratio of the isopropanol to the acetonitrile to the water is 4: 2;
the mass spectrum in the ultra-high performance liquid chromatography tandem mass spectrum adopts a multi-reaction monitoring mode and a positive ion mode for analysis: the ion source is an electrospray ion source, the temperature of dry gas is 300 ℃, the flow of the dry gas is 11L/min, the pressure of the atomizing gas is 20psi, the voltage of a capillary tube is 4000V, the temperature of sheath gas is 400 ℃, the flow of the sheath gas is 11L/min, and the voltage of a nozzle is 1000V;
the chromatographic column of the ultra-high performance liquid chromatography in the ultra-high performance liquid chromatography tandem mass spectrum is a BEH C18 chromatographic column; mobile phase a was (acetonitrile and water 6: 4, containing 10mM ammonium acetate) and phase B was (isopropanol and acetonitrile 9: 1, containing 10mM ammonium acetate);
the mobile phase elution gradient of the ultra-high performance liquid chromatography is as follows:
TABLE 5 ultra performance liquid chromatography mobile phase elution gradient
Example 4 application of the detection method of alpha-hydroxyceramide based on LC-MS/MS in serum of septic mice
1. Animal experiments and sample Collection
Selecting C57B/L6 mice with male non-specific pathogenic bacteria, weighing 22-25 g, purchasing from Shanghai Ricius experimental animal center, (Shanghai, China), placing under constant temperature condition, breeding for a week in an adaptive manner, circulating for 12h day and night, and feeding food and water.
Mice were randomized into 2 groups: normal control group (CON group) and LPS induced sepsis group (LPS group). Intraperitoneal injection of LPS (5mg/kg) induced the onset of sepsis. CON group mice were injected with equal amounts of saline. After 3h of LPS injection, blood samples were collected from both CON group and LPS group mice by orbital puncture, after the blood was allowed to stand in a refrigerator at 4 ℃ for 2h, the blood was centrifuged at 4 ℃ and 3500rpm for 15min, and the supernatant serum was taken and stored in a refrigerator at-80 ℃ for further experiments. All animal experiments were performed exactly according to the animal experimental principles.
2. Evaluation of mouse animal models of sepsis
Evaluation of sepsis mouse animal models was performed from mouse serum biochemical indicators. Serum cytokines (IL-1. beta., TNF-. alpha.) are important indicators for the degree of sepsis in mice. Mice in CON and LPS groups were sacrificed after abdominal anesthesia. The chest of the mouse was cut along the manubrium to expose the heart. Blood was drawn using a 1ml syringe, stored in an EP tube, centrifuged at (4000rpm, 4 ℃) for 15min, and the supernatant was collected and stored at-80 ℃. IL-1. beta. and TNF-. alpha.were detected using enzyme-linked immunosorbent assay (ELASA).
3. The method is applied to the detection of serum ceramide in sepsis mice and normal control mice, the change of the content of ceramide in mice of a CON group and an LPS group is analyzed, and the pathogenesis of sepsis is discussed.
The specific detection steps are as follows:
(1) preparation of alpha-hydroxyceramide standard substance stock solution and working solution
Preparation of stock solution: accurately weighing appropriate amount of ceramide standard, respectively, adding appropriate amount of methanol solution for dissolving, preparing into 1.0mg/mL standard stock solution for subsequent experiment, and storing in a refrigerator at-20 deg.C.
Preparation of working solution: precisely sucking the stock solution of the standard substance of 1.0mg/mL, diluting with methanol step by step to obtain a series of required working solutions, and storing in a refrigerator at-20 ℃.
(2) Preparation of surrogate matrix (5% BSA):
500mg of bovine serum albumin (BSA, > 95%) was weighed accurately, and 10mL of Phosphate Buffered Saline (PBS) was added to prepare a 5% BSA substitute matrix.
(3) Pretreatment of serum samples
Preparing an Internal Standard (IS) solution: an appropriate amount of internal standard Cer (d18:1-d7/16:0) standard is precisely weighed, and methanol is added for dissolution to prepare 100ng/mL methanol solution of Cer (d18:1-d7/16: 0).
(4) Thawing the serum obtained in the step 2 in a refrigerator at 4 ℃, taking 40 mu L of serum, adding 160 mu L of extracting solution (chloroform: methanol IS 4: 2), carrying out vortex oscillation for 30 seconds, standing for 10min at room temperature, centrifuging for 15min, taking 90 mu L of lower layer to a new 1.5mLEP tube to obtain a material 1, mixing the material 1 with 10 mu L of Internal Standard (IS)100ng/mL of Cer (d18:1-d7/16:0) solution, volatilizing by using a vacuum centrifugal concentrator to obtain a material 2, mixing the material 2 with 50 mu L of complex solvent (isopropanol, acetonitrile and water IS 4: 2), standing for 10min, taking supernatant, putting the supernatant into a sample injection vial, transferring to an automatic sample injector plate, and waiting for detection on an upper computer;
(5) performing ultra-high performance liquid chromatography tandem mass spectrometry on the material 3 obtained in the step (4);
the alpha-hydroxyceramide is Cer (d18:1/16:0(2OH)), Cer (d18:1/18:0(2OH)), Cer (d18:1/20:0(2OH)), Cer (d18:1/22:0(2OH)), Cer (d18:1/18:1(2OH)) and Cer (d18:1/24:1(2 OH));
the temperature of the centrifugation is 4 ℃; the rotating speed of the centrifugation is 13000 rpm;
the mass spectrum in the ultra-high performance liquid chromatography tandem mass spectrum adopts a multi-reaction monitoring mode and a positive ion mode for analysis: the ion source is an electrospray ion source, the temperature of dry gas is 300 ℃, the flow of the dry gas is 11L/min, the pressure of the atomizing gas is 20psi, the voltage of a capillary tube is 4000V, the temperature of sheath gas is 400 ℃, the flow of the sheath gas is 11L/min, and the voltage of a nozzle is 1000V;
the parent-child ion pairs of the alpha-hydroxyceramide standard and the corresponding mass spectrum parameters are as follows
TABLE 6 Mass Spectrometry parameters
The chromatographic column of the ultra-high performance liquid chromatography in the ultra-high performance liquid chromatography tandem mass spectrum is a BEH C18 chromatographic column; mobile phase a was (acetonitrile and water 6: 4, containing 10mM ammonium acetate) and phase B was (isopropanol and acetonitrile 9: 1, containing 10mM ammonium acetate);
the mobile phase elution gradient of the ultra-high performance liquid chromatography is as follows:
TABLE 7 ultra performance liquid chromatography mobile phase elution gradient
4. Data processing and statistical analysis
Using Agilent Mashunter Workstation Data Acquisition software to collect Data, and performing Qualitative Analysis by Agilent Masshunter Qualitative Analysis software; quantitative Analysis is carried out by Agilent QQ Quantitative Analysis (version 7.0.457.0) software; statistical Ttest analysis was performed using the software Graphpad Prism 7.0.
5. Results and discussion
The serum inflammatory factors (IL-1. beta. and TNF-. alpha.) were measured by ELISA in mice of CON group and LPS group. The results are shown in FIG. 5, and show that the level of proinflammatory cytokines (IL-1. beta. and TNF-. alpha.) is significantly increased in the serum of mice in LPS group (p <0.001) compared to CON group.
The optimization method is applied to the measurement of alpha-hydroxy ceramide in the serum of CON group mice (n ═ 3) and LPS group mice (n ═ 3). Analyzing the change of ceramide content in mice of CON group and LPS group, and discussing the pathogenesis of sepsis. The changes in the serum concentration of alpha-hydroxyceramide in mice of CON group and LPS group are shown in FIG. 6. The average Cer (d18:1/24:1(2OH)) concentration in LPS group was 10.83ng/mL, which was significantly higher than 6.65ng/mL (p <0.0001) in CON group. Serum Cer (d18:1/16:0(2OH)) and Cer (d18:1/20:0(2OH)) levels in mice in the LPS group were also significantly higher than those in the CON group (p < 0.05). The changes in the serum concentrations of alpha-hydroxyceramide in mice in the CON group and LPS group are shown in FIG. 6, and the LPS group showed an overall upward trend compared to the CON group, consistent with the results reported previously (Delogu G, Famularo G, Amati F et al. central associations in therapeutic properties: A porous marker of multiple organ dysfunction syndrome. CRITICAL CARE MEDICINE 27(11),2413-2417 (1999)), indicating that the onset and treatment of sepsis may be related to the metabolism of alpha-hydroxyceramide. In contrast, the differences between Cer (d18:1/18:0(2OH)), Cer (d18:1/18:1(2OH)) and Cer (d18:1/22:0(2OH)) in the LPS group and the control group are not statistically significant, and may be caused by the small number of samples and the large differences in the groups.
Example 5 verification of the methodology of the detection method of the invention:
1. selectivity of the process
Interference of the "5% BSA substituted matrix" with the internal standard Cer (d18:1-d7/16:0) was observed by comparing the chromatograms of 6 "5% BSA substituted matrix" double blank samples (without internal standard and alpha-hydroxyceramide mixed standard) and single blank samples (with internal standard, without alpha-hydroxyceramide standard). The effect of "5% BSA surrogate matrix" on the alpha-hydroxyceramide to be tested was observed by comparing the chromatograms of 6 "5% BSA surrogate matrix" double blank samples (without internal standard and alpha-hydroxyceramide standard) and single blank samples (without internal standard, with alpha-hydroxyceramide mixed standard). The serum sample was pretreated by the serum treatment method described in the present invention. If the 5% BSA instead of the matrix is not interfered by the internal standard and the alpha-hydroxy ceramide to be detected, the method has good selectivity and specificity.
2. Residue(s) is (are)
The residue was estimated by a method of sequentially injecting a blank sample, a high concentration sample, and a blank sample. The residue in the blank sample after the high concentration sample should not exceed 20% of the lower limit of quantitation and not exceed 5% of the internal standard.
3. Lower limit of quantitation (LOQ)
The lower limit of quantification refers to the lowest concentration that can be reliably quantified when the method detects a sample, generally refers to the lowest point of a standard curve, has a signal-to-noise ratio (S/N) ≧ 10, and has acceptable precision and accuracy. And continuously carrying out parallel analysis on the lowest point of the standard curve for 5 times within the same day, and calculating the accuracy and precision, wherein the precision of the lower limit of the quantification is less than 20%, and the accuracy is 80-120%.
4. Preparation of alpha-hydroxy ceramide standard curve
Preparation of alpha-hydroxyceramide standard curve series stock solution: precisely sucking each substance of alpha-hydroxyceramide with a proper volume of 1.0mg/mL, adding methanol with a certain volume, uniformly mixing by vortex, preparing a mixed standard solution with 100 mu g/mL, and diluting by methanol step by step into a series of concentrations, wherein 8 mass concentrations are respectively 62.5ng/mL, 125ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, 4000ng/mL and 8000 ng/mL.
Preparation of alpha-hydroxyceramide standard curve series working solution: firstly, precisely sucking 50 mu L of each alpha-hydroxyceramide standard curve series stock solution, then adding 950 mu L of 5% BSA to replace a substrate, uniformly mixing by vortex to prepare an alpha-hydroxyceramide standard curve series working solution, wherein 8 mass concentrations are respectively 3.125ng/mL, 6.25ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 200ng/mL and 400ng/mL, finally, processing and analyzing according to the method of the invention, the concentration of ceramide is taken as an abscissa, the peak area ratio of ceramide to an internal standard Cer (d18:1-d7/16:0) is taken as an ordinate to obtain a linear regression equation, and the weighting coefficient is 1/X.
5. Preparation of alpha-hydroxy ceramide Quality Control (QC) samples
According to the method of the invention, low, medium and high concentration QC samples are prepared by replacing the matrix with 5% BSA, wherein the concentrations are respectively low concentration (LQC) at 9.375ng/mL, medium concentration (MQC) at 150ng/mL and high concentration (HQC) at 300ng/mL, and the samples are used for subsequent methodological verification experiments.
6. Precision and accuracy of alpha-hydroxyceramide
Precision and accuracy were examined by QC samples at low, medium and high three concentrations (LQC, MQC, HQC). The accuracy is the consistency between the measured value of the mass concentration of the substance to be measured and the known real value of the measured value under three concentrations (LQC, MQC and HQC), and is usually represented by Relative Error (RE), the numerical value is between 85% and 115%, and the RE value of the lower limit of quantification is between 80% and 120%. Precision refers to the consistency of multiple test results among samples of the same lot at three concentrations (LQC, MQC), usually expressed as Relative Standard Deviation (RSD), and the RSD value should be less than 15% and the RSD value at the lower limit of quantitation should be less than 20%. The precision and accuracy within a day were evaluated by parallel detection 6 times of QC samples at three concentrations (LQC, MQC, HQC) within the same day. Intra-day precision was assessed by performing 5 replicates of QC samples at three concentrations (LQC, MQC, HQC) per day for three consecutive days.
7. Extraction recovery and matrix effect of alpha-hydroxy ceramide
In order to evaluate the extraction recovery rate of the sample, preparing QC samples with low, medium and high concentrations (LQC, MQC and HQC) by using 5% BSA to replace a matrix before the sample is extracted, performing sample injection analysis after pretreatment to obtain a peak area (A) of a standard before the sample is extracted, and performing parallel detection for three times; after a 5% BSA (bovine serum albumin) substitute matrix sample is pretreated, a standard solution is added to prepare a solution with consistent QC sample concentration of low, medium and high concentrations (LQC, MQC and HQC), sample injection analysis is carried out, a standard added peak area (B) is obtained after the sample is extracted, and parallel detection is carried out for three times; and taking the mixed standard substance methanol solution, preparing a solution with the same concentration as the QC sample, carrying out sample injection analysis to obtain a response value (C) of the compound to be detected, and carrying out parallel detection for 3 times. Calculation formula of extraction recovery rate and matrix effect: the extraction recovery rate is A/B multiplied by 100 percent; the matrix effect is B/C × 100%. The extraction recovery rate should be more than 50%, and the matrix effect should be between 80-120%.
Results and discussion
1. Selectivity of the process
The method for pretreating the serum sample is characterized in that a '5% BSA (bovine serum albumin) alternative matrix' double blank sample (without an internal standard and a mixed ceramide standard), a single blank sample (with an internal standard and without a ceramide standard) and a single blank sample (without an internal standard and with a ceramide standard) are prepared respectively according to the method for pretreating the serum sample, and the results show that the '5% BSA alternative matrix' has no obvious interference peaks at the peaks of the internal standard and each substance to be detected, so that the method has better selectivity.
2. Residue is remained
The residual effect in the blank after each high concentration of alpha-hydroxyceramide sample was no more than 20% of the lower quantitative effect, no more than 5% of the internal standard. The results show that the process is not affected by residual effects.
3. Lower limit of quantification
The results of the lower limit of the quantification are shown in the table, the precision of the lower limit of the quantification of the alpha-hydroxyceramide is less than 20 percent, the accuracy is between 80 and 120 percent, and the results are in an acceptable range and meet the requirements of methodology verification.
4. Standard curve of alpha-hydroxy ceramide
The ratio of the peak area of the alpha-hydroxyceramide to the area of the IS peak IS used AS a vertical coordinate, the concentration of the alpha-hydroxyceramide IS used AS a horizontal coordinate, a linear regression equation IS constructed, linear equations, linear correlation coefficients (R2) and weights of all substances of the alpha-hydroxyceramide are shown in the table, all substances of the AS show good linear relations in a detection range, and the linear correlation coefficients (R2) are all larger than 0.99.
TABLE 8 Linear equation, Linear correlation coefficient and weight for each substance of alpha-hydroxyceramide
5. Precision and accuracy
TABLE 9 detection precision and accuracy of alpha-hydroxyceramide
6. Extraction recovery and matrix compound name
TABLE 10 recovery of the extraction of each substance of alpha-hydroxyceramide
Mean: an average value; SD: standard deviation; RSD: relative Standard Deviation (SD)
Therefore, the methodological verification results of the detection method meet the requirements of biological analysis method guidelines.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The alpha-hydroxyceramide detection method based on the ultra performance liquid chromatography-mass spectrometry combined technology is characterized by comprising the following steps of:
(1) mixing the serum and the extracting solution, performing first standing, centrifuging to obtain the lowest layer to obtain a material 1, mixing the material 1 with an internal standard solution, volatilizing to obtain a material 2, mixing the material 2 with a double solvent, performing second standing, and taking the supernatant to obtain a material 3;
(2) carrying out ultra-high performance liquid chromatography tandem mass spectrometry on the material 3 obtained in the step (1);
the alpha-hydroxyceramides are Cer (d18:1/16:0(2OH)), Cer (d18:1/18:0(2OH)), Cer (d18:1/20:0(2OH)), Cer (d18:1/22:0(2OH)), Cer (d18:1/18:1(2OH)) and Cer (d18:1/24:1(2 OH)).
2. The method for detecting alpha-hydroxyceramide based on ultra-high performance liquid chromatography-mass spectrometry as claimed in claim 1, wherein the volume ratio of the serum to the extracting solution in the step (1) is 3-5: 14-18; the extracting solution is chloroform and methanol; the volume ratio of the chloroform to the methanol is 2-6: 1-3.
3. The method for detecting alpha-hydroxyceramide according to claim 1, wherein the step (1)
The first standing time and the second standing time are independent for 8-12 min; the centrifugation time is 13-17 min; the centrifugation temperature is 3-5 ℃; the centrifugal rotating speed is 12000-14000 rpm.
4. The method for detecting alpha-hydroxyceramide based on ultra-high performance liquid chromatography-mass spectrometry combined technology as claimed in claim 1, wherein the volume ratio of the material 1 to the internal standard solution in the step (1) is 80-100: 8-12; the internal standard solution is a Cer (d18:1-d7/16:0) solution with the concentration of 90-110 ng/mL.
5. The detection method of alpha-hydroxyceramide based on ultra performance liquid chromatography-mass spectrometry as claimed in claim 1, wherein the volume ratio of the material 1 to the double solvent in the step (1) is 8-10: 4-6; the double solvent is isopropanol, acetonitrile and water; the volume ratio of the isopropanol to the acetonitrile to the water is 2-6: 1-3.
6. The method for detecting alpha-hydroxyceramide according to claim 1, wherein the mass spectrum in the ultra high performance liquid chromatography-mass spectrometry in the step (2) adopts a multi-reaction monitoring mode and a positive ion mode for analysis: the ion source is an electrospray ion source, the temperature of the dry gas is 250-350 ℃, the flow rate of the dry gas is 9-13L/min, the pressure of the atomization gas is 18-22 psi, the voltage of a capillary tube is 3800-4200V, the temperature of the sheath gas is 380-420 ℃, the flow rate of the sheath gas is 9-13L/min, and the voltage of a nozzle is 800-1200V.
7. The method for detecting alpha-hydroxyceramide based on the combined ultra performance liquid chromatography-mass spectrometry technology according to claim 1, wherein the chromatographic column of the ultra performance liquid chromatography in the ultra performance liquid chromatography tandem mass spectrometry of the step (2) is a BEH C18 chromatographic column; the mobile phase A is acetonitrile and water, and the mobile phase B is isopropanol and acetonitrile.
8. The method for detecting the alpha-hydroxyceramide based on the ultra-high performance liquid chromatography-mass spectrometry combined technology as claimed in claim 7, wherein the volume ratio of the acetonitrile to the water in the phase A is 5-7: 3-5; the volume ratio of the isopropanol to the acetonitrile in the phase B is 8-10: 1-2.
9. The method for detecting alpha-hydroxyceramide according to claim 7, wherein the phase A and the phase B independently contain 8-12 mM ammonium acetate.
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CN112180018A (en) * | 2019-11-20 | 2021-01-05 | 南京品生医学检验实验室有限公司 | Liquid chromatography-mass spectrometry method for detecting 13 ceramides in plasma |
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