A chromatographic feature prediction and qualitative analysis method for ceramide based on LC-MS / MS

Abstract

The present invention relates to the field of biological detection technology, and provides a method for predicting and qualitatively determining the chromatographic characteristics of ceramides based on LC-MS / MS. The method comprises the following steps: dividing ceramides into three major categories according to their structures; constructing a quadratic equation of retention time and XlogP for each major category based on existing SphCer standards, and obtaining theoretical equations for the three major categories of sphingosine ceramides; the retention time of ceramides in the sample to be tested can be predicted based on the theoretical equation, and a qualitative study of the ceramides in the sample to be tested can be performed. This method can summarize a complete and accurate RT prediction curve for SphCer that can be successfully qualitatively verified under the MS / MS theoretical database. The qualitative method based on the prediction model of existing standard information and target molecule XlogP, combined with MS / MS information to verify the structure, has been proven to be highly feasible for sphingosine ceramides.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a ceramide chromatographic feature prediction and qualitative method based on LC-MS / MS. Background Art

[0002] Ceramide (Cer) is a class of lipid bioregulatory molecules classified as sphingolipids. It is composed of a long-chain base (LCB) linked to a fatty acid (FA) via an amide bond. Sphingosine (d18:1) ceramides (Sph Cer) are the most common type of ceramide in mammals. The structure of Sph Cer consists of a molecule of d18:1 sphingosine (a long-chain base) condensed with a molecule of a saturated / unsaturated / hydroxy-substituted / non-hydroxy-substituted fatty acid amide. Clearly, the structure of Sph Cer exhibits a strong regularity. The structure of Sph Cer is strongly correlated with its lipid-water partition coefficient (LogP), and different LogPs significantly affect the chromatographic retention behavior of Sph Cer. In conventional qualitative methods, the experiment primarily relies on the peak elution time of the target compound in the standard and sample, supplemented by qualitative comparison of MS / MS information. For target molecules that lack standards, such as a series of lipid small molecules represented by Sph Cer, reasonable inference of their peak time will effectively compensate for the qualitative defects caused by the lack of standards and reduce experimental costs.

[0003] The concept of lipidomics, first proposed in 2003, primarily refers to the systematic analysis of the overall lipid composition and interactions of a target, using technical means to understand lipid structure and function, identify key lipid biomarkers, and thus reveal the mechanisms of lipid interaction within metabolic networks. During the 20 years of lipidomics' development, a significant number of studies have focused on ceramides. However, the intense interest in their biological activities and applications is not matched by the lack of mature technology for determining the content and species of ceramides in living organisms. Traditional LC-MS / MS qualitative and quantitative methods, which rely heavily on comparison with standards, are unable to meet the current requirements for bioanalysis of a range of lipid molecules, particularly ceramides. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for predicting and qualitatively analyzing the chromatographic characteristics of ceramides based on LC-MS / MS. The method of the present invention requires less ceramide standards and is low in cost, which can make up for the shortcomings of inaccurate metabolite qualitative analysis caused by the lack of standards in current metabolite analysis.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A method for predicting and qualitatively analyzing ceramide chromatographic characteristics based on LC-MS / MS, comprising the following steps:

[0007] (1) According to the structure of ceramide, the sphingosine ceramides in the ceramide database are divided into three categories: saturated non-hydroxy Sph Cer; unsaturated hydroxy Sph Cer; α-hydroxy substituted Sph Cer;

[0008] (2) Using the LogP online calculation software XlogP3, enhanced atomic XlogP calculations were performed on the molecular formula of each sphingosine ceramide molecule in the database;

[0009] (3) performing LC-MS / MS analysis based on existing Sph Cer standards to obtain the retention time of each standard;

[0010] (4) The Sph Cer standards were divided into a test set and a validation set. A quadratic equation of retention time and XlogP was constructed for the Sph Cer standards in the test set according to each major category. The quadratic equation was verified using the retention time and XlogP of the Sph Cer standards in the validation set to obtain the theoretical equations for the three major categories of sphingosine ceramides.

[0011] (5) The sample to be tested is pretreated and then subjected to LC-MS / MS analysis. The XlogP of the target ceramide is substituted into the theoretical equation in step (4) to predict the retention time of the target ceramide. The retention time of the target ceramide is compared with the actual retention time of the various ceramides in the sample to be tested, and the various ceramides in the sample to be tested can be qualitatively identified.

[0012] Preferably, the chromatographic parameters in the LC-MS / MS analysis are:

[0013] Chromatographic column: BEH C18 column;

[0014] Flow rate: 0.2-0.3 mL / min;

[0015] Mobile phase: Phase A is an acetonitrile-water mixture containing 10 mM ammonium acetate, with a volume ratio of acetonitrile to water of 6:3-5; Phase B is an isopropanol-acetonitrile mixture containing 10 mM ammonium acetate, with a mass ratio of isopropanol to acetonitrile of 5-12:1;

[0016] Gradient elution program: 0-1.5 min, 68% phase A and 32% phase B; 1.5-15.5 min, 68% phase A and 32% phase B; 15.5-15.6 min, 15% phase A and 85% phase B; 15.6-18 min, 3% phase A and 97% phase B; 18-18.1 min, 3% phase A and 97% phase B; 18.1-20 min, 68% phase A and 32% phase B.

[0017] Preferably, the mass spectrometry parameters in the LC-MS / MS are:

[0018] electrospray ion source;

[0019] Capillary temperature: 320℃;

[0020] Spray voltage: 3.8kV;

[0021] Maximum spray current: 100μA;

[0022] Sheath gas flow rate: 30L / min;

[0023] Auxiliary gas flow: 5L / min;

[0024] Probe heating temperature: 300℃.

[0025] Preferably, the mass spectrometer adopts Full Scan mode and Full MS-ddMS2 mode.

[0026] Preferably, the theoretical equations of the three major types of sphingosine ceramides in step (4) are:

[0027] Saturated non-hydroxyl Sph Cer: y = -0.0576x 2 +2.4467x-8.428;

[0028] S-configured α-hydroxyl substituted Sph Cer: y = -0.0606x 2 +2.4962x-8.5615;

[0029] R-configured α-hydroxy substituted Sph Cer: y = -0.0587x 2 +2.4185x-7.5653;

[0030] Unsaturated hydroxyl Sph Cer: y = -0.0484x 2 +2.1378x-6.0519;

[0031] Where y is the retention time and x is XlogP.

[0032] Preferably, the sample to be tested in step (5) includes serum.

[0033] Preferably, the pretreatment method of the test sample is: after the serum and the extract are mixed and incubated, potassium hydroxide solution is added for hydrolysis, water is added for neutralization, and centrifugation is performed to obtain an upper extract, and the upper extract is blown dry and redissolved.

[0034] Preferably, the extract is a mixture of methyl tert-butyl ether and methanol, and the volume ratio of methyl tert-butyl ether to methanol is 5 to 6:1;

[0035] The volume ratio of the serum to the extract is 1:8-15;

[0036] The incubation temperature is 40-50° C., and the incubation time is 1-3 hours.

[0037] Preferably, the concentration of the solute in the potassium hydroxide solution is 0.5 to 2 M, and the mass ratio of the serum to the potassium hydroxide solution is 4:1 to 4;

[0038] The hydrolysis temperature is 35-40° C., and the hydrolysis time is 1-3 hours.

[0039] Preferably, the re-dissolving solvent is a mixture of isopropanol and methanol, the volume ratio of isopropanol to methanol is 1:1-2, and the volume ratio of the upper layer extract to the re-dissolving solvent is 8-15:1.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The method of the present invention is a reliable and scientific qualitative method for Sph Cer. This method uses the molecular descriptor XlogP, which is directly related to the molecular structure information, to predict RT, and summarizes a complete, accurate, and successfully qualitative RT prediction curve for Sph Cer under the verification of the MS / MS theoretical database. This method is a universal research method. It can be applied to the separation and identification of a variety of small molecule homologues. The qualitative method based on the existing standard information and the prediction model of the target molecule XlogP, combined with MS / MS information to verify the structure, has been proven to have high feasibility on sphingosine ceramide objects. The method of the present invention requires less ceramide standards and is low in cost, which can make up for the serious deficiency of inaccurate metabolite qualitative analysis due to the lack of standards in current metabolite analysis. This method is expected to be expanded to more lipidomics research contents in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the extracted ion chromatogram of ceramide standard;

[0043] Figure 2 This is the XlogP-RT graph of the standard non-hydroxyl saturated ceramide;

[0044] Figure 3 This is the verification diagram of the ceramide parent-daughter ion pair;

[0045] Figure 4 This is a diagram showing the effect of serum matrix on RT. DETAILED DESCRIPTION

[0046] The present invention provides a method for predicting and qualitatively analyzing ceramide chromatographic characteristics based on LC-MS / MS, comprising the following steps:

[0047] (1) According to the structure of ceramide, the sphingosine ceramides in the ceramide database are divided into three categories: saturated non-hydroxy Sph Cer; unsaturated hydroxy Sph Cer; α-hydroxy substituted Sph Cer;

[0048] (2) Using the LogP online calculation software XlogP3, enhanced atomic XlogP calculations were performed on the molecular formula of each sphingosine ceramide molecule in the database;

[0049] (3) performing LC-MS / MS analysis based on existing Sph Cer standards to obtain the retention time of each standard;

[0050] (4) The Sph Cer standards were divided into a test set and a validation set. A quadratic equation of retention time and XlogP was constructed for the Sph Cer standards in the test set according to each major category. The quadratic equation was verified using the retention time and XlogP of the Sph Cer standards in the validation set to obtain the theoretical equations for the three major categories of sphingosine ceramides.

[0051] (5) The sample to be tested is pretreated and then subjected to LC-MS / MS analysis. The XlogP of the target ceramide is substituted into the theoretical equation in step (4) to predict the retention time of the target ceramide. The retention time of the target ceramide is compared with the actual retention time of the various ceramides in the sample to be tested, and the various ceramides in the sample to be tested can be qualitatively identified.

[0052] In the present invention, sphingosine ceramides in the ceramide database are first divided into three categories according to the structure of ceramide: saturated non-hydroxy Sph Cer; unsaturated hydroxy Sph Cer; α-hydroxy substituted Sph Cer; the ceramide database includes lipidmaps (https: / / www.lipidmaps.org / ), lipidbank (https: / / lipidbank.jp / ), lipidblast (https: / / fiehnlab.ucdavis.edu / projects / LipidBlast), scifinder (https: / / scifinder.cas.org / scifinder / view / scifinder / scifinderExplore.jsf), and chemspider (https: / / www.chemspider.com / StructureSearch.aspx), and Sph CeR that is not involved in the database but theoretically exists is supplemented to obtain 115 Sph CeR, which are divided into three categories according to their structure, including 34 saturated non-hydroxy Sph CeR; 27 unsaturated Sph CeR; 54 α-hydroxy substituted Sph CeR. Cer (containing 27 R configurations and 27 S configurations).

[0053] In the present invention, XlogP calculations were performed on the molecular formula of each sphingosine ceramide molecule in the database using the online LogP calculation software XlogP3 (http: / / www.sioc-ccbg.ac.cn / ?p=42&software=xlogp3). The online LogP calculation software XlogP3 (http: / / www.sioc-ccbg.ac.cn / ?p=42&software=xlogp3) is a modification of the atomic LogP system and a fast table lookup technique. This hybrid algorithm considers the state of each atom in the molecule and characterizes the contribution of atomic characteristics to LogP.

[0054] In the present invention, the structural regularity of the same type of ceramide is extremely strong, and its XlogP is also affected by the different effects of the fatty acid carbon chain. Therefore, it is reasonable to infer that the RT of ceramide under fixed chromatographic conditions can be calculated. In traditional theory, the retention time of ceramide has a certain linear relationship with its structure (such as the carbon number of the fatty acid chain and the degree of unsaturation), that is, when the degree of unsaturation is the same, the retention time increases with the increase of the carbon number of the fatty acid chain; when the carbon number of the fatty acid chain is the same, the retention time decreases with the increase of the unsaturation. However, structural information such as the position of the double bond and the hydroxyl substitution site cannot be expressed by the carbon number. Therefore, it is necessary to comprehensively consider the impact of different functional groups on the overall molecule. XlogP is a modification of the atomic LogP system and is a fast table lookup technology. This hybrid algorithm takes into account the state of each atom in the molecule and characterizes the contribution of atomic characteristics to LogP. Therefore, the significance of different molecular structures can be accurately located.

[0055] In the present invention, LC-MS / MS analysis is performed based on existing Sph Cer standards to obtain the retention time of each standard. The Sph Cer standard adopts an existing ceramide standard. The ceramide standard is prepared into a standard stock solution with a concentration of 1.0 mg / mL using a methanol solution. The standard stock solution is then diluted with a methanol solution to obtain a standard working solution of 100 ng / mL for LC-MS / MS analysis.

[0056] In the present invention, the chromatographic parameters in the LC-MS / MS analysis are:

[0057] Chromatographic column: BEH C18 column (2.1 mm × 50 mm, 2.5 μm, Waters, Milford, MA, USA);

[0058] Flow rate: 0.2-0.3 mL / min, preferably 0.22-0.28 mL / min, more preferably 0.24-0.26 mL / min;

[0059] Mobile phase: Phase A is an acetonitrile-water mixture containing 10 mM ammonium acetate, with a volume ratio of acetonitrile to water of 6:3-5, preferably 6:3.5-4.5, and more preferably 6:3.8-4.2; Phase B is an isopropanol-acetonitrile mixture containing 10 mM ammonium acetate, with a mass ratio of isopropanol to acetonitrile of 5-12:1, preferably 6-10:1, and more preferably 7-9:1;

[0060] Gradient elution program: 0-1.5 min, 68% phase A and 32% phase B; 1.5-15.5 min, 68% phase A and 32% phase B; 15.5-15.6 min, 15% phase A and 85% phase B; 15.6-18 min, 3% phase A and 97% phase B; 18-18.1 min, 3% phase A and 97% phase B; 18.1-20 min, 68% phase A and 32% phase B.

[0061] In the present invention, the mass spectrometry parameters in the LC-MS / MS are:

[0062] electrospray ion source;

[0063] Capillary temperature: 320℃;

[0064] Spray voltage: 3.8kV;

[0065] Maximum spray current: 100μA;

[0066] Sheath gas flow rate: 30L / min;

[0067] Auxiliary gas flow: 5L / min;

[0068] Probe heating temperature: 300℃.

[0069] In the present invention, the mass spectrometry adopts Full Scan mode and Full MS-ddMS2 mode.

[0070] In the present invention, Sph Cer standards are divided into a test set and a validation set. A quadratic equation of retention time and XlogP is constructed for the Sph Cer standards in the test set according to each major category. The quadratic equation is verified using the retention time and XlogP of the Sph Cer standards in the validation set to obtain the theoretical equations for the three major categories of sphingosine ceramides.

[0071] In the present invention, the theoretical equations of the three major types of sphingosine ceramides in step (4) are:

[0072] Saturated non-hydroxyl Sph Cer: y = -0.0576x 2 +2.4467x-8.428;

[0073] S-configured α-hydroxyl substituted Sph Cer: y = -0.0606x 2 +2.4962x-8.5615;

[0074] R-configured α-hydroxy substituted Sph Cer: y = -0.0587x 2 +2.4185x-7.5653;

[0075] Unsaturated hydroxyl Sph Cer: y = -0.0484x 2 +2.1378x-6.0519;

[0076] Where y is the retention time and x is XlogP.

[0077] In the present invention, the sample to be tested is pretreated and then subjected to LC-MS / MS analysis. The XlogP of the target ceramide is substituted into the theoretical equation in step (4) to predict the retention time of the target ceramide. The retention time of the target ceramide is compared with the actual retention time of multiple ceramides in the sample to be tested, so that multiple ceramides in the sample to be tested can be qualitatively identified.

[0078] In the present invention, the sample to be tested preferably includes serum.

[0079] In the present invention, the pretreatment method of the sample to be tested is: after the serum and the extract are mixed and incubated, potassium hydroxide solution is added for hydrolysis, neutralization is performed, water is added and centrifuged to obtain an upper extract, the upper extract is blown dry and redissolved; the extract is a mixture of methyl tert-butyl ether and methanol, and the volume ratio of methyl tert-butyl ether to methanol is 5 to 6:1, preferably 5.2 to 5.8:1, and more preferably 5.4 to 5.6:1; the volume ratio of serum to extract is 1:8 to 15, preferably 1:9 to 14, and more preferably 1:10 to 12; the incubation temperature is 40 to 50°C, preferably 42 to 48°C, and more preferably 44 to 46°C; the incubation time is 1 to 3 hours, preferably 1.5 to 2.5 hours, and more preferably 2 hours.

[0080] In the present invention, the concentration of the solute in the potassium hydroxide solution is 0.5-2M, preferably 0.8-1.8M, and more preferably 1-1.5M; the mass ratio of the serum to the potassium hydroxide solution is 4:1-4, preferably 4:2-3; the hydrolysis temperature is 35-40°C, preferably 36-39°C, and more preferably 37-38°C; the hydrolysis time is 1-3h, preferably 1.5-2.5h, and more preferably 2h.

[0081] In the present invention, the neutralization adopts acetic acid with a volume percentage concentration of 15-25%, and the volume percentage concentration of acetic acid is preferably 17-23%, more preferably 19-21%; the amount of water added after the neutralization is 1-3 times the volume of the serum, preferably 1.5-2.5 times, more preferably 2 times; the centrifugal speed is 3000-4000 rpm, preferably 3200-3800 rpm, more preferably 3400-3600 rpm; the centrifugal time is 20-30 min, preferably 22-28 min, more preferably 24-26 min.

[0082] In the present invention, the reconstitution solvent is a mixture of isopropanol and methanol, and the volume ratio of isopropanol to methanol is 1:1-2, preferably 1:1.2-1.8, and more preferably 1:1.4-1.6; the volume ratio of the upper layer extract to the reconstitution solvent is 8-15:1, preferably 9-14:1, and more preferably 10-12:1.

[0083] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0084] Example 1

[0085] This example provides a method for predicting and qualitatively analyzing ceramide chromatographic characteristics based on LC-MS / MS, comprising the following steps:

[0086] (1) The ceramide databases were searched: lipidmaps (https: / / www.lipidmaps.org / ), lipidbank (https: / / lipidbank.jp / ), lipidblast (https: / / fiehnlab.ucdavis.edu / projects / Lipid Blast), scifinder (https: / / scifinder.cas.org / scifinder / view / scifinder / scifinderExplore.jsf), and chemspider (https: / / www.chemspider.com / StructureSearch.aspx). Sph Cers that were not included in the database but theoretically existed were supplemented, and 115 Sph Cers were obtained. The 115 Sph Cers were divided into three categories according to their structures, including 34 saturated non-hydroxyl Sph Cers, 27 unsaturated Sph Cers, and 54 α-hydroxyl substituted Sph Cers (including 27 R configurations and 27 S configurations).

[0087] (2) XlogP calculation was performed on the molecular formula of sphingosine ceramide obtained above using the LogP online calculation software XlogP3 (http: / / www.sioc-ccbg.ac.cn / ?p=42&software=xlogp3).

[0088] (3) LC-MS / MS analysis was performed on 29 existing Sph Cer standards. The 29 existing ceramide standards were dissolved in methanol to prepare a standard stock solution with a concentration of 1.0 mg / mL. The standard stock solution was then diluted with methanol to obtain a standard working solution with a concentration of 100 ng / mL. Information on the standards used is shown in Table 1:

[0089] Table 1. Information of various ceramide standards

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] The chromatographic parameters in the LC-MS / MS analysis were:

[0097] Chromatographic column: BEH C18 column (2.1 mm × 50 mm, 2.5 μm, Waters, Milford, MA, USA); flow rate: 0.26 mL / min; mobile phase: Phase A: acetonitrile-water mixture containing 10 mM ammonium acetate, acetonitrile:water volume ratio 6:4; Phase B: isopropanol-acetonitrile mixture containing 10 mM ammonium acetate, isopropanol:acetonitrile mass ratio 9:1. Gradient elution was used; the chromatographic mobile phase elution gradient is shown in Table 2:

[0098] Table 2. Elution gradient

[0099]

[0100] The mass spectrometry parameters in LC-MS / MS are:

[0101] The full scan mode was used to obtain the peak time and separation of each parent ion in the standard mixed solution.

[0102] The theoretical parent ion information was included in the inclusion list, and the secondary scanning mode (Full MS-ddMS2) of positive and negative ion modes was used to obtain the parent-daughter ion pair information to accurately identify the Sph Cer molecule.

[0103] In Full Scan mode, positive ion mode was used for acquisition, electrospray ion source; capillary temperature: 320°C; spray voltage: 3.8 kV; maximum spray current: 100 μA; sheath gas flow: 30 L / min; auxiliary gas flow: 5 L / min; probe heating temperature: 300°C.

[0104] In Full MS-ddMS2 mode, acquisition was performed in positive and negative ion modes, using an electrospray ion source; capillary temperature: 320°C; spray voltage: 3.8 kV; maximum spray current: 100 μA; sheath gas flow: 30 L / min; auxiliary gas flow: 5 L / min; and probe heating temperature: 300°C. For inclusions, refer to Table 1.

[0105] 29 Sph Cer standard working solutions were mixed and analyzed by LC-MS / MS. The standard [M+H] was extracted using ThermoFisher QualBrowser software under Full Scan conditions. + Ion current. Mass accuracy deviation is 5ppm. Extracted ion current is shown in Figure 1 .Depend on Figure 1 It can be seen that the method of the present invention has a strong separation ability for the three major types of ceramide standards and is suitable for the detection and analysis of ceramide.

[0106] (4) According to the XlogP-RT plot of the non-hydroxy saturated ceramide with the largest number of standard products, it can be seen that ( Figure 2 ), the RT of ceramide conforms to the trend of a quadratic equation. Therefore, short, medium, and long carbon chain ceramides among the standard ceramides were selected as the test set as the source for the XlogP-RT equation estimation, and the standards used for estimation were prepared into a mixed standard solution of 100 ng / mL. The chromatographic and mass spectrometric parameters in step (3) were applied, and the standard [M+H] was extracted under Full Scan conditions using ThermoFisher QualBrowser software analysis. + Ion current. The list of standards used in the theoretical equation calculation, RT, and theoretical RT equation are shown in Table 3 (where y = RT and x = XlogP):

[0107] Table 3. Theoretical equations of standards

[0108]

[0109]

[0110] *The underlined ceramides in the compound column are theoretical ceramides and are not standard substances.

[0111] The remaining 17 ceramide standards were used as a validation set to verify the accuracy of the above RT equation. The remaining 17 ceramide standards were prepared into a 100 ng / mL mixed standard solution and the chromatographic and mass spectrometric parameters in step (3) were applied. The standard [M+H] was extracted under Full Scan conditions and analyzed with ThermoFisher QualBrowser software. + Ion current, record the peak time of each standard and compare it with the theoretical value. The results are shown in Table 4:

[0112] Table 4. Analysis of ceramide standards in the validation set

[0113]

[0114]

[0115] As shown in Table 4, the absolute error in the RT inferred by this method for the aforementioned Sph Cer is less than 0.3 min. With the exception of Cer (d18:1 / 2:0), the RT deviations for all other substances are within 0.2 min. The RT deviations for 14 (82.4%) ceramides are within 0.1 min. This demonstrates the feasibility of the RT prediction model of the present method in practical applications.

[0116] (5) The parent-daughter ion pair was verified on the ceramide standard. This method obtained the fragmentation pattern of Sph Cer under positive and negative ions. Currently, most quantitative methods for Sph Cer use a single positive ion mode, and qualitative methods use a single negative ion mode. This method adopts both positive and negative ion modes to qualitatively characterize the target metabolite from multiple angles. Among them, considering the possibility of hydrogen rearrangement, the fragment ions of -H and -3H should be considered in the negative ion mode and classified as the same daughter ion. Figure 3 Shown is the MS / MS spectrum of a representative ceramide standard, Cer (d18:1 / 18:0), and its corresponding fragmentation pattern. In positive ion mode, focus on the characteristic daughter ions with mass-to-charge ratios of 264, 252, and 282. In negative ion mode, focus on the characteristic daughter ions of parent ions -298, -239, -283, and -326. These seven characteristic ions serve as the basis for MS / MS qualitative analysis, accurately confirming that the parent ions captured within the target RT range are the target Sph Cer. Based on the fragmentation pattern described above, after spectral comparison, all ceramide parent ions captured within the theoretical RT range exhibit the characteristic ions shown above. Therefore, this RT estimation method offers reliable qualitative prediction accuracy from a chromatographic perspective and can be used for qualitative analysis of Sph Cer in real samples.

[0117] Example 2

[0118] This example provides an application of a chromatographic feature prediction and qualitative method for ceramides based on LC-MS / MS in rat serum, comprising the following steps:

[0119] (1) Animal experiments and sample collection

[0120] Female SD rats, weighing 300 g and free of specific pathogens, were purchased from the Shanghai Southern Model Laboratory Animal Center (Shanghai, China) and housed under constant temperature for one week with a 12-hour day / night cycle. Food and water were provided. After the acclimatization period, blood was collected from the abdominal aorta. After 2 hours of refrigeration at 4°C, the blood was centrifuged at 3500 rpm for 15 minutes at 4°C. The supernatant serum was pooled from five rats and stored at −80°C until further experimentation.

[0121] (2) Serum pretreatment

[0122] Place 100 μL of rat serum thawed at 4°C in a glass centrifuge tube, add 1000 μL of extraction solution (V:MTBE:Methanol = 5.5:1), vortex for 30 seconds, and incubate at 48°C for 2 hours to fully extract lipids. Add 75 μL of 1 M potassium hydroxide and mix at 37°C for 2 hours to hydrolyze neutral lipids. Add 25 μL of 20% acetic acid to neutralize the potassium hydroxide. Add 200 μL of water to induce phase separation, vortex for 30 seconds, and centrifuge at 3500 rpm for 25 minutes. Remove 400 μL of the upper layer, air dry, and reconstitute with 40 μL of reconstitution solution (V:isopropanol:Methanol = 1:1). Transfer the supernatant into a vial and place it on the autosampler tray for analysis.

[0123] (3) Data processing

[0124] The LC-MS / MS parameters in Example 1 were applied, ThermoFisher Xcalibur software was used for data acquisition, and ThermoFisher QualBrowser software was used for qualitative analysis.

[0125] (4) Retention time (RT) deviation between actual sample and standard

[0126] To clarify the effect of serum matrix on the chromatographic retention behavior of metabolites, the RT difference between ceramides (Cer (d18:1 / 16:0), Cer (d18:1 / 18:0, Cer (d18:1 / 24:0)) that are present at high concentrations in actual samples and ceramide standard solutions was further compared to determine whether the serum matrix has an effect on RT. The comparison results are shown in Figure 4 ,Depend on Figure 4As can be seen, the RT of ceramide in the sample is close to that of the standard, with a delta RT of less than 0.1 min. Therefore, matrix interference on RT can be ignored, demonstrating that the RT of this chromatographic method for ceramide is stable and feasible.

[0127] Finally, 55 SphCer species were identified in rat serum (26 without commercial standards and 29 with commercial standards). The types of ceramides in serum are shown in Table 5, where the "Number of Characteristic Ions" column corresponds to the number of 7 characteristic ions described in the "Parent-Daughter Ion Pair Verification" section of step (5) of Example 1 after the parent ion is broken down within the theoretical RT range. As shown in Table 5, compared with the RT of SphCer in the actual sample, 32 SphCer species have |ΔRT|≤0.1min; 41 SphCer species have more than 4 MS / MS daughter ion qualitative information. This demonstrates that this method has good operability and accuracy in rat serum samples.

[0128] Table 5. Detection of ceramide in rat serum

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] As can be seen from the above examples, the present invention provides a method for predicting and qualitative analysis of ceramide chromatographic characteristics based on LC-MS / MS. The method of the present invention requires less ceramide standard and is low in cost, which can make up for the shortcomings of inaccurate metabolite qualitative analysis caused by the lack of standard substances in current metabolite analysis.

[0135] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for predicting and qualitatively analyzing ceramide chromatographic characteristics based on LC-MS / MS, characterized in that: The following steps are involved: (1) According to the structure of ceramide, the sphingosine ceramides in the ceramide database are divided into three categories: saturated non-hydroxy Sph Cer; unsaturated hydroxy Sph Cer; α-hydroxy substituted Sph Cer; (2) Use the LogP online calculation software XlogP3 to perform XlogP calculation on the molecular formula of each sphingosine ceramide molecule in the database; (3) Perform LC-MS / MS analysis based on the existing Sph Cer standards to obtain the retention time of each standard; (4) The Sph Cer standards were divided into a test set and a validation set. A quadratic equation of retention time and XlogP was constructed for the Sph Cer standards in the test set according to each major category. The quadratic equation was verified using the retention time and XlogP of the Sph Cer standards in the validation set to obtain the theoretical equations for the three major categories of sphingosine ceramides. (5) Pre-treating the sample to be tested, and then performing LC-MS / MS analysis, substituting the XlogP of the target ceramide into the theoretical equation in step (4) to predict the retention time of the target ceramide, and comparing it with the actual retention time of multiple ceramides in the sample to be tested, so as to qualitatively identify multiple ceramides in the sample to be tested; The chromatographic parameters in the LC-MS / MS analysis were: Chromatographic column: BEH C18 column; Flow rate: 0.2~0.3mL / min; Mobile phase: Phase A is an acetonitrile-water mixture containing 10 mM ammonium acetate, with a volume ratio of acetonitrile to water of 6:3-5; Phase B is an isopropanol-acetonitrile mixture containing 10 mM ammonium acetate, with a mass ratio of isopropanol to acetonitrile of 5-12:1; Gradient elution program: 0-1.5 min, 68% phase A and 32% phase B; 1.5-15.5 min, 68% phase A and 32% phase B; 15.5-15.6 min, 15% phase A and 85% phase B; 15.6-18 min, 3% phase A and 97% phase B; 18-18.1 min, 3% phase A and 97% phase B; 18.1-20 min, 68% phase A and 32% phase B; The mass spectrometry parameters in the LC-MS / MS are: electrospray ion source; Capillary temperature: 320℃; Spray voltage: 3.8 kV; Maximum spray current: 100μA; Sheath gas flow rate: 30 L / min; Auxiliary gas flow rate: 5 L / min; Probe heating temperature: 300°C; In step (5), the sample to be tested includes serum; The pretreatment method of the test sample is as follows: serum and extract are mixed and incubated, potassium hydroxide solution is added for hydrolysis, water is added for neutralization, and centrifugation is performed to obtain an upper extract, the upper extract is blown dry and redissolved; The extract is a mixture of methyl tert-butyl ether and methanol, and the volume ratio of methyl tert-butyl ether to methanol is 5-6:1; The volume ratio of the serum to the extract is 1:8-15; The incubation temperature is 40-50° C., and the incubation time is 1-3 hours.

2. The ceramide chromatographic feature prediction and qualitative analysis method based on LC-MS / MS according to claim 1, characterized in that: The mass spectrometry adopted Full Scan mode and Full MS-ddMS2 mode.

3. The ceramide chromatographic feature prediction and qualitative analysis method based on LC-MS / MS according to claim 1, characterized in that: The theoretical equations for the three major types of sphingosine ceramides in step (4) are: Saturated non-hydroxyl Sph Cer: y = -0.0576x 2 +2.4467x-8.428; S-configuration α-hydroxyl substituted Sph Cer: y = -0.0606x 2 +2.4962x-8.5615; R-configured α-hydroxy substituted Sph Cer: y = -0.0587x 2 +2.4185x-7.5653; Unsaturated hydroxyl Sph Cer: y = -0.0484x 2 +2.1378x-6.0519; Where y is the retention time and x is XlogP.

4. The ceramide chromatographic feature prediction and qualitative analysis method based on LC-MS / MS according to claim 1, characterized in that: The concentration of the solute in the potassium hydroxide solution is 0.5~2M, and the mass ratio of the serum to the potassium hydroxide solution is 4:1~4; The hydrolysis temperature is 35-40° C., and the hydrolysis time is 1-3 hours.

5. The ceramide chromatographic feature prediction and qualitative analysis method based on LC-MS / MS according to claim 1, characterized in that: The reconstitution solvent is a mixture of isopropanol and methanol, the volume ratio of isopropanol to methanol is 1:1-2, and the volume ratio of the upper layer extract to the reconstitution solvent is 8-15:1.

Images