MALDI-FT-ICR mass spectrometry probe for single-chain sphingolipid detection and methods of making and using the same

Abstract

The application discloses a MALDI-FT-ICR mass spectrum probe for single-chain sphingolipid detection and a preparation method and a use method thereof, and relates to the technical field of analytical testing. The MALDI-FT-ICR mass spectrum probe for single-chain sphingolipid detection has the general structure of: the preparation method comprises the following steps: 4-pyridine propionic acid or pyridine 4-propenoic acid is reacted with salicylaldehyde or a salicylaldehyde derivative in a protective atmosphere. The use method comprises the following steps: different concentrations of sphingosine methanol solutions are prepared, the stock solution of the probe is added into each concentration of the sphingosine methanol solution, and a mixed solution is obtained; the mixed solution is mixed with a matrix methanol solution, is spotted on a target plate, is dried at room temperature, and then is subjected to MALDI analysis; and a standard curve is drawn with different concentrations of the sphingosine in the step S1 as the horizontal coordinate and the MALDI ion signal intensity of the product obtained in the step S1 as the vertical coordinate. The mass spectrum probe provided by the application has high sensitivity, low cost and high throughput.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical testing technology, more particularly, to a MALDI-FT-ICR mass spectrometry probe for detecting single-chain sphingolipids and a preparation method and use method thereof. BACKGROUND

[0002] Sphingolipids are a class of amphipathic lipids containing a sphingosine backbone, which have unique contributions to eukaryotic membrane structure and function, and are also important signal molecules. Some sphingolipids such as ceramide, sphingomyelin, sphingosine-1-phosphate and sphingosine have been confirmed to be involved in cell growth, apoptosis, programmed cell death, inflammatory response and cell migration processes. Due to the important role of sphingolipids, changes in sphingolipid metabolism can have a significant impact on the biological effects of the body. Single-chain sphingolipids such as sphingosine, dihydrosphingosine and 3-ketodihydrosphingosine not only serve as a source of synthesis of other more complex sphingolipids, but also have the function of biological signal transduction, and play a key role in cancer and Niemann-Pick disease type C. In addition, 3-ketodihydrosphingosine will accumulate as a toxic substance in tumor cells and destroy the function of the endoplasmic reticulum.

[0003] At present, most of the single-chain sphingolipid detection is based on LC-MS / MS method and fluorescent probe method. The LC-MS / MS method is accompanied by certain limitations such as the need for a longer pretreatment process and analysis time; the fluorescent probe method also has problems such as the need for fluorescent or chromophore group tags, long incubation time, difficult synthesis, and self-fluorescence interference of the probe.

[0004] Another alternative is MALDI mass spectrometry, which has the following analytical advantages compared with LC-MS / MS method or fluorescent probe method: 1. simple operation, no need for complex sample pretreatment; 2. qualitative and quantitative analysis is completed by accurately determining the mass-to-charge ratio of the analyte, avoiding the complex probe synthesis process; 3. can simultaneously detect multiple compounds, including substrates and products; 4. without the limitations of self-fluorescence interference and other factors in the fluorescent probe method, these advantages can reduce false positive analysis results. Due to the high sensitivity, high pollution resistance and high throughput analysis characteristics of matrix-assisted laser desorption ionization (MALDI) mass spectrometry, MALDI mass spectrometry has become an important tool for screening endogenous substances and drugs. Therefore, it is necessary to develop a high-sensitivity, low-cost, high-throughput MALDI mass spectrometry probe for the detection and quantitative analysis of single-chain sphingolipids. SUMMARY

[0005] The purpose of the present application is to provide a MALDI-FT-ICR mass spectrometry probe for detecting single-chain sphingolipids and a preparation method and use method thereof, in order to solve the above problems.

[0006] According to a first aspect of the present application, a MALDI-FT-ICR mass spectrometry probe for detecting single-chain sphingolipids is provided, having a general structure of:

[0007]

[0008] wherein R is H, F or -N(Et)2, R1 is and when R1 is R is H.

[0009] Specifically, the structures of formula I to formula VII are involved, and are specifically as follows:

[0010]

[0011] According to a second aspect of the present application, a preparation method of a MALDI-FT-ICR mass spectrometry probe for detecting single-chain sphingolipids is provided, comprising:

[0012] reacting 4-pyridine propionic acid or pyridine 4-propenoic acid with salicylaldehyde or a salicylaldehyde derivative under a protective atmosphere to obtain the MALDI-FT-ICR mass spectrometry probe for detecting single-chain sphingolipids;

[0013] when R1 is R is H, first reacting to obtain and then reacting with halogenated methane to obtain the MALDI-FT-ICR mass spectrometry probe for detecting single-chain sphingolipids.

[0014] According to a third aspect of the present application, a use method of the MALDI-FT-ICR mass spectrometry probe for detecting single-chain sphingolipids is provided, comprising:

[0015] S1. preparing sphingosine methanol solutions with different concentrations, adding a stock solution of the MALDI-FT-ICR mass spectrometry probe for detecting single-chain sphingolipids to each of the sphingosine methanol solutions with different concentrations to obtain a mixed solution; mixing the mixed solution with a matrix methanol solution, spotting on a target plate, drying at room temperature, and then performing MALDI analysis;

[0016] The specific reaction equation is as follows:

[0017]

[0018] wherein R2 is a corresponding substituent group of sphingosine.

[0019] S2. taking the different concentrations of sphingosine in step S1 as the horizontal coordinates, and taking the MALDI ion signal intensity of the product obtained in step S1 as the vertical coordinates, to draw a standard curve.

[0020] Preferably, the sphingosine comprises sphingosine, dihydrosphingosine or 3-ketodihydro sphingosine.

[0021] Preferably, the solvent of the stock solution is methanol and / or acetonitrile.

[0022] Preferably, the solvent of the stock solution is methanol.

[0023] Preferably, the concentration of the stock solution is 100 µM-10 mM.

[0024] Preferably, the concentration of the stock solution is 1 mM.

[0025] Preferably, the matrix in the matrix methanol solution comprises α-4-cyanocinnamic acid and / or 2,5-dihydroxybenzoic acid.

[0026] Preferably, the matrix in the matrix methanol solution comprises α-4-cyanocinnamic acid.

[0027] According to the technical content disclosed in the present application, the following beneficial effects are achieved:

[0028] The MALDI-FT-ICR mass spectrometry probe for detecting single-chain sphingolipids provided by the present application has a fast and efficient reaction with the substrate initiated by the MALDI laser. Compared with the prior art, the synthesis cost of the molecular probe is reduced, the problems such as long pretreatment process and analysis time, and self-fluorescence interference of the probe are avoided, fast and accurate analysis of several single-chain sphingolipids can be achieved on the MALDI target plate, and the analysis speed and efficiency are greatly improved.

[0029] The mass spectrometry probe provided by the present application has the technical characteristics of simple synthesis, stability under mass spectrometry conditions, and strong ionization, and the sensitivity is several times higher than that of the single-chain sphingolipid itself. The reaction of the probe with the substrate is catalyzed by the MALDI laser, and the reaction is carried out in a very short time, with high conversion rate and without catalyst.

[0030] The mass spectrometry probe is structurally modified and a permanent positive charge is introduced, so that the alkali metal adduct mass spectrometry peak is inhibited, and a purer background signal is obtained.

[0031] The mass spectrometry probe provided by the present application can quantitatively detect several single-chain sphingolipids such as sphingosine, dihydrosphingosine and 3-ketodihydro sphingosine within a certain concentration range, and has good sensitivity and substrate selectivity.

[0032] The mass spectrometry probe and method provided by the present application are used in combination with MALDI mass spectrometry, and can realize analysis of several single-chain sphingolipids on the target plate, and are easy to popularize and apply.

[0033] Other features and advantages of the present application will become apparent from the following detailed description of exemplary embodiments thereof, with reference to the accompanying drawings. Attached Figure Description

[0034] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0035] Figure 1 The MALDI spectra of Formula I and sphingosine of this invention are shown below.

[0036] Figure 2 The MALDI spectrum of sphingosine is shown.

[0037] Figure 3 This is a comparison of the substrate conversion rates of the compound shown in Formula I of the present invention with sphingosine under solution conditions and MALDI conditions;

[0038] Figure 4 The MALDI spectra of formulas I-VI and sphingosine are shown below;

[0039] Figure 5 The MALDI spectra and mass spectrometry responses of formulas I and VII of this invention and sphingosine were compared and evaluated.

[0040] Figure 6 The present invention provides quantitative MALDI mass spectrometry response curves for single-chain sphingolipids of different concentrations using the molecular probe method of this invention. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0042] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use.

[0043] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and equipment should be considered part of the specification.

[0044] In all the examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0045] It should be noted that similar labels and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be discussed further in subsequent figures.

[0046] The matrix assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometer used in the following examples was a ScimaX FT-ICR MRMS (Bruker Daltonics, Germany) with a 355 nm wavelength Nd:YAG laser. The mass spectrometry test parameters were: acceleration voltage: 20.000 kv; delayed extraction voltage: 18.000 kv; delayed extraction time: 150 ns; reflectron voltage: 20.000 kv; lens voltage: 6.000 kv; frequency: 1000 Hz; laser energy: 0-100%; spatial resolution of 100 μιη per pixel analyzed; 2M data points transient acquisition data were used with an ion accumulation time of 0.03 s. The test mode was a reflected positive ion mode.

[0047] Examples 1-6

[0048] This example provides a MALDI-FT-ICR mass spectrometry probe for single chain sphingolipid detection, which is prepared as follows:

[0049] 4-pyridine propionic acid (300 mg, 2.0 mmol) / pyridine 4-propenoic acid (300 mg, 2.0 mmol) and dicyclohexyl carbodiimide (DCC) (409 mg, 2.0 mmol) were dissolved in anhydrous dichloromethane under argon protection, and after stirring at room temperature for 15 min, the corresponding salicylaldehyde (2.4 mmol) and pyridine (16 μΐ, 0.2 mmol) were added, and the reaction was continued for 8 hours under argon atmosphere. After the reaction was completed as monitored by TLC, the insoluble matter was filtered. The filtrate was dried with anhydrous sodium sulfate and concentrated, and column chromatography was used for separation (petroleum ether: ethyl acetate = 10: 1).

[0050] The specific reaction equation is shown below:

[0051]

[0052] Formula I-VI were obtained, and the structure confirmation results are as follows.

[0053] Formula I: white solid (460 mg, 92%). 1 H NMR (500 MHz, Chloroform-d) δ 10.01 (s, 1H), 8.58 - 8.53 (m, 2H), 7.87 (dd, J = 7.6, 1.8 Hz, 1H), 7.63 (td, J = 7.8, 1.8 Hz, 1H), 7.42 (td, J = 7.5, 1.0 Hz, 1H), 7.25 - 7.20 (m, 2H), 7.10 (dd, J = 8.1, 1.1 Hz, 1H), 3.12 (dd, J = 8.7, 6.9 Hz, 2H), 3.04 (dd, J = 7.9, 6.1 Hz, 2H). 13C NMR (126 MHz, Chloroform-d) δ 188.86, 170.66, 150.89, 149.99, 148.99, 135.37, 132.22, 127.98, 126.63, 123.77, 123.43, 34.25, 29.80. MS (ESI) m / z: 256.1 [M+H] + ;

[0054] Formula II: White solid (465 mg, 91%). 1 H NMR (500 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.73 - 8.68 (m, 2H), 7.98 (dd, J = 7.7, 1.7 Hz, 1H), 7.90 (d, J = 16.1 Hz, 1H), 7.81 (qd, J = 5.6, 4.9, 1.7 Hz, 3H), 7.56 (t, J = 7.5 Hz, 1H), 7.43 (d, J = 7.1 Hz, 1H), 7.23 (d, J = 16.1 Hz, 1H). 13 C NMR (126 MHz, DMSO-d6) δ 190.38, 164.74, 151.07 (d, J = 22.5 Hz), 144.61, 141.39, 136.26, 131.80, 128.38, 127.35, 124.22, 122.84, 122.04. MS (ESI) m / z: 254.1 [M+H] + ;

[0055] Formula III: White solid (589 mg, 91%). 1 H NMR (500 MHz, Chloroform-d) δ 9.66 (s, 1H), 8.53 (d, J = 6.1 Hz, 2H), 7.61 (d, J = 8.9 Hz, 1H), 7.27 - 7.21 (m, 2H), 6.54 (dd, J = 8.9, 2.5 Hz, 1H), 6.16 (d, J = 2.6 Hz, 1H), 3.40 (t, J = 7.1 Hz, 4H), 3.12 (t, J = 7.6 Hz, 2H), 3.01 (t, J = 7.3 Hz, 2H), 1.19 (t, J = 7.1 Hz, 6H). 13 C NMR (126 MHz, Chloroform-d) δ 186.80, 170.76, 153.06, 149.62 (d, J = 48.2 Hz), 134.93, 123.90, 116.02, 108.41, 104.50, 44.80, 34.32, 33.92, 29.86, 12.41. MS (ESI) m / z: 327.2 [M+H]+ ;

[0056] Formula IV: White solid (587 mg, 90%). 1 H NMR (500 MHz, Chloroform-d) δ 9.79 (s, 1H), 8.69 - 8.64 (m, 2H), 7.80 (d, J = 16.1 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.43 - 7.38 (m, 2H), 6.86 (d, J = 16.0 Hz, 1H), 6.56 (dd, J = 8.9, 2.5 Hz, 1H), 6.37 (d, J = 2.5 Hz, 1H), 3.39 (q, J = 7.1 Hz, 4H), 1.19 (t, J = 7.2 Hz, 6H). 13 C NMR (126 MHz, Chloroform-d) δ 186.29, 164.30, 153.52, 153.05, 150.57, 143.79, 141.22, 133.59, 121.93, 121.69, 116.03, 108.65, 104.32, 44.77, 12.40. MS (ESI) m / z: 325.2 [M+H] + ;

[0057] Formula V: White solid (493 mg, 91%). 1 H NMR (500 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.50 (d, J = 4.9 Hz, 2H), 8.03 - 7.94 (m, 1H), 7.37 (t, J = 7.4 Hz, 3H), 3.11 (t, J = 7.5 Hz, 2H), 3.02 (t, J = 7.5 Hz, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 189.12, 171.06, 153.18 (d, J = 12.4 Hz), 150.27, 133.63 (d, J = 11.1 Hz), 125.59 (d, J = 3.1 Hz), 124.28 (d, J = 3.6 Hz), 114.54, 111.97, 33.68, 29.37. MS (ESI) m / z: 274.1 [M+H] + ;

[0058] Formula VI: White solid (463 mg, 85%). 1H NMR(500MHz,DMSO-d6)δ10.07(s,1H),8.70(d,J=5.0Hz,2H),8.06(dd,J=8.6,6.5Hz,1H), 7.91(d,J=16.1Hz,1H),7.81(d,J=5.1Hz,2H),7.51–7.39(m,2H),7.22(d,J=16.2Hz,1H). 13 C NMR(126MHz,DMSO-d6)δ189.16,167.05,164.30,152.78,151.00,144.96,141.30,13 4.16,125.62(d,J=3.2Hz),122.85,121.75,114.82,112.37.MS(ESI)m / z:272.1[M+H] + .

[0059] Example 7

[0060] Iodomethane (24 μL, 0.4 mmol) was added to an acetonitrile solution of the compound of formula I (100 mg, 0.4 mmol) at room temperature. The reaction was heated to 50 °C for 12 hours, cooled to room temperature, and quenched with a saturated aqueous solution of sodium thiosulfate. The mixture was then concentrated under reduced pressure and purified by preparative high-performance liquid chromatography (acetonitrile:water = 10:1) to obtain formula VII, with the following reaction equation:

[0061]

[0062] The structural confirmation results are as follows:

[0063] Formula VII: Yellow oily substance (140 mg, 90%). 1 H NMR (500MHz, DMSO-d6) δ10.06(s,1H),8.91(d,J=6.1Hz,2H),8.12(d,J=6.1Hz,2H),7.93(d,J=7.7Hz ,1H),7.77(t,J=7.8Hz,1H),7.52(t,J=7.6Hz,1H),7.29(d,J=8.1Hz,1H),4.31(s,3H),2.08(s,2H). 13C NMR (126 MHz, DMSO-d6) δ 188.47, 168.92, 158.33, 149.02, 143.14, 134.10, 129.57, 126.12, 125.66, 125.17, 121.99, 45.57, 38.69 - 37.56 (m), 37.43 (d, J = 21.0 Hz), 30.44, 27.71. MS (ESI) m / z: 270.1 [M+H] + .

[0064] Example 8

[0065] MALDI mass spectrometry of sphingosine and the molecular probe of Formula I:

[0066] Prepare a methanol solution of the probe of Formula I and a sphingosine standard in methanol. Mix 10 μL of the probe solution with 10 μL of the sphingosine solution, and then add 20 μL of CHCA matrix (10 mg / mL, 70% acetonitrile in water, 0.1% trifluoroacetic acid) or 20 μL of DHB matrix (15 mg / mL, 80% acetonitrile in water, 0.1% trifluoroacetic acid) solution. The final concentration of the probe of Formula I is 1 mM, and the final concentration of sphingosine is 100 μM. Take 0.5 μL of the mixture and spot it directly on a 384 ground steel target plate. After drying at room temperature, perform MALDI mass spectrometry.

[0067] As shown in Figure 1 , the MALDI test spectrum of the probe and 100 μM sphingosine is shown. By analyzing the labeled probe of Formula I, a hydrogenated peak at m / z = 537 with high peak degree can be detected, and only a small amount of dehydrated peak signal (m / z = 282) of sphingosine itself remains. As shown in Figure 2 , the MALDI spectrum of 100 μM sphingosine in a traditional matrix (CHCA matrix, 10 mg / mL, 70% acetonitrile in water, 0.1% trifluoroacetic acid) is shown. Compared with this, the ion signal intensity of the reaction product of the probe and sphingosine is increased by several times, the spectrum is easy to identify, and the interference of small molecule matrix background is avoided. In addition, the reaction solutions of the probe and sphingosine incubated for 0 h, 0.5 h, and 2 h under solution conditions are monitored by LC-MS method, and the substrate conversion rate is calculated; compared with this, the conversion rate of sphingosine in MALDI detection is more than 90% in a very short time.

[0068]

[0069] As shown in Figure 3 , the huge difference in substrate conversion rate under the two conditions indicates that the reaction between the probe and sphingosine is initiated by the MALDI laser, and this reaction is named as MALDI laser-induced condensation reaction.

[0070] Examples 9-13

[0071] Prepare methanol solutions of probes of types I-VI by mixing 10 μL of the I-VI probe methanol solution with 10 μL of sphingosine standard methanol solution. Finally, add 20 μL of CHCA matrix (10 mg / mL, 70% acetonitrile aqueous solution, 0.1% trifluoroacetic acid) or 20 μL of DHB matrix (15 mg / mL, 80% acetonitrile aqueous solution, 0.1% trifluoroacetic acid). The final probe concentration is 1 mM, and the final sphingosine concentration is 100 μM. Spot 0.5 μL of the above mixture onto a 384 ground steel target plate, dry at room temperature, and then perform MALDI mass spectrometry analysis.

[0072] like Figure 4 The images show the MALDI spectra of the six molecular probes represented by formulas I-VI and sphingosine. All of them can undergo MALDI laser-induced condensation with sphingosine, with the probe represented by formula I exhibiting the highest product ion signal intensity.

[0073] Example 14

[0074] MALDI mass spectrometry analysis was performed on the molecular probe of formula VII with sphingosine:

[0075] Prepare methanol solutions of probes shown in Formula I and Formula VII, and a methanol solution of sphingosine standard. Take 10 μL of methanol solutions of probes I and VII and mix them separately with 10 μL of sphingosine solution. Finally, add 20 μL of CHCA matrix (10 mg / mL, 70% acetonitrile aqueous solution, 0.1% trifluoroacetic acid) or 20 μL of LHB matrix (15 mg / mL, 80% acetonitrile aqueous solution, 0.1% trifluoroacetic acid) to make the final probe concentration 1 mM and the final sphingosine concentration 100 μM. Spot 0.5 μL of the above mixture onto a 384 ground steel target plate, dry at room temperature, and then perform MALDI mass spectrometry analysis.

[0076] from Figure 5 As can be seen, the molecular probe shown in Formula VII has a higher product ion signal intensity than the probe shown in Formula I. At the same time, the introduction of permanent charge significantly suppresses the sodium ion mass spectrometry addition peak, resulting in a purer mass spectrometry signal background.

[0077] Example 15

[0078] Evaluation, quantitative analysis, and standard curve plotting of the detection limits of the molecular probes and several single-chain sphingolipids of this invention:

[0079] The 10 μL of different concentrations of 3-keto-sphinganine, sphingosine and sphinganine in methanol solution were added with 10 μL of the molecular probe in methanol solution shown in formula VII, and finally 20 μL of CHCA matrix (10 mg / mL, 70% acetonitrile aqueous solution, 0.1% trifluoroacetic acid) or 20 μL of DHB matrix (15 mg / mL, 80% acetonitrile aqueous solution, 0.1% trifluoroacetic acid) was added to make the final concentration of the probe 1 mM, and the final concentration of 3-keto-sphinganine, sphingosine and sphinganine 1 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 5 μM, 10 μM and 100 μM. 0.5 μL of the above mixture was spotted on a 384 ground steel target plate, and after drying at room temperature, MALDI mass spectrometry was performed. The standard curve was plotted with the different concentration values of 3-keto-sphinganine, sphingosine and sphinganine as the abscissa and the MALDI ion signal intensity of the product as the ordinate. As shown in Figure 6 the ion signal intensity of the molecular probe product showed a good linear relationship with the concentration of several single-chain sphingolipids (in the range of 30 nM-10 μM), showing good accuracy and reproducibility in the determination of single-chain sphingolipids.

[0080] Although some specific embodiments of the present application have been described in detail by examples, those skilled in the art should understand that the above examples are only for illustration, but not for limiting the scope of the present application. Those skilled in the art should understand that the above embodiments can be modified without departing from the scope and spirit of the present application. The scope of the present application is defined by the appended claims.

Claims

1. A MALDI-FT-ICR mass spectrometry probe for single-chain sphingolipid detection, characterized in that, A structural general formula of which is: wherein R is H, F or -N(Et)2, and R1is and when R1is R is H.

2. A method for the preparation of a MALDI-FT-ICR mass spectrometry probe for the detection of single-chain sphingolipids according to claim 1, characterized in that, The method comprises the following steps: The 4-pyridine propionic acid or pyridine 4-propenoic acid is reacted with salicylaldehyde or a salicylaldehyde derivative under a protective atmosphere to obtain the MALDI-FT-ICR mass spectrum probe for single-chain sphingolipid detection; When R1 is R is H, first reacting to obtain Then using methyl iodide to react with it, to obtain the MALDI-FT-ICR mass spectrometry probe for single-chain sphingolipid detection.

3. A method of using the MALDI-FT-ICR mass spectrometry probe for single-chain sphingolipid detection according to claim 1, characterized in that, The method comprises the following steps: S1. Prepare sphingosine methanol solutions with different concentrations, and add a stock solution of the MALDI-FT-ICR mass spectrum probe for single-chain sphingolipid detection to each of the sphingosine methanol solutions to obtain a mixture; mix the mixture with a matrix methanol solution, spot on a target plate, dry at room temperature, and then perform MALDI analysis; S2. Take the different concentrations of sphingosine in step S1 as the horizontal coordinates, and take the MALDI ion signal intensity of the product obtained in step S1 as the vertical coordinates to draw a standard curve.

4. The method of use of claim 3, wherein, The sphingosine comprises sphingosine, dihydrosphingosine or 3-ketodihydrosphingosine.

5. The method of use of claim 3, wherein, The solvent of the stock solution is methanol and / or acetonitrile.

6. The method of use of claim 5, wherein, The solvent of the stock solution is methanol.

7. The method of use of claim 3, wherein, The concentration of the stock solution is 100 μM-10 mM.

8. The method of use of claim 7, wherein, The concentration of the stock solution is 1 mM.

9. The method of use according to any one of claims 3-8, wherein, The matrix in the matrix methanol solution is α-4-cyanocinnamic acid and / or 2,5-dihydroxybenzoic acid.

10. The method of use of claim 9, wherein, The matrix in the matrix methanol solution is α-4-cyanocinnamic acid.

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