TW201443432A - Method for detecting compound by thin-layer chromatograph mass spectrometry - Google Patents

Abstract

The invention provides a method for detecting compounds by thin-layer chromatograph mass spectromery, comprising: (a) providing a sample, wherein the sample is dissolved in a separation solution and comprises a plurality of compounds; (b) dipping the sample on a substrate plate to separate the sample by the thin layer chromatography (TLC) method and to obtain a plurality of analysis spots, wherein the analysis comprises at least one compound; (c) dipping a nano-particles solution on the analysis separated spots, wherein the compounds are absorbed by the nano-particles solution; (d) drying the substrate; and (e) placing the substrate plate with the absorbed compounds directly in to a mass spectrometry to for the analysis of analyze the separated compounds.

Description

Method for analyzing compounds by thin layer chromatography mass spectrometry

[0001] The present invention relates to an analytical method, and in particular to an analytical method combining thin layer chromatography (TLC) and mass spectrometry (MS).

[0002] In a chemical reaction process, a chemical reaction is usually monitored using thin layer chromatography (TLC) to determine whether a chemical reaction is completed or not. Generally, the chemical structure of the substance to be tested can be judged by comparing the analyte to a standard sample. However, in the absence of an appropriate standard, or when the chemical reaction step or the intermediate product is to be monitored, it is not easy to judge the structure of the analyte by thin layer chromatography alone. Therefore, other analytical methods are needed to help determine the chemical structure of the analyte.
[0003] Mass spectrometry (MS) determines the chemical structure of a test object by analyzing the molecular weight of the analyte. Combined with thin layer chromatography and mass spectrometry, the structural data of the analyte can be separated and obtained. However, analytical techniques are limited due to cumbersome sample preparation and interference with organic acid matrices in areas of small molecular weight (less than 500 Da).
Therefore, it is extremely necessary in the industry to develop a method for analyzing a compound by thin layer chromatography mass spectrometry, which is simple and rapid, and does not require an external matrix, and can effectively analyze a low molecular weight analyte.

The present invention provides a method for analyzing a compound by thin layer chromatography mass spectrometry, comprising the steps of: (a) providing a sample, wherein the sample is dissolved in a separation solution and having a plurality of compounds in the sample; (b) Attaching the sample spot to a plate, separating the sample by a thin layer chromatography to obtain a plurality of analysis points, wherein each analysis point includes at least one compound; (c) Dropping a nanoparticle solution onto the analysis points to adsorb the compounds in the nanoparticle solution; (d) performing a drying step on the substrate; and (e) adsorbing the substrate on the substrate Directly placed in a mass spectrometer for mass spectrometry analysis.
The above and other objects, features and advantages of the present invention will become more <RTIgt;

12. . . Substrate

14. . . dropper

16. . . Nanoparticle solution

16a. . . Metal nanoparticle

16b. . . Organic acid

S1, S2, S3. . . Analysis point

【0007】
1A to 1D are a series of cross-sectional views for explaining a flow chart of the method for analyzing a compound by thin layer chromatography mass spectrometry of the present invention.
Fig. 2A-2H shows the mass spectrum of Example 2 of the present invention, respectively.
Figure 3 shows the mass spectrum of Example 3.
Figures 4A to 4F show mass spectra of Comparative Example 1, Comparative Example 2, and Example 4, respectively.
Fig. 5A is a view showing the mass spectrum of the intermediate product (intermediate III) of Example 5 of the present invention.
Figure 5B shows a mass spectrum of the product (product IV) of Example 5 of the present invention.
Figures 6A-6B show mass spectra of natural matter detected by an embodiment of the present invention.

The present invention provides a method for analyzing a compound by thin layer chromatography mass spectrometry comprising the following steps (a) to (e). 1A to 1D are a series of cross-sectional views for explaining a flow chart of the method for analyzing a compound by thin layer chromatography mass spectrometry of the present invention. First, step (a) is carried out to provide a sample in which the sample is dissolved in the separation solution and the sample has a plurality of compounds. The "sample" here is the sample to be tested. The sample to be tested usually has many different compounds, which can be separated by subsequent analysis methods.
Further, the sample of step (a) may be derived from a reactant, an intermediate or a product during a chemical reaction, and the chemical reaction is monitored at a real time.
[0010] The separation solution includes dichloromethane (DCM), methanol, ethyl acetate (EA), n-hexane, acetone, chloroform, toluene (chloroform). Toluene) or water. However, the separation solution is not limited to the above-mentioned solvents, and those skilled in the art can select the desired separation solution depending on the needs of the actual application.
[0011] Thereafter, step (b) is performed, the sample point is attached to a plate, and the sample is separated by thin layer chromatography to obtain a plurality of analysis points, wherein each analysis point Include at least one compound. Referring to Figure 1A, it is shown that after thin layer chromatography, the substrate 12 has a plurality of analysis points S1, S2, S3 thereon.
[0012] The substrate 12 comprises an aluminum, silica gel or glass substrate. In a preferred embodiment, a commercially available TLC plate (e.g., tantalum film) can be used directly as the substrate of the present invention, and the substrate need not be specially modified.
[0013] Thin layer chromatography (TLC) is a chromatography method, mainly using silica gel or aluminum as a stationary phase, and attaching sample points thereto. In the thin layer static phase, the liquid developing agent is used as a mobile phase, and the capillary action is moved from bottom to top to separate various components. In addition, thin layer chromatography (TLC) can also be used to track the extent of chemical reactions.
[0014] In the step (b), since the sample itself may be transparent and colorless, and the analysis points S1, S2, and S3 may also be transparent and colorless, the method for analyzing a compound by thin layer chromatography mass spectrometry of the present invention includes using The analysis points S1, S2, and S3 are observed by ultraviolet light (UV) or staining buffer.
[0015] Next, step (c) is performed. Referring to FIG. 1B, the nanoparticle solution 16 is dropped on the analysis points S1, S2, and S3 by the dropper 14, and the compounds are adsorbed to the nanoparticle solution. in. Referring to FIG. 1C, the nanoparticle solution 16 includes a plurality of metal nanoparticles 16a and an organic acid 16b modified on the metal nanoparticles 16a.
The metal nanoparticles 16a include iron (Fe), cobalt (Co), nickel (Ni), gold (Au), silver (Ag), or a combination thereof. The organic acids 16b include 2,5-dihydroxybenzoic acid (DHB) and 3,5-dimethoxy-4-hydroxycinnamic acid (3,5-dimethoxy-4-hydroxycinnamic acid). , SA), α-Cyano-4-hydroxycinnamic acid (α-CHC), 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid)[( E)-3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid, Ferulic acid], pyridine-2-carboxylic acid, Picolinic acid, 3-hydroxy-2-picolinic acid ( 3-hydroxypyridine-2-carboxylic acid, 3-HPA), 2-(4'-Hydroxybenzeneazo)benzoic acid (HABA).
[0017] In a preferred embodiment, the metal nanoparticles 16a have magnetic properties such as iron (Fe), cobalt (Co), and nickel (Ni). The advantage of using magnetic metal nanoparticles is that the magnetic metal nanoparticles can be recycled and reused to save process costs.
[0018] In one embodiment, 2,5-dihydroxybenzoic acid (DHB) may be first modified onto iron oxide (Fe ₃ O ₄ ) nanoparticles, and then appropriate Solvent to form nanoparticle solution 16.
[0019] It should be noted that when the nanoparticle solution 16 is dropped on the analysis points S1, S2, and S3, the surface of the metal nanoparticle 16a has a specific function because the organic acid 16b is modified on the metal nanoparticle 16a. Bases, such as aromatic rings, carboxylates, hydroxyl groups, which can generate charge-charge interactions, hydrogen bonds between the functional groups and the compound to be analyzed. Hydrogen bonding and a hydrophobic effect, so that the compound is adsorbed on the metal nanoparticles 16a in the nanoparticle solution 16, thereby achieving a concentration step of extraction and improving detection sensitivity. In addition, the choice of solvent affects the effect of extraction concentration, and the solvent having a polarity close to that of the compound to be analyzed can be selected according to the needs of the actual application to enhance the concentration effect.
[0020] Thereafter, step (d) is performed to perform a drying step on the substrate 12. The drying step purges the substrate 12, for example, using nitrogen gas.
[0021] Next, step (e) is performed. Referring to FIG. 1C, the substrate 12 on which the compounds are adsorbed is directly placed in a mass spectrometer for mass spectrometry. The mass spectrometer includes a matrix-assisted laser desorption ionization (MALDI). Figure 1D shows the mass spectrum, with the X-axis representing the mass-to-charge ratio (m/z) of the compound and the Y-axis representing the signal intensity.
[0022] It should be noted that before the step (e), since the modified organic acid 16b on the metal nanoparticle 16a can function as a matrix, that is, it can help energy transfer, therefore, no additional matrix is required (matrix) ) to the substrate. In addition, the present invention allows the substrate to be placed "directly" in the mass spectrometer without the need for a complicated sample extraction process, which saves process cost and time.
[0023] In the prior art, when performing matrix-assisted laser desorption ionization (MALDI), when detecting low molecular weight (molecular weight less than 500 Da), it is usually subjected to a matrix. The interference affects the analysis results. Compared with the prior art, the organic acid used in the present invention is modified on the metal nanoparticles in the nanoparticle solution. Therefore, although the organic acid can play the role of a matrix (which can help proton transfer), it does not occur. The interference of the matrix allows the method of the present invention to be effectively applied to detect low molecular weight compounds and achieve better detection sensitivity.
[0024] The compounds detected by the present invention have a molecular weight of from about 100 to about 2000 Da, preferably from about 100 to about 500 Da. The detection method of the present invention has a detection sensitivity of pmole (10 ^-15 mole).
[0025] A method of analyzing a compound by thin layer chromatography mass spectrometry as disclosed herein, wherein the compound comprises an organic molecule, a biomolecule or an inorganic molecule. Organic molecules include acid, ester, aldehyde, alkane, alkene, alkyne, and amine. , amide, aromatic hydrocarbons, azide, heterocyclic compounds, sulfides, silanes, natural products Or an organic catalyst intermediate (intermediate).
In addition, organic molecules include various drugs such as opium, amphetamine, morphine, heroin, marijuana, cocaine, and ecstasy (3,4). -methylenedioxy-methamphetamine, MDMA), ketamine, etc.
[0027] Biomolecules include proteins, peptides, deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) and its metabolism, lipid, cholesterol ( Cholesterol) with its metabolites, saccharides, disaccharides, glycolipids, glycopeptides, vitamins, biotins, neurotransmitters Antibiotics.
[0028] The inorganic molecules include a metal complex or a catalyst intermediate.
[0029] It should be noted that in the prior art, since the metabolites usually contain salts (such as sodium and potassium), these salts usually interfere with the results of mass spectrometry. Compared with the prior art, the embodiments of the present invention are preferably applicable to detecting small molecular weight metabolites, because the present invention performs thin layer chromatography (TLC), salts are not dissolved in the separation solution. Therefore, the interference of the salt can be removed.
[0030] In one embodiment, the method of the present invention for analyzing a compound by thin layer chromatography mass spectrometry can be applied to the process of monitoring a nucleophilic substitution reaction when the starting material methoxytriethylene glycol ( Methoxyl triethylene glycol, C ₇ H ₁₆ O ₄ , molecular weight 164.1), after reaction with 2-chloro-2-phenylacetyl chloride, gives the ester product (C ₁₅ H ₂₁ ClO ₅ , molecular weight 316.1). The starting materials and products can be detected by matrix-assisted laser desorption ionization (MALDI).
[0031] In another embodiment, the method of the present invention for analyzing a compound by thin layer chromatography mass spectrometry can be applied to monitor the acylation catalyzed by 4-dimethylaminopyridine (DMAP). The intermediate product can be detected by the method disclosed in the present invention, and the chemical reaction can be monitored in real time, and whether the reaction is completed or not can be judged.
[0032] In summary, the present invention provides a method for analyzing a compound by thin layer chromatography mass spectrometry, which combines thin layer chromatography with mass spectrometry, uses thin layer chromatography to separate, and combines mass spectrometry. The molecular weight of the compound is detected, and the nanoparticle solution is dropped on the substrate to extract the concentrated compound, and the substrate is directly subjected to mass spectrometry without additional matrix addition. The method can be applied to analyze various organic molecules. , biomolecules or inorganic molecules, especially for small molecules with low molecular weight (100-500 Da). In addition, the method for analyzing a compound by thin layer chromatography mass spectrometry provided by the present invention can be applied to monitor a chemical reaction in a real time, and can detect a reactant, an intermediate or an intermediate during a chemical reaction. Product to judge whether the reaction is complete or not.
[0033] [Embodiment]
[0034] Example 1 Preparation of acid-modified metal nanoparticles (DHB@MNPs)
(1) FeCl ₂ ‧4H ₂ O and FeCl _{3 were} sequentially added to an aqueous solution of hydrochloric acid (HCl) (deoxidized), and the mixture was stirred and reacted for 2 hours to obtain a mixture A.
(2) The mixture A of the step (1) is slowly added to an aqueous solution of sodium hydroxide (NaOH) (deoxidized) to obtain a mixture B.
(3) The mixture B is dispensed into a centrifuge tube, and secondary deionized water (dd-water) is added to the centrifuge tube, and after shaking uniformly, the aqueous layer is removed.
(4) Slowly drip 0.01 N hydrochloric acid aqueous solution (HCl) into the centrifuge tube and stir for 2 minutes, remove the supernatant (supermatnat), and finally add secondary deionized water to dissolve back to obtain iron oxide nanoparticle solution (Fe). ₃ O ₄ @MNP).
(5) The iron oxide nanoparticle solution was placed in a single-necked flask, and then propanol was added thereto, and shaken at high speed for 30 minutes to obtain a mixture C.
(6) Tetraethylorthosilicate (TEOS) and 28-30% aqueous ammonia (NH ₄ OH) were added to the mixture C, and reacted at 60 ° C for 2 hours to obtain a mixture D.
(7) The mixture D was washed sequentially with propanol and secondary deionized water, and the supernatant was removed by high speed centrifugation, and this step was repeated 3 times to obtain a mixture E.
(8) Preparation of organic acid: 2,5-dihydroxybenzoic acid (DHB) was dissolved in 3 ml of secondary deionized water, and 0.01 NHC1 was added to obtain a mixture F.
(9) The mixture E and the mixture F were reacted at room temperature for 12 hours to obtain a mixture G. The mixture G was washed sequentially with propanol and secondary deionized water, and the supernatant was removed by high speed centrifugation to obtain a mixture H. Further, 0.1 N NaOH was added to the mixture H, followed by washing with propanol and secondary deionized water, and finally dried under high vacuum to obtain metal nanoparticles.
Example 2 Detecting different kinds of compounds The metal nanoparticles of Example 1 were used to re-dissolve the metal nanoparticles in a solvent to prepare a 1000 ppm solution of nano particles, such as secondary deionized water and methanol. , acetonitrile (ACN) or 1,4-dioxane.
The various sample points of Table 1 were attached to TLC plates, and the compounds were separated by thin layer chromatography and a plurality of analytical points were obtained. The separated solutions are shown in Table 1.
Thereafter, a 1000 ppm nanoparticle solution was dropped on the analysis points to adsorb the compounds in the nanoparticle solution. Thereafter, a drying step is performed on the TLC plate.
Next, the conductive paste was desorbed on the back side of the TLC plate and adhered to a stainless steel sample tray, and the TLC plate was directly placed in a mass spectrometer for mass spectrometry analysis. Figures 2A-2H show mass spectra of sample numbers 1-8, respectively.

Table 1

*Note: The ratio of the height of the compound rising in the TLC plate to the rising height of the separation solution is called the R _f value.
As can be seen from Fig. 2G, the sensitivity of the analytical method of the present invention for detecting cholesterol is 100 fg and 0.26 fmol (S/N ratio = 4).
As can be seen from Fig. 2H, the sensitivity of the analytical method of the present invention for detecting sulfonamides is 1 ng, 2 pmol (S/N ratio = 3).
Embodiment 3
The metal nanoparticles of Example 3 were similar to Example 1, except that the organic acid was changed to α-Cyano-4-hydroxycinnamic acid (α-CHC).
The nano-solution of Example 3 was back-dissolved in a solvent to prepare a 1000 ppm nanoparticle solution.
Thereafter, the sample spots were attached to a TLC plate, and the compounds were separated by thin layer chromatography and a single analysis point was obtained.
Thereafter, a 1000 ppm nanoparticle solution was dropped on the analysis points to adsorb the compounds in the nanoparticle solution. Thereafter, a drying step is performed on the TLC plate.
Next, a conductive adhesive was adhered to the back surface of the TLC plate, and adhered to a stainless steel sample tray, and the TLC plate was directly placed in a mass spectrometer for mass spectrometry.
Figure 3 shows the mass spectrum of sorbitol detected.
[0037] Example 4
See response mechanism 1, starting with 2-chloro-2-methoxy-phenyl ethylene chlorotrifluoroethylene (2-chloro-2- yl triethylene glycol _{(methoxyl triethylene glycol, C 7 H} 16 O 4, molecular weight 164.1) After the phenylacetyl chloride reaction, an ester product (C ₁₅ H ₂₁ ClO ₅ , molecular weight 316.1) is obtained.



Reaction mechanism 1
See Table 2, which shows the mass spectrometric structures detected by different analytical methods for the compound I and the compound II.

Table 2

Please refer to FIG. 4A and FIG. 4D, which respectively show the signals obtained by mass spectrometry (MALDI MS) after separation of the starting materials and products by thin layer chromatography (TLC) without adding any matrix. Figure. It is known from Figures 4A and 4D that no signal can be detected since no substrate is used.
Please refer to FIG. 4B and FIG. 4E, which respectively show signal diagrams obtained by mass spectrometry after separation of the starting materials and products by thin layer chromatography under the condition of adding DHB as a matrix. It is known from Figures 4B and 4E that using DHB as a substrate, since the fragments of DHB generate a lot of signals, the background signal interferes with the signal of the compound to be analyzed.
Please refer to FIG. 4C and FIG. 4F, respectively, showing that the starting material and the product are separated by thin layer chromatography, and the nanoparticle solution (DHB@MNPs) is dropped on the starting material and the product, and analyzed by mass spectrometry. The resulting signal map. From page 4C, a signal for the binding of sodium to the starting material (m/z = 187.1) was observed, and a signal for the product to bind sodium (m/z = 339.1) was observed from Figure 4F.
See Table 3 for the detection sensitivity of Compound I and Compound II.

table 3

Example 5 Real Time Detection of Chemical Reactions




Reaction mechanism 2
Reaction Scheme 2 shows the acylation catalyzed by 4-dimethylaminopyridine (DMAP), which can be monitored by the analytical method of the present invention, and the intermediate product is monitored and the product monitored. (product IV).
Figure 5A shows the mass spectrum of the intermediate product (intermediate III), and Figure 5B shows the mass spectrum of the product (product IV). It can be seen that the analytical method of the compound disclosed in the present invention can be applied to monitor the chemical reaction in real time to judge whether the reaction is completed or not.
Example 6 Detection of natural products The rhizomes of the natural product Ceylon seven-fin fern (Helminthostachys zeylanica) were analyzed to obtain a series of anti-inflammatory flavonoid families. Includes flavonoids (ugonins JX).
Figure 6A shows the mass spectrum of ugonins J. Figure 6B shows the mass spectrum of ugonins L.
The present invention has been described in terms of several preferred embodiments, and is not intended to limit the scope of the present invention, and may be made by those skilled in the art without departing from the spirit and scope of the invention. Any changes and modifications are intended to be included in the scope of the invention as defined by the appended claims.

12. . . Substrate

14. . . dropper

16. . . Nanoparticle solution

S1, S2, S3. . . Analysis point

Claims (13)

A method for analyzing a compound by thin layer chromatography mass spectrometry, comprising the steps of:
(a) providing a sample, wherein the sample is dissolved in a separation solution and the sample has a plurality of compounds;
(b) attaching the sample spot to a plate, and separating the sample by a thin layer chromatography to obtain a plurality of analysis points, wherein each of the analysis points includes at least one compound;
(c) dropping a nanoparticle solution onto the analysis points to adsorb the compounds in the nanoparticle solution;
(d) performing a drying step on the substrate;
(e) The substrate on which the compounds are adsorbed is placed directly into a mass spectrometer for mass spectrometry. A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 1, wherein the nanoparticle solution comprises a plurality of metal nanoparticles and an organic acid modified on the metal nanoparticles. A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 2, wherein the metal nanoparticles comprise iron (Fe), cobalt (Co), nickel (Ni), gold (Au), silver. (Ag) or a combination of the above. A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 2, wherein the organic acid comprises 2,5-dihydroxybenzoic acid (DHB), 3,5- 3,5-dimethoxy-4-hydroxycinnamic acid (SA), α-Cyano-4-hydroxycinnamic acid (α-CHC), 3-(4-hydroxy-3-methoxyphenyl)-2-acrylic acid) [(E)-3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid, Ferulic acid] picolinic acid ( Pyridine-2-carboxylic acid, Picolinic acid), 3-hydroxypyridine-2-carboxylic acid (3-HPA) or 2-(p-hydroxyphenylazo)benzoic acid [2-(4) '-Hydroxybenzeneazo)benzoic acid, HABA]. A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 1, wherein the separation solution comprises dichloromethane (DCM), methanol, ethyl acetate (EA), N-hexane, acetone, chloroform, toluene or water. A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 1, wherein the substrate comprises an aluminum sheet, a silica gel or a glass substrate. A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 1, wherein the mass spectrometer comprises a matrix-assisted laser desorption ionization mass (MALDI MS). A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 1, wherein the compounds have a molecular weight of about 100 to 2000 Da. A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 1, wherein the compounds include an acid molecule, an ester molecule, an aldehyde molecule, and an alkane molecule ( Alkane), alkene, alkyne, amine, amide, aromatic hydrocarbons, azide, heterocyclic compounds (heterocyclic compound), sulfide, silane, natural product or organic catalyst intermediate. A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 1, wherein the compound comprises a protein, a peptide, a deoxyribonucleic acid (DNA)/ribonucleic acid (ribonucleic acid, RNA) and its metabolism, lipid, cholesterol and its metabolites, saccharides, disaccharides, glycolipids, glycosides ( Glycopeptide), vitamin, biotin, neurotransmitter, antibiotic. A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 1, wherein the compounds comprise a metal complex or a catalyst intermediate. The method of analyzing a compound by thin layer chromatography mass spectrometry as described in claim 1 of the patent application does not require the addition of a matrix to the substrate before performing step (e). A method for analyzing a compound by thin layer chromatography mass spectrometry as described in claim 1, wherein the sample of the step (a) is derived from a reactant, an intermediate product in a chemical reaction process. Or a product, monitoring the chemical reaction in real time.