TW201443432A - Method for detecting compound by thin-layer chromatograph mass spectrometry - Google Patents
Method for detecting compound by thin-layer chromatograph mass spectrometry Download PDFInfo
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【0001】本發明係有關於一種分析方法,且特別係有關於一種結合薄層層析法(thin layer chromatography, TLC)與質譜法(Mass spectrometry, MS)的分析方法。
[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】於化學反應過程中,通常使用薄層層析法(TLC)監控(monitor)化學反應,以判斷化學反應完成與否。一般可藉由將待測物與標準樣品(reference)作比對,以判斷待測物質的化學結構。然而,在無適當標準品,或欲監控化學反應步驟或是中間產物較為複雜時,不容易僅由薄層層析法判斷待測物的結構。因此,需要其他的分析方法,以幫助判斷待測物的化學結構。
【0003】質譜分析法(MS)藉由分析待測物的分子量以判斷待測物的化學結構。結合薄層層析法與質譜分析法可達到分離與得知待測物的結構資料。然而,由於繁瑣的樣品製備與有機酸基質在小分子量(小於500 Da)的區域造成干擾,使得分析技術上受限。
【0004】因此,業界極須發展一種利用薄層層析質譜分析化合物的方法,此方法簡單且快速,且不需要外加基質,能有效分析低分子量的待測物。
[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.
【0005】本發明提供一種利用薄層層析質譜分析化合物的方法,包括以下步驟:(a) 提供一樣品,其中該樣品溶於一分離溶液中且該樣品中具有複數個化合物;(b) 將該樣品點附於一基板(plate)上,藉由一薄層層析法(thin layer chromatography)分離該樣品,以得到複數個分析點,其中每一個分析點包括至少一個化合物;(c) 將一奈米粒子溶液滴於該些分析點,使該些化合物吸附於該奈米粒子溶液中;(d) 對該基板進行一乾燥步驟;以及(e)將吸附上該些化合物的該基板直接置入一質譜儀中,以進行質譜分析。
【0006】為讓本發明之上述和其他目的、特徵、和優點能更明顯易懂,下文特舉出較佳實施例,並配合所附圖式,作詳細說明如下:
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...基板12. . . Substrate
14...滴管14. . . dropper
16...奈米粒子溶液16. . . Nanoparticle solution
16a...金屬奈米粒子16a. . . Metal nanoparticle
16b...有機酸16b. . . Organic acid
S1、S2、S3...分析點S1, S2, S3. . . Analysis point
【0007】
第1A~1D圖為一系列剖面圖,用以說明本發明之利用薄層層析質譜分析化合物的方法之流程圖。
第2A-2H圖分別顯示本發明實施例2的質譜圖。
第3圖顯示實施例3之質譜圖。
第4A ~4F圖分別顯示比較例1、比較例2與實施例4之質譜圖。
第5A圖顯示本發明實施例5之中間產物(intermediate III)之質譜圖。
第5B圖顯示本發明實施例5之產物(product IV)之質譜圖。
第6A~6B圖顯示本發明實施例偵測天然物之質譜圖。
【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.
【0008】 本發明提供一種利用薄層層析質譜分析化合物的方法,包括以下步驟(a)-步驟(e)。第1A~1D圖為一系列剖面圖,用以說明本發明之利用薄層層析質譜分析化合物的方法之流程圖。首先,進行步驟(a),提供樣品,其中樣品溶於分離溶液中且樣品中具有複數個化合物。此處所謂「樣品」即為待測的樣品,待測樣品中通常具有許多不同的化合物,可藉由後續的分析方法將其分離。
【0009】此外,步驟(a)之樣品可來自於化學反應過程中的反應物(reactant)、中間產物(intermediate)或產物(product),以及時(real time)監控化學反應。
【0010】 分離溶液包括二氯甲烷(dichloromethane, DCM)、甲醇(methanol)、乙酸乙酯(ethyl acetate, EA)、正己烷(n-hexane)、丙酮(acetone)、氯仿(chloroform)、甲苯(toluene)或水。然而,分離溶液並不限於上述提及之溶劑,本領域人士可依據實際應用之需求,選擇所需的分離溶液。
【0011】之後,進行步驟(b),將樣品點附於基板(plate)上,藉由薄層層析法(thin layer chromatography)分離該樣品,以得到複數個分析點,其中每一個分析點包括至少一個化合物。請參見第1A圖,其顯示經過薄層層析法(thin layer chromatography)後,基板12之上具有複數個分析點S1, S2, S3。
【0012】基板12包括鋁片(alumina)、矽膠片(silica gel)或玻璃基板。於一較佳實施例中,可直接使用市售TLC板(例如矽膠片)作為本發明之基板,並不需要特別修飾此基板。
【0013】薄層層析法(TLC)是一種層析法(chromatography),主要利用矽膠(silica gel)或鋁片(alumina)作為層析靜相(stationary phase),再將樣品點附在此薄層靜相上,以液體展開劑作為流動相(mobile phase),透過毛細作用由下往上移動,以分離出各種不同成分。此外,薄層層析法(TLC) 還可以用於追蹤化學反應的進行程度。
【0014】於步驟(b)中,由於樣品本身可能是透明無色,而分析點S1、S2、S3也可能是透明無色,因此,本發明之利用薄層層析質譜分析化合物的方法尚包括利用紫外光(UV)或是染劑(staining buffer)觀察該些分析點S1、S2、S3。
【0015】接著,進行步驟(c),請參見第1B圖,利用滴管14將奈米粒子溶液16滴於該些分析點S1、S2、S3,使該些化合物吸附於奈米粒子溶液16中。請參見第1C圖,奈米粒子溶液16中包括複數個金屬奈米粒子16a與修飾於該些金屬奈米粒子16a上的有機酸16b。
【0016】 該些金屬奈米粒子16a包括鐵(Fe)、鈷(Co)、鎳(Ni)、金(Au)、銀(Ag)或上述之組合。該些有機酸16b包括2,5-二羥基苯甲酸(2,5-dihydroxybenzoic acid, DHB)、3,5-二甲氧基-4-羟基苯丙烯酸(3,5-dimethoxy-4-hydroxycinnamic acid, SA)、α-氰基-4-羟基肉桂酸(α-Cyano-4-hydroxycinnamic acid, α-CHC)、3-(4-羥基-3-甲氧苯基)-2-丙烯酸)[(E)-3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid,Ferulic acid] 、吡啶甲酸(pyridine-2-carboxylic acid, Picolinic acid)、3-羟基-2-吡啶甲酸(3-hydroxypyridine-2-carboxylic acid, 3-HPA)、2-(對羥基苯偶氮)苯甲酸[2-(4'-Hydroxybenzeneazo)benzoic acid, HABA]。
【0017】 於一較佳實施例中,金屬奈米粒子16a具有磁性,例如鐵(Fe)、鈷(Co)、鎳(Ni)。使用磁性金屬奈米粒子之優點在於,後續可回收再利用磁性金屬奈米粒子,以節省製程成本。
【0018】於一實施例中,可先將2,5-二羥基苯甲酸(2,5-dihydroxybenzoic acid, DHB)修飾到氧化鐵(Fe3O4)奈米粒子上,之後,添加適當的溶劑,以形成奈米粒子溶液16。
【0019】 須注意的是,當奈米粒子溶液16滴於該些分析點S1、S2、S3時,因為金屬奈米粒子16a上修飾上有機酸16b,使金屬奈米粒子16a表面具有特定官能基,例如芳香環(aromatic rings)、羧酸鹽(carboxylate)、羥鹽(hydroxyl group),這些官能基與欲分析的化合物之間可產生電荷-電荷作用力(charge-charge interaction)、氫鍵(hydrogen bonding)與疏水效應(hydrophobic effect),因此,使化合物被吸附於奈米粒子溶液16中的金屬奈米粒子16a上,因而達到萃取濃縮步驟,進行提高偵測靈敏度。另外,溶劑的選擇會影響萃取(extraction)濃縮的效果,可依據實際應用之需求,選擇極性與所欲分析的化合物極性接近的溶劑,以提高濃縮的效果。
【0020】之後,進行步驟(d),對基板12進行一乾燥步驟。乾燥步驟例如使用氮氣吹乾(purge)基板12。
【0021】接著,進行步驟(e),請參見第1C圖,將吸附上該些化合物的基板12直接置入質譜儀中,以進行質譜分析。質譜儀包括基質輔助雷射脫附離子化質譜儀(matrix-assisted laser desorption ionization, MALDI)。第1D圖顯示質譜圖,X軸代表化合物的質量電荷比(m/z),Y軸代表訊號強度(intensity)。
【0022】須注意的是,進行步驟(e)之前,由於金屬奈米粒子16a上修飾的有機酸16b可扮演基質的作用,亦即,能幫助能量轉移,因此,不需要額外添加基質(matrix)到基板上。另言之,本發明可”直接”將基板置於質譜儀中,不需要經過複雜的樣品萃取過程,可節省製程成本與時間。
【0023】於習知技術中,進行基質輔助雷射脫附離子化質譜儀(matrix-assisted laser desorption ionization, MALDI)時,偵測低分子量時(分子量小於500 Da)通常會受到基質(matrix)的干擾,而影響分析結果。相較於習知技術,本發明所使用的有機酸修飾於奈米粒子溶液中的金屬奈米粒子上,因此,有機酸雖然可扮演基質的角色(可幫助質子轉移),但是卻不會產生基質的干擾,因此,可使本發明之方法有效應用於偵測低分子量之化合物,且可達到較佳的偵測靈敏度。
【0024】本發明所偵測的該些化合物之分子量為約100-2000 Da,較佳為約100-500 Da。本發明之分析方法其偵測靈敏度可達到pmole(10-15mole)等級。
【0025】本發明所揭露之利用薄層層析質譜分析化合物的方法,其中化合物包括有機分子、生物分子或無機物分子。有機分子包括酸類分子(acid)、酯類分子(ester)、醛類分子(aldehyde)、烷類分子(alkane)、烯類分子(alkene)、炔類分子(alkyne)、胺類分子(amine)、醯胺類分子(amide)、芳香烴化合物(aromatic hydrocarbons)、疊氮化合物(azide)、雜環化合物(heterocyclic compound)、硫化物(sulfide)、矽氧化合物(silane)、天然物(natural product)或有機催化劑中間體(intermediate)。
【0026】此外,有機分子亦包括各種毒品,例如鴉片(opium)、安非他命(amphetamine)、嗎啡(morphine)、海洛因(heroin)、大麻(marijuana)、古柯鹼(cocaine)、搖頭丸(3,4-methylenedioxy-methamphetamine, MDMA)、K他命(ketamine)等。
【0027】生物分子包括蛋白質(protein)、胜肽(peptide)、去氧核醣核酸(deoxyribonucleic acid, DNA)/核醣核酸(ribonucleic acid, RNA)與其代謝物(metabolism)、脂質(lipid)、膽固醇(cholesterol)與其代謝物、單醣分子(saccharide)、雙醣分子(disaccharide)、醣脂質(glycolipid)、醣胜肽(glycopeptide)、維他命(vitamine)、生物素(biotin)、神經傳導分子(neurotransmitter)、抗生素(antibiotic)。
【0028】 無機物分子包括金屬錯合物(metal complex)或金屬催化劑中間體(catalyst intermediate)。
【0029】須注意的是,習知技術中,由於代謝物中通常含有鹽類(如鈉、鉀),這些鹽類通常會干擾質譜分析結果。相較於習知技術,本發明之實施例較佳可應用於偵測小分子量的代謝物,因為本發明進行薄層層析法(thin layer chromatography, TLC),鹽類不會溶解於分離溶液中,因此,可去除鹽類的干擾。
【0030】於一實施例中,本發明之利用薄層層析質譜分析化合物的方法可應用於監控親核取代反應(nucleophilic substitution reaction)的過程,當起始物甲氧基三乙二醇(methoxyl triethylene glycol, C7H16O4,分子量164.1)與2-氯-2-苯基乙烯氯(2-chloro-2-phenylacetyl chloride)反應後,會得到酯類產物(C15H21ClO5,分子量316.1)。可由基質輔助雷射脫附離子化質譜儀(matrix-assisted laser desorption ionization, MALDI)偵測到起始物與產物。
【0031】於另一實施例中,本發明之利用薄層層析質譜分析化合物的方法可應用於監控4-二甲氨基吡啶(4-dimethylaminopyridine, DMAP)所催化的醯化反應(acylation),可藉由本發明所揭露之方法偵測到中間產物(intermediate),以及時(real time)監控化學反應,並判斷反應完成與否。
【0032】綜上所述,本發明提供一種利用薄層層析質譜分析化合物的方法,其結合薄層層析法(thin layer chromatography)與質譜法,利用薄層層析法進行分離,結合質譜偵測化合物的分子量,並藉由將奈米粒子溶液滴於基板上,以萃取濃縮化合物,於不需要額外添加基質的條件下,直接將基板進行質譜分析,此方法可應用於分析各種有機分子、生物分子或無機物分子,特別是針對低分子量(100-500 Da)的小分子。此外,本發明所提供之利用薄層層析質譜分析化合物的方法可應用於及時(real time)監控化學反應,於化學反應過程中,可偵測反應物(reactant)、中間產物(intermediate)或產物(product),以判斷反應進行完全與否。
【0033】【實施例】
【0034】實施例1製備修飾上酸的金屬奈米粒子(DHB@MNPs)
(1) 將FeCl2‧4H2O與FeCl3依序加入鹽酸(HCl)水溶液中(經過去氧處理),攪拌混合並反應2小時,以得到混合物A。
(2) 將步驟(1)之混合物A緩慢加入氫氧化鈉(NaOH)水溶液中(經過去氧處理),得到混合物B。
(3) 將混合物B分裝到離心管(centrifuge tube),並加入二次去離子水(dd-water)到離心管中,震盪均勻後,去除水溶液層。
(4) 將0.01 N鹽酸水溶液(HCl)緩慢滴入離心管並攪拌2分鐘,去除上清液(supermatnat)後,最後再加入二次去離子水回溶,得到氧化鐵奈米粒子溶液(Fe3O4@MNP)。
(5) 取氧化鐵奈米粒子溶液置於單頸瓶中,再加入丙醇,以高速震盪30分鐘,以得到混合物C。
(6) 將四乙氧基矽烷(Tetraethylorthosilicate, TEOS)與28-30%的氨水(NH4OH)加入混合物C中,於60℃下反應2小時,以得到混合物D。
(7) 將混合物D依序以丙醇和二次去離子水進行清洗,並用高速離心去除上清液,重複此步驟3次,以得到混合物E。
(8) 製備有機酸:將2,5-二羥基苯甲酸(2,5-dihydroxybenzoic acid, DHB)溶於3 ml二次去離子水中,並加入 0.01 NHC1,以得到混合物F。
(9) 將混合物E與混合物F於室溫下反應12小時,以得到混合物G。將混合物G依序以丙醇和二次去離子水進行清洗,並用高速離心去除上清液,以得到混合物H。再加入0.1 N NaOH於混合物H中,依序以丙醇和二次去離子水進行清洗,最後以高真空進行乾燥,以得到金屬奈米粒子。
【0035】 實施例2 偵測不同種類的化合物
取實施例1之金屬奈米粒子,以溶劑回溶金屬奈米粒子,製作成1000 ppm的奈米粒子溶液,溶劑例如二次去離子水、甲醇、乙晴(acetonitrile, ACN)或1,4-二氧雜環己烷(1,4-dioxane)。
將表1的各種不同樣品點附於TLC板上,藉由薄層層析法(thin layer chromatography)分離該些化合物並得到複數個分析點,分離溶液顯示於表1中。
之後,將1000 ppm的奈米粒子溶液滴於該些分析點,使該些化合物吸附於奈米粒子溶液中。之後,對TLC板進行一乾燥步驟。
接著,以導電膠脫附於TLC板背面,並黏著於不鏽鋼(stainless)樣品盤上,將TLC板直接置入一質譜儀中,以進行質譜分析。第2A-2H圖分別顯示樣品標號1-8的質譜圖。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.
表1
Table 1
※註:化合物在TLC板上升的高度與分離溶液上升高度的比值,稱為Rf值。
由第2G圖可知,本發明之分析方法用於偵測膽固醇時,其靈敏度為100 fg,0.26 fmol(S/N ratio=4)。
由第2H圖可知,本發明之分析方法用於偵測磺胺類時,其靈敏度為1 ng,2 pmol(S/N ratio=3)。
【0036】 實施例3
實施例3之金屬奈米粒子類似於實施例1,差別僅在於有機酸改為α-氰基-4-羟基肉桂酸(α-Cyano-4-hydroxycinnamic acid, α-CHC)。
將實施例3之奈米溶液以溶劑回溶,製作1000 ppm的奈米粒子溶液。
之後,將樣品點附於TLC板上,藉由薄層層析法(thin layer chromatography)分離該些化合物並得到單一個分析點。
之後,將1000 ppm的奈米粒子溶液滴於該些分析點,使該些化合物吸附於奈米粒子溶液中。之後,對TLC板進行一乾燥步驟。
接著,以導電膠黏貼於TLC板背面,並黏著於不鏽鋼(stainless)樣品盤上,將TLC板直接置入一質譜儀中,以進行質譜分析。
第3圖顯示偵測到山梨醣醇(sorbitol)的質譜圖。
【0037】實施例4
請參見反應機制1,起始物為甲氧基三乙二醇(methoxyl triethylene glycol, C7H16O4,分子量164.1)與2-氯-2-苯基乙烯氯(2-chloro-2-phenylacetyl chloride)反應後,會得到酯類產物(C15H21ClO5,分子量316.1)。
反應機制1
請參見表2,其顯示起始物(compound I)與產物(compound II)使用不同分析方法所偵測到的質譜分析結構。*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.
表2
Table 2
請參見第4A圖與第4D圖,其分別顯示起始物與產物經由薄層層析法(TLC)分離之後,於不添加任何基質的條件下,由質譜分析(MALDI MS)所得到之訊號圖。由第4A圖與第4D圖中得知,由於並未使用任何基質,所以無法偵測到任何訊號。
請參見第4B圖與第4E圖,其分別顯示起始物與產物經由薄層層析法分離之後,於添加DHB作為基質的條件下,由質譜分析所得到之訊號圖。由第4B圖與第4E圖中得知,使用DHB作為基質,由於DHB的碎片(fragments)會產生很多訊號,因此,背景訊號會干擾所欲分析的化合物的訊號。
請參見第4C圖與第4F圖,其分別顯示起始物與產物經由薄層層析法分離之後,將奈米粒子溶液(DHB@MNPs)滴於起始物與產物之上,由質譜分析所得到之訊號圖。由第4C圖中可觀察到起始物結合鈉的訊號(m/z=187.1),由第4F圖中可觀察到產物結合鈉的訊號(m/z=339.1)。
請參見表3,其顯示化合物I與化合物II之偵測靈敏度。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.
表3
table 3
【0038】 實施例5 及時(real time)偵測化學反應
反應機制2
反應機制2顯示由4-二甲氨基吡啶(4-dimethylaminopyridine, DMAP)所催化的醯化反應(acylation),可藉由本發明之分析方法監控反應過程中的中間產物(intermediate III),以及監控產物(product IV)。
第5A圖顯示中間產物(intermediate III)之質譜圖,第5B圖顯示產物(product IV)之質譜圖。由此可知,本發明所揭露之化合物之分析方法可應用於及時(real time)監控化學反應,以判斷反應完成與否。
【0039】 實施例6 偵測天然物
取天然物錫蘭七指蕨(Helminthostachys zeylanica)的根莖(rhizomes)進行分析,可得到一系列的抗發炎的類黃酮素家族(anti-inflammatory flavonoid family),包括黃酮類化合物(ugonins J-X)。
第6A圖顯示ugonins J之質譜圖。第6B圖顯示ugonins L之質譜圖。
【0040】雖然本發明已以數個較佳實施例揭露如上,然其並非用以限定本發明,任何所屬技術領域中具有通常知識者,在不脫離本發明之精神和範圍內,當可作任意之更動與潤飾,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。
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...基板12. . . Substrate
14...滴管14. . . dropper
16...奈米粒子溶液16. . . Nanoparticle solution
S1、S2、S3...分析點S1, S2, S3. . . Analysis point
Claims (13)
(a)提供一樣品,其中該樣品溶於一分離溶液中且該樣品中具有複數個化合物;
(b) 將該樣品點附於一基板(plate)上,藉由一薄層層析法(thin layer chromatography)分離該樣品,以得到複數個分析點,其中每一個分析點包括至少一個化合物;
(c) 將一奈米粒子溶液滴於該些分析點,使該些化合物吸附於該奈米粒子溶液中;
(d) 對該基板進行一乾燥步驟;以及
(e) 將吸附上該些化合物的該基板直接置入一質譜儀中,以進行質譜分析。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.
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CN106932524A (en) * | 2015-12-30 | 2017-07-07 | 中国科学院化学研究所 | Thin layer of liquid chromatograph-mass spectrometer coupling device, purposes and detection method |
CN111060645A (en) * | 2019-12-25 | 2020-04-24 | 安徽中科赛飞尔科技有限公司 | SERS detection method for adulterated drugs |
CN111189954A (en) * | 2019-12-28 | 2020-05-22 | 安徽中科赛飞尔科技有限公司 | Method for detecting drugs in hair by TLC-SERS |
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CN106932524A (en) * | 2015-12-30 | 2017-07-07 | 中国科学院化学研究所 | Thin layer of liquid chromatograph-mass spectrometer coupling device, purposes and detection method |
CN106932524B (en) * | 2015-12-30 | 2018-11-27 | 中国科学院化学研究所 | Thin layer of liquid chromatograph-mass spectrometer coupling device, purposes and detection method |
CN111060645A (en) * | 2019-12-25 | 2020-04-24 | 安徽中科赛飞尔科技有限公司 | SERS detection method for adulterated drugs |
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