201245711 * 六、發明說明： • 【發明所屬之技術領域】 本發明是有關於一種樣品成分的分析方法，特別是指 一種以直接電喷灑游離質譜法鑑別樣品成分的分析方法。 【先前技術】 藉由質譜分析技術’人們可獲知一樣品中待測物 (analytes)的分子量，繼而配合進一步比對而確認該待測物 的真貫身为’因此自2 0世紀初期發展以來，用以實施該質 譜分析技術的質譜儀，因為具有操作簡便且可快速獲得摘 測結果之優勢’已然成為一廣為各領域使用之鑑定工具。 申請人一直致力於質譜分析之領域中，於1999年已發 展出直接電喷嚨游離探針（Direct Electrospray pr〇be，DPE)， 其做法是以銅線圍繞成一具尖端的銅線圈，將待分析之液 態樣品直接滴在銅線圈上，並施加高電壓，讓樣品在銅線 圈尖端產生電喷灑現象，進而游離分析物。另於同年將前 述技術進行改良，其做法是將兩根光纖並排，並在末端纏 繞金屬絲’再將液態樣品置於金屬絲上，因液體表面張力 的關係’樣品會附著在金屬絲上，並施加高電壓使樣品受 到電動力的驅使而延著兩光纖所形成的管道前進，最後在 末端產生電喷灑並游離分析物。於2〇〇1年本案申請人亦提 出以尖端的界面可以有效率進行電噴灑游離概念，並發展 出適用在微晶片（microchip)上的電喷灑游離界面。本案申請 人另於2003年及2005年也分別提出與電喷灑游離相關之 蜗艾研九（Rapid Commun. Mass Spectrom. ·\709-\Ί\3 201245711 ;以及 2005，77，8170-8173) 〇 另外’與直接電喷嚨游離探針技術相關的分析方法， 最著名的便是2007年由Hiraoka教授所發展的探針式電喷 灑游離法（Probe Electrospray Ionization，PESI)，此分析方法 疋先以金属針沾黏液態的分析物，再移至質譜入口前端並 施加一高電壓’使金屬針尖端的分析物產生電喷灑，進而 讓分析物游離。 然而，上述技術皆需要使用液態的分析物，也就是說 ，若分析物為固態，則需要先將分析物進行萃取，讓待分 析之成分溶於液體中，才能進行游離分析，需要繁複的前 置作業，使用上相當不便。 【發明内容】 因此，本發明之目的，即在提供一種操作便利，用能 快速更換樣品之以直接電喷灑游離質譜法鑑別樣品成分的 分析方法。 於是，本發明以直接電喷灑游離質譜法鑑別樣品成分 的分析方法，包含一準備步驟，及一分析步驟。 該準備步驟將-待測樣品裁切出一尖端，並將該待測 樣品固定於一設置有電極的載台纟，使該待測樣品與電極 接觸’且尖端是朝向一質譜儀的入口。 “該分析步驟是將萃取液滴》該待測樣品及電極上，使 卒取液將㈣測樣品内的成分溶出，再經由電極施加高壓 電’使在該待測樣品尖端的萃取液因高電場而產生電噴灑 ’讓含有該待測樣品成分的萃取液由該人口進人質譜儀中 201245711 進行分析。本發明之功效在於，不需要將待測樣品進行前 處理’只要將該待測樣品裁切出一尖端，就能直接進行成 刀分析，且待測樣品的更換快速，能應用於大量樣。 檢測。 时级二 【實施方式】 有關本發明之前述及其他技術内容、特點與功效，在 以下配合參考圖式之一個較佳實施例的詳細說明中，將可 清楚的呈現。 參閱圖1，A本發明以直接電喷灌游離質譜法鑑別樣品 成分的分析方法之較佳實施例，包含一準備步驟21，及一 分析步驟22。 參閱圖2、3並配合圖！，該準備步驟21將一待測樣品 3。裁切出一尖# 31 ’並將該待測樣品3固定於一設置有電 極4的載台5 Λ ’使該待測樣品3與電極*接觸，且尖端 ?是朝向-質譜儀6的入口 61。於本實施例中，是以兩片 昼克力板作為載台5，但不以此為限，另外，於本實施例中 該待測樣品3是以植物葉片為例說明。 1析步驟22是將萃取液滴於該待測樣品3及電極4 使萃取液將该待測樣品3内的成分溶出，再經由電極* ▲门壓電（6KV) ’使在該待測樣品3之尖端3 i的萃取液 nr電％而產生電噴灑’讓含有該待測樣& 3成分的萃取 k入口 61進入質譜儀6中進行分析。參閱圖4，是使 的50 /。甲醇水溶液作為萃取液直接於甜菊葉 片上進仃電喷灑所得的配醣體之質譜圖，其中圖私及圖朴 201245711 是分別以質譜正離子及負離子模式進行掃描所得之離子訊 號，所偵測到配醣體正離子分別有m/z 1〇〇5 3、m/z843 3、 m/z 767.4、m/z 725.4及m/z68l 3等，而負離子訊號則有 m/z 1051.3、m/z 1001.3、m/z 958 4、_ 9〇1、_ 889 2、 m/z 839.2 ^ m/z 809.2 ^ m/z 772.6 > m/z 667.1 A m/z 506.0 〇 參閱圖5,為直接於甜菊葉片上進行電喷灑所的配醣體 之質譜/質譜（MS/MS)圖，其中圖5a是以分析物離子m/z 843進行MS/MS分析結果，可得m/z 681 2之碎片離子，而 圖5b則是分析物離子m/z 1〇〇5進行MS/MS分析結果可 得m/Z 843_1及m/z 681」之碎片離子。要說明的是，一次 質譜（MS)分析可提供分析物質量資訊，而質譜/質譜 (MS/MS)分析可提供質量碎片的資訊，因此可做為分析物結 構鑑定的依據，此部分為熟知該項技藝者所能輕易理解， 不再予以贅述。 參閱圖6a，是使用1〇吣的8〇 %甲醇水溶液作為萃 取液，直接於夹竹桃葉片上進行電喷灑所得之質譜圖，可 獲知其内郤成分包含〇d〇r〇side(m/z 555.2及m/z 573.2)、 〇leandrm(m/z 599.2 及 m/z 615·2)及 Neritaloside (m/z 631.2) 專離子Λ號。而圖6b是〇leandrin分析物離子（m/z 599)進 行質譜質譜分析所得之圖譜，可獲得m/z 539·1及m/z 455.ι 之碎月離子訊號圖。 參閱圖7，本分析方法亦探討不同溶液組成對於直接於 爽竹桃葉片上進行電喷灑所得訊號之影響，所選用的溶液 組成分別有（a)5〇 %乙腈（acet〇nitrUe，ACN)水溶液 6 201245711 (ACN/H20)、（b)50 % 丙酮水溶液（Acetone/H20)，及（c)50 % 異丙醇（isopropanol，IPA)水溶液（IPA/H20)。如圖7所示， 為分別以lOpL的上述溶液萃取並分析夾竹桃葉片所得之總 離子層析圖（Total i〇n chromatogram，TIC)，離子訊號持續的 時間分別為圖7a : 3.7分、圖7b : 4_0分，及圖7c : 4.7分 ，其中，如圖7c所示，以50%IPA/H2〇所得分析時間持續 最長且離子訊號也相較其他二者穩定。 參閱圖8，則是本分析方法的再現性測試。分別以相同 溶劑體積（10μί)的50%甲醇水溶液（Me〇H/H2〇)，並直接以 電喷灑游離質譜法連續分析夾竹桃葉片五次，由所得之總 離子層析圖可成功獲得連續五次的離子強度變化。 參閱圖9直接分析同一片葉片（_£>少 vw.pzha)但不同部位葉片組織所得之離子訊號，圖知是分 析葉面、綠色部位’圖9b則是分析葉面白色部位，確實獲得 不同離子訊號之分布，而圖9e則是同_片葉子枯萎後之葉 面所獲付的分析結果，由圖9a〜e之訊號分析比較可知，不 同位、不同生長狀態，均能由内部分子組成分布區別。 參閱圖10,為本分析方法在區別植物病蟲害的測試， 圖l〇a所取的分析樣品是正常芒果葉片，而冑鳩則為有病 蟲害的亡果葉片’ *質譜訊號就可區別出其差異性，如圖 l〇b所不，有病蟲害的芒果葉片在〜之間— 全沒有離子訊號分布。 疋 參閱圖11，為本分析方法測試植物表面是否有農藥殘 ;圖11巾是以陶斯松（Chl〇rpyrif〇s)為例測試挪試方 201245711 法是將農藥稀釋1000倍後以霧化器均勻噴灑於植物表面， 乾燥後再取樣測試。圖11 a為陶斯松訊號，圖i lt)為植物無 喷藥部位，圖11c為喷藥部位。由此可知，喷灑於植物表面 的農藥乾燥後仍能測得其存在。 參閱圖12，為本分析方法測試植物内是否有農藥殘留 ，於圖12中是以嘉磷塞（Glyphosate)為例測試’測試方法是 將農藥稀釋1000倍後讓植物吸收3小時及6小時後分別採 樣測試。圖12a為嘉磷塞訊號，圖12b為背景值，圖12c為 3小時後取樣，圖12d為6小時後取樣。由此可知，即使農 藥不是在植物表面而是被植物吸收之内部，仍能測出農藥 的存在。 另外要特別說明的是，待測樣品3為植物葉片時，可 以不需要由枝幹上取下，直接裁剪出該尖端31後，使待測 樣品3能财置於該載台5上就能測試分析，甚至是若待 測樣叩3纟身已有尖銳處，也可不需由枝幹上取下或裁剪 而直接被夾置於該載台5就能測試分析。 综上所述，本發明以直接電喷灑游離質譜法鑑別樣 成分的分析方法，只要將待測樣& 3直接裁剪出—尖端3 並夾置於該載台5上而與電極4接觸，再滴上萃取液』 通電1 4直接於待測樣品3之尖端3ι產生尖端放電， 而供質-曰儀6分析樣品成分’故確實能達成本發明之目# 〇 ^ …惟以上所述者，僅為本發明之較佳實施例而已，當不 月b以此限定本發明實 β貫施之範ϋ，即大凡依本發明申請專利 8 201245711 範圍及發明說明内容所作之簡單的等效變化與修飾，皆仍 屬本發明專利涵蓋之範圍内。 【圖式簡單說明】 圖1是一流程圖，說明本發明以直接電喷灑游離質譜 法鑑別樣品成分的分析方法之較佳實施例； 圖2是一俯視示意圖，說明該較佳實施例的操作方法 圖3是一侧視示意圖，輔助說明圖2 ; 圖4a、4b是質譜圖，分別以正離子及負離子模式於甜 菊葉片上進行電喷灑所得之離子訊號； 圖5a、5b是質譜/質譜圖，分別以分析物離子m/z 843 及m/z 1005進行分析； 圖6a是一質譜圖，於夾竹桃葉片上進行電喷灑所得之 離子訊號； 圖6b是一質谱/質譜圖，以分析物離子進行分 析； 圖7a〜c疋質谱目，說明不同溶液組成對於直接於夹竹 桃葉片上進行電喷灑所得訊號之影響； 圖8是一質谱圖，是於夾竹桃葉片上進行電喷灌所得 訊號之再現性測試； 不同部位及不同生長 圖9a〜c是質譜圖，說明同一葉片 時期之内部成分的差異； 圖 訊號； l〇a、l〇b是質譜圖 說明正常與病蟲害葉片之質譜 201245711 圖11 a〜c是質譜圖，說明測試待測物品表面之農藥的結 果；及 圖12a〜d是質譜圖，說明測試吸收於待測物品内部之 農藥的結果。 10 201245711 【主要元件符號說明】 21 準備步驟 4 電極 22 分析步驟 5 載台 3 待測樣品 6 質譜儀 31 尖端 61 入口201245711 * VI. Description of the invention: • Technical field to which the invention pertains The present invention relates to a method for analyzing a sample component, and more particularly to an analytical method for identifying a component of a sample by direct electrospray ionization mass spectrometry. [Prior Art] By mass spectrometry technology, one can know the molecular weight of an analyte (analytes) in a sample, and then cooperate with further comparison to confirm that the true cross-section of the analyte is 'so since the early 20th century The mass spectrometer used to carry out the mass spectrometry technique has become an identification tool widely used in various fields because of its advantages of being easy to operate and quick to obtain the result of the measurement. Applicants have been working in the field of mass spectrometry. In 1999, Direct Electrospray pr〇be (DPE) was developed, which is a copper wire wound around a tipped copper coil. The analyzed liquid sample was dropped directly onto the copper coil and a high voltage was applied to cause the sample to electrospray at the tip of the copper coil, thereby freeing the analyte. In the same year, the above technology was improved by arranging two fibers side by side and winding the wire at the end and then placing the liquid sample on the wire. The sample will adhere to the wire due to the surface tension of the liquid. And applying a high voltage causes the sample to be driven by the electrodynamic force to advance along the tube formed by the two fibers, and finally an electric spray is generated at the end and the analyte is released. In 2001, the applicant also proposed that the concept of electrospraying can be efficiently carried out with a sophisticated interface, and an electrospray free interface suitable for use on a microchip is developed. In 2003 and 2005, the applicant also proposed the ventilator Ai Jiujiu (Rapid Commun. Mass Spectrom. · \709-\Ί\3 201245711; and 2005, 77, 8170-8173) 〇In addition, the analytical method related to the direct electrospray ion free probe technology, the most famous is the probe electrospray Ionization (PESI) developed by Professor Hiraoka in 2007. The analyte is first viscous with a metal needle and then moved to the front end of the mass spectrometer and a high voltage is applied to electrospray the analyte at the tip of the metal needle to free the analyte. However, all of the above techniques require the use of a liquid analyte, that is, if the analyte is solid, the analyte needs to be extracted first, and the component to be analyzed is dissolved in the liquid before the free analysis can be performed. It is quite inconvenient to use the homework. SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide an analytical method for identifying a sample component by direct electrospray ionization mass spectrometry using a rapid exchange of samples. Thus, the present invention analyzes a sample component by direct electrospray ionization mass spectrometry, and includes a preparation step and an analysis step. The preparation step cuts the sample to be tested out of a tip, and fixes the sample to be tested to a stage provided with an electrode such that the sample to be tested is in contact with the electrode and the tip is toward the entrance of a mass spectrometer. "The analysis step is to extract the droplets" on the sample to be tested and the electrode, so that the stroke liquid will dissolve the components in the sample (4), and then apply high voltage electricity through the electrode to make the extract at the tip of the sample to be tested The electric field is generated by a high electric field. The extract containing the sample component to be tested is analyzed by the population into the mass spectrometer 201245711. The effect of the present invention is that the sample to be tested does not need to be pretreated as long as the sample is to be tested. When the sample is cut out, the cutting analysis can be directly performed, and the sample to be tested can be quickly replaced and can be applied to a large number of samples. Detection. Time 2 [Embodiment] The foregoing and other technical contents and features of the present invention are related to The effect will be clearly shown in the following detailed description of a preferred embodiment of the reference drawings. Referring to Figure 1, a preferred embodiment of the present invention for analyzing sample components by direct electrospray ionization mass spectrometry , comprising a preparation step 21 and an analysis step 22. Referring to Figures 2 and 3 and in conjunction with the figure!, the preparation step 21 will take a sample to be tested 3. Cut a tip # 31 ' and The sample to be tested 3 is fixed to a stage 5 provided with an electrode 4 Λ 'to make the sample 3 to be tested in contact with the electrode *, and the tip ? is an inlet 61 toward the mass spectrometer 6. In this embodiment, two are The sheet of the sheet is used as the stage 5, but is not limited thereto. In addition, in the embodiment, the sample to be tested 3 is described by taking a plant leaf as an example. The sample 3 and the electrode 4 cause the extract to dissolve the components in the sample 3 to be tested, and then generate the nr electric % of the extract at the tip 3 i of the sample to be tested 3 via the electrode * ▲ gate piezoelectric (6 kV) Electrospray 'The extraction k inlet 61 containing the sample to be tested & 3 is entered into the mass spectrometer 6 for analysis. Referring to Figure 4, the 50 / methanol aqueous solution is used as an extract directly onto the stevia leaf. The mass spectrum of the glycoside obtained by sprinkling, in which Tutu and Tupu 201245711 are ion signals scanned by mass spectrometry positive ion and negative ion modes, respectively, the detected glycoside positive ions have m/z 1〇〇, respectively. 5 3, m / z 843 3, m / z 767.4, m / z 725.4 and m / z 68l 3, etc., while the negative ion signal has m / z 1051.3, m/z 1001.3, m/z 958 4, _ 9〇1, _ 889 2, m/z 839.2 ^ m/z 809.2 ^ m/z 772.6 > m/z 667.1 A m/z 506.0 〇 Figure 5 is a mass spectrometry/mass spectrometry (MS/MS) image of a glycoside directly electrophoresed on stevia leaves, wherein Figure 5a is the result of MS/MS analysis of analyte ion m/z 843, available The fragment ion of m/z 681 2, and the fragment ion of m/Z 843_1 and m/z 681” can be obtained by MS/MS analysis of the analyte ion m/z 1〇〇5. It should be noted that primary mass spectrometry (MS) analysis provides information on analyte quality, while mass spectrometry/mass spectrometry (MS/MS) analysis provides information on mass fragmentation and can therefore be used as a basis for analyte structure identification. This artist can easily understand and will not repeat them. Referring to Fig. 6a, a mass spectrum obtained by electrospraying directly on an oleander leaf using an aqueous solution of 8 〇% methanol as a extract, can be found that the internal component contains 〇d〇r〇side (m/). z 555.2 and m/z 573.2), 〇leandrm (m/z 599.2 and m/z 615·2) and Neritaloside (m/z 631.2). Figure 6b shows the spectrum of the 〇leandrin analyte ion (m/z 599) by mass spectrometry. The fragmentation signal of m/z 539·1 and m/z 455.ι can be obtained. Referring to Figure 7, the analysis method also investigates the effect of different solution compositions on the signals directly electrophoresed on the leaves of the saplings. The selected solution consists of (a) 5% acetonitrile (ACN) aqueous solution 6 201245711 (ACN/H20), (b) 50% aqueous acetone solution (Acetone/H20), and (c) 50% isopropanol (IPA) aqueous solution (IPA/H20). As shown in Fig. 7, the total ion chromatogram (TIC) obtained by extracting and analyzing the oleander leaves with the above solution of lOpL, respectively, the ion signal duration is as shown in Fig. 7a: 3.7, Fig. 7b 4_0 points, and Fig. 7c: 4.7 points, wherein, as shown in Fig. 7c, the analysis time with 50% IPA/H2 持续 lasts the longest and the ion signal is stable compared to the other two. Referring to Figure 8, the reproducibility test of the analytical method is shown. The oleander leaves were continuously analyzed five times with 50% aqueous methanol solution (Me〇H/H2〇) in the same solvent volume (10 μί), and directly by electrospray ionization mass spectrometry. The total ion chromatogram obtained was successfully obtained. Five times the change in ionic strength. Refer to Figure 9 to directly analyze the same leaf (_£> less vw.pzha) but the ion signal obtained from the leaf tissue of different parts, the figure shows that the leaf surface and the green part are analyzed. Figure 9b is the analysis of the white part of the leaf surface. The distribution of different ion signals, and Figure 9e is the analysis result obtained from the leaves of the leaves after wilting. The signal analysis of Fig. 9a~e shows that different bits and different growth states can be composed of internal molecules. Composition distribution differences. Referring to Figure 10, the analysis method is used to distinguish the plant pests and diseases. The analysis sample taken in Figure l〇a is normal mango leaves, and the cockroaches are the diseased leaves of the diseased fruit. The mass spectrometry signal can distinguish the difference. Sex, as shown in Figure l〇b, the mango leaves with pests and diseases are between ~ and there is no ion signal distribution.疋 Refer to Figure 11 to test whether there is pesticide residue on the surface of the plant for this analytical method; Figure 11 shows that the test is carried out by using the example of Chl〇rpyrif〇s. 201245711 The method is to dilute the pesticide 1000 times and then use the atomizer to evenly Spray on the surface of the plant, dry and sample again. Figure 11a shows the Taosson signal, Figure i lt) shows the spray-free part of the plant, and Figure 11c shows the sprayed part. It can be seen that the pesticide sprayed on the surface of the plant can still be detected after drying. Refer to Figure 12 to test whether there is pesticide residue in the plant for this analytical method. In Figure 12, Glyphosate is used as an example. The test method is to dilute the pesticide 1000 times and allow the plants to absorb for 3 hours and 6 hours. Sampling tests separately. Fig. 12a shows the Jiaphoser signal, Fig. 12b shows the background value, Fig. 12c shows the sample after 3 hours, and Fig. 12d shows the sample after 6 hours. It can be seen that even if the pesticide is not absorbed on the surface of the plant but absorbed by the plant, the presence of the pesticide can be detected. In addition, when the sample 3 to be tested is a plant leaf, the tip 31 can be directly cut out without being removed from the stem, so that the sample 3 to be tested can be placed on the stage 5 to be tested. Analysis, even if the sample to be tested has a sharp point, it can be tested and analyzed by being directly placed on the stage 5 without being removed or cut by the branches. In summary, the present invention analyzes the sample components by direct electrospray ionization mass spectrometry, as long as the sample to be tested & 3 is directly cut out from the tip 3 and placed on the stage 5 to be in contact with the electrode 4. , and then the extract is dripped. The electric current is directly applied to the tip 3 of the sample to be tested 3 to generate a tip discharge, and the donor-fuge 6 analyzes the sample component, so that the object of the present invention can be achieved. It is only a preferred embodiment of the present invention, and the simple equivalent of the scope of the present invention and the description of the invention is limited by the fact that the present invention is not limited to the present invention. Variations and modifications are still within the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart showing a preferred embodiment of the present invention for analyzing a sample component by direct electrospray ionization mass spectrometry; FIG. 2 is a top plan view showing the preferred embodiment of the preferred embodiment. Fig. 3 is a side view showing an auxiliary view of Fig. 2; Figs. 4a and 4b are mass spectra of ion signals obtained by electrospraying on stevia leaves in positive ion and negative ion modes, respectively; Fig. 5a, 5b are mass spectrometry/ The mass spectrum is analyzed by analyte ions m/z 843 and m/z 1005 respectively; Figure 6a is a mass spectrum diagram of the ion signal obtained by electrospraying on the oleander leaves; Figure 6b is a mass spectrometry/mass spectrometer, Analyze ion analysis; Figure 7a ~ c 疋 mass spectrometer, showing the effect of different solution composition on the signal directly electrophoresed on the oleander leaves; Figure 8 is a mass spectrum, on the oleander leaves Reproducibility test of signals obtained by electrospray irrigation; different parts and different growth patterns 9a~c are mass spectra showing the difference of internal components in the same blade period; Figure signal; l〇a, l〇b is the mass spectrogram FIG pests blade with mass 201 245 711 11 a~c the spectrum is described results of testing pesticides article surface to be measured; and FIG 12a~d spectrum is, results of the tests of pesticide absorbed inside the test article. 10 201245711 [Explanation of main component symbols] 21 Preparation steps 4 Electrode 22 Analysis procedure 5 Stage 3 Sample to be tested 6 Mass spectrometer 31 Tip 61 Entrance