TW201702592A - Method of analysis - Google Patents
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- TW201702592A TW201702592A TW105111925A TW105111925A TW201702592A TW 201702592 A TW201702592 A TW 201702592A TW 105111925 A TW105111925 A TW 105111925A TW 105111925 A TW105111925 A TW 105111925A TW 201702592 A TW201702592 A TW 201702592A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/622—Ion mobility spectrometry
- G01N27/623—Ion mobility spectrometry combined with mass spectrometry
Abstract
Description
本發明之一實施形態係關於一種對試樣中所包含之成分進行定性或定量分析之方法。本發明之具體實施形態係關於一種迅速並且簡便地對角質層成分進行分析之方法。 One embodiment of the present invention relates to a method for qualitative or quantitative analysis of components contained in a sample. DETAILED DESCRIPTION OF THE INVENTION A specific embodiment of the present invention relates to a method for rapidly and easily analyzing a component of a stratum corneum.
先前，對於少量有機化合物、肽、蛋白質、及合成聚合物等各種分析物，作為對試樣進行定性或定量分析之方法，已知有HPLC(high performance liquid chromatography，高效液相層析法)法、氣相層析法、NMR(Nuclear Magnetic Resonance，核磁共振)法、或質量分析法等，但該等分析方法需要對分析物進行預處理，不可謂簡便之方法。 Previously, HPLC (high performance liquid chromatography) method was known as a method for qualitative or quantitative analysis of a sample for various analytes such as organic compounds, peptides, proteins, and synthetic polymers. , gas chromatography, NMR (Nuclear Magnetic Resonance), or mass analysis, etc., but these methods require pretreatment of the analyte, which is not a simple method.
尤其是於將分析對象設為皮膚之情形時，由於皮膚狀態會因季節或哺乳動物(例如人類)之身體狀況或年齡、或於皮膚塗佈化妝品之前後等各種因素而時時刻刻產生變化，故而正尋求迅速且簡便之分析方法。為了改善皮膚狀態或保持成分(例如，水分、蛋白質)，重要的是塗佈化妝品或藥品，並瞬時地對其效果是否已達成進行測定。迄今為止，已知：因皮膚狀態之變化，角質層中所包含之NMF(天然保濕因子)之量會產生變化。作為NMF之成分，可列舉：胺基酸、礦物質、PCA(吡咯啶酮羧酸)、乳酸鹽、脲等。因此，期待迅速且簡便地對某種皮膚狀態下之該等成分進行定性或定量地分析之方法。 In particular, when the object to be analyzed is set to the skin, the skin condition may change constantly due to various factors such as the season or the physical condition or age of the mammal (for example, a human), or the skin before and after the application of the cosmetic. Therefore, a quick and easy analysis method is being sought. In order to improve the skin condition or to maintain ingredients (for example, moisture, protein), it is important to coat a cosmetic or a drug and to instantaneously determine whether the effect has been achieved. Heretofore, it has been known that the amount of NMF (natural moisturizing factor) contained in the stratum corneum changes due to changes in skin condition. Examples of the component of NMF include an amino acid, a mineral, PCA (pyrrolidone carboxylic acid), lactate, and urea. Therefore, a method for qualitatively or quantitatively analyzing such components in a certain skin state is expected to be quickly and easily performed.
於專利文獻1中揭示有皮膚存在物質之分析方法，其係對採取自 皮膚之試樣使用熱分解質譜法或熱脫附質譜法，對存在於皮膚表面或皮膚內部之特定物質進行定量。此時，於自皮膚採取試樣時，使用黏著性膠帶。然而，存在如下問題：無法精度良好地對角質層中所包含之成分之分佈狀態進行定量。 Patent Document 1 discloses an analysis method for a skin-existing substance, which is taken from The skin sample is quantified by thermal decomposition mass spectrometry or thermal desorption mass spectrometry for specific substances present on the skin surface or inside the skin. At this time, an adhesive tape is used when taking a sample from the skin. However, there is a problem that the distribution state of the components contained in the stratum corneum cannot be accurately quantified.
另一方面，作為對採取自皮膚之試樣中所包含之各成分進行分析之方法，質量分析成為有效之方法。質量分析係將試樣進行離子化，並將所產生之離子藉由電場或磁場之作用分成m/z值而獲得質量光譜之分析法。於此情形時，重要的是根據試樣之狀態而選擇最佳之離子化法。作為質量分析之離子化法，已知有各種方法，近年來，DESI(Desorption Electrospray Ionization，脫附電噴灑游離)、或DART(Direct Analysis in Real Time，實時直接分析)一直受到關注(專利文獻2及3)。DESI係使經離子化之溶劑附著於試樣而使離子脫離之方法。又，作為DART，已知有對試樣加成使電子激發狀態之原子或分子與大氣中之水碰撞使其進行潘寧離子化所產生之質子而進行離子化之方法。 On the other hand, mass spectrometry is an effective method for analyzing the components contained in the sample taken from the skin. Mass analysis is a method of ionizing a sample and dividing the generated ions into m/z values by the action of an electric field or a magnetic field to obtain a mass spectrum. In this case, it is important to select the optimum ionization method depending on the state of the sample. Various methods have been known as an ionization method for mass analysis. In recent years, DESI (Desorption Electrospray Ionization) or DART (Direct Analysis in Real Time) has been attracting attention (Patent Document 2) And 3). DESI is a method in which an ionized solvent is attached to a sample to detach ions. Further, as DART, there is known a method in which a sample is added to ionize an atom or a molecule in an electron-excited state to collide with water in the atmosphere to perform protonation by Penning ionization.
進而，於先前之離子化法中，作為容易製備質量分析用之試樣之方法，已知有紙噴霧離子化法(paper spray ionization method)。該方法由於係將試樣載置於濾紙使其滲入，故而試樣之操作較容易，固體試樣或液體試樣之任一者均能夠應對。又，具有所使用之濾紙亦可被用作分離場等優勢(專利文獻4)。然而，必須施加高電壓以進行離子化，於試樣測定中伴隨危險。如此，DESI、DART、紙噴霧離子化法由於需要用以將試樣進行離子化之機器，故而成為複雜之構成之裝置。進而，雖亦存在能夠於低電壓(~3V左右)下安全地進行紙噴霧離子化法之方法，但需要含浸有奈米碳管(CNT)之濾紙，測定成本提高，含浸濾紙之製作耗費工夫(非專利文獻1)。 Further, in the prior ionization method, a paper spray ionization method is known as a method for easily preparing a sample for mass analysis. In this method, since the sample is placed on the filter paper to be infiltrated, the operation of the sample is easy, and either the solid sample or the liquid sample can be handled. Further, the filter paper to be used can also be used as an separation field or the like (Patent Document 4). However, it is necessary to apply a high voltage for ionization, which is accompanied by danger in the measurement of the sample. As described above, the DESI, DART, and paper spray ionization methods are complicated devices because they require a device for ionizing a sample. Further, there is a method in which a paper spray ionization method can be safely performed at a low voltage (about 3 V), but a filter paper impregnated with a carbon nanotube (CNT) is required, and the measurement cost is increased, and the production of the impregnated filter paper takes time and effort. (Non-Patent Document 1).
又，亦已知有嵌入離子化法，且於試樣之離子化時無需電壓、 熱、及氣體，因(高)電壓、高熱、氣體而引起之危險性較少。具有能夠將裝置小型化、初期投資或運轉費用低廉等優勢(非專利文獻2、非專利文獻3)。然而，嵌入離子化法由於需要預先使試樣溶解於液體中，故而難以對固體試樣或其表面上之成分進行分析。又，非專利文獻2、非專利文獻3中記載之分析裝置由於在將溶解於液體之試樣注入裝置時使用移液管等注入工具，故而於對其他試樣進行分析時需要更換注入工具，而耗費工夫，並非簡便之方法。 Moreover, an intercalation ionization method is also known, and no voltage is required for ionization of a sample. Heat, and gas, are less dangerous due to (high) voltage, high heat, and gas. There is an advantage that the apparatus can be downsized, the initial investment, or the operation cost is low (Non-Patent Document 2 and Non-Patent Document 3). However, since the intercalation ionization method requires the sample to be dissolved in the liquid in advance, it is difficult to analyze the solid sample or the components on its surface. Further, in the analysis device described in Non-Patent Document 2 and Non-Patent Document 3, when the sample injected into the liquid is injected into the device, the tool is injected using a pipette or the like. Therefore, it is necessary to replace the injection tool when analyzing other samples. It takes a lot of work and is not an easy way.
如以上所述，對試樣中之成分進行分析之方法中，裝置變得複雜，或即便使裝置變得簡便，亦無法於短時間內容易地進行分析。 As described above, in the method of analyzing the components in the sample, the apparatus becomes complicated, or even if the apparatus is made simple, the analysis cannot be easily performed in a short time.
[專利文獻1]日本專利特開平9-243636號公報 [Patent Document 1] Japanese Patent Laid-Open No. Hei 9-243636
[專利文獻2]日本專利特開2008-180659號公報 [Patent Document 2] Japanese Patent Laid-Open Publication No. 2008-180659
[專利文獻3]日本專利特開2014-206488號公報 [Patent Document 3] Japanese Patent Laid-Open Publication No. 2014-206488
[專利文獻4]美國專利申請公開第2012/0119079號說明書 [Patent Document 4] US Patent Application Publication No. 2012/0119079
[非專利文獻1]R. Narayanan, et al., Angew. Chem. Int. Ed., 53, 5936-5940(2014) [Non-Patent Document 1] R. Narayanan, et al., Angew. Chem. Int. Ed., 53, 5936-5940 (2014)
[非專利文獻2]V. S. Pagnotti, et al., Anal. Chem., 83, 3981-3985(2011) [Non-Patent Document 2] V. S. Pagnotti, et al., Anal. Chem., 83, 3981-3985 (2011)
[非專利文獻3]B. Wang, et al., Anal. Chem., 86, 1000-1006(2014) [Non-Patent Document 3] B. Wang, et al., Anal. Chem., 86, 1000-1006 (2014)
本發明係鑒於上述先前技術所具有之問題，目的在於提供一種能夠於短時間內容易地對試樣中所包含之成分、尤其是角質層中所包含之成分及其分佈狀態進行分析之分析方法及能夠實現該分析方法之 分析裝置。 The present invention has been made in view of the problems of the prior art described above, and an object of the invention is to provide an analysis method capable of easily analyzing components contained in a sample, particularly components contained in a stratum corneum, and distribution states thereof in a short time. And capable of implementing the analytical method Analytical device.
本發明者等人發現了一併具有上述紙噴霧離子化法及嵌入離子化法之優勢之新穎之分析方法。即，本發明者等人實現了使包含溶解於溶劑之試樣之試樣溶液含浸於載體(例如濾紙)，於該載體與質量分析裝置之分析部之抽吸口不相接之狀態下自抽吸口抽吸試樣溶液，無需電壓、熱及氣體等之情況下於數秒以內進行質量分析，從而完成了本發明。 The present inventors have found a novel analytical method which has the advantages of the above-described paper spray ionization method and embedded ionization method. That is, the inventors of the present invention have realized that a sample solution containing a sample dissolved in a solvent is impregnated into a carrier (for example, a filter paper), and the carrier is not in contact with the suction port of the analysis portion of the mass spectrometer. The sample solution was sucked at the suction port, and mass analysis was performed within a few seconds without the need of voltage, heat, gas, etc., thereby completing the present invention.
即，本發明如下。 That is, the present invention is as follows.
[1]一種分析方法，其特徵在於：其係對試樣中所包含之成分進行定性或定量分析之方法，且包含如下步驟：自接收包含溶解於溶劑之試樣之試樣溶液之液體接收部，將該試樣溶液供給至於至少一端形成有導引該試樣溶液之微小空間群之載體；於質量分析裝置之分析部之抽吸該試樣溶液之抽吸口與該載體不相接之狀態下，自該抽吸口抽吸到達該載體之一端之該試樣溶液；及進行該成分之質量分析。 [1] An analytical method characterized in that it is a method for qualitatively or quantitatively analyzing a component contained in a sample, and comprising the steps of: receiving a liquid from a sample solution containing a sample dissolved in a solvent; And supplying the sample solution to a carrier having at least one end formed with a small space group for guiding the sample solution; and suctioning the suction port of the sample solution to the analysis portion of the mass spectrometer without contacting the carrier In the state, the sample solution reaching the one end of the carrier is sucked from the suction port; and mass analysis of the component is performed.
[2]一種分析方法，其特徵在於：其係對試樣中所包含之成分進行定性或定量分析之方法，且包含如下步驟：將該試樣自塗佈該試樣之塗佈部塗佈至直至至少一端形成有微小空間群之載體；對該塗佈部供給溶劑；於質量分析裝置之分析部之抽吸包含溶解於該溶劑之該試樣之試樣溶液之抽吸口與該載體不相接之狀態下，自該抽吸口抽吸到達於該載體之一端之該試樣溶液；及 進行該成分之質量分析。 [2] An analysis method characterized in that it is a method for qualitatively or quantitatively analyzing a component contained in a sample, and comprising the steps of: coating the sample from a coating portion to which the sample is coated. a carrier having a minute space group formed at at least one end; a solvent is supplied to the coating portion; and a suction port of the sample solution containing the sample dissolved in the solvent is sucked into the analysis portion of the mass spectrometer and the carrier In a state of no connection, the sample solution reaching the one end of the carrier is sucked from the suction port; and Perform a quality analysis of the ingredients.
[3]一種分析方法，其特徵在於：其係對試樣中所包含之成分進行定性或定量分析之方法，且包含如下步驟：將該試樣自供轉印該試樣之部分轉印至直至至少一端地形成有微小空間群之載體；將溶劑供給至轉印有該試樣之部分；於質量分析裝置之分析部之抽吸包含溶解於該溶劑之該試樣之試樣溶液之抽吸口與該載體不相接之狀態下，自該抽吸口抽吸到達至該載體之一端之該試樣溶液；及進行該成分之質量分析。 [3] An analysis method, characterized in that it is a method for qualitatively or quantitatively analyzing a component contained in a sample, and comprising the steps of: transferring the sample from a portion for transferring the sample until a carrier having a minute space group formed at least at one end; supplying a solvent to a portion to which the sample is transferred; and suctioning a sample solution containing the sample dissolved in the solvent in the analysis portion of the mass spectrometer The sample solution that reaches the one end of the carrier is sucked from the suction port in a state where the port is not in contact with the carrier; and mass analysis of the component is performed.
[4]一種分析方法，其特徵在於：其係對角質層中所包含之成分進行定性或定量分析之方法，且包含如下步驟：將表面具有黏著層之片體以該黏著層側成為皮膚之方式貼附，並將該片體自該皮膚剝下，藉此，將角質層剝離；將附著於該片體之該角質層利用該片體與形成有微小空間群之載體夾住；對該載體供給溶劑；於質量分析裝置之分析部之抽吸包含溶解於該溶劑之該角質層之試樣溶液之抽吸口與該載體不相接之狀態下，自該抽吸口抽吸到達至該載體之一端之該試樣溶液；及進行該成分之質量分析。 [4] An analysis method characterized in that it is a method for qualitatively or quantitatively analyzing a component contained in a stratum corneum, and comprises the steps of: a sheet having an adhesive layer on the surface and a skin layer on the side of the adhesive layer; Attaching the sheet, and peeling the sheet from the skin, thereby peeling off the stratum corneum; and the stratum corneum attached to the sheet is sandwiched by the sheet and the carrier forming the microscopic space group; The carrier is supplied with a solvent; and the suction port of the sample solution containing the stratum corneum dissolved in the solvent is not in contact with the carrier in the analysis portion of the mass spectrometer, and is sucked from the suction port to reach The sample solution at one end of the carrier; and performing mass analysis of the component.
[5]一種分析方法，其特徵在於：其係對角質層中所包含之成分進行定性或定量分析之方法，且包含如下步驟：將形成有微小空間群且表面具有黏著層之載體以該黏著層側成為皮膚之方式貼附，並將該載體自該皮膚剝下，藉此，將角質層剝離； 對該載體供給溶劑；於質量分析裝置之抽吸包含溶解於該溶劑之該角質層之試樣溶液之抽吸口與該載體不相接之狀態下，自該抽吸口抽吸到達至該載體之一端之該試樣溶液；及進行該成分之質量分析。 [5] An analysis method characterized by: a method for qualitatively or quantitatively analyzing a component contained in a stratum corneum, and comprising the steps of: forming a carrier having a minute space group and having an adhesive layer on the surface thereof; The layer side is attached to the skin and the carrier is peeled off from the skin, thereby peeling off the stratum corneum; Supplying a solvent to the carrier; in a state in which the suction port of the sample solution containing the stratum corneum dissolved in the solvent is not in contact with the carrier, the suction from the suction port reaches the The sample solution at one end of the carrier; and performing mass analysis of the component.
[6]如上述[1]~[5]之分析方法，其特徵在於：將該試樣溶液導引至該載體之一端之導引機構安裝於該載體。 [6] The analysis method according to [1] to [5] above, characterized in that the guiding mechanism for guiding the sample solution to one end of the carrier is attached to the carrier.
[7]如上述[1]~[6]之分析方法，其特徵在於：該載體之一端之角之角度之至少一者並非未達90°。 [7] The analysis method according to [1] to [6] above, characterized in that at least one of the angles of the corners of one end of the carrier is not less than 90°.
[8]如上述[1]、[6]及[7]之分析方法，其特徵在於：於對該載體供給溶劑之後，供給該試樣溶液。 [8] The analysis method according to the above [1], [6], and [7], wherein the sample solution is supplied after the solvent is supplied to the carrier.
[9]如上述[2]、[3]、[6]及[7]之分析方法，其特徵在於：於塗佈或轉印該試樣之前及/或後，對該載體滴加溶劑。 [9] The analysis method according to the above [2], [3], [6] and [7], characterized in that a solvent is added to the carrier before and/or after coating or transferring the sample.
[10]如上述[4]~[7]之分析方法，其特徵在於：於使該角質層附著之前或後，對該載體滴加溶劑。 [10] The analysis method according to the above [4] to [7], characterized in that a solvent is added to the carrier before or after the attachment of the stratum corneum.
[11]一種質量分析裝置，其特徵在於：其係對試樣中所包含之成分進行定性或定量分析者，且具備：載體，其形成有微小空間群；保持部，其保持該載體；供給部，其對該載體供給溶劑或試樣溶液；分析部，其具有抽吸口，且對該成分進行定性或分析；且於該載體與該抽吸口不相接之狀態下，該載體由該保持部保持，自該供給部供給之包含溶解於該溶劑之試樣之該試樣溶液到達載體之一端，並自該抽吸口抽吸該到達之試樣溶液。 [11] A mass spectrometer characterized in that it qualitatively or quantitatively analyzes a component contained in a sample, and comprises: a carrier formed with a minute space group; and a holding portion that holds the carrier; a portion that supplies a solvent or a sample solution to the carrier; an analysis portion that has a suction port and that characterizes or analyzes the component; and in a state where the carrier is not in contact with the suction port, the carrier is The holding portion holds the sample solution containing the sample dissolved in the solvent supplied from the supply portion to one end of the carrier, and sucks the sample solution that has arrived from the suction port.
根據本發明，能夠於短時間內容易且精度良好地對試樣中所包 含之成分，具體而言，角質層中所包含之成分(例如，NMF、脂質、肽、蛋白質等)之分佈狀態或組成進行定量。又，根據本發明，能夠提供一種用以對經皮吸收至皮膚中之藥劑、合成高分子等進行定性或定量地分析之方法。 According to the present invention, it is possible to easily and accurately package the sample in a short time. The component, specifically, the distribution state or composition of the components (for example, NMF, lipid, peptide, protein, etc.) contained in the stratum corneum is quantified. Moreover, according to the present invention, it is possible to provide a method for qualitatively or quantitatively analyzing a drug, a synthetic polymer or the like which is percutaneously absorbed into the skin.
1‧‧‧質量分析裝置 1‧‧‧Quality analysis device
10‧‧‧導引機構 10‧‧‧Guiding agency
10a‧‧‧導引機構 10a‧‧‧Guiding agency
10b‧‧‧導引機構 10b‧‧‧Guiding agency
10c‧‧‧導引機構 10c‧‧‧Guiding agency
10d‧‧‧導引機構 10d‧‧‧Guiding agency
10d'‧‧‧導引機構 10d'‧‧‧Guide
10e‧‧‧導引機構 10e‧‧‧Guiding agency
10f‧‧‧導引機構 10f‧‧‧Guiding agency
10g‧‧‧導引機構 10g‧‧‧Guide
100‧‧‧載體 100‧‧‧ Carrier
100'‧‧‧載體 100'‧‧‧ Carrier
100"‧‧‧載體 100"‧‧‧ carrier
110‧‧‧載體之一端 110‧‧‧ one end of the carrier
120‧‧‧液體接收部 120‧‧‧Liquid receiving department
200‧‧‧保持部 200‧‧‧ Keeping Department
300‧‧‧供給部 300‧‧‧Supply Department
310‧‧‧試樣溶液 310‧‧‧ sample solution
320‧‧‧溶劑 320‧‧‧Solvent
400‧‧‧分析部 400‧‧‧Analysis Department
402‧‧‧殼體 402‧‧‧Shell
410‧‧‧抽吸口 410‧‧ ‧ suction port
500‧‧‧片體 500‧‧‧ tablets
500'‧‧‧片體 500'‧‧‧ tablets
600‧‧‧黏著片體 600‧‧‧Adhesive sheet
700‧‧‧角質層 700‧‧‧ horny layer
800‧‧‧黏著層 800‧‧‧Adhesive layer
圖1係表示本發明之質量分析裝置之典型例之圖。 Fig. 1 is a view showing a typical example of the mass spectrometer of the present invention.
圖2(A)係自上方觀察本發明之質量分析裝置之載體之一端110及抽吸口410之周邊之圖。(B)表示a-a'方向之剖視圖。 Fig. 2(A) is a view showing the vicinity of one end 110 and the suction port 410 of the carrier of the mass spectrometer of the present invention from above. (B) shows a cross-sectional view in the a-a' direction.
圖3表示(A)具備導引機構10a之載體100'、及(B)b-b'方向之剖視圖。 Fig. 3 is a cross-sectional view showing the direction of the carrier 100' and the (B)b-b' of the guide mechanism 10a.
圖4(A)~(D)表示具備各種導引機構(10b、10c、10d、10d')之載體之剖視圖。 4(A) to 4(D) are cross-sectional views showing a carrier having various guiding mechanisms (10b, 10c, 10d, 10d').
圖5(A)表示於載體100"之緣側具備導引機構(階差)之載體。(B)表示c-c'方向之剖視圖。 Fig. 5(A) shows a carrier having a guiding mechanism (step) on the edge of the carrier 100", and (B) is a cross-sectional view in the c-c' direction.
圖6係表示(A)將2片片體500載置於載體100上，(B)形成有導引機構10f之一例之圖。(C)表示d-d'方向之剖視圖。 Fig. 6 is a view showing an example in which (A) two sheets 500 are placed on the carrier 100, and (B) is formed with a guiding mechanism 10f. (C) shows a cross-sectional view in the d-d' direction.
圖7表示(A)將1片片體500'載置於載體100上，(B)於該片體之緣側形成有導引機構10g之例。(C)表示e-e'方向之剖視圖。 Fig. 7 shows an example in which (A) one sheet 500' is placed on the carrier 100, and (B) a guide mechanism 10g is formed on the edge side of the sheet. (C) shows a cross-sectional view in the e-e' direction.
圖8表示將藉由黏著片體600而剝離之角質層700夾於該黏著片體600與載體100之間之例。 FIG. 8 shows an example in which the stratum corneum layer 700 peeled off by the adhesive sheet 600 is sandwiched between the adhesive sheet body 600 and the carrier 100.
圖9表示使已剝離之角質層700擔載於被保持於載體100之黏著層800上之例。 FIG. 9 shows an example in which the exfoliated stratum corneum layer 700 is carried on the adhesive layer 800 held on the carrier 100.
圖10係將17個胺基酸標準混合物作為試樣進行分析，而表示本發明之分析方法之再現性之圖。表示使用3個載體(濾紙小片)分別對該試樣進行分析之結果。 Fig. 10 is a graph showing the reproducibility of the analytical method of the present invention by analyzing 17 amino acid standard mixtures as samples. The results of analysis of the sample using three carriers (filter paper pieces) are shown.
圖11係將17個胺基酸標準混合物作為試樣進行分析，而表示本發 明之分析方法之線性及敏感性之圖。 Figure 11 shows the analysis of 17 amino acid standard mixtures as samples. A diagram showing the linearity and sensitivity of the analytical method.
圖12係將17個胺基酸標準混合物作為試樣進行分析，而表示本發明之分析方法之線性之圖。 Figure 12 is a graph showing the linearity of the analytical method of the present invention by analyzing 17 amino acid standard mixtures as samples.
圖13係將血管收縮素(angiotensin)II作為試樣進行分析，而對本發明之分析方法之實施可能性進行實際驗證之圖。 Fig. 13 is a view showing the actual verification of the possibility of carrying out the analysis method of the present invention by analyzing angiotensin II as a sample.
圖14係將胰島素作為試樣進行分析，而對本發明之分析方法之實施可能性進行實際驗證之圖。 Fig. 14 is a diagram showing the actual verification of the possibility of carrying out the analysis method of the present invention by analyzing insulin as a sample.
圖15係將市售之化妝品作為試樣進行分析，而對本發明之分析方法之有效性進行實際驗證之圖。 Fig. 15 is a view showing the actual verification of the effectiveness of the analysis method of the present invention by analyzing a commercially available cosmetic as a sample.
圖16係將人類皮膚(角質層)作為試樣進行分析，而對本發明之分析方法之有效性進行實際驗證之圖。 Fig. 16 is a diagram showing actual analysis of the effectiveness of the analysis method of the present invention by analyzing human skin (stratum corneum) as a sample.
本發明者等人新開發了用以對試樣中所包含之成分定性或定量地進行分析之提取微小液滴導入質量分析(Extractive micro-Droplet Introduction Mass Spectrometry，EmDI-MS)。本發明係關於一種使用該EmDI-MS對試樣中所包含之成分(例如，對角質層中所包含之成分)進行定性或定量分析之方法。於本申請案中，本發明之分析方法之概念及詳細情況首次係與超高速地對固體試樣及液體試樣進行直接分析之應用例一併呈現。本說明書中，示出EmDI-MS由於一併具有有效性已得到確認之紙噴霧離子化法及嵌入離子化法之特徵，故而被確立為獨特之分析方法。於EmDI-MS中，積層(載置)於載體之固體試樣及液體試樣之成分係藉由一次所供給之數μl之溶劑而提取。包含所提取之成分之試樣溶液自載體之一端瞬時地導入質量分析裝置之分析部，且質量分析於數秒以內完成。此處，應該特別記載的是本發明之EmDI-MS一併具有紙噴霧離子化法及嵌入離子化法之優勢，例如可列舉如下方面：無需高電壓、雷射、熱或氣體來將分析物離子化；為 高速分析，且試樣之操作容易；進而能夠分離。藉由該EmDI-MS，得以消除先前視為缺點之(高)電壓施加(紙噴霧離子化法)、及固體試樣之處理之困難性(嵌入離子化法)。本發明者等人能夠於不對試樣進行預處理之情況下對少量有機化合物、肽、蛋白質、及合成聚合物、角質層成分等各種分析物進行超高速(例如，<1秒鐘)且定性及定量分析。 The inventors of the present invention have newly developed an extractive micro-Droplet Introduction Mass Spectrometry (EmDI-MS) for qualitatively or quantitatively analyzing a component contained in a sample. The present invention relates to a method for qualitatively or quantitatively analyzing a component contained in a sample (for example, a component contained in a stratum corneum) using the EmDI-MS. In the present application, the concept and details of the analysis method of the present invention are first presented together with an application example for direct analysis of a solid sample and a liquid sample at an ultrahigh speed. In the present specification, the characteristics of the paper spray ionization method and the embedded ionization method in which the EmDI-MS has been confirmed to be effective have been described, and thus it has been established as a unique analysis method. In the EmDI-MS, the components of the solid sample and the liquid sample which are laminated (mounted) on the carrier are extracted by a solvent of several μl supplied at a time. The sample solution containing the extracted component is instantaneously introduced from the one end of the carrier into the analysis portion of the mass spectrometer, and the mass analysis is completed in a few seconds. Here, it should be particularly noted that the EmDI-MS of the present invention has the advantages of a paper spray ionization method and an intercalation ionization method, and for example, the following aspects can be cited: no high voltage, laser, heat or gas is required to carry the analyte. Ionization; High-speed analysis, and the operation of the sample is easy; With the EmDI-MS, it is possible to eliminate the (high) voltage application (paper spray ionization method) which is previously considered to be a disadvantage, and the difficulty in processing a solid sample (embedded ionization method). The inventors of the present invention can perform ultra-high speed (for example, <1 second) and qualitative analysis on various analytes such as a small amount of an organic compound, a peptide, a protein, a synthetic polymer, and a stratum corneum component without pretreating a sample. And quantitative analysis.
根據本發明，提供一種分析方法等，該分析方法之特徵在於：其係對試樣中所包含之成分進行定性或定量分析之方法，且包含如下步驟：自接收包含溶解於溶劑之試樣之試樣溶液之液體接收部，將該試樣溶液供給至於至少一端形成有導引該試樣溶液之微小空間群之載體；於質量分析裝置之抽吸該試樣溶液之抽吸口與該載體不相接之狀態下，自該抽吸口抽吸到達該載體之一端之該試樣溶液；及進行該成分之質量分析。 According to the present invention, there is provided an analysis method or the like which is characterized in that it is a method for qualitatively or quantitatively analyzing a component contained in a sample, and comprises the steps of: receiving a sample containing a solvent dissolved therein; a liquid receiving portion of the sample solution, the sample solution is supplied to a carrier having at least one end formed with a small space group for guiding the sample solution; and a suction port for sucking the sample solution with the carrier by the mass spectrometer In the non-contact state, the sample solution reaching the one end of the carrier is sucked from the suction port; and mass analysis of the component is performed.
1.分析方法(概念) 1. Analysis method (concept)
本發明本質上而言係使用質量分析裝置之分析方法，但無需電壓、熱及氣體來將分析物離子化，能夠在2~3秒鐘以內(根據情況，未達1秒鐘)完成定性或定量分析。更具體而言，本發明之分析方法之特徵在於：一併具有先前之紙噴霧離子化法及嵌入離子化法之各者之優點。典型而言，如圖1及2所示，本發明之分析方法包含：步驟(1)，其係自接收包含溶解於溶劑之試樣之試樣溶液310之液體部120將該試樣溶液310供給至於至少一端形成有導引該試樣溶液之微小空間群之載體100；步驟(2)，其係於質量分析裝置1之分析部400之抽吸該試樣溶液之抽吸口410與該載體100不相接之狀態下，自抽吸口410抽吸到達該載體100之一端110之該試樣溶液；及步驟(3)，其係於分析部400獲得質量光譜。 The present invention essentially uses an analytical method of a mass spectrometer, but does not require voltage, heat, and gas to ionize the analyte, and can be qualitatively or within 2 to 3 seconds (less than 1 second, depending on the situation). Quantitative analysis. More specifically, the analysis method of the present invention is characterized by having the advantages of both the previous paper spray ionization method and the embedded ionization method. Typically, as shown in FIGS. 1 and 2, the analysis method of the present invention comprises the step (1) of receiving the sample solution 310 from a liquid portion 120 that receives a sample solution 310 containing a sample dissolved in a solvent. Provided to the carrier 100 having at least one end formed with a small space group for guiding the sample solution; and the step (2) of the suction port 410 for sucking the sample solution of the analysis unit 400 of the mass spectrometer 1 and the In the state where the carrier 100 is not connected, the sample solution reaching the one end 110 of the carrier 100 is sucked from the suction port 410; and the step (3) is obtained by the analyzing portion 400 to obtain a mass spectrum.
又，作為其他態樣，步驟(1)中，亦可將試樣直接塗佈於載體 100，其後供給溶劑320，於載體100中使目標成分溶解於溶劑320而製作試樣溶液，以代替預先使試樣溶解而成之試樣溶液。 Further, as another aspect, in the step (1), the sample may be directly coated on the carrier. 100. Thereafter, the solvent 320 is supplied, and the target component is dissolved in the solvent 320 in the carrier 100 to prepare a sample solution, instead of the sample solution obtained by dissolving the sample in advance.
作為另一態樣，步驟(1)中，亦可將試樣轉印於載體100，對轉印有試樣之部分供給溶劑320，於載體100中使目標成分溶解於溶劑320而製作試樣溶液。 In another aspect, in the step (1), the sample may be transferred to the carrier 100, the solvent 320 may be supplied to the portion to which the sample is transferred, and the target component may be dissolved in the solvent 320 in the carrier 100 to prepare a sample. Solution.
亦可使用對載體上之試樣供給數μL之溶劑而獲得之試樣溶液。 A sample solution obtained by supplying a solvent of several μL to a sample on a carrier can also be used.
本發明中，作為其他實施態樣，例如於如圖8所示般對角質層中所包含之成分進行定性或定量分析之情形時，亦可將表面具有黏著層之片體600以黏著層側成為皮膚之方式貼附，並自皮膚剝下片體600，藉此將角質層700剝離，並將附著於片體600之角質層700利用片體600及形成有微小空間群之載體100夾住，對載體100供給溶劑320使角質層700中所包含之成分溶解於溶劑320而製作試樣溶液。 In the present invention, as another embodiment, for example, when qualitative or quantitative analysis is performed on the components contained in the stratum corneum as shown in FIG. 8, the sheet 600 having the adhesive layer on the surface may be adhered to the adhesive layer side. The skin is attached, and the sheet 600 is peeled off from the skin, whereby the stratum corneum 700 is peeled off, and the stratum corneum layer 700 attached to the sheet 600 is sandwiched by the sheet 600 and the carrier 100 formed with the micro space group. The solvent 320 is supplied to the carrier 100 to dissolve the components contained in the stratum corneum 700 in the solvent 320 to prepare a sample solution.
又，於對角質層中所包含之成分進行定性或定量分析之情形時，亦可如圖9所示般以具有黏著層800之載體100之黏著層800側成為皮膚之方式進行貼附，並自皮膚將載體剝下，藉此將角質層700剝離，其後，對載體100供給溶劑320，使角質層700溶解於溶劑320而製作試樣溶液。 Further, in the case of qualitatively or quantitatively analyzing the components contained in the stratum corneum, as shown in FIG. 9, the adhesive layer 800 side of the carrier 100 having the adhesive layer 800 may be attached as a skin, and The horny layer 700 is peeled off from the skin by peeling off the carrier, and then the solvent 320 is supplied to the carrier 100, and the horny layer 700 is dissolved in the solvent 320 to prepare a sample solution.
步驟(2)中，供給至載體100之試樣溶液310自抽吸口410被引入至分析部400。 In the step (2), the sample solution 310 supplied to the carrier 100 is introduced from the suction port 410 to the analysis portion 400.
步驟(3)中，利用分析部400對藉由步驟(2)中因分析部400之真空壓產生之抽吸力而被抽吸之試樣溶液中所包含之成分之質量進行測定。 In the step (3), the analysis unit 400 measures the mass of the component contained in the sample solution that is sucked by the suction force generated by the vacuum pressure of the analysis unit 400 in the step (2).
本發明之分析方法係除將預先使試樣溶解而成之試樣溶液310引入至分析部400以外，將溶劑320供給至載體100上之試樣而製成提取出目標成分後之試樣溶液，其後，將該試樣溶液瞬時地引入至真空下之分析部400，由此能夠對目標成分定性或定量地進行分析。藉此， 由於削減了預處理之操作，測定時間亦成為瞬時，而且能夠大幅提高一次引入至分析裝置之目標成分之量，故而能夠提高絕對感度。 In the analysis method of the present invention, the sample solution 310 obtained by dissolving the sample in advance is introduced into the sample other than the analysis unit 400, and the solvent 320 is supplied to the sample on the carrier 100 to prepare a sample solution obtained by extracting the target component. Thereafter, the sample solution is instantaneously introduced into the analysis unit 400 under vacuum, whereby the target component can be qualitatively or quantitatively analyzed. With this, Since the pre-processing operation is reduced, the measurement time is instantaneous, and the amount of the target component introduced into the analysis device can be greatly increased, so that the absolute sensitivity can be improved.
若根據分析部400之內部之真空壓及溫度而計算所抽吸之液滴之汽化體積，則一滴約6μL之水成為約12L之體積。實際上，由於對壓力之影響較輕微，而且會瞬時地恢復至原先之真空壓，故而確認對測定無影響。由於分子量較小之水之汽化體積最大，但本發明之分析方法實際驗證了於將溶劑設為水之情形時能夠進行測定，故而可謂能夠使用其他任一溶劑。如此，能夠變更溶劑種類係能夠藉由改變溶劑之極性而自載體上所載置之試樣進行粗區分(能夠按順序對目標成分進行分析)。於該方面而言，與先前之紙噴霧離子化法相同，具有即便於干擾成分較多之血液等情形時亦能夠迅速地進行分析之優勢。 When the vaporization volume of the aspirated droplet is calculated based on the vacuum pressure and temperature inside the analysis unit 400, a drop of about 6 μL of water becomes a volume of about 12 L. In fact, since the influence on the pressure is slight and instantaneously returns to the original vacuum pressure, it is confirmed that there is no influence on the measurement. Since the vaporization volume of water having a small molecular weight is the largest, the analysis method of the present invention actually proves that the measurement can be performed when the solvent is made into water, and thus any other solvent can be used. In this way, the type of the solvent can be changed, and the sample placed on the carrier can be roughly divided by changing the polarity of the solvent (the target component can be analyzed in order). In this respect, similarly to the conventional paper spray ionization method, it is advantageous in that analysis can be performed promptly even in the case of blood having a large amount of interference components.
2.試樣製備 2. Sample preparation
根據本發明，能夠藉由本發明之分析方法進行測定之試樣並無限定，可為固體試樣或半固體試樣或液體試樣之任一者，包含存在於任意物體表面上之成分。於一態樣中，亦可將使試樣溶解於溶劑中而成之試樣溶液310用於分析。於另一態樣中，可對預先塗佈於載體100之液體或(半)固體之試樣供給適當之溶劑320，而作為試樣溶液使用。於進而又一態樣中，可對轉印於載體100之試樣供給適當之溶劑320，而作為試樣使用。又，作為其他態樣，如圖9所示，可將使用具有黏著層800之載體100而自皮膚剝離之角質層700作為試樣而使用，亦可對角質層700供給適當之溶劑320而作為試樣溶液使用。或可將使用表面具有黏著層之片體(黏著片體600)而自角質層剝離之角質層700作為試樣，進而供給適當之溶劑320，而製成試樣溶液。如上所述，任一試樣均可使用，但需要有用於塗佈試樣使試樣溶液含浸、或夾住使已剝離之角質層黏著之片材之載體。 According to the present invention, the sample which can be measured by the analysis method of the present invention is not limited, and may be any of a solid sample, a semi-solid sample or a liquid sample, and includes a component present on the surface of any object. In one aspect, the sample solution 310 obtained by dissolving the sample in a solvent may also be used for analysis. In another aspect, a suitable solvent 320 may be supplied to a liquid or (semi)solid sample previously coated on the carrier 100 for use as a sample solution. In still another aspect, a suitable solvent 320 can be supplied to the sample transferred to the carrier 100 to be used as a sample. Further, as another aspect, as shown in FIG. 9, the stratum corneum layer 700 peeled from the skin using the carrier 100 having the adhesive layer 800 may be used as a sample, or the keratin layer 700 may be supplied with a suitable solvent 320 as a sample. The sample solution is used. Alternatively, the horny layer 700 exfoliated from the stratum corneum using a sheet having an adhesive layer on the surface (adhesive sheet 600) may be used as a sample, and a suitable solvent 320 may be supplied to prepare a sample solution. As described above, any of the samples may be used, but a carrier for coating the sample to impregnate the sample solution or sandwiching the sheet which adheres the exfoliated stratum corneum is required.
於試樣製備中所使用之溶劑只要為使試樣溶解並提取試樣中所 包含之成分者，則並無限定，可為水、有機溶劑(例如，乙醇、甲醇、乙腈、己烷)、或其等之混合物。亦可加入酸或鹼、微量之離子對試劑或鹽。供給之量可為1滴，亦可為相當於約2~20μL之量。再者，於試樣含浸於載體100中之情形時，如上所述，較佳為對載體100供給溶劑320之態樣，但作為另一態樣，於試樣為包含目標成分之試樣溶液之情形時，可對載體100直接供給試樣溶液310，自分析部400之抽吸口410抽吸試樣溶液而進行分析。該情形之試樣溶液之量以上述供給量為標準。 The solvent used in the preparation of the sample is as long as the sample is dissolved and extracted in the sample. The components included are not limited, and may be water, an organic solvent (for example, ethanol, methanol, acetonitrile, hexane), or a mixture thereof. An acid or a base, a trace amount of an ion pair reagent or a salt may also be added. The amount of supply may be one drop, or may be equivalent to about 2 to 20 μL. Further, in the case where the sample is impregnated in the carrier 100, as described above, it is preferable to supply the solvent 100 to the carrier 100, but as another aspect, the sample is a sample solution containing the target component. In this case, the sample solution 310 may be directly supplied to the carrier 100, and the sample solution may be aspirated from the suction port 410 of the analysis unit 400 for analysis. The amount of the sample solution in this case is based on the above-mentioned supply amount.
作為一態樣，亦可於對上述載體100供給試樣溶液310之前，對該載體100滴加上述任一溶劑作為預浸潤。作為另一態樣，亦可於塗佈或轉印試樣之前及/或後，對該載體100滴加上述任一溶劑。作為其他態樣，亦可於使角質層700附著之前或後，對該載體100滴加上述任一溶劑。 As one aspect, any of the above solvents may be added to the carrier 100 as a pre-wetting before the sample solution 310 is supplied to the carrier 100. As another aspect, any of the above solvents may be added dropwise to the carrier 100 before and/or after coating or transferring the sample. As another aspect, any of the above solvents may be added dropwise to the carrier 100 before or after the horny layer 700 is attached.
3.載體 3. Carrier
本發明之分析方法之特徵在於：使用用以積層(載置)試樣等之載體100。如後文所述，典型而言，本發明之分析方法係於本發明之質量分析裝置1之分析部400具有之抽吸口410與被供給有試樣溶液310之載體100不相接之狀態下進行質量分析者。此處，可使用之載體100較佳為自接收所供給之試樣溶液310或溶劑320之液體接收部120至該載體100之至少一端形成有用以將試樣溶液向分析部400之抽吸口410導引之微小空間群。 The analysis method of the present invention is characterized in that a carrier 100 for laminating (mounting) a sample or the like is used. As will be described later, the analysis method of the present invention is generally in a state in which the suction port 410 of the analysis unit 400 of the mass spectrometer 1 of the present invention is not in contact with the carrier 100 to which the sample solution 310 is supplied. Under the quality analyst. Here, the carrier 100 that can be used preferably forms a suction port for receiving the sample solution to the analysis portion 400 from the liquid receiving portion 120 that receives the supplied sample solution 310 or solvent 320 to at least one end of the carrier 100. 410 guided small space group.
於在本說明書中使用時，所謂「液體接收部」(120)係指接收所供給之試樣溶液310或溶劑320之載體100上之任意區域，並不意圖明確地區分液體接收部120與其以外之區域。若為試樣載置於載體100之狀態，則係指供給用以使試樣中所包含之成分溶解之溶劑320之部分(或構造)，又，若試樣為液體試樣，則係指供給該試樣溶液310之部 分(或構造)。再者，液體接收部亦可僅為暫時地接收自供給部300所供給之試樣溶液310或溶劑320之載體100上之任一區域，但於載體100具備導引機構10之情形時，較佳為與導引機構相接或為其附近之區域。又，於一態樣中，於在載體100中溶解或提取試樣之情形時，液體接收部120可為距抽吸口410較遠之載體100上之一端附近(未圖示)。 As used herein, the term "liquid receiving portion" (120) refers to any region on the carrier 100 that receives the supplied sample solution 310 or solvent 320, and is not intended to clearly distinguish the liquid receiving portion 120 from The area. If the sample is placed on the carrier 100, it means a portion (or structure) to which the solvent 320 for dissolving the components contained in the sample is supplied, and if the sample is a liquid sample, it means Supplying the portion of the sample solution 310 Points (or construction). Further, the liquid receiving portion may only temporarily receive any region on the carrier 100 of the sample solution 310 or the solvent 320 supplied from the supply portion 300, but when the carrier 100 is provided with the guiding mechanism 10, It is connected to or in the vicinity of the guiding mechanism. Further, in a case where the sample is dissolved or extracted in the carrier 100, the liquid receiving portion 120 may be near one end of the carrier 100 (not shown) which is far from the suction port 410.
於利用保持部200(例如，夾具)保持載體100之情形時，於載體上需要用以形成液體接收部之餘白。此種餘白例如於圖2中相當於S1及S2，但可不必設置處於前端側之餘白部分(S2)，保持部200可與載體之一端110重疊。另一方面，沿保持部200之側面之餘白部分(S1)可為保持部200之兩側，或亦可為單側。 In the case where the carrier 100 is held by the holding portion 200 (for example, a jig), a margin for forming the liquid receiving portion is required on the carrier. Such a margin is equivalent to S 1 and S 2 in FIG. 2, for example, but it is not necessary to provide a margin portion (S 2 ) on the front end side, and the holding portion 200 may overlap with one end 110 of the carrier. On the other hand, the remaining white portion (S 1 ) along the side surface of the holding portion 200 may be both sides of the holding portion 200 or may be one side.
於本發明中所使用之載體100較佳為形成有微小空間群。此處，於本說明書中使用時，所謂「微小空間群」係指具有如產生毛細現象之構造之物體，發揮將含浸之試樣溶液通過微小空間群而導引至載體100之至少一端110之作用。作為可於本發明中使用之載體，只要為具有上述液體接收部及微小空間群之載體，則並無限定，例如可為濾紙、纖維、樹脂、布、薄層層析板(將二氧化矽凝膠、氧化鋁、聚醯胺樹脂等吸附劑呈薄膜狀固定於玻璃、鋁等之板上而成之薄層板)。 The carrier 100 used in the present invention is preferably formed with a minute space group. Here, in the present specification, the term "microscopic space group" means an object having a structure in which a capillary phenomenon is generated, and the impregnated sample solution is guided to the at least one end 110 of the carrier 100 through the minute space group. effect. The carrier which can be used in the present invention is not limited as long as it is a carrier having the liquid receiving portion and the minute space group, and may be, for example, a filter paper, a fiber, a resin, a cloth, or a thin layer chromatography plate (a cerium oxide) An adsorbent such as a gel, an alumina or a polyamide resin is a thin layer plate which is fixed in a film form on a plate of glass or aluminum.
本發明之分析方法中所使用之載體100之形狀較佳為適合能夠將含浸於載體100之試樣溶液於載體100之至少一端110抽吸至分析部400之抽吸口410之形狀。作為載體100之形狀，較佳為距抽吸口410較近但不尖銳，例如較佳為此種載體之一端之角之角度為非未達90°之形狀。作為載體100之形狀，例如可列舉正方形、長方形、梯形等。作為較佳之態樣，若以使用有濾紙之情形為例，則例如可使用市售之定性濾紙(GE Healthcare Japan公司製造；圓形濾紙：最大500mm直徑；方型濾紙：最大600mm)作為濾紙。繼而，較佳為使用對照抽吸 口410之狀態(形狀、大小等)而將濾紙切取成具有適當之形狀與大小之濾紙小片。例如，濾紙小片之形狀及大小並無限定，典型而言，可為長方形(例如，短邊2.5mm×長邊10mm)、梯形(上底2.5mm×下底10mm)。此種濾紙小片亦可於將試樣積層於切斷前之濾紙之後自濾紙切取。又，如上所述，載體100之一端之角之角度較佳為非未達90°。作為此種角度，較佳為90°以上且未達180°，更佳為90°以上且未達150°，進而更佳為90°。 The shape of the carrier 100 used in the analysis method of the present invention is preferably adapted to be capable of drawing a sample solution impregnated with the carrier 100 to the suction port 410 of the analysis portion 400 at at least one end 110 of the carrier 100. The shape of the carrier 100 is preferably closer to the suction port 410 but not sharp. For example, it is preferred that the angle of one end of the carrier is not less than 90°. Examples of the shape of the carrier 100 include a square, a rectangle, a trapezoid, and the like. In a preferred embodiment, for example, a commercially available qualitative filter paper (manufactured by GE Healthcare Japan; round filter paper: maximum 500 mm diameter; square filter paper: maximum 600 mm) can be used as the filter paper. Then, preferably using a control suction The state of the port 410 (shape, size, etc.) is cut into filter paper pieces having an appropriate shape and size. For example, the shape and size of the filter paper piece are not limited, and are typically rectangular (for example, short side 2.5 mm x long side 10 mm) and trapezoidal (upper bottom 2.5 mm x lower bottom 10 mm). Such a filter paper piece can also be cut out from the filter paper after the sample is laminated on the filter paper before cutting. Further, as described above, the angle of the corner of one end of the carrier 100 is preferably not less than 90°. As such an angle, it is preferably 90 or more and less than 180, more preferably 90 or more and less than 150, and still more preferably 90.
如上所述，本發明所使用之載體100較佳為形成有液體接收部120及微小空間群，但亦可進而於該載體100設置用以輔助使試樣溶液310或溶劑320自分析部400之抽吸口410抽吸之導引機構。此處，「導引機構」係於業者中普遍使用之用語，於本發明中，如上所述係指用以輔助使試樣溶液310或溶劑320自分析部400之抽吸口410抽吸之構造，且材質及大小並無限定。作為發揮此種導引機構之作用者，例如如圖2所示，可為兼任保持濾紙小片之保持部200(例如，夾具)之側緣10。又，本發明由於無需施加高電壓來將試樣離子化，故而所使用之保持部200可為絕緣性者。 As described above, the carrier 100 used in the present invention is preferably formed with the liquid receiving portion 120 and the minute space group, but may be further provided in the carrier 100 to assist the sample solution 310 or the solvent 320 from the analysis portion 400. The guiding mechanism of the suction port 410 is sucked. Here, the "guiding mechanism" is a term commonly used by the practitioner, and in the present invention, as described above, is used to assist in sucking the sample solution 310 or the solvent 320 from the suction port 410 of the analysis portion 400. Structure, and the material and size are not limited. As a function of the guide mechanism, for example, as shown in FIG. 2, the side edge 10 of the holding portion 200 (for example, a jig) for holding the filter paper piece may be used. Further, in the present invention, since the sample is ionized without applying a high voltage, the holding portion 200 used can be insulated.
例如，於使用夾具將濾紙小片固定之態樣中，濾紙小片較佳為不由夾具覆蓋其整體，而以於濾紙小片殘留有餘白之方式被固定。該餘白之中，夾具側面之餘白部分相當於供給試樣溶液310或溶劑320之液體接收部120。該餘白部分之寬度並無限定，只要為約0.5~2mm即可。於供給後，試樣溶液310或溶劑320一面含浸一面朝濾紙之一端110即分析部400之抽吸口410流動，如此，夾具側面10發揮作為導引機構之作用。另一方面，如上所述，存在於濾紙小片之餘白之中，距抽吸口410較近之側之餘白部分(圖2中之S2)可不必設置，但於設置餘白部分之情形時，只要為足夠形成試樣溶液之微小液滴(儲液)之大小即可。此種餘白部分之面積並無限定，較佳為約0.001~10mm2。 For example, in the case of fixing the filter paper piece by using a jig, the filter paper piece is preferably not covered by the jig, and is fixed in such a manner that the filter paper piece remains white. In the margin, the remaining white portion of the side surface of the jig corresponds to the liquid receiving portion 120 to which the sample solution 310 or the solvent 320 is supplied. The width of the margin portion is not limited, and may be about 0.5 to 2 mm. After the supply, the sample solution 310 or the solvent 320 is impregnated while flowing toward the suction port 410 of the analysis unit 400, which is one end of the filter paper. Thus, the side surface 10 of the jig functions as a guiding mechanism. On the other hand, as described above, it exists in the margin of the filter paper piece, and the margin portion (S 2 in FIG. 2 ) on the side closer to the suction port 410 may not necessarily be provided, but the margin portion is provided. In the case, it is sufficient to form a small droplet (reservoir) of the sample solution. The area of such a white portion is not limited, but is preferably about 0.001 to 10 mm 2 .
再者，為了使業者理解，可使用保持部200之側緣作為導引機構10，或亦可使用設置於載體上之槽作為導引機構(10a、10b、10c、10d、10d')。例如，圖3所示之載體中，所設置之槽之形狀如剖視圖所示，為具有傾斜面及曲面者，但關於槽之深度及位置均無限定。因此，只要達成本發明之目的，作為導引機構而利用之載體上之槽之形狀、深度、及位置並無限定，例如可例示圖4所示之導引機構(10b、10c、10d、10d')。又，如圖5所示，可藉由在載體100"之一側面設置階差，將該階差作為導引機構10e而利用(圖5(A))。具有導引機構10e之載體100"之剖面如圖5(B)所示。 Furthermore, in order to make the understanding of the industry, the side edge of the holding portion 200 may be used as the guiding mechanism 10, or the groove provided on the carrier may be used as the guiding mechanism (10a, 10b, 10c, 10d, 10d'). For example, in the carrier shown in FIG. 3, the shape of the groove provided as shown in the cross-sectional view is an inclined surface and a curved surface, but the depth and position of the groove are not limited. Therefore, as long as the object of the present invention is achieved, the shape, depth, and position of the groove on the carrier used as the guiding means are not limited, and for example, the guiding mechanism (10b, 10c, 10d, 10d shown in Fig. 4) can be exemplified. '). Further, as shown in Fig. 5, the step can be used as the guiding mechanism 10e by setting a step on one side of the carrier 100" (Fig. 5(A)). The carrier 100" having the guiding mechanism 10e" The cross section is shown in Fig. 5(B).
另一方面，於載體100不設置槽，而於載體100之表面上載置片體500，藉此亦可設置導引機構。例如，如圖6般，可藉由在載體100上相互空開間隔地載置2片片體500而於該等2片片體500之間設置導引機構10f。又，如圖7般，可藉由將1片片體500'載置於載體100，與圖5之情形同樣地利用片體500'之側緣於載體100上形成階差，將該階差作為導引機構10g而利用。此處，可使用之片體500並無特別限定，可為任一種素材，亦可為具有不吸收溶劑之性質者。載置於載體之片體具有抑制溶劑之蒸發之效果，且於在載體上提取成分之情形時，具有提高提取效率之作用。 On the other hand, the carrier 100 is not placed on the carrier 100, and the sheet 500 is placed on the surface of the carrier 100, whereby a guiding mechanism can also be provided. For example, as shown in FIG. 6, the guide body 10f can be provided between the two sheets 500 by placing two sheets 500 on the carrier 100 at intervals. Further, as shown in Fig. 7, by placing one sheet 500' on the carrier 100, a step is formed on the carrier 100 by the side edge of the sheet 500' as in the case of Fig. 5, and the step is formed. It is used as the guide mechanism 10g. Here, the sheet body 500 that can be used is not particularly limited, and may be any material or may have a property of not absorbing a solvent. The sheet placed on the carrier has an effect of suppressing evaporation of the solvent, and has an effect of improving extraction efficiency when the component is extracted on the carrier.
以試樣為皮膚之角質層之情形為例，對試樣及載體之製備進行說明。於在本說明書中使用時，所謂「角質層」係指位於皮膚之最外層，且表皮角化細胞已角化之扁平之角質層細胞重疊之層。細胞-細胞間被角質層細胞間脂質填滿，且角質層之角質層細胞重疊有約15層，平均具有約10~20μm厚。角質層之層厚因部位而不同，於臉頰相對較薄，另一方面，於腳後跟等，角質層細胞之重疊達到約50層。 利用本發明之方法能夠測定之層厚並無特別限定，將約5~15層設為分析對象。為將角質層剝離，膠帶剝離法較有效。能夠用於將角質層 剝離之表面具有黏著層之片體(黏著片體600等)並無特別限定，可列舉：D-Squame(CuDerm公司製造)、Sellotape(註冊商標)(Nichiban公司製造)、PPS膠帶(Nichiban公司製造)等。例如，使用此種片材採取角質層之後，使用形成有該黏著片體600及微小空間群之載體100夾住附著於該黏著片體600之角質層700(圖8)。可對夾住角質層之該載體供給溶劑，而作為溶解有該角質層之成分之該溶劑試樣溶液使用。又，亦能夠使用表面具有黏著層800且形成有微小空間群之載體100。於此情形時，由於在載體100上保持有已被剝離之角質層700，故而可藉由對該載體供給溶劑而製作試樣溶液(圖9)。 The preparation of the sample and the carrier will be described by taking the case where the sample is the stratum corneum of the skin. As used herein, the term "stratum corneum" refers to a layer of flattened stratum corneum cells that are located at the outermost layer of the skin and whose keratinocytes have been keratinized. The cell-cell is filled with intercellular lipids in the stratum corneum, and the stratum corneum cells of the stratum corneum overlap by about 15 layers, and have an average thickness of about 10-20 μm. The thickness of the stratum corneum varies from site to site, and is relatively thin on the cheeks. On the other hand, on the heel, the overlap of the stratum corneum cells reaches about 50 layers. The layer thickness which can be measured by the method of the present invention is not particularly limited, and about 5 to 15 layers are used as analysis targets. In order to peel off the stratum corneum, the tape peeling method is effective. Can be used to cut the stratum corneum The sheet having the adhesive layer on the surface to be peeled off (the adhesive sheet 600 and the like) is not particularly limited, and examples thereof include D-Squame (manufactured by CuDerm Co., Ltd.), Sellotape (registered trademark) (manufactured by Nichiban Co., Ltd.), and PPS tape (manufactured by Nichiban Co., Ltd.). )Wait. For example, after the stratum corneum is used using such a sheet, the stratum corneum layer 700 attached to the adhesive sheet body 600 is sandwiched by the carrier 100 on which the adhesive sheet body 600 and the minute space group are formed (FIG. 8). The carrier may be supplied with a solvent to the carrier sandwiching the stratum corneum, and used as a solvent sample solution in which the component of the stratum corneum is dissolved. Further, it is also possible to use the carrier 100 having the adhesive layer 800 on its surface and having a small space group formed therein. In this case, since the exfoliated layer 700 having been peeled off is held on the carrier 100, a sample solution can be prepared by supplying a solvent to the carrier (Fig. 9).
4.質量分析裝置 4. Mass analysis device
典型而言，本發明係使包含溶解於溶劑之試樣之試樣溶液310含浸於載體100，並於該載體100與分析部400之抽吸口410不相接之狀態下，自抽吸口410抽吸該試樣溶液310，無需電壓、熱及氣體等而於數秒以內進行質量分析(參照圖1)。更具體而言，本發明可提供一種質量分析裝置1，其特徵在於：其係對試樣中所包含之成分進行定性或定量分析者，且具備：載體100，其形成有微小空間群；保持部200，其保持該載體；供給部300，其對該載體供給溶劑320或試樣溶液310；及分析部400，其具有抽吸口410，且對該成分進行定性或分析；且於該載體100與該抽吸口410不相接之狀態下，該載體100由該保持部200保持，自該供給部300所供給之包含溶解於該溶劑之試樣之試樣溶液310到達一端110，並自該抽吸口410抽吸該已到達之試樣溶液。 Typically, in the present invention, the sample solution 310 containing the sample dissolved in the solvent is impregnated into the carrier 100, and the carrier 100 is not in contact with the suction port 410 of the analysis unit 400, and is self-suction port. The sample solution 310 is sucked by 410, and mass analysis is performed within a few seconds without requiring voltage, heat, gas, or the like (refer to FIG. 1). More specifically, the present invention can provide a mass spectrometer 1 characterized in that it qualitatively or quantitatively analyzes components contained in a sample, and includes: a carrier 100 formed with a minute space group; a portion 200 that holds the carrier; a supply portion 300 that supplies a solvent 320 or a sample solution 310 to the carrier; and an analysis portion 400 that has a suction port 410 and that characterizes or analyzes the component; When the 100 is not in contact with the suction port 410, the carrier 100 is held by the holding portion 200, and the sample solution 310 supplied from the supply portion 300 containing the sample dissolved in the solvent reaches the end 110, and The sample solution that has arrived is sucked from the suction port 410.
本發明中所使用之「保持部」(200)係指於分析部400之抽吸口 410之前方保持載體100之部分或構件(參照圖1及2)。保持部200由於無需高電壓來將試樣離子化，故可為絕緣性之材質，只要為具有足夠保持載體之構造者，則並無限定。對於保持部200並無限定，可為絕緣性夾具、或導電性夾具等。 The "holding portion" (200) used in the present invention refers to the suction port of the analyzing portion 400. A portion or member of the carrier 100 is held before 410 (refer to Figures 1 and 2). Since the holding portion 200 does not require a high voltage to ionize the sample, it can be made of an insulating material, and is not limited as long as it has a structure sufficient to hold the carrier. The holding portion 200 is not limited, and may be an insulating jig or a conductive jig.
本發明中所使用之「供給部」(300)係為了對載體100供給試樣溶液310或溶劑320而具有貯存該試樣溶液或溶劑之空間及噴出該試樣溶液等之構件者，例如可列舉移液管、注射器等。再者，供給部300並無特別限定，可為如移液管之使用般具有獨立之構造者，或亦可使其配備於保持部200，還可固定於載體100。 The "supply portion" (300) used in the present invention is a member for storing a sample solution 310 or a solvent 320 for the carrier 100, and a member for storing the sample solution or solvent and for ejecting the sample solution, for example, List pipettes, syringes, etc. Further, the supply unit 300 is not particularly limited, and may have an independent structure such as a pipette, or may be provided in the holding unit 200 or may be fixed to the carrier 100.
本發明中所使用之「分析部」(400)係除用以抽吸試樣溶液之抽吸口410以外，使試樣溶液體中之成分供於分析之部分，對根據成分之性狀而產生之測定訊號進行檢測。此種分析部400並無限定，可使用：混合型串聯質量分析機器(例如，LTQ-Orbtrap(註冊商標)(Thermo Fisher Scientific,Inc.,San Jose,CA))、6430qTOF(Agilent Technologies,Inc.,CA)、LCMS-8050(島津製作所，京都))、離子遷移率-TOF型質量分析儀(例如，Synapt G2 SI(Nihon Waters K.K.))、qTOF型質量分析儀(例如，6530qTOF(Agilent Technologies)、三級四極型質量分析儀(例如，6430QQQ(Agilent Technologies))、小型單四極型質量分析儀(例如，QDa(Nihon Waters K.K.))、Q-Orbitrap型質量分析儀(QExactive(Thermo Fisher SCIENTIFIC))等。 The "analytical unit" (400) used in the present invention is a part of the sample solution which is supplied to the analysis portion in addition to the suction port 410 for sucking the sample solution, and is produced according to the properties of the component. The measurement signal is detected. The analysis unit 400 is not limited, and a hybrid tandem mass spectrometer (for example, LTQ-Orbtrap (registered trademark) (Thermo Fisher Scientific, Inc., San Jose, CA)), 6430qTOF (Agilent Technologies, Inc.) can be used. , CA), LCMS-8050 (Shimadzu Corporation, Kyoto), ion mobility-TOF type mass analyzer (for example, Synapt G2 SI (Nihon Waters KK)), qTOF type mass analyzer (for example, 6530qTOF (Agilent Technologies) , three-stage quadrupole mass analyzer (for example, 6430QQQ (Agilent Technologies)), small single quadrupole mass analyzer (for example, QDa (Nihon Waters KK)), Q-Orbitrap type mass analyzer (QExactive (Thermo Fisher SCIENTIFIC) )Wait.
本發明之特徵在於：於載體100之一端110與抽吸口410不相接之狀態下，試樣溶液自載體之一端110被抽吸至抽吸口410，但根據試樣溶液之黏性、因分析部400內之真空壓而產生之於抽吸口410附近之抽吸力等條件，對該載體之一端110與抽吸口410之距離及高度進行調整，以抽吸形成於該載體之一端110之微小液滴。該載體之一端110與抽吸口410之距離並無限定，較佳為0.1~0.5mm。又，分析部400內 之真空壓並無限定，較佳為0.1hPa以下。 The present invention is characterized in that, in a state in which one end 110 of the carrier 100 is not in contact with the suction port 410, the sample solution is sucked from the one end 110 of the carrier to the suction port 410, but according to the viscosity of the sample solution, The distance and height of one end 110 of the carrier and the suction port 410 are adjusted by suction pressure generated by the vacuum pressure in the analysis unit 400 in the vicinity of the suction port 410, and are formed by suction on the carrier. A tiny droplet at one end 110. The distance between one end 110 of the carrier and the suction port 410 is not limited, and is preferably 0.1 to 0.5 mm. Moreover, the analysis unit 400 The vacuum pressure is not limited, and is preferably 0.1 hPa or less.
典型而言，使用本發明之質量分析裝置1之試樣之分析以下。將載體100固定於保持部200，以間隔成為0.1~0.5mm之方式進行調整、並固定，以使該載體之一端110不與分析部400之抽吸口410相接。繼而，例如於使用液體作為試樣之情形時，將所製備之試樣溶液310自供給部300供給至載體100上之液體接收部120。所供給之試樣溶液一面含浸於載體100一面貯存於載體之一端110，並迅速地自分析部400之抽吸口410被抽吸至分析部400。可將所抽吸出之試樣溶液利用分析部400進行測定，而獲得測定訊號(參照實施例1~4)。 Typically, the analysis of the sample using the mass spectrometer 1 of the present invention is as follows. The carrier 100 is fixed to the holding portion 200, and is adjusted and fixed so as to have an interval of 0.1 to 0.5 mm so that one end 110 of the carrier is not in contact with the suction port 410 of the analysis portion 400. Then, for example, when a liquid is used as the sample, the prepared sample solution 310 is supplied from the supply unit 300 to the liquid receiving portion 120 on the carrier 100. The supplied sample solution is stored on one side of the carrier 110 while being impregnated on the carrier 100, and is quickly sucked from the suction port 410 of the analysis unit 400 to the analysis unit 400. The sample solution to be aspirated can be measured by the analysis unit 400 to obtain a measurement signal (see Examples 1 to 4).
[實施例] [Examples]
以下，列舉製造例及實驗例對本發明之構成及效果進一步明確地進行說明。但是，本發明並不受該等實施例任何限制。 Hereinafter, the constitution and effects of the present invention will be further clearly described by way of production examples and experimental examples. However, the invention is not limited by the examples.
於以下對在本實施例中具體所使用之材料及裝置進行說明。 The materials and devices specifically used in the present embodiment will be described below.
(1)材料 (1) Materials
自和光純藥股份有限公司(日本大阪)購入LC/MS級之甲醇(MeOH)及乙腈(ACN)。使用MilliQ水純化系統型號MQ Advantage獲得新鮮的水。該裝置係由Elix RO/IEX淨化器、SP-TOF淨化器、及ASM自動淨化模組(MilliporeInc.)而構成。自Nacalai Tesque日本(日本大阪)取得甲酸(99%)。 LC/MS grade methanol (MeOH) and acetonitrile (ACN) were purchased from Wako Pure Chemical Co., Ltd. (Osaka, Japan). Fresh water was obtained using the MilliQ water purification system model MQ Advantage. The device consists of an Elix RO/IEX purifier, an SP-TOF purifier, and an ASM automatic purification module (Millipore Inc.). Formic acid (99%) was obtained from Nacalai Tesque Japan (Osaka, Japan).
自味之素股份有限公司(日本東京)取得鹽酸離胺酸。自Sigma-Aldrich公司購入甘胺酸(d2)。 Amino acid hydrochloride was obtained from Ajinomoto Co., Ltd. (Tokyo, Japan). Glycine (d2) was purchased from Sigma-Aldrich.
自Whatman(GE Healthcare Life Sciences,Inc.,UK)取得濾紙42(55mm直徑)。如後文所述，不進行處理而使用，但於分析中切斷成適當尺寸之小片。於對角質層成分進行分析之情形時，使用能夠自Promotool,Inc.(日本東京)取得之黏著皮膚剝離膠帶(Skinchecker(註冊商標))。 Filter paper 42 (55 mm diameter) was taken from Whatman (GE Healthcare Life Sciences, Inc., UK). As described later, it was used without treatment, but was cut into small pieces of an appropriate size in the analysis. In the case of analyzing the stratum corneum component, an adhesive skin release tape (Skinchecker (registered trademark)) which can be obtained from Promotool, Inc. (Tokyo, Japan) was used.
自Agilent Technologies,Inc.(Paloalto,CA)以0.1M鹽酸中之1mM(nmol/μL)之形式取得實施例1中所使用之包含17個胺基酸之標準混合物(產品編號5061-3330；L-天冬胺酸、甘胺酸、L-丙胺酸、L-纈胺酸、L-白胺酸、L-異白胺酸、L-絲胺酸、L-蘇胺酸、L-酪胺酸、L-脯胺酸、L-精胺酸、L-組胺酸、L-麩胺酸、L-胱胺酸、L-苯基丙胺酸、L-離胺酸、及L-甲硫胺酸)。 A standard mixture of 17 amino acids used in Example 1 was obtained from Agilent Technologies, Inc. (Paloalto, CA) in 1 mM (nmol/μL) in 0.1 M hydrochloric acid (Product No. 5061-3330; L - Aspartic acid, glycine, L-alanine, L-proline, L-leucine, L-isoleucine, L-serine, L-threonine, L-tyramine Acid, L-proline, L-arginine, L-histamine, L-glutamic acid, L-cystine, L-phenylalanine, L-lysine, and L-methyl sulfide Amino acid).
自Sigma-Aldrich取得實施例2中所使用之試劑級之血管收縮素II及胰島素。又，自Shiseido Co.,Ltd(日本東京)取得化妝品(IPSA METABOLIZER Moist White W1)。 The reagent grade angiotensin II and insulin used in Example 2 were obtained from Sigma-Aldrich. Further, cosmetics (IPSA METABOLIZER Moist White W1) were obtained from Shiseido Co., Ltd (Tokyo, Japan).
(2)載體及其使用 (2) Carrier and its use
將濾紙(55mm直徑)(Whatman(GE Healthcare Life Sciences,Inc.,UK)切斷成長方形片(典型而言為2.5×10mm)。形狀未必為長方形，只要微小液滴可被面向質量分析機器之毛細管入口之濾紙一端抽吸，則可為任意之形狀。同樣，濾紙小片之大小並無限定，但必需為能夠充分將供於質量分析之試樣保持於濾紙之大小。繼而，將濾紙小片夾於利用任意之材料(金屬、塑膠等)所製作之夾具。其後，使濾紙小片之前端部接近於質量分析機器之毛細管入口之開口部(抽吸口)而配置。以毛細管入口之位置為基準，固定於充分由向質量分析機器內之抽吸力抽吸形成於濾紙小片之一端之微小液滴之高度，將毛細管入口與濾紙小片間之距離設為約0.5mm。距離可根據提取溶劑之組成而最佳化，可進行適當調整。較佳為使毛細管入口之中心線與濾紙小片之中心線一致。然而，只要微小液滴於濾紙小片之一端形成，且恰好地被抽吸至質量分析機器內，則無需使軸一致。 Filter paper (55 mm diameter) (Whatman (GE Healthcare Life Sciences, Inc., UK) was cut into rectangular pieces (typically 2.5 x 10 mm). The shape is not necessarily rectangular as long as the tiny droplets can be oriented to the mass analysis machine The filter paper at the capillary inlet may be of any shape, and the size of the filter paper is not limited, but it is necessary to sufficiently maintain the sample for mass analysis in the size of the filter paper. Then, the filter paper clip is clamped. A jig made of any material (metal, plastic, etc.). Thereafter, the front end of the filter paper piece is placed close to the opening (suction port) of the capillary inlet of the mass spectrometer. The reference is fixed to the height of the minute droplet formed at one end of the filter paper piece by suction into the mass spectrometer, and the distance between the capillary inlet and the filter paper piece is set to about 0.5 mm. The distance can be determined according to the extraction solvent. The composition is optimized and can be appropriately adjusted. It is preferable to make the center line of the capillary inlet coincide with the center line of the filter paper piece. However, as long as the minute droplets are on the filter paper One end of the sheet is formed, and just being drawn into the machine mass analysis, it is not necessary to consistent shaft.
供給之提取溶劑之體積典型而言為5~20μL，但亦可對照濾紙小片之尺寸而進行變化。一般而言，若增大濾紙小片之尺寸，則必需相應地增大應供給之提取溶劑之體積。其原因在於：因過量之溶劑會於 濾紙小片形成「圓頂」，並於濾紙小片之一端形成微小液滴(儲液)，但並非必要條件。 The volume of the extraction solvent supplied is typically 5 to 20 μL, but it can also be changed in comparison with the size of the filter paper. In general, if the size of the filter paper piece is increased, it is necessary to correspondingly increase the volume of the extraction solvent to be supplied. The reason is that the excess solvent will The filter paper pieces form a "dome" and form tiny droplets (reservoir) at one end of the filter paper piece, but this is not a requirement.
(3)分析部 (3) Analysis Department
於全部實施例中，使用作為混合型串聯質量分析機器之LTQ-Orbtrap(註冊商標)(Thermo Fisher Scientific,Inc.,SanJose,CA)，而取得質量分析資料。作為用以導入試樣溶液之三維操作階段，將Nanospray Flex離子供給源(註冊商標)安裝於質量分析機器之源極歧管。由於EmDI-MS實驗中不需要電壓，故而未使用用於供給高電壓(典型而言為±2~5kV)之連接電纜。藉由使用附鱷魚嘴夾之導電電纜連接主要之導電面/非導電面，而將試樣區域之接地電位固定。 In all the examples, mass analysis data was obtained using LTQ-Orbtrap (registered trademark) (Thermo Fisher Scientific, Inc., San Jose, CA) as a hybrid tandem mass spectrometer. As a three-dimensional operation stage for introducing a sample solution, a Nanospray Flex ion source (registered trademark) was attached to a source manifold of a mass spectrometer. Since no voltage is required in the EmDI-MS experiment, a connection cable for supplying a high voltage (typically ±2 to 5 kV) is not used. The ground potential of the sample area is fixed by connecting the main conductive surface/non-conductive surface with a conductive cable attached to the crocodile mouth clip.
只要無特別指示，於各實驗中，將毛細管入口之溫度設定成350℃。為了避免對出口施加意外的電壓，而將毛細管電壓設為0kV。對質量分析之大部分使用線性型四極離子捕捉質量分析器LTQ(註冊商標)而進行。將機器特有之設定設為如下：微掃描數：1；最大射出時間：10ms；掃描速度：普通或高速；AGC離子靶設定(全掃描)：3.00e+4。使用Xcalibur 2.1軟體(Thermo Fisher Scientific,CA)控制質量分析機器，進而，進行資料分析。 The temperature of the capillary inlet was set to 350 ° C in each experiment unless otherwise specified. In order to avoid applying an unexpected voltage to the outlet, the capillary voltage is set to 0 kV. Most of the mass analysis was performed using a linear quadrupole ion trap mass analyzer LTQ (registered trademark). The machine-specific settings are set as follows: micro-scan count: 1; maximum shot time: 10 ms; scan speed: normal or high speed; AGC ion target setting (full scan): 3.00e+4. The mass analysis machine was controlled using Xcalibur 2.1 software (Thermo Fisher Scientific, CA), and further, data analysis was performed.
實施例1：本發明之分析方法之驗證Example 1: Verification of the analytical method of the present invention
本發明之分析方法之驗證係使用17個胺基酸標準混合物自(a)再現性、(b)線性、及(c)感度之觀點而進行。 The verification of the analytical method of the present invention was carried out using the 17 amino acid standard mixtures from the viewpoints of (a) reproducibility, (b) linearity, and (c) sensitivity.
(a)再現性 (a) Reproducibility
使用銅製鱷魚嘴夾，分別夾住同一尺寸及同一形狀之3張濾紙小片(具體而言，於2.5mm×10mm之長方形之濾紙小片上積層10nmol之各胺基酸)。濾紙小片於室溫(約25℃)下放置約15分鐘進行乾燥。使中心軸一致，並將濾紙小片配置於質量分析機器之毛細管入口前，以手動暫時地供給10μL之MeOH-純化水(1：1)之總量。同時，使用質量 分析機器開始資料收集，但預先將取得質量射程設定為m/z 50-500。將各試樣之執行時間設定為30秒，但實際之分析時間均為數秒以內。進而，為了對再現性進行評價，對3張濾紙小片重複進行相同之方法。平均之質量光譜能夠根據各資料之TICC(全離子電流層析圖)而獲得，根據表觀之峰強度比(y軸之高度)進行定性地比較(圖10)。 Using a copper crocodile mouth clip, three pieces of filter paper of the same size and the same shape were respectively sandwiched (specifically, 10 nmol of each amino acid was laminated on a 2.5 mm × 10 mm rectangular filter paper piece). The filter paper pieces were allowed to stand at room temperature (about 25 ° C) for about 15 minutes for drying. The center axis was made uniform, and the filter paper pieces were placed in front of the capillary inlet of the mass spectrometer to temporarily temporarily supply 10 μL of MeOH-purified water (1:1). At the same time, use quality The analysis machine starts data collection, but the mass range is set to m/z 50-500 in advance. The execution time of each sample was set to 30 seconds, but the actual analysis time was within a few seconds. Further, in order to evaluate the reproducibility, the same method was repeated for three sheets of filter paper. The average mass spectrum can be obtained from the TICC (full ion current chromatogram) of each data, and is qualitatively compared based on the apparent peak intensity ratio (the height of the y-axis) (Fig. 10).
(b)線性 (b) Linear
藉由使用17個胺基酸標準與甘胺酸(d2)之混合物之內部標準法，對本發明之EmDI-MS法之線性進行評價。將混合物溶液(後文所述)積層於4片各濾紙小片。此處，17個胺基酸量係每張濾紙小片(2.5mm×10mm)設為1、5、10及50ng(以下，分別稱為「Std1」、「Std2」、「Std3」及「Std4」)。為了作為內部標準發揮功能，甘胺酸(d2)量設為固定量(10ng)。 The linearity of the EmDI-MS method of the present invention was evaluated by an internal standard method using a mixture of 17 amino acid standards and glycine acid (d2). A mixture solution (described later) was laminated on 4 sheets of each filter paper. Here, the amount of 17 amino acids is set to 1, 5, 10, and 50 ng per filter paper piece (2.5 mm × 10 mm) (hereinafter, referred to as "Std1", "Std2", "Std3", and "Std4", respectively. ). In order to function as an internal standard, the amount of glycine (d2) was set to a fixed amount (10 ng).
Std1、Std2及Std3藉由使3種標準溶液之分注液積層於3張濾紙小片上而製備。為了製備Std3，積層包含10mM之標準溶液之母液(各1mM之胺基酸)。Std1及Std2分別使用包含0.01mM及0.1M之胺基酸之溶液(任一者均包含10mM之內部標準)。該等係藉由使用甲醇-純化水(1：1)使母液連續稀釋而製備。Std之製備係將以1mM之內部標準進行過量供給(spike)後之母液之分注液積層於另一濾紙小片。分注液係藉由重複積層-乾燥循環而於濾紙小片上積層10次。乾燥處理係於室溫下在平穩之空氣流下進行。 Std1, Std2, and Std3 were prepared by laminating three standard solution dispersions onto three sheets of filter paper. To prepare Std3, a mother liquor containing 10 mM of a standard solution (1 mM each of the amino acids) was laminated. Std1 and Std2 each used a solution containing 0.01 mM and 0.1 M amino acid (each containing an internal standard of 10 mM). These were prepared by serially diluting the mother liquor using methanol-purified water (1:1). Std was prepared by layering the mother liquor after a spike in an internal standard of 1 mM onto another filter paper. The dispensing solution was laminated 10 times on the filter paper piece by repeating the build-up-drying cycle. The drying treatment is carried out at room temperature under a steady stream of air.
Std1~Std4之濾紙小片係與上述順序相同地供於EmDI-MS分析。根據Std1~Std4校準標準之平均質量光譜計算各胺基酸與內部標準間之波峰之高度，並進行比較。繼而，將積層於濾紙小片上之胺基酸量設為x軸進行繪圖，製作校準曲線。根據藉由最小平方法而產生之校準線之R2(決定係數)及切片對本發明之分析方法之線性進行評價(圖11)。 The filter paper pieces of Std1 to Std4 were supplied to the EmDI-MS analysis in the same manner as described above. The height of the peak between each amino acid and the internal standard was calculated and compared according to the average mass spectrum of the Std1~Std4 calibration standard. Then, the amount of amino acid accumulated on the filter paper piece was plotted on the x-axis to prepare a calibration curve. The linearity of the analytical method of the present invention was evaluated based on the R 2 (determination coefficient) and the slice of the calibration line generated by the least squares method (Fig. 11).
(c)敏感性 (c) Sensitivity
敏感性係根據Std1之質量光譜中之胺基酸訊號之訊號對雜訊(SN)比而推定(圖12)。檢測下限(LLOD)係以SN>3之推定濃度進行定義。 Sensitivity is estimated based on the signal of the amino acid signal in the mass spectrum of Std1 versus the noise (SN) ratio (Figure 12). The lower limit of detection (LLOD) is defined by the estimated concentration of SN > 3.
實施例2：使用標準試樣之分析方法之驗證Example 2: Verification of analytical methods using standard samples
使用肽對本發明之分析方法之實施可能性進行驗證。於本實施例中，使用血管收縮素II及胰島素作為肽，分別溶解於包含1mM之0.1%甲酸及2%之CAN之純化水中。將10μL各溶液積層於各濾紙小片(2.5mm×10mm之長方形)，並使濾紙小片於室溫下進行乾燥。於各濾紙小片積層有肽10nmol。按照實施例1之順序進行本發明之EmDI-MS分析，但於本實施例中，將提取溶劑變更為含有0.1%甲酸之ACN-純化水(1：1)。進而，為了藉由「預濕潤」提高肽之提取效率，且使微小液滴容易吸收至質量分析機器內，於濾紙小片應用2次提取溶劑。預濕潤中應用包含0.1甲酸之ACN-純化水(1：1)5μL。為了提取及形成微小液滴，於剛進行預濕潤處理之後(約10秒後)應用上述溶劑(10μL)。將血管收縮素II及胰島素之分析之結果(質量光譜)分別示於圖13及14。如由該等質量光譜所明確，由於得以觀察到血管收縮素II及胰島素之各者特有之質量光譜，故而可實際驗證本發明之分析方法亦能夠對使用肽之試樣進行分析。 The possibility of implementation of the analytical method of the invention was verified using peptides. In the present example, angiotensin II and insulin were used as peptides, respectively, and dissolved in purified water containing 1 mM of 0.1% formic acid and 2% of CAN. 10 μL of each solution was layered on each filter paper piece (2.5 mm × 10 mm rectangle), and the filter paper pieces were dried at room temperature. The peptide was laminated on each filter paper to have a peptide of 10 nmol. The EmDI-MS analysis of the present invention was carried out in the same manner as in Example 1, but in the present example, the extraction solvent was changed to ACN-purified water (1:1) containing 0.1% formic acid. Further, in order to increase the extraction efficiency of the peptide by "pre-wetting" and to easily absorb the fine droplets into the mass spectrometer, the solvent is applied twice to the filter paper. A pre-wet was applied with ACN-purified water (1:1) of 5 μL of 0.1 formic acid. In order to extract and form minute droplets, the above solvent (10 μL) was applied immediately after the pre-wetting treatment (about 10 seconds later). The results of the analysis of angiotensin II and insulin (mass spectrum) are shown in Figs. 13 and 14, respectively. As is clear from these mass spectra, since the mass spectrum unique to each of angiotensin II and insulin can be observed, it can be actually verified that the analysis method of the present invention can also analyze the sample using the peptide.
實施例3：使用化妝品之分析方法之驗證Example 3: Verification of analytical methods using cosmetics
繼而，使用化妝品對本發明之分析方法之實施可能性進行驗證。將化妝品(原型之化妝液，無預處理)之一部分(1μL)積層於濾紙小片(2.5mm×10mm之長方形)。使濾紙小片於室溫下乾燥約10分鐘，其後，按照實施例1之順序進行本發明之EmDI-MS分析。將結果示於圖15。化妝品中雖包含多種成分，但能夠主要明確地分離成合成聚合物、界面活性劑、及有效成分，即便於使用半固形物之情形時，本發明之分析方法之有效性亦得到實際驗證。 Then, the possibility of implementation of the analytical method of the present invention is verified using cosmetics. A part (1 μL) of a cosmetic (prototype lotion without pretreatment) was laminated on a filter paper piece (2.5 mm × 10 mm rectangle). The filter paper pieces were allowed to dry at room temperature for about 10 minutes, after which the EmDI-MS analysis of the present invention was carried out in the same manner as in Example 1. The results are shown in Fig. 15. Although the cosmetic contains a plurality of components, it can be mainly separated into a synthetic polymer, a surfactant, and an active ingredient, and the effectiveness of the analysis method of the present invention is actually verified even in the case of using a semi-solid.
實施例4：使用人類皮膚(角質層)之分析方法之驗證Example 4: Verification of analytical methods using human skin (stratum corneum)
使用人類皮膚，藉由EmDI-MS法對角質層中所包含之成分進行分析。藉由黏著帶剝離法，使用黏著帶裝置(Skinchecker(註冊商標))自健康之男性志願者(48歲)之內側前臂採取角質層作為試樣。將典型之順序示於以下。 The components contained in the stratum corneum were analyzed by the EmDI-MS method using human skin. The stratum corneum was taken as a sample from the medial forearm of a healthy male volunteer (48 years old) by an adhesive tape device (Skinchecker (registered trademark)) by an adhesive tape peeling method. The typical sequence is shown below.
(1)利用肥皂將內側前臂溫和地洗淨。 (1) Wash the inner forearm gently with soap.
(2)為使平衡而等待30分鐘。 (2) Wait 30 minutes for balance.
(3)藉由使用Skinchecher(註冊商標)之膠帶剝離回收角質層(10層)。 (3) The stratum corneum (10 layers) was peeled off by using a tape of Skinchecher (registered trademark).
(4)將所回收之角質層置於濾紙上，對接著區域平穩地施壓以使其固定。 (4) The recovered stratum corneum was placed on a filter paper, and the pressing area was smoothly pressed to fix it.
(5)將角質層所接著之濾紙切斷成小片(2.5mm×10mm之長方形)。 (5) The filter paper next to the stratum corneum was cut into small pieces (rectangular shape of 2.5 mm × 10 mm).
(6)使用鱷魚嘴夾(金屬、塑膠及其他)夾住濾紙小片，設置於質量分析機器之毛細管入口附近。 (6) Clamp the filter paper piece with a crocodile mouth clip (metal, plastic, and others) and set it near the capillary inlet of the mass spectrometer.
(7)對濾紙供給5~10μL之提取溶劑，迅速地進行EmDI-MS分析。 (7) 5 to 10 μL of the extraction solvent was supplied to the filter paper, and the EmDI-MS analysis was quickly performed.
(8)1次EmDI-MS實驗於數秒以內完成。 (8) One time EmDI-MS experiment was completed in a few seconds.
將使用角質層進行分析之結果示於圖16。如由該結果所明確，出現作為角質層中所包含之NMF(天然保濕因子)之成分之胺基酸、礦物質、PCA(吡咯啶酮羧酸)、乳酸鹽、脲等之質量光譜，即便於將人類皮膚作為試樣之情形時，本發明之分析方法之有效性亦得到實際驗證。 The results of analysis using the stratum corneum are shown in Fig. 16. As is clear from the results, mass spectra of amino acids, minerals, PCA (pyrrolidone carboxylic acid), lactate, urea, etc., which are components of NMF (natural moisturizing factor) contained in the stratum corneum, appear even The effectiveness of the analytical method of the present invention is also practically verified in the case of using human skin as a sample.
圖1表示實施上文所述之本發明之方法之分析裝置。於圖1中，分析裝置具備分析部410、保持形成有微小空間群之載體100之保持部200、對載體100供給試樣之供給部300作為主要之構成要素。分析部 400可包含作為離子源之真空容器、於該容器內形成真空之真空泵、對上述容器內施加高電壓之高電壓源、與作為離子源之上述真空容器連通而將已被離子化之試樣分離之分析部、對在該分析部中所篩選出之離子進行增感並進行檢測之檢測部等，且該等構成要素收容於殼體402內。於殼體402之一側面設置有構成試樣導入部之抽吸口410。 Figure 1 shows an analytical device for carrying out the method of the invention described above. In FIG. 1, the analysis device includes an analysis unit 410, a holding unit 200 that holds the carrier 100 in which the micro space group is formed, and a supply unit 300 that supplies the sample to the carrier 100 as main components. Analysis Department 400 may include a vacuum vessel as an ion source, a vacuum pump that forms a vacuum in the vessel, a high voltage source that applies a high voltage to the vessel, and a vacuum vessel that is an ion source to communicate the ionized sample. The analysis unit and the detection unit that sensitize and detect the ions selected by the analysis unit, and the components are housed in the casing 402. A suction port 410 constituting the sample introduction portion is provided on one side surface of the casing 402.
載體100於抽吸口410之附近，被保持部200保持於能夠在載體之一端110不接觸於抽吸口410之情況下將試樣溶液自載體之一端110抽吸之位置。載體100例如可設為濾紙、纖維、樹脂、布、薄層層析板(將二氧化矽凝膠、氧化鋁、聚醯胺樹脂等吸附劑呈薄膜狀固定於玻璃、鋁等之板上之薄層板)。保持部200亦可使用支架(未圖示)或框架(未圖示)等固定構件而固定於抽吸口410之前方之特定位置。供給部300可藉由移液管或注射器等構成。供給部300配置於能夠將溶劑320或試樣溶液310供給至載體100之位置。供給部300可使用支架(未圖示)或框架(未圖示)等固定構件而固定於載體100之上方之特定位置。 The carrier 100 is held in the vicinity of the suction port 410 by the holding portion 200 at a position where the sample solution can be sucked from one end 110 of the carrier without the one end 110 of the carrier contacting the suction port 410. The carrier 100 can be, for example, a filter paper, a fiber, a resin, a cloth, or a thin layer chromatography plate (the adsorbent such as cerium oxide gel, alumina, or polyamide resin is fixed in a film form on a plate of glass, aluminum, or the like. Thin layer board). The holding portion 200 may be fixed to a specific position in front of the suction port 410 by using a fixing member such as a bracket (not shown) or a frame (not shown). The supply unit 300 can be constituted by a pipette or a syringe or the like. The supply unit 300 is disposed at a position where the solvent 320 or the sample solution 310 can be supplied to the carrier 100. The supply unit 300 can be fixed to a specific position above the carrier 100 using a fixing member such as a bracket (not shown) or a frame (not shown).
應注意存在實現本說明書所揭示之實施形態之代替方法。因此，應認為：本實施形態為例示，而並非限制性者。進而，申請專利範圍並非限定於本說明書所提供之詳細情況，對其整個範圍及等效發明具有權利。 It should be noted that there are alternative ways of implementing the embodiments disclosed herein. Therefore, the present embodiment is considered to be illustrative and not restrictive. Further, the scope of the patent application is not limited to the details provided in the specification, and the scope of the invention and the equivalent invention.
1‧‧‧質量分析裝置 1‧‧‧Quality analysis device
100‧‧‧載體 100‧‧‧ Carrier
110‧‧‧載體之一端 110‧‧‧ one end of the carrier
200‧‧‧保持部 200‧‧‧ Keeping Department
300‧‧‧供給部 300‧‧‧Supply Department
310‧‧‧試樣溶液 310‧‧‧ sample solution
320‧‧‧溶劑 320‧‧‧Solvent
400‧‧‧分析部 400‧‧‧Analysis Department
402‧‧‧殼體 402‧‧‧Shell
410‧‧‧抽吸口 410‧‧ ‧ suction port
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