JP2012145351A - Substrate for mass spectrometry - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a substrate for mass spectrometry, on which a sufficient S/N ratio with high resolution can be obtained in a laser desorption ionization mass spectrometry (LDI-MS) method to accurately perform mass spectrometry of a sample of even a trace amount of less than several fmol.SOLUTION: A substrate for mass spectrometry includes: a base material having conductivity; and a measurement spot for sample holding provided on a surface of the base material. The substrate is used in laser desorption ionization mass spectrometry by attaching an analysis sample on the measurement spot. The measurement spot includes a metal oxide and an ion supply organic substance supplying a proton and/or a cation by laser irradiation, and a height of the measurement spot is 1 μm to 100 μm.

Description

  The present invention relates to a substrate for mass spectrometry used for holding a sample to be analyzed in a laser desorption ionization mass spectrometry (LDI-MS) method.

  Mass spectrometry (MS) ionizes and separates sample molecules to be measured (analyzed) (biologically-related natural polymer compounds and synthetic polymer compounds such as proteins, peptides, polysaccharides) according to their mass, This is an analysis method that enables structural analysis by dissociation products (fragment ions). In this method, sample molecules are ionized, and separation and detection are performed based on the difference between the mass (m) and the quotient (m / z) of the valence (z) of the ions. Mass spectrometry has high detection sensitivity and selectivity, and is used in many fields such as analysis of biological substances, environmentally regulated substances, and drugs. Here, ionization refers to taking electrons from sample molecules into an ion state, or adding protons or alkali metal ions to a sample to form an ion state. There is a laser desorption / ionization (LDI) method in which a laser is irradiated and ionized. Ions ionized by the LDI method are separated at a difference of m / z by passing through a mass separation unit such as a time-of-flight (TOF) and observed by a detector. Further, the intensity appears at the peak depending on the molecular ion concentration at the time of detection. By analyzing the obtained mass spectrum, the structure of the sample molecule to be measured can be analyzed (LDI-MS).

This LDI-MS includes nicotinic acid, a combination of ultrafine cobalt powder and glycerol as an ionization aid (matrix) in the analysis sample in order to achieve efficient ionization of sample molecules, Fe ₃ O ₄ nano Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS: see Non-Patent Documents 1 to 3) or a matrix-derived method using a combination of particles and polyacrylic acid. In order to eliminate the complex peaks, a surface-assisted laser desorption / ionization method (SALDI: Surface Assisted Laser Desorption / Ionization; Patent Documents 1 to 5) Patent Document 4) is known, and the above-mentioned MALDI-MS method and porous silicon which is one of the above-mentioned SALDI methods. A method combining a DIOS method (desorption / ionization on (porous) silicon: DIOS) using a template is also known (Non-patent Document 5).

  Furthermore, in Patent Document 6 by the present inventors, mass spectrometry in which a metal oxide layer made of an oxide of one or more metals selected from Fe, Co, and Cu is provided on a substrate. In addition, Patent Document 7 discloses a substrate for mass spectrometry in which metal fine particles such as cobalt are coated on a base material, and a metal oxide layer and a metal of these mass spectrometry substrates are disclosed. It is also known to perform laser desorption ionization mass spectrometry by attaching an analysis sample to the fine particle coating surface.

  However, in the above non-patent documents 1 to 5 and patent documents 1 to 7 and methods, in any case, in order to promote ionization of sample molecules, an organic acid or an organic acid metal is contained in the analysis sample every time it is measured. It is necessary to add ionization aids (matrix) and ionizing agents such as salt and ultrafine metal powder, which complicates the measurement work, and the ionizing agent itself causes noise, and depending on the amount of addition Sensitivity may change.

  In recent years, the need for mass spectrometry for biological samples and polymer samples has been increasing, and under the circumstances described above, there has been a need to further improve the performance of matrix-free ionization methods. In particular, there is a strong demand to improve ionization efficiency without adding an ionizing agent to the analysis sample.

  Therefore, as a result of intensive studies to meet these demands, the present inventors have previously been able to supply a predetermined metal oxide and protons and / or cations onto a conductive substrate. We have developed and proposed a matrix-free substrate for mass spectrometry that does not require the addition of an ionizing agent to the analysis sample during mass analysis, by providing a measurement spot that coexists with other ion-feeding organic substances (PCT / JP2010) / 61684). This mass spectrometry substrate not only eliminates the need to add an ionizing agent to the analysis sample during mass analysis, but also eliminates problems such as noise and reduced detection sensitivity. Can be analyzed with high resolution and high accuracy.

U.S. Pat.No. 6,288,390 JP 2008-107,209 JP U.S. Patent No. 7,122,792 JP 2008-204,654 JP2009-009,811 JP 2010-151,727 JP-A-62-284,256

Karas, M .; Hillenkamp, F. Anal. Chem. 1988, 60, 2299. Tanaka, K .; Eaki, H .; Ido, Y .: Akita, S .; Yoshida, Y .; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151. CHIU Yu-Chih, et al., Anal Lett, Vol.41, No.1 / 3, pp260-267 (2008.01.01) Wei, J .; Buriak, J .; Siuzdak, G. Nature 1999, 401, 243. R. Arakawa, N. Miyake, S. Okuno, H. Yamaoka, Rapid Commun. Mass Spectrom. 2006, 20, 2063.

  The inventors of the present invention are further discussing the previously proposed matrix-free mass analysis substrate that does not require the addition of an ionizing agent in the analysis sample. The height of the measurement spot where the ion-providing organic substance coexists affects the detection sensitivity. By setting the spot height within a predetermined range, a sufficient S / N ratio (sample peak intensity and baseline peak intensity can be obtained. (Signal to Noise ratio), and as a result, the inventors have found that mass analysis can be performed with high accuracy even with a very small amount of sample lower than several fmol, and the present invention has been completed.

  Therefore, an object of the present invention is to obtain a sufficient S / N ratio with high resolution in a laser desorption ionization mass spectrometry (LDI-MS) method, and to perform mass spectrometry with high accuracy even in a very small amount of sample lower than several fmol. An object of the present invention is to provide a substrate for mass spectrometry that can perform the above.

  That is, the present invention has a conductive substrate and a measurement spot for holding a sample provided on the surface of the substrate, and an analysis sample is attached to the measurement spot in laser desorption ionization mass spectrometry. It is a substrate for mass spectrometry to be used, and the measurement spot includes a metal oxide and an ion supplying organic substance that supplies protons and / or cations by laser irradiation, and the spot height is 1 to 100 μm. It is the board | substrate for mass spectrometry characterized by these.

  The substrate for mass spectrometry of the present invention is an ion supply that supplies a predetermined metal oxide and protons and / or cations into a measurement spot having a spot height of 1 to 100 μm provided on the surface of a conductive substrate. When an analytical sample is attached to this measurement spot and irradiated with a laser, protons and cations are supplied from the ion-providing organic material and the metal oxide, respectively. Ionization of sample molecules is promoted.

  In the present invention, the base material having conductivity is not particularly limited as long as it has conductivity necessary for the mass spectrometry technique. For example, silicon (n-type, p-type) which is an impurity semiconductor, Examples thereof include SUS304 and a base material made of metal such as a plate in which they are laminated. In general, if a base material made of a material with a relatively low thermal conductivity is used, the energy from the laser is difficult to dissipate to the substrate for analysis, so the sample can be analyzed with a relatively low laser energy. This is advantageous in that the destruction of the sample to be analyzed can be suppressed.

  Further, the shape of the substrate used in the present invention is not particularly limited as long as it can be fixed to the sample stage of the mass spectrometer, and even if the entire surface is a flat plate-like substrate, the analysis sample is dropped on the surface. A plurality of recesses having an appropriate cross-sectional shape (for example, a closed parenthesis closed shape of vertical braces, a closed parenthesis shape of vertical brackets, a closed parenthesis shape of vertical turtle shell brackets, a closed parenthesis shape of vertical angle brackets, etc.) The base material provided with (well) may be sufficient. The maximum depth of the well is usually 100 μm or more and 2000 μm or less, preferably 120 μm or more and 300 μm or less. When the well depth is less than 100 μm, when the metal dispersion is applied, the liquid leaks around the well and it is difficult to form a uniform measurement spot. When the well depth is deeper than 2000 μm, an error tends to occur in the flight distance of the ionized sample, and the measurement error of the molecular weight tends to increase.

In the present invention, the metal oxide constituting the measurement spot for holding the sample provided on the surface of the substrate is not particularly limited as long as it promotes ionization of the sample. Is preferably an oxide of one or more metals selected from the group consisting of Fe, Co and Cu, and can efficiently desorb and ionize the sample to be analyzed in laser desorption ionization mass spectrometry Therefore, Fe ₂ O ₃ (diiron trioxide) is more preferable, and α-Fe ₂ O ₃ is more preferable. For example, when Fe ₂ O ₃ is used as iron oxide, it may be used alone, or may be used in combination with another iron oxide. Further, if necessary, iron oxide such as Co or Cu may be used. You may use it in combination with the oxide of another metal.

  The metal oxide used in the present invention may have various shapes such as a spherical shape, a needle shape, a polyhedron, an amorphous shape, a hierarchical shape, a leaf shape, a snowflake shape, etc. Or it may be amorphous. As for the particle diameter of the metal oxide, when preparing a measurement point on the surface of the base material to prepare a substrate for mass spectrometry, the metal oxide is a metal oxide dispersion liquid dispersed in a predetermined dispersant. Therefore, the primary particle diameter is preferably 1 nm or more and 100 nm or less, more preferably 20 nm or more and 60 nm or less. When the primary particle diameter of the metal oxide is less than 1 nm, the measurement spot is formed by laser irradiation. There is a possibility that the metal oxide itself may be easily detected as noise because it is easily broken or scattered. On the contrary, if the particle diameter exceeds 100 nm, the particles are likely to aggregate in the dispersion liquid. There is a possibility that a problem that it becomes difficult to hold the measurement spot is caused.

Furthermore, in the present invention, an ion-feeding organic substance that constitutes a measurement spot for holding a sample provided on the surface of the substrate and can supply protons and cations by laser irradiation is preferably as follows. Can be mentioned.
(1) An organic acid salt having at least two carboxy groups and at least one hydroxy group, wherein hydrogen of some carboxy groups is substituted with an alkali metal, (2) at least two carboxy groups An organic acid salt having a group and at least one hydroxy group, wherein all of the hydrogens of the carboxy group are replaced by alkali metals, (3) having at least two carboxy groups and at least one hydroxy group The hydrogen of the carboxy group and the hydroxy group is not substituted, and (4) has at least two carboxy groups, and the hydrogen of the carboxy group is not substituted, and An organic acid having no hydroxy group.

Among these, as the organic acid salt of (1), for example, monosodium dihydrogen citrate [NaOOCCH ₂ C (OH) (COOH) CH ₂ COOH], disodium monohydrogen citrate [NaOOCCH ₂ C (OH) (COONa ) CH ₂ COOH], monosodium monohydrogen tartrate [NaOOCCH (OH) CH (OH) COOH], monosodium monohydrogen succinate [NaOOC (CH ₂ ) ₂ COOH] and the like. Of these organic acid salts, from the viewpoint of performing mass spectrometry with higher resolution and higher accuracy, monosodium dihydrogen citrate or disodium monohydrogen citrate is preferably used.

Examples of the organic acid salt of (2) include trisodium citrate [NaOOCCH ₂ C (OH) (COONa) CH ₂ COONa], disodium tartrate [NaOOCCH (OH) CH (OH) COONa], and succinic acid Sodium [NaOOC (CH ₂ ) ₂ COONa] and the like can be exemplified. Of these organic acid salts, trisodium citrate is preferable from the viewpoint of performing mass spectrometry with higher resolution and higher accuracy.

Further, as the organic acid (3), for example, citric acid [HOOCCH ₂ C (OH) (COOH) CH ₂ COOH], tartaric acid [HOOCCH (OH) CH (OH) COOH], malic acid [HOOCCH ₂ CH (OH) ) COOH] and the like. Of these organic acid salts, citric acid is preferable from the viewpoint of performing mass spectrometry with higher resolution and higher accuracy.

Furthermore, examples of the organic acid (4) include succinic acid [HOOC (CH ₂ ) ₂ COOH], oxalic acid [(COOH) ₂ ], cinnamic acid [HOOCCH = CHC ₆ H ₄ OH], and diglycolic acid. [HOOCCH ₂ OCH ₂ COOH], sebacic acid [HOOC (CH ₂ ) ₈ COOH] and the like can be exemplified. Of these, succinic acid is preferable from the viewpoint of accurate analysis with higher resolution.

  These ion-providing organic substances may be used by mixing two or more of the same organic acid salts and organic acids of (1) to (4), and (1) to (4) of organic acids. Two or more different types of salts and organic acids may be mixed and used.

In mass spectrometry using the substrate for mass spectrometry of the present invention, by laser irradiation, from a metal oxide and an ion-providing organic substance (organic acid, organic acid salt, etc.) attached thereto, protons ([H ⁺ ]) and Since ions such as cations (for example, [Na ⁺ ]) are supplied, citric acid and citric acid that have been added to the analysis sample so as to promote ionization of the sample molecules each time each analysis sample is analyzed. The sample molecules in the analysis sample can be efficiently ionized without using an ionizing agent such as sodium. In addition, the case where the ionizing agent used so far and the ion supplying organic substance to be attached to the metal oxide in the present invention are made of the same compound is included, but the conventional ionizing agent is used as noise during measurement. In contrast to the detection, the ion-providing organic substance added in the present invention is attached to the metal oxide in advance, so that the ionization of the organic substance itself acting as an ionizing agent is less likely to occur than in the sample. , It becomes difficult to be detected as background noise.

Here, the means for adhering the ion supplying organic substance as described above to the metal oxide is not particularly limited, but preferably a metal oxide obtained by dispersing these ion supplying organic substances in advance and atomizing them. A dispersion is prepared, and this metal oxide dispersion is applied onto a substrate and dried to obtain a substrate for mass spectrometry.
Hereinafter, although the example of the method of obtaining the board | substrate for mass spectrometry using a metal oxide dispersion liquid is demonstrated, this invention is not limited to this.

  When obtaining a metal oxide dispersion, for example, the above-mentioned ion-feeding organic substance is dissolved in a solvent as a dispersant, and the metal oxide is added and dispersed. Of course, these three components of the ion supplying organic substance, the metal oxide, and the necessary solvent may be mixed and dispersed at the same time. These dispersing operations may be performed by a conventional method, and for example, can be performed using a paint shaker, a ball mill, a sand mill, a centrimill, a three roll, etc. in addition to a normal stirring operation. Preferably, dispersion beads such as glass beads, zircon beads, zirconia beads, steel balls and the like are mixed with the metal oxide in a solvent and kneaded.

  About the addition ratio of the metal oxide and ion supply organic substance used when obtaining a metal oxide dispersion liquid, 0.003 mol or more and 0.3 mol or less of ion supply organic substance with respect to 1 mol of metal oxides, Preferably it is 0.03 mol or more and 0.2 mol or less. A dispersion in which the metal oxide is dispersed in a particle diameter of 1 to 100 nm can be suitably obtained by dispersing the metal oxide under the conditions adjusted so as to achieve this addition ratio (laser diffraction type particle size measuring instrument. (Measure particle size with Honeywell, Microtrac UPA).

  The solvent used to obtain the metal oxide dispersion is a volatile solvent, and the metal oxide is stably dispersed while the ion-supplying organic substance is sufficiently dissolved and adsorbed on the metal oxide. There is no particular limitation as long as it can be made, for example, water, alcohols such as methanol, ethanol, propanol, 2-propanol, butanol and diacetone alcohol, and ketones such as acetone, 2-butanone and acetylacetone. Esters such as ethyl acetate, ethers such as diethyl ether and tetrahydrofuran (THF), aliphatic hydrocarbons such as hexane and petroleum ether, aliphatic and aromatic halogenated groups such as chloroform, methylene chloride and chlorobenzene Hydrocarbons, aromatic hydrocarbons such as benzene, toluene, etc. These can be used alone or in admixture of two or more.

  The amount of the solvent used in the metal oxide dispersion is prepared so that the viscosity of the dispersion finally obtained by dispersing the metal oxide is suitable for coating or printing on the substrate. It is preferable that the dispersion be prepared so that the viscosity of the dispersion falls within the range of 2 to 10000 cps (E-type viscometer: 20 ° C.). In the present invention, from the viewpoint of preventing the appearance of peaks other than the sample to be analyzed as much as possible, the metal oxide dispersion is composed of only three components of a metal oxide, an ion supply organic substance, and a solvent. It is desirable to consist of However, if necessary, a surfactant other than the ion-feeding organic substance, a polymer, a silane coupling agent, or the like may be used on the particle surface to the extent that the analysis of the sample to be analyzed is not greatly affected.

  Next, the metal oxide dispersion prepared in this way is applied to the surface of the substrate and dried, whereby a measurement spot made of a metal oxide having an ion-providing organic substance attached to the surface of the substrate is formed. Then, a substrate for mass spectrometry is prepared. Here, there is no restriction | limiting in particular about the application | coating means at the time of apply | coating a metal oxide dispersion liquid to the surface of a base material, A well-known method is employable. For example, brush coating, roll coating, gravure coater, knife coater, roll coater, comma coater, spin coater, bar coater, dipping coating, spray coating, etc., gravure printing, offset printing, screen printing, silk printing, inkjet Printing methods such as printing can be used. Further, application (or printing) to the base material may be performed by selecting a portion to which the sample to be analyzed is attached. Moreover, there is no restriction | limiting in particular also about the drying means after apply | coating a metal oxide dispersion liquid to a base material, What is necessary is just to be able to evaporate a solvent by drying (heating as needed).

  In the present invention, the mass spectrometric substrate thus prepared needs to have a spot height of usually 1 μm to 100 μm, preferably 5 μm to 50 μm. When the spot height of the measurement spot is lower than 1 μm, it is difficult to obtain a sufficient S / N ratio. For example, when the sample in the analysis sample is testosterone which is a kind of hormone, the target number fmol (1 fmol = 0.001 pmol), it is difficult to obtain detection sensitivity on the order, and conversely, even if the spot height is higher than 100 μm, improvement in S / N ratio and improvement in detection sensitivity (higher sensitivity) are not recognized.

  Here, regarding the spot height of the measurement spot in the substrate for mass spectrometry of the present invention, in the measurement spot made of a metal oxide provided on the surface of the base material and attached with an ion supplying organic substance, This means the height dimension from the surface to the surface of the measurement spot. If the substrate has a plurality of recesses (wells) on the surface, the measurement spots are formed in the recesses (wells). In this case, it means the height dimension from the bottom surface of the recess (well) to the top surface of the measurement spot. The spot height of the measurement spot may be adjusted by any method, but preferably in a metal oxide dispersion for forming the measurement spot with a constant measurement spot size. It is preferable to adjust the concentration of the metal oxide.

When the metal oxide dispersion is applied to the surface of the substrate in this way, at least a part of the metal oxide to which a predetermined ion-feeding organic substance is adhered is immobilized on the surface of the substrate as an aggregate. it is conceivable that. At this time, the BET value of the metal oxide to which the ion supplying organic substance is attached on the substrate has a specific surface area of 0.1 m ² / g or more, preferably 1 m ² / g or more and 1000 m ² / g or less. It is good. If the BET value of the immobilized metal oxide is 0.1 m ² / g or more, the particle size (aggregate size) of the metal oxide does not become too large, and the sample to be analyzed can be ionized efficiently. Thus, the possibility of fragmentation can be eliminated. Further, if the BET value is in the range of 1 m ² / g or more and 1000 m ² / g or less, mass analysis can be performed with higher resolution. Although the relationship between the specific surface area of the metal oxide and the detection sensitivity at the time of mass spectrometry has not yet been fully elucidated, the present invention has a BET value in the above range, so that a measurement molecule that is a sample to be analyzed It is presumed that they can maintain a certain distance from each other and are efficiently desorbed and ionized.

  Mass spectrometry using the substrate for mass spectrometry of the present invention can be performed in the same manner as a known method. For example, an analysis sample is prepared by dissolving a sample to be analyzed in a volatile solvent at a desired concentration, and an appropriate amount of the analysis sample is dropped, applied, printed, etc. on the substrate for mass spectrometry of the present invention. Can be attached to perform mass spectrometry. Here, as the solvent for dissolving the sample, for example, the same solvent as used for obtaining the metal oxide dispersion described above can be exemplified.

  In this way, the mass spectrometry substrate to which the analysis sample is attached is prepared. The mass analysis substrate to which the analysis sample is attached can be used for analysis using a known mass spectrometer. The analysis conditions can be set as appropriate. For example, as a laser to be irradiated, a laser beam is irradiated by irradiating a pulse laser beam (wavelength: 337 nm, 520 nm, 1020 nm, etc.) of about 3 to 10 ns. Is absorbed by the ion-providing organic substance or metal oxide, causing a rapid temperature rise, and the sample to be analyzed (measurement molecule) attached thereto is softly ionized. At this time, ionization is further promoted by protons and cations supplied from the ion supplying organic substance and the metal oxide. The generated ions are separated and detected by the difference in m / z by the action of the mass separation unit comprising, for example, a time-of-flight type, a quadrupole type, an ion trap type, a sector type, a Fourier transform type, or a composite type thereof. Observed with a vessel. As a result, structure analysis and mass calculation of the target molecule can be performed from the molecular ion peak corresponding to each m / z.

  The substrate for mass spectrometry of the present invention can be used for analyzing various polymers, and is suitable for analysis of proteins, synthetic polymers and the like, for example. In particular, if the substrate for mass spectrometry of the present invention is used, the detection sensitivity in mass spectrometry is improved, so that angiotensin I, angiotensin II, polyvinyl alcohol, polyacrylic acid, polymethyl methacrylate, polyethylene glycol, verapamil hydrochloride, testosterone, par It is suitable for analysis of fluorooctanyl sulfonic acid, insulin (human), propranolol hydrochloride, etc., among them, molecules that could not be detected conventionally, polyvinyl alcohol, polyacrylic acid, etc. that were difficult to detect Mass spectrometry is also possible for polymer compounds.

  Since the mass spectrometry substrate of the present invention can perform mass analysis with high sensitivity, analysis can be performed with a very small amount of sample that is lower than conventional mass spectrometry. For example, in the case of testosterone which is a kind of hormone, it can be analyzed with fmol order (1 fmol = 0.001 pmol) which is a practical detection sensitivity, as well as amol order (1 amol = 0.001 fmol), and further zmol order ( 1 zmol = 0.001 amol), for example, as shown in the examples, accurate analysis is possible without being affected by noise even if the sample is 50 zmol or less. The sample to be analyzed can be mixed with the metal oxide dispersion and dropped onto the base material to obtain a substrate for mass spectrometry, and at the same time, the sample to be analyzed can be attached.

  According to the substrate for mass spectrometry of the present invention, the sample to be analyzed can be efficiently desorbed and ionized, and an analysis with a sufficient S / N ratio can be performed with high resolution. In addition, mass analysis of a polymer compound having a large molecular weight distribution can be performed easily and with high accuracy, and it can also be used for measurement of partial structure analysis, molar distribution, molecular weight distribution, etc. of a low molecular compound. In addition, it is possible to analyze a sample that is impossible or difficult to detect with a conventional analysis substrate. In addition, since the substrate for mass spectrometry of the present invention can ionize a sample under relatively mild conditions and hardly changes in quality, for example, even after opening the one contained in a vacuum package or the like, It can be used reliably over a long period of time.

1 is a mass spectrum measured for a 50 pmol testosterone-containing analytical sample using the mass spectrometric substrate of Reference Example 1. FIG. FIG. 2 is a mass spectrum measured for an analytical sample containing 5 pmol of testosterone using the mass spectrometry substrate of Reference Example 1. FIG. 3 is a mass spectrum measured for a 50 fmol testosterone-containing analytical sample using the substrate for mass spectrometry of Reference Example 1.

FIG. 4 is a mass spectrum measured for a 50 pmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 1. FIG. 5 is a mass spectrum measured for a 5 pmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 1.

6 is a mass spectrum measured for a 50 pmol testosterone-containing analytical sample using the mass spectrometric substrate of Example 2. FIG. FIG. 7 is a mass spectrum measured for an analytical sample containing 5 pmol of testosterone using the substrate for mass spectrometry of Example 2. FIG. 8 is a mass spectrum measured for a 50 fmol testosterone-containing analytical sample using the mass spectrometric substrate of Example 2.

FIG. 9 is a mass spectrum measured for a 50 pmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 3. FIG. 10 is a mass spectrum measured for a 5 pmol testosterone-containing analytical sample using the mass spectrometric substrate of Example 3. FIG. 11 is a mass spectrum measured for a 50 fmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 3.

FIG. 12 is a mass spectrum measured for a 5 fmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 3. FIG. 13 is a mass spectrum measured for a 50 amol testosterone-containing analytical sample using the mass spectrometry substrate of Example 3.

FIG. 14 is a mass spectrum measured for a 50 zmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 3. FIG. 15 is a mass spectrum measured for a 5 zmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 3.

FIG. 16 is a mass spectrum measured for a 50 pmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 4. FIG. 17 is a mass spectrum measured for a 5 pmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 4. FIG. 18 is a mass spectrum measured for a 50 fmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 4.

FIG. 19 is a mass spectrum measured for a 50 pmol testosterone-containing analytical sample using the mass spectrometry substrate of Example 5.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a reference example, this invention is not limited to the content of these Examples. In addition, "part" used in an Example and a reference example represents a weight part unless there is particular notice.

[Reference Example 1]
Add distilled water (9.98 parts), monosodium dihydrogen citrate (0.394 parts), commercially available spherical α-Fe ₂ O ₃ (2.5 parts), and zirconia beads (40 parts) to a 50 mL container, and add to the paint shaker. For 12 hours to obtain a liquid. The obtained liquid was diluted about 100 times with the same distilled water used for dispersion to obtain a metal oxide dispersion having an iron oxide concentration of 0.78% by weight.
When the particle size of α-Fe ₂ O _{3 in} the obtained metal oxide dispersion was measured using a laser diffraction particle size analyzer (Honeywell: Microtrac UPA), the average particle size (D50) was 40 nm. .

  Next, on the sample plate for AXIMA series (manufactured by Shimadzu Corporation, product name “AXIMA target”, groove depth around the spot 40 μm, product number: 223-25579-17; hereinafter referred to as “genuine plate”) 2 μL of the metal oxide dispersion obtained above was dropped with a micropipette and dried to form a measurement spot with a diameter of 2.5 mm, whereby a substrate for mass spectrometry according to Reference Example 1 was obtained. When the spot height of the obtained measurement spot was measured using an ultra-depth measurement microscope VK-8510 (manufactured by Keyence Corporation), the spot height (average value) was 0.73 μm.

Using the mass spectrometry substrate obtained above, mass spectrometry of testosterone was performed as follows.
First, testosterone is dissolved in acetonitrile to prepare a sample solution having a testosterone concentration of 83 μM, and 0.6 μL of this sample solution is dropped on a measurement spot of a substrate for mass spectrometry and dried, so that the sample solution corresponds to 50 pmol. The concentration of each was adjusted. Next, the analysis substrate to which the sample is attached is fixed to a sample stage of a MALDI-TOF / MS apparatus (AXIMA-CFR manufactured by Shimadzu Corporation) and irradiated using an N ₂ pulse laser (3 ns) of 337 nm as an excitation light source, Measurement was performed using a time-of-flight mass spectrometer.
The obtained mass spectrum is shown in FIG.

  Next, in the mass spectrum of FIG. 1, the baseline in the range of m / z 800 to 1000 is the baseline peak intensity (N: Noise), and the intensity of the sample testosterone peak (m / z: 311.0) (S: Signal) ) And the baseline peak intensity (N), the S / N ratio was found to be 0.1 mV and 3.5 mV as shown in Table 1, and the S / N ratio was 35. It was.

  As is clear from the mass spectrum in FIG. 1, although the sodium ion peak (m / z: 22.9) is detected largely, other background noise is hardly detected and sodium ion is detected with high resolution. The isotope peak of the adduct molecule and its sample was detected, and the ionization of the sample was good. Moreover, almost no peaks derived from fragment ions were detected, confirming that mass spectrometry of testosterone can be performed with high accuracy.

Next, 0.6 μL of the sample solution having a testosterone concentration of 8.3 μM prepared above is dropped on the measurement spot of the substrate for mass spectrometry of Reference Example 1 prepared in advance, and dried so as to correspond to 5 pmol. The concentration of the solution was adjusted, mass spectrometry was performed in the same manner as described above, and the S / N ratio was determined in the same manner as described above.
The results are shown in Table 1, and the obtained mass spectrum is shown in FIG.

Further, 0.6 μL of the sample solution having the testosterone concentration of 0.083 μM prepared above is dropped on the measurement spot of the substrate for mass spectrometry of Reference Example 1 prepared in advance, and dried, so that the sample solution corresponds to 50 fmol. The concentration of each was adjusted, mass spectrometry was performed in the same manner as described above, and the S / N ratio was determined in the same manner as described above.
The results are shown in Table 1, and the obtained mass spectrum is shown in FIG.

  As is clear from the results shown in Table 1 of the mass spectrum shown in FIG. 1 to FIG. 3, the analysis sample corresponding to 50 pmol has an S / N ratio of 35 and a sufficiently large value with high accuracy. Testosterone can be mass-analyzed, and when it becomes an analysis sample corresponding to 5 pmol or 50 fmol, although mass spectrometry of testosterone is sufficiently possible, its S / N ratio is 9 or 10, respectively. Although it cannot be said that the value is sufficiently large, it is possible to analyze a very small amount of sample that is impossible or difficult to detect with a conventional analysis substrate. However, when the height of the measurement spot is less than 1 μm, it is difficult to control the film thickness and uneven coating after film formation occurs, so that it is difficult to detect the sample stably, and the S / N ratio tends to decrease significantly. There is.

[Example 1]
In the same manner as in Reference Example 1, a metal oxide dispersion having an iron oxide concentration of 6.25% by weight and an average particle diameter (D50) of 42 nm was prepared, and the spot of Example 1 having a measurement spot with a spot height of 5.2 μm was prepared. A substrate for mass spectrometry was obtained.
The obtained measurement spot on the mass spectrometry substrate of Example 1 was subjected to SEM observation at a magnification of 3,000. As a result, the diameter of about 100 nm to 10 μm, which was not observed on the measurement spot of the mass spectrometry substrate of Reference Example 1, was observed. Relatively large spherical fine particles were observed.

For the obtained mass spectrometry substrate of Example 1, the concentration of the sample solution was adjusted to correspond to 50 pmol and 5 pmol in the same manner as in Reference Example 1, and mass spectrometry was performed in the same manner as above. The S / N ratio was determined in the same manner as described above.
The results are shown in Table 1, and the obtained mass spectra are shown in FIGS. 4 and 5, respectively.

[Example 2]
In the same manner as in Reference Example 1, a metal oxide dispersion having an iron oxide concentration of 10.0% by weight and an average particle size (D50) of 41 nm was prepared, and Example 2 having a measurement spot with a spot height of 16.2 μm was used. A substrate for mass spectrometry was obtained. As a result of SEM observation at a magnification of 3,000, spherical fine particles similar to those in Example 1 were observed.
For the obtained mass spectrometry substrate of Example 2, the concentration of the sample solution was adjusted to correspond to 50 pmol, 5 pmol, and 50 fmol in the same manner as in Reference Example 1, and mass spectrometry was performed in the same manner as above. The S / N ratio was determined in the same manner as described above.
The results are shown in Table 1, and the obtained mass spectra are shown in FIGS.

Example 3
In the same manner as in Reference Example 1, a metal oxide dispersion having an iron oxide concentration of 15.0% by weight and an average particle diameter (D50) of 45 nm was prepared, and the spot of Example 3 having a measurement spot with a spot height of 18.1 μm was prepared. A substrate for mass spectrometry was obtained. As a result of SEM observation at a magnification of 3,000, spherical fine particles similar to those in Example 1 were observed.
For the obtained mass spectrometric substrate of Example 3, the sample solution concentrations were adjusted to correspond to 50 pmol, 5 pmol, 50 fmol, 5 fmol, 50 amol, 50 zmol, and 5 zmol, respectively, in the same manner as in Reference Example 1 above. Then, mass spectrometry was performed in the same manner as described above, and the S / N ratio was determined in the same manner as described above.
The results are shown in Table 1, and the obtained mass spectra are shown in FIGS.

Example 4
In the same manner as in Reference Example 1, a metal oxide dispersion having an iron oxide concentration of 20.0% by weight and an average particle size (D50) of 42 nm was prepared, and the sample of Example 4 having a measurement spot with a spot height of 29.5 μm was prepared. A substrate for mass spectrometry was obtained. As a result of SEM observation at a magnification of 3,000, spherical fine particles similar to those in Example 1 were observed.
For the mass spectrometric substrate obtained in Example 4, the concentration of the sample solution was adjusted so as to correspond to 50 pmol, 5 pmol and 50 fmol in the same manner as in Reference Example 1, and the mass was determined in the same manner as above. Analysis was performed and the S / N ratio was determined in the same manner as described above.
The results are shown in Table 1, and the obtained mass spectra are shown in FIGS.

Example 5
In the same manner as in Reference Example 1, a metal oxide dispersion having an iron oxide concentration of 25.0% by weight and an average particle diameter (D50) of 40 nm was prepared, and the spot of Example 5 having a measurement spot with a spot height of 45.8 μm was prepared. A substrate for mass spectrometry was obtained. As a result of SEM observation at a magnification of 3,000, spherical fine particles similar to those in Example 1 were observed.
For the obtained mass spectrometry substrate of Example 5, the concentration of the sample solution was adjusted to correspond to 50 pmol in the same manner as in Reference Example 1, and mass spectrometry was performed in the same manner as described above. The S / N ratio was determined in the same manner as described above.
The results are shown in Table 1, and the obtained mass spectrum is shown in FIG.

Claims (6)

A substrate for mass spectrometry having a conductive base material and a measurement spot for holding a sample provided on the surface of the base material, and using the analytical sample attached to the measurement spot in laser desorption ionization mass spectrometry And
The said measurement spot contains a metal oxide and the ion supply organic substance which supplies a proton and / or a cation by laser irradiation, The spot height is 1-100 micrometers, The board | substrate for mass spectrometry characterized by the above-mentioned.   The mass according to claim 1, wherein the metal oxide is an oxide of one or more metals selected from the group consisting of Fe, Co, and Cu, and has a primary particle diameter of 1 to 100 nm. Analysis board. The substrate for mass spectrometry according to claim 2, wherein the metal oxide is Fe ₂ O ₃ .   2. The ion-providing organic material is an organic acid salt having at least two carboxy groups and at least one hydroxy group, and hydrogen of a part of the carboxy groups is substituted with an alkali metal. The substrate for mass spectrometry as described in any one of -3.   The organic acid salt is one or a mixture of two or more selected from the group consisting of monosodium dihydrogen citrate, disodium monohydrogen citrate, monosodium monohydrogen tartrate, and monosodium monohydrogen succinate. The mass spectrometry substrate according to claim 4.   The substrate for mass spectrometry according to claim 4 or 5, wherein the measurement spot contains 10 to 20 parts by weight of the organic acid salt with respect to 100 parts by weight of the metal oxide.

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