JP2006010681A - Method of producing sample for matrix-assisted laser desorption/ionization mass spectrometry - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of producing a sample for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, with which a signal higher than that in the conventional methods can be obtained, using the MALDI mass spectrometry technique. <P>SOLUTION: In the method of producing the sample for the matrix-assisted laser desorption/ionization mass spectrometry, a liquid containing a solvent, a sample material which is an object of analysis and a matrix for the matrix-assisted laser desorption/ionization mass spectrometry is applied to the upper surface of a target, by using a spin coat method, under the conditions that its rotation rate be not less than 3,000 rpm, whereby a membrane is formed and made dried. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a sample for matrix-assisted laser desorption (hereinafter referred to as “MALDI”) mass spectrometry.

  In a method for producing a sample for MALDI mass spectrometry in matrix-assisted laser desorption mass spectrometry using a synthetic polymer as an analysis target, conventionally, a liquid containing a sample material to be analyzed, a matrix for MALDI mass spectrometry, and a solvent is generally used. A method of dropping and drying on a measurement sample stage (hereinafter referred to as “target”) called a target has been used (for example, see Non-Patent Document 1).

  On the other hand, in MALDI mass spectrometry using biopolymers as an analysis target, a liquid containing the analysis sample material, a matrix for MALDI mass spectrometry, and a solvent is applied onto the target using a spin coating method with a rotation speed of 500 rpm. A method for producing a sample for MALDI mass spectrometry by coating is known (see, for example, Non-Patent Document 2). However, the signal intensity was not sufficient.

  In MALDI mass spectrometry, there has been a demand for further increasing the intensity of the detected signal, and there has been a demand for a method for producing a sample for MALDI mass spectrometry that can provide a signal with a higher intensity than before.

Chemical Reviews, 2001, Vol. 101, p. 527-569 Rapid Communication in Mass Spectrometry (RAPID COMMUNICATION IN MASS SPECTROMETRY), 1995, Vol. 9, p. 180-187

  An object of the present invention is to provide a method for producing a sample for MALDI mass spectrometry, which can obtain a signal having a higher intensity than before in MALDI mass spectrometry.

  In order to solve the above-mentioned problems, the present inventor has continued to intensively study a method for producing a sample for MALDI mass spectrometry. A liquid containing a sample material to be analyzed, a matrix for MALDI mass spectrometry, and a solvent is added to the spin coater. It is surprisingly found that the signal intensity in MALDI mass spectrometry is increased when a sample for analysis is produced by coating on a target at a rotation speed of a certain level or more, forming a film, and drying to complete the present invention. It came to.

That is, the present invention is to apply a liquid containing a sample material to be analyzed, a matrix for MALDI mass spectrometry, and a solvent on a target at a rotational speed of 3000 rpm or more by a spin coating method to form a film, and to dry the film. A method of producing a sample for MALDI mass spectrometry is provided.
Furthermore, the present invention provides a MALDI mass spectrometric method characterized by using the MALDI mass spectrometric sample produced by the analytical sample production method described above.
Furthermore, the present invention provides a quality control method for a synthetic polymer characterized by using the above analysis method.
Furthermore, the present invention provides an analysis system characterized by including a spin coater and a matrix-assisted laser desorption mass spectrometer using the mass spectrometry method or quality control method described above.
Furthermore, the present invention includes a step of polymerizing one or more raw material monomers, obtaining data by an analysis method for analyzing the resultant synthetic polymer as an analysis target sample material, and controlling the polymerization reaction based on the data. A method for producing a synthetic polymer, the method for producing a synthetic polymer using the above analysis method as the analysis method.

  If the method for producing a sample for MALDI mass spectrometry of the present invention is used, a high signal intensity can be obtained in MALDI mass spectrometry, and it becomes an analytical method particularly suitable for development and quality control of synthetic polymers. Very useful.

The present invention is described in detail below.
In the method for producing a sample for MALDI mass spectrometry of the present invention, a liquid containing a sample material to be analyzed, a matrix for MALDI mass spectrometry, and a solvent (hereinafter sometimes referred to as “sample liquid”) is prepared by spin coating. It is applied on the target and dried at a rotational speed of 3000 rpm or more.

  The spin coating method is used in the method for producing an analytical sample of the present invention. Spin coating is a method of applying a liquid to an object. The object to be applied is fixed on a rotating disk and can be rotated, and after the liquid is attached to the object, the spin coating method is applied. By rotating the object or rotating the object and then dropping the liquid onto the object, the liquid is spread and applied on the object using a centrifugal force to form a film. The rotation speed indicates the rotation speed of the rotating disk.

  In order to deposit the sample solution on the target before coating by spin coating, the sample solution is collected with a micropipettor, a microsyringe, etc., and a small amount of liquid is ejected onto the target (referred to as “attachment”). .), Or the sample solution may be dropped directly on the target.

Next, the sample stage is rotated by a spin coater to form a film. As the spin coater, any device that can fix the sample stage and can rotate to about 5000 rpm may be used, and specific examples include 1H-D7 manufactured by Mikasa Corporation. The rotation speed in the spin coating in the present invention is 3000 rpm or more, and preferably 4000 rpm or more. Moreover, it is preferable that the rotational speed of a spin coat is 10,000 rpm or less.
When the target is rotated first, the sample solution may be directly dropped on the target. It is preferable to start the rotation of the target immediately after attaching or dripping.

  The film obtained by this spin coating is dried before the target is inserted into the analysis chamber of the MALDI mass spectrometer. Drying may be performed during spin coating, for example, while heating at 30 ° C. to 110 ° C., or may be performed using a vacuum drier, or may be performed in combination. .

  Here, the target used in the method for producing an analytical sample of the present invention is not particularly limited as long as it is an object made of a non-volatile substance having a size and weight that can be mounted on a spin coater. The target used is made of a metal such as stainless steel, copper, or nickel, and has a flat portion for applying the sample solution. In particular, when the spin coater is small, a plate having a thickness of 0.005 mm to 10 mm is used as a target, the sample solution is applied by the spin coat method, and then attached to another object and inserted into the MALDI mass spectrometer. May be.

  As a method for preparing a sample liquid used in the present invention, a liquid containing a MALDI mass spectrometry matrix and a liquid containing a sample material to be analyzed may be mixed, or a MALDI mass spectrometry matrix and a sample material to be analyzed may be prepared. May be prepared by dissolving them in a solvent for dissolving them. Alternatively, the sample material to be analyzed may be added to a liquid containing the matrix for MALDI mass spectrometry, or may be prepared by adding the matrix for MALDI mass spectrometry to a liquid containing the sample material to be analyzed.

  The sample material to be analyzed used in the method for producing an analytical sample of the present invention may be any material as long as it has low volatility. However, MALDI mass spectrometry is suitable for analyzing a material having a high molecular weight, and is a matrix for MALDI mass spectrometry. Therefore, it is preferable that the molecular weight of the sample to be analyzed is 500 or more, and more preferable that the molecular weight is 1000 or more. When the molecular weight of the sample to be analyzed has a distribution, the weight average molecular weight in terms of polystyrene measured by size exclusion chromatography is preferably 500 or more, and more preferably a compound having a weight average molecular weight of 1000 or more. .

  MALDI mass spectrometry is an analysis method that can be suitably applied when a synthetic polymer is used as a sample material to be analyzed. Examples of the synthetic polymer include polyethylene, polypropylene, polystyrene, polymethacrylic acid, polymethylmethacrylate, polyethersulfone (PES), polyetheretherketone (PEEK), polyester, polyphenylene, and the like.

  Specific examples of the matrix for MALDI mass spectrometry that can be used in the method for producing an analytical sample of the present invention include 2,5-dihydroxybenzoic acid, trans-3-indoleacrylic acid, dithranol, and 2- (4 -Hydroxyphenylazo) benzoic acid (HABA), all-trans-retinol (for example, see Non-Patent Document 1), 1,4-diphenylbutadiene, 1,1,4,4-tetraphenyl-1,3-butadiene and the like And those having a purity of 90% or more are preferred, and those having a molecular weight of 1000 or more are more preferably 5% or less. Since the effects of the present invention are remarkably exhibited, disulanol and / or 1,1,4,4-tetraphenyl-1,3-butadiene is preferable as the matrix for MALDI mass spectrometry, and 1,1,4,4-tetraphenyl is preferable. -1,3-butadiene is more preferred.

  The amount ratio of the sample to be analyzed and the matrix for MALDI mass spectrometry is not particularly limited, but the sample material to be analyzed: matrix for MALDI mass spectrometry = 1: 1 (weight ratio) to 1: 2000000 (weight ratio) is preferable. : 2 (weight ratio) to 1: 100000 (weight ratio) is more preferable.

  In the method for producing an analytical sample of the present invention, a solvent that can be used for preparing a liquid containing a matrix for MALDI mass spectrometry and a sample material to be analyzed is appropriately selected depending on the matrix for MALDI mass spectrometry and the sample material to be analyzed. However, it is desirable to be able to dissolve the MALDI mass spectrometry matrix and the sample to be analyzed. Also, those having a high boiling point of the solvent and difficult to dry are not suitable. On the other hand, if the boiling point is low and the volatility is too high, there is a problem that the spin coating method is difficult to operate. The boiling point of the liquid is preferably in the range of 25 ° C to 80 ° C. Specific examples include tetrahydrofuran, n-propyl alcohol, acetone, methyl ethyl ketone, methanol, ethanol, isopropyl alcohol, ethyl acetate, chloroform, methylene chloride, carbon tetrachloride, hexafluoroisopropyl alcohol, diethyl ether, ethyl propyl ether, and the like. Can do. These liquids can be used alone or in combination of two or more. The concentration when these MALDI mass spectrometry matrix or sample material to be analyzed is dissolved in a solvent is preferably about 1 to 100 mg / ml.

The matrix for MALDI mass spectrometry can contain an ionization initiator. Specific examples of the ionization initiator include metal salts such as silver trifluoroacetate, copper nitrate, sodium chloride, and potassium chloride, ammonium salts, and organic salts.
The amount ratio of the ionization initiator to be contained and the matrix for MALDI mass spectrometry is preferably 1: 2 (weight ratio) to 1: 4000000 (weight ratio), and 1: 4 (weight ratio). Ratio) to 1: 200000 (weight ratio) is more preferable.
The ionization initiator can be dissolved in a solvent and then mixed with the matrix for MALDI mass spectrometry. In this case, the concentration of the ionization initiator when dissolved in the solvent is preferably about 1 to 100 mg / ml.

  Since the sample for MALDI mass spectrometry produced by the method for producing an analytical sample of the present invention gives high signal intensity, the sample for MALDI mass spectrometry produced by the method for producing an analytical sample of the present invention is used. The MALDI mass spectrometry method performed in this manner can be suitably used for quality control of synthetic polymers.

  In order to perform quality control of the synthetic polymer by the analysis method of the present invention, the sample for analysis of the present invention is randomly sampled from the produced synthetic polymer, and the sample of the synthetic polymer obtained by sampling is used. A sample for MALDI mass spectrometry is produced by the production method, and the sample is inserted into a MALDI mass spectrometer, and each sample is subjected to mass spectrometry in order to obtain data. From the obtained signal intensity data for each m / z, the standard deviation of the population was estimated using the statistical quality control method, the average value of the population was estimated, etc. And the difference test can be performed. In addition, a control chart can be created and managed to keep the process in a stable state. Further, if the obtained data is data indicating molecular weight, the data is compared with a management value of molecular weight, and if the data is the management value, the produced synthetic polymer is regarded as an acceptable product, and the data If is not the control value, the produced synthetic polymer can be rejected. FIG. 9 shows a flowchart of one embodiment of the synthetic polymer quality control method of the present invention.

  As described above, in order to perform the analysis method of the present invention, particularly in order to perform quality control of the synthetic polymer using the analysis method of the present invention, an analysis system including a spin coater and a MALDI mass spectrometer is required. It is preferable to use it. According to the analysis system of the present invention, since a high signal intensity of MALDI mass spectrometry can be obtained from a synthetic polymer sample, quality control of the synthetic polymer can be performed efficiently. One embodiment of the analysis system of the present invention is shown in FIG. The dryer in FIG. 10 dries the film obtained by spin coating.

  Further, the synthetic polymer production method of the present invention obtains data by an analysis method in which one or more raw material monomers are subjected to a polymerization reaction, and the resulting synthetic polymer is analyzed as a sample material to be analyzed. A method for producing a synthetic polymer comprising a step of controlling a polymerization reaction, wherein the analytical method is a method for producing a synthetic polymer. Examples of the raw material monomer include, for example, polyethylene, polypropylene, polystyrene, polymethacrylic acid, polymethylmethacrylate, polyethersulfone (PES), polyetheretherketone (PEEK) by polymerization reaction. ), A raw material monomer that can be a synthetic polymer such as polyester and polyphenylene, and specifically, propylene, styrene, methacrylic acid, methyl methacrylate, 4,4′-dichlorodiphenylsulfone, bisphenol-S, Examples include hydroquinone, 4,4′-difluorobenzoyl, dimethyl terephthalate, ethylene glycol, maleic anhydride, fumaric acid, phthalic anhydride, isophthalic acid, terephthalic acid, and the like. Further, as a step of controlling the polymerization reaction based on the data, specifically, if the data obtained by analysis is data indicating molecular weight, the data is compared with a management value of molecular weight, and the data is When it is the control value, a synthetic polymer product is obtained, and when the data is not the control value, a step of controlling the polymerization reaction is exemplified. Examples of the control factor for the polymerization reaction include temperature, pressure, raw material monomer concentration, catalyst concentration, polymerization time, and the like. In the method for producing a synthetic polymer of the present invention, a synthetic polymer having a narrow molecular weight distribution can be produced. FIG. 11 shows a flowchart of one embodiment of the method for producing a synthetic polymer of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.

Example 1
Tosoh Corporation with 1,1,4,4-tetraphenyl-1,3-butadiene (PFAALTZ & BAUER reagent, purity 98% or more, molecular weight 1000 or more, less than 2% by weight) as a matrix for MALDI mass spectrometry A polystyrene standard product F-1 (trade name, molecular weight 10,000) manufactured by company was used as a sample, and silver trifluoroacetate was used as an ionization initiator. The mixing ratio of the matrix for MALDI mass spectrometry, the sample to be analyzed, and the ionization initiator is described in J. Org. Am. Soc. Mass Spectrom. 1996, 7, 11-24 and Rapid Commun. Mass Spectrom. 2001, 15, 675-678, 5% of each THF solution prepared at a concentration of 20 mg / ml for MALDI mass spectrometry, 10 mg / ml for the sample material to be analyzed, and 10 mg / ml for silver trifluoroacetate: Mixing 2: 1 (volume ratio). A matrix for MALDI mass analysis, a sample material to be analyzed, and an ionization initiator mixed solution were attached to a target for MALDI mass spectrometry by 7.5 μl with a micropipette and rotated at a speed of 4500 rpm for 10 seconds in a spin coater. The target was inserted into a Reflex III MALDI mass spectrometer manufactured by Bruker Daltonics. Then, the measurement was performed in the positive ionization mode with an acceleration voltage of 25.0 kV and an integration of 300 laser shots. The signal intensity was evaluated by using signal intensities of m / z = 10268 and 10372 indicating 97-mer and 98-mer of styrene monomer in the obtained mass spectrum. The signal intensities when using the MALDI mass spectrometric sample production method of Example 1 were 1954 and 1912 counts, respectively.

Comparative Example 1
About the manufacturing method of the sample for MALDI mass spectrometry used in Example 1, measurement and evaluation were performed under the same conditions as in Example 1 except that the solvent was evaporated as it was after applying the sample solution to the target without using the spin coating method. went. The signal intensities when using the MALDI mass spectrometry sample production method of Comparative Example 1 were 797 and 838 counts, respectively.

  The results of Example 1 and Comparative Example 1 are summarized in Table 1. When the method for producing a sample for MALDI mass spectrometry used in Example 1 was used, a stronger signal intensity was obtained as compared with the conventional method for producing a sample for MALDI mass spectrometry used in Comparative Example 1. It was found that stronger signal intensity can be obtained by using the MALDI mass spectrometric sample production method.

Comparative Example 2
About the manufacturing method of the sample for MALDI mass spectrometry used in Example 1, it measured and evaluated on the same conditions as Example 1 except having made the rotational speed into 500 rpm when using a spin coat method. The signal intensities when using the method for producing a sample for MALDI mass spectrometry in Comparative Example 2 were 813 and 774 counts, respectively.

  The results of Example 1 and Comparative Example 2 are summarized in Table 1. When the method for producing a sample for MALDI mass spectrometry used in Example 1 is used, a method for producing a sample for MALDI mass spectrometry by spin coating using a rotational speed described in non-patent literature as in Comparative Example 2 Since a stronger signal intensity was obtained, it was found that stronger signal intensity was obtained using the MALDI mass spectrometry sample production method of Example 1.

Example 3
About the manufacturing method of the sample for MALDI mass spectrometry used in Example 1, it measured and evaluated on the same conditions as Example 1 except having made the rotational speed into 3000 rpm when using a spin coat method. The signal intensities when using the MALDI mass spectrometric sample production method of Example 3 were 2174 and 2104 counts, respectively.

Comparative Example 5
About the manufacturing method of the sample for MALDI mass spectrometry used in Example 1, it measured and evaluated on the same conditions as Example 1 except having made the rotational speed into 2000 rpm when using a spin coat method. The signal intensities when using the method for producing a sample for MALDI mass spectrometry in Example 3 were 797 and 762 counts, respectively.

Example 2
About the matrix for MALDI mass spectrometry used in Example 1, 1,1,4,4-tetraphenyl-1,3-butadiene (reagent manufactured by PFALTZ & BAUER) was replaced with disulanol (reagent manufactured by Aldrich, purity of 98% or more, Measurement and evaluation were performed under the same conditions as in Example 1 except that the component having a molecular weight of 1000 or more was changed to less than 2% by weight. The signal intensities when using the method of Example 2 were 653 and 644 counts, respectively.

Comparative Example 3
About the manufacturing method of the sample for MALDI mass spectrometry used in Example 2, it measured and evaluated on the same conditions as Example 2 except having evaporated the solvent as it was after attaching to the target without using a spin coater. The signal intensities when using the method for producing a sample for MALDI mass spectrometry in Comparative Example 3 were 239 and 208 counts, respectively.

  The results of Example 2 and Comparative Example 3 are summarized in Table 2. When the method for producing the sample for MALDI mass spectrometry used in Example 2 was used, a stronger signal intensity was obtained compared to the method for producing the sample for MALDI mass spectrometry used in Comparative Example 3. It was found that stronger signal intensity can be obtained by using the method for producing a sample for MALDI mass spectrometry.

Comparative Example 4
About the manufacturing method of the sample for MALDI mass spectrometry used in Example 2, it measured and evaluated on the same conditions as Example 2 except having made the rotational speed into 500 rpm when using a spin coat method. The signal intensities when using the MALDI mass spectrometry sample production method of Comparative Example 4 were 224 and 228 counts, respectively.

  The results of Example 2 and Comparative Example 4 are summarized in Table 2. When the method for producing a sample for MALDI mass spectrometry used in Example 2 is used, as compared with the method for producing a sample for MALDI mass spectrometry by spin coating using a rotational speed described in non-patent literature as in Comparative Example 4. Thus, it was found that stronger signal intensity was obtained by using the method for producing a sample for MALDI mass spectrometry in Example 2.

It is a chart which shows the result of Example 1, and uses 1,1,4,4-tetraphenyl-1,3-butadiene (PFAALTZ & reagent made by BAUER) as a matrix for MALDI mass spectrometry, and the rotational speed is 4500 rpm. The measurement result of Tosoh Co., Ltd. polystyrene standard product F-1 (molecular weight 10,000) obtained by the manufacturing method of the sample for MALDI mass spectrometry which used the spin coat method was shown. The scale of the vertical axis is an arbitrary unit. It is a chart which shows the result of the comparative example 1, Tosoh Co., Ltd. polystyrene standard product F-1 obtained by the manufacturing method of the sample for MALDI mass spectrometry which evaporated the solvent as it was after attaching to a target without using a spin coater ( The measurement result of molecular weight 10,000) is shown. Although the scale of the vertical axis is an arbitrary unit, it is the same display range as FIG. It is a chart which shows the result of the comparative example 2, using 1,1,4,4-tetraphenyl-1,3-butadiene (a reagent made by PFALTZ & BAUER) as a matrix for MALDI mass spectrometry, and the rotation speed is 500 rpm. The measurement result of Tosoh Co., Ltd. polystyrene standard product F-1 (molecular weight 10,000) obtained by the manufacturing method of the sample for MALDI mass spectrometry which used the spin coat method was shown. Although the scale of the vertical axis is an arbitrary unit, it is the same display range as FIG. It is a chart which shows the result of Example 2, and obtained by the manufacturing method of the sample for MALDI mass spectrometry using the spin coat method which used the disanol (Aldrich reagent) as a matrix for MALDI mass spectrometry, and set the rotational speed to 4500 rpm. The measurement result of the polystyrene standard product F-1 (molecular weight 10,000) manufactured by Tosoh Corporation is shown. The scale of the vertical axis is an arbitrary unit. It is a chart which shows the result of the comparative example 3, Comprising: Tosoh Co., Ltd. polystyrene standard goods F-1 obtained by the manufacturing method of the sample for MALDI mass spectrometry which evaporated the solvent as it was after attaching to a target without using a spin coater ( The measurement result of molecular weight 10,000) is shown. The scale of the vertical axis is an arbitrary unit, but is the same display range as FIG. It is a chart which shows the result of the comparative example 4, and is obtained by the manufacturing method of the sample for MALDI mass spectrometry using the spin coat method which used disulanol (Aldrich reagent) as a matrix for MALDI mass spectrometry, and set the rotational speed to 500 rpm. The measurement result of the polystyrene standard product F-1 (molecular weight 10,000) manufactured by Tosoh Corporation is shown. The scale of the vertical axis is an arbitrary unit, but is the same display range as FIG. 10 is a chart showing the results of Example 3. 10 is a chart showing the results of Comparative Example 5. The flowchart which shows one Embodiment of the quality control method of the synthetic polymer of this invention. The figure which shows one Embodiment of the analysis system of this invention. The flowchart which shows one Embodiment of the manufacturing method of the synthetic polymer of this invention.

Claims (6)

  A liquid containing a sample material to be analyzed, a matrix for matrix-assisted laser desorption / mass spectrometry, and a solvent is applied onto a target at a rotational speed of 3000 rpm or more by a spin coating method to form a film and then dried. A method for producing a sample for matrix-assisted laser desorption mass spectrometry.   The production method according to claim 1, wherein disranol and / or 1,1,4,4-tetraphenyl-1,3-butadiene is used as a matrix for matrix-assisted laser desorption mass spectrometry.   A matrix-assisted laser desorption / mass spectrometry method using the matrix-assisted laser desorption / mass spectrometry sample produced by the production method according to claim 1 or 2.   A method for quality control of a synthetic polymer, wherein the analysis method according to claim 3 is used.   An analysis system using the method according to claim 3, comprising a spin coater and a matrix-assisted laser desorption mass spectrometer.   Production of a synthetic polymer comprising a step of polymerizing one or more raw materials monomers, obtaining data by an analysis method for analyzing the resultant synthetic polymer as a sample material to be analyzed, and controlling the polymerization reaction based on the data A method for producing a synthetic polymer using the analysis method according to claim 3 as the analysis method.

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