WO2020055131A1

WO2020055131A1 - Method for producing test pieces of water-insoluble material for maldi mass spectrometry and method for quantitative analysis of water-insoluble material using maldi mass spectrometry

Info

Publication number: WO2020055131A1
Application number: PCT/KR2019/011775
Authority: WO
Inventors: 배용진; 임영희; 윤여영; 정주은; 박병현; 유현식
Original assignee: 주식회사 엘지화학
Priority date: 2018-09-11
Filing date: 2019-09-11
Publication date: 2020-03-19

Abstract

The present invention relates to a method for producing test pieces in which the thickness of the samples of homogeneous water-insoluble material is uniform, and to a method for quantitative analysis of water-insoluble material by analyzing such test pieces using MALDI mass spectrometry. Specifically, the test pieces can be produced by: adding a volatile solvent to a mixture of water-insoluble material and matrix; placing, in a pellet cup made of water-soluble material, and pressing, with even pressure, the sample obtained by mixing the mixture of water-insoluble material and matrix until the solvent has evaporated; and melting the pellet cup with water.

Description

Manufacturing method of water-insoluble substance specimen for MALDI mass spectrometry and quantitative analysis method of water-insoluble substance using MALDI mass spectrometry

This application claims the benefit of priority based on Korean Patent Application No. 10-2018-0108097 filed on September 11, 2018 and Korean Patent Application No. 10-2019-0112140 filed on September 10, 2019, above All contents disclosed in the patent document are included as part of the present specification.

The present invention relates to a method for manufacturing a water-insoluble material specimen for MALDI mass spectrometry and a quantitative analysis method using the same specimen, and more specifically, a method for manufacturing a water-insoluble material specimen for homogeneous and small thickness variation for MALDI mass spectrometry and This method relates to a method for obtaining a reproducible MALDI spectrum using a commercial MALDI-TOF MS instrument.

In order to obtain quantitative information through the mass spectrum, it is necessary to obtain a reproducible spectrum for the same sample and to prepare a linear calibration curve according to the concentration of the sample.

MALDI-TOF mass spectrometry (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry, hereinafter simply referred to as “MALDI”) is a mass spectrometry capable of analyzing insoluble materials. However, MALDI is used for accurate quantitative analysis because the crystal made by mixing the sample and matrix is heterogeneous and the ion signal is not stable due to the laser shot-to-shot variability and the spectrum reproducibility is poor. It is known as a difficult technique. Recently, it has been reported that the reproducibility of the MALDI spectrum for peptides and biomolecular substances has been reported, but the quantitative method of synthetic polymers is not standardized and it is difficult to obtain a consistently effective result using commercial MALDI-TOF MS equipment.

The present inventors have prepared polymer specimens of uniform thickness by electrospray through a mask with holes for soluble polymer samples in order to increase the reproducibility of the MALDI spectrum. 10-2017-0130010, Application date: 2017.10.11, Applicant: LG Chemical Co., Ltd.). In addition, the present inventors discovered that the thickness of the specimen affects the reproducibility of the spectrum and obtained a reproducible MALDI spectrum using polymer specimens by concentrations with a small thickness variation, and then used a linear calibration curve using an internal standard. Has developed a technology to write (Korea Application No. 10-2017-0157161, filing date: 2017.11.23, Applicant: LG Chem). All contents disclosed in the above patent documents are incorporated herein by reference. However, these methods could not be applied to insoluble (or poorly soluble) substances.

In the case of an insoluble material in the prior art, attempts to secure the homogeneity of the sample have been reported using pestles, mortars, ball mills, etc. to secure reproducibility of the MALDI spectrum, but this is a skill Depending on the degree of homogeneity, the sample is difficult to handle due to static electricity. In addition, there is a method of preparing a specimen in the form of pellets by mixing a matrix and an insoluble material and then pressing it, but it is difficult to control the thickness of the specimen.

In addition, CS technique (compressed-sample technique) is known as a method for preparing a specimen of an insoluble material. CS technology is a method of manufacturing a specimen by drilling a hole in a plate on which a specimen is placed, inserting a sample in a state where one side of the hole is blocked, and pressing. However, according to the CS technique, the electric field is not uniformly formed in the specimen due to the hole in the plate, so the m / z of the peak appears differently in the spectrum obtained at the center and edge of the hole.

Thereafter, attempts have been made to minimize the distortion of the electric field by reducing the size of the pores and adding a conductive material (eg, powder) to the sample to overcome the above disadvantages. It is difficult and it is expected that the reproducibility of the specimen production may be reduced because it is not always constant to ensure the homogeneity of the sample by mixing the sample with the conductive material. No method has been developed to obtain specimens that provide reproducible MALDI spectra for such insoluble materials.

In order to secure the reproducibility of the MALDI spectrum for insoluble materials using commercial MALDI-TOF MS equipment, the task of homogeneously mixing the insoluble material and the matrix and the preparation of a specimen with a small thickness deviation must be solved.

The problem to be solved by the present invention is to provide a method for preparing a specimen of uniform thickness in which a sample of a water-insoluble substance is homogeneously distributed in order to obtain a reproducible MALDI spectrum of a water-insoluble substance.

Another problem to be solved by the present invention is to obtain a reproducible MALDI spectrum for a specimen of a water-insoluble material having a uniform thickness and to quantitatively analyze the water-insoluble material therefrom.

In order to solve the above problems, the present invention is a method for preparing a specimen of water-insoluble material for MALDI mass spectrometry,

(S1) mixing the water-insoluble substance and the matrix, and then adding a volatile solvent to disperse the water-insoluble substance and the matrix until the volatile solvent evaporates to obtain a sample of the water-insoluble substance;

(S2) After filling the sample into the groove of the pellet cup made of a water-soluble material, and inverting and pressing it, a method is provided by dissolving the pellet cup with water to obtain a water-insoluble material specimen.

In addition, the present invention is a method for quantitative analysis of a water-insoluble substance using a water-insoluble substance specimen prepared by the above method,

Obtaining a MALDI mass spectrum for the water-insoluble material specimen; And

It provides a quantitative analysis method of a water-insoluble substance comprising the steps of calculating a signal intensity ratio of the water-insoluble substance and the matrix from the peak results of the spectrum and plotting it as a weight ratio of the water-insoluble substance and the matrix. .

The present invention by producing a water-insoluble material specimen having a homogeneous and small thickness deviation. The reproducible MALDI spectrum can be acquired, enabling quantitative analysis of water-insoluble materials using commercial MALDI-TOF MS equipment.

1 illustrates a process for obtaining a homogeneous water-insoluble material sample in a method for preparing a MALDI mass spectrometer according to an embodiment of the present invention.

Figure 2 illustrates the process of obtaining a specimen from a water-insoluble material sample in the method of manufacturing a specimen for MALDI mass spectrometry according to an embodiment of the present invention.

Figure 3 illustrates the manufacturing process of the KBr pellet cup used in the manufacturing method of the specimen for MALDI mass spectrometry according to an embodiment of the present invention.

Figure 4 shows the manufacturing process of the specimen, including the production of pellet cups.

5 is a photomicrograph of a water-insoluble material specimen obtained by a method of manufacturing a MALDI mass spectrometer according to an embodiment of the present invention.

6 is a reproducibility evaluation result of a spot-to-spot MALDI mass spectrum of water-insoluble material specimens prepared according to Examples and Comparative Examples of the present invention.

7 is a MALDI mass spectrum obtained for each of the three water-insoluble material specimens prepared under the same conditions according to an embodiment of the present invention.

Figure 8 shows the signal strength ratio of the analyte (water-insoluble substance) and the matrix from the results of the MALDI mass spectrum obtained for the water-insoluble substance specimen prepared according to the embodiment of the present invention as a weight ratio of the analyte and the matrix It shows the linear quantitative test line created by.

The present invention can be applied to various conversions and can have various embodiments, and specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to a specific embodiment, it should be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In the description of the present invention, when it is determined that a detailed description of known technologies related to the present invention may obscure the subject matter of the present invention, the detailed description will be omitted.

As used herein, the term “water insoluble” is meant to include anything that is not soluble or difficult to dissolve in water.

As used herein, the term “homogeneity” or “homogeneity” refers to a state in which components contained in a sample or a specimen are uniformly distributed to each other.

Hereinafter, the present invention will be described in more detail.

One embodiment of the present invention relates to a method for preparing a specimen of uniform thickness in which a sample of a water-insoluble substance is homogeneously distributed in order to obtain a MALDI spectrum of a reproducible water-insoluble substance, wherein the method is (S1) water-insoluble Obtaining a material sample; And (S2) obtaining a specimen from the sample using a pellet cup.

In the step (S1), after mixing the water-insoluble substance and the matrix, a volatile solvent is added to disperse the water-insoluble substance and the matrix until the volatile solvent evaporates, thereby obtaining a water-insoluble substance sample.

Figure 1 specifically illustrates the process of obtaining a homogeneous water-insoluble material sample in the method of manufacturing a specimen for MALDI mass spectrometry according to an embodiment of the present invention.

Referring to FIG. 1, in the present invention, a water-insoluble substance and a matrix to be analyzed are mixed using a pestle and a mortar (a), and a volatile solvent is added to the mixture to dissolve the matrix, thereby generating it. A water-insoluble substance can be obtained by dispersing the water-insoluble substance in the solution (b), and then stirring the dispersion with a spatula (c and c ') until all the volatile solvent used is evaporated.

That is, the present invention is added to the step (a) of mixing the analyte (a water-insoluble substance) and the matrix using a pestle and a pestle to increase the homogeneity of the sample in the prior art, and the solution is dissolved using a volatile solvent Dispersing the water-insoluble substance in step (b) and while maintaining the dispersion state is characterized by performing the step (c and c ') of homogeneously mixing the matrix and the water-insoluble substance through solvent evaporation. According to the sample obtaining process of the present invention, the homogeneity of the sample is improved and static electricity is not generated in the sample, thereby improving the handling property of the sample.

The degree to which each component is uniformly distributed in the sample obtained according to the present invention can be evaluated from the error range of the MALDI spectrum result of the specimen prepared therefrom, and the smaller the error range, the more uniform the distribution of the sample, i.e. homogeneity. It can be evaluated as excellent.

In one embodiment, the water-insoluble substance is an anthraquinone pigment, such as Pigment Red 177 (PR177, 4,4'-diamino [1,1'-bianthracen] -9,9 ', 10,10'-tetraone), Copper phthalocyanic pigments, perylene pigments, diketopyrrolopyrrole pigments, benzimidazole pigments, isoindoline pigments, dioxazine pigments, and the like, but are not limited thereto.

In one embodiment, the matrix mixed with the water-insoluble substance is DCTB (trans-2- [3- (4-tert-butylphenyl) -2-methyl-2-propenylidene] malononitrile) as a substance having low solubility in water. , DPF (α, β-diphenylfumaronitrile), α-cyano-4-hydroxycinnamic acid, dithranol (1,8,9-trihydroxyanthracene), etc. It can, but is not limited to.

The content of the water-insoluble substance and the matrix is not particularly limited, but may be mixed in a weight ratio of 1:20 to 1: 100, such as 1:20.

In one embodiment, the water-insoluble substance and the volatile solvent added to the matrix may be one or more selected from tetrahydrofuran (THF), chloroform, and the like, but is not limited thereto.

In one embodiment, in addition to the pestle and pestle when mixing the water-insoluble material and the matrix, a ball mill may be used.

Next, in the step (S2), the previously obtained sample is filled into a groove of a pellet cup made of a water-soluble material, and then inverted and pressed, the pellet cup is dissolved with water to obtain a water-insoluble material specimen.

Figure 2 specifically illustrates the process of obtaining a specimen from a water-insoluble material sample in a method for preparing a specimen for MALDI mass spectrometry according to an embodiment of the present invention.

Referring to FIG. 2, in the present invention, a water-soluble material pellet cup is prepared to fill a sample of the water-insoluble material obtained before the groove thereof (d), the pellet cup filled with the sample is turned over (e), and the press is pressed in that state. After performing (f), the used pellet cup was dissolved with water (g) and further dried (h) to obtain a water-insoluble specimen. At this time, the press may be performed for 10 to 20 seconds at a pressure of 1 to 5 bar using a hot press.

In the present invention, in order to press a homogeneous water-insoluble material sample obtained according to the above process at the same time between samples in a certain area at the same pressure during sample preparation, the same amount of sample can be limited to a certain area. As a means, a pellet cup is used. The pellet cup may be used without limitation as long as it is a water-soluble material soluble in water. For example, a pellet cup made of KBr, NaBr, MgBr ₂ , NaCl or a mixture thereof may be used.

Referring to FIG. 3, the KBr pellet cup manufacturing apparatus is placed on a flat square plate 6, a square frame 1 with a circular hole in the middle placed on the plate 6, and a circular hole of the square frame 1 The paper can use circular members (2), (3) and (4) and a circular member (5) which is placed on the circular member (4) and then pressed.

The manufacturing process of a specific KBr pellet cup is as follows.

First, the frame 1 with a circular hole in the center is placed on the plate 6, and the circular member 2 is inserted into the circular hole of the frame 1 (step i). The plate 6 is a plate in which the entire process is performed from KBr pellet cup production to production of the specimen, and the shape, dimension, and material are not particularly limited, and the entire specimen production process can be easily performed. Subsequently, the circular member 3 is put on the circular member 2 (step ii). At this time, the circular member 2 has a convex portion in the center, and the circular member 3 has a small circular hole in the center. Then, KBr is filled in the center hole of the circular member 3 (step iii). The convex portion of the circular member 2 and its size and the hole in the center of the circular member 3 and its size correspond to the shape and dimensions of the KBr pellet cup later. When KBr is completely filled in the center hole of the circular member 3, the circular member 4 is placed on it (step iv). The circular member 4 has a convex portion at the center. Thereafter, after placing the circular member 5 on the circular member 4 (step v), a press is performed (step vi). At this time, the same pressure is applied over the entire area of the circular member 5. The circular members (2), (3) and (4) are placed in order in the circular holes of the frame (1), so they must be exactly the size of the circular holes of the frame (1). After the circular member 5 is sufficiently pressed, the

circular members

5 and 4 are removed, and the circular member 2 and the frame 1 are removed, leaving only the circular member 3 on the plate 6. KBr is tightly filled in the center hole of the circular member 3 by a press. Finally, the circular member 3 is removed to complete the KBr pellet cup (step vii). The finished pellet cup has a grooved shape in the center by the convex portion of the circular member 2.

In the present invention, the shape and dimensions of the frame 1 are not particularly limited, but it is preferable that all the

circular members

2, 3, and 4 enter the circular hole of the frame 1, so that the frame 1 The thickness of can be equal to or greater than the combined thickness of the

circular members

2, 3 and 4, respectively. The circular member 5 should be smaller than the size of the circular member 4 to be easy to press. The material of the circular members (1) to (5) is not particularly limited, for example, stainless steel (stainless steel), aluminum (aluminium) or the like can be used.

In one embodiment, the press can be performed using a hot press. For example, the press may be performed for 10 to 20 seconds at a pressure of 1 to 5 bar.

The size of the circular hole in the frame 1 and the hole in the center of the circular member 3 is not particularly limited.

In one embodiment, the pellet cup may have a circular or polygonal shape.

Figure 4 schematically shows the manufacturing process of the specimen including the production of the pellet cup, the pellet cup manufacturing apparatus 10 is filled with a pellet cup material such as KBr and then pressed to produce a pellet cup (the specific method is FIG. 3), filling the pellet cup with a sample of a water-insoluble substance, turning it over, mounting it on a MALDI sample plate, and pressing, and dissolving the pellet cup.

When the pellet cup is used in this way, it is not necessary to drill a hole in the plate on which the specimen is placed, as in the case of specimen preparation by a conventional CS technique (compressed sample technique). The disadvantages can be overcome. That is, since the pellet cup applied in the present invention can press the same pressure per unit volume in a state in which a sample is limited therein, it is possible to manufacture a specimen having a uniform thickness. Also, by dissolving only the pellet cup with water for the sample pressed together with the pellet cup, it is possible to prevent the specimen surface from being damaged by the press.

The water-insoluble material specimen of the present invention prepared in the above manner may have a thickness of 20 μm or less, for example 10 μm or less. In addition, when the thickness of the specimen is measured at a plurality of points, for example, three or more points, and the standard deviation of the average value is calculated, the thickness deviation may be 30% or less.

When a water-insoluble material specimen with a small thickness variation is used for MALDI mass spectrometry while securing a thickness capable of analyzing as described above, a reproducible MALDI spectrum can be obtained, from which a quantitative analysis of a water-insoluble material sample is possible.

Therefore, the present invention provides a method for quantitative analysis of a water-insoluble substance using MALDI mass spectrometry of a water-insoluble substance specimen prepared using a pellet cup.

Specifically, the analysis method includes obtaining a MALDI mass spectrum for a water-insoluble material specimen prepared using a pellet cup; And calculating a signal intensity ratio of the water-insoluble substance and the matrix from the peak results of the spectrum, and plotting the ratio of the signal strength of the water-insoluble substance and the matrix to create a quantitative calibration curve.

The MALDI mass spectrum can be obtained using commercial MALDI-TOF MS equipment. At this time, in order to obtain a spectrum having a good S / N ratio (signal-to-noise ratio), multiple points, for example, 50 to 200 shots per point in the specimen, for example, 3 or more, Alternatively, an average value is obtained by obtaining one spectrum for each point from five or more spots.

In one embodiment, the MALDI mass spectrum of the water-insoluble material specimen has a relative standard deviation (RSD) indicating the error of the result when three or more points are measured (spot-to-spot) in the same specimen. 40% or less, specifically 35% or less, such as 25 to 35% or 25 to 30%. In addition, RSD of sample-to-sample MALDI mass spectrum results measured on three or more specimens prepared under the same conditions is 40% or less, specifically 30% or less, such as 20 to 30% Or 25 to 30%. This RSD range is to demonstrate the reproducibility of the MALDI spectrum obtained from specimens manufactured according to the present invention to ensure thickness uniformity.

In addition, the quantitative calibration curve created using the results of the reproducible MALDI spectrum, that is, the quantitative calibration curve depicting the signal intensity ratio of the water-insoluble substance and the matrix as the weight ratio of the water-insoluble substance and the matrix are all linear (see FIG. 8). ), From which it is possible to quantitatively analyze water-insoluble substances.

Hereinafter, the present invention will be described in detail as specific examples so that those skilled in the art to which the present invention pertains can easily practice. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

<실시예 1><Example 1>

Step 1: Preparation of the sample

Pigment Red 177 (PR177, C ₂₈ H ₁₆ N ₂ O ₄ , Exact Mass 444.11) represented by the following formula as a water-insoluble substance to be analyzed and DCTB (trans-2- [3- (4-tert-butylphenyl)) as a matrix -2-methyl-2-propenylidene] malononitrile) was used. As shown in Fig. 2, the water-insoluble substance and the matrix were mixed in a mortar with a weight ratio of 1:20 and grinded with a pestle. Here, THF (2 mL) was added as a volatile solvent for dispersing the mixture, and mixing was continued. Subsequently, stirring was continued while stirring with a spatula until the THF evaporated to obtain a sample in which the water-insoluble substance and the matrix were uniformly distributed.

Step 2: Preparation of the specimen

The sample prepared in the above step was filled into the groove of the KBr pellet cup prepared as shown in FIG. 4. The KBr pellet cup filled with the sample was turned over and mounted on the MALDI sample plate, and then 5 bar of pressure was applied to the KBr pellet cup for 20 seconds. Subsequently, KBr was dissolved in water to prepare a PR177 specimen having a thickness of about 10 μm.

5 shows a micrograph of the prepared specimen, from which it can be confirmed that there is no surface damage of the sample.

<비교예 1><Comparative Example 1>

Step 1: Preparation of the sample

A sample was prepared by adding PR 177 as a water-insoluble substance and DCTB as a matrix in a weight ratio of 1:20 in a mortar and grinding while changing to a pestle.

Step 2: Preparation of the specimen

Specimens were prepared in the same manner as in Step 2 of Example 1.

<비교예 2><Comparative Example 2>

Step 1: Preparation of the sample

Samples were prepared in the same manner as in Step 1 of Example 1.

Step 2: Preparation of the specimen

Specimens were prepared by placing the sample obtained in step 1 on a MALDI sample plate without using a KBr pellet cup and then simply pressing it.

실험예 1Experimental Example 1 : 스팟-투-스팟(spot-to-spot) 재현성 평가: Evaluation of spot-to-spot reproducibility

Using a MALDI-TOF mass spectrometer (UltrafleXtreme, Bruker Daltonics, Germany), MALDI mass spectra were obtained for 5 spots of PR177 specimens prepared according to the examples and comparative examples, respectively. From the spectral results, the intensity ratio (A ^{+ ·} ) of the basic peak of the PR177 ion (A ^{+ ·} ) at each spot (spot) and the ion (M ⁺ ) having the greatest intensity (intensity) among DCTB-derived ions / M ⁺ intensity ratio). The intensity ratio for each of the five points of the PR177 specimen prepared according to Comparative Example 1 is shown in FIG. 6 (a), and the strength ratio for each of the five points of the PR177 specimen prepared according to Comparative Example 2 is shown in FIG. As b), the strength ratio for each of the five points of the PR177 specimen prepared according to Example 1 is shown in FIG. 6 (c).

In (a) and (b) of Figure 6, the intensity ratio (A ^{+ ·} / M ⁺ intensity ratio) at each point of the specimens prepared in Comparative Examples 1 and 2 can be seen to deviate significantly from the average (green arrow Reference). Particularly, in the case of Comparative Example 2, in which a pellet cup was not used to prepare a specimen after preparing a sample in the same manner, a signal was not detected at all when the position of the specimen was changed even under a condition in which a signal was well displayed at a specific location. However, this phenomenon is considered to be caused by a large variation in the thickness of the specimen, so that the positional relationship between the laser and the specimen is greatly changed.

On the other hand, in the results for the specimen prepared by the method according to Example 1 of Figure 6 (c) it can be seen that the intensity ratio (A ^{+ ·} / M ⁺ intensity ratio) at each point is less than the average deviation (In the case of Example 1, the RSD between intensity ratios was 30% or less).

As described above, when a sample obtained by improving the dispersibility of a mixture using a volatile solvent in preparing a sample in which a water-insoluble substance and a matrix are mixed, as in the present invention, is prepared in a specimen using a pellet cup, the water-insoluble substance Compared to Comparative Example 1 in which a sample was obtained by performing only simple mixing using a pestle and a pestle and a pestle, and Comparative Example 2 in which a pellet cup was not used in preparing the specimen, the homogeneity of the specimen was improved to spot the MALDI mass spectrum. The spot-to-spot reproducibility is high.

실험예 2Experimental Example 2 : 샘플-투-샘플(sample-to-sample) 재현성 평가: Evaluation of sample-to-sample reproducibility

Three specimens were prepared under the same conditions according to the above examples. The MALDI mass spectra obtained for each specimen are shown in Fig. 7 as (a), (b) and (c).

From FIG. 7, the intensity pattern of the peaks in the three specimens and the reproducibility of the peak position can be confirmed. In addition, the RSD between the base peak of the PR177 ion (A ^{+ ·} ) and the base peak of the ion (M ⁺ ) having the greatest intensity among DCTB-derived ions (A ^{+ ·} / M ⁺ intensity ratio) is 25 %. Therefore, the MALDI mass spectrum of the specimen prepared according to the present invention also has high sample-to-sample reproducibility.

실험예 3Experimental Example 3 : 정량 검정선의 평가: Evaluation of quantitative assay line

The signal intensity ratio of the analyte (water-insoluble substance) and the matrix was calculated using the mass spectrum peak of FIG. 7, and a quantitative calibration curve was prepared by plotting this as the weight ratio of the analyte and the matrix, and the results are shown in FIG. 8. Shown.

Specifically, FIG. 8 is a quantitative test line prepared using matrix peaks corresponding to m / z 332 (a) and m / z 250 (b), all of which are linear (R ² = 0.99 or more).

Since the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims

(S1) mixing the water-insoluble substance and the matrix, and then adding a volatile solvent to disperse the water-insoluble substance and the matrix until the volatile solvent evaporates to obtain a sample of the water-insoluble substance;

(S2) Filling the sample into the groove of a pellet cup made of a water-soluble material, turning it over and pressing it, and then dissolving the pellet cup with water to obtain a water-insoluble substance specimen, MALDI mass spectrometry for quantitative analysis of the water-insoluble substance Method for manufacturing a test specimen.
The method of claim 1, wherein the water-insoluble material is an anthraquinone pigment, copper phthalocyanic pigment, perylene pigment, diketopyrrolopyrrole pigment, benzimidazole pigment, isoindoline pigment, dioxazine pigment Or a mixture of these, the method of manufacturing a specimen for MALDI mass spectrometry.
The method according to claim 2, wherein the water-insoluble anthraquinone pigment is Pigment Red 177 (PR177, 4,4'-diamino [1,1'-bianthracen] -9,9 ', 10,10'-tetraon). Method of manufacturing specimen for MALDI mass spectrometry.
According to claim 1, The matrix is mixed with the water-insoluble substance is DCTB (trans-2- [3- (4-tert-butylphenyl) -2-methyl-2-propenylidene] malononitrile), DPF (α, β- diphenylfumaronitrile, α-cyano-4-hydroxycinnamic acid, dithranol (1,8,9-trihydroxyanthracene) or a mixture thereof Method for preparing a specimen for mass spectrometry.
The method of claim 1, wherein the water-insoluble substance and the volatile solvent injected into the matrix include tetrahydrofuran (THF), chloroform, or a mixture thereof.
The method of claim 1, wherein the pellet cup is capable of limiting the same amount of samples per unit volume.
The method of claim 1, wherein the pellet cup is made of KBr, NaBr, MgBr 2 , NaCl or mixtures thereof.
A method for quantitative analysis of a water-insoluble substance using a water-insoluble substance specimen prepared by the method according to claim 1,

Obtaining a MALDI mass spectrum for the water-insoluble material specimen; And

Quantitative analysis method of a water-insoluble substance comprising the steps of calculating the signal intensity ratio of the water-insoluble substance and the matrix from the peak results of the spectrum and plotting it as a weight ratio of the water-insoluble substance and the matrix.
The method of claim 8, wherein the relative standard deviation (RSD) of the MALDI mass spectrum measured at different points of the specimen is 40% or less.
The method of claim 8, wherein the RSD of the MALDI-TOF mass spectrum measured by applying a plurality of the specimens under the same conditions is 40% or less.