Method for preparing sample for analysis and method for analyzing cyclic oligomer

Abstract

A method for preparing a sample for analysis according to the present invention comprises: a step for preparing a sample solution that contains phenylethyl alcohol and a cyclic-oligomer-containing sample, and a matrix solution that contains a matrix; and a step for drying the sample solution and the matrix solution on a sample plate of a MALDI mass spectrometer. Specifically, as shown in, e.g., fig. 3, a cyclic-oligomer-containing sample 101 is dissolved in phenylethyl alcohol (PEA) 102 to prepare a sample solution 10, and the sample solution 10 is mixed with a matrix solution 20 obtained by dissolving a matrix 201 in a prescribed solvent 202 to prepare a sample / matrix / phenylethyl alcohol (PEA) mixed solution 30. The mixed solution (sample / matrix / phenylethyl alcohol (PEA) mixed solution) 30 is dropped into individual wells 401 of a sample plate 40 of a MALDI mass spectrometer and is dried in the wells 401. According to this method, the peak of the cyclic oligomer can be detected with high sensitivity, preferentially, and with excellent reproducibility through MALDI-MS.

Description

Method for preparing analytical samples and method for analyzing cyclic oligomers

[0001] The present invention relates to a method for preparing analytical samples for mass spectrometry of cyclic oligomers such as cyclic oligosaccharides and cyclic peptides, and to a method for analyzing cyclic oligomers.

[0002] Cyclodextrins (CDs), which are oligosaccharides in which glucose molecules are linked in a ring, have vacancies within their cyclic structure. The exterior of the CD cyclic structure is hydrophilic due to the OH groups of glucose, while the interior is hydrophobic as it is mainly composed of carbon and hydrogen. Therefore, CDs readily incorporate other hydrophobic molecules (guest molecules) into their vacancies to form inclusion compounds. Because inclusion compounds improve the stability and solubility of guest molecules, CDs are widely used in various fields such as food, pharmaceuticals, and cosmetics. Furthermore, CD derivatives have been synthesized by chemically modifying the basic structure of CDs in order to increase the solubility of CDs themselves or to adjust their inclusion ability.

[0003] A method for analyzing cyclic oligosaccharides, including CD and CD derivatives, is known to be mass spectrometry using MALDI-MS. For example, Non-Patent Literature 1 (BUNSEKI KAGAKU 2005_54_983) reports an example of analyzing γ-CD and maltosyl-α-CD with a glycosylation branching structure using MALDI-MS. In the same document, an analytical sample was prepared using DHB as the matrix, and the analytical sample was analyzed using MALDI-MS in positive mode. 2 By analyzing the [M+Na] of γ-CD and maltosyl-α-CD, + [M-Glcn] + It is stated that (n=1 to 5) was detected.

[0004] Furthermore, Non-Patent Document 2 (JMSSJ2002_50_204) reports an example of analyzing α-CD, γ-CD, and glycosyl-β-CD with a glycosylated branching structure using MALDI-MS. In this document, the analytical sample was prepared using norHarman as the matrix, and the analytical sample was subjected to MS in negative mode of MALDI-MS. 2By analysis, [M-H] of α-CD, γ-CD, and glycosyl-β-CD - , [(M-C₄H₈O₄)-Glcn-H] - (n = 0 to 2) has been reported to be detected.

[0005] BUNSEKI KAGAKU, Vol. 54, No. 10, pp. 983 - 990 (2005); J. Mass Spectrom. Soc. Jpn, Vol. 50, No. 4, pp. 204 - 207 (2002)

[0006] In the analysis of cyclic oligosaccharides using MALDI-MS, cyclic oligosaccharides tend to form adducts with the matrix, and molecular weight-related ions such as [M+Na] + , [M-H] - etc. are difficult to be detected as main peaks. Also, even if peaks of molecular weight-related ions are detected, there is a problem that the sensitivity is low. Furthermore, 2,5-dihydroxybenzoic acid (2,5-Dihydroxybenzoic Acid (DHB)), which is known as a matrix for sugar chains, forms crystals with bias and tends to form so-called sweet spots, and formation of a more uniform matrix / sample mixed crystal and highly reproducible peak detection are desired.

[0007] Also, it has been reported that cyclic oligosaccharides can be structurally analyzed by obtaining fragments in which monosaccharides are successively removed from cyclic oligosaccharides by MS n analysis. In order to more accurately and reliably perform molecular weight analysis and structural analysis of the entire cyclic oligosaccharide, in addition to MS analysis, it is necessary to improve the detection sensitivity of MS n analysis, and further, in both positive and negative modes, it is desired that molecular weight-related ion peaks and MS n fragment peaks can be detected with high reproducibility and high sensitivity.

[0008] So far, it has been necessary to prepare analytical samples according to the analysis in each of the positive and negative modes, which has been a time-consuming task. That is, MS analysis of cyclic oligosaccharides, MS nThe development of a common method for preparing analytical samples to enable highly sensitive and easy analysis is desirable.

[0009] The above describes MS analysis of cyclic oligosaccharides. n While the problems in analysis have been explained, compounds containing cyclic structures in which basic units (monomers) are linked cyclically exist other than cyclic oligosaccharides. For example, cyclic peptides have a backbone in which amino acids are linked cyclically. Compounds with cyclic structures such as cyclic oligosaccharides and cyclic peptides (oligomers) are referred to as cyclic oligomers in this specification. Cyclic peptides are widely used, especially as peptide pharmaceuticals, and well-known cyclic peptide pharmaceuticals include cyclosporine, polymyxin, and daptomycin. LC-ESI-MS is mainly used for the analysis of cyclic peptides, but there are few reports using MALDI-MS. MALDI-MS exhibits a simple mass spectrum that detects monovalent ions without an LC step, and therefore has the potential to enable easier and faster analysis compared to LC-ESI-MS. The development of a simpler and faster analytical method for cyclic peptides using MALDI-MS is desired.

[0010] The problem that this invention aims to solve is to enable easy and rapid MALDI-MS analysis of cyclic oligomers. Specifically, it aims to enable MS analysis in either the positive mode or the negative mode by MALDI-MS, MS n In the analysis, the goal is to detect peaks derived from cyclic glycans with high sensitivity and reproducibility. Furthermore, the goal is to enable the detection of molecular weight-related ion peaks of cyclic peptides with higher sensitivity, preference, and reproducibility using MALDI-MS.

[0011] A method for preparing an analytical sample according to one aspect of the present invention, which was made to solve the above problems, comprises the steps of: preparing a sample solution which is a solution containing a sample containing a cyclic oligomer and phenylethyl alcohol; preparing a matrix solution which is a solution containing a matrix; and drying the sample solution and the matrix solution on a sample plate of a MALDI mass spectrometer.

[0012] Furthermore, a method for analyzing cyclic oligomers according to another aspect of the present invention, which was developed to solve the above-mentioned problems, comprises the steps of: preparing an analytical sample by the analytical sample preparation method according to the above-described aspect of the present invention; and subjecting the prepared analytical sample to MALDI mass spectrometry.

[0013] In the present invention, cyclic oligomers include cyclic oligosaccharides and cyclic peptides. According to the present invention, MS analysis in either positive mode or negative mode, and MS n In analysis, peaks derived from cyclic oligosaccharides (i.e., molecular weight-related ions and fragment ions of cyclic oligosaccharides) can be detected with high sensitivity and reproducibility. In addition, according to the present invention, molecular weight-related ion peaks of cyclic peptides can be detected with higher sensitivity, preferentially, and reproducibly.

[0014] Figures showing the chemical structures of α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), and γ-cyclodextrin (γ-CD), which are representative examples of cyclic oligosaccharides. Figures showing the chemical structures of cyclosporine A (a) and daptomycin (b), which are representative examples of cyclic peptides. A conceptual diagram showing the flow of the method for preparing analytical samples according to the embodiment of the present invention. Mass spectra of β-CD when (a) CHCA, (b) DHB, (c) norHarman, and (d) SA were used as matrices in Experimental Example 1. Mass spectra of β-CD in negative mode when CHCA was used as the matrix in Experimental Example 1. 2 Mass spectrum at the time of analysis. 2,4 A or 0,2 Figure showing ring cleavage of X (see Non-Patent Literature 2 (J. Mass Spectrom. Soc. Jpn, Vol. 50, No. 4, pp. 204-207 (2002))). In Experimental Example 1, CHCA was used as the matrix and β-CD was MS in negative mode. 3Mass spectrum obtained by analysis. Mass spectrum obtained by MS analysis of β-CD in positive mode using CHCA as the matrix in Experimental Example 2. 2 Mass spectrum obtained through analysis. In Experimental Example 2, CHCA was used as the matrix, and β-CD was analyzed by MS in positive mode. 3 Mass spectra obtained by analysis. Photograph of the appearance of a sample plate on which analytical samples (sample / matrix mixed crystals) prepared in Example 1 were placed, using (a) water, (b) (R)-(+)-1-Phenylethyl Alcohol ((R)-(+)-PEA), and (c) (S)-(-)-1-Phenylethyl Alcohol ((S)-(-)-PEA) as solvents and a matrix solution containing CHCA. Mass spectra obtained by MS analysis in positive and negative modes for analytical samples prepared in Example 1 using various β-CD sample solutions with water, (R)-(+)-PEA, and (S)-(-)-PEA as solvents and a matrix solution containing CHCA. Mass spectra obtained by MS analysis in positive and negative modes for β-CD in Example 1 using a β-CD sample solution with (S)-(-)-PEA as a solvent and a matrix solution containing CHCA. 2 Analysis and MS 3Mass spectra obtained by performing MS analysis in positive and negative modes on analytical samples prepared in Example 2 using sample solutions of α-CD, β-CD, and γ-CD with (S)-(-)-PEA as the solvent, and a sample solution containing CHCA. Mass spectra obtained by performing MS analysis in positive and negative modes on analytical samples prepared in Example 3 using a sample solution of β-CD with (S)-(-)-PEA as the solvent, and a matrix solution containing CHCA, a matrix solution containing DHB, and a matrix solution containing norHarman, respectively. Mass spectra obtained by performing MS analysis in positive and negative modes on analytical samples prepared in Example 4 using various sample solutions of β-CD with water, (R)-(+)-PEA, and (S)-(-)-PEA as the solvents, respectively, and a matrix solution containing CHCA. Mass spectra obtained by MS analysis of analytical samples prepared in Example 5 using various sample solutions containing cyclosporine A with 100% ACN, 50% ACN aqueous solution, and (S)-(-)-PEA as solvents, respectively, and matrix solutions containing CHCA and DHB, respectively. Mass spectra obtained by MS analysis of analytical samples prepared in Example 5 using various sample solutions containing cyclosporine A with 100% ACN, (S)-(-)-PEA, and (R)-(+)-PEA as solvents, respectively, and matrix solutions containing CHCA and DHB, respectively. Photograph of a sample plate on which analytical samples (sample / matrix mixed crystals) prepared in Example 5 using various sample solutions with 100% ACN, (S)-(-)-PEA, and (R)-(+)-PEA as solvents, respectively, and matrix solutions containing CHCA and DHB, respectively. In Example 6, mass spectra were obtained by MS analysis of sample solutions containing daptomycin with water, (S)-(-)-PEA, and (R)-(+)-PEA as solvents, respectively, and analytical samples prepared using a matrix solution containing CHCA or a matrix solution containing DHB.In the reference example, mass spectra obtained by MS analysis of analytical samples prepared with Glu-Fib sample solutions using (S)-(-)-PEA and 50% ACN 0.1% TFA aqueous solutions as solvents, and a matrix solution containing CHCA. In the reference example, mass spectra obtained by MS analysis of analytical samples prepared with Cyt C sample solutions using (S)-(-)-PEA and 50% ACN 0.1% TFA aqueous solutions as solvents, and a matrix solution containing SA.

[0015] The following describes embodiments for carrying out the method for preparing analytical samples and the method for analyzing cyclic oligomers according to the present invention.

[0016] The method for preparing an analytical sample according to this embodiment includes the steps of: preparing a sample solution which is a solution containing a cyclic oligomer and phenylethyl alcohol; preparing a matrix solution which is a solution containing a matrix; and drying the sample solution and the matrix solution on a sample plate of a MALDI mass spectrometer.

[0017] The sample is not particularly limited as long as it is a cyclic oligomer or contains a cyclic oligomer. Examples of samples containing cyclic oligomers include samples derived from food, pharmaceuticals, cosmetics, and detergents.

[0018] A cyclic oligomer refers to an oligomer that contains a cyclic structure formed by linking together basic units (monomers) such as sugars and peptides. The cyclic structure may be present in either the main chain or the side chain of a cyclic oligomer. Representative examples of cyclic oligomers include cyclic oligosaccharides and cyclic peptides.

[0019] Cyclodextrins are oligosaccharides in which glucose molecules are linked in a ring by α-1,4-glycosidic bonds. They are also called cyclodextrins. There are several types of cyclic oligosaccharides depending on the size of the ring (degree of polymerization of glucose). Representative examples shown in Figure 1 include α-cyclodextrin (α-CD) (a cyclic oligosaccharide composed of 6 glucose units), β-cyclodextrin (β-CD) (a cyclic oligosaccharide composed of 7 glucose units), and γ-cyclodextrin (γ-CD) (a cyclic oligosaccharide composed of 8 glucose units). The degree of polymerization of the cyclic oligosaccharides contained in the sample is not particularly limited. In addition, chemically modified cyclic oligosaccharides are also included.

[0020] Cyclic peptides are peptides that have a structure in which amino acids are linked together in a ring by peptide bonds. Representative examples of cyclic peptides include cyclosporine (a) and daptomycin (b), shown in Figure 2. The number of peptides constituting a cyclic peptide in a sample is not particularly limited. Furthermore, cyclic peptides may include chemically modified forms.

[0021] The matrix can be any known matrix used in MALDI mass spectrometry. Among these, α-Cyano-4-hydroxycinnamic acid (CHCA) is particularly preferred.

[0022] Phenylethyl alcohol comprises two structural isomers (1-phenylethyl alcohol and 2-phenylethyl alcohol). 1-phenylethyl alcohol further comprises two optical isomers ((R)-(+)-1-phenylethyl alcohol and (S)-(-)-phenylethyl alcohol). The phenylethyl alcohol used in the present invention may be any of the above isomers, but 1-phenylethyl alcohol (R-isomer, S-isomer, or racemic mixture) is preferably used. Among these, (S)-(-)-phenylethyl alcohol is particularly preferred because it can detect peaks derived from cyclic oligosaccharides with high sensitivity.

[0023] In the method for preparing analytical samples according to this embodiment, first, a solution containing a cyclic oligomer or a sample containing a cyclic oligomer and phenylethyl alcohol (sample solution) and a solution containing a matrix (matrix solution) are prepared. For example, as shown in Figure 3, phenylethyl alcohol 102 is added to a tube containing a sample 101 consisting of a cyclic oligosaccharide or a sample 101 containing a cyclic oligosaccharide to prepare a sample solution 10 in which the sample 101 is dissolved in phenylethyl alcohol 102. Also, a predetermined solvent 202 is added to a tube containing a matrix 201 to prepare a matrix solution 20 in which the matrix 201 is dissolved in solvent 202. Then, the sample solution 10 is mixed with the matrix solution 20 to prepare a sample / matrix / phenylethyl alcohol (PEA) mixed solution 30. Next, the mixed solution (sample / matrix / phenylethyl alcohol (PEA) mixed solution) 30 is dropped into each well 401 of the sample plate 40 of a MALDI mass spectrometer and dried on the well 401 (this method is called the pre-mix method).

[0024] Alternatively, for example, the sample solution and matrix solution may be added separately to each well of the sample plate to prepare a sample / matrix mixed solution on the wells, and then the mixed solution may be dried (this method is called the on-target mix method). In both the pre-mix method and the on-target mix method, a sample / matrix mixed crystal (analytical sample) is formed on each well of the sample plate.

[0025] In the analytical method for cyclic oligomers according to this embodiment, the analytical sample prepared by the above method is subjected to MALDI mass spectrometry. The MALDI mass spectrometer is not particularly limited as long as it has a MALDI ion source. For example, there is MALDI-TOFMS, which is a combination of a MALDI ion source and a time-of-flight mass spectrometer (TOFMS), and MALDI-ITMS, which is a combination of a MALDI ion source and an ion trap mass spectrometer (ITMS). MALDI-ITMS may trap ions by utilizing an electric field generated by applying a sinusoidal high-frequency voltage to the electrodes, or by utilizing an electric field generated by applying a rectangular wave voltage, which is produced by rapidly switching two different voltages, to the electrodes. The latter is called a digital ion trap mass spectrometer (DITMS).

[0026] MALDI mass spectrometers may have a raster scan function. The raster scan function is a function that acquires measurement data by irradiating each of a number of pre-set measurement points at different positions on a single well of a sample plate with a predetermined number of laser beams, and then derives the final measurement data by integrating all of these measurement data. When the mixed crystal formed on the sample plate is non-uniform, the detection sensitivity may vary depending on the laser irradiation position. Since the measurement data obtained by the raster scan function is integrated data, the variation in measurement results due to differences in laser irradiation position is reduced. In addition, with raster scan measurements, the laser irradiation position is automatically selected, so there is no intervention from human intervention in selecting the measurement position, making it possible to acquire more objective and reproducible data.

[0027] The MALDI mass spectrometer performs MS analysis to detect ions that provide molecular weight information for sample molecules (also called molecular weight-related ions). In addition to MS analysis, the MALDI mass spectrometer also performs MS to detect fragment ions generated by the cleavage of the binding site from these ions. n It may also be a method that performs analysis (where n is an integer greater than or equal to 2).

[0028] In the method for preparing analytical samples according to this embodiment, analytical samples prepared using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix are subjected to MS analysis in positive mode using a MALDI mass spectrometer. 2 Analysis, and MS 3 When analyzing using any of the analysis methods, or when analyzing MS analysis in negative mode, MS 2 Analysis, and MS 3 When analyzed using any of the analytical methods, peaks derived from cyclic oligosaccharides can be detected with high sensitivity and reproducibility. Therefore, samples containing cyclic oligosaccharides, α-cyano-4-hydroxycinnamic acid (CHCA), and phenylethyl alcohol are useful as standard samples for performance evaluation or mass calibration of MALDI mass spectrometers.

[0029] The performance evaluation of MALDI mass spectrometers is, for example, MS. 2 For performance verification of the analysis, the analytical sample, prepared using the specified method with the specified sample concentration and phenylethyl alcohol, is subjected to MS of the specified precursor ion under the specified instrument conditions. 2 This is done by performing an analysis to confirm whether the signal-to-noise ratio (S / N) and peak intensity (mV) of the specified product ion meet predetermined criteria. At this time, [M+Na] is measured using MS analysis. + When molecular weight-related ions such as these are detected with high intensity, these ions can be reliably selected as precursor ions, and MS 2 Analysis can be performed. Also, MS 2 When the specified product ions are generated with sufficient sensitivity and reproducibility during the analysis, the signal-to-noise ratio (S / N) and peak intensity (mV) of these product ions are compared with pre-set reference values ​​using MS. 2It is possible to evaluate the condition of the equipment during analysis.

[0030] Therefore, the performance evaluation kit or mass calibration kit for the MALDI mass spectrometer according to this embodiment may include a cyclic oligomer, α-cyano-4-hydroxycinnamic acid (CHCA), and phenylethyl alcohol. This kit may include consumables necessary for the operation of performance evaluation or mass calibration of the MALDI mass spectrometer, a performance evaluation sample prepared using the cyclic oligomer, α-cyano-4-hydroxycinnamic acid (CHCA), and phenylethyl alcohol, and an application program for performing performance evaluation or mass calibration by analyzing the results of analyzing the mass calibration sample with the MALDI mass spectrometer.

[0031] The present invention will be described below with reference to several examples, but these are merely illustrative and the invention is not limited thereto. First, Experiments 1 and 2, which were conducted to select the matrix to be used in the examples, will be described.

[0032] [Experimental Example 1] In Experimental Example 1, β-cyclodextrin (β-CD), a cyclic oligosaccharide, was used as the cyclic oligomer. In addition, CHCA (α-Cyano-4-hydroxycinnamic Acid) and SA (3,5-dimethoxy-4-hydroxycinnamic acid), which are compatible with proteins and peptides, and DHB (2,5-Dihydroxybenzoic Acid) and norHarman (9H-Pyrido[3,4-b]indole), which are compatible with sugar chains, were used as the matrix.

[0033] [1] Preparation of sample solution A 1 mg / mL aqueous solution of β-CD (Sigma-Aldrich) was prepared as the sample solution.

[0034] [2] Preparation of Matrix Solutions As matrix solutions, 10 mg / mL 50% acetonitrile (ACN) aqueous solutions of CHCA (LaserBio), DHB (LaserBio), norHarman (Sigma-Aldrich), and SA (LaserBio) were prepared, respectively.

[0035] [3] Preparation of the sample for analysis: 0.5 μL of the sample solution prepared in [1] was dropped onto the sample plate, followed by 0.5 μL of the matrix solution prepared in [2], and then air-dried (on-target mix method).

[0036] [4] Mass Spectrometry The sample plate on which the analytical sample prepared in [3] is placed is then subjected to an improved MALDImini, a MALDI-DITMS equipped with a negative mode. TM Insert into -1 (Shimadzu Corporation) and perform MS analysis in negative mode. 2 Analysis and MS 3 An analysis was conducted.

[0037] [5] Results Figures 4(a) to (d) show the mass spectra of β-CD obtained by negative mode MS analysis using CHCA, DHB, norHarman, and SA as matrices. The horizontal axis of the mass spectrum represents the mass-to-charge ratio (m / z), and the vertical axis represents the relative intensity (Int%). As shown in Figure 4, regardless of whether CHCA, DHB, norHarman, or SA is used, the [MH] of β-CD - A peak (m / z 1133) was detected (where M is the sample molecule and H is the hydrogen atom). However, the overall sensitivity of peak detection was low when using SA. When using CHCA, DHB, and norHarman, [MH] - In addition to the peak, there is also the matrix-related ion adduct ([M+mH] - Peaks originating from (M is the sample molecule, m is the matrix-related molecule, and H is the hydrogen atom) were detected. When using DHB and norHarman, [MH] - [M+mH] - The peak was detected with high intensity. On the other hand, when using CHCA, [M+mH] - [MH] - The peak was highly intense and therefore detected preferentially.

[0038] Figure 5 shows a negative mode MS using CHCA as the matrix. 2 This is a mass spectrum obtained through analysis. As shown in Figure 5, MS 2In the mass spectrum analysis, a precursor ion peak was detected at m / z 1133, and peaks of multiple fragment ions derived from the precursor ion were detected at m / z 365, m / z 407, m / z 527, m / z 569, m / z 689, m / z 851, and m / z 1013, respectively.

[0039] The intervals between peaks m / z 365, m / z 527, m / z 689, m / z 851, and m / z 1013 were 162 Da, which was thought to be due to the elimination of glucose, a monosaccharide constituting β-CD. Furthermore, the intervals between peaks m / z 407 and m / z 527, m / z 568 and m / z 689, m / z 731 and m / z 851, and m / z 1013 and m / z 1133 were 120 Da. This is due to the elimination of glucose at the reducing or non-reducing end of β-CD, as disclosed in Non-Patent Literature 2 (J. Mass Spectrom. Soc. Jpn, Vol. 50, No. 4, pp. 204-207 (2002)). 2,4 A or 0,2 This was thought to originate from a ring cleavage of X (see Figure 6).

[0040] Figure 7 shows a negative mode MS using CHCA as the matrix. 3 This is a mass spectrum obtained through analysis. As shown in Figure 7, MS 3 In the mass spectrum analysis, multiple fragment ion peaks originating from the precursor ion (m / z 1013) were detected at m / z 365, m / z 527, m / z 689, and m / z 851, respectively. The intervals between these peaks (m / z 365, m / z 527, m / z 689, m / z 851, and m / z 1013) were all 162 Da.

[0041] [Experimental Example 2] [1] Preparation of sample solution A 1 mg / mL aqueous solution of β-CD (manufactured by Sigma-Aldrich) was prepared as the sample solution.

[0042] [2] Preparation of matrix solution A 10 mg / mL 50% acetonitrile (ACN) aqueous solution of CHCA (LaserBio) was prepared as the matrix solution. [3] Preparation of sample for analysis 0.5 μL of the sample solution prepared in [1] was dropped onto the sample plate, then 0.5 μL of the matrix solution prepared in [2] was dropped onto the plate and allowed to air dry (on-target mix method).

[0043] [4] Mass Spectrometry The sample plate on which the analytical sample prepared in [3] is placed is then subjected to a conventional MALDImini, which is a MALDI-DITMS equipped with a positive mode. TM Insert into -1 (manufactured by Shimadzu Corporation) and perform MS analysis. 2 Analysis and MS 3 An analysis was conducted.

[0044] [5] Results Figures 8, 9, and 10 show MS analysis of β-CD in positive mode using CHCA as the matrix. 2 Analysis and MS 3 The analysis was performed and the resulting mass spectrum is shown. As shown in Figure 8, in positive mode MS analysis, the [M+Na] of β-CD was observed. + A peak (m / z 1158) of [M+Na] (where M is the sample molecule and Na is the sodium atom) was detected. As shown in Figure 9, [M+Na] + MS with (m / z 1158) as the precursor ion 2 Analysis revealed multiple fragment ion peaks originating from the precursor ion (m / z 1158) at m / z 347, m / z 509, m / z 671, m / z 833, and m / z 995, respectively. These peaks were detected at intervals of 162 Da from the precursor ion's m / z 1158.

[0045] As shown in Figure 10, an MS with an ion of m / z 833 as the precursor ion was used. 3 Analysis revealed multiple fragment ion peaks originating from the precursor ion (m / z 833) at m / z 347 (346.47), m / z 508, and m / z 671, respectively. These peaks were detected at intervals of 162 Da from the precursor ion's m / z.

[0046] Based on the results of Experimental Example 1 and Experimental Example 2, by using CHCA as the matrix, MS analysis in both positive and negative modes can be performed. 2 Analysis and MS 3 In the analysis, it was found that peaks originating from β-CD could be detected. Furthermore, when CHCA was used as the matrix, MS analysis, MS 2 Analysis and MS 3 The mass spectra obtained by either analysis showed simple peak patterns, making mass spectral analysis easy. On the other hand, DHB and norHarman have been used as matrices for MALDI mass spectrometry of cyclic oligosaccharides, but mass spectrometry using DHB and norHarman as matrices yields molecular weight information for β-CD ([MH] - Rather than the peak of the matrix-related ion adduct ([M+mH]), - The peak of ) showed a higher detection intensity.

[0047] On the other hand, in mass spectrometry using CHCA as a matrix, [MH] - This was detected as the main peak. CHCA is a matrix commonly used in peptide mass spectrometry, and it has lower affinity for sugars compared to DHB and norHarman. From the results of Experiments 1 and 2, it was confirmed that using CHCA as a matrix results in low detection sensitivity and variability of ions derived from β-CD, making it difficult to reproduce the measurement results.

[0048] Next, specific examples of the present invention will be described. Examples 1 to 4 are examples of samples containing cyclic oligosaccharides, and Examples 5 and 6 are examples of samples containing cyclic peptides.

[0049] [Example 1] [1] Preparation of sample solution β-CD (manufactured by Sigma-Aldrich) was dissolved in water, (R)-(+)-1-Phenylethyl Alcohol ((R)-(+)-PEA), and (S)-(-)-1-Phenylethyl Alcohol ((S)-(-)-PEA), respectively, to prepare a 1 mg / mL solution of β-CD.

[0050] [2] Preparation of the matrix solution A 10 mg / mL 50% acetonitrile (ACN) aqueous solution of CHCA (LaserBio) was prepared as the matrix solution.

[0051] [3] Preparation of the sample for analysis The sample solution prepared in [1] and the matrix solution prepared in [2] were mixed in a mixing ratio of 1:1 (v / v) to prepare a sample / matrix mixed solution. 0.5 μL of the sample / matrix mixed solution was dropped onto a sample plate, and the sample plate was dried by placing it in a desiccator and applying a vacuum (pre-mix method). As a result, a sample / matrix mixed crystal was formed on the sample plate.

[0052] [4] Mass Spectrometry The sample plate on which the sample / matrix mixed crystal obtained in [3] is placed is subjected to a conventional MALDImini equipped with positive mode. TM -1 (manufactured by Shimadzu Corporation) or an improved MALDImini equipped with negative mode. TM Insert into -1 (manufactured by Shimadzu Corporation) and perform MS analysis. 2 Analysis and MS 3 An analysis was conducted.

[0053] [5] Results Figure 11 is a photograph of a sample plate on which the sample / matrix mixed crystals prepared in this example were placed. Figures 11(a), (b), and (c) show photographs of sample plates on which analytical samples (sample / matrix mixed crystals) prepared with β-CD sample solutions and matrix solutions containing CHCA, respectively, using water, (R)-(+)-PEA, and (S)-(-)-PEA as solvents. As shown in Figure 11, in the analytical sample ((a)) prepared with a sample solution using water as the solvent, sample / matrix mixed crystals were formed that spread uniformly over a portion of the wells on the sample plate. On the other hand, in the analytical samples ((b), (c)) prepared with sample solutions using phenylethyl alcohol ((R)-(+)-PEA and (S)-(-)-PEA) as the solvent, the sample / matrix mixed crystals were formed more thinly and uniformly, covering the entire well.

[0054] Figure 12 shows the mass spectra obtained by MS analysis in positive and negative modes for analytical samples prepared with various sample solutions and a matrix solution containing CHCA. All mass spectra shown in Figure 12 were obtained using the same laser power. The values ​​indicated on the mass spectra represent [M+Na]. + [MH] - This is the peak intensity (mV). As shown in Figure 12, in all of the mass spectra (a) to (f), the detection of adduct peaks such as the matrix is ​​suppressed, and [M+Na] + or [MH] - The peak was detected as the main peak. Furthermore, in both positive and negative modes, the detection sensitivity (peak intensity) was higher when the sample solution was prepared using phenylethyl alcohol ((R)-(+)-PEA, (S)-(-)-PEA) ((c) to (f)) than when it was prepared using water ((a) and (b)). In particular, the improvement in detection sensitivity was significant when the sample solution was prepared using (S)-(-)-PEA ((e) and (f)), with a 100-fold improvement in detection sensitivity in positive mode and a 10-fold improvement in negative mode compared to water.

[0055] Figure 13 shows the analytical samples prepared in Example 1 using a β-CD sample solution with (S)-(-)-PEA as the solvent and a matrix solution containing CHCA, analyzed by MS in both positive and negative modes. 2 Analysis and MS 3 This is the mass spectrum obtained when the analysis was performed. In MS analysis, [M+Na] + or [MH] - With the improved detection sensitivity of MS, 2 Analysis and MS 3 In the analysis as well, [M+Na] + or [MH] - The detection sensitivity of fragment ions generated from this has been improved.

[0056] [Example 2] [1] Preparation of sample solutions α-CD, β-CD, and γ-CD (all manufactured by Sigma-Aldrich) were dissolved in (S)-(-)-PEA to prepare 1 mg / mL solutions of α-CD, β-CD, and γ-CD, respectively.

[0057] [2] Preparation of the matrix solution A 10 mg / mL 50% acetonitrile (ACN) aqueous solution of CHCA (LaserBio) was prepared as the matrix solution.

[0058] [3] Preparation of analytical samples Similar to Example 1, a sample / matrix mixed crystal was formed on a sample plate by the pre-mix method.

[0059] [4] Mass Spectrometry The sample plate on which the sample / matrix mixed crystal obtained in [3] is placed is subjected to a conventional MALDImini equipped with positive mode. TM -1 (manufactured by Shimadzu Corporation) or an improved MALDImini equipped with negative mode. TM MS analysis was performed after inserting it into -1 (manufactured by Shimadzu Corporation).

[0060] [5] Results Figure 14 shows the mass spectra obtained by MS analysis in positive and negative modes of analytical samples prepared with sample solutions of α-CD, β-CD, and γ-CD using (S)-(-)-PEA as a solvent, and a sample solution containing CHCA. As shown in Figure 14, when any of the cyclic oligosaccharides α-CD, β-CD, and γ-CD were measured, [M+Na] + or [MH] - However, it was detected with high sensitivity, while the detection of the adduct ion peak was suppressed.

[0061] [Example 3] [1] Preparation of sample solution β-CD (manufactured by Sigma-Aldrich) was dissolved in (S)-(-)-PEA to prepare a 1 mg / mL solution of β-CD.

[0062] [2] Preparation of Matrix Solutions As matrix solutions, 10 mg / mL 50% ACN aqueous solutions of CHCA (LaserBio), DHB (LaserBio), and norHarman (Sigma-Aldrich) were prepared, respectively.

[0063] [3] Preparation of Analytical Sample Similar to Example 1, sample / matrix mixed crystals were formed on the sample plate by the pre-mix method.

[0064] [4] Mass Spectrometry The sample plate on which the sample / matrix mixed crystals obtained in [3] were placed was inserted into a conventional MALDImini TM -1 (manufactured by Shimadzu Corporation) and a modified MALDImini TM -1 (manufactured by Shimadzu Corporation) equipped with a negative mode, respectively, and MS analysis was performed.

[0065] [5] Results Fig. 15 shows mass spectra when analytical samples prepared with a sample solution of β-CD using (S)-(-)-PEA as a solvent and matrix solutions containing CHCA, DHB, and norHarman were subjected to MS analysis in positive and negative modes. In the positive mode, the peak of [M+Na] + was detected as the main peak in all cases using the matrices of CHCA, DHB, and norHarman. On the other hand, in the negative mode, the peak of [M-H] - was detected as the main peak when using CHCA, but when using DHB and norHarman, adduct ion peaks of the matrix etc. were detected with high sensitivity.

[0066] From the results of Examples 1 to 3, by preparing an analytical sample using a sample solution in which a cyclic oligosaccharide is dissolved in phenylethyl alcohol and a matrix solution containing CHCA, it was found that in each of the positive and negative modes of MS analysis, MS 2 analysis and MS 3 analysis, peaks derived from cyclic oligosaccharides can be detected with high sensitivity and good reproducibility in a state where the detection of adduct ion peaks is suppressed. In particular, it was found that using (S)-(-)-PEA as phenylethyl alcohol can detect peaks derived from cyclic oligosaccharides with higher sensitivity than using (R)-(+)-PEA.

[0067] Because sugars containing cyclic oligosaccharides have high solubility in water, water is usually used as the solvent for sample solutions when mass spectrometry is performed on sugars. However, the results from Examples 1 to 3 show that using phenylethyl alcohol as the solvent for the sample solution, rather than water, yields a more uniform analytical sample suitable for MALDI mass spectrometry and improves the detection sensitivity of peaks derived from cyclic oligosaccharides. This finding was obtained through diligent research by the inventors of the present invention.

[0068] [Example 4] [1] Preparation of sample solution β-CD (manufactured by Sigma-Aldrich) was dissolved in water, (R)-(+)-PEA, and (S)-(-)-PEA, respectively, to prepare a 1 mg / mL solution of β-CD.

[0069] [2] Preparation of the matrix solution A 10 mg / mL 50% acetonitrile (ACN) aqueous solution of CHCA (LaserBio) was prepared as the matrix solution.

[0070] [3] Preparation of analytical samples Similar to Example 1, a sample / matrix mixed crystal was formed on a sample plate by the pre-mix method.

[0071] [4] Mass Spectrometry The sample plate containing the sample / matrix mixed crystal obtained in [3] was inserted into a MALDI-TOFMS, the MALDI-8030 (manufactured by Shimadzu Corporation), and MS analysis was performed in positive and negative modes.

[0072] Figure 16 shows the mass spectra obtained by MS analysis in positive and negative modes of analytical samples prepared in Example 4 using various β-CD sample solutions with water, (R)-(+)-PEA, and (S)-(-)-PEA as solvents, respectively, and a matrix solution containing CHCA. The upper part of Figure 16 shows the mass spectra in positive mode, and the lower part shows the mass spectra in negative mode. The mass spectra on the left, center, and right of Figure 16 are the mass spectra when the solvent of the sample solution was water, (R)-(+)-PEA, and (S)-(-)-PEA, respectively. All mass spectra shown in Figure 16 were obtained by MS analysis performed with the same laser power.

[0073] The peak intensity values (mV) of [M+Na] + , [M-H] - are shown. As can be seen from these numerical values, by using a sample solution containing phenylethyl alcohol ((R)-(+)-PEA, (S)-(-)-PEA), the detection sensitivity of the peak of [M+Na] + or [M-H] - was improved. Also, in the mass spectra ((a), (c) to (f)) in which the peak of [M+Na] + or [M-H] - was detected, the detection of adduct ions was suppressed, and the peak of [M+Na] + or [M-H] - was detected as the main peak. In particular, when (S)-(-)-PEA was used ((e), (f)), the effect of improving the detection sensitivity was large.

[0074] From the above, even when using not only MALDI-ITMS but also MALDI-TOFMS as MALDI mass spectrometry, by the method for preparing an analytical sample according to the present invention, in MS analysis in each of the positive mode and the negative mode, the detection of adduct ion peaks was suppressed, and it was found that the peak derived from cyclic oligosaccharide could be detected with high sensitivity and good reproducibility.

[0075] [Example 5] [1] Preparation of sample solution Cyclosporin A (MW = 1202.6, MedChemExpress), which is a cyclic peptide, was dissolved in 100% acetonitrile (ACN) to prepare a 10 mM solution of cyclosporin A. Then, the 10 mM solution of cyclosporin A was serially diluted with 100% ACN, 50% ACN aqueous solution, (S)-(-)-PEA, and (R)-(+)-PEA, respectively, to prepare a 0.01 mM solution of cyclosporin A.

[0076] [2] Preparation of matrix solution As matrix solutions, 5 mg / mL 50% ACN 0.1% TFA aqueous solutions of CHCA (LaserBio) and DHB (LaserBio) were prepared, respectively.

[0077] [3] Preparation of the sample for analysis The sample solution prepared in [1] and the matrix solution prepared in [2] were mixed in a mixing ratio of 1:1 (v / v) to prepare a sample / matrix mixed solution, and 0.5 μL of the sample / matrix mixed solution was dropped onto a sample plate (pre-mix method). The sample plate was dried by vacuuming it with a vacuum pump.

[0078] [4] Mass Spectrometry The sample plate on which the analytical sample (sample / matrix mixed crystal) prepared in [3] was placed was inserted into a MALDImini-1 (Shimadzu Corporation), which is a MALDI-DIT-MS, and MS analysis was performed.

[0079] [5] Results Figure 17 shows the mass spectra obtained by MS analysis of sample solutions prepared with 100% ACN, 50% ACN aqueous solution, and (S)-(-)-PEA as solvents, respectively, and analytical samples prepared with a matrix solution containing CHCA and a matrix solution containing DHB, respectively. The upper and lower panels of Figure 17 show the mass spectra obtained using the matrix solution containing CHCA and the matrix solution containing DHB, respectively. The mass spectra on the left, center, and right of Figure 17 are the mass spectra of the sample solutions prepared with 100% ACN, 50% ACN aqueous solution, and (S)-(-)-PEA as solvents, respectively.

[0080] As can be seen from Figure 17, when 100% ACN or a 50% ACN aqueous solution is used as the solvent for the sample solution, [M+H] + [M+Na] + In addition, multiple adduct peaks and elimination peaks were detected, whereas when (S)-(-)-PEA was used, [M+Na] + It was confirmed that only a simple peak was detected. Furthermore, peak detection was particularly sensitive when using a matrix solution containing CHCA.

[0081] Figure 18 shows the mass spectra obtained by MS analysis of sample solutions prepared with 100% ACN, (S)-(-)-PEA, and (R)-(+)-PEA as solvents, respectively, and analytical samples prepared with matrix solutions containing CHCA and DHB, respectively. Figure 18 shows the mass spectra obtained by MS analysis of the analytical solutions on a different day than Figure 17. As can be seen from Figure 18, when (S)-(-)-PEA and (R)-(+)-PEA are used as solvents in the sample solutions, [M+Na] + Only a simple peak was detected. In particular, it was confirmed that the peak could be detected with higher sensitivity when (S)-(-)-PEA was used as the solvent for the sample solution and a matrix solution containing CHCA was used.

[0082] Figure 19 shows a photograph of a sample plate on which analytical samples (sample / matrix mixed crystals) prepared with sample solutions using 100% ACN, (S)-(-)-PEA, and (R)-(+)-PEA as solvents, respectively, and matrix solutions containing CHCA and DHB, respectively. As shown in Figure 19, very uniform sample / matrix mixed crystals were formed throughout the wells of the sample plate from each analytical sample prepared with the sample solution using (S)-(-)-PEA as a solvent, and the sample solution using (R)-(+)-PEA as a solvent, along with the matrix solution containing CHCA. Furthermore, when these sample / matrix mixed crystals were actually subjected to MS analysis, very uniform mass spectra were obtained.

[0083] From the above, it was confirmed that when phenylethyl alcohol (PEA) is used as the sample solvent for cyclosporine A, and especially when (S)-(-)-PEA is used and CHCA is used as the matrix, the peaks of molecular weight-related ions can be detected with high sensitivity, preferentially, and easily.

[0084] [Example 6] [1] Preparation of sample solution A 10 mM solution of daptomycin (MW 1620.67; MedChemExpress), a cyclic peptide, was prepared by dissolving it in water (100% water). Then, the 10 mM solution of daptomycin was serially diluted with water, (S)-(-)-PEA, and (R)-(+)-PEA to prepare 0.01 mM sample solutions.

[0085] [2] Preparation of Matrix Solutions As matrix solutions, 5 mg / mL aqueous solutions of DHB (LaserBio) and CHCA (LaserBio) in 50% ACN and 0.1% TFA were prepared, respectively.

[0086] [3] Preparation of the sample for analysis The sample solution prepared in [1] and the matrix solution prepared in [2] were mixed in a mixing ratio of 1:1 (v / v) to prepare a sample / matrix mixed solution, and 0.5 μL of this sample / matrix mixed solution was dropped onto a sample plate (Pre-mix method). The sample plate was dried by vacuuming it with a vacuum pump.

[0087] [4] Mass Spectrometry The sample plate on which the analytical sample (sample / matrix mixed crystal) obtained in [3] is placed is subjected to MALDImini, a MALDI-DIT-MS. TM -1 (manufactured by Shimadzu Corporation) was inserted, and MS analysis was performed.

[0088] [5] Results Figures 2(a) and (b) show the chemical structures of cyclosporine A and daptomycin. As can be seen from these chemical structures, cyclosporine A is a cyclic peptide consisting of 11 amino acids, while daptomycin is a cyclic lipopeptide with a cyclic portion consisting of 10 amino acids, a side chain consisting of 3 amino acids, and a decanoyl group at its end. Compared to cyclosporine A, daptomycin has fewer amino acids constituting the ring, and the ring is slightly smaller.

[0089] Figure 20 shows the mass spectra obtained by MS analysis of daptomycin sample solutions using water, (S)-(-)-PEA, and (R)-(+)-PEA as solvents, respectively, and analytical samples prepared with a matrix solution containing CHCA and a matrix solution containing DHB.

[0090] As can be seen from Figure 20, when CHCA is used as the matrix, the [M+H] ratio is the same whether water or PEA is used as the solvent for the sample solution. + and [M+Na] + [M+H] was detected, but when PEA was used as the solvent, [M+H] + and [M+Na] + , or [M+H] + A tendency for more preferential ionization was observed. The sensitivity was similar when water was used as the solvent and when PEA was used. However, when PEA was used as the solvent, molecular weight-related ions [M+H] were more abundant. + and [M+Na] + , or [M+H] + It was thought that the preferential ionization and detection of daptomycin would facilitate its analysis.

[0091] Furthermore, when DHB is used as the matrix, and when water is used as the solvent for the sample solution, molecular weight-related ions [M+H] + and [M+Na] + Although not confirmed otherwise, the molecular weight-related ions were detected when PEA was used as the solvent for the sample solution. In particular, the molecular weight-related ions were detected with high sensitivity when (S)-(-)-PEA was used as the solvent.

[0092] Based on the above, the effect of the solvent PEA was confirmed not only with cyclosporine A in Example 5, but also with daptomycin, a cyclic peptide. In that case, using CHCA as the matrix and (S)-(-)-PEA as the solvent for the sample solution allowed for more effective detection of molecular weight-related ions.

[0093] [Reference Example] The following is a reference example of MALDI mass spectrometry analysis performed on a sample containing the acyclic peptides [Glu1]-fibrinopeptide B ([Glu-Fib]) and cytochrome C (Cyt C).

[0094] [1] Preparation of sample solutions As sample solutions, Glu-Fib (Sigma-Aldrich) was dissolved in a 50% ACN 0.1% TFA aqueous solution or (S)-(-)-PEA to prepare sample solutions of 20 fmol / μL and 200 fmol / μL, respectively. Similarly, Cyt C (Sigma-Aldrich) was dissolved in a 50% ACN 0.1% TFA aqueous solution or (S)-(-)-PEA to prepare a sample solution of 5 pmol / μL.

[0095] [2] Preparation of Matrix Solution As the matrix solution, a 5 mg / mL aqueous solution of CHCA (LaserBio) or SA (LaserBio) 50% ACN 0.1% TFA was prepared.

[0096] [3] Preparation of analytical samples The sample solution from [1] and the matrix solution from [2] were mixed in a mixing ratio of 1:1 (v / v), and 0.5 μL of the resulting sample / matrix mixture was dropped onto a sample plate (pre-mix method). The sample plate was dried by vacuuming with a vacuum pump. CHCA was used as the matrix for Glu-Fib, and SA was used as the matrix for Cyt C.

[0097] [4] Mass Spectrometry The sample plate containing the analytical sample (sample / matrix mixed crystal) obtained in [3] was inserted into the MALDImini-1 (Shimadzu Corporation), a MALDI-DIT-MS, and a prototype MALDImini-1 equipped with a negative mode, and MS analysis was performed.

[0098] [5] Results Figure 21 shows the mass spectra obtained by MS analysis of Glu-Fib sample solutions prepared with (S)-(-)-PEA and 50% ACN 0.1% TFA aqueous solutions as solvents, and analytical samples prepared with a matrix solution containing CHCA. 50% ACN 0.1% TFA aqueous solution is commonly used as a solvent for Glu-Fib sample solutions. When a Glu-Fib sample solution is prepared using 50% ACN 0.1% TFA aqueous solution as a solvent, the [M+H] of Glu-Fib + Or [MH]- While (S)-(-)-PEA was detected, when (S)-(-)-PEA was used as the solvent, molecular weight-related ion peaks were either not detected or detected with very low sensitivity.

[0099] Figure 22 shows the mass spectra obtained by MS analysis of Cyt C sample solutions prepared using (S)-(-)-PEA and 50% ACN 0.1% TFA aqueous solutions as solvents, and analytical samples prepared with a matrix solution containing SA. When a Cyt C sample solution is prepared using a 50% ACN 0.1% TFA aqueous solution, which is commonly used when analyzing Cyt C, the [M+H] of Cyt C is observed. + Or [MH] - While molecular weight-related ion peaks were detected, no molecular weight-related ion peaks were detected when (S)-(-)-PEA was used as the solvent.

[0100] The results from Examples 5 and 6 and the Reference Example show that solvent PEA is effective in detecting cyclic peptides with high sensitivity and preference, but it is ineffective in detecting acyclic peptides. In fact, it was confirmed that ionization of acyclic peptides themselves becomes difficult. This property of solvent PEA is considered a very unique characteristic, suggesting the possibility of preferentially ionizing only cyclic peptides or cyclic peptide pharmaceuticals from samples containing acyclic peptides, which are said to be common in living organisms and in nature.

[0101] [Embodiments] It will be apparent to those skilled in the art that the exemplary embodiments described above are specific examples of the following embodiments.

[0102] (Section 1) A method for preparing an analytical sample according to one aspect of the present invention comprises the steps of: preparing a sample solution which is a solution containing a sample containing a cyclic oligomer and phenylethyl alcohol; preparing a matrix solution which is a solution containing a matrix; and drying the sample solution and the matrix solution on a sample plate of a MALDI mass spectrometer.

[0103] According to the method for preparing analytical samples in paragraph 1, for example, MS analysis in either positive mode or negative mode, and MS n In analysis, peaks derived from cyclic oligosaccharides (i.e., molecular weight-related ions and fragment ions of cyclic oligosaccharides) can be detected with high sensitivity and reproducibility. Furthermore, molecular weight-related ion peaks of cyclic peptides can be detected with higher sensitivity, preferentially, and reproducibly.

[0104] (Paragraph 2) The method for preparing the analytical sample in Paragraph 2 is the same as the method for preparing the analytical sample in Paragraph 1, but with the matrix being α-cyano-4-hydroxycinnamic acid (CHCA).

[0105] (Paragraph 3) The method for preparing the analytical sample relating to Paragraph 3 is the method for preparing the analytical sample relating to Paragraph 1 or Paragraph 2, wherein the phenylethyl alcohol is replaced with 1-phenylethyl alcohol.

[0106] (Paragraph 4) The method for preparing the analytical sample relating to Paragraph 4 is the same as the method for preparing the analytical sample relating to Paragraph 3, but with the phenylethyl alcohol replaced by (S)-(-)-1-phenylethyl alcohol.

[0107] (Paragraph 5) The method for preparing the analytical sample relating to Paragraph 5 is the same as the method for preparing the analytical sample relating to Paragraph 3, but with the phenylethyl alcohol replaced by (R)-(+)-1-phenylethyl alcohol.

[0108] According to the method for preparing analytical samples in paragraph 2, 3, 4, or 5, when a sample containing cyclic oligomers is analyzed with a MALDI mass spectrometer, peaks originating from cyclic oligomers can be detected with even greater sensitivity, reproducibility, and preferentiality.

[0109] (Paragraph 6) The method for preparing an analytical sample relating to Paragraph 6 is the method for preparing an analytical sample relating to any of Paragraphs 1 to 5, wherein the MALDI mass spectrometer is MALDI-ITMS or MALDI-TOFMS.

[0110] (Paragraph 7) The method for preparing an analytical sample relating to Paragraph 7 is the method for preparing an analytical sample relating to any of Paragraphs 1 to 6, wherein the MALDI mass spectrometer is a MALDI-DITMS.

[0111] According to the method for preparing analytical samples in paragraph 6 or 7, peaks derived from cyclic oligomers can be detected with greater sensitivity and reproducibility.

[0112] (Paragraph 8) The method for preparing an analytical sample relating to Paragraph 8 is the method for preparing an analytical sample relating to any of Paragraphs 1 to 7, wherein the cyclic oligomer is a cyclic oligosaccharide.

[0113] (Paragraph 9) The method for preparing an analytical sample relating to Paragraph 9 is the method for preparing an analytical sample relating to Paragraph 8, wherein the cyclic oligosaccharide is cyclodextrin.

[0114] (Paragraph 10) The method for preparing an analytical sample according to Paragraph 10 is the method for preparing an analytical sample according to any of Paragraphs 1 to 7, wherein the cyclic oligomer is a cyclic peptide.

[0115] (Paragraph 11) The method for preparing an analytical sample according to paragraph 11 is the method for preparing an analytical sample according to paragraph 10, wherein the cyclic peptide is cyclosporine.

[0116] (Section 12) A method for analyzing cyclic oligomers according to another aspect of the present invention comprises the steps of: preparing an analytical sample by a method for preparing an analytical sample according to any of Sections 1 to 11; and subjecting the prepared analytical sample to MALDI mass spectrometry.

[0117] By using the analytical method for cyclic oligomers described in paragraph 12, cyclic oligomers in a sample can be easily, sensitively, and reproducibly detected.

[0118] (Section 13) A kit for evaluating the performance of a MALDI mass spectrometer according to yet another aspect of the present invention comprises a cyclic oligomer, α-cyano-4-hydroxycinnamic acid (CHCA), and phenylethyl alcohol.

[0119] By using the performance evaluation kit for MALDI mass spectrometers described in paragraph 13, the performance evaluation of MALDI mass spectrometers can be easily performed.

[0120] 10...Sample solution 20...Matrix solution 30...Sample / matrix / phenylethyl alcohol (PEA) mixed solution 40...Sample plate

Claims

1. A method for preparing an analytical sample, comprising the steps of: preparing a sample solution which is a solution containing a cyclic oligomer and phenylethyl alcohol; preparing a matrix solution which is a solution containing a matrix; and drying the sample solution and the matrix solution on a sample plate of a MALDI mass spectrometer.

2. The method for preparing an analytical sample according to claim 1, wherein the matrix is ​​α-cyano-4-hydroxycinnamic acid (CHCA).

3. The method for preparing an analytical sample according to claim 1 or 2, wherein the phenylethyl alcohol is 1-phenylethyl alcohol.

4. The method for preparing an analytical sample according to claim 3, wherein the phenylethyl alcohol is (S)-(-)-1-phenylethyl alcohol.

5. The method for preparing an analytical sample according to claim 3, wherein the phenylethyl alcohol is (R)-(+)-1-phenylethyl alcohol.

6. The method for preparing an analytical sample according to claim 1 or 2, wherein the MALDI mass spectrometer is MALDI-ITMS or MALDI-TOFMS.

7. The method for preparing an analytical sample according to claim 1 or 2, wherein the MALDI mass spectrometer is a MALDI-DITMS.

8. The method for preparing an analytical sample according to claim 1, wherein the cyclic oligomer is a cyclic oligosaccharide.

9. The method for preparing an analytical sample according to claim 8, wherein the cyclic oligosaccharide is a cyclodextrin.

10. The method for preparing an analytical sample according to claim 1, wherein the cyclic oligomer is a cyclic peptide.

11. The method for preparing an analytical sample according to claim 10, wherein the cyclic peptide is cyclosporine.

12. A method for analyzing a cyclic oligomer, comprising the steps of: preparing an analytical sample by the method for preparing an analytical sample described in claim 1 or 2; and subjecting the prepared analytical sample to MALDI mass spectrometry.

13. A kit for evaluating the performance of a MALDI mass spectrometer, containing a cyclic oligomer, α-cyano-4-hydroxycinnamic acid (CHCA), and phenylethyl alcohol.

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