Pretreatment method, analysis method, program, and analysis system

Abstract

This pretreatment method of a biological cell-containing sample for infrared spectroscopic analysis comprises: a step for preparing a first acidic solution containing an organic acid; and a step for bringing cells into contact with the first acidic solution.

Description

Preprocessing method, analysis method, program and analysis system

[0001] The present invention relates to a pretreatment method, an analysis method, a program, and an analysis system, and more particularly to a pretreatment method, an analysis method, a program, and an analysis system for a sample containing cells.

[0002] Infrared spectroscopy plays an important role in the chemical industry in the analysis of materials and the quality control of finished products. In recent years, research has also been conducted into the application of infrared spectroscopy to the field of biology. Non-Patent Document 1 discloses a study in which cultured Staphylococcus aureus is suspended in an aqueous ethanol solution contained in a tube together with a sterilized metal rod, and the cells are disrupted by vortex mixing, followed by identification of the strain by infrared spectroscopy.

[0003] Performance Evaluation of the IR Biotyper(R) System for Clinical Microbiology: Application for Detection of Staphylococcus aureus Sequence Type 8 Strains, Jung Sung Hong et al., Antibiotics, 7 July 2022, 11(7), 909.

[0004] As described above, conventional cell disruption methods for infrared spectroscopic analysis require adding sterilized metal rods (also known as beads) to a tube containing cells and then vortexing the tube, which is time-consuming and labor-intensive.

[0005] The present disclosure has been made to solve such problems, and its purpose is to provide a method for pretreating a sample containing biological cells for infrared spectroscopic analysis, which enables cell disruption in a simple manner.

[0006] A pretreatment method according to a first aspect of the present disclosure is a method for pretreatment of a sample containing biological cells for infrared spectroscopic analysis, comprising the steps of preparing a first acidic solution containing an organic acid and contacting the cells with the first acidic solution.

[0007] An analytical system according to another aspect of the present disclosure includes an infrared spectrophotometer and a mass spectrometer. The infrared spectrophotometer performs infrared spectroscopic analysis of an analytical sample obtained by pretreating a sample containing biological cells by contacting the cells with a first acidic solution containing an organic acid. The mass spectrometer performs mass analysis of the analytical sample.

[0008] According to the present invention, it is possible to provide a pretreatment method for a sample containing biological cells for infrared spectroscopic analysis, which allows for cell disruption in a simple manner.

[0009] FIG. 1 is a schematic diagram showing the configuration of an analysis system according to an embodiment. FIG. 2 is a diagram showing an example of an infrared spectrum of Experiment 1. FIG. 3 is a diagram showing the results of microorganism discrimination based on the infrared spectrum of Experiment 1. FIG. 4 is a diagram showing an example of an infrared spectrum of Experiment 2. FIG. 5 is a diagram showing the results of microorganism discrimination based on the infrared spectrum of Experiment 2. FIG. 6 is a diagram showing an example of an infrared spectrum of Experiment 3. FIG. 7 is a diagram showing the results of microorganism discrimination based on the infrared spectrum of Experiment 3. FIG. 8 is a diagram showing an example of an infrared spectrum of Experiment 4. FIG. 9 is a diagram showing the results of microorganism discrimination based on the infrared spectrum of Experiment 4. FIG. 10 is a diagram showing the mass spectrum of Experiment 5.

[0010] An embodiment of the present disclosure (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0011] In this specification, "%" of a solution means "volume %" unless otherwise specified. Hereinafter, this embodiment will be described in detail with reference to the drawings. In the following, the same or corresponding parts in the drawings will be denoted by the same reference numerals, and their description will not be repeated in principle.

[0012] [1. Configuration of the Analysis System] First, an example of an analysis system 100 that performs the analysis method according to this embodiment is shown. The analysis method according to this embodiment includes the pretreatment method according to this embodiment. In this specification, unless otherwise specified, "pretreatment" refers to the preparation of a sample containing biological cells for infrared spectroscopic analysis.

[0013] 1, an analysis system 100 includes an analysis unit 10 and a control device 4. The analysis unit 10 includes a pre-processing unit 1, an infrared spectrophotometer 2, and a mass spectrometer 3.

[0014] The pretreatment unit 1 is a unit that pretreats samples containing biological cells. In one embodiment, the pretreatment unit 1 is implemented by an analyst using laboratory equipment. In another embodiment, the pretreatment unit 1 is an automatic pretreatment unit (not shown) controlled by the control unit 4. The automatic pretreatment unit may include an autosampler (not shown). Hereinafter, a sample containing biological cells cultured in an incubator or the like (not shown) and prior to pretreatment by the pretreatment unit 1 will be referred to as a "culture sample." In this specification, the culture sample corresponds to an example of a "sample." Furthermore, a sample that is subjected to analysis after pretreatment by the pretreatment unit 1 will be referred to as an "analysis sample." At least a portion of the analysis sample prepared by the pretreatment unit 1 is subjected to infrared spectroscopy in the infrared spectrophotometer 2. A portion of the analysis sample prepared by the pretreatment unit 1 may be subjected to mass spectrometry in the mass spectrometer 3.

[0015] In one embodiment, the sample containing biological cells includes microbial cells. This allows for microbial analysis, which is useful in fields such as industry, manufacturing, and medicine. In another embodiment, the sample containing biological cells may include cells of organisms other than microorganisms (e.g., animals and plants). This may allow for analytical results that are useful in fields such as industry, manufacturing, and medicine, depending on the type of cells.

[0016] In one embodiment, the sample containing biological cells is a culture sample of cultured biological cells. In another embodiment, the sample containing biological cells comprises biological cells collected from the environment. In yet another embodiment, the sample containing biological cells is a portion of tissue collected from a living animal or plant.

[0017] Unless otherwise specified, in this specification, the type of microorganism includes at least one systematic taxonomic classification of the genotype, strain, subspecies, species, genus, and family of the microorganism. Furthermore, the determination of the type of microorganism includes classification, identification, and differentiation of the type of the microorganism. Hereinafter, the determination of the type of microorganism will also be simply referred to as the determination of the microorganism.

[0018] The infrared spectrophotometer 2 performs infrared spectroscopic analysis of the analytical sample. Specifically, the infrared spectrophotometer 2 irradiates the analytical sample with infrared light and separates the transmitted or reflected light to obtain an infrared spectrum. In one embodiment, the infrared spectrophotometer 2 is a Fourier transform infrared spectrophotometer (FT-IR). The infrared spectrum exhibits a shape corresponding to the substances contained in the analytical sample. Microbial cells contain different types of substances or different amounts of the same type of substance depending on the type of microorganism. Hereinafter, the combination of the types and / or amounts of substances contained in such cells is referred to as the "composition." As the cellular composition differs for each type of microorganism, different types of microorganisms exhibit infrared spectra with different shapes. The infrared spectrophotometer 2 transmits the acquired infrared spectrum to the control device 4.

[0019] The mass spectrometer 3 performs mass analysis of the analytical sample. Specifically, the mass spectrometer 3 ionizes substances contained in the analytical sample, separates the ions S according to their time of flight, which correlates with their mass-to-charge ratio (m / z), and then detects them to obtain a mass spectrum. In one embodiment, the mass spectrometer 3 ionizes substances in the analytical sample using a matrix-assisted laser desorption / ionization (MALDI) method. The mass spectrometer 3 is, for example, a MALDI-TOF MS (Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry) instrument. In the mass spectrum, substances contained in the analytical sample are detected as peaks corresponding to their m / z values. Therefore, different types of microorganisms exhibit mass spectra with peaks at different positions. The mass spectrometer 3 transmits the acquired mass spectrum to the control device 4.

[0020] The control device 4 includes a processor 41 and a memory 42. In one embodiment, the control device 4 is a computer. The processor 41 is typically an arithmetic processing unit such as a CPU (Central Processing Unit) or an MPU (Micro Processing Unit). The processor 41 controls the operation of the analysis system 100 by reading and executing a program stored in the memory 42. For example, the processor 41 can automatically perform the analysis method described in this embodiment by executing a program stored in the memory 42 that causes the analysis method described in this embodiment to be performed.

[0021] The memory 42 is realized by a non-volatile memory such as a random access memory (RAM), a read only memory (ROM), a flash memory, etc. The memory 42 stores programs executed by the processor 41, data used by the processor 41, etc.

[0022] The display 43 is configured by, for example, a liquid crystal panel capable of displaying images. The display 43 displays images related to the reception of user operation inputs and the results of processing by the processor 41.

[0023] The control device 4 receives the infrared spectrum from the infrared spectrophotometer 2 via an input / output interface (not shown). The control device 4 determines the type of microorganism based on the shape of the infrared spectrum. The control device 4 receives the mass spectrum from the mass spectrometer 3 via an input / output interface (not shown). The control device 4 determines the type of microorganism based on the position (m / z) of the peak in the mass spectrum.

[0024] The control device 4 may be configured with multiple computers. Furthermore, some or all of the functions of the control device 4 described above may be located in an electronic computer, server, or the like that is physically separated from the analysis unit 10.

[0025] [2. Conventional Pretreatment Methods] Infrared spectroscopy has traditionally been used in the field of materials, for example, for the analysis of chemical products (e.g., plastic products). In this field, pretreatment of a sample (e.g., a chemical product) prior to infrared spectroscopy involves, for example, wiping the sample interface. However, analyzing the composition of cells using infrared spectroscopy requires treatments such as lysing the sample cells and extracting the contents of the sample cells. For example, Non-Patent Document 1 discloses a method in which cells suspended in an aqueous ethanol solution are disrupted using beads and then subjected to infrared spectroscopy.

[0026] However, conventional pretreatment methods using beads (hereinafter also referred to as "bead-breaking methods") require analysts to perform delicate and skilled procedures, such as inserting and removing beads. Furthermore, depending on the type of cells in a sample, bead-breaking may need to be repeated several times (e.g., two or three times) for a single sample, further complicating the process. Therefore, pretreatment of a single sample takes a long time (e.g., 20 to 30 minutes). Furthermore, components derived from the disrupted cells adhere to the beads and are lost during bead recovery. Therefore, a large number of cells must be prepared for analysis using the components remaining in the ethanol solution. For example, preparing a sample for infrared spectroscopy requires adding a heaping amount of cells to the loop of a platinum loop. This requires, for example, culturing the cells until a sufficient number of colonies form on solid medium and / or picking up enough colonies to fill the loop of a platinum loop. As described above, conventional bead-breaking methods are laborious and time-consuming.

[0027] As described above, the bead crushing method has the problem that it takes time and effort to obtain a sample for analysis, which is particularly noticeable when processing multiple samples.

[0028] Furthermore, since the bead crushing method uses beads, it is not easy to implement it in a general automatic analyzer that performs operations such as dispensing and adding liquids.

[0029] 3. Pretreatment Method According to the Embodiment In view of the above-mentioned problems, the inventors conducted trial and error in pretreatment of samples and discovered a method for efficiently disrupting cells by contacting the cells with an organic acid.

[0030] The pretreatment method according to this embodiment is a method for pretreatment of a sample containing biological cells for infrared spectroscopic analysis, and includes a "preparation step" of preparing a first acidic solution containing an organic acid, and a "contact step" of contacting the cells with the first acidic solution.

[0031] (3-1. Cell Types) The sample may contain cells derived from prokaryotes or eukaryotes. The sample is typically derived from microorganisms and may contain unknown microorganisms. Prokaryotes include bacteria and archaea. Bacteria include Escherichia bacteria (such as Escherichia coli), Bacillus bacteria (such as Bacillus subtilis), Lactobacillus bacteria (such as Lactic acid bacteria), Synechocystis bacteria (such as Cyanobacteria), and Mycobacteria (such as Actinomycetes). Archaea include Methanophilus, Methanococcus, Thermococcus, and Phyllococcus. Eukaryotes include animals, plants, fungi, and protists. Fungi include filamentous fungi, yeast, mushrooms, molds, and the like, and include the phyla Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota, Glomeromycota, and Microsporidia. The Ascomycota includes the genus Aspergillus (such as Aspergillus mold), the genus Penicillium (such as blue mold), the genus Saccharomyces (such as budding yeast), etc. In one embodiment, the sample is a cell (bacterial body) of a fungus in the broad sense, including the bacteria, archaea, and fungi described above.

[0032] These cells are disrupted and cytoplasmic components are eluted in two steps:

[0033] (3-2. Preparation Step) The sample to be contacted with the first acidic solution may be in liquid or solid form. More specifically, the sample may be, for example, a liquid containing a bacterial culture solution, or a bacterial colony scooped with a toothpick or the like from a bacterial culture on a solid medium. The bacterial culture solution contains bacteria and a medium for culturing the bacteria. The amount of bacteria in the sample may be smaller than that in the conventional bead-crushing method.

[0034] The acid may be, for example, an organic acid such as formic acid, trifluoroacetic acid, or lactic acid, which is commonly used in biological experiments. These organic acids are readily available, and even if those skilled in the art use them for pretreatment of infrared spectroscopy and mass spectrometry, they will not adversely affect the measurement results. Other examples of acids include acids with an acid dissociation constant (pKa) of 0.2 to 5, more preferably 0.2 to 4. Acids with a dissociation constant of 0.2 to 5 partially overlap with the examples of acids listed above, but include, for example, at least one of trifluoroacetic acid, oxalic acid, glycine, salicylic acid, formic acid, lactic acid, benzoic acid, acetic acid, and butyric acid. Of course, the examples of acids listed above are not limited to these. In one embodiment, the organic acid is formic acid. As described below, inorganic acids can also be used. The acid contained in the first acidic solution may be a single acid (pure substance) or a mixture of multiple acids.

[0035] In this specification, "acid" refers to a pure substance (e.g., 100% formic acid). A solution obtained by diluting an acid with a solvent to a predetermined concentration is referred to as a "first acidic solution." The solvent typically contains water. The water is preferably pure water, ultrapure water, or ion-exchanged water. The solvent may contain an organic solvent in addition to or instead of water, as long as the effects of the pretreatment method according to this embodiment are achieved. The organic solvent is, for example, a polar organic solvent. The organic solvent is, for example, acetonitrile, methanol, or ethanol.

[0036] The preparation step may be a step of preparing a first acidic solution by diluting an acid with a solvent to a predetermined concentration, or a step of placing an already prepared first acidic solution on a laboratory bench or injecting it into an automatic pretreatment device. As described above, the preparation step is not particularly limited in terms of its form, as long as it can prepare a first acidic solution for contacting cells with the first acidic solution in the subsequent contact step.

[0037] (3-3. Contacting Step) A specific means for the "contacting step" typically involves mixing the sample with a first acidic solution. In this specification, the mixed solution of the sample and the first acidic solution is referred to as the "second acidic solution." By mixing the sample with the first acidic solution, the cells can be dispersed in the first acidic solution. This ensures that the cells are in contact with the first acidic solution, thereby improving the efficiency of the treatment of the cells with the first acidic solution.

[0038] The mixing ratio of the first acidic solution to the sample is not limited as long as the effects of the pretreatment method according to this embodiment are achieved. For example, the sample may be mixed in a volume ratio of 1 / 2 or less to 1 / 1000 or more of the first acidic solution. However, generally, when mixing with the first acidic solution, the sample is prepared so that the amount of non-cellular components (e.g., liquid medium used for cell culture) contained in the sample is minimized. Specifically, for example, the sample before mixing with the first acidic solution is centrifuged, the supernatant is removed, the remaining precipitate (cells) is recovered, and the recovered precipitate is mixed with the first acidic solution. It is also preferable to further centrifuge and wash the precipitate with ultrapure water or the like. For these reasons, the acid content (concentration) and pH of the second acidic solution are almost unchanged from those of the first acidic solution. Hereinafter, the first acidic solution and the second acidic solution may be collectively referred to as "acidic solutions."

[0039] The concentration of the acid in the acidic solution is, for example, 50 to 90% by volume, preferably 60 to 80% by volume, and more preferably 65 to 75% by volume.

[0040] In one embodiment, the first acidic solution is 50-90% formic acid, preferably 60-80% formic acid, and more preferably 65-75% formic acid. In this embodiment, the second acidic solution is formic acid at the above concentrations containing cells.

[0041] In one embodiment, the first acidic solution is 50-90% trifluoroacetic acid, preferably 60-80% trifluoroacetic acid, and more preferably 65-75% trifluoroacetic acid, and in this embodiment, the second acidic solution is trifluoroacetic acid at the above-mentioned concentrations containing cells.

[0042] In one embodiment, the first acidic solution is 50-90% lactic acid, preferably 60-80% lactic acid, more preferably 65-75% lactic acid, and in this embodiment, the second acidic solution is lactic acid at the above concentrations containing cells.

[0043] By using an organic acid at this concentration, cytoplasm can be extracted particularly efficiently. Whether the acidic solution has been mixed properly can be confirmed by measuring the hydrogen ion exponent (pH) of the acidic solution using, for example, a pH meter.

[0044] Another example of a specific means for the "contact step" is a means for contacting (e.g., placing) the cells with an object (e.g., a cloth) impregnated with the first acidic solution. Thus, the specific means for the "contact step" is not limited as long as the effect of the pretreatment method according to this embodiment is achieved.

[0045] According to the pretreatment method of this embodiment, cell disruption for infrared spectroscopic analysis can be performed without using beads by contacting cells with a first acidic solution containing an organic acid, thereby enabling cell disruption for infrared spectroscopic analysis to be performed in a simple manner.

[0046] More specifically, the pretreatment method according to this embodiment allows for the pretreatment of a wide variety of samples in a short period of time using simple experimental techniques such as centrifugation and stirring with a vortex mixer (hereinafter simply referred to as "vortexing"). Furthermore, a single formic acid treatment can disrupt cell walls and cell membranes. Therefore, pretreatment can be completed in a few minutes (e.g., 5 minutes). Furthermore, since there is no loss of cell-derived components due to adhesion to the beads, the amount of bacterial cells required for analysis is smaller than with the bead-breaking method. Therefore, even when culturing bacteria in a solid medium, it is not necessary to scoop out as many bacteria as with the bead-breaking method. Furthermore, for example, it is possible to pretreat a portion of a culture sample cultured to a predetermined volume in a liquid medium by dispensing it. Therefore, culture samples can be prepared easily and quickly.

[0047] As described above, the pretreatment according to this embodiment differs from general pretreatment for infrared spectroscopic analysis in that the sample (cells) are disrupted by contacting them with an organic acid. This allows cells to be efficiently disrupted by simple operations such as mixing and dispensing liquids, without using beads. As a result, the pretreatment according to this embodiment can also be performed with a general automatic pretreatment device. Using a general automatic pretreatment device makes it even easier to analyze cells from multiple types of organisms at the same time.

[0048] (3-4. Other Aspects) The second acidic solution preferably further contains acetonitrile. The concentration of acetonitrile is, for example, 30% to 70%, preferably 40% to 60%, and more preferably about 50%, based on the second acidic solution. As described above, the inventors have discovered that including acetonitrile in the second acidic solution further improves the efficiency of cell disruption. Furthermore, by including acetonitrile in the second acidic solution, the analytical sample can be dried uniformly and in a short time after being dropped onto the sample plate. This improves analytical accuracy.

[0049] The pretreatment method according to this embodiment preferably further includes a step of centrifuging the second acidic solution and removing precipitate. In one embodiment, an analyst centrifuges the second acidic solution contained in a container such as a tube, collects the resulting supernatant in another container, and subjects the supernatant to infrared spectroscopy and / or mass spectrometry. The inventors discovered that the step of removing the precipitate as described above sharpens the peaks of the infrared spectrum, thereby improving the accuracy of organism classification based on the shape of the infrared spectrum. The reason why removing the precipitate improves the results of infrared spectroscopy as described above can be explained as follows: The second acidic solution contains not only cytoplasmic components but also cell wall components. Cell wall components are less diverse than cytoplasmic components and are therefore not suitable for distinguishing microorganisms. If the second acidic solution were subjected to infrared spectroscopy directly, not only the cytoplasmic components but also the cell wall components would be reflected in the infrared spectrum. Therefore, by precipitating the cell wall components by centrifugation and collecting only the supernatant containing the cytoplasmic components, spectral components corresponding to the cell wall components are removed, while spectral components corresponding to the cytoplasmic components remain. This results in sharper peaks in the infrared spectrum, improving the accuracy of organism classification.

[0050] The pretreatment method according to this embodiment preferably further includes a step of dispersing cells in an ethanol solution prior to the "contact step." The ethanol concentration in the ethanol solution is, for example, 70% to 90%, preferably 75% to 85%, and more preferably approximately 80%, based on the ethanol solution. Using an ethanol solution of this concentration allows efficient inactivation and washing of bacterial cells. Specifically, ethanol disrupts secretions on the cell surface, dispersing the cells and shrinking them, further enhancing cell disruption by the organic acid treatment. Therefore, the efficiency of elution of cytoplasmic components can be further improved. The pretreatment method according to this embodiment preferably includes a step of centrifuging the cells and removing the supernatant (ethanol) after the step of dispersing the cells in an ethanol solution.

[0051] The pretreatment method according to the embodiment may be performed by an analyst using ordinary laboratory equipment, or may be performed by a computer. For example, the processor 41 may execute a predetermined program stored in the memory 42 to control the pretreatment unit 1, which is an automatic pretreatment device, and perform the pretreatment method according to the embodiment. When the automatic pretreatment device performs pretreatment, the analyst's efforts are reduced compared to when the analyst performs pretreatment manually. Furthermore, it becomes easier to perform pretreatment accurately and to pretreat a large amount of samples.

[0052] [4. Analytical Method According to the Embodiment] The analytical method according to the embodiment includes a "drying step" in which a predetermined amount of the analytical sample obtained by the pretreatment method according to the embodiment is dropped onto a sample plate and dried to produce a dried product, and an "analyzing step" in which an infrared spectrum is obtained by performing infrared spectroscopic analysis of the dried product using Fourier transform infrared spectroscopy. This makes it possible to accurately analyze cytoplasmic components contained in the analytical sample.

[0053] In one embodiment of the drying step, a predetermined amount of analytical sample is dropped into each well of the sample plate. The predetermined amount is, for example, a value between 1 μL and 15 μL, preferably between 8 μL and 12 μL, and more preferably about 10 μL. By placing such an amount of analytical sample on the sample plate, infrared spectra capable of identifying organisms can be obtained.

[0054] The analytical method according to the embodiment may further include a step of classifying the organisms from which the analytical sample is derived (organisms contained in the culture sample) by cluster analysis of the infrared spectrum. This allows for a simple method of classifying the organisms from which the analytical sample is derived based on the infrared spectrum. As shown in the experimental example described below, the analytical method according to the embodiment allows for simple pretreatment while obtaining infrared spectra with sufficient accuracy to distinguish microorganisms through cluster analysis. This allows for the pretreatment and organism identification processes to be completed in a short time.

[0055] The analytical method of this embodiment may further include the steps of adding a matrix substance to the analytical sample and performing matrix-assisted laser desorption / ionization mass spectrometry on the analytical sample to which the matrix substance has been added to obtain a mass spectrum. This configuration allows the analytical sample pretreated for infrared spectroscopic analysis to also be subjected to mass spectrometry. This eliminates the need to prepare a new sample for mass spectrometry. Furthermore, the results of infrared spectroscopic analysis and mass spectrometry for the same analytical sample pretreated under the same conditions from the same culture sample can be compared.

[0056] In one embodiment, an analyst drops a portion of an analytical sample prepared in a container such as a tube onto a sample plate for infrared spectroscopic analysis and performs infrared spectroscopic analysis. The analyst then adds a matrix solution containing a matrix substance to at least a portion of the analytical sample remaining in the tube, and drops a portion of the analytical sample in the tube onto a sample plate for mass spectrometry to perform mass spectrometry. The analyst may add the matrix solution directly to at least a portion of the analytical sample remaining in the tube, or may dispense a portion of the analytical sample remaining in the tube and add the matrix solution to the dispensed analytical sample. Alternatively, the analyst may add the matrix solution to a dried material prepared on a sample plate for mass spectrometry and perform mass spectrometry. In particular, if the sample plate for infrared spectroscopic analysis can also be used for mass spectrometry, in other words, if the sample plate for the infrared spectrophotometer 2 and the mass analyzer 3 can be shared, the dried material prepared on the shared sample plate may be subjected to infrared spectroscopic analysis, followed by addition of a matrix solution and mass spectrometry. In this case, mass spectrometry can be performed simply by adding the matrix solution to the dried material after use in infrared spectroscopic analysis. Therefore, it is possible to eliminate the need to drop new analytical samples onto a sample plate for mass spectrometry.

[0057] The analytical method according to the embodiment may further include a step of classifying the organism from which the analytical sample is derived based on the mass spectrum. This allows for a simple method of classifying the organism from which the analytical sample is derived based on the mass spectrum. Furthermore, the classification results of the organisms obtained by infrared spectroscopy and those obtained by mass spectrometry can be compared.

[0058] The analytical method according to the embodiment further includes a step of displaying the results of the infrared spectroscopy analysis and the results of the mass spectroscopy analysis. In one example, the processor 41 displays the infrared spectrum, the organism classification results based on the infrared spectrum, the mass spectrum, the organism classification results based on the mass spectrum, etc. on the display 43. The organism classification results are displayed, for example, in the form of a dendrogram (phylogenetic tree). This configuration allows the analysis results of the infrared spectroscopy analysis and the analysis results of the mass spectroscopy analysis to be easily compared and examined.

[0059] The analytical method according to the above-described embodiment may be implemented by an analyst operating the infrared spectrophotometer 2, the mass spectrometer 3, and / or the control device 4. Alternatively, the analytical method may be implemented by the control device 4 controlling the entire analytical system as an automated analytical system. For example, the processor 41 may execute a predetermined program stored in the memory 42 to inject an analytical sample prepared in the pretreatment unit 1 into the infrared spectrophotometer 2 and the mass spectrometer 3, and control the infrared spectrophotometer 2 and the mass spectrometer 3 to perform the analysis. Using an automated analytical system reduces the analyst's workload compared to manually preparing an analytical sample, injecting it into the infrared spectrophotometer 2 and the mass spectrometer 3, and operating the infrared spectrophotometer 2 and the mass spectrometer 3. Furthermore, it becomes easier to perform accurate analyses and analyze large quantities of samples.

[0060] [5. Example of Pretreatment Method and Analysis Method According to This Embodiment] An example of the pretreatment method and analysis method according to this embodiment will be described below. (1) 200 μL of distilled water is added to a microtube (volume of approximately 1.5 mL) containing bacterial cells, and the bacterial cells are dispersed to an OD of 1. (2) 800 μL of ethanol is added and mixed. (3) The bacterial cells are precipitated by centrifugation, and the supernatant is removed. (4) Approximately 70% formic acid is prepared (preparation step). 25 μL of the formic acid is added to disperse the bacterial cells (contact step). (5) 25 μL of acetonitrile is added and dispersed. (6) 2 to 10 μL of the analysis sample prepared in (5) is dropped onto a sample plate and dried (drying step). (7) The dried product prepared in (6) is subjected to infrared spectroscopy using infrared spectrophotometer 2 (analysis step). (8) 1 μL of the analysis sample prepared in (5) is dropped onto a sample plate and dried. After drying, the matrix solution is further dropped and dried to prepare a mixed crystal of the sample and the matrix substance. (9) The mixed crystal prepared in (8) is subjected to mass analysis using the mass spectrometer 3. (10) The results of the infrared spectroscopic analysis and the mass analysis are displayed on the display 43, and the analysis results are confirmed.

[0061] 6. Experimental Examples Next, the pretreatment method according to this embodiment and its effects will be described in more detail using experimental examples, but the pretreatment method according to this embodiment is not limited to these experimental examples.

[0062] In the experimental examples, the following four strains of lactic acid bacteria were used: Lacticaseibacillus paracasei subsp. paracasei NBRC 15889, Lacticaseibacillus paracasei subsp. paracasei NBRC 3953, Lacticaseibacillus paracasei subsp. tolerans JCM 1171, and Lacticaseibacillus paracasei subsp. tolerans NBRC 15906T. Hereinafter, the above four strains will also be simply referred to as paracasei NBRC 15889, paracasei NBRC 3953 tolerans JCM 1171, and tolerans NBRC 15906T.

[0063] Paracasei subsp. paracasei and Paracasei subsp. tolerans are subspecies. Paracasei NBRC 15889 and Paracasei NBRC 3953 are different strains of the same subspecies. Paracasei tolerans JCM 1171 and Paracasei tolerans NBRC 15906T are the same strain but purchased from different sources. Specifically, Paracasei tolerans JCM 1171 was purchased through the RIKEN Institute, and Paracasei tolerans NBRC 15906T was purchased through the National Institute of Technology and Evaluation. Therefore, the difference between Paracasei tolerans JCM 1171 and Paracasei tolerans NBRC 15906T is due to the genetic mutations that occurred during passage between the two institutions.

[0064] In the following experiments, cells were used that had been cultured on MRS agar medium at 35° C. for 24 hours.

[0065] (6-1. Experiment 1) Pretreatment and analysis in Experiment 1 were performed as follows. (1) Bacterial cells were dispersed in 200 μL of distilled water to an OD of approximately 1. (2) 800 μL of ethanol was added to disperse the bacterial cells, and the resulting supernatant was removed by centrifugation. (3) 50 μL of 70% formic acid solution was added to re-disperse the precipitated bacterial cells. (4) 50 μL of acetonitrile was further added and mixed to prepare a formic acid / acetonitrile mixed solution. In Experiment 1, this formic acid / acetonitrile mixed solution corresponds to one example of an "analytical sample." (5) 10 μL of the formic acid / acetonitrile mixed solution for each bacterial strain was dropped into three wells of a sample plate to prepare a dried product. (6) The dried product obtained in (5) was analyzed using an infrared spectrophotometer. Steps (1) to (4) took approximately 5 minutes.

[0066] The analytical conditions for infrared spectroscopy in Experiment 1 were as follows: Number of differential points: 31 points Wavenumber range: 650 to 1300 cm －１ Normalization: Vector Normalization Similarity: Euclidean Cluster analysis: Ward method Figure 2 shows an example of an infrared spectrum obtained as a result of Experiment 1. Figure 2 shows an infrared spectrum obtained by analyzing one dried product of B. tolerans NBRC 15906T. The horizontal axis of Figure 2 represents the frequency (wavenumber) of infrared light. The vertical axis represents absorbance. Referring to Figure 2, an infrared spectrum reflecting the composition of the substances in the analyzed sample was obtained. However, in the wavenumber range indicated by box 91, the absorbance was relatively uniformly high compared to the subsequent Experiments 2 to 4, and the peaks were not clear. Therefore, it was thought that the spectrum was saturated in this wavenumber range due to the excessive amount of protein in the dried product. In other words, it was thought that too much light was absorbed in this wavenumber range, making the peaks characteristic of the microorganism difficult to see.

[0067] Figure 3 is a dendrogram showing the microbial discrimination results obtained by cluster analysis of the infrared spectra in Figure 2. The horizontal axis in Figure 3 represents the distance between each cluster. Each cluster was classified at the level of line L1. The three infrared spectra obtained for each strain are referred to as strain name-1, strain name-2, and strain name-3, respectively.

[0068] Referring to Figure 3, all three infrared spectra of C. paracasei NBRC 3953 were classified into cluster C1, all three infrared spectra of C. paracasei NBRC 15889 were classified into cluster C3, and C. tolerans JCM 1171 and C. tolerans NBRC 15906T were classified into cluster C2 (see box 92).

[0069] As described above, Paracasei subsp. paracasei and Paracasei subsp. tolerans were separated into different clusters. Furthermore, Paracasei subsp. paracasei NBRC 3953 and Paracasei NBRC 15889 were separated into different clusters. As described above, in Experiment 1, subspecies and different strains were successfully separated.

[0070] On the other hand, paracasei subsp. tolerans could not be separated from tolerans JCM 1171 and tolerans NBRC 15906T.

[0071] (6-2. Experiment 2) The pretreatment in Experiment 2 was a modification of (5) of the pretreatment in Experiment 1. Specifically, the amount of the formic acid / acetonitrile mixed aqueous solution (analytical sample) dropped onto the sample plate in (5) was reduced from 10 μL to 2 μL.

[0072] The pretreatment in Experiment 2 was performed as follows: (1) Bacterial cells were dispersed in 200 μL of distilled water to an OD of approximately 1. (2) 800 μL of ethanol was added to disperse the bacterial cells, and the resulting supernatant was removed by centrifugation. (3) 50 μL of 70% formic acid solution was added to re-disperse the precipitated bacterial cells. (4) 50 μL of acetonitrile was further added and mixed to prepare a formic acid / acetonitrile mixed aqueous solution. In Experiment 2, this formic acid / acetonitrile mixed aqueous solution corresponds to one example of the "analytical sample." (5) 2 μL of the formic acid / acetonitrile mixed aqueous solution was dropped onto a sample plate to prepare a dried product. (6) The dried product obtained in (5) was analyzed using an infrared spectrophotometer. The time required for steps (1) to (4) was approximately 5 minutes.

[0073] The analytical conditions for infrared spectroscopy in Experiment 2 were as follows: Number of differential points: 31 points Wavenumber range: 650 to 1300 cm －１ Normalization: Vector Normalization Similarity: Euclidean Cluster analysis: Ward method Figure 4 shows an example of an infrared spectrum obtained as a result of Experiment 2. Figure 4 shows an infrared spectrum obtained by analyzing one dried product of B. tolerans NBRC 15906T. The horizontal axis of Figure 4 indicates the frequency (wavenumber) of infrared light. The vertical axis indicates absorbance. Referring to Figure 4, for the wavenumber range indicated by box 93, the saturation observed in Figure 2 is resolved and a peak is detected. This is thought to reflect a decrease in the amount of protein in the dried product.

[0074] Figure 5 is a dendrogram showing the microbial discrimination results obtained as a result of cluster analysis of the infrared spectra in Figure 4. The horizontal axis in Figure 5 represents the distance between each cluster. Each cluster was classified at the level of line L2. The three infrared spectra obtained for each strain are referred to as Strain Name-1, Strain Name-2, and Strain Name-3, respectively.

[0075] Referring to Figure 5, all three infrared spectra of C. paracasei NBRC 3953 were classified into cluster C4, and all three infrared spectra of C. paracasei NBRC 15889 were classified into cluster C3. The infrared spectra of C. tolerans JCM 1171 and C. tolerans NBRC 15906T were classified into cluster C1 or cluster C2 (see box 94). As described above, in Experiment 2, subspecies and different strains could be separated.

[0076] On the other hand, paracasei subsp. tolerans could not be isolated from JCM 1171 and NBRC 15906T.

[0077] (6-3. Experiment 3) The pretreatment in Experiment 3 was a modification of the pretreatment (4) in Experiment 2. Specifically, in Experiment 2 (4), the sample mixed with acetonitrile was dropped directly onto the sample plate, whereas in Experiment 4 (4), the sample mixed with acetonitrile was centrifuged and the supernatant was dropped onto the sample plate.

[0078] The pretreatment in Experiment 3 was performed as follows: (1) Bacterial cells were dispersed in 200 μL of distilled water to an OD of approximately 1. (2) 800 μL of ethanol was added to disperse the bacterial cells, and the resulting supernatant was removed by centrifugation. (3) 50 μL of 70% formic acid aqueous solution was added to re-disperse the precipitated bacterial cells. (4) 50 μL of acetonitrile was further added and mixed to prepare a formic acid / acetonitrile mixed solution. The formic acid / acetonitrile mixed solution was then centrifuged (10,000 g, 1 minute) to obtain a supernatant. In Experiment 3, the supernatant of the formic acid / acetonitrile mixed solution corresponds to an example of the "analytical sample." (5) 2 μL of the supernatant of the formic acid / acetonitrile mixed solution was dropped onto a sample plate to prepare a dried product. (6) The dried product obtained in (5) was analyzed using an infrared spectrophotometer. The time required for steps (1) to (4) was approximately 5 minutes.

[0079] The analytical conditions for infrared spectroscopy in Experiment 3 were as follows: Number of differential points: 3 Wavenumber range: 650 to 1300 cm －１Normalization: Vector Normalization Similarity: Euclidean Cluster analysis: Ward method Figure 6 shows an example of an infrared spectrum obtained as a result of Experiment 3. Figure 6 shows an infrared spectrum obtained by analyzing one dried sample of subsp. tolerans NBRC 15906T. The horizontal axis of Figure 6 represents the frequency (wavenumber) of infrared light. The vertical axis represents absorbance. Referring to Figure 6, the peak in the wavenumber range indicated by box 95 is sharper than in Figure 4. This is thought to be because in Experiment 4, cell walls and the like were removed as precipitates in pretreatment (4).

[0080] On the other hand, the absorbance in Figure 6 is weaker than in Figure 4. For example, the maximum absorbance in Figure 4 is approximately 0.35, while the maximum absorbance in Figure 6 is approximately 0.12. The difference in absorbance between Figure 4 and Figure 6 is thought to be due to the reduction in the amount of protein in the analytical sample caused by centrifugation in pretreatment (4). Therefore, the infrared spectrum in Figure 6 may contain a relatively large amount of noise, as the absorbance is generally low.

[0081] Figure 7 is a dendrogram showing the results of microbial discrimination obtained as a result of cluster analysis of the infrared spectra in Figure 6. The horizontal axis in Figure 7 represents the distance between each cluster. Each cluster was classified at the level of line L3. The three infrared spectra obtained for each strain are referred to as Strain Name-1, Strain Name-2, and Strain Name-3, respectively.

[0082] Referring to Figure 7, all three infrared spectra of S. paracasei NBRC 3953 were classified into cluster C2, and all three infrared spectra of S. paracasei NBRC 15889 were classified into cluster C1. Furthermore, all three infrared spectra of S. tolerans NBRC 15906T were classified into cluster C3, and all three infrared spectra of S. tolerans NBRC 15906T were classified into cluster C4. As described above, in Experiment 3, not only were subspecies and different strains separated, but also the same strain purchased from different sources. In other words, it was possible to classify strains with slight genetic differences that arose during the passage of the same strain.

[0083] (6-4. Experiment 4) The pretreatment in Experiment 4 was a modification of (5) of the pretreatment in Experiment 3. Specifically, the amount of the supernatant (analytical sample) of the formic acid / acetonitrile mixed aqueous solution dropped onto the sample plate in (5) was increased from 2 μL to 10 μL.

[0084] The pretreatment in Experiment 4 was performed as follows: (1) Bacterial cells were dispersed in 200 μL of distilled water to an OD of approximately 1. (2) 800 μL of ethanol was added to disperse the bacterial cells, and the resulting supernatant was removed by centrifugation. (3) 50 μL of 70% formic acid aqueous solution was added to re-disperse the precipitated bacterial cells. (4) 50 μL of acetonitrile was further added and mixed to prepare a formic acid / acetonitrile mixed solution. The formic acid / acetonitrile mixed solution was then centrifuged (10,000 g, 1 minute) to obtain a supernatant. In Experiment 4, the supernatant of the formic acid / acetonitrile mixed solution corresponds to an example of the "analytical sample." (5) 10 μL of the supernatant of the formic acid / acetonitrile mixed solution was dropped onto a sample plate to prepare a dried product. (6) The dried product obtained in (5) was analyzed using an infrared spectrophotometer. The time required for steps (1) to (4) was approximately 5 minutes.

[0085] The analytical conditions for infrared spectroscopy in Experiment 4 were as follows: Number of differential points: 3 Wavenumber range: 650 to 1300 cm －１Normalization: Vector Normalization Similarity: Euclidean Cluster analysis: Ward method Figure 8 shows an example of an infrared spectrum obtained as a result of Experiment 4. Figure 8 shows an infrared spectrum obtained by analyzing one dried sample of subsp. tolerans NBRC 15906T. The horizontal axis of Figure 8 represents the frequency (wavenumber) of infrared light. The vertical axis represents absorbance. Referring to Figure 8, the peaks in the wavenumber range indicated by box 96 are even sharper than those in Figure 6. This is thought to reflect the fact that in Experiment 4, the amount of analytical sample (supernatant of the formic acid / acetonitrile mixed aqueous solution from which cell walls and other components have been removed) added dropwise to the sample plate in pretreatment (5) was increased, resulting in an increase in the amount of cytoplasmic proteins and other components contained in the analytical sample. As a result, sharper peaks with reduced noise than those in Figure 6 were observed in Figure 8.

[0086] Figure 9 is a dendrogram showing the microbial discrimination results obtained as a result of cluster analysis of the infrared spectra in Figure 8. The horizontal axis in Figure 9 represents the distance between each cluster. Each cluster was classified at the level of line L4. The three infrared spectra obtained for each strain are referred to as Strain Name-1, Strain Name-2, and Strain Name-3, respectively.

[0087] Referring to Figure 9, all three infrared spectra of C. paracasei NBRC 3953 were classified into cluster C3, and all three infrared spectra of C. paracasei NBRC 15889 were classified into cluster C4. All three infrared spectra of C. tolerans NBRC 15906T were classified into cluster C2, and all three infrared spectra of C. tolerans NBRC JCM 1171 were classified into cluster C1. As described above, in Experiment 4, we were able to separate not only subspecies and different strains, but also the same strain purchased from different sources. In other words, we were able to classify strains with slight genetic differences that arose during the passage of the same strain.

[0088] Furthermore, in Experiment 4, the distance between the clusters of B. tolerans NBRC 15906T and B. tolerans JCM 1171 (the distance between clusters of the same subspecies) was smaller than the distance between the clusters of B. paracasei NBRC 3953 and B. paracasei NBRC 15889 (the distance between clusters of different strains of the same subspecies). On the other hand, in Experiment 3 (Figure 7), the distance between the clusters of B. tolerans NBRC 15906T and B. tolerans JCM 1171 (the distance between clusters of the same subspecies) was greater than the distance between the clusters of B. paracasei NBRC 3953 and B. paracasei NBRC 15889 (the distance between clusters of different strains of the same subspecies). Because the difference in protein sequences between the same strains of the same subspecies is generally smaller than the difference in protein sequences between different strains of the same subspecies, the dendrogram in Experiment 4 is considered to represent the actual situation more accurately than the dendrogram in Experiment 3. In other words, referring to FIGS. 7 and 9, the pretreatment method of Experiment 4 was considered to be a method capable of classifying microorganisms more accurately than the pretreatment method of Experiment 3.

[0089] As described above, infrared spectroscopic analysis of the analytical samples prepared by each pretreatment in Experiments 1 to 4 showed that microorganisms could be distinguished at the subspecies level. This is thought to reflect the fact that cell disruption for infrared spectroscopic analysis could be performed appropriately using a simple method that does not require bead crushing, and that as a result, infrared spectra with different shapes were obtained for each subspecies.

[0090] (6-5. Experiment 5) In Experiment 5, a matrix solution was added to the supernatant (analysis sample) of the formic acid / acetonitrile mixed aqueous solution prepared in Experiment 4(4), and mass spectrometry was performed.

[0091] The analysis in Experiment 5 was carried out as follows. (1) 1 μL of the supernatant (analysis sample) of the formic acid / acetonitrile mixture prepared in Experiment 4 was dropped onto a sample plate and dried. (2) 1 μL of CHCA solution was dropped onto the dried product obtained in (1) and dried to obtain a mixed crystal of the sample and matrix substance. The CHCA solution was prepared by dissolving CHCA in a 40% acetonitrile / 10% ethanol aqueous solution containing 1% TFA to a CHCA concentration of 10 mg / mL. (3) The mixed crystal obtained in (2) was analyzed using a MALDI-8020 (Shimadzu Corporation) to obtain a mass spectrum. Microorganisms were then identified based on the peaks in the mass spectrum corresponding to ribosomal proteins.

[0092] Figure 10 shows the mass spectra obtained as a result of Experiment 5. The horizontal axis of Figure 10 represents m / z, and the vertical axis represents signal intensity. Referring to Figure 10, the mass spectra of the same subspecies of S. paracasei NBRC 15889, S. paracasei NBRC 3953, S. tolerans JCM 1171, and S. tolerans NBRC 15906T showed similar patterns.

[0093] Furthermore, we attempted to identify each sample based on the peaks corresponding to ribosomal proteins in the mass spectrum, and were able to correctly identify them at the subspecies level.

[0094] As described above, the pretreatment procedure described in Experiment 4 resulted in the preparation of analytical samples that could be accurately identified at the subspecies level by both infrared spectroscopy and mass spectrometry. Therefore, it is believed that appropriate pretreatment for infrared spectrometry and mass spectrometry could be performed using a simple method that does not require bead crushing.

[0095] Aspects It will be understood by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0096] (Item 1) A pretreatment method according to one aspect is a method for pretreatment of a sample containing biological cells for infrared spectroscopic analysis, and includes the steps of preparing a first acidic solution containing an organic acid and contacting the cells with the first acidic solution.

[0097] According to the pretreatment method described in paragraph 1, cells can be disrupted by contacting them with an organic acid without using beads, thereby providing a pretreatment method for a sample containing biological cells for infrared spectroscopic analysis that allows for cell disruption in a simple manner.

[0098] (Item 2) In the pretreatment method according to item 1, the organic acid includes at least one selected from the group consisting of formic acid, trifluoroacetic acid, and lactic acid.

[0099] According to the pretreatment method described in Section 2, the pretreatment method can be carried out using the organic acid described in Section 2, which is easily available and which does not adversely affect the measurement results when used by a person skilled in the art for pretreatment of infrared spectroscopic analysis and mass spectrometry.

[0100] (Item 3) In the pretreatment method described in items 1 or 2, the step of contacting the sample with the first acidic solution includes a step of preparing a second acidic solution, which is a mixed solution of the sample and the first acidic solution.

[0101] According to the pretreatment method described in the third aspect, the cells can be reliably brought into contact with the first acidic solution, thereby improving the efficiency of treatment of the cells with the first acidic solution.

[0102] (Item 4) In the pretreatment method described in item 3, the concentration of the organic acid is 50% by volume or more and 90% by volume or less, based on the first acidic solution or the second acidic solution.

[0103] According to the pretreatment method described in item 4, by using the organic acid at the above concentration, cytoplasm can be extracted particularly efficiently.

[0104] (Item 5) In the pretreatment method described in item 3 or 4, the second acidic solution further contains acetonitrile.

[0105] The efficiency of cell disruption is further improved by the pretreatment method described in item 5. Furthermore, after the analytical sample is dropped onto the sample plate, the analytical sample can be dried uniformly and in a short time.

[0106] (Item 6) The pretreatment method according to any one of Items 3 to 5, further comprising the step of centrifuging the second acidic solution to remove precipitate.

[0107] According to the preprocessing method described in paragraph 6, the peaks of the infrared spectrum become sharper, improving the accuracy of classification of organisms based on the shape of the infrared spectrum.

[0108] (Item 7) The pretreatment method according to any one of Items 1 to 6, further comprising a step of dispersing the cells in an aqueous ethanol solution prior to the step of contacting the cells with the first acidic solution, wherein the concentration of ethanol in the aqueous ethanol solution is 70% by volume or more and 90% by volume or less, based on the aqueous ethanol solution.

[0109] According to the pretreatment method described in item 7, by using an aqueous ethanol solution of the above concentration, the inactivation and washing of the bacterial cells can be carried out efficiently.

[0110] (Item 8) In the pretreatment method according to any one of items 1 to 7, the organism is a microorganism.

[0111] According to the pretreatment method described in item 8, it is possible to carry out microorganism analysis, which is useful in the fields of industry, manufacturing, medicine, etc.

[0112] (Item 9) An analytical method comprising the steps of: preparing a dried product by dropping a predetermined amount of an analytical sample obtained by pretreating a sample using the pretreatment method described in any one of items 1 to 8 onto a sample plate and drying the drop; and obtaining an infrared spectrum by performing infrared spectroscopic analysis of the dried product using Fourier transform infrared spectroscopy.

[0113] According to the analytical method described in item 9, it is possible to analyze cytoplasmic components contained in an analytical sample with high accuracy.

[0114] (Item 10) In the analytical method described in Item 9, the predetermined volume is 1 μL or more and 15 μL or less.

[0115] According to the pretreatment method described in paragraph 10, an infrared spectrum capable of identifying living organisms can be obtained.

[0116] (Item 11) The preprocessing method according to item 9 or 10 further comprises a step of classifying organisms by cluster analysis of the infrared spectra.

[0117] According to the analytical method described in item 11, the organism from which the analytical sample is derived can be classified in a simple manner based on the infrared spectrum.

[0118] (Item 12) The analytical method according to any one of items 9 to 11 further includes the steps of adding a matrix substance to the analytical sample, and obtaining a mass spectrum by performing matrix-assisted laser desorption / ionization mass analysis on the analytical sample to which the matrix substance has been added.

[0119] According to the analytical method described in paragraph 12, an analytical sample pretreated for infrared spectroscopic analysis can also be subjected to mass spectrometry. This eliminates the need to prepare a new sample for mass spectrometry. Furthermore, the results of infrared spectroscopic analysis and mass spectrometry can be compared for the same analytical sample pretreated under the same conditions from the same culture sample.

[0120] (Item 13) The analytical method according to item 12, wherein the step of adding a matrix substance includes the step of adding a matrix solution containing the matrix substance to the dried material on the sample plate.

[0121] According to the analytical method described in item 13, it is possible to eliminate the need to newly drop analytical samples onto a sample plate for mass spectrometry.

[0122] (Item 14) The analytical method according to item 12 or 13 further comprises a step of displaying the analytical results of the infrared spectroscopy and the analytical results of the mass spectroscopy.

[0123] According to the analytical method described in paragraph 14, the analytical results obtained by infrared spectroscopy and the analytical results obtained by mass spectrometry can be easily compared.

[0124] (Item 15) A program that, when executed by a computer, causes the computer to carry out the analysis method described in any one of items 9 to 14.

[0125] According to the program described in paragraph 15, the analysis method described in this embodiment can be automatically performed.

[0126] (Item 16) An analytical system according to another aspect includes an infrared spectrophotometer and a mass spectrometer. The infrared spectrophotometer performs infrared spectroscopic analysis of an analytical sample obtained by pre-treating a sample containing biological cells by contacting the cells with a first acidic solution containing an organic acid. The mass spectrometer performs mass analysis of the analytical sample.

[0127] According to the analytical system described in paragraph 16, cells can be disrupted for infrared spectroscopic analysis by a simple method of contacting them with an organic acid without using beads. Furthermore, the analytical sample pretreated for infrared spectroscopic analysis can also be subjected to mass spectrometry. This eliminates the need to prepare a new sample for mass spectrometry. Furthermore, the results of infrared spectroscopic analysis and mass spectrometry can be compared for the same analytical sample pretreated under the same conditions from the same culture sample.

[0128] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims, not by the above description, and is intended to include all modifications within the meaning and scope of the claims.

[0129] 1 Pretreatment unit, 2 Infrared spectrophotometer, 3 Mass spectrometer, 4 Control unit, 10 Analysis unit, 41 Processor, 42 Memory, 43 Display, 100 Analysis system, C1, C2, C3, C4 Cluster, S ion.

Claims

1. A method for pretreating a sample containing biological cells for infrared spectroscopic analysis, comprising the steps of: preparing a first acidic solution containing an organic acid; and contacting the cells with the first acidic solution.

2. The pretreatment method according to claim 1, wherein the organic acid includes at least one selected from the group consisting of formic acid, trifluoroacetic acid and lactic acid.

3. A pretreatment method as described in claim 1 or 2, wherein the step of contacting the sample with the first acidic solution includes a step of preparing a second acidic solution which is a mixed solution of the sample and the first acidic solution.

4. The pretreatment method according to claim 3, wherein the concentration of the organic acid is 50% by volume or more and 90% by volume or less, based on the first acidic solution or the second acidic solution.

5. The pretreatment method according to claim 3, wherein the second acidic solution further contains acetonitrile.

6. The pretreatment method of claim 3, further comprising the step of centrifuging the second acidic solution to remove precipitate.

7. The pretreatment method according to claim 1 or 2, further comprising a step of dispersing the cells in an aqueous ethanol solution prior to the step of contacting the cells with the first acidic solution, wherein the concentration of ethanol in the aqueous ethanol solution is 70% by volume or more and 90% by volume or less based on the aqueous ethanol solution.

8. A pretreatment method according to claim 1 or 2, wherein the organism is a microorganism.

9. An analytical method comprising the steps of: dropping a predetermined amount of an analytical sample obtained by pretreating the sample using the pretreatment method described in claim 1 onto a sample plate and drying to prepare a dried product; and performing infrared spectroscopic analysis of the dried product using Fourier transform infrared spectroscopy to obtain an infrared spectrum.

10. The analytical method according to claim 9, wherein the predetermined amount is between 1 μL and 15 μL.

11. The method of claim 9 or 10, further comprising a step of classifying the organisms by cluster analysis of the infrared spectra.

12. The analytical method according to claim 11, further comprising the steps of: adding a matrix substance to the analytical sample; and obtaining a mass spectrum by performing matrix-assisted laser desorption / ionization mass spectrometry on the analytical sample to which the matrix substance has been added.

13. The analytical method according to claim 12, wherein the step of adding a matrix material includes the step of adding a matrix solution containing the matrix material to the dried material on the sample plate.

14. The analysis method according to claim 12, further comprising a step of displaying the results of the analysis by infrared spectrometry and the results of the analysis by mass spectrometry.

15. A program which, when executed by a computer, causes the computer to carry out the analysis method according to claim 12.

16. An analytical system comprising: an infrared spectrophotometer; and a mass spectrometer, wherein the infrared spectrophotometer performs infrared spectroscopy analysis of an analytical sample obtained by pretreating a sample containing biological cells by contacting the cells with a first acidic solution containing an organic acid; and the mass spectrometer performs mass analysis of the analytical sample.

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