Simultaneous analysis method of polypeptides and anionic low molecular weight compounds

Abstract

To provide an analysis method capable of easily detecting polypeptides and anionic low molecular weight compounds in a sample simultaneously and with high accuracy.SOLUTION: A disclosed analysis method is an analysis method of a sample using a liquid chromatograph-mass spectrometer (LC-MS) using a reversed-phase column, in which weak anion exchange groups and hydrophobic groups are individually bonded to the surface of the support as a column in the liquid chromatograph. With this, polypeptides and anionic low molecular weight compounds can be simultaneously analyzed. The polypeptide may be an allergen protein, and the anionic low molecular weight compound may be a sulfite derivative. The weak anion exchange group is preferably a third amino group, and the hydrophobic group is preferably an octadecyl group.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for simultaneously analyzing multiple components using liquid chromatography. [Background technology]

[0002] Food allergies cause symptoms such as dermatitis, asthma, gastrointestinal dysfunction, and anaphylactic shock due to immune responses caused by food. Even a small amount of allergen contamination is dangerous for food allergy patients. Therefore, a highly sensitive allergen detection method capable of detecting trace amounts of allergens in samples such as food is required. Patent Document 1 describes a method in which a sample is treated with a protease and a target allergen-derived polypeptide in the enzyme-treated sample is detected by liquid chromatography, and a reverse phase liquid chromatograph is described as an example of a liquid chromatograph used for such detection.

[0003] Patent Document 2 describes an ion chromatography analysis method capable of simultaneously quantifying chloride ions, phosphate ions, and sulfate ions in a sample, such as wine, that contains sulfite ions or organic acids, such as tartaric acid or malic acid, in which a carbonyl compound, such as acetone, is added to the mobile phase and an ion chromatography column for anion analysis is used.

[0004] Patent Document 3 describes a method for measuring sulfurous acid in wine, which uses a reverse-phase column as a separation column and adds an ion pair reagent to the mobile phase to separate stable hydroxymethanesulfonic acid derived from free sulfurous acid in wine from acetaldehyde-bound sulfurous acid in wine, thereby measuring free sulfurous acid.

[0005] Patent Document 4 describes the use of analytical results of multiple sulfur compounds and multiple sulfur metabolism-related compounds to evaluate the freshness of test substances such as agricultural products, and describes liquid chromatography-mass spectrometry as an example of the analytical method. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 16551 [Patent Document 2] JP 2008-96258 A [Patent Document 3] JP 2003-75423 A [Patent Document 4] International Publication No. 2018 / 207879 Summary of the Invention [Problem to be solved by the invention]

[0007] Conventionally, sodium sulfite, sodium hyposulfite, sulfur dioxide, potassium pyrosulfite, and sodium pyrosulfite (hereinafter referred to as "sulfites, etc.") have been added to foods for the purpose of bleaching, preventing oxidation, etc., but since sulfites, etc. may contain salts that induce allergy-like symptoms in the human body depending on the amount ingested, Western countries are required to label on food packaging that sulfites, etc. have been added to the food, in the same way as allergen proteins such as wheat and eggs. Under these circumstances, there is a demand for highly accurate analysis of the presence or absence of sulfites, etc. in food samples, in the same way as allergen proteins.

[0008] Polypeptides with relatively large molecular weights, such as allergen proteins, and anionic low molecular weight compounds with relatively small molecular weights, such as sulfites, have different physicochemical properties. Therefore, when analyzing both components of a food-derived sample using liquid chromatography, it has been common to prepare a liquid chromatograph corresponding to each component and analyze them individually, which requires a lot of time and effort for analysis. No technology has yet been provided that can easily analyze polypeptides and anionic low molecular weight compounds in a sample with high accuracy using liquid chromatography.

[0009] An object of the present invention is to provide an analytical method capable of simultaneously and easily analyzing a polypeptide and an anionic low-molecular-weight compound in a sample with high accuracy. [Means for solving the problem]

[0010] The present invention relates to a method for simultaneously analyzing polypeptides and low molecular weight anionic compounds in sample analysis using a liquid chromatograph-mass spectrometer, in which a reverse-phase column having weak anion exchange groups and hydrophobic groups individually bonded to the surface of a carrier is used as the column in the liquid chromatograph. Effect of the Invention

[0011] According to the analytical method of the present invention, it is possible to simultaneously analyze a polypeptide and an anionic low molecular weight compound in a sample, and to simultaneously and specifically detect both components with high sensitivity under a single analytical condition. [Brief description of the drawings]

[0012] [Figure 1] 1(a) to (h) show the LC-MS analysis results (chromatograms) of Example 1, respectively. [Diagram 2] 2(a) to (h) show the LC-MS analysis results (chromatograms) of Comparative Example 1, respectively. [Diagram 3] 3(a) to (h) show the results (chromatograms) of LC-MS analysis of Comparative Example 2, respectively. [Figure 4] 4(a) to (h) show the LC-MS analysis results (chromatograms) of the reference example, respectively. [Diagram 5] 5(a) to (h) show the LC-MS analysis results (chromatograms) of the control example, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The analytical method of the present invention involves simultaneously analyzing a polypeptide and an anionic low molecular weight compound in a sample. In the present invention, "simultaneous analysis" is synonymous with "simultaneous analysis" and typically refers to combining the analysis results for the polypeptide and the analysis results for the anionic low molecular weight compound into a single result output from an analytical device (e.g., a liquid chromatograph-mass spectrometer). The "single result" is specifically, for example, a chromatogram output by the analytical device, such as the graph shown in FIG. 1.

[0014] In the present invention, the term "polypeptide" refers to a compound in which a plurality of amino acids are bound by peptide bonds. Generally, a polypeptide having about 10 or less amino acids (number of amino acid residues) is called an "oligopeptide", a polypeptide having about 10 to 50 amino acids is called a "polypeptide", and a polypeptide having 50 or more amino acids is called a "protein". The term "polypeptide" in the present invention is a general term for these, and the number of amino acid residues may be 2 or more, preferably 50 or less, more preferably 40 or less, and even more preferably 30 or less.

[0015] In the present invention, the polypeptide to be analyzed (target substance in LC-MS) (target polypeptide) is not particularly limited, and includes, for example, polypeptides derived from living organisms such as various tissues, cells, bacteria, and viruses; polypeptides synthesized by known synthesis methods; polypeptides obtained by known genetic engineering techniques; and polypeptide fragments of proteins generated by enzymatic degradation. The isoelectric point, function, structure, etc. of the target polypeptide are not particularly limited.

[0016] The analysis method of the present invention is particularly useful for analyzing allergen proteins among polypeptides. In the present invention, "allergen protein" refers to any immunogenic protein or peptide that specifically reacts with the antibodies of a person with an allergic disease and can induce an immune response. The type of allergen protein is not particularly limited, and examples thereof include food-derived allergens, pollen-derived allergens, house dust-derived allergens, and animal hair-derived allergens, and may be derived from plants or animals.

[0017] The analytical method of the present invention is particularly useful for the analysis of foods, and therefore is particularly useful when the allergenic protein to be analyzed is derived from a food allergen. Food allergens can be broadly divided into plant food allergens and animal food allergens. Examples of plant food allergens include grains such as wheat, rye, barley, oats, corn, rice, buckwheat, millet, foxtail millet, and barnyard millet; beans or nuts such as almonds, coconuts, peanuts, walnuts, soybeans, peas, green beans, hazel nuts, and Brazil nuts; fruits and vegetables such as strawberries, oranges, kiwis, potatoes, celery, onions, tomatoes, parsley, carrots, garlic, mangoes, melons, apples, pumpkins, grapefruits, cherries, pears, sweet potatoes, bamboo shoots, and spinach; and other examples include sesame and mustard. Examples of animal food allergens include crustaceans such as shrimp and crab; fish such as salmon, mackerel, abalone, and squid; meats such as beef, pork, and chicken; milk, and chicken eggs.

[0018] In the present invention, the term "anionic low molecular weight compound" refers to a compound that has anionic properties, i.e., that dissociates in water and bears a negative charge, and that is a low molecular weight compound. Specifically, the term "low molecular weight" refers to a compound having a weight average molecular weight of preferably 1000 or less, more preferably 700 or less, and even more preferably 500 or less. The acid dissociation constant pKa of the anionic low molecular weight compound in water is preferably 7 or less, more preferably 6 or less, from the viewpoint of improving retention in a column having a weak anion exchange group when a mobile phase containing a volatile acid is used as the mobile phase of the liquid chromatograph in the analytical method of the present invention.

[0019] In the present invention, specific examples of anionic low molecular weight compounds (target anionic low molecular weight compounds) to be analyzed (target substances in LC-MS) include sulfite derivatives such as sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium hyposulfite, potassium bisulfite, potassium pyrosulfite, and ammonium bisulfite; pesticides such as glyphosate, N-acetylglyphosate, glufosinate, N-acetylglufosinate, AMPA (aminomethylphosphonic acid), N-acetylAMPA (N-acetylaminomethylphosphonic acid), MPPA (3-methylphosphinicopropionic acid), HEPA (3-methylphosphinicopropionic acid), and ethephone, as well as their metabolites, sodium erythorbate, benzoic acid, sorbic acid, and dehydroacetic acid. All of these anionic low molecular weight compounds can be used in food or food ingredients. Specifically, sulfites, which are a type of sulfurous acid derivative, are used as food bleaches and antioxidants, glyphosate, glufosinate, etc. are used as herbicides, sodium erythorbate is used as a food antioxidant, and benzoic acid, sorbic acid, and dehydroacetic acid can be used as food preservatives. Sulfites and the like can exist in food as free sulfurous acid, as well as as sulfurous acid derivatives bound to components in food, and the analysis method of the present invention can easily analyze the content of any form with high accuracy.

[0020] In the analytical method of the present invention, the object to be analyzed is not particularly limited, and examples thereof include foods, cosmetics, and pharmaceuticals; raw materials thereof; and equipment used in the manufacturing process thereof. In the analysis method of the present invention, the object to be analyzed may be used as a sample as it is, or the object to be analyzed may be used as a sample after being pretreated. Examples of the pretreatment include crushing, dissolving, suspending, and extracting, and a combination of two or more of these may be used. When the object to be analyzed is an instrument, the sample may be a cleaning liquid generated by cleaning the instrument with a liquid, or the cleaning liquid that has been pretreated as described above. Alternatively, the surface of the instrument to be analyzed may be rubbed with a sheet of paper, fabric, or the like, and the sheet may be subjected to the pretreatment as necessary as a sample.

[0021] In the analytical method of the present invention, the prepared sample may be treated with a protease. That is, in the present invention, the polypeptide (target polypeptide) to be analyzed (the target substance in LC-MS) may be a peptide generated by enzymatic degradation of a protein. Specific examples of the protease include trypsin, chymotrypsin, elastase, thermolysin, etc., and trypsin is preferred. The conditions for the enzymatic treatment of the sample may be appropriately selected depending on the type of enzyme, and in the case of trypsin, for example, the enzyme concentration is preferably 1000 to 20000 U, 25 to 45°C, pH 7 to 9, and 4 to 24 hours. When a sample contains a target polypeptide, the polypeptide contained in the sample after enzymatic treatment has fewer amino acid residues than the target polypeptide, but is a polypeptide derived from the target polypeptide. On the other hand, when a sample does not contain a target polypeptide, the sample after enzymatic treatment does not contain a polypeptide derived from the target polypeptide. Therefore, the presence or absence of the target polypeptide in a sample treated with a protease can be detected by detecting the presence or absence of the target polypeptide in the sample. In this regard, Patent Document 1 discloses that food allergens such as buckwheat, crustaceans (crab, shrimp), milk, eggs, and peanuts can be detected with high sensitivity by detecting a specific amino acid sequence. In the present invention, the technology disclosed in Patent Document 1 (allergen detection method) can be appropriately utilized.

[0022] The analytical method of the present invention uses a liquid chromatograph (LC)-mass spectrometer (MS) (hereinafter also referred to as "LC-MS"). The LC-MS combines an LC section that separates components of a sample with an MS section that detects the components after separation by the LC section. The LC-MS may further include a data processing section and a recording section as means for processing the detection results by the MS section.

[0023] The LC section typically includes a column for separating components in a sample, a pump for delivering a mobile phase to the column, and a sample introduction device for introducing the mobile phase delivered from the pump into the column. As the LC section, a high performance liquid chromatograph (HPLC) is preferred.

[0024] The MS section typically includes an ion source that ionizes components, a mass separation section that separates ions according to differences in mass / charge ratio (m / z), and an ion detection section that detects the separated ions. As a method for ionizing components, any method conventionally used in LC-MS can be used without particular limitation, and examples thereof include electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). The mass separator may be any known type without any particular limitation, and examples of such types include magnetic sector type, quadrupole type, and time-of-flight type. The mass separator may be a tandem type in which a plurality of mass separators of the same type are connected, or a hybrid type in which a plurality of mass separators of different types are connected. Specific examples of tandem mass separators include a tandem quadrupole (MS / MS) type and a tandem time-of-flight (TOF / TOF) type. Specific examples of hybrid mass separators include a quadrupole-time-of-flight (MS / TOF) type, an ion trap-time-of-flight (IT / TOF) type, and a quadrupole ion cyclotron resonance (MS / ICR) type. In mass spectrometry using the MS section, target substances (polypeptides, anionic low molecular weight compounds) separated in the LC section are separated in the mass separation section according to their mass / charge ratio (m / z). Therefore, by storing the m / z values ​​of the target substances in a database in advance, the presence or absence of the target substance in a sample can be detected based on the measured m / z values ​​of the precursor ions and product ions of the target substance. As the measurement mode of mass spectrometry by the MS section, multiple reaction monitoring (MRM) is preferable because it has high measurement accuracy (S / N ratio) and is capable of detecting multiple types of target substances at once.

[0025] For example, in the analysis of a sample using an LC-MS / MS equipped with a tandem quadrupole mass separation section, the LC section separates the sample into components based on differences in affinity, and then the first mass separation section in the MS section further dissociates and fragments only the components with specific masses, and then the second mass separation section in the MS section detects (quantifies) specific ions.

[0026] The analytical method of the present invention is characterized in that a reversed-phase column in which a weak anion exchange group and a hydrophobic group are individually bonded to the surface of a carrier is used as a column in a liquid chromatograph, i.e., a column constituting the LC part of an LC-MS. That is, the reversed-phase column used in the present invention includes a carrier and a weak anion exchange group and a hydrophobic group bonded to the surface of the carrier, and both functional groups are each bonded to the surface of the carrier, and as in the column of Comparative Example 2 described below, one of the two functional groups is bonded to the surface of the carrier, and the other functional group is not bonded to the one functional group. By performing LC-MS analysis using such a specific reversed-phase column, it is possible to simultaneously analyze polypeptides and anionic low molecular weight compounds in a sample.

[0027] Examples of the weak anion exchange group of the reversed-phase column used in the present invention include primary amino groups, secondary amino groups, tertiary amino groups, and quaternary ammonium groups. Among these, tertiary amino groups are particularly preferred as the weak anion exchange group of the reversed-phase column, since they can sufficiently retain the target substance (anionic low molecular weight compound) and can be eluted from the column with a low salt concentration solution suitable for LC-MS analysis. Specific examples of the tertiary amino group include dimethylamino group and diethylamino group.

[0028] Examples of the hydrophobic group that the reversed-phase column used in the present invention has include octadecyl, octyl, ethyl, methyl, docosyl, triacontyl, phenyl, and pentafluorophenyl groups. Among these, the octadecyl group is particularly preferred as the hydrophobic group of the reversed-phase column, since it can adequately retain the target substance (polypeptide) and perform good separation and elution depending on the organic solvent concentration.

[0029] The reversed-phase column used in the present invention typically comprises a stationary phase and a container packed with the stationary phase, and the stationary phase comprises the support and weak anion exchange groups and hydrophobic groups individually bound to the surface of the support. The carrier can be any carrier that has been conventionally used as a carrier for a reverse phase column, without any particular limitation. Examples of the carrier include inorganic porous carriers such as silica gel, alumina, porous glass, and carbon particles; and polymer gels (organic porous carriers) such as polystyrene gel, poly(meth)acrylic acid gel, and polyvinyl alcohol gel. The volume average particle size of the carrier is preferably 1 to 10 μm, and more preferably 1.5 to 7 μm.

[0030] In the present invention, a commercially available reversed phase column can also be used. Suitable commercially available reversed phase columns include those under the trade name "InertSustain AX-C18" (manufactured by GL Sciences Inc.) and those under the trade name "Atlantis Premier BEH C18 AX" (manufactured by Waters Corporation).

[0031] The mobile phase (eluent) in liquid chromatography is not particularly limited as long as it can separate the target substance (polypeptide, anionic low molecular weight compound) from the sample. From the viewpoint of elution of anionic low molecular weight compounds and improving the detection sensitivity of the target substance, however, a solution consisting of water containing a volatile acid and an organic solvent is preferred.

[0032] Examples of the "volatile acid" include formic acid, acetic acid, trifluoroacetic acid, difluoroacetic acid, etc., and these may be used alone or in combination of two or more. Among these, formic acid is particularly preferred. The pH of the "water containing a volatile acid" is not particularly limited, but from the viewpoint of retaining and separating the target substance (polypeptide, anionic low molecular weight compound) and improving the detection sensitivity, it is preferably 5 or less, more preferably 1.5 to 4.5. The content of the volatile acid in the "water containing a volatile acid" is preferably 0.01 to 5 mass %, more preferably 0.05 to 2 mass %, based on the total mass of the water.

[0033] Examples of the organic solvent constituting the gradient solution together with the "water containing a volatile acid" include acetonitrile, methanol, ethanol, propanol, isopropanol, acetone, and tetrahydrofuran, and these can be used alone or in combination of two or more. Among these, acetonitrile is particularly preferred. The organic solvent may contain the "volatile acid". That is, an "organic solvent containing a volatile acid" can be used as the organic solvent constituting the gradient solution. The content of the volatile acid in the "organic solvent containing a volatile acid" is preferably 0.01 to 5% by mass, more preferably 0.05 to 2% by mass, based on the total mass of the organic solvent.

[0034] In the gradient solution, the volume ratio of the volatile acid-containing water (A) to the organic solvent (B), A:B, is preferably 100:0 to 0:100, and more preferably 99:1 to 1:99.

[0035] If necessary, one or more volatile salts such as ammonium formate and ammonium acetate may be added to the gradient solution. This provides the effect of improving the peak shape and increasing the detection sensitivity for some of the target substances (polypeptides, anionic low molecular weight compounds). The concentration of the volatile salts in the gradient solution is preferably about 0.1 to 100 mM. In addition, in Patent Document 3, the detection sensitivity of sulfurous acid in a sample (wine) is improved by adding an ion pair reagent such as triethylamine or tributylamine to the mobile phase, but in the present invention, it is not necessary to add an ion pair reagent to the mobile phase. EXAMPLES

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0037] (reagent) Acetonitrile (FUJIFILM Wako Pure Chemical Corporation, special grade, for HPLC) Methanol (FUJIFILM Wako Pure Chemical Corporation, special grade, for HPLC) · Trypsin (Sigma-Aldrich Co. LLC, derived from porcine pancreas, Bio Reagent) Iodoacetamide (IAA) (Sigma-Aldrich Co. LLC, Bio Ultra) Dithiothreitol (DTT) (Sigma-Aldrich Co. LLC, molecular biology grade) Urea (Fujifilm Wako Pure Chemical Corporation, for molecular biology) Trifluoroacetic acid (TFA) (Junsei Chemical Co., Ltd., special grade) Formic acid (Fujifilm Wako Pure Chemical Corporation, for LC-MS) Trishydroxymethylaminomethane (Tris) (Fujifilm Wako Pure Chemical Industries, biochemical grade) Ammonium bicarbonate (Fujifilm Wako Pure Chemical Industries, for proteomics) Ammonium acetate (Junsei Chemical Co., Ltd., special grade) Formaldehyde (Junsei Chemical Co., Ltd., special grade) Sodium bisulfite (Fujifilm Wako Pure Chemical Industries, for molecular biology)

[0038] (buffer) ·Buffer A: 0.1MDTT, 4M urea containing_0.1M Tris-HCl buffer (pH8.2) Buffer B: 5 mM ammonium acetate buffer (pH 4.5) containing 0.2% formaldehyde by mass

[0039] (Preparation of food to be analyzed) Buffer A was added to each of the seven types of food allergens (shrimp, wheat flour, buckwheat flour, chicken egg, milk, peanuts, and walnuts) to extract the seven types of allergen proteins. The seven types of allergen proteins were added to a separately prepared allergen-free curry roux (food) so that the concentration of each allergen protein in the final food to be analyzed was 10 μg / g, and an aqueous sodium hydrogen sulfite solution was added so that the concentration of sulfur dioxide in the final food to be analyzed was 10 μg / g, to prepare a food to be analyzed containing the seven types of allergen proteins and sulfite.

[0040] (Sample preparation) Using the food to be analyzed, sample precursor A containing the seven types of allergen protein-derived polypeptides and sample precursor B containing hydroxymethanesulfonic acid (an anionic low molecular weight compound), a type of sulfurous acid derivative, were prepared by the following method, and the two precursors A and B were mixed at a mixing mass ratio of the former: the latter = 1: 1 to prepare the samples. The prepared samples were dispensed into polypropylene vials and subjected to analysis by LC-MS described below. The polypeptides in the sample were peptides generated by enzymatic hydrolysis of proteins, and the number of amino acid residues was 8 to 13. The anionic low molecular weight compounds in the sample had a weight-average molecular weight of 112 and an acid dissociation constant (pKa) in water of 6.9.

[0041] (Preparation of Sample Precursor A) 1.0 g of the food to be analyzed was collected in a 15 mL conical tube, 10 mL of buffer A was added, and the mixture was extracted by shaking at 37°C for 3 hours. The supernatant after centrifugation (2000×g, 5 minutes) was used as a protein extract. 1 mL of the protein extract was collected in another conical tube, 4 mL of 50 mM ammonium bicarbonate solution and 200 μL of 4% IAA solution were added, and the mixture was shaken at 37°C for 1 hour in the dark to alkylate the free thiol groups in the protein. 100 μL of 1% trypsin solution was further added to the reaction solution and shaken at 37°C for 16 hours to decompose the protein. 50 μL of TFA was added to the solution after protein decomposition to inactivate trypsin. The solution after trypsin inactivation was centrifuged (2000×g, 5 minutes), and the supernatant after centrifugation was collected and used in the subsequent desalting and purification process. In the desalting and purification process, OASIS HLB (60 mg, 3 mL, manufactured by Waters) was used as a solid-phase extraction column, and conditioning was performed with 5 mL of methanol and 5 mL of water. The entire amount of the supernatant after centrifugation was added to the column after conditioning, and after washing with 8 mL of 0.5 mass% TFA solution, elution was performed with 6 mL of 70 mass% acetonitrile solution. The entire amount of the solution after elution was collected in a pear-shaped flask, dried on a rotary evaporator, redissolved in 1 mL of 5 mass% acetonitrile solution containing 0.1 mass% formic acid, and filtered with a filter (pore size 0.22 μm) to prepare sample precursor A.

[0042] (Preparation of Sample Precursor B) 1.0 g of the food to be analyzed was collected in a 15 mL conical tube, 10 mL of buffer B was added, and the mixture was shaken and extracted at room temperature (around 25°C) for 1 hour. The supernatant after centrifugation (2000 x g, 5 minutes) was used as the sulfite extract. 2 mL of the sulfite extract was collected in a screw-type glass test tube and heated on an aluminum block at 80°C for 30 minutes to react the sulfite with formaldehyde to produce hydroxymethanesulfonic acid (sulfite derivative). After cooling the heated solution to room temperature, it was diluted 10 times with buffer B and filtered through a filter (pore size 0.22 μm) to prepare sample precursor B.

[0043] Example 1 The allergen protein-derived polypeptide and the sulfurous acid derivative (hydroxymethanesulfonic acid) in the sample were simultaneously analyzed using a tandem quadrupole mass spectrometer (LC-MS / MS). The analysis conditions in Example 1 are as follows. The analysis results (chromatogram) of Example 1 are shown in FIG. 1. (Analysis conditions for Example 1) (1) LC-MS instrument configuration LC section: HPLC, product name "NexeraX2" (manufactured by Shimadzu Corporation) ·MS section: MS / MS (tandem quadrupole mass spectrometer), product name "LC-MS8060" (manufactured by Shimadzu Corporation) (2) HPLC conditions Column: A reversed-phase mixed-mode column with tertiary amino groups (weak anion exchange groups) and octadecyl groups (hydrophobic groups), product name "InertSustain AX-C18" (volume average particle size 3 μm, inner diameter 2.1 mm, length 150 mm, manufactured by GL Sciences Inc.) Column oven temperature: 50℃ ·Mobile phase flow rate: 0.3mL Mobile phase: A gradient solution of an aqueous solution of formic acid (A) with a formic acid concentration of 0.1% by mass and an acetonitrile solution (B) with a formic acid concentration of 0.1% by mass Mobile phase gradient conditions: First, a mobile phase with a volume ratio of A:B = 99:1 was supplied to the column until 4 minutes after the start of measurement, then the proportion of mobile phase B was increased so that the volume ratio of A:B = 40:60 was reached until 16.5 minutes after the start of measurement, then the proportion of mobile phase B was increased so that the volume ratio of A:B = 5:95 was reached until 20 minutes after the start of measurement, then the mobile phase with a volume ratio of A:B = 5:95 was supplied until 25 minutes after the start of measurement, and finally the mobile phase with a volume ratio of A:B = 99:1 was supplied until 30 minutes after the start of measurement. (3) Mass spectrometry conditions Ionization method: ESI method Polarity: Positive if the target substance is a polypeptide, negative if the target substance is an anionic low molecular weight compound Interface voltage: 4.5 kV if the target substance is a polypeptide, -1.2 kV if the target substance is an anionic low molecular weight compound Interface temperature: 300℃ ·DL temperature: 250℃ Heat block temperature: 400℃ Nebulizer gas flow rate: 3L / min Heating gas flow rate: 10L / min Drying gas flow rate: 10L / min Measurement mode: MRM MRM transitions: see Table 1 below

[0044] [Table 1]

[0045] Comparative Example 1 The HPLC column used was a column having a positive charge on the silica gel surface and an octadecyl group (product name "bioZen Peptide PS-C18", manufactured by Phenomenex). The column used in Comparative Example 1 differs from the column in Example 1 having a weak anion exchange group (tertiary amino group) in that it does not have a weak anion exchange group. Other than the above, the polypeptide derived from the allergen protein and the sulfurous acid derivative (hydroxymethanesulfonic acid) in the sample were simultaneously analyzed in the same manner as in Example 1. The analysis results (chromatogram) of Comparative Example 1 are shown in FIG. 2.

[0046] Comparative Example 2 A column having a functional group in which a tertiary amino group is bonded to the end of an octadecyl group (product name "Acclaim MixedMode WAX-1", manufactured by Thermo Fisher Scientific Co., Ltd.) was used as the HPLC column. The column used in Comparative Example 2 has a weak anion exchange group (tertiary amino group) and a hydrophobic group (octadecyl group), but differs from the column in Example 1 in which both functional groups are individually bonded to the surface of the carrier in that a functional group in which a weak anion exchange group is further bonded to the end of the hydrophobic group is bonded to the carrier (silica gel). Except for the above points, the polypeptide derived from the allergen protein and the sulfurous acid derivative (hydroxymethanesulfonic acid) in the sample were simultaneously analyzed in the same manner as in Example 1. The analysis results (chromatogram) of Comparative Example 2 are shown in FIG. 3.

[0047] [Reference example] A reversed-phase triple mixed-mode column (product name "Scherzo SM-C18MF", manufactured by Intact Co., Ltd.) having a weak cation exchange group, a weak anion exchange group, and an octadecyl group (hydrophobic group) was used as the HPLC column. The column used in the Reference Example differs from the column in Example 1, which does not have a weak cation exchange group, in that it has a weak cation exchange group. Apart from the above, the simultaneous analysis of the allergen protein-derived polypeptide and the sulfurous acid derivative (hydroxymethanesulfonic acid) in the sample was carried out in the same manner as in Example 1. The analysis results (chromatogram) of the Reference Example are shown in FIG. 4.

[0048] [Control Example] Except for using the allergen-free curry roux used in the above (Preparation of food to be analyzed) as the sample, the analysis was carried out in the same manner as in Example 1. The analytical results (chromatogram) of the control example are shown in FIG.

[0049] In Figures 1 to 5, (a) is a chromatogram for polypeptides derived from crustacean (specifically, shrimp) allergens, (b) is a chromatogram for polypeptides derived from wheat (specifically, wheat flour) allergens, (c) is a chromatogram for polypeptides derived from buckwheat (specifically, buckwheat flour) allergens, (d) is a chromatogram for polypeptides derived from egg (specifically, chicken egg) allergens, (e) is a chromatogram for polypeptides derived from milk (specifically, cow's milk) allergens, (f) is a chromatogram for polypeptides derived from walnut allergens, and (g) is a chromatogram for hydroxymethanesulfonic acid, a type of sulfurous acid derivative. As shown in Figures 1(a) to (h), the analytical method of Example 1 was able to detect all target substances with high accuracy, demonstrating that the method is suitable for the simultaneous analysis of polypeptides and sulfite derivatives in a sample. The analytical method of Comparative Example 1 was able to detect the sulfite derivatives well (see Figure 2(h)), but was unable to retain a portion of the polypeptide derived from the allergen protein (see Figures 2(b), (d), and (g)), indicating that the method is unsuitable for the simultaneous analysis of polypeptides and sulfite derivatives in a sample. As shown in Figures 3(a) to (h), the analytical method of Comparative Example 2 is unable to retain either the polypeptide derived from the allergen protein or the sulfite derivative, and is therefore unsuitable for the simultaneous analysis of both components. The analytical method of the Reference Example was able to detect the sulfite derivatives well (see FIG. 4(h)), but was unable to retain the polypeptide derived from the allergen protein or exhibited significant peak tailing (see FIGS. 4(a)-(g)), indicating that the method is unsuitable for the simultaneous analysis of both components. It is presumed that the analytical method of the Reference Example resulted in this result because a column having a weak cation exchange group in addition to a weak anion exchange group and an octadecyl group was used for an acidic mobile phase containing a volatile acid. It is presumed that the column used in the Reference Example may be capable of simultaneous analysis of both components depending on the mobile phase used.

Claims

1. A method for simultaneously analyzing a polypeptide and an anionic low molecular weight compound in sample analysis using a liquid chromatograph-mass spectrometer, in which a reversed-phase column having weak anion exchange groups and hydrophobic groups individually bonded to the surface of a carrier is used as the column in the liquid chromatograph.

2. 2. The analytical method according to claim 1, wherein the polypeptide has 50 or less amino acid residues, and the anionic low-molecular-weight compound has a weight-average molecular weight of 1,000 or less.

3. 3. The analytical method according to claim 1, wherein the polypeptide is a peptide produced by enzymatic degradation of a protein, and the anionic low-molecular-weight compound has an acid dissociation constant pKa in water of 7 or less.

4. 3. The analysis method according to claim 1, wherein the polypeptide is an allergen protein and the anionic low-molecular-weight compound is a sulfite derivative.

5. 3. The analytical method according to claim 1, wherein the weak anion exchange group is a tertiary amino group.

6. The analytical method according to claim 1 or 2, wherein the hydrophobic group is an octadecyl group.

7. 3. The analytical method according to claim 1, wherein the mobile phase in the liquid chromatograph is a solution comprising water containing a volatile acid and an organic solvent.

Images