Patent Application
20190360983
The present invention relates to a method for determining the origin of glutamic acid in a sample and, in a broader sense, relates to a method for determining the origin of an amino acid.
An amino acid is a one of organic compound having both an amino group and a carboxyl group in one molecule and is contained in many foods. Above all, salts of glutamic acid and aspartic acid are known as typical umami components, and, in particular, glutamates have become to be used in processed food products all over the world with the spread of Japanese food.
Glutamates are industrially produced usually by a fermentation process in which molasses, starch, and so on of sugar cane are fermented to produce glutamic acid, and the glutamic acid is crystallized as monosodium glutamate (MSG) to be easily dissolved in water. MSG and glutamates obtained by hydrolyzing protein contained in, for example, kelp differ from each other in the point that the former (MSG) contains almost no minerals other than sodium, whereas the latter contains minerals (e.g., calcium and potassium) in addition to sodium.
By the way, in recent years, the traceability of food is regarded as important, and there is also a demand for amino acids derived from meat to be removed by religious reasons. In addition, as described above, since MSG contains almost no minerals other than sodium, sodium is prone to be overdosed. Accordingly, there is a demand for suppressing the amount of use of industrial products, such as MSG.
As a method for determining the origin (such as raw material and original production area) of food, a method for determining the origin of glucosamine by elemental analysis-isotope ratio mass spectrometry (EA-IRMS) has been disclosed (Patent Literature 1). However, this method is not supposed to analyze organic compounds having both an amino group and a carboxyl group in one molecule, such as amino acids. Consequently, there is no disclosure about the process of purifying a sample, and it is difficult to accurately measure the stable isotope ratio of an amino acid.
In addition, as a method for measuring the stable isotope ratio of an amino acid, a method in which an amino acid is derivatized to pivaloyl/isopropyl ester for measuring the stable isotope ratio of the amino acid by gas chromatography-isotope ratio mass spectrometry (GC-IRMS) has been disclosed (Non Patent Literature 1 and Patent Literature 2). This method can measure the isotope ratios of multiple amino acids at a time, and the accuracy thereof is high. Therefore, it is a very excellent method.
However, in this method, carbon not derived from amino acid but derived from pivaloyl chloride and isopropanol are introduced in the process of derivatization. Accordingly, the stable carbon isotope ratio cannot be accurately measured.
The present invention provides, in view of importance of traceability, a method for determining the origin of an amino acid and a method for measuring the same.
The present inventors have made it possible to measure the stable isotope ratio of an amino acid, with a considerably higher accuracy than that of conventional methods, by measuring the stable carbon isotope ratio (hereinafter referred to as “δ13C”) of the amino acid by elemental analysis-stable isotope ratio mass spectrometry (hereinafter referred to as “EA-IRMS”) and measuring the stable nitrogen isotope ratio (hereinafter referred to as “δ15N”) of the amino acid by gas chromatography-stable isotope ratio mass spectrometry (hereinafter referred to as “GC-IRMS”), and also made it possible to determine the origin of an amino acid.
According to the present invention, it is possible to accurately measure the stable isotope ratio of an amino acid and to determine the origin of an amino acid.
The present invention makes it possible to measure the stable isotope ratio of an amino acid with a considerably higher accuracy than that of conventional methods by measuring the 613C of the amino acid by EA-IRMS and measuring the δ15N by GC-IRMS. In addition, the origin of an amino acid is determined by comparing the stable isotope ratio of the amino acid whose origin is unclear with the stable isotope ratios of amino acids whose origins are clear.
In the present specification, the term “amino acid” refers to a one of organic compound having both an amino group and a carboxyl group as functional groups, and examples thereof include aspartic acid, glutamic acid, and glycine. In addition, the term “amino acid” in the present invention includes amino acid salts unless otherwise stated. Specifically, for example, monosodium glutamate, calcium glutamate, and potassium glutamate are treated as amino acids.
The present invention is characterized in that δ13C is measured by elemental analysis-stable isotope ratio mass spectrometry (EA-IRMS) and δ15N is measure by gas chromatography-stable isotope ratio mass spectrometry (GC-IRMS). Methods for measuring δ13C and δ15N will now be specifically described.
The sample of the present invention is amino acid-containing food (including umami seasoning, reagent, and so on). When protein contained in a sample is not decomposed into amino acids, the protein is required to be hydrolyzed in advance as pretreatment. The protein may be hydrolyzed by any method, and a known method can be used. For example, a method in which an appropriate amount of hydrogen chloride is added to a sample and the sample is heated to decompose the protein into amino acids can be used.
The stable isotope ratio in a sample is defined by per mil deviation (δ value, unit: ‰ (per mil)) against the stable isotope ratio of an international standard substance shown in the following expression 1:
δ value=[(R sample/R international standard substance)−1]×1000(‰) Expression 1:
As the standard substances, Cretaceous Pee Dee layer belemnite carbonate is used for carbon, and nitrogen gas in the atmosphere is used for nitrogen, and the δ values are written as δ13C and δ15N, respectively.
As described above, the δ15N of an amino acid can be measured by gas chromatography-stable isotope ratio mass spectrometry (GC-IRMS) (see Non Patent Literatures 1 and 2). GC-IRMS can measure stable isotope ratios of multiple amino acids at a time, and the accuracy thereof is very high. Therefore, it is a very useful method.
The δ15N is preferably measured through an extraction step (A1), a degreasing step (A2), a cation exchange step (A3), a derivatization step (A4), and a GC-IRMS step (A5). However, the extraction step (A1) and the degreasing step (A2) are not essential steps. For example, the degreasing step (A2) is unnecessary for a sample containing almost no lipids.
First, the extraction step (A1) will be described. In the extraction step, a hydrogen chloride solution or the like is added to a sample, the mixture is stirred and centrifuged, and the precipitated insoluble components are removed. If the sample contains a large amount of insoluble components, a trouble, such as column clogging, may be caused in the subsequent steps. Accordingly, it is preferable to remove most insoluble components in advance by centrifugation.
Next, in the degreasing step (A2), the sample is degreased by liquid-liquid extraction using water and an organic solvent (for example, “dichloromethane/n-hexane”).
Next, the cation exchange step (A3) will be described. In the cation exchange step, the amino acid in the sample is adsorbed to a cation-exchange column, the components (impurities) not adsorbed to the column are washed out, and the amino acid is desorbed from the column and is collected. The cation-exchange column (column-like shape) can be replaced by a cation-exchange resin (amorphous shape) or a cation-exchange membrane (membrane-like shape).
The cation exchange step (A3) will be described in more detail. First, for example, hydrogen chloride is added to a sample for cationization of an amino acid. Subsequently, the sample (containing the cationized amino acids) is passed through a cation-exchange column. Consequently, the cationized amino acid is adsorbed to the column, and uncationized components (such as carbohydrates) are not adsorbed to the column. Accordingly, the components not adsorbed to the column can be removed by passing, for example, distilled water through the column.
Next, weakly basic aqueous ammonia is added to the column to desorb and collect the amino acid from the column. On this occasion, since the amino acid is dissolved in the aqueous ammonia, the solution is evaporated to dryness by a known method, such as nitrogen-blowing high-speed concentration in order to remove the aqueous ammonia.
Next, the derivatization step (A4) will be described. In order to perform accurate measurement by gas chromatography (GC), generally, a sample is required to be gasified at about 300° C. or less and converted to a derivative that is not thermally decomposed. In this regard, amino acids are organic compounds having both an amino group and a carboxyl group in one molecule and therefore have strong ionic bonds and low volatility. Accordingly, in order to obtain accurate data by GC-IRMS, amino acids are required to be derivatized to increase the volatility.
Examples of a known method for derivatization include a tert-butyl dimethyl silyl derivative method, a trifluoroacyl/isopropyl ester derivative method, a pentafluoroacyl/isopropyl ester derivative method, a pivaloyl/isopropyl ester derivative method, and an ethoxycarbonyl/ethyl ester derivative method.
In the present invention, among these derivatization methods, preferred is the pivaloyl/isopropyl ester derivative method. In the pivaloyl/isopropyl ester derivative method, since the derivative group does not contain silicon, fluorine, and the like and has a relatively large number of carbon atoms, the derivatized amino acid is relatively stable and is hardly thermally decomposed, and the method is therefore suitable for measurement by gas chromatography.
Finally, the GC-IRMS step (A5) will be described. Typical specification and measurement procedure of GC-IRMS will be described here, but they are not limited thereto. First, the amino acid derivative described above is adjusted to an appropriate concentration with, for example, dichloromethane and is poured into a column together with a carrier gas (e.g., helium gas). The amino acid derivative is carried inside the column together with the carrier gas, and the moving speed is different among compounds. Therefore the arrival times of the components (for example, a glutamic acid derivative and an aspartic acid derivative) at the outlet of the column are different from each other to allow separation of the components from one another. The separated amino acid derivatives are continuously introduced into a reactor (combustion furnace/reduction furnace) directly connected to the column.
The introduced amino acid derivatives are each oxidatively decomposed to N2, NxOy (nitrogen oxide), H2O, and CO2 in the combustion furnace (temperature: 800° C. to 1150° C.), and the NxOy is further reduced to nitrogen gas (N2) in the reduction furnace (temperature: 550° C. to 650° C.). Furthermore, H2O is removed by a water-permeable filter, CO2 is removed by liquid-nitrogen trap, N2 is then introduced into an IRMS system together with a carrier gas, and the isotope ratio of N2 is measured. Since CO2 generates CO+ (m/z28) by ionization, it is necessary to remove CO2 before the measurement.
A reactor that simultaneously performs oxidation and reduction of amino acid derivatives may be used. In such a case, the temperature of the reactor (reactor temperature) is about 800° C. to 1150° C.
From the viewpoint of accurately measuring an isotope ratio, the temperature of the combustion furnace is preferably 950° C. to 1050° C. Insufficient oxidation causes generation of CO gas of the same mass as N2 gas, which becomes an obstacle to accurate measurement of the isotope ratio. In contrast, since peroxidation increases the rate of generation of nitrogen oxides other than N2, there is a risk that not all peroxides are reduced in the reduction furnace, which becomes an obstacle to accurate measurement of the isotope ratio.
As described above, the δ15N of an amino acid can be measured by GC-IRMS. However, the measurement of the stable isotope ratio of an amino acid by GC-IRMS requires derivatization of the amino acid and introduction of carbon derived from a derivatizing reagent (such as pivaloyl group). Accordingly, the result of δ13C measurement is affected by carbon derived from the derivatizing reagent, and the accuracy thereof is reduced. A reduction in the accuracy becomes significant with an increase in the molecular weight of the derivatizing reagent, in other words, the accuracy is reduced by increasing the thermal stability of the amino acid derivative. Accordingly, GC-IRMS cannot accurately measure the δ13C.
Due to the above-described reasons, in the present invention, it is necessary to measure the δ13C of an amino acid by elemental analysis-stable isotope ratio mass spectrometry (EA-IRMS). The measurement by EA-IRMS will now be described in detail.
In the measurement by GC-IRMS, even if the sample contains impurities that do not volatilize at about 300° C., the isotope analysis is not affected, and a specific amino acid (e.g., glutamic acid) can be isolated by gas chromatography (GC). Accordingly, the burden by purification of the sample is low.
In contrast, the measurement by EA-IRMS is largely affected by impurities that burn at about 1000° C. and amino acids other than the amino acid (e.g. glutamic acid) as the measurement target. Accordingly, the measurement of the δ13C of a specific amino acid by EA-IRMS is preferably performed through an extraction step (B1), a degreasing step (B2), a column purification step (B3), a cation exchange step (B4), a separation step (B5), and an EA-IRMS step (B6).
First, the extraction step (B1) will be described. In the extraction step, a hydrogen chloride solution or the like is added to a sample, the mixture is stirred and centrifuged, and the precipitated insoluble components are removed. If the sample contains a large amount of insoluble components, a trouble, such as column clogging, may be caused in the subsequent steps. Accordingly, it is preferable to remove most insoluble components in advance by centrifugation.
Next, the degreasing step (B2) will be described. In the degreasing step, the sample is degreased by liquid-liquid extraction using water and an organic solvent (for example, “dichloromethane/n-hexane”).
Next, the column purification step (B3) will be described. The column purification step (B3) is composed of a reverse phase chromatography purification step (B3-1) and a decolorization step (B3-2).
In the reverse phase chromatography purification step (B3-1), low-polar substances, such as lipids, are separated by reverse phase chromatography. Since the polarity of lipids is lower than that of glutamic acid, lipids can be efficiently separated by reverse phase chromatography (the elution speed increases with an increase in the polarity). The conditions for the reverse phase chromatography are, for example, an ODS column as the stationary phase and water or alcohol as the mobile phase.
Next, in the decolorization step (B3-2), the dissolved sample is passed through activated carbon to remove a compound having a cyclic structure, such as a pigment, contained in the sample. Since compounds having a cyclic structure are often colored, the term “decolorization” is used. However, a compound having a cyclic structure is removed by this step, even if the compound is colorless. Although the activated carbon may have any shape (such as, amorphous, column-like, or membrane-like shape), a column-like shape is preferred in consideration of workability and so on.
Next, the cation exchange step (B4) will be described. The cation exchange step (B4) is a purification step in which the amino acid in the sample is adsorbed to a cation-exchange column, the impurities not adsorbed to the column is washed out, and the amino acid is desorbed and collected from the column finally. Specifically, the purification method is as described in the “cation exchange step (A3)”.
Next, the separation step (B5) will be described. In the separation step (B5), only a specific amino acid is separated and purified from the extracted and degreased sample by hydrophilic interaction liquid chromatography (HILIC). Since the polarity of amino acids is too high, separation and purification by reverse phase chromatography are difficult. Accordingly, it is preferable to separate and purify a specific amino acid by HILIC. The conditions for the HILIC are, for example, a gel prepared by binding an amino group to a polymer as the stationary phase and an aqueous ammonium hydrogen carbonate solution/methanol mixture solution as the mobile phase.
Finally, the EA-IRMS step (B6) will be described. Here, typical specification and measurement procedure of EA-IRMS will be described, but they are not limited thereto.
It is repetition, but since the EA-IRMS system does not include a mechanism for separating amino acids, it is necessary to use a specific amino acid separated and purified from a sample in advance for the measurement. In the following description about the
EA-IRMS step (B6), the amino acid and the amino acid salt to be used for EA-IRMS are collectively referred to as “amino acid sample”.
The amino acid sample introduced into an elemental analyzer (EA) is oxidatively decomposed to N2, NxOy (nitrogen oxide), H2O, and CO2 in the combustion furnace, and NxOy is further reduced to nitrogen gas (N2) in the reduction furnace (temperature: 550° C. to 700° C.). H2O is removed by water trap (Mg(ClO4)2), and CO2 and N2 are then introduced into an IRMS system together with a carrier gas, and the stable isotope ratio of CO2 is measured.
Here, the reasons that δ13C cannot be accurately measured by GC-IRMS and the reasons that δ15N cannot be accurately measured by EA-IRMS will be collectively described.
(Reasons that δ13C Cannot be Accurately Measured by GC-IRMS)
As described above, in order to measure the stable isotope ratio of an amino acid by GC-IRMS, derivatization of the amino acid is required, and a carbon atom derived from a derivatizing reagent (such as pivaloyl group) is introduced into the amino acid. Accordingly, when the δ13C of an amino acid is measured by GC-IRMS, correction calculation is required, resulting in a reduction in the accuracy. In addition, isotope fractionation of δ13C may occur during the derivatization or separation with a GC column.
(Reasons that δ15N Cannot be Accurately Measured by EA-IRMS)
Ammonia derived from ammonium hydrogen carbonate used as the mobile phase is added to the carboxyl group of amino acid crystals obtained in a pretreatment step to form a salt, resulting in introduction of a nitrogen atom. Accordingly, correction calculation is required, resulting in a reduction in the accuracy. In addition, isotope fractionation of δ15N may occur during the purification by reverse phase chromatography in the pretreatment step or the separation step by HILIC.
The isotope fractionation is a change in the stable isotope ratio through chemical or physical process. In order to accurately analyze the stable isotope ratio of a sample, the pretreatment method needs to be selected to cause isotope fractionation as low as possible.
A procedure for determining the origin of an amino acid will be described using glutamic acid as an example. The origin of glutamic acid can be determined by (1) measuring the stable isotope ratios (δ13C and δ15N) of glutamic acid whose origin is clear, then (2) measuring the stable isotope ratios of glutamic acid whose origin is unclear contained in a sample, and finally (3) comparing the stable isotope ratios of both samples. Since the isotope ratios differ depending on the production area and the raw material, it is preferable to accumulate data of glutamic acid whose origin is clear as much as possible.
A method for measuring the stable isotope ratio of an amino acid and a method for determining the origin of the amino acid will now be described using glutamic acid as an example, but the present invention is not limited to the following examples.
Distilled water and 12 M hydrogen chloride were added to a sample, followed by treatment at 110° C. for 12 to 24 hours to decompose the protein contained in the sample into amino acids. Subsequently, the sample was centrifuged, and the supernatant was collected. The amounts of the sample, distilled water, and hydrogen chloride were appropriately changed according to the amount of glutamic acid that can be separated and purified from a sample.
The process for measuring the stable nitrogen isotope ratio (δ15N) of glutamic acid is as follows.
Distilled water and 1 M hydrogen chloride were added to a sample, followed by stirring for 10 minutes and then centrifugation. The precipitated insoluble components were removed.
To the aqueous solution from which the insoluble components were removed was added a dichloromethane/n-hexane mixed solvent to perform liquid-liquid extraction. On this occasion, since an amino acid and fat or oil are selectively dissolved in the aqueous layer and the organic layer, respectively, the fat or oil was removed by collecting the aqueous layer only.
The amino acid was extracted under the following conditions:
Sample-cationizing agent: hydrogen chloride,
Stationary phase: “AG50W-X8 200-400 mesh Resin” manufactured by Bio-Rad Laboratories, Inc., and
Mobile phase: distilled water for removing impurities, and 10% ammonia solution for collecting the amino acid.
2-Propanol/thionyl chloride (4:1) was added to the sample, followed by heating at 110° C. for 2 hours to esterify the carbonyl group of the amino acid. Subsequently, pivaloyl chloride/dichloromethane (1:4) was added thereto, followed by heating at 110° C. for 2 hours to pivaloylate the amino group to obtain an amino acid derivative.
The amino acid derivative was introduced into a GC-IRMS system, and the MSN was measured. The specification and the measurement conditions of the GC-IRMS system (manufactured by Thermo Fisher Scientific) were as shown in Table 1.
The stable carbon isotope ratio (δ13C) of glutamic acid contained in a sample was measured by the following procedure.
Distilled water and 1 M hydrogen chloride were added to a sample, followed by stirring for 10 minutes and then centrifugation. The precipitated insoluble components were removed.
TO the aqueous solution from which the insoluble components were removed was added a dichloromethane/n-hexane mixed solvent to perform liquid-liquid extraction. On this occasion, since an amino acid and fat or oil are selectively dissolved in the aqueous layer and the organic layer, respectively, the fat or oil was removed by collecting the aqueous layer only.
In the present invention, in order to simplify the purification step, the reverse phase chromatography step (B3-1) and the decolorization step (B3-2) were simultaneously performed. Specifically, porous spherical silica gel whose surface was modified with an octadecylsilyl group was placed in a polypropylene chromatographic column and was leveled until the silica gel interface became horizontal. Subsequently, column activated carbon was placed in the column to prepare a column for purification.
A sample was added to this column, and water was further added to the column as the mobile phase to remove lipids and pigments.
The amino acid was extracted under the following conditions:
Sample-cationizing agent: hydrogen chloride,
Stationary phase: “Amberlite IR120BH” manufactured by The Dow Chemical Company, and
Mobile phase: distilled water for removing impurities, and 10% ammonia solution for collecting the amino acid.
The resulting extract was vacuum-concentrated (60° C.) and was then dissolved in a distilled water/methanol mixture solution, followed by clarification by centrifugation and a hydrophilic PTFE filter (“Millex-LH” manufactured by MilliporeSigma).
Ammonium glutamate only was separated and purified by hydrophilic interaction liquid chromatography (HILIC). The conditions for HILIC were as follows:
Apparatus: “LC20AP” manufactured by Shimadzu Corporation,
Stationary phase: “Asahipak NH2P” manufactured by Showa Denko K.K., and
Mobile phase: aqueous ammonium hydrogen carbonate solution/methanol.
The δ13C was measured by EA-IRMS using 0.5 mg of ammonium glutamate placed on tin foil. The specification and the measurement conditions of the EA-IRMS system (manufactured by Thermo Fisher Scientific) were as shown in Table 2.
The δ13C and δ15N of glutamic acid contained in samples 1 to 34 were measured according to the conditions shown in Tables 3 to 5.
First, samples 1 to 13 will be described. MSG 1 to 4 were monosodium glutamate (MSG) produced by fermenting carbohydrate contained in sugar cane and were acquired in Japan or Brazil. Similarly, MSG 5 to 10 were produced from maize, as a raw material, acquired in Brazil, China, Taiwan, or Thailand; and MSG 11 to 13 were produced from tapioca, as a raw material, acquired in India, Vietnam, or Thailand.
Samples 1 to 13 were monosodium glutamate (MSG) that had been separated and purified and were therefore not required to be purified again. Furthermore, unlike ammonium glutamate, there is no risk of decreasing the accuracy due to ammonia during the measurement. Accordingly, the δ13C and δ15N of samples 1 to 13 were measured by EA-IRMS only.
Next, samples 14 to 22 will be described. Samples 14 to 22 were seasonings containing glutamates obtained by hydrolysis of protein contained in food. The raw material of seasonings 1 to 3 was maize, that of seasonings 4 to 6 was soybean, that of seasoning 7 was wheat, that of seasoning 8 was beet, and that of seasoning 9 was bonito. Since the samples had already been hydrolyzed, hydrolysis was not performed again.
Lastly, samples 23 to 34 will be described. Samples 23 to 34 were tomato, Chinese cabbage, broccoli, dried shiitake mushroom, pork, kelp, cheese, chicken, sardine, and scallops. The degreasing step was performed or not performed depending on the amount of lipids.
In
According to the analysis results of measurement examples, glutamic acid was roughly classified into (1) glutamic acid prepared by fermenting carbohydrate derived from C3 plant (such as tapioca and wheat), (2) glutamic acid prepared by fermenting carbohydrate derived from C4 plant (such as sugar cane and maize), (3) glutamic acid prepared by hydrolyzing protein contained in C3 plant, (4) glutamic acid prepared by hydrolyzing protein contained in C4 plant or meat obtained by feeding with C4 plant, (5) glutamic acid prepared by hydrolyzing protein contained in kelp, and (6) glutamic acid prepared by hydrolyzing protein contained in fish or shellfish.
The δ13C and δ15N of glutamic acid in samples 101 to 113 in which the origins of the raw materials were unclear were measured. Unlike the cases of analysis examples 1 to 34, since the impurities (such as lipids and amino acids other than glutamic acid) contained in samples 101 to 113 were not clear, all purification steps were performed.
In
The origins of Examples 1 to 13 predicted by comparison with analysis examples 1 to 34 were as follows.
In general, since large fish or shellfish, which are higher in the food chain, tend to have higher δ15N, it was predicted that the sample of Example 4 was glutamic acid derived from small fish or shellfish, such as shellfish and sardine, and that the sample of Example 8 was glutamic acid derived from large fish or shellfish, such as bonito and tuna.
In Example 11, since the δ15N was −3.5 to 5‰ and the δ13C was −15‰ or more, it was predicted that the sample was not MSG but glutamic acid derived from C4 plant (such as maize and sugar cane) or meat. In livestock industry, since maize is often used as feed, meat is apt to have an isotope ratio close to those of C4 plant, and it is probably difficult to discriminate the both.
The isotope fractionation of δ13C in the purification steps (B1) to (B5) was verified as follows.
Table 7 shows the results of measurement of δ13C of a reagent, L(+)-monosodium glutamate hydrate (manufactured by FUJIFILM Wako Pure Chemical Corporation, 98%-102%), by EA-IRMS when no purification was performed, when only the extraction step (B1) was performed, when only the degreasing step (B2) was performed, when only the column purification step (B3) was performed, when only the cation exchange step (B4) was performed, when only the separation step (B5) was performed, and when all the purification steps were performed.
According to Table 7, carbon isotope fractionation hardly occurs when the extraction step (B1), the degreasing step (B2), the column purification step (B3), the separation step (B5), and all the purification steps were performed. Accordingly, “liquid-liquid extraction”, “removal of fat or oil component by reverse phase chromatography”, “removal of pigment component with activated carbon column”, and “separation and purification of amino acid by hydrophilic interaction liquid chromatography” are purification methods that hardly cause isotope fractionation and can be used in arbitrary combination thereof for purification of an amino acid.
In contrast, carbon isotope fractionation readily occurs in the cation exchange step. Accordingly, the cation exchange step is preferably performed in combination with another purification step, without being performed alone.
Since nitrogen isotope fractionation hardly occurs in the cation exchange step (Non Patent Literature 3), in the measurement of δ15N, it is not necessary to note isotope fractionation by cation exchange.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2017-019817 | Feb 2017 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2018/003082 | 1/31/2018 | WO | 00 |
