FLUORESCENCE DERIVATIZATION REAGENT AND AMINE ANALYSIS METHOD

Abstract

The present invention relates to a method of analyzing a polyamine having a plurality of amino groups in the same molecule or a monoamine having an amino group and a phenolic hydroxyl group in the same molecule on the basis of the measurement result of the intensity of excimer fluorescence emitted as a result of the derivatization of the polyamine or the monoamine with a fluorescence derivatization reagent having a pyrene group. The method is characterized in that the fluorescence derivatization reagent comprises 2-chloro-4-methoxy-6-(4-(pyren-1-yl) butoxy)-1, 3, 5-triazine represented by the formula (2).

##STR00001##

Claims

1. A fluorescence derivatization reagent for fluorescently derivatizing a polyamine having a plurality of amino groups in a same molecule or a monoamine having an amino group and a phenolic hydroxyl group in a same molecule, the fluorescence derivatization reagent comprising a compound represented by the following general formula (1): ##STR00008## wherein X represents a halogen element, R.sub.1 represents an alkyl group, and R.sub.2 represents an alkyl chain.

2. The fluorescence derivatization reagent as recited in claim 1, wherein the compound is 2-chloro-4-methoxy-6-(4-(pyren-1-yl) butoxy)-1, 3, 5-triazine represented by the following formula (2). ##STR00009##

3. An amine analysis method of analyzing a polyamine having a plurality of amino groups in a same molecule or a monoamine having an amino group and a phenolic hydroxyl group in a same molecule on a basis of a measurement result of intensity of excimer fluorescence emitted as a result of derivatization of the polyamine or the monoamine with a fluorescence derivatization reagent having a pyrene group, wherein the fluorescence derivatization reagent comprises a compound represented by the general formula (1): ##STR00010## wherein X represents a halogen element, R.sub.1 represents an alkyl group, and R.sub.2 represents an alkyl chain.

4. The amine analysis method as recited in claim 3, wherein the fluorescence derivatization reagent comprises a 2-chloro-4-methoxy-6-(4-(pyren-1-yl) butoxy)-1, 3, 5-triazine represented by the following general formula (2). ##STR00011##

5. A compound represented by the following general formula (1): ##STR00012## wherein X represents a halogen element, R.sub.1 represents an alkyl group, and R.sub.2 represents an alkyl chain.

6. The compound as recited in claim 5, wherein the compound is represented by the following formula (2). ##STR00013##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a reaction formula of 2, 4-dichloro-6-methoxy-1, 3, 5-triazine and 1-pyrenebutanol showing a synthesis example of CMPT according to one embodiment of the present invention.

[0023] FIG. 2 is a graph showing a relationship between a reaction time and fluorescence intensity when excimer fluorescence intensity was detected after fluoresamine derivatization of a histamine by dissolving a histamine ACN solution in a DMF solution containing pyridine and CMPT.

[0024] FIG. 3 is a graph showing a relationship between a concentration of CMPT and fluorescence intensity when excimer fluorescence intensity was detected after fluoresamine derivatization of a histamine by dissolving a histamine ACN solution in a DMF solution containing pyridine and CMPT.

[0025] FIGS. 4(a), 4(b) and 4(c) show HPLC analysis results (FIG. 4(a)), HPLC conditions (FIG. 4(c)) and MS results (FIG. 4(b)) when histamine was fluorescently derivatized with CMPT at a CMPT concentration of 20 mM and a reaction time of 20 minutes.

[0026] FIG. 5 is a graph showing a relationship between excimer fluorescence intensity and a storage time.

[0027] FIG. 6(a) shows an image showing a state in which soy sauce is separated into an aqueous layer and an acetonitrile layer, FIG. 6(b) shows a graph showing a relationship between a sodium hydroxide concentration and a recovery rate, and FIG. 6(c) shows a graph showing a difference in recovery rate when using heated ACN and unheated ACN.

[0028] FIG. 7 shows pretreatment conditions of a histamine analysis method according to this example.

[0029] FIG. 8 is a chromatogram showing a result of histamine detection using the histamine analysis method according to this example.

[0030] FIG. 9 shows a reaction between histamine and CMPT.

EMBODIMENT FOR CARRYING OUT THE INVENTION

[0031] A compound according to the present invention is a compound useful as an excimer fluorescence derivatization reagent represented by the following general formula

##STR00005##

[0032] In the general formula (1), X represents a halogen element, R.sub.1 represents an alkyl group, R.sub.2 represents an alkyl chain. In particular, 2-chloro-4-methoxy-6-(4-(pyren-1-yl) butoxy)-1, 3, 5-triazine (CMPT) represented by the following formula (2) in which X denotes Cl (chlorine), R.sub.1 denotes a methyl group, and R.sub.2 denotes a butyl chain) is excellent in reactivity with a polyamine and in storage stability.

##STR00006##

[0033] Hereinafter, examples of a polyamine analysis method using CMPT will be described. In the following, the case in which a histamine is analyzed will be mainly described, which is also applicable to a polyamine other than a histamine, or a monoamine having an amino group and a phenolic hydroxyl group in the same molecule such as a tyramine.

EXAMPLES

(1) Production Method of CMPT

[0034] As shown in FIG. 1, 1.2 equivalents of Collidine was added to 1 equivalent of 2, 4-dichloro-6-methoxy-1, 3, 5-triazine and 1 equivalent of 1-pyrenebutanol, and the mixture was left at room temperature for 24 hours. This solution was separated by a high performance liquid chromatograph (HPLC apparatus) and then purified by recrystallization. When the obtained product was confirmed by an NMR analysis and a mass spectrometry (MS), it was identified as a compound (CMPT) represented by the following formula.

##STR00007##

[0035] In CMPT, the pyrene group is a fluorescence site and the Cl group is a reaction site.

(2) Consideration of Solution for CMPT

[0036] To search for bases and solvents suitable for an excimer fluorescence derivatization reaction of a histamine using CMPT, the state of a solution in which CMTP was dissolved in three kinds of bases (potassium carbonate, Collidine, Pyridine) and seven solvents (DMF (N, N-dimethylformamide, DMSO (Dimethyl sulfoxide), THF (tetrahydrofuran), ACN (acetonitrile), ethyl acetate, chloroform, acetone) was observed. The results are shown in Table 1. x in Table 1 indicates a combination of a base and a solvent in which CMPT was not completely dissolved.

TABLE-US-00001 TABLE 1 Base/ Ethyl Chloro- solvent DMF DMSO THF ACN acetate form Acetone Potassium X X X X X X carbonate Collidine X X X X X X Pyridine X X X X X X

[0037] From Table 1, it is understood that DMF is excellent as a solvent for CMPT. There was no difference in solubility of CMPT in any of potassium carbonate, collidine, and pyridine. However, in a solution using potassium carbonate, precipitation of CMPT was observed when water was added. Considering that water is added by a pretreatment for separating a histamine in soy sauce as described later, it is not preferable to use potassium carbonate. Further, the excimer fluorescence intensity when a histamine was fluorescently derivatized with a solution using a pyridine as a base and a solution using collidine was measured, the intensity of excimer fluorescence was larger in pyridine than in collidine. Therefore, in this example, pyridine was adopted as a base used for the histamine analysis.

(3) Consideration of Reaction Time of Dimerization of Excimer Fluorescence and Concentration of CMPT

[0038] FIGS. 2 and 3 show the results of detecting the excimer fluorescence intensities by dissolving 10 L of a histamine ACN solution in 3 L of pyridine and 20 L of DMF solution of CMPT and fluorescently derivatizing the histamine and then separating the fluorescence derivatization histamine by HPLC. FIG. 2 shows the relationship between the reaction time and the fluorescence intensity (area) when the concentration of CMPT was set to 20 mM and the reaction temperature was set to 50 C. FIG. 3 shows the relationship between the concentration of CMPT and the fluorescence intensity when the reaction temperature was set to 50 C. and the reaction time was set to 20 minutes. As can be seen from FIG. 2, the fluorescence intensity gradually increased until the reaction time reached 20 minutes, but the fluorescence intensity hardly changed after 20 minutes. As can be seen from FIG. 3, the fluorescence intensity gradually increased in the concentration range up to 20 mM, but the fluorescence intensity hardly changed in the concentration range larger than 20 mM. From the viewpoint of shortening the time required for the histamine analysis and reducing the amount of CMPT used for the histamine analysis, the reaction time for excimer fluorescence derivatization in the histamine analysis according to this example was set to 20 minutes and the CMPT concentration was set to 20 mM.

[0039] The results of HPLC analysis of the histamine (standard product of 50 ppm) performed under the conditions are shown in FIG. 4(a), and the conditions of HPLC at this time are shown in FIG. 4(b). Further, from FIG. 4(c) showing the results of the MS analysis of substances in which excimer fluorescence was detected by HPLC analysis, it was confirmed that substances in which 2 molecules of CMPT bound to 1 molecule of histamine were generated.

(4) Confirmation of Storage Stability of CMPT in Solution State

[0040] CMPT was dissolved in DMF to prepare a solution. After storing this solution at room temperature, a histamine was fluorescently derivatized with this solution. The resulting fluorescence derivatization histamine was separated using HPLC and the intensity of the excimer fluorescence was obtained. As a result, the relationship between the excimer fluorescence intensity and the storage time became as shown in FIG. 5. As can be seen from FIG. 5, even in the state of a DMF solution, CMPT did not deteriorate in reactivity with a histamine for more than 40 days, and was capable of fluorescently derivatizing. From this, it is understood that CMPT is superior to a conventional excimer fluorescence derivatization reagent in storage stability in a solution state.

(5) Consideration of Pretreatment Conditions

[0041] When amines contained in soy sauce are analyzed by LC-MS/MS, a pretreatment for separating amines from other components (salt, organic acid, protein, melanoidin, amino acids, etc.) is necessary. As this pretreatment method, the method described in Non-Patent Document 4 is known. In this method, 0.1 M of magnesium sulfate (20 L), 1M of sodium hydroxide (20 L, pH 10), and 320 L of acetonitrile were added to soy sauce (20 L), and centrifuged to separate an acetonitrile layer containing amines and an aqueous layer containing other components (See FIG. 6(a)). Magnesium sulfate is added to salt out protein, etc., and sodium hydroxide is added to adjust the pH and increase the amount of molecule type histamine. By reference to this pretreatment method, the present inventors considered pretreatment conditions suitable for fluorescently derivatizing a histamine with CMPT.

[0042] Specifically, the proportion (recovery rate (%)) of the histamine recovered in the acetonitrile layer when a sample containing a histamine having a known concentration was separated into an acetonitrile layer and an aqueous layer was investigated for both of a case in which the concentration of the sodium hydroxide was changed to 1 to 4M FIG. 6(b)) and a case in which the unheated acetonitrile and heated acetonitrile were used (FIG. 6(c)).

[0043] As can be understood from FIG. 6(b), when the sodium hydroxide concentration was 1M, the recovery rate of the histamine was 25%, but in the range of 2M to 4M, the recovery rate of the histamine was 45%. From the above, it seems that in the sodium hydroxide used for a pretreatment of soy sauce, the suitable concentration range is 2M to 4M.

[0044] Further, as can be understood from FIG. 6(c), when the heated ACN was used, the peak shape of the fluorescence derivatization histamine was in a bilaterally symmetrical shape, but when the unheated ACN was used, the peak shape of the fluorescence derivatization histamine was in a bilaterally asymmetrical shape. This is probably because the protein in the sample could not be sufficiently removed when using the unheated ACN and the fluorescence peak of the substance derived from the protein overlapped with the fluoresce peak derived from the histamine. Therefore, it seems preferable to use the heated ACN rather than the unheated ACN for pretreatment of soy sauce.

[0045] On the basis of the results of the aforementioned (1) to (5), the pretreatment conditions of soy sauce in the histamine analysis according to this example were determined as contents shown in FIG. 7. Here, octanediamine was used as the internal standard (Internal Standard (I.S)). Further, a DMF solution (20 mM) of CMPT was added to the acetonitrile layer separated to the aqueous layer by this pretreatment to fluorescently derivatize the amine (histamine), and thereafter excimer fluorescence intensity detection was detected by the HPLC analysis. The results is shown in FIG. 8. From this result, it was confirmed that histamine was fluorescently derivatized by the reaction shown in FIG. 9 and pyrene excimer was formed. Further, the detection limit (Limit of detection, LOD), the quantitation lower limit (Limit of quantitation, LOQ), linearity, and calibration curve range of the histamine analysis at this time are shown in Table 2. From Table 2, it was found that the histamine analysis according to this example can obtain sufficient linearity and sensitivity for analyzing the histamine in soy sauce.

TABLE-US-00002 TABLE 2 LOD Calibration S/N = 3 LOQ curve Compound (ppm) (ppm) Linearity range Histamine 7.5 25 0.9993 25 to 500

[0046] Next, for a plurality of samples different in histamine concentration, the intra-day variation of the histamine analysis and the inter-day variation of the histamine analysis were examined. The results are shown in Table 3.

TABLE-US-00003 TABLE 3 Addition concentration Precision (%) Accuracy (%) 25 ppm Intra-day 2.05 120.7 Inter-day 6.01 123.5 50 ppm Intra-day 4.85 98.0 Inter-day 100 ppm Intra-day 2.76 96.0 Inter-day 3.07 92.6 250 ppm Intra-day 5.61 95.1 Inter-day 500 ppm Intra-day 3.26 96.9 Inter-day 5.04 93.1 Intra-day n = 6, Inter-day n = 2 6 days

[0047] As can be seen from Table 3, the intra-day fluctuations were 2.05 to 5.61%, and the inter-day fluctuations was 3.07% to 6.01%. Further, the accuracy (trueness) was 92.6 to 123.5%. From the above, the histamine analysis under the aforementioned pretreatment conditions was found to be excellent both in reproducibility and accuracy.

[0048] Next, a quantitative analysis of a histamine was carried out by the histamine analysis method according to this example, using soy sauce of the same type as the soy sauce described in Non-Patent Document 4 as a sample. The results are shown in Table 4. The column at the right end of Table 4 shows the histamine quantification value obtained by the MS analyzer described in Non-Patent Document 4.

TABLE-US-00004 TABLE 4 Him quantification Him value Sample Manufacturing quantification (Document No. Type company value value) 1 Dark soy sauce A 1.34 2.0 2 Dark soy sauce B 241.0 234.5 3 Dark soy sauce C 11.0 14.1 4 Dark soy sauce D 34.1 32.6 5 Dark soy sauce E 768.05 774 6 Dark soy sauce F 1.45 1.2 7 Light soy sauce B 362.1 363.7 8 Light soy sauce G 1.24 1.9 9 Light soy sauce H 9.91 11.1 10 Refermented B 65.7 62.5 soy sauce 11 Saishikomi I 172.3 173 (refermented) soy sauce 12 Saishikomi G 47.0 46.2 (refermented) soy sauce 13 Tamari (rich) J 138.9 131.1 soy sauce

[0049] As can be understood from Table 4, the histamine analysis method according to the present example also yielded the same degree of quantitative value as that of the MS analysis method. From this, it was found that the histamine analysis method according to this example is an effective method of measuring the amount of histamine in soy sauce.

[0050] It should be noted that the aforementioned examples are merely examples of the present invention, and even if appropriate modifications and additions are made within the scope of the gist of the present invention, it is encompassed within the claim of the present application.