JPH0511572B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a fluorescence detection method using a reagent with a new composition. More specifically, the present invention relates to an improvement in a fluorescence detection method in which a primary amine is converted into an isoindole-type fluorescent compound by the o-phthalaldehyde method, and the resulting fluorescence is detected to quantify the primary amine. [Prior Art] Conventional techniques such as absorption analysis have been used for quantitative analysis of substances, especially trace substances, and although these are simple and excellent methods, their drawback is that their sensitivity is rather low. In contrast, in fluorescence analysis, sensitivity can be easily improved by increasing the sensitivity of the detector and the strength of the light source, and the concentration that can be measured by absorption analysis is generally 10 ^-6 M.
However, it is possible to measure samples up to about 10 ^-9 to ^{10 -10} M. Fluorescence analysis also has excellent selectivity because it is possible to select optimal values for two parameters: excitation wavelength, fluorescence wavelength, and fluorescence wavelength. In this way, fluorescence analysis is excellent for detecting and quantifying minute amounts of substances, but it also requires methods that not only detect the fluorescence of the luminescent substance itself, but also chemically modify non-fluorescent substances or add fluorescent groups to molecules. It is an excellent analysis method that can be easily applied to various substances, such as introducing a fluorophore into a fluorophore and detecting the fluorescence. As a reagent to induce non-fluorescent substances to fluorophores,
There are OPAs that react with primary amines to give fluorophores. OPA, like fluorescein, reacts with primary amines to give a fluorophore, but the presence of 2-mercaptoethanol (hereinafter abbreviated as ME) has been considered essential for the production of fluorophores (M .
Roth: Anal.Chem. vol. 43, p. 880 (1971); SS
Simons and DF Johnson: Jr.Org.Chem.43 volumes
2886 pages (1978)). Application examples include α-amino acids, their metabolic products serotonin, physiologically active amines such as histamine, catecholamines such as norepinephrine, epinephrine, and dopamine, polyamines such as putrescine, spermidine, and spermine, and primary amines such as peptides and amino sugars. An example of quantitative determination using high performance liquid chromatography and fluorescence analysis has been reported. The reagent is water-soluble and does not form a precipitate even when mixed with the column eluent, and can be easily incorporated into automatic analyzers such as automatic amino acid analysis. Sensitivity is also higher than fluorescein [JRBenson and
PEHare: Proc.Nat.Acad.Sci., USA Volume 72 619
Page (1975)]. Reagents used for the reaction are commercially available, such as
0.700 g of OPA, 2.0 ml of ME, 15% Brij35 [polyoxyethylene lauryl ether; Kao Atlas Co., Ltd.
Contains 2.0ml of product name] and 10.0ml of methanol,
Combine these and make potassium borate buffer (PH10.4).
The surfactant Brij35 improves the sensitivity [JRBenson
and PEHare: Proc.Nat.Acad.Sci.USA 72 volumes
619 (1975)], methanol is a solubilizing agent for OPA, and the pH during the reaction must be alkaline, so a potassium borate buffer (PH 10.4) is used. [Problems to be solved by the invention] As described above, although the detection sensitivity of the reagent is high, for example at the nmol to pmol level in the quantification of amino acids, and the quantification is easy, it has a serious drawback in terms of stability of the reagent. The deterioration of the reagent progresses rapidly, and due to this deterioration, the reagent itself interferes with the measurement, and the reality is that no fluorescence is observed at all after about two months of reagent preparation. Furthermore, if a degraded reagent is used, the target phosphor obtained may deteriorate in a short period of time, and the inconvenience of having to complete the reaction to measurement extremely quickly is unavoidable. For this reason, it is virtually impossible to apply a pre-column method in which column separation is performed after labeling, for example, when combining column separation with high performance liquid chromatography or fluorescence analysis. [Means for Solving the Problems] As a result of extensive research into the aging deterioration of the above reagents and the negative effects that amplify them, the present inventors converted primary amines into isoindole-type fluorescent compounds using the OPA method. The present invention has been completed based on the discovery that the problems associated with conventional reagents can be solved at once by formulating a reagent such that the reaction is carried out in the presence of TG. The present invention converts a primary amine into an isoindole type fluorescent compound by the o-phthalaldehyde method,
In a fluorescence detection method for quantifying primary amines by detecting the fluorescence, the fluorescence detection method is characterized in that the reaction for producing a fluorescent compound is carried out in the presence of thioglycerol (α-monothioglycerol). It is an invention. In other words, whether it is mixed with OPA or mixed for the first time, when OPA acts on the primary amine, the reagent is formulated so that TG coexists, and the rest is operated in the same way as before. Accordingly, the present invention can be easily put into practice. The reagents of the method of the present invention, whether mixed or mixed at the time of use, are stable over long periods of storage, and the fluorescent compounds obtained by reacting with primary amines are also stable.
Since the detected fluorescence is stable, there is no time constraint for performing the detection reaction. It is conventional to provide highly sensitive detection methods. [Function] The mechanism by which the reagents of the method of the present invention are stable after mixing when all the reagents are mixed and stored or mixed before use is still unknown.
Between OPA and TG, for example, the previous OPA and ME
No mutual reaction was observed as in the case with, and no undesired side reactions were observed in contrast to the desired reaction with the sample. In the case of the method of the present invention, an isoindole type fluorescent compound is obtained by the following reaction. The fluorescent compounds obtained are stable and e.g.
This is completely different from the case where OPA and ME are used, which gradually undergo a rearrangement reaction and change into a non-fluorescent dihydroisoindolone compound even at room temperature or lower temperatures, and have stable fluorescence. is detected,
Moreover, its stability is such that it can withstand continuous irradiation at the excitation wavelength. [Effects of the Invention] As already mentioned, when the method of the present invention is carried out, the reagents are stable, the obtained fluorophore is also stable, and the operation and sensitivity are the same as those obtained by ordinary fluorescence detection. However, there are no time constraints on the detection procedure; for example, when combining this with column separation such as high-performance liquid chromatography or fluorescence analysis, primary amines are converted to isoindole-type fluorescent compounds by OPA before entering the column. , a pre-column method in which the particles are then separated using a column is also fully possible. Note that TG generally does not have the strong bad odor that thio compounds have, and has suitable solubility for practical purposes. [Examples] Examples are shown below, but the present invention is not limited in any way by these Examples. Example 1 Add 0.700 g of OPA and methanol to a solution of 30.9 g of boric acid and 21.5 g of potassium hydroxide dissolved in distilled water.
10.0 ml, TG 2.5 ml, and 2.0 ml of 15% Brij35 were added and diluted with distilled water to make a total volume of 1 to prepare an OPA test solution of the present invention (PH 10.4). Separately, to a solution of 30.9 g of boric acid and 21.5 g of potassium hydroxide dissolved in distilled water, 0.700 g of OPA, 10.0 ml of methanol, 2.0 ml of ME, and 2.0 ml of 15% Brij35 were added.
Dilute with distilled water to make a total volume of 1, and compare OPA
A test solution (PH10.4) was prepared. Next, add 10.0 ml of both OPA test solutions to each 10 ml stoppered test tube, and add 50 ppmL-α of the sample.
- 20μ of aqueous alanine solution was added to each, and the fluorescence value of the reaction solution due to L-α-alanine was measured. Measurement was performed using a spectrofluorophotometer (Hitachi, MPE-4 model)
Excitation wavelength Ex338nm, fluorescence wavelength Em425nm
I went there. The results are shown in FIG. 1 (present invention) and FIG. 2 (comparative example). In the figure, the horizontal axis represents the passage of time (unit: minutes) from the time of OPA sample preparation to 0 minutes, and the subordinate axis represents the zero adjustment with distilled water and the cell (1 cm).
Take 0.5 ppm quinine sulfate (N/10 sulfuric acid solution), and the fluorescence intensity of the same solution is 74% of the scale of the recording paper.
The height of the peak on the recording paper (reading of the recording paper) with the sensitivity set so that In addition, the × mark indicates the sample (L-α-
Shows the fluorescence value (fluorescence measurement value) at each elapsed time (unit = minutes) of the reaction solution added and reacted with alanine (containing alanine).
- a sample containing no α-alanine was added;
Shows the fluorescence value (blank value) of the OPA test solution at each elapsed time (unit = minutes). Therefore, it is calculated as the fluorescence value due to L-α-alanine=fluorescence measurement value−blank value−blank value. As is clear from FIGS. 1 and 2, the present invention
In the OPA reagent solution, the fluorescent compounds produced are particularly stable, and the detected fluorescence is also extremely stable. This suggests that there are no time constraints from reaction to detection, and that the pre-column method can be easily applied. Example 2 The OPA test solution of the present invention in Example 1 and the comparative OPA test solution were stored at 5°C, and the fluorescence value due to L-α-alanine was measured over time in the same manner as in Example 1. The test solution deteriorated rapidly over time, and the blank value exceeded 100 after 2 months, and no sample fluorescence value was observed.However, the OPA test solution of the present invention was stable, and the blank value did not increase at all after 2 months. This was not observed, and the fluorescence value (31.7) remained the same as at the time of preparation. Example 3 Using the OPA test solution of the present invention from Example 1 and using aqueous solutions containing 10 nmol of each of the various amino acids listed in Table 1 as samples, the fluorescence value was measured for 20μ of the sample in the same manner as in Example 1, and the results in Table 1 were obtained. Obtained.

【table】

【table】 [Brief explanation of the drawing]

Figure 1 shows the fluorescence measurement value (x mark) and blank value (• mark) using the OPA reagent solution of the present invention in Example 1, and Figure 2 shows the fluorescence measurement value (x mark) using the comparative example OPA reagent solution of Example 1. and a blank value (・mark). In both cases, the horizontal axis represents the passage of time (unit = minutes),
The vertical axis represents the height of the peak (reading on the recording paper).

Claims (1)

[Claims] 1 In a fluorescence detection method in which a primary amine is converted into an isoindole-type fluorescent compound by the o-phthalaldehyde method and the resulting fluorescence is detected to quantify the primary amine, the reaction for producing the fluorescent compound is compared with thioglycerol (α - monothioglycerol).