JPH0513255B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a stable fluorescence detection reagent. More specifically, a method for quantifying primary amines by reacting o-phthalaldehyde (OPA) with primary amines in the presence of a thiol compound to generate isoindole-type fluorescent compounds and detecting the fluorescence. The present invention relates to the stabilization of fluorescence detection reagents used for fluorescence detection, and relates to fluorescence detection methods using stabilized reagents.

[Conventional technology]

Conventional methods for quantitative analysis of substances, especially trace substances, include absorption spectroscopy, which is a simple and excellent method, but has the disadvantage of somewhat low sensitivity.

On the other hand, in fluorescence analysis, the sensitivity can be easily improved by increasing the sensitivity of the detector and the intensity of the light source, and the concentration that can be measured by absorption analysis etc. is generally 10 ^-6 M.
However, it is possible to measure samples up to about 10 ^-9 to ^{10 -10} M. In addition, fluorescence analysis allows the two parameters of excitation wavelength and fluorescence wavelength to be optimally selected, so selectivity is significantly superior.

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. Like fluorescein, OPA reacts with a primary amine to give a fluorophore, but the presence of 2-mercaptoethanol (hereinafter abbreviated as ME) is essential for the production of the fluorophore. It is well known that isoindole-type fluorescent compounds are produced by this reaction, as described in M. Roth: Anal. Chem. vol. 43, p. 880 (1971), SSSimons and DF Johnson: Jr.
It is described in Org.Chem.Volume 43, Page 2886 (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. Examples of high performance liquid chromatography and fluorescence analysis quantification have 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
(1975)).

Reagents used for the reaction are commercially available, such as
0.700g OPA, 2.0ml 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).
For example, it is diluted to 1.

The reagent directly involved in the reaction with the primary amine is
They are OPA and ME, and ME is used in molar excess relative to OPA. Surfactant Brij35 improves sensitivity (JRBenson and PEHare: Proc. Nat. Acad.
Sci. USA Vol. 72, p. 619 (1975)). Methanol is also a solubilizing agent for OPA. The pH during the reaction must be alkaline, and a potassium borate buffer (PH10.4) is used.

[Problem that the invention seeks to solve]

In this way, the detection sensitivity of reagents is high, for example, at the nmol to pmol level for the quantification of amino acids, and while they have the advantage of being easy to quantify, they have a serious drawback in terms of stability, and reagents deteriorate rapidly. However, due to its deterioration, the reagent itself interferes with the measurement, and in reality, 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 problems]

As a result of intensive research into the deterioration of the above reagent over time and the negative effects that amplify it, the present inventors found that the cause was that ME was mixed with other reagents and was present in the alkaline potassium borate buffer. The present invention was completed based on these findings. The present invention is o
- A method for quantifying a primary amine by reacting phthalaldehyde with a primary amine in the presence of a thiol compound to generate an isoindole type fluorescent compound and detecting the fluorescence. Fluorescence detection of primary amines, characterized in that 2-mercaptoethanol, which is used as It is an invention of a method.

That is, if ME among the reagents is dissolved in water or an aqueous boric acid solution and separated as other reagents, the conventional problems can be smoothly solved.

The reagent of the present invention may be prepared using the same composition ratio as before, but it must be prepared separately from the ME and mixed with the necessary water (of course, for practical purposes, it is distilled water) or It is prepared and stored as a two-part solution separated from other reagents by dissolving it in an aqueous boric acid solution.

[Effect]

The details of the reaction mechanism of deterioration during reagent storage are unknown, but ME remains stable until it is mixed before use.
Interaction between OPA and ME is not possible until the time of their mixing, and undesired side reactions with respect to the reaction with the sample are significantly suppressed.

〔Effect of the invention〕

In the reagent of the present invention, deterioration of the reagent over time is effectively suppressed, and when mixed before use, fluorescence from the sample is expressed in the absence of any inhibitory factors.
There will be no hindrance to analysis.

That is, the fluorescence detection method of the present invention has the inventiveness of providing the conventional high sensitivity without any slight disadvantages and the reliability of the measurement results is extremely high. Application to pre-column methods is also fully possible.

〔Example〕

Examples are shown below.

Example 1 30.9g of boric acid and 21.5g of potassium hydroxide were added to distilled water.
Dissolved in 473ml, 495ml potassium borate buffer (PH
10.8). Add OPA0.700g and methanol
10.0 ml and 2.0 ml of 15% Brij35 were dissolved to prepare solution A. Next, 2.0 ml of ME was dissolved in 505 ml of distilled water to obtain liquid B.

When solutions A and B were stored at 5°C and the change in fluorescence for L-α-alanine was observed over time, it was found that 6
No increase in the blank value was observed even after a month had passed, and the fluorescence value (31.2) was also not significantly different from the initial value.

For this measurement, 10.0 ml of OPA reagent, which was prepared by mixing equal amounts of liquid A and B at the time of use, was placed in a test tube with a stopper, and 50 ppmL of the sample - 20 μ of an α-alanine aqueous solution was added thereto. The fluorescence value of the reaction solution due to L-α-alanine was measured. A spectrofluorometer (Hitachi, MDF-4 model) was used for the measurement, and the excitation wavelength was
The measurement was performed at Ex338nm and fluorescence wavelength Em425nm.

Exactly the same effect was observed for primary amines other than L-α-alanine.

Example 2 27.8g of boric acid and 21.5g of potassium hydroxide in distilled water
Dissolved in 474ml, 495ml potassium borate buffer (PH
11.0). Add OPA0.700g and methanol
10.0 ml and 2.0 ml of 15% Brij35 were dissolved to prepare solution A. Next, add 2.0ml of ME, 3.1g of boric acid, and 504ml of distilled water.
The solution was dissolved in 505 ml of an aqueous solution of boric acid to prepare solution B.

When solutions A and B were stored at 5° C. and measured in the same manner as in Example 1, there was no increase in the blank value and the fluorescence value was the same as in Example 1.

Comparative example: In 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 and 10.0 g of methanol were added.
ml, 2.0 ml of ME, and 2.0 ml of 15% Brij35, diluted with distilled water to make a total volume of 1, and prepare an OPA test solution with a pH of 10.4.

When this OPA test solution (one solution) was stored at 5° C. and the change in fluorescence over time was measured in the same manner as in Example 1, no fluorescence value was observed in the sample after 2 months.

Claims (1)

[Claims] 1. In a method for quantifying a primary amine by reacting o-phthalaldehyde with a primary amine in the presence of a thiol compound to generate an isoindole type fluorescent compound and detecting the fluorescence, 2-Mercaptoethanol used as a thiol compound is dissolved in water or an aqueous boric acid solution, separated from other reagents, and used by mixing with all other reagents before use. Fluorescence detection method.