JP3470345B2 - Filler for chemiluminescence detection and analysis method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for analyzing a substance by chemiluminescence detection method in liquid chromatography and flow injection.

[0002]

2. Description of the Related Art Luminol, lucigenin, acridine derivatives and the like have been known as chemiluminescent reagents which give strong luminescence by reacting with hydrogen peroxide under alkaline conditions (clinical examination, p. 153, Vol. 29, No. .2, 1985
Year). Among them, when analyzing and detecting oxidants using high performance liquid chromatography, the method of adding luminol or lucigenin to an alkaline solution and sending it as a reaction solution, mixing it with a substance to be analyzed and emitting light is another method. High sensitivity compared to many reports (Bunseki, 442)
Page, No. 6, 1989). However, since the acridine derivative is more expensive than other chemiluminescent reagents, there are hardly any examples of the acridine derivative used in the above-mentioned waste-type detection method such as continuous liquid feeding. On the other hand, acridine derivatives have a low luminescence yield, such as the ability to measure low concentrations of blood thyroid stimulating hormone (TSH), such as Graves' disease, which was previously unmeasurable by utilizing proteins as labeling agents in immunoassays. It is known to be high ("Bioluminescence and chemiluminescence" 87
Page, Hirokawa Shoten, 1988), if it can be used in the same manner as luminol and lucigenin, higher sensitive analysis is possible compared to other chemiluminescent reagents.

[0003]

[Problems to be Solved by the Invention] In liquid chromatography and flow injection analysis using acridine derivative chemiluminescence detection method, expensive acridine derivative is efficiently used and saved, and at the same time, background fluctuation is reduced and high sensitivity is achieved. It is an object of the present invention to enable various analyzes.

[0004]

It has been found that the above object can be achieved by using a filler having an acridine derivative according to the present invention immobilized thereon (hereinafter referred to as a filler according to the present invention). That is, by filling the flow cell in the chemiluminescence detector with the packing material according to the present invention and using it, a redox substance (a reducing agent in the presence of an oxidizing agent and dissolved oxygen) can be obtained without sending an acridine derivative solution. It has been found that can be quantified with high sensitivity.

That is, the present invention provides (1) 10 of acridine rings.
The position (nitrogen atom) is substituted with an electron donating group such as an alkyl group, an aralkyl group or an aryl group, and is quaternized,
A chemiluminescent detection filler in which an acridine derivative whose 9-position is substituted with a carboxy group or a group derived therefrom is immobilized on a solvent-insoluble carrier, (2) a liquid using the filler according to (1) above The present invention relates to a method for analyzing a redox substance by chromatography and (3) a method for analyzing a redox substance by flow injection using the filler according to (1) above.

The acridine derivative used in the present invention is
A compound capable of emitting light via a dioxetane structure by redox, wherein the 10-position (nitrogen atom) of the acridine ring is substituted with an electron-donating group such as an alkyl group, an aralkyl group or an aryl group to be quaternized, and the 9-position is carboxy. It is substituted with a group or a group derived therefrom. Also,
Optionally, the other carbon atom of the acridine ring is one or more organic chemically feasible groups such as alkyl groups, aralkyl groups, aryl groups, nitro groups, amino groups, substituted alkylamino groups, acyl groups, alkoxy groups, It may be substituted with a halogen atom or the like. As the 10-position substituent,
A methyl group, an ethyl group, a benzyl group, a phenyl group and the like are preferable, and a carboxyl group, an alkoxycarbonyl group and the like are preferable as the 9-position substituent. As the anion of the acridinium salt, tosylate ion, halogen ion, fluorinated sulfate ion and the like are suitable.

The solvent-insoluble carrier used in the filler according to the present invention is not particularly limited as long as it is insoluble in water or an aqueous solution containing a salt or an organic solvent, nylon, polystyrene, polyacrylamide, polyacrylate. ,
Synthetic polymers such as polycarbonate, polyester, polyurethane, copolymers thereof and cross-linked products thereof,
Examples thereof include natural polymers such as cellulose and agarose, and inorganic substances such as silica, alumina and titania.
The carrier insoluble in these solvents needs to have a functional group such as a hydroxyl group, an amino group, a carboxyl group, an epoxy group, or a halogen for binding the acridine derivative in the carrier. Also, these synthetic polymers may have any shape as long as they can be filled in the flow cell, but considering the pressure resistance performance of the flow cell while being filled with a high density, they are spherical with a diameter of several tens of μm to several hundreds of μm. Is advantageous. Further, since the luminescence reaction of the acridine derivative is carried out under alkaline conditions, it is preferably insoluble in an alkaline solution.

There are no particular restrictions on the immobilization of the acridine derivative, and it may be used directly or directly with a bifunctional compound such as propanediamine, glutaraldehyde, 1,4-butanediol diglycidyl ether or 4-aminobutyric acid. It can be performed by using it as a spacer. The immobilization reaction is carried out in the presence of a catalyst such as an acid, a base or the like, or a condensing agent such as carbodiimides, N-hydroxysuccinimide or carbonyldiimidazole in an appropriate solvent depending on each functional group. , Or in the absence thereof, it can be carried out according to a conventional method.

The thus-obtained packing material according to the present invention is filled in the flow cell in the chemiluminescence detector, and the shape of the flow cell used at this time is not particularly limited, and may be cubic or flat. Although it may be shaped like a tube, it is usually a tube made of quartz glass or fluorinated resin processed into a spiral shape or a zigzag total internal volume of 100 to
About 200 μl is often used to increase the light receiving efficiency (Fig. 1). The light emission detection method at this time may be either the analog mode or the counting mode.

A redox substance is analyzed using a liquid chromatography system and a flow injection system (hereinafter, referred to as a system according to the present invention) using a chemiluminescence detector in which a packing material according to the present invention is filled in a flow cell. In this case, as shown in FIG. 2, in addition to the eluent, an alkaline solution such as a phosphate buffer solution may be sent. In some cases, if the eluent is an alkaline solution, a pump for sending the eluent. It can be analyzed only. The pH of the alkaline solution used here is not particularly limited as long as it is 7 or more, but in order for the acridine derivative to emit light, pH 9 to 1
3 is preferable.

Especially, the analysis of hydrogen peroxide as an oxidizing agent in the redox substances is useful not only in general analysis but also in analysis of bio-related substances. At this time, the substance to be analyzed is separated by the separation column, and then converted into hydrogen peroxide by the post column on which the hydrogen peroxide-generating enzyme is immobilized and detected. The enzyme to be used is appropriately selected depending on the substance to be analyzed, but in some cases, not only the one-step reaction but also a plurality of reactions can be combined to detect the finally obtained hydrogen peroxide. There are many reports on the immobilized enzyme column technology, but for example, Imai Kazuhiro, "Bioluminescence and Chemiluminescence", page 233, Hirokawa Shoten (1988) can be referred to. The present invention can also be applied to analysis of ascorbic acid, reducing sugars, etc. as reducing substances.

As the analytical conditions for performing liquid chromatography and flow injection using the system according to the present invention, an eluent containing a salt or a buffer, a temperature range, a flow rate, etc., which are usually used, can be selected.

[0013]

EXAMPLES A typical example of an analysis method using the packing material according to the present invention and the chromatography system according to the present invention will be shown below, which will be described more specifically. However, it goes without saying that these are merely examples for explanation and the present invention is not limited thereto.

Example 1 Amino group-containing gel copolymer obtained by reacting an epoxy-containing gel copolymer obtained from glycidyl methacrylate and glycerin dimethacrylate with propanediamine (amino group: 217 μmol / g dry gel) 1 0.0 g
Was dispersed in 2 ml of 0.1 M sodium hydrogen carbonate aqueous solution,
Acridine derivative (4- (2-succinimidyloxycarbonylethyl) phenyl-10-methylacridinium-9-
Carboxylic acid fluorosulfate 4- (2-Succinimidyloxycarbony
lethyl) phenyl-10-methylacridinium-9-carboxylate fl
2 ml of 50% methanol solution containing 10 mg of uorosulfonate) was added, and the mixture was shaken at room temperature for 4 hours. The gel was collected by filtration to remove unreacted substances, washed successively with methanol and distilled water and dried to obtain an acridine derivative-immobilized gel. Unreacted
Quantification by HPLC confirmed that 7 mg was immobilized.

Example 2 The packing material obtained in Example 1 was used to fill a flow cell (internal diameter 1.0 mm, length 600 mm, fluorinated resin tube) in a chemiluminescence detector, and the system according to the present invention was used. Aqueous hydrogen peroxide solutions of various concentrations were analyzed.The results showed a high linear relationship between the hydrogen peroxide concentration and the chemiluminescence intensity as shown in Fig. 3. Analytical conditions Eluent H ₂ O flow rate 0.5 ml / min Reaction solution 0.1M NaHPO ₄ -NaOH solution (pH 11.0) Flow rate 0.5 ml / min The chromatogram under the above analysis conditions is shown in FIG.

Example 3 The epoxy group of an epoxy group-containing gel copolymer obtained from glycidyl methacrylate and glycerin dimethacrylate was ring-opened modified with water, and then 1,4-butanediglycidyl ether was reacted to give 1, 1.0 g of an amino group-containing gel-like copolymer reacted with 3-propanediamine (amino group: 70 μmol per 1 g of dry gel) was added to 2 ml of a dimethylformamide solution in which 10 mg of the acridine derivative used in Example 1 was dissolved and added at room temperature. It was shaken for 30 minutes. The gel was collected by filtration to remove unreacted substances, washed successively with methanol and distilled water and dried to obtain an acridine derivative-immobilized gel. The unreacted material was quantified by HPLC and it was confirmed that 9 mg was immobilized.

Example 4 In Example 2, the filler obtained in Example 3 was used instead of the filler obtained in Example 1, and an aqueous hydrogen peroxide solution was analyzed in the same manner. As a result, as shown in FIG. 4, a high linear relationship was obtained between the hydrogen peroxide concentration and the chemiluminescence intensity.

Example 5 Instead of the acridine derivative used in Example 3, 10
A similar reaction was carried out using an acridine derivative whose position was substituted with a phenyl group. The gel was collected by filtration to remove unreacted substances, washed successively with methanol and distilled water and dried to obtain an acridine derivative-immobilized gel. 8 mg of unreacted material was determined by HPLC
It was confirmed that it was fixed.

Example 6 In Example 2, the hydrogen peroxide aqueous solution was similarly analyzed using the filler obtained in Example 5 instead of the filler obtained in Example 1. As a result, a high linear relationship was obtained between the concentration of hydrogen peroxide and the chemiluminescence intensity, as in the case of using other acridine derivative-immobilized gels.

Comparative Example A gel in which an acridine derivative is not immobilized can be prepared according to Example 1. Using the obtained packing material, a column chromatogram was obtained in the same manner as in Example 2 (Fig. 6). However, the acridine derivative was always sent as a reaction solution.

[0021]

EFFECTS OF THE INVENTION Chemiluminescence by an acridine derivative allows highly sensitive analysis, but the reagent is expensive. The filler according to the present invention is one in which an acridine derivative is bound to a solvent-insoluble carrier by a stable covalent bond, and should be used sufficiently stably under alkaline conditions that are the optimum pH of the acridine derivative chemiluminescent system. In addition, since it is not necessary to constantly feed the acridine derivative, not only the waste of the reagent can be prevented, but also the background fluctuation caused by the waste does not occur and the noise level can be lowered.

[Brief description of drawings]

FIG. 1 shows an example of the shape of a flow cell.

FIG. 2 is a diagram of a chromatography system according to the present invention.

FIG. 3 shows the relationship between hydrogen peroxide and chemiluminescence intensity when an acridine derivative is immobilized (Example 2).

FIG. 4 shows the relationship between hydrogen peroxide and chemiluminescence intensity when an acridine derivative is immobilized (Example 4).

FIG. 5 is a chromatogram obtained by analyzing an aqueous hydrogen peroxide solution when a packing material having an acridine derivative immobilized thereon is used.

FIG. 6 is a chromatogram obtained by analyzing an aqueous hydrogen peroxide solution when an acridine derivative was used as a reaction solution and was constantly fed.

[Explanation of symbols]

1 Light-transmissive tube 1A light transmissive tube 1B light transmissive tube 2 reflective surface 3 protrusions 4 window boards 5 cell plate 6 long groove 6A long groove 6B long groove 7 Eluent 8 pumps 9 injectors 10 columns 11 Chemiluminescence detector 12 recorder 13 Alkaline solution

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Bibliographical references 3-17564 (JP, U) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 30/48

Claims (3)

(57) [Claims] 1. A group substituted with an electron-donating group such as an alkyl group, an aralkyl group or an aryl group at the 10-position (nitrogen atom) of an acridine ring to be quaternized, and a 9-position is a carboxy group or a group derived therefrom. A chemiluminescent detection packing material in which an acridine derivative substituted with is immobilized on a carrier insoluble in a solvent. 2. A method for analyzing a redox substance by liquid chromatography, which uses the packing material according to claim 1. 3. A method for analyzing a redox substance by flow injection, which uses the filler according to claim 1.