JP3216203B2 - Catecholamine measurement reagent and catecholamine determination method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an arylaminomethyl derivative, a novel fluorogenic substrate, a pyrrolobenzoxazole derivative, and a method for determining catecholamines using the compound.

[0002]

BACKGROUND ART Dopamine, norepinephrine, and epinephrine are known as catecholamines, which are biologically active compounds having a 3,4-dihydroxyphenyl skeleton and are biologically active. They are involved in a wide range of biological regulatory mechanisms as neurotransmitters or adrenal medulla hormones. Therefore, analysis of catecholamines is indispensable in studies of the endocrine and nervous systems. However, the catecholamine content in tissues is extremely low,
Highly sensitive analytical methods are required. Recently, a fluorescence analysis method that is easy to use and has high sensitivity has attracted attention as a microanalysis method of catecholamine, and the analysis of catecholamine has been performed by a fluorescence method as one of clinical tests. Among them, 1,2-developed by the present inventors
Fluorescent quantification of catecholamines using diphenylethylenediamine as a fluorescent derivatization reagent is known as a more sensitive and simpler method of analyzing catecholamines than conventional fluorescent analysis methods (JP-A-60-205262).

[0003]

The above prior art in which 1,2-diphenylethylenediamine is used as a fluorescent derivatization reagent for catecholamines uses the above technique in which a metal oxide ion such as ammonium molybdate is used as a catalyst in order to shorten the reaction time. Depending on its concentration, the reaction solution may become cloudy and a white substance may be precipitated after the fluorescent reaction, which has been a major problem in quantifying catecholamine. In addition, the synthesis cost of 1,2-diphenylethylenediamine, which is a fluorescent derivatization reagent, was rather complicated, so that the reagent cost was high.

[0004]

DISCLOSURE OF THE INVENTION The present inventor has developed a fluorescent reaction mechanism of 1,2-diphenylethylenediamine and catecholamine and a reaction product thereof for the purpose of developing a catecholamine fluorescent reagent having higher sensitivity and lower cost. As a result of the determination of the structure of
An arylaminomethyl derivative in which diphenylethylenediamine is more simply designed has been found as a new fluorescent derivatization reagent for catecholamines, and the present invention has been completed.

That is, the present invention provides an arylaminomethyl derivative which is a specific fluorescent derivatization reagent for catecholamine represented by the general formula [I]:

[0006]

Embedded image

(Wherein, Ar represents an aryl group and L represents a leaving group) Pyrrolobenz, a fluorogenic substrate represented by the general formula [II], which is a fluorescent reaction product of an arylaminomethyl derivative and catecholamine Oxazole derivatives,

[0008]

Embedded image

(Wherein Ar represents an aryl group, R ₁ and R ₂ are the same or different and each represent a hydrogen atom, a hydroxyl group or a methyl group) and an arylaminomethyl derivative represented by the general formula [I]. It is intended to provide a method for quantifying catecholamine used as a fluorescent derivatization reagent.

[0010]

Hereinafter, the present invention will be described in detail.

The aryl group represented by Ar in the arylaminomethyl derivative of the present invention represented by the general formula [I] includes a phenyl group, a substituted phenyl group, a naphthyl group,
Examples include a substituted naphthyl group (eg, a methylnaphthyl group, a methoxylnaphthyl group), a pyridyl group, a furyl group, a biphenylyl group, and the like. Examples of the substituent of the substituted phenyl group include a 2-, 3- or 4-position methoxyl group and ethoxyl. Group, hydroxyl group, methyl group, ethyl group, chloro group, fluoro group, dimethylamino group, cyano group, nitro group, 3,4-dimethoxyl group, dichloro group, methylenedioxyl group and the like.

The leaving group represented by L in the arylaminomethyl derivative of the present invention represented by the general formula [I] includes a hydrogen atom, a cyano group, a nitro group and a halogen group (for example, chloro, fluoro, bromo, A substituted acyl group, a substituted sulfonyl group, a hydroxyl group, a thiol group, or an arylaminomethyl group. Examples of the substituent of the substituted acyl group and the substituted sulfonyl group include an alkyl group (for example, a methyl group, an ethyl group, and the like). ), An amino group, and an amino substituent (for example, a carboxylalkyl group, an alkoxycarbonyl group, an aminoalkylcarbonyl group, an aminoarylsulfonyl group, a sulfoaryl group, an aminocarboxyaryl group, or an aminoalkoxycarbonylalkyl group). Examples of the aryl group of the arylaminomethyl group include a phenyl group, a substituted phenyl group, a naphthyl group, a substituted naphthyl group (such as a methylnaphthyl group and a methoxylnaphthyl group), a pyridyl group, a furyl group, and a biphenylyl group. Examples of the substituent of the group include a methoxyl group at the 2-, 3- or 4-position, an ethoxyl group, a hydroxyl group, a methyl group, an ethyl group, a chloro group, a fluoro group, a dimethylamino group, a cyano group, a nitro group, and a 3,4 group. -Position dimethoxyl group, dichloro group, methylenedioxyl group and the like.

The aryl group represented by Ar in the pyrrolobenzoxazole derivative of the present invention represented by the general formula [II] includes a phenyl group, a substituted phenyl group, a naphthyl group and a substituted naphthyl group (for example, a methylnaphthyl group, a methoxylnaphthyl group) Etc.), a pyridyl group, a furyl group or a biphenylyl group. Examples of the substituent of the substituted phenyl group include a 2-, 3- or 4-position methoxyl group, an ethoxyl group, a hydroxyl group, a methyl group, an ethyl group, and a chloro group. Group, fluoro group, dimethylamino group, cyano group, nitro group, 3,4-dimethoxyl group, dichloro group, methylenedioxyl group and the like. Further, as R ₁ and R ₂ in the formula, a hydrogen atom, a hydroxyl group, a methyl group and the like are exemplified.

The reaction between the arylaminomethyl derivative and catecholamine proceeds rapidly at pH 4-9 in the presence of a catalyst, an oxidizing agent and a reaction accelerator, as in the case of the conventional 1,2-diphenylethylenediamine reaction. As the catalyst, metal oxide ions such as ammonium molybdate, tungstate, niobate and chromate are effective, and preferably, ammonium molybdate is used. As the oxidizing agent, KIO ₄ , Na
IO ₄ , NaIO ₃ , KIO ₃ , K ₃ Fe (CN) ₆ ,
H ₂ O _{2 and the} like are effective, preferably KIO ₄ . As the reaction accelerator, a water-soluble organic solvent such as methanol, ethanol, acetonitrile and 2-propanol is effective, and preferably, methanol and ethanol are exemplified.

The fluorescence reaction mechanism of catecholamine and arylaminomethyl derivative according to the present invention is as shown in FIG. 1, and the reaction product has exactly the same structure as the reaction product of 1,2-diphenylethylenediamine and catecholamine. It is a pyrrolobenzoxazole derivative. This derivative is a very strong fluorescent substrate and is extremely stable.

[0016]

FIG.

This fluorescence analysis method is an excellent method which can be applied not only to manual techniques but also to pre-labeling and post-labeling methods of high performance liquid chromatography.

[0018]

The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

The catecholamines used in the examples are norepinephrine (NE), epinephrine (E), dopamine (DA) and isoproterenol (IP) used as an internal standard, and the fluorescent derivatizing reagent is an arylaminomethyl derivative. 2-phenylglycinonitrile (α-aminobenzyl cyanide) (P
GN) and the conditions were examined. When multiple components such as a biological sample are mixed, high performance liquid chromatography (HPLC) is an effective separation and analysis method.

FIG. 2 shows a standard operation method of precolumn fluorescent derivatization HPLC of catecholamine using PGN devised in the present invention.

[0021]

FIG. 2

As reaction aids coexisting with the fluorescent derivatizing reagent, ammonium molybdate (catalyst), which gave the best results for catecholamine, KIO ₄
(Oxidizing agent) and methanol (reaction accelerator).

FIG. 3 shows the height of the fluorescent peak obtained when the concentration of ammonium molybdate was changed from 0 mM to 30 mM according to the standard operation method of FIG. 2 on the vertical axis. It is clear that the addition of ammonium molybdate increased the fluorescence peak height for each catecholamine. The concentration of ammonium molybdate was preferably 10-30 mM. In addition to ammonium molybdate, metal oxide ions such as tungstate, niobate and chromate were also effective as catalysts.

[0024]

FIG. 3

FIG. 4 shows the effect of KIO ₄ concentration on the fluorescence peak height. It is clear that KIO ₄ increases the fluorescence peak height for all catecholamines. The concentration of KIO ₄ was preferably 7.5 to 15 mM. Other oxidizing agents examined include NaIO ₄ , KIO ₃ , NaIO ₃ , K ₃ Fe
(CN) ₆ and H ₂ O ₂ were also effective. But,
With respect to K ₃ Fe (CN) ₆ , cloudiness occurred after the reaction at 50 mM or more.

[0026]

FIG. 4

FIG. 5 is a diagram showing the effect of the methanol concentration in the reaction solution on the fluorescence peak height. It can be seen that methanol gives a high fluorescence peak for all catecholamines. The methanol concentration in the reaction solution was preferably in the range of 52-62%. As other reaction accelerators, water-soluble organic solvents such as ethanol, acetonitrile and 2-propanol were effective.

[0028]

FIG. 5

FIG. 6 shows the effect of the PGN concentration on the fluorescence peak height. It is clear that the fluorescence peak height increases for all catecholamines in the range of 5-50 mM. The PGN concentration was preferably 30-45 mM.

[0030]

FIG. 6

FIG. 7 shows Britton-Robi in buffer.
FIG. 3 is a diagram showing the effect of pH on the fluorescence peak height when using an nson buffer. It is clear that all the catecholamines give high fluorescence peaks in the pH range of 4.5 to 7.5. Preferably pH 5
The range was -5.5.

[0032]

FIG. 7

FIG. 8 shows the effect of the reaction time at 50 ° C. This indicates that the reaction time was completed in 15-20 minutes.

[0034]

FIG. 8

FIG. 9 shows a chromatogram obtained according to the standard operation method of precolumn fluorescent derivatization HPLC of catecholamine using PGN according to the method of the present invention (FIG. 2). The reagent 1,2-diphenylethylenediamine (DP
The chromatogram obtained by using E) is shown (the chromatogram of PGN is a chromatogram obtained by setting the detection sensitivity to 5 times that of DPE).

[0036]

FIG. 9

The HPLC conditions are as follows. Column: TSKgel ODS-80TM Column temperature: room temperature (20 to 25 ° C.) Eluent: CH ₃ CN—CH ₃ OH (50 mM) -Tri
s-HCl (5: 1: 5, v / v) buffer (pH 7.
0) Eluent flow rate: 0.8 ml / min Fluorescence detection: Ex 350 nm, Em 475 nm Each peak can be confirmed by PGN at the same retention time as DPE, and the detection limit of catecholamine by PGN (S / N = 3) , 10fmol (NE), 3f
mol (E), 7 fmol (DA), 7 fmol (I
P).

Although the sensitivity is slightly inferior to the case where the conventional DPE is used, it can be seen that the sensitivity is still very high for all catecholamines. The concentrations of DPE and PGN used here were 18.2 pmol / 5
0 μl. Furthermore, it was confirmed that the arylaminomethyl derivative according to the present invention selectively reacts only with a compound having a catechol skeleton and does not react with biological components such as glucose and amino acids at all, and is a selective reagent for catecholamine.

[0039]

As described above, the catecholamine quantification method using the arylaminomethyl derivative of the present invention as a fluorescent derivatization reagent is a highly sensitive and simple catecholamine quantification method as compared with the conventional method. The reagent cost can be considerably reduced as compared with the case where diphenylethylenediamine is used for the fluorescent derivatization reagent. Therefore, the present invention is useful in the field of catecholamine secretion and nervous system research and clinical examination.

[Brief description of the drawings]

FIG. 1 is a diagram showing a fluorescence reaction mechanism of a catecholamine and an arylaminomethyl derivative.

FIG. 2 is a diagram showing a standard operation method of precolumn fluorescent derivatization HPLC of catecholamine using PGN.

FIG. 3 is a graph showing the influence of the concentration of ammonium molybdate.

FIG. 4 is a graph showing the effect of KIO ₄ concentration.

FIG. 5 is a graph showing the effect of methanol concentration.

FIG. 6 is a graph showing the effect of PGN concentration.

FIG. 7 is a graph showing the influence of pH.

FIG. 8 is a graph showing the effect of reaction time at 50 ° C.

FIG. 9 is a diagram showing chromatograms obtained by the present example and a conventional method.

Continuation of front page (56) References JP-A-1-265158 (JP, A) JP-A-4-48263 (JP, A) JP-A-61-88148 (JP, A) JP-A-60-113152 (JP JP-A-59-42450 (JP, A) JP-A-60-143766 (JP, A) JP-A-60-205262 (JP, A) JP-A-3-53165 (JP, A) 3-11071 (JP, A) JP-A-63-14777 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/50 G01N 21/78 G01N 31/00 G01N 31 / 22 122

Claims (8)

(57) [Claims] 1. An arylaminomethyl derivative which is a specific fluorescent derivatization reagent of catecholamine represented by the general formula [I]. Embedded image (Wherein, Ar represents an aryl group and L represents a leaving group) 2. A pyrrorobenzoxazole derivative of a fluorescent substrate represented by the general formula [II], which is a product of a fluorescent reaction between an arylaminomethyl derivative and catecholamine. Embedded image (In the formula, Ar represents an aryl group, and R ₁ and R ₂ are the same or different and represent a hydrogen atom, a hydroxyl group or a methyl group.) 3. A method for quantifying catecholamines, wherein the arylaminomethyl derivative represented by the general formula [I] according to claim 1 is used as a fluorescent derivatization reagent. 4. The method according to claim 1, wherein the arylaminomethyl derivative represented by the general formula [I] is used as a precolumn fluorescent derivatization reagent or a postcolumn fluorescent derivatization reagent. Method for catecholamines. 5. The method for quantifying catecholamine according to claim 3, wherein a catalyst, an oxidizing agent, and a reaction accelerator are used as a reaction auxiliary coexisting with the fluorescent derivatizing reagent. 6. The method according to claim 5, wherein a metal oxide ion such as ammonium molybdate is used as the catalyst.
The method for quantifying catecholamines described in the above section. 7. KIO ₄ , NaIO ₄ , Na as an oxidizing agent
_{IO 3, KIO 3, K 3} Fe (CN) 6 or H ₂ 0 ₂ Determination method of catecholamines according to claim 5 wherein, wherein the use of. 8. The method according to claim 5, wherein methanol, ethanol, acetonitrile or 2-propanol is used as the reaction accelerator.