JP2557121B2 - Novel quinoxalinone derivative and method for quantifying carboxylic acid using this compound - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel quinoxalinone derivative and a new method for quantifying carboxylic acid using this compound.

(Prior Art) Regarding the quantification of carboxylic acids using high performance liquid chromatography (HPLC), various fluorescent derivatization reagents have been proposed so far. Most of these reagents have atomic groups such as coumarin, phenanthrene, anthracene, pyrene, and dimethylaminonaphthalene as fluorophores, and halogenomethyl and halogenoacyl, amino, and hydrazino as reactive groups for carboxylic acid. It has groups such as, hydroxy, and diazomethyl.

For coumarin as a fluorophore, see Dnges, W.
"Anal. Chem." 1970, 49, pp.442-445 (reference 1), Farin
otti, R.; Siard, P.; Bourson, J.; Kirkiacharian, S.; Valeu
r, B .; Mahuzier, G. "J. Chromatogr." 1983, 269, pp. 81-9.
0 (Reference 2), Tsuchiya, H .; Hayashi, T .; Naruse, H .; Taka
gi, N. "J. Chromatogr." 1982, 234, pp. 121-130 (Reference 3), Goya, S .; Takadate, A .; Fujino, H .; Irikura, M. "Pharmaceutical magazine" 1980, 100. , pp.744-748 (Reference 4) and Takadat
e, A.; Tahara, T.; Fujino, H.; Goya, S .; `` Chem.Pharm.Bul
l. ”1982, 30, pp. 4120-4125 (Reference 5) and the like.

For phenanthrene, Watkins, WD; Peterson,
MB “Anal. Biochem.” 1982, 125.pp. 30-40 (Reference 6), Lloyd, JBF “J. Chromatogr.” 1980, 189, pp. 359.
-373 (Reference 7) and Ikeda, M .; Shimada, K .; Sakaguchi,
T .; Matsumoto, U. "J. Chromatogr." 1984, 305, pp. 261-2
70 (reference 8) and the like.

For anthracene, Korte, WD “J. Chromatog
r. ”1982, 243, pp. 153-157 (Reference 9), Lingeman, H .; Hu
lshoff, A.; Underberg, WJM; Offermann, FBJM `` JC
hromatogr. ”1984, 290, pp.215-222 (Reference 10) and Nimu.
ra, N .; Kinoshita, T. "Anal. Lett." 1980, 13, pp. 191-19.
3 (Reference 11) and the like.

For pyrene, Kamada, S; Maeda, M .; Tsuji, A. `` J.
Chromatogr. "1983, 272, pp. 29-41 (Reference 12) and Nimur.
a, N.; Kinoshita, T.; Yoshida, T; Uetake, A; Nakai, C.
l. Chem. "1988, 60, pp. 2067-2070 (Reference 13) and the like.

For dimethylaminonaphthalene, Goto, J.; Got
o, N.; Hikichi, A; Nishimaki, T.; Nambara T. `` Anal. Chem.
Acta '' 1980, 120, pp.187-192 (Reference 14), Ryan, PJ; Ho
neyman, TW `` J. Chromatogr. '' 1984, 312, pp.461-466
(Reference 15) and Lee, YM; Nakamura, H; Nakajima, T. "Ana
l.Sci. "1989, 5, pp.209-210 (Reference 16) and the like.

Regarding halogenomethyl and halogenoacyl as a reactive group for a carboxylic acid, the above-mentioned references 1 to 3, 6, 9 and 1
It is described in 2nd grade. For amino, see References 7 and 8 above
And 16 etc. The hydrazino is described in the above-mentioned Document 14 and the like. For hydroxy,
It is described in the above Documents 4, 10 and 15. Diazomethyl is described in the above-mentioned documents 5, 11 and 13 and the like.

The present inventors have shown in the past that 6,7-dimethoxy-1-methyl-2 (1H) -quinoxalinone (hereinafter abbreviated as “DMEQ”) emits an extremely strong fluorescence as compared with a conventional fluorophore. We have discovered 3-bromomethyl-DMEQ as a highly sensitive fluorescent labeling reagent for the determination of carboxylic acids by HPLC (Yamaguchi, M .; Hara, S .; Matsunaga, R .; Nakamura, M .;
Ohkura, Y. "J. Chromatogr." 1985, 346, pp.227-236).

However, most of the above reagents required dry neutral solvent, relatively high temperature and long heating time in the labeling reaction. To overcome these problems, a UV labeling reagent, 2,4-dinitrophenylhydrazine, was recently developed (Miwa, H .; Hiyama, C .; Ya
mamoto, M. "J. Chromatogr." 1985, 321, pp.165-174).
This reagent is a coupling agent, 1-ethyl-3-
It reacts with various carboxylic acids in an aqueous solution of (3-dimethylaminopropyl) carbodiimide (hereinafter abbreviated as "EDC") to give an acid hydrazide. However, when the carboxylic acid was quantified by the HPLC method using this reagent, the detection sensitivity was not good.

(Problems to be Solved by the Invention) As described above, a number of carboxylic acid labeling reagents have been proposed so far, but it is necessary to use a specific solvent or to react at a high temperature, and It had drawbacks to be overcome such as long heating time and poor detection sensitivity. Therefore, development of a labeling reagent that does not have such drawbacks and can detect carboxylic acid sensitively under ordinary conditions has been awaited. In other words, it reacts with a carboxylic acid in an aqueous solution at room temperature, and the resulting reaction product with the carboxylic acid exhibits a large fluorescence quantum yield (ie,
Research and development of labeling reagents with high sensitivity have been desired.

(Means for Solving the Problems) As a result of earnest research on a carboxylic acid labeling reagent satisfying all of the above conditions, the present inventors have found that a certain novel quinoxalinone derivative is carboxylic acid at room temperature in an aqueous solution. The present invention was completed based on this finding by reacting with an acid and finding that the fluorescence quantum yield of the reaction product is large.

The present invention relates to a method for quantifying a carboxylic acid, which comprises using a novel compound represented by the following formula (I) as a labeling reagent and the novel compound.

In the formula, R ₁ , R ₂ and R ₃ represent C _1-6 lower alkyl. Particularly preferred is when R ₁ , R ₂ and R ₃ are C _1-3 lower alkyl. R ₄ is a group-(CH ₂ ) _n CONHNH ₂
In this case, n may be any integer from 0 to 6.

The quantification of carboxylic acid according to the present invention is carried out as follows. The fluorescent labeling reagent of formula (I) is reacted with a carboxylic acid to produce the corresponding quinoxalinone derivative (reaction product). After completion of the reaction, the fluorescent labeling reagent represented by the formula (I) and the quinoxalinone derivative which is a reaction product are separated to measure the fluorescence intensity of the latter. This chemical reaction is expressed by the following formula.

(Note) In the above reaction formula, R ₁ , R ₂ and R ₃ represent C _1-6 lower alkyl, n represents an integer from 0 to 6, and R represents any group.

As is clear from the above reaction formula, the carboxylic acid that can be quantified by the method of the present invention is not limited to a specific range, and any carboxylic acid may be used.

The content of the present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

〔Example〕

The equipment sample was placed in a 1 cm quartz cell and the uncorrected fluorescence spectrum and intensity were measured using a Hitachi 650-60 type spectrofluorometer. A spectral bandwidth of 10 nm was used in both the excitation monochromator and the emission monochromator.

¹ H-NMR spectrum was measured using Hitachi R-90H type spectrometer.
It was measured at 90 MHz. Solution used in the measurement is about 5 N, N- dimethylformamide -d ₇ and dimethylsulfoxide -d ₆ containing tetramethylsilane as internal standard
% Solution (in the following NMR spectrum data, s is singlet, t is triplet, m is multiple, and br is
each abbreviation of broad). Mass spectrum (MS) is J
It measured using the EOLDX-300 type | mold spectrometer.

Chemicals Unless otherwise noted, all chemicals were of analytical reagent grade. As the water, deionized distilled water was used. Valeric acid (C ₅ ), capron (C ₆ ), caprylic acid (C ₈ ), capric acid (C ₁₀ ), lauric acid (C ₁₂ ), myristic acid (C ₁₄ ), palmitic acid (used in this Example) C ₁₆ ), mangalinic acid (C ₁₇ ), stearic acid (C ₁₈ ), arachidic acid (C ₂₀ ), thromboxane B ₂ (thro
mboxane B ₂ ), prostaglandin B ₂ and leukotriene B ₄ (leukotriene B ₄ ) were all purchased from Sigma, USA (St. Louis, MO). 1,2-Diamino-4,5-dimethoxybenzene is described in Nakamura, M .; Toda,
M .; Saito, H .; Ohkura, Y. "Anal. Chim. Acta" 1982, 134, p.
Prepared by the method described on pages 39-45, this compound is now commercially available.

Synthesis of labeling reagent A Known direction described in literature (Hara, S .; Yamaguchi, M .; Nakamur
a, M .; Ohkura, Y. "Chem. Pharm. Bull." 1985, 33, pp. 3493
-3498), α-ketoglutaric acid was reacted with 1,2-diamino-4,5-dimethoxybenzene to produce a compound (II) represented by the following formula.

1.5 g of the compound (II) was dissolved in 100 ml of anhydrous methanol and treated with an ethereal diazomethane solution (note that ethereal diazomethane can be obtained by, for example, Schlenk, H .; Gellerm.
An, JL "Anal. Chem." 1960, 32, pp. 1412-1415. ). The reaction mixture was evaporated to dryness under reduced pressure. The residue dissolved in 30 ml of chloroform was used as a carrier for silica gel 60 (about 130 g, 70-230
Mesh; manufactured by Nippon Merck & Co., Inc.) and purified by column chromatography (25 x 3.5 (inner diameter) cm) using n-hexane-ethyl acetate (1: 1) as an eluent to obtain a compound (III) of the following formula (0.92 g, 62% yield).

Physical property data of compound (III): colorless needle crystal.mp 178-179 ° C, NMR (Me ₂ NCHO-d ₇ ) (p
pm) δ 2.85 (t, J = 7Hz, 2H), 3.13 (t, J = 7Hz, 2H), 3.66
(S, 3H), 3.72 (s, 3H), 3.92 (s, 3H), 4.02 (s, 3H), 7.
09 (s, 1H), 7.27 (s, 1H). Elemental analysis: Calculated value (C ₁₅ H ₁₈ N
₂ O ₅ ): C, 58.82; H, 5.92; N, 9.15. Measured value: C, 58.94; H, 5.87;
N, 9.19.MS m / e, 306. 0.9 g of compound (III) in 45% aqueous solution of hydrazine hydrate 1
Dissolve in 00 ml and heat the mixture at 100 ° C. for about 60 minutes. The resulting precipitate was recrystallized from ethanol to obtain a labeling reagent A (new substance) of the following formula (0.72 g, yield 80%).

Physical data of labeling reagent A: colorless needle crystals.mp 205-206 ° C. NMR (Me ₂ NCHO-d ₇ ) (pp
m) δ2.63 (t, J = 7Hz, 2H), 3.12 (t, J = 7Hz, 2H), 3.71
(S, 3H), 3.92 (s, 3H), 4.02 (s, 3H), 3.4−4.4 (br, 2
H), 7.08 (s, 1H), 7.28 (s, 1H), 8.9-9.1 (br, 1H).
Elemental analysis: Calculated value (C ₁₄ H ₁₈ N ₄ O ₄ ): C, 54.90; H, 5.92; N, 1
8.29. Measured value: C, 55.01; H, 5.73; N, 18.27.MS m / e, 306. Synthesis of labeling reagent B Known direction described in literature (Iwata, T .; Yamaguchi, M .; Hara,
S .; Nakamura, M .; Ohkura, Y. "J. Chromatogr." 1986, 362,
pp.209-216), a compound (IV) represented by the following formula was produced by reacting α-ketomalonic acid with 1,2-diamino-4,5-dimethoxybenzene.

1.5 g of the compound (IV) is treated in the same manner as in the case of producing the compound (III) in the synthesis of the labeling reagent A to obtain a compound (V) represented by the following formula, and further compound (V) is labeled with the above The labeling reagent B (new substance) represented by the following formula was synthesized by treating in the same manner as in the case of synthesis of the reagent A. The synthesized labeling reagent B was recrystallized from ethanol (0.9 g, yield 60%).

Physical property data of compound (V): pale yellow needle crystal.mp 163-164 ° C. NMR (Me ₂ SO-d ₆ ) (pp
m) δ 3.80 (s, 3H), 3.96 (s, 3H), 4.03 (s, 3H), 4.07
(S, 3H), 6.72 (s, 1H), 7.38 (s, 1H). Elemental analysis: Calculated value (C ₁₃ H ₁₄ N ₂ O ₅ ): C, 56.12; H, 5.04; N, 10.07. Measured value: C, 5
9.13; H, 5.04; N, 9.98.MS m / e, 278. Physical data of labeling reagent B: orange powder.mp 276-278 ° C. NMR (Me ₂ NCHO-d ₇ ).
(Ppm) δ3.7−3.9 (br, 2H), 3.75 (s, 3H), 3.97 (s, 3
H), 4.09 (s, 3H), 7.47 (s, 1H), 7.90 (s, 1H), 9.0-9.
2 (br, 1H). Elemental analysis: Calculated value (C ₁₂ H ₁₄ N ₄ O ₄ ): C, 51.8
0; H, 5.05; N, 20.13.Measured value: C, 51.70; H, 5.15; N, 20.18.MS
m / e, 278. The labeling reagents A and B showed a stable crystalline state for one year or more even under direct sunlight. Solutions of these reagents in dimethylformamide could be used for at least 2 weeks.

Reaction of labeling reagent A or B with stearic acid Labeling reagent A or B (500 mg), stearic acid (462 mg)
And EDC (314 mg) with 2% pyridine in ethanol (10
0 ml) and the mixtures were left at room temperature for 3 hours. Then the mixture was evaporated to dryness under reduced pressure.
The residue dissolved in a small amount of ethyl acetate was subjected to silica gel 60 column chromatography (25 × 2.5 cm, silica gel amount about 75
g, 70-230 mesh) and separated with the same solvent.
The main fraction was evaporated to dryness and the residue was recrystallized from ethanol to give the product (VI) represented by the formula below (0.4 g, yield 42%) or product (VII) (0.4 g, yield 44%). ) Got.

It was confirmed by the following physical property data that the reaction products of stearic acid and the labeling reagents A and B were the corresponding DMEQ derivatives, that is, the products (VI) and (VII).

Physical property data of product (VI): colorless needle crystal.mp 194-196 ° C. NMR (Me ₂ NCHO-d ₇ ) (pp
m) δ 0.76 (t, J = 7Hz, 3H), 1.16 (s, 28H), 1.3-1.7
(M, 2H), 2.11 (t, J = 7Hz, 2H), 2.53 (t, J = 7Hz, 2
H), 3.01 (t, J = 7Hz, 2H), 3.60 (s, 3H), 3.81 (s, 3H),
3.90 (s, 3H), 6.97 (s, 1H), 7.24 (s, 1H), 9.4-9.6 (b
r, 2H). Elemental analysis: Calculated value (C ₃₂ H ₅₂ N ₄ O ₅ ): C, 67.10; H,
9.15; N, 9.78.Measured: C, 67.02; H, 9.30; N, 9.73.MS m / s, 57
2. Physical property data of product (VII): orange powder.mp 192-194 ° C. NMR (Me ₂ NCHO-d ₇ ).
(Ppm) δ 0.76 (t, J = 7Hz, 3H), 1.1-1.8 (m, 32H), 3.6
1 (s, 3H), 3.82 (s, 3H), 3.92 (s, 3H), 6.88 (s, 1H),
7.25 (s, 1H), 9.4-9.6 (br, 2H). Calcd _{(C 30 H 48 N 4 O} 5):. C, 65.38; H, 9.08; N, 10.52 measurements: C, 65.
28; H, 9.00; N, 10.34.MS m / e.544.

Labeling with a labeling reagent Add 2 to the test solution (10μ) of carboxylic acid to be quantified
4.9 mmol reagent (1 μmol of EDC aqueous solution and 10% pyridine aqueous solution each dissolved in dimethylformamide)
00 μ) was added. The resulting mixture is at room temperature (15-30 ° C)
It was left for 15 minutes to react. Reaction mixture (100μ
) Was injected into the chromatograph. As a blank, 10 μm of water was used instead of 100 μm of the test aqueous solution and treated in the same manner as above.

HPLC equipment and conditions Water with U6K universal injector (10μ loop)
A Hitachi F1000 Fluorescence Spectrometer equipped with a s510 high performance liquid chromatograph and a 12μ flow cell operating at an excitation wavelength of 365 nm and an emission wavelength of 447 nm was used. The column temperature of the chromatograph was room temperature (20-27 ° C). Gradient elution was performed using a Hitachi 833A solvent gradient system to separate the DMEQ derivative of the carboxylic acid. Column is Biofine RP
C-SC18B (250 x 4.6 (inner diameter) mm, 5 μm; made by Jusco)
And TSK gel ODS-120T (250 × 4.6 (inner diameter) mm, 5 μm;
Tosoh Corporation) was used.

Results and Discussion 1. Study carboxylic acid in fluorescence properties using a reaction product of stearic acid labeling reagents A, is B, particularly preferred moved upon separating DMEQ derivatives of saturated fatty acids of C ₅ -C ₂₀ with HPLC In order to find the phase, the stearic acid obtained above and the labeling reagents A, B
Reaction products with, ie products (VI) and (VII)
Fluorescence characteristics of the same in methanol, acetonitrile, water and their mixtures (all of which are widely used as mobile phases in reverse phase chromatography) were investigated.

Figure 1 shows the product (VI) in methanol. 2 shows the fluorescence excitation spectrum (A) and the fluorescence emission spectrum (B) in FIG. The fluorescence excitation spectrum (maximum, 365 nm) and fluorescence emission spectrum (maximum, 447 nm) of the product (VI) in methanol were almost the same as those in water and acetonitrile. The maximum wavelength of the spectrum when using the aqueous methanol solution and the aqueous acetonitrile solution was independent of the concentration of water. On the other hand, the fluorescence intensity when using the aqueous methanol solution was higher than when using the aqueous acetonitrile solution in which the content rate of water was varied. The fluorescence intensity is almost maximum when the water concentration in the methanol aqueous solution is 0-70%, and the water concentration is 70%.
%, It decreased in proportion to the concentration of water. It was in neutral and alkaline solutions that the product (VI) fluoresced most strongly (Fig. 2;
I) (1.0 nmol / ml) in citrate buffer (0.1 mol,
pH 3 to 6) and phosphate buffer (0.1 mol, pH 6 to 9) showing the effect of pH on the fluorescence intensity). These results suggest that an aqueous solution of methanol is suitable as a mobile phase for reverse phase chromatography separation of DMEQ derivatives of carboxylic acids such as fatty acids by gradient elution.

On the other hand, the maximum wavelengths of the fluorescence excitation spectrum and the fluorescence emission spectrum of the product (VII) in methanol, acetonitrile, and water are 415 nm and 485 nm, respectively, which are slightly longer than those of the product (VI). It was

Regarding the reactivity of the labeling reagents A and B, that is, the reaction time, the reaction temperature and the yield of the reaction product, no difference was found between them.

2. Separation of DMEQ Derivatives of Carboxylic Acids DMEQ of C ₅ -C ₂₀ fatty acids on a reverse phase column (Biofine RPC-18B) by gradient elution with aqueous methanol.
Simultaneous separation of the derivatives was possible. That is, 100 μl of a solution of a standard mixture of saturated fatty acids was taken, labeled with the method described above, and subjected to an HPLC column (Biofine RPC-SC 18
Separated over B). Gradient elution of aqueous methanol was used as the mobile phase (0-6 min, 40% methanol; 6-
30 minutes, 40-100% methanol; 30-50 minutes, 100% methanol; flow rate 1.0 ml / min). Results are shown in FIG. Figure 3 shows
A typical chromatogram when the methanol concentration gradient in the mobile phase is changed between 40% and 100% is shown.
All peaks were completely resolved within 42 minutes. The number of each peak represents the next fatty acid.

1 ... Valeric acid 2 ... Caproic acid 3 ... Caprylic acid 4 ... Capric acid 5 ... Lauric acid 6 ... Myristic acid 7 ... Palmitic acid 8 ... Margaric acid 9 ... Stearic acid 10 ... Arachidic acid 11 ... Blank Separation of DMEQ derivative of the above fatty acid In, changes in the concentration of methanol had practically no effect on the maximum wavelengths of fluorescence excitation and fluorescence and the fluorescence intensity of the DMEQ derivatives of all these carboxylic acids. The spectrum of the DMEQ derivative was substantially the same as the spectrum of the product (VI) with the labeling reagent A.

3. Labeling conditions Labeling conditions were investigated using a mixture of various fatty acids of 1.0 nmol / ml each.

The labeling reagent A was dissolved in dimethylformamide to prepare a reaction solution. For all the above fatty acids, the strongest peaks were obtained when the concentration of labeling reagent A in the reaction solution was greater than about 2.0 mmol. In this example, it was 4.0 mmol.

EDC and pyridine were used to facilitate labeling of the carboxylic acid with labeling reagent A. Maximum and constant peak heights were achieved when the concentration of pyridine in the solution was in the range of 7-15%. The peak height of each fatty acid was maximum and constant when the concentration of EDC was higher than 1.0 mol. In this example, the concentration of pyridine was 10
%, EDC concentration was 2.0 mol.

The labeling reaction between stearic acid and the labeling reagent A was possible even at 0 ° C. FIG. 4 is a graph showing the influence of reaction time and reaction temperature on the peak height, in which 100 μ each of stearic acid (1.0 nmol / ml) solution was taken and labeled with the labeling reagent A by the method described above. The result of having separated what was done by HPLC is shown. (■) is 0 ℃, (○)
Is 15 to 30 ℃, (●) is 37 ℃, (□) is 60 ℃, and (▲) is 80
Indicates the peak height in the case of ° C. As is clear from FIG. 4, the fluorescence intensity, that is, the peak height, reached the maximum value faster as the temperature increased. However, when the labeling reaction temperature was 60 ° C., the peak height decreased. At room temperature (15 to 30 ° C.), the peak heights for all fatty acids reached a maximum after being left for 10 minutes (in this example, they were left at room temperature for 15 minutes). The DMEQ derivative in the final mixture was stable at room temperature under sunlight for at least 24 hours. By comparing the peak height value for stearic acid with that of product (VI), the yield of the fluorescent derivative of stearic acid under the above reaction conditions was calculated to be 74.2%.

4. Accuracy, calibration curve and detection limit The accuracy of the carboxylic acid quantification method of the present invention was determined by repeatedly measuring using a standard mixture of fatty acids (containing 1.0 nmol / ml of each fatty acid). The coefficient of variation is 1.2 for all fatty acids measured (n = 10 in each case).
It did not exceed%.

The relationship between the peak height and the amount of individual fatty acids is 10
There was a proportional relationship in the range of 30 femtomoles per μ to at least 5.0 picomoles. The correlation coefficient between the peak height and the amount of each fatty acid is 0 for all measured fatty acids.
It was 992 or higher.

To confirm the sensitivity, standard mixtures (100 μ) each containing 1.0 nmol / ml C ₁₄ -C ₂₀ fatty acids were treated by the labeling method described above, and the reaction mixtures were subsequently diluted 50-fold with water and subjected to HPLC. A 93% aqueous methanol solution was used as the mobile phase of HPLC, and the other HPLC conditions were the same as in the case of FIG. Results are shown in FIG. FIG. 5 is a chromatogram in which each peak corresponds to 50 femtomoles of fatty acid, and each peak number means the next fatty acid.

1 ... Palmitic acid 2 ... Margaric acid 3 ... Stearic acid 4 ... Arachidic acid 5 ... Blank FIG. 5 shows that the detection limit of fatty acid by the method of the present invention is 3-6 femtomoles at the signal-noise ratio = 3. There is.

5. Reaction of compound other than linear saturated fatty acid with labeling reagent A Unsaturated fatty acid (myristooleic acid, palmitooleic acid, oleic acid, linoleic acid, arachidonic acid, eicosabentaenoic acid, etc.), hydroxycarboxylic acid Carboxylic acids such as (lactic acid, malic acid, etc.), dicarboxylic acids (oxalic acid, malonic acid, succinic acid, adipic acid, etc.) and aromatic carboxylic acids (benzoic acid, salicylic acid, cinnamic acid, etc.) are also labeled under the above labeling conditions. And reacted with the labeling reagent A to produce a fluorescent derivative.

Separation of DMEQ derivatives of arachidonic acid metabolites by HPLC was performed as follows. Arachidonic acid metabolites (each 1.
100 μl of standard mixture (0 nmol / ml) was taken and processed by the method described above to make DMEQ derivative. The reaction mixture was injected onto an HPLC column (TSK gel 0DS-120T column),
Other HPLC conditions were separated as in the case of FIG. The chromatogram of the DMEQ derivative of the arachidonic acid metabolite thus obtained is shown in FIG. The detection limit (signal-noise ratio = 3) of arachidonic acid metabolite was about 5-10 femtomoles. Of the four peaks in FIG. 6, (1) indicates thromboxane B ₂ , (2) indicates prostaglandin B ₂ , (3) indicates leukotriene B ₄ , and (4) indicates blank.

The α-keto acids (pyruvic acid, α-ketoglutarate, phenylpyruvic acid, etc.) and 17 different α-amino acids did not fluoresce under the above labeling conditions. Other substances such as alcohols, sugars, aminos, aldehydes, ketones, phenols and sulfhydryl compounds also failed to give fluorescent derivatives under the above labeling conditions.

From these experimental results, it can be seen that the reagents used in the present invention act selectively on carboxylic acids.

(Effect of the invention) By using the novel reagent of the present invention represented by the formula (I), it becomes possible for the first time to selectively label a carboxylic acid in an aqueous solution. Moreover, this labeling reaction proceeds rapidly even at a moderate temperature such as room temperature. Furthermore, the above reagents are extremely sensitive and can detect carboxylic acids on the order of a few femtomoles.

Therefore, the method of the present invention is useful for the quantitative fluorometric analysis of biologically important carboxylic acids, in particular, thermally labile carboxylic acids such as arachidonic acid metabolites by HPLC.

[Brief description of drawings]

FIG. 1 is a diagram showing a fluorescence excitation spectrum and a fluorescence emission spectrum of a product (VI) (1.0 mmol / ml) in methanol, acetonitrile and water. Figure 2 shows the product (VI) in citrate buffer (0.1M, pH
3 to 6) and a graph showing the influence of pH on the fluorescence intensity in a phosphate buffer (0.1 M, pH 6 to 9). FIG. 3 is a diagram showing a chromatogram of a DMEQ derivative of saturated fatty acid. FIG. 4 is a graph showing the influence of the reaction time and the reaction temperature on the peak height in the labeling reaction. FIG. 5 is a diagram showing a chromatograph of a DMEQ derivative of C ₄ -C ₂₀ carboxylic acid. FIG. 6 is a diagram showing a chromatogram of a DMEQ derivative of an arachidonic acid metabolite.

Claims (2)

(57) [Claims] 1. A novel quinoxalinone derivative represented by the following general formula. (Wherein R ₁ , R ₂ and R ₃ represent a C _1-6 lower alkyl group, and R ₄ represents a group — (CH ₂ ) _n CONHNH ₂ (n is an integer from 0 to 6) means) 2. A method for quantifying carboxylic acid, which comprises using a compound represented by the following formula as a reagent. (Wherein R ₁ , R ₂ and R ₃ represent a C _1-6 lower alkyl group, and R ₄ represents a group — (CH ₂ ) _n CONHNH ₂ (n is an integer from 0 to 6) means)