JP2549744B2 - Method for quantifying α-keto acid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for quantifying α-keto acid, which comprises using 4,5-diaminophthalhydrazide as a highly sensitive chemiluminescent reagent.

(Prior Art) Some labeling reagents (derivatization reagents) used for quantifying α-keto acid by spectrophotometric detection or fluorometric detection using liquid chromatography have been reported so far.

Colorimetric determination of α-keto acids by transfer of 2,4-dinitrophenylhydrazine was performed by C. Hemming and CJ Gubler.
Proposed by (Anal.Biochem.92 (1979) 3
1) has poor detection sensitivity and lacks selectivity for α-keto acids. On the other hand, for the determination of α-keto acid by a fluorometric method, a method using 4′-hydrazino-2-stilbazole as a reagent (T. Hirata, M. Kai, K. Kohashi and Y.
Ohkura, J. Chromatogr., 226 (1981) 25), and Ortho-
Method using phenylenediamine (JCLioa, NEHoffm
an, JJ Barboriak and DARoth, Clin. Chem., 23 (1977)
802, and T. Hayashi, H. Tsuchiya and H. Naruse, J. Chrom
atogr., 273 (1983) 245), respectively.
Both methods have fairly good sensitivity, but cannot measure α-keto acid at the femtomole (fmol: 10 ⁻¹⁵ mol) level.

The present inventors have previously reported 1,2-diamino-, as a highly sensitive and selective fluorescent labeling reagent for α-keto acids.
4,5-Dimethoxybenzene and 1,2-diamino-4,5-methylenedioxybenzene were synthesized (for the former reagent, S. Hara, Y. Takemori, T. Iwata, M. Yamaguchi, M. Naka).
mura and Y. Ohkura, Anal. Chim. Acta, 172 (1985) 167 and S. Hara, M. Yamaguchi, M. Nakamura and Y. Ohkura, Chem.P.
harm.Bull., 33 (1985) 3493, and for the latter reagent, M. Nakamura, S. Hara, M. Yamaguchi, Y. Takemori and Y.
Ohkura, Chem. Pharm. Bull., 35 (1987) 687, respectively. ). The present inventors have applied these reagents to liquid chromatography, and as a result, have made it possible to quantify α-keto acid at the femtomole level. These reagents have been used for the determination of α-keto acids in human urine and serum (S. Hara, Y. Takemori, M.
Yamaguchi, M. Nakamura and Y. Ohkura, J. Chromatogr., 344
(1985) 33).

Recently, the technique of chemiluminescence detection has been introduced into an analytical method by liquid chromatography, and several reagents that react with amino acids, amines, and carboxylic acids to give chemiluminescent derivatives have been reported. SRSpurlin and MM Cooper, Anal. Lett., 19 (1
986) 2277, for reagents for amines, see T. Kawas.
aki, M. Maeda and A. Tsuji, J. Chromatogr., 328 (1985) 12
1.For reagents for carboxylic acids, see T. Kawasaki,
M. Maeda and A. Tsuji, J. Chromatogr., 328 (1985) 121, H.
Yuki, H. Azuma, M. Maeda and T. Kawasaki, Chem. Pham. Bul
l., 36 (1988) 1905, and H. Karatani, J. Takano, S. Mor.
ishita, M. Yoshida and M. Sato, Analytical Chemistry, 38 (1989) 59.
Are reported respectively. ).

(Problems to be Solved by the Invention) As described above, many attempts have been made to quantify α-keto acid, but a very small amount of α-
So far, no report has been made on the reagents necessary for sensitive and selective quantification of keto acid. For example, extremely small amounts of various α-keto acids are present in the living body, and it has been desired to develop reagents necessary for quantifying such small amounts of α-keto acids by chemiluminescence.

(Means for Solving the Problem) As a result of intensive studies by the present inventor, a reagent capable of quantifying a very small amount of α-keto acid with high sensitivity by using chemiluminescence , 3,4- and 4,5-diaminophthalhydrazide dihydrochloride (hereinafter referred to as "3,4-DPH.
2HCl ”and“ 4,5-DPH · 2HCl ”), especially
It was discovered that 4,5-DPH.2HCl selectively reacts with α-keto acid in dilute hydrochloric acid to produce a compound exhibiting chemiluminescence, and the present invention was completed based on this finding.

The determination of α-keto acid according to the present invention is performed as follows. 4,5-DPH.2HCl is reacted with α-keto acid to make the corresponding quinoxalinone derivative. Then, this derivative is reacted with hydrogen peroxide in the presence of potassium hexacyanoferrate (III) in an alkaline medium, and the reaction product emits light. This luminescence is quantified by a detector. The above chemical reaction is expressed by the following equation.

(Note) R means any substituent.

As can be seen from the above reaction formula, the quantifiable α-keto acid by the method of the present invention is not limited to a specific range,
Any α-keto acid may be used. As used herein, the term "α-keto acid" means all α-keto acids. Examples of α-keto acids include α-ketobutyric acid, pyruvic acid, phenylpyruvic acid, o (m, p) -hydroxyphenylpyruvic acid, benzoylformic acid, α-ketovaleric acid, α-ketoisovaleric acid, α- Ketocaproic acid,
Examples include α-ketoisocaproic acid and α-keto-β-methylvaleric acid.

Examples of the present invention will be shown below, but the present invention is not limited thereto.

(Examples) Chemicals and Solvents All chemicals and solvents used were the purest ones available. For all aqueous solutions, Milli-QII system (Millipore: Millipor
Distilled water purified in e) was used. Hydrogen peroxide (30%, v /
For v), a product manufactured by Mitsubishi Mutual Chemical Company was used.

Synthesis of reagents 3,4- and 4,5-diaminophthalhydrazide (hereinafter referred to as "3,
“4-DPH” and “4,5-DPH” are each synthesized from 4-nitrophthalic acid by the Williams-Sharaby method (for the Williams-Shallaby method, RL Williams and SW Shalaby, J. Hetero).
cycl. Chem., 10 (1973) 891. ). That is, 4-nitrophthalic acid was refluxed in methanol-concentrated sulfuric acid to obtain dimethyl 4-nitrophthalate. The ester was reduced to dimethyl 4-aminophthalate and subsequently converted to the acetamide derivative. This acetamide derivative was nitrated to obtain a mixture of dimethyl 4-acetamido-5-nitrophthalate and dimethyl 4-acetamido-3-nitrophthalate. These compounds were separated by fractional crystallization and column chromatography. Each of the separated acetamide derivatives is hydrolyzed to obtain a corresponding nitramine derivative, and each nitramine derivative is catalytically reduced to obtain two diaminophthalates (that is, 4,5- and 3,4-diamino) in an isomer relationship. Dimethyl phthalate) was obtained. Each of these diaminophthalates was condensation reacted with hydrazine hydrate and triethylamine in methanol to produce 4,5-DPH and 3,4-DPH.
4,5-DPH and 3,4-DPH were each separately mixed with a small amount of concentrated hydrochloric acid and the resulting precipitate was recrystallized from ethanol to give the corresponding dihydrochloride salt. The chemical structures of 4,5-DPH and 3,4-DPH, and their respective dihydrochlorides were confirmed by MS, IR and NMR data. The thus obtained 4,5-DPH
Both 2HCl and 3,4-DPH · 2HCl maintained a stable crystalline state at room temperature for 6 months or longer in a desiccator containing silica gel.

Preparation of reagent solution Using 0.6M hydrochloric acid containing 0.6M mercaptoethanol 3,
Solutions of 4- and 4,5-DPH.2HCl (1.2 mM) were prepared. These solutions were used within 5 hours after preparation. Water and 2.0M
A hydrogen peroxide solution (50 mM) and a potassium hexacyanoferrate (III) solution (30 mM) were prepared using sodium hydroxide.

Liquid Chromatography Conditions and Chemiluminescence Detection System FIG. 1 is a flow chart of a liquid chromatography / chemiluminescence quantification system. In this figure, each symbol has the following meaning.

_{P 1, P 2, P 3} ; Liquid Chromatography Pump I: Injection Valve D: Detector G: guard column M _1, M _2: mixing apparatus Rec: recording device A: Stainless steel tube E: mobile phase R ₁ : Hydrogen peroxide solution R ₂ : Potassium hexacyanoferrate (III) solution Chromatography was performed using Hitachi 635 solution chromatograph. This chromatograph shows Rheodyne
7125 injector loaded sample injector valve (Rheody
It is equipped with ne 7125 syringe-loadingg sample injetor valve, I) in Fig. 1 (20-μ loop). α-
The diaminophthalhydrazide (DPH) derivative of keto acid is TSK
Separation was performed on a gel ODS-120T (5 μm) phase-transfer column (250 × 4.6 mm inner diameter) (Tosoh Corporation, Tokyo). Separation is performed using acetonitrile-50 mM phosphate buffer (pH 7.0) (1
3:87, v / v) and a flow rate of 1 ml / min. The column temperature was room temperature (23 ± 1 ° C.).

The eluate from the column is first mixed with a hydrogen peroxide solution,
It was then mixed with a potassium hexacyanoferrate (III) solution. The hydrogen peroxide solution has a flow rate of 1.0 ml / min, and the potassium hexacyanoferrate (III) solution has a flow rate of 2.0 ml / min.
It was sent to each mixing device (M ₁ , M _{2 in} FIG. 1) by a pump (Jasco, Tokyo) (P ₂ , P _{3 in} FIG. ₁ ). Regarding chemiluminescence of the column eluate after mixing with each of the above solutions,
AC-2 with 60μ flow cell
The detection was carried out by a 200 luminescence detection device (ATTO, Tokyo) (D in FIG. 1). In this liquid chromatography system, a stainless steel tube (inner diameter 0.5 m
m, A) in FIG. 1 was used.

Labeling treatment with α-keto acid 100 μm of multiple test solutions of α-keto acid placed in a tube (100 × 15 mm inner diameter) sealed with a screw cap
A 100 mM 1.2 mM DPH (diaminophthalhydrazide) solution was added. Seal this tube tightly and 100
Heat at 45 ° C. for 45 minutes. A 20 μl portion of the final reaction mixture was injected onto the chromatograph. As a blank, the same treatment was applied to 100 μ of water instead of the test solution.

Evaluation of diaminophthalhydrazides and their dihydrochlorides as chemiluminescent labeling reagents 3,4- and 4,5-DPH and their dihydrochloride suitability as a chemiluminescent labeling reagent for α-keto acids Was evaluated. The α-keto acids used in this evaluation were α-ketobutyric acid, ρ-hydroxyphenylpyruvic acid, α-ketovaleric acid, α-ketoisovaleric acid, α-ketocaproic acid, α-ketoisocaproic acid, α-keto. It was -β-methylvaleric acid and phenylpyruvic acid. For all these α-keto acids tested, 4,5-D
The chemiluminescence intensity obtained using PH ・ 2HCl is 3,4-D.
The value was about 30 times higher than the chemiluminescence intensity when PH ・ 2HCl was used. One of the reasons why the chemiluminescence is weak when the 3,4-DPH derivative is used is considered to be the steric hindrance effect at the C-3 position (KDGundermann and M. Drawert, Che.
m.Ber., 95 (1962) 2018). The diaminophthalhydrazides used are only slightly soluble in water and dilute hydrochloric acid,
It gave a small interfering peak in the chromatogram. The interference peak was due to impurities in the reagent.

From this test result, it was found that 4,5-DPH.2HCl was the most suitable reagent. 4,5-DPH ・ 2HCl itself showed very weak chemiluminescence under the test conditions (equimolar concentrations of α-ketoisovaleric acid and 4,5-DPH ・ 2H
About 2% of chemiluminescence of the reaction derivative with Cl).

Conditions for liquid chromatography α-ketobutyric acid, phenylpyruvic acid, p-hydroxyphenylpyruvic acid, α-ketovaleric acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-ketocaproic acid and α- The DPH derivative of α-keto acid consisting of 8 kinds of ketoisocaproic acid was separated. Separation of these α-keto acid derivatives was carried out by TSK gel ODS-120T reverse phase column and acetonitrile-50 mM phosphate buffer (pH 7.
0). The concentration of acetonitrile in the mobile phase affected the separation of blank and α-keto acids peaks. When the concentration of acetonitrile was higher than 15%, the peak for α-ketobutyric acid overlapped with the blank. On the other hand, when the concentration of acetonitrile was less than 10%, the elution was delayed, and as a result, the peak width was widened. Particularly in the case of phenylpyruvic acid, the broadening of the peak width was large. When the concentration of the phosphate buffer in the mobile phase was 10-100 mM and the pH was 2.5-7.5, there was no effect on the separation of the α-keto acids and the chemiluminescence luminosity. The mobile phase is aqueous methanol
methanol) was used, the peaks for the above eight derivatives and the blank could not be separated no matter how the concentration of methanol was changed.

Therefore, liquid chromatography was performed on the DPH derivatives of the eight α-keto acids using acetonitrile-50 mM phosphate buffer (pH 7.0) (13:87, v / v). These derivatives were successfully isolated within 50 minutes.
The obtained chromatogram is shown in FIG. The numbers 1 to 9 mean the following α-keto acids.

1: α-ketobutyric acid 2: p-hydroxyphenylpyruvic acid 3: α-ketovaleric acid 4: α-ketoisovaleric acid 5: α-ketoisocaproic acid 6: α-keto-β-methylvaleric acid 7: α-ketocapron Acid 8: Phenylpyruvate 9: Blank As is apparent from FIG. 2, each α-keto acid gave a single peak in the chromatogram. This indicates that 4,5-DPH.2HCl can be used as a labeling reagent for α-keto acids.

Labeling of α-keto acids with 4,5-DPH · 2HCl α-keto acids and 4,5-DPH · 2HCl reacted in dilute hydrochloric acid but not in neutral or alkaline solutions. In order to maximize the peak height of the chromatogram, the concentration of hydrochloric acid in the DPH solution needs to be 0.4 to 0.8 molar, and the concentration of 4,5-DPH2HCl in the reagent solution is about 1 .
0 mmol or more was required. In this example, the former concentration was 0.6 mol and the latter concentration was 1.2 mmol.
Β-mercaptoethanol was used as a stabilizer of 4,5-DPH · 2HCl during the labeling reaction. When this stabilizer was not used, the area of the blank (peak 9) in FIG. 2 was large, which prevented the detection of α-ketobutyric acid (peak 1) and p-hydroxyphenylpyruvic acid (peak 2). Therefore, it is preferable to use β-mercaptomethanol (0.6 M in DPH solution).

The labeling reaction between 4,5-DPH.2HCl and α-keto acid became faster as the reaction temperature increased. Examples of p-hydroxyphenylpyruvic acid and α-ketovaleric acid are shown in FIG. According to this, after heating at 100 ° C for 40 minutes, the peak height becomes maximum and becomes constant. Therefore, in this reaction, heating at 100 ° C. for about 45 minutes is desirable.

Chemiluminescence reaction The optimum conditions for chemiluminescence were investigated by setting the flow rates of hydrogen peroxide solution and potassium hexacyanoferrate (III) solution to 1.0 ml / min and 2.0 ml / min, respectively.

The concentrations of hydrogen peroxide, potassium hexacyanoferrate (III) and sodium hydroxide are very important in measuring the intensity of chemiluminescence. The maximum intensities were investigated by varying the concentrations of these three reagents separately. The results are shown in FIGS.
Figure 4 shows hydrogen peroxide, and Figure 5 shows hexacyanoiron (II
FIG. 6 shows the concentration of sodium hydroxide and the intensity of chemiluminescence, respectively, for I) potassium acid. In addition,
In FIGS. 4 to 6, 1 is p-hydroxyphenylpyruvic acid and 2 is α-ketovaleric acid. As is clear from these figures, the maximum chemiluminescence intensity was 50 mM for hydrogen peroxide, 30 mM for potassium hexacyanoferrate (III), and 2.0 M for sodium hydroxide. Become.

Chemiluminescence was observed immediately after mixing the eluate obtained by the above liquid chromatography with the potassium hexacyanoferrate (III) solution. Therefore, it was considered that the flow of the tube between the second mixing device (M _{2 in} FIG. 1) and the detector affected the chemiluminescence reaction, and the chemiluminescence intensity was investigated for various tube lengths. The results are shown in Fig. 7. In the figure, 1 represents p-hydroxyphenylpyruvic acid, and 2 represents changes in peak height when α-ketovaleric acid was used. As is clear from FIG. 7, the height of the chemiluminescence peak for α-keto acid increases as the length of the tube decreases. This indicates that the chemiluminescent reaction occurs very quickly and ends in a short time.

The relationship between the calibration curve, the accuracy and the detection limit peak height and the concentration of each α-keto acid was examined, and it was found that the former is proportional to the latter from 20 femtomoles to at least 100 picomoles per 20 μ injection amount. This concentration range of α-keto acids corresponds to 200 femtomolar to 1 nanomolar in a 100 μ test solution.

8 kinds of α-keto acids (concentration of each: 500 pmol / ml)
The detection accuracy was obtained by repeatedly measuring (total 6 times) using the mixture of. The coefficient of variation did not exceed 4.0% for any α-keto acid.

The detection limits of 8 kinds of α-keto acids (content per 10 μm injection amount (unit: femtomoles), signal-noise ratio = 3) were as follows.

α-keto acid: 5.0, p-hydroxyphenylpyruvic acid: 22.
0, α-ketovaleric acid: 4.0, α-ketoisovaleric acid: 15.0, α-
Ketoisocaproic acid: 12.0, α-keto-β-methylvaleric acid: 50.0, α-ketocaproic acid: 60, phenylpyruvic acid:
5.0 The sensitivity of the method of the present invention is about 2 to 20 times higher than that of the fluorescence analysis method using conventional liquid chromatography using 1,2-diamino-4,5-methylenedioxybenzene (N. Nakamura, S. Hara, M. Yamaguchi, Y. Takemori and
Y. Ohkura, Chem. Pharm. Bull., 35 (1987) 687). In particular,
The method of the present invention is useful for the quantification of p-hydroxyphenylpyruvic acid, which can be measured only on the order of 0.5 to 1.0 picomoles by the most sensitive fluorescence analysis method.

Reaction of other substances with 4,5-DPH.2HCl Ascorbic acid and 4,5-D under the preferred reaction conditions described above.
When PH.2HCl was reacted, several peaks appeared in the chromatogram (retention time 7-13 minutes). However, the intensity of these peaks is 1/3 of that of the α-keto acid mentioned above.
Was less than. Other biologically important substances were also examined under the above-mentioned preferable reaction conditions at a concentration of 10 nmol / ml, but none of them emitted chemiluminescence.
The compounds used in this test are as follows.

2-, 3- and 4-methoxybenzaldehyde: 4-methylbenzaldehyde; 17 1-amino acids; histamine;
Tyramine; tryptamine; 2-phenylethylamine; glutathione; thiamine; uracil; adenine; uric acid; urea; bilirubin; acetone; acetophenone; lactic acid; malic acid; butyric acid; inositol; d-glucose; d-xylose; d-fructose; d- Mannose; d-ribose; d-
Lactose; aldosterone; epiandrosterone;
Cortisone; Cholesterol These results show that the labeling method of the present invention has selectivity for α-keto acid and is extremely useful for detecting α-keto acid.

(Effects of the Invention) The method of the present invention using 4,5-DPH · 2HCl as a chemiluminescent labeling reagent is selective for α-keto acids, and has a femtomolar level of α which was not possible by conventional methods. -It is possible to detect keto acids. Therefore, the present invention can be applied to quantify a very small amount of various α-keto acids contained in blood or urine. Particularly, the method of the present invention exerts a great effect on the detection of p-hydroxyphenylpyruvic acid which is present in an extremely small amount in normal urine.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a liquid chromatograph-chemiluminescence detection system used in practicing the method of the present invention. FIG. 2 is a diagram showing a chromatogram of a DPH derivative of α-keto acid. FIG. 3 is a graph showing the influence of reaction time and reaction temperature on the labeling reaction of α-keto acid with 4,5-DPH · 2HCl. FIG. 4 is a graph showing the effect of hydrogen peroxide concentration on the height of the chemiluminescence peak. FIG. 5 is a graph showing the influence of the concentration of potassium hexacyanoferrate (III) on the height of the chemiluminescence peak. FIG. 6 is a graph showing the effect of sodium hydroxide concentration on the height of the chemiluminescence peak. FIG. 7 is a graph showing the effect of the length of the tube between the second mixing device and the chemiluminescence detector on the height of the chemiluminescence peak.

Claims (1)

(57) [Claims] 1. A method for quantifying α-keto acid, which comprises using 4,5-diaminophthalhydrazide as a reagent.