JPS6182167A - Analysis of amino acid - Google Patents

Abstract

PURPOSE:To make possible the easy analysis of amino acids, more particularly, secondary amino acid with high accuracy and high sensitivity by subjecting a sample contg. amino acids having the secondary amino group to liquid chromatography, oxidizing electrochemically the elutate in alkalinity and adding a fluorescence emitting reagent thereto. CONSTITUTION:The elutate 2 obtd. after making a sodium citrate soln. acidic by a hydrochloric acid or citric acid, etc. is fed from a vessel 1 by a liquid feed pump 3 and is mixed with the sample injected into a sample injecting part 4. The mixture thereof is fed to a separating column 5. A cation exchange resin is packed in the column 5 in the case of the acidic elutate. The eluted liquid in the sample is held at 9-12pH by an alkali buffer soln. 7 in a vessel 6 and is mixed with an o-phthalaldehyde reagent 12 in a vessel 11. The mixture is fed to a reacting part 14 where the mixture is brought into reaction with the amino acids in the sample. The luminous compd. thereof is measured by a fluorophotometer 15. The analysis of the cyclic amino acids such as proline and hydroxyproline having the secondary amino group which are heretofore difficult to analyze is thus made possible always with the high accuracy and high sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to a method for analyzing amino acids, and in particular to a method for analyzing cyclic amino acids having a secondary amino group such as proline, hydroxyproline, etc.

(B) Prior Art Conventionally, the analysis of amino acids containing cyclic amino acids with secondary amino groups such as glolin and hydroxyproline involves first treating these amino acids with an oxidizing agent such as sodium hypochlorite. After converting the 2a amino group into a primary amino group, O-phthalo-μ-dehyde (hereinafter OPA
This method is carried out by reacting with a fluorescent substance (abbreviated as ``Sho 57-135'') to form a fluorescent substance, and measuring the fluorescence intensity.
Publication No. 355).

However, in the above analytical method, the oxidizing agent used is generally unstable, and once the oxidizing agent reagent is prepared, it can only be used for about a week, and the titer of the reagent must be measured about once a day. The problem is that it doesn't.

(79 Purpose) This invention was made in order to solve the above problems, and by performing electrochemical oxidation without using an oxidizing agent, it is possible to easily and always achieve high precision and high sensitivity in a stable state. The purpose is a method for analyzing amino acids.

2) Structure This invention subjects a sample containing an amino acid having a secondary amino group to liquid chromatography that separates it using an acidic or alkaline mobile phase, electrochemically oxidizes the obtained eluate under alkaline conditions, and generates fluorescence. The present invention provides an amino acid analysis method comprising adding a chemical reagent and reacting with the amino acid to convert it into a fluorescent compound, and performing fluorescence analysis of the resulting reaction solution.

The following combinations of mobile phase and separation column are used in the liquid chromatograph 7 parts of the present invention. The mobile phase may be either acidic or alkaline. The acidic mobile phase used is a sodium citrate solution acidified with hydrochloric acid, citric acid, perchloric acid, etc. In this case, the separation column is one packed with a cation exchange resin (for example, manufactured column, engineering 5C-O)/5150
4) etc. are used. On the other hand, as the alkaline mobile phase, an aqueous disodium hydrogen phosphate solution, a boric acid buffer, etc. are used, and the separation column in this case is one packed with anion exchange resin (for example, Shimabara column, 15A-07/
52504) etc. are used.

The eluent obtained by the above liquid chromatography is p
After being maintained at 7μ potency of H9-12, the amino acid having a secondary amino group is oxidized by electrochemical oxidation, and the amino group is converted into a primary amine group.

If the pH is less than 9, electrochemical oxidation is insufficient.

When the 9% eluent obtained using an acidic mobile phase is less than 9, an alkaline buffer is added to bring the pH to 9-12. As this buffer solution, one prepared by adding potassium hydroxide to an aqueous boric acid solution, an aqueous solution of a mixture of sodium podate, boric acid, and potassium sulfate, an aqueous solution of sodium hyponate, one sodium bicarbonate, and the like are used.

Electrochemical oxidation is performed by inserting at least one pair of positive and negative electrodes and applying voltage. For example, a 2-electrode EC detector 5100A 7μ for high-performance liquid chromatographs manufactured by Couloque Gosha, which is equipped with a Bohusgoofite electrode, is used, and in this case, the appropriate voltage to be applied is LO to λ4v.

Next, for example, OPA f: is added as a fluorescent reagent and reacted with amino acids to convert it into a fluorescent substance, and the fluorescence intensity is measured to analyze the amino acids.

Amino acids can be analyzed in the same manner as in the present invention by using ninhydrin as a reaction reagent instead of a fluorogenic reagent such as 0PAO, and by performing DK absorbance analysis instead of fluorescence analysis of the reaction solution.

(e) Examples This invention will be explained by examples, but this invention is not limited to the following.

FIG. M1 and FIG. 2 are diagrams illustrating the configuration of an embodiment of an analyzer for carrying out the present invention.

In Fig. 1 and Fig. 2, (1, 1') is a mobile phase container, (2, 2 is a mobile phase, (3, 3') is a mobile phase liquid pump, and (4, 4') is a mobile phase container. Sample injection part, (5,5') is separation column, (6) is alkaline buffer container, () is alkaline buffer, (8) is alkaline buffer pump, (9
) is the mixing part, (10,10') is the electrochemical oxidation part,
(ml, 11') is OPA reagent container, (12, 12'
) is a PA reagent, (13, 13 is an OPA reagent pump, (14, 14') is a reaction section, and (15, 15') is a fluorometer.

Example (when acidic mobile phase was used) 1. Examination of pH of electrochemical oxidation part Buffer solutions of various pHs were added to the eluent and analyzed under the following conditions.

a0 analyzer (device shown in Figure 1) Separation column: Shimabara 5C-0F/51504 (5
5℃) Mixing part carp /&/: inner diameter approx. 0.5 mm, length approx. 0.2 m
Stainless steel pipe (55℃) Electrochemical oxidation section: Cell reaction section for high-performance liquid chromatography manufactured by Coulochem Co., Ltd. /I/: Stainless steel pipe with an inner diameter of about 0.5 m and a length of about zm (about 65℃) ) Fluorometer: Shimabara RF-530 (excitation light wavelength 348 nm, measurement fluorescence improvement 45 onm) b, sample solution (aqueous solution) 3al injection concentration hydroxyglolin (pro) 1 μmol/
m Glolin (Pro) // Aspartic acid (Asp) l fimo1/li
t-threonine (Thr) tt-serine (Ser)
ttgutamic acid (Glu) tt
Green (GD) II Afnin (Ala) ttC1 mobile phase (flow rate 0.3 ml/min) A 0.2N aqueous sodium citrate solution (containing 7 thietanoμ) was adjusted to pH 3.20 with perchloric acid. .

d, OPA reagent (flow rate 0.3 yd/win) OP
A O, Bf, Engineering 1 No A/'711j%n-7 Seti/I/\Stein 1f and 20% Br1j 35 To a mixture of 4d of aqueous solution, 122klf of sodium carbonate, 40. Total amount of fluoride and potassium sulfate 56.4
-1io aqueous solution (carbonic acid, -borate buffer, pH 10,
2) Added to bring the total amount to 1.

e. Added buffer solution and electrochemical oxidation conditions The chromadogume obtained by the above analysis (112 and 3)K are shown in FIGS. 3 to 5. As a result, it is clear that proline and hydroxyproline cannot be detected when the pH during electrochemical oxidation is acidic, such as 4.3. When fF-X)H is alkaline such as 9.4 or 9.7, proline and hydroxyproline are detected even when the above-mentioned amino acids having a primary amino group coexist.

Examination of applied voltage to electrochemical oxidation section Electrochemical oxidation was performed and analyzed by changing the applied voltage under the following conditions.

a0 analyzer (same as above 1), sample liquid
〃C0 mobile phase // d, OPA reagent 〃e,
Applied buffer solution 1.8 above (D analysis? & L3 same f, applied voltage of electrochemical oxidation section Voltage CV) 4 0.85 5 LO 62,0 72,4 Obtained by analysis of 4 to 7 above The chromatograms obtained are shown in Figures 6 to 9, and the relationship between the applied voltage and the concentration of proline and hydroxyproline in the chromatograms is shown in Figures 6-9.

FIG. 12 shows a graph showing the relationship between the height and the height.

The results show that the analysis sensitivity for proline and hydroxyproline is better when the applied voltage exceeds the LOV.

Example n (using alkaline mobile phase) Twenty-eight samples were analyzed under the following conditions.

a0 analyzer: System separation force shown in Figure 2:
Shimabara Corporation 5A-07/52504 (60°C) Electrochemical oxidation section: Example i, 1. Same reaction to a carp /l/:
I/Fluorometer:
〃 b, Sample solution 10-Injection analysis inhibition 7 Hydroxyproline Approximately 2 fi I! mol
/111O91N hydrochloric acid solution 8 Proline approximately 2 μmol, /dO,1N hydrochloric acid solution C1 Mobile phase (flow rate 0.5 m/min) 50mM sodium dihydrogen phosphate aqueous solution d, OPA reagent (flow rate 0.3
rd/min) Same as in Example I above. Applied voltage in electrochemical oxidation section: 0.9V.
It is shown in Fig. and Fig. 11. This shows that hydroxyproline and glolin can be analyzed with good sensitivity.

(F) Effects According to the present invention, amino acids, especially amino acids having a secondary amine group, can be quantitatively analyzed in a simple and stable manner with high accuracy and sensitivity.

[Brief explanation of the drawing]

Figures 1 and 2 are explanatory diagrams of the structure of a companion analysis table for carrying out the unpacking of this invention, Figures 3 to 11 are the results obtained by analysis under various analysis conditions, and Figure 12 is This is a graph showing the relationship between the voltage applied to the electrochemical oxidation section and the peak height of amino acids in chromium. (1%1...mobile phase container, (2,2')...mobile phase, (3,3', 8.13, 13')...liquid feeding bonde, (4,4') ...Sample injection part, (5,5')...
・Separation column, (6)...alkaline buffer container, ()
)... Alkaline buffer, (9)... Mixing section, (lO
110')...Chemical oxidation part, (11,11')...
・0-lid μAμdehyde reagent container, (12,12′)
... 0-FtaμAμdehyde reagent% (14,14'
)...Reaction part, (15,15')...Fluorometer.

Claims (1)

[Claims] 1. A sample containing an amino acid with a secondary amino group is subjected to liquid chromatography that is separated using an acidic or alkaline mobile phase, the resulting eluate is electrochemically oxidized under alkaline conditions, and a fluorescent reagent is added. An amino acid analysis method comprising reacting with the amino acid to convert it into a fluorescent compound, and performing fluorescence analysis of the resulting reaction solution.