JPS6314910B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and apparatus for analyzing sugars. More specifically, the present invention involves adding a reaction reagent consisting of a boric acid aqueous solution containing a basic amino acid to a sample containing sugars or a column eluate obtained by subjecting the same to liquid chromatography, and performing a heating reaction. Fluorophotometric analysis of saccharides, characterized in that the saccharides are qualitatively and/or quantitatively analyzed by cooling the saccharides, irradiating them with excitation light and measuring their fluorescence intensity, and/or irradiating them with ultraviolet rays and measuring their absorbance. law and/or
The present invention also relates to an ultraviolet absorption analysis method and an analysis device suitable for implementing this method.

Conventionally, sugars have been detected using a liquid chromatograph using a differential refractometer. However, since this differential refractometer cannot employ the gradient elution method, it is not easy to analyze samples containing multi-component sugars such as biological samples. In other words, in liquid chromatography, even if sugars are eluted by continuously changing the solvent composition, there is usually a large difference in the refractive index of each solvent. It was almost impossible to analyze sugars due to the large influence of Furthermore, differential refractometers have inherently low sensitivity, making it difficult to detect trace amounts of sugars.

In this regard, the inventors of the present invention have previously developed a method and apparatus for reacting saccharides with a specific reaction reagent to generate fluorescence in saccharides that do not themselves emit fluorescence, and detecting the fluorescence intensity to analyze saccharides. (Refer to Japanese Patent Application Laid-open No. 70739/1983). As a result of further research, the inventors of this invention discovered that by adding and reacting a boric acid aqueous solution containing basic amino acids to saccharides, they were able to analyze saccharides with higher sensitivity and excellent linearity by fluorescence measurement. The present invention was achieved by discovering that it is possible to perform a similar analysis of saccharides by ultraviolet absorbance measurement.

The reagent for reacting with sugars used in this invention is
A boric acid aqueous solution containing basic amino acids,
Examples of basic amino acids include arginine, lysine, histidine, and the like. Usually, it is appropriate that the concentration of basic amino acids in the reaction reagent be 0.5-4% and the concentration of boric acid 1-6%, and the pH of the reaction reagent obtained at this time is approximately 7-9. Such reaction reagents are respectively added to a column eluate obtained by subjecting a sample containing sugars to liquid chromatography. In some cases, the sample may be added without being separated and used for measuring total sugars.

As the column eluent used in liquid chromatography, it is appropriate to use water or a boric acid aqueous solution (usually 0.1M to 0.5M) whose pH is adjusted to neutral to weakly basic, and the above reaction to this column eluent The mixing ratio of the reagents is preferably in the range of 50 to 150%. Note that known columns and devices can be used for liquid chromatography.

On the other hand, the reaction is preferably carried out at 140°C to 180°C for 3 to 5
It takes place for a minute. After the reaction, the reaction mixture is cooled to room temperature and introduced into a fluorometer and/or an ultraviolet absorption photometer for measurement, thereby making it possible to qualitatively and/or quantitatively analyze saccharides based on the fluorescence intensity and/or absorbance.

On the other hand, one of the main structural features of the analyzer of the present invention is that the mixture of column eluent and reaction reagent is configured to move through a reaction liquid flow path to a heating tank and then to a cooling tank. As a result, it is possible to perform a reaction to perform fluorescence analysis and/or ultraviolet absorption analysis, and it is also possible to employ a gradient elution method in liquid chromatography. Multi-component sugars can be analyzed at once with high precision.

Another structural feature of the analyzer of the present invention is that the flow of the sample liquid from the supply of the column eluent and reaction reagent to the discharge of the reaction liquid is continuous and one-time flow. This makes high-speed analysis possible and suitable for complete automation.

Examples of sugars that can be analyzed using the analytical method and apparatus of the present invention include monosaccharides such as glucose, mannose, galactose, fructose, and rhamnose;
Examples include oligopolysaccharides such as maltose, lactose, and maltotriose, amino sugars such as glucosamine and galactosamine, and uronic acids such as glucuronic acid and galactulonic acid, and can be applied to all reducing sugars. It is particularly suitable for analyzing biological samples that contain multiple components of these saccharides.

The present invention will be described in detail below based on embodiments shown in the figures. Note that this invention is not limited by this.

In FIG. 1, an automatic saccharide analyzer 1 includes a high-performance liquid chromatograph main body 25 and a reaction liquid pipe 9 extending to an eluent pipe 3 of a column 2.
, a reaction reagent supply pipe 4 which is connected to the reaction liquid pipe, and a heating tank 6, a cooling tank 7, through which the reaction liquid pipe 9 passes in order after the joint connection part (mixing part) 5. It mainly consists of an ultraviolet absorption photometer and/or a fluorophotometer 8, and a reaction liquid discharge pipe 10 further extending from a reaction liquid pipe 9.

The reaction reagent supply line 4 is provided with a pump 11 and a damper 12, and supplies a basic amino acid-containing boric acid aqueous solution as a reaction reagent to the reaction liquid line 9, where it is mixed with the column eluent. Normally, a three-way conduit is used for the merging connection part 5 of both flow paths.

The heating tank 6 is filled with tin, which has good heat transfer, as a heating medium, and an electric heater 17 and a reaction liquid pipe 9 as a heat source are inserted into this heating medium. The insertion portion 13 of this reaction liquid conduit is formed into a coil shape with a predetermined length. In addition, 18 is a heat sensitive body for temperature control, 19 is an inner tank part, 20 is an outer tank part, and 21 is a heat insulating material filled between both tanks.

The cooling tank 7 introduces circulating cooling water, and the reaction liquid pipe 9 is inserted into this cooling water. The insertion portion 14 of this reaction liquid conduit is also formed into a coil shape with a predetermined length.

Said fluorometer and/or ultraviolet absorption photometer 8
A recorder 16 and the like are attached, and a resistance tube 22 is interposed in the reaction liquid discharge conduit 10.

Next, automatic saccharide analyzer 1 consisting of the above configuration
We will explain how to analyze sugars using

First, the pump 11 of the reaction reagent supply line 4 and the pump 23 of the high performance liquid chromatography apparatus main body 25 are activated in advance, and a reaction reagent consisting of a boric acid aqueous solution containing a basic amino acid and a column eluent are mixed into the reaction solution. It flows into the conduit 9 and maintains a steady state. Next, the sample is transferred from the main body injection part 24 to the column 2.
When injected into the column, the sample is separated into components by column,
It is eluted from the column and enters the reaction liquid pipe 9 through the eluent pipe 3, where it is mixed with a reaction reagent and heated to 150°C for about 5 minutes to react in a heating tank 6, and then passes through a cooling tank 7. It is then cooled down to room temperature. Each sample component (sugar) separated by this passage is converted into an ultraviolet absorbing and fluorescent substance, and its concentration is detected by an ultraviolet absorption photometer and/or a fluorometer 8.

In this way, sugar analysis becomes possible with high sensitivity. Furthermore, after mixing the column eluent with the reaction reagent,
Automatically reacts while being moved and discharged in a transitory manner,
Since cooling, ultraviolet absorbance and/or fluorescence measurements are performed, it is suitable for complete automation. Of course, there is no point where the movement is interrupted, so analysis can be performed at high speed.

More importantly, since a gradient elution method can be employed in a liquid chromatograph, it is also possible to analyze multi-component saccharides such as saccharides in living organisms at once with high precision.

Hereinafter, this invention will be explained in more detail with reference to Examples.

Example 1 Saccharides were analyzed using an automatic analyzer 1 as shown in FIG. The conditions are shown below.

Liquid chromatograph device: Shimadzu High Performance Liquid Chromatograph LC-3A Analytical column: Shimadzu Gel SCR-101N Mobile phase: Purified water Mobile phase flow rate: 0.5 ml/min Column temperature: 60°C Detector: Fluorometer (Shimadzu RF-530; (excitation wavelength 320 nm; fluorescence wavelength 430 nm) Reaction reagent: 2.0% arginine/5.0 boric acid aqueous solution Reaction reagent flow rate: 0.5 ml/min Reaction temperature: 150°C Sample: glucose, maltose, fructose, xylose Sample injection amount: 0.25 to 7.5 μg each As described above, when arginine was used as a reaction reagent, the linearity between the injection amount of each sugar and the analytical value was investigated. The results are shown in FIG. In this way, excellent linearity was obtained over a wide range for all sugars, indicating that highly accurate and accurate measurements are possible.

Example 2 Analysis was carried out in the same manner as in Example 1, except that the detector sensitivity was increased and a sample (20μ) containing 2n (nano) moles each of glucose, maltose, and maltotriose was used. The results are shown in FIG. In this way, excellent sensitivity can be obtained even in small amounts.

Example 3 An ultraviolet absorption photometer (Shimadzu UVD-
Analysis was carried out in the same manner as in Example 2, except that 2; detector sensitivity: 0.01 AUFS; wavelength used: 254 nm). The results are shown in FIG. As described above, excellent sensitivity was also obtained with respect to ultraviolet absorption, and it can be seen that saccharides that do not substantially absorb ultraviolet rays by themselves come to strongly absorb ultraviolet rays through the treatment of the present invention.

Example 4 Automatic analyzer 1 in which a fluorometer (RF-530; excitation wavelength 320 nm, fluorescence wavelength 430 nm; detector sensitivity 16 times) and an ultraviolet absorption photometer (UVD-2; wavelength 254 nm) are connected as detectors. The analysis was performed using The conditions are as follows.

Liquid chromatography device: Shimadzu high performance liquid chromatograph LC-3A Analysis column: Shimazu LC column ISA-07/S2504 Mobile phase: Adjusted to PH8.5 with potassium hydroxide
0.3M boric acid aqueous solution Mobile phase flow rate: 0.5ml/min Column temperature: 65℃ Reaction reagent: 2.0% arginine/5.0% boric acid aqueous solution Reaction reagent flow rate: 0.5ml/min Reaction temperature: 150℃ Sample: Glucose 5, Maltose 1 , a mixture of mannose 2, fructose 3, galactose 4, and xylose 6. Sample injection amount: 1 μg or more of each. Fluorescence and absorbance were measured separately in parallel, and charts were drawn simultaneously based on the results. It is shown in FIG. In FIG. 5, a peak projecting downward from the top indicates ultraviolet absorbance, and a peak projecting upward from the bottom indicates fluorescence intensity.

In this way, excellent sensitivity has been demonstrated for each sugar, both in terms of fluorescence and absorption.

Example 5 A 0.4M boric acid aqueous solution adjusted to pH 9.0 with potassium hydroxide was used as the mobile phase, and the mobile phase flow rate was 0.6.
ml/min, Shimadzu FLD-1 as a fluorometer;
Excitation wavelength 300-400nm (360nm maximum), measurement wavelength
The procedure was the same as in Example 4, except that a wavelength of 430 nm or higher (using a filter) was used, a 2% histidine/3.0% boric acid aqueous solution was used as the reaction reagent, the reaction reagent flow rate was 0.6 ml/min, and the sample injection amount was 2 μg each. conducted an analysis. The results are shown in FIG.

[Brief explanation of the drawing]

FIG. 1 is a configuration explanatory diagram showing a specific example of the analyzer of the present invention, and FIG. 2 is a straight line between the measured intensity (peak height) obtained by the method of the present invention and the actual concentration. It is a graph showing the relationship, and the third to
FIG. 6 is a diagram showing an example of a chromatograph obtained by the method of the present invention. 1... Automated saccharide analyzer, 2... High performance liquid chromatography column, 3... Eluent pipe line, 4...
...Reaction reagent supply pipe line, 5...Mixing section, 6...Heating tank, 7...Cooling tank, 8...Ultraviolet absorption photometer and/or fluorometer, 9...Reaction liquid pipe line, 10...
...Reaction liquid discharge part, 11... Pump, 12... Damper, 13... Reaction liquid pipe line part, 14... Reaction liquid pipe line part, 25... Chromatography apparatus main body.

Claims (1)

[Claims] 1. A reaction reagent consisting of a boric acid aqueous solution containing a basic amino acid is added to a sample containing sugars or a column eluate obtained by subjecting the same to liquid chromatography, and a heating reaction is performed. 1. A method for analyzing saccharides, characterized in that the saccharides are analyzed by cooling the saccharides after cooling, irradiating them with excitation light and measuring their fluorescence intensity, and/or irradiating them with ultraviolet rays and measuring their absorbance. 2. A reaction solution flow path is extended from the column eluent flow path to the main body of the liquid chromatograph apparatus, a supply flow path for a boric acid aqueous solution containing a basic amino acid is connected to this flow path, and then a heating section and a cooling section are connected. A saccharide analysis device characterized in that a saccharide analyzer is provided with a fluorescence measurement section and/or an ultraviolet absorption measurement section in this order.