JP7384360B2 - Albumin analysis method and albumin analysis device - Google Patents

Description

The present invention relates to a method and apparatus for analyzing albumin, which is one of proteins.

Albumin is a protein with a molecular weight of approximately 66,000 that accounts for 50-70% of blood proteins. Albumin has a total of 35 cysteine (Cys) residues, but only the 34th Cys residue (Cys34) from the N-terminus is free (has a free thiol (SH) group). All other Cys residues form disulfide (SS) bonds within the molecule. Albumin in which no substance is bound to the SH group of Cys34 is called reduced albumin. On the other hand, the SH group of Cys34 may form a covalent bond with glutathione in the blood or cysteine, a free amino acid, and such albumin is called oxidized albumin.

Various studies have shown that the ratio of oxidized/reduced forms of albumin in human serum (human serum albumin) (also called the "degree of albumin oxidation") increases with age, and that it also increases in various pathological conditions. It is becoming clear that there is a correlation with diseases. Therefore, it is thought that the oxidized/reduced form ratio of human serum albumin can be used as a diagnostic marker or prognostic marker for specific diseases, and research is underway to clinically apply this. In addition, since albumin in the urine increases in kidney diseases, etc., there is hope for early detection of diseases using albumin in the urine.

Era S, et al., ``Further Studies on the Resolution of Newman Mercapto- and Non-Mercapto Albumin and on Human Serum Albumin in the Elderly.'' Further studies on the resolution of human mercapt- and nonmercaptalbumin and on human serum albumin in the elderly by high-performance liquid chromatography,” International Journal of Peptide Protein Research ( International journal of peptide and protein research), 1988, Vol.31, No.5, pp.435-442 M. Sogami and 4 others, “Resolution of Human Mercapt-and Nonmercaptalbumin by High-Performance Liquid Chromatography” "Performance Liquid Chromatography", International journal of peptide and protein research, 1984, Vol. 24, pp. 96-103 Funk W., and 5 others, "Enrichment of cysteinyl adducts of human serum albumin", Analytical Biochemistry, 2010, Vol.400, No.1, pp.61-68

Conventionally, liquid chromatography (LC) using an ion exchange column has been used to separate reduced albumin and oxidized albumin, and an ultraviolet-visible spectrometer (UV-VIS) has been used to detect albumin separated by the column. detector) is used (see Non-Patent Documents 1 and 2). In this method, the reduced form and the oxidized form can be separated and quantified, and the oxidized/reduced form ratio can be determined from the results.

However, in LC separation using an ion exchange column, the peaks are relatively broad, so when other components are present, the peaks of these components tend to overlap, which may impede accurate quantification. Furthermore, since the detection sensitivity of this method is relatively low, if the content of one or both of the oxidized and reduced forms is minute, the quantitative accuracy may be reduced or detection may not be possible.

On the other hand, a method for relatively accurately quantifying oxidized albumin is known to convert oxidized albumin into reduced albumin by applying a low concentration of DTT (Dithiothreitol) to the Cys34 bond of oxidized albumin. (See Non-Patent Document 3). Reduced albumin can be quantified without DTT treatment and with DTT treatment, and the absolute amount of oxidized albumin can be calculated from the difference, and the oxidized/reduced ratio can be calculated based on the results. You can also do that.

However, with this method, unless treatment conditions such as the concentration of DTT are appropriately set, there is a possibility that not all of the oxidized Cys34 will necessarily convert to the reduced form, or that DTT will act on Cys residues other than Cys34. , there is a risk that the conversion from oxidized form to reduced form may not be carried out appropriately. Therefore, analysis work is complicated, and it is not suitable for applications that require efficiency and low cost, such as screening.

The present invention has been made to solve the above problems, and its purpose is to improve the detection sensitivity and quantitative accuracy of oxidized albumin and reduced albumin, even when albumin is present in trace amounts. An object of the present invention is to provide an albumin analysis method and analyzer that can accurately determine the oxidized/reduced ratio.

An albumin analysis method according to one aspect of the present invention, which has been made to solve the above problems, includes:
After separating albumin from other components in the target sample using liquid chromatography using a reversed-phase column, fluorescence due to specific amino acid residues contained in albumin is detected, and based on the detection results, the concentration of albumin in the target sample is detected. a first measurement step of quantifying albumin;
a second measurement step of selectively fluorescently labeling reduced albumin in the target sample to detect fluorescence, and quantifying reduced albumin in the target sample based on the detection result;
a calculation step of calculating an oxidized/reduced ratio of albumin in the target sample based on the quantitative results obtained in the first measurement step and the second measurement step;
It is intended to carry out the following.

Furthermore, an albumin analyzer according to one aspect of the present invention, which has been made to solve the above problems, includes:
a reverse phase column that separates albumin from other components in the target sample; a first fluorescence due to a specific amino acid residue contained in albumin in the eluate from the reverse phase column; a liquid chromatograph section having a detector that respectively detects second fluorescence due to a substance used for fluorescent labeling of reduced albumin;
Quantifying albumin in the target sample based on the first fluorescence detection result, quantifying reduced albumin in the target sample based on the second fluorescence detection result, and based on the quantification results, a calculation processing unit that calculates the oxidized/reduced ratio of albumin in the target sample;
It is equipped with the following.

In the albumin analysis method and analyzer according to the above aspects of the present invention, liquid chromatography using a reverse phase column rather than an ion exchange column is used to separate albumin from other components (proteins, etc.). By using a reversed phase column, albumin can be better separated from other components, and the influence of overlapping of other components can be reduced. Unlike an ion exchange column, it is difficult to separate reduced albumin and oxidized albumin using a reversed phase column, but this is not necessary in the present invention.

In addition, to detect albumin, which has been temporally separated from other components by liquid chromatography using a reversed-phase column, we use fluorescence detection based on natural fluorescence from specific amino acids contained in albumin, rather than an ultraviolet-visible spectrometer. Some amino acids that make up proteins emit relatively strong natural fluorescence, and albumin also contains these amino acids, so by detecting this fluorescence, albumin can be detected with higher sensitivity than conventional methods. can be detected. Thereby, the total amount of albumin in the target sample can be calculated with high sensitivity and with almost no influence from other components.

As mentioned above, it is difficult to separate reduced albumin and oxidized albumin by liquid chromatography using a reversed phase column. Therefore, in the present invention, a target sample is subjected to a labeling treatment using a substance that selectively fluorescently labels reduced albumin, and the absolute amount of reduced albumin is calculated by detecting the fluorescence caused by the substance. The total amount of oxidized albumin can be determined from the quantitative results of reduced albumin and the total amount of albumin, and the ratio of oxidized/reduced albumin in the target sample can be calculated with high accuracy.

According to the albumin analysis method and analyzer according to the above aspects of the present invention, the oxidized/reduced form ratio can be calculated with high accuracy even when the amount of albumin contained in the target sample is small. Further, since there is no need for sample pretreatment that requires setting of complicated processing conditions such as the above-mentioned DTT treatment, analysis work can be performed easily and efficiently. Further, according to the albumin analyzer according to the above aspect of the present invention, both the total amount of albumin and the reduced albumin in the target sample are quantified in one liquid chromatography analysis of the target sample. The time required can be shortened.

FIG. 1 is a diagram explaining the principle of an embodiment of the albumin analysis method according to the present invention. 1 is a schematic configuration diagram of an albumin analyzer that is an embodiment of the present invention. FIG. 2 is a diagram showing a chromatogram obtained by analyzing an albumin-containing sample with a liquid chromatograph using a conventional ion exchange column and a fluorescence detector. FIG. 2 is a diagram showing a chromatogram based on the fluorescence intensity of a fluorescent labeling substance obtained by analyzing an albumin-containing sample with the albumin analyzer of the present embodiment. FIG. 3 is a diagram showing a chromatogram based on the fluorescence intensity of natural fluorescence obtained by analyzing an albumin-containing sample with the albumin analyzer of the present embodiment. FIG. 3 is a diagram showing a comparison of chromatogram waveforms with and without DAABD modification in the albumin analyzer of the present embodiment.

EMBODIMENT OF THE INVENTION Hereinafter, one embodiment of the albumin analysis method and albumin analysis apparatus according to the present invention will be described with reference to the accompanying drawings.

[Explanation of the principle of the albumin analysis method according to the present embodiment]
FIG. 1 is an explanatory diagram of the principle of the albumin analysis method according to this embodiment. Here, it is assumed that blood (serum) or urine of a subject is used as a sample and albumin in the sample is analyzed. In the albumin analysis method of this embodiment, two types of measurements are performed: one is to quantify the total amount of albumin in the target sample, and the other is to quantify reduced albumin in the target sample. do. However, as will be described in detail later, these two measurements do not necessarily mean two measurements, and the two measurements can be performed substantially simultaneously by one measurement on the target sample.

The measurement for quantifying the total amount of albumin in the target sample involves separating albumin from other components (contaminant components in the sample) by LC using a reversed phase column, and natural fluorescence caused by specific amino acids contained in albumin. This is carried out in combination with fluorescence detection targeting.

As mentioned above, conventional albumin analysis methods utilize LC using an ion exchange column. Although it is possible to separate oxidized albumin and reduced albumin by LC using an ion exchange column, the peaks corresponding to each albumin are quite broad. Therefore, if a contaminant component (for example, another protein) with a similar retention time is present, there is a possibility that such a contaminant component overlaps with albumin and elutes. On the other hand, although it is difficult to separate oxidized albumin and reduced albumin using LC using a reversed phase column, the peaks corresponding to these albumins are considerably sharper than when using an ion exchange column. Therefore, even if a contaminant component whose retention time is close to that of albumin is present, such contaminant component and albumin can be accurately separated. Furthermore, since the peak height increases as the peak width becomes narrower, sensitivity can be improved.

In addition, in conventional albumin analysis methods, an ultraviolet-visible spectroscopic detector was used to detect albumin, but the inventor of the present application utilized natural fluorescence derived from amino acids contained in albumin, compared to an ultraviolet-visible spectroscopic detector. We experimentally found that higher sensitivity can be obtained using fluorescent detection. Generally, it is known that three types of aromatic amino acids can be used as fluorescent probes in protein molecules, but in albumin, among these, tryptophan residues (excitation wavelength 295 nm → fluorescence wavelength 340 nm) and tyrosine residues are used as fluorescent probes. Natural fluorescence originating from the group (excitation wavelength 280 nm→fluorescence wavelength 340 nm) is useful. In particular, one molecule of albumin contains only one tryptophan residue but 18 tyrosine residues. Therefore, when considering the total fluorescence intensity of each amino acid, tyrosine is more useful, and it was experimentally confirmed that tyrosine-derived fluorescence can be detected with higher sensitivity .

FIG. 3 is a diagram showing a chromatogram obtained by analyzing a sample containing albumin using LC that combines an ion exchange column and fluorescence detection using natural fluorescence. It can be seen that although the reduced albumin and oxidized albumin are sufficiently separated, each peak is quite broad.

FIG. 4 is a diagram showing a chromatogram obtained by analyzing a sample containing albumin using LC using a reversed phase column. It can be seen that by using a reversed phase column, the peak derived from albumin is observed sharply. However, when detected using an ultraviolet-visible spectrometer, the baseline is generally high and a large drift occurs, which also causes the peak to be low. On the other hand, by performing fluorescence detection, the baseline is lowered and stabilized, and the peak becomes higher. According to experiments conducted by the present inventors, by changing from LC using an ion exchange column to LC using a reversed phase column, the sensitivity is improved by about 200 times. Furthermore, by changing from ultraviolet-visible spectroscopic detection to fluorescence detection, the sensitivity is improved by about 4 times. As described above, the albumin analysis method of this embodiment achieves sufficiently high sensitivity compared to conventional methods by combining LC separation using a reversed-phase column and fluorescence detection that targets the natural fluorescence derived from amino acids contained in albumin. With this, the total amount of albumin can be determined.

FIG. 5 is a diagram showing chromatograms obtained by detection using an ultraviolet-visible spectroscopic detector, fluorescence detection for natural fluorescence derived from tyrosine, and fluorescence detection for natural fluorescence derived from tryptophan. From the results of this experiment, it can be seen that the use of tyrosine is more advantageous than tryptophan in terms of detection sensitivity, as described above.

On the other hand, as mentioned above, it is difficult to separate the reduced form and oxidized form by LC separation using a reversed phase column (although it is not impossible, it is quite difficult to set the conditions for separation). By detecting only reduced albumin using a fluorescent reagent that reacts only with reduced Cys, reduced albumin and oxidized albumin were separated upon detection. As a fluorescent reagent, DAABD-Cl (excitation wavelength 395 nm→fluorescence wavelength 505 nm) , which was personally developed and commercialized by Kazuhiro Imai, one of the inventors of the present invention, can be used. Since DAABD-Cl reacts only with reduced albumin and does not react with oxidized albumin, reduced albumin can be quantified with high sensitivity by detecting fluorescently labeled reduced albumin. Of course, it is not limited to DAABD-Cl, but any fluorescent labeling substance that reacts only with reduced albumin, more specifically, that specifically binds to the SH group of free Cys34, can be used.

Once the absolute amount of total albumin (oxidized + reduced) in the target sample and the absolute amount of reduced albumin in the target sample are known, the amount of oxidized albumin in the target sample can be calculated by subtracting the latter from the former. Absolute quantities can be calculated. Therefore, from the calculation result, it is possible to further calculate the reduced/oxidized albumin ratio in the target sample.

Fluorescence detection is used for both of the above two measurements, but regardless of whether tyrosine or tryptophan is used to detect natural fluorescence, detection of DAABD-labeled reduced albumin depends on the excitation light and fluorescence wavelength. are different. Therefore, we experimentally investigated the effect of DAABD modification on the fluorescence intensity of tyrosine and tryptophan.

FIG. 6 is a diagram showing an actual measurement example of a comparison of peak shapes with and without DAABD modification, and FIG. 6(b) is an enlarged time axis diagram of FIG. 6(a). In the figure, +DAABD indicates DAABD modification, -DAABD indicates no DAABD modification, FR395>505 nm indicates fluorescence detection of DAABD, FR280>340 nm indicates fluorescence detection of tyrosine, and FR295>340 nm indicates fluorescence detection of tryptophan. In this experiment, the sample was a standard human serum albumin (HSA) (1 mM).

When the peak area of each peak was calculated and the change in peak area due to the presence or absence of DAABD modification was investigated, in fluorescence detection of tyrosine, when DAABD modification was performed, the peak area was 103.15% of that without DAABD modification. On the other hand, in fluorescence detection of tryptophan, when DAABD modification was performed, the peak area was 112.61% of that without DAABD modification. That is, it was found that the fluorescence detection of tryptophan was more affected by the DAABD modification than the fluorescence detection of tyrosine. On the other hand, it was also found that almost no influence by DAABD modification was observed in fluorescence detection of tyrosine. This means that by using a detector that can simultaneously detect fluorescence of two or more wavelengths, it is possible to simultaneously detect reduced albumin using fluorescence derived from DAABD modification and detection of the entire albumin using fluorescence derived from tyrosine. It means that. That is, the two measurements described above can be performed simultaneously in substantially one LC analysis.

As mentioned above, when using tyrosine-derived fluorescence to detect albumin as a whole, there is no need to substantially consider the influence of DAABD modification, but if you want to quantify albumin with higher accuracy, DAABD Arithmetic processing may be performed to correct the effect of modification (increase in peak area). Further, as long as calculation processing to correct the influence of such DAABD modification is performed, tryptophan-derived fluorescence may be used to detect albumin as a whole. By performing simultaneous detection of two wavelengths in a single LC analysis in this way, the quantification of the total amount of albumin and the quantification of only reduced albumin can be performed in parallel, reducing the time and effort required for analysis. Efficient analysis is possible.

[Configuration and operation of albumin analyzer of this embodiment]
FIG. 2 is a schematic configuration diagram of an embodiment of an albumin analyzer for carrying out the albumin analysis method described above.

This albumin analyzer includes a liquid chromatograph section 10, a data processing section 20, and a display section 30. The liquid chromatograph section 10 includes a mobile phase container 11 in which a mobile phase is stored, a pump 12 that sucks and supplies the mobile phase from the mobile phase container 11, an injector 13 that injects a sample into the mobile phase, and a reversed phase column. 14, a column oven 15 that maintains the column 14 at a constant temperature, and a fluorescence detector 16 that can detect two wavelengths simultaneously. The data processing unit 20 includes a data storage unit 21, a chromatogram creation unit 22, a peak detection unit 23, a quantitative calculation unit 24, and a reduced type/oxidized type ratio calculation unit 25 as functional blocks.

Generally, the entity of the data processing unit 20 is a computer called a personal computer or a higher-performance workstation, and by running data processing software preinstalled on the computer, the functions of each of the functional blocks described above are implemented. can be realized.

The excitation wavelength and fluorescence wavelength of the fluorescence detector 16 are excitation wavelength: 280 nm and fluorescence wavelength: 340 nm, which can detect natural fluorescence due to tyrosine in albumin, and fluorescence due to DAABD, which is used for fluorescent labeling of reduced albumin. Excitation wavelength: 395 nm, fluorescence wavelength: 505 nm.

The sample to be analyzed by the liquid chromatograph unit 10 is a target sample containing albumin subjected to a DAABD-Cl reaction treatment. Through this reaction treatment, only reduced albumin in the sample is labeled with DAABD. Pump 12 supplies mobile phase to reversed phase column 14 at a constant flow rate. A target sample is injected into the mobile phase from the injector 13 at a predetermined timing, and pushed by the mobile phase, the target sample is introduced into the reverse phase column 14. Regardless of whether the albumin is in the oxidized or reduced form, the albumin in the target sample is separated from other components (such as other proteins) in the time direction while passing through the reversed phase column 14 and is eluted from the outlet of the reversed phase column 14 .

The fluorescence detector 16 simultaneously detects the two fluorescence wavelengths described above and sends detection data corresponding to the respective fluorescence intensities to the data processing section 20. That is, detection data that reflects the absolute amount of albumin as a whole in the target sample and detection data that reflects the absolute amount of only reduced albumin in the same target sample are sent in parallel to the data processing unit 20, and the data is stored. It is temporarily stored in the section 21. The chromatogram creation unit 22 creates chromatograms based on the two systems of detection data stored in the data storage unit 21. This chromatogram creation process can be performed even before the LC analysis is completed.

Since the time at which albumin elutes from the column outlet is roughly known depending on the LC separation conditions such as the mobile phase flow rate, the peak detection section 23 detects two chromatograms in a predetermined time range before and after the albumin elution time. Perform peak detection for each. Then, a peak derived from whole albumin and a peak derived from reduced albumin are detected, and the peak area (or peak height) is calculated for each. The quantitative calculation unit 24 refers to previously prepared calibration curves (calibration curve for total amount of albumin and calibration curve for reduced albumin), and calculates the absolute amount of total albumin and reduced form from the peak area (or peak height). Calculate the absolute amount of albumin.

The reduced/oxidized form ratio calculation unit 25 calculates the absolute amount of oxidized albumin by subtracting the absolute amount of reduced albumin from the absolute amount of albumin as a whole. Then, the reduced/oxidized ratio is calculated from the absolute amount of reduced albumin and the absolute amount of oxidized albumin, and is output to the display unit 30 together with the calculation results of these absolute amounts. Thereby, information such as the reduced/oxidized albumin ratio and absolute amount contained in the target sample can be presented to the user.

Note that, as described above, when performing quantification from the peak area, a process may be performed to correct the influence of DAABD modification. Furthermore, instead of detecting natural fluorescence due to tyrosine in albumin in the fluorescence detector 16, the excitation wavelength and fluorescence wavelength may be set so that natural fluorescence due to tryptophan can be detected. Furthermore, the fluorescence detector 16 capable of simultaneous detection of three wavelengths is used to detect natural fluorescence caused by tryptophan and natural fluorescence caused by tyrosine, and based on the detection data of both, the absolute value of albumin as a whole is determined. You may also ask for the quantity.

Moreover, although it is preferable that the fluorescence detector 16 simultaneously detects fluorescence of a plurality of wavelengths, it may be one that detects each fluorescence by sequentially switching a plurality of fluorescence wavelengths.

As described above, according to the albumin analyzer of the present embodiment, by performing one LC analysis on the target sample and performing data processing based on the data obtained in the LC analysis, the target sample The ratio of reduced/oxidized albumin in the albumin and the absolute amount of each albumin can be obtained with high accuracy. In addition, as mentioned above, the detection sensitivity for albumin as a whole and reduced albumin is high, so even if the absolute amount of albumin contained in the target sample is small, which cannot be analyzed using conventional methods, reduced/oxidized albumin can be detected. The ratio can be calculated satisfactorily.

It should be noted that the above-mentioned embodiment is an example of the present invention, and it is clear that modifications, changes, and additions made as appropriate within the scope of the spirit of the present invention are encompassed within the scope of the claims of the present application.

[Various aspects]
It will be apparent to those skilled in the art that the exemplary embodiments described above are specific examples of the following aspects.

(Section 1) One embodiment of the albumin analysis method according to the present invention is
After separating albumin from other components in the target sample using liquid chromatography using a reversed-phase column, fluorescence due to specific amino acid residues contained in albumin is detected, and based on the detection results, the concentration of albumin in the target sample is detected. a first measurement step of quantifying albumin;
a second measurement step of selectively fluorescently labeling reduced albumin in the target sample to detect fluorescence, and quantifying reduced albumin in the target sample based on the detection result;
a calculation step of calculating an oxidized/reduced ratio of albumin in the target sample based on the quantitative results obtained in the first measurement step and the second measurement step;
It is intended to carry out the following.

(Section 5) One embodiment of the albumin analyzer according to the present invention is
a reverse phase column that separates albumin from other components in the target sample; a first fluorescence due to a specific amino acid residue contained in albumin in the eluate from the reverse phase column; a liquid chromatograph section having a detector that respectively detects second fluorescence due to a substance used for fluorescent labeling of reduced albumin;
Quantifying albumin in the target sample based on the first fluorescence detection result, quantifying reduced albumin in the target sample based on the second fluorescence detection result, and based on the quantification results, a calculation processing unit that calculates the oxidized/reduced ratio of albumin in the target sample;
It is equipped with the following.

According to the albumin analysis method described in Section 1 and the albumin analyzer described in Section 5, even when the amount of albumin contained in the sample to be analyzed is small, the oxidized/reduced form ratio can be accurately determined. It can be calculated. Further, since there is no need for sample pretreatment such as DTT treatment, which requires setting of complicated processing conditions, analysis work can be performed easily and efficiently. In particular, according to the albumin analyzer described in Section 5, both the total amount of albumin and the reduced albumin in the target sample are quantified in one liquid chromatography analysis of the target sample. The time required for this can be reduced.

(Paragraph 2) In the albumin analysis method described in Paragraph 1, reduced albumin is selectively labeled with fluorescence, and albumin and other components are separated by liquid chromatography using the reversed phase column. The first measurement step and the second measurement step are performed substantially simultaneously by respectively detecting the fluorescence due to a specific amino acid residue contained in the fluorescent label and the fluorescence due to the substance used for the fluorescent label. can be taken as a thing.

Detection of fluorescence due to a specific amino acid residue contained in albumin and detection of fluorescence due to a substance used for a fluorescent label may be performed simultaneously, or may be performed alternately by switching wavelengths. According to the albumin analysis method described in Section 2, both the total amount of albumin and the reduced albumin in the target sample are quantified in one liquid chromatography analysis of the target sample. The time required for analysis can be shortened, and the effort required for analysis work can also be reduced.

(Section 3) In the albumin analysis method according to Item 1 or 2, the fluorescent label may use DAABD-Cl.

According to the albumin analysis method described in Section 3, reduced albumin can be detected with high sensitivity.

(Section 4) In the albumin analysis method according to any one of Items 1 to 3, the specific amino acid residue is at least one of tyrosine, tryptophan, or phenylalanine. It can be assumed that there is. In particular, since albumin contains a large amount of tyrosine, it is advantageous to use natural fluorescence due to tyrosine to increase detection sensitivity.

10...Liquid chromatograph section 11...Mobile phase container 12...Pump 13...Injector 14...Reverse phase column 15...Column oven 16...Fluorescence detector 20...Data processing section 21...Data storage section 22...Chromatogram creation section 23...Peak Detection unit 24...Quantitative calculation unit 25...Reduced type/oxidized type ratio calculation unit 30...Display unit

Claims (4)

Albumin in the target sample is separated from other components by liquid chromatography using a reversed phase column , fluorescence due to specific amino acid residues contained in albumin is detected, and based on the detection results, albumin in the target sample is separated. a first measurement step of quantifying;
Selectively fluorescently label reduced albumin in the target sample using DAABD-Cl (4-[2-(dimethylamino)ethylaminosulfonyl]-7-chloro-2,1,3-benzoxadiazole). a second measurement step of detecting fluorescence and quantifying reduced albumin in the target sample based on the detection result;
a calculation step of calculating the oxidized/reduced ratio of albumin in the target sample based on the quantitative results obtained in the first measurement step and the second measurement step;
An albumin analysis method characterized by performing the following. Prior to performing liquid chromatography using the reversed phase column, reduced albumin in the target sample is selectively labeled with fluorescence , and then albumin and other components are separated by liquid chromatography using the reversed phase column. , the first measurement step and the second measurement step are substantially performed by respectively detecting the fluorescence due to the specific amino acid residue contained in the albumin and the fluorescence due to the substance used for the fluorescent label. The albumin analysis method according to claim 1, which is carried out simultaneously. The albumin analysis method according to claim 1 or 2 , wherein the specific amino acid residue is at least one of tyrosine, tryptophan, and phenylalanine. A reverse phase column that separates albumin from other components in the target sample, a first fluorescence due to a specific amino acid residue contained in albumin in the eluate from the reverse phase column, and a first fluorescence in the eluate. The second fluorescence caused by DAABD-Cl (4-[2-(dimethylamino)ethylaminosulfonyl]-7-chloro-2,1,3-benzoxadiazole), which was used for fluorescent labeling of reduced albumin, was a liquid chromatograph section having a detector for each detection;
Quantifying albumin in the target sample based on the first fluorescence detection result, quantifying reduced albumin in the target sample based on the second fluorescence detection result, and based on the quantification results, a calculation processing unit that calculates the oxidized/reduced ratio of albumin in the target sample;
An albumin analyzer equipped with.

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