JPH03255956A - Analysis of hemoglobin - Google Patents

Abstract

PURPOSE:To analyze the components of saccharogenic Hbs (hemoglobins) in a short period of time by eluting the saccharogenic Hbs adsorbed in an affinity column and introducing the saccharogenic Hbs to an ion exchange column by a selecting means, then separating and measuring the components of the respective saccharogenic Hbs. CONSTITUTION:The flow passage of a 6-way valve 10 is set at I to inject a sample into the system from an auto-sampler 8 while the moving phase C liquid in an eluant tank 3 is fed by a liquid feed pump 6. The sample is introduced together with the C liquid to the affinity column 9. The non-saccharogenic Hbs which are not held in the column 9 are introduced through the connecting end a.f of the valve 10 to a detector 13 and are discharged from a drain 15. The valve 10 is thereafter changed over to a flow passage II and the moving phase is changed with the A liquid in an eluant tank 1. The saccharogenic Hbs held in the column 9 are eluted by this A liquid and are introduced via the connecting end a.b of the valve 10 to the ion exchange column 11 where the saccharogenic Hbs are separated to respective Hb components. The components are introduced in order of the elution through the connecting end e.f of the valve 10 to the detector 13. The saccharogenic Hb components are detected by the detector 13 and are then discharged from the drain 15.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for analyzing hemoglobin. More specifically, the present invention relates to a method for analyzing glycated hemoglobin and/or non-glycated hemoglobin in blood, particularly hemoglobin A1° in glycated hemoglobin, in a short time and with high precision using liquid chromatography.

(Prior Art) Glycated hemoglobin in the blood, particularly hemoglobin A1°, is known as one of the indicators of diabetes, and its measurement is being carried out. Hemoglobin A1゜ is a complex in which one molecule of glucose is bound non-enzymatically to the amino group of valine present at the N-terminus of the β chain of hemoglobin (hereinafter hemoglobin is referred to as Hb), as shown in the following formula (1〉). body.Hb
When glucose binds to the amino group of
(L-HbA+J (I) is formed: red glucose (I)
(TI) In the above formula (1), β-A-NH2 is i
JH2 (indicated by b) represents the amino group of valine at the N-terminus of the β chain of the Hb. This L-BbA+. (1
) is a reversible reaction, and the equilibrium is L-HbAs depending on the glucose concentration. (I) tends toward the production direction or dissociation direction.

(I) further undergoes Amatoli rearrangement and irreversibly changes to stable HbA1° (S-HbAI.) (■).

This 5-HbAs accounts for the total amount of Hb in the blood. It is known that the relative percentage of glucose in the blood (blood sugar level) is not affected by temporal changes and reflects the patient's average blood sugar status over the past 1 to 3 months. . Therefore, 5-HbAI. By measuring the value, it is possible to screen for diabetes and manage blood sugar in diabetic patients. In this specification, unless otherwise stated, HbA
l. This is 5-HbAl.

This refers to

HbAI. Various methods are known for measuring , such as liquid chromatography using ion exchange columns, affinity columns, electrophoresis, etc., and are used in the field of clinical testing.

In particular, HbAI by liquid chromatography methods. The measurement of is widely used, and high-performance liquid chromatography (
Dedicated measuring devices using IIPLc) technology have also been developed.

A column packed with ion exchange resin is used to measure HbA 1° by ion exchange chromatography.
Each Hb acid component is separated by its charge difference. HbAI obtained by this method. Values vary depending on the type of ion exchange resin used, column size, and other analysis conditions. For example, under certain analytical conditions, L-HbAI. and/or fetal hemoglobin (HbF) is HbAt
. eluted at the same time and therefore HbAl. The value is Hb
F and/or L-HbA +. The price increases by the amount of overlap. The above HbAs. Even when using a dedicated measuring device, HbAI. Although it is possible to separate L-)1bA1° from HbF, it is difficult to completely separate L-)1bA1°.

Furthermore, E. C. Abraham et al. (J. Lab.
, Cl1n, Med.

1983, ■2.18? -197) is an unknown non-glycated Hb in the analysis of Hb by ion-exchange chromatography.
b is HbAl. It is pointed out that they are eluted at the same time.

and Llrsula Turpeinen et al. (CI
in, Chew, 1989, 35.33-3
6) reported that this unknown non-glycated Hb may be acetylated Hb. In other words, Hb obtained by conventional ion-exchange chromatography-based Hb analysis
AI. The values include the above HbF and/or L-HbA
It is believed that in addition to 1°, an unknown value of non-glycated Hb is also added. Thus, it is difficult to accurately measure HbA 1° using only ion exchange chromatography.

On the other hand, in the above-mentioned affinity chromatography measurement method, the dihydrobixiboronyl group of the filler and the glycated H
By utilizing the interaction with the 1,2-cis diol group of the sugar chain of b, it is possible to separate glycated Hb from non-glycated Hb in the sample. A kit for measuring glycated Hb using an oven column method using this method is also commercially available. According to this method, the degree of glycation of Hb can be measured as the relative percentage of the amount of glycated Hb to the total amount of Hb in the sample. However, in addition to HbAIo, glycated Hb separated by affinity chromatography also contains Hb bound to sugars other than glucose, and Hb bound to glucose other than the valine residue at the N-terminus of the β chain of Hb. These are known as HbAs. cannot be separated.

In order to eliminate the above drawbacks, E, C, Abraha+
(mentioned above) proposed a method in which Hb is separated into glycated Hb and non-glycated Hb by batch affinity chromatography, and then these are separated into each component (b) by ion exchange chromatography. ing.

According to this method, 1bA1° can be measured without being influenced by the unknown non-glycated Hb. However, in this method, after separating Hb into glycated Hb and non-glycated Hb by affinity chromatography, it is not possible to separate each glycated Hb acid component by, for example, directly subjecting the glycated Hb to ion exchange liquid chromatography. This is because the glycated Hb sample separated by the affinity chromatography has too high a pl and salt concentration, and cannot be sufficiently separated by ion exchange chromatography as it is. A pretreatment step to adjust the pH is usually required. If necessary, the sample may be dialyzed to lower the salt concentration before adjusting the pHg. Furthermore, the composition, salt concentration, and pH of the mobile phase for desorbing glycated Fib in the separation process by affinity chromatography are different from those of the mobile phase used for separation by ion exchange liquid chromatography.
The affinity chromatography step and the ion exchange chromatography step cannot be performed continuously. In this way, the method of E. C. Abraham et al.
Processing is complicated, automation is difficult, and measurement requires a long time.

Therefore, this method is not suitable as a method for routine clinical testing. Accuracy in less time <HbAl. It is desired to develop a method that can analyze the

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional problems, and its purpose is to obtain a desired type of H in a short time and with high precision.
b. In particular, it is an object of the present invention to provide an Hb analysis method that is capable of analyzing HbAtc and is easy to automate.

(Means for Solving the Problems) The present invention provides a flow path in which a sample supply means, an affinity column, an ion exchange column are connected to a switching means for the flow path, and a detector are provided in series, and the switching means is a state in which the ion exchange column is separated from the flow path, and
A method for analyzing hemoglobin in a sample using a measurement system configured to be able to switch between a state in which the ion exchange column is connected to the flow path, the method comprising: (b) separating the ion exchange column from the flow path, guiding the sample from the sample providing means to the affinity column, adsorbing glycated hemoglobin in the sample to the affinity column, and excluding non-glycated hemoglobin from the system; By switching the switching means,
(c) connecting the ion exchange column to the flow path, eluting the glycated hemoglobin adsorbed on the affinity column, guiding it to the ion exchange column and separating it into each glycated hemoglobin component; measuring components of hemoglobin with the detector, thereby achieving the above object.

In the present invention, a sample supply means, a first switching means to which an affinity column is connected, a second switching means to which an ion exchange column is connected, and a detector are provided in series in a flow path, and the first switching means is provided in series in a flow path. The switching means is configured to be able to switch between a state in which the affinity column is separated from the flow path and a state in which the affinity column is connected to the flow path, and the second switching means is configured to switch between a state in which the affinity column is separated from the flow path and a state in which the affinity column is connected to the flow path. A method for analyzing hemoglobin in a sample using a measurement system configured to be switchable between a state where the ion exchange column is separated from a flow path and a state where the ion exchange column is connected to the flow path, the method comprising: The first switching means connects the affinity column to the flow path, the second switching means disconnects the ion exchange column from the flow path, and the sample from the sample providing means is connected to the flow path. a step of introducing the glycated hemoglobin in the sample to the affinity column, adsorbing the glycated hemoglobin in the sample to the affinity column, and excluding non-glycated hemoglobin from the system; (b) by switching the second switching means, the glycated hemoglobin adsorbed on the affinity column (c) measuring each glycated hemoglobin component with the detector; The above objectives are achieved.

The present invention provides a hemoglobin analysis method using the measurement system, wherein (a) the first switching means connects the affinity column to the flow path, and the second switching means: The ion exchange column is separated from the flow path, the sample from the sample providing means is guided to the affinity column, and the glycated hemoglobin in the sample is adsorbed to the affinity column,
Step of excluding non-glycated hemoglobin from the system; (b) By switching the second switching means, the glycated hemoglobin adsorbed on the affinity column is eluted and guided to the ion exchange column, and each glycated hemoglobin component is (C) step of measuring each glycated hemoglobin component with the detector; (d) separating the affinity column from the flow path by the first switching means; With the switching means, the ion exchange column is connected to the flow path, and a sample having the same composition as in item (a) is guided from the sample providing means to the ion exchange column without being applied to the affinity column. , a step of separating hemoglobin into each hemoglobin component; (e
) measuring each hemoglobin component separated in the above (d) using the detector; and (f) measuring the measured value of each hemoglobin component obtained in the above (e) and the measured value obtained in the above (c). The above object is achieved by calculating the amount of each non-glycated hemoglobin component from the difference between the measured value of each glycated hemoglobin component.

Connection diagrams showing an example of an analysis device used to carry out the method of the present invention are shown in FIGS. 1 and 2.

1 and 2 show a configuration in which devices used for general-purpose liquid chromatography are combined. The analyzer used in the present invention generally employs a method called column switching method, in which two types of columns are connected via a six-way valve, and the flow path can be changed arbitrarily using the six-way valve. be.

Here, the analysis method of the present invention will be explained using the apparatus shown in FIG. 1 as an example. As shown in FIG. 1, this device includes an affinity column 9, a six-way valve 10 having connection ends a to f, and a detector 13 in a flow path 20 through which an eluent flows.
are provided in this order, and the ion exchange column 11
is connected to the flow path 20 via the connecting ends b-e of the six-way valve 10. Affinity column 9 is connected to connection end a of six-way valve 10, and detector 13 is connected to connection end f. Upstream of the affinity column 9, eluent tanks 1.2 and 3, a liquid feed pump 6, and an autosampler 8 are connected in the above order.
Mixer 5 is installed between 3 and the liquid pump as necessary.
is provided. By switching the connection of this six-way valve 10, it is possible to select flow path (1) or (2) as shown in FIG. The flow path ■ has connection ends a and f,
b and c, d and e are connected, respectively, and become a, c
and dS e and f are respectively occluded. In the flow path ■, connecting ends a & b, c, dle, and f communicate with each other, and a and f
, b and cS d and e are occluded, respectively. Eluent tanks 1.2 and 3 contain mobile phases A, B, and C, respectively.

A specific example of another apparatus for carrying out the invention is shown in FIG. This device includes a six-way valve 12 having a connection end g-1 having the same configuration as above, a six-way valve 10 having connection ends a to f, and a detector 13, which are provided in this order in a flow path 20. Column 9 is connected via connection end hk of six-way valve 12, and ion exchange column 11 is connected via connection end b-e of six-way valve 10. Upstream of the six-way valve 12 are eluent tanks 1.2 and 3.
, a liquid feeding pump 6, and an autosampler 8 are connected in the above order. Auto sampler 8 has six-way valve 1
A mixer 5 is connected to the connecting end g of 2, and a mixer 5 is provided between the eluent tanks 1 to 3 and the liquid feeding pump as necessary. Furthermore, the eluent tank 4 and the liquid feed pump 7 are connected to the connection end 1 of the six-way valve 12 in this order. This six-way valve 1
By switching the connection of 2, the channel ■ or ■ as shown in FIG. 2 can be selected, and by switching the connection of the six-way valve 10, the channel construction or ■ as shown in FIG. is possible. Eluent tank 1.2.3
and 4 contain mobile phases A, B, C, and D, respectively.

Next, a method for measuring glycated hemoglobin will be explained using the apparatus shown in FIG. 1 as an example. First, with the flow path of the hexagonal valve 10 set to ■, the eluent tank 3 is
A sample is injected into the system from the autosampler 8 while feeding the mobile phase C solution inside. The sample is guided to the affinity column 9 together with the mobile phase C solution. The mobile phase C solution is Hb
This is an eluent that allows the affinity column 9 to retain only glycated Hb among all Hb in the sample without denaturing the Hb.

Non-glycated Hb not retained on affinity column 9
is guided to the detector 13 via the connection ends a-f of the six-way valve 10, and is discharged from the drain 15. Non-glycated Hb is
If necessary, the amount is measured by the detector 13.

After all non-saccharified 111b is detected, the flow path of the six-way valve 10 is switched to ■, and the mobile phase is changed from liquid C to liquid A in the eluent tank 1. Solution A is an eluent that can elute glycated Hb retained in the affinity column 9 without denaturing Hb, and can separate each Hb acid component from the glycated Hb in the ion exchange column 11. By flowing liquid A,
Glycated Hb retained in the affinity column 9 is eluted, and together with the mobile phase A, the connection end a-
b to the ion exchange column 11. In the ion exchange column 11, the glycated Hb is separated into each Hb acid component, and guided to the detector 13 through the connecting ends e-f of the six-way valve 10 in the order in which they are eluted. Glycated Hb acid component detector 13
It is discharged from the drain 15 after being detected. Separation in this ion exchange column 11 is performed by using liquid B in the eluent tank 2, which has a higher ability to separate glycated Hb than liquid A, together with Ad, and changing the mobile phase from liquid A to liquid B stepwise or linearly. It is recommended to change to This increases the ability to separate each Hb acid component, and HbAo (here, non-glycated 1
Since 1b has already been removed, the measurement time can be shortened by speeding up the elution of glycated Hb belonging to WbAo.

The above mobile phase solution A can be any liquid as long as it does not denature Hb, can elute glycated Hb retained in the affinity column, and can separate each Hb acid component from the glycated Hb with an ion exchange column. The composition may be as follows. For example, it contains 0 to 10 hM of a metal chelating agent such as ethylenediaminetetraacetic acid (EDTA), or 1 to 500+ iM of a substance having a 1,2-cis-diol group, and the pH
A buffer solution with a concentration of 1 to 50 hM that is less than or equal to neutrality is preferred. Examples of buffer bases include Tris [) lis(hydroxymethyl)aminomethane], B15tris [
Bis-tris(2-hydroxyethyl)imino-tris(hydroxymethyl)methane], HEPES[N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid], and the like are used. As the substance having a 1,2-cis-diol group, for example, nrubitol, mannitol, etc. are used.

Generally, a compound having a large number of cis-diol groups has better elution power than a compound having one cis-diol group.

The mobile phase B solution satisfies the same conditions as the mobile phase A solution, and has a higher glycation concentration than the mobile phase A solution in the ion exchange column.
A buffer solution having a high elution capacity for b is used. In order to make the elution ability of glycated Hb higher than that of the mobile phase A solution, the concentration of the A solution may be increased. For example, by increasing the concentration of the buffer base in the mobile phase A solution or adding salts such as sodium chloride or sodium acetate to the A solution, a buffer with a higher elution ability for glycated Hb than the mobile phase A solution can be used. A liquid is obtained.

The mobile phase C solution does not denature flb, does not contain a substance having a 1,2-cis-diol group, and contains all 11 in the sample.
Any composition may be used as long as the eluent can retain only glycated Hb in the affinity column. The dihydroxyboronyl group and the sugar chain of glycated Hb, which are suitable as functional groups for the affinity column packing material, are:
Since the bonding is performed under weakly alkaline conditions, a buffer solution having a slightly alkaline pH (pH 8.0 to 10.0) is preferable as the liquid C. For example, a 10-5001M buffer (pH 8,0
~10.0) is preferably used. Ammonium acetate, HEPES, etc. are used as the buffer base.

When magnesium chloride is not added to the mobile phase C solution, a phosphate buffer can be used for the mobile phase A solution and B solution.

The packing material packed in the affinity column is Hb
can be separated into glycated Hb and non-glycated Hb, has sufficient mechanical strength to prevent the packing material from collapsing when the affinity column is connected in series with an ion exchange column, and has low non-specific adsorption of proteins. Any filler may be used. In particular, a filler into which a dihydroxyboronyl group is introduced as a functional group is preferably used. Examples of columns packed with such a packing material include TSKg
Examples include el Baronate-5PW Glass (Higashi-N Co., Ltd.), 5hodex AFpakPB-894 or FB-894L (Showa Denko ■). Although the shape of the column is not particularly limited, a column having a length to diameter ratio of 20:1 to 50:1 is preferred. Furthermore, in order to reduce non-specific adsorption of Hb and the packing material, it is preferable to use the column at a temperature of 5 to 10°C.

As the packing material packed in the ion exchange column, any packing material capable of separating glycated Hb into each component can be used. In particular, HbAl. A packing material that can separate Hb from other components in glycated Hb and has low nonspecific adsorption of proteins is suitable. Cation exchange resins, particularly weak cation exchange resins, are preferably used. Examples of columns packed with such a weak cation exchange resin include MICRONEX A
le-HSn (Tanezu Chemical Industry Co., Ltd.), TSKgal
CM-2SW or TSKgel CM-3SW
(Tosoh ■), etc. The shape of the column is not particularly limited.

As the sample, for example, a sample containing at least red blood cells and/or Hb is used. Red blood cells can be obtained, for example, by collecting blood in a blood collection tube containing an anticoagulant such as EDTA, and washing the blood with a solution that is isotonic with red blood cells, such as physiological saline. Distilled water, deionized water, or a surfactant solution is added to the washed red blood cells to cause hemolysis, thereby obtaining a hemolyzed sample.

A chromatogram of the Hb elution pattern analyzed in this manner is shown in FIG. In Figure 3, Pl represents non-glycated Hb, Pl represents glycated Hb, and P3 represents HbA.
l. The peaks caused by are shown. The amount of each Hb is calculated from the peak area of the -chromatogram. Here, HbA + in the method of the present invention. The method of calculating the value (%) is explained in the third section.
This will be explained using the figure as an example. HbA 1° value (%) is Hb
A+. The area of peak P3 of glycated Hb corresponding to
It can be calculated by dividing by the b peak area (the sum of the areas of PL and Pl). This calculation is shown by the following equation (2).

Furthermore, in the method of the present invention, the degree of saccharification (%) of Hb can also be
The area of peak P2 in b is calculated as the total Hb peak area (PI and Pl
It can be easily calculated by dividing by the sum of the areas of

This calculation is shown by the following equation (3).

Using the apparatus shown in Fig. 2 above, the saccharification H
A measurement of b can be made. First, the six-way valves 12 and 1
0 to form the flow paths ■ and I, the sample is injected from the autosampler 8 as in the case of FIG. 1 above, and the affinity column 9 separates glycated Hb and non-glycated Hb. Next, the six-way valve 10 is switched to form flow paths (1) and (2).

Glycated Hb is eluted from the affinity column 9, separated by the ion exchange column 11, and detected by the detector 13.

Furthermore, using this device, it is possible to analyze non-glycated Hb. For example, after the above steps are completed, the flow path of the six-way valve 12 is set to ■, and the flow path of the six-way valve 10 is set to ■, and the mobile phase A in the eluent phase 1 is sent by the liquid sending pump 6. In this state, a sample is injected into the system from the autosampler 8. This sample passes through the connection end g-1 of the six-way valve 12 together with the mobile phase A liquid, and passes through the connection end g-1 of the six-way valve 12.
0 to the ion exchange column 11 through connection ends a-b.

In this ion exchange column 11, all the Hb in the hemolyzed sample is separated into each Hb acid component under the conditions where the mobile phase A solution is fed. Then, the hexagonal valve 10 is placed in the order of elution of each Hb acid component.
is led to the detector 13 through the connecting ends e-f of and is discharged to the drain 15 after being detected. In this way, a total Bb chromatogram is obtained.

Each of the above Hb acid component detection steps and non-glycated and glycated Fib
The detection step and the detection step may be performed in any order. First, total Hb is detected using the ion exchange column 11, and then the affinity column 9 and the ion exchange column 11 are combined to detect non-glycated and glycated 1
A chromatogram obtained when measuring 1b is shown in FIG. In Figure 4, P4 is total Hb and P5 is non-glycated Hb.
, and P6 indicates the peak of glycated Hb. All Hb above
From the difference between the chromatogram of (P4) and the chromatogram of glycated Wb (P6), the chromatogram of non-glycated Hb can be determined by calculation.

Compared to the method using the apparatus shown in Figure 1, the method using the apparatus shown in Figure 2 takes longer measurement time because it is necessary to inject the same amount of sample twice, and requires one more six-way valve. . However, since the calculation yields a chromatogram of non-glycated Hb, HbA. Ursula elutes at the same time as
It is also possible to measure acetylated Hb as reported by Turpeinen et al. (supra). Furthermore, while performing separation on the ion exchange column, the six-way valve 12
Using the other flow path (2), the mobile phase solution can be flowed by the liquid feed pump 7 to wash the affinity column 9 in preparation for the next analysis. As the D solution, an acetate buffer of 1M or less is preferably used. In this way, analysis can be performed continuously using the above device.

(Example) The invention is illustrated by the following examples.

01 (i) Blood of a healthy person was collected using a vacuum blood collection tube containing EDTA, 5 times the volume of physiological saline was added, and then centrifuged to obtain red blood cells. This operation was repeated twice to wash the red blood cells, and 20 times the volume of deionized water was added to the resulting washed red blood cells to cause hemolysis, and this hemolysate was used as a sample.

The Wb in the sample obtained in the above (1) was analyzed using the apparatus shown in FIG. The equipment used, its settings, and measurement conditions (sample injection amount, column type, and mobile phase) are as follows.

Note that the conditions for feeding the mobile phase and the switching conditions for the six-way valve were set as shown in Table 1 below.

<Equipment used and its settings> Controller: 5CL-6A liquid pump manufactured by Shimadzu Corporation = LC-6A manufactured by Shimadzu Corporation (used at a flow rate of 1.0+gl/min) Auto sampler: Shimadzu Corporation Seisakusho 5IL-6A hexagonal valve: Sekisui Chemical Co., Ltd. MSC-620
Detector: Sekisui Chemical Co., Ltd. UV-Visible Detector UVD-121 (used between wavelengths 415) <Measurement conditions> Sample injection amount: 50 μl Affinity column: Tosoh Corporation TSKgel Boronate-5PW Glass
5.0mm1DX 5. Column temperature lO℃ Ion exchange column: Sekisui Chemical Co., Ltd. MICRONEX Ale-HSI [6,0mm1DX? , 5e+* Ram temperature 40°C Mobile phase A solution: 21M B15tris-hydrochloric acid buffer (
pH 6,0) + 100i + M sorbitol mobile phase solution D: 30+aM B15tris-HCl buffer (pH 6,0) + 100mM sorbitol + 300mM sodium acetate Mobile phase solution C: 250mM ammonium acetate-hydrochloride buffer (pH 8,5) + 10mM magnesium chloride The obtained chromatogram is shown in FIG. In Figure 3, Pl is non-glycated Hb, P2 is glycated Hb, and P
3 is HbA+. The beak is shown. The measurement result is HbA+
. The value was 3.0%, and the degree of saccharification was 5.7%. This result was almost the same as the result obtained by the conventional method in the comparative example described below.

Table 1 Time Mobile phase distribution ratio (%) Hexagonal rub (min) (A/B/C) flow path 0-10 10-11 11-20 20-30 30-31 31-40 010/100 5015010 →30/7010 30/7010→0/10010 0/10010 Ilb in a sample having the same composition as in Example 1 was analyzed using the apparatus shown in FIG. As shown below, the analysis adopted a method in which the total Hb was first separated into each component using the ion exchange column 11 and then measured, and then the affinity column 9 and the ion exchange column 11 were combined for measurement. . The equipment and measurement conditions used were the same as in Example 1. As the liquid feeding pump 7 and the six-way valve 12 shown in FIG. 2, one liquid feeding pump and one six-way valve of the same type as in Example 1 were additionally used. O, 1M as mobile phase solution
An acetic acid aqueous solution was used. The mobile phase feeding conditions and the six-way valve switching conditions were set as shown in Table 2 below.

The flow rate of the liquid pump 6 is always 1. o+ol/min, and the flow rate of the liquid pump 7 is O when the flow path of the hexagonal valve 12 is ■.
The flow rate was set to 1.0 ml/min when the flow path was ■.

The obtained chromatogram is shown in FIG. In FIG. 4, P4 shows the peak of total Hb, P5 shows the peak of non-glycated Hb, and P6 shows the peak of glycated Hb. The measurement result was HbA+ from the chromatogram when measuring all [1b]. The value is 3.
8%, and the chromatogram measured using a combination of affinity column and ion exchange column shows that H
bAl. The value is 3.2%, and the degree of saccharification is 5.5%
Met. This result was almost the same as the result of Example 1.

(Margin below) 0-1 1-10 10-20 20-21 21-24 24-34 34-35 35-44 44-54 54-55 55-60 Table 2 5015010-30/7010 30/7010→O/ 10010 0/10010 010/100 5015010→30/7010 30/7010-0/10010 0/10010 Salt tUX I in the same hemolyzed sample (referred to as sample a) as in Example 1 using the conventional batch method. (The analysis of b was conducted as follows.

First, using Glyphafin GHb (Seikagaku Kogyo Co., Ltd.) according to the method described in the instruction manual,
b was fractionated into non-glycated 11b and glycated Hb. These fractions were divided into sample b (non-glycated Hb fraction) and sample C (
saccharification) 1 to fractions), and each sample was divided into 2 volumes of dialysate (5+nM !J acid buffer, pH 7,3) at 4.
Dialyzed for 5 hours at °C. Each dialysed sample was then
Measurement was performed using an automatic glycated hemoglobin measuring device HA-8121 (Kyoto Daiichi Kagaku Co., Ltd.). The results are shown in FIG. In FIG. 5, FIG. 5A shows the chromatogram of sample a1, FIG. 5A shows the chromatogram of sample b1, and FIG. 5C shows the chromatogram of sample C. The shaded area is HbAl. Indicates the position of the peak.

According to the chromatogram of the sample (Figure 5b), the non-glycated Hb fraction contained HbA. It can be seen that there is a peak eluted at the same position. Therefore, in the conventional method of measuring glycated 11b in a hemolyzed sample without removing the non-glycated Hb fraction, this peak is added to the HbAtc peak.
True HbAtc value cannot be obtained. This non-glycated Hb acid component HbAl. The peak eluting at the same position as Ursu
According to the report by Turpeinen et al. (supra), it is considered to be acetylated Hb.

Comparing the chromatogram of glycated Hb of Example 1 (Figure 3) and the chromatogram of glycated Hb of Example 2 (Figure 4, P6) with the chromatogram of Figure 5C, it is found that
In Jc, the measurement time is as short as 4 minutes, so each glycated Hb15
It looks like 11E has been compressed, but the overall pattern is HbAI. Judging from the ratio between the amount (area) and the amount (area) of HbA, it can be determined that they are almost the same. HbAI relative to the total amount of glycated Hb. Calculating the relative percentage of the amount, it is 29.2% in Figure 3, 29.8% in Figure 4, and 29.8% in Figure 5C.
The result was 28.4%, indicating that there was almost no difference. Furthermore, the degree of saccharification in this comparative example was 5.1%, which was almost the same as the results of Examples 1 and 2.

(Effects of the Invention) According to the Hb analysis method of the present invention, by using a measurement system in which two types of columns are connected, it is possible to measure glycated Hb and/or non-glycated Hb 1b in a short time and with good reproducibility. HbAI, especially non-glycated) without being affected by the 1b component. measurements can be made. Since the measurement system used in the method of the present invention can be automated, it can be expected to be applied to daily use in the field of clinical testing. Furthermore, according to the method of the present invention, Hb
A.I. Since the degree of glycation of Ib can be measured simultaneously with the measurement of HbAI. Together with the value, it can be used for diabetes screening and blood sugar management for diabetic patients.

4. Don't worry! Figures 1 and 2 are connection diagrams showing an example of an analytical device used to carry out the method of the present invention; Figures 3 and 4 are chromatograms obtained in an example of the present invention; and Figure 5 is a chromatogram when TLB was analyzed by the conventional batch method, and Figure 5a is a hemolyzed sample containing total Hb;
Figure 5 shows the non-glycated Hb fraction, and Figure 5 C shows the glycated) I
This is a chromatogram when fraction b is used as a sample.

1.2.3.4... Eluent tank, 5... Mixer 6
．． 7...Liquid pump, 8...Auto sampler 9
...Affinity column, IQ, 12...Six-way valve, If...Ion exchange column, 13...Detector, 14.15...Drain.

that's all

Claims (1)

[Claims] 1. A flow path is provided with a sample supply means, an affinity column, a switching means for the flow path connected to an ion exchange column, and a detector in series, and the switching means is connected to the ion exchange column. A method for analyzing hemoglobin in a sample using a measurement system configured to be able to switch between a state where the column is separated from the flow path and a state where the ion exchange column is connected to the flow path, the method comprising: (a) By switching the switching means, the ion exchange column is separated from the flow path, the sample from the sample providing means is guided to the affinity column, glycated hemoglobin in the sample is adsorbed to the affinity column, and non-glycated hemoglobin is absorbed. (b) By switching the switching means, the ion exchange column is connected to the flow path, and the glycated hemoglobin adsorbed on the affinity column is eluted and guided to the ion exchange column, and each A method for measuring hemoglobin, comprising: separating glycated hemoglobin into components; and (c) measuring each glycated hemoglobin component with the detector. 2. A sample supply means, a first switching means connected to an affinity column, a second switching means connected to an ion exchange column, and a detector are provided in series in the flow path, and the first switching means is configured to be able to switch between a state in which the affinity column is separated from the flow path and a state in which the affinity column is connected to the flow path, and the second switching means connects the ion exchange column to the flow path. A method for analyzing hemoglobin in a sample using a measurement system configured to be switchable between a state in which the ion exchange column is separated from the ion exchange column and a state in which the ion exchange column is connected to a flow path, the method comprising: The switching means connects the affinity column to the flow path, and the second switching means disconnects the ion exchange column from the flow path, so that the sample from the sample providing means is connected to the flow path. (b) by switching the second switching means, the glycated hemoglobin adsorbed on the affinity column is removed from the glycated hemoglobin adsorbed on the affinity column; A method for measuring hemoglobin, comprising the steps of: eluting the glycated hemoglobin and introducing it into the ion exchange column to separate each glycated hemoglobin component; and (c) measuring each glycated hemoglobin component with the detector. 3. A hemoglobin analysis method using the measurement system according to claim 2, wherein: (a) the first switching means connects the affinity column to the flow path, and the second switching means The means separates the ion exchange column from the flow path, guides the sample from the sample providing means to the affinity column, adsorbs glycated hemoglobin in the sample onto the affinity column, and directs non-glycated hemoglobin to the system. (b) a step of eluting the glycated hemoglobin adsorbed on the affinity column by switching the second switching means, guiding it to the ion exchange column and separating it into each glycated hemoglobin component; ( c) measuring each glycated hemoglobin component with the detector; (d) using the first switching means to separate the affinity column from the flow path; and using the second switching means, With the ion exchange column connected to the flow path, a sample having the same composition as in section (a) is guided from the sample providing means to the ion exchange column without being applied to the affinity column, and the hemoglobin is separated from each hemoglobin. (e) measuring each hemoglobin component separated in the above (d) with the detector; and (f) measuring values of each hemoglobin component obtained in the above (e). A method for measuring hemoglobin, comprising the step of calculating the amount of each non-glycated hemoglobin component from the difference between the measured value of each glycated hemoglobin component obtained in the above (c). 4. The hemoglobin analysis method according to any one of claims 1 to 3, wherein the packing material of the affinity column has a dihydroxybonyl group as a functional group. 5. The hemoglobin analysis method according to any one of claims 1 to 3, wherein the packing material of the ion exchange column is a cation exchange resin.