JP2021092516A - Method for correcting setting flow rate value of buffer - Google Patents

Abstract

To provide a method for performing stable step gradient in liquid chromatography for glycosylated hemoglobin.SOLUTION: A method injects a blood sample into a column with a buffer with highest elution output passed through, and corrects a setting flow rate value of the buffer from a ratio of an elution time of a peak eluted at a void position to a reference elution time determined in advance.SELECTED DRAWING: Figure 4

Description

The present invention relates to a method of correcting a set flow rate value of a buffer.

Diagnosis is often made based on the proportion of glycated hemoglobin (SA1c) in the blood as an index for determining diabetes. As a measurement method of SA1c%, a plurality of measurement methods having different measurement principles are used. The typical method is by liquid chromatography. The liquid chromatography method also includes an ion exchange chromatography method and an affinity chromatography method depending on the difference in separation mode. In either method, a plurality of buffers having different salt concentrations and compositions are switched (step gradient) to separate the target glycated hemoglobin, and SA1c% is calculated from the proportion of the whole. The step gradient performs buffer switching according to a preset timetable for the elution capacity of each buffer.

With the progress of technology in recent years, a pump capable of delivering liquid with high accuracy has become easily available. Therefore, it has become the mainstream to perform qualitative quantification by replacing "elution capacity" with "elution time". Therefore, if the amount of liquid sent by the pump fluctuates due to some factor, the analysis accuracy will be reduced.

For example, a sample composed of two components, component A and component B, is adsorbed on the column by component B, and component A passes through the column to elute the buffer 1, and the component B adsorbed on the column desorbs and elutes the buffer 2. Is assumed to be used. It is assumed that the step gradient for switching the buffer is performed in 0.5 minutes based on the flow rate of 1.0 mL / min, and the component A elutes in 0.3 minutes and the component B elutes in 0.7 minutes. When the actual flow rate drops to 0.8 mL / min for some reason, the elution time changes as shown in FIG.
That is, the change in the actual flow rate in the step gradient affects the measurement result.

An object of the present invention is to provide a method of calculating the actual flow rate of a liquid feed pump by a simple method and correcting a set flow rate value of a buffer so that a stable step gradient can be performed.

In order to solve the above problems, the present inventors have reached the present invention as a result of repeated diligent studies.

That is, the present invention
In liquid chromatographic analysis of glycated hemoglobin by step gradient
Inject the blood sample into the column with the buffer with the highest dissolution output passed through, and then inject it into the column.
The present invention relates to a method of correcting a set flow rate value of a buffer from the ratio of the elution time of a peak eluted at a void position to a predetermined reference elution time.

Hereinafter, the present invention will be described in detail.

In the case of liquid chromatography, there is a certain amount of void volume (vacancy volume) between the sample injection section, the analysis column, and the detection section. Generally, in liquid chromatography, the injected sample undergoes some physical / scientific interaction with the gel in the analytical column, causing a difference in the passing speed and separation. However, if there is no physical / scientific interaction between the sample and the gel, the sample will pass through and elute at the position where the void volume buffer has been fed.
That is, under such conditions, the relationship of void capacity = elution time × actual flow rate is established.

First, a case where it is applied to "measurement of glycated hemoglobin using affinity chromatography" which is one aspect of the present invention will be described. The present invention is applicable to liquid chromatography, and of course, ion exchange chromatography in which three types of buffers are switched and used.

In actual sample measurement, glycated hemoglobin is adsorbed on the column in buffer 1 and other components are eluted. The buffer 2 then desorbs the adsorbed glycated hemoglobin from the column (see FIGS. 2a and 3a).

When a sample is injected with the "buffer 2" used to elute the glycated hemoglobin adsorbed on the column, the glycated hemoglobin and the non-glycated hemoglobin are not separated, and 1 at the position of the void volume of the system. It will elute as a book peak (hereinafter sometimes referred to as "void peak") (see FIGS. 2b and 3b).

Therefore, from the elution time (E0) of the void peak obtained when the reference flow rate (f0) is reached and the elution time (R0) of the void peak obtained at the timing when the correction is desired, the reference flow rate and the flow rate before the correction are obtained. Assuming that the set values are the same, the corrected flow rate f can be calculated by the following equation (1) and corrected to the set flow rate according to the actual flow rate.

The reference flow rate (f0) needs to be measured and obtained in advance. In particular, when correction is performed in order to eliminate the difference between devices and the difference between facilities, it is desirable to measure more accurately. It is desirable that the flow rate is accurately measured by a gravimetric method using an electronic balance, a capacitance method using a measuring container such as a graduated cylinder, or the like. Also. It is better to calculate the reference flow rate not only once but also again at the timing when the condition of the apparatus changes significantly, for example, after the column is replaced or after the maintenance of the apparatus.

In the buffer 2, all the components are eluted at the void position regardless of the sample type. Therefore, the sample used here may be a blood-derived sample and is not particularly limited. Preferably, it is desirable to use a calibrator (Low Level, High Level) used for preparing a calibration curve, or a control sample (Low Level, High Level) used for daily management. Alternatively, one of the patient specimens prepared for the actual measurement may be used.

Further, the timing of performing the flow rate correction based on the elution time of the void peak of the present invention is not particularly limited as long as it is before the measurement of the actual sample, and one of the standard samples is selected at the timing of creating the calibration curve. It is also effective to use one of the standard samples at the timing of measuring the control, or to use some sample at an arbitrary timing by the operator.

In liquid chromatographic analysis of glycated hemoglobin, stable step gradient can be performed.

It is the figure which showed the separation by affinity chromatography schematically, and shows the theoretically correct behavior when the actual flow rate changes. It is a figure which showed the measurement of the glycated hemoglobin measurement by affinity chromatography schematically. FIG. A shows the case where the actual step gradient is performed, and FIG. B shows the case where the saccharified hemoglobin desorption buffer (buffer 2) is passed through the liquid. It is a figure which showed typically the chromatogram when the measurement of glycated hemoglobin by affinity chromatography was performed. FIG. A shows the case where the actual step gradient is performed, and FIG. B shows the case where the saccharified hemoglobin desorption buffer (buffer 2) is passed through the liquid. It is a figure which showed the system structure used in the glycated hemoglobin analysis by the affinity chromatography method of Examples 1 and 2. FIG. 5 is a diagram showing the relationship between the reciprocal of the set flow rate and the elution time of the void peak in Example 1. In Example 2, it is the figure which showed the difference between the systems and the effect of the correction by this invention at the time of measurement under the same conditions. FIG. A shows the A0 peak in the step gradient, and FIG. B shows the variation in the elution time of the A1c peak in the step gradient. It is a figure which showed in Example 2 that there is no difference by the sample used for the correction of the method of this invention. The horizontal axis shows the sample species, and the vertical axis shows the elution time of the void peak. It is a figure which showed the system structure used in the glycated hemoglobin analysis by the ion exchange chromatography method of Example 3. FIG. 5 is a diagram showing the relationship between the reciprocal of the set flow rate and the elution time of the void peak in Example 3. FIG. 3 is a diagram showing the difference between systems and the effect of correction according to the present invention when measured under the same conditions in Example 3. FIG. A shows a case where all measurements are made at the same set flow rate, and FIG. B shows a case where the flow rate is corrected.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

First, it was verified by a system for measuring glycated hemoglobin by affinity chromatography, which is the first uniform state of the present invention.
FIG. 4 shows the system configuration used in this verification.
A liquid feed pump (8), a sample injection mechanism (9), an analysis column (12), and a visible light detector (13) were connected. In addition, switching valves (5) and (6) were arranged on the suction side of the liquid feed pump (8) so that the SA1c adsorption eluent (1) and the SA1c desorption eluent (2) could be selected.
All components use 8020 series HPLC manufactured by Tosoh Corporation, and the analysis column is TSKgel Boronate-5PW (manufactured by Tosoh Corporation, particle size 10 μm) using m-aminophenylboronic acid as a ligand. A column tube having an inner diameter of 2.5 mm and a length of 10 mm was filled.
The liquid feed pump (8) has a built-in pressure gauge for the purpose of monitoring / control, but in this verification, the pressure gauge was not used (passed through), and an independent pressure gauge (21) was used. It was connected to the outlet side of the pump and used for pressure monitoring.

As the SA1c adsorption eluent (pH 8.8), one prepared with the following composition was used.
Glycine 50 mM
Magnesium chloride hexahydrate 5 mM
Sodium chloride 50 mM
The SA1c desorption eluent (pH 8.8) used was prepared with the following composition.
Glycine 50 mM
Sodium chloride 50 mM
D-sorbitol 100 mM
Other measurement conditions are as follows.
Column temperature: 45 ° C
Detection wavelength: 415 nm (single wavelength)
Detector response: 0.15s
Data acquisition Sampling pitch: 100ms

As a sample, a calibrator manufactured by Tosoh Corporation (Cal_1: HbA1c 5.9% [NGSP], Cal_2: HbA1c 10.9% [NGSP]), a control (Ctrl_1: HbA1c 5.0 ± 0.3% [NGSP]) , Ctl_2: HbA1c 10.1 ± 0.5% [NGSP]) was used. When using it, it was dissolved and diluted according to the procedure in the same instruction manual. At the same time, an actual blood sample was also used. When used, it was dissolved and diluted with a hemolytic / washing solution dedicated to a glycohemoglobin analyzer (HLC-723 series) manufactured by Tosoh Corporation.

(Example 1)
First, the time change of the void peak due to the set flow rate was confirmed.
The flow path switching valve (5) is closed, the flow path switching valve (6) is opened, the buffer 2 for desorbing glycated hemoglobin is passed through the analysis column, the sample is injected, and all the components are in the void position. It was eluted and its temporal change was confirmed (Sample: Ctrl_2).
Table 1 is a table showing the elution time of the void peak (representing the average value of 5 times) with respect to the set flow rate. Further, FIG. 5 is a diagram showing the reciprocal of the set flow rate and the elution time of the void peak.

As can be seen from these, a good linear relationship can be seen between the elution time of the void peak and the reciprocal of the set flow rate. The result of approximation by the linear equation (y = ax) without an intercept is shown.
The regression equation and R2 coefficient are as follows.
y = 0.2053 x R2 = 0.9897
The R2 coefficient is very good at 0.99, and it can be seen that there is a good correlation between the two. That is, it is shown that when the liquid was sent by the buffer 2, the sample had almost no interaction with the gel and was eluted at the void volume position.
That is, in the state where the buffer 2 is fed, there is almost no interaction with the separation gel, and the buffer 2 is eluted at the void position of the measurement system, which means that the flow rate can be calculated from the elution time of the void peak.

(Example 2)
Next, in order to verify the effectiveness of the method of the present invention, it was applied to a method of reducing the difference between devices.
By replacing the liquid feed pump A of the system used in Example 1 with other pumps B to F and using the present invention, it was verified whether the measurement result became a closer value. The equipment configuration and measurement conditions were the same except for the liquid feed pump. Hereinafter, the system using the pump A will be referred to as "system A", and the system using the pumps B to F will be referred to as systems B to F, respectively.
First, the set flow rate was set to the same 0.750 mL / min, separation by step gradient, and void confirmation by buffer 2 were performed (sample: Ctrl_2). The step gradient was buffer 1 from the start of measurement to 0.583 minutes, buffer 2 from 0.583 minutes to 2.500 minutes, and buffer 1 after 2.500 minutes. The results are shown in Table 2. Each measurement is performed 5 times, and the value of the void peak in the table represents the average value.

From Table 2, it can be seen that there is a difference in the elution time of each peak between the systems.
Next, an attempt was made to correct the set flow rate based on the elution time of the void peak. Here, the system A was used as a standard machine, and the flow rate corrections of the other systems B to F were performed.
The flow rate correction value is calculated as follows.
System B flow rate correction value:
(Set flow rate of system A) × (Elution time of system B) / (Elution time of system A)
0.750 x 0.2778 / 0.2848 = 0.732
System C flow correction value:
(Set flow rate of system A) × (Elution time of system C) / (Elution time of system A)
0.750 x 0.2784 / 0.2848 = 0.733
Similarly, the flow rate correction value of the system D is calculated as 0.731, the flow rate correction value of the system E is 0.746, and the flow rate correction value of the system F is 0.735.

FIG. 6 is a diagram comparing the result of measuring the set flow rate at 0.750 mL / min and the result of measuring by resetting the set flow rate to the flow rate correction value. It can be seen that the identity of the chromatogram is enhanced by performing the correction.

Table 3 is a table showing the reproducibility of the elution time between the systems of A0 peak and A1c peak when the actual step gradient is performed and the correction is not performed and when the correction is performed. It can be seen that the Cv% of each peak decreased and the variation was small.

Up to this point, verification has been carried out using control samples of freeze-dried products. Next, in the present invention, it was verified whether or not there was a difference depending on the sample used.

The samples used for verification were the lyophilized products used for preparing the calibration curve (Cal_1, Cal_2), the freeze-dried products used for quality control (Ctrl_1, Ctrl_2), and whole blood patient samples (# 1 to # 1). There are a total of 14 types of # 10). The values of sA1c% are shown in Table 4. This value is a value measured by the Glycohemoglobin Analyzer GHb11 of Tosoh Corporation.

Further, FIG. 7 is a diagram showing the relationship between the elution time of the sample and the void peak. The measurement was performed in the order of Cal_1, 2, Ctrl_1, 2, and # 1 to # 10. That is, the horizontal axis of FIG. 7 also has the meaning of elapsed time. As can be seen from this, the elution time (peak top time) of the void peak in the state where the buffer 2 is fed is almost the same regardless of the type of the sample and the form of the sample (whole blood, lyophilized product). That is, it can be seen that the present invention may be any blood sample used for measuring glycated hemoglobin and does not need to be particularly limited.

(Example 3)
It was verified by a saccharified hemoglobin measurement system by ion exchange chromatography.
FIG. 8 shows the system configuration used.
A liquid feed pump (8), a sample injection mechanism (9), an analysis column (12), and a visible light detector (13) were connected. In addition, switching valves (5), (6), and (7) are arranged on the suction side of the liquid feed pump (8) so that three types of eluents (1), (2), and (3) having different salt concentrations can be selected. I made it.
All components used 8020 series HPLC manufactured by Tosoh Corporation, and the analysis column and eluent used were those for the glycohemoglobin analyzer GHbVIII manufactured by Tosoh Corporation.
The liquid feed pump (8) has a built-in pressure gauge for the purpose of monitoring / control, but in this verification, the pressure gauge was not used (passed through), and an independent pressure gauge (21) was used. It was connected to the outlet side of the pump and used for pressure monitoring.
Other measurement conditions are as follows.
Column temperature: 25 ° C
Detection wavelength: 415 nm (single wavelength)
Detector response: 0.15s
Data acquisition Sampling pitch: 100ms

First, the time change of the void peak due to the set flow rate was confirmed.
Assuming that the flow path switching valves (5) and (6) are closed and the flow path switching valve (7) is opened, the buffer 3 having the highest elution output is passed through the analysis column, a sample is injected, and all the components are voided. It was eluted at the position and its time change was confirmed (Sample: Ctrl_2).
Table 5 is a table showing the elution time of the void peak (representing the average value of 5 times) with respect to the set flow rate. Further, FIG. 9 is a diagram showing the reciprocal of the set flow rate and the elution time of the void peak.

As can be seen from these, a good linear relationship can be seen between the elution time of the void peak and the reciprocal of the set flow rate. That is, even in the saccharified hemoglobin measurement system by ion exchange chromatography, it means that the flow rate actually sent can be calculated from the elution time of the void peak.

Next, the liquid feed pump A of the system was replaced with other pumps B to E, and it was verified whether the measurement result became a closer value by using the present invention. The equipment configuration and measurement conditions were the same except for the liquid feed pump. Hereinafter, the system using the pump A will be referred to as "system A", and the systems using the pumps B to E will be referred to as systems B to E.
First, the set flow rate was set to the same 1.25 mL / min, and the elution time of the void peak by the buffer 3 having the highest dissolution output was measured (sample: Ctrl_2). Next, an attempt was made to correct the set flow rate based on the elution time of the void peak. Here, the system A was used as a standard machine, and the flow rate corrections of the other systems B to E were performed. The results are shown in Table 6. Each measurement is performed 5 times, and the value of the void peak in the table represents the average value.

FIG. 10 is a diagram comparing the result of measurement by the three-component step gradient at a set flow rate of 1.25 mL / min and the result of measurement by the three-component step gradient after resetting the set flow rate to the flow rate correction value. Yes (Sample: Ctl_2). The step gradient is buffer 1 from the start of measurement to 0.333 minutes, buffer 2 from 0.333 minutes to 0.700 minutes, buffer 3 from 0.700 minutes to 1.333 minutes, and buffer 1 after 1.333 minutes. did. It can be seen that the identity of the chromatogram is enhanced by performing the correction.

1 buffer 1
2 buffer 2
3 buffer 3
4 Degassing device 5 Switching valve for buffer 1 6 Switching valve for buffer 2 7 Switching valve for buffer 3 8 Liquid feed pump A
9 Sample injection mechanism 10 Pre-filter 11 Pre-heat coil 12 Analytical column 13 Visible light detector 14 Column oven 16 Liquid feed pump B
17 Liquid feed pump C
18 Liquid transfer pump D
19 Liquid transfer pump E
20 Liquid feed pump F
21 Pressure gauge

Claims (6)

In liquid chromatographic analysis of glycated hemoglobin by step gradient
Inject the blood sample into the column with the buffer with the highest dissolution output passed through, and then inject it into the column.
A method of correcting the set flow rate value of the buffer from the ratio of the elution time of the peak eluted at the void position to the predetermined reference elution time. The first aspect of the present invention is to correct the set flow rate value by multiplying the set flow rate value of the buffer by a value obtained by dividing the elution time of the peak eluted at the void position by a predetermined reference elution time. The method described. The method according to claim 1 or 2, wherein the blood sample is either a standard sample used for preparing a calibration curve, a control sample used for confirming the operating state of the device, or the blood of a patient. .. The method according to any one of claims 1 to 3, wherein the separation principle of the liquid chromatography analysis is based on ion exchange. The method according to any one of claims 1 to 3, wherein the separation principle of the liquid chromatography analysis is based on affinity. A method for analyzing glycated hemoglobin in a blood sample, which comprises measuring an actual sample after performing the method according to any one of claims 1 to 5.

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