JP7413671B2 - Analysis method using liquid chromatography - Google Patents

Description

The present invention relates to an analysis method using a liquid chromatograph.

Diagnosis is often made based on the percentage of glycated hemoglobin (sA1c) in the blood as an index for determining diabetes. As a method for measuring sA1c%, several testing methods are known based on different measurement principles, one of which is a testing method using liquid chromatography.

Among the testing methods using liquid chromatography, there is also a method based on the principle of "affinity chromatography." Specifically, after blood is hemolyzed and diluted, it is introduced into a column filled with a gel containing aminophenylboronic acid groups, and the first eluent is used to adsorb glycated hemoglobin to the gel and elute only non-glycated hemoglobin. , switch to the second eluent to elute the glycated hemoglobin. When measuring glycated hemoglobin by affinity chromatography, the column temperature needs to be 40 to 45°C. This method has a problem in that when there is a large difference between the temperature of the analytical column and the room temperature, thermal equilibrium is disrupted due to the start/stop of liquid feeding, and it takes time to re-equilibrate.

An object of the present invention is to provide an analysis method that can suppress consumption of eluent and maintain thermal equilibrium even when there is a large difference between the temperature of an analytical column and room temperature.

In order to solve the above-mentioned problems, an analysis method using a liquid chromatograph according to the present invention includes a liquid feeding pump for feeding a mobile phase, an injection part for injecting a specimen, and a combination of the liquid feeding pump and the injection part. a column in fluid communication, a detector downstream of the column, a first flow path with an outlet of the detector in fluid communication with a drain, and an outlet of the detector on the suction side of the liquid pump. and a second flow path in fluid communication with the liquid chromatograph, using a liquid chromatograph equipped with a switching unit capable of switching to the first flow path during measurement and switch to the second flow path during non-measurement. It is characterized by

Hereinafter, one embodiment of the present invention will be described in detail.

FIG. 1 is a flow path configuration diagram showing one embodiment of a chromatograph used in the present invention, and assumes affinity chromatography of glycated hemoglobin, but the present invention is not limited thereto. Note that in this specification, the mobile phase may be referred to as a buffer or an eluent.

A tank 1 storing a glycated hemoglobin adsorption buffer (hereinafter sometimes referred to as buffer A) and a tank 2 storing a glycated hemoglobin desorption buffer (hereinafter sometimes referred to as buffer B) are arranged in parallel. It is connected to degassing unit 3. Communication of buffer A to the flow path is controlled by a flow path opening/closing mechanism 4, and communication of buffer B to the flow path is controlled by a flow path opening/closing mechanism 5. The discharge side of the liquid pump 6 communicates with an injection section 7, an analysis column 8, and a detector 9. A prefilter 12 and a preheating coil 13 are provided upstream of the analytical column 8 and housed in a constant temperature bath 10 together with the analytical column 8 and detector 9.

For the purpose of switching the flow path of the buffer discharged from the detector 9, a switching unit 11 that can take two flow paths is arranged. The structure of the switching unit 11 is not particularly limited as long as it can have a flow path through which the buffer discharged from the detector 9 flows to the drain and a flow path through which the buffer flows to the suction side of the liquid feeding pump 6. It may also be a three-way valve or the like. As shown in Figure 1, it is preferable to use a 4-way, 2-position switching valve with two grooves on the rotor, one groove carved at 60° and the other groove at 120°. be.
Port A is connected to the tank 1 side, port B is connected to the liquid sending pump 6 side, drained liquid from the detector 9 is connected to port C, and port D is connected to a drain (liquid drainage section).

The state is as shown in FIG. 1a during measurement and as shown in FIG. 1b when not measuring. During measurement, the waste liquid from the detector 9 is directly led to the drain. When not measuring, the waste liquid from the detector 9 is connected to the suction side of the liquid pump 6. As a result, the liquid sending pump 6 is in a state where it can suck both the buffer A stored in the tank 1 and the waste liquid from the detector 9, but since the amount of waste liquid and the amount of liquid sent are almost the same, almost 100% is recycled. It will be done.

The point at which the waste liquid from the detector 9 is connected to the suction side of the liquid feeding pump 6 may be anywhere between the liquid feeding pump 6 and the tank 1, and is not limited to any particular point. FIG. 1 shows an example of recycling between a point downstream from the confluence (P1) of Buffer A and Buffer B and a liquid transfer pump. As shown in FIG. 2, between the flow path opening/closing mechanism 4 and the confluence point (P1) (P2 in FIG. 1) and the liquid pump 6, as shown in FIG. 3, between the tank 1 and the opening/closing mechanism 4. There is no problem in recycling between the point (P3 in FIG. 1) and the liquid feeding pump 6.

In the general sequence of affinity chromatography, liquid feeding is started by pressing the measurement start button, and after operations such as column initialization are performed as necessary, the first analyte is introduced into the column, and buffer A Glycated hemoglobin is separated and quantified using a step gradient of Buffer B and Buffer B. This process is repeated for a specified number of samples, and after all samples have been sampled, post-processing such as washing the column is performed as necessary, and then liquid feeding is stopped and the system enters a stand-by state.

Since the heat capacity of the temperature-controlled portion, including the analytical column, differs depending on whether the liquid feeding is continued or when the liquid feeding is stopped, the actual temperature of the temperature-controlled portion differs depending on the liquid feeding condition.
Therefore, when measuring a sample using a typical sequence, the actual temperature drops from T1, which has been stabilized, to T2 when the liquid transfer starts, and it takes a long time to reach thermal equilibrium (see Figure 4). . If the sample is measured during this time, the results will be unreliable. The only way to avoid this is to lengthen the time from the start of liquid feeding until the actual injection of the sample and wait for thermal restabilization, but this increases the amount of buffer consumed.

In the present invention, the waste liquid is directly poured into the drain during measurement, and the waste liquid is recycled during non-measurement, so that almost no buffer is consumed and there is no need to stop liquid feeding. Regardless of whether it is during measurement or not, if the liquid is being fed from the liquid pump, the column temperature (this is the actual temperature of the column, not the set temperature of the thermostat; for example, 35°C). -45° C.) is always stable and does not require time for thermal restabilization, allowing efficient and reliable results to be obtained (see Figure 5).

The analytical method of the present invention suppresses the consumption of eluent and makes it possible to maintain thermal equilibrium even when there is a large difference between the temperature of the analytical column and room temperature.

FIG. 2 is a flow path diagram showing one aspect of a method of recycling buffer A according to the present invention. FIG. 1a shows the state when recycle is OFF, and FIG. 1b shows the state when recycle is ON. 2 is a flow path diagram showing another aspect of the method of recycling buffer A of the present invention (a mode of recycling at point (P2) in FIG. 1). 2 is a flow path diagram showing another aspect of the method of recycling buffer A of the present invention (a mode of recycling at point (P3) in FIG. 1). FIG. 2 is a schematic diagram showing the actual temperature of an analytical column in a general glycated hemoglobin measurement sequence using two buffers. FIG. 2 is a schematic diagram showing the actual temperature of an analytical column in a glycated hemoglobin measurement sequence using two buffers according to the present invention. This is the basic flow path of the glycated hemoglobin analyzer HLC-723GVIII manufactured by Tosoh Corporation, which was used to verify the effects of the present invention. In addition, in this verification, the dashed line area was left unused. FIG. 2 is a diagram showing in detail the analytical column section/detection section of the glycated hemoglobin analyzer HLC-723GVIII manufactured by Tosoh Corporation, which was used to verify the effects of the present invention. FIG. 7a is a view with the column oven cover closed, and FIG. 7b is a view with the column oven cover removed. FIG. 3 is a diagram showing changes in actual temperature near the inlet of an analytical column in Example 1 when recycling is not performed and when recycling is performed. FIG. 8a shows the case where recycling is not performed, and FIG. 8b shows the case where recycling is performed. FIG. 3 is a diagram plotting the change in the actual temperature near the inlet of the analytical column and the resulting sA1c area % versus elapsed time in Example 2, when recycling is not performed and when recycling is performed. FIG. 9a shows the case where recycling is not performed, and FIG. 9b shows the case where recycling is performed.

In order to clarify the effects of the present invention, the system shown in FIG. 1 was constructed and verified.
Tosoh Corporation's "Automatic Glycated Hemoglobin Analyzer HLC-723GVIII" was used, but three types of ion exchange eluents were used, and two types of eluents for affinity separation (sA1c adsorption eluent (buffer A), sA1c The analytical column was changed to one for affinity chromatography (eluent for desorption) and used as a ``glycohemoglobin device using affinity chromatography'' (see Figure 6).
In addition, when recycling buffer A, as shown in FIG. It was configured to connect. Note that the recycle valve used was HPV-RcA manufactured by GL Sciences, Inc.
The recycle valve is a two-position rotor valve, and when recycling is not performed, the liquid pump is connected to buffer A, and the liquid that has passed through the detector is guided to the drain. During recycling, the liquid pump can take a flow path that connects both the buffer A and the liquid that has passed through the detector.

FIG. 7 is a diagram showing details of the analytical column and visible light detection section.
The analytical column and visible light detection section are one temperature control unit. A column storage section 29 and a detector unit 16 are arranged on one aluminum plate 14, and a thermo module 30 is arranged below it, so that the temperature is controlled as one block. A temperature sensor 18, which is the basis of temperature control, is placed inside the aluminum plate at the bottom of the column storage section, and is controlled so that the temperature at this point is constant. In this embodiment, in order to measure the actual temperature near the inlet of the analytical column 21, another temperature sensor 22 is disposed on the inlet surface of the analytical column so that the actual temperature can be measured.
The analytical column used was a column tube with an inner diameter of 2.5 mm and a length of 10 mm filled with TSKgel Boronate-5PW (manufactured by Tosoh Corporation, particle size 10 μm) with m-aminophenylboronic acid as a ligand.

The eluent for sA1c adsorption (pH 8.8) was adjusted to have the following composition.
HEPES 10mM
Magnesium chloride hexahydrate 50mM
Sodium chloride 500mM
Sodium azide 0.1%
The sA1c desorption eluent (pH 8.8) was adjusted to have the following composition.
HEPES 10mM
Sodium chloride 500mM
D-Sorbitol 30mM
Sodium azide 0.1%

Other measurement conditions are as follows.
Column temperature 40℃~45℃
Flow rate 1.0mL/min
Detection wavelength 415/500nm

As a specimen, a freeze-dried product (0.5% [NGSP]) was first dissolved in 0.5 mL of purified water, left to stand at room temperature for 30 minutes, and then 0.5 mL of purified water was added to 30 μL of the first solution. Level_2 (HbA1c 10.0±) of A1c control (manufactured by Tosoh Corporation) to which 5 mL was added was used.

The area of glycated hemoglobin (sA1c) corresponds to the difference between the area of peak 1 and the area of peak 2, so the area % of sA1c is
(sA1c peak area)*100/(total peak area)
Calculated with.

(Example 1)
First, temperature changes in the analytical column were measured when the buffer was being fed and when it was stopped. The column constant temperature bath is controlled by PID control so that the value measured by the temperature sensor is 45°C. Whether the buffer is being fed or not, the temperature is maintained at 45°C within a certain precision range. However, in reality, the point where the temperature sensor is installed is always maintained at 45°C, and not the entire interior of the column constant temperature chamber is maintained at 45°C. Generally, the further away from the temperature control point, the lower the actual temperature tends to be.

FIG. 8a is a diagram showing the results of measuring the temperature near the inlet of the analytical column, and is the result of observing actual temperature changes due to ON/OFF of the liquid feeding pump of the present device. When the liquid feeding was stopped, the actual temperature at the inlet of the analytical column was 38.5°C, but when the liquid feeding was started, the actual temperature dropped rapidly and became constant around 32.5°C. When the liquid feeding was stopped, the actual temperature rose rapidly and became constant at the original temperature of 38.5°C.
This is because when liquid feeding starts, the buffer placed at around room temperature (around 25°C) flows into the column, which has reached thermal equilibrium at a constant temperature, and the actual temperature drops. . For this reason, the actual temperature of the column will not become constant until 15 minutes or more have passed after the start of liquid feeding, and during this time, even if you measure the sample, you will not be able to obtain highly reliable measured values. .

FIG. 8b is a diagram showing the actual temperature when the liquid is constantly fed from the liquid feeding pump and the recycle valve is switched. Before starting measurement, turn on the recycle valve to initialize, turn on the recycle valve again 20 minutes after the start of measurement (recycling state), and turn off the recycle valve after 30 minutes (normal flow path, liquid effluent from the detector). (to the drain).
Since liquid feeding has continued since before the start of measurement, the actual temperature of the column remains constant at around 32.5 degrees. Measure the sample with the recycle valve OFF (normal flow path, the effluent from the detector goes to the drain), and when not measuring the sample (Stand-by), do not stop the liquid feeding and turn the recycle valve ON. By keeping it in the recycle state, the temperature of the analytical column can be kept constant.

(Example 2)
We verified the effects on the sA1c measurement results when liquid feeding was continued and recycled in a stand-by state and when liquid feeding was stopped in a stand-by state as in the past. After pressing [Start] to start measurement, the specimen was continuously measured 20 times, and the sA1c area % was calculated. FIG. 9 is a diagram in which the sA1c area % and the actual column inlet vicinity temperature are plotted on the horizontal axis of the elapsed time from the start of measurement. FIG. 9a shows the results of measurement without recycling (conventional method), and the actual temperature drops from the start of the measurement, during which time the sA1c area % gradually drops. The variation in sA1c area % decreases from the fifth sample onwards, when the actual column temperature has completely decreased and re-equilibration has been completed.

FIG. 9b shows the results measured with recycling (method of the present invention). Before starting measurement, the recycle valve is turned on to perform initialization, and at the same time as the start of measurement, the recycle valve is turned off. Since the liquid is constantly being fed, the temperature equilibrium is not disturbed, and the sA1c area % is stably calculated from the first sample.

Table 1 is a table showing variations in sA1c area % when recycling is performed (method of the present invention) and when recycling is not performed (conventional method). Cv% was calculated with n=1 to 10 and n=11 to 20. As you can see, without recycling (conventional method), the result is 3.217% for n = 1 to 10, and 1.353% for n = 11 to 20, indicating that there is a large variation in data after the start of measurement. .
With recycling (method of the present invention), it was 0.337% for n = 1 to 10 and 0.610% for n = 11 to 20, indicating that stable measurement results were obtained from the start of the measurement. .

Next, according to the present invention, the influence on the buffer consumption amount is calculated based on the following assumptions.
(assumption)
・With a flow rate of 1.00 mL/min, a step gradient of flowing buffer A for 0.6 minutes from sample injection, buffer B until 1.6 minutes, and buffer A until 3.0 minutes, continuous in a 3.0 minute cycle. and measure.
・In the case of no recycling When you press the [START] key to start measurement, the flow of buffer A starts at the same time, and the actual temperature of the column decreases until it becomes thermally stable at about 15 It's a minute later. After that, the sample is injected.
- When recycling is enabled When the [START] key is pressed to start measurement, recycling is turned off at the same time. Since buffer A is being fed before the [START] key is pressed, the sample can be injected after approximately 1 minute of equilibration time.
・100 sample measurements

Without recycling, when 100 samples are divided into two batches and measured, 15 mL of buffer A is consumed while waiting for stability (time until it becomes thermally stable) and 100 mL is consumed in 50 measurements. If two batches are measured, 230 mL will be consumed twice. With recycling, 1 mL of buffer A is consumed while waiting for stability (equilibration time) and 100 mL is consumed during 50 measurements. If two batches are measured, 202 mL will be consumed twice. The difference between the two is 28 mL. Note that there is no difference between the two regarding the consumption amount of buffer B.
When measuring 100 samples divided into 5 batches, 275 mL of buffer A is consumed without recycling. With recycling, 205 mL is consumed. The difference between the two increases to 70 mL.

As explained above, in the measurement system that uses recycling according to the present invention, it is possible to significantly shorten the time from the instruction to start measurement until the actual sample can be injected for the second and subsequent batches. In addition, it is possible to significantly reduce the amount of buffer used, and it can be a useful means in a glycated hemoglobin measurement system where running costs are important.

1. Tank (buffer for glycated hemoglobin adsorption)
2. Tank (buffer for desorption of glycated hemoglobin)
3. Deaeration unit 4. Channel opening/closing mechanism 5. Channel opening/closing mechanism6. Liquid pump7. Sample injection mechanism 8. Analytical column 9. Detector 10. Constant temperature bath11. Switching unit 12. Prefilter 13. Preheat coil 14. Aluminum plate 15. Cover 16. Detector unit 17. Heat radiation fin 18. Control temperature sensor 19. Preheat coil 20. Preheat coil cover 21. Analytical column 22. Temperature sensor for measuring actual temperature 23. buffer 1
24. buffer 2
25. buffer 3
26. Channel opening/closing mechanism 27. Channel opening/closing mechanism 28. Channel opening/closing mechanism 29. Column storage section

Claims (4)

a liquid sending pump that sends a mobile phase;
an injection part for injecting the sample;
a column in fluid communication with the liquid pump and the inlet;
a detector downstream of the column;
a constant temperature bath that houses the column and the detector;
a switching unit capable of switching between a first flow path in which an outlet of the detector is in fluid communication with a drainage part; and a second flow path in which an outlet of the detector is in fluid communication with a suction side of the liquid feeding pump; ,
An analysis method using a liquid chromatograph equipped with
Switch to the first flow path when measuring, switch to the second flow path when not measuring ,
Sending the mobile phase from the liquid feeding pump during both measurement and non-measurement,
The method as described above, characterized in that the waste liquid is not directly returned to the mobile phase tank. The method according to claim 1, wherein the actual temperature of the column is maintained within a range of 35°C to 45°C. 3. The method according to claim 1, wherein the column is a column filled with an affinity gel. 4. The method according to claim 1 , wherein glycated hemoglobin is analyzed in a blood sample.