JPH1010107A - Sample analysis method by liquid chromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sample method for preventing the contamination of the passage of an analysis system by remaining materials or the like derived from a sample when many samples are measured by liquid chromatograph. SOLUTION: In this method, after a plurality of samples are continuously measured by liquid chromatograph by using the same measuring conditions, the measurement is stopped and the other sample is measured again under the same measuring conditions described above. The analysis of the sample by the liquid chromatography is performed in this way. At this time, after the end of the continuous sample measurement, cleaning liquid is sent into the passage of the analysis system, and the remaining material caused by the sample is removed. Thereafter, cleaning eluant is sent. Furthermore, this method may be applied to the measurement of the saccharified hemoglobin in the hemolysis sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for analyzing a sample by liquid chromatography.

[0002]

2. Description of the Related Art Liquid chromatographs are widely used for analyzing components in samples in fields such as organic chemistry, biochemistry, and medicine. This liquid chromatograph system is usually configured as shown in FIG. The eluent 1 includes a switching mechanism 2 (e.g., a solenoid valve, and eluents 1a, 1b,
1c etc.), and the liquid feed pump 3
The sample enters the separation column 5 filled with the filler via the sample introduction device 4, and the components in the sample are separated by the separation column 5. Each of the separated components is detected by the detector 6, for example, by measuring the absorbance.
The result is recorded in the recorder 7, and the eluent 1 after detection is stored in the waste liquid reservoir 8.

In the analysis of a sample by liquid chromatography using such a liquid chromatograph, after a plurality of samples are continuously measured under the same measurement conditions,
The measurement is interrupted (for example, after the end of the working hour of the day) and then again (for example, the next day, the case of restarting the next day)
When processing a large number of samples so that other samples are measured using the same measurement conditions as described above, if the flow path of the liquid chromatograph is contaminated with substances derived from the sample to be measured, the analysis results Influences, shortens the life of each device or component, or increases costs due to increased maintenance.

[0004] With regard to the cleaning of the flow path, usually in an automatic sample introduction device 4 or the like, the flow path of the sample in the sample introduction device 4 is washed with a dedicated cleaning liquid every time one sample is measured.
However, in the analysis system flow path as shown in FIG. 3, no special cleaning is usually performed. The reason for not washing the analysis system channel is that the eluent is always passed during the measurement, and usually, after the measurement of one sample, residual substances derived from the sample flow into the analysis system channel such as a separation column. This is because the elution conditions are set so as not to cause the generation.

[0005] However, in practice, there are residual substances from the sample on the analytical data that do not affect the analysis in the short term, and these are accumulated in the long term and affect the analysis. There was something. For example, clogging occurs due to adsorption or accumulation in the filter installed upstream of the separation column or in the separation column itself, or deterioration in detection sensitivity due to adsorption or accumulation in the detection unit (photometric cell, etc.) of the detector. Cause

[0006] As a method for cleaning such residual substances,
After long-term analysis, measures have been taken such as removing the filters and performing ultrasonic cleaning, and washing the detector cells and the like with a detergent.

As a countermeasure to prevent the accumulation of the residual substance as much as possible, the accumulation of the residual substance is most likely to occur when the eluent is not flowing, that is, when the liquid supply pump is stopped. When a chromatograph is not used, a flow path other than the separation column is replaced with ion-exchanged water or the like, and the separation column is detached and stored.

However, even if these measures are taken,
The following problems remain. Washing of the packing material in the separation column where residual substances have been adsorbed and accumulated can be washed and removed with reagents, but usually, adsorption and accumulation of residual substances often occur irreversibly. In many cases, such fillers cannot be used. This is particularly likely when a biological sample such as blood is used as the sample. In addition, in this cleaning, if a too strong cleaning operation (such as using a cleaning liquid having a composition far from the use conditions or using a reagent that degrades the material of the filler itself) is performed, the filling is performed rather than the removal of the residual substance. Such a cleaning is also difficult to use because it degrades the agent itself.

If the entire flow path is replaced with ion-exchanged water or the like, labor for re-operation is troublesome, and this replacement method is not suitable for a very long time except when stopping.

[0010] For this reason, it is necessary to prevent contamination of the analysis system flow path by the sample remaining substance (particularly, contamination of the filler) in order to minimize the contamination more than in the washing process. In addition, there is a need for a method that can be performed simply, in some cases automatically, and in which the re-operation work can be performed smoothly, instead of a complicated method such as liquid replacement of the flow path.

As an example of the analysis of a sample by liquid chromatography as described above, measurement of glycated hemoglobin in a hemolyzed sample can be mentioned. Glycated hemoglobin is the product of sugar in the blood, which enters the red blood cells in proportion to its concentration and then binds to hemoglobin, and the concentration is the average sugar concentration in the blood for the past one to two months. It is said to reflect. Since the blood sugar level and the urinary sugar level are less susceptible to physiological factors, the concentration of glycated hemoglobin in the blood is used as an optimal index for diagnosis of diabetes or follow-up of diabetic patients. For this reason, the measurement of glycated hemoglobin by liquid chromatography is widely performed in the field of clinical testing, and at measurement sites such as clinical testing centers, multiple samples are continuously measured using the same measurement conditions and then measured. , And another sample is measured again under the same measurement conditions as described above, so that many samples are measured every day.

The above-mentioned glycated hemoglobin is measured by using a hemolyzed sample obtained by lysing blood to remove glycated hemoglobin in erythrocytes from erythrocytes and performing liquid chromatography using a separation column filled with a cation exchange resin. A dedicated measuring device for glycated hemoglobin for this measurement is commercially available and widely used. In this measuring device, measurement is performed by a three-liquid step gradient method using three types of eluents described later as eluents. After measuring a large number of samples, the device is stopped, and then the device is operated again. Due to the repeated effect of this, the retention time of glycated hemoglobin may change due to contamination of the analysis system flow path, such as adsorption of residual substances derived from the sample mainly to the packing material, or if the operation is continued. Therefore, prior to the start of use of the apparatus every day, the retention time was checked to finely adjust the measurement system, and in some cases, the separation column was discarded and replaced with a new one.

[0013]

SUMMARY OF THE INVENTION The invention described in claim 1 (hereinafter, the invention described in claim 1 is referred to as "invention 1") has been made in view of the above problems, and has as its object the purpose of liquid chromatography. When measuring many samples by graph,
It is an object of the present invention to provide a simple method for preventing the analysis system flow path from being contaminated by a residual substance derived from a sample. The object of the invention according to claim 2 (hereinafter, the invention according to claim 2 is referred to as the present invention 2) is when the sample is hemolyzed and the measurement target component is glycated hemoglobin, and the invention according to claim 1 is performed. To achieve the same purpose as.

[0014]

According to the method for analyzing a sample by liquid chromatography of the present invention, a plurality of samples are continuously measured by a liquid chromatograph under the same measuring conditions, and then the measurement is interrupted. In the method for analyzing a sample by liquid chromatography in which another sample is measured again under the same measurement conditions as described above, after the continuous measurement of the sample is completed, the washing solution is sent to the analysis system flow path to derive the sample. After removing the remaining substances, the washing eluent is sent.

The method for analyzing a sample by liquid chromatography according to the second aspect of the present invention is the method for analyzing a sample by liquid chromatography according to the first aspect of the present invention, wherein the sample is hemolyzed and the target component is glycated hemoglobin.

In the following description, the present invention refers to the present inventions 1 and 2.

In the method for analyzing a sample by liquid chromatography according to the present invention, after the continuous measurement of the sample is completed, a washing solution is sent to an analysis system flow path to remove residual substances derived from the sample, and then a washing eluent is used. Is sent.

[0018] The continuous measurement means that the measurement is repeated while changing the sample under the same conditions.

The above analysis system flow paths refer to all flow paths through which an eluent flows for analysis in a liquid chromatograph system configuration (for example, as shown in FIG. 3).

The above-mentioned washing liquid refers to a liquid that can remove residual substances derived from a sample and does not adversely affect materials constituting a flow path of an analysis system including a filler. To remove the residual substance derived from the sample means to remove the residual substance by removing or decomposing it from the material constituting the analysis channel including the filler. Therefore,
The type of the cleaning liquid differs depending on the type of the sample to be measured and the type of the filler. Further, since the washing solution is partially mixed with the eluent for analysis in the flow channel, a substance which adversely affects the analysis is generated by mixing with the eluent for analysis (for example, precipitation of salt). Things are not preferred.

As the above-mentioned washing solution, a washing solution selected according to the above conditions may be specially prepared as a dedicated washing solution. For example, a sample is analyzed by a gradient method (eluent for analysis is not limited to one kind). , An analytical method using a plurality of eluents having different dissolution powers)
When the eluent having the highest solubility among the eluents used for analysis has the washing effect of the present invention, it is simple and preferable to use this eluent.

The time for sending the above-mentioned washing liquid needs to be a time for removing the residual substances derived from the sample, and differs depending on the sample to be measured.

The washing eluent is sent to prevent the washing solution from adversely affecting the analysis, and does not adversely affect the materials constituting the analysis system flow path, including the filler. If present, it can be arbitrarily selected, but usually, the eluent first sent during analysis is preferable.

It is necessary that the time and the amount of the eluent for washing be sent so that the influence of the washing solution on the analysis is eliminated.

The liquid chromatography method to which the present invention 1 is applied may be any of the conventionally used liquid chromatography methods, and the filler may be any of various inorganic or organic modes (for example, ion exchange). , Reversed-phase, normal-phase mode, etc.)
As the eluent, although it differs depending on the filler or the sample to be measured, for example, an alcohol-based or hydrocarbon-based organic solvent, a buffer containing an inorganic acid salt or an organic acid salt, or a mixture thereof is used.

The liquid chromatography system used in the present invention may be any of conventionally used systems, for example, the system shown in FIG. 3 described in the section of the prior art.

In the sample analysis method of the present invention, the washing method for sending the washing solution to the analysis system flow path includes an automatic washing method and a manual washing method.

As the above automatic cleaning method, for example, FIG.
Can be performed by the system shown in FIG. FIG. 1 is obtained by adding a control unit 9 to the liquid chromatograph system shown in FIG. The control unit 9 is a controller that controls the entire system. The function of the control section 9 is to perform a series of controls such that the cleaning liquid is supplied for a certain period of time after the continuous measurement, the cleaning liquid eluent is supplied, and then the liquid supply is stopped. Therefore, the control unit 9
Is a sample introduction device 4 for detecting the end of the continuous measurement.
In addition, the switching mechanism 2 for automatically switching the liquid sending,
It is connected to the liquid sending pump 3 to stop the liquid sending. In addition, after a series of continuous measurements is completed and the analysis is temporarily stopped,
When the washing operation is unnecessary, for example, when the measurement is immediately restarted, it is preferable that the washing operation can be controlled so that the washing operation can be canceled.

The control unit 9 may be an independent device. For example, if the liquid sending pump 3 or the sample introduction device 4 is provided with a control unit function, the control unit 9 may be controlled using it. Good.

The above-described automatic cleaning method is particularly preferable in the case of continuous operation at night or the like.

The manual cleaning method differs depending on the system used as a manual operation part, but it is necessary to confirm the end of continuous measurement and to input the start of the supply of the cleaning liquid at a minimum.

In the present invention 2, the liquid chromatography method is the same as that of the present invention 1 except that the sample is blood lysate and the target component is glycated hemoglobin. This is the same as the method for analyzing a sample by liquid chromatography. .
And the measurement mode of this liquid chromatography is
This is the same as the method described in the section of the prior art.

[0033]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to examples and comparative examples of the present invention, but the present invention is not limited thereto.

In the following Examples and Comparative Examples, glycated hemoglobin A1c in the sample was measured by liquid chromatography as a measurement target component, using the sample as hemolyzed blood. The measuring device and measuring method, the hemolytic agent and the hemolyzed sample are as follows.

Measuring device and method for measuring glycated hemoglobin The measurement of glycated hemoglobin was performed by using Hi-
This was performed by selecting appropriate conditions using Auto A1c HA-8121. This HA-8121 is a dedicated apparatus for measuring glycated hemoglobin by liquid chromatography, uses a separation column filled with a cation exchange resin, and separates and elutes each hemoglobin component in 4 minutes by cation exchange. Things. As eluents, eluent A (0.10 M phosphate buffer, pH 6), eluent B
(0.15M phosphate buffer, pH 7), eluent C (0.
Measurement by a three-liquid step gradient method using 10 M phosphate buffer (pH 6) (eluent A is sent for 130 seconds, eluent B is sent for 20 seconds, and eluent C is sent for 90 seconds)
The chromatogram was obtained by measuring the absorbance at 415 nm of each component eluted from the separation column.

Hemolyzing agent Hemolysis of 0.05 M phosphate buffer (pH 7.0) in which Triton X-100 (polyethylene glycol octyl phenyl ether, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved at 0.1% by weight as a hemolytic reagent. Used as an agent.

Hemolyzed blood sample A blood sample of a healthy person was used as a blood sample. Immediately after blood collection, 1 ml of whole blood was added with 10 mg of sodium fluoride as a blood anticoagulant at a rate of 3 μl. Was diluted 150-fold with the above hemolytic agent to obtain a hemolyzed sample.

Example 1 First, 20 samples of the above hemolyzed blood sample were prepared.
Zero samples were continuously measured, and after completion, the eluent B having the strongest eluting power among the above eluents was sent as a washing solution for 60 seconds, and then the eluent A was sent as a washing eluent for 60 seconds and sent. Stopped. On the next day, 200 samples of the lysed blood sample were continuously measured in the same manner. After the completion, the eluent B was similarly sent for 60 seconds, the eluent A was sent for 60 seconds, and the sending was stopped. further,
The same operation was performed on the following day, and this was repeated for 8 days including the first measurement day.
Repeated for days. The representative chromatogram obtained is shown in FIG. Each peak in FIG. 2 shows 11 glycated hemoglobins HbA1a and HbA1b, 12 fetal hemoglobin HbF, and 13 unstable glycated hemoglobin HbA1.
c, 14 stable glycated hemoglobin HbA1c and 15 normal hemoglobin HbA0. Stable glycated hemoglobin HbA1 in the chromatogram of the first sample of each day
The retention time (sec) of c was measured and is shown in Table 1. Day 1 ~
Until the eighth day, the retention time (seconds) hardly changed.

(Comparative Example 1) In Example 1, "200 samples were continuously measured on each day, and after completion, eluent B was sent for 60 seconds, eluent C was sent for 60 seconds, and Has stopped "
Instead of saying, "Continuously measure 200 samples,
Example 1 except that the liquid supply was stopped (that is, the measurement of the 200th sample was performed in the order of eluent A, eluent B, and eluent C). Glycated hemoglobin was measured in the same manner, and the stable glycated hemoglobin HbA1c in the chromatogram of the first sample of each day was measured.
Was measured and the results are shown in Table 1. From the fourth day, the retention time (seconds) of HbA1c was shortened, and deterioration of the filler was observed.

[0040]

[Table 1]

[0041]

The structure of the method for analyzing a sample by liquid chromatography of the present invention 1 is as described above, and the analysis method of the present invention 1 eliminates contamination of the flow path of the analysis system by residual substances derived from the sample. It provides a simple way to prevent
When the analysis method of the present invention 1 is used, adsorption of a sample to an analysis system flow path (particularly, a separation column) is prevented, and the life of the analysis system flow path (particularly, a separation column) can be extended. The configuration of the method for analyzing a sample by liquid chromatography of the present invention 2 is as described above. When the analysis method of the present invention 2 is used, in the measurement of glycated hemoglobin in lysed blood, an analysis system flow path (particularly a separation column) ) Can be prevented from adsorbing the sample, and the life of the analysis system flow path (particularly, the separation column) can be extended.

[Brief description of the drawings]

FIG. 1 shows a control unit 9 for automatic cleaning added.
It is an explanatory view showing a system of a liquid chromatograph.

FIG. 2 is a chromatogram obtained in Example 1.

FIG. 3 is an explanatory diagram showing a liquid chromatograph system.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Eluent 2 Switching mechanism 3 Liquid sending pump 4 Sample introduction device 5 Separation column 6 Detector 7 Recorder 8 Waste liquid storage 9 Control part

Claims (2)

[Claims] A liquid chromatograph in which a plurality of samples are continuously measured under the same measurement conditions, the measurement is interrupted, and another sample is measured again under the same measurement conditions. In the method for analyzing a sample by chromatography, after the continuous measurement of the sample is completed, it is preferable to send a washing solution to an analysis system flow path to remove a residual substance derived from the sample, and then send an eluent for washing. Characteristic method of analyzing a sample by liquid chromatography. 2. The method for analyzing a sample by liquid chromatography according to claim 1, wherein the sample is hemolyzed blood, and the measurement target component is glycated hemoglobin.