JPH04366762A - Device and method for analysis using liquid chromatography - Google Patents

Abstract

PURPOSE:To provide a method and device for analysis using liquid chromatography whereby the result of measurement is analyzed and samples conforming to requirements for re-measurement can be re-measured while conditions of measurement are automatically changed. CONSTITUTION:An analysis device using liquid chromatography includes a sample introducing means 2, a liquid chromatograph 1 to which a column 3 and a detection means 4 are connected in series, and a detected data comparison means 5 to which electric signals corresponding to the amount of detection of samples measured from the detection means 4 are given and which then performs A/D conversion of the electric signals and then compares chromatogram pattern data obtained by the conversion with normal chromatogram pattern data given in advance. When the sample chromatogram pattern data obtained by the conversion cannot be recognized as the normal chromatogram pattern data, the same sample can be re-measured by a control means 6 under different analysis conditions from predetermined ones.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to an apparatus and method for analyzing samples using liquid chromatography, and relates to an analyzer and method that enables samples showing abnormal measurement results to be re-measured efficiently. Regarding analysis methods.

0002

BACKGROUND OF THE INVENTION Liquid chromatography (hereinafter abbreviated as LC) is one of the separation and analysis methods used in analytical chemistry, medicine, pharmacy, biochemistry, and various other fields. In systems where LC is applied to daily analysis, the analysis conditions for LC are usually fixed. As such analysis systems, there are those that give priority to LC separation over measurement time, and those that give priority to measurement time over separation.

[0003] As an example of the latter, Japanese Patent Laid-Open No. 63-29806
There is a measurement of hemoglobin A1c (hereinafter referred to as HbA1c) as disclosed in No. 3 and JP-A-2-309253. HbA1c is one of the test items for diabetes in the field of clinical testing. As a dedicated measuring device for HbA1c,
Devices using high performance liquid chromatography (hereinafter referred to as HPLC) are widely used. Using these specialized measuring machines, it is now possible to measure one sample in about 4 minutes, but until a few years ago it took about twice as long.

[0004] The reason why the measurement time can be shortened in the measurement of HbA1c is because the performance of the separation column has been significantly improved. In this way, dedicated HbA1c measuring instruments are being developed with the aim of shortening the measurement time to meet market demands. The fact that HbA1c measurement is an analysis system that prioritizes measurement time over separation becomes clear when examining the separation conditions in a dedicated measuring instrument. The measured value of HbA1c is
It is defined as the relative ratio (percentage) of the amount of HbA1c to the total amount of hemoglobin. Specifically, H
bA1a, HbA1b, HbF, HbA1c, HbA0
The measured value is calculated based on the peak area values of five hemoglobins eluted in this order.

Among the hemoglobin components mentioned above, HbA0 contains many components, but by using a mobile phase with strong elution ability, these many components almost form a single peak. separated into Normally, when measuring HbA1c, it is not necessary to separate many hemoglobin components contained in HbA0, and it is desired to shorten the measurement time rather than separating HbA0 finely.

[0006] However, when HbA1c is measured, some samples sometimes exhibit abnormal chromatograms or abnormal measured values. The reason why such samples are generated is ■
■ Because the sample has changed over time, ■ Because it is affected by drugs, ■ Because there is an abnormality in the hemoglobin component ratio, ■ Because there is an abnormality in the amino acid sequence of the hemoglobin subunit (i.e., abnormal hemoglobin). ).

By the way, when an abnormality occurs in the measurement results as described above, in order to find out which of the above-mentioned causes is the cause, repeat the measurement by changing the measurement conditions so as to increase the degree of separation. There is something I want to do. In such cases, conventionally, one of the following two methods has generally been implemented. The first method is to perform the measurement again using the same analyzer under different analysis conditions, and the second method is to perform the measurement again using a different analyzer with a higher resolution. In either method, the measurer makes a judgment based on the first measurement result and performs the measurement again.

[0008]

[Problems to be Solved by the Invention] As mentioned above, the first,
Both of the second methods have the following problems because the measurer makes a judgment based on the first measurement result and performs the measurement again. ■The criteria for deciding whether to re-measure varied depending on the measurer. ■When re-measuring with the same analyzer under different measurement conditions, that is, in the first method above, the measurer must change the measurement conditions themselves, and it is complicated to return to the original analysis conditions after re-measurement. work was required. - After obtaining the initial measurement results, preparations for re-measurement are made, which wastes time, and it takes a considerable amount of time to obtain the re-measurement results.

[0009] Therefore, an object of the present invention is to solve the problems in the prior art as described above, and to analyze measurement results and adapt them to re-measurement conditions in an analysis device and analysis method using LC. It is an object of the present invention to provide an analysis device and method that enable re-measurement of a sample by automatically changing the measurement conditions.

0010

[Means for Solving the Problems] The analyzer of the present invention is an apparatus for analyzing a sample by liquid chromatography, and includes a sample introduction means, a column, and a detected amount of the sample separated and eluted from the column, which are connected in series. a liquid chromatography device having a detection means for outputting an electric signal according to the amount detected; detection data comparison means for comparing the chromatogram pattern data of the sample sample and normal chromatogram pattern data given in advance, and electrically connected to the liquid chromatography and the detection data comparison means, When a sample is separated under predetermined analysis conditions and the detection data comparison means cannot recognize that the chromatogram pattern data of the sample is a normal chromatogram pattern, liquid chromatography
Utilizing liquid chromatography, characterized in that it comprises a control means for controlling the liquid chromatography and the detection data comparison means so as to cause the same sample to be remeasured under analysis conditions different from the predetermined analysis conditions. This is an analytical device that has been developed.

[0011] Furthermore, the analysis method of the present invention analyzes a sample under predetermined analysis conditions using the above-mentioned analyzer using liquid chromatography, and generates electricity according to the detected amount of the sample obtained from the detection means. The signal is A/D converted by the detection data comparison means, and the obtained chromatogram pattern data of the sample is compared with normal chromatogram pattern data given in advance, and the chromatogram pattern data of the sample is determined to be a normal chromatogram. This is an analysis method using liquid chromatography that is characterized in that when a pattern cannot be recognized, the same sample is automatically analyzed again under analysis conditions different from the predetermined analysis conditions.

0012

[Operation] An outline of the analysis apparatus and analysis method using liquid chromatography of the present invention will be explained with reference to FIG. The liquid chromatography 1 includes at least a sample introduction means 2, a column 3 connected in series to the sample introduction means 2,
Further, a detection means 4 is connected to the downstream side of the column 3, and the detection means 4 detects the sample separated and eluted from the column 3, and outputs an electric signal according to the detected amount of the sample.

A detected data comparing means 5 is electrically connected to the detecting means 4 . The detection data comparison means is configured to compare the normal chromatogram pattern data of the sample given in advance and the chromatogram pattern data according to the detected amount of the sample given from the detection means 4, and output the result. has been done. The control means 6, the liquid chromatography 1, and the detected data comparison means 5 are electrically connected, and the control means 6 controls the liquid chromatography 1 and the detected data to perform the following analysis method. The comparison means 5 is configured to be controllable.

First, based on a command signal from the control means 6, a sample is separated in the liquid chromatography 1 under predetermined analysis conditions, and the detected amount of the sample is converted into an electrical signal by the detection means 4. In the detection data comparing means 5, the electrical signal corresponding to the detected amount of the sample obtained by the detecting means 4 is A/D converted, thereby obtaining chromatogram pattern data of the sample, and comparing it with the chromatogram pattern data of the sample. , and normal chromatogram pattern data given in advance are compared, and an output indicating the result is given to the control means 6. When the detection data comparison means 5 outputs an output to the control means 6 indicating that the chromatogram pattern data of the sample cannot be recognized as a normal chromatogram pattern, the control means 6 detects that the chromatogram pattern data of the sample is different from the original analysis conditions. The liquid chromatography 1 is operated under the analysis conditions and the measurement is performed again. As described above, in the analysis apparatus and analysis method of the present invention, a sample determined to be abnormal under the initial analysis conditions is automatically re-measured under different analysis conditions.

[0015]

[Description of Examples] Hereinafter, the present invention will be clarified by describing examples of the present invention. FIG. 2 is a schematic diagram of an analysis device using liquid chromatography according to an embodiment of the present invention.

In the analyzer of this embodiment, the liquid chromatography 10 includes a mobile phase switching means 11, a liquid feeding means 12,
It has a structure in which sample introduction means 13, column 14, and detection means 15 are connected in series. Two types of mobile phases a and b are arranged upstream of the mobile phase switching means 11. The mobile phase switching means 11 is configured so that either mobile phase a or b can be selected, or both mobile phases a and b can be mixed at a specified ratio. This mobile phase switching means 11 is specifically composed of a switching valve that can operate as described above. The liquid feeding means 12 is provided to feed the mobile phases a and b selected by the mobile phase switching means 11, and specifically realizes a flow rate suitable for analysis by the liquid chromatography 10. It consists of a pump that can be used. The sample introducing means 13 has a structure that functions to inject the sample to be measured into the mobile phase in the flow path between the liquid feeding means 12 and the column 14. Further, the column 14 is constituted by an appropriate column used in ordinary liquid chromatography.

The detection means 15 is configured to output an electric signal corresponding to the detected amount of the sample separated and eluted by the column 14. Detection data comparison means 16 is electrically connected to detection means 15 . The detection data comparison means 16 A/D converts the electrical signal corresponding to the detected amount of the sample outputted from the detection means 15, and the sample chromatogram pattern data obtained by the conversion and the normal data given in the memory in advance. It is configured to compare the chromatogram pattern data and provide an output to the control means 17 according to the comparison result. Further, in the analyzer of this embodiment, a sample detection sensor 18 is connected to the sample introducing means 13. The sample detection sensor 18 is provided to detect whether or not a sample to be measured is present in the sample introduction means 13.

The control means 17 is electrically connected to the detection data comparison means 16 and the sample detection sensor 18, as well as the mobile phase switching means 11 and liquid feeding means 12 of the liquid chromatography 10, and performs the analysis method described below. The mobile phase switching means 11, the liquid feeding means 12, the detection means 1
5 and a computer that controls the detected data comparison means 16.

Next, an analysis method using the analyzer of this embodiment shown in FIG. 2 will be explained with reference to the flowcharts of FIGS. 3 and 4. FIG. 3 shows a flowchart of the analysis method of the present invention, and is an example of a case where re-measurement is performed immediately when the measurement result is determined to be abnormal.

Referring to FIG. 3, in step S1,
Start measurement. Next, in step S2, the sample detection sensor 18 compares whether a sample is present on the sample introducing means 13 or not. The control means 17 includes a sample detection sensor 18
When informed that the sample exists, the liquid chromatography 10 performs measurement according to step S3. On the other hand, if the sample does not exist in step S2, the process proceeds to step S6 and ends the measurement. Specifically, the presence or absence of the sample is determined by the sample detection sensor 1 detecting whether or not a sample cup is prepared in the sample introducing means 13.
This is done by checking 8.

The above step S3 is a step in which a normal measurement is performed, and the mobile phase switching means 11, the liquid feeding means 12, and the sample introducing means of the liquid chromatography 10 are controlled in accordance with the normal analysis conditions according to a command from the control means 17. 13 is operated, and the detection means 15 outputs an electrical signal corresponding to the detected amount of the sample eluted from the column 14.

Next, in step S4, it is determined whether or not there is an abnormality in the measurement results. Specifically, the chromatogram pattern data of the sample obtained by A/D converting the electrical signal corresponding to the detected amount of the sample given from the detection means 15, and the normal chromatogram pattern data given in advance. are compared in the detected data comparing means 16, and the results are given to the control means 17. If the result is determined to be abnormal, the process proceeds to step S5, and if the result is determined to be normal, the process returns to step S2.

Step S5 is a step for re-measuring, in which the mobile phase switching means 11, liquid feeding means 12 and sample introduction means 13 perform analysis conditions different from the initial analysis conditions, according to a command from the control means 17. That is, the same sample is measured again under analysis conditions that provide higher separation. An electrical signal corresponding to the detected amount of the sample at the time of re-measurement is output from the detection means 15, the result is given to the control means 17, and the result of the re-measurement is output.

Next, when the re-measurement is completed, the control means 17
changes the analysis conditions from the remeasurement conditions to the original analysis conditions. Next, in step S2, the next sample is measured. Note that in step S6, all the samples have been measured,
This step is reached when there is no longer a sample to be measured in the sample introduction means 13.

FIG. 3 is a flowchart showing a method for immediately re-measuring a sample when it is determined to be abnormal after measuring the sample. On the other hand, FIG. The flowchart shows a case where only the samples determined to be abnormal are continuously re-measured after the measurement of all the samples has been completed.

Referring to FIG. 4, measurement is started in step S11. Specifically, the control means 17 applies normal analysis conditions to the mobile phase switching means 11, the liquid feeding means 12, and the sample introduction means 13, and the analysis is started. next,
In step S12, it is determined whether a sample exists in the sample introduction means 13. If the sample is present,
Proceeding to step S13, measurement is performed according to normal analysis conditions. That is, the detection amount corresponding to the sample is converted into an electrical signal in the detection means 15, and the detection data comparison means 1
6, the data is compared with the normal chromatogram pattern data, and the comparison result is given to the control means 17.
saves the comparison result in step S14.

After step S14 is completed, the process returns to step S12 again and measurement of the next sample is started. In this way, steps S12 to S14 are repeated to measure all the samples. Then, when there are no more samples, that is, after the measurement of all samples is completed, steps S12 to S
15, the measurement results saved in step S14 are read out in the order of measurement, and if there is a sample determined to be abnormal, the process advances to step S16. Step S16 is a step of performing re-measurement, and is a step of re-measuring the sample determined to be abnormal under analysis conditions that provide better separation than the initial analysis conditions. After the re-measurement is completed, the process returns to step S15, and if there is no sample to be re-measured, the process proceeds to step S17, where the measurement ends.

As mentioned above, in any of the analysis methods explained with reference to the flowcharts shown in FIGS. 3 and 4, samples determined to be abnormal under normal analysis conditions are separated more than under the original analysis conditions. It can be seen that re-measurement is automatically performed under analysis conditions that improve the results.

Note that the standard for performing the above-mentioned re-measurement, that is, the normal chromatogram pattern data given in advance to the detection data comparison means 16, can be determined arbitrarily depending on the object to be measured. For example, in the case of measuring HbA1c, , the following criteria may be mentioned. ■HbA1c value is lower than the normal range ■HbA
1c peak cannot be detected ■ There are two or more peaks near where the HbA1c peak elutes (usually one) ■ HbF value is higher than the normal range ■ HbA1a
The sum of the HbA1b value and the HbA1b value is higher than the normal range ■The HbA0 peak is not a single peak

[0030] If any of the above items apply, it is determined that the chromatogram pattern data does not fall within the normal chromatogram pattern data, and if the measurement is performed again, the cause of the abnormal HbA1c value can be investigated. .

In the following examples, an analyzer was prepared using a high performance liquid chromatograph manufactured by Sekisui Chemical Co., Ltd. under the trade name SSLC-20 so as to have the same analysis system as the analyzer shown in FIG. The specific configurations are as follows. Mobile phase switching means: SGR-720 liquid feeding means
:LCP-320 (Flow rate 2.2m
l/min) Sample introduction means: ASU-420 (injection volume 10 μl) Detection means: UVD-421 (
Wavelength: 415 nm) Detection data comparison means: CAS-220 control means
:CAS-220 column
:MICRONEX-HS2,6
φ×75mm (manufactured by Sekisui Chemical Co., Ltd.) Mobile phase a
;100mM potassium phosphate buffer (pH6.0) Mobile phase b;320mM potassium phosphate buffer (pH6.0)

[0032] The samples were prepared in the following manner. Using a vacuum blood collection tube (manufactured by Sekisui Chemical Co., Ltd., trade name: Incepac P) that uses NaF as an anticoagulant, 2 ml of blood was collected from a healthy adult, 5 μl of this blood was dispensed into a sample cup, and then, 1 ml of hemolytic reagent (21 L of hemolytic washing solution manufactured by Sekisui Chemical Co., Ltd.) was added to prepare a sample.

Further, the initial analysis conditions were as shown in FIG. 5, and the analysis conditions for re-measurement, that is, the conditions for improving separation than the normal analysis conditions, were as shown in FIG. Furthermore, Figure 5
And the vertical axis of FIG. 6 shows the proportion of mobile phase b.

Example 1 Nine normal samples and HbS (abnormal hemoglobin) sample 1
The books were measured according to the flowchart shown in FIG. As a result, the HbS sample met the abnormality criterion (2) and was automatically re-measured following the normal measurement. Normal sample, HbS
, chromatograms of re-measurement of HbS are shown in FIG. 7, FIG. 8, and FIG. 9, respectively.

Example 2 The same sample as in Example 1 was measured according to the flowchart shown in FIG. As a result, after continuous measurement of all samples,
The test was completed by re-measuring only the HbS sample.

[0036]

[Effects of the Invention] As described above, according to the analysis device and analysis method using liquid chromatography of the present invention, standards for re-measurement when there is a sample showing an abnormal measurement result are set in advance. and re-measurement will be performed automatically. Therefore, the consistency of the re-measurement standard is ensured, so
Even if the measurer is a beginner, he or she will not miss a sample showing an abnormal measurement result. Furthermore, since re-measurement is automatically performed, it becomes possible to omit the troublesome work of switching analysis conditions by the measurer. In addition, since re-measurement is automatically performed, measurement time is also shortened.

[0037] Therefore, if the analyzer and analysis method of the present invention are used, for example, to measure HbA1c, it is possible to reliably and quickly obtain information about other diseases from the results of measurements for the purpose of testing for diabetes. It becomes possible.

[Brief explanation of drawings]

FIG. 1 is a schematic block diagram for explaining an analysis device of the present invention.

FIG. 2 is a schematic configuration diagram of an analysis device according to an embodiment of the present invention.

FIG. 3 is a diagram showing a flowchart for explaining one embodiment of the analysis method of the present invention.

FIG. 4 is a diagram showing a flowchart of an analysis method according to another embodiment of the present invention.

FIG. 5 is a diagram showing analysis conditions for normal measurement.

FIG. 6 is a diagram showing analysis conditions when there is a sample showing an abnormal measurement result.

FIG. 7 is a diagram showing a chromatogram of a normal sample measured in Example 1.

FIG. 8 is a diagram showing a chromatogram of HbS measured in Example 1.

FIG. 9 is a diagram showing a chromatogram when HbS was remeasured in Example 1.

Claims (2)

[Claims] Claim 1: An apparatus for analyzing a sample by liquid chromatography, comprising a sample introduction means, a column, and a detection means for outputting an electric signal according to the detected amount of the sample separated and eluted from the column, which are connected in series. chromatogram pattern data of a sample obtained by A/D converting and converting an electrical signal corresponding to the detected amount, which is electrically connected to the detection means, and A detection data comparison means for comparing given normal chromatogram pattern data is electrically connected to the liquid chromatography and the detection data comparison means, and the liquid chromatography separates the sample under predetermined analysis conditions. and when the detected data comparing means cannot recognize that the chromatogram pattern data of the sample is a normal chromatogram pattern, the liquid chromatography is used to re-measure the same sample under analysis conditions different from the predetermined analysis conditions. An analytical device using liquid chromatography, characterized in that it is provided with a control means for controlling the liquid chromatography and the detected data comparison means so as to perform the analysis. 2. A sample is analyzed under predetermined analysis conditions using the analyzer using liquid chromatography according to claim 1, and an electric signal corresponding to the detected amount of the sample obtained from the detection means is Chromatogram pattern data of the sample obtained by A/D conversion in the detection data comparison means,
When the chromatogram pattern data of the sample cannot be recognized as a normal chromatogram pattern by comparing it with the normal chromatogram pattern data given in advance,
An analysis method using liquid chromatography, characterized in that the same sample is automatically analyzed again under analysis conditions different from the predetermined analysis conditions.