JP6856434B2 - Liquid chromatographic measurement method, liquid chromatography measurement device, and liquid chromatography measurement program - Google Patents

Description

The present invention relates to a liquid chromatography measuring method, a liquid chromatography measuring device, and a liquid chromatography measuring program, and in particular, a liquid chromatography measuring method, a liquid chromatography measuring device, and a liquid chromatography measuring program used for analyzing a biological sample such as blood. Regarding.

Conventionally, a method of measuring glycohemoglobin (HbA1c) and mutant Hb of a measurement sample such as blood by a liquid chromatography method has been proposed (see, for example, Patent Document 1 and Non-Patent Document 1).

Conventionally, when measuring glycohemoglobin (HbA1c) of a measurement sample, when mutant hemoglobin is detected and separated or removed to measure both HbA1c and mutant hemoglobin (variant mode), hemoglobin A1c is mainly measured. In the case of (fast mode), it was usual to measure using a separate measuring device or to replace the analysis column for measurement. This is because conditions such as the concentration of the eluent in the pipe affect the accuracy of the measurement, so that it is necessary to measure using a different eluent in each case. For this reason, conventionally, even if the liquid chromatography apparatus can measure the fast mode and the variant mode, when the measurement mode is changed, a cleaning step and an equilibration step in the column and the pipe are required. there were. Specifically, when changing the measurement mode, the measurement mode is changed after cleaning the column and the inside of the pipe by an end sequence at the end of the measurement mode. Then, in the changed measurement mode, a start sequence for equilibrating the inside of the column and the pipe is performed. Conventionally, it has not been performed to switch a plurality of measurement modes for a plurality of measurement samples and perform continuous measurement without adding an extra step.

Japanese Unexamined Patent Publication No. 2009-236768

Bio-Rad Laboratories "Variant II Dual Program" Catalog

As described above, when changing the measurement mode, a cleaning step and an equilibrium step in the column and the pipe are required, so that the amount of eluent used increases and the measurement time becomes long. .. Further, when the present inventors continuously measure a plurality of measurement samples by switching between the variant mode and the fast mode in one liquid chromatography apparatus, the eluent used only in the variant mode is next. It has been found that the measurement of HbA1c may be affected and the retention time (retention time) of HbA1c may be deviated. This problem is not so problematic when mainly performing only one of the variant mode and the fast mode in one liquid chromatography apparatus.

However, when both measurement modes are switched and continuously executed by one liquid chromatography device, if there is a difference in the measured retention time of HbA1c, the user can obtain a peak of HbA1c based on the retention time of HbA1c. There are problems such as confusion in the case of identification and complicated process of identifying the peak of HbA1c in the liquid chromatography apparatus. Therefore, when switching and executing both measurement modes with one liquid chromatography device, it is necessary to provide a sufficient switching time, and it is difficult to shorten the measurement time.

The present invention provides a liquid chromatography measuring method, a liquid chromatography measuring apparatus, and a liquid chromatography measuring apparatus capable of shortening the measuring time while suppressing a deviation in the holding time of HbA1c when measuring HbA1c by switching a plurality of measuring modes. An object of the present invention is to provide a liquid chromatography measurement program.

In one aspect of the liquid chromatography measurement method of the present invention, the first component separation eluate, the second component separation eluate, and the cleaning eluate are sequentially sent to the analysis column using the liquid chromatography method. By sequentially feeding the first measurement mode for measuring the hemoglobin A1c and the mutant hemoglobin of the measurement sample, the first component separation eluate and the cleaning eluate to the analysis column, the hemoglobin A1c can be obtained. A step of switching between a second measurement mode for measurement, a first holding time of the hemoglobin A1c in the first measurement mode, and a second holding time of the hemoglobin A1c in the second measurement mode. After the step of feeding the cleaning eluate and after the cleaning eluate before the influence of the second component separating eluate disappears in the first measurement mode, The step of feeding the first component separating eluate is included.

According to this, in one aspect, when HbA1c is measured by switching a plurality of measurement modes, it is possible to shorten the measurement time while suppressing a deviation in the holding time of HbA1c. In addition, "the same holding time" is included in "same" not only when both are completely the same, but also when there is a difference in holding time, if the difference is within ± 5%.

Further, in one aspect of the present invention, the liquid feeding time for feeding the first component separating eluate after feeding the cleaning eluate in the first measurement mode is the second measurement. It is longer than the liquid feeding time for feeding the first component separating eluate after feeding the cleaning eluate in the mode.

Further, in one aspect of the present invention, the holding means capable of holding the measurement sample for at least two measurements holds one of the low-concentration calibrator and the high-concentration calibrator for two measurements. The measurement mode and the second measurement mode are calibrated, and the holding means holds the other calibrator of the low concentration calibrator and the high concentration calibrator for two measurements, and the first measurement mode and the first measurement mode are held. Calibrate the measurement mode of 2.

Further, in one aspect of the liquid chromatography measuring apparatus of the present invention, the first component separation eluate, the second component separation eluate, and the cleaning eluate are sequentially fed to the analysis column by using the liquid chromatography method. The first measurement mode for measuring the hemoglobin A1c and the mutant hemoglobin of the measurement sample by liquid, and the hemoglobin by sequentially feeding the first component separation eluate and the cleaning eluate to the analysis column. A switching means for switching between a second measurement mode for measuring A1c, a first holding time of the hemoglobin A1c in the first measurement mode, and a second holding time of the hemoglobin A1c in the second measurement mode. The first liquid feeding means for feeding the cleaning eluate before the influence of the second component separating eluate disappears in the first measurement mode so that the holding time is the same. After the cleaning eluate, a second liquid feeding means for feeding the first component separating eluate is included.

According to this, in one aspect, when HbA1c is measured by switching a plurality of measurement modes, it is possible to shorten the measurement time while suppressing a deviation in the holding time of HbA1c.

Further, in one aspect of the present invention, the liquid feeding time for feeding the first component separating eluate after feeding the cleaning eluate in the first measurement mode is the second measurement. It is longer than the liquid feeding time for feeding the first component separating eluate after feeding the cleaning eluate in the mode.

In one aspect of the present invention, the measurement sample, the first component separation eluate, the second component separation eluent, and the prefilter for filtering the cleaning eluate are the same as the analysis column. It is integrated.

According to this, in one aspect, for example, the dead volume at the connection portion of the pre-filter and the capacity of the piping can be reduced, and the difference between the devices can be reduced. When the measurement mode is not switched, such a difference between devices does not cause any problem even if left unattended, but in a device that switches the measurement mode, such a difference between devices is a difference in holding time. Affects. Therefore, integrating the pre-filter with the analysis column is advantageous from the viewpoint of reducing the deviation of the holding time.

In the liquid chromatography measurement program of one aspect of the present invention, the first component separation eluate, the second component separation eluate, and the cleaning eluate are sequentially placed on an analysis column using a liquid chromatography method on a computer. The first measurement mode for measuring the hemoglobin A1c and the mutant hemoglobin of the measurement sample by sending the liquid, and the eluate for separating the first component and the eluate for cleaning are sequentially sent to the analysis column. The second measurement mode for measuring the hemoglobin A1c is switched between the first retention time of the hemoglobin A1c in the first measurement mode and the second retention time of the hemoglobin A1c in the second measurement mode. In the first measurement mode, the cleaning eluate is sent before the influence of the second component separating eluate disappears, and after the cleaning eluate, the cleaning eluate is used. The process of sending the first component separation eluate is executed.

According to this, in one aspect, when HbA1c is measured by switching a plurality of measurement modes, it is possible to shorten the measurement time while suppressing a deviation in the holding time of HbA1c.

According to the present invention, in one aspect, when HbA1c is measured by switching a plurality of measurement modes, it is possible to shorten the measurement time while suppressing a deviation in the holding time of HbA1c. ..

It is a schematic block diagram of the HPLC apparatus. It is a block diagram of the control system of an HPLC apparatus. It is a flowchart of a liquid chromatography measurement program. It is a diagram which shows an example of a chromatogram. (A) is a diagram for explaining the liquid feeding timing of each eluent when the fast mode is continuous, and (B) is a diagram for explaining the liquid feeding timing of each eluent when switching from the variant mode to the fast mode. (C) is a diagram for explaining the case where the liquid A is sent after the liquid C in the variant mode, and (D) is the figure for explaining the case where the liquid A is sent after the liquid B in the variant mode. It is a figure for demonstrating the case where the liquid feeding time of liquid A is lengthened. (A) is a diagram for explaining the case where feedback control is not performed based on the deviation of the holding time, and (B) is a diagram for explaining the case where feedback control is performed based on the deviation of the holding time. It is a schematic block diagram which shows the modification of the HPLC apparatus.

Hereinafter, embodiments of the present invention will be described.

FIG. 1 shows a schematic configuration diagram of an HPLC apparatus X using high performance liquid chromatography (HPLC).

The HPLC apparatus X is configured to set the blood collection tube 11 and automatically measure the concentration of glycohemoglobin (HbA1c) in whole blood. The HPLC apparatus X includes an apparatus main body 2 including a plurality of eluent bottles 12A, 12B, 12C, 12D, 12E (five in FIG. 1) and the like.

Each eluent bottle 12A to 12E holds eluents A to E to be supplied to the analysis column 60 described later. Each eluent has a different composition, component ratio, pH, osmotic pressure, etc., depending on the application.

The apparatus main body 2 has a sample preparation unit 5, an analysis unit 6, and a photometric unit 7.

The blood collection tube 11 is, for example, housed in a rack (not shown) and is configured to move to a position where it can be collected by a nozzle 51 in the sample preparation unit 5 described later.

The sample preparation unit 5 is for preparing a sample to be introduced into the analysis column 60 from the blood collected from the blood collection tube 11. The sample preparation unit 5 has a nozzle 51 and a dilution tank 53.

The nozzle 51 is for collecting various liquids including the blood sample 13 of the blood collection tube 11, and is capable of sucking and discharging the liquid and moving in the vertical direction and the horizontal direction. .. The operation of the nozzle 51 is controlled by the control unit 100, which will be described later.

The analysis unit 6 controls the adsorption and desorption of biological components to the filler of the analysis column 60, and supplies various biological components to the photometric unit 7. The set temperature in the analysis unit 6 is, for example, about 40 ° C. The analysis column 60 holds a filler for selectively adsorbing hemoglobin in the sample. As the filler, for example, a methacrylic acid-methacrylic acid ester copolymer is used.

The analysis unit 6 includes a manifold 61, a liquid feed pump 62, and an injection valve 63 in addition to the analysis column 60.

The manifold 61 is for selectively supplying the eluate to the analysis column 60 from a specific eluent bottle among the plurality of eluent bottles 12A to 12E. The manifold 61 is connected to the eluent bottles 12A, 12B, 12C, 12D, and 12E via the pipes 80A to 80E, and is connected to the injection valve 63 via the pipe 84.

The liquid feed pump 62 is for applying power for moving the eluent to the injection valve 63, and is provided in the middle of the pipe 84.

The injection valve 63 collects a certain amount of introduction sample and enables the introduction sample to be introduced into the analysis column 60, and includes a plurality of introduction ports and discharge ports (not shown). An injection loop 64 is connected to the injection valve 63. The injection loop 64 can hold a fixed amount (for example, several μL) of liquid, and by appropriately switching the injection valve 63, the injection loop 64 communicates with the diluting tank 53 and is communicated with the diluting tank 53 from the diluting tank 53 to the injection loop 64. Select a state in which the introduction sample is supplied to the injection loop 64, and a state in which the injection loop 64 communicates with the analysis column 60 via the prefilter PF and the pipe 85 and the introduction sample is introduced into the analysis column 60 from the injection loop 64. Can be done. As such an injection valve 63, for example, a hexagonal valve can be used. The pre-filter PF is a filter for filtering a sample or an eluent.

The photometric unit 7 is for optically detecting hemoglobin contained in the desorbed liquid from the analysis column 60, and is connected to the waste liquid tank 88 for discharging the desorbed liquid from the analysis column 60 via the pipe 87. It is connected.

FIG. 2 shows a block diagram of the control system of the HPLC apparatus X. As shown in FIG. 2, the HPLC apparatus X includes a control unit 100. The control unit 100 includes a CPU (Central Processing Unit) 100A, a ROM (Read Only Memory) 100B, a RAM (Random Access Memory) 100C, a non-volatile memory 100D, and an input / output interface (I / O) 100E via a bus 100F. Each is connected. Further, the HPLC apparatus X includes an operation unit (not shown) that receives an input from the operator.

A sample preparation unit 5, an analysis unit 6, and a photometric unit 7 are connected to the I / O 100E.

The liquid chromatography measurement program described later is stored in advance in the non-volatile memory 100D as an example in this embodiment. The CPU 100A reads and executes the liquid chromatography measurement program stored in the non-volatile memory 100D. Alternatively, the liquid chromatography measurement program may be recorded on a recording medium such as a CD-ROM and executed by reading the program with a CD-ROM drive or the like.

Next, the operation of this embodiment will be described.

FIG. 3 shows a flowchart of the liquid chromatography measurement process executed by the CPU 100A of the control unit 100.

First, the operator sets the blood collection tube 11 containing the blood sample 13. By moving the rack containing the plurality of blood collection tubes 11, the blood collection tubes 11 are transported to the collection position.

When the operator instructs the start of measurement, the CPU 100A reads and executes the liquid chromatography measurement program stored in the non-volatile memory 100D.

In step S10, it is determined whether the blood sample 13 to be measured is measured in the variant mode or the fast mode (also referred to as the normal mode), and when the measurement is performed in the fast mode, the process proceeds to step S12 and the measurement is performed in the variant mode. If so, the process proceeds to step S26. Here, the variant mode (first measurement mode) is the HbA1c of the blood sample 13 by sequentially feeding a plurality of types of eluates, which will be described later, to the analysis column 60 in a predetermined order by using a liquid chromatography method. And a measurement mode for measuring mutant hemoglobin. Further, in the fast mode (second measurement mode), among a plurality of types of eluents, a plurality of common eluates common to the eluent used in the variant mode are sequentially sent to the analysis column 60 in a predetermined order. This is a measurement mode for measuring HbA1c.

Which of the measurement modes, the variant mode and the fast mode, is to be measured is preset for each blood sample 13 by, for example, the user. For example, the measurement mode can be identified and set by reading the barcode information attached to the blood collection tube 11, receiving an instruction signal from the host computer, or inputting from the operation unit. Even if the rack contains a mixture of blood collection tubes 11 measured in the fast mode and the variant mode, it can be accommodated.

Here, the plurality of types of eluents are liquids A to C in this embodiment. The liquid A (first component separation eluent) is an eluent for elution of HbA1c and for equilibrating the analysis column 60. The liquid B (cleaning eluent) is an eluent for elution of all the hemoglobin remaining on the analysis column 60, that is, an eluent for washing the analysis column 60. Liquid C (second component separation eluent) is an eluent for eluting hemoglobin other than HbA1c after HbA1c is eluted. In addition, the solution B, the solution C, and the solution A are in descending order of the dissolution output of hemoglobin.

Then, in the variant mode, solutions A, B, and C are sent to the analysis column 60 in a predetermined order for measurement, and in the fast mode, solutions A and B are sent to the analysis column 60 in a predetermined order. Liquid is sent to and measured. That is, liquid A and liquid B are common eluents that are commonly specified in variant mode and fast mode, and liquid C is a non-common eluent that is used only in variant mode.

In step S12, the blood sample 13 is introduced. Specifically, first, a blood sample 13 is collected from the blood collection tube 11. That is, the blood sample 13 is collected from the blood collection tube 11 by operating the nozzle 51. The blood sample 13 collected by the nozzle 51 is supplied to the dilution tank 53 by operating the nozzle 51.

Then, the sample held in the injection loop 64 by switching the injection valve 63 is filtered by the pre-filter PF and introduced into the analysis column 60. When the sample is introduced into the analysis column 60, HbA1c, mutant Hb, and the like are adsorbed on the filler.

In step S14, the solution A is supplied to the analysis column 60 for a predetermined time. As a result, HbA1c is eluted from the analysis column 60.

In step S16, the liquid B is supplied to the analysis column 60 for a predetermined time. As a result, all the hemoglobin remaining in the analysis column 60 is eluted, and the analysis column 60 is washed.

In step S18, the solution A is supplied to the analysis column 60 for a predetermined time. As a result, the analysis column 60 is equilibrated.

As described above, in the fast mode, the liquid is sent to the analysis column 60 in the order of liquid A → liquid B → liquid A.

The desorbing liquid containing various hemoglobin discharged from the analysis column 60 is supplied to the photometric unit 7 via the pipe 86. This desorbing liquid is guided to the waste liquid tank 88 via the pipe 87.

In the photometric unit 7, light is continuously irradiated to the desorbing liquid, and the light receiving result (absorbance) is output to the control unit 100. Then, the control unit 100 calculates the chromatogram.

As shown in FIG. 4, the chromatogram is a graph showing the relationship between the elapsed time from the start of measurement and the absorbance as a result of light reception. It is possible to know which hemoglobin was detected by the position where the peak of this chromatogram appears, and also by the integrated value of the absorbance at the peak portion (mountainous portion), that is, the size of the area of the peak portion. You can know the concentration of.

In the present embodiment, for example, when the measurement sample contains HbA1c and HbA0, some hemoglobin, a specific mutant hemoglobin, for example, mutant hemoglobin C, and mutant hemoglobin S, the eluent is switched and sequentially fed to the analysis column 60. As shown in FIG. 4, it is assumed that peaks appear at each time point of ta, tb, tc, tf, and tg in the case of liquid. In this case, it is possible to determine whether or not any hemoglobin has been detected by determining whether or not a peak appears at the time of ta seconds from the calculated chromatogram. Similarly, it can be determined whether or not HbA1c is detected by determining whether or not a peak appears at the time of tb, and HbA0 by determining whether or not a peak appears at the time of tc. It is possible to determine whether or not HbS is detected, and by determining whether or not a peak appears at the time of tf, it is possible to determine whether or not HbS is detected, and a peak appears at the time of tg. It is possible to determine whether or not the mutant hemoglobin C has been detected by determining whether or not the mutant hemoglobin C has been detected. If a peak appears at a time other than ta, tb, tc, tf, and tg, it can be determined that hemoglobin that was not expected in advance was detected. In addition, the concentration of each hemoglobin can be measured from the magnitude of the absorbance at each time point.

In the present embodiment, in the fast mode, for example, when the measurement sample contains HbA1c, HbA0, some hemoglobin, mutant hemoglobin C, and mutant hemoglobin S, the eluent is switched according to the above control sequence to analyze column 60. As shown in FIG. 4, it is assumed in advance that the peak of HbA1c appears at tb from the start of measurement when the liquids are sequentially sent to. In this case, it can be determined whether or not HbA1c is detected by determining whether or not a peak appears in the vicinity of tb from the calculated chromatogram.

Therefore, in step S20, HbA1c is measured based on the calculated chromatogram. That is, as described above, it is determined whether or not a peak appears in the vicinity of tb, and if a peak appears in the vicinity of tb, it is determined that HbA1c has been detected. Then, the concentration of HbA1c is measured from the integrated value of the absorbance at the peak portion near tb.

In step S24, the measurement result is stored in the non-volatile memory 100D. That is, the concentration of HbA1c and the presence / absence of detection of mutant hemoglobin are stored in the non-volatile memory 100D.

In step S44, it is determined whether or not the above measurement has been completed for all the blood collection tubes 11, and if the measurement has been completed for all the blood collection tubes 11, this routine is terminated, and the measurement has not been completed for the blood collection tubes 11. If is present, the process returns to step S10 to determine whether the blood sample 13 to be measured is measured in the variant mode or the fast mode.

If it is determined in step S10 that the measurement is performed in the variant mode, the process proceeds to step S26.

In step S26, the blood sample 13 is introduced in the same manner as in step S12.

In step S28, the solution A is supplied to the analysis column 60 for a predetermined time. As a result, HbA1c is eluted from the analysis column 60.

In step S30, the liquid C is supplied to the analysis column 60 for a predetermined time. As a result, hemoglobin other than HbA1c is eluted after HbA1c is eluted from the analysis column 60.

In step S32, the solution A is supplied to the analysis column 60 for a predetermined time. As a result, the analysis column 60 is equilibrated.

In step S34, the liquid B is supplied to the analysis column 60 for a predetermined time. As a result, all the hemoglobin remaining in the analysis column 60 is eluted, and the analysis column 60 is washed.

In step S36, the solution A is supplied to the analysis column 60 for a predetermined time. As a result, the analysis column 60 is equilibrated.

As described above, in the variant mode, the liquid is sent to the analysis column 60 in the order of liquid A → liquid C → liquid A → liquid B → liquid A.

In step S38, HbA1c is measured in the same manner as in step S20.

In step S40, the mutant hemoglobin is measured. That is, as described above, it is determined whether or not the mutant hemoglobin is detected by determining whether or not the peak appears at a time point other than tb and tc. Then, when a peak appears at a time point other than tb and tc, the concentration of mutant hemoglobin is measured from the integrated value of the peak portion.

In step S42, the measurement result is stored in the non-volatile memory 100D. That is, the concentration of HbA1c and the concentration of mutant hemoglobin are stored in the non-volatile memory 100D.

In this way, in the present embodiment, the measurement can be performed by switching between the fast mode and the variant mode with one device.

Next, referring to FIG. 5 (A), when the measurement in the fast mode is continuous, the liquid feed timing of the liquid A and the liquid B, the timing at which the peak of HbA1c appears, and the liquid B are transferred to the analysis column 60. The residual amount of the liquid B in the analysis column 60 after the liquid is sent will be described.

In FIG. 5A, the waveform W1 represented by hatching represents the waveform of the peak absorbance of HbA1c. Further, the waveform W2 represents the liquid feeding timing of the liquid B. Further, the waveform W3 shows the residual amount of the liquid B in the analysis column 60. That is, the vertical axis in FIG. 5A has two meanings: the absorbance of HbA1c and the residual amount of the liquid B.

For example, as shown in FIG. 5 (A), in the nth measurement in the fast mode, the liquid A is sent from the time t1 to the time t2, the liquid B is sent from the time t2 to the time t3, and the time point t3 to the time t4. Liquid A is sent up to. Here, when the liquid B is sent from the time t2 to the time t3, the liquid B mixes with the liquid A in the pipe until it enters the analysis column 60, so that the residual amount (concentration) of the liquid B in the analysis column 60). Is not actually like the waveform W2, but is a waveform that gradually increases like the waveform W3 and then gradually decreases. The peak of HbA1c _emerges at _{t pn} time. That the start of measurement at which t1 peak of HbA1c from point _t time to _pn time T seconds retention time appearing (retention time).

Then, from the time t4, the liquid A is started to be sent at the (n + 1) th measurement, but the liquid B sent at the nth measurement remains a little on the analysis column 60, but (n + 1). The liquid A liquid feeding at the measurement eye is started. This is to shorten the time for one measurement as much as possible, but if the next (n + 2) measurement is also a fast mode measurement, the residual liquid B remaining in the analysis column 60 at the start of the (n + 2) measurement is left. The amount is substantially the same as the residual amount of the liquid B remaining on the analysis column 60 at the start of the (n + 1) measurement eye. As described above, when the fast mode is continuous, the residual amount of the liquid B at the start of the measurement is substantially the same. Therefore, for example, the holding time at the (n + 1) measurement eye, that is, the time from the t1 time point to the _tpn time point, and the holding time at the (n + 1) measurement eye, that is, the time from the t4 time point to the tp _(n-1) time point. , Are almost the same, and there is no particular problem.

Next, when the variant mode is switched to the fast mode, the timing of sending the liquids A to C, the timing at which the peak of HbA1c appears, and the C in the analysis column 60 after the liquid C is sent to the analysis column 60. The residual amount of the liquid and the residual amount of the liquid B in the analysis column 60 after the liquid B is sent to the analysis column 60 will be described.

For example, as shown in FIG. 5 (B), in the nth measurement in the variant mode, the liquid A is sent from the time t1 to the time t2, the liquid C is sent from the time t2 to the time t3, and the liquid C is sent from the time t3 to the time t4. Liquid A is sent from t4 to t5, and liquid A is sent from t5 to t6. The time points from t4 to t6 in FIG. 5 (B) correspond to the time points from t2 to t4 in FIG. 5 (A), respectively. That is, the liquid feeding time of the liquid B in FIG. 5 (B) (time from t4 to t5) is the same as the liquid feeding time of the liquid B in FIG. 5 (A) (time from t2 to t3), and FIG. The liquid feeding time of the liquid A (time from t5 to t6) in (B) is the same as the liquid feeding time (time from t3 to t4) of the liquid A in FIG. 5 (A).

Here, the waveform W4 represents the liquid feeding timing of the C liquid. Further, the waveform W5 indicates the residual amount of the liquid C in the analysis column 60. When the liquid C is sent from the time t2 to the time t3, the residual amount of the liquid C in the analysis column 60 does not actually become the waveform W4, but the waveform W5, as in the case of the liquid B described above. The waveform gradually increases and then gradually decreases. Therefore, at the time of t6 when the (n + 1) measurement th is started, a small amount of the C liquid sent at the nth measurement remains in the analysis column 60. Further, as in FIG. 5A, a small amount of liquid B remains.

As a result, as shown in FIG. 5 (B), the timing at which the peak of HbA1c appears at the (n + 1) measurement eye is advanced by a second as compared with the case of FIG. 5 (A), and the holding time is (TA). ) Seconds. This is because, at the time of t6, the residual amount of the liquid B is the same as that in the case of FIG. 5 (A), but the liquid C remains.

Therefore, in the present embodiment, as shown in FIG. 5 (C), the liquid feeding time of the liquid A to be fed after the liquid C is lengthened as compared with the case of FIG. 5 (B). That is, the amount of the liquid A to be fed after the liquid C is increased as compared with the case of FIG. 5 (B). In the example of FIG. 5 (C), the liquid feeding time (time from t3 to t4) of the liquid A to be fed after the liquid C is longer than that in the case of FIG. 5 (B). That is, the liquid feeding time of the liquid A to be fed in step S32 is lengthened. The liquid feeding time of the liquid B and the liquid A to be fed after the liquid C and the liquid A is the same as in the case of FIG. 5 (B).

As a result, as shown in FIG. 5C, at the time of t6 when the (n + 1) measurement th is started, the C liquid sent at the nth measurement disappears, and the influence of the C liquid disappears. Since the residual amount of the liquid B is the same as in the case of FIG. 5 (A), the holding time of HbA1c at the (n + 1) measurement eye is T seconds, and the fast mode is continuous as shown in FIG. 5 (A). It is the same as the holding time of HbA1c. The liquid feeding time of the liquid A to be fed after the liquid C does not necessarily have to be set to the time when the liquid C disappears from the analysis column 60, and even if a small amount of the liquid C remains in the analysis column 60, the holding time It suffices if the time is set so that there is no deviation.

Further, as shown in FIG. 5 (D), instead of lengthening the liquid feeding time of the liquid A to be fed after the liquid C, the liquid feeding time of the liquid A to be fed after the liquid B is shown in FIG. 5 (B). ) May be longer than in the case of b seconds. That is, the liquid feeding time of the liquid A to be fed in step S36 is lengthened. As a result, the measurement time of the nth measurement (time from t1 to t6) can be shortened as compared with the case of FIG. 5C. This is because the remaining amount of the liquid B has decreased by the amount of the remaining amount of the liquid C.

As described above, in the present embodiment, when HbA1c is measured in the variant mode, the HbA1c is fed to the analysis column 60 after the liquid C is fed so that the retention time of HbA1c is the same as the retention time of HbA1c measured in the fast mode. The liquid feeding time of at least one of the liquid A to be liquid, that is, the liquid A to be fed immediately after the liquid C and the liquid A to be fed immediately after the liquid B is controlled. Thereby, at the start of the measurement in the fast mode and the variant mode, the influences of the residual components and the residual amount of the liquid B and the liquid C on the elution of HbA1c can be set to be substantially the same. Therefore, when HbA1c is measured by switching between the variant mode and the fast mode, it is possible to suppress a deviation in the holding time of HbA1c.

In the present embodiment, as one aspect of controlling the elution condition in the analysis column 60, the eluent feeding time is controlled so that the retention time of HbA1c measured in the variant mode and the fast mode is the same. However, the mode for controlling the elution conditions in the analysis column 60 is not limited to this. For example, other aspects of controlling the elution conditions in the analysis column 60 include controlling the eluent feeding timing, controlling the eluent feeding amount, controlling the eluent flow velocity, and erasing. This includes adding a liquid and changing the concentration of the eluate. By making such an elution condition different from the elution condition when the variant mode and the fast mode are not switched, the retention time of HbA1c measured in the variant mode and the fast mode may be controlled to be the same.

When the first holding time measured in the variant mode and the second holding time measured in the fast mode are different, B in the variant mode depends on the difference between the first holding time and the second holding time. The liquid feeding timing may be different.

Generally, in the variant mode, since more types of eluents are used than in the fast mode, the influence of the C solution remains in the next measurement, and the retention time of HbA1c is usually shortened in the next measurement. .. Since the liquid B is used in both the variant mode and the fast mode, the difference in the holding time is not affected, and it is considered that the difference in the residual amount of the liquid C affects the difference in the holding time.

Therefore, for example, as shown in FIG. 6B, feedback control for advancing the liquid feeding timing of the B liquid by d seconds according to the difference (c seconds) between the first holding time and the second holding time is performed. Do. As a result, the remaining amount of the synthesis of the C and B solutions at the nth measurement is adjusted, and as shown in FIG. 6 (B), the first holding time T1 second measured in the next variant mode is measured in the fast mode. It becomes the same as the second holding time T2 seconds. In the example of FIG. 6B, since the first holding time measured in the variant mode is c seconds shorter than the second holding time measured in the fast mode, the liquid B feeding timing is set to d seconds. Although it is made earlier, for example, if the first holding time measured in the variant mode is c seconds later than the second holding time measured in the fast mode, the liquid B feeding timing should be delayed by d seconds. Just do it. The degree to which the liquid B feeding timing is adjusted according to the difference between the first holding time and the second holding time, that is, the correspondence between c seconds and d seconds, is obtained by experiment in advance. Alternatively, the measurement may be actually repeated and obtained from the measurement result.

The above feedback control is performed, for example, at the time of measurement of the variant mode in which the residual amount of the liquid C affects the retention time, but the present invention is not limited to this.

Further, in the present embodiment, the case where the pre-filter PF is provided between the injection valve 63 and the analysis column 60 has been described, but the present invention is not limited to this, and the pre-filter PF is used as an analysis column, for example, as shown in FIG. The configuration may be integrated with 60. As a result, the capacity of the pipe 85 can be reduced, and the residual amount of the C liquid can be suppressed as much as possible. Therefore, it is possible to suppress the deviation of the holding time of HbA1c as much as possible.

Further, in the liquid chromatography apparatus, when there is a possibility that the measurement conditions may be changed, such as when the analysis column is replaced or when the liquid chromatography apparatus is maintained, a sample for calibration (hereinafter referred to as a calibrator) is used. It is usual to calibrate the liquid chromatography apparatus (hereinafter referred to as calibration). Further, even if there is no possibility that the measurement conditions are changed, it is usual to carry out calibration regularly to maintain the measurement accuracy.

In this case, it is necessary to prepare two types of calibrators, a low-concentration Low calibrator and a high-concentration High calibrator, and perform calibration for each calibrator.

When switching between the variant mode and the fast mode with one liquid chromatography apparatus as in this embodiment, it is necessary to calibrate each measurement mode. For example, in the variant mode, calibration is performed using a Low calibrator and a High calibrator. If you switch to the fast mode and then perform calibration using a low-concentration and high-concentration calibrator, cleaning the device and preparing the calibrator become complicated, and the user can set the order and number of calibrators to be placed. There is a high possibility of making a mistake. That is, in the case of the above example, the measurement is executed in the order of Low (variant mode) → High (variant mode) → Low (fast mode) → High (fast mode), and the calibrator is switched three times. , The channel cleaning must be performed 3 times. In addition, a Low calibrator and a High calibrator must be prepared for each measurement mode of the variant mode and the fast mode.

Therefore, the pipe 83 connecting the diluting tank 53 and the injection valve 63 is a pipe having a capacity capable of holding at least two measurement samples, and for example, a Low calibrator for two measurements is held in the pipe 83. Then, calibrate with the Low calibrator in each measurement mode of the variant mode and the fast mode, record the measurement result of each measurement mode, and then hold the high calibrator for two measurements in the pipe 83 to hold the high calibrator for two measurements in the variant mode and the fast mode. In each measurement mode of the above, calibration by a high calibrator may be performed and the measurement result of each measurement mode may be recorded.

As a result, the measurement is executed in the order of Low (variant mode) → Low (fast mode) → High (variant mode) → High (fast mode), the calibrator is switched once, and the flow path is cleaned once. I'm done. In this way, the calibration work can be simplified, the cost can be reduced, and the possibility that the user makes a mistake in the order in which the calibrators are placed, the number of the calibrators, and the like can be reduced. This method can be used not only for calibration but also for measurement (control measurement) of quality-controlled substances (specimens), which reduces the amount of reagents used and the amount of quality-controlled substances used. In addition to being able to contribute, it is possible to reduce misplacement of quality control substances by the user.

Further, when the pipe 83 is a pipe having a capacity capable of holding two measurement samples, the measurement is first performed in the fast mode using half of the measurement samples, and when some abnormality occurs. For example, if the measured HbA1c value is abnormally low, the measurement may be performed in the variant mode using the other half of the measurement samples.

The present invention is not limited to the above-described embodiment, and can be modified in various ways. For example, the present invention is applied not only to an HPLC device for measuring the hemoglobin concentration in blood, but also to a sample other than blood, a component other than the hemoglobin concentration, or a liquid chromatography device other than the HPLC device. can do.

X HPLC apparatus 5 Sample preparation unit 6 Analysis unit 7 Metering unit 11 Blood collection tube 13 Blood sample 60 Analysis column 100 Control unit

Claims (8)

Using the liquid chromatography method, the hemoglobin A1c and the mutant hemoglobin of the measurement sample are measured by sequentially feeding the first component separation eluate, the second component separation eluate, and the washing eluate to the analysis column. A step of switching between the first measurement mode for measuring the hemoglobin A1c and the second measurement mode for measuring the hemoglobin A1c by sequentially feeding the first component separation eluate and the cleaning eluate to the analysis column. When,
In the first measurement mode, the first retention time of the hemoglobin A1c in the first measurement mode and the second retention time of the hemoglobin A1c in the second measurement mode are the same. The step of sending the first component separation eluate after the second component separation eluate is sent, and the step of sending the first component separation eluate.
A step of feeding the cleaning eluate after the first component separating eluent before the influence of the second component separating eluate disappears.
A step of feeding the first component separation eluate after the cleaning eluate, and
Only including,
After the second component separation eluate is sent, the time for sending the first component separation eluate and the cleaning eluate before the start of the next measurement is the time at the start of the next measurement. It is set so that the influence of the second component separation eluent is eliminated.
Liquid chromatography measurement method. Using the liquid chromatography method, the hemoglobin A1c and the mutant hemoglobin of the measurement sample are measured by sequentially feeding the first component separation eluate, the second component separation eluate, and the washing eluate to the analysis column. A step of switching between the first measurement mode for measuring the hemoglobin A1c and the second measurement mode for measuring the hemoglobin A1c by sequentially feeding the first component separation eluate and the cleaning eluate to the analysis column. When,
In the first measurement mode, the first retention time of the hemoglobin A1c in the first measurement mode and the second retention time of the hemoglobin A1c in the second measurement mode are the same. The step of feeding the cleaning eluate before the influence of the second component separating eluent disappears, and
A step of feeding the first component separation eluate after the cleaning eluate, and
Including
The liquid feeding time for feeding the first component separation eluate after feeding the cleaning eluate in the first measurement mode is the cleaning eluent in the second measurement mode. It is longer than the liquid feeding time for feeding the first component separation eluent after liquid feeding.
Liquids chromatographic measurement method. The first measurement mode and the second measurement mode are obtained by holding one of the low-concentration calibrator and the high-concentration calibrator for two measurements in a holding means capable of holding at least two measurement amounts of the measurement sample. Is executed, and the holding means holds the other calibrator of the low-concentration calibrator and the high-concentration calibrator for two measurements, and calibrates the first measurement mode and the second measurement mode. The liquid chromatography measurement method according to claim 1 or 2. Using the liquid chromatography method, the hemoglobin A1c and the mutant hemoglobin of the measurement sample are measured by sequentially feeding the first component separation eluate, the second component separation eluate, and the washing eluate to the analysis column. Switching between the first measurement mode for measuring the hemoglobin A1c and the second measurement mode for measuring the hemoglobin A1c by sequentially feeding the first component separation eluate and the cleaning eluate to the analysis column. Means and
A liquid feeding means for feeding the first component separating eluate, the second component separating eluent, and the cleaning eluent.
Including
The liquid feeding means is such that the first holding time of the hemoglobin A1c in the first measurement mode and the second holding time of the hemoglobin A1c in the second measurement mode are the same. In the first measurement mode, after the second component separation eluate is sent, the first component separation eluate is sent, and then the influence of the second component separation eluate disappears. Before, the cleaning eluate was sent to the solution.
After the cleaning eluate, the first component separating eluate is sent .
After the liquid feeding means feeds the second component separating eluate, the time for sending the first component separating eluate and the cleaning eluent before the start of the next measurement is as follows. It is set so that the influence of the second component separating eluent is eliminated at the start of measurement.
Liquid chromatography measuring device. Using the liquid chromatography method, the hemoglobin A1c and the mutant hemoglobin of the measurement sample are measured by sequentially feeding the first component separation eluate, the second component separation eluate, and the washing eluate to the analysis column. Switching between the first measurement mode for measuring the hemoglobin A1c and the second measurement mode for measuring the hemoglobin A1c by sequentially feeding the first component separation eluate and the cleaning eluate to the analysis column. Means and
In the first measurement mode, the first retention time of the hemoglobin A1c in the first measurement mode and the second retention time of the hemoglobin A1c in the second measurement mode are the same. Before the influence of the second component separation eluate disappears, the first liquid feeding means for feeding the cleaning eluent and the first liquid feeding means.
After the cleaning eluent, a second liquid feeding means for feeding the first component separating eluent and a second liquid feeding means.
Including
The liquid feeding time for feeding the first component separation eluate after feeding the cleaning eluate in the first measurement mode is the cleaning eluent in the second measurement mode. It is longer than the liquid feeding time for feeding the first component separation eluent after liquid feeding.
Liquids chromatographic measurement apparatus. A claim in which the measurement sample, the first component separation eluate, the second component separation eluate, and a prefilter for filtering the cleaning eluent are integrated with the analysis column. 4 or the liquid chromatography measuring apparatus according to claim 5. On the computer
Using the liquid chromatography method, the hemoglobin A1c and the mutant hemoglobin of the measurement sample are measured by sequentially feeding the first component separation eluate, the second component separation eluate, and the washing eluate to the analysis column. The first measurement mode is switched between the first measurement mode in which the hemoglobin A1c is measured and the second measurement mode in which the hemoglobin A1c is measured by sequentially feeding the first component separation eluate and the cleaning eluate to the analysis column.
In the first measurement mode, the first retention time of the hemoglobin A1c in the first measurement mode and the second retention time of the hemoglobin A1c in the second measurement mode are the same. After the second component separation eluate is sent, the first component separation eluate is sent, and after the first component separation eluate is sent, the second component separation is performed. Before the influence of the eluent for cleaning disappears, the eluate for cleaning is sent.
It is a liquid chromatography measurement program for executing a process of sending the first component separation eluate after the cleaning eluate.
After the second component separation eluate is sent, the time for sending the first component separation eluate and the cleaning eluate before the start of the next measurement is the time at the start of the next measurement. It is set so that the influence of the second component separation eluent is eliminated.
Liquid chromatography measurement program. On the computer
Using the liquid chromatography method, the hemoglobin A1c and the mutant hemoglobin of the measurement sample are measured by sequentially feeding the first component separation eluate, the second component separation eluate, and the washing eluate to the analysis column. The first measurement mode is switched between the first measurement mode in which the hemoglobin A1c is measured and the second measurement mode in which the hemoglobin A1c is measured by sequentially feeding the first component separation eluate and the cleaning eluate to the analysis column.
In the first measurement mode, the first retention time of the hemoglobin A1c in the first measurement mode and the second retention time of the hemoglobin A1c in the second measurement mode are the same. Before the influence of the second component separation eluent disappears, the cleaning eluent is sent.
It is a liquid chromatography measurement program for executing a process of sending the first component separation eluate after the cleaning eluate.
The liquid feeding time for feeding the first component separation eluate after feeding the cleaning eluate in the first measurement mode is the cleaning eluent in the second measurement mode. It is longer than the liquid feeding time for feeding the first component separation eluent after the liquid feeding.
Liquid chromatography measurement program.

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