JP6897267B2 - Peak identification method that is not affected by fluctuations in elution time - Google Patents

Description

The present invention relates to a method that enables accurate peak identification even when the elution time of each peak changes with time in a chromatogram having a plurality of peaks.

Chromatography is a method of separating and quantifying a sample containing a plurality of components with a column. In general, qualification is performed from the elution time of each component peak, and quantification is performed based on the output degree of the detector. To perform qualitative analysis, measure a standard sample containing the target component in advance, set the elution time of each component as the reference elution time, and create an "identification table" that defines the identification range with the allowable range for it. I do.

When identifying an unknown sample, compare the elution times of multiple component peaks appearing in the chromatogram of the unknown sample with the identification table to determine whether or not it falls within the identification range, and if it falls within the identification range. It is a common technique to identify the corresponding component.

However, in actual chromatography, it is often seen that the composition of the eluent fluctuates slightly over time and the elution time increases or decreases over time. In addition, the column oven and liquid feed pump that make up the chromatograph also lead to fluctuations in the elution time. Depending on the factors of the installation environment, the temperature of the column oven may fluctuate slightly, or the amount of liquid sent by the pump may fluctuate slightly, and the elution time may fluctuate.

FIG. 1 is a diagram showing a case where the elution time is delayed with time when measuring an unknown sample. In this case, the elution time of each component of the unknown sample 1 is within the range of -3% to + 3%, and can be identified as Comp_1, Comp_2, Comp_3, Comp_4, Comp_5, and Comp_6, respectively, but the unknown samples 2 and 3 are each component. Since the elution time of is not within the range of -3% to + 3%, it cannot be identified and is judged to be an "unknown component".

Even in such a case, the reference elution time of the identification table is replaced with the elution time of the identified component, the permissible width is recalculated accordingly, and the identification table is updated so that the identification can be performed correctly. A method such as using it for identification of an unknown sample has also been proposed, but as shown in FIG. 2, when the components registered in the identification table cannot be identified at the time of measurement of an unknown sample, or when some components are not originally contained. Then, there was a problem that the update of the identification table did not function normally.

An object of the present invention is that component identification can be performed accurately even if the elution time fluctuates with time, and in particular, even when a component registered in the identification table is undetected, it affects a series of identifications. It provides a peak identification method that does not reach the above.

The present invention relates to a method for continuously identifying two or more unknown samples by liquid chromatography.
For the first unknown sample, a specific component in the unknown sample was identified based on an identification table created based on the elution time of the standard sample.
Based on the identification result, the identification table is corrected.
Based on the corrected identification table, components other than the specific component are identified, and
For the nth unknown sample (n = an integer of 2 or more), the specific component was identified in the corrected identification table used in the (n-1) th case.
Based on the identification result, the corrected identification table used in the (n-1) case is further corrected by the same method as in the first case.
It is characterized in that components other than the specific component are identified based on the corrected identification table.

The details of the present invention will be described below.

FIG. 3a is a chromatogram of a standard sample and unknown samples 1 to 3, and FIG. 3b is a plot of elution times of each component (Comp_1 to Comp_6) of unknown samples 1 to 3. The standard sample contains 6 components, Comp_1 to Comp_6, and is also registered in the identification table. Similarly, the unknown samples 2 and 3 contain 6 components corresponding to Comp_1 to Comp_6, but the unknown sample 1 contains only 5 components corresponding to Comp_1 to Comp_5.

First, as in the general identification method, the elution time as a reference for identification and the identification range thereof are specified from the elution time of each component (Comp_1 to Comp_6) of the standard sample (creation of an identification table). The identification range may be specified by the absolute time for each component, but it is easier to set by specifying the ratio to the reference elution time.

Hereinafter, a method of calculating the ratio to the reference elution time will be described.

First, a component that is always detected in a standard sample and an unknown sample is designated as a specific component (hereinafter, may be referred to as an internal standard component). As the internal standard component, it is desirable to specify one that can be completely separated from other components and has a relatively high content (large output). Therefore, Comp_5, which elutes fifth, was used as the internal standard component.

Regarding the identification range of each component, the internal standard component is preferably set to a value larger than that of the other components. Specifically, it is desirable that the identification range (η%) of the internal standard is about 2 to 5 times the identification range (γ%) of other components. As a result, when measuring an unknown sample, even if the elution time fluctuates, the internal standard component can be identified more reliably. In FIGS. 3 and 1, the allowable identification range of the internal standard component (Comp_5) was ± 15% with respect to the reference elution time, and ± 3% for the other components.

The unknown sample 1 is measured, at least the elution time of each peak (Table 2) is detected, and the internal standard is compared with the identification table (Table 1), depending on whether or not it is at least within the allowable range of the internal standard component. Perform component identification.

Next, the elution time (8.670) of the internal standard component of the unknown sample 1 obtained above is divided by the reference elution time (8.500) of the internal standard component registered in the identification table, and the elution time is obtained. The correction coefficient (f) of is calculated. Here, the correction coefficient (f) is calculated as 8.670 / 8.500 = 1.020.

Next, the identification table is updated by the correction coefficient (f).
The identification table is updated as a new basic elution time by multiplying the reference elution time of the identification table (Table 1) by a correction coefficient of 1.020. The identification range is also recalculated accordingly, and the identification table is updated (Table 3).

Next, the elution times of Peak_1 to Peak_4 of the unknown sample 1 are compared with the updated identification table (Table 3), and peaks other than the internal standard component are identified depending on whether or not they are within the permissible range. As a result, it can be seen that Peak_1 corresponds to Comp_1, Peak_2 corresponds to Comp_2, Peak_3 corresponds to Comp_3, and Peak_1 corresponds to Comp_4.

When measuring the next unknown sample, first, the internal standard component (Comp_5) is identified based on the identification table (Table 3) updated above, the correction coefficient (f) is calculated, and the identification table is used. Update again to identify peaks other than internal standard components.

By using the identification method of this method, the identification table can be updated for all components even when the components registered in the identification table are not detected in the unknown sample as shown in FIG. , Even if a component not detected above is detected in the subsequent measurements, it is possible to reliably identify the component.

In the present invention, when two or more unknown samples are continuously identified by liquid chromatography, the components can be accurately identified even if the elution time fluctuates with time, and in particular, the components registered in the identification table. Does not affect a series of identifications, even if is undetected.

It is a figure which showed how the identification did not go well by the general method performed without changing the identification condition at all. It is a figure which showed how the identification did not go well by the method which involves the update of the identification condition. It is a figure which showed the appearance which does not affect the subsequent measurement and identification even when the component to be identified is not detected by the identification method of this invention. It is a figure which showed the liquid chromatogram system used in Example 1. FIG. It is a figure identified by the chromatogram in Example 1 and the identification method of this invention. The broken line in the figure indicates the identification range. It is a figure which was identified by the chromatogram in Example 1 and the conventional method which did not update the identification condition. The broken line in the figure indicates the identification range. It is a figure which showed the liquid chromatogram system used in Example 2. It is a figure identified by the chromatogram in Example 2 and the identification method of this invention. The broken line in the figure indicates the identification range. FIG. A is an enlarged view of the main component portion, and FIG. B is an overall view. It is the chromatogram in Example 2 and the figure which was identified by the conventional method which did not update the identification condition. The broken line in the figure indicates the identification range. FIG. A is an enlarged view of the main component portion, and FIG. B is an overall view.

The effect of the present invention was verified using an actual chromatogram. The present invention is not construed as being limited to the contents of the following examples.

(Example 1)
Verification was carried out in a non-suppressed ion chromatography system. The actual measurement was performed using the liquid chromatogram system shown in FIG. The system consists of a solvent degassing device (SD-8020) 2, a sample side liquid feed pump (DP-8020) 3, a reference side liquid feed pump (DP-8020) 10, a sample injection device (AS-8020) 4, and a column oven. It was composed of (CO-8020) 6, an electric conductivity meter (CM-8020) 12, and a data processing device (LC-8020II) 9 (all manufactured by Toso Co., Ltd.). The analytical column 5, using the Tosoh Corp. TSKgel IC-Anion-PW XL ( 4.6mmI.D. × 5cm), anions _{^{(NO 2 -, Br -,}} NO 3 -, PO 4 2- were separated _SO ^{4 2-).}
Other conditions are as follows.
Injection volume: 30 uL, column temperature: 40 ° C., sample side flow velocity: 0.600 mL / min, reference side flow velocity: 0.3 mL / min,
Eluent: Boric acid (360 mg), sodium tetraborate (575 mg), glycerin (5.0 g), potassium gluconate (350 mg), acetonitrile (40 mL), n-butanol (30 mL) in 1 L of pure water. Up In order to make the effect of the present invention easy to understand, verification was carried out by slightly changing the flow velocity and changing the number of components containing anions to be measured. The anion mixing ratio and flow velocity are shown in Tables 4 and 5, and the measurement combinations are shown in Table 6.

FIG. 5 is a chromatogram measured with the above combination.
It can be seen that as the flow velocity decreases (A_7 to B_1), the elution time of each ion becomes slower with time, and B_7 returns to the initial elution time. Symbols represented by the following in the chromatogram arrows (like SO ₄ ^2-component of D_4) which are representing a portion missing the ion component.

First, the standard sample (A_7) was identified and an identification table (initial value) was prepared (Table 7).

In this embodiment, the phosphoric acid ion (PO 4 ^_2-) as an internal standard components eluted in the fourth.
The tolerance of the internal standard component _(PO ^{4 2-)} and ± 12% of the reference elution time, other components _{^{(NO 2 -, Br -,}} NO 3 -, SO 4 2-) standard elution time to tolerance of Identification was performed as ± 4% of.

Table 8 shows the elution times of unknown samples (A_7 to C_6).

First, a result of measuring an unknown sample A_7, since peak eluting in 9.217 minutes within the identification range of phosphate ions in Table 7 _(PO ^{4 2-)} (8.116~10.197 min), phosphorus acid ion _(PO ^{4 2-)} to be identified. Next, the correction coefficient f of the identification condition is calculated. Divided by the phosphate ions identified in the _(PO ^{4 2-)} elution time (9.217 min) an internal standard and the phosphate ion _(PO ^{4 2-)} standard elution time of (9.223 min) The value is the correction coefficient f. In this case, 9.217 / 9.223 = 0.9993. The correction coefficient was multiplied by the reference elution time and allowable width of the basic identification table (Table 7) to update the identification range. The updated identification table was compared with the elution time of the unknown sample, and each peak was identified.

When the above-mentioned work is performed in the same manner for B_6 to C_6, the identification table is updated as shown in Table 9. As a result of correcting the identification table, it was found that identification can be performed reliably and accurately.

For comparison, FIG. 6 shows the results of identification by a method that does not update the identification table. The broken line in the figure indicates the identification range, but when the flow rate changes and the elution time fluctuates by this amount, it can be seen that peaks exceeding the identification range frequently occur, causing a problem.

Such a delay in elution time with time and returning to the original state at a certain timing is a phenomenon that is often seen when the eluent is readjusted, and in the identification method of the present invention, such a phenomenon is observed. Even in this case, it is not necessary to recreate the identification table from scratch, and the identification table can be used continuously.

(Example 2)
Verification was carried out using a glycohemoglobin analyzer system.
The actual measurement was performed using the liquid chromatogram system shown in FIG. The device used was an automatic glycohemoglobin analyzer HLC-723GVIII (manufactured by Tosoh Corporation), and the eluent used was the same eluent. The sample used was the Low level (A1c: about 5.4%) of the same dedicated HbA1c calibrator set.
In order to make the effect of the present invention easy to understand, the sample was measured by slightly changing the Flow Factor (relative flow velocity). Flow Factor is a parameter in which the absolute flow velocity increases as the value increases. The Flow Factor was verified in seven patterns in the order of 0.960, 0.980, 0.990, 1.000, 1.010, 1.020, 1.0400.
First, a standard sample (Flow Factor = 0.960) was identified, and an identification table (initial value) was prepared (Table 10).

In this example, the A0 peak contained most was used as the internal standard peak, and the permissible range of identification was ± 10% with respect to the reference elution time at the A0 peak and 5% at the other peaks.

The results of the elution time at each Flow Factor are shown in Table 11.

First, as a result of measurement under the condition of Flow Factor = 0.980, the peak eluted at 0.800 minutes was within the identification range of A0 in Table 10 (0.7350 to 0.8983 minutes), so that it was identified as A0. To. Next, the correction coefficient f of the identification condition is calculated. The correction coefficient f is obtained by dividing the elution time of A0 identified above (0.8000 minutes) by the reference elution time of A0 (0.8167 minutes) with the internal standard as the internal standard. In this case, 0.800 / 0.8167 = 0.9796. The correction coefficient was multiplied by the reference elution time and allowable width of the basic identification table (initial value) to update the identification range. The updated identification table was compared with the elution time of the unknown sample, and each peak was identified.

When the above-mentioned work is performed in the same manner, the identification table is updated as shown in Table 12. As a result of correcting the identification table, it was found that identification can be performed reliably and accurately.

FIG. 8 is a diagram showing the identification by the method of the present invention in which the identification table based on the elution time of the peak, which is an internal standard, is updated. As can be seen from FIG. 8, as the Flow Factor increases, the elution time of each peak increases.

Even in such a state, since the permissible width of the internal standard peak A0 is set larger than that of other components, the A0 peak can be reliably identified, and the identification table based on the obtained correction coefficient can be updated to obtain other components. All peaks have also been identified.

FIG. 9 shows the result of identification without updating the identification table. The identification range was ± 5% of the reference elution time. When the Flow Factor is 0.960 to 1.000, all peaks can be identified correctly. However, when the Flow Factor is 1.010, the peak of LA1C + is identified, and when the Flow Factor is 1.020 or more, all the peaks are in the identification range. It becomes impossible to identify because it exceeds.

1. 1. Eluent 2. Degassing device 3. Liquid feed pump 4. Sample injection valve 5. Analytical column 6. Column constant temperature bath 7. Electrical conductivity meter 8. Waste liquid 9. System control and data processing equipment 10. Eluent A
11. Eluent B
12. Eluent C
13. Washing solution / dissolved blood 14. Open / close valve 15. Visible detector 16. Line filter

Claims (3)

A method for continuously identifying two or more unknown samples by liquid chromatography.
For the first unknown sample, a specific component in the unknown sample was identified based on an identification table created based on the elution time of the standard sample.
Based on the identification result, the identification table is corrected.
Based on the corrected identification table, components other than the specific component are identified, and
For the nth unknown sample (n = an integer of 2 or more), the specific component was identified in the corrected identification table used in the (n-1) th case.
Based on the identification result, the corrected identification table used in the (n-1) case is further corrected by the same method as in the first case.
A method characterized by identifying components other than the specific component based on the corrected identification table. The identification method according to claim 1, wherein the permissible width of the specific component with respect to the reference elution time in the identification table is 2 to 5 times the permissible width of the components other than the specific component. The correction method of the identification table is to divide the elution time of the specific component by the reference elution time of the specific component registered in the identification table used for identification to calculate a coefficient, and to calculate the coefficient of each component in the identification table. The identification method according to claim 1 or 2, wherein the identification table is corrected by multiplying the reference elution time and the permissible width thereof by the coefficient.

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