JP2018179853A - Peak identification method unaffected by variation in elution time - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peak identification method, even when there is temporal variation in elution times, capable of accurately identifying components and especially, even when a component registered in an identification table is undetected, not affecting a series of identification.SOLUTION: The method of continuously identifying two or more unknown samples by liquid chromatography, includes: identifying a specific component in a first unknown sample on the basis of an identification table created on the basis of an elution time of a standard sample; correcting the identification table on the basis of the identification result; and identifying components other than the specific component on the basis of the identification result: and identifying a specific component of an nth (n=an integer equal to or more than 2) unknown sample on the basis of a corrected identification table having been used in the (n-1)th one; further correcting the corrected identification table having been used in the (n-1)th one by the same method as the first one, on the basis of the identification result; and identifying the components other than the specific component using the corrected identification table.SELECTED DRAWING: None

Description

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

  Chromatography is a method of separating and quantifying a sample containing a plurality of components on a column. In general, qualitative analysis is performed from the elution time of each component peak, and quantitative analysis is performed according to the output degree of the detector. In order to conduct qualitative analysis, a standard sample containing the target component is measured in advance, the elution time of each component is determined as the reference elution time, and an "identification table" is created with an identification range in which the tolerance range is set. I do.

  When identifying an unknown sample, compare the elution time 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 practice to identify the corresponding component.

  However, in actual chromatography, it is often seen that the composition of the eluent changes slightly with time, and the elution time also increases or decreases with time. In addition, the column oven and the liquid transfer pump that constitute the chromatograph also lead to fluctuations in the elution time. The temperature of the column oven may slightly fluctuate due to factors of the installation environment, or the amount of pump delivery may slightly fluctuate, which may cause the elution time to fluctuate.

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

  Even in such a case, the standard elution time of the identification table is replaced with the elution time of the identified component so that correct identification can be made, the tolerance width is recalculated accordingly, and the identification table is updated. There is also a proposal that this method is used to identify unknown samples, but when the components registered in the identification table can not be identified at the time of unknown sample measurement as shown in FIG. 2 or some components are not originally included However, there was a problem that the update of the identification table would not function properly.

  It is an object of the present invention to accurately identify components even if the elution time varies with time, and in particular, to affect a series of identification even when components registered in the identification table are not detected. Provides a peak identification method that does not exert

The present invention relates to a method for continuously identifying two or more unknown samples by liquid chromatography,
The first unknown sample identifies a specific component in the unknown sample based on an identification table created based on the elution time of the standard sample,
Correcting the identification table based on the identification result;
Identify components other than the specific component based on the corrected identification table,
The n-th (n = 2 or more integer) unknown sample identifies the specific component in the corrected identification table used in the (n-1) -th sample,
Based on the identification result, the corrected identification table used in the (n-1) -th case is further corrected in the same manner as 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 standard samples and unknown samples 1 to 3, and FIG. 3b is a diagram in which the elution time of each component (Comp_1 to Comp 6) of unknown samples 1 to 3 is plotted. The standard sample contains six components Comp_1 to Comp_6, and is also registered in the identification table. The unknown samples 2 and 3 similarly include six components corresponding to Comp_1 to Comp_6, but the unknown sample 1 includes only five components corresponding to Comp_1 to Comp_5.

  First, from the elution time of each component (Comp_1 to Comp_6) of the standard sample, as in the general identification method, the elution time serving as a reference for identification and its identification range are designated (creation of an identification table). The identification range may be specified in absolute time for each component, but setting can be performed more easily if it is specified in terms of the ratio to the standard elution time.

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

  First, a component which is necessarily detected in the standard sample and the unknown sample is designated as a specific component (hereinafter sometimes referred to as an internal standard component). The internal standard component was completely separated from the other components, and it is desirable to designate one having a relatively high content (large output), so the fifth eluting Comp_5 was used as the internal standard component.

  For the identification range of each component, the internal standard component is preferably set to a larger value than the other components. Specifically, the identification range (同 定%) of the internal standard is desirably about 2 to 5 times the identification range (γ%) of the other components. This makes it possible to more reliably identify the internal standard component even when the elution time varies during measurement of an unknown sample. In FIG. 3 and Table 1, the identification tolerance range of the internal standard component (Comp — 5) was ± 15% with respect to the standard elution time, and ± 3% for the other components.

  Measure the unknown sample 1, detect the elution time (Table 2) of at least each peak, and compare with the identification table (Table 1) to determine whether the internal standard at least falls within the tolerance range of the internal standard component. Conduct component identification.

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

Next, the identification table is updated with this correction coefficient (f).
The standard elution time of the identification table (Table 1) is multiplied by the correction factor 1.020 to update the identification table as a new basic elution time. In accordance with this, the identification range is recalculated 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 they fall within the allowable range. As a result, it is understood that Peak_1 corresponds to Comp_1, Peak_2 corresponds to Comp_2, Peak_3 corresponds to Comp_3, and Peak_4 corresponds to Comp_4.

  When the next unknown sample is to be measured, 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 calculated. Update again to identify peaks other than internal standard components.

  By using the identification method of this method, as shown in FIG. 3, even if the component registered in the identification table is not detected in the unknown sample, the identification table can be updated for all the components. Even in the subsequent measurement, even if a component not detected above is detected, identification can be performed reliably.

  In the present invention, when two or more unknown samples are continuously identified by liquid chromatography, accurate component identification is possible even if the elution time varies with time, and in particular, components registered in the identification table Even if not detected, it does not affect the series of identification.

It is the figure which showed a mode that identification did not go well by the general method performed without changing identification conditions at all. It is the figure which showed a mode that identification did not go well by the method with the update of identification conditions. Even when the component of identification object is not detected by the identification method of this invention, it is the figure which showed a mode that the subsequent measurement and identification are not shown influence. FIG. 2 shows a liquid chromatogram system used in Example 1. 1 is a chromatogram in Example 1 and a diagram identified by the identification method of the present invention. The broken line in the figure indicates the identification range. It is the chromatogram in Example 1, and the figure identified by the conventional method which does not update identification conditions. The broken line in the figure indicates the identification range. FIG. 6 is a view showing a liquid chromatogram system used in Example 2. FIG. 2 is a chromatogram in Example 2 and a diagram identified by the identification method of the present invention. The broken line in the figure indicates the identification range. FIG. A is an enlarged view of the main component, and FIG. B is an overall view. It is the chromatogram in Example 2, and the figure identified by the conventional method which does not update identification conditions. The broken line in the figure indicates the identification range. FIG. A is an enlarged view of the main component, 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 performed on a non-suppressed ion chromatography system. The actual measurement was performed using a liquid chromatogram system as shown in FIG. The system includes a solvent degassing apparatus (SD-8020) 2, a sample side liquid feed pump (DP-8020) 3, a reference side liquid feed pump (DP-8020) 10, a sample injection unit (AS-8020) 4, a column oven (CO-8020) 6, electric conductivity meter (CM-8020) 12, and data processor (LC-8020 II) 9 (all are manufactured by Tosoh Corp.). As analysis column 5, TSKgel IC-Anion-PW XL (4.6 mm ID × 5 cm) manufactured by Tosoh Corp. is used, and anions (NO ₂ ⁻ , Br ⁻ , NO ₃ ⁻ , PO ₄ ^{2 −} , SO ₄ ^2- ) were separated.
Other conditions are as follows.
Injection volume: 30 uL, column temperature: 40 ° C., sample side flow rate: 0.600 mL / min, reference side flow rate: 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 In addition, in order to make the effect of the present invention intelligible, the flow rate was slightly changed, and the number of components of the anion to be measured was changed to carry out the verification. The anion mixing ratio and the flow rate are as shown in Tables 4 and 5, and the combination of the measurements are as shown in Table 6.

FIG. 5 is a chromatogram measured by the above combination.
As the flow rate decreases (A_7 to B_1), it can be seen that the elution time of each ion becomes slower with time and returns from B_7 to the initial elution time again. In addition, the symbol shown by the down arrow in the chromatogram represents the location where the ion component is missing (SO ₄ ^2- component of D_4, etc.).

  First, identification of a standard sample (A_7) was performed, and an identification table (initial value) was created (Table 7).

In this example, the phosphate ion (PO ₄ ^2- ) eluted fourth was used as an internal standard component.
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

  The elution times of unknown samples (A_7 to C_6) are shown in Table 8.

First, as a result of measuring unknown sample A_7, the peak eluted at 9.217 minutes is within the identification range of phosphate ion (PO ₄ ^2- ) in Table 7 (8.116 to 10.197 minutes), so It is identified as acid ion (PO ₄ ^2- ). Next, the correction coefficient f of the identification condition is calculated. The elution time of the above-identified phosphate ion (PO ₄ ^2- ) (9.217 minutes) was divided by the standard elution time of the phosphate ion (PO ₄ ^2- ) (9.223 minutes). The value is the correction coefficient f. In this case, 9.217 / 9.223 = 0.9993. The reference elution time and tolerance range of the basic identification table (Table 7) were multiplied by the correction factor to update the identification range. The identification table after updating and the elution time of the unknown sample were collated to identify each peak.

  When the above-described work is similarly performed on 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 it is possible to perform identification reliably and accurately.

  The result of having identified by the method which does not update an identification table in FIG. 6 for a comparison is shown. The broken line in the figure indicates the identification range, but if the flow rate changes and the elution time fluctuates, it can be seen that peaks exceeding the identification range occur frequently, causing a failure.

  Such a time-lapse delay of the elution time occurs, and returning to the original state at a certain timing is a phenomenon often seen when the elution solution is readjusted etc. In the identification method of the present invention, such a phenomenon Even if this is the case, the identification table does not have to be recreated from 1 and can be used subsequently.

(Example 2)
Verification was performed on a glycated hemoglobin analyzer system.
The actual measurement was performed using a liquid chromatogram system as shown in FIG. The device used was an automatic glycohemoglobin analyzer HLC-723GVIII (manufactured by Tosoh Corp.), and the eluent used was the same dedicated eluent. The sample used was the Low level (A1c: about 5.4%) of the same dedicated HbA1c calibrator set.
In addition, in order to make the effect of this invention intelligible, the flow factor (relative flow velocity) was changed slightly, and the measurement of the sample was implemented. 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 and 1.0400.
First, identification of a standard sample (Flow Factor = 0.960) was performed, and an identification table (initial value) was created (Table 10).

In this example, the A0 peak contained most as an internal standard peak, and the allowable range of identification was ± 10% to the reference elution time for the A0 peak, and 5% for the other peaks.

  The results of elution time for 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 is in the identification range of A0 in Table 10 (0.7350 to 0.8983 minutes), so it is identified as A0. Ru. Next, the correction coefficient f of the identification condition is calculated. A value obtained by dividing the elution time of A0 identified above (0.8000 minutes) by the standard elution time of A0 (0.8167 minutes) with the internal standard as a correction coefficient is f. In this case, 0.800 / 0.8167 = 0.9796. The reference elution time and the tolerance range of the basic identification table (initial value) were multiplied by the correction factor to update the identification range. The identification table after updating and the elution time of the unknown sample were collated to identify each peak.

  If the above-described work is similarly performed, the identification table is updated as shown in Table 12. As a result of correcting the identification table, it was found that it is possible to perform identification reliably and accurately.

  FIG. 8 is a diagram showing the identification by the method of the present invention for updating the identification table based on the elution time of the peak serving as the internal standard. As can be seen from FIG. 8, the elution time of each peak is earlier as the Flow Factor is larger.

  Even in such a state, the allowable range of the internal standard peak A0 is set larger than other components, so that the A0 peak can be identified with certainty, and the identification table based on the obtained correction coefficient is updated to the other All peaks can also be identified.

  FIG. 9 shows the result of identification without updating the identification table. The identification range was ± 5% with respect to the standard elution time. With Flow Factor of 0.960 to 1.000, all peaks were correctly identified, but at Flow Factor of 1.010, the peak of LA1C + is identified, and at Flow Factor of 1.020 or more, all peaks are in the identification range Can not be identified because

1. Eluent 2. Degassing device Transfer pump 4. Sample injection valve 5. Analysis column 6. Column thermostat 7. Electrical conductivity meter 8. Waste liquid 9. System control and data processor 10. Eluent A
11. Eluent B
12. Eluent C
13. Washing solution / blood transfusion 14. Opening and closing valve 15. Visible detector 16. Line filter

Claims (3)

A method of continuously identifying two or more unknown samples by liquid chromatography, comprising:
The first unknown sample identifies a specific component in the unknown sample based on an identification table created based on the elution time of the standard sample,
Correcting the identification table based on the identification result;
Identify components other than the specific component based on the corrected identification table,
The n-th (n = 2 or more integer) unknown sample identifies the specific component in the corrected identification table used in the (n-1) -th sample,
Based on the identification result, the corrected identification table used in the (n-1) -th case is further corrected in the same manner as 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 tolerance width of the specific component in the identification table with respect to the standard elution time is 2 to 5 times the tolerance width of the component other than the specific component.   The correction method of the identification table divides the elution time of the specific component by the standard elution time of the specific component registered in the identification table used for identification to calculate a coefficient, and the coefficients of each component in the identification table are calculated. The identification method according to claim 1 or 2, wherein the identification table is corrected by multiplying the reference elution time and the tolerance range thereof by the coefficient.

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