JP2018080981A - Peak identification method based on peak area ratio - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peak identification method which is adaptable to changes of peak shape when analyzing a standard sample for creating a calibration curve when the density range is extremely wide.SOLUTION: The method includes the steps of, with respect to chromatogram of standard sample containing 2 or more quantitative object components: calculating the peak area ratio obtained by dividing a peak area by total sum of peak areas as standard peak area ratio for each quantitative object component; and with respect to a chromatogram of a sample in which the standard sample is diluted at a certain rate, identifying quantitative components of an object on the basis of the standard peak area ratio.SELECTED DRAWING: None

Description

  The present invention relates to a method for easily and reliably creating a calibration curve used for quantitative calculation in chromatography.

  When preparing a calibration curve, generally, a mixed standard sample stock solution containing a plurality of components for quantitative purposes is diluted with a diluent in multiple stages to prepare a standard sample solution having a plurality of concentrations. Next, a plurality of standard samples having different concentrations are subjected to a chromatograph, peaks corresponding to the respective components are identified, and output values (area, height, absorbance, etc.) are calculated. Finally, it is a general procedure to create an output value (area, height, absorbance, etc.), that is, a calibration curve with respect to the concentration for each component. Although such a series of calibration curve creation operations can be performed manually, in recent years, it is often created automatically by a data processing apparatus.

  The premise that this series of flows is accurately executed is that the elution time of each component is substantially constant even for standard samples having different concentrations. FIG. 1 is a diagram schematically showing the influence of time variation due to concentration. FIG. 1a shows a case where the elution time does not vary depending on the concentration, FIG. 1b shows a case where the elution time is delayed depending on the concentration, and FIG. 1c shows a case where the elution time is advanced depending on the concentration. In ideal chromatography, the elution time is constant without being affected by the concentration as shown in FIG. 1a. However, in reality, as shown in FIGS. 1b and 1c, as the concentration increases, the peak often causes a tailing or reading phenomenon to some extent.

  In this case, even if the components are the same, they do not fall within the identification time tolerance specified in the identification conditions, and cannot be identified, and values can be passed to the output values (area, height, absorbance) for the calibration curve. Disappear. In addition, when there is a peak derived from an impurity in the vicinity of the target component, the peak derived from the impurity is misidentified as the target component, and the output value (area, height, absorbance) for the calibration curve is incorrect. It will also be handed over. Although it is conceivable to widen the identification tolerance as a solution, the probability of being able to identify accurately to a certain degree increases, but the risk of misidentifying the peak derived from the impurity as the target component is also increased.

  The subject of this invention is providing the peak identification method which can respond to a peak shape change at the time of analyzing the standard sample for calibration curve preparation with a very wide density | concentration range.

  As described above, when preparing a standard curve for liquid chromatography, a standard sample mixed with multiple components is diluted in sequence, and multiple standard samples with different concentrations are prepared and subjected to chromatography. It will take a similar shape. Even in standard samples having different concentrations, the mixing ratio of a plurality of components contained therein is theoretically a constant value. Even if the peak shape changes, the peak area ratio becomes a substantially constant value within the quantitative range of the detector. Even if the standard sample contains unexpected impurities, the mixing ratio with respect to the whole is very small and does not have a significant effect on the target standard sample component ratio (area ratio). .

  Accordingly, the present inventor has found that the identification accuracy of each component can be improved by incorporating the above-described characteristics into at least a part of the identification method.

  That is, the present invention calculates the peak area ratio obtained by dividing the peak area by the sum of all peak areas, as the standard peak area ratio for each quantification target component, for the chromatogram of a standard sample containing two or more quantification target components. In the chromatogram of the sample obtained by diluting the standard sample at a certain ratio, the component to be quantified is identified based on the standard peak area ratio. The details will be described below.

  First, a chromatogram of a standard sample containing two or more quantitative target components serving as a reference is acquired, and a peak area ratio obtained by dividing the peak area by the sum of all peak areas is calculated. The chromatogram may be acquired only for this purpose, but if the peak area ratio is known in advance, the value may be used. This peak area ratio is defined as a standard peak area ratio.

Next, the component to be quantified is identified based on the standard peak area ratio in a chromatogram of a sample obtained by diluting the standard sample at a certain ratio. As a criterion for determining whether or not identification is possible, there is a method of simply determining by setting a threshold, a method of determining by comparing the area difference ratios for all components, or a method of determining by combining both, There is no particular limitation. For example, the area difference ratio is calculated as follows: Area difference ratio = absolute value (1-peak area ratio / standard peak area ratio)
However, any expression can be used as long as the difference in area can be determined. When the above formula is used, the closer the area difference ratio is to zero, the more similar items to be compared are.

  In order to explain the effect of the present invention in an easy-to-understand manner, it is performed based on pseudo-chromatograms using a plurality of normal functions having different concentrations. FIG. 2b is a chromatogram serving as a reference for identification, and FIG. 2a is a verification chromatogram having different concentrations. The broken line in the figure indicates a range of ± 5% with respect to the elution time of three peaks (AAAA, BBBB, CCCC) to be identified.

  FIG. 3 is an enlarged view of the verification chromatogram with different concentrations in FIG. 2a. In addition to the three peaks (# 2, # 4, # 6) to be identified, four impurity components (# 1, # 3, # 5, # 7) are intentionally added to each verification chromatogram. .

The flow of identification is as follows.
(Step 1)
The peak area of the target component for identification is obtained from the reference chromatogram, and the ratio of the peak area to the sum of all peak areas is calculated.
(Step 2)
For a sample diluted at a fixed ratio, peak detection is performed by a method generally used in chromatography, and at least the peak area is obtained, and the peak area ratio with respect to the sum of all peak areas is calculated. In order to make the following explanation easy to understand, the elution time of each peak is also described here.
(Step 3)
For the detected peak, an area difference ratio (calculated by the above-described method) with all peak area ratios of the identification conditions with respect to the obtained area ratio is calculated, and a component having the closest peak area ratio is specified. That is, the tabulated standard peak area ratio is compared with each peak area ratio, and the component with the closest value is identified as a specific quantitative target component.

  Verification chromatograms 1 and 4 were identified by the method described above. Identification conditions are shown in Table 1, and identification results of verification chromatograms 1 and 4 are shown in Tables 2 and 3, respectively.

一方、従来の溶出時間の許容幅により同定した。同定条件を表４、検証クロマトグラム１、４の同定結果をそれぞれ表５、６に示す。なお、溶出時間の許容幅は基準となる溶出時間の±２．０％とした。

On the other hand, it identified by the tolerance | permissible_range of the conventional elution time. Identification conditions are shown in Table 4, and identification results of verification chromatograms 1 and 4 are shown in Tables 5 and 6, respectively. The allowable range of elution time was ± 2.0% of the standard elution time.

このように、従来の溶出時間を基にした同定法では、溶出時間が大きく変化する場合は正確に同定することができない。これを回避するため、同定条件の溶出時間許容幅を広げる方策もあるが、許容幅を広げた場合、ピーク＃１、３、５、７のような目的外のピークを同定対象のピークと誤判定するリスクが高くなり、最善の方策ではない。

Thus, the identification method based on the conventional elution time cannot be accurately identified when the elution time changes greatly. In order to avoid this, there is a measure to widen the elution time tolerance of the identification conditions. However, when the tolerance is widened, peaks other than the target such as peaks # 1, 3, 5, and 7 are mistakenly identified as peaks to be identified. Judgment risk increases and is not the best policy.

  As described above, the identification method based on the area ratio of the present invention enables accurate identification even when the elution time varies depending on the concentration and the like, and the superiority of the present invention is clear. It is.

  In the present invention, in addition to the above identification by comparison of peak area ratios, the reference elution time of the standard sample may be further compared with the elution time of the diluted sample chromatogram. In the case of the verification chromatogram described above, the area ratios of the identification target components are greatly different from each other, so that the identification can be performed with high accuracy only by the area ratio. It becomes difficult to identify with high accuracy only by the ratio. Therefore, it is possible to easily perform identification by combining the allowable range of elution time, which is a conventional identification method, and the identification method based on the area ratio of the present invention.

  Specifically, the target component is first narrowed down by the elution time. At this time, it is preferable to set it to be larger than a general allowable range in consideration of variation in elution time depending on the sample concentration. In the case of identification based only on a general elution time, the allowable range is about ± 2 to 5%, but in the present invention, it is preferable to set a value of ± 5 to 20%. Thereby, the component which satisfy | fills conditions is narrowed down to multiple. Next, the identification method based on the area ratio described above is applied to the plurality of components narrowed down by the elution time, and final identification is performed.

  As described above, the present invention can identify the component to be quantified based on the standard peak area ratio for a chromatogram of a sample obtained by diluting a standard sample at a certain ratio. By applying the present invention to a sample group obtained by diluting a standard sample, it is possible to suitably create a calibration curve.

It is the figure which showed typically the change of the peak shape by a density | concentration change. FIG. 1a shows an ideal case, FIG. 1b shows a case where reading is caused by concentration, and FIG. 1c shows a case where tailing is caused by concentration. It is a pseudo-chromatogram. FIG. 2a shows two types of verification chromatograms for preparing calibration curves having different concentrations, and FIG. 2b shows a chromatogram serving as a reference for identification. FIG. 3 is an enlarged view of each verification chromatogram in FIG. 2. It is the figure which showed the system configuration used in the Example. 2 is a chromatogram for explaining Example 1. FIG. 2 is a verification chromatogram for preparing a calibration curve in Example 1. FIG. # In the figure indicates the peak number detected by executing peak detection by data processing. 2 is a chromatogram for explaining Example 2. FIG. 6 is an example of a verification chromatogram for creating a calibration curve in Example 2. FIG. # In the figure indicates the peak number detected by executing peak detection by data processing. The ← → range in the figure indicates the allowable range of the elution time of the identification target component.

  The effect of the present invention was verified using an actual chromatogram.

The actual measurement was performed using the liquid chromatogram system shown in FIG. The system includes a solvent degassing device (SD-8020) 2, a liquid feed pump (DP-8020) 3, a sample injection device (AS-8020) 4, a column oven (CO-8020) 6, an ultraviolet-visible detector (UV- 8020) 7 and a data processing device (LC-8020II) 9 (both manufactured by Tosoh Corporation). As the analytical column 5, TSKgel ODS-100Z (5 μm, 4.6 mm ID × 15 cm) manufactured by Tosoh Corporation was used, and p-hydroxybenzoic acids were separated. Other conditions are as follows.
Injection volume: 30 uL, column temperature: 40 ° C., flow rate: 1.0 mL / min
Eluent: CH ₃ CN / H 2 O Mixture sample: Methyl p-Hydroxybenzoate (2.50 ug / 1 mL) Ethyl p-Hydroxybenzoate (1.67 ug / 1 mL) 0.83 ug / 1 mL) Hexyl p-Hydroxybenzoate (1.33 ug / 1 mL) Heptyl p-Hydroxybenzoate (3.33 ug / 1 mL) Mixture The above mixture was designated as Standard Sample 1, a 1/2 diluted sample was Standard Sample 2, and 1 more The sample diluted 1/2 was designated as standard sample 3, the sample further diluted 1/2 was designated as standard sample 4, and the sample further diluted 1/2 was designated as standard sample 5. The eluent composition was measured by changing CH ₃ CN: 65.0%, 64.5%, 64.0%, 63.5%, 63.0% in order to intentionally change the elution time. .

Example 1
In order to clarify the effect of the present invention, as described below, the effect of the present invention was verified based on a chromatogram obtained by slightly changing the concentration of acetonitrile in the eluent for each standard sample concentration.

  The reference chromatogram and the chromatograms of standard samples 1 to 5 for preparing a calibration curve are shown in FIG.

  First, the standard chromatogram of FIG. 5 is obtained by a general peak detection method, peak areas are obtained for six components to be identified, the area ratio to the total area is calculated, and identification is performed as shown in Table 7. The basic identification conditions were determined. For simplicity, Peak_1 to Peak_6 were used in order of elution time.

表８〜１２に検量線作成用の標準試料１から５の計算結果および同定結果を示す。面積差比率は、基準クロマトグラムでの各成分の面積比率（標準ピーク面積比率）と、それぞれのピークの面積比率の差の割合を示している。
面積差比率＝絶対値（１−ピーク面積比率／標準ピーク面積比率）
つまり面積差比率が小さい成分が、基準のクロマトグラムの成分と類似性が高いと判断できる。ここでは、面積差比率が最も小さい成分を合致する成分と判定する。また、面積差比率が１．０に近いピークは、面積値が極端に小さいことを意味するため、面積差比率が０．５を超えるピークは同定対象でないと判断した（ｕｎｋｎｏｗｎ 表記）。

Tables 8 to 12 show the calculation results and identification results of standard samples 1 to 5 for preparing a calibration curve. The area difference ratio indicates the ratio of the area ratio (standard peak area ratio) of each component in the reference chromatogram and the area ratio of each peak.
Area difference ratio = absolute value (1-peak area ratio / standard peak area ratio)
That is, it can be determined that a component having a small area difference ratio is highly similar to a component of the reference chromatogram. Here, the component with the smallest area difference ratio is determined as the matching component. Moreover, since the peak whose area difference ratio is close to 1.0 means that the area value is extremely small, the peak whose area difference ratio exceeds 0.5 was determined not to be an identification target (unknown notation).

以上のように、本発明の同定方法では、クロマトグラムで溶出時間が大きく変動する場合であっても、何の障害もなく確実に同定することができる。

As described above, according to the identification method of the present invention, even when the elution time varies greatly in the chromatogram, it can be reliably identified without any obstacles.

(Example 2)
The chromatogram used as the reference for identification and the chromatograms of the standard samples 1 to 5 were the same as those used in Example 1, and an identification method combining the elution time and the area ratio was performed.

  FIG. 7 shows the reference chromatogram, the chromatograms of the standard samples 1 to 5, and the elution time tolerance of the peak to be identified. The broken line in the figure indicates the standard elution time of each peak, and ← → indicates the allowable elution time width.

  First, as shown in Table 13, identification conditions that are the basis of identification are created. An allowable range of elution time as a first criterion is set, and an area ratio to the total area as a second criterion is calculated. The tolerance of identification was defined as ± 14% with respect to the elution time of each peak of the reference chromatogram. As an example, in Peak_1, a peak that falls within the reference elution time range of 2.327 min ± 2.327 * 14/100 is provisionally identified as a candidate for “Peak_1”.

表１４〜１８に標準試料１から５の計算結果および同定結果を示す。なお、同定結果は溶出時間による第一の同定法で絞り込まれた成分から、面積比率による第二の同定法により決定した成分（最終同定結果）を記している。

Tables 14 to 18 show the calculation results and identification results of standard samples 1 to 5. In addition, the identification result has described the component (final identification result) determined by the 2nd identification method by area ratio from the component narrowed down by the 1st identification method by elution time.

以上のように、実施例１と同様の結果が得られた。

As described above, the same result as in Example 1 was obtained.

(Comparative example)
The identification method based on the elution time which is a conventional method was performed. The tolerance of identification was defined as ± 2% with respect to the elution time of each peak of the reference chromatogram. For example, in Peak_1, a peak that falls within the reference elution time range of 2.327 min ± 2.327 * 2/100 is identified as “Peak_1”. Details are as shown in Table 19.

表２０〜２４に標準試料１から５の同定結果を示す。

Tables 20 to 24 show the identification results of standard samples 1 to 5.

このように、検量線作成時において、溶出時間に大きな変動がある場合、溶出時間を基にした従来の同定方法では確実な同定ができないことが分かる。

As described above, when the elution time varies greatly during the calibration curve creation, it can be seen that the conventional identification method based on the elution time cannot be reliably identified.

1. 1. Eluent 2. Deaeration device Liquid feed pump (sample side)
4). 4. Sample injection valve Analysis column 6. 6. Column constant temperature bath UV-visible detector 8. Waste liquid9. System control and data processing equipment

Claims (4)

  For a chromatogram of a standard sample containing two or more quantification target components, the peak area ratio obtained by dividing the peak area by the sum of all peak areas is calculated as the standard peak area ratio for each quantification target component, and the standard sample is A method for identifying a component to be quantified based on the standard peak area ratio in a chromatogram of a sample diluted at a constant ratio.   The standard peak area ratio tabulated is compared with each peak area ratio of a chromatogram of a diluted sample, and the component having the closest value is identified as the specific component to be quantified. The method described.   The method according to claim 1, further comprising comparing a reference elution time of the standard sample with an elution time of a chromatogram of a diluted sample.   A method for preparing a calibration curve by identifying a component to be quantified by a method according to any one of claims 1 to 3 in a sample group in which the standard sample is diluted so as to have a plurality of concentrations.

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