JP6862780B2 - Peak identification method based on peak area ratio - Google Patents

Description

The present invention relates to a method for easily and surely producing 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 quantification purposes is diluted with a diluent in multiple steps 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 to identify peaks corresponding to each component, and output values (area, height, absorbance, etc.) are calculated. Finally, the general procedure is to create an output value (area, height, absorbance, etc.) for each component, that is, a calibration curve. Such a series of operations for creating a calibration curve can be performed manually, but in recent years, it is often created automatically by a data processing device.

The premise that this series of steps is carried out accurately is that the elution time of each component is almost constant even for standard samples having different concentrations. FIG. 1 is a diagram schematically showing the effect of time variation due to concentration. FIG. 1a shows a case where the elution time does not fluctuate 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 shortened depending on the concentration. In ideal chromatography, the elution time is constant, unaffected by 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 a greater or lesser extent.

In such a case, even if the components are the same, they do not fall within the identification time allowable range specified in the identification conditions, identification becomes impossible, and the values can be passed to the output values (area, height, absorbance) for the calibration curve. It disappears. In addition, when a peak derived from an impurity exists in the vicinity of the target component, the peak derived from the impurity is erroneously identified as the target component, and the output value (area, height, absorbance) for the calibration curve is erroneously set. It will also be handed over. As a solution, it is possible to widen the permissible range of identification, but the probability of being able to identify accurately to a certain extent increases, but at the same time, the risk of misidentifying the peak derived from impurities as the target component also increases.

An object of the present invention is to provide a peak identification method capable of responding to a change in peak shape when a standard sample for preparing a calibration curve having a very wide concentration range is analyzed.

As described above, when preparing a calibration curve for general liquid chromatography, a standard sample in which a plurality of components are mixed is sequentially diluted, a plurality of standard samples having different concentrations are prepared, and these are subjected to chromatography. It will take a similar shape. Even in standard samples with different concentrations, the mixing ratio of the plurality of components contained therein is theoretically a constant value. Even if the peak shape changes, the peak area ratio remains almost constant as long as it is within the quantification range of the detector. Even if the standard sample contains unexpected impurities, the mixing ratio to the whole is very small and does not significantly affect the abundance ratio (area ratio) of the target standard sample component. ..

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

That is, in the present invention, for a chromatogram of a standard sample containing two or more components to be quantified, 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 component to be quantified. Then, the chromatogram of the sample obtained by diluting the standard sample at a constant ratio is characterized in that 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 reference components to be quantified is obtained, and the peak area ratio obtained by dividing the peak area by the sum of all peak areas is calculated. The chromatogram may be obtained only for this purpose, but if the peak area ratio is known in advance, that value may be used. This peak area ratio is defined as the standard peak area ratio.

Next, with respect to the chromatogram of the sample obtained by diluting the standard sample at a constant ratio, the component to be quantified is identified based on the standard peak area ratio. Criteria for determining whether or not identification is possible include a method of simply setting a threshold value, a method of comparing the area difference ratios for all components, and a method of determining by combining both. It is not particularly limited. For example, as a calculation method of the area difference ratio, the area difference ratio = absolute value (1-peak area ratio / standard peak area ratio).
However, any formula may be used as long as the difference in area can be discriminated. When the above formula is used, the closer the area difference ratio is to zero, the more similar the items to be compared are.

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

FIG. 3 is an enlarged view of verification chromatographs having different concentrations in FIG. 2a. In addition to the three peaks to be identified (# 2, # 4, # 6), 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 peak area ratio to the total peak area is calculated.
(Step 2)
Peak detection is performed on a sample diluted at a constant ratio by a method generally used in chromatography, the minimum peak area is obtained, and the peak area ratio to the total sum of all peak areas is calculated. In order to make the following explanation easier to understand, the elution time of each peak is also described here.
(Step 3)
For the detected peak, the area difference ratio (calculated by the above method) with all the peak area ratios of the identification conditions with respect to the obtained area ratio is calculated, and the 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 having the closest value is identified as a specific quantification target component.

Verification chromatograms 1 and 4 were identified by the methods described above. The identification conditions are shown in Table 1, and the identification results of the 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 was identified by the conventional allowable range of elution time. The identification conditions are shown in Table 4, and the identification results of the 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。

As described above, the conventional identification method based on the elution time cannot accurately identify the case where the elution time changes significantly. In order to avoid this, there is a measure to widen the elution time permissible range of the identification condition, but when the permissible range is widened, unintended peaks such as peaks # 1, 3, 5, and 7 are mistaken for the peaks to be identified. The risk of judgment is high and it 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 fluctuates depending on the concentration or the like, and the superiority of the present invention is clear. Is.

Further, in the present invention, in addition to the above-mentioned identification by comparison of the peak area ratio, the reference elution time of the standard sample and the elution time of the chromatogram of the diluted sample may be further compared. In the case of the above-mentioned verification chromatograph, since the area ratios of the components to be identified are greatly different from each other, identification can be performed accurately only by the area ratios, but if the area ratios of the components to be identified are close, the area It is difficult to identify accurately only by the ratio. Therefore, by combining the permissible range of elution time, which is a conventional identification method, with the identification method based on the area ratio of the present invention, identification can be easily performed.

Specifically, first, the target components are narrowed down according to the elution time. At this time, it is preferable to set the elution time larger than the general allowable range in consideration of the fluctuation of the elution time depending on the sample concentration. In the case of identification based only on a general elution time, the permissible range is about ± 2 to 5%, but in the present invention, it is preferable to set a value of ± 5 to 20%. As a result, the components that satisfy the conditions are narrowed down to a plurality. 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, in the present invention, it is possible to identify the component to be quantified based on the standard peak area ratio in the chromatogram of the sample obtained by diluting the standard sample at a constant ratio. By applying the present invention to a sample group obtained by diluting a standard sample, it is possible to suitably prepare a calibration curve.

It is the figure which showed the change of the peak shape by the density change typically. FIG. 1a shows the ideal case, FIG. 1b shows the case where the reading is caused by the concentration, and FIG. 1c shows the case where the tailing is caused by the 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 as a reference for identification. It is an enlarged view of each of the verification chromatograms of FIG. It is a figure which showed the system configuration used in an Example. It is a chromatogram for explaining Example 1. 6 is a verification chromatogram for preparing a calibration curve in Example 1. The # in the figure indicates the number of the detected peak when peak detection is executed by data processing. It is a chromatogram for explaining Example 2. This is an example of a verification chromatogram for preparing a calibration curve in Example 2. The # in the figure indicates the number of the detected peak when peak detection is executed by data processing. The ← → range in the figure indicates the permissible range of 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 consists of 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, and an ultraviolet-visible detector (UV-). It was composed of 8020) 7 and a data processing device (LC-8020II) 9 (both manufactured by Toso Co., Ltd.). As the analysis column 5, TSKgel ODS-100Z (5 μm, 4.6 mm ID × 15 cm) manufactured by Tosoh Corporation was used to separate p-hydroxybenzoic acids. Other conditions are as follows.
Injection volume: 30 uL, column temperature: 40 ° C., flow rate: 1.0 mL / min
Eluent: CH ₃ CN / H2O Mixture Sample: Methyl p-Hydroxybendoate (2.50 ug / 1 mL) Ethyl p-Hydroxybenzoate (1.67 ug / 1 mL) Propyl p-Hydroxybendozate (0.33ug / 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 used as standard sample 1, and the 1/2 diluted sample was used as standard sample 2, and further 1 The / 2-diluted sample was designated as standard sample 3, the further 1/2-diluted sample was designated as standard sample 4, and the further 1/2-diluted sample 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, the effect of the present invention was verified based on the chromatogram obtained by slightly changing the concentration of acetonitrile in the eluent for each concentration of the standard sample as described below.

FIG. 5 shows a reference chromatogram and a chromatogram of standard samples 1 to 5 for preparing a calibration curve.

First, the reference chromatogram of FIG. 5 is identified by a general peak detection method to obtain the peak area for the 6 components to be identified, calculate the area ratio to the total area, and identify as shown in Table 7. The basic identification conditions for For simplicity, Peek_1 to Peek_6 were used in ascending 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 the standard samples 1 to 5 for preparing the calibration curve. The area difference ratio indicates the ratio of the difference between the area ratio of each component (standard peak area ratio) 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 judged that the component having a small area difference ratio has high similarity to the component of the reference chromatogram. Here, the component having the smallest area difference ratio is determined to be a matching component. Further, since a peak having an area difference ratio close to 1.0 means that the area value is extremely small, it was determined that a peak having an area difference ratio exceeding 0.5 is not 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, in the identification method of the present invention, even when the dissolution time fluctuates greatly in the chromatogram, it can be reliably identified without any trouble.

(Example 2)
As the chromatograms used as the criteria for identification and the chromatograms of the standard samples 1 to 5, the ones used in Example 1 were used, and the identification method was performed by combining the elution time and the area ratio.

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

First, as shown in Table 13, identification conditions that are the basis of identification are created. The permissible range of elution time, which is the first criterion, is set, and the area ratio to the total area, which is the second criterion, is calculated. The allowable range of identification was defined as ± 14% of the elution time of each peak of the reference chromatogram. As an example, in Peak_1, a peak within the range of the reference dissolution time of 2.327 min ± 2.327 * 14/100 is tentatively identified as a candidate for “Peek_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 the standard samples 1 to 5. As for the identification results, the components determined by the second identification method based on the area ratio (final identification result) are described from the components narrowed down by the first identification method based on the 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 results as in Example 1 were obtained.

(Comparison example)
An identification method based on the elution time, which is a conventional method, was performed. The allowable range of identification was defined as ± 2% with respect to the elution time of each peak of the reference chromatogram. As an example, in Peak_1, a peak within the range of the reference elution time 2.327 min ± 2.327 * 2/100 is identified as “Peek_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 fluctuates greatly at the time of preparing the calibration curve, it can be seen that reliable identification cannot be performed by the conventional identification method based on the elution time.

1. 1. Eluent 2. Degassing device 3. Liquid feed pump (sample side)
4. Sample injection valve 5. Analytical column 6. Column constant temperature bath 7. Ultraviolet-visible detector 8. Waste liquid 9. System control and data processing equipment

Claims (4)

For a chromatogram of a standard sample containing two or more components to be quantified, the peak area ratio obtained by dividing the peak area by the sum of all peak areas is defined as the standard peak area ratio for each component to be quantified, and the concentration of the component to be quantified A method characterized by identifying the component to be quantified based on the standard peak area ratio of a chromatogram of a sample obtained by calculating without being based on a ratio and diluting the standard sample at a constant ratio. The tableized standard peak area ratio is compared with each peak area ratio of the chromatogram of the sample obtained by diluting the standard sample, and the component having the closest value is identified as the specific component to be quantified. The method according to claim 1. The method according to claim 1 or 2, further comprising comparing the reference elution time of the standard sample with the elution time of a chromatogram of a sample obtained by diluting the standard sample. How the sample group obtained by diluting the standard samples so that the multiple levels, to identify the quantified ingredients in the process according to any of claims 1 to 3, to create a multi-point calibration curve of the quantitative target component.

Images