Liquid chromatograph device and quantitative analysis device

Abstract

In order to improve analysis accuracy while reducing analysis labor, a liquid chromatograph device 100 for quantifying a prescribed substance to be measured comprises a control unit 9 that, on the basis of RMS coefficients that are respective ratios of response ratios Rr of the substance to be measured and a plurality of prescribed standard substances different from the substance to be measured to substance quantity ratios Rn of the substance to be measured and the standard substances, substance quantities of the standard substances, and detection response ratios based on detection results for the substance to be measured and the standard substances, quantifies the substance to be measured with respect to each of the standard substances and averages results of quantification of the substance to be measured with respect to each of the standard substances, thereby determining final quantification results.

Description

Liquid chromatograph and quantitative analyzer

[0001] The present invention relates to a chromatographic apparatus and a quantitative analysis apparatus for quantifying a target substance contained in an unknown sample.

[0002] A technique is known in which a first internal standard and a second internal standard are used in a chromatograph, each of which is quantified by the internal standard method based on the respective calibration curves for the analyte, and the respective quantified values ​​are finally averaged (see, for example, Patent Document 1).

[0003] Japanese Patent Application Laid-Open No. 2020-51827

[0004] In methods based on internal standards, a standard substance containing both the analyte and the internal standard must be injected when creating a calibration curve. Furthermore, analyzing unknown samples also requires the time-consuming task of adding an internal standard to each sample.

[0005] The present invention has been made in view of the above points, and aims to enable improvement in analytical accuracy while reducing the time and effort required for analysis.

[0006] In order to achieve the above object, the present invention provides a liquid chromatograph apparatus for quantifying a predetermined target substance, characterized in that it has a control unit that quantifies the target substance corresponding to each standard substance based on each RMS coefficient, which is the ratio of the response ratio Rr between the target substance and each standard substance to the substance amount ratio Rn between the target substance and a plurality of predetermined standard substances different from the target substance, the substance amount of each standard substance, and each detection response ratio based on the detection results of the target substance and each standard substance, and that averages the quantification results of the target substance corresponding to each standard substance to determine a final quantification result.

[0007] As a result, when applying the RMS coefficient, the amount of work required can be easily reduced compared to when a calibration curve is calculated, and multiple types of standard substances can be easily applied. Therefore, the amount of work required for analysis can be reduced while the accuracy of analysis can be easily improved.

[0008] The present invention makes it possible to improve analytical accuracy while reducing the amount of work required for analysis.

[0009] FIG. 1 is a block diagram showing a schematic configuration of a liquid chromatograph.

[0010] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings.

[0011] (General configuration of liquid chromatograph) The liquid chromatograph 100 quantifies a predetermined measurement target, and as shown in FIG. 1 , is equipped with a data processing device 7 for overall control, a mobile phase (eluent or a mixed solution with a solvent, etc.) 1, a pump 2 for delivering the mobile phase 1, an autosampler 3 for injecting the sample, a column 4 for separating components, a column oven 5 for maintaining a constant temperature for the column 4, a detector 6 for detecting the separated components, and an operation display unit 10.

[0012] The data processing device 7 is composed of a computer having a control unit 9 (CPU or the like) that executes analysis and analyzes the analysis results, and performs calculations related to the RMS coefficients described below and averaging of quantitative values, and a storage unit 8 (hard disk or the like) that stores the analysis results, calibration curve information described below, and conversion information (RMS coefficients). The operation and display unit 10 accepts various operation inputs and displays the analysis results.

[0013] The detector 6 is an absorptiometry detector, a fluorescence detector, a mass detector, or a three-dimensional photodiode array detector that has multiple elements for detecting signal intensity and can simultaneously obtain signal intensity over time at multiple wavelengths.

[0014] A sample is injected from an injector (not shown) of an autosampler 3, passes through a column 4 together with a mobile phase 1 delivered by a pump 2, and is separated into various components in the sample.

[0015] The sample separated into its components is detected by a detector 6. The signal from the detector 6 is sent to a data processing device 7 where data processing is performed.

[0016] The column 4 is a device generally used as a separation section for separating components of a sample present in the mobile phase 1. Examples of the column 4 include a packed column and a monolith column. Various types of column packing materials can be used for the column 4, such as adsorption type, partition type, and ion exchange type. The column 4 is preferably installed in a column oven 5 so that the column 4 can be kept at a constant temperature and sample separation can be performed with good reproducibility.

[0017] (Quantitative Method) First, quantitative determination using the RMS coefficient will be described.

[0018] In the liquid chromatograph apparatus 100, for example, quantification can be performed using the general absolute calibration curve method and the internal standard method. In addition, as shown in the following (Table 1) (Equation 1), quantification can also be performed using the RMS coefficient (RMS: Relative Molar Sensitivity), which is defined as the ratio of the response ratio Rr between the target substance and the standard substance to the substance amount ratio Rn between the target substance and the standard substance (which may be a mass ratio, volume ratio, concentration ratio, or the like, in addition to a substance amount ratio in the strict sense).

[0019]

[0020]

[0021] Here, the subscript "anal" represents the analyte and "ref" represents the reference. Furthermore, for the analyte and reference, A represents the peak area or peak height as a response amount, and n represents the amount of substance (e.g., moles). As shown in Equation 1, for example, a sample solution containing the analyte and the reference, each with a known amount of substance, is injected into an HPLC, and the resulting peak areas are input as the respective response amounts, allowing the RMS coefficient to be calculated.

[0022] (Quantification using external standard substance using RMS coefficient) For example, if the response amount (Aref) and substance amount (nref) are obtained by measuring glycine as the standard substance (external standard substance) shown in (a) in (Table 1) once every morning, for example, for alanine (RMS coefficient = 1.74), glutamic acid (RMS coefficient = 1.87), and aspartic acid (RMS coefficient = 2.02) as the measurement target substances, if the response amounts (Aanal) for these substances are measured, the respective substance amounts (nanal) can be calculated as shown in the following (Equation 2). Therefore, if the external standard substance shown in (a) in (Table 1) is measured, the measurement target substance can be quantified without measuring the measurement target substance shown in (b).

[0023]

[0024] (Quantification using an internal standard using RMS coefficients) When an internal standard is used, by measuring an unknown sample containing a known amount of a standard (internal standard) and measuring the response amount (Aref) of the standard as well as the response amount (Aanal) of the substance to be measured, the substance amount (nanal) of the substance to be measured can also be determined as in the above (Equation 1). Therefore, the substance to be measured can be quantified without measuring the substance to be measured or the standard for calibrating the day-to-day variation as shown in (c) and (d) of (Table 1).

[0025] As described above, the RMS method is a quantitative analysis method that uses the RMS coefficient. Because the known RMS coefficient can be considered a constant, by inputting the measured response ratio Rr into (Equation 1), the substance amount ratio Rn is output. Because the substance amount nref of the standard substance is known, the substance amount nana of the substance to be measured can be determined. The RMS method is not a relative method for convenience, but is positioned as a reliable quantitative analysis method that uses the substance amount as a reference.

[0026] The amount of substance (mol) of the quantitative value can be converted to mass (g) using the molecular weight. Furthermore, if the sample injection volume, such as 10 μL, is input as a volume, it can also be converted to a concentration, such as mol / L or g / L. More specifically, for example, the memory unit 8 may store a conversion coefficient for converting the quantitative result into a predetermined concentration unit, and the operation and display unit 10 may accept the specification of the concentration unit and convert the quantitative result into the specified concentration unit for output.

[0027] (Quantification using multiple reference materials) By performing the above-described RMS quantification using two or more types of reference materials (standard materials), it is possible to ensure a higher level of mathematically and statistically reliable quantitative value data. That is, even if the variability and bias of the RMS coefficients affect the reliability of the quantitative value data, the use of two or more types of reference materials can improve the mathematically and statistically reliable data, and can demonstrate the robustness of the analysis from the viewpoint of quality control.

[0028] For example, two or more types of reference substances refA, refB, refC, etc. are quantitatively calculated in the same manner as above. Then, the amounts of the two or more types of substances (e.g., molar concentrations ManrlA, ManrlB, ManrlC) obtained are averaged, thereby improving the reliability of the quantitative values.

[0029] Here, the quantitative value to be averaged may be the final quantitative result obtained by averaging the results of quantification by the absolute calibration curve method and / or the internal standard method and the results of quantification of the substance to be measured corresponding to each standard substance using the RMS coefficient.

[0030] Furthermore, although the molar concentrations ManrlA, ManrlB, ManrlC, etc. are theoretically consistent, for example, if their ratio (e.g., ratio to the average value) is not within the range of 100±10%, the reliability of the data is deemed to be significantly low, and an error or alert may be issued, or the peak may be excluded from the calculation of the average value. Similarly, an alert may be issued for insufficiently separated peaks, or the peak may be excluded from the calculation of the average value. Furthermore, statistical indicators such as the relative standard deviation (RSD) may be calculated and the above-mentioned processing may be performed.

[0031] Furthermore, isocratic elution or gradient elution may be used in measurements to determine the peak area or peak height as the response amount A of the analyte (anal) and the reference (ref). That is, the RMS quantification method may be applied to peak area ratios not only between peaks eluted by isocratic elution but also between peaks eluted by gradient elution. This is based on the idea that peak areas are largely preserved even with minute fluctuations in gradient elution. Gradient elution here also includes stepwise elution. Furthermore, the area ratio between a peak eluted by gradient elution and a peak eluted by isocratic elution may also be used.

[0032] Furthermore, the detection wavelengths do not necessarily need to be the same when calculating the peak area ratio. Note that, by using responses at multiple detection wavelengths for one standard substance, similar averaging processing can be performed even when there is only one standard substance.

[0033] (Example of an Amino Acid Analysis Application) Quantification of multiple target substances may be performed using a set of multiple predetermined standard substances as described above. Specifically, for example, 17 components, such as Asp (aspartic acid), Thr (threonine), Ser (serine), etc., of the protein hydrolysate PH method can be specified as the standard substances, and approximately 20 components, such as Tau (taurine), Orn (ornithine), GABA (γ-aminobutyric acid), etc., specific to the biofluid analysis PF method can be assigned as the analyte. Furthermore, difficult-to-obtain components (such as homocysteine-cysteine ​​disulfide, argininosuccinic acid, 2-aminoadipic acid, and aminoethylcysteine) can also be used as target substances. Since each component of the PH method is quantified using the absolute calibration curve method, the absolute calibration curve method and a method using multiple standard substances as described above may be used in combination.

[0034] As described above, when RMS coefficients are applied, the amount of work required can be reduced compared to when a calibration curve is calculated, and multiple types of standard substances can be easily applied. Therefore, the amount of work required for analysis can be reduced while the analytical accuracy can be easily improved.

[0035] Various application examples of the combined method and other methods are explained in detail below. In the combined PF method, only 17 components are injected as external standard substances. There is no need to prepare approximately 20 other standard substances, such as Tau. Conventionally, it was necessary to prepare these approximately 20 components, but the advantage of this combined PF method is that this is no longer necessary.

[0036] Typically, for components injected with an external standard such as Asp, a calibration curve can be obtained for that component itself, so the absolute calibration curve method (and / or internal standard method) is used. On the other hand, for components such as Tau that do not have a standard injected, the RMS method can be used. For example, using one component such as Asp as an external standard, the quantitative value of Tau is calculated using the RMS coefficient with Tau. This is the basic combined method of the PF method (Table 2 (1)).

[0037] Here, the RMS quantification method using multiple standards (multiple standard quantification method) can be applied. That is, in order to quantify Tau and the like by the RMS method together with the quantification of Asp by the absolute calibration curve method or the like, a method can be adopted that uses not only one component, Asp, but also multiple external standards, such as Thr and Ser, that were actually injected. Since Tau has its own RMS coefficient with Thr, Ser, etc., the RMS method can calculate the quantitative values ​​of Tau for multiple external standards. Finally, these quantitative values ​​can be averaged to obtain the quantitative value of Tau. In this way, the multiple standard quantification method can be used for components for which no standard is prepared. Furthermore, this method can be called a combined method involving the multiple standard quantification method (Table 2 (2)). It should be noted that the effect of improving accuracy can be obtained not only when Asp is quantified by the absolute calibration curve method or the like, but also when only Tau and the like are averaged using the RMS method with multiple external standards, such as Asp, Thr, and Ser (Table 2 (3)). Furthermore, multiple substances to be measured, such as other substances to be measured, such as Orn, may be quantified in the same manner as Tau (Table 2(4)).

[0038] As mentioned above, it would be natural to think that for components injected with external standards, such as Asp, the absolute calibration curve method based on the calibration curve of the component itself would be sufficient. However, if the accuracy of the quantitative value needs to be improved, the multiple standard quantification method can be applied even to components injected with external standards. That is, since Asp also has its own RMS coefficient with Thr and Ser, for which external standards other than Asp are prepared, multiple RMS quantitative values ​​for Asp can be calculated. These averages can be used as the overall RMS quantitative value. For example, an RMS quantitative method compares the ratio of the peak area of ​​Thr as the external standard to the peak area of ​​Asp as an unknown component with the RMS coefficient. In this case, it is unusual that the peak area of ​​Asp as the external standard is not referenced. While the desired accuracy may vary from case to case, a method of averaging the quantitative value results of the Asp absolute calibration curve method and the overall RMS quantitative value of Asp with some weighting, as necessary, may be employed. This method is simply called the fusion method, meaning that it is a method that combines the absolute calibration curve method or the like with the multiple standard substance quantification method (Table 2(5)). The above is an explanation of the combined method, the multiple standard substance quantification method, the combined method with the multiple standard substance quantification method, and the fusion method. Note that the number of RMS method quantification values ​​to be averaged with the quantitative value results of the Asp absolute calibration curve method or the like is not limited to multiple values; for example, one RMS method quantification value for Thr may be averaged with the quantitative value results of the Asp absolute calibration curve method or the like (Table 2(6)).

[0039] In addition, we will add a note about error processing. Whether using the multi-standard substance quantification method or the fusion method, the quantitative values ​​of the elements before calculating the average value should be approximately equal. Therefore, outlier processing can be done based on this idea. Outliers can be simply detected, or statistical tests such as the Smirnoff-Grubbs test or the Thompson test can be used. If an outlier is detected, the user is notified and the outlier is removed and the average processing is performed again. The calculation result with the outlier removed is output as the quantitative value from the multi-standard substance quantification method or the fusion method. This can also be called the outlier-excluded quantitative value.

[0040] It should be noted that the standard substance (reference substance) used in the RMS quantification method in the above-mentioned combination method or fusion method may be one or more types. For example, in the above-mentioned fusion method, the quantitative value of Asp determined by the absolute calibration curve method and the quantitative value of Asp determined by the RMS quantification method using Thr as the standard substance (reference substance) can be averaged with some weighting as necessary. In other words, the quantitative value determined by a method other than the RMS quantification method, such as the absolute calibration curve method, can be compared (averaged, etc.) with the quantitative value determined by the RMS quantification method, and in this case, the RMS coefficient used in the RMS quantification method may be one or more. Even in this case, it is possible to reduce the analytical effort while improving the analytical accuracy.

[0041]

[0042] (Further Quantification) While the above description has been given using a liquid chromatograph as an example, the same technique can be applied to various quantitative analysis devices. That is, as long as it is possible to calculate the ratio (RMS coefficient) of the response ratio Rr between a target substance and each of a plurality of predetermined standard substances different from the target substance to the respective substance amount ratio Rn, the same calculation and averaging technique can be applied. Specifically, for example, the same calculation and averaging technique can be applied to the measurement results of analytical devices capable of quantifying substances, such as mass spectrometers and spectrophotometers, in addition to chromatographs.

[0043] As described above, the present invention is useful for a chromatographic device that quantifies a target substance contained in an unknown sample.

[0044] REFERENCE SIGNS LIST 1 Mobile phase 2 Pump 3 Autosampler 4 Column 5 Column oven 6 Detector 7 Data processing device 8 Memory unit 9 Control unit 10 Operation display unit 100 Liquid chromatograph

Claims

1. A liquid chromatograph for quantifying a predetermined target substance, characterized in that it has a control unit that quantifies the target substance corresponding to each standard substance based on each RMS coefficient, which is the ratio of the response ratio Rr between the target substance and each standard substance to the substance amount ratio Rn between the target substance and each of a plurality of predetermined standard substances different from the target substance, the substance amount of each standard substance, and the respective detection response ratios based on the detection results of the target substance and each standard substance, and that averages the quantification results of the target substance corresponding to each standard substance to determine a final quantification result.

2. The liquid chromatograph of claim 1, wherein a plurality of types of target substances are quantified for a set of a plurality of predetermined standard substances.

3. A liquid chromatograph according to claim 1, wherein the control unit further calculates a final quantification result by averaging the results of quantification using the absolute calibration curve method and / or the internal standard method and the results of quantification of the substance to be measured corresponding to each standard substance using the RMS coefficient.

4. A liquid chromatograph apparatus for quantifying a first analyte and a second analyte, comprising: a control unit for quantifying the first analyte by an absolute calibration curve method; and determining the second analyte corresponding to the standard substance, using the first analyte as a standard substance, based on an RMS coefficient, which is the ratio of the response ratio Rr of the second analyte and the standard substance to the substance amount ratio Rn of the second analyte and the standard substance, the substance amount of the standard substance, and a detection response ratio based on the detection results of the second analyte and the standard substance.

5. A liquid chromatograph according to claim 4, wherein there are a plurality of types of standard substances, the second substance to be measured is quantified for each standard substance, and the results of the quantification of the second substance to be measured for each standard substance are averaged to obtain a final quantification result.

6. A liquid chromatograph for quantifying a predetermined target substance, comprising: a liquid chromatograph that quantifies the target substance by an absolute calibration curve method; and quantifies the target substance corresponding to the standard substance based on an RMS coefficient, which is the ratio of the response ratio Rr of the target substance to the standard substance to the substance amount ratio Rn of the target substance to a predetermined standard substance different from the target substance, the substance amount of the standard substance, and a detection response ratio based on the detection results of the target substance and the standard substance; and a control unit that averages the results of the quantification of the target substance by the absolute calibration curve method and the results of the quantification of the target substance corresponding to the standard substance to determine a final quantification result.

7. The liquid chromatograph according to claim 6, wherein the averaging is performed using weighting.

8. A liquid chromatograph according to any one of claims 1 to 7, wherein the control unit determines abnormal values ​​in each quantitative result, and averages the results after excluding the abnormal values.

9. A liquid chromatograph according to any one of claims 1 to 7, characterized in that the detection results of the measurement target substance and each standard substance are obtained by gradient elution and / or isocratic elution, respectively.

10. A quantitative analysis device for quantifying a predetermined target substance, characterized in that it has a control unit that quantifies the target substance corresponding to each standard substance based on each RMS coefficient, which is the ratio of the response ratio Rr between the target substance and each standard substance to the substance amount ratio Rn between the target substance and each of a plurality of predetermined standard substances different from the target substance, the substance amount of each standard substance, and the respective detection response ratios based on the detection results of the target substance and each standard substance, and that averages the quantification results of the target substance corresponding to each standard substance to determine a final quantification result.

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