Liquid chromatography device and quantitative analysis device

Abstract

In order to reduce the analysis burden and improve the analysis accuracy, a liquid chromatograph (100) that quantifies a prescribed measurement target substance has a control section (9) that quantifies the measurement target substance in correspondence with each standard substance based on the ratio of the response ratio Rr of the measurement target substance to each of the mass amounts Rn of a plurality of prescribed standard substances that are different from the measurement target substance, i.e., the respective RMS coefficients, the mass amounts of each standard substance, and the respective detection response ratios based on the detection results of the measurement target substance and each standard substance, and averages the results obtained by quantifying the measurement target substance in correspondence with each of the standard substances, thereby obtaining a final quantification result.

Description

Technical Field

[0001] This invention relates to a chromatographic apparatus and a quantitative analysis apparatus for quantifying the analyte contained in an unknown sample. Background Technology

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

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Application Publication No. 2020-51827 Summary of the Invention

[0006] The problem that the invention aims to solve

[0007] In methods based on internal standards, a standard substance containing both the analyte and the internal standard must be injected when constructing the calibration curve. Furthermore, analysis of unknown samples also requires effort, such as adding an internal standard to each sample.

[0008] The present invention was made in view of the above circumstances, and its purpose is to reduce the analytical burden and improve the analytical accuracy.

[0009] Methods for solving problems

[0010] To achieve the above objectives, the present invention relates to a liquid chromatography apparatus for quantifying a specified analyte, characterized in that the liquid chromatography apparatus has a control unit that quantifies the analyte corresponding to each standard substance based on the ratio of the response ratio Rr of the analyte to each standard substance relative to the molar ratio Rn of the analyte and a plurality of specified standard substances different from the analyte, i.e., their respective RMS coefficients, the molar amounts of each standard substance, and the respective detection response ratios based on the detection results of the analyte and each standard substance, and averages the results obtained by quantifying the analyte corresponding to each standard substance to obtain a final quantitative result.

[0011] Therefore, when using RMS coefficients, the burden can be easily reduced compared to calculating calibration curves, thus making it easier to use a variety of standard substances, thereby easily reducing the analytical burden and improving analytical accuracy.

[0012] Invention Effects

[0013] This invention reduces the analytical burden and improves analytical accuracy. Attached Figure Description

[0014] Figure 1 This is a block diagram showing the general configuration of a liquid chromatography apparatus. Detailed Implementation

[0015] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

[0016] (Brief outline of a liquid chromatography apparatus)

[0017] The liquid chromatography apparatus 100 quantifies the specified analyte, such as... Figure 1 As shown, the system includes a data processing device 7 for controlling the entire system, a mobile phase (eluent or a mixture with solvent, etc.) 1, a pump 2 for delivering the mobile phase 1, an autosampler 3 for injecting the sample, a chromatographic column 4 for separating components, a column oven 5 for maintaining the temperature of the chromatographic column 4, a detector 6 for detecting the separated components, and an operation display unit 10.

[0018] The data processing device 7 is composed of a computer, which includes: a control unit 9 (CPU, etc.) that performs analysis and parses the analysis results, performs calculations related to the RMS coefficients (described later), and performs averaging of quantitative values; and a storage unit 8 (hard disk, etc.) that stores the analysis results, parsing results, calibration curve information (described later), and conversion information (RMS coefficients). The operation display unit 10 accepts various operation inputs and displays the analysis results and parsing results.

[0019] Detector 6 has multiple absorbance detectors, fluorescence detectors, quality detectors, or elements for detecting signal intensity. It is a three-dimensional photodiode array detector capable of simultaneously acquiring signal intensity relative to time at multiple wavelengths.

[0020] The sample is injected from the injector (not shown) of the autosampler 3 and passes through the chromatographic column 4 together with the mobile phase 1 delivered from the pump 2, where it is separated into various components of the sample.

[0021] The sample after component separation is detected by detector 6. The signal from detector 6 is sent to data processing device 7 for data processing.

[0022] The chromatographic column 4 is a device typically used as a separation unit to separate components of a sample present in the mobile phase 1. Chromatographic columns 4 can be packed columns, monolithic columns, etc. Various types of column packing materials, such as adsorption-type, partition-type, and ion-exchange-type packing materials, can be used as the column packing material for the chromatographic column 4. In order to maintain the chromatographic column 4 at a constant temperature and to perform sample separation with good reproducibility, the chromatographic column 4 is preferably housed within a column oven 5.

[0023] (Quantitative methods)

[0024] First, the quantitative method using RMS coefficients will be explained.

[0025] In the liquid chromatography apparatus 100, for example, in addition to quantification based on the general absolute calibration curve method and internal standard method, as shown in Table 1 (Equation 1) below, quantification using the RMS coefficient (RMS: Relative Molar Sensitivity) can also be performed. The RMS coefficient is defined as the ratio of the response ratio Rr of the analyte to the standard substance to the molar ratio Rn of the analyte to the standard substance (in addition to the molar ratio in the narrow sense, it can also be the mass ratio, volume ratio, concentration ratio, etc.).

[0026]

[0027] [Formula 1]

[0028] Here, the suffix "anal" indicates the analyte, and "ref" indicates the reference. Furthermore, "A" for both the analyte (anal) and reference (ref) represents the peak area and peak height as the response quantities, respectively, and "n" represents the mass (e.g., mol). As shown in Equation 1, for example, by injecting a sample solution containing a known mass of both the analyte and the reference into an HPLC system, and inputting the resulting peak areas as the respective response quantities, the RMS coefficient can be calculated.

[0029] (Quantification using RMS coefficients and external standards)

[0030] Therefore, when the response amount (Aref) and substance amount (nref) are obtained by measuring glycine, for example, as a standard substance (external standard) represented in (Table 1) (a), once a day, if the response amount (Aanal) is measured for alanine (RMS coefficient = 1.74), glutamic acid (RMS coefficient = 1.87), and aspartic acid (RMS coefficient = 2.02), which are the analytes, the substance amount (nanal) can be calculated as follows (Equation 2). Therefore, if the measurement is performed for the external standard substance represented in (Table 1) (a), the analyte substance represented in (b) can be quantified without performing the measurement.

[0031] [Formula 2]

[0032] (Quantification using RMS coefficients and internal standard)

[0033] When using an internal standard, as shown in (e) of Table 1, by including a known amount of the standard (internal standard) in the unknown sample for determination, if the response amount (Aanal) to the analyte is measured and the response amount (Aref) to the standard is measured simultaneously, the amount of the analyte (nanal) can still be calculated as described in (Equation 1) above. Therefore, the analyte can be quantified without measuring the analyte or standard for correction of daily variation as shown in (c) and (d) of Table 1.

[0034] As described above, the RMS method refers to a quantitative analysis method that utilizes RMS coefficients. Since known RMS coefficients can be treated as constants, the mass ratio Rn is output by inputting the response ratio Rr determined in (Equation 1). Because the mass nref of the standard substance is known, the mass nanal of the analyte can be calculated. The RMS method is not a convenient relative method, but rather a reliable quantitative analysis method based on mass.

[0035] The amount of substance (mol) in a quantitative value can be converted to mass (g) using molecular weight. Furthermore, if a sample injection volume of 10 μL or similar is input, it can also be converted to a concentration such as mol / L or g / L. More specifically, for example, the storage unit 8 may store conversion factors for converting the quantitative result into a specified concentration unit, and the operation display unit 10 may accept the specified concentration unit, convert the quantitative result into the specified concentration unit, and output it.

[0036] (Quantitative analysis using multiple standard reference materials)

[0037] By using two or more standard substances (reference materials) for the aforementioned RMS-based quantification, the reliability of the quantitative data can be ensured to a higher degree from a mathematical and statistical perspective. That is, even if the deviation and bias of the RMS coefficients affect the reliability of the quantitative data, the reliability of the data can be improved from a mathematical and statistical perspective by using two or more standard substances, and the robustness of the analysis can be demonstrated from the point of view of accuracy management.

[0038] For example, for two or more standard substances refA, refB, refC, ..., quantitative calculations are performed in the same manner as described above. Then, by averaging the obtained amounts of the two or more substances (e.g., molar concentrations ManrlA, ManrlB, ManrlC), the reliability of the quantitative values ​​can be improved.

[0039] Here, as the quantitative value for the above averaging, the final quantitative result can also be obtained by averaging the results of quantification by the absolute calibration curve method and / or internal standard method with the results of quantification of the analyte by the RMS coefficient corresponding to each standard substance.

[0040] Furthermore, while the molar concentrations ManrlA, ManrlB, and ManrlC mentioned above are theoretically consistent, if their ratios (e.g., their ratios relative to the average) are not within the range of 100 ± 10%, the reliability of the data may be considered significantly low, triggering an error or alarm, or the data may be excluded from the calculation of the average. Similarly, alarms may be triggered for poorly separated peaks, or they may be excluded from the calculation of the average. Alternatively, statistical indicators such as the relative standard deviation (RSD) can be calculated to perform the aforementioned processing.

[0041] Furthermore, in the measurement of peak area and peak height used to determine the response A of the analyte *anal* and the standard *ref*, isocratic elution and gradient elution can be used. That is, the ratio of peak areas can be calculated not only between peaks dissolved by isocratic elution but also between peaks dissolved by gradient elution. This is based on the consideration that the peak area is roughly preserved even with small variations in gradient elution. Here, gradient elution also includes step gradient elution. Furthermore, the ratio of the area of ​​peaks dissolved by gradient elution to that dissolved by isocratic elution can also be used.

[0042] Furthermore, the detection wavelengths used to determine the peak area ratio do not necessarily need to be the same. It should be noted that for a single standard substance, multiple detection wavelengths can be used to obtain the response, thus allowing for the same averaging process even when only one standard substance is used.

[0043] (Examples of applications in amino acid analysis)

[0044] The quantification of various target substances can be performed using a combination of the specified standard substances as described above. Specifically, for example, 17 components such as Aspartic acid, Threonine, and Serine from the protein hydrolysate pH method can be designated as standard substances, and approximately 20 components specific to the biofluid PF method, such as Tau (taurine), Orn (ornithine), and GABA (gamma-aminobutyric acid), can be assigned as analytes. Furthermore, components that are difficult to obtain (such as homocysteine-cysteine ​​disulfide, arginine-succinic acid, 2-aminoadipic acid, and aminoethylcysteine) can also be used as analytes. It should be noted that the components in the pH method are quantified using an absolute calibration curve method; therefore, the absolute calibration curve method and the method using multiple standard substances as described above can also be used in combination.

[0045] As mentioned above, when applying RMS coefficients, the workload is significantly reduced compared to determining calibration curves, thus allowing for the easy application of various standard substances. Therefore, the analytical burden is reduced, and analytical accuracy is easily improved.

[0046] The following details various application examples, including the combined use method. In the combined use method of the PF method, only 17 components are injected as external standards. It is not necessary to prepare other standard substances such as Tau (approximately 20 other components). Previously, these approximately 20 components had to be prepared; therefore, the advantage of this combined use method of PF lies in this omission.

[0047] Typically, for components injected with external standards such as Asp, the absolute standard curve method (and / or internal standard method) is used because a standard curve can be obtained from the component itself. On the other hand, for components without injected standards such as Tau, the RMS method can be used. For example, using Asp as an external standard, the quantitative value of Tau is calculated by the RMS coefficient with Tau. This is a combination of the basic PF method (Table 2(1)).

[0048] Here, the RMS quantitative method using multiple standard substances (multiple standard substance quantitative method) can be applied. That is, in order to quantify Tau and other substances by RMS method together with the quantification of Asp based on absolute calibration curve method, a method can be adopted that uses not only one component of Asp, but also multiple external standard substances such as Thr and Ser that are actually injected. Tau and Thr, Ser, etc. each have their own RMS coefficients, so the quantitative value of Tau corresponding to the number of different types of external standard substances can be calculated by using RMS method. Finally, these quantitative values ​​can be averaged to obtain the quantitative value of Tau. In such components where no standard substances are prepared, the multiple standard substance quantitative method can be used. In addition, this method can be called the combined method of multiple standard substance quantitative method (Table 2(2)). It should be noted that it is not limited to quantifying Asp by absolute calibration curve method, etc., but even if only Tau and other substances are averaged by RMS method using multiple external standard substances such as Asp, Thr, Ser, etc., the effect of improving accuracy can be obtained (Table 2(3)). In addition, similar to Tau, it can also be used to quantify other substances, such as Orn (Table 2(4)).

[0049] As mentioned earlier, for components with injected external standards, such as Asp, the absolute calibration curve method based on the component's own calibration curve should naturally be considered. However, when it is desirable to improve the accuracy of the quantitative value, even for components with injected external standards, multiple standard reference quantitative methods can be applied. That is, for Asp, there are also RMS coefficients for Asp and Thr, Ser, etc., for which external standards have been prepared, so multiple RMS quantitative values ​​for Asp can be calculated. These averages can be used as the comprehensive RMS quantitative value. For example, this is an RMS quantitative method that compares the ratio of the peak area of ​​Thr (as an external standard) to the peak area of ​​Asp (an unknown component) with the RMS coefficient. In this case, the peak area of ​​Asp itself (as an external standard) is not referenced. As for which method has better accuracy, it may vary depending on the situation, but a method of averaging the quantitative values ​​from the Asp absolute calibration curve method and the comprehensive RMS quantitative value of Asp can be used as needed. This method can be simply referred to as the "fusion method," which means the fusion method of absolute calibration curve method and other quantitative methods of multiple reference materials (Table 2(5)). The above are the explanations of the combined method, the quantitative method of multiple reference materials, the combined method of multiple reference materials and the fusion method. It should be noted that the RMS quantitative value that is averaged with the quantitative value results of Asp absolute calibration curve method, etc. is not limited to multiple values. For example, the RMS quantitative value obtained from Thr can be averaged with the quantitative value results of Asp absolute calibration curve method, etc. (Table 2(6)).

[0050] Furthermore, error handling is supplemented. Regardless of whether it's a multi-reference material quantification method or a fusion method, the quantitative values ​​of the elements before calculating the average should be approximately equal. Therefore, outlier handling can be based on this idea. Outliers can be easily identified, or statistical tests such as the Smirnov-Grubbs test and Thompson's test can be used. When outliers are found, they are notified to the user, and the averaging process is re-executed after excluding the outliers. The calculation result after excluding outliers is output as the quantitative value for the multi-reference material quantification method or the fusion method. Specifically, it can also be referred to as the outlier-excluded quantitative value.

[0051] It should be noted that in the RMS quantification method of the above-mentioned combined or fusion methods, only one or more standard substances (reference materials) are required. For example, in the fusion method, the Asp quantification value obtained by the absolute calibration curve method and the Asp quantification value obtained by the RMS quantification method using Thr as the standard substance (reference material) can be averaged by applying some weighting as needed. In other words, the quantification value obtained by non-RMS quantification methods such as the absolute calibration curve method can be compared and studied with the quantification value obtained by the RMS quantification method (averaging, etc.). In this case, only one or more RMS coefficients are required in the RMS quantification method. In this case, the analytical burden can be reduced while the analytical accuracy can be improved.

[0052]

[0053] (Other quantitative methods)

[0054] The above description uses a liquid chromatography apparatus as an example, but the present invention is not limited to this and can be applied to various quantitative analysis devices using the same method. That is, as long as the ratio (RMS coefficient) of the response ratio Rr of the analyte to each of the various specified standard substances different from the analyte can be calculated relative to their respective molar ratios Rn, the same calculation and averaging method can be applied. Specifically, in addition to chromatographs, this method can also be applied to the measurement results of mass spectrometers, spectrophotometers, and other analytical devices capable of quantitative analysis of substances.

[0055] Industrial applicability

[0056] As explained above, the present invention is useful for chromatographic apparatus for quantifying analytes contained in unknown samples.

[0057] Explanation of reference numerals in the attached figures

[0058] 1. Mobile phase

[0059] 2 pumps

[0060] 3. Autosampler

[0061] 4. Chromatographic column

[0062] 5. Column oven

[0063] 6 detectors

[0064] 7. Data processing device

[0065] 8. Storage Section

[0066] 9. Control Department

[0067] 10. Operation Display Section

[0068] 100 Liquid Chromatography Apparatus

Claims

1. A liquid chromatography apparatus for quantifying a specified analyte, characterized in that, The liquid chromatography apparatus has a control unit that quantifies the analyte according to each standard substance based on the ratio of the response ratio Rr of the analyte to each standard substance relative to the molar ratio Rn of the analyte and a plurality of prescribed standard substances different from the analyte, i.e., their respective RMS coefficients, the molar amounts of each standard substance, and the respective detection response ratios based on the detection results of the analyte and each standard substance. The control unit then averages the results obtained from quantifying the analyte according to each standard substance to obtain the final quantitative result.

2. The liquid chromatography apparatus according to claim 1, characterized in that, Quantitative analysis of various target substances is performed using the aforementioned sets of standard substances.

3. The liquid chromatography apparatus according to claim 1, characterized in that, The aforementioned control unit will also average the results obtained by quantification using the absolute calibration curve method and / or internal standard method, as well as the results obtained by quantifying the analyte using the aforementioned RMS coefficients and corresponding standard substances, to obtain the final quantitative result.

4. A liquid chromatography apparatus for quantifying a first analyte and a second analyte, characterized in that, The liquid chromatography apparatus has a control unit that quantifies the first analyte using an absolute calibration curve method and uses the first analyte as a standard substance. Based on the RMS coefficient (the ratio of the response ratio Rr of the second analyte to the standard substance to the respective mass ratio Rn of the second analyte to the standard substance), the mass of the standard substance, and the detection response ratio based on the detection results of the second analyte and the standard substance, the second analyte is determined corresponding to the standard substance.

5. The liquid chromatography apparatus according to claim 4, characterized in that, There are several types of the aforementioned standard substances. The second analyte is quantified in accordance with each standard reference material, and the results obtained by quantifying the second analyte in accordance with each standard reference material are averaged to obtain the final quantitative result.

6. A liquid chromatography apparatus for quantifying a specified analyte, characterized in that, The aforementioned liquid chromatography apparatus includes a control unit that quantifies the analyte using an absolute calibration curve method. The control unit quantifies the analyte in accordance with the standard substance based on the ratio of the response ratio Rr of the analyte to the standard substance relative to the mass ratio Rn of the analyte and a specified standard substance different from the analyte (i.e., the RMS coefficient), the mass of the standard substance, and the detection response ratio based on the detection results of the analyte and the standard substance. The control unit then averages the quantification results obtained by the absolute calibration curve method and the quantification results obtained by the standard substance to obtain the final quantification result.

7. The liquid chromatography apparatus according to claim 6, characterized in that, The above averaging is performed using weighted averages.

8. The liquid chromatography apparatus according to any one of claims 1 to 7, characterized in that, The aforementioned control unit identifies outliers in each quantitative result, removes these outliers, and then performs averaging.

9. The liquid chromatography apparatus according to any one of claims 1 to 7, characterized in that, The detection results of the above-mentioned target substances and standard substances are based on gradient elution and / or isocratic elution, respectively.

10. A quantitative analysis device for quantifying a specified analyte, characterized in that, The quantitative analysis device has a control unit that quantifies the analyte according to each standard substance based on the ratio of the response ratio Rr of the analyte to each standard substance relative to the mass ratio Rn of the analyte and each of the specified standard substances different from the analyte, i.e., their respective RMS coefficients, the mass of each standard substance, and the respective detection response ratio based on the detection results of the analyte and each standard substance. The control unit then averages the results obtained by quantifying the analyte according to each standard substance to obtain the final quantitative result.

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