Quantitative method and chromatographic apparatus

Abstract

This reduces the effort required to quantify the target substance contained in an unknown sample. [Solution] A quantitative method for quantifying a predetermined target substance using a chromatograph apparatus 100 comprises: a standard substance measurement step of performing measurements to detect the actual retention time of the standard substance and the quantitative index of the standard substance for the standard substance; a target substance measurement step of performing measurements to detect the actual retention time of the target substance and the quantitative index of the target substance for the target substance for the target substance; a target substance identification step of identifying the target substance based on the above measurement results; and a quantitative method for quantifying the target substance.

Description

Technical Field

[0001] The present invention relates to a quantification method for quantifying a measurement target substance contained in an unknown sample, and a chromatograph device.

Background Art

[0002] As a quantitative analysis of chromatography such as a liquid chromatograph device, a relative molar sensitivity method that enables quantification of an unknown sample without necessarily requiring a standard sample of an analysis target substance is known. For example, Non-Patent Document 1 describes that "in the relative molar sensitivity method of each instrumental analysis, from the obtained chromatogram, the ratio of the peak area or peak height of the test component to the reference substance is determined, and this ratio is divided by the relative molar sensitivity defined separately to obtain the molar ratio of the test component to the reference substance. Next, the mass ratio can be obtained by multiplying this molar ratio by the molecular weight ratio of the test component to the reference substance."

[0003] Also, a technique for normalizing a chromatogram signal by an internal standard method or a relative retention time method is known in order to suppress fluctuations in analysis conditions due to changes in the operating environment of a liquid chromatograph device or the like. For example, Patent Document 1 describes that "when obtaining a chromatogram signal, a standard sample whose retention time (standard retention time) or the like is known is mixed into an unknown sample. Then, the reference peak for the above standard sample is identified from the obtained chromatogram waveform, and the chromatogram signal is normalized by the ratio of the actually measured retention time for the identified reference peak to the predetermined standard retention time." (See paragraph 0002).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] To quantify the target substance contained in an unknown sample, the time axis of the chromatogram is normalized based on the retention time measured for a standard sample to identify the target substance. Then, the ratio of the peak area or peak height of the target substance to a predetermined reference substance is determined, and the amount of the target substance is calculated using the relative molar sensitivity corresponding to the identified target substance.

[0007] Therefore, it is necessary to perform measurements on a standard sample to normalize the time axis of the chromatogram, and to perform measurements to determine the peak area or peak height of a predetermined reference substance for quantification, which requires a great deal of effort in the measurement process.

[0008] This invention has been made in view of the above points, and aims to reduce the effort required to quantify the target substance contained in an unknown sample. [Means for solving the problem]

[0009] To achieve the above objectives, The present invention A quantitative method using a liquid chromatograph to quantify an unknown target substance, A standard material measurement process is performed to measure a predetermined known amount of a standard material different from the above-mentioned target substance, in order to detect the actual retention time of the standard material and the quantitative index of the standard material. The above-mentioned measurement target substance includes a measurement process for which measurements are taken to detect the actual retention time of the measurement target substance and the quantitative index of the measurement target substance, The measured retention time of the above standard material, The measured retention time of the above-mentioned substance, The standard retention time of the above-mentioned standard material is the standard retention time of the standard material, and A process for identifying the target substance, based on the standard retention time of the target substance, which is the standard retention time of the target substance, The RMS coefficient is the ratio of the response ratio Rr between the above-mentioned substance and the above-mentioned standard substance to the respective molar ratio Rn between the above-mentioned substance and the above-mentioned standard substance. The above known amount of the above standard substance, and A quantitative step for quantifying the target substance based on the detection response ratio derived from the detection results of the quantitative index of the target substance and the quantitative index of the standard substance, It is characterized by having the following features.

[0010] This allows for the quantification of target substances in unknown samples by measuring the retention time of the standard substance when measuring the standard substance, thereby reducing the effort required for quantification. [Effects of the Invention]

[0011] This invention makes it easy to reduce the effort required to quantify the target substance contained in an unknown sample. [Brief explanation of the drawing]

[0012] [Figure 1] This is a block diagram showing the schematic configuration of a liquid chromatography apparatus. [Figure 2] This is a chromatogram of a measurement example. [Modes for carrying out the invention]

[0013] Embodiments of the present invention will be described in detail below with reference to the drawings.

[0014] (Schematic configuration of a liquid chromatography apparatus) The liquid chromatograph device 100 quantifies a predetermined measurement substance. As shown in FIG. 1, it includes a data processing device 7 that controls the whole, a mobile phase 1 (a mixed solution with an eluent or a solvent, etc.), a pump 2 that feeds the mobile phase 1, an autosampler 3 that injects a sample, a column 4 that separates components, a column oven 5 that keeps the column 4 at a constant temperature, a detector 6 that detects the separated components, and an operation display unit 10.

[0015] The data processing device 7 is composed of a computer having a control unit 9 (such as a CPU, etc.) that executes analysis and analyzes the analysis results, and a storage unit 8 (such as a hard disk, etc.) that stores the analysis results, the analysis results, calibration curve information, and conversion information (RMS coefficient) described later. The operation display unit 10 accepts various operation inputs, etc., and displays the analysis results, analysis results, etc. The data processing device 7 and the operation display unit 10 do not necessarily have to be incorporated into the liquid chromatograph device 100 and may be an external computer.

[0016] The detector 6 has a plurality of elements that detect signal intensity, and is a three-dimensional detector that can simultaneously acquire the signal intensity with respect to time at a plurality of wavelengths, or an absorption photometric detector, a fluorescence detector, a mass detector, etc.

[0017] The sample is injected from an injector (not shown) of the autosampler 3, passes through the column 4 together with the mobile phase 1 fed from the pump 2, and is separated into various components in the sample.

[0018] The sample separated into components is detected by the detector 6. The signal of the detector 6 is sent to the data processing device 7 for data processing.

[0019] The column 4 is a device generally used as a separation unit that separates the components of the sample present in the mobile phase 1. Examples of the column 4 include a packed column and a monolithic column. As the column packing material of the column 4, various types such as adsorption type, distribution type, and ion exchange type can be used. It is desirable that the column 4 be installed in the column oven 5 so that the column 4 is kept at a constant temperature and the sample can be separated with good reproducibility.

[0020] (quantification method) First, for convenience, we will explain the quantitative analysis using the RMS coefficient.

[0021] In the liquid chromatograph apparatus 100, in addition to the ability to perform quantitative analysis using general absolute calibration curve methods and internal standard methods, as shown in (Table 1) below, quantitative analysis 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 substance to be measured and the standard substance to the molar ratio Rn (which can be a molar ratio in the narrow sense, but also a mass ratio, volume ratio, concentration ratio, etc.) between the substance to be measured and the standard substance, as shown in (Equation 1) below.

[0022] [Table 1]

[0023]

number

[0024] Here, the subscript `anal` represents the analyte, and `ref` represents the reference material. Furthermore, for both the analyte (anal) and the reference material (ref), `A` represents the peak area or peak height as a response quantity (quantitative indicator), and `n` represents the amount of substance (e.g., moles). As shown in (Equation 1), for example, a sample solution containing the analyte and reference material in known amounts can be injected into an HPLC, and the resulting peak area or peak height can be input as the respective response quantities to calculate the RMS coefficient.

[0025] (Quantitative analysis using external standards with RMS coefficients) Therefore, for example, if the response amount (Aref) and amount of substance (nref) are obtained for glycine, which is a standard substance (external standard substance) shown in (a) of (Table 1) by measurement once every morning, then for alanine (RMS coefficient = 1.74), glutamic acid (RMS coefficient = 1.87), and aspartic acid (RMS coefficient = 2.02), which are substances to be measured, if the response amount (Aanal) for each can be measured, the amount of substance (nanal) for each can be determined as shown in (Equation 2) below. Thus, by performing measurements on the external standard substance shown in (a) of (Table 1), the quantitative determination of the substance to be measured can be performed without performing measurements on the substance to be measured shown in (b). In other words, the standard substance measurement process is performed on the sample containing the standard substance, and then the substance to be measured process is performed on the sample containing the substance to be measured.

[0026]

number

[0027] (Quantitative determination using internal standard substance with RMS coefficient) When using an internal standard, as shown in (e) in (Table 1), by including a known amount of the standard (internal standard) in the unknown sample and measuring it, the response amount (Aref) for the standard (Aanal) along with the response amount for the target substance can be measured, and then, as shown in (Equation 1) above, the amount of substance (nanal) for the target substance can be determined. Therefore, the quantitative determination of the target substance can be performed without measuring the target substance or standard substance used for calibration of daily variation, as shown in (c) and (d) in (Table 1). In other words, for the standard substance and the sample containing the target substance, the standard substance measurement process and the target substance measurement process are performed in the same process.

[0028] As described above, the RMS method is a quantitative analysis method that utilizes the RMS coefficient. Since known RMS coefficients can be treated as constants, the molar ratio Rn is output by inputting the measured response ratio Rr into (Equation 1). Since the molar amount nref of the standard substance is known, the molar amount nanal of the substance being measured can be determined. The RMS method is positioned not as a convenient relative method, but as a reliable quantitative analysis method based on the amount of substance.

[0029] The amount of substance (mol) in the quantitative value can be converted to mass (g) based on the molecular weight. Furthermore, if the sample injection volume, such as 10 μL, is input, it can also be converted to concentrations such as mol / L or g / L. More specifically, for example, the storage unit 8 may store conversion coefficients for converting the quantitative result to a predetermined concentration unit, and the operation display unit 10 may accept the specification of the concentration unit and output the quantitative result converted to the specified concentration unit.

[0030] (Identification of the substance to be measured) The quantitative analysis described above is performed by first identifying the target substance contained in the unknown sample, and then using an RMS coefficient corresponding to that identified target substance. Furthermore, the identification of the target substance can be made possible without performing separate measurements for identification by measuring the retention time of the standard substance (measured retention time of the standard substance) when the standard substance is measured as an external or internal standard substance for the quantitative analysis, and normalizing the chromatogram obtained when the unknown sample is measured.

[0031] More specifically, by normalizing the time axis of the chromatogram obtained by measuring an unknown sample containing the target substance, based on the measured retention time of the standard substance and the standard retention time of the standard substance, the target substance can be identified based on the normalized chromatogram and the standard retention time of the target substance. Note that the normalization may be performed assuming a proportional relationship between the standard retention time and the measured retention time, or it may be performed assuming a predetermined functional relationship.

[0032] The specific data processing method for the above identification is not particularly limited. For example, in a normalized chromatogram, a time window corresponding to the standard retention time of the substance to be measured can be set, and the substance to be measured having a retention time (peak) that falls within the time window can be identified. Alternatively, in a normalized chromatogram, the substance to be measured may be identified if its retention time is within a predetermined range (or minimum) of the standard retention time of the substance to be measured.

[0033] As described above, when measuring a standard substance as an external or internal standard substance for quantitative analysis, the retention time of that standard substance (actual retention time of the standard substance) is also measured and used to identify the target substance, thereby reducing the effort required to quantify the target substance contained in an unknown sample.

[0034] Furthermore, if the quantitative measurement and the retention time measurement are performed simultaneously as described above, the standard substance used to detect the measured retention time of the standard substance and the standard substance used to detect the quantitative index of the standard substance may be different standard substances.

[0035] (Confirmation of normal operation) As described above, based on measurements of standard substances for quantification and identification, these standard substances may also be used as so-called QC samples to confirm the operational normality of the equipment (normality identification). In this case, the efficiency of quantification can be particularly increased by performing normality identification, identification of the target substance, and quantification in sequence.

[0036] (Details of the RMS internal standards method) The details of this invention will first be explained using the RMS internal standard method. As shown in the last row of Table 1, the internal standard substance (e) is added to each unknown sample. Because this addition process is complicated, the RMS internal standard method is generally avoided. However, once the RMS internal standard method is accepted, the retention time tref of (e) is measured for each sample injection, which offers the advantage of compensating for variations in the measured retention time of the internal standard substance between injections. There are advantages to this method, even if it requires extra effort.

[0037] The retention time tanal of the substance to be measured can be estimated for each injection, based on the measured retention time tref of the internal standard substance. As mentioned above, the relationship between the measured retention time tref of the internal standard substance and the estimated retention time tanal of the substance to be measured may be proportional or a predetermined functional relationship (Equation 3). This relationship may be treated as a time axis, as in Patent Document 1, and called chromatogram normalization, or the perspective may be changed, the time axis may be fixed, and the two retention times may be recognized as being in a relative relationship, as shown in (Equation 3).

[0038]

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[0039] In the RMS internal standard method, it is usually assumed that a standard substance of the substance to be measured is not readily available, so one challenge is how to estimate the retention time (tanal) of the substance to be measured. In this invention, we have devised a method of using the measured retention time (tref) of the internal standard substance as one solution. Normally, rather than estimating the tanal, it is more common to directly inject the substance to be measured and measure the tanal, even if the peak area of ​​the substance to be measured is unstable.

[0040] For example, the function in (Equation 3) can be expressed as a linear equation as in (Equation 4). A proportional relationship occurs when the intercept b is zero. It is desirable to determine these coefficients a and b experimentally beforehand and use them as predetermined values ​​thereafter. Note that in the case of isocratic elution and stepwise elution, the function can be expressed as a roughly linear equation as in (Equation 4), but in the case of gradient elution, it is thought that the function will be expressed as a curved function.

[0041]

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[0042] Furthermore, the function in (Equation 3) can also be treated as a two-variable function, as in (Equation 5) and (Equation 6). Equation (5) corresponds to a method of estimating tanal by injecting two internal standard substances and using their respective measured retention times, tref1 and tref2. For example, it represents a case where the tanal of the target substance is estimated by sandwiching the peak of the target substance's component between the peak of internal standard substance 1 and the peak of internal standard substance 2. In the case of (Equation 6), although there is only one internal standard substance, this method estimates the retention time tanal of the target substance by taking into account not only the measured retention time tref but also the peak area Aref. This has a strong connotation of secondary correction, but it is intended to correct phenomena such as the measured retention time being slightly correlated depending on the size of the internal standard substance's peak.

[0043]

number

[0044]

number

[0045] Summarizing equations (3) through (6), there may be cases where the substance to be measured is available, but its amount and concentration are unknown. In other words, there is no reference material for the quantitative analysis of the substance to be measured. Even in such cases, if the coefficients of the function are determined in advance, it is possible to obtain a function with those predetermined coefficients, at least for the retention time. That is, by injecting an internal standard, the tref and other parameters can be determined, and the retention time tanal of the substance to be measured can be estimated. If tanal can be estimated, the advantage of the RMS internal standard method is that, since the RMS coefficients are known, a reference material for quantitative calculation of the substance to be measured is not necessary, and quantitative analysis can be performed simply by measuring the peak area of ​​the substance to be measured.

[0046] (Details of the RMS-external standard method) Let's reconsider the RMS external standard method, keeping in mind the concept of the RMS internal standard method. Looking at (Table 1), the difference is that there is no need to add (e) to the unknown sample; for example, an external standard substance (a) is injected once in the morning as a reference substance. The characteristic of the RMS external standard method is that this external standard substance is a reference substance that is not the substance being measured itself. If the external standard substance were equal to the substance being measured, then the absolute calibration curve method could simply be used.

[0047] The RMS external standard method, like the RMS internal standard method, requires that the amount of substance nref of the external standard substance (the reference substance) is known. As shown in (Equation 2), for example, it is necessary to measure Aref once in the morning. Since the RMS coefficient is known, as long as the Aanal of the substance to be measured can be measured for each injection of the unknown sample, the quantitative value nanal can be calculated using a common Aref. In the RMS internal standard method, it was necessary to add an internal standard substance, which is a reference substance with a known nref, for each injection, but the RMS external standard method differs in that this does not need to be done for unknown samples. In addition, in the RMS internal standard method, the response Aref of the internal standard substance is also measured for each injection. For example, if an interfering peak appears in the unknown sample, it is expected that it will affect the measurement of Aref, but in the RMS external standard method, as shown in (Table 1), no reference substance is added to the unknown sample, so this concern is not present. For example, if there are concerns about interference from interfering peaks, such as in crude animal-derived samples, it is preferable to use the RMS external standard method, as the magnitude of interfering peaks can be relatively suppressed in calibration samples, such as those prepared once in the morning.

[0048] By the way, in the RMS external standard method, how is the retention time tanal of the substance being measured estimated? In the RMS external standard method, it is possible to continuously use the measured retention time tref of an external standard substance, for example, if it is injected once in the morning, rather than the substance being measured itself. In other words, the retention time tanal of the substance being measured is estimated once in the morning using the measured retention time tref of the external standard substance, according to a certain relational equation (Equation 3), such as a proportional relationship. Of course, the retention time will vary slightly with each injection, so a certain tolerance range, such as a time window, is added to absorb the variation in retention time.

[0049] An important aspect of the RMS external standard method is that, for example, once in the morning, the response Aref, such as peak area, and the retention time tref are measured from an external standard substance that is not the substance being measured itself. In this invention, since two types of variables are obtained in a single measurement, first the retention time tanal of the substance being measured is estimated from the measured retention time tref of the external standard substance (Equation 3). Then, for each injection of the unknown sample, the response Aanal of the substance being measured, identified using a time window centered on the estimated retention time tanal, is measured, and the quantitative value nanal is obtained by (Equation 2).

[0050] (Use of QC samples) QC samples are injected at appropriate times to confirm the normal operation of an analytical instrument, including quality control components. Generally, retention time and the magnitude of the response, such as peak area, are checked to assess normal operation. For example, to observe the range of the response, three types of QC samples, H, M, and L, are prepared by dividing the QC component into three concentration levels: high, medium, and low. This control method is used to determine that the instrument is operating normally if each sample falls within a certain tolerance.

[0051] Strictly speaking, a QC sample is not a reference substance for quantitative analysis and therefore should not be used as a reference substance for quantitative analysis. However, in the case of the RMS external standard method, if a very high level of quantitative accuracy is not required, it is possible to substitute a certain QC component in the QC sample for the reference substance. That is, the QC sample is injected once in the morning and the chromatogram is measured (Table 1). First, the measured retention time tref can be determined by positioning the QC component as the reference substance. Then, the other response Aref is also measured by positioning the QC component as the reference substance. In this way, the tref and Aref of the two variables required for the RMS external standard method can be obtained, for example, with a single measurement in the morning. After that, the response Aanal of the target substance can be measured sequentially from each injection using an unknown sample. If a slight improvement in quantitative accuracy is expected, the representative ratio Aref / nref can also be calculated from the average value considering the amount of each substance by measuring three types of QC samples H, M, and L once each in the morning for a total of three measurements (Equation 7). Using this ratio, it is also possible to quantify the target substance using (Equation 2). The subscripts H, M, and L indicate the respective concentration levels.

[0052]

number

[0053] In the case of the RMS external standard method, the discussion of measured retention time is the same as in the RMS internal standard method, and QC samples can be effectively utilized. For example, it is sufficient to measure the retention time tref of the reference substance once in the morning. As mentioned above, the retention time tanal of the substance to be measured can be easily estimated using (Equation 3).

[0054] (Cases where there are two types of reference substances) As a variation of the RMS external standard method, it is conceivable to use two types of external standards, one for quantitative calibration and the other for measuring retention time (Table 2). Table 2 is an extended version of Table 1, showing the quantitative calibration material and the measuring retention time material in separate columns. By presenting it as in Table 2, it is emphasized that the peak area measurement process and the retention time measurement process can be handled independently. To accurately measure the peak area, an isolated peak is desirable for external standard 1 used for quantitative calibration. On the other hand, there may be circumstances such as wanting the peak of external standard 2 used for measuring retention time to appear at a certain time interval from the peak of the substance being measured. Of course, as explained above, it is also fine for external standard 1 and external standard 2 to be the same. This is also true for the RMS internal standard method, where it is possible to use two types of reference materials, internal standard 1 and internal standard 2. As an extension of this, it is possible to use the RMS external standard method for quantitative analysis and the RMS internal standard method for measuring retention time, or vice versa (see Table 2).

[0055] [Table 2]

[0056] (summary) The entire scope of the present invention will be summarized again using Figure 2.

[0057] The RMS internal standard method involves adding an internal standard to an unknown sample containing the target substance (Glu), as shown in Figure 2(a). Typically, a single-component reference substance, ref1 (Asp: aspartic acid), is measured as the internal standard. In this invention, both tref and Aref are measured from the chromatogram. The retention time tanal of the target substance is estimated from tref using (Equation 3), and then the target substance is identified. The quantitative value of the target substance (Glu) is then calculated from (Equation 2) using the RMS coefficient. Figure 2(b) also shows a case where the reference substance has two components. Ala (alanine) was selected as the reference substance ref2. In the case of two components, (Equation 5) is used to calculate the retention time tanal of the target substance. Furthermore, (Equation 2) is used for quantitative analysis, and the quantitative values ​​of ref1 and ref2 are calculated from their respective peak areas, for example. Then, the quantitative values ​​are averaged to obtain a more accurate quantitative value. Figure 2(b) shows an example with two components, but three or four components are also acceptable. Furthermore, while Asp and Ala are selected as reference components in this example, it is undesirable if the unknown sample before addition contains Asp or Ala, as this may interfere with the quantitative analysis. This example is for illustrative purposes only.

[0058] Next, let's summarize the RMS external standard method. As shown in Figure 2(c), the external standard is injected once in the morning. Of course, measurements can be taken two or three times and averaged. Typically, the external standard is a single-component ref1. The retention time and peak area are measured from this ref1. The retention time tanal of the target substance is estimated from (Equation 3), and the chromatogram of the unknown sample shown in Figure 2(d) is awaited. In the RMS external standard method, internal standards (Asp or Ala) as shown in Figures 2(a) and (b) are not added. After identifying the analyte from the unknown sample using the above tanal, only the peak area of ​​the target substance is measured. Then, the RMS quantitative calculation is performed using (Equation 2). Furthermore, as shown in Figure 2(e), the external standard can be made into two components, and the peak identification can be strengthened or the accuracy of the quantitative calculation can be improved, similar to the RMS internal standard method described above. It is also possible to expand this discussion to three or four components.

[0059] (Insertion of RMS-external standard method) If quantitative results are not immediately required after measuring the chromatogram of an unknown sample, a so-called "sandwich" method can be employed, using both morning and evening chromatograms. Figure 2(f) shows the evening chromatogram. Typically, there is one external standard substance. Comparing the morning and evening chromatograms, the retention time of external standard substance ref1 changes to some extent. The average of these morning and evening retention times can be used as tref, or, interpreting this as a trend, a method can be adopted in which tref is changed at different times of the day, such as 10 AM, noon, and 3 PM. If this trend is to be captured as a more reliable phenomenon, the retention time of external standard substance ref1 can be measured more frequently at noon and other times in addition to morning and evening. In any case, the retention time tanal of the substance being measured can be estimated using the tref recognized by this sandwich method.

[0060] For an unknown sample (Figure 2(d)), quantitative calculation is performed using (Equation 2) with the peak area of ​​the target substance identified by this tanal. Here, several methods can be considered for determining the response Aref of the external standard substance ref1. Simply put, the peak area in the morning and the peak area in the evening can be averaged to obtain Aref. Alternatively, it is possible to consider the trend, similar to the retention time. Increasing the measurement frequency of the peak area, similar to the retention time, is also effective.

[0061] Furthermore, by adding external standard material ref2, the external standard material can be made into two components, as shown in Figures 2(e) and (f). In the case of two components, both the calculation of retention time and the quantification calculation are simplified by averaging.

[0062] As an alternative, as explained in (Table 2), retention time and peak area can be considered generally independent. For example, it is possible to use RMS internal standard ref1 for measuring the retention time of the reference substance (Figure 2(a)) and RMS external standard ref2 for measuring the peak area of ​​the reference substance (Figure 2(e), etc.). Furthermore, depending on the user's approach, it is possible to reverse the roles of internal and external standards. The number of components in the reference substance is not limited to one component; it can be increased to two or more components depending on the user's approach. A reasonable retention time estimation method and quantitative calculation method must be adopted for each case.

[0063] Furthermore, if real-time quantitative results are not required for each injection, secondary processing such as offline processing can be performed as described above. Component identification and RMS quantitative calculation will be performed at the end of the sequential continuous analysis. While expressions such as "once in the morning" or "twice in the evening" are symbolic, in extreme processing steps, it is also possible to perform continuous analysis of an unknown sample (Figure 2(d)) without specifically performing the morning injection (Figure 2(c)(e)), and then use the RMS external standard method from the chromatogram (Figure 2(f)) obtained by injecting only the reference substance in the evening.

[0064] Table 2 shows that the roles of the reference material can be treated independently for peak area measurement and retention time measurement. Furthermore, Figure 2 re-examines the time series of the injection process in an offline processing manner, emphasizing that there is no problem even if the reference material is injected after the unknown sample. The reference material for peak area measurement and the reference material for retention time measurement can be treated independently, and it is also optional whether the reference material for each application is used as an RMS internal standard or an RMS external standard. If there are two or more types of reference materials, the method of adding and injecting the reference material—whether as an RMS internal standard or an RMS external standard—is also optional, and can span both internal and external applications. In some cases, it is possible to inject the same reference material as an RMS external standard while also adding it as an RMS internal standard.

[0065] Returning to the topic of offline processing, the independence of the applications of peak area and retention time, as well as the arbitrariness of using RMS internal and external standards, can be further considered in addition to the scalability of the time series of the injection process. For example, a reference substance for measuring retention time as an RMS internal standard is added to an unknown sample, and in the evening, a reference substance for measuring peak area as an RMS external standard is injected. These reference substances may be the same compound or different. Of course, it is also possible to inject the external standard in the morning and evening. Again, it is possible to use multiple types of reference substances, and it is also arbitrary to inject them multiple times. Appropriate and rational statistical numerical processing is desirable. There is no distinction between RMS internal and external standards in (Equation 2) for peak area measurement and (Equation 3) and (Equation 5) for retention time measurement. Furthermore, from the perspective of the time series of the injection process as shown in Figure 2, (Equation 2) and (Equation 3), etc., can be treated without distinction.

[0066] Finally, as a supplement, (Equation 8) and (Equation 9) are given as examples of specific linear expressions for (Equation 5) and (Equation 6).

[0067]

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[0068]

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[0069] It should be noted that the present invention is not limited to the embodiments described above, and various modifications are included. For example, the embodiments described above are explained in detail to make the present invention easier to understand, and are not necessarily limited to those having all the configurations described. Also, although the embodiments described above were explained using a liquid chromatograph as an example, they are applicable to chromatographs in general, such as gas chromatographs. [Explanation of Symbols]

[0070] 1 Mobile phase 2 pumps 3 Autosampler 4 columns 5-column oven 6 Detectors 7 Data Processing Devices 8 Memory section 9. Control Unit 10 Operation display section 100 Liquid Chromatography Apparatus

Claims

1. A quantitative method using a chromatographic apparatus to quantify an unknown target substance, A standard material measurement process is performed to measure a predetermined known amount of a standard material different from the above-mentioned target substance, in order to detect the actual retention time of the standard material and the quantitative index of the standard material. The above-mentioned measurement target substance includes a measurement process for which measurements are taken to detect the actual retention time of the measurement target substance and the quantitative index of the measurement target substance, The measured retention time of the above standard material, The measured retention time of the above-mentioned substance, The standard retention time of the above-mentioned standard material is the standard retention time of the standard material, and A process for identifying the target substance, based on the standard retention time of the target substance, which is the standard retention time of the target substance, The RMS coefficient is the ratio of the response ratio Rr between the above-mentioned substance and the above-mentioned standard substance to the respective molar ratio Rn between the above-mentioned substance and the above-mentioned standard substance. The above known amount of the above standard substance, and A quantitative step for quantifying the target substance based on the detection response ratio derived from the detection results of the quantitative index of the target substance and the quantitative index of the standard substance, A quantitative method characterized by having the following features.

2. A quantitative method according to claim 1, A quantitative method characterized in that, after the above-mentioned standard substance measurement step is performed on a sample containing the above-mentioned standard substance, the above-mentioned target substance measurement step is performed on a sample containing the above-mentioned target substance.

3. A quantitative method according to claim 1, A quantitative method characterized in that, for a sample containing the above-mentioned standard substance and the above-mentioned substance to be measured, the above-mentioned standard substance measurement step and the above-mentioned substance to be measured step are performed in the same step.

4. A quantitative method according to claim 1, A quantitative method characterized in that, in the above-described standard material measurement process, the standard material from which the measured retention time of the standard material is detected and the standard material from which the quantitative index of the standard material is detected are different standard materials.

5. A quantitative method according to claim 1, The above-described method for identifying a target substance is characterized by normalizing the time axis of the chromatogram obtained by the above-described method for measuring a target substance based on the measured retention time of the standard substance and the standard retention time of the standard substance, and identifying the target substance based on the normalized chromatogram and the standard retention time of the target substance.

6. A quantitative method according to claim 5, The above-described step for identifying the substance to be measured is a quantitative method characterized by setting a time window in the normalized chromatogram corresponding to the standard retention time of the substance to be measured, and identifying the substance to be measured that has a retention time that falls within the time window.

7. A quantitative method according to claim 5, The above-described step for identifying the substance to be measured is a quantitative method characterized by identifying, in the normalized chromatogram, the substance to be measured having a retention time within a predetermined range from the standard retention time of the substance to be measured.

8. A quantitative method according to claim 1, further, A quantitative method characterized by having a normality identification step for identifying the normality of the chromatograph apparatus based on the measurement results in the standard material measurement step described above.

9. A quantitative method according to claim 8, A quantitative method characterized in that the above-mentioned normality identification step, the above-mentioned target substance identification step, and the above-mentioned quantitative analysis step are performed in order.

10. A chromatograph apparatus used in the quantitative method of claim 1, A chromatograph unit that separates and measures the components contained in the sample, A control unit that performs the above-mentioned process of identifying the substance to be measured and the above-mentioned process of quantitative analysis, A chromatograph apparatus characterized by having the following features.

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