阅读说明：本技术 色谱质谱分析方法及色谱质谱分析装置 (Method and apparatus for chromatographic mass spectrometry ) 是由 前川彰 伊藤伸也 山下博教 于 2017-06-12 设计创作，主要内容包括：本发明提供一种色谱质谱分析方法,其具有下述工序：将具有与分析对象成分类似的保留时间并且质荷比不同的内标物质添加至试样的工序,利用色谱质谱分析装置测定试样,获得分析对象成分的色谱图(101)和内标物质的色谱图(102)的工序,由内标物质的色谱图检测峰(113),求出该峰的峰开始时间和峰结束时间的工序,以及将求得的峰开始时间和峰结束时间应用于分析对象成分的色谱图的峰开始时间和峰结束时间的工序。(chromatographic mass spectrometry methods include a step of adding an internal standard substance having a retention time similar to that of a target component and a different mass-to-charge ratio to a sample, a step of measuring the sample with a chromatographic mass spectrometer to obtain a chromatogram (101) of the target component and a chromatogram (102) of the internal standard substance, a step of detecting a peak (113) from the chromatogram of the internal standard substance to determine a peak start time and a peak end time of the peak, and a step of applying the determined peak start time and peak end time to the peak start time and peak end time of the chromatogram of the target component.)

The method for mass spectrometry of , comprising the steps of:

a step of adding an internal standard substance having a retention time similar to that of the component to be analyzed and a different mass-to-charge ratio to the sample,

measuring the sample with a chromatographic mass spectrometer to obtain a chromatogram of the analysis target component and a chromatogram of the internal standard substance,

a step of detecting a peak from the chromatogram of the internal standard substance and determining a peak start time and a peak end time of the peak, and

and applying the peak start time and the peak end time obtained to the peak start time and the peak end time of the chromatogram of the analysis target component.

2. The method of chromatographic mass spectrometry of claim 1,

adding a plurality of different internal standard substances as the internal standard substances to a sample,

obtaining a plurality of chromatograms as chromatograms of the internal standard substance,

detecting peaks from the plurality of chromatograms, determining peak start time and peak end time of each peak,

selecting or calculating 1 set of peak start time and peak end time from the obtained peak start times and peak end times,

applying the set of peak start time and peak end time to a peak start time and a peak end time of a chromatogram of the analysis target component.

3. The chromatographic mass spectrometry method according to claim 2, wherein the peak start time and the peak end time of the chromatogram having the largest signal-to-noise ratio among the plurality of chromatograms are selected as the set of peak start time and peak end time.

4. The method for chromatographic mass spectrometry according to claim 2, wherein an average value of peak start times and an average value of peak end times of chromatograms in which signal-to-noise ratios in the plurality of chromatograms are equal to or greater than a threshold value are calculated, and the respective average values are defined as the set of peak start times and peak end times.

5. The method of chromatographic mass spectrometry according to claim 2, wherein the earliest peak start time and the latest peak end time among the plurality of peak start times and the plurality of peak end times determined from the plurality of chromatograms are selected as the set of peak start times and peak end times.

6. The method of chromatographic mass spectrometry of claim 2,

when the difference between the signal intensities corresponding to the same retention time is obtained from the data obtained by normalizing the signal intensities of the chromatogram of the analysis target component and the chromatogram of 1 internal standard substance, and the dispersion is used as a noise value, the peak start time and the peak end time of the internal standard substance having the largest peak height to noise value ratio are selected as the set of the peak start time and the peak end time.

7. The method for chromatographic mass spectrometry according to claim 1, wherein a value of the square of the signal intensity at each point of the chromatogram of the internal standard substance is obtained, and the peak start time and the peak end time are obtained from the chromatogram with the value as a data point.

8. The chromatography-mass spectrometry method according to claim 1, wherein the internal standard substance is a stable isotope-labeled compound having a similar retention time to that of the analysis target component.

9, A chromatograph mass spectrometer for simultaneously measuring an analysis target component and an internal standard substance and obtaining a chromatogram of the analysis target component and a chromatogram of the internal standard substance, comprising:

a peak detection unit for determining a peak start point and a peak end point from a chromatogram of an internal standard substance,

a peak range detection section that extracts a peak start time and a peak end time from the peak start point and the peak end point detected by the peak detection section, an

And a peak range application unit that applies the peak start time and the peak end time extracted by the peak range detection unit to a peak start point and a peak end point of the chromatogram of the analysis target component.

10. The chromatographic mass spectrometry apparatus of claim 9,

the peak detection unit determines a peak start point and a peak end point of each peak from a plurality of chromatograms for different internal standard substances,

the peak range detecting unit selects or calculates 1 set of peak start time and peak end time from the plurality of peak start points and the plurality of peak end points obtained by the peak detecting unit,

the peak range application unit applies the set of the peak start time and the peak end time to a peak start point and a peak end point of a chromatogram of the analysis target component.

11. The chromatography-mass spectrometry apparatus according to claim 9, wherein the internal standard substance is a stable isotope-labeled compound having a similar retention time to that of the analysis target component.

12. The chromatographic mass spectrometry apparatus of claim 9, wherein the chromatogram is a liquid chromatogram.

Technical Field

The present invention relates to a chromatography-mass spectrometry method and a chromatography-mass spectrometry apparatus.

Background

In recent years, a quantitative analysis method using a liquid chromatography mass spectrometer has been widely used for analysis of drug components, metabolites, residues in environmental samples, and the like in biological samples, and liquid chromatography uses a high-speed type in which separation time by a column is a maximum of several tens of minutes, or a super high-speed type in which separation is performed for a maximum of minutes, and so on, and is a step further, mass spectrometers for detecting components after separation are used, for example, in a quadrupole mass spectrometer, an ion trap mass spectrometer, and a time-of-flight mass spectrometer, and they are used individually for analysis purposes, but in many cases, a quadrupole mass spectrometer is used for quantitative analysis.

The quadrupole mass spectrometer can measure a sample by scanning and Selective Ion Monitoring (SIM). Scanning is used for qualitative analysis of an unknown sample, and detects an ion amount with respect to a mass-to-charge ratio (m/z) within a predetermined range of the mass-to-charge ratio as a signal. The SIM is a device that selectively detects the amount of ions relative to a pre-specified mass-to-charge ratio. In triple quadrupole mass spectrometers and the like, Selective Reaction Monitoring (SRM) is also used, which selectively detects the amount of specific ionic ions generated from a component to be analyzed. When the mass-to-charge ratio of an ion derived from a component to be analyzed and the daughter ion is known, quantitative analysis can be performed with high sensitivity by these methods.

In order to confirm a drug component, a metabolite, a residue in an environmental sample, and the like in a biological sample, a chromatogram showing a temporal change in ion amount is obtained by SIM or SRM of a liquid chromatography mass spectrometer, peaks corresponding to an analysis target component and an internal standard substance are detected, and a measured value is obtained, in peak detection, generally, a peak area and a peak height are calculated as a measured value after determining a start point and an end point of a peak, and an analysis target component and an internal standard substance are determined by a retention time of a peak, but the retention time depends on a kind, a state, and a separation condition of a column of a liquid chromatography, and a peak detection method includes a method of detecting a change amount of data by gradient, and as a method of improving the method, there are a shoulder detection method as shown in patent document 1, and further a method of detecting by fitting an arbitrary function as shown in patent document 2, and the like.

Disclosure of Invention

Problems to be solved by the invention

When identifying a peak from a chromatogram and calculating the area and height of the peak, the peak is detected using, for example, the methods shown in patent documents 1 and 2. However, in the analysis of a drug component, a metabolite, a residue in an environmental sample, and the like in a biological sample, there are cases where an analysis target component is extremely small. In this case, a clear peak does not appear on the chromatogram, and it is necessary to detect the analysis target component by combining noise removal and advanced signal processing, which makes it difficult to detect the peak.

In the analysis by chromatography, there is a problem that the retention time varies. The retention time of the analysis target component is affected by clogging of the flow path, deterioration of the column, and subtle differences in the ambient temperature and the column temperature. Therefore, even if the retention time of the analysis target component under the predetermined analysis condition is known, the peak position is usually searched by setting a time range. In addition to the above-described situation in which peak detection is difficult, calculation of the measurement value of the analysis target component becomes more difficult in consideration of the retention time variation.

As described above, even when the analysis target component is a trace amount, a method capable of accurately identifying the peak position and calculating the measurement value is required.

Means for solving the problems

The chromatographic mass spectrometry method of the present invention comprises, as an mode, a step of adding an internal standard substance having a retention time similar to that of a target component and a different mass-to-charge ratio to a sample, a step of measuring the sample with a chromatographic mass spectrometer to obtain a chromatogram of the target component and a chromatogram of the internal standard substance, a step of detecting a peak from the chromatogram of the internal standard substance to determine a peak start time and a peak end time of the peak, and a step of applying the determined peak start time and peak end time to the peak start time and peak end time of the chromatogram of the target component.

The chromatograph mass spectrometer of the present invention, as the mode, is a chromatograph mass spectrometer that simultaneously measures an analysis target component and an internal standard substance and obtains a chromatogram of the analysis target component and a chromatogram of the internal standard substance, and includes a peak detection unit that obtains a peak start point and a peak end point from the chromatogram of the internal standard substance, a peak range detection unit that extracts a peak start time and a peak end time from the peak start point and the peak end point detected by the peak detection unit, and a peak range application unit that applies the peak start time and the peak end time extracted by the peak range detection unit to the peak start point and the peak end point of the chromatogram of the analysis target component.

Effects of the invention

According to the present invention, it is possible to calculate a measurement value without affecting the quality of a chromatogram of a component to be analyzed.

The problems, configurations, and effects other than those described above will be apparent from the following description of the embodiments.

Drawings

Fig. 1 is a conceptual diagram showing an example in which retention times at the starting point and the ending point of a component peak of an internal standard substance chromatogram are applied to the starting point and the ending point of a peak of an analysis target component chromatogram.

Fig. 2 is a schematic diagram showing a configuration example of a liquid chromatography mass spectrometer.

Fig. 3 is a conceptual diagram showing an example of a sample prepared by the internal standard method and an example of signal intensity when the sample is measured by a mass spectrometer.

Fig. 4 is a diagram showing an example of a calibration curve created by the internal standard method.

Fig. 5 is a graph showing an example of measurement result data of SRM in an unknown sample of testosterone and testosterone 13C.

Fig. 6 is a diagram illustrating an example of the graphical user interface.

Fig. 7 is a graph showing an example of applying the retention times of the peak onset and end points of testosterone d3 to the peak onset and end points of testosterone, testosterone 13C.

Fig. 8 is a functional block diagram of examples showing a configuration related to peak detection.

Fig. 9 is a flowchart showing a flow of processing related to peak detection.

Detailed Description

Embodiments of the present invention will be described below with reference to the drawings. The embodiments of the present invention are not limited to the examples described below, and various modifications can be made within the scope of the technical idea.

Here, a liquid chromatography mass spectrometer is given as an example of a chromatography mass spectrometer, but the present invention can also be applied to a gas chromatography mass spectrometer as long as the analysis target component and the internal standard substance are simultaneously measured.

[ example 1]

An example of detecting testosterone, which is a male hormone, will be described using the simplest apparatus configuration among ordinary liquid chromatography mass spectrometry apparatuses, and fig. 2 is a schematic diagram showing a configuration example of a liquid chromatography mass spectrometry apparatus.

The solvent transfer unit 201 transfers a solvent such as water or acetonitrile, and the solvent transfer unit 201 can perform isocratic transfer in which two or more solvents are provided and a ratio is fixed, and gradient transfer in which the composition of the solvent is changed by a time program, in general, , and the solvent transfer unit 201 often transfers the solvent other than during analysis, thereby achieving the balance and stabilization of the sample separation unit 204.

The sample introduction unit 202 sets a sample to be analyzed by an analyst, and suctions the sample in sequence by a sample introduction mechanism called an auto-sampler. In addition, each sample aspiration requires washing of the site such as the needle or the sample introduction channel. Therefore, although not shown, a washing mechanism is generally mounted separately.

The sample injection unit 203 is a site for injecting the sample aspirated by the sample introduction unit into a flow path from the solvent delivery unit. These are generally used in a hexagonal valve, and are combined with a device called a sample loop. The sample loop is usually connected to the side of the sample introduction section. When the sample is sucked, the sample sucked by the sample introduction section is introduced into the sample loop, and after the introduction is completed, the sample loop filled with the sample is switched to the flow path to the sample separation section, thereby constructing a structure in which the sample is injected from the solvent transfer section 201 into the flow path of the sample separation section 204.

The sample separation section 204 is provided with a column for separating each component of a sample, the so-called column is a column in which a filler such as silica gel or a polymer is filled in a metal cylinder, and if a sample is injected from one side thereof, and a solvent is continuously transported in step , components move in the column at a speed corresponding to the affinity between the components and the filler.

The mass spectrometer 205 ionizes the components separated by the sample separator 204 and detects the mass-to-charge ratio. Examples of the ionization method include electrospray ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI). In addition, the mass spectrometer section generally uses a quadrupole type, an ion trap type, a time-of-flight type, or the like. In the quadrupole type, there is a triple quadrupole type in which 3 stages are arranged, except for the case where 1 stage of quadrupole is arranged. In some cases, the ion selected in the 1 st stage is split in the 2 nd stage, and a substance having a specific mass-to-charge ratio is selected from the generated ion in the 3 rd stage and measured. Such an assay is referred to as SRM.

further includes a step of transmitting the signal of the amount of ions detected by the mass spectrometer 205 to the analyzer 206, and performing various data processing such as smoothing processing, peak detection processing, area and height calculation processing as chromatograms of the SIM and SRM.

When the content and concentration of a specific component in an unknown sample are to be analyzed, a plurality of standard samples of concentration series prepared in a target concentration range are first analyzed by an analyzer, and a graph showing the relationship between the measured value and the concentration is derived from the correlation between the calculated measured value and the known concentration. This graph is referred to as the standard curve. Next, the unknown sample is measured, and after the measurement value of the component to be analyzed is calculated, the concentration value suitable for the calibration curve is calculated. Thus, a sample of unknown concentration can be measured by preparing a calibration curve, and the concentration can be estimated from the calibration curve for the measured value.

In the case of quantitative analysis by a liquid chromatography mass spectrometer, is used as a general method, and in the case of the internal standard method, for example, an internal standard substance having a retention time similar to that of a target component and a different mass-to-charge ratio is added to a sample collected from a living body, fig. 3 is a conceptual diagram showing an example of a sample produced by the internal standard method and an example of a signal intensity when the sample is measured by the mass spectrometer, and standard samples 311 to 313 and an unknown sample 314 in which amounts of the internal standard substance 302 are added to a plurality of concentrations of the target component 301 corresponding to a series of measured concentrations are measured, and in data processing, first, a standard curve is created by performing peak detection on each of the peaks 321 of the target component and the peak 322 of the internal standard substance for the standard samples 311 to 313, and correlating the ratio of the measured values, that is (measured value of the target component)/(measured value of the internal standard substance) with a known concentration.

Fig. 4 is a diagram showing an example of a calibration curve created by the internal standard method. A represents the measured value of the component to be analyzed in the reference samples 311 to 313₁～a₃The measured value of the internal standard substance is represented by i₁～i₃The concentration c of the analyte component contained in each standard sample can be determined based on the known concentration c₁～c₃A standard curve 401 is drawn. Then, the unknown sample 314 is measured, and the measurement value a of the component to be analyzed can be obtained_uAnd inMeasurement value i of target substance_uCalculating the concentration c of the component to be analyzed contained in the unknown sample_u. That is, the value (a) of the ratio can be adjusted_u/i_u) Concentration c was determined by applying to calibration curve 401_u。

In this way, the internal standard method, which calculates the ratio of the measurement values of the analysis target component and the internal standard substance, has an advantage that error factors such as an error in the amount of sample to be introduced and an error due to volatilization of the solvent are canceled out by the ratio calculation without affecting the result. Therefore, it is necessary to select the internal standard substance so that the chemical behavior of the analysis target component and the internal standard substance are as identical as possible.

Specifically, a stable isotope labeled compound in which parts of atoms constituting the analysis target component are replaced with a nitrogen stable isotope 15N, a carbon stable isotope 13C, an oxygen stable isotope 18O, a hydrogen stable isotope 2H, or the like is used as the internal standard substance, and testosterone (hereinafter, referred to as testosterone 13C) labeled with 13C, for example, can be exemplified as testosterone, and the concentration thereof can be set to an arbitrary amount depending on the sensitivity and accuracy of the mass spectrometer, and 500fmol, for example, can be exemplified.

Fig. 1 is a conceptual diagram showing an example in which retention times of component peak start points and end points of an internal standard substance chromatogram are applied to peak start points and end points of an analysis target component chromatogram.

When an analysis target component and an internal standard substance are simultaneously measured by SRM, two chromatograms, that is, a chromatogram 101 of the analysis target component and a chromatogram 102 of the internal standard substance are obtained as shown in fig. 1.

In such a case, conventionally, (a group of) a measurement value and a retention time of a peak top or the like in a predetermined region is obtained by detecting a peak (group) in a chromatogram 101 of an analysis target component, the measurement value and the retention time of the peak top or the like are obtained. From the peak (group) thus obtained, the peak 123 closest to the retention time of the peak of the internal standard substance was selected as the analysis target component. In the case where a threshold value of the signal intensity is set to detect the peak, the peak 123 cannot be detected by the threshold value. Therefore, if the threshold is lowered, although there is insufficient signal intensity, a method of detecting the gradient of the amount of change in signal intensity and a complicated process of peak detection such as fitting to a gaussian function are repeated, and it is difficult to obtain reliable results.

However, as described above, since the internal standard substance is selected so as to have the same chemical behavior as the analysis target component as much as possible, the retention times in the two chromatograms are substantially equal, that is, the retention times at the peak start point and the peak end point in the chromatogram 101 of the analysis target component are considered to be similar to the retention times at the peak start point and the peak end point of the internal standard substance.

It is desirable that the retention time of the internal standard substance is the same as that of the analysis target component, but if the retention time of the analysis target component and the retention time of the internal standard substance are different to such an extent that the influence on the calculation of the peak area and the peak height of the analysis target component can be ignored, there is no problem.

Fig. 8 is a functional block diagram of examples showing a configuration relating to peak detection provided in the analysis unit 206 of the liquid chromatography mass spectrometer of the present embodiment, and describes peak determination of the present embodiment with reference to fig. 8 .

Peak determination is performed by a peak detection unit 801 on the chromatogram of an internal standard substance obtained from a SIM or SRM, and a peak start point 806 and a peak end point 809 and internal standard peak fraction data 808 are output, a peak start time 813 and a peak end time 814 are extracted from the peak start point and the peak end point in a peak range detection unit 802, a component peak fraction data 810 is output from an analysis target component chromatogram by using the peak start time and the peak end time in a peak range application unit 803, and an internal standard measurement value (area, height) and a component measurement value (area, height) are respectively obtained from the internal standard peak fraction data 808 and the component peak fraction data 810 in a peak calculation unit 804, and it is described in example 1 that the peak range detection unit 802 extracts the peak start time 813 from only the peak start point 806 and the peak end time 814 from only the peak end point 809, and the function of the peak range detection unit 802 at step is described in example 2.

After data collection (S11), peak detection of the chromatogram of the internal standard substance is performed in the peak detection unit 801 (S12), further peak range detection is performed by the peak range detection unit 802 (S13), peak range application to the chromatogram of the analysis target component is performed by the peak range application unit 803 based on the peak start time 813 and the peak end time 814 obtained by the peak range detection unit 802 (S14), and peak calculation is performed in the peak calculation unit 804 (S15).

By following the above procedure, it is possible to calculate a precise measurement value without being affected by the presence or absence of a signal derived from a component to be analyzed and the signal intensity, and it is possible to calculate a measurement value even in a situation where the peak detection of a component to be analyzed is difficult by calculating the area and height from the chromatogram of a component to be analyzed using the retention time at the peak start point and the peak end point of the internal standard substance by including an internal standard substance quantified by in a total sample and determining the peak in the chromatogram of SIM or SRM.

Further, even when the problem of the retention time fluctuation as shown in the problem section occurs, it is assumed that the fluctuation showing the same behavior occurs in both the analysis target component and the internal standard substance. Therefore, even when the retention time varies in a situation where a peak of the analysis target component is not detected as described above, the measurement value can be accurately calculated by the method of the present embodiment.

Conventionally, there are cases where the peak detection is difficult due to a trace amount of the analysis target component, and cases where the peak detection is impossible due to noise determination due to the influence of a large amount of impurity components. However, by determining the peak of the analysis target component from the retention time at the peak start point and the peak end point of the internal standard substance, even in such a situation, the measurement value of the target component can be obtained, and robustness (robustness) is excellent.

In this example, testosterone 13C as an internal standard substance was used for testosterone as an analysis target component, that is, testosterone 13C quantified by as an internal standard substance was added to a sample, and the sample was measured by a liquid chromatography mass spectrometer, thereby obtaining a chromatogram of the analysis target component and a chromatogram of the internal standard substance.

Examples of analytical conditions for testosterone and testosterone 13C are shown. In the liquid chromatography, a solvent was a water/acetonitrile solution at a flow rate of 0.2mL/min, and a C18 column (particle diameter: 5 μm, tube diameter: 2.0 mm. times.50 mm) was used as the column. The retention time under this condition can be exemplified by, for example, 64 seconds. The separated sample is ionized by, for example, ESI ion source and measured by SRM using a triple quadrupole mass spectrometer.

Fig. 5 is a graph showing an example of measurement result data of SRM in an unknown sample of testosterone and testosterone 13C. This figure shows an example of applying the retention times of the peak onset and end points of testosterone 13C to the peak onset and end points of testosterone. The chromatogram 501 of testosterone is the result of the separation of components and the measurement of SRM under the above analysis conditions, and Q1 and Q3 should be set to have mass-to-charge ratios of 289 and 97, respectively. Furthermore, the chromatogram 502 of testosterone 13C is a result obtained by measuring testosterone 13C by SRM similarly, and Q1 and Q3 should be set to have mass-to-charge ratios of 292 and 100, respectively.

In the analysis result example, since the concentration of testosterone as an analysis target component was low and the peak 505 of testosterone in the chromatogram 501 was unclear, accurate peak detection was difficult, and in addition, , the signal-to-noise ratio (S/N) of the peak 506 of testosterone 13C added in a sufficient amount as an internal standard substance was good, and a peak that could be identified was easily obtained.

In this example, peak detection was first performed for peak 506 of testosterone 13C. Thereby, the peak start point 511 and the peak end point 512 are detected. These 2 points were connected and a baseline 504 for testosterone 13C was drawn and the area and height of the peak 506 of testosterone 13C was calculated. Next, the retention times of the peak onset 511 and the peak end 512 detected for the peak 506 of testosterone 13C were applied to the chromatogram 501 of testosterone as the peak onset and the peak end of the peak 505 of testosterone. The signal intensity at each point was set to a value corresponding to the retention time at the peak start point 511 and the peak end point 512 in the arrangement of the signal intensities in the chromatogram 501 of testosterone. The 2 points are connected and a baseline 503 of testosterone is drawn and the area and height of the testosterone peak 505 is calculated. Finally, the area ratio and height ratio of testosterone to testosterone 13C were calculated, and the concentration was calculated from the standard curve.

In addition, since SRM detects ion amounts corresponding to a plurality of mass-to-charge ratios in a time division manner, retention times of data points in a chromatogram 501 of testosterone and a chromatogram 502 of testosterone 13C may not be , and in such a case, data points in a chromatogram 501 of testosterone that are closest to retention times of a peak start point 511 and a peak end point 512 detected with respect to a peak 506 of testosterone 13C may be set as a peak start point and a peak end point of a peak 505 of testosterone.

By using the above method, peak detection of peak 505 of testosterone under conditions that are difficult to identify is not performed, but it is possible to draw a baseline and calculate the area and height. That is, even when the peak recognition is difficult due to a large amount of noise, and otherwise, when the problem of variation in retention time occurs, the area and height of the analysis target component can be calculated more accurately.

In addition, as another effect, since peak detection of the analysis target component is not necessary, the load of the data analysis process is reduced. In particular, since the internal standard substance is usually measured by a detector in a detectable concentration range with sufficient S/N, it is possible to accurately calculate the peak start point and the peak end point by a relatively simple algorithm. That is, the retention time at the peak start point and the peak end point is applied to the chromatogram of the analysis target component, and therefore, the peak of the analysis target component whose concentration region is unknown and whose retention time varies can be detected with certainty, and the load of the wide-range data analysis processing can be reduced.

Fig. 6 is a diagram showing examples of a graphical user interface in the analysis unit 206 for instructing the peak detection method in the present embodiment, and it is noted that an analysis target component and an internal standard substance which are targets of peak detection are set as detection target components.

In fig. 6, "ID" is a mark that designates the number of a detection target component in order from 1, and the number of ID corresponds to the measurement channel of the SRM of the mass spectrometer. They may employ automatic input according to the setting conditions of the detection target components. "name" is the name of the detection target component. For this item, the user can freely input a name, but cannot set two or more identical names. "expected RT" specifies the retention time of the peak top of the detected target component. The "RT range" specifies a range of peaks determined as detection target components. Within the range of retention time specified by the column, the peak whose peak top is closest to the expected RT is determined as the peak of the detection target component. In this example, the peak of testosterone 13C with ID ═ 2 was determined over a range from (expected RT-RT range) up to (expected RT + RT range), i.e. retention times of 64 ± 10 seconds.

In "IS ID", the ID of the internal standard substance IS specified. In the case of the content material itself, the column sets 0 as a special value. In addition, in the case of a component to be analyzed, the ID of the internal standard substance is specified as the ratio of the area to the height of the component to be analyzed. In the example of fig. 6, testosterone with ID-1 requires testosterone 13C with ID-2 to be set as an internal standard, and IS ID IS therefore set to 2. In addition, testosterone 13C is an internal standard substance and is therefore set to a particular value of 0.

In the present embodiment, the input point of the target method IS selected from among a plurality of peak detection methods, and as an example of the selection items, the input point IS selected from, for example, a gradient detection method (selected item name Delta), a peak detection method (selected item name Fitting) by Fitting, a peak start/end time (With IS) using an internal standard substance, and the like.

As described above, the peak detection method can be set in the analysis target component and the internal standard substance when the peak detection process is performed on the chromatogram obtained by the SRM of the liquid chromatography mass spectrometer.

[ example 2]

In the SRM using the mass spectrometer, a plurality of channels can be usually set, and therefore, testosterone 13C as an internal standard substance is measured at the same time in the measurement of testosterone in example 1, in this example, in addition to testosterone 13C shown in example 1, an example in which testosterone d3 obtained by substituting part of hydrogen atoms contained in testosterone with deuterium is added as an internal standard substance to a sample and these are measured at the same time is shown in step .

Fig. 7 is a graph showing an example of simultaneous measurement of testosterone, testosterone d3, and testosterone 13C. Here is shown an example of applying the retention time of the peak onset and end points of testosterone d3 to the peak onset and end points of testosterone, testosterone 13C. The chromatogram 701 of testosterone, the chromatogram 702 of testosterone d3, and the chromatogram 703 of testosterone 13C were simultaneously and individually determined by SRM using a mass spectrometer.

The mass-to-charge ratio to be set in the SRM conditions for the testosterone chromatogram 701 and the testosterone 13C chromatogram 703 was the same as in example 1. The mass to charge ratios that should be set in the chromatogram 702 of testosterone d3 were 292 and 97 for Q1 and Q3. Under this assay condition, testosterone d3, and testosterone 13C were measured simultaneously, and peak 711 of testosterone, peak 712 of testosterone d3, and peak 713 of testosterone 13C were detected.

For testosterone d3, testosterone 13C as internal standard substances, peaks were clearly detected, but peak 712 of testosterone d3 compared with peak 713 of testosterone 13C, the signal to noise ratio (S/N) was good. In such a situation, the peak 712 of testosterone d3, which is the best S/N internal standard substance, for example, can be used to detect the peak start 721 and peak end 722, and the quantitative values of testosterone and testosterone 13C can be calculated from their retention times.

In this example, 2 kinds of internal standard substances were used, but 3 kinds or more than 3 kinds may be used. In the case of using 2 or more internal standard substances, a plurality of chromatograms as chromatograms of the internal standard substances are obtained. Peaks are detected from each of these chromatograms, and if the peak start time and peak end time of each peak are found, a plurality of peak start times and a plurality of peak end times are obtained. From these plural peak start times and plural peak end times, 1 set of peak start times and peak end times is selected or calculated, and this set of peak start times and peak end times is applied to the peak start times and peak end times of the chromatogram of the analysis target component. Examples of a method for determining a set of a peak start time and a peak end time to be applied to a chromatogram of a component to be analyzed include the following methods.

(1) For example, a method of selecting substances with the best S/N.

In the chromatogram of the internal standard substance, a section near the peak of the internal standard substance is defined as a noise determination region, a half value of the difference between the maximum value and the minimum value of the signal intensity in the section is defined as N, and the height of the peak of the internal standard substance is defined as S. The S/N is obtained from each of the chromatograms of the plurality of internal standard substances, and the set of the peak start time and the peak end time of the peak of the internal standard substance among the substances having the largest values among the values is applied as the peak start time and the peak end time in the chromatogram of the analysis target component. This enables more stable peak detection, and can suppress errors and variations in calculation of the quantitative value.

(2) For example, a method of selecting a substance having S/N of a value equal to or greater than a threshold value and using the peak start time and peak end time of each selected peak as the average value of the peak start time and peak end time is used.

The chromatogram of an internal standard substance having S/N not less than a threshold is selected, and the average value of peak start times and the average value of peak end times in these chromatograms are calculated. These are used as a set of peak start time and peak end time to be applied to a chromatogram of an analysis target component. This can average the influence of the impurity components and noise, and contributes to stable peak detection.

(3) For example, a method of performing peak detection of all internal standard substances, and selecting a peak start point of the earliest retention time and a peak end point of the latest retention time.

The earliest peak start time and the latest peak end time among the plurality of peak start times and the plurality of peak end times obtained from the chromatogram of the plurality of internal standard substances are selected as a set of peak start times and peak end times to be applied to the chromatogram of the analysis target component. By selecting the earliest peak start time and the latest peak end time, a wider range is considered as a peak, and stable peak determination is possible with respect to fluctuations in the peak start point and the peak end point of the component to be analyzed.

(4) For example, in the case of obtaining the difference between the signal intensities corresponding to the same retention time in the data obtained by normalizing the intensity of the chromatogram of the analysis target component and the chromatogram of any internal standard substance, and setting the dispersion as a noise value, an internal standard substance having the highest ratio of the peak height to the noise value of the internal standard substance is selected, and the peak start time and the peak end time are selected.

The maximum value and the minimum value of each signal intensity in the chromatogram of the analysis target component are normalized by 1 and 0, and the signal intensities in the chromatogram of the internal standard substance at step are also normalized by 1 and 0, the difference between the signal intensities corresponding to the same retention time is obtained, and the dispersion is set as a noise value.

(5) For example, a method of determining a square value of signal intensity at each point of a plurality of chromatograms, performing peak detection of the chromatograms using the calculated value as a data point, and determining a peak start time and a peak end time.

Peak determination is performed on a chromatogram obtained by squaring the signal intensity at each retention time of the chromatogram of the internal standard substance, thereby enabling peak determination with noise suppressed. This method, which is related to the selection of the peak start time and the peak end time, can be used in combination with the above-described method.

When 2 or more internal standard substances are used, the peak detection unit 801 in the block diagram of fig. 8 is applied a plurality of times, and the peak range detection unit 802 determines the peak start time 813 and the peak end time 814 by referring to the plurality of peak start points 806, the plurality of peak end points 809, the chromatogram of each internal standard substance, and the chromatogram of the analysis target component, further .

By the above method, the peak start point and the peak end point of the analysis target component can be accurately detected from the peak start point and the peak end point of the internal standard substance.

For example, the above-described embodiments are described in detail to facilitate understanding of the present invention, and it is not limited to the case where the present invention includes all of the described configurations, and , it is also possible to replace portion of the configuration of a certain embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of a certain embodiment, and it is also possible to add, delete, and replace portion of the configuration of each embodiment with another configuration.

Description of the symbols

101: chromatogram of analysis target component, 102: chromatogram of internal standard substance, 111: peak start point, 112: peak end point, 121: peak start point, 122: peak end point, 201: solvent conveying section, 202: sample introduction portion, 203: sample injection section, 204: sample separation unit, 205: mass spectrometry section, 206: analysis unit, 511: peak start point, 512: peak end point, 721: peak start point, 722: peak end point.