JP2008256667A - Quantitative analysis method using mass spectrometer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for evaluating reliability of analysis data in the case where a quantitative analysis is carried out without using any isotopic labeling method. <P>SOLUTION: An internal standard substance detected simultaneously with an analytical component is mixed in a mobile phase or an eluate of a liquid chromatograph, and a mass chromatogram of an ion derived from the internal standard substance is acquired and recorded by using a data analyzing section. Then, an analysis sample is mixed to obtain analysis data of the sample, and the strength of the ion derived from the internal standard substance is compared with that of a blank sample at an analysis actual time in the data analyzing section. If a miscompare is detected, such the judgement is made that a quantitative analysis inhibitory factor is generated, and its analysis mode is changed from a quantitative analysis mode in which the priority of a tandem mass spectrometry is low, to a qualitative analysis mode in which a high priority is put on the tandem mass spectrometry. If the strength of the ion derived from the internal standard substance matches with that of the blank sample in the analysis actual time, the analysis mode is changed into the quantitative analysis mode again. In a matching time zone, the analysis data are collected, and analyzing is carried out efficiently and precisely. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a mass spectrometry system for bio-related substances and organic substances, and more particularly to a quantitative mass spectrometry method and a mass spectrometry system for pharmacokinetics or metabolite analysis in drug discovery, protein analysis, and disease marker search.

  When searching for markers for disease diagnosis, it is important to compare the analysis results of samples from patients with diseases and samples from healthy individuals, and extract the components with significant differences from the detected components. . Furthermore, it is necessary to identify the fluctuation component. In such analysis, a liquid chromatograph / mass spectrometer (LC / MS) is often used. LC / MS is an on-line separation / analysis system that can separate samples containing multiple components with LC and mass-analyze the separated components with MS, and is an analyzer widely used in fields other than marker search. . In the mass spectrometer (MS) portion, a mass spectrometer having a high mass resolution capable of performing tandem mass spectrometry such as MS / MS analysis, which can secure a dynamic range of 3 digits or more, is often used. This makes it possible to accurately perform a series of analyzes such as extraction of variable components by quantitative analysis, identification (qualitative analysis) of many unknown components in the variable components, and measurement of content by quantitative analysis.

  As is well known, tandem mass spectrometry is a technique that selects ions of a certain component from the results of mass analysis and decomposes them by colliding them with gas molecules, and further performs mass analysis of the ions that have been decomposed. It is common to carry out identification (qualitative analysis) of substances. On the other hand, in quantitative analysis, a mass spectrum (mass spectrum) that does not use tandem mass spectrometry is often acquired. If qualitative analysis by tandem mass spectrometry is performed focusing on ions of a certain substance, data of mass spectrometry that does not use tandem cannot be acquired during that time, and the accuracy of quantitative analysis is substantially reduced. . Therefore, it is necessary to control not to perform tandem analysis when performing quantitative analysis. Therefore, conventionally, first, qualitative analysis for performing tandem mass spectrometry for identification is performed, and then quantitative analysis for acquiring a mass spectrum is performed.

  According to the conventional quantitative analysis procedure, first, a standard substance is analyzed at several concentrations. And the time change (mass chromatogram) of ionic strength is acquired with respect to m / z (mass / charge ratio) of the ion derived from the standard substance, and the peak area of the mass chromatogram is obtained. A calibration curve is created from the relationship between the area and the sample substance concentration. Next, the same substance whose concentration is unknown is analyzed to determine the peak area of the mass chromatogram. Then, based on the prepared calibration curve, the substance concentration corresponding to the peak area of the mass chromatogram is determined. Since this method is premised on obtaining a standard substance in advance and creating a calibration curve, it is difficult to apply this method to unknown components that are targets for marker search.

  Patent Document 1 describes a method of searching for a marker by performing a relative quantitative analysis on an unknown component for which a calibration curve cannot be created. According to this method, analysis data is first acquired with a standard sample containing various components, then analysis data is acquired with another sample that is expected to contain the same component, and the ionic strength for each component is acquired. The ratio (or area ratio) is calculated. And a standard ionic strength ratio is calculated | required and the ionic strength ratio of each component is normalized using the value. By the above, the relative ionic strength ratio of each component can be determined, and the component (marker candidate) with the remarkable fluctuation | variation of ionic strength can be specified. In order to identify a marker candidate, an analysis in which tandem mass spectrometry is preferentially performed separately is necessary.

  In addition, relative quantitative analysis can be performed by using an isotope labeling method for an unknown component for which a calibration curve cannot be created. That is, relative quantitative analysis is possible by mixing a sample containing various components and a standard sample labeled with an isotope (Non-Patent Document 1). Isotope-labeled components are detected at the same time as unlabeled components, but the m / z of detected ions differs by a predetermined value, so compare the ion intensity (or peak area) of the mass chromatogram for that ion pair. Thus, the concentration ratio of each component can be determined. If this method is adopted, quantitative analysis can be performed without problems. However, this method has limitations on the types of samples that can be applied, and is laborious and costly. Therefore, the isotope labeling method is often not adopted in marker search.

  As the LC / MS interface, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), etc. are used. It is known that a quantitative analysis inhibiting factor called matrix effect or ion suppression occurs particularly in an interface using ESI. The quantitative analysis inhibiting factor is a phenomenon in which the ionic strength fluctuates due to the following contention of charges between components, and even when performing relative quantitative analysis as described in Patent Document 1, If suppression occurs, the analysis result of analysis data may impair reliability. Incidentally, the matrix effect is a phenomenon in which the ionic strength is reduced when a sample contains a large amount of ionic components, and can be avoided if desalting is sufficiently performed in sample preparation.

  Ion suppression occurs when the ionization target component is equal to or greater than the maximum ion amount that can be generated at the interface (ionization unit). When this phenomenon occurs, charge competing occurs between various components, and the ionization efficiency varies depending on the chemical nature and amount of each component. As a result, the linearity is lost in the relationship between the detected ion intensity and the component concentration (Non-Patent Document 2). The maximum amount I of ions that can be generated has the following relationship when the liquid flow rate Q, the liquid electrical conductivity κ, and the surface tension γ are used.

I = β (ε) (Qκγ / ε) ^1/2 (1)
Here, β is a constant, and ε is the dielectric constant of the liquid. From formula (1), it is shown that it is effective to increase the electrical conductivity κ of the liquid in order to prevent the occurrence of ion suppression. However, if the electric conductivity κ is too high, the ion generation efficiency is reduced. Therefore, it is desirable to set κ within a range in which ions can be efficiently generated when the amount of acid or the like added to the mobile phase. In other words, since the electrical conductivity κ needs to satisfy two conflicting needs, it cannot actually be set to a high enough value to reduce ion suppression. Therefore, it is difficult to effectively prevent the ion suppression phenomenon regardless of conditions.

  The above problems may occur not only in ESI but also in other LC / MS interfaces such as atmospheric pressure chemical ionization (APCI) and in the ionization section of GC / MS. This is because there is an upper limit on the maximum amount of ions that can be generated in the interface (ionization section).

  As a method for detecting the occurrence of a quantitative analysis-inhibiting factor such as ion suppression, there is an ion intensity monitor using an internal standard substance. For example, Non-Patent Document 3 uses isocratic LC (flow rate 0.25 mL / min) with a constant mobile phase component, and infuses an internal standard substance from the downstream side of the separation column (flow rate 5 μL / min). A method for evaluating sample preparation by introduction is described. If the sample is not sufficiently purified, a quantitative analysis inhibition factor occurs due to the influence of the salt contained in the sample immediately after the introduction of the sample, and the intensity of the ion derived from the internal standard substance is reduced. By monitoring this ionic strength, a quantitative analysis inhibiting factor can be detected. However, since this method ionizes after passing a relatively long path after LC separation, the separation accuracy tends to decrease when the LC flow rate is low, and it is effective for semi-micro LC and general-purpose LC with high LC flow rate. It is difficult to apply to separation means such as micro LC and nano LC (capillary LC) with low LC flow rate. Furthermore, the internal standard has not been optimized.

  If the occurrence of a quantitative analysis-inhibiting factor is detected, review the sample preparation to increase the purity of the sample and eliminate impurities that may interfere with quantitative analysis in the sample, or review the LC separation conditions. The quantitative analysis is performed again, for example, by reducing the types of various components contained in the separated components by separating over time.

USP6835927 Y. Ishihara, T. Sato, T. Tabata, N. Miyamoto, K. Sagane, T. Nagasu, Y. Oda, Quantitative mouse brain proteomics using culture-derived isotope tags as internal standard, Nature Biotechnology 23 (2005) 617- 621. K. Tang, J. S. Page, R. D. Smith, Charge competition and the linear dynamic range of detection in electrospray ionization mass spectrometry, Journal of American Society for Mass Spectrometry 15 (2004) 1416-1423. R. Bonfiglio, R. C. King, T. V. Olah, K. Merkle, The effects of sample preparation methods on variability of the electrospray ionization response for model drug compounds, Rapid Communications in Mass Spectrometry 13 (1999) 1175-11885.

  As described above, in marker search, samples from variable patients and healthy individuals are compared to extract variable components, and the unknown component substances consisting of many types that make up the variable components are identified accurately. Therefore, it is required to perform quantitative analysis of component substances with high sensitivity and high accuracy without being affected by factors that hinder quantitative analysis such as ion suppression phenomenon. Moreover, it is required to perform this series of analyzes in as short a time as possible, that is, high throughput. It is also necessary to perform a series of analyzes at the lowest possible cost.

  In response to this requirement, analysis using a calibration curve, which is a conventional quantitative analysis technique, needs to acquire calibration curve data from a standard material in advance, and cannot be applied to quantitative analysis including unknown substances. The analysis using the isotope labeling method as described in Non-Patent Document 1 is also limited in the types of samples, takes time, and is expensive. In addition, the relative quantitative analysis method described in Patent Document 1 has a problem that the accuracy of the analysis result is lowered because the influence of the fluctuation of the ion intensity due to the ion suppression phenomenon cannot be avoided.

  Therefore, an issue for marker search is to perform an analysis that does not use an isotope labeling method and that is not affected by factors that inhibit quantitative analysis such as ion suppression. However, when quantitative analysis is performed without using the isotope labeling method, it is necessary to perform the analysis again when a quantitative analysis-inhibiting factor occurs. Therefore, it is important in terms of cost and speed to minimize the number of qualitative analyzes and quantitative analysis.

  High throughput has the following problems. In marker search, quantitative analysis that acquires a mass spectrum without performing tandem mass analysis and qualitative analysis that performs tandem mass analysis for identification are required. Moreover, in many cases, a sample composed of a very large number of components is analyzed, but in that case, it is often difficult to perform a qualitative analysis on all components detected in one analysis (Patent Document 2). . This is because the throughput of tandem mass spectrometry is limited. Therefore, a large number of tandem mass spectrometry is required, and the throughput of a series of marker searches cannot be increased.

  In addition, as a method for detecting a quantitative analysis inhibition factor, the method described in Non-Patent Document 3 cannot ensure accuracy in quantitative analysis using a low liquid flow rate LC or a gradient mode LC, and therefore the liquid flow rate is low. There is a problem that high sensitivity analysis by LC and analysis of a small amount of sample cannot be performed. When the LC flow rate is low, it is necessary to introduce the internal standard material from the upstream of the separation column. For this purpose, it is necessary that the internal standard material is not adsorbed on the separation column. This is a very important problem not only in the isocratic mode in which the mixing ratio of the organic solvent in the LC mobile phase is constant, but also in the gradient mode in which the ratio changes with time. Therefore, it is necessary to fully consider the chemical properties of the internal standard.

  An object of the present invention is to solve the above problems, and to perform qualitative and quantitative analysis with high sensitivity and high throughput without using the isotope labeling method and without being affected by factors that inhibit quantitative analysis. Provided is an analysis method for acquiring reliable data with a minimum number of analyzes for qualitative and quantitative analysis without using an isotope labeling method.

  The further subject of this invention is providing the internal standard substance which detects generation | occurrence | production of the quantitative analysis inhibition factor.

JP2005-091344

  In order to solve the above problems, the following analysis methods are provided. That is, first, an internal standard substance detected at the same time as the analysis component is mixed with the mobile phase or eluate of a liquid chromatograph, and a mass chromatogram of ions derived from the internal standard substance is obtained under conditions that do not cause quantitative analysis inhibition. And recorded in the data analysis unit. Typically, a blank sample containing no analysis sample is analyzed. Next, the analysis sample is mixed to obtain analysis data of the sample. At this time, the data analysis unit compares the intensity of the internal standard substance-derived ions with that at the time of blank sample analysis in the analysis real time. At this time, if a mismatch is detected in the ionic strength, it is determined that a quantitative analysis inhibition factor has occurred during mixing of the analysis sample, and in this case, the accuracy of the qualitative analysis results decreases due to the quantitative analysis inhibition factor. Is changed from the quantitative analysis mode in which the priority of tandem mass spectrometry is low to the qualitative analysis mode in which priority is given to tandem mass spectrometry. Then, if the intensity of the internal standard substance-derived ion coincides with that of the blank sample in the actual analysis time, for example, by reducing the amount of the analysis sample mixed, the quantitative analysis mode is changed again. Further, when there is a time zone in which the intensities of the ions derived from the internal standard substance match in the mass chromatogram, the analysis data of the sample in this matching time zone is acquired as effective analysis data. In this analysis method, a substance having the property of being stably detected during the actual analysis time is used as the internal standard substance.

  In addition, in the case of positive ion analysis, the isoelectric point is not significantly lower than the pH (hydrogen ion concentration) of the mobile phase as a substance that reacts sensitively with the internal standard substance to quantitative analysis inhibition factors such as ion suppression. Provide a material with low hydrophobicity. On the other hand, in the case of negative ion analysis, a substance having high basicity and low hydrophobicity is provided.

  In order to further improve efficiency, two types of internal standard substances with greatly different sensitivity to analysis inhibition factors are introduced, analysis samples are mixed and analyzed, and mass chromatograms of ions from both internal standard substances are compared. This also provides a method for detecting the occurrence of an analysis inhibiting factor in a single analysis. Select two types of internal reference materials with different isoelectric points as two types of internal reference materials with greatly different sensitivities to analysis-inhibiting factors.

  In addition, one type of internal standard substance is introduced, and other substances that exist in the analysis solution and can become the second internal standard substance are searched for as the second internal standard substance, and their mass chromatograms are compared. This also provides a method for detecting the occurrence of an analysis-inhibiting factor in a single analysis.

  Furthermore, as an analysis apparatus for solving the above-mentioned problems, a mobile phase introduction unit that mixes an internal standard substance and introduces a mobile phase, a sample introduction unit, a separation unit, an ionization / mass analysis unit, a data analysis unit, and an image Means for acquiring and storing a first mass chromatogram of ions derived from the internal standard substance without mixing the analysis sample, and a second part of the ions derived from the internal standard substance in a state where the analysis sample is mixed; An analysis apparatus having means for acquiring and comparing the mass chromatograms and means for collecting analysis data when the degree of mismatch between the first and second mass chromatograms is below a certain level is provided.

  Also provided is an apparatus having means for acquiring and comparing mass chromatograms of both in the state where the first and second internal standard substances are mixed in the mobile phase and collecting analysis data according to the comparison result. In addition, with the first internal standard substance mixed in the mobile phase, there is also an analytical data monitor means for searching for substances that can become the second internal standard substance in the analysis solution, and obtained by searching. An apparatus for acquiring and comparing mass chromatograms of the second internal standard substance and the first internal standard substance and collecting analysis data according to the comparison result is also disclosed.

  An internal standard is mixed with the LC mobile phase and eluate, and a blank sample is first analyzed under conditions that do not cause an analysis-inhibiting factor. Next, the analysis sample is analyzed. At that time, the intensity of the ion derived from the internal standard substance is measured in the analysis real time and compared with the analysis result of the blank sample in the analysis real time. It is possible to accurately detect whether or not an obstruction factor has occurred. Furthermore, the error of quantitative data can be evaluated based on the degree of mismatch.

  Comparing the mass chromatograms of the internal standard substance-derived ions at the time of mixing the blank sample and the analysis sample, the data in the matching time zone is determined not to be affected by the analysis inhibiting factor, and the time zone in which the ion intensity matches By acquiring the analysis data of the analysis sample in as the effective analysis data, it is possible to eliminate the waste of analysis time and improve the analysis efficiency.

  Also, by introducing two types of internal standard substances and acquiring and comparing mass chromatograms in a single analysis, it is possible to detect the occurrence of analysis-inhibiting factors within a single analysis real time. Can be further shortened.

  In addition, by mixing an internal standard in advance in the mobile phase of LC, micro LC or nano LC (capillary LC) having a low liquid flow rate can be used for quantitative mass spectrometry. This is advantageous for high-sensitivity analysis and enables analysis of a small amount of sample.

  Quantitative analysis-inhibiting factors occur, so many components are often detected simultaneously, so qualitative analysis can be prioritized to reduce the total number of analyzes and increase throughput. Is possible.

  Furthermore, it is necessary to keep the mass accuracy of the mass spectrometer high in order to identify the detection component. However, in data acquisition using LC, a change in m / z value may be detected during analysis. Therefore, by monitoring the m / z value of ions derived from the internal standard substance (known substance) that is always detected, it is possible to correct the detected ion to a correct m / z value. As a result, analytical data with extremely high mass accuracy can be obtained.

  Moreover, if the intensity | strength (mass chromatogram area) of a detection ion is normalized based on the intensity | strength of internal standard substance origin ion, the comparison between data can be performed with high precision. This is because the ionic strength may vary somewhat from analysis to analysis.

  FIG. 1 shows a configuration diagram of an embodiment of the mass spectrometry system of the present invention. The apparatus includes a mobile phase introduction unit 101, a sample introduction unit 102, a separation unit 103, an ionization / mass analysis unit 104, a data analysis unit 105, a screen display unit 106, and an analysis mode control unit 107. The data analysis unit 105, the screen display unit 106, and the analysis mode control unit 107 are collectively referred to as a control system 108. The mobile phase introduction unit 101, the sample introduction unit 102, the separation unit 103, the ionization / mass analysis unit 104, and the control system 108 are comprehensively controlled by a system control unit that controls the entire apparatus. The desired operation is realized while exchanging information on the control state. A mobile phase is introduced from the mobile phase introduction unit 101, and a sample 205 made of various components is introduced by the sample introduction unit 102 and separated by a separation unit 103 made of a separation device such as a liquid chromatograph (LC). In the mobile phase introduction unit 101, not only mobile phases A (201) and B (202) but also C (203) are prepared. Mobile phases A and B are those used in ordinary reverse phase chromatographs. Typical mobile phase A (201) is 2% acetonitrile water (0.1% formic acid) and mobile phase B (202) is 98%. Acetonitrile water (formic acid 0.1%).

  In addition, mobile phase C (203) is obtained by adding a certain amount of internal standard substance 204 to mobile phase A. From the beginning, when internal standard substance is added to mobile phases A and B, mobile phase C is It is unnecessary. The important point is that the internal standard substance 204 is always introduced into the separation unit 103 at a constant concentration. Samples 205 (blank samples, analysis samples, etc.) introduced into the sample introduction unit 102 are separated by the separation unit 103 and sequentially introduced into the ionization / mass analysis unit 104 as separation components for ionization and mass analysis. . The output of the mass analysis unit is introduced into the data analysis unit 105, where it is stored and analyzed as analysis data. The data analysis unit 105 shown in FIG. 1 is provided with a screen display unit 106, and information indicating the priority in tandem mass spectrometry, such as “quantitative analysis priority mode” or “qualitative analysis priority mode” in the analysis real time. Is displayed. In addition, the time dependence of the total ion intensity (total ion chromatogram) and the analysis status such as the latest mass spectrum or tandem mass spectrometry spectrum are displayed. Control of the mass analysis unit 104 may be performed by the data analysis unit 105 or may be performed by another information processing facility (analysis mode control unit 107) as indicated by a broken line. Alternatively, as will be described later, the screen display unit 106 may have an analysis mode switching button and can be switched by the operator.

  The outline of the analysis procedure is as follows. First, the mobile phases A, B, and C as described above are prepared, and the mixing ratio of the mobile phase A and the mobile phase B is set to an initial value while the ratio of the mobile phase C containing the internal standard substance is kept constant, such as 3%. Set the value to change the mixing ratio over time. Typically, for example, the mixing ratio of mobile phases A and B is 92% and 5% at the start (C is fixed at 3%), and the ratio is 47% and 50% at the end after 60 minutes. Change the mixing ratio. The initial value of the mixing ratio, the numerical value at the end, the mixing time, and the like are examples, and can be changed as appropriate. The gradient mode in which the mixing ratio of the mobile phases A and B is changed with time is a technique often used in LC separation.

  As the internal standard substance, a desired substance is selected and prepared according to the conditions described later. The internal standard is selected so that it is always detected within the LC retention time range that is detected simultaneously with the analysis component. This is mixed with the mobile phase or mobile phase component, and a blank sample is introduced from the sample introduction unit 102. Then, a mass chromatogram of ions derived from the internal standard substance is acquired and recorded in the data analysis unit 105. The data analysis unit 105 is provided with data storage means 113 configured by a data storage medium or the like. At this time, it is necessary to acquire data under conditions that do not cause a quantitative analysis inhibiting factor. For this reason, the same material as the mobile phase A or pure water is introduced as a blank sample to be introduced by the sample introduction unit 102, and other substances than those existing in advance in the mobile phase are not mixed. Care must be taken that the blank sample does not contain impurities. In addition, since the internal standard itself does not cause ion suppression, it is necessary to include only the minimum amount necessary for ion detection. Next, an analysis sample is introduced by the sample introduction unit 102 instead of the blank sample, and analysis data of the sample mixed with the same amount of the internal standard substance is acquired and recorded in the data analysis unit.

  A mass chromatogram of typical internal standard substance-derived ions is shown in FIG. The horizontal axis represents the LC retention time, and the vertical axis represents the ionic strength. This data was obtained using a liquid chromatograph / mass spectrometer (LC / MS). Usually, in LC in LC / MS, a reverse phase column is used as a separation column. Therefore, it is desirable that this internal standard substance has a high hydrophilicity enough to pass through the reverse phase column. When the hydrophilicity is very high, the ions derived from the internal standard substance contained in the mobile phase are always stably detected in the retention time of the separation, so that the occurrence of the quantitative analysis inhibiting factor can always be monitored. In the example of FIG. 2, a synthetic peptide having an amino acid sequence of SSSSSSK was used as the internal standard substance. And the pure water was used for the blank sample. In this example, when pure water of the blank sample was introduced at the timing of holding time 0, pure water eluted after 12.6 minutes. For this reason, the ionic strength 1201 of the internal standard substance-derived ion in FIG. 2 drops at a retention time of 12.6 minutes, and the ion derived from the internal standard substance is not detected. In this time range, components that are not separated in the sample are eluted, and if it is considered that they are not subject to quantitative analysis, there is no problem even if it is not possible to monitor the occurrence of quantitative analysis inhibiting factors. On the other hand, in other time ranges, the dependence of ionic strength on retention time is very low and changes only smoothly. This is explained that the ionization efficiency of the internal standard substance does not change significantly due to the change of the mobile phase component.

  The principle of ion suppression, which is a quantitative analysis inhibiting factor, is briefly described below. In a spray ionization method such as an electrospray ionization method, charged droplets having a size of about a micron are first generated by spraying a liquid. In this charged droplet, liquid phase ions are distributed on the droplet surface by electrostatic repulsion. Next, when the solvent molecules are evaporated from the charged droplets, gaseous ions are generated from the charged droplets by an ion evaporation process or a charge residual process. Therefore, the main source of gaseous ions detected by a mass spectrometer is liquid phase ions present on the surface of a charged droplet. The charged droplets are reduced in size as the solvent molecules evaporate, and the surface charge density is increased. As a result, ionization (such as proton addition) also proceeds on the analyte to be analyzed that is not charged near the droplet surface. When the amount of the analysis target substance is sufficiently small relative to the charge on the droplet surface, the ionization efficiency of the analysis target substance is constant. When this condition is satisfied, the relationship between the detected ion intensity and the sample amount is constant. However, if the amount of the analyte is equal to or greater than the charge on the surface of the droplet, the supply of charge to the analyte is partially insufficient, and the contention of charges between the analyte occurs on the droplet surface. . As a result, the efficiency with which gaseous ions are generated from the charged droplets varies (reduces). That is, the occurrence of ion suppression. The reduction rate of ionization efficiency depends on the physicochemical properties of the analysis target substance. According to the above ion generation process, the main factors that characterize the analyte subject to ion suppression are 1) low ionization in the liquid phase, and 2) easy access to the droplet surface (surface). It is considered that there are two points: low activity). On the other hand, a component that is completely ionized in the liquid phase can exist on the surface of the charged droplet as ions, and is considered to be hardly affected by ion suppression.

  Therefore, in selecting an internal standard substance for monitoring the occurrence of a quantitative analysis inhibiting factor, the inventors have indicated the isoelectric point (or acidic / basic) and hydrophobicity (or hydrophilicity) of the internal standard substance as indices. I found it useful for the first time. The isoelectric point is the pH when the charge average of the entire compound is zero. It was confirmed that the internal standard substance having an isoelectric point lower than 7 is acidic and reacts sensitively to the quantitative analysis inhibiting factor in positive ion analysis, which is advantageous for detection of the quantitative analysis inhibiting factor. However, when the isoelectric point is sufficiently lower than the pH of the mobile phase, the generation efficiency of positive ions becomes very low. Usually, an acid such as formic acid is added to the mobile phase used in reversed-phase LC / MS for the purpose of balancing LC separation and ionization, and the pH is adjusted to about 3. On the other hand, with regard to hydrophobicity (or hydrophilicity), it is necessary to have lower hydrophobicity (or higher hydrophilicity) than G or A, which is an amino acid having average hydrophobicity (or hydrophilicity). Become.

  When the ionization efficiency is low, it is necessary to mix the internal standard substance into the mobile phase at a high concentration, which is not preferable as the internal standard substance. This is because the occurrence of the quantitative analysis inhibiting factor depends on the amount of the ionized substance, so that the addition of the internal standard substance itself may cause the quantitative analysis inhibition. From the above viewpoint, when analyzing positive ions, synthetic peptides with high hydrophilicity and isoelectric points of 3 to 8 (acidic peptides such as DSSSSS and SSSSSS, isoelectric points are 3.8 and 5.2 respectively) Are most suitable for internal standard substances. On the other hand, basic peptides having an isoelectric point of 8 or more (SSSSSK and SSKSSK, isoelectric points are 8.5 and 10.0, respectively) can also be used as internal standard substances. However, basic peptides are less affected by quantitative analysis-inhibiting factors than acidic peptides and are disadvantageous for detection, and tend to generate not only monovalent protonated molecules but also polyvalent protonated molecules. Multivalent protonated molecules (multivalent ions) can be deprotonated by gas phase ion molecule reaction and become monovalent ions. This corresponds to a decrease in ionic strength and means that it can be difficult to distinguish from detection of quantitative analysis inhibition factors.

Therefore, a basic substance having an isoelectric point of 8 or more that easily generates multivalent ions is not suitable for detecting a quantitative analysis inhibiting factor. In summary, when analyzing positive ions, the hydrophobicity is low (high hydrophilicity), it is acidic, has a low isoelectric point of 3 or more, and only monovalent protonated molecules. A substance to be detected, that is, a substance having an isoelectric point of 3 or more (or an isoelectric point not lower than the pH of the mobile phase) and 8 or less is most suitable for the internal standard substance. Taking a peptide as an example, since the amino acid having an isoelectric point of 3 or less is only D (isoelectric point 2.8), most components have an isoelectric point of 3 or more. Therefore, if an internal standard substance having a property with an isoelectric point of about 3 is used, it can be considered that the ions derived from the internal standard substance are most strongly affected by quantitative analysis inhibiting factors such as ion suppression. Peptides that can be used for such internal standard substances can be selected by selecting amino acids that contain typical acidic amino acids D and E, and S, Q, and N that are highly hydrophilic and difficult to ionize. Good. (D and E are highly hydrophilic, and pK _R (side chain) is about 4.) On the other hand, when analyzing negative ions, they are highly hydrophilic (low hydrophobicity) and basic. A substance having a high isoelectric point is most suitable for the internal standard substance. As a peptide that can be used for such an internal standard substance, a peptide containing typical basic amino acids R and K and highly hydrophilic S, Q, N and the like in the amino acid sequence may be selected. R and K are also highly hydrophilic and have an isoelectric point of 9 or more and 10 or more in pK _R (side chain). On the other hand, S, Q, and N not only have high hydrophilicity, but also have the property of being difficult to ionize in the liquid phase. Examples of such peptides include KNNNNN and RNNNNN, with isoelectric points of 8.75, 9.75, and 8 or more, respectively. Of course, there is no reason why the internal standard must be a peptide, and any compound having the above properties can be used as well.

  Finally, it is necessary to study the molecular weight of the internal standard. If the m / z of the internal standard-derived ion overlaps with the m / z of other detection ions to the extent that it cannot be identified by the mass resolution of the mass spectrometer, it may be erroneously recognized that the intensity of the internal standard-derived ion has increased Sexuality occurs. In this case, as described later, it becomes difficult to detect ion suppression. Therefore, for example, in peptide analysis, if the m / z of ions derived from an internal standard is 600 or more or 350 or less, it is empirically expected that the possibility of overlapping with m / z of other detection ions is very low. Moreover, when analyzing a low molecular weight compound, those having an m / z of 500 or more are rare, and if m / z of ions derived from an internal standard is 400 or more, it is considered that there is no problem. In general, since the internal standard substance tends to become more hydrophobic as the molecular weight increases, the molecular weight is desirably 1000 or less.

  In the analysis using such an internal standard substance, the intensity of ions derived from the internal standard substance is reduced due to the occurrence of quantitative analysis inhibiting factors. At the same time, the intensity of other ions may also be reduced, but the maximum value of the intensity reduction rate is the intensity reduction rate of ions derived from the internal standard substance. By using this property, the ionic strength reduction rate of ions derived from the internal standard substance can be included in the error as the maximum reduction rate in the measured intensity of other components, and reflected in the statistical processing of various data. Is possible. In practice, measurement data always includes measurement errors, so a threshold is set based on that error, and if the ionic strength reduction rate of ions derived from the internal standard is lower than that, the threshold is adopted as the error. In the opposite case, the ionic strength reduction rate of the internal standard substance-derived ions may be adopted as the error. The numerical values of the isoelectric points described here are all rough guidelines, and an empirical error of about 10% may be present.

  In the past, analysis using ESI was premised, but in the case of using APCI, the proton affinity that governs the gas phase ion molecule reaction becomes an important physical quantity instead of the isoelectric point. That is, by setting the proton affinity of the internal standard substance low, it is possible to make it most susceptible to quantitative analysis inhibition factors. For example, since the proton affinity of amino acids and the like is 200 kcal / mol or more, a component having a lower proton affinity and higher hydrophilicity is a candidate for the internal standard substance. Use of water (about 165 kcal / mol), methanol (about 180 kcal / mol), acetonitrile (about 187 kcal / mol) contained in LC mobile phase solvent is convenient. The reduction rate of ionic strength due to the occurrence of quantitative analysis inhibition factors can be handled in the same manner as in ESI. Finally, the third requirement for the internal standard is that the ion m / z is different from that of the analyte.

  FIG. 12 shows an analysis example of a plasma sample. The total ion chromatogram when the injection amount is changed by two digits from 0.5 μg to 0.005 μg is shown superimposed on (a). In addition, the mass chromatogram of ions derived from the internal standard substance (DSSSSS) is shown superimposed on (b). From this result, when the injection amount is 0.005 μg, no reduction is observed in the intensity of ions derived from the internal standard substance (DSSSSS). Therefore, it is considered to be equivalent to the analysis result of the blank sample, and this analysis data shows that there is no problem even if the quantitative analysis is performed. However, when the injection amount is 0.05 μg, the intensity of the ion derived from the internal standard substance is reduced by about 20 to 40% after the retention time of 50 minutes. Therefore, after the holding time of 50 minutes, it is considered that other ionic strengths are also possibly reduced to the same extent. When the injection amount is 0.5 μg, the intensity of the internal standard substance-derived ions is remarkably reduced in almost all retention time regions in which the separated components are detected.

  FIG. 13 and FIG. 14 show a comparison of mass spectra obtained in the retention times indicated by (1) and (2) in FIG. It is indicated that the pattern of the mass spectrum acquired in (c) when the injection amount is the maximum is different from the result when the injection amount is low ((a) and (b)). This difference is explained by the occurrence of ion suppression. Focusing on the ions detected in FIGS. 13 and 14 (m / z = 637.97 and 588.96, respectively), the area of these ion peaks (the peak area in the mass spectrum integrated in the retention time direction) and the injection amount The relationship is shown in FIG.

  Since this figure is a log-log plot, quantitative analysis is possible when fitting to a straight line with a slope of 1. As shown in FIG. 15, for the above two types of ions, when the injection amounts are 0.005 and 0.05 μg, the data points can be plotted on a straight line with a slope of 1, but the injection amount is 0.5 μg. In some cases, the data points are clearly off the straight line with slope 1. These ions are described as having received significant ion suppression at 0.5 μg. Then, it is shown that the deviation from the straight line is less than the reduction in the intensity of the internal standard substance-derived ions. This is consistent with the explanation that the reduction rate of ionic strength is less than that of ions derived from the internal standard substance.

  When quantitative analysis is performed, detected ions are processed as a data set by collecting the retention time, m / z (or m and z), peak area, and peak area error of the ions. By including not only the error in sample preparation and the measurement error in the mass spectrometer, but also the effect of ion suppression, this peak area error is considered to be important for improving the accuracy of the data analysis results.

  In FIG. 3, the example of the display data in the screen display part of a data analysis part is shown. FIG. 9 shows the flow of the analysis process in the present embodiment, and the analysis process will be described in detail below with reference to FIGS. FIG. 9 shows a basic process for analyzing a specimen of a diseased patient or a healthy person. Before entering the analysis of FIG. 9, a fluctuation component having a significant difference between the two is extracted. In FIG. 9, only the fluctuation component may be analyzed, or each specimen may be analyzed in the process of FIG. 9 for the purpose of analyzing all the components without specifying the fluctuation component yet.

  Figure 3 shows the ionic strength 1201b (data b, solid black line) of the internal standard substance-derived ion when analyzing the tryptic enzyme digestion product of BSA (bovine serum albumin) as an analysis sample and the internal standard when analyzing a blank sample. It is an example of ionic strength 1201a (data a, gray solid line) of substance-derived ions. As an internal standard substance, a synthetic peptide having an amino acid sequence of SSSSSSK was used as in FIG. As shown in FIG. 9, after starting analysis 1001, mobile phases A and B and mobile phase C containing an internal standard substance are first mixed at a predetermined mixing ratio (initial value) and introduced from the mobile phase introduction part. A blank sample is introduced from the sample introduction part almost simultaneously (1002). The analysis is started with this blank sample introduction timing as time zero. Next, with the mixing ratio of the mobile phase C kept constant, the mobile phase was introduced while changing the mixing ratio of the mobile phases A and B with a predetermined change amount over time (1003). It is controlled to be constant and sequentially separated by LC (1004), ionization by ionization unit (1005), mass analysis by mass analysis unit, ion intensity of ions derived from internal standard substance, that is, data of mass chromatogram (data a ) Acquisition is performed (1006). When analysis data is acquired by changing the mixing ratio of the mobile phase to the mixing ratio at the end over a predetermined time, the process ends (1007) and the data is stored in the data storage unit as data a (1008). Through the above steps, a mass chromatogram (data a) of the reference internal standard substance obtained using the blank sample was obtained (1050).

  Next, an analysis sample is introduced, and mass chromatogram data of ions derived from the internal standard substance are obtained in the same manner. First, as with the acquisition of data a, the mixing ratio of mobile phases A, B, and C is set to an initial value and introduced into the mobile phase, and the analysis sample is introduced almost simultaneously (1009). Next, with the mixing ratio of the mobile phase C kept constant, the mixing ratio of the mobile phases A and B is changed over time in the same way as when acquiring the data a (1010), and sequentially separated as in steps 1004 to 1006 (1011). ), Ionization (1012), and mass analysis of detected ions and mass chromatogram data (data b) of ions derived from the internal standard substance are obtained (1013). At this time, during analysis, data analysis / analysis (1021) is performed while performing real-time analysis control (1051) as described later. Through the above steps, the analysis sample is introduced, and mass chromatogram acquisition of the internal standard substance-derived ions and analysis of the sample are simultaneously performed (1009 to 1023).

  The LC / MS interface uses electrospray ionization (ESI), but the ionic strengths for each analysis do not always match. Therefore, the ionic strength is normalized, that is, the level is adjusted in the holding time zone (time zone I in FIG. 3) immediately before the unseparated components contained in the sample are eluted. In the time zone III in the figure, the intensities of the ions derived from the internal standard substance are almost the same, indicating that there is no quantitative analysis inhibiting factor in a wide range. On the other hand, in the time zone indicated by time zone IV in FIG. The occurrence of this discrepancy is interpreted to indicate that the intensity of ions derived from the internal standard substance fluctuates when data b is acquired (that is, when the analysis sample is mixed), causing quantitative analysis inhibition factors such as ion suppression. it can. In data normalization and comparison, data is processed so that normalization and comparison are performed based on the average level in each holding time without being affected by high-frequency noise components. For example, the low frequency component is extracted by removing the high frequency component of the data, and the data in the same holding time are compared. For example, in the normalization process, the display is adjusted so that the size of the data a is 100% and the data b is 100% ± 5%. This ± 5% is an example, and is stored in advance in the control system 8 as a standardization (level adjustment) allowable range, or can be input by the operator. Also in the comparison process, the data a is set to 100% in the same manner, and the percentage of the data b with respect to a is calculated. For example, when data b is 97% of a, it is determined that there is a difference of 3%. The numerical value obtained in this way (here, 3%) is hereinafter referred to as a difference ratio and is used as an index indicating the degree of mismatch between the data a and b.

  When the data is viewed in detail, it can be seen that the data a and the data b have an error of about several percent even in the time zone III. This error of several percent is well known as a measurement variation of mass chromatogram by ordinary LC / MS. Since there is an error in ionic strength in such a mass chromatogram, if the difference between data a and b is a small deviation within a few percent that comes from the measurement error of the instrument, it is allowed as a measurement variation, and the difference between data a and b When a certain threshold is exceeded, it is necessary to determine that a difference due to the quantitative analysis inhibition factor has occurred. Therefore, a difference ratio (mismatch degree) threshold value that serves as a determination criterion is determined in advance, data is acquired, a difference ratio (mismatch degree) is calculated in real time, and compared with the mismatch degree threshold value. This inconsistency threshold is stored in the control system 8 in advance, or can be input by the operator, in the same manner as the standardization tolerance. In this embodiment, since the error of the apparatus has a measurement error of about 5 to 10% as a standard level, the difference ratio (inconsistency) threshold is set to 15%. That is, when the difference ratio d between the data a and b exceeds 15%, the determination is made as a quantitative analysis inhibition factor. Such an error is considered to depend on the mass spectrometer, and an error corresponding to the apparatus can be input to the data analysis unit 105. The data analysis unit 105 of FIG. 1 calculates the level adjustment means 114 for performing the level adjustment between the data a and b and the image display control described above, and the degree of inconsistency between the data and stores it in advance. The calculation means 115 for comparing with the mismatch threshold value is incorporated.

  Now, the detected discrepancy in time zone IV in FIG. 3 indicates that a quantitative analysis-inhibiting factor such as ion suppression has occurred. In time zone IV, the accuracy of the analysis data of the sample decreases and quantitative analysis is difficult. is there. On the other hand, the data in time zone III has sufficient accuracy and can be quantitatively analyzed. Therefore, by extracting only the data of the analysis sample in time zone III, quantitative analysis of the analysis sample in this time zone is performed. If quantitative analysis of the analysis sample can be sufficiently performed in this time zone III, highly reliable quantitative analysis can be performed as it is. As a result, even when a quantitative analysis inhibition factor occurs during analysis, data in a time zone that is not affected by the inhibition factor can be used effectively, and waste of analysis can be eliminated. In addition, in time zone IV, quantitative evaluation impediments occur, and the accuracy of the quantitative evaluation results decreases, making it impossible to use the data. The analysis mode may be switched as described above. In addition, if the data cannot be obtained in a single analysis, the above analysis may be repeated.

  Also, when the occurrence of quantitative analysis inhibition factors such as ion suppression is detected, it is possible to prepare a sample that does not cause quantitative analysis inhibition factors by reducing the concentration, purifying, and separating the sample. is there. In this case as well, if no discrepancy is observed in the mass chromatogram of the ions derived from the internal standard substance as a result of re-preparing the sample preparation in the screen display section of the data analysis section, there is no cause for quantitative analysis inhibition. Can be confirmed. Change the sample preparation conditions by repeatedly changing the sample preparation conditions, and if there is no quantitative analysis-inhibiting factor or if there is a certain period of time that can be quantitatively analyzed without being affected, the sample preparation conditions are changed. You may control to stop.

  FIG. 4 shows an example in which a quantitative analysis inhibiting factor occurs remarkably. In this example, it was shown that the ionic strength of ions derived from the internal standard substance was inconsistent over a very wide range shown in the time zone III in the figure, causing a quantitative analysis inhibition factor. This data is not suitable for quantitative analysis. It is shown that there is. In this case, 1) reduce the amount of analysis sample to about 1/10, 2) perform separation / fractionation in advance in sample preparation, 3) remove ionic impurities due to desalting in sample preparation, It is necessary to carry out reanalysis after taking such measures. Thus, depending on the degree of ionic strength mismatch, effective quantitative analysis data may not be obtained unless measures are taken on the sample. The control system is also configured so that the reference value is set in advance in the time of the inconsistent area and the inconsistent area of the ionic strength, and the sample preparation is sequentially repeated when the time of the inconsistent area of the ionic strength is shorter than the reference time. did. This reference time may also be stored in the control unit 8 in advance or input by the operator.

  In the retention time zone indicated by the dashed ellipse in FIG. 4, the intensity of the internal standard substance-derived ions (data b) in the sample analysis data increases and is equivalent to that of the blank sample (data a). . However, when the mass spectrum in this case was examined, ions different from those derived from the internal standard substance but having an equivalent m / z were detected. Therefore, the quantitative analysis inhibiting factor does not occur even in this holding time zone. As described above, when analyzing a very complicated sample, an ion whose m / z almost coincides with the ion derived from the internal standard substance may be observed, which adversely affects the detection of the quantitative analysis inhibiting factor. there is a possibility. Therefore, it is convenient to compare mass chromatograms of ions derived from internal standard substances in real time of analysis, and to perform tandem mass spectrometry such as MS / MS when a mismatch occurs. By investigating the relationship between the ionic strength of ions derived from the internal standard substance and the intensity of dissociated ions detected by the tandem mass spectrometry spectrum in advance, This is because the ionic strength is required.

  In proteome analysis, metabolomic analysis, and marker search, since components contained in a sample are not necessarily known, both qualitative (identification) analysis and quantitative (variation) analysis are required. However, in qualitative (identification) analysis, it is necessary to acquire not only ordinary mass spectra but also tandem mass spectrometry spectra such as MS / MS at high throughput, whereas quantitative (variation) analysis is performed as much as possible. It is necessary to obtain normal mass spectra with high throughput without performing. That is, the priority order of data acquired by the mass spectrometer differs depending on the purpose.

  Conventionally, for example, a method in which a qualitative analysis is performed first and a quantitative analysis is performed after all components have been identified has been common. Specifically, a file for instructing an analysis procedure is stored in the control unit 8 of the analyzer. When performing a qualitative analysis, the control unit 8 refers to the analysis procedure instruction file and performs, for example, a normal mass analysis once. After performing the analysis, the top 10 types of spectral peaks are extracted and tandem analyzed, the components are confirmed by performing normal mass spectrometry again, and the next top 10 types are extracted from the spectrum again to perform tandem analysis. Qualitative analysis of all components was performed. At this time, when there are few types of components to be qualitatively analyzed, for example, there are only about 15 types of components, qualitative analysis of all the components is completed in the second tandem analysis, and then quantitative analysis by normal mass spectrometry is performed. There was also a control to do. This was sometimes referred to as a qualitative analysis priority mode.

  When such a qualitative analysis is completed and a quantitative analysis is performed, another analysis procedure instruction file is stored in the control unit 8 in advance, and the control unit 8 refers to it to perform a normal mass analysis. High-precision quantitative analysis was performed. In other words, analysis procedure instruction files (first and second analysis procedure instruction files) for qualitative and quantitative analysis are stored in qualitative and quantitative analysis, respectively, and control is performed based on these files, and analysis is performed in the order of qualitative analysis → quantitative analysis. It was common to be executed. However, since data acquisition for the purpose of qualitative (identification) analysis is performed in advance and data acquisition for the purpose of quantitative (variation) analysis is performed in advance, high-throughput analysis suited to the situation cannot be performed. On the other hand, if there is a function for changing the analysis mode in real time, it is advantageous from the viewpoint of high throughput when analyzing a sample containing multiple components.

  Therefore, as shown in FIGS. 1 and 6, in the present invention, the mass chromatograms of ions derived from the internal standard substance are compared in the real time of analysis, and if a mismatch occurs, qualitative (identification) analysis priority is given. The control system was configured to change the analysis mode, such as switching to a mode. That is, the order of performing qualitative / quantitative analysis is controlled in real time according to the data to be acquired. In the present embodiment, for example, first, quantitative analysis is preferentially performed (quantitative analysis priority mode). Then, the presence / absence of a quantitative analysis inhibiting factor is determined by the above-described method, and the quantitative analysis is continued in a time zone not affected by the inhibiting factor, and the analysis data is stored in the control unit 8. When it is detected that an inhibitory factor has occurred and the effect has become apparent, the accuracy of quantitative analysis is considered to decrease, so switching to give priority to qualitative analysis (qualitative analysis priority mode). As a result, it has become possible to perform qualitative analysis efficiently even during a time period that is affected by factors that hinder quantitative analysis.

  The quantitative analysis priority mode will be described in more detail. Conventionally, in the quantitative analysis mode, it is common to perform control without performing qualitative analysis by tandem. In contrast, in this example, control was newly performed so that qualitative analysis could be performed depending on spectral characteristics even during quantitative analysis. This is called the quantitative analysis priority mode. For example, when a component that is expected to be present in the analysis sample is stored in the control unit 8 in advance during a time period when no quantitative analysis inhibition factor is detected, and a spectrum of a component that was not expected by quantitative analysis is obtained. Alternatively, a qualitative analysis may be performed during the quantitative analysis. Utilizing this function, for example, the component analysis result of the analysis sample is obtained from the specimen of a healthy person, stored in the control unit 8, and is referred to when analyzing the specimen of a sick patient. It was also possible to switch to qualitative analysis and identify only the components that were not detected. By controlling the analysis procedure in this way, the accuracy and efficiency (that is, throughput) can be dramatically improved in marker search and the like.

  In this case, the internal standard substance may be one type or two types as described later. Then, as shown in the figure, the analysis state is displayed on the screen of the data analysis unit or the control unit of the mass spectrometer in real time in the “quantitative analysis (priority) mode” or “qualitative analysis (priority) mode”. Etc. so that the operator can visually confirm that the device is operating normally. In this mode switching, a third analysis procedure instruction file or the like is stored in the control unit 8, and the qualitative analysis priority mode "and the" quantitative analysis priority mode "are set based on the sequentially acquired data. As shown in FIG. 6, a qualitative / quantitative analysis mode selection button 109 is provided on the screen display unit, and the pointing device 112 is monitored as necessary while the operator sequentially monitors the analysis results. For example, the mode may be switched by the pointer 110 based on the above. Determination criteria for determining switching may also be stored in the control unit 8 or input by an operator.

  As described above, real-time analysis control (1051) shown in FIG. 9 includes (1) real-time normalization and level adjustment of data a and data b at the initial stage of analysis (1014, 1015), (2) Determination of presence / absence of real-time quantitative analysis inhibiting factor (inconsistency between intensity data a and b of internal standard substance-derived ions) (1016), and extraction of data of effective time period in which quantitative analysis inhibiting factor is not generated based on the determination result (1017) ), (3) Depending on the degree of influence of the quantitative analysis obstruction factor (such as the length of time that data a and b match), sample separation when the degree of influence is large (the length of matching time is below the reference value) Review preparation conditions (1018, 1019), (4) Select priority mode switching (1020) for quantitative / qualitative (tandem) analysis in the time zone affected by quantitative analysis inhibition factors Configure your system, quantification by analysis of acquired data, and controls to perform the qualitative analysis (1021). In addition, for example, in quantitative analysis priority mode, analysis is performed in real time so that a significant feature in the spectrum, for example, an unexpected component peak compared to the predicted component of the analysis sample stored in advance, is detected. Control mode. When the analysis is completed and the analysis data has not been removed, the analysis is repeated to acquire all necessary data.

  The data analysis unit 105 includes means 116 for detecting a time zone in which the degree of mismatch between the data a and b is less than or greater than the threshold value of the mismatch level, and a time zone with a low mismatch level, that is, a high match level (match time zone). And a means 117 for collecting the analysis data in FIG. The analysis procedure instruction file storage unit and file reading control unit are provided.

  Next, as a second embodiment, an analysis method using two types of internal standard substances will be described with reference to FIGS. As shown in FIG. 10, when two types of internal standard substances are used, the presence or absence of occurrence of a quantitative analysis inhibiting factor is not performed in one analysis, instead of performing the analysis twice as in the first embodiment. Can be evaluated. Thereby, the analysis time can be further shortened. The two internal standard substances are substances that are always detected as ions, that is, hydrophilic substances. Furthermore, one is acidic, the other is basic, the former changes sensitively to quantitative analysis inhibiting factors, and the latter Select a substance that hardly changes.

  That is, a substance having an isoelectric point of approximately 3 or more and 8 or less and one having an isoelectric point of approximately 8 or more is selected for one of the internal standard substances. Here, a substance having high hydrophilicity and acidity is defined as a first internal standard substance, and a substance having high hydrophilicity and basicity is defined as a second internal standard substance. The upper diagram of FIG. 5 shows the mass chromatogram (1202a, solid black line) of ions derived from the first internal standard substance and the mass chromatogram (1202b, gray broken line) of ions derived from the second internal standard substance obtained in this example. ). In this embodiment, in the time zone I of FIG. 5, the intensity levels of the first and second internal standard substance-derived ions are adjusted in real time in order to normalize the intensity. Displayed on the screen. In addition, when analyzing by LC / MS, there is a possibility that an unexpected subtle variation in the amount of ions caused by the apparatus may occur. However, when the analysis is performed twice as in Example 1, random variations occur each time. Since this can happen, the effects of this unexpected random ion content variation cannot be avoided. However, in this embodiment, two data can be acquired and compared at the same time, so when an unexpected change in the amount of ions caused by the device occurs, the same ion fluctuation occurs in both data, and in the difference between the two, It is not affected by ion fluctuation. As a result, the present embodiment has an advantage that the degree of mismatch can be accurately evaluated with higher accuracy than the first embodiment.

  The lower diagram of FIG. 5 is a total ion chromatogram, and the analysis data in the retention time region (time zone III) different from the retention time (time zone IV) where the quantitative analysis inhibition factor occurs can be quantitatively analyzed. Therefore, only the analysis data in the holding time region (time zone III) is extracted and stored in a data analysis unit or the like with another name. In this way, only analysis data that can be quantitatively analyzed can be analyzed. In this example, as described above, the first analysis point is that the target analysis sample is analyzed while determining the occurrence of quantitative analysis inhibition factors in one analysis using two types of internal standard substances. Unlike the example, the other parts are the same as in the first embodiment. That is, the degree of inconsistency between the two internal standard substances is calculated from the mass chromatograms of the two internal standard substances in the same manner as in the first embodiment, and compared with a predetermined inconsistency threshold, and the analysis time zone when the inconsistency is smaller than the threshold. And analysis data in this analysis time zone is collected. Two internal standards are introduced into the mobile phase at a constant concentration so that they can be detected stably over the entire analysis time.

  As in the first embodiment, when the disparity between the two mass chromatograms is large, the quantitative / qualitative analysis priority mode is switched to give priority to the qualitative analysis (tandem analysis), or the switching state is displayed. If the length of the time zone in which the discrepancy is sufficiently small is shorter than the reference time set in advance, the sample preparation state is reviewed and control is repeated until the discrepancy is reduced to achieve high efficiency and high accuracy. Realized the analysis. The configuration of the apparatus used in the second embodiment is the same as that of the first embodiment shown in FIG. 1 except that the details of the calculation contents of the calculation means in the data analysis unit 105 and the control system 8 are partially different. Is almost the same.

  As a third embodiment, one kind of internal standard substance is introduced as the first internal standard substance, and the second internal standard substance is not actively introduced, such as impurities that have been mixed in unintentionally. A method for searching for a component that can be a second internal standard material from various component materials and comparing the mass chromatograms of both components at the same time will be described. As shown in FIG. 11, the analysis process of this embodiment includes a step of searching and selecting a substance that can be used as the second internal standard substance as compared with the second embodiment of FIG. In this case, the first internal standard substance is a hydrophilic and acidic substance similar to the first internal standard substance in the second embodiment, and a substance that reacts sensitively to the quantitative analysis inhibiting factor is selected in advance. .

  A second internal standard substance search monitor is provided to detect substances that are present in the analyzer and are stably detected as ions in a wide retention time region and that are hardly affected by quantitative analysis inhibiting factors. And detect while changing the sample. For example, ions of a kind of impurity such as siloxane were also observed stably in a wide holding time region, and the ionic strength hardly changed with respect to quantitative analysis inhibiting factors such as ion suppression. Such a substance is selected as the second internal standard substance, and the mass chromatograms of the first and second internal standard substances are obtained and compared at the same time as in the second embodiment to detect the quantitative analysis inhibiting factor. To do. Other parts are the same as those in the first and second embodiments. Compared with the apparatus configuration of the first embodiment, the apparatus of the present embodiment searches for monitoring means for monitoring analysis data of a plurality of substances in order to search for substances that can be the second internal standard substance. An input unit for selecting and inputting the found substance as the second internal standard substance, that is, an input device using a pointing device or a keyboard, and an image display menu such as a selection menu, a selection button, and a numeric input field It is a device configuration.

  Next, as a fourth embodiment, a method of detecting a quantitative analysis inhibiting factor only by an analysis result in which one type of internal standard substance is introduced and a blank sample is not analyzed and an analysis sample is introduced will be described. As shown in FIG. 2, the mass chromatogram of the internal standard substance shows a tendency not to change rapidly with respect to time. Therefore, it is possible in principle to detect the occurrence of the quantitative inhibition factor without using the analysis result of the blank sample. Therefore, only when the analysis sample is introduced, a mass chromatogram of ions derived from the internal standard substance is obtained, and a quantitative analysis inhibition factor is detected by examining whether or not there is a rapid change with respect to time. In this analysis method, it is necessary to pay attention to the point that it is difficult to detect the occurrence of an analysis inhibition factor when the intensity reduction of ions derived from the internal standard substance due to the analysis inhibition factor is not rapid.

  In the configuration diagram of another embodiment of the mass spectrometry system of the present invention shown in FIG. 7, unlike the embodiment of FIG. 1, the internal standard substance 204 is introduced from the syringe pump 111 downstream of the separation unit 103. As a result, the internal standard substance 204 is mixed into the eluate of the liquid chromatograph at a constant ratio. Of course, the internal standard substance 204 only needs to be mixed with the analysis liquid, and the mixing location may be anywhere as long as it is upstream of the interface (ion source). When a general-purpose liquid chromatograph, a semi-micro liquid chromatograph, or a micro liquid chromatograph with a liquid chromatograph flow rate higher than 1 microliter / minute is used, such a configuration is more advantageous than the configuration shown in FIG. is there. This is because the exchange of the solution containing the internal standard substance is easy. This embodiment can be implemented in combination with the first to fourth embodiments.

  FIG. 8 shows the retention time dependence of the mass shift using detection of ions derived from the internal standard substance in another embodiment of the mass spectrometry system of the present invention. When the analysis using a liquid chromatograph takes several minutes or more, the mass accuracy at the ppm level may be reduced due to a temperature change or the like. Therefore, by examining the measured value (1402 in FIG. 8) of the m / z of the ion derived from the internal standard substance whose theoretical mass (1401 in FIG. 8) is known with respect to the holding time, the ions detected at each holding time The measured value of m / z (1404 in FIG. 8) can be corrected by proportional distribution (1403 in FIG. 8). This makes it possible to identify substances with extremely high accuracy, particularly in database searches in qualitative analysis. This embodiment can be implemented in combination with the first to fifth embodiments.

  So far, we have described detection methods for quantitative analysis inhibition factors and mass spectrometry systems using them, mainly for the field of marker search for disease diagnosis. On the other hand, in a field such as pharmacokinetics in which tandem mass spectrometry called Multiple Reaction Monitoring (MRM) is used, tandem mass spectrometry is used not only for qualitative analysis of substances but also for quantitative analysis. In such a case, it is necessary to perform tandem mass spectrometry of ions derived from the internal standard substance in advance and register one or more kinds of main fragment ions in the mass spectrometer. Thereby, a mass-mass chromatogram of main fragments of ions derived from the internal standard substance can be obtained. Then, in the mass chromatogram of the fragment ions, it is possible to detect the occurrence of the quantitative analysis inhibiting factor based on the fluctuation (reduction) in intensity. The specific detection method is the same as described above.

The block diagram of one Example in the mass spectrometry system of this invention. Mass chromatogram of ions derived from internal standard in typical blank sample analysis data. The detection example of comparison of the mass chromatogram of the ion derived from an internal standard substance in the analysis data of a blank sample and an analysis sample in one Example in the mass spectrometry system of the present invention, and generation detection of a quantitative analysis inhibition factor. Comparison of mass chromatograms of ions derived from internal standard substances in analysis data of blank samples and analysis samples and generation of quantitative analysis inhibition factors on a large scale. In one Example in the mass spectrometry system of the present invention, two types of internal standard substances are used, and mass chromatograms of ions derived from internal standard substances in the analysis data of the analysis sample are compared, and the occurrence detection examples of quantitative analysis inhibition factors are shown. The screen of the data analysis part in one Example in the mass spectrometry system of this invention, or the screen of the control part of a mass spectrometer. The block diagram of one Example in another mass spectrometry system of this invention. The example of the time dependence of the measurement m / z of the ion derived from an internal standard substance in the mass spectrometry system of this invention. The figure explaining the analysis process in 1st Example of this invention. The figure explaining the analysis process in the 2nd Example of this invention. The figure explaining the analysis process in the 3rd Example of this invention. Comparison of (a) total ion chromatogram obtained using the mass spectrometry system of the present invention and (b) mass chromatogram of ions derived from the internal standard substance. Comparison of mass spectra acquired at retention time (1) using the mass spectrometry system of the present invention. Comparison of mass spectra acquired at retention time (2) using the mass spectrometry system of the present invention. Relationship between peak area and injection amount for ions detected at retention times (1) and (2).

Explanation of symbols

101: Mobile phase introduction unit, 102: Sample introduction unit, 103: Separation unit, 104: Ionization / mass analysis unit, 105: Data analysis unit, 106: Screen display unit, 107: Analysis mode control unit, 108: Control system, 109: Analysis mode selection button, 110: Pointer, 111: Syringe pump, 112: Pointing device, 113: Data storage means, 114: Level adjustment means, 115: Inconsistency calculation / comparison means, 116: Matching time zone detection means, 117: Analytical data collection means, 201: Mobile phase A, 202: Mobile phase B, 203: Mobile phase C, 204: Internal standard, 205: Blank sample or analytical sample, 1001: Analysis start step, 1002: Mobile phase, Blank sample introduction step, 1003: Mobile phase introduction continuation step, 1004: Separation step, 1005: Ionization step, 1006: Mass spectrometry, Mass chromatogram data (data a) acquisition step, 1007: Completion determination step, 1008: Data Memory step, 1009: mobile phase, sample introduction step, 1010: mobile phase introduction step, 1011: separation step, 1012: ionization step, 1013: mass spectrometry, mass chromatogram data acquisition step, 1014: analysis Initial stage judgment step, 1015: Data level preparation (standardization) step, 1016: Data mismatch degree judgment step, 1017: Quantitative analysis data acquisition step, 1018: Data coincidence time judgment step, 1019: Sample preparation condition review Step, 1020: Quantitative / qualitative analysis mode switching step, 1021: Data analysis / analysis step, 1022: Termination determination step, 1023: Termination step, 1024: Second internal standard substance search / selection step, 1050: Blank sample Analysis step, 1051: Real-time analysis control step, 1201: Ionic strength of ions derived from internal standard, 1201a: Bra Ion intensity of internal standard substance-derived ions during sample analysis (data a), 1201b: Ion intensity of internal standard substance-derived ions during analysis sample analysis (data b), 1202a: Ions of ions derived from the first internal standard substance Intensity, 1202b: Ion intensity of ions derived from the second internal standard, 1401: The m / z theoretical value of the internal standard, 1402: The measured m / z of the internal standard, 1403: m / z correction of the analysis sample Value, 1404: m / z measured value of the analytical sample.

Claims (33)

In an analysis method using a liquid chromatograph / mass spectrometer,
Mixing the standard substance into the analysis solution;
Obtaining a first mass chromatogram of ions derived from the standard substance without mixing an analysis sample with the analysis solution;
Obtaining a second mass chromatogram of ions derived from the standard substance in a state where the analysis sample is mixed with the analysis solution;
Adjusting the levels of the first and second mass chromatograms;
Calculating a degree of inconsistency between the first and second mass chromatograms, and comparing with a pre-stored inconsistency degree threshold;
Detecting an analysis time zone in which the degree of inconsistency is smaller than the inconsistency degree threshold;
A method of collecting analysis data of an analysis sample acquired in the analysis time zone. The analysis method according to claim 1,
The reference material has hydrophilicity,
An analysis method comprising mixing the standard substance into a mobile phase or eluate of the liquid chromatograph at a constant concentration. The analysis method according to claim 2,
The standard method has an isoelectric point of approximately 3 or more and 8 or less. The analysis method according to claim 1,
When obtaining the first mass chromatogram, any one of the components constituting the mobile phase is used as a blank sample. The analysis method according to claim 1,
An analysis method characterized by using pure water as a blank sample when acquiring the first mass chromatogram. The analysis method according to claim 1,
An analysis method comprising a switching step of switching between a quantitative analysis priority mode and a qualitative analysis priority mode according to analysis data of the analysis sample or data of the mass chromatogram. The analysis method according to claim 6,
The analysis method characterized in that the qualitative analysis is a tandem analysis. The analysis method according to claim 6,
An analysis method comprising a step of displaying a switching state of the analysis mode. The analysis method according to claim 1,
Storing the procedure for switching the analysis mode in advance and the switching criteria;
Comparing the analysis data of the analysis sample with the switching criteria according to the switching procedure;
An analysis characterized by having a switching step of switching between the quantitative analysis priority mode and the qualitative analysis priority mode according to the magnitude relationship between the mismatch level and the mismatch level threshold or the comparison result between the analysis data and the switching criterion. Method. The analysis method according to claim 1,
Comparing the length of the analysis time zone in which the inconsistency is smaller than the inconsistency threshold with a reference time stored in advance,
An analysis method characterized in that the analysis is performed by changing the preparation conditions of the analysis solution according to the result of the comparison. The analysis method according to claim 10,
According to the magnitude relationship between the analysis time zone and the reference time, the step of repeating the change in the preparation conditions of the analysis solution or the analysis,
And a step of stopping the change in the preparation conditions of the analysis solution when the analysis time period is longer than the reference time or the mismatch is lower than the mismatch threshold. . The analysis method according to claim 1,
Storing in advance a theoretical value of the ratio of the mass and charge of the reference material;
Measuring the mass to charge ratio of the reference material relative to the analysis time;
And a step of correcting the actual measurement value of the mass-to-charge ratio of the analysis sample based on the relationship between the theoretical value and the actual measurement value.   2. The analysis method according to claim 1, wherein the standard substance is introduced from a downstream side of the separation column of the liquid chromatograph. In an analysis method using a liquid chromatograph / mass spectrometer,
Mixing the first and second standard substances having different isoelectric points into the analysis solution;
Obtaining a first mass chromatogram of ions derived from the first standard substance and a second mass chromatogram of ions derived from the second standard substance;
Adjusting the levels of the first and second mass chromatograms;
Calculating a degree of inconsistency between the first and second mass chromatograms, and comparing with a pre-stored inconsistency degree threshold;
Detecting an analysis time zone in which the degree of inconsistency is smaller than the inconsistency degree threshold;
A method of collecting analysis data of an analysis sample acquired in the analysis time zone. The analysis method according to claim 14,
The first and second standard substances have hydrophilicity,
An analysis method comprising mixing the standard substance into a mobile phase or eluate of the liquid chromatograph at a constant concentration. The analysis method according to claim 15, wherein
The first and second standard substances have an isoelectric point of one standard substance of about 3 to 8 and the other standard substance has an isoelectric point of about 8 or more. The analysis method according to claim 16, wherein
The analysis method characterized in that the first and second standard substances are hydrophilic. In an analysis method using a liquid chromatograph / mass spectrometer,
Mixing the first standard with the analysis solution;
Monitoring mass chromatograms of a plurality of substances to search for a second standard substance;
Obtaining a first mass chromatogram of ions derived from the first standard substance and a second mass chromatogram of ions derived from the second standard substance;
Adjusting the levels of the first and second mass chromatograms;
Calculating a degree of inconsistency between the first and second mass chromatograms, and comparing with a pre-stored inconsistency degree threshold;
Detecting an analysis time zone in which the degree of inconsistency is smaller than the inconsistency degree threshold;
A method of collecting analysis data of an analysis sample acquired in the analysis time zone. A mobile phase introduction part for mixing the standard substance and introducing the mobile phase into the analysis solution;
A sample introduction part for introducing a sample into the analysis solution;
A separation unit for separating the analysis solution using a liquid chromatograph;
An ionization / mass spectrometry unit for ionizing / mass analyzing the separated substance;
In a liquid chromatograph / mass spectrometer comprising a data analysis unit for analyzing the data acquired by the analysis and a screen display unit for displaying the status of the analysis,
The data analysis unit
Means for acquiring and storing a first mass chromatogram of ions derived from the standard substance without mixing an analysis sample in the analysis solution;
Level adjusting means for obtaining a second mass chromatogram of ions derived from the standard substance in a state in which the analysis sample is mixed with the analysis solution and adjusting the level with the first mass chromatogram;
A computing means for calculating the degree of inconsistency between the first and second mass chromatograms and comparing with a pre-stored inconsistency degree threshold;
Means for detecting an analysis time period in which the degree of inconsistency is smaller than the inconsistency degree threshold;
And an analyzer for collecting analysis data of the analysis sample acquired in the analysis time zone. The analyzer according to claim 19,
An analyzer comprising switching means for switching between a quantitative analysis priority mode and a qualitative analysis priority mode according to analysis data of the analysis sample or data of the mass chromatogram. The analyzer according to claim 20,
The image display unit displays the switching state. In an analysis method using a liquid chromatograph / mass spectrometer,
Mixing the standard substance into the analysis solution;
Obtaining a first mass chromatogram of ions derived from the standard substance in a state where mixing of the analysis sample into the analysis solution can be ignored;
Obtaining a second mass chromatogram of ions derived from the standard substance in a state where the analysis sample is mixed with the analysis solution;
Adjusting the levels of the first and second mass chromatograms;
Calculating a degree of inconsistency between the first and second mass chromatograms, and comparing with a pre-stored inconsistency degree threshold;
And a step of reflecting the inconsistency as an error of a peak area in the analysis data of the analysis sample. The positive ion analysis method according to claim 22,
The analytical method, wherein the standard substance contains an acidic amino acid. The negative ion analysis method according to claim 22,
The analysis method characterized in that the standard substance contains a basic amino acid.   23. The analysis method according to claim 22, wherein ion peak data is acquired as information on the peak area and its error, retention time, and peak m / z (or m or z) based on analysis sample data. An analysis method characterized by: A standard substance introduction part for introducing a standard substance into the analysis solution;
A sample introduction part for introducing a sample into the analysis solution;
A separation unit for separating the analysis solution using a liquid chromatograph;
An ionization / mass spectrometry unit for ionizing / mass analyzing the separated substance;
A data analysis unit for analyzing the data obtained by the analysis, a screen display unit for displaying the status of the analysis,
In a liquid chromatograph / mass spectrometer consisting of
The data analysis unit
Means for acquiring and storing a first mass chromatogram of ions derived from the standard substance in a state in which mixing of the analysis sample can be ignored in the analysis solution;
Level adjusting means for obtaining a second mass chromatogram of ions derived from the standard substance in a state in which the analysis sample is mixed with the analysis solution and adjusting the level with the first mass chromatogram;
Computing means for calculating the degree of inconsistency between the first and second mass chromatograms;
An analyzer having means for adding the degree of inconsistency to the analysis data of the analysis sample. Means for storing a first mass chromatogram of ions derived from the standard obtained in a state where the mixing of the analysis sample is negligible in the analysis solution;
Level adjusting means for adjusting the level of the second mass chromatogram of ions derived from the standard substance obtained in a state where the analysis sample is mixed with the analysis solution, with the first mass chromatogram;
Computing means for calculating the degree of inconsistency between the first and second mass chromatograms;
And a means for adding the discrepancy to the analysis data of the analysis sample. In positive ion analysis using a liquid chromatograph / mass spectrometer,
It is an internal standard substance used to detect quantitative analysis inhibition factors,
An internal standard substance, wherein the internal standard substance is hydrophilic and acidic. The internal standard substance according to claim 28,
The internal standard substance is hydrophilic and has an isoelectric point of 3 or more and 8 or less. In negative ion analysis using a liquid chromatograph / mass spectrometer,
It is an internal standard substance used to detect quantitative analysis inhibition factors,
An internal standard substance, wherein the internal standard substance is hydrophilic and basic. The internal standard substance according to claim 30,
The internal standard substance is hydrophilic and has an isoelectric point of 8 or more.   The analysis method using the internal standard substance of Claim 30.   The analysis method using the internal standard substance of Claim 30.

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