Method for calculating the lower limit of quantification of mass spectrometric analysis

Abstract

A method for determining the minimum concentration of a first analyte to be mass-analyzed in a mass spectrometer is disclosed, the method comprising: (i) performing mass analysis on the first analyte at a known concentration using a mass spectrometer according to a first mass analysis method to obtain an ion signal having a peak; and (ii) determining the number N of ions N detected by the mass spectrometer during the first mass analysis method to form the peak. 峰 (iii) Determine the number N of ions that need to be detected in the peak in order to achieve a pre-selected relative standard deviation for the ion peak. req ; and (iv) determine the relative standard deviation C for achieving the preselected standard deviation based on the values ​​of the following: RSD Simultaneously, the minimum concentration of the first analyte that can be analyzed according to the first mass analysis method: the known concentration, N 峰 and N req .

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to UK Patent Application No. 2404411.7, filed on 27 March 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates generally to mass spectrometers, and more particularly to a method and mass spectrometer for determining the minimum concentration of an analyte that can be mass-analyzed while enabling reliable quantification of the analyte. Background Technology

[0004] In mass spectrometry, analytes in a sample are analyzed by ionization, and the resulting precursor ions or fragment or product ions derived therefrom are then detected in a mass analyzer. For any given analytical method, the amount of an analyte can only be reliably quantified if the analyte in the sample exceeds a threshold amount. This means that for a given volume of sample to be analyzed by mass, the amount of the analyte can only be reliably quantified if the analyte in the sample is present at or above a concentration known as the lower limit of quantitation (LLOQ). The concentration of an analyte as the LLOQ is the concentration of the analyte that satisfies the following criteria: (i) a preselected signal-to-noise ratio (S / N) value of the ion signal detected during the mass analysis of the analyte; and (ii) a preselected relative standard deviation (RSD%) value of the ion signal. Typically, determining the minimum concentration of an analyte that satisfies the RSD% requirement is difficult and time-consuming. Summary of the Invention

[0005] According to a first aspect, the present invention provides a method for determining a minimum concentration of a first analyte to be mass-analyzed in a mass spectrometer, the method comprising: (i) performing mass analysis on a known concentration of the first analyte using a mass spectrometer according to a first mass analysis method to obtain an ion signal having a peak; and (ii) determining the number N of ions N detected by the mass spectrometer during the first mass analysis method to form the peak. 峰 (iii) Determine the number N of ions that need to be detected in the peak in order to achieve a pre-selected relative standard deviation for the ion peak. req ; and (iv) determine the relative standard deviation C in achieving the preselected standard deviation based on the values ​​of the following: RSD Simultaneously, the minimum concentration of the first analyte that can be analyzed according to the first mass analysis method: the known concentration, N 峰 and N req .

[0006] As described elsewhere in this document, a reliable quantification of the amount of an analyte can only be achieved if the analyte is analyzed in quantities exceeding a threshold in the sample to be analyzed for quality. The minimum concentration is the concentration of the first analyte required in the sample to achieve a pre-selected relative standard deviation when the volume of the sample being analyzed is the same as the volume of the sample analyzed when the known concentration was determined.

[0007] If the volume of the actual sample to be analyzed is greater than the volume of the sample analyzed when the known concentration was determined, the concentration of the analyte in the actual sample to be analyzed can be lower than the minimum concentration while still supplying the mass analyzer with a threshold amount of analyte, in order to reliably quantify the amount of analyte in the sample. Conversely, if the volume of the actual sample to be analyzed is less than the volume of the sample analyzed when the known concentration was determined, the concentration of the analyte in the actual sample to be analyzed may need to be higher than the minimum concentration in order to supply the mass analyzer with a threshold amount of analyte, in order to reliably quantify the amount of analyte in the sample.

[0008] In instances where the volume of the actual sample to be analyzed differs from the volume of the sample analyzed when the known concentration was determined, the minimum concentration of the analyte in the actual sample to be analyzed can be determined in order to reliably quantify the analyte by multiplying the known concentration by the volume of the sample analyzed when the known concentration was determined, and then dividing the product by the volume of the actual sample to be analyzed.

[0009] Step (ii) may include using relationships To calculate the number of ions N 峰 Where A is the area of ​​the peak; P is the number of instances of performing the first quality analysis method during the peak period; T is the duration of analysis of the first analyte according to the first quality analysis method during each instance of performing the first quality analysis method; and W is the width of the peak at its bottom.

[0010] Step (ii) may include, according to the following To calculate the number of ions N 峰 Where G(m) is the value of N 峰 and The relevant mass correlation function, wherein the value of G(m) depends on the mass or mass-to-charge ratio of the ionic substance detected at the ion detector during the first mass analysis method.

[0011] Step (iii) may include, according to To calculate the number of ions N reqWhere A(m) is a mass-related function, and the value of A(m) depends on the mass or mass-to-charge ratio of the ionic substance detected at the ion detector during the first mass analysis method, and where RSD c It is the value of the pre-selected relative standard deviation.

[0012] For a given mass or mass-to-charge ratio, the value of A(m) is the relative standard deviation (RSD) of the pulse height distribution of ions for that mass or mass-to-charge ratio using a given detector. PHD Related, and their relationship is as follows: .

[0013] The known concentration of the first analyte is C. inj And step (iv) includes determining the minimum concentration C from the following. RSD : .

[0014] The method may include determining the signal-to-noise ratio of a peak; selecting a minimum acceptable signal-to-noise ratio value for the ion peak when analyzing a first analyte according to a first mass analysis method; and estimating the minimum acceptable signal-to-noise ratio C by: S / N Simultaneously, the minimum concentration of the first analyte that can be used for quality analysis according to the first quality analysis method is: multiply the known concentration of the first analyte by the minimum acceptable signal-to-noise ratio value, and then divide this by the determined signal-to-noise ratio.

[0015] The mass spectrometer can automatically determine C RSD and C S / N The higher of the two is the minimum concentration of the first analyte to be analyzed according to the first quality analysis method.

[0016] The mass spectrometer can automatically display the determined minimum concentration on an electronic display.

[0017] The method may include performing a mass analysis on a sample containing the first analyte using a mass spectrometer or the mass spectrometer according to the first mass analysis method, wherein: (i) the sample contains a lower concentration of the first analyte compared to the known concentration; or (ii) the volume of the sample for which the mass analysis is performed multiplied by the concentration of the first analyte in the sample is less than the known concentration multiplied by the volume in which the mass analysis was performed when the known concentration was determined.

[0018] A sample can only be analyzed in this manner if it is known that the sample contains a first analyte at a concentration higher than the minimum concentration determined in the method described above (or if the volume of the sample being analyzed multiplied by the concentration of the first analyte in the sample is greater than the minimum concentration multiplied by the volume of the sample analyzed when the known concentration was determined).

[0019] Mass spectrometers can be based on C RSD and / or C S / N The mass spectrometer can automatically adjust one or more operating conditions based on a predetermined value, and use one or more adjusted operating conditions to perform a mass analysis of the sample; or the mass spectrometer can be based on C RSD and / or C S / N The determined value is used to automatically display information or options for adjusting one or more operating conditions.

[0020] Mass spectrometers can be based on C RSD The definite value of C S / N The definite value or C RSD and C S / N The mass spectrometer can automatically adjust one or more operating conditions based on a predetermined higher value. For example, the mass spectrometer can compare any one of these predetermined values ​​with a target value, and the mass spectrometer can automatically adjust the one or more operating conditions based on this comparison.

[0021] The step of automatically adjusting one or more operating conditions of the mass spectrometer may include: a) adjusting the duration of the time for mass analysis of the analyte according to the first mass analysis method during the step of mass analysis of the sample; and / or b) adjusting the voltage or other operating conditions of one or more ion optical elements of the mass spectrometer applied during the step of mass analysis of the sample.

[0022] For example, the duration of the time for mass analysis of the first analyte according to the first mass analysis method during the step of mass analysis of the sample can be increased.

[0023] The method may include: selecting a target concentration of a first analyte to be analyzed according to a first mass analysis method; determining the concentration C. RSD And optional location C S / N ; and a) if C RSD And optional location C S / N If the concentration is below the target concentration, it is determined that the first analyte can be analyzed according to the first mass analysis method; and / or b) if C RSD and / or C S / N If the concentration is higher than the target concentration, it is determined that the first analyte cannot be reliably analyzed according to the first mass analysis method.

[0024] The mass spectrometer may have a processor configured to perform the determination automatically. The mass spectrometer may be configured to output the determination results on an electronic display.

[0025] This method assumes that the volume of the sample to be analyzed is equal to the volume of the sample at a given concentration C. RSD and / or C S / N The volumes analyzed were the same. As described with respect to claim 1, if the volumes were different, the concentrations described above could be replaced by a value representing the amount of the first analyte, obtained by multiplying each of the concentrations described above by the corresponding volume of the sample used in determining that concentration.

[0026] The method may include using a chromatographic apparatus to separate a sample containing different analytes, such that the different analytes are eluted from the chromatographic apparatus during different corresponding chromatographic peaks; wherein step (i) is performed during the chromatographic peak of the first analyte to obtain the ion signal having the peak.

[0027] The chromatographic apparatus can be a liquid chromatograph or another type of separator (such as a gas chromatograph).

[0028] Although a method for determining the minimum concentration of an analyte to be analyzed according to a first mass analysis method has been described, it is also expected that the minimum concentration of the first analyte to be analyzed can be determined when the first analyte is analyzed according to a different second mass analysis method.

[0029] Therefore, a method corresponding to the method described above can be performed, except that the first analyte is analyzed by a second, different quality analysis method. The first and second quality analysis methods can be performed on the first analyte during the same chromatographic peak or alternatively in separate experiments.

[0030] For example, the method may include: (i) performing a mass analysis on the known concentration of the first analyte during the chromatographic peak of the first analyte according to a second different mass analysis method to obtain an ion signal having a peak; (ii) determining the number N of ions N that form the peak detected by a mass spectrometer during the second mass analysis method. 峰 (iii) Determine the number N of ions that need to be detected in the peak in order to achieve a pre-selected relative standard deviation for the ion peak. req ; and (iv) determine the relative standard deviation C that still achieves the pre-selected standard deviation based on the values ​​of the following: RSD Simultaneously, the minimum concentration of the first analyte that can be analyzed according to the second mass analysis method: the known concentration, N 峰 and N req .

[0031] The analyte can be analyzed by tandem mass spectrometry in the first mass analysis method and / or the second mass analysis method.

[0032] For example, the first analyte can be analyzed by tandem mass spectrometry, wherein the mass spectrometer monitors a first multiple reaction monitoring (MRM) transition in the first mass analysis method and a second MRM transition in the second mass analysis method.

[0033] In other words, in the first mass analysis method, the mass spectrometer can control a first mass filter to transmit only a single precursor ion, control a fragmentation or reaction device to fragment or react the ion to form fragment or product ions, and control a second mass filter to transmit a single fragment or product ion from the fragment or product ions to an ion detector. In the second mass analysis method, the mass spectrometer can control the first mass filter to transmit only a single precursor ion (which may be the same as or different from the single precursor ion transmitted in the first mass analysis method), control a fragmentation or reaction device to fragment or react the ion to form fragment or product ions, and control the second mass filter to transmit a single fragment or product ion from the fragment or product ions to an ion detector, wherein the fragment or product ions transmitted by the second mass filter are different in the second mass analysis than in the first mass analysis.

[0034] The mass spectrometer can be repeatedly switched between the first mass analysis method and the second mass analysis method during the chromatographic peak of the first analyte.

[0035] Although a method for determining the minimum concentration of an analyte to be analyzed according to a first (and optionally a second) quality analysis method has been described, it may be desirable to determine such a minimum concentration for quality analysis of each of a variety of different analytes. This can be accomplished in a manner corresponding to that described above.

[0036] Therefore, this method can determine the minimum concentration of a second different analyte to be mass analyzed, including: (i) performing mass analysis on the second analyte at a known concentration using a mass spectrometer according to another mass analysis method to obtain an ion signal with a peak; and (ii) determining the number N of ions N that form the peak detected by the mass spectrometer during the other mass analysis method. 峰 (iii) Determine the number N of ions that need to be detected in the peak in order to achieve a pre-selected relative standard deviation for the ion peak. req ; and (iv) determine the relative standard deviation C that still achieves the pre-selected standard deviation based on the values ​​of the following: RSD Simultaneously, the minimum concentration of a second analyte that can be analyzed using other quality analysis methods: the known concentration, N 峰 and N req .

[0037] It may also be desirable to determine the minimum concentration of the second analyte to be analyzed when performing a quality analysis on the second analyte using another different quality analysis method. This can be accomplished in a manner corresponding to the above description of the quality analysis of the (first) analyte using a second quality analysis method.

[0038] It will be understood that different analytes can be co-eluted from the chromatographic apparatus, that is, they can have overlapping chromatographic peaks.

[0039] Therefore, the method may include determining the minimum concentration of a second different analyte to be mass-analyzed in a mass spectrometer, wherein the method includes: (i) performing mass analysis of the second analyte at a known concentration using a mass spectrometer according to an additional mass analysis method to obtain an ion signal with a peak; and (ii) determining the number N of ions N detected by the mass spectrometer during the additional mass analysis method to form the peak. 峰 (iii) Determine the number N of ions that need to be detected in the peak in order to achieve a pre-selected relative standard deviation for the ion peak. req ; and (iv) determine the relative standard deviation C that still achieves the pre-selected standard deviation based on the values ​​of the following: RSD Simultaneously, the minimum concentration of a second analyte that can be analyzed using other quality analysis methods: the known concentration, N 峰 and N req .

[0040] The first and second analytes can be analyzed by tandem mass spectrometry, wherein the mass spectrometer monitors a first multiple reaction monitoring (MRM) transition in a first mass analysis method and monitors different multiple reaction monitoring (MRM) transitions in another mass analysis method; and wherein the mass spectrometer repeatedly switches between the first mass analysis method and the other mass analysis method during at least a portion of the time during which the chromatographic peaks of the first and second analytes overlap.

[0041] The present invention also provides a mass spectrometer arranged and configured to perform the methods described herein.

[0042] Therefore, a first aspect of the present invention provides a mass spectrometer configured to perform the methods described above.

[0043] This invention provides a mass spectrometer configured to: (i) perform mass analysis on the concentration of an analyte according to a first mass analysis method to obtain an ion signal with a peak; and (ii) determine the number N of ions N that form the peak detected by the mass spectrometer during the first mass analysis method. 峰 (iii) Determine the number N of ions that need to be detected in the peak in order to achieve a pre-selected relative standard deviation for the ion peak. req; and (iv) determine the relative standard deviation C for achieving the preselected standard deviation based on the values ​​of the following: RSD Simultaneously, the minimum concentration of the first analyte that can be analyzed according to the first quality analysis method: the concentration, N 峰 and N req .

[0044] The present invention also provides a computer-readable medium comprising instructions for causing a mass spectrometer to perform any of the methods described above.

[0045] In a second aspect, the present invention provides a mass spectrometry analysis method, comprising: receiving an input indicating that a first analyte and a second analyte have been selected for mass analysis in a mass spectrometer using a chromatographic separator to separate the analytes; determining that the first analyte is to be monitored during a first time window and the second analyte is to be monitored during a second time window; determining that the first time window and the second time window overlap within a first time period; and determining a minimum duration for which each of the first analyte and the second analyte needs to be analyzed during the first time period to obtain a corresponding ion peak of each of the first analyte and the second analyte satisfying a corresponding preselected condition.

[0046] This method allows control of a user interface, such as an electronic display, to instruct an operator to adjust the mass spectrometer so that it analyzes the first and second analytes for a defined minimum duration during a first time period. Alternatively, the method can automatically control the mass spectrometer to do so.

[0047] The method may include: using a chromatographic separator to separate a first analyte and a second analyte; and analyzing each of the first analyte and the second analyte for at least their respective minimum duration during the first time period in order to obtain a corresponding ion peak of each of the first analyte and the second analyte satisfying their preselected conditions.

[0048] The first analyte and the second analyte cannot be simultaneously mass-analyzed (i.e. detected) by the mass spectrometer. Therefore, during the first time period, the mass spectrometer can repeatedly switch between monitoring a first multiple reaction monitoring (MRM) transition when mass-analyzing the first analyte and monitoring a second different multiple reaction monitoring (MRM) transition when mass-analyzing the second analyte.

[0049] The method may include: receiving an input indicating that an additional analyte has been selected for mass analysis in a mass spectrometer; determining that the additional analyte needs to be monitored during an additional time window; determining that the additional time window overlaps with a first time window and a second time window in a second time period; and determining the minimum duration for which each of the first analyte, the second analyte, and the additional analyte needs to be analyzed during the second time period in order to obtain a corresponding ion peak of each of the first analyte, the second analyte, and the additional analyte satisfying a corresponding preselected condition.

[0050] This method allows control of a user interface, such as an electronic display, to instruct the operator to adjust the mass spectrometer so that it analyzes the first analyte, the second analyte, and other analytes for a defined minimum duration during a second time period. Alternatively, the method can automatically control the mass spectrometer to do so.

[0051] The method may include: using a chromatographic separator to separate a first analyte, a second analyte, and a further analyte; and analyzing each of the first analyte, the second analyte, and the further analyte for at least their respective minimum duration during a second time period in order to obtain a corresponding ion peak of each of the first analyte, the second analyte, and the further analyte satisfying its preselected conditions.

[0052] According to the implementation plan, the first analyte, the second analyte, and other analytes cannot be simultaneously analyzed (i.e. detected) by the mass spectrometer.

[0053] During the second time period, the mass spectrometer can repeatedly switch between monitoring a first multiple reaction monitoring (MRM) transition when performing mass analysis on a first analyte; monitoring a second different multiple reaction monitoring (MRM) transition when performing mass analysis on a second analyte; and monitoring a third different multiple reaction monitoring (MRM) transition when performing mass analysis on another analyte.

[0054] This method determines that adding an additional analyte to the analysis will reduce the duration of time available for analyzing the first and / or second analyte during the second time interval. Therefore, relative to the corresponding time interval before the addition of the additional analyte, this method reduces the duration of analysis of the first and / or second analyte during the second time interval. However, the duration of analysis of the first and / or second analyte during the second time interval is reduced to a time still sufficiently long for the corresponding ion peak to satisfy its preselection criteria.

[0055] This method can use one or more computer processors to perform each of the defined steps in the described determination process. The method can be implemented on a mass spectrometer equipped with such a processor. A user can use an electronic user interface to select a first analyte and a second analyte, the electronic user interface providing the input in response. The user can also use the user interface to select additional analytes.

[0056] One or more pre-selected conditions can be the pre-selected signal-to-noise ratio of the ion peak; one or more pre-selected conditions can be the relative standard deviation of the ion peak.

[0057] The present invention also provides a computer-readable medium comprising instructions that, when executed on a computer, cause a mass spectrometer to perform the methods described above.

[0058] The computer can be the computer of the mass spectrometer.

[0059] A second aspect of the invention also provides a mass spectrometer arranged and configured to perform the methods described with respect to the second aspect of the invention.

[0060] Therefore, the present invention provides a mass spectrometer comprising a chromatographic separator and a mass analyzer, wherein the mass spectrometer is configured to: receive an input indicating that a first analyte and a second analyte have been selected for mass analysis in the mass spectrometer; determine that the first analyte is to be monitored during a first time window and the second analyte is to be monitored during a second time window; determine that the first time window and the second time window overlap within a first time period; and determine the minimum duration for which each of the first analyte and the second analyte needs to be analyzed during the first time period to obtain a corresponding ion peak of each of the first analyte and the second analyte satisfying a corresponding preselection condition.

[0061] The mass spectrometer includes one or more processors and electronic circuitry configured to perform the described determination steps.

[0062] In a third aspect, the present invention provides a computer-implemented method for determining whether or not a mass spectrometer can be reliably used to perform mass analysis on an analyte, the method comprising: receiving input indicating that a first analyte has been selected for mass analysis in a mass spectrometer using a chromatographic separator; and determining whether or not a further analyte can be used for mass analysis.

[0063] Whether the mass spectrometer is capable of reliably performing mass analysis on the other analyte: i) determining whether to monitor the first analyte during a first time window and the other analyte during a second time window; ii) determining whether the first and second time windows overlap within a first time period; iii) if mass analysis of both the first analyte and the other analyte is selected, determining the duration of time available during the first time period for mass analysis of each of the first analyte and / or the other analyte; iv) determining the minimum amount or minimum concentration of the first analyte and / or the other analyte required to obtain corresponding ion peaks with one or more preselected conditions based on the determined duration of time available during the first time period for mass analysis of the first analyte and / or the other analyte, respectively; and v) determining whether to perform mass analysis on the other analyte or whether the mass spectrometer is capable of reliably performing mass analysis on the other analyte based on a comparison of the determined minimum amount or minimum concentration of the first analyte and / or the other analyte with the preselected target amount or concentration of each of the first analyte and / or the other analyte, respectively.

[0064] For example, it can be determined that if a quality analysis is performed on another analyte, the minimum amount or concentration of the first analyte will increase to a level higher than the target amount or concentration of the first analyte (because less time is available for analyzing the first analyte). Additionally or alternatively, it can be determined that if a quality analysis is performed on another analyte, the minimum amount or concentration of the other analyte will increase to a level higher than the target amount or concentration of the other analyte (because even less time is available for analyzing the other analyte). In this case, it is determined that the other analyte cannot be reliably analyzed, or that the other analyte should not be analyzed.

[0065] This method can use one or more computer processors to perform each of the defined steps in the described determination process. The method can be implemented on a mass spectrometer equipped with such a processor. A user can use an electronic user interface to select a first analyte in response to the input provided. The user can also use the user interface to select additional analytes.

[0066] One or more pre-selected conditions can be the pre-selected signal-to-noise ratio of the ion peak; one or more pre-selected conditions can be the relative standard deviation of the ion peak.

[0067] The method described herein with respect to the first aspect of the invention may be used, for example, based on C. RSD and / or C S / N The calculation determines the minimum amount or concentration of the first analyte and / or other analytes required for mass analysis to obtain corresponding ion peaks with one or more preselected conditions.

[0068] According to the implementation plan, the first analyte and other analytes cannot be analyzed for quality at the same time.

[0069] In step iii), the duration available for mass analysis of the first analyte and / or the other analyte may be determined based on mass analysis of the first analyte and the other analyte by tandem mass spectrometry, wherein the mass spectrometer repeatedly switches between monitoring a first multiple reaction monitoring (MRM) transition when mass analyzing the first analyte and monitoring different multiple reaction monitoring (MRM) transitions when mass analyzing the other analyte.

[0070] It should be understood that the time available for quality analysis of each of the first and other analytes will be reduced by selecting the other analytes to be analyzed.

[0071] The present invention provides a computer-readable medium comprising instructions that, when executed on a computer, cause a mass spectrometer to perform the methods described above.

[0072] The computer can be the computer of the mass spectrometer.

[0073] The present invention provides a method for mass spectrometry analysis, the method comprising: performing the method described above; and then separating a sample comprising the first analyte and the additional analyte in a chromatographic separator; and a) if it is determined in step v) that mass analysis of the additional analyte is to be performed, the method of mass spectrometry analysis repeatedly switches between mass analysis of the first analyte and mass analysis of the additional analyte during a first time period; or b) if it is determined in step v) that mass analysis of the additional analyte is not to be performed, the method of mass spectrometry analysis performs mass analysis of the first analyte without performing mass analysis of the additional analyte during the first time period.

[0074] The duration of elution of the first analyte from the chromatographic separator may overlap with the duration of elution of the other analyte from the chromatographic separator.

[0075] In step a), the first analyte and the other analytes may be based on mass analysis by tandem mass spectrometry, wherein the mass spectrometer repeatedly switches between monitoring a first multiple reaction monitoring (MRM) transition when performing mass analysis on the first analyte and monitoring different multiple reaction monitoring (MRM) transitions when performing mass analysis on the other analytes.

[0076] Although a method has been described in which a first analyte has been selected for quality analysis and it is determined whether to perform quality analysis on other analytes, it will be understood that the method may select multiple analytes to be performed for quality analysis and then determine whether to perform quality analysis on other analytes. In such embodiments, the method may determine that other analytes will be monitored during a time window that overlaps with the time window for monitoring multiple selected analytes. The method described above may then be performed for each of the multiple selected analytes.

[0077] A third aspect of the invention also provides a mass spectrometer arranged and configured to perform the methods described herein. Therefore, a third aspect of the invention provides a mass spectrometer configured to perform the methods described above.

[0078] The mass spectrometer includes one or more processors and electronic circuitry configured to perform the described determination steps. Attached Figure Description

[0079] The various embodiments will now be described by way of example only, with reference to the accompanying drawings, wherein:

[0080] Figure 1 An embodiment of the mass spectrometer according to the present invention is shown;

[0081] Figure 2 An overview of an embodiment of the present invention for calculating the concentration required to achieve the RSD% defined in LLOQ for an analyte is illustrated;

[0082] Figure 3 An example of a chromatographic peak that has been sampled at multiple points is shown;

[0083] Figure 4 Examples of pulse height distributions for ions of several different corresponding masses are shown;

[0084] Figure 5 It shows Figure 4 The probability density function of the pulse height distribution is shown.

[0085] Figure 6 The ion signals detected for two different MRM transitions are shown;

[0086] Figure 7 A table illustrating the analysis of several different analytes is shown; and

[0087] Figure 8 An example is shown of how adding an analyte to an analytical method affects the LLOQ concentration of each of the other analytes in certain other analytes. Detailed Implementation

[0088] In mass spectrometry, analytes in a sample are analyzed by ionization, and the resulting precursor ions or fragment or product ions derived therefrom are then detected in a mass analyzer. For any given analytical method, the amount of an analyte can only be reliably quantified if the analyte is analyzed in a quantity exceeding a threshold in the sample. This means that for a mass analysis of a given volume of sample, the amount of the analyte can only be reliably quantified if the analyte is present in the sample at a concentration at or above a level known as the lower limit of quantitation (LLOQ). The concentration of an analyte as the LLOQ is the concentration of the analyte that satisfies the following criteria: (i) a preselected signal-to-noise ratio (S / N) value of the ion signal detected during the mass analysis of the analyte; and (ii) a preselected relative standard deviation (RSD%) value of the ion signal. The relative standard deviation discussed herein refers to the standard deviation of the area of ​​the ion peak detected for the same ion substance in multiple different analyses of the same sample divided by the average of those areas (and multiplied by 100 to obtain a percentage value). For example, in MRM experiments, the relative standard deviation is the standard deviation of the area of ​​the ion peak detected for the same MRM transition in multiple different analyses of the same sample, divided by the average of those areas (and multiplied by 100 to obtain a percentage value). Users can define the S / N and RSD% values ​​they need the LLOQ concentration to meet.

[0089] Typically, when developing methods for analyzing samples, the LLOQ concentration for each analyte of interest is determined to ensure that the sample being analyzed contains that analyte at a concentration that satisfies the LLOQ concentration for each analyte of interest, thus enabling accurate quantification. For example, a user can define the LLOQ concentration of an analyte as a concentration that achieves both a 5:1 S / N ratio and 10% RSD%.

[0090] Currently, in order to calculate the S / N value of the LLOQ concentration of an analyte, the user injects a sample containing a relatively high concentration of the analyte into a mass spectrometer for mass analysis, determines the peak height in the ion signal detected for the analyte, determines the baseline noise with respect to the peak, determines the S / N value of the peak, and then uses the S / N value to estimate the concentration of the analyte when the ion peak will be detectable only when it is above the background noise or the concentration of the analyte that will meet the predetermined S / N value.

[0091] For example, the S / N value required to meet LLOQ conditions can be defined by the user as 10:1. If a mass analysis of an analyte produces an ion peak with an S / N value of 1000:1, then a mass analysis of the analyte at a concentration 100 times lower is expected to provide an ion peak with an S / N value of approximately 10:1, which is the S / N value required by LLOQ. Therefore, the concentration of the analyte required to meet the S / N value defined by LLOQ is determined to be one percent of the concentration of the analyte in the sample for mass analysis. Similarly, if a mass analysis of an analyte produces an ion peak with an S / N value of 100:1, then a ion peak with an S / N value of approximately 10:1 will be expected when the sample is diluted 10 times. Similarly, if the height of the ion peak is determined to be 10... 5 The count was 10 and the baseline noise was determined to be 10. 3 By counting the analyte, the user will be able to determine if they will be unable to inject the analyte at a concentration 100 times lower and still be able to distinguish the analyte's ion peak from noise. Therefore, it is relatively easy to determine the minimum S / N value of the compound that will provide a satisfactory LLOQ.

[0092] However, users typically find it difficult to estimate the minimum concentration of an analyte that will satisfy the LLOQ's RSD% requirement. Typically, to do this, users must prepare multiple dilutions of a sample, each containing a different concentration of the analyte, perform multiple mass analyses of the analyte in each of these dilutions, and then determine the RSD% of the ion signal of the analyte in each of the dilutions. This must be done until a concentration with an RSD% that satisfies the RSD% defined in the LLOQ is found. This concentration is then determined as the minimum concentration of the analyte that can be reliably analyzed in future experiments while still expected to meet the desired LLOQ's RSD% requirement. It will be understood that this routine process is extremely time-consuming, especially considering that it may be desirable to analyze hundreds of different analytes, and possibly also to analyze multiple different ion peaks for each analyte, such as by monitoring multiple MRM transitions for each analyte.

[0093] Embodiments of the present invention provide an improved method for calculating the LLOQ concentration of an analyte for a given method of quality analysis.

[0094] According to an embodiment of the invention, the sample is separated by a chromatographic apparatus such as a liquid chromatography apparatus, such that different analytes in the sample are eluted from the apparatus at different corresponding time periods. The eluted analytes are then ionized, and the resulting analyte ions are analyzed by tandem mass spectrometry (also known as MS / MS).

[0095] Figure 1An embodiment of a mass spectrometer according to the present invention is shown. The spectrometer includes a liquid chromatography apparatus 2, an ion source 4, a first mass filter 6, a fragmentation or reaction apparatus 8, a second mass filter 10, and an ion detector 12.

[0096] As described above, the sample is injected into a liquid chromatography apparatus, causing different analytes in the sample to elute at different corresponding time intervals. The eluted analytes are ionized by an ion source (e.g., an electrospray ionization source) to generate precursor ions. It will be understood that other types of ion sources may be used alternatively. The precursor ions are passed to a first mass filter, which is configured to transmit only a narrow range of mass-to-charge ratios at any given time, e.g., corresponding to a single precursor ion. This mass filter selects the analytes to be mass-analyzed. The precursor ions transmitted by the first mass filter are passed to a fragmentation or reaction apparatus and undergo fragmentation or reaction (e.g., with reactant ions or molecules) to form fragments or product ions. These fragments or product ions are then passed to a second mass filter, which is configured to transmit only a narrow range of mass-to-charge ratios at any given time, e.g., corresponding to a single fragment or product ion. The ions transmitted by the second mass filter bombard an ion detector and generate an ion signal.

[0097] An operable mass spectrometer is used to monitor one or more multiple reaction monitoring (MRM) transitions. When monitoring an MRM transition, a first mass filter is configured to isolate a specific mass-to-charge ratio corresponding to a particular precursor ion (i.e., from a particular analyte) and to transport that specific precursor ion to the fragmentation or reaction apparatus. A second mass filter is configured to isolate a specific mass-to-charge ratio corresponding to a particular fragment or product ion and to transport that ion to an ion detector. If an ion is detected when both mass filters are configured in this manner, the precursor ion and therefore the analyte can be determined with relatively high confidence.

[0098] As mentioned above, different analytes in the sample elute from the chromatographic apparatus within different corresponding time ranges, i.e., as different chromatographic peaks. It may be desirable to monitor more than one MRM transition for each analyte. When monitoring more than one MRM transition during the chromatographic peak of an analyte, a first mass filter is configured to isolate and transfer precursor ions of the analyte, and a second mass filter switches between isolating / transferring different fragment / product ions corresponding to different corresponding MRM transitions. As will be described in more detail below, each MRM transition is monitored multiple times (e.g., 10 to 15 times) during each chromatographic peak. Therefore, the second mass filter is configured to transfer fragment ions for a specific MRM transition only during a short period of time (referred to as residence time) before switching to monitoring the next MRM transition.

[0099] Figure 2An overview of an embodiment of the present invention for calculating the concentration of an analyte required to achieve an RSD% as defined by a user in the LLOQ for a given method of analysis is illustrated. To calculate this concentration, the embodiment uses: (i) a user-defined RSD% value (RSD%) for the method of analysis to satisfy the LLOQ. c The number of ions (N) that need to be detected in the ion peak. req (ii) The number of ions (N) detected in the ion peak by the method described in the analysis. 峰 (iii) the known concentration C of the analyte in the sample being analyzed (i.e., injected into the mass spectrometer); inj This method can be combined with the methods described above to determine whether the LLOQ concentration is limited by baseline noise, S / N, or RSD% and optionally displayed to the user.

[0100] from Figure 2 As can be seen, the implementation plan uses various parameters to calculate in order to achieve RSD. c The value of the number of ions N to be detected. req The number of ions N to be detected. req It is determined based on a combination of pulse height distribution and ion arrival statistics, as will be discussed in more detail below.

[0101] As mentioned above, in order to determine the concentration of the analyte that needs to be analyzed to meet the user-defined RSD% value of LLOQ, for a specific analytical method, such as for a specific MRM transition, the implementation calculates the number N of ions detected for that MRM transition during the chromatographic peak of the analyte. 峰 In simple scenarios, the ion number N can be calculated from the ion peak based on the peak area. 峰 For example, a mass spectrometer can sample the ion signal by monitoring MRM transitions at multiple points during analyte elution to ensure that the chromatographic peak is sufficiently limited for accurate integration, for example, at 10 to 15 points. The ion signal reported for each data point of the peak is proportional to the ion count per second.

[0102] Figure 3 An example of a chromatographic peak sampled at 13 points above the peak, as described above, is shown, as depicted by the vertical line. If the mass spectrometer is operating to continuously analyze only a single MRM transition during the chromatographic peak, the entire area under the peak can be determined and directly converted to the ion number N. 峰However, it is often desirable to analyze multiple different MRM transitions within overlapping time ranges. For example, it may be desirable to alternate between monitoring different MRM transitions of the same analyte during its chromatographic peak. Additionally or alternatively, different analytes may be co-eluted from the chromatographic separation device, and it may be desirable to alternate between monitoring different MRM transitions of the different corresponding analytes during the co-elution periods. In such instances, the ion detected for any given MRM transition is only detected during a portion of its analyte's chromatographic peak.

[0103] Therefore, in order to calculate the actual number N of ions detected for a given MRM transition of an analyte during its chromatographic peak. 峰 It is necessary to take into account the number of times the ions used for MRM transitions are detected during the chromatographic peak and the duration of each of these transitions. Figure 3 The shaded vertical bars within the peak represent the ion signal detected for a single MRM transition during the chromatographic peak. In this particular example, 13 MRM transitions were monitored during the peak, but it will be understood that fewer or more could be monitored.

[0104] As we will understand from the above, the number N of ions detected during the MRM transition during the chromatographic peak is... 峰 It is related to the area of ​​the chromatographic peak, the number of points during which MRM transitions are monitored during the peak, and the duration of each of these MRM transitions (i.e., the residence time of the second filter).

[0105] If the mass spectrometer uses a non-pulse counting detector, the contribution of the ion signal to a single ion may differ for ions with different masses or mass-to-charge ratios. Therefore, it may be advantageous to take this into account when calculating the number of detected ions by applying an adjustment factor that depends on the mass or mass-to-charge ratio of the detected ion.

[0106] Therefore, the following equation can be used to determine the actual number of ions N for the MRM transition count during the chromatographic peak. 峰 :

[0107]

[0108] Where G(m) is the quality-related adjustment factor; A is the area of ​​the chromatographic peak; P is the number of points at which the MRM transition is monitored on the peak; T is the time at which the MRM transition is monitored at each of these points (i.e., the residence time of the second mass filter), in seconds; and W is the width of the chromatographic peak at its bottom, in seconds.

[0109] As described above, in addition to calculating the number of ions N 峰 In addition, the implementation scheme also calculates the RSD% to achieve the RSD defined by LLOQ, i.e., to achieve RSD. CThe minimum number of ions N that needs to be detected in the chromatographic peak req To achieve a given RSD C The theoretical minimum number of ions N that needs to be detected. req It is defined by a combination of the effects of ion arrival statistics and pulse height distribution, as will be described below.

[0110] The number of ions arriving at the detector in any given peak is based on ion arrival statistics and follows a Poisson distribution. The RSD of the Poisson distribution is given by [reference to RSD]. Arr It is known and given by the following equation, where N is the number of ions sampled:

[0111]

[0112] However, not all ions produce the same detector response when they strike the detector. More specifically, the ion detector generates output pulses in response to ion impacts, but these output pulses do not have a fixed intensity; instead, they follow a pulse height distribution known as the pulse height.

[0113] Figure 4 Examples of pulse height distributions for several ions of different corresponding masses detected by a detector are shown. Each pulse height distribution shows the number of ions hitting the detector (y-axis) as a function of the detector pulse intensity (x-axis) they produce. As can be seen, each distribution is a peak, indicating that a relatively large proportion of the ions each produce a similar output at the detector. Figure 4 It is shown that ions with different masses can have different pulse height distributions, and this is preferably true in calculating N. req This was taken into consideration.

[0114] Figure 5 It shows Figure 4 The probability density function for the pulse height distribution is shown. The probability density function for each mass represents the probability that an ion impact at the detector will produce each pulse intensity along the x-axis.

[0115] RSD due to pulse height distribution PHD The following equation gives the result, where A(m) is the mass correlation function and N is the number of ions sampled:

[0116]

[0117] As those skilled in the art will understand, parameter A(m) may vary depending on the detector type and detector gain, but is expected to be consistent for each polarity of the ion on a given detector.

[0118] Therefore, the total RSD of the peak is determined by RSD. Arrand RSD PHD Both are defined and obtained by orthogonally adding these values. Therefore, the RSD defined by LLOQ... C The value of RSD Arr and RSD PHD The following are related:

[0119]

[0120] Using the RSD given above Arr and RSD PHD The equations that replace each of them are provided below in the RSD. C The equation:

[0121]

[0122] Therefore, the RSD is provided. C Required number of ions N req Given from the following:

[0123]

[0124] As described above, in order to determine the LLOQ concentration of an analyte, for example for a specific MRM transition, this implementation uses: (i) to achieve RSD c The value needs to be the number of ions N detected during the analyte peak. req (ii) The number of ions N detected during the peak of this MRM transition. 峰 ; and (iii) the known concentration C of the analyte injected to produce the peak. inj .

[0125] More specifically, since it can be assumed that the number N of ions counted in the chromatographic peak is... 峰 The RSD is proportional to the concentration of the injected analyte, therefore it is still expected to meet the LLOQ. C The maximum factor D required to dilute the analyte can be determined as follows:

[0126]

[0127] Therefore, the concentration C of the injected analyte can be used as follows: inj The RSD that satisfies LLOQ is calculated using the dilution factor D. c The required concentration C LLOQ :

[0128]

[0129] As mentioned above, reliable quantification of an analyte's quantity is only possible when the analyte in the sample to be analyzed exceeds a threshold amount. It will be understood that the LLOQ concentration described herein is the ratio of the volume of the analyte in the sample to be analyzed to the volume of the sample at which the known concentration C is determined. inj The concentration required in the sample to meet the LLOQ requirement when the volume of the sample to be analyzed is the same. If the volume of the sample to be analyzed is higher than the volume required to determine the known concentration C... inj If the volume of analyte used for mass analysis is specified, then the concentration of the analyte in the sample to be analyzed can be lower than C while still supplying the mass analyzer with the threshold amount of analyte. LLOQ This is to ensure reliable quantification of the amount of analyte in the sample. Conversely, if the volume of the sample to be analyzed is less than the volume required to determine the known concentration C... inj If the volume of analyte analyzed at that time is such that, in order to provide the mass analyzer with the threshold amount of analyte, the concentration of the analyte in the sample to be analyzed may need to be higher than C. LLOQ This is to enable reliable quantification of the amount of analyte in the sample.

[0130] The volume of the sample to be analyzed and the concentration C to be determined. inj In instances where different volumes of samples were subjected to mass analysis, the minimum concentration of the analyte in the sample to be analyzed can be determined in order to reliably quantify the analyte: [The text then abruptly shifts to a different topic:] ...to determine the minimum concentration of the analyte in the sample to be analyzed, in order to reliably quantify the analyte: ... [The text then abruptly shifts again:] ...with a known concentration C... inj Multiply by the known concentration C to determine inj The volume of the sample for which mass analysis was performed was then calculated, and the product was then divided by the volume of the sample to be analyzed.

[0131] Now refer to Figure 6 An example illustrating an embodiment of the invention is described below. A tandem quadrupole mass spectrometer was used to perform tandem mass analysis of an analyte at a concentration of 1000 ppb in the manner already described above. The MRM transition from the precursor ion of the analyte with a mass-to-charge ratio of 305 Da to its fragment ion with a mass-to-charge ratio of 127 Da was monitored during the chromatographic peak of the analyte. In other words, a first mass filter was configured to isolate / transmit the ion with a mass-to-charge ratio of 305 Da, and a second mass filter was configured to isolate / transmit the ion with a mass-to-charge ratio of 127 Da. The spectrometer also analyzed the MRM transition from the precursor ion to its fragment ion with a mass-to-charge ratio of 121 Da during the chromatographic peak. In other words, a first mass filter was configured to isolate / transmit the ion with a mass-to-charge ratio of 305 Da, and a second mass filter was configured to isolate / transmit the ion with a mass-to-charge ratio of 121 Da.

[0132] Figure 6The ion signals detected for fragment ion material with a mass-to-charge ratio of 127 Da and for fragment ion material with a mass-to-charge ratio of 121 Da are shown.

[0133] Mass analysis of 127Da ions was performed at 13 points during the chromatographic peak by setting a second mass filter for the mass spectrometer at each of these points to transmit only 127Da ions to the detector. This... Figure 6 The peak is illustrated by 13 intensity points located above the baseline. At each of these points in the chromatographic peak, a second mass filter was configured to transport ions with a residence time of 0.003 seconds. The peak area of ​​the 127 Da ion was determined to be 6.44 × 10⁻⁶. 5 The value of A(m) was determined, and the peak width (at the bottom) was determined to be 7.08 seconds. Parameters A(m) and G(m) were calculated using a simple quadratic equation (with different values ​​for each ion polarity). For the 127 Da ion, the value of A(m) was 5.76 × 10⁻⁶. -1 And the value of G(m) is 8.25 × 10 -1 .

[0134] Using the above N 峰 The equation is used to determine the number of 127Da ions detected during the chromatographic peak. As is clear from the above, N 峰 The value of the parameter in the equation is: G(m) = 8.25 × 10 -1 A = 1.045 x 10 5 One unit; P=13; T=0.003s; and W=7.08s. Therefore, N 峰 The value was determined to be 475 ions.

[0135] Using the above N req The equation is used to determine the RSD to satisfy the user-defined equation. C The required value is the number of 127 Da ions that need to be detected during the chromatographic peak. As is clear from the above, the value of A(m) to be used in the equation is 5.76 × 10⁻⁶. -1 RSD C The value of is defined by the user as 0.1 (i.e., 10%). Therefore, using the above N... req The equations determined that in order to satisfy RSD C The required value is 133 ions of 127 Da that need to be detected during the chromatographic peak.

[0136] Then calculate N. 峰 The value divided by N req The value of is used to determine the dilution factor D. As described above, the dilution factor is determined while still satisfying the user-defined RSD. CThe value can also be used to determine the dilution factor of the analyzed sample. Therefore, based on the above equation, the dilution factor D was determined to be 3.57.

[0137] The analyte concentration that produced a 305 Da precursor ion (and a final 127 Da fragment ion) in the analyzed sample was 1000 ppb. Therefore, the RSD of the analyte was obtained by dividing the analyzed sample concentration (i.e., 1000 ppb) by 3.57 to provide a concentration of 280 ppb. C The required concentration C LLOQ .

[0138] The minimum concentration of the analyte required in the sample was also determined while still meeting the desired S / N ratio set by the user. Figure 6 The signal-to-noise ratio of the 127 Da peak was determined to be 28.3 (the noise level has been reduced from...). Figure 6 (Removed from the middle). The S / N value defined by the user to meet the LLOQ is S / N=10. Since the S / N value determined for the injected sample is 28.3, it is recognized that the sample can be diluted 2.8 times before the S / N value will reach the value required for the LLOQ. Therefore, the minimum concentration of analyte required in the sample to meet the S / N required for the LLOQ is determined to be 353 ppb (i.e., 1000 ppb divided by 2.8).

[0139] Therefore, for 127Da fragment ions, one user defines LLOQ as requiring an RSD of 0.1 (i.e., 10%). C In this example with an S / N value of 10, the limiting factor for LLOQ concentration is the required S / N value because it needs to be higher than the required RSD. C The required higher concentration of analyte is needed to achieve the LLOQ concentration.

[0140] As mentioned above, Figure 6 The ion signals detected for fragment ions with a mass-to-charge ratio of 121 Da are also shown. Mass analysis of 121 Da ions was performed at 16 points during the chromatographic peak by setting a second mass filter of the mass spectrometer at each of these points to transfer the 121 Da ions to the detector. This... Figure 6 The peak is illustrated by 16 intensity points located above the baseline. At each of these points in the chromatographic peak, a second mass filter was configured to transport ions with a residence time of 0.003 seconds. The peak area of ​​the 121 Da ion was determined to be 7.23 × 10⁻⁶. 6 The value of the peak (at the bottom) was determined to be 8.82 seconds. Parameters A(m) and G(m) were calculated using a simple quadratic equation (with different values ​​for each ion polarity). For the 121 Da ion, the value of A(m) was 5.74 × 10⁻⁶.-1 And the value of G(m) is 8.23 ​​× 10 -1 .

[0141] Using the above N 峰 The equation is used to determine the number of 121Da ions detected during the chromatographic peak. As is clear from the above, N 峰 The value of the parameter in the equation is: G(m) = 8.23 ​​× 10 -1 A = 1.1737 x 10 6 One unit; P=16; T=0.003s; and W=8.82s. Therefore, N 峰 The value was determined to be 5.25 × 10. 3 One ion.

[0142] Using the above N req The equation is used to determine the RSD to satisfy the user-defined equation. C The required value is the number of 121 Da ions that need to be detected during the chromatographic peak. As is clear from the above, the value of A(m) to be used in the equation is 5.74 × 10⁻⁶. -1 RSD C The value of is defined by the user as 0.1 (i.e., 10%). Therefore, using the above N... req The equation determines that, in order to satisfy RSD C The required value is 133 121Da ions that need to be detected during the chromatographic peak.

[0143] Then calculate N. 峰 The value divided by N req The value of is used to determine the dilution factor D. As described above, the dilution factor is determined while still satisfying the user-defined RSD. C The value can also be calculated by the dilution factor of the analyzed sample. Therefore, based on the above equation, the dilution factor D was determined to be 39.5.

[0144] The analyte concentration that produced a 305 Da precursor ion (and a final 121 Da fragment ion) in the analyzed sample was 1000 ppb. Therefore, the RSD of the analyte was obtained by dividing the analyzed sample concentration (i.e., 1000 ppb) by 39.5 to provide an LLOQ concentration of 25 ppb. C The required concentration C LLOQ .

[0145] The minimum concentration of the analyte required in the sample was also determined while still meeting the desired S / N ratio set by the user. Figure 6 The signal-to-noise ratio of the 121 Da peak was determined to be 82.2 (the noise level has been reduced from...). Figure 6(Removed from the middle). The S / N value defined by the user to meet the LLOQ is S / N=10. Since the S / N value determined for the injected sample is 82.2, it is recognized that the sample can be diluted 8.22 times before the S / N value will reach the value required by the LLOQ. Therefore, the minimum concentration of analyte in the sample required to meet the S / N defined in the LLOQ is determined to be 122 ppb (i.e., 1000 ppb divided by 8.22).

[0146] Therefore, for 121 Da fragment ions, one user defines LLOQ as requiring an RSD of 0.1 (i.e., 10%). C In this example with an S / N value of 10, the limiting factor for LLOQ concentration is the required S / N value because it needs to be higher than the required RSD. C To achieve the required LLOQ concentration, a higher concentration of sample is needed.

[0147] Embodiments of the present invention have various applications. For example, a user may require the LLOQ value of an analyte to be at or below a desired target value. For example, for pesticide analysis, a user may want the LLOQ concentration of the pesticide analyte to be at or below a specific value, which could be, for example, five times the minimum residue limit (MRL) of the pesticide. Embodiments of the present invention predict the achievement of a user-defined RSD. C The values ​​and / or the LLOQ concentration required to achieve the user-defined S / N values. Then, one or both of these values ​​can be displayed to the user and / or compared with a target value, for example, as shown regarding... Figure 7 As described.

[0148] Figure 7 A table illustrating analyses of several different analytes is shown. The first column indicates the analyte in the sample being analyzed. The second column indicates the type of analysis, i.e., whether it is quantitative or qualitative. The third column indicates the mass-to-charge ratio of the precursor ion of the analyte to be analyzed. During analysis, the precursor ion is fragmented to form fragment ions, and one or more fragment ions are analyzed, i.e., MRM transitions are monitored. The fourth column indicates the mass-to-charge ratio of the fragment ions used for mass analysis. The fifth column indicates the average retention time of the analyte in the liquid chromatography separation device. The sixth column indicates the concentration of the analyte in the sample being analyzed. The seventh column indicates the signal-to-noise ratio of the ion signal detected for the product ion during the chromatographic peak of the analyte. The eighth column indicates the baseline noise in the ion signal of the product ion. The ninth column indicates the peak area of ​​the product ion. The tenth column indicates the analyte's peak area based on RSD. C Or a concentration that is independent of the S / N ratio and has been set as the desired threshold concentration and must satisfy the LLOQ. For example, this desired threshold concentration may be set to be five times lower than the minimum residue limit for each pesticide analyte.

[0149] The second-to-last column indicates the concentration of the analyte required to meet the S / N value defined by the user in LLOQ. For the example shown in the table, the user-defined S / N value is 3:1, and the concentrations in the second-to-last column are calculated according to the implementation described herein.

[0150] The last column indicates the analyte to meet the RSD already defined by the user in LLOQ. C The minimum concentration required. For the implementation shown in the table, the RSD is defined by the user. C The value is 0.1 (i.e., 10%), and the concentration in the last column is calculated according to the implementation scheme described herein.

[0151] For each row in the table, i.e., for each MRM transition of the analyte, it can be seen that the concentration values ​​in the last two columns can be lower than, equal to, or higher than the concentration values ​​in the third-to-last column. This allows the user to determine the concentration values ​​while still meeting the S / N and RSD settings already set by the user. C The LLOQ requirement specifies whether the analyte is detectable at the concentration (e.g., minimum residue limit) in the third-to-last column.

[0152] For example, the data in the first row of the table indicates that for the quantitative analysis of acephate, using an MRM transition from 184.1 Da to 142.6 Da would be expected to analyze acephate concentrations as low as 0.1 ppb (see the third-to-last column). The last two columns of the table indicate that the S / N and RSD settings already configured by the user in LLOQ are still met. C The minimum concentration of acephate that can be analyzed simultaneously is required to be 0.01 ppb. Therefore, it is clear that the MRM transition of acephate at concentrations as low as the desired minimum can be analyzed while still meeting the LLOQ already set by the user.

[0153] In contrast, the data in row 7 of the table indicates that it is not possible to analyze exemplary MRM transitions for flonicamid concentrations as low as the desired minimum concentration while still satisfying the LLOQ already set by the user. More specifically, the data in row 7 of the table shows that for quantitative analysis of flonicamid, using MRM transitions from 230 Da to 67.7 Da would be expected to analyze flonicamid concentrations as low as 0.01 ppb (see the third to last column). However, the last two columns of the table indicate that it is not possible to analyze flonicamid concentrations as low as the desired minimum concentration while still satisfying the S / N and RSD already set by the user in the LLOQ. C The minimum concentrations of fipronil required for simultaneous analysis are 1.76 ppb and 0.53 ppb, respectively. These values ​​are significantly higher than the expected minimum concentrations, and therefore indicate that analysis at the expected minimum concentrations will not provide detectable or reliable results, and such analysis cannot be reliably performed.

[0154] A mass spectrometer can be configured to automatically adjust its analytical method for any given analyte (e.g., for any given MRM transition) so that its LLOQ value is at or below the target concentration of that analyte. Alternatively, the mass spectrometer can be configured to automatically display to the operator information on how to adjust the spectrometer's operation to achieve an LLOQ value at or below the target concentration of the analyte. For example, in any of these scenarios, if the LLOQ value of a first analyte (or a first MRM transition) is determined to be above its target concentration, the mass filter residence time used to analyze the first analyte (or the first MRM transition) can be increased. If a second analyte (or a second MRM transition, e.g., for the same analyte) is analyzed as part of the method, and the LLOQ value of the second analyte (or the second MRM transition) is below the corresponding target concentration of the second analyte, the mass filter residence time used to analyze the second analyte (or the second MRM transition) can be reduced to compensate for the increased residence time when analyzing the first analyte (or the first MRM transition). Choose to reduce the residence time to avoid increasing the LLOQ concentration of the second analyte (or the second MRM transition) above its target value. Although the individual residence times of the two analytes (or the two MRM transitions) may vary, this can be controlled so that their combined duration does not increase and / or decrease.

[0155] As mentioned above, different analytes in a sample typically elute from the chromatographic separator as different chromatographic peaks. It may be desirable to monitor one or more MRM transitions for each analyte. When monitoring more than one MRM transition during a chromatographic peak, a second mass filter switches between monitoring different MRM transitions, allowing each MRM transition to be monitored multiple times during each peak (typically 10 to 15 times as described above). Each time an MRM transition is monitored, the second mass filter is configured to deliver fragment ions for that MRM transition within a period known as the residence time. The length of the usable residence time depends on the number of different MRM transitions that must be monitored during a chromatographic peak. For example, if more MRM transitions must be monitored during each chromatographic peak, less time is available to monitor each MRM transition; that is, the mass filter residence time must be shorter.

[0156] Furthermore, different analytes can elute from the chromatographic separator during overlapping time periods, i.e., these different analytes elute with overlapping chromatographic peaks. It will be necessary to monitor different MRM transitions for these different analytes. Therefore, during elution times where there is more chromatographic peak overlap, a greater number of MRM transitions will need to be monitored, while during elution times where there is less or no chromatographic peak overlap, a smaller number of MRM transitions will need to be monitored. Therefore, it should be understood that these factors affect the duration of residence time for any given instance of an MRM transition that can be analyzed by the second mass filter.

[0157] When performing an analytical method, it may be desirable to change the number of MRM transitions being monitored, for example, by adding a new MRM transition to monitor or removing an MRM transition so that it is no longer monitored. For instance, it may be desirable to analyze an additional analyte, and therefore it will be necessary to monitor at least one MRM transition of that new analyte. As described above, changing the number of MRM transitions that must be monitored during a given time range changes the time available to monitor each MRM transition, and thus changes the residence time of the second mass filter for any given instance of a given MRM transition that can be analyzed. This affects the sensitivity of the mass analysis because changing the residence time of the mass filter changes the number of ions transported for the MRM transition being analyzed. This, in turn, will affect the concentration of the analyte required to meet the user-defined LLOQ parameter.

[0158] Due to the number N ions detected during a given MRM transition during the chromatographic peak. 峰 The effect of changing the residence time on LLOQ concentration is proportional to the residence time used to analyze this MRM transition; therefore, the effect of changing the residence time can be expressed as follows:

[0159]

[0160] LLOQ 新 The new LLOQ concentration is due to changes in residence time; LLOQ 旧 The LLOQ concentration was determined before the change in residence time; Dwell 新 This refers to the length of stay after the change in the length of stay; and Dwell 旧 This refers to the length of stay before the change in the length of stay.

[0161] A mass spectrometer can be configured to add additional MRM transitions to an existing analytical method that analyzes other MRM transitions. To avoid increasing the total analysis time for an MRM transition, the residence time of at least one MRM transition from the existing method must be reduced. However, as can be seen from the above relationship, this will increase the LLOQ concentration of that MRM transition, and it may be desirable to keep the LLOQ concentration below a target value for that MRM transition. The spectrometer can be configured such that, when a new MRM transition is added to the analytical method, it automatically calculates, for example, using the above relationship, a reduced residence time for one or more other MRM transitions, such that the new MRM transition is added to the analytical method without increasing the total analysis time, and the LLOQ concentration of each MRM transition does not increase above its corresponding target value. The spectrometer can be configured to automatically adjust the residence time of the MRM transitions based on this calculation during analysis, or the spectrometer can be configured to automatically display information to the operator on how to adjust the spectrometer's operation to do so.

[0162] Alternatively, the methods described above can be used to assess the potential effects of adding or removing an analyte from an analytical method. A decision can then be made regarding whether an analyte should be added to the analysis, based on the change this would cause to the LLOQ concentration of that analyte or to one or more other analytes being analyzed in the method. For example, if adding an analyte to the analysis would result in a corresponding LLOQ concentration of that analyte or another analyte being analyzed exceeding its target value, the spectrometer can indicate this, and optionally, the analyte should not be added to the analysis.

[0163] Even if the LLOQ of the analyte is recommended to be added to the analytical method, 旧 The value is unknown, but this technique can still be used to determine the factors that would cause the expected change in LLOQ for each of the other analytes in other analytes (i.e., Dwell). 旧 Divide by Dwell 新 (Factors).

[0164] Figure 8 An example is shown of how adding an analyte to an analytical method affects the LLOQ concentration of each of the other analytes in certain other analytes. Figure 8A list of MRM transitions for which the method is set up for analysis is shown. Each row relates to a different analyte; for example, the first row relates to phenytoin, and the second row relates to metformin. Each row also indicates the number of MRM transitions to be monitored for the analyte; for example, two MRM transitions are monitored for phenytoin. Each row indicates the elution time of the analyte from the chromatographic apparatus; for example, the first row indicates that phenytoin eluted over a period of 0.7 min to 2.50 min. The elution time of the analyte is also... Figure 8 The right side is shown graphically with horizontal bars, and it can be seen that many analytes have overlapping elution times, i.e., overlapping chromatographic peaks.

[0165] from Figure 8 As can be seen, adding an analyte to an analysis that has an elution time range that overlaps with the elution time ranges of one or more other analytes will affect the number of MRM transitions that must be monitored during that overlapping period. Similarly, removing an analyte from the analysis that has an elution time range that overlaps with the elution time ranges of one or more other analytes will also affect the number of MRM transitions that must be monitored during that overlapping period.

[0166] For example, from Figure 8 As can be seen, if ibuprofen is added to the analytical method, this will affect the time available for analyzing all analytes covered by the vertical arrows, since they all have elution times that overlap with the elution time of ibuprofen. For this subset of analytes, the residence time of the second mass spectrometer used in each instance of each MRM transition analysis will be automatically reduced, for example, to maintain a given number of analytical points for that analyte during each chromatographic peak. This reduces the number of ions counted for each MRM transition in each chromatographic peak, which changes the LLOQ concentration of each analyte in the analytes. The effect of this on the LLOQ concentration of each analyte can be determined as described above, and this is used to determine whether the analysis of ibuprofen should be added to the method. Alternatively, as described above, the mass spectrometer can automatically reduce the residence time of one or more analytes in a subset of analytes in a manner that prevents its LLOQ concentration from rising above its corresponding target value.

[0167] Although the invention has been described with reference to preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made without departing from the scope of the invention as set forth in the appended claims.

[0168] For example, although this paper has described the use of N-based 峰 and N reqThis document describes techniques for determining the LLOQ concentration of an analyte to meet the RSD% requirement. However, when determining the concentration of an analyte to be analyzed, additional factors affecting the RSD% can also be considered. For example, variability in sample injection from the chromatographic system to the mass spectrometer can increase the LLOQ concentration. Therefore, the techniques described herein can be used as guidance, along with other considerations, when determining the minimum concentration of the analyte to be analyzed (for a given sample volume) during method development.

[0169] Additionally or alternatively, although the analytes have been described as being analyzed by tandem mass spectrometry, it is envisioned that other methods of mass analysis may be used as alternatives.

Claims

1. A method for determining the minimum concentration of a first analyte to be mass-analyzed in a mass spectrometer, the method comprising: (i) Using a mass spectrometer, the first analyte at a known concentration is analyzed according to a first mass analysis method to obtain an ion signal with a peak; (ii) Determine the number N of ions N that formed the peak as detected by the mass spectrometer during the first mass analysis method. 峰 ; (iii) Determine the number N of ions N that need to be detected in the ion peak in order to achieve a preselected relative standard deviation. req ;as well as (iv) Determine the relative standard deviation C for achieving the preselected standard deviation based on the values ​​of the following: RSD Simultaneously, the minimum concentration of the first analyte that can be analyzed according to the first quality analysis method: the known concentration, N 峰 and N req .

2. The method of claim 1, wherein step (ii) includes using a relationship To calculate the number of ions N 峰 Where A is the area of ​​the peak; P is the number of instances in which the first quality analysis method is performed during the peak. T is the duration during which the first analyte is analyzed according to the first quality analysis method during each instance of the execution of the first quality analysis method; And W is the width of the peak at its bottom.

3. The method according to claim 1 or 2, wherein step (iii) comprises according to To calculate the number of ions N req Where A(m) is a mass-related function, and the value of A(m) depends on the mass or mass-to-charge ratio of the ionic substance detected at the ion detector during the first mass analysis method, and where RSD c It is the value of the pre-selected relative standard deviation.

4. The method according to claim 1, 2 or 3, wherein the known concentration of the first analyte is C. inj And step (iv) includes determining the minimum concentration C from the following. RSD : .

5. The method according to any preceding claim, the method comprising: determining the signal-to-noise ratio of the peak; selecting a minimum acceptable signal-to-noise ratio value for the ion peak when analyzing the first analyte according to the first mass analysis method; and estimating the minimum acceptable signal-to-noise ratio C by: S / N Simultaneously, the minimum concentration of the first analyte that can be analyzed according to the first quality analysis method is: multiply the known concentration of the first analyte by the minimum acceptable signal-to-noise ratio value, and then divide this by the determined signal-to-noise ratio.

6. The method of claim 5, wherein the mass spectrometer automatically determines C RSD and C S / N The higher of the two is the minimum concentration of the first analyte to be analyzed according to the first quality analysis method.

7. The method according to any of the preceding claims, wherein the mass spectrometer automatically displays the determined minimum concentration on an electronic display.

8. The method according to any preceding claim, further comprising subsequently performing a mass analysis on the sample containing the first analyte using a mass spectrometer or the mass spectrometer according to the first mass analysis method, wherein: (i) The sample contains a lower concentration of the first analyte compared to the known concentration; or (ii) The volume of the sample for which the mass analysis was performed, multiplied by the concentration of the first analyte in the sample, is less than the known concentration multiplied by the volume of the sample in which the mass analysis was performed when the known concentration was determined.

9. The method of claim 8, wherein the mass spectrometer is based on C RSD and / or C S / N The determined values ​​are used to automatically adjust one or more operating conditions in its operating conditions, and one or more adjusted operating conditions are used to perform the steps of quality analysis of the sample; or The mass spectrometer mentioned above is based on C RSD and / or C S / N The determined value is used to automatically display information or options for adjusting one or more operating conditions.

10. The method of claim 9, wherein the step of automatically adjusting one or more operating conditions of the mass spectrometer comprises: a) Adjusting the duration of the time for performing quality analysis of the analyte according to the first quality analysis method during the step of performing quality analysis of the sample; And / or b) adjusting the voltage or other operating conditions of one or more ion optical elements that transmit ions through the mass spectrometer during the steps of mass analysis of the sample.

11. The method according to any preceding claim, wherein the method comprises: Select the target concentration of the first analyte for which quality analysis is desired according to the first quality analysis method; Determine the concentration C RSD And optional location C S / N ;as well as a) If C RSD And optional location C S / N If the concentration is below the target concentration, then it is determined that the first analyte can be analyzed according to the first mass analysis method; And / or b) If C RSD and / or C S / N If the concentration is higher than the target concentration, it is determined that the first analyte cannot be reliably analyzed according to the first mass analysis method.

12. The method according to any preceding claim, the method comprising using a chromatographic apparatus to separate a sample containing different analytes, such that the different analytes are eluted from the chromatographic apparatus during different corresponding chromatographic peaks; wherein step (i) is performed during the chromatographic peak of the first analyte to obtain the ion signal having the peak.

13. The method according to claim 12, wherein the method comprises: (i) The known concentration of the first analyte is analyzed by a second different mass analysis method during the chromatographic peak of the first analyte in order to obtain an ion signal with a peak; (ii) Determine the number N of ions N that formed the peak as detected by the mass spectrometer during the second mass analysis method. 峰 ; (iii) Determine the number N of ions that need to be detected in the ion peak in order to achieve a preselected relative standard deviation. req ;as well as (iv) Determine the relative standard deviation C that still achieves the preselected standard deviation based on the values ​​of the following items. RSD Simultaneously, the minimum concentration of the first analyte that can be analyzed according to the second quality analysis method: the known concentration, N 峰 and N req .

14. The method of claim 13, wherein the first analyte is analyzed by tandem mass spectrometry, wherein the mass spectrometer monitors a first multiple reaction monitoring (MRM) transition in the first mass analysis method and a second MRM transition in the second mass analysis method.

15. The method of claim 14, wherein the mass spectrometer repeatedly switches between the first mass analysis method and the second mass analysis method during the chromatographic peak of the first analyte.

16. The method according to any one of claims 12 to 15, the method comprising determining a minimum concentration of a second distinct analyte to be mass-analyzed in the mass spectrometer, the method comprising: (i) Using the mass spectrometer, the second analyte at a known concentration is analyzed by another mass analysis method to obtain an ion signal with a peak; (ii) Determine the number N of ions N that formed the peak as detected by the mass spectrometer during the additional mass analysis method. 峰 ; (iii) Determine the number N of ions that need to be detected in the ion peak in order to achieve a preselected relative standard deviation. req ;as well as (iv) Determine the relative standard deviation C that still achieves the preselected standard deviation based on the values ​​of the following items. RSD Simultaneously, the minimum concentration of the second analyte that can be analyzed according to the other quality analysis method: the known concentration, N 峰 and N req .

17. The method of claim 16, wherein the first analyte and the second analyte are analyzed by tandem mass spectrometry, and the mass spectrometer monitors a first multiple reaction monitoring (MRM) transition in the first mass analysis method and monitors different multiple reaction monitoring (MRM) transitions in the additional mass analysis method; and wherein the mass spectrometer repeatedly switches between the first mass analysis method and the additional mass analysis method during at least a portion of the time during which the chromatographic peak of the first analyte overlaps with the chromatographic peak of the second analyte.

18. A mass spectrometer configured to perform the method according to any of the preceding claims.

19. A computer-readable medium comprising instructions for causing a mass spectrometer according to claim 18 to perform the method according to any one of claims 1 to 17.

20. A method for mass spectrometry analysis, the method comprising: The input indicating that the first and second analytes have been selected for mass analysis in a mass spectrometer using a chromatographic separator to separate the analytes is received. It is determined that the first analyte should be monitored during a first time window and the second analyte should be monitored during a second time window; Determine that the first time window and the second time window overlap within the first time period; as well as Determine the minimum duration for which each of the first analyte and the second analyte needs to be analyzed during the first time period in order to obtain the corresponding ion peak of each of the first analyte and the second analyte satisfying the corresponding preselected conditions.

21. The method of claim 20, the method comprising using the chromatographic separator to separate the first analyte and the second analyte; and analyzing each of the first analyte and the second analyte for at least their respective minimum duration during the first time period to obtain a corresponding ion peak of each of the first analyte and the second analyte satisfying their preselected conditions.

22. The method of claim 21, wherein during the first time period, the mass spectrometer repeatedly switches between: monitoring a first multiple reaction monitoring (MRM) transition when performing mass analysis on the first analyte; and monitoring a second different multiple reaction monitoring (MRM) transition when performing mass analysis on the second analyte.

23. The method according to claim 20, 21 or 22, wherein the method further comprises The input indicating that another analyte has been selected for mass analysis in the mass spectrometer is received; It was determined that the additional analyte should be monitored during an alternate time window; Determine that the additional time window overlaps with the first time window and the second time window within the second time period; and Determine the minimum duration for which each of the first analyte, the second analyte, and the additional analyte needs to be analyzed during the second time period in order to obtain the corresponding ion peak of each of the first analyte, the second analyte, and the additional analyte that satisfies the corresponding preselected conditions.

24. The method of claim 23, the method comprising using the chromatographic separator to separate the first analyte, the second analyte, and the additional analyte; and analyzing each of the first analyte, the second analyte, and the additional analyte for at least their respective minimum duration during the second time period to obtain a corresponding ion peak of each of the first analyte, the second analyte, and the additional analyte satisfying its preselected conditions.

25. The method of claim 24, wherein during the second time period, the mass spectrometer repeatedly switches between: monitoring a first multiple reaction monitoring (MRM) transition when performing a mass analysis on the first analyte; monitoring a second different multiple reaction monitoring (MRM) transition when performing a mass analysis on the second analyte; and monitoring a third different multiple reaction monitoring (MRM) transition when performing a mass analysis on the additional analyte.

26. The method according to any one of claims 20 to 25, wherein the one or more preselected conditions are preselected signal-to-noise ratios of the ion peaks; and / or wherein the one or more preselected conditions are relative standard deviations of the ion peaks.

27. A computer-readable medium comprising instructions that, when executed on a computer, cause the mass spectrometer to perform the method according to any one of claims 20 to 26.

28. A computer-implemented method for determining whether to perform mass analysis on an analyte in a mass spectrometer or whether a reliable mass analysis of the analyte can be performed in a mass spectrometer, the method comprising: The input indicating that the first analyte has been selected for mass analysis in a mass spectrometer using a chromatographic separator to separate the analyte is received. The following methods are used to determine whether to perform quality analysis on additional analytes or Whether the mass spectrometer is capable of reliably analyzing the other analytes: i) Determine whether the first analyte should be monitored during a first time window and whether the additional analyte should be monitored during a second time window; ii) Determine that the first time window and the second time window overlap within the first time period; iii) If quality analysis of both the first analyte and the other analyte is selected, the duration of time available during the first time period for quality analysis of each of the first analyte and / or the other analyte is determined. iv) Determine the minimum amount or minimum concentration of the first analyte and / or the other analyte required for quality analysis to obtain corresponding ion peaks with one or more pre-selected conditions, based on a determined duration of time during the first time period that would be available for quality analysis of the first analyte and / or the other analyte, respectively; and v) Determine whether to perform mass analysis on the additional analyte or whether the mass spectrometer is capable of reliably performing mass analysis on the additional analyte by comparing the determined minimum amount or minimum concentration of the first analyte and / or the additional analyte with a preselected target amount or concentration of each of the first analyte and / or the additional analyte.

29. The method of claim 28, wherein the one or more preselected conditions are preselected signal-to-noise ratios of the ion peaks; and / or wherein the one or more preselected conditions are relative standard deviations of the ion peaks.

30. The method of claim 28 or 29, wherein in step iii), the duration for which mass analysis of the first analyte and / or the other analyte will be available is determined based on mass analysis of the first analyte and the other analyte by tandem mass spectrometry, wherein the mass spectrometer is repeatedly switched between monitoring a first multiple reaction monitoring (MRM) transition during mass analysis of the first analyte and monitoring different multiple reaction monitoring (MRM) transitions during mass analysis of the other analyte.

31. A computer-readable medium comprising instructions that, when executed on a computer, cause the mass spectrometer to perform the method according to any one of claims 28 to 30.

32. A method for mass spectrometry analysis, the method comprising: Perform the method according to claim 28, 29 or 30; And then The sample containing the first analyte and the other analyte is separated in a chromatographic separator; as well as a) If it is determined in step v) that the additional analyte is to be subjected to mass analysis, the method of mass spectrometry repeatedly switches between performing mass analysis on the first analyte and performing mass analysis on the additional analyte during the first time period. or b) If it is determined in step v) that the additional analyte will not be subjected to quality analysis, then the method of mass spectrometry performs quality analysis on the first analyte during the first time period without performing quality analysis on the additional analyte.

33. The method of claim 32, wherein in step a), the first analyte and the other analyte are analyzed based on mass analysis by tandem mass spectrometry, and the mass spectrometer is repeatedly switched between monitoring a first multiple reaction monitoring (MRM) transition when performing mass analysis on the first analyte and monitoring different multiple reaction monitoring (MRM) transitions when performing mass analysis on the other analyte.

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