A method for optimizing the parameter settings of at least one mass spectrometer.

Abstract

A method for optimizing at least one parameter setting of at least one mass analyzer (110) operating at unit resolution is disclosed. The method includes: a) determining at least one analyte detection window for detecting an analyte of interest using a mass analyzer (110), the analyte detection window being defined by a central mass-to-charge ratio value of the analyte and a predefined width, the central mass-to-charge ratio value of the analyte being set to a theoretical mass-to-charge ratio value of the analyte of interest having two or fewer decimal places and / or a mass-to-charge ratio value of the analyte of interest determined by a high-resolution mass spectrometric measurement having two or fewer decimal places; and b) determining at least one internal standard detection window for detecting an internal standard using the mass analyzer (110), the internal standard detection window being defined by a central mass-to-charge ratio value of the internal standard and a predefined width, the central mass-to-charge ratio value of the internal standard being set to a mass-to-charge ratio value of the internal standard calculated for the analyte of interest having two or fewer decimal places and / or a mass-to-charge ratio value of the internal standard determined by a high-resolution mass spectrometric measurement having two or fewer decimal places.

Description

Technical Field

[0001] The present invention relates to a method, a mass spectrometer, a computer program, and a computer program product for optimizing at least one parameter setting of at least one mass spectrometer.

Background Art

[0002] In order to perform an assay using a mass spectrometry (MS) device such as liquid chromatography-mass spectrometry (LC-MS) or tandem mass spectrometry (LC-MS / MS) assay combined with mass spectrometry, the setting of assay parameters needs to be defined and / or set and optimized. Optimization may include adjusting and / or regulating several parameters. For example, the parameters to be optimized may be mass scales such as ion source gas, curtain gas, ion spray voltage, temperature, declustering potential, entrance potential, focusing lens, prefilter, ion energy, collision energy, collision gas, cell exit potential, MS resolution, source gas flow rate, source gas pressure, etc. in the case of tandem MS multiple reaction monitoring (MRM) transitions. Specifically, in the case of quantitative mass spectrometry where the experiment requires quantitative data regarding one or more specific ions, the mass spectrometry method needs to be optimized for a specific analyte standard.

[0003] Most mass spectrometry systems use low-resolution mass filters and operate at a unit resolution, i.e., sufficient to separate two peaks by one mass unit. In low-resolution mass spectrometry systems, such as triple quadrupole MS deployments, it is common to use semi-automatic optimization techniques where analyte-specific parameter settings are determined. Typically, the parameter to be optimized is the mass axis or the detection window. Mass axis adjustment is usually performed using an implanted adjustment compound with ions having known m / z values ​​at the required level of precision. Typically, for mass-scale optimization, a unit mass resolution (±0.35 amu) with a mass axis precision of 0.1–0.2 amu is acceptable, so the mass-to-charge ratio of ionic species is used to one decimal place. More precise settings cannot be applied because experimental bias and errors strongly affect the m / z ratio.

[0004] In quantitative mass spectrometry assays, the use of internal standards is strongly recommended to achieve accurate results.

[0005] Although analytes and internal standards may have only slight differences in their physical and chemical properties, slight variations in their ratios occur, directly affecting the accuracy of results within the calibration interval. The reasons for these variations are not fully understood, and it is generally accepted to compensate for them by recalibrating the assay. Calibration makes the ratio between the analyte and the internal standard a mathematical relationship with respect to the volume of the analyte. Therefore, firstly, the instrument's mass axis may be adjusted to ensure the instrument functions properly. Secondly, adjustments and / or optimizations of the method may be performed to establish the method for the analyte and internal standard and to ensure sensitivity and selectivity. Subsequently, the method and / or assay may be calibrated to ensure accurate results.

[0006] Issues to be resolved Accordingly, an object of the present invention is to provide a method, a mass spectrometer, a computer program, and a computer program product for optimizing at least one parameter setting of at least one mass spectrometer, avoiding the aforementioned drawbacks of known methods, apparatus, computer programs, and computer program products. In particular, the method and apparatus shall improve optimization stability so that the calibration interval can be extended. [Overview of the project]

[0007] overview This problem is addressed by a method, mass spectrometer, computer program, and computer program product for optimizing at least one parameter setting of at least one mass spectrometer having the features of an independent claim. Advantageous embodiments, which may be realized individually or in any combination, are described in the dependent claims and throughout the specification.

[0008] Where used below, the terms “have,” “comprise,” or “include,” or any grammatical variations thereof, are used inclusively. Therefore, these terms may refer to both situations in which the entity described in this context has no further features beyond those introduced by these terms, and situations in which one or more additional features exist. For example, the expressions “A has B,” “A has B,” and “A includes B” may both refer to situations in which A has no other elements besides B (i.e., A consists solely of B and exclusively of B), and situations in which entity A has one or more additional elements besides B, such as element C, elements C and D, or even further elements.

[0009] Furthermore, it should be noted that the terms “at least one,” “one or more,” or similar expressions indicating that a feature or element may exist once or more times are usually used only once when introducing each feature or element. In most cases below, when referring to each feature or element, the expressions “at least one” or “one or more” will not be repeated, despite the fact that each feature or element may exist once or more than once.

[0010] Furthermore, where used below, the terms “preferably,” “more preferably,” “particularly,” “more especially,” “specifically,” “more specifically,” or similar terms are used in conjunction with any feature without limiting the possibility of alternatives. Thus, the features introduced by these terms are arbitrary features and are not intended to limit the scope of the claims in any way. The present invention may be carried out by using alternative features as will be recognized by those skilled in the art. Similarly, features introduced by “in embodiments of the present invention” or similar expressions are intended to be arbitrary features without any limitation with respect to alternative embodiments of the present invention, without any limitation with respect to the scope of the present invention, and without any limitation with respect to the possibility of combining such features with other arbitrary or non-arbitrary features of the present invention.

[0011] A first aspect of the present invention discloses a method for optimizing at least one parameter setting of at least one mass spectrometer operating at unit resolution. Preferably, the method is a computer-implemented method.

[0012] As used herein, the term “computer implementation method” is a broad term and should be given its usual customary meaning to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to a method comprising, but not limited to, at least one computer and / or at least one computer network. The computer and / or computer network may comprise at least one processor configured to perform at least one of the method steps of the method according to the present invention. Preferably, each of the method steps is performed by the computer and / or computer network. The method may be performed entirely automatically, specifically without user interaction. As used herein, the term “automatically” is a broad term and should be given its usual customary meaning to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to a process performed entirely by, but not limited to, at least one computer and / or computer network and / or machine, particularly without manual action and / or user interaction.

[0013] As used herein, the term “mass spectrometry” is a broad term and should be given its usual customary meaning to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to, but not limited to, an analytical technique for determining the mass-to-charge ratio of ions. Mass spectrometry may be performed using at least one mass spectrometer and / or at least one mass spectrometer comprising at least one mass spectrometer. As used herein, the term “mass spectrometer” is a broad term and should be given its usual customary meaning to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to, but not limited to, an analyzer configured to detect at least one analyte based on its mass-to-charge ratio (m / z).

[0014] A mass spectrometer may be or may comprise at least one quadrupole analyzer. As used herein, the term “quadrupole mass spectrometer” is a broad term and should be given its usual customary meaning to those skilled in the art, and not limited to any special or customized meaning. Specifically, the term may refer to a mass spectrometer comprising at least one quadrupole as a mass filter, but not limited to any specific meaning. As used herein, the term “mass filter” is a broad term and should be given its usual customary meaning to those skilled in the art, and not limited to any special or customized meaning. Specifically, the term may refer to an apparatus configured to select ions to be injected into the mass filter according to a mass-to-charge ratio m / z, but not limited to any specific meaning. The mass filter comprises two pairs of electrodes. The electrodes may be rod-shaped, and especially cylindrical. Ideally, the electrodes may be hyperbolic. The electrodes may be designed to be identical. The electrodes may be arranged to extend parallel to a common axis, for example, the z-axis. A quadrupole mass spectrometer may comprise at least one power supply circuit configured to apply at least one direct current (DC) voltage and at least one alternating current (AC) voltage between two pairs of electrodes in a mass filter. The power supply circuit may be configured to maintain each opposing electrode pair at the same potential. The power supply circuit may be configured to periodically change the sign of the charge on the electrode pairs so that stable trajectories are possible only for ions within a constant mass-to-charge ratio m / z. The trajectories of ions in the mass filter can be described by the Matthew differential equation. To measure ions with different m / z values, the DC and AC voltages may be changed over time so that ions with different m / z values ​​can be transmitted to the detector.

[0015] A mass spectrometer may be configured for multiple reaction monitoring. As used herein, the term “multiple reaction monitoring (MRM)” is a broad term, and its usual customary meaning should be given to those skilled in the art, and not limited to any special or customized meaning. Specifically, the term may refer to, but not limited to, a method used in mass spectrometry, specifically tandem mass spectrometry, in which multiple product ions from one or more precursor ions are monitored. As used herein, the term “monitoring” is a broad term, and its usual customary meaning should be given to those skilled in the art, and not limited to any special or customized meaning. Specifically, the term may refer to, but not limited to, the determination and / or detection of multiple product ions. A quadrupole mass spectrometer may have multiple quadrupoles. A mass spectrometer may include a triple quadrupole mass spectrometer having three quadrupoles.

[0016] A mass spectrometer may be equipped with at least one ionization source. The term “ionization source,” as used herein, also indicated as “ion source,” is a broad term and should be given its usual, customary meaning to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to, but is not limited to, an apparatus configured to generate ions from, for example, neutral gas molecules. The ionization source may be at least one source selected from the group consisting of at least one gas-phase ionization source such as at least one electron shock (EI) source or at least one chemical ionization (CI) source, at least one plasma desorption (PDMS) source, at least one fast atomic shock (FAB) source, at least one secondary ion mass spectrometry (SIMS) source, at least one laser desorption (LDMS) source, and at least one matrix-assisted laser desorption (MALDI) source, at least one thermospray (TSP) source, at least one atmospheric pressure chemical ionization (APCI) source, at least one electrospray (ESI) source, and at least one atmospheric pressure ionization (API) source, or may include at least one source.

[0017] A mass spectrometer may have at least one detector. As used herein, the term “detector” is a broad term, and its usual, customary meaning should be given to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to an apparatus configured to detect incoming ions, but is not limited to these. A detector may be configured to detect charged particles. A detector may be, or comprise, at least one electron multiplier.

[0018] The mass spectrometer may be or comprise a liquid chromatography-mass spectrometer. The mass spectrometer may be connected to and / or comprise at least one liquid chromatography apparatus, also called a liquid chromatograph. The liquid chromatograph may be used for sample preparation for the mass spectrometer. Other embodiments of sample preparation may be possible, such as at least one gas chromatograph. As used herein, the term “liquid chromatography-mass spectrometer” is a broad term, and its usual customary meaning should be given to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to a combination of liquid chromatography and mass spectrometry, but is not limited to this. The mass spectrometer may comprise at least one liquid chromatograph. The liquid chromatography-mass spectrometer may comprise at least one high-performance liquid chromatography (HPLC) apparatus or at least one microliquid chromatography (μLC) apparatus. A liquid chromatography-mass spectrometer may comprise a liquid chromatography (LC) instrument and a mass spectrometer (MS) instrument, in this case a mass filter, wherein the LC instrument and the mass filter are connected via at least one interface. The interface connecting the LC instrument and the MS instrument may include an ionization source configured to generate molecular ions and move the molecular ions into the gas phase. The interface may further include at least one ion mobility module positioned between the ionization source and the mass filter. For example, the ion mobility module may be a high-field asymmetric waveform ion mobility spectroscopy (FAIMS) module.

[0019] As used herein, the term “liquid chromatography (LC) apparatus” is a broad term and should be given its usual and customary meaning to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to an analytical module configured to separate one or more analytes of interest from other components of a sample in order to detect one or more analytes using a mass spectrometer. An LC apparatus may comprise at least one LC column. For example, an LC apparatus may be a single-column LC apparatus or a multi-column LC apparatus having multiple LC columns. The LC column may have a stationary phase through which a mobile phase is pumped to separate and / or elute and / or transition the analytes of interest. A liquid chromatography-mass spectrometer may further comprise a sample preparation station for automated sample preparation and preparation of samples, each containing at least one analyte of interest.

[0020] A mass spectrometer is configured to operate with unit resolution, also known as unit mass resolution. As used herein, the term “resolution” may refer to a measure of the mass spectrometer’s ability to distinguish between two peaks with different mass-to-charge ratios. As used herein, the term “operating with unit resolution” is a broad term and should be given its usual, conventional meaning to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to the suitability of a mass spectrometer for separating two ions that differ by only one mass unit, but not limited to this. The term “unit resolution” may refer to a mass resolution in the range of ±0.1 to ±0.4 amu, preferably ±0.2 to ±0.35. Specifically, a mass spectrometer may have a mass resolution of approximately ±0.35 amu. Specifically, a mass spectrometer may be a so-called low-resolution mass spectrometer. In known low-resolution mass spectrometers for optimizing the mass scale, a unit mass resolution of, for example, ±0.35 amu with a mass axis accuracy of 0.1–0.2 amu is acceptable, and therefore the mass-to-charge ratio of ionic species is given to one decimal place. For known instruments and methods where experimental bias and errors strongly affect the m / z ratio, more precise settings are considered. As outlined in detail below, this application proposes using two decimal places or more. It has been found that using two decimal places or more for the m / z ratio results in a significant difference in signal intensity and improved accuracy for the peak area ratio (analyte / internal standard). In particular, the actual area ratio is less susceptible to fluctuations, resulting in improved calibration and / or robustness and stability. Therefore, by applying this methodological feature, the calibration interval can be extended.

[0021] As used herein, the term “parameter setting” is a broad term and should be given its usual and customary meaning to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to the values ​​and / or ranges of parameters that define at least one assay performed by a mass spectrometer, though not limited to those specified. The assay may be a quantitative assay. For example, parameter setting may include one or more parameters such as mass scale, MRM transition in tandem MS, cell exit potential, MS resolution, source gas flow rate / pressure, temperature source gas temperature, ion source gas, curtain gas, ion spray voltage, temperature, declustering potential, incident potential, focusing lens, prefilter, ion energy, collision energy, and collision gas.

[0022] As used herein, the term “optimize” parameter settings is a broad term and should be given its usual, conventional meaning to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to the process of solving at least one optimization problem, but is not limited to these. Optimization may include evaluating and / or adjusting and / or selecting at least one parameter of the parameter settings that define an assay. Optimization may include selecting the best parameter value with respect to several criteria, such as the minimum intensity loss of a measured peak. Optimization may include determining extreme values, such as maximization and / or minimization. Optimization may include defining the most robust parameter settings, such as a plateau in a graph, as optimal. Specifically, this method may include optimizing the analyte detection window and the internal standard detection window, in particular their central mass-to-charge ratio values.

[0023] This method, as an example, involves the following steps that can be performed in a given order: a) A step of determining at least one analyte detection window for detecting a target analyte using a mass spectrometer, wherein the analyte detection window is defined by the central mass-to-charge ratio of the analyte and a predefined width, and the central mass-to-charge ratio of the analyte is set to the theoretical mass-to-charge ratio of the target analyte having two decimal places and / or the mass-to-charge ratio of the target analyte determined by a high-resolution mass spectrometry measurement having two decimal places, b) A step of determining at least one internal standard detection window for detecting an internal standard substance using a mass spectrometer, wherein the internal standard detection window is defined by the central mass-to-charge ratio of the internal standard substance and a predefined width, and the central mass-to-charge ratio of the internal standard substance is set to the mass-to-charge ratio of the internal standard substance having two decimal places calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard substance determined by a high-resolution mass spectrometry measurement having two decimal places. Includes.

[0024] However, it should be noted that different orders are possible. Furthermore, one or more method steps can be performed once or repeatedly. In addition, two or more method steps can be performed simultaneously or in overlapping order. This method may include further method steps not described.

[0025] The mass spectrometer may be configured to analyze at least one sample containing the analyte of interest. As used herein, the term "sample" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. This term may specifically refer to any test sample, including but not limited to, biological samples and / or internal standard samples. A sample may contain one or more analytes of interest. For example, the sample may be selected from the group consisting of physiological fluids including blood, serum, plasma, saliva, aqueous humor, cerebrospinal fluid, sweat, urine, milk, ascitic fluid, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells, etc. The sample may be used directly as obtained from each source, or may be subjected to a pretreatment and / or sample preparation workflow. For example, analytes of interest may generally be vitamins, vitamin D, dependence drugs, therapeutic drugs, hormones, and metabolites.

[0026] For further details regarding the sample, see, for example, European Patent Application Publication No. 3,425,369, the entire disclosure of which is incorporated herein by reference. Other analytes of interest are also possible.

[0027] The internal standard substance may be a compound structurally similar to the analyte of interest. For example, the sample may be pretreated by adding at least one internal standard substance. The sample may contain at least one internal standard substance at a known concentration. The internal standard substance may be a structurally similar compound or may contain it. The internal standard substance may be an isotope-labeled version of the analyte, preferably an isotopologue of the analyte of interest. The higher the structural similarity, the better the performance.

[0028] As used herein, the term "analyte detection window" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. This term may specifically refer to, but is not limited to, the range or frame of mass-to-charge ratios in which the detection and / or measurement of the analyte of interest is performed.

[0029] As used herein, the term "internal standard detection window" is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. Specifically, without limitation, this term may refer to the range or frame of mass-to-charge ratios within which the detection and / or measurement of an internal standard substance is performed.

[0030] Each of the analyte detection window and / or the internal standard detection window may have, for example, initial settings stored in at least one database of a mass spectrometer. As used herein, the term "settings" of a detection window is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be limited to a special or customized meaning. Specifically, without limitation, this term may refer to the limits of one or both of the detection windows, particularly the values of the lower and upper mass-to-charge ratio limits. Specifically, the settings may include one or both of the lower limit of the detection window, i.e., the mass-to-charge ratio value at which detection begins, and the upper limit of the detection window, i.e., the mass-to-charge ratio value at which detection stops. The analyte detection window is defined by the central mass-to-charge ratio value of the analyte and a predefined width. The predefined width may also be expressed as an MS resolution. The internal standard detection window is defined by the central mass-to-charge ratio value of the internal standard substance and a predefined width. The term "central mass-to-charge ratio value" may refer to the center of a range or frame of mass-to-charge ratios. The predefined width may be 0.7 amu (i.e., ±0.35 amu). However, other widths may also be possible.

[0031] The central mass-to-charge ratio (CPR) of the analyte is set to the theoretical CPR of the analyte having two decimal places and / or the CPR of the analyte determined by high-resolution mass spectrometry having two decimal places. The central mass-to-charge ratio of the internal standard is set to the CPR of the internal standard calculated for the analyte having two decimal places and / or the CPR of the internal standard determined by high-resolution mass spectrometry having two decimal places. As used herein, the term “set to” may refer to the process of adjusting and / or selecting the initial central mass-to-charge ratio to a value more suitable for the assay. For example, the theoretical mass-to-charge ratio of the analyte may be a calculated mass-to-charge ratio and / or obtained from at least one database. Additionally or alternatively, the CPR of the analyte may be set to an experimental value. As used herein, the term “high-resolution mass spectrometry” is a broad term and should be given its usual customary meaning to those skilled in the art, and should not be limited to any special or customized meaning. This term specifically refers to measurements performed using a high-resolution mass spectrometer, but is not limited to such measurements. The high-resolution mass spectrometer may be configured to perform m / z measurements with at least four decimal places, and at least three decimal places are important. The high-resolution mass spectrometer may be configured for the elucidation of molecular formulas and / or the measurement of molecular mass defects. In contrast to known optimization techniques, the methods according to the present invention propose a combination of experimental and theoretical methods. For example, the mass-to-charge ratio of the analyte may be calculated, or at least determined by high-resolution MS measurement, and the m / z value for an internal standard may be calculated for the analyte. Other parameters for setting the parameters of the mass spectrometer, such as voltage, may be determined experimentally.

[0032] The theoretical mass-to-charge ratio of the analyte of interest has two decimal places, and / or the mass-to-charge ratio of the analyte of interest determined by high-resolution mass spectrometry has two decimal places. Preferably, the theoretical mass-to-charge ratio of the analyte of interest has two decimal places, and / or the mass-to-charge ratio of the analyte of interest determined by high-resolution mass spectrometry has two decimal places. The mass-to-charge ratio of the internal standard calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard determined by high-resolution mass spectrometry may have two decimal places. More preferably, the theoretical mass-to-charge ratio of the analyte of interest has three decimal places, and / or the mass-to-charge ratio of the analyte determined by high-resolution mass spectrometry has three decimal places. The mass-to-charge ratio of the internal standard calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard determined by high-resolution mass spectrometry may have three decimal places.

[0033] Although a low-resolution MS system was used, it was surprisingly found that using the m / z ratio to two decimal places or more made the peak area ratio (analyte / internal standard) less susceptible to fluctuations, resulting in improved calibration stability. Furthermore, improved accuracy of signal intensity can be observed. Therefore, by applying this methodological feature, the calibration interval can be extended. Further evaluation showed that the two molecular species differ in their mass defects, which typically causes deviations in the m / z ratio to one or two decimal places. For isotope-labeled internal standards, a difference to two decimal places typically occurs. The resulting signal intensity is covered by the initial calibration, but the fluctuating mass axis results in a fluctuating peak area ratio due to the nonlinearity of the intensity function. This deviation causes an "uncalibrated" deviation, and therefore bias in the test results. Using the m / z ratio to two decimal places or more can reduce the performance difference between the analyte and the internal standard, and can significantly reduce the bias that occurs in the area ratio.

[0034] As outlined above, a mass spectrometer may have three quadrupoles. In the first quadrupole Q1, a precursor ion, also called the parent ion, can be isolated. The precursor ion may be the ion of interest that can be pre-selected by the first quadrupole Q1. In the second quadrupole Q2, it can be fragmented into daughter ions, also called fragment ions or product ions. The third quadrupole Q3 may be used to filter and / or select the fragment ions. Determination of the analyte detection window and the internal standard detection window may be performed for the first quadrupole and the third quadrupole. This method may include determining the analyte detection window and the internal standard detection window for each of the first quadrupole Q1 and / or the third quadrupole Q3 of a triple quadrupole mass spectrometer.

[0035] The method may include optimizing at least one additional parameter for the detection of the analyte and an internal standard using at least one analyte sample containing the analyte of interest. The additional parameter may, in particular, be at least one transition-specific parameter. The additional parameter for the detection of the internal standard may be harmonized with the additional parameter for the detection of the analyte determined using the analyte sample. As used herein, the term “harmonized” is a broad term and should be given its usual customary meaning to those skilled in the art, and should not be limited to any special or customized meaning. Specifically, the term may refer to setting the additional parameter for the detection of the internal standard to the value of the additional parameter for the detection of the analyte, but is not limited to this. The additional parameter may be at least one parameter selected from the group consisting of ion source gas, spray gas, probe position, ion spray voltage, dry gas, declustering potential, curtain gas pressure or flow, incident potential, focusing lens, ion prefilter, Q1 m / z value and resolution, ion energy, exit lens, impact energy, impact gas, impact cell exit potential, Q3 m / z value and resolution, and detector setting / voltage. For example, further parameters may be optimized in the following order: 1. Ion source gas; 1a. Atomized gas, quality / identity, temperature, flow rate / pressure, 1b. Probe position (x, y, z position), 1c. Ion atomization voltage, 1d. Dry gas, quality / identity, temperature, flow rate / pressure, 2. Declustering potential, 3. Curtain gas pressure / flow rate, 4. Incident potential, 5. Focusing lens, 6. Ion prefilter, 7. Q1 m / z value and resolution, 8. Ion energy, 9. Exit lens, 10. Focusing lens, 11. Ion prefilter, 12. Collision energy, 13. Collision gas; quality / identity, pressure / flow rate, 14. Collision cell exit potential, 15. Focusing lens, 16. Ion prefilter, 17. Q3 m / z value and resolution, 18. Ion energy, 19. Exit lens, 20. Detector setting / voltage. Further parameters may be optimized in the order listed. However, other orders are also possible. For example, preliminary values ​​1a to 1d can be used to optimize values ​​2 to 19. Value 20 may be non-analyte specific.The declustering potential (DP) can be the voltage applied to the orifice through which ions enter a mass spectrometer configured to prevent ions from clustering together. An incident potential (EP) is applied to the inlet of the mass spectrometer. The collision energy (CE) can refer to the acceleration rate of ions as they enter the second quadrupole Q2. Higher collision energy results in greater fragmentation. The collision cell potential (CXP) can be applied to focus and accelerate ions from the second quadrupole Q2 to the third quadrupole Q3. The ion spray voltage (IS) refers to the voltage applied to the tip of the ion spray needle through which the sample is ionized.

[0036] This method may include optimizing multiple parameters, particularly the analyte detection window and the internal standard detection window. The optimization of these multiple parameters can be carried out as an optimization procedure. The optimization procedure consists of the following steps: i) A step of determining the initial mass-to-charge ratio parameter of the parent ion of the analyte by operating the first quadrupole of the mass spectrometer in mass scanning mode using at least one analyte sample, ii) A step of optimizing the declustering potential by using the analyte sample and repeating the determination of the mass-charge ratio of the parent ion to determine the final mass-charge ratio parameter of the parent ion, iii) A step of determining the initial mass-to-charge ratio parameter for the product ion by operating the third quadrupole of the mass spectrometer in mass scanning mode using the analyte sample. iv) A step of optimizing the collision energy for product ions using the analyte sample, v) A step of optimizing the cell ejection potential for product ions using an analyte sample. vi) Repeat step iii) thereby determining the final mass-to-charge ratio parameter for the product ion. vii) A step of optimizing the final mass-charge ratio parameters determined for the parent ion and the final mass-charge ratio parameters for the product ions by performing step a), wherein the final mass-charge ratio parameters for the parent ion are set to the theoretical mass-charge ratio value of the parent ion having two decimal places and / or the mass-charge ratio value of the parent ion determined by high-resolution mass spectrometry having two decimal places, and the final mass-charge ratio parameters for the product ions are set to the respective theoretical mass-charge ratio values ​​of the product ions having two decimal places and / or the respective mass-charge ratio values ​​of the product ions determined by high-resolution mass spectrometry having two decimal places. viii) A step of optimizing the initial mass-charge ratio parameters of the parent ion and product ion of an internal standard by performing step b), wherein the initial mass-charge ratio parameter of the parent ion of the internal standard is set to a mass-charge ratio value having two decimal places calculated for the analyte of interest, and the initial mass-charge ratio parameter of the product ion of the internal standard is set to a mass-charge ratio value having two decimal places calculated for the analyte of interest. ix) A step of harmonizing one or more of the declustering potential, collision energy, and cell exit potential for the product ions of the internal standard material with the collision energy for the initial product ions of the analyte and the cell exit potential for the product ions of the analyte, respectively. It may include.

[0037] Step i) may be omitted, and theoretical values ​​may be used. Alternatively, step i) may be performed to determine whether the optimization is working properly, as it is required for step ii). For all other parameters, default values ​​may be used. By performing the method steps outlined above, all other parameters can be optimized directly or indirectly.

[0038] The term "mass-to-charge ratio parameter" can refer to the detection window, particularly the central mass-to-charge ratio value.

[0039] In a further aspect of the present invention, a method for quantitative multiple reaction monitoring is disclosed. This method comprises performing at least one quantitative assay on at least one mass spectrometer operating at unit resolution using at least one parameter setting optimized by a method for optimizing at least one parameter setting according to the present invention. Accordingly, with respect to definitions and embodiments, refer to the definitions and embodiments outlined with respect to the method for optimizing at least one parameter setting.

[0040] In a further embodiment, a mass spectrometer is disclosed. The mass spectrometer comprises at least one control unit configured to perform a method for optimizing at least one parameter setting and / or a method for quantitative multiple reaction monitoring according to the present invention. Accordingly, with respect to definitions and embodiments, refer to the definitions and embodiments outlined with respect to the method for optimizing at least one parameter setting.

[0041] Furthermore, as used herein, the term “control unit” generally refers to any device adapted to perform the method steps described above, preferably by using at least one data processing device, more preferably by using at least one processor and / or at least one application-specific integrated circuit. Thus, as an example, at least one control unit may comprise at least one data processing device storing software code containing several computer commands. The control unit may provide one or more hardware elements for performing one or more of the specified operations, and / or provide one or more processors with software to be executed for performing one or more of the specified operations.

[0042] The mass spectrometer may comprise at least one liquid chromatography-mass spectrometer configured for monitoring multiple reactions. The mass spectrometer, in particular the control unit, may comprise at least one database configured to store at least one parameter setting, in particular an initial parameter setting and / or an optimized parameter setting. The mass spectrometer may further comprise at least one evaluation device, which is configured to evaluate the measurement signal and thereby determine at least one measurement, in particular a concentration. The evaluation device may be part of the control unit or may be a separate device.

[0043] When the program is executed on a computer or computer network, computer programs comprising computer-executable instructions for performing the method according to the present invention are further disclosed and proposed in one or more embodiments included herein. Specifically, the computer programs may be stored on a computer-readable data carrier and / or computer-readable storage medium.

[0044] As used herein, the terms “computer-readable data carrier” and “computer-readable storage medium” may specifically refer to non-temporary data storage means such as hardware storage media on which computer executable instructions are stored. Specifically, a computer-readable data carrier or storage medium may be, or include, storage media such as random-access memory (RAM) and / or read-only memory (ROM).

[0045] Therefore, specifically, one, two or more, or all of the method steps described above can be performed using a computer or computer network, preferably using a computer program.

[0046] When the program is executed on a computer or computer network, in one or more embodiments contained herein, computer program products having program code means for performing the method according to the present invention are further disclosed and proposed herein. Specifically, the program code means may be stored in a computer-readable data carrier and / or computer-readable storage medium.

[0047] Further disclosed and proposed herein is a data carrier storing data structures that, after being loaded into a computer or computer network, such as the working memory or main memory of a computer or computer network, can perform methods according to one or more of the embodiments disclosed herein.

[0048] Further disclosed and proposed herein are computer program products having program code means stored in a machine-readable carrier to perform one or more of the embodiments included herein when the program is executed on a computer or computer network. As used herein, a computer program product refers to a program as a tradable product. The product may generally exist in any format, such as paper format, or on a computer-readable data carrier and / or computer-readable storage medium. Specifically, a computer program product may be delivered over a data network.

[0049] Finally, disclosed and proposed herein are modulated data signals containing instructions readable by a computer system or computer network for performing one or more of the embodiments disclosed herein.

[0050] Referring to the computer implementation aspects of the present invention, one or more method steps or all method steps of the methods relating to one or more embodiments disclosed herein may be performed using a computer or computer network. Therefore, generally, any method step involving data provision and / or manipulation may be performed using a computer or computer network. Generally, these method steps may include any method step, except typically method steps requiring manual intervention, such as certain embodiments that perform sample provision and / or actual measurements.

[0051] Specifically, in this specification, the following: - A computer or computer network comprising at least one processor adapted to perform a method according to one of the embodiments described herein, - A computer-loadable data structure adapted to perform a method according to one of the embodiments described herein while the data structure is being executed on a computer. - A computer program adapted to perform a method according to one of the embodiments described herein while the program is running on a computer. - A computer program comprising programming means for performing a method according to one of the embodiments described herein while the computer program is running on a computer or on a computer network, - A computer program comprising the program means according to a prior embodiment, wherein the program means is stored on a storage medium readable by a computer. - A data structure is stored in a storage medium, and after the data structure is loaded into the main memory and / or working memory of a computer or computer network, the storage medium is adapted to perform the method according to one of the embodiments described herein. - A computer program product having program code means, wherein the program code means may be stored on a storage medium, or is stored therein, in order to perform a method according to one of the embodiments described herein when the program code means is executed on a computer or computer network. Further information will be disclosed.

[0052] In summary, without excluding further possible embodiments, the following embodiments can be envisioned.

[0053] Embodiment 1 A method for optimizing the setting of at least one parameter of at least one mass spectrometer operating at unit resolution, a) A step of determining at least one analyte detection window for detecting a target analyte using a mass spectrometer, wherein the analyte detection window is defined by the central mass-to-charge ratio of the analyte and a predefined width, and the central mass-to-charge ratio of the analyte is set to the theoretical mass-to-charge ratio of the target analyte having two decimal places and / or the mass-to-charge ratio of the target analyte determined by a high-resolution mass spectrometry measurement having two decimal places, b) A step of determining at least one internal standard detection window for detecting an internal standard substance using a mass spectrometer, wherein the internal standard detection window is defined by the central mass-to-charge ratio of the internal standard substance and a predefined width, and the central mass-to-charge ratio of the internal standard substance is set to the mass-to-charge ratio of the internal standard substance having two decimal places calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard substance determined by a high-resolution mass spectrometry measurement having two decimal places. Methods that include...

[0054] Embodiment 2 The method according to the preceding embodiment, wherein the theoretical mass-to-charge ratio of the analyte of interest has two decimal places, and / or the mass-to-charge ratio of the analyte of interest determined by high-resolution mass spectrometry has two decimal places, and the mass-to-charge ratio of the internal standard calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard determined by high-resolution mass spectrometry has two decimal places.

[0055] Embodiment 3 The method according to any one of the preceding embodiments, wherein the theoretical mass-to-charge ratio of the analyte of interest has at least three decimal places, and / or the mass-to-charge ratio of the analyte of interest determined by high-resolution mass spectrometry has at least three decimal places, and the mass-to-charge ratio of an internal standard calculated for the analyte of interest and / or the mass-to-charge ratio of an internal standard determined by high-resolution mass spectrometry has at least three decimal places.

[0056] Embodiment 4 The method according to any one of the prior embodiments, wherein the internal standard is a compound structurally similar to the analyte of interest.

[0057] Embodiment 5: The method according to the preceding embodiment, wherein the internal standard is an isotopically labeled version of the analyte.

[0058] Embodiment 6 The method according to any one of the preceding embodiments, wherein the predefined width is 0.7amu.

[0059] Embodiment 7 The method according to any one of the prior embodiments, wherein the mass spectrometer is configured for monitoring multiple reactions.

[0060] Embodiment 8: The method according to the prior embodiment, wherein the mass spectrometer comprises a triple quadrupole mass spectrometer having three quadrupoles.

[0061] Embodiment 9: The method according to a prior embodiment, wherein the method includes determining an analyte detection window and an internal standard detection window for each of the first quadrupole Q1 and / or third quadrupole Q3 of a triple quadrupole mass spectrometer.

[0062] Embodiment 10 The method according to any one of the preceding embodiments, wherein the method comprises optimizing at least one further parameter of the parameter setting, wherein the further parameter for the detection of an internal standard is harmonized with the further parameter for the detection of an analyte determined using an analyte sample.

[0063] Embodiment 11 The method according to the preceding embodiment, wherein further parameters are at least one parameter selected from the group consisting of ion source gas, spray gas, probe position, ion spray voltage, dry gas, declustering potential, curtain gas pressure or flow, incident potential, focusing lens, ion prefilter, Q1 m / z value and resolution, ion energy, exit lens, impact energy, impact gas, impact cell exit potential, Q3 m / z value and resolution, and detector setting / voltage.

[0064] Embodiment 12 The method includes optimizing a plurality of parameters, and optimizing the plurality of parameters is according to an optimization procedure, and the optimization procedure is i) Determining the initial mass-to-charge ratio parameter of the parent ion of the analyte by operating the first quadrupole of the mass spectrometer in mass scanning mode using at least one analyte sample, ii) A step of optimizing the declustering potential by using the analyte sample and repeating the determination of the mass-charge ratio of the parent ion to determine the final mass-charge ratio parameter of the parent ion, iii) The step of determining the initial mass-to-charge ratio parameter for the product ion by operating the third quadrupole of the mass spectrometer in mass scanning mode using the analyte sample, iv) A step of optimizing the collision energy for product ions using the analyte sample, v) A step of optimizing the cell ejection potential for product ions using an analyte sample, vi) Repeat step iii) thereby determining the final mass-to-charge ratio parameter for the product ion, vii) A step of optimizing the final mass-charge ratio parameters determined for the parent ion and the final mass-charge ratio parameters for the product ions by performing step a), wherein the final mass-charge ratio parameters for the parent ion are set to the theoretical mass-charge ratio value of the parent ion having two decimal places and / or the mass-charge ratio value of the parent ion determined by high-resolution mass spectrometry having two decimal places, and the final mass-charge ratio parameters for the product ions are set to the respective theoretical mass-charge ratio values ​​of the product ions having two decimal places and / or the respective mass-charge ratio values ​​of the product ions determined by high-resolution mass spectrometry having two decimal places, viii) A step of optimizing the initial mass-charge ratio parameters of the parent ion and product ion of an internal standard by performing step b), wherein the initial mass-charge ratio parameter of the parent ion of the internal standard is set to a mass-charge ratio value having two decimal places calculated for the analyte of interest, and the initial mass-charge ratio parameter of the product ion of the internal standard is set to a mass-charge ratio value having two decimal places calculated for the analyte of interest, ix) A step of determining at least one internal standard detection window for detecting an internal standard substance using a mass spectrometer, wherein the internal standard detection window is defined by the central mass-to-charge ratio of the internal standard substance and a predefined width, and the central mass-to-charge ratio of the internal standard substance is set to the mass-to-charge ratio of the internal standard substance having two decimal places calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard substance determined by a high-resolution mass spectrometry measurement having two decimal places, x) A step of harmonizing one or more of the declustering potential, collision energy, and cell exit potential for the product ions of the internal standard material with the collision energy and cell exit potential for the product ions of the analyte, respectively. A method according to any one of the prior embodiments, including:

[0065] Embodiment 13 A method for quantitative multiple reaction monitoring, comprising performing at least one quantitative assay on at least one mass spectrometer operating at unit resolution using at least one parameter setting optimized by a method for optimizing at least one parameter setting described in any one of the preceding embodiments.

[0066] Embodiment 14 A mass spectrometer comprising at least one control unit configured to perform a method for optimizing at least one parameter setting as described in Embodiments 1 to 12 and / or a method for quantitative multiple reaction monitoring as described in Embodiment 13.

[0067] Embodiment 15 A computer program including instructions, wherein when the instructions are executed by a control unit of a mass spectrometer described in a preceding embodiment, the control unit causes the control unit to perform a method for optimizing at least one parameter setting as described in Embodiments 1 to 12 and / or a method for quantitative multiple reaction monitoring as described in Embodiment 13.

[0068] Embodiment 16 A computer-readable storage medium containing instructions, wherein when the instructions are executed by a control unit of a mass spectrometer described in any one of the preceding embodiments, the control unit causes the control unit to execute a method for optimizing at least one parameter setting as described in Embodiments 1 to 12 and / or a method for quantitative multiple reaction monitoring as described in Embodiment 13. [Brief explanation of the drawing]

[0069] Further optional features and embodiments are disclosed in more detail in subsequent descriptions of embodiments, preferably in conjunction with dependent claims. Here, each optional feature may be implemented independently and in any viable combination, as will be understood by those skilled in the art. The scope of the present invention is not limited by preferred embodiments. Embodiments are schematically shown in the figures, where the same reference numerals in these figures refer to identical or functionally equivalent elements.

[0070] The diagram is as follows:

[0071] [Figure 1] This document describes an embodiment of a method for optimizing at least one parameter setting. [Figure 2] This shows an embodiment of the mass spectrometer according to the present invention. [Figure 3] This is a theoretical effect of using the first and second decimal places. [Figure 4A] This shows experimental data regarding the first and third decimal places. [Figure 4B] This shows experimental data regarding the first and third decimal places. [Figure 4C] This shows experimental data regarding the first and third decimal places. [Modes for carrying out the invention]

[0072] Detailed description of the invention Figure 1 shows a flowchart of a method a, in particular an embodiment of a computer implementation method, for optimizing at least one parameter setting of at least one mass spectrometer 110 operating at unit resolution.

[0073] An embodiment of the mass spectrometer 110 according to the present invention is shown in Figure 2. The mass spectrometer 110 may be an analyzer configured to detect at least one analyte based on the mass-to-charge ratio (m / z).

[0074] The mass spectrometer 110 may be or comprise at least one quadrupole analyzer 112. The quadrupole mass analyzer 112 is at least one mass analyzer comprising at least one quadrupole as a mass filter. The mass filter may be configured to select ions injected into the mass filter according to a mass-to-charge ratio m / z. The mass filter comprises two pairs of electrodes. The electrodes may be rod-shaped, and in particular cylindrical. Ideally, the electrodes may be hyperbolic. The electrodes may be designed identically. The electrodes may be arranged to extend parallel along a common axis, for example, the z-axis. The quadrupole mass analyzer 112 may comprise at least one power supply circuit configured to apply at least one direct current (DC) voltage and at least one alternating current (AC) voltage between the two pairs of electrodes of the mass filter. The power supply circuit may be configured to hold each opposing electrode pair at the same potential. The power supply circuit may be configured to periodically change the sign of the charge on the electrode pair so that stable orbits are possible only for ions within a constant mass-to-charge ratio (m / z). The ion orbits in the mass filter can be described by the Matthew differential equation. To measure ions with different m / z values, the DC and AC voltages may be changed over time so that ions with different m / z values ​​can be transmitted to the detector 114.

[0075] The mass spectrometer 110 may be configured for monitoring multiple reactions. The quadrupole mass spectrometer 112 may have multiple quadrupoles. The mass spectrometer 110 may be a triple quadrupole mass spectrometer having three quadrupoles, as shown in Q1, Q2, and Q3 in Figure 2.

[0076] The mass spectrometer 110 may include at least one ionization source 116. The ionization source 116 may be configured to generate ions from, for example, neutral gas molecules. The ionization source 116 may be or include at least one source selected from the group consisting of at least one gas-phase ionization source such as at least one electron shock (EI) source or at least one chemical ionization (CI) source; at least one desorption ionization source such as at least one plasma desorption (PDMS) source, at least one fast atomic shock (FAB) source, at least one secondary ion mass spectrometry (SIMS) source, at least one laser desorption (LDMS) source and at least one matrix-assisted laser desorption (MALDI) source; at least one thermospray (TSP) source, at least one atmospheric pressure chemical ionization (APCI) source, at least one electrospray (ESI) source and at least one atmospheric pressure ionization (API) source.

[0077] Ions enter the mass spectrometer 110 in the Karr template 118 and orifice plate 120. The mass spectrometer 110 may be equipped with a quadrupole ion guide in the first vacuum stage. The ions then pass through an opening (IQ0) and reach a second vacuum stage having an additional quadrupole ion guide (Q0) and an additional opening (IQ1, or ST1). In the first quadrupole Q1, a precursor ion, also called the parent ion, can be isolated. The precursor ion may be the ion of interest that can be pre-selected by the first quadrupole Q1. In the collision cell of the second quadrupole Q2, in particular the second quadrupole Q2, the precursor ion can be fragmented into daughter ions, also called fragment ions or product ions. A third quadrupole Q3 may be used to filter and / or select the fragment ions. The Q2 quadrupole is separated from the first quadrupole Q1 and the third quadrupole Q3 by intermediate lenses IQ2 (or ST2) and IQ3 (or ST3). Fragment ions may pass through the exit lens 122 and collide with the detector 114. The detector 114 may be configured to detect incoming ions. The detector 114 may be configured to detect charged particles. The detector 114 may be or may comprise at least one electron multiplier tube.

[0078] The mass spectrometer 110 may be, or comprise, a liquid chromatography mass spectrometer, not shown in Figure 2. The mass spectrometer 110 may be connected to and / or comprise at least one liquid chromatography apparatus, also called a liquid chromatograph. The liquid chromatograph may be used for sample preparation for the mass spectrometer 110. Other embodiments of sample preparation may be possible, such as at least one gas chromatograph. The liquid chromatography mass spectrometer may be, or comprise at least one high-performance liquid chromatography (HPLC) apparatus or at least one microliquid chromatography (μLC) apparatus. The liquid chromatography mass spectrometer may comprise a liquid chromatography (LC) apparatus and a mass spectrometer (MS) apparatus, in this case a mass filter, where the LC apparatus and the mass filter are connected via at least one interface. The interface connecting the LC instrument and the MS instrument may include an ionization source 116 configured to generate molecular ions and move the molecular ions into the gas phase. The interface may further include at least one ion mobility module positioned between the ionization source 116 and the mass filter. For example, the ion mobility module may be a high-field asymmetric waveform ion mobility spectroscopy (FAIMS) module.

[0079] The mass spectrometer 110 is configured to operate at a unit resolution, also known as unit mass resolution. The mass spectrometer 110 is configured to separate two ions that differ by only one mass unit. The unit resolution may be in the range of ±0.1 to ±0.4 amu, preferably ±0.2 to ±0.35. Specifically, the mass spectrometer 110 may have a mass resolution of about ±0.35 amu. Specifically, the mass spectrometer 110 may be a so-called low-resolution mass spectrometer. In known low-resolution mass spectrometers for optimizing the mass scale, a unit mass resolution with a mass axis accuracy of 0.1 to 0.2 amu, e.g., ±0.35 amu, is a common setting, so the mass-to-charge ratio of ion species is used to the first decimal place. For known instruments and methods that cannot be applied, more precise settings are considered because experimental bias and errors strongly affect the m / z ratio. As outlined in detail below, this application proposes using two decimal places or more. Using two or more decimal places for the m / z ratio was found to result in a significant difference in signal intensity and improved accuracy regarding the peak area ratio (analyte / internal standard). In particular, the actual area ratio is less susceptible to variability, which leads to improved calibration and / or robustness and stability. Therefore, by applying this methodological feature, the calibration interval can be extended.

[0080] The parameter setting may include values ​​and / or ranges of parameters that define at least one assay performed by the mass spectrometer 110. The assay may be a quantitative assay. For example, the parameter setting may include one or more of the following parameters: mass scale, MRM transition of tandem MS, cell exit potential, MS resolution, source gas flow rate / pressure, temperature source gas temperature, ion source gas, curtain gas, ion spray voltage, temperature, declustering potential, incident potential, focusing lens, pre-filter, ion energy, collision energy, collision gas.

[0081] Optimizing parameter settings may involve the process of solving at least one optimization problem. Optimization may involve evaluating and / or adjusting and / or selecting at least one parameter of the parameter settings that define the assay. Optimization may involve selecting the best parameter value with respect to several criteria, such as the minimum intensity loss of the measured peak. Optimization may involve determining extreme values, such as maximization and / or minimization. Optimization may involve defining the most robust parameter settings, such as the plateau in the graph, as optimal. Specifically, this method may involve optimizing the analyte detection window and the internal standard detection window, in particular their central mass-to-charge ratio values.

[0082] As shown in Figure 1, this method, for example, can be performed in the following steps in a given order: a) (Reference numeral 124) A step of determining at least one analyte detection window for detecting a target analyte using a mass spectrometer 110, wherein the analyte detection window is defined by the central mass-to-charge ratio of the analyte and a predefined width, and the central mass-to-charge ratio of the analyte is set to the theoretical mass-to-charge ratio of the target analyte having two decimal places and / or the mass-to-charge ratio of the target analyte determined by a high-resolution mass spectrometry measurement having two decimal places, b) (Reference numeral 126) A step of determining at least one internal standard detection window for detecting an internal standard substance using a mass spectrometer 110, wherein the internal standard detection window is defined by the central mass-to-charge ratio of the internal standard substance and a predefined width, and the central mass-to-charge ratio of the internal standard substance is set to the mass-to-charge ratio of the internal standard substance having two decimal places calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard substance determined by a high-resolution mass spectrometry measurement having two decimal places. Includes.

[0083] As outlined above, the mass spectrometer 110 may have three quadrupoles. The determination of the analyte detection window and the internal standard detection window may be performed for the first quadrupole and the third quadrupole. This method may include determining the analyte detection window and the internal standard detection window for each of the first quadrupole Q1 and / or the third quadrupole Q3 of the triple quadrupole mass spectrometer 110.

[0084] The mass spectrometer 110 may be configured to analyze at least one sample containing the analyte of interest. The sample may be any test sample, such as a biological sample and / or an internal standard sample. The sample may contain one or more analytes of interest. For example, the sample may be selected from the group consisting of physiological fluids, including blood, serum, plasma, saliva, lens fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, mucus, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells, etc. The sample may be used directly as obtained from its respective source, or it may be subjected to a pretreatment and / or sample preparation workflow. For example, the analytes of interest may generally be vitamins, vitamin D, addictive drugs, therapeutic agents, hormones, and metabolites.

[0085] For further details regarding the sample, see, for example, European Patent Application Publication No. 3 425 369, the full disclosure of which is included herein by reference. Analytes for other purposes are also possible.

[0086] The internal standard may be a compound structurally similar to the analyte of interest. For example, the sample may be pretreated by adding at least one internal standard. The sample may contain at least one internal standard at a known concentration. The internal standard may be or may contain a structurally similar compound. The internal standard may be an isotopically labeled version of the analyte, preferably an isotopolog of the analyte of interest. The higher the structural similarity, the better the performance.

[0087] The analyte detection window may be a range or frame of mass-to-charge ratios in which the detection and / or measurement of the analyte of interest is performed. The internal standard detection window is a range or frame of mass-to-charge ratios in which the detection and / or measurement of the internal standard substance is performed. Each of the analyte detection window and / or the internal standard detection window may have an initial setting stored in, for example, at least one database of the mass spectrometer 119. The setting of each detection window may include limits for one or both of the detection window, in particular the lower and upper mass-to-charge ratio limits. Specifically, the setting may include one or both of the lower limit of the detection window, i.e., the mass-to-charge ratio value at which detection begins, and the upper limit of the detection window, i.e., the mass-to-charge ratio value at which detection stops. The analyte detection window is defined by the central mass-to-charge ratio value of the analyte and a predefined width. The internal standard detection window is defined by the central mass-to-charge ratio value of the internal standard substance and a predefined width. The central mass-to-charge ratio value may be the center of the mass-to-charge ratio range or frame. The predefined width may be 0.7 amu (i.e., ±0.35 amu). However, other widths may be possible.

[0088] The central mass-to-charge ratio (CPR) of the analyte is set to the theoretical CPR of the analyte having two decimal places and / or the CPR of the analyte determined by a high-resolution mass spectrometry measurement having two decimal places. The central mass-to-charge ratio of the internal standard is set to the CPR of the internal standard calculated for the analyte having two decimal places and / or the CPR of the internal standard determined by a high-resolution mass spectrometry measurement having two decimal places. For example, the theoretical mass-to-charge ratio of the analyte may be a calculated mass-to-charge ratio and / or obtained from at least one database. Additionally or alternatively, the CPR of the analyte may be set to an experimental value. High-resolution mass spectrometry measurements may include measurements performed using a high-resolution mass spectrometer. The high-resolution mass spectrometer may be configured to perform m / z measurements having at least four decimal places, with at least three decimal places being important. The high-resolution mass spectrometer may be configured for elucidating molecular formulas and / or measuring molecular mass defects. In contrast to known optimization methods, the method according to the present invention proposes a combination of experimental and theoretical methods. For example, the mass-to-charge ratio of the analyte may be calculated, or at least determined by high-resolution MS measurement, and the m / z value for an internal standard may be calculated for the analyte. Other parameters for setting the parameters of the mass spectrometer 110, such as voltage, may be determined experimentally.

[0089] The theoretical mass-to-charge ratio of the analyte of interest has two decimal places, and / or the mass-to-charge ratio of the analyte of interest determined by high-resolution mass spectrometry has two decimal places. The theoretical mass-to-charge ratio of the analyte of interest has two decimal places, and / or the mass-to-charge ratio of the analyte of interest determined by high-resolution mass spectrometry has two decimal places. The mass-to-charge ratio of the internal standard calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard determined by high-resolution mass spectrometry may have two decimal places. The theoretical mass-to-charge ratio of the analyte of interest has three decimal places, and / or the mass-to-charge ratio of the analyte determined by high-resolution mass spectrometry has three decimal places. The mass-to-charge ratio of the internal standard calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard determined by high-resolution mass spectrometry may have three decimal places. The theoretical mass-to-charge ratio of the analyte of interest has four decimal places, and / or the mass-to-charge ratio of the analyte of interest determined by high-resolution mass spectrometry has four decimal places. The mass-to-charge ratio of the internal standard calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard determined by high-resolution mass spectrometry may have four decimal places.

[0090] Although a low-resolution MS system was used, it was surprisingly found that using the m / z ratio to two decimal places or more made the peak area ratio (analyte / internal standard) less susceptible to fluctuations, resulting in improved calibration stability. Furthermore, improved accuracy of signal intensity can be observed. Therefore, by applying this methodological feature, the calibration interval can be extended. Further evaluation showed that the two molecular species differ in their mass defects, which typically causes deviations in the m / z ratio to one or two decimal places. For isotope-labeled internal standards, a difference to two decimal places typically occurs. The resulting signal intensity is covered by the initial calibration, but the fluctuating mass axis results in a fluctuating peak area ratio due to the nonlinearity of the intensity function. This deviation causes an "uncalibrated" deviation, and therefore bias in the test results. Using the m / z ratio to two decimal places or more can reduce the performance difference between the analyte and the internal standard, and can significantly reduce the bias that occurs in the area ratio.

[0091] Figure 3 shows the results of two upper plots of simulations of relative signal intensity versus m / z using a precisely calibrated mass axis for analytes (left plot) using one decimal place (square) and two decimal places (triangle) and internal standards (right plot) using one decimal place (square) and two decimal places (triangle). No difference in relative signal intensity is observed for two decimal places. For one decimal place only, deviations covered by assay calibration can be found. Figure 3 also shows the results of simulations of intensity versus m / z after a 0.01 amu mass axis shift for analytes (left plot) using one decimal place (square) and two decimal places (triangle) and internal standards (right plot) using one decimal place (square) and two decimal places (triangle) in the two lower plots. Again, no difference is observed for two decimal places. However, using only the first decimal place can lead to larger deviations. The fluctuating mass axis results in a fluctuating peak area ratio due to the nonlinearity of the intensity function. This deviation causes an "uncalibrated" deviation, and therefore a bias in the test results. This simulation shows that using the second decimal place or further for the m / z ratio can reduce the performance difference between the analyte and the internal standard, and significantly reduce the bias that occurs in the area ratio.

[0092] Figures 4A to 4C show experimental data for the first and third decimal places for the following analytes: testosterone (Figure 4A), cyclosporine A (Figure 4B), and phenytoin (Figure 4C). For each experiment, 3 samples × 6 replicates × 12 blocks were provided, and 216 samples were processed within 66 hours with assay calibration at t=0. Bias analysis was performed, including calculating the relative recovery rate of each sample against a known target value. Furthermore, precision analysis was performed, including calculating the coefficient of variation (cv).

[0093] The following experimental results were obtained regarding testosterone.

[0094] [Table 1]

[0095] The following experimental results were obtained regarding cyclosporine A.

[0096] [Table 2]

[0097] The following experimental results were obtained regarding phenytoin.

[0098] [Table 3]

[0099] Therefore, it has been shown that robustness can be significantly improved for each analyte by using the third decimal place instead of the first decimal place for the MRM transition. The bias decreases over time, which improves calibration stability and reduces the optimization frequency. Furthermore, using the third decimal place instead of the first decimal place for the MRM transition can slightly improve accuracy. A smaller cv results in higher accuracy. [Explanation of symbols]

[0100] 110 Mass spectrometer 112 Quadrupole Mass Spectrometer 114 detectors 116 Ionization source 118 Car Templates 120 Car Template 118 and Orifice Plate 122 Emission Lens 124 Step a) 126 Step b)

Claims

1. A method for optimizing the setting of at least one parameter of at least one mass spectrometer (110) operating at unit resolution, a) A step of determining at least one analyte detection window for detecting a target analyte using the mass spectrometer (110), wherein the analyte detection window is defined by the central mass-to-charge ratio of the analyte and a predefined width, and the central mass-to-charge ratio of the analyte is set to the theoretical mass-to-charge ratio of the target analyte having two decimal places and / or the mass-to-charge ratio of the target analyte determined by a high-resolution mass spectrometry measurement having two decimal places; b) A step of determining at least one internal standard detection window for detecting an internal standard substance using the mass spectrometer (110), wherein the internal standard detection window is defined by the central mass-to-charge ratio of the internal standard substance and the predefined width, and the central mass-to-charge ratio of the internal standard substance is set to the mass-to-charge ratio of the internal standard substance having two decimal places calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard substance determined by a high-resolution mass spectrometry measurement having two decimal places. Methods that include...

2. The method according to claim 1, wherein the theoretical mass-to-charge ratio of the target analyte has two decimal places, and / or the mass-to-charge ratio of the target analyte determined by high-resolution mass spectrometry has two decimal places, and the mass-to-charge ratio of the internal standard calculated for the target analyte and / or the mass-to-charge ratio of the internal standard determined by high-resolution mass spectrometry has two decimal places.

3. The method according to claim 1 or 2, wherein the theoretical mass-to-charge ratio of the target analyte has at least three decimal places, and / or the mass-to-charge ratio of the target analyte determined by high-resolution mass spectrometry has at least three decimal places, and the mass-to-charge ratio of the internal standard calculated for the target analyte and / or the mass-to-charge ratio of the internal standard determined by high-resolution mass spectrometry has at least three decimal places.

4. The method according to any one of claims 1 to 3, wherein the internal standard is a compound structurally similar to the analyte of the object.

5. The method according to claim 4, wherein the internal standard is an isotopically labeled version of the analyte.

6. The method according to any one of claims 1 to 5, wherein the mass spectrometer (110) is configured for monitoring multiple reactions.

7. The method according to claim 6, wherein the mass spectrometer (110) comprises a triple quadrupole mass spectrometer having three quadrupoles.

8. The method according to claim 7, wherein the method includes determining an analyte detection window and an internal standard detection window for each of the first quadrupole (Q1) and / or the third quadrupole (Q3) of the triple quadrupole mass spectrometer.

9. The method according to any one of claims 1 to 8, comprising optimizing at least one further parameter of the parameter setting, wherein the further parameter for the detection of the internal standard substance is harmonized with the further parameter for the detection of the analyte determined using an analyte sample.

10. The method according to claim 9, wherein the further parameters are at least one parameter selected from the group consisting of ion source gas, spray gas, probe position, ion spray voltage, dry gas, declustering potential, curtain gas pressure or flow, incident potential, focusing lens, ion prefilter, first quadrupole m / z value and resolution, ion energy, exit lens, collision energy, collision gas, collision cell exit potential, third quadrupole m / z value and resolution, and detector setting / voltage.

11. The method includes optimizing a plurality of parameters, and optimizing the plurality of parameters is done according to an optimization procedure, and the optimization procedure is i) Determining the initial mass-to-charge ratio parameter of the parent ion of the analyte by operating the first quadrupole of the mass spectrometer (110) in mass scanning mode using at least one analyte sample; ii) A step of optimizing the declustering potential by using the analyte sample and repeating the determination of the mass-charge ratio of the parent ion to determine the final mass-charge ratio parameter of the parent ion, iii) The steps of determining the initial mass-to-charge ratio parameter for the product ion by operating the third quadrupole of the mass spectrometer (110) in mass scanning mode using the analyte sample, iv) A step of optimizing the collision energy for product ions using the analyte sample, v) A step of optimizing the cell ejection potential for product ions using the analyte sample, vi) Repeat step iii) thereby determining the final mass-to-charge ratio parameter for the product ion, vii) A step of optimizing the final mass-charge ratio parameters for the parent ion and the final mass-charge ratio parameters for the product ions by performing step a), wherein the final mass-charge ratio parameters for the parent ion are set to the theoretical mass-charge ratio value of the parent ion having two decimal places and / or the mass-charge ratio value of the parent ion determined by high-resolution mass spectrometry having two decimal places, and the final mass-charge ratio parameters for the product ions are set to the respective theoretical mass-charge ratio values ​​of the product ions having two decimal places and / or the respective mass-charge ratio values ​​of the product ions determined by high-resolution mass spectrometry having two decimal places, viiii) A step of optimizing the initial mass-charge ratio parameters of the parent ion and product ion of the internal standard by performing step b), wherein the initial mass-charge ratio parameter of the parent ion of the internal standard is set to a mass-charge ratio value having two decimal places calculated for the target analyte, and the initial mass-charge ratio parameter of the product ion of the internal standard is set to a mass-charge ratio value having two decimal places calculated for the target analyte, ix) A step of determining at least one internal standard detection window for detecting an internal standard substance using the mass spectrometer, wherein the internal standard detection window is defined by the central mass-to-charge ratio of the internal standard substance and the predefined width, and the central mass-to-charge ratio of the internal standard substance is set to the mass-to-charge ratio of the internal standard substance having two decimal places calculated for the analyte of interest and / or the mass-to-charge ratio of the internal standard substance determined by a high-resolution mass spectrometry measurement having two decimal places; x) A step of harmonizing one or more of the declustering potential, collision energy, and cell exit potential of the product ion of the internal standard material with the collision energy and cell exit potential of the product ion of the analyte, respectively. The method according to any one of claims 1 to 10, including the method described in any one of claims 1 to 10.

12. A method for quantitative multiple reaction monitoring, comprising performing at least one quantitative assay on at least one mass spectrometer (110) operating at unit resolution using at least one parameter setting optimized by a method for optimizing at least one parameter setting according to any one of claims 1 to 11.

13. A mass spectrometer (110) comprising at least one control unit configured to perform a method for optimizing at least one parameter setting according to any one of claims 1 to 11 and / or a method for quantitative multiple reaction monitoring according to claim 12.

14. A computer program including instructions, wherein, when the program is executed by the control unit of the mass spectrometer (110) described in claim 13, the instructions cause the control unit to execute a method for optimizing at least one parameter setting as described in any one of claims 1 to 11 and / or a method for quantitative multiple reaction monitoring as described in claim 12.

15. A computer-readable storage medium containing instructions, wherein, when a program is executed by the control unit of a mass spectrometer (110) according to claim 13, the instructions cause the control unit to execute a method for optimizing at least one parameter setting according to any one of claims 1 to 11 and / or a method for quantitative multiple reaction monitoring according to claim 12.

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