METHOD FOR OPTIMIZING A PARAMETER SETTING OF AT LEAST ONE MASS SPECTROMETRY DEVICE

Abstract

A method for optimizing at least one parameter setting of at least one mass spectrometry device (110) operating at unit resolution is disclosed. The method comprises the following steps: a) determining at least one analyte detection window for detecting an analyte of interest with the mass spectrometry device (110), wherein the analyte detection window is defined by a central mass to charge ratio value of the analyte and a predefined width, wherein the central mass to charge ratio value of the analyte is set to a theoretical mass to charge ratio value of the analyte of interest having more than one decimal place and/or a mass to charge ratio value of the analyte of interest determined by a high resolution mass spectrometry measurement having more than one decimal place; b) determining at least one internal standard detection window for detecting an internal standard substance with the mass spectrometry device (110), wherein the internal standard detection window is defined by a central mass to charge ratio value of the internal standard substance and the pre-defined width, wherein the central mass to charge ratio value of the internal standard substance is set to a mass to charge ratio value of the internal standard substance calculated relative to the analyte of interest and having more than one decimal place and/or to a mass to charge ratio value of the internal standard substance determined by a high resolution mass spectrometry measurement having more than one decimal place.

Claims

1. A method for optimizing at least one parameter setting of at least one mass spectrometry device operating at unit resolution, the method comprising: a) determining at least one analyte detection window for detecting an analyte of interest with the mass spectrometry device, wherein the analyte detection window is defined by a central mass to charge ratio value of the analyte and a pre-defined width, wherein the central mass to charge ratio value of the analyte is set to a theoretical mass to charge ratio value of the analyte of interest having more than one decimal place and/or a mass to charge ratio value of the analyte of interest determined by a high resolution mass spectrometry measurement having more than one decimal place; b) determining at least one internal standard detection window for detecting an internal standard substance with the mass spectrometry device, wherein the internal standard detection window is defined by a central mass to charge ratio value of the internal standard substance and the pre-defined width, wherein the central mass to charge ratio value of the internal standard substance is set to a mass to charge ratio value of the internal standard substance calculated relative to the analyte of interest and having more than one decimal place and/or to a mass to charge ratio value of the internal standard substance determined by a high resolution mass spectrometry measurement having more than one decimal place.

2. The method according to claim 1, wherein the theoretical mass to charge ratio value of the analyte of interest has two decimal places and/or the mass to charge ratio value of the analyte of interest determined by a high resolution mass spectrometry measurement has two decimal places, wherein the mass to charge ratio value of the internal standard substance calculated relative to the analyte of interest and/or the mass to charge ratio value of the internal standard substance determined by a high resolution mass spectrometry measurement has two decimal places.

3. The method according to claim 1, wherein the theoretical mass to charge ratio value of the analyte of interest has at least three decimal places and/or the mass to charge ratio value of the analyte of interest determined by a high resolution mass spectrometry measurement has at least three decimal places, wherein the mass to charge ratio value of the internal standard substance calculated relative to the analyte of interest and/or the mass to charge ratio value of the internal standard substance determined by a high resolution mass spectrometry measurement has at least three decimal places.

4. The method according to claim 1, wherein the internal standard substance is a structurally similar compound to the analyte of interest.

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

6. The method according to claim 1, wherein the mass spectrometry device is configured for multiple reaction monitoring.

7. The method according to claim 6, wherein the mass spectrometry device comprises a triple quadrupole mass spectrometry device comprising three quadrupoles.

8. The method according to claim 7, wherein the method comprises determining analyte detection windows and internal standard detection windows for each of a first quadrupole Q1 and/or a third quadrupole Q3 of the triple quadrupole mass spectrometry device.

9. The method according to claim 1, wherein the method comprises optimizing at least one further parameter of the parameter setting, wherein the further parameter for the detection of the internal standard substance is harmonized to the further parameter for detection of the analyte determined using the analyte sample.

10. The method according to claim 9, wherein the further parameter is at least one parameter selected from the group consisting of ion source gas, spray gas, probe position, ion spray voltage, drying gas, declustering potential, curtain gas pressure or flow, entrance potential, focusing lenses, ion pre-filter, Q1 m/z value and resolution, ion energy, exit lenses, collision energy, collision gas, collision cell exit potential, Q3 m/z value and resolution, and detector settings/voltage.

11. The method according to claim 1, wherein the method comprises optimizing a plurality of parameters, wherein optimizing of the plurality of parameters follows an optimization procedure, wherein the optimization procedure comprises; i) determining an initial mass to charge ratio parameter of a parent ion of the analyte by running a first quadrupole of the mass spectrometry device in a mass scan mode using at least one analyte sample; ii) optimizing a declustering potential by using the analyte sample and repeating the determining of the mass to charge ratio of the parent ion for determining a final mass to charge ratio parameter of the parent ion; iii) determining initial mass to charge ratio parameters for product ions by running a third quadrupole of the mass spectrometry device in a mass scan mode using the analyte sample; iv) optimizing a collision energy for product ions using the analyte sample; v) optimizing cell exit potential for product ions using the analyte sample; vi) repeating step iii) thereby determining final mass to charge ratio parameters for product ions; vii) performing step a) thereby optimizing the determined final mass to charge ratio parameter of the parent ion and final mass to charge ratio parameters for product ions, wherein the final mass to charge ratio parameter of the parent ion is set to a theoretical mass to charge ratio value of the parent ion having more than one decimal place and/or a mass to charge ratio value of the parent ion determined by a high resolution mass spectrometry measurement having more than one decimal place, wherein the final mass to charge ratio parameters for product ions are set to the respective theoretical mass to charge ratio values of the product ions having more than one decimal place and/or respective mass to charge ratio values of the product ions determined by a high resolution mass spectrometry measurement having more than one decimal place; viii) performing step b) thereby optimizing initial mass to charge ratio parameters of a parent ion and product ions of the internal standard substance, wherein the initial mass to charge ratio parameter of the parent ion of the internal standard substance is set to a mass to charge ratio value calculated relative to the analyte of interest and having more than one decimal place and the initial mass to charge ratio parameters of the product ions of the internal standard substance are set to mass to charge ratio values calculated relative to the analyte of interest and having more than one decimal place; ix) determining at least one internal standard detection window for detecting an internal standard substance with the mass spectrometry device, wherein the internal standard detection window is defined by a central mass to charge ratio value of the internal standard substance and the pre-defined width, wherein the central mass to charge ratio value of the internal standard substance is set to a mass to charge ratio value of the internal standard substance calculated relative to the analyte of interest and having more than one decimal place and/or to a mass to charge ratio value of the internal standard substance determined by a high resolution mass spectrometry measurement having more than one decimal place; x) harmonizing one or more of declustering potential, collision energy and cell exit potential for product ions of the internal standard substance to the collision energy for product ions and the cell exit potential for product ions of the analyte, respectively.

12. A method for quantitative multiple reaction monitoring, wherein the method comprises performing at least one quantitative assay on at least one mass spectrometry device operating at unit resolution using at least one parameter setting optimized by a method for optimizing at least one parameter setting according to claim 1.

13. A mass spectrometry device, wherein the mass spectrometry device comprises at least one control unit configured for performing a method for optimizing at least one parameter setting according to claim 1.

14. A computer program comprising instructions which, when the program is executed by a control unit of the mass spectrometry device according to claim 1, causes the control unit to perform a method for optimizing at least one parameter setting according to claim 1.

15. A computer-readable storage medium comprising instructions which, when the program is executed by a control unit of the mass spectrometry device according to claim 1, causes the control unit to perform a method for optimizing at least one parameter setting according to claim 1.

Description

SHORT DESCRIPTION OF THE FIGURES

[0099] Further optional features and embodiments will be disclosed in more detail in the subsequent description of embodiments, preferably in conjunction with the dependent claims. Therein, the respective optional features may be realized in an isolated fashion as well as in any arbitrary feasible combination, as the skilled person will realize. The scope of the invention is not restricted by the preferred embodiments. The embodiments are schematically depicted in the Figures. Therein, identical reference numbers in these Figures refer to identical or functionally comparable elements.

[0100] In the Figures:

[0101] FIG. 1 shows an embodiment of a method a method for optimizing at least one parameter setting;

[0102] FIG. 2 shows an embodiment of a mass spectrometry device according to the present invention;

[0103] FIG. 3 theoretical impact of using one vs. two decimal places.

[0104] FIG. 4A to 4C show experimental data tor one vs. three decimal places.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0105] FIG. 1 shows a flow chart of an embodiment of a method a, in particular computer-implemented, method for optimizing at least one parameter setting of at least one mass spectrometry device 110 operating at unit resolution.

[0106] An embodiment of the mass spectrometry device 110 according to the present invention is shown in FIG. 2. The mass spectrometry device 110 may be an analyzer configured for detecting at least one analyte based on a mass-to-charge ratio (m/z).

[0107] The mass spectrometry device 110 may be or may comprise at least one quadrupole analyzer 112. The quadrupole mass analyzer 112 is or comprises at least one mass analyzer comprising at least one quadrupole as mass filter. The mass filter may be configured for selecting ions injected to the mass filter according to their mass-to-charge ratio m/z. The mass filter comprises two pairs of electrodes. The electrodes may be rod-shaped, in particular cylindrical. In ideal case, the electrodes may be hyperbolic. The electrodes may be designed identical. The electrodes may be arranged in parallel extending along a common axis, e.g. a z axis. The quadrupole mass analyzer 112 may comprise at least one power supply circuitry configured for applying 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 circuitry may be configured for holding each opposing electrode pair at identical potential. The power supply circuitry may be configured for changing sign of charge of the electrode pairs periodically such that stable trajectories are only possible for ions within a certain mass-to-charge ratio m/z. Trajectories of ions within the mass filter can be described by the Mathieu differential equations. For measuring ions of different m/z values DC and AC voltage may be changed in time such that ions with different m/z values can be transmitted to a detector 114.

[0108] The mass spectrometry device 110 may be configured for multiple reaction monitoring. The quadrupole mass analyzer 112 may comprise a plurality of quadrupoles. The mass spectrometry device 110 may comprise a triple quadrupole mass spectrometry device comprising three quadrupoles, denoted Q1, Q2, Q3 in FIG. 2.

[0109] The mass spectrometry device 110 may comprise at least one ionization source 116. The ionization source 116 may be configured for generating ions, e.g. from neutral gas molecules. The ionization source 116 may be or may comprise at least one source selected from the group consisting oft at least one gas phase ionization source such as at least one electron impact (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 atom bombardment (FAB) source, at least one secondary ion mass spectrometry (SIMS) source, at least one laser desorption (IDMS) source, and at least one matrix assisted laser desorption (MALDI) source; at least one spray ionization source such as at least one thermospray (TSP) source, at least one atmospheric pressure chemical ionization (APCI) source, at least one electrospray (EST), and at least one atmospheric pressure ionization (API) source.

[0110] The ions enter the mass spectrometry device 110 at a curtain plate 118 and at an orifice plate 120. The mass spectrometry device 110 may comprise a quadrupole ion guide in a first vacuum stage. Subsequent, the ions pass through an aperture (IQ0) and reach a second vacuum stage having an additional quadrupole ion guide (Q0), and an additional aperture (IQ1, or ST1). In a first quadrupole Q1 a precursor ion, also denoted parent ion, may be isolated. The precursor ion may be an ion of interest which may be preselected with the first quadrupole Q1. Within a second quadrupole Q2, in particular a collision cell of the second quadrupole Q2, the precursor ion may be fragmented into daughter ions, also denoted fragment ions or product ions. A third quadrupole Q3 may be used for filtering and/or selecting said fragment ions. The Q2 quadrupole is separated from the first quadrupole Q1 and the third quadrupole Q3 by interquad lenses IQ2 (or ST2) and IQ3 (or ST3). The fragment ions may pass an exit lens 122 and impinge on the detection 114. The detector 114 may be configured for detecting incoming ions. The detector 114 may be configured for detecting charged particles. The detector 114 may be or may comprise at least one electron multiplier.

[0111] The mass spectrometry device 110 may be or may comprise a liquid chromatography mass spectrometry device, not shown in FIG. 2. The mass spectrometry device 110 may be connected to and/or may comprise at least one liquid chromatography device, also denoted liquid chromatograph. The liquid chromatograph may be used as sample preparation for the mass spectrometry device 110. Other embodiments of sample preparation may be possible, such as at least one gas chromatograph. The liquid chromatography mass spectrometry device may be or may comprise at least one high performance liquid chromatography (HPLC) device or at least one micro liquid chromatography (μLC) device. The liquid chromatography mass spectrometry device may comprise a liquid chromatography (LC) device and a mass spectrometry (MS) device, in the present case the mass filter, wherein the LC device and the mass filter are coupled via at least one interface. The interface coupling the LC device and the MS device may comprise the ionization source 116 configured for generating of molecular ions and for transferring of the molecular ions into the gas phase. The interface may further comprise at least one ion mobility module arranged between the ionization source 116 and the mass filter. For example, the ion mobility module may be a high-field asymmetric waveform ion mobility spectrometry (FAIMS) module.

[0112] The mass spectrometry device 110 is configured for operating at unit resolution, also denoted unit mass resolution. The mass spectrometry device 110 is configured for separating two ions differing by one mass unit. The unit resolution may be in a range of ±0.1 to ±0.4 amu, preferably ±0.2 to ±0.35. Specifically, the mass spectrometry device 110 may have a mass resolution of about ±0.35 amu. Specifically, the mass spectrometry device 110 may be a so-called low resolution mass spectrometry device. In known low resolution mass spectrometry devices for the optimization of the mass scale one decimal number is used for a mass to charge ratio of the ion species since unit mass resolution, e.g. ±0.35 amu, with a mass axis accuracy of 0.1-0.2 amu is a common setting. A more accurate setting is considered for known devices and methods as not applicable as experimental bias and error strongly affects the m/z ratios. As will be outlined in detail below, the present application proposes to use more than one decimal number. It was found that the usage of more than one decimal number for the m/z ratios lead to significant differences in signal intensity and improved precision for peak area ratios (analyte/internal standard). In particular real area ratios were less affected by drifts what leads to an improved calibration and/or robustness and stability. Thus, calibration intervals can be prolonged by applying this methodical feature.

[0113] The parameter setting may comprise values and/or ranges of parameters defining at least one assay performed by the mass spectrometry device 110. The assay may be a quantitative assay. For example, the parameter setting may comprise one or more of the following parameters mass scale, MRM transition for tandem MS, cell exit potentials. MS resolution, source gas flow/pressure, with temperature source gas temperature, ion source gas, curtain gas, ion spray voltage, temperature, declustering potential, entrance potential, focusing lens, pre-filter, ion energy, collision energy, collision gas, and the like.

[0114] The optimizing of the parameter setting may comprise a process of solving at least one optimization problem. The optimizing may comprise evaluating and/or tuning and/or selecting at least one parameter of the parameter setting defining the assay. The optimizing may comprise selecting a best parameter value with regard to some criterion, e.g. minimal intensity loss of a measured peak or the like. The optimizing may comprise determining an extremum such as a maximization and/or minimization. The optimizing may comprise defining as optimum the most robust parameter setting, e.g. a plateau in the graph. Specifically, the method may comprise optimizing an analyte detection window and an internal standard detection window, in particular their central mass to charge ratio values.

[0115] As shown in FIG. 1, the method comprises the following steps which, as an example, may be performed in the given order. [0116] a) (reference number 124) determining at least one analyte detection window for detecting an analyte of interest with the mass spectrometry device 110, wherein the analyte detection window is defined by a central mass to charge ratio value of the analyte and a pre-defined width, wherein the central mass to charge ratio value of the analyte is set to a theoretical mass to charge ratio value of the analyte of interest having more than one decimal place and/or a mass to charge ratio value of the analyte of interest determined by a high resolution mass spectrometry measurement having more than one decimal place, [0117] b) (reference number 126) determining at least one internal standard detection window for detecting an internal standard substance with the mass spectrometry device 110, wherein the internal standard detection window is defined by a central mass to charge ratio value of the internal standard substance and the pre-defined width, wherein the central mass to charge ratio value of the internal standard substance is set to a mass to charge ratio value of the internal standard substance calculated relative to the analyte of interest and having more than one decimal place and/or to a mass to charge ratio value of the internal standard substance determined by a high resolution mass spectrometry measurement having more than one decimal place.

[0118] As outlined above, the mass spectrometry device 110 may comprise three quadrupoles. The determining of the analyte detection window and the internal standard detection window may be performed for the first quadrupole and the third quadrupole. The method may comprise determining analyte detection windows and internal standard detection windows for each of the first quadrupole Q1 and/or the third quadrupole Q3 of the triple quadrupole mass spectrometry device 110.

[0119] The mass spectrometry device 110 may be configured for analyzing at least one sample comprising the analyte of interest. The sample may be an arbitrary test sample such as a biological sample and/or an internal standard sample. The sample may comprise one or more analytes of interest. For example, the sample may be selected from the group consisting of a physiological fluid, including blood, serum, plasma, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cells or the like. The sample may be used directly as obtained from the respective source or may be subject of a pretreatment and/or sample preparation workflow. For example, analytes of interest may be vitamins, vitamin D, drugs of abuse, therapeutic drugs, hormones, and metabolites in general.

[0120] For further details with respect to the sample, reference is made e.g. to EP 3 425 369 A1, the full disclosure is included herewith by reference. Other analytes of interest are possible.

[0121] The internal standard substance may be a structurally similar compound to the analyte of interest. For example, a sample may be pretreated by adding the at least one internal standard substance. Said sample may comprise the at least one internal standard substance with a known concentration. The internal standard substance may be or may comprise structurally similar compounds. The internal standard substance may be an isotopically labeled versions of the analyte, preferably an isotopologue of the analyte of interest. The higher the structural similarity the better the performance.

[0122] The analyte detection window may be a mass to charge ratio range or frame in which the detection and/or measurement of the analyte of interest is performed. The internal standard detection window is a mass to charge ratio range or frame in which the detection and/or measurement of the internal standard substance is performed. The each of the analyte detection window and/or the internal standard detection window may have an initial setting, e.g. stored in at least one database of the mass spectrometry device 119. The setting of the respective detection window may comprise values for one or both limits, in particular a lower mass to charge ratio limit and an upper mass to charge ratio limit, of the detection window. Specifically, the setting may comprise one or both of a value for a lower limit of the detection window, i.e. a mass to charge ratio at which the detection starts, and a value for an upper limit of the detection window, i.e. mass to charge ratio at which the detection stops. The analyte detection window is defined by a central mass to charge ratio value of the analyte and a pre-defined width. The internal standard detection window is defined by a central mass to charge ratio value of the internal standard substance and the pre-defined width. The central mass to charge ratio value may be a center of the mass to charge ratio range or frame. The pre-defined width may be 0.7 amu (i.e. ±0.35 amu). However, other width may be possible.

[0123] The central mass to charge ratio value of the analyte is set to a theoretical mass to charge ratio value of the analyte of interest having more than one decimal place and/or a mass to charge ratio value of the analyte of interest determined by a high resolution mass spectrometry measurement having more than one decimal place. The central mass to charge ratio value of the internal standard substance is set to a mass to charge ratio value of the internal standard substance calculated relative to the analyte of interest having more than one decimal place and/or to a mass to charge ratio value of the internal standard substance determined by a high resolution mass spectrometry measurement having more than one decimal place. For example, the theoretical mass to charge ratio of the analyte may be a calculated mass to charge ratio and/or may be obtained from at least one database. Additionally or alternatively, the charge ratio value of the analyte may be set to an experimental value. The high resolution mass spectrometry measurement may comprise a measurement performed using a high resolution mass spectrometry device. The high resolution mass spectrometry device may be configured for performing m/z-measurements having at least four decimal places, wherein at least three decimal places are significant. The high resolution mass spectrometry device may be configured for elucidating molecular formulas and/or measuring mass defect of molecules. In contrast to known optimization approaches, the method according to the present invention proposes a combination experimental and theoretical approaches. For example, mass to charge ratio values of the analyte may be calculated or at least determined by a high resolution MS measurement and the m/z values for the internal standard substance may be calculated relative to the analyte. Other parameters of the parameter setting of the mass spectrometry device 110 like voltages may be determined experimentally.

[0124] The theoretical mass to charge ratio value of the analyte of interest has more than one decimal place and/or the mass to charge ratio value of the analyte of interest determined by a high resolution mass spectrometry measurement has more than decimal place. The theoretical mass to charge ratio value of the analyte of interest has two decimal places and/or the mass to charge ratio value of the analyte of interest determined by a high resolution mass spectrometry measurement has two decimal places. The mass to charge ratio value of the internal standard substance calculated relative to the analyte of interest and/or the mass to charge ratio value of the internal standard substance determined by a high resolution mass spectrometry measurement may have two decimal places. The theoretical mass to charge ratio value of the analyte of interest has three decimal places and/or the mass to charge ratio value of the analyte of interest determined by a high resolution mass spectrometry measurement has three decimal places. The mass to charge ratio value of the internal standard substance calculated relative to the analyte of interest and/or the mass to charge ratio value of the internal standard substance determined by a high resolution mass spectrometry measurement may have three decimal places. The theoretical mass to charge ratio value of the analyte of interest has more than three decimal places and/or the mass to charge ratio value of the analyte of interest determined by a high resolution mass spectrometry measurement has more than three decimal places. The mass to charge ratio value of the internal standard substance calculated relative to the analyte of interest and/or the mass to charge ratio value of the internal standard substance determined by a high resolution mass spectrometry measurement may have more than three decimal places.

[0125] Although low resolution MS systems were used, it was surprisingly fond that the usage of more than one decimal number for the m/z ratios leads to that peak area ratios (analyte/internal standard) were less affected by drifts what leads to an improved calibration stability. Moreover, improved precision in signal intensity can be observed. Thus, calibration intervals can be prolonged by applying this methodical feature. Further evaluation showed that both molecular species differ in their mass defect what typically causes a deviation of the m/z ratio in the first or second decimal place. For isotopically labeled internal standards typically differences in the second decimal place occur. Although the resulting signal intensity is covered by initial calibration, a drifting mass axis leads to a drifting peak area ratio due to nonlinearity of the intensity function. This deviation causes an “uncalibrated” deviation, thus, a bias of the test result. Using more than one decimal number for m/z ratios, may reduce the performance difference between analyte and internal standard and significantly can decrease the occurring bias of the area ratio.

[0126] FIG. 3 shows in the two upper plots results of a simulation of relative signal intensity vs m/z using a correctly adjusted mass axis for (left plot) an analyte using one decimal place (square) and two decimal places (triangle) and for (right plot) an internal standard substance using one decimal place (square) and two decimal places (triangle). In case of two decimal places no difference in relative signal intensity can be observed. In case of only one decimal place a deviation can be found which is covered by assay calibration. FIG. 3 shows in the lower two plots results of a simulation of intensity vs m/z after mass axis shift of 0.01 amu for (left plot) an analyte using one decimal place (square) and two decimal places (triangle) and for (right plot) an internal standard substance using one decimal place (square) and two decimal places (triangle). Again, in case of two decimal places no difference can be observed. However, for only one decimal place a larger deviation can be observed. The drifting mass axis leads to a drilling peak area ratio due to nonlinearity of the intensity function. This deviation causes an “uncalibrated” deviation, thus a bias of the test result. This simulation shows that using more than one decimal number for m/z ratios, may reduce the performance difference between analyte and internal standard and significantly can decrease the occurring bias of the area ratio.

[0127] FIGS. 4A to 4C show experimental data for one vs. three decimal places for the following analytes Testosterone (FIG. 4A), Cyclosporin A (FIG. 4B), Phenytoin (FIG. 4C). For each experiment three sample x six replicates x twelve blocks giving 216 sample processings within 66 h were performed with assay calibration at t=0 h. A bias analysis was performed comprising calculating of the relative recovery of each sample to its known target value. In addition a precision analysis was performed comprising calculating of coefficients of variations (cv).

[0128] For Testosterone the following experimental results were obtained:

TABLE-US-00001 3 decimal numbers 1 decimal number recovery after 66 h 100.7%  101.7% drift (delta recovery) −0.1%   +1% cv 2.00%  2.18%

[0129] For Cyclosporin A the following experimental results were obtained:

TABLE-US-00002 3 decimal numbers 1 decimal number recovery after 66 h 90.3% 104.2% drift (delta recovery) +1.1%  +4.0% cv 3.34%  4.14%

[0130] For Phenytoin the following experimental results were obtained:

TABLE-US-00003 3 decimal numbers 1 decimal number recovery after 66 h 92.3% 81.7% drift (delta recovery) −3.9% −10.0%  cv 4.31% 4.37%

[0131] Thus, for each analyte it is shown that robustness can be significantly increased by using three decimal numbers for MRM transitions instead of one. The bias is smaller over the time such that it is possible to increase calibration stability and to reduce optimization frequency. Moreover, precision can be slightly improved using three decimal numbers for MRM transitions instead of one. The cv is smaller resulting in higher precision.

LIST OF REFERENCE NUMBERS

[0132] 110 mass spectrometry device [0133] 112 quadrupole mass analyzer [0134] 114 detector [0135] 116 ionization source [0136] 118 curtain plate [0137] 120 curtain plate 118 and at an orifice plate [0138] 122 exit lens [0139] 124 step a) [0140] 126 step b)