Quality control tools for LC-MS

Abstract

This invention relates to a method for identifying and / or verifying at least one analyte peak in a chromatogram of a sample for an analyte from a liquid chromatography-mass spectrometry (LC-MS) apparatus, the method comprising the steps of: a) determining a chromatogram of the sample by acquiring multiple data points of quantification factor signal intensity and / or qualitative factor signal intensity over time; b) determining a ratio of the analyte quantification factor to the analyte qualitative factor for at least a portion of the data points acquired in step a); c) comparing the ratio determined in step b) with a benchmark; and d) identifying and / or verifying at least one analyte peak in the chromatogram based on the comparison in step c). The invention also relates to a method for identifying and / or verifying at least one analyte peak in a chromatogram of a sample containing an internal standard for an analyte, and to related systems and computer program products.

Description

[0001] This invention relates to a method for identifying and / or verifying at least one analyte peak in a chromatogram of a sample for an analyte from a liquid chromatography-mass spectrometry (LC-MS) apparatus, the method comprising the steps of: a) determining a chromatogram of the sample by acquiring multiple data points of quantitative factor signal intensity and / or qualitative factor signal intensity over time; and, optionally, acquiring multiple data points of internal standard quantitative factor signal intensity and / or internal standard qualitative factor signal intensity over time, if the sample contains an internal standard; b) determining a ratio type for at least a portion of the data points acquired in step a), the ratio type being selected from a list consisting of: (i) the ratio of analyte quantitative factor to analyte qualitative factor, (ii) the ratio of internal standard quantitative factor to internal standard qualitative factor, (iii) the ratio of analyte quantitative factor to internal standard quantitative factor, and (iv) the ratio of analyte qualitative factor to internal standard qualitative factor; c) comparing the ratio determined in step b) with a benchmark; and d) identifying and / or verifying at least one analyte peak in the chromatogram based on the comparison step c). The present invention also relates to a method for identifying and / or verifying at least one analyte peak in a chromatogram of a sample containing an internal standard from a liquid chromatography-mass spectrometry device for an analyte, the method comprising the steps of: a) determining a chromatogram of the sample by acquiring multiple data points of the signal intensity of a quantitative factor over time, and optionally acquiring multiple data points of the signal intensity of a qualitative factor over time; b) determining a ratio type for at least a portion of the data points acquired in step a), the ratio type being selected from a list consisting of: (i) the ratio of the analyte quantitative factor to the analyte qualitative factor, (ii) the ratio of the internal standard quantitative factor to the internal standard qualitative factor, (iii) the ratio of the analyte quantitative factor to the internal standard quantitative factor, and (iv) the ratio of the analyte qualitative factor to the internal standard qualitative factor; c) comparing the ratio determined in step b) with a benchmark; and d) identifying and / or verifying at least one analyte peak in the chromatogram based on the comparison in step c); and relating to methods, systems, and computer program products associated therewith. Technical Field

[0002] This invention relates to a method for identifying and / or verifying at least one analyte peak in a chromatogram of a sample from a liquid chromatography-mass spectrometry device, to a quality control method applying the aforementioned method, and to related systems and computer program products. Background Technology

[0003] For LC-MS assays, peak area ratios are a common method for calculation or validation. They are part of several international guidelines for validating mass spectrometry assays, such as those developed by CLSI (Clinical and Laboratory Standards Institute), EMA (European Medicines Agency), or GTFCh (German Society for Toxicology and Forensic Chemistry). To ensure the quality of the analytical assay, the non-extraction system suitability test of the spiked compound measured before the analytical run must meet acceptability requirements, such as the minimum absolute peak area or maximum retention time deviation from the target value. During the analytical run, quality control (QC) samples are subsequently tested at a certain frequency, and the calculated results are checked against acceptable ranges. Furthermore, retention time, peak width (given by the difference in retention time between peak boundaries), absolute peak area of ​​the internal standard (ISTD), and the quantification factor / qualification factor peak area ratio of the analyte are typically monitored in each sample, and should meet acceptability requirements for maximum deviation or specific cutoff values. However, most of these parameters are based on peak integrals:

[0004] Peak boundaries, interference detection, and result calculations all depend directly on peak integration. However, background noise (e.g., due to an aged MS source) or tailing peaks (e.g., due to an aged LC column) can lead to improper peak integration. Peak boundaries are typically defined as the out-of-range point of the retention time of the integrated peak and are therefore susceptible to improper peak integration. The peak area ratio of two different transitions of the same analyte (e.g., the ratio of quantification factor to qualitative factor) is used to verify peak identity and eliminate interference. This principle stems from the fact that the peak area ratio of different analyte transitions deviates from a fixed value, independent of analyte concentration. However, this value can change not only due to detection artifacts (i.e., interference) of individual mass transitions but also due to improper peak integration. These two issues cannot be clearly distinguished simply by monitoring these peak area ratios, thus often requiring manual peak inspection by experts. In the case of peak areas of the analyte and internal standard (ISTD), their ratio (analyte / ISTD ratio) yields the result, i.e., the desired concentration. Since result calculations depend directly on peak integration, its failure will directly affect the results.

[0005] Furthermore, due to the immaturity of the integration procedure, peak integration failures are very common, and these failures cannot be automatically distinguished from the corresponding system faults. Therefore, peak checks usually require manual verification by experts. On the other hand, such peak checks largely depend on the operator's experience, increasing the human workload. Therefore, alternative procedures with parameters independent of peak integration are needed for verification.

[0006] Problems to be solved

[0007] Nevertheless, there is still a need in the field for ways and methods to help ensure the correctness of peak identification and result calculation in LC-MS measurements. Summary of the Invention

[0008] This problem is solved by methods, systems, computer program products, computers or computer networks, computer-loadable data structures, computer programs, and storage media having the features of the independent claims. Advantageous embodiments that may be implemented individually or in any arbitrary combination are listed in the dependent claims.

[0009] Therefore, the present invention relates to a method for identifying and / or verifying at least one analyte peak in a chromatogram of a sample for an analyte from a liquid chromatography-mass spectrometry apparatus, the method comprising the following steps:

[0010] a) Provided by acquiring multiple data points of quantitative factor signal intensity and / or qualitative factor signal intensity over time, in one embodiment for determining the chromatogram of the sample; and, optionally provided over time, in one embodiment for acquiring multiple data points of internal standard quantitative factor signal intensity and / or internal standard qualitative factor signal intensity, when the sample contains an internal standard;

[0011] b) For at least a portion of the data points collected in step a), determine the ratio type, which is selected from a list of the following: (i) the ratio of analyte quantification factor to analyte qualitative factor, (ii) the ratio of internal standard quantification factor to internal standard qualitative factor, (iii) the ratio of analyte quantification factor to internal standard quantification factor, and (iv) the ratio of analyte qualitative factor to internal standard qualitative factor;

[0012] c) Compare the ratio determined in step b) with the benchmark; and

[0013] d) Based on the comparison step c) Identify and / or verify at least one analyte peak in the chromatogram.

[0014] As those skilled in the art will understand, the internal standard quantification factor and / or internal standard qualitative factor, and the ratios containing these values, can only be determined if the sample contains an internal standard. Therefore, the foregoing method can be a method for identifying and / or validating at least one analyte peak in a chromatogram of a sample from a liquid chromatography-mass spectrometry device, the method comprising the following steps:

[0015] a) Provided by acquiring multiple data points of quantitative and qualitative factor signal intensity over time, in one embodiment for determining the chromatogram of the sample;

[0016] b) Determine the ratio of the analyte quantification factor to the analyte qualitative factor for at least a portion of the data points collected in step a);

[0017] c) Compare the ratio determined in step b) with the benchmark; and

[0018] d) Based on the comparison step c) Identify and / or verify at least one analyte peak in the chromatogram.

[0019] Furthermore, the aforementioned method can also be a method for identifying and / or verifying at least one analyte peak in a chromatogram of a sample containing an internal standard from a liquid chromatography-mass spectrometry device for an analyte, the method comprising the following steps:

[0020] a) Provided by acquiring multiple data points of quantitative factor signal intensity and / or qualitative factor signal intensity over time, in one embodiment for determining the chromatogram of the sample; and optionally provided over time, in one embodiment for acquiring multiple data points of internal standard quantitative factor signal intensity and / or internal standard qualitative factor signal intensity;

[0021] b) For at least a portion of the data points collected in step a), determine the ratio type, which is selected from a list of the following: (i) the ratio of analyte quantification factor to analyte qualitative factor, (ii) the ratio of internal standard quantification factor to internal standard qualitative factor, (iii) the ratio of analyte quantification factor to internal standard quantification factor, and (iv) the ratio of analyte qualitative factor to internal standard qualitative factor;

[0022] c) Compare the ratio determined in step b) with the benchmark; and

[0023] d) Based on the comparison step c) Identify and / or verify at least one analyte peak in the chromatogram.

[0024] As used herein, the terms are broad and are given common and customary meanings to those skilled in the art; therefore, unless otherwise stated, the terms used herein are not limited to specific or customary meanings. As used below, the terms “having,” “comprising,” or “including,” or any of their arbitrary grammatical variations, are used in a non-exclusive manner. Thus, these terms can refer either to a situation where no other features exist in the entity described in the context besides those introduced by these terms, or to a situation where one or more other features exist. For example, the expressions “A has B,” “A includes B,” and “A includes B” can refer to a situation where no other elements exist in A besides B (i.e., where A is solely and uniquely composed of B), or to a situation where one or more other elements (such as element C, element D, or even other elements) exist in entity A besides B. Furthermore, as will be understood by those skilled in the art, the expressions “comprising one” and “comprising a” preferably mean “comprising one or more,” that is, equivalent to “comprising at least one.” Similarly, the term "determine an X" means determining one X, as well as determining more than one X, such as two, three, or four Xs. Furthermore, the term "multiple" refers to a large number of indicated items; in one embodiment, at least two indicated items; in a further embodiment, at least three indicated items; in a further embodiment, at least four indicated items; and in a further embodiment, at least five indicated items.

[0025] Furthermore, as used below, the terms “preferred,” “more preferably,” “most preferably,” “particularly,” “more particularly,” “specifically,” “more specifically,” or similar terms are used in combination with optional features without limiting further possibilities. Therefore, features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As those skilled in the art will recognize, the invention can be practiced using alternative features. Similarly, features introduced by “in one embodiment” or similar expressions are intended to be optional features without limiting further embodiments of the invention, without limiting the scope of the invention, and without limiting the possibility of combining features introduced in this manner with other optional or non-optional features of the invention.

[0026] As used herein, the term "about" refers to an indication value having a technical precision generally accepted in the relevant field, preferably ±20%, more preferably ±10%, and most preferably ±5%. Furthermore, the term "substantially" means that there is no deviation affecting the indication result or use, i.e., potential deviations will not cause the indication result to deviate from the specified value by more than ±20%, more preferably ±10%, and most preferably ±5%. Therefore, "substantially composed of..." means including the specified components, but excluding other components, except for: materials present as impurities, unavoidable materials present due to the process used to provide said components, and components added for purposes other than achieving the technical effects of the invention. For example, compositions defined using the phrase "substantially composed of..." include any known acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition substantially composed of one set of components will contain less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of unspecified components. As described herein, the measured and calculated parameters are presented on an exemplary basis; as those skilled in the art will understand, the parameters can be modified by standard mathematical operations, particularly by multiplication, division, addition, subtraction, reciprocal formation, scaling, and other operations known to those skilled in the art; in one embodiment, the baseline is adjusted accordingly, particularly by applying the same mathematical operations. The measured and calculated parameters can also be used for score calculation, which can be based on one or more parameter values, optionally by weighting, and / or by further mathematical operations, particularly as described above, such as scaling.

[0027] The method for identifying and / or validating at least one analyte peak is an in vitro method. Furthermore, it may include steps other than those explicitly mentioned above. For example, further steps may involve, for instance, providing a sample for step a), or further calculations in steps b) and / or c). Additionally, the method may include step a1) determining the expected lower peak boundary and / or expected upper peak boundary corresponding to the analyte, and optionally determining the expected lower peak boundary and / or expected upper peak boundary corresponding to the internal standard. Furthermore, one or more of these steps may be performed by automated equipment. In one embodiment, specifically, steps b) and c) are performed by a processor, specifically a computer, which may be configured as an evaluation device as specified elsewhere herein. In one embodiment, the method for identifying and / or validating at least one analyte peak in the chromatogram of a sample containing an internal standard further includes, in step a), acquiring multiple data points of the internal standard quantification factor signal intensity and / or internal standard qualitative factor signal intensity over time. In one embodiment, the signal intensity or combination thereof (quantitative factor signal intensity, qualitative factor signal intensity, internal standard quantitative factor signal intensity, and / or internal standard qualitative factor signal intensity, if any) to be determined is independently selected for each data point. In one embodiment, the signal intensity is determined such that at least one of the ratios specified in step b) can be determined. In one embodiment, for at least all data points known or suspected to be related to the elution time of the analyte, at least the quantitative factor signal intensity and the qualitative factor signal intensity are determined. In a further embodiment, for at least all data points known or suspected to be related to the elution time of the analyte, the quantitative factor signal intensity, the qualitative factor signal intensity, the internal standard quantitative factor signal intensity, and the internal standard qualitative factor signal intensity are determined.

[0028] The term "liquid chromatography-mass spectrometry device," abbreviated as "LC-MS device," is understood by those skilled in the art. In one embodiment, the term refers to a device configured for combining a liquid chromatography (LC) device and a mass spectrometer (MS). Thus, in one embodiment, the device includes at least one LC unit and at least one MS unit, wherein the LC unit and the MS unit are coupled via at least one interface. As used herein, the term "liquid chromatography (LC) unit," in one embodiment, refers to an analytical module configured to separate one or more target analytes from other components of a sample by liquid chromatography, and in one embodiment, to detect said one or more analytes using a mass spectrometer. LC can be based on any separation principle deemed appropriate by a person skilled in the art; in one embodiment, LC is reversed-phase chromatography, hydrophobic interaction chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, or chiral chromatography; in a further embodiment, LC is reversed-phase chromatography. An LC device may include at least one LC column. For example, an LC device may be a single-column LC device or a multi-column LC device 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 transfer a target analyte. The LC unit may be or may include at least one high-performance liquid chromatography (HPLC) unit and / or at least one microfluidic liquid chromatography (µLC) device. As used herein, in one embodiment, the term "mass spectrometry unit" refers to a mass analyzer configured for detecting at least one analyte based on the mass-to-charge ratio of the analyte or a fragment thereof. The mass spectrometry unit may be or may include at least one quadrupole mass spectrometer. The interface coupling the LC unit and the MS unit may include at least one ionization source configured to generate molecular ions and for transferring the molecular ions to the gas phase. In one embodiment, the MS unit is a tandem mass spectrometry (MS / MS) unit; in a further embodiment, it is a triple quadrupole MS / MS; and in a further embodiment, it is in multiple reaction monitoring (MRM) mode.

[0029] The term "chromatogram" is well known to those skilled in the art. In one embodiment, the term refers to a correlation graph of the quantitative measure of one or more signals obtained from a sample by an MS detector with the chromatographic separation process, in one embodiment, over time, such as retention time and / or elution volume. In one embodiment, the quantitative measure of the signal is correlated with the concentration of at least a portion of the sample component, particularly with an analyte or internal standard; thus, the quantitative measure of the signal may more specifically be signal intensity. Thus, in one embodiment, the chromatogram is an MS chromatogram, and in a further embodiment, an MS / MS chromatogram. In one embodiment, the quantitative measure of the signal includes the analyte signal intensity and / or the internal standard signal intensity. In one embodiment, the quantitative measure of the signal includes an analyte quantification factor, an internal standard quantification factor, an analyte qualitative factor, and / or an internal standard qualitative factor. Thus, in one embodiment, determining at least one chromatogram includes measuring at least one of the analyte quantification factor, the internal standard quantification factor, the analyte qualitative factor, and the internal standard qualitative factor over time and / or elution time, as described above; in this case, in one embodiment, the MS is a tandem MS. As those skilled in the art will understand, the foregoing representation may be, but is not necessarily, a graphical representation; however, the representation may also be provided as, for example, a list of value pairs (e.g., elution time / quantitative factor value pairs and / or elution time / qualitative factor value pairs) or a mathematical model. As described above, a chromatogram may represent more than one signal; in one embodiment, a chromatogram represents two signals, such as the quantitative factor signal intensity and the qualitative factor signal intensity; in a further embodiment, a chromatogram represents three signals, and in a further embodiment, a chromatogram represents four signals, such as the quantitative factor signal intensity and the qualitative factor signal intensity of the analyte and the internal standard, respectively. It should be understood that a chromatogram may also represent additional signals; however, the aforementioned large number of signals may also be represented by multiple chromatograms, each representing one signal. As those skilled in the art will further understand, elution time may be replaced by any other measure of LC progress that those skilled in the art deem appropriate, particularly by elution volume or retention time. A chromatogram may include data points throughout the entire LC-MS run of the sample; in one embodiment, particularly where the position of the analyte peak in the chromatogram can be predicted, for example, from previous runs, the chromatogram may include data points on the predicted analyte peak width, for example, from the presumed lower peak boundary to the presumed upper peak boundary, optionally further including data representing 1%, 5% in one embodiment, 10% in a further embodiment, 50% in a further embodiment, and 100% in a further embodiment of the corresponding presumed boundary value extending downstream and / or upstream of the analyte peak.

[0030] The term "peak" is well known to those skilled in the art and, in one embodiment, refers to at least one local maximum value in a chromatogram. Accordingly, the term "analyte peak" refers to a peak associated with an analyte and, in one embodiment, is used to represent an identified peak of a target analyte. Peak integration methods are known in the art, and in one embodiment, the term "peak integration" refers to at least one mathematical operation and / or mathematical algorithm for determining the peak area enclosed by the peaks of a chromatogram. Specifically, peak integration may include the identification and / or measurement of curve features of a chromatogram. Peak integration may include one or more of the following: peak detection, peak discovery, peak identification, peak fitting, peak evaluation, determination of lower peak boundaries and / or upper peak boundaries, determination of background, and determination of baseline. Peak integration may allow the determination of one or more of the following: peak area, retention time, peak height, and peak width. In one embodiment, peak detection and / or integration are performed automatically, i.e., without manual operation or user interaction. In particular, peak identification and / or peak detection and / or determination of peak area may be performed non-manually and without manual operation or user interaction.

[0031] As used herein, the term "analyte peak identification" refers to any measure for determining at least one parameter of a peak in a chromatogram. In one embodiment, identification includes identifying lower and / or upper peak boundaries, identifying peak identity and / or peak purity, and / or identifying the ratio of the analyte peak area to the internal standard peak area (peak area ratio).

[0032] As used herein, the term “validate analyte peak” includes any measure that confirms a pre-established measurement of an analyte by means of a chromatogram; thus, in one embodiment, the validation is validating the putative lower peak boundary and / or the putative upper peak boundary of the peak, validating peak identity and / or peak purity, and / or validating the ratio of the analyte peak area to the internal standard peak area (peak area ratio).

[0033] As used herein, the term "proposed peak boundary" refers to a peak boundary that has not been verified by the methods specified herein. Thus, in one embodiment, a proposed peak boundary is a preliminary peak boundary and can be determined by conventional methods, such as peak detection and / or peak integration, based on a chromatogram specified elsewhere herein; a proposed peak boundary can also be a peak boundary determined based on a different chromatogram, such as an LC-UV / VIS-chromatogram; furthermore, a proposed peak boundary can be a predetermined peak boundary, determined, for example, based on a previous LC-MS run using the same or similar samples or using substantially pure analytes and / or internal standards. In one embodiment, a proposed peak boundary can also be a predicted peak boundary, for example, predicted based on a previous run using a chemically similar compound to the analyte. Accordingly, a proposed peak boundary can be identified by specific values ​​or ranges, and in further embodiments, by specific values, such as retention time or elution volume.

[0034] As described above, the term "expected analyte peak area to internal standard peak area ratio," also known as "expected peak area ratio," refers to the ratio of the analyte peak area to the internal standard peak area that has not been validated by the methods specified herein; therefore, in one embodiment, the expected peak area ratio is a preliminary peak area ratio and can be determined by conventional methods based on chromatograms specified elsewhere herein. As those skilled in the art will understand, peak area ratios as described above are commonly used to quantify analytes in LC-MS methods, particularly when using calibration curves.

[0035] As used herein, the term "sample," also known as "test sample," refers to any type of composition of substances; therefore, the term can refer to, but is not limited to, any arbitrary sample, such as a biological sample. In one embodiment, the sample is a liquid sample, and in a further embodiment, an aqueous sample. In one embodiment, the test sample may be selected from the group consisting of: physiological fluids, including blood, serum, plasma, saliva, lens fluid, tears, cerebrospinal fluid, sweat, urine, emulsion, ascites, mucus, synovial fluid, peritoneal fluid, amniotic fluid; lavage fluid; tissues, cells, etc. In one embodiment, the sample is a blood, plasma, serum, saliva, or urine sample, and in a further embodiment, a blood, plasma, or serum sample. However, the sample may also be a natural or industrial liquid, particularly surface water or groundwater, sewage, industrial wastewater, processing fluid, soil wash, etc. In one embodiment, the sample contains or is suspected of containing at least one target chemical compound, i.e., a chemical substance to be identified, which is referred to as an "analyte." The sample may contain one or more other chemical compounds, which do not need to be identified and are generally referred to as a "matrix." Samples may be used directly from their respective sources or may undergo one or more pretreatment and / or sample preparation steps. Therefore, samples may be pretreated by physical and / or chemical methods, in one embodiment by centrifugation, filtration, mixing, homogenization, chromatographic analysis, precipitation, dilution, concentration, contact with binders and / or detection reagents, and / or any other method deemed appropriate by a technician. That is, one or more internal standards may be added to the sample before, during, and / or after the sample preparation steps. Samples may be doped with internal standards. For example, internal standards may be added to the sample at a predefined concentration. Internal standards may be selected such that they are readily identifiable under normal operating conditions of the mass spectrometry apparatus. The concentration of the internal standard may be predetermined and significantly higher than the concentration of the analyte.

[0036] As described above, and as used herein, the term "analyte" refers to any compound or compound group that should be identified in a sample. In one embodiment, the analyte is a macromolecule, i.e., a compound having a molecular weight greater than 1000 u (i.e., greater than 1 kDa). In a further embodiment, the analyte is a biological macromolecule, particularly a polypeptide, polynucleotide, polysaccharide, or any of the above fragments. In one embodiment, the analyte is a small molecule compound, i.e., a compound having a molecular weight of up to 1000 u (1 kDa). In a further embodiment, the analyte is a compound metabolized by the body of a subject, particularly a human subject, or a compound administered to a subject to induce metabolic changes in the subject. Thus, in one embodiment, the analyte is an abused drug or its metabolites.

[0037] In one embodiment, the analyte is a therapeutic agent, such as valproic acid; clonazepam; methotrexate; voriconazole; mycophenolic acid (total); mycophenolic acid-glucuronide; acetaminophen; salicylic acid; theophylline; digoxin; immunosuppressants, especially cyclosporine, everolimus, sirolimus and / or tacrolimus; analgesics, especially pethidine, norpethidine, tramadol and / or O-desmethyltramadol; antibiotics, especially gentamicin, tobramycin, amikacin, vancomycin-resistant, piperacillin (tazobactam), meropenem and / or linezolid; antiepileptic drugs, especially phenytoin sodium, valproic acid, free phenytoin sodium, free valproic acid, levetiracetam, carbazine, carbazine-10,11-epoxide, phenobarbital, primidone, gabapentin, zonisamide, lamotrigine and / or topiramate. In one embodiment, the analyte is a hormone, particularly cortisol, estradiol, progesterone, testosterone, 17-hydroxyprogesterone, aldosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), dihydrotestosterone, and / or cortisone; in one embodiment, the sample is a serum or plasma sample and the analyte is cortisol, DHEA-S, estradiol, progesterone, testosterone, 17-hydroxyprogesterone, aldosterone, DHEA, dihydrotestosterone, and / or cortisone; in one embodiment, the sample is a saliva sample and the analyte is cortisol, estradiol, progesterone, testosterone, 17-hydroxyprogesterone, androstenedione, and / or cortisone; in one embodiment, the sample is a urine sample and the analyte is cortisol, aldosterone, and / or cortisone. In one embodiment, the analyte is a vitamin, specifically vitamin D, particularly ergocalciferol (vitamin D2) and / or cholecalciferol (vitamin D3) or derivatives thereof, such as 25-hydroxy-vitamin D2, 25-hydroxy-vitamin D3, 24,25-dihydroxy-vitamin D2, 24,25-dihydroxy-vitamin D3, 1,25-dihydroxy-vitamin D2 and / or 1,25-dihydroxy-vitamin D3. In a further embodiment, the analyte is a metabolite of the subject.

[0038] As used herein, in one embodiment, the term "internal standard" refers to an analyte present in a sample at a defined concentration. Thus, in one embodiment, the concentration of the internal standard is known; however, it is also conceivable that the concentration of the standard is unknown, but is identical at least for the target sample and at least one calibration sample; in this case, in one embodiment, the concentration of the internal standard is identical for all analyzed samples. In one embodiment, the internal standard is structurally similar to the analyte; in a further embodiment, it is structurally identical to the analyte. Particularly in the latter case, in one embodiment, the internal standard is an isotopically labeled molecule, particularly an isotopically labeled form of the analyte, such as... 2 H (deuterated) 15 N and / or 13 C-labeled derivatives. Internal standard samples may be samples containing at least one internal standard substance in a predetermined amount. For more details on standard samples, please refer to, for example, EP 3 425 369 A1.

[0039] As used herein, the term "provide" refers to making indicated information or objects available. Thus, in the case of providing a chromatogram, the chromatogram may be provided as data, such as a graphical representation, as a list of measurements, such as value pairs, like elution time / quantitative factor value pairs and / or elution time / qualitative factor value pairs, or as a mathematical model. The chromatogram may be provided by any medium that a person skilled in the art would deem suitable, particularly via an operational connection between the MS apparatus and the evaluation apparatus, via a data connection such as a data network, or via a data storage medium. In one embodiment, provision is determined as specified below.

[0040] As understood by those skilled in the art, the term "determine" means to decide, draw conclusions, or ascertain facts and / or data. Therefore, in one embodiment, determination involves quantitative determination. Furthermore, "determining a chromatogram" involves measuring parameter values ​​of a chromatogram as specified elsewhere herein, specifically recording and optionally storing data points on a suitable storage device that represent the signal of the LC-MS apparatus recorded during an LC-MS run. Similarly, in one embodiment, "determining a ratio" is establishing a value for a ratio as specified elsewhere herein. As those skilled in the art will understand, typically, ratios are calculated from values ​​measured simultaneously during chromatographic analyses, for example, with the same elution time or elution volume.

[0041] In the context of this specification, those skilled in the art will understand the terms "quantitative factor" and "qualitative factor." In one embodiment, these terms refer to the signal of a fragment of an analyte generated in a tandem MS, wherein the fragment that is typically the most abundant and / or most reliably detected is used to quantify the analyte (analyte quantitative factor), while a second fragment is used to confirm the identity of the analyte (analyte qualitative factor). The corresponding terms are often abbreviated as AQN (analyte quantitative factor) and AQL (analyte qualitative factor). In one embodiment, particularly if used to calculate the ratio of quantitative factor to qualitative factor, the value is a single-cycle value, i.e., the intensity determined in one scan cycle, selected ion monitoring (SIM) cycle, or multiple reaction monitoring (MRM) cycle. In a further embodiment, particularly if used to calculate the ratio of quantitative factor to qualitative factor, the value is derived from the single-cycle intensity value through standard mathematical calculations, including, in particular, normalization, standardization, calculation of the average or median of multiple single-cycle values, smoothing of multiple single-cycle value data, etc. Therefore, in one embodiment, each value used as a quantitative or qualitative factor value is derived from a maximum of ten, in one embodiment a maximum of five, in a further embodiment a maximum of two single-cycle intensity values, and in one embodiment a continuous single-cycle intensity value. The values ​​of these two parameters are typically determined as the signal intensity of an ion with a corresponding m / z value and can be correlated with the LC process to generate the chromatogram of this specification; correspondingly, the AQN or AQL value can be determined as a single-cycle value. The statement "determining the ratio of the quantitative factor to the qualitative factor" involves calculating this ratio by dividing the value of the qualitative factor by the value of the quantitative factor. In one embodiment, the AQN / AQL ratio is a compound-specific and measurement method-specific parameter of the analyte. It should be understood that the above method, with necessary modifications, is applicable to the measurement of internal standard quantitative and qualitative factors.

[0042] As used herein, the term "a portion of a data point" refers to a sub-section of a determined data point. As will be understood from the disclosure herein, the recorded and / or selected portion of a data point may depend on the parameters to be identified or verified. Thus, in cases where a presumed (upper or lower) peak boundary should be identified or verified, in one embodiment, a portion of a data point includes data points upstream and / or downstream of the presumed peak boundary. Thus, in one embodiment, a portion of a data point may include 50, in a further embodiment 25, in a further embodiment ten, in a further embodiment five containing the presumed peak boundary, and in one embodiment symmetrically containing data points of the presumed peak boundary. Accordingly, where peak identity, peak purity, and / or peak area ratio should be identified and / or verified, a portion of the data points, in one embodiment, includes appropriately discrete data points, in one embodiment, regularly discrete data points on the peak width, such as data points 1-3, 7-9, 13-15, etc., or uniformly discrete data points on the peak width, such as every second or every third data point; in this case, in one embodiment, data points corresponding to at least 1 / 4, in a further embodiment at least 1 / 3 of the peak width, and in a further embodiment at least half of the data points corresponding to the peak width are analyzed; in a further embodiment, neighboring data points are analyzed; in this case, in one embodiment, at least 50%, in a further embodiment at least 75%, in a further embodiment at least 90%, in a further embodiment at least 95%, and in a further embodiment at least 99% of the data points corresponding to the peak width are analyzed; in a further embodiment, all data points of the peak are analyzed.

[0043] As used herein, the term "benchmark" refers to a discriminator that allows identification of relevant parameters and / or their validation. Such a discriminator may be a target ratio value, such as the AQN / AQL ratio indicating substantially pure analytes. In one embodiment, a benchmark is a quantitative indicator, in further embodiments a value (e.g., a threshold), a range (e.g., a range of values), a score, or any other value or range deemed appropriate by a technician. In one embodiment, depending on the type of benchmark selected, a benchmark is an analyte-specific, apparatus-specific, and / or method-specific parameter, in further embodiments an analyte-specific parameter. Methods for establishing appropriate benchmarks are provided elsewhere herein based on examples. In one embodiment, in the case of analyzing the quantitative factor / qualitative factor ratio of an analyte, the benchmark is a value of the quantitative factor / qualitative factor ratio determined for substantially pure analytes; correspondingly, in one embodiment, in the case of analyzing the quantitative factor / qualitative factor ratio of an internal standard, the benchmark is a value of the quantitative factor / qualitative factor ratio determined for substantially pure internal standards. In a further embodiment, when analyzing the quantitative factor / qualitative factor ratio of an analyte, the benchmark is a range derived from the quantitative factor / qualitative factor ratio determined for a substantially pure analyte, for example, ±100% for a substantially pure analyte, ±50% in one embodiment, and ±30% in a further embodiment. Correspondingly, in one embodiment, when analyzing the quantitative factor / qualitative factor ratio of an internal standard, the benchmark is a range derived from the quantitative factor / qualitative factor ratio determined for a substantially pure internal standard, for example, ±100% for a substantially pure internal standard, ±50% in one embodiment, ±25% in a further embodiment, and ±10% in a further embodiment. The above method can be applied to other ratios, such as the ratio of the analyte quantitative factor to the internal standard quantitative factor, with necessary modifications.

[0044] In one embodiment, identifying and / or verifying at least one analyte peak includes identifying and / or verifying at least one presumed peak boundary of the analyte peak. In this case, in one embodiment, analysis is particularly performed on data points containing the presumed peak boundary. In a further embodiment, it may be sufficient to analyze only data points close to the presumed peak boundary and within the analyte peak. In one embodiment, an analyte peak is identified and / or verified if the determined ratio meets a predefined acceptance criterion based on a benchmark, particularly within a predefined benchmark range; in a further embodiment, the ratio lies within a predefined benchmark range for a predefined portion of the data points, for example, 3 out of at least 5 data points. In one embodiment, a peak boundary is identified and / or verified if the average of the ratios over a plurality of n data points meets a predefined acceptance criterion; in one embodiment, n is at least two data points, in a further embodiment at least three data points, in a further embodiment at least four data points, in a further embodiment at least five data points, in a further embodiment at least seven data points, and in a further embodiment at least ten data points. As used herein, the expression “average” of a value refers to any parameter that indicates a reasonable average value, particularly the median or average.

[0045] In one embodiment, identifying and / or verifying at least one analyte peak includes identifying and / or verifying peak identity and / or peak purity. In this case, in one embodiment, a portion of the data points for which the ratio is determined in step b) includes data points between an assumed lower peak boundary and an assumed upper peak boundary. In one embodiment, peak identity and / or peak purity are verified if the determined ratio conforms to predefined acceptance criteria based on a benchmark, particularly within a predefined benchmark range. In a further embodiment, peak identity is verified if the average of the ratio over a plurality of n data points is within a predefined benchmark range, where n is at least two data points in one embodiment, at least three data points in a further embodiment, at least four data points in a further embodiment, at least five data points in a further embodiment, at least seven data points in a further embodiment, and at least ten data points in a further embodiment. In a further embodiment, peak purity is verified when the ratio is within a predefined baseline range for a predefined portion of the data points, wherein the predefined portion of the data points in one embodiment is at least 80% of the analyzed data points located between the presumed lower peak boundary and / or the presumed upper peak boundary; in another embodiment, at least 90% of the analyzed data points located between the presumed lower peak boundary and / or the presumed upper peak boundary; in another embodiment, at least 95% of the analyzed data points located between the presumed lower peak boundary and / or the presumed upper peak boundary; in another embodiment, at least 80% of the data points located between the presumed lower peak boundary and / or the presumed upper peak boundary; in another embodiment, at least 90% of the data points located between the presumed lower peak boundary and / or the presumed upper peak boundary; and in yet another embodiment, at least 95% of the data points located between the presumed lower peak boundary and / or the presumed upper peak boundary.

[0046] In one embodiment, identifying and / or verifying at least one analyte peak includes identifying and / or verifying an estimated peak area ratio. In one embodiment, the method further includes determining the estimated peak area ratio based on the estimated peak boundary identified in step a1), and further includes determining the ratio of the analyte quantification factor to the internal standard quantification factor, or the ratio of the analyte qualitative factor to the internal standard qualitative factor. In this case, in one embodiment, a portion of the data points for which the ratio is determined in step b) includes data points between the estimated lower peak boundary and the estimated upper peak boundary, as specified above in one embodiment. Further, in this case, the estimated peak area ratio is verified if the average of the ratios calculated on at least 10% of the data points between the estimated lower peak boundary and / or the estimated upper peak boundary, in one embodiment on at least 50% of the data points between the estimated lower peak boundary and / or the estimated upper peak boundary, and in another embodiment on at least 90% of the data points between the estimated lower peak boundary and / or the estimated upper peak boundary, lies within the range defined by the peak area ratio ± X, where X is 100% of the peak area ratio, 50% in one embodiment, and 30% in a further embodiment.

[0047] In one embodiment, the method is a method for verifying at least one putative peak boundary, peak purity, and / or putative peak area ratio of an analyte peak. In one embodiment, the method includes determining the ratio of the analyte quantification factor to the internal standard quantification factor, the ratio of the analyte qualitative factor to the internal standard qualitative factor, the ratio of the analyte quantification factor to the analyte qualitative factor, the ratio of the internal standard quantification factor to the internal standard qualitative factor, the ratio of the analyte quantification factor to the internal standard qualitative factor, and the ratio of the internal standard quantification factor to the analyte qualitative factor, and further includes determining the distribution of said ratios. As used herein, the term "distribution" refers to any parameter indicating the statistical dispersion of values ​​that a person skilled in the art would consider appropriate. In one embodiment, the distribution is a representation of the frequency of a particular ratio value, for example, it may be graphically represented as a graph of the ratio value and its frequency. In one embodiment, the distribution value is skewness or kurtosis. In one embodiment, the benchmark is a value or range of said distribution predetermined with respect to samples containing at least partially pure analytes, in one embodiment, samples where the analyte is determined to be separated from potential interfering substances, and in a further embodiment, samples containing substantially pure analytes. In a further embodiment, the sample is one whose average peak resolution with respect to recent interference is determined to be at least about 0.7, in one embodiment at least about 0.8, and in a further embodiment at least about 0.9. In a further embodiment, if the distribution value corresponds to a reference value or reference range, at least one putative peak boundary, peak purity, and / or putative peak area ratio of the analyte peak is verified. In one embodiment, the reference range is a distribution value determined for at least partially pure analytes ±100%, in one embodiment ±75%, and in a further embodiment ±50%.

[0048] As those skilled in the art will understand from this specification, methods for identifying and / or verifying at least one analyte peak may include identifying and / or verifying at least one presumed peak boundary, peak identity, peak purity, and / or peak area ratio of the analyte peak; therefore, the method may include one or more of the above-described identifications and / or verifications; furthermore, the method may include one or more identifications combined with one or more verifications, for example, the identification of at least one peak boundary may be combined with the verification of the peak area ratio. It will also be understood that, for example, analyzing data points located within the presumed peak boundary may be used to identify and / or verify the lower and upper presumed peak boundaries, peak identity, peak purity, and peak area ratio of the analyte peak. In one embodiment, such analysis is performed in a hierarchical manner, for example by first identifying and / or verifying at least one putative peak boundary, and only proceeding to identify and / or verify peak identity, peak purity, and / or peak area ratio if at least one putative peak boundary can be identified / verified; and / or first identifying and / or verifying at least one putative peak boundary, peak identity, and / or peak purity of the analyte peak, and only proceeding to identify and / or verify peak area ratio if at least one putative peak boundary, peak identity, and / or peak purity can be identified / verified.

[0049] Advantageously, in the foundational work of this invention, it was discovered that single-cycle mass transition parameters can be used to identify and / or validate key parameters for LC-MS analysis, particularly peak boundaries, peak identity, peak purity, and peak area ratio. Advantageously, the data points used for this purpose can be retrieved from the raw LC-MS data of the initial analytical run, and no additional tests or reagents are required.

[0050] The definitions given above, with necessary modifications to the details, apply to the following. Further additional definitions and explanations, with necessary modifications to the details, also apply to all embodiments described in this specification.

[0051] This invention also relates to a quality control method for liquid chromatography-mass spectrometry (LC-MS) measurement of analytes in samples, the method comprising the following steps:

[0052] A) Use a liquid chromatography-mass spectrometry apparatus to measure the analyte in the sample and determine at least one chromatogram;

[0053] B) Performing steps b) to d) of the method of the present invention for identifying and / or verifying at least one analyte peak, and

[0054] C) Evaluate the quality of the LC-MS measurement based on the results of step B).

[0055] The quality control method is an in vitro method. Furthermore, it may include steps other than those explicitly mentioned above. For example, further steps may involve, for instance, providing a sample for step a), or further calculations in steps b) and / or c). Additionally, if at least one of the estimated peak boundaries, peak identity, peak purity, and peak area ratio is not verified in step B), the method may include further steps that are one or more of the following: i) marking the result of step A) as unreliable; ii) not outputting the result of step A), and (iii) initiating a rerun of the sample, optionally using a different LC-MS protocol. This method may be assisted or performed by automated equipment, such as the evaluation unit described below.

[0056] As used herein, the term "quality control" is well known to those skilled in the art. In one embodiment, quality control is the process of ensuring that a process performed by an entity and / or a product manufactured conforms to predefined quality standards. In a further embodiment, quality control in sample measurements, particularly in the measurement of medical samples such as patient samples, for example in clinical diagnostics and / or clinical chemistry, includes ensuring that analytical results obtained using a particular measurement method correspond to results obtainable using a gold standard method, and thus, in one embodiment, to results theoretically obtainable within a pre-specified range. Therefore, in one embodiment, the method for quality control includes, as a further step, evaluating the quality of the LC-MS measurement based on the results of step B), and optionally taking appropriate measures, as specified above in one embodiment.

[0057] Those skilled in the art will understand the term "measurement of an analyte in a sample." In one embodiment, the term relates to a qualitative, semi-quantitative, or quantitative determination of the amount of an analyte in a sample, and in another embodiment, it relates to a quantitative determination of the amount of an analyte in a sample. As used herein, the term "amount" of an analyte relates to any quantitative measure of the analyte and is equivalent to other corresponding measures, such as mass fraction and concentration, which can be calculated from the amount given the mass or volume of the sample. Therefore, the measurement result of an analyte in a sample can be expressed in any unit that a person skilled in the art would consider appropriate, including arbitrary units, measurements of weight, mass fraction, concentration, etc., or measurements derived therefrom, such as those in predefined SI units.

[0058] Methods for determining the amount of an analyte using LC-MS are known in principle to those skilled in the art. In one embodiment, the method includes quantitatively determining the amount of an analyte in a sample by determining the peak area of ​​the analyte (analyte peak area); in a further embodiment, the peak area of ​​an internal standard (IS peak area) is further determined and the ratio of the analyte peak area to the IS peak area is determined; and in one embodiment, the ratio is compared with a calibration function to determine a concentration value. The corresponding methods are known to those skilled in the art.

[0059] The present invention also relates to a system for determining the concentration of at least one analyte in a sample, comprising:

[0060] (I) At least one liquid chromatography-mass spectrometry (LC-MS) apparatus, wherein the LC-MS apparatus is configured to measure analytes in a sample and to acquire data points over time, in one embodiment, for performing step a) of the method of the present invention for identifying and / or verifying at least one analyte peak; and

[0061] (II) At least one evaluation device, wherein the evaluation device is configured to perform at least steps b), c) and / or d), in one embodiment, for performing all steps b) to d) of the method of the present invention for identifying and / or verifying at least one analyte peak.

[0062] As used herein, the term "system" refers to different devices operatively interconnected with each other. These devices may be implemented in a single physical unit or may be physically separate units operatively interconnected with each other. Suitable components and their characteristics are described elsewhere below and above in the context of methods for identifying and / or verifying at least one analyte peak. Therefore, the methods of the present invention can be implemented by the system specified herein. Thus, in one embodiment, the device is configured to perform methods for identifying and / or verifying at least one analyte peak as specified elsewhere herein, and / or quality control methods as specified elsewhere. The system may include other devices or units, particularly data collectors, output units, communication interfaces, and / or any other devices or units deemed suitable by a person skilled in the art.

[0063] The LC-MS apparatus and method for determining at least one chromatogram have been described above in the context of the method of the present invention for identifying and / or verifying at least one analyte peak.

[0064] The term "evaluation apparatus" generally refers to any apparatus suitable for performing the method steps described above, in one embodiment by using at least one data processing device, and in further embodiments by using at least one processor and / or at least one application-specific integrated circuit. Thus, as an example, the at least one evaluation apparatus may include at least one data processing unit having software code stored thereon, the software code comprising a plurality of computer commands. The evaluation apparatus may provide one or more hardware elements for performing one or more specified operations, and / or may provide software running thereon to one or more processors for performing one or more method steps.

[0065] As used herein, the term "data collector" refers to any arbitrary storage unit configured for storing data, particularly data points determined by an LC-MS apparatus, chromatograms, determined ratios, identification and / or verification results, and / or recommendations and / or decisions made regarding further processing of the sample. In one embodiment, a data collector includes at least one database configured to receive and / or store at least one chromatogram. In another embodiment, a data collector includes at least one database comprising one or more benchmark values.

[0066] As used herein, the term "output unit" refers to any arbitrary unit configured to transfer information from the system to other entities, which may be other data processing devices and / or users. Thus, an output device may include a user interface, such as a suitably configured display, or it may be a printer. However, an output unit may also be an indicator, such as a light source, indicating, for example, residual detection.

[0067] Those skilled in the art will understand that the term "communication interface" refers to any arbitrary interface configured for information exchange, particularly data exchange. Such data exchange can be achieved through permanent or temporary physical connections, such as coaxial cables, fiber optic cables, optical fiber or twisted-pair cables, 10 BASE-T cables, storage unit connectors, such as USB, FireWire, and similar connectors. Alternatively, it can be achieved through temporary or permanent wireless connections using, for example, radio waves such as Wi-Fi, LTE, LTE Upgrade, or Bluetooth.

[0068] The present invention further discloses and proposes a computer program comprising computer-executable instructions, which, when executed on a computer or computer network, are used to perform the method according to the invention in one or more embodiments appended herein. Specifically, the computer program may be stored on a computer-readable data carrier. Thus, specifically, one, more than one, or even all of the method steps indicated above can be performed by using a computer or computer network, preferably by using a computer program.

[0069] The present invention further discloses and proposes a computer program product having program code tools so that, when the program is executed on a computer or computer network, the method according to the invention is performed in one or more embodiments appended herein. Specifically, the program code tools may be stored on a computer-readable data carrier.

[0070] The present invention further discloses and proposes a data carrier having a data structure stored thereon, which, after being loaded into a computer or computer network, such as after being loaded into the working memory or main memory of the computer or computer network, can perform methods according to one or more embodiments disclosed herein.

[0071] This invention further proposes and discloses a computer program product having program code tools stored on a machine-readable carrier, so that when the program is executed on a computer or computer network, it performs methods according to one or more embodiments disclosed herein. As used herein, a computer program product refers to a program that is a tradable product. This product can generally exist in any format (such as in paper format) or on a computer-readable data carrier. Specifically, the computer program product can be distributed on a data network.

[0072] The present invention further proposes and discloses a modulated data signal comprising instructions readable by a computer system or computer network for performing a method according to one or more embodiments disclosed herein.

[0073] In one embodiment, referring to the computer implementation aspect of the invention, one or more method steps, or even all method steps, of the methods according to one or more embodiments disclosed herein can be performed using a computer or computer network. Therefore, generally speaking, any method steps including providing and / or processing data can be performed using a computer or computer network. Generally, these method steps can include any method steps other than those typically requiring manual operation (such as providing samples and / or performing certain aspects of actual measurements).

[0074] Specifically, the present invention further discloses:

[0075] A computer or computer network including at least one processor, wherein the processor is adapted to perform a method according to one of the embodiments described in this specification.

[0076] A computer-loadable data structure is provided that, when executed on a computer, performs a method according to one of the embodiments described in this specification.

[0077] A computer program, wherein the computer program is adapted, when executed on a computer, to perform a method according to one of the embodiments described in this specification.

[0078] A computer program, including program tools, for performing a method according to one of the embodiments described herein when the computer program is executed on a computer or on a computer network.

[0079] A computer program, comprising program means according to the foregoing embodiments, wherein such program means are stored on a computer-readable storage medium.

[0080] A storage medium, wherein a data structure is stored on the storage medium and wherein the data structure is adapted to perform a method according to one of the embodiments described herein after being loaded into the main memory and / or working memory of a computer or computer network.

[0081] A computer program product having program code tools, wherein the program code tools can be stored or stored on a storage medium for executing a method according to one of the embodiments described in this specification when the program code tools are executed on a computer or computer network.

[0082] The present invention also relates to a method for optimizing the analysis method for analytes on an LC-MS apparatus, the method comprising the following steps:

[0083] a) Provided by acquiring multiple data points of quantitative and qualitative factor signal intensities over time, in one embodiment for determining the chromatogram of the sample;

[0084] b) Determine the estimated lower peak boundary, estimated upper peak boundary, estimated peak maximum value, peak identity and / or peak purity;

[0085] c) Verify the putative lower peak boundary, putative upper peak boundary, putative peak maximum value, peak identity and / or peak purity of step (b) by means of the method of the present invention for identifying and / or verifying at least one analyte peak.

[0086] d) If at least one of the proposed lower peak boundary, proposed upper peak boundary, proposed peak maximum value, peak identity, and / or peak purity is not verified in step (c), then modify the sample pretreatment parameters, LC parameters, and / or MS parameters; and

[0087] e) This optimizes the analytical methods for the analytes.

[0088] The method for optimizing the analytical method is an in vitro method. The method may include steps other than those described above; for example, a further step may involve providing a sample for analysis. In the method for optimizing the analytical method, the sample may include a partially or substantially pure analyte; however, in one embodiment, the sample is a body fluid sample or a sample derived from somatic cells, and in another embodiment, it is a body fluid sample. The method may be assisted by automated equipment. In one embodiment, the method is a fully automated method.

[0089] In principle, the term "optimized analytical method" is understood by those skilled in the art. In one embodiment, the term refers to a process including modifying the parameters of an analytical LC-MS method to allow for substantially interference-free measurement of the target analyte, in one embodiment, in the presence of a matrix component, and in a further embodiment, in the presence of a matrix component expected to be present in the sample. Thus, in one embodiment, method steps a) through e) are repeated until step d), where the estimated lower peak boundary, estimated upper peak boundary, estimated peak maximum, peak identity, and / or peak purity are verified.

[0090] Those skilled in the art will understand that the term "sample pretreatment parameter" refers to any pretreatment parameter known or assumed to have an impact on the results of an analytical LC-MS run of the sample. In one embodiment, this parameter relates to sample pretreatment after receipt by the analytical laboratory. Thus, in one embodiment, a sample pretreatment parameter is a parameter of sample processing, such as temperature; a parameter of physical sample pretreatment, such as centrifugation, filtration, heating and / or cooling; or a parameter of chemical sample pretreatment, such as sample precipitation and / or extraction and the solvents used for this purpose, derivatization of sample components, etc.

[0091] Those skilled in the art will understand that the term "LC parameter" refers to any LC parameter known or assumed to have an impact on the results of an analytical LC-MS run of a sample. In one embodiment, the LC parameter is selected from sample dilution, sample volume, separation principle and LC column selection, selection of one or more solvents, gradient configuration, column temperature, etc.

[0092] Those skilled in the art will understand that the term "MS parameter" refers to any MS parameter known or assumed to have an impact on the results of an analytical LC-MS run of a sample. In one embodiment, the MS parameter is selected from ionization method, detector type, number of fragments, etc.

[0093] As used herein, the term "parameter modification" refers to any change to a parameter that is known or assumed to have an impact on the results of an LC-MS analysis run. Therefore, the modification is typically to the extent that the expected results would change. In one embodiment, the modified parameter is an LC parameter. In one embodiment, the parameter is modified by an operator of the LC-MS device; in further embodiments, the parameter is modified automatically, particularly according to an optimization algorithm. Suitable algorithms are known in the art. Thus, in one embodiment, the method for optimizing the analysis approach is implemented on a system as specified above; as those skilled in the art will understand, in this case, the system may include multiple LC-MS devices operatively connected to at least one designated evaluation device.

[0094] The present invention also relates to a method for purifying a target compound from a preparative sample on an LC apparatus, the method comprising:

[0095] a) The components of the prepared sample are separated into fractions using the LC device, and one or more fractions are initially allocated for pooling;

[0096] b) Record the MS chromatogram of at least one fraction of the fraction initially allocated for pooling in step a);

[0097] c) Verify the allocation of at least one fraction for pooling in step b) by means of a method for identifying and / or verifying at least one analyte peak;

[0098] d) Including the sample with validated dispensing in a pooling process containing the fraction of the compound; and

[0099] e) The target compound is thus purified.

[0100] The method used to purify the compound is an in vitro method, and in one embodiment, a preparative method. Furthermore, the method may include further steps beyond those described above. For example, further steps may involve sample pretreatment and / or sample prepurification. Thus, in one embodiment, the sample is a prepurified sample. Additionally, LC-separated chromatograms, such as UV / VIS chromatograms, may be recorded. In one embodiment, this method is the final LC step in the preparative purification of the compound.

[0101] As used herein, the term "target compound" includes any chemical molecule that a person skilled in the art would find interesting to purify or further purify. In one embodiment, the target compound is a compound derived from one of the groups indicated above as an analyte, or one of the compounds indicated above as an analyte. Thus, in one embodiment, the target compound is a low molecular weight compound having a molecular weight of at most 1 kDa. In a further embodiment, the target compound is a macromolecule, in one embodiment a biomacromolecule, in another embodiment having a molecular weight greater than 1 kDa. In one embodiment, the target compound is a peptide, in a further embodiment a therapeutic peptide.

[0102] As used herein, the term "preparative sample" refers to a sample containing an amount of target compound sufficient for preparative purification. As those skilled in the art will understand, the amount sufficient for preparative purification depends largely on the type of target compound contained; in one embodiment, the preparative sample contains at least 1 mg, in another embodiment at least 10 mg, in a further embodiment at least 100 mg, in a further embodiment at least 1 g, in a further embodiment at least 10 g, in a further embodiment at least 100 g, and in a further embodiment at least 1 kg of target compound. As mentioned above, in one embodiment, the preparative sample is a pre-purified sample; therefore, in one embodiment, the target compound is contained in the preparative sample with a purity of at least 10%, in a further embodiment at least 25%, in a further embodiment at least 50%, in a further embodiment at least 75%, in a further embodiment at least 85%, in a further embodiment at least 90%, in a further embodiment at least 95%, and in a further embodiment at least 99%. As those skilled in the art will understand, the volume of a preparative sample depends largely on the type of target compound, its concentration, and the LC to be performed; therefore, the volume of a preparative sample can range from a few microliters to several thousand liters. Typically, preparative samples do not contain an internal standard.

[0103] Those skilled in the art will understand that the term "fraction" refers to a sub-part of a material composition, in one embodiment, a sub-part of the eluent from LC separation as described herein, produced by applying a separation step to a preparative sample. In one embodiment, at least one fraction is known or suspected to contain the target compound. Particularly when a smaller number of fractions are obtained, particularly three or two fractions, which need not have the same volume. In one embodiment, a fraction as specified herein is a virtual fraction, i.e., produced by formally dividing the eluent from the separation step into sub-parts, which may have the same or different volumes. In a further embodiment, the fraction is a physical fraction, which may have the same or different volumes, contained in separate containers. In one embodiment, at least a portion of the fraction is a fraction known or suspected to contain the target compound, for example from prior instances of performing the method.

[0104] As used herein, the term “amplified sample of a fraction” refers to a sub-part of a fraction that is typically a smaller sub-part compared to the volume of the fraction, such as one-tenth, one-hundredth, one-thousandth, or even smaller than the volume of the fraction.

[0105] Those skilled in the art will understand that the term "at least one fraction" refers to at least one fraction of all fractions provided. In one embodiment, at least one fraction is at least two, in one embodiment at least five, and in a further embodiment at least ten. In a further embodiment, at least one fraction involves at least 1%, in one embodiment at least 5%, in a further embodiment at least 10%, in a further embodiment at least 25%, and in a further embodiment at least 50% of all fractions provided. In one embodiment, at least a portion of the fractions includes all fractions provided. As described above, in one embodiment, at least a portion of the fractions are known or suspected to contain the target compound, for example from prior instances of performing the method; furthermore, it is known that purity is only critical to a portion of the fractions, for example, near the start and / or end of the product peak in the LC chromatogram. Therefore, in a method for purifying a target compound, it is sufficient to record MS chromatograms only for the fractions for which the purity of the target compound needs to be determined, i.e., the fractions that need to be validated for pooling, as described below. Therefore, in one embodiment, the method for purifying the target compound may include identifying and / or verifying at least one peak boundary of the target compound peak as specified above, and / or identifying and / or verifying the peak identity and / or peak purity of the target compound peak as specified above.

[0106] As used herein, the term "allocation of sample for pooling" refers to allocating fractions to become part of a pool of target compounds for future purification or further purification. In one embodiment, pooling includes the act of physically combining at least two separate fractions to form a pool; however, given the foregoing, the term also includes redirecting a sub-portion of the eluent from an LC run that may contain several dummy fractions to the pool. In one embodiment, the number of fractions to be pooled may also be one. As used herein, the term "preliminary allocation" refers to a non-final allocation of fractions; such preliminary allocations may be based on an earlier instance of the same LC run, analysis of the UV / VIS chromatogram of the LC run, and / or any other method deemed appropriate by a technician. After preliminary allocation, the fraction needs to be confirmed for inclusion in the pool; in particular, such fractions may be fractions close to the expected start or end point of the peak of the target compound. In one embodiment, fractions may exist that do not require validation of the allocation, for example if such fractions are known to be insignificant in terms of interference; such samples may ultimately be allocated for pooling without validation. In this case, the resulting pool typically includes both the final allocated fraction and the validated allocated fraction. As those skilled in the art will understand, pooling can also create more than one target compound pool; for example, a high-purity pool and a second lower-purity pool can be generated, the second pool being used, for example, to repeat the same LC run or for another different separation method. According to the foregoing, the term "validated allocation" refers to a non-initial allocation of the fraction; in one embodiment, the fraction is incorporated into the target compound pool after a validated allocation.

[0107] In summary, the following specific implementation is envisioned:

[0108] Example 1. A method for identifying and / or verifying at least one analyte peak in a chromatogram of a sample for an analyte from a liquid chromatography-mass spectrometry device, the method comprising the following steps:

[0109] a) A chromatogram of the sample is provided by acquiring multiple data points of the quantitative factor signal intensity and / or the qualitative factor signal intensity over time; and, in the case where the sample contains an internal standard, optionally, over time, in one embodiment, multiple data points of the internal standard quantitative factor signal intensity and / or the internal standard qualitative factor signal intensity are acquired.

[0110] b) For at least a portion of the data points collected in step a), determine the ratio type, which is selected from a list of the following: (i) the ratio of analyte quantification factor to analyte qualitative factor, (ii) the ratio of internal standard quantification factor to internal standard qualitative factor, (iii) the ratio of analyte quantification factor to internal standard quantification factor, and (iv) the ratio of analyte qualitative factor to internal standard qualitative factor;

[0111] c) Compare the ratio determined in step b) with the benchmark; and

[0112] d) Based on the comparison step c) Identify and / or verify at least one analyte peak in the chromatogram.

[0113] Example 2. The method according to Example 1, wherein the sample contains an internal standard and wherein the method includes the following steps:

[0114] a) Provide a chromatogram of the sample by acquiring multiple data points of the quantitative factor signal intensity and / or the qualitative factor signal intensity over time; and, optionally, over time, in one embodiment, provide multiple data points of the internal standard quantitative factor signal intensity and / or the internal standard qualitative factor signal intensity.

[0115] b) For at least a portion of the data points collected in step a), determine the ratio type, which is selected from a list of the following: (i) the ratio of analyte quantification factor to analyte qualitative factor, (ii) the ratio of internal standard quantification factor to internal standard qualitative factor, (iii) the ratio of analyte quantification factor to internal standard quantification factor, and (iv) the ratio of analyte qualitative factor to internal standard qualitative factor;

[0116] c) Compare the ratio determined in step b) with the benchmark; and

[0117] d) Based on the comparison step c) Identify and / or verify at least one analyte peak in the chromatogram;

[0118] or

[0119] The sample does not contain an internal standard and the method includes the following steps:

[0120] a) Provide a chromatogram of the sample by acquiring multiple data points of the signal intensity of quantitative and qualitative factors over time;

[0121] b) Determine the ratio of the analyte quantification factor to the analyte qualitative factor for at least a portion of the data points collected in step a);

[0122] c) Compare the ratio determined in step b) with the benchmark; and

[0123] d) Based on the comparison step c) Identify and / or verify at least one analyte peak in the chromatogram.

[0124] Example 3. The method according to Example 1 or 2, wherein providing a chromatogram is determining the chromatogram.

[0125] Example 4. The method according to any one of Examples 1 to 3, wherein the method further comprises a further step a1), the further step a1) determining the presumed lower peak boundary and / or presumed upper peak boundary corresponding to the analyte, and optionally determining the presumed lower peak boundary and / or presumed upper peak boundary corresponding to the internal standard.

[0126] Example 5. The method according to any one of Examples 1 to 4, wherein the presumed lower peak boundary and / or the presumed upper peak boundary are predefined presumed peak boundaries, and / or wherein the presumed lower peak boundary and / or the presumed upper peak boundary are determined based on the chromatogram, in one embodiment by performing automated peak identification.

[0127] Example 6. The method according to any one of Examples 1 to 5, wherein the benchmark is a predetermined benchmark value, benchmark range or benchmark score, and in one embodiment a predefined benchmark range.

[0128] Example 7. The method according to any one of Examples 1 to 6, wherein the benchmark is a predetermined ratio for purified analytes and / or purified internal markers in one embodiment, or calculated based on such ratio.

[0129] Example 8. The method according to Example 7, wherein the baseline is the predetermined ratio ± X, in one embodiment it is a predetermined ratio ± X for the purified analyte, X is 100% of the predetermined ratio, in one embodiment it is 50%, and in a further embodiment it is 30%.

[0130] Example 9. The method according to any one of Examples 1 to 8, wherein the identification and / or verification of at least one analyte peak includes identifying and / or verifying at least one presumed peak boundary of the analyte peak.

[0131] Example 10. The method according to Example 9, wherein the portion of the data points for which the ratio is determined in step b) includes data points downstream and / or upstream of the peak boundary to be identified or verified.

[0132] Example 11. The method according to Example 9 or 10, wherein the peak boundary is identified and / or verified when the ratio is within a predefined reference range.

[0133] Example 12. The method according to any one of Examples 9 to 11, wherein the peak boundary is identified and / or verified when the ratio is within a predefined reference range for a predefined portion of the data points, in one example when at least 3 out of 5 consecutive data points.

[0134] Example 13. The method according to any one of Examples 9 to 12, wherein the peak boundary is identified and / or verified when the average of the ratios over a plurality of n data points meets a predefined acceptance criterion, wherein in one embodiment n is at least two data points, in a further embodiment at least three data points, in a further embodiment at least four data points, in a further embodiment at least five data points, in a further embodiment at least seven data points, and in a further embodiment at least ten data points.

[0135] Example 14. The method according to any one of Examples 1 to 13, wherein the identification and / or verification of at least one analyte peak includes identifying and / or verifying peak identity and / or peak purity.

[0136] Example 15. The method according to any one of Examples 1 to 14, wherein the portion of the data points for which the ratio is determined in step b) includes data points between the estimated lower peak boundary and the estimated upper peak boundary.

[0137] Example 16. The method according to any one of Examples 13 to 15, wherein peak identity and / or peak purity are verified when the ratio is within a predefined reference range.

[0138] Example 17. Verify peak identity when the ratio is within a predefined reference range for a predefined portion of the data points, according to any one of Examples 13 to 16.

[0139] Example 18. The method according to any one of Examples 13 to 17, wherein peak identity is verified when the average of the ratios over a plurality of n data points is within a predefined baseline range, wherein in one embodiment n is at least two data points, in a further embodiment at least three data points, in a further embodiment at least four data points, in a further embodiment at least five data points, in a further embodiment at least seven data points, and in a further embodiment at least ten data points.

[0140] Example 19. The method according to any one of Examples 13 to 16, wherein peak purity is verified when the ratio is within a predefined baseline range for a predefined portion of the data points, wherein the predefined portion of the data points in one embodiment is at least 80% of the analyzed data points located between the presumed lower peak boundary and / or the presumed upper peak boundary, in one embodiment is at least 90% of the analyzed data points located between the presumed lower peak boundary and / or the presumed upper peak boundary, in one embodiment is at least 95% of the analyzed data points located between the presumed lower peak boundary and / or the presumed upper peak boundary, in one embodiment is at least 80% of the data points located between the presumed lower peak boundary and / or the presumed upper peak boundary, in one embodiment is at least 90% of the data points located between the presumed lower peak boundary and / or the presumed upper peak boundary, and in one embodiment is at least 95% of the data points located between the presumed lower peak boundary and / or the presumed upper peak boundary.

[0141] Example 20. The method according to any one of Examples 13 to 16 or 19, wherein peak purity is verified when: the average of the ratios over a plurality of n data points meets a predefined acceptance criterion for at least 80% of the data points between the presumed lower peak boundary and / or the presumed upper peak boundary, in one embodiment at least 90% of the data points between the presumed lower peak boundary and / or the presumed upper peak boundary, in one embodiment at least 95% of the data points between the presumed lower peak boundary and / or the presumed upper peak boundary; in one embodiment n is at least two data points, in a further embodiment at least three data points, in a further embodiment at least four data points, in a further embodiment at least five data points, in a further embodiment at least seven data points, and in a further embodiment at least ten data points.

[0142] Example 21. The method according to any one of Examples 2 to 20, wherein the method further comprises determining the ratio of the estimated analyte peak area to the internal standard peak area (peak area ratio), wherein the method comprises determining the ratio of the analyte quantification factor to the internal standard quantification factor, or the ratio of the analyte qualitative factor to the internal standard qualitative factor, and wherein the identification and / or verification of at least one analyte peak comprises identifying and / or verifying the peak area ratio.

[0143] Example 22. The method according to Example 21, wherein the portion of the data points for which the ratio is determined in step b) includes data points between the estimated lower peak boundary and the estimated upper peak boundary.

[0144] Example 23. The method according to Example 21 or 22, wherein the estimated peak area ratio is verified by: the average of the ratio calculated for at least 10% of the data points between the estimated lower peak boundary and / or the estimated upper peak boundary, in one embodiment at least 50% of the data points between the estimated lower peak boundary and / or the estimated upper peak boundary, in another embodiment at least 90% of the data points between the estimated lower peak boundary and / or the estimated upper peak boundary, falling within the range defined by the peak area ratio ± X, where X is 100% of the peak area ratio, in one embodiment 50%, and in a further embodiment 30%.

[0145] Example 24. The method according to any one of Examples 2 to 23, wherein the method includes identifying and / or verifying at least one presumed peak boundary, peak purity and / or presumed peak area ratio of the analyte peak.

[0146] Example 25. The method according to Example 24, wherein the method includes determining the ratio of the analyte quantification factor to the internal standard quantification factor or the ratio of the analyte qualitative factor to the internal standard qualitative factor, and further includes determining the distribution of the ratio, in one embodiment including determining the skewness and / or peak state of the data points, in one embodiment, wherein the method is a method for verifying at least one putative peak boundary, peak purity and / or putative peak area ratio of the analyte peak.

[0147] Example 26. The method according to any one of Examples 23 to 25, wherein at least one putative peak boundary, peak purity, and / or putative peak area ratio of the analyte peak is identified and / or verified when the distribution of said ratios over a plurality of n data points conforms to predefined acceptance criteria, wherein in one embodiment n is at least two data points, in a further embodiment n is at least three data points, in a further embodiment n is at least four data points, in a further embodiment n is at least five data points, in a further embodiment n is at least seven data points, and in a further embodiment n is at least ten data points.

[0148] Example 27. A quality control method for liquid chromatography-mass spectrometry (LC-MS) measurement of a sample, the method comprising the following steps:

[0149] A) Use a liquid chromatography-mass spectrometry apparatus to measure the analyte in the target sample and determine at least one chromatogram;

[0150] B) Performing steps b) to d) of the method according to any one of Examples 1 to 23, and

[0151] C) Evaluate the quality of the LC-MS measurement based on the results of step B).

[0152] Example 28. The method according to Example 27, wherein if at least one of the estimated peak boundary, peak identity, peak purity and peak area ratio is not verified in step B), then at least one of the following is performed: i) marking the result of step A) as unreliable; ii) not outputting the result of step A), and (iii) initiating a rerun of the sample, optionally using a different LC-MS protocol.

[0153] Example 29. The method according to any one of the foregoing embodiments, wherein method steps b), c) and / or d), in one embodiment, steps b) to d) are executed by a computer.

[0154] Example 30. The method according to any one of the preceding examples, wherein the chromatographic mass spectrometer apparatus includes a tandem mass spectrometer (MS / MS) unit.

[0155] Example 31. A system for determining the concentration of at least one analyte in a sample, the system comprising:

[0156] (I) At least one liquid chromatography-mass spectrometry (LC-MS) apparatus, wherein the LC-MS apparatus is configured to measure analytes in a sample and to acquire data points over time, in one embodiment, for performing step a) of the method according to any one of Examples 1 to 28; and

[0157] (II) At least one evaluation device, wherein the evaluation device is configured to perform at least steps b), c) and / or d) of the method according to any one of Embodiments 1 to 28, wherein in one embodiment, it is configured to perform all steps b) to d).

[0158] Example 32. The apparatus according to the foregoing embodiments, wherein the apparatus is configured to perform the quality control method according to any one of Examples 27 to 30.

[0159] Example 33. A computer or computer network including at least one processor, wherein the processor is adapted to perform at least steps b), c) and / or d) of the method according to any one of Examples 1 to 28, wherein in one embodiment, all steps b) to d) are performed.

[0160] Example 34. A computer-loadable data structure, when executed on a computer, is adapted to perform at least steps b), c), and / or d) of the method according to any one of Examples 1 to 28, wherein in one embodiment, all steps b) to d) are performed.

[0161] Example 35. A computer program, wherein the computer program is adapted to perform at least steps b), c) and / or d) of the method according to any one of Examples 1 to 28 when the program is executed on a computer, wherein in one embodiment, all steps b) to d) are performed.

[0162] Example 36. A computer program comprising program means, when executed on a computer or computer network, performs at least steps b), c), and / or d) of the method according to any one of Examples 1 to 28, wherein in one embodiment, all steps b) to d) are performed.

[0163] Example 37. A computer program comprising program means according to the foregoing embodiments, wherein such program means are stored on a computer-readable storage medium.

[0164] Example 38. A storage medium, wherein a data structure is stored on the storage medium and wherein the data structure is adapted to perform at least steps b), c) and / or d) of the method according to any one of Examples 1 to 28 after being loaded into the main storage device and / or working storage device of a computer or computer network, wherein in one embodiment, all steps b) to d) are performed.

[0165] Example 39. A computer program product having a program code tool, wherein the program code tool can be stored on or on a storage medium, the computer program product being configured to perform at least steps b), c), and / or d) of the method according to any one of Examples 1 to 28 when the program code tool is executed on a computer or on a computer network, wherein in one embodiment, all steps b) to d) are performed.

[0166] Example 40. A method for optimizing an analytical method for an analyte on an LC-MS apparatus, the method comprising the following steps:

[0167] a) Provided by acquiring multiple data points of quantitative and qualitative factor signal intensities over time, in one embodiment for determining the chromatogram of the sample;

[0168] b) Determine the presumed lower peak boundary, the presumed upper peak boundary, peak identity and / or peak purity;

[0169] c) Verify the estimated lower peak boundary, estimated upper peak boundary, peak identity and / or peak purity of step (b) by the method according to any one of Examples 1 to 28;

[0170] d) If at least one of the proposed lower peak boundary, proposed upper peak boundary, peak identity, and / or peak purity is not verified in step (c), then modify the sample pretreatment parameters, LC parameters, and / or MS parameters; and

[0171] e) This optimizes the analytical methods for the analytes.

[0172] Example 41. A method for purifying a target compound from a preparative sample on an LC apparatus, the method comprising:

[0173] a) The components of the prepared sample are separated into fractions using the LC device, and one or more fractions are initially allocated for pooling;

[0174] b) Record the MS chromatogram of at least one fraction of the fraction initially allocated for pooling in step a);

[0175] c) Verify the allocation of at least one fraction for pooling in step b) by means of a method for identifying and / or verifying at least one analyte peak;

[0176] d) Including the sample with validated dispensing in a pooling process containing the fraction of the compound; and

[0177] e) The target compound is thus purified.

[0178] Example 42. The subject matter of any of the foregoing examples, wherein the values ​​of the quantitative factor signal strength, the qualitative factor signal strength, the internal standard quantitative factor signal strength and / or the internal standard qualitative factor signal strength are single-cycle signal strengths or values ​​derived therefrom by standard mathematical operations, in one example being single-cycle signal strengths.

[0179] All disclosures in all references cited in this specification, and all disclosures specifically mentioned herein, are incorporated herein by reference.

[0180] Attached illustrations

[0181] Figure 1 shows the chromatograms of clonazepam (A) and EDDP (B) and their quantitative / qualitative factor ratios.

[0182] Figure 2 shows the chromatogram of benzoyl sphagine and its quantitative / qualitative factor ratio blot.

[0183] Figure 3 shows the skewed imprint of the analyte quantification factor of testosterone versus the internal standard single-cycle ratio at different peak resolutions of testosterone under isotope interference.

[0184] The following examples are merely illustrative of the invention. In any case, they should not be construed as limiting the scope of the invention.

[0185] Example 1: LC-MS measurement of serum samples doped with mixtures containing benzoylmacin, clonazepam, and EDDP, and their ISTDs (internal standards) benzoylmacin-13C6, clonazepam-d4, and EDDP-d3. Individual data points were read from the raw MS data, and retention times of transitions were aligned for each analyte / ISTD combination. Table 1 below summarizes the calculations of peak integrals and single-cycle ratios, their deviations from their respective target values, and the acceptable ranges.

[0186] Table 1: Peak boundaries, quantitative / qualitative factor ratios, and analyte / internal standard (ISTD) ratios calculated by peak integral and single-cycle ratio, and their deviations from target values; bold indicates unvalidated results.

[0187] Target value Peak integral By single cycle ratio Δ is the deviation from the target value or the verification status. Benzoyl spore alkaloid Peak boundary start point 100±30% 8,078 ms 0 / 5 Unverified Peak boundary endpoint 100±30% 12,590ms 4 / 5 Verified Quantitative factor / qualitative factor ratio 4.03±10% 4.12 4.05 2.2%0.5% Analyte / ISTD ratio 42.53±10% 46.62 42.27 12.0%-0.6% Clonazepam Peak boundary start point 100±30% 11,956 ms 5 / 5 Verified Peak boundary endpoint 100±30% 18,557 ms 4 / 5 Verified Quantitative factor / qualitative factor ratio 1.15±10% 1.16 1.14 0.9%-0.9% Analyte / ISTD ratio 4.30±10% 4.03 4.63 -6.3%7.7% EDDP Peak boundary start point 100±30% 43,664ms 4 / 5 Verified Peak boundary endpoint 100±30% 56,774ms 4 / 5 Verified Quantitative factor / qualitative factor ratio 4.64±10% 4.75 4.54 2.4%-2.2% Analyte / ISTD ratio 14.08±10% 14.14 14.28 0.4%1.4%

[0188] Example 2: Calculate the quantitative factor / qualitative factor single-cycle ratio of Example 1 relative to their target values. Figure 1 Figure 1 shows the chromatograms of clonazepam and EDDP and their quantitative / qualitative factor ratios. Figure 2 shows the chromatogram of benzoylmacin and its quantitative / qualitative factor ratio. To validate peak boundaries, peak boundaries were detected at 8,078 and 12,590 ms for benzoylmacin, 11,956 and 18,557 ms for clonazepam, and 43,664 and 56,774 ms for EDDP. The ratios of five consecutive single cycles around their detected peak boundaries were monitored. The number of smoothed data points within an acceptable range of ±30% error of the target value was counted. Validation was achieved if at least three of the five data points were within the acceptable range. Except for the significant difference in the peak initiation boundary of benzoylmacin, the peak boundaries determined by peak integrals were all validated.

[0189] Example 3: The significant deviation of the peak initiation boundary of benzoyl sphagnum in Example 2, along with the sharp change in the single-cycle ratio of the quantitative to qualitative factors in that measurement, led to the hypothesis that there is potential interference from co-eluting compounds near the peak initiation boundary. Such co-eluting interference cannot be detected by any peak shape parameters because it does not significantly affect the peak profile. However, the median of the single-cycle ratio within the peak boundary, calculated separately, can be used to verify interference near the peak initiation point by comparing it with the corresponding target value.

[0190] Example 4: Determine the analyte / ISTD single-cycle ratio and calculate its median within the peak boundaries. Comparison with the peak area ratio determined by peak integration verifies the amount determined for all compounds except benzoylmacin. This result again leads to the assumption that there is interfering substance below the benzoylmacin peak, thus consistent with the assumption described in Example 3. This interference directly affects the final result, i.e., the measured concentration of benzoylmacin, and cannot be monitored by traditional parameters such as the quantitative / qualitative peak area ratio, thus leading to the risk of ignoring the interference.

[0191] Example 5: Measurement of serum samples containing mixtures of testosterone and epitestosterone, as well as ISTD (internal standard) testosterone-13C3, in six different LC protocols and six analytical replicates by LC-MS. Individual data points were read from the raw MS data, and retention times of transitions were aligned for the analyte / ISTD combination.

[0192] Example 6: Calculate and monitor the skewness of the analyte / ISTD single-cycle ratio in Example 5 to verify peak identity and / or purity of the samples. Figure 3 shows the skewness imprint of the analyte / ISTD single-cycle ratio of testosterone at different peak resolutions in the isotope interference table, along with the respective target values ​​and acceptable ranges. Validation is achieved when the skewness value is within the acceptable range of ±100% error of the target value. Peak identity and / or purity can be verified for samples measured in LC protocols one, two, and three. For samples measured in LC protocols four, five, and six, the skewness values ​​exceed the acceptable range, therefore peak identity and / or purity in these samples cannot be verified.

[0193] Example 7: The samples in Example 6 that were not validated by deviation from the analyte / ISTD single-cycle ratio skewness in their distribution led to the hypothesis that a co-eluting compound might be interfering. Furthermore, the average peak resolution between testosterone and epitestosterone (as interference) for each LC protocol and sample was estimated. Peak identity and / or purity could be validated by the corresponding skewness in all samples with an average peak resolution equal to or greater than 0.9, while peak identity and / or purity could not be validated in samples with values ​​below 0.9. These observations confirm the hypothesis that a co-eluting interference exists in these samples, in this example, epitestosterone.

[0194] literature:

[0195] EP 3 425 369 A1

Claims

1. A method for identifying and / or verifying at least one analyte peak in a chromatogram of a sample for an analyte from a liquid chromatography-mass spectrometry apparatus, the method comprising the steps of: a) The chromatogram of the sample is determined by acquiring multiple data points of the quantitative factor signal intensity and / or the qualitative factor signal intensity over time; and, if the sample contains an internal standard, multiple data points of the internal standard quantitative factor signal intensity and / or the internal standard qualitative factor signal intensity are acquired over time. b) Determine the ratio type for at least a portion of the data points collected in step a), the ratio type being selected from a list of the following: (i) the ratio of analyte quantification factor to analyte qualitative factor, (ii) the ratio of internal standard quantification factor to internal standard qualitative factor, (iii) the ratio of analyte quantification factor to internal standard quantification factor, and (iv) the ratio of analyte qualitative factor to internal standard qualitative factor; c) Compare the ratio determined in step b) with the benchmark; as well as d) Based on the comparison step c) Identify and / or verify at least one analyte peak in the chromatogram.

2. The method of claim 1, wherein the sample comprises an internal standard and wherein the method comprises the following steps: a) Provide a chromatogram of the sample by acquiring multiple data points of quantitative factor signal intensity and / or qualitative factor signal intensity over time; Furthermore, multiple data points of the internal standard quantitative factor signal intensity and / or internal standard qualitative factor signal intensity are collected over time; b) Determine the ratio type for at least a portion of the data points collected in step a), the ratio type being selected from a list of the following: (i) the ratio of analyte quantification factor to analyte qualitative factor, (ii) the ratio of internal standard quantification factor to internal standard qualitative factor, (iii) the ratio of analyte quantification factor to internal standard quantification factor, and (iv) the ratio of analyte qualitative factor to internal standard qualitative factor; c) Compare the ratio determined in step b) with the benchmark; as well as d) Based on the comparison step c) Identify and / or verify at least one analyte peak in the chromatogram; or The sample does not contain an internal standard and the method includes the following steps: a) Provide a chromatogram of the sample by acquiring multiple data points of the signal intensity of quantitative and qualitative factors over time; b) Determine the ratio of the analyte quantification factor to the analyte qualitative factor for at least a portion of the data points collected in step a); c) Compare the ratio determined in step b) with the benchmark; and d) Based on the comparison step c) Identify and / or verify at least one analyte peak in the chromatogram.

3. The method according to claim 1 or 2, wherein the method includes the further step a1) determining the estimated lower peak boundary and / or estimated upper peak boundary corresponding to the analyte, and determining the estimated lower peak boundary and / or estimated upper peak boundary corresponding to the internal standard.

4. The method according to claim 1 or 2, wherein the benchmark is a predetermined ratio for the analyte and / or internal markers or is calculated based on the ratio.

5. The method according to claim 1 or 2, wherein the benchmark is a predetermined ratio for the purified analyte and / or the purified internal marker, or is calculated based on the ratio.

6. The method according to claim 1 or 2, wherein identifying and / or verifying at least one analyte peak includes identifying and / or verifying at least one presumed peak boundary of the analyte peak.

7. The method of claim 6, wherein the portion of the data points for which the ratio is determined in step b) includes data points downstream and / or upstream of the presumed peak boundary to be identified or verified, and / or wherein the presumed peak boundary is identified and / or verified if the ratio is within a predefined reference range for a predefined portion of the data points.

8. The method of claim 7, wherein, if the ratio is located at least 3 out of 5 consecutive data points, the presumed peak boundary is identified and / or verified.

9. The method according to claim 1 or 2, wherein identifying and / or verifying at least one analyte peak includes identifying and / or verifying peak identity and / or peak purity.

10. The method of claim 9, wherein peak identity and / or peak purity are verified when the ratio is within a predefined baseline range.

11. The method of claim 1 or 2, wherein the method further comprises determining the ratio of the estimated analyte peak area to the internal standard peak area, wherein the method comprises determining the ratio of the analyte quantification factor to the internal standard quantification factor, or the ratio of the analyte qualitative factor to the internal standard qualitative factor, and wherein identifying and / or verifying at least one analyte peak comprises identifying and / or verifying the ratio of the analyte peak area to the internal standard peak area.

12. The method of claim 1 or 2, wherein the portion of the data points for which the ratio is determined in step b) comprises data points between the assumed lower peak boundary and the assumed upper peak boundary.

13. The method according to claim 1 or 2, wherein the method includes identifying and / or verifying at least one presumed peak boundary, peak purity, and / or the ratio of the presumed analyte peak area to the internal standard peak area, including determining the ratio of the analyte quantification factor to the internal standard quantification factor, or the ratio of the analyte qualitative factor to the internal standard qualitative factor, and including determining the distribution of said ratio.

14. The method of claim 10, wherein at least one presumed peak boundary, peak purity, and / or presumed ratio of analyte peak area to internal standard peak area of ​​the analyte peak are identified and / or verified when the distribution of said ratio over a plurality of n data points conforms to a predefined acceptance criterion.

15. The method of claim 14, wherein the n data points are at least two data points.

16. The method of claim 14, wherein the n data points are at least three data points.

17. The method of claim 14, wherein the n data points are at least four data points.

18. The method of claim 14, wherein the n data points are at least five data points.

19. The method of claim 14, wherein the n data points are at least seven data points.

20. The method of claim 14, wherein the n data points are at least ten data points.

21. The method of claim 13, wherein the ratio of the estimated analyte peak area to the internal standard peak area is verified when the average of the ratio calculated from at least 10% of the data points between the estimated lower peak boundary and / or the estimated upper peak boundary is within the range defined by the ratio of the baseline analyte peak area to the internal standard peak area ±30%.

22. The method according to claim 1 or 2, wherein the values ​​of the quantitative factor signal intensity, the qualitative factor signal intensity, the internal standard quantitative factor signal intensity, and / or the internal standard qualitative factor signal intensity are single-cycle signal intensities.

23. A method for quality control of liquid chromatography-mass spectrometry measurements of a sample, the method comprising the following steps: A) Use a liquid chromatography-mass spectrometry apparatus to measure the analyte in the sample of interest and determine at least one chromatogram; B) Performing steps b) to d) of the method according to any one of claims 1 to 22, and C) Evaluate the quality of the liquid chromatography-mass spectrometry measurement based on the results of step B).

24. A system for determining the concentration of at least one analyte in a sample, the system comprising: (I) At least one liquid chromatography-mass spectrometry device, wherein the liquid chromatography-mass spectrometry device is configured to measure the analyte in the sample and to acquire data points over time; as well as (II) At least one evaluation device, wherein the evaluation device is configured to perform at least steps b), c) and / or d) of the method according to any one of claims 1 to 22.

25. The system of claim 24, wherein the liquid chromatography-mass spectrometry apparatus is configured to measure the analyte in the sample and to perform step a) of the method according to any one of claims 1 to 22.

26. The system of claim 24, wherein the evaluation device is configured to perform all steps b) to d) of the method of any one of claims 1 to 22.

27. A computer program product having a program code tool, wherein the program code tool is capable of being stored on or on a storage medium, the computer program product being configured to perform at least steps b), c), and / or d) of the method according to any one of claims 1 to 22 when the program code tool is executed on a computer or on a computer network.

28. A computer program product having a program code tool, wherein the program code tool is capable of being stored on or on a storage medium, the computer program product being configured to perform all steps b) to d) of the method according to any one of claims 1 to 22 when the program code tool is executed on a computer or on a computer network.

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