Universal Calibration for Quantitative Mass Spectrometry

Abstract

The present invention relates to a method for determining an analyte in a sample by mass spectrometry (MS), the method comprising: (a) mixing a predetermined amount of an internal calibrator with the sample, the internal calibrator comprising a predetermined amount of at least two non-identical isotopologues of an analyte, (b) determining MS signals of ions generated from the analyte (analyte signal) and from the at least two isotopologues (isotopologue signal), (c) providing a calibration based on the analyte signal and the isotopologue signal determined in step (b), and (d) determining the analyte based on the calibration provided in step (c).Furthermore, the present invention relates to devices, systems and uses related thereto.

Description

[Technical field]

[0001] The present invention relates to a method for determining an analyte in a sample by mass spectrometry (MS), comprising: (a) mixing a predetermined amount of an internal calibrator with the sample, the internal calibrator comprising a predetermined amount of at least two non-identical isotopologues of an analyte; (b) determining MS signals of ions generated from the analyte (analyte signal) and MS signals of ions generated from the at least two isotopologues (isotopologue signal); (c) providing a calibration based on the analyte signal and the isotopologue signal determined in step (b); and (d) determining the analyte based on the calibration provided in step (c). Furthermore, the present invention relates to devices, systems and uses related thereto. [Background technology]

[0002] The quality of absolute quantification by analytical methods such as mass spectrometry (MS) depends heavily on the quality of the calibration process used to convert the measured signal to analyte concentration. MS signals are in principle amenable to external and internal calibration methods. For example, in liquid chromatography mass spectrometry (LC-MS), it may be possible to avoid matrix effects by using a suitable LC method that in the best case can remove all matrix effects from the analyte of interest. Only certain matrix effects can be compensated for by external calibration if the matrix is ​​coeluted with the analyte signal. If the sample matrix is ​​changing (e.g. caused by different sample types or different ionization properties), external calibration is less accurate.

[0003] Nevertheless, state-of-the-art methodologies for MS quantification rely on the concept of stable isotope dilution and external calibration curves generated from reference standards by using the signal response (analyte signal / internal standard signal). By increasing the calibration frequency, data quality and reliability can be improved, but this increases the measurement time and therefore the cost. Also, while external calibration is widely adapted in LC-MS environments for batch mode assays, it is less suitable to deal with dynamic multiplexed systems that can run many different assays with random access. The calibration demands in this case are too high to allow regular recalibration in this case.

[0004] To avoid the aforementioned drawbacks, especially matrix effects, standard addition methods have been devised in analytical chemistry where standards are added to samples at various concentrations, thereby ensuring that potential matrix effects affect the standards to the same extent as the analytes.

[0005] A different approach is the use of an internal standard to compensate for matrix effects, but requires an additional standard curve to calibrate the analyte / internal standard ratio. All the aforementioned methods are calibration methods known in the art and described in standard textbooks.

[0006] A further method, multipoint internal standard regression, has been described, for example, in Hoffman et al. (2020), Clin Chem 66(3):474 and WO 2017 / 178453. However, similar to external calibration, this method requires that the concentration of the analyte be within a range fixed by the concentration range of the calibrator used.

[0007] Issues to be resolved In view of the above, there remains a need in the art for improved means and methods for determining analytes by MS. Summary of the Invention

[0008] overview This problem is addressed by a method, an apparatus, a system and a use with the features of the independent claims. Advantageous embodiments, which may be realized in one way or in any arbitrary combination, are set out in the dependent claims.

[0009] The present invention therefore provides a method for determining an analyte in a sample by mass spectrometry (MS), comprising the steps of: (a) mixing a predetermined amount of an internal calibrator with the sample, the internal calibrator comprising a predetermined amount of at least two non-identical isotopologues of an analyte; (b) determining MS signals of ions generated from the analyte (analyte signal) and MS signals of ions generated from the at least two isotopologues (isotopologue signals); (c) providing a calibration based on the analyte and isotopologue signals determined in step (b); (d) determining said analyte based on the calibration provided in step (c); The present invention relates to a method comprising the steps of:

[0010] Generally, the terms used herein should be given their ordinary and customary meanings to those skilled in the art, and should not be limited to special or customized meanings unless otherwise indicated. The terms "have", "comprise", or "include" used below, or any grammatical variations thereof, are used inclusively. Thus, these terms may refer to both the situation where no further features are present in the entity described in this context, in addition to the features introduced by these terms, and the situation where one or more additional features are present. As an example, the expressions "A has B", "A comprises B", and "A includes B" may both refer to the situation where no other elements are present in A other than B (i.e., the situation where A consists solely and exclusively of B), and the situation where one or more further elements are present in entity A other than B, such as element C, elements C and D, or further elements. Also, as will be appreciated by those skilled in the art, the terms "comprising a" and "comprising an" refer, in one embodiment, to "comprising one or more," i.e., equivalent to "comprising at least one." Thus, a phrase referring to one item of a plurality, unless otherwise indicated, in one embodiment refers to at least one such item, and in further embodiments, to a plurality thereof; thus, for example, identifying a "cell" refers to identifying at least one cell, and in one embodiment, to identifying a multiplicity of cells.

[0011] Furthermore, when used hereinafter, the terms "preferably," "more preferably," "most preferably," "particularly," "more particularly," "specifically," "more specifically," or similar terms are used in conjunction with any feature without restricting further possibilities. Features introduced by these terms are therefore optional features and are not intended to constrain the scope of the claims in any manner. The invention may be implemented using alternative features, as one skilled in the art will recognize. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any limitations on further embodiments of the invention, without any limitations on the scope of the invention, and without any limitations on the possibility of combining the feature so introduced with other optional or non-optional features.

[0012] In one embodiment, the method specified herein below is an in vitro method. The method steps may in principle be performed in any order deemed appropriate by the skilled artisan, but in one embodiment are performed in the specified order, and one or more, in one embodiment all, of the steps may be assisted or performed by automated equipment. Furthermore, the method may include steps other than those explicitly mentioned above.

[0013] As used herein, the term "standard conditions" refers to IUPAC standard ambient temperature and pressure (SATP) conditions, unless otherwise specified, i.e., in one embodiment, a temperature of 25° C. and an absolute pressure of 100 kPa, and in one embodiment, the standard conditions include a pH of 7. Furthermore, unless otherwise indicated, the term "about" refers to the indicated value with a technical precision generally accepted in the relevant field, in one embodiment, to the indicated value ±20%, in a further embodiment, to ±10%, and in a further embodiment, to ±5%. Furthermore, the term "essentially" indicates that there is no deviation that affects the indicated result or use, i.e., potential deviations do not cause the indicated result to deviate by more than ±20%, in a further embodiment, to ±10%, and in a further embodiment, to ±5%. Thus, "consisting essentially of" means including the specified components, but excluding other components, except for materials present as impurities, unavoidable materials present as a result of the process used to provide the components, and components added for purposes other than achieving the technical effect of the present invention. For example, a composition defined using the phrase "consisting essentially of" encompasses any known acceptable excipients, additives, diluents, carriers, etc. In one embodiment, a composition consisting essentially of a set of components contains less than 5% by weight, in a further embodiment less than 3% by weight, in a further embodiment less than 1% by weight, and in a further embodiment less than 0.1% by weight of an unspecified component(s).

[0014] As mentioned above, the method of determining an analyte specified herein may include steps in addition to those specified herein above. The additional steps may in particular concern specific MS steps deemed appropriate by the skilled person, such as providing a sample for step (a), sample pretreatment as specified herein below, mixing desorption matrix, ionization, etc. Furthermore, after step (d), the result of the determination may be output to a user, may be stored in a database, may be used in standard mathematical calculations, etc.

[0015] The term "determining" is understood by those skilled in the art to refer to ascertaining, concluding, or confirming facts and / or data. Thus, in one embodiment, the determining is a qualitative, semi-quantitative, or quantitative determination, and in one embodiment relates to a quantitative determination. A qualitative determination may be determining whether the value of the parameter is above a predefined threshold, such as a detection limit or a physiologically relevant threshold. A semi-quantitative determination is assigning the measurement to a pre-established category, such as a "low", "medium", or "high" concentration. In one embodiment, the determining is quantitative, i.e. determining the value of a quantitative measure of the parameter.

[0016] The term "determining an analyte in a sample" is understood by those skilled in the art. 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 one embodiment 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 that can be calculated from the amount when the sample mass or sample volume is known. Thus, the measurement result of an analyte in a sample can be expressed in any unit, measure such as weight, mass fraction, concentration, or a measure derived therefrom, including any unit considered appropriate by the skilled artisan, for example the international unit according to a given definition. Methods for determining the amount of an analyte by MS are in principle known to those skilled in the art.

[0017] As mentioned above, the term "analyte" as used herein relates to any chemical compound or group of compounds to be determined in a sample. In one embodiment, the analyte is a macromolecule, i.e. a compound with a molecular mass of more than 2500 u (i.e. more than 2.5 kDa). In a further embodiment, the analyte is a biological macromolecule, in particular a polypeptide, a polynucleotide, a polysaccharide, or a fragment of any of the above. In one embodiment, the analyte is a small molecule chemical compound, i.e. a compound with a molecular mass of up to 2500 u (2.5 kDa), in one embodiment up to 1.5 kDa, in a further embodiment up to 1 kDa. The analyte may be any chemical compound of interest, in one embodiment, the analyte is a chemical compound that is metabolized by the body of a subject, a compound that is administered to a subject to induce a change in the metabolism of the subject, a chemical compound of interest, e.g. an educt, intermediate or product, in a technical process, a chemical compound of interest in an environmental sample, etc. In one embodiment, the analyte is a chemical compound that is metabolized by the body of a subject, in particular a human subject, or a compound that is administered to a subject to induce a change in the metabolism of the subject. Thus, in one embodiment, the analyte is a drug of abuse or a metabolite thereof, such as amphetamine, cocaine, methadone, ethyl glucuronide, ethyl sulfate, an opiate, particularly buprenorphine, 6-monoacatylmorphine, codeine, dihydrocodeine, morphine, morphine-3-glucuronide, and / or tramadol, and / or an opioid, particularly acetylfentanyl, carfentanyl, fentanyl, hydrocodone, norfentanyl, oxycodone, and / or oxymorphone.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, particularly cyclosporine, everolimus, sirolimus, and / or tacrolimus, analgesics, particularly meperidine, normeperidine, tramadol, and / or O-desmethyl-tramadol. , antibiotics, in particular gentamicin, tobramycin, amikacin, vancomycin, piperacillin (tazobactam), meropenem, and / or linezolid, antiepileptic drugs, in particular phenytoin, valporic acid, free phenytoin, free valproic acid, levetiracetam, carbamazepine, carbamazepine-10,11-epoxide, phenobarbital, primidone, gabapentin, zonisamide, lamotrigine, and / or topiramate. In one embodiment, the analyte is a hormone, in particular 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 analytes are 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 analytes are cortisol, estradiol, progesterone, testosterone, 17-hydroxyprogesterone, androstenedione, and / or cortisone. In one embodiment, the sample is a urine sample and the analytes are cortisol, aldosterone, and / or cortisone.In one embodiment, the analyte is a vitamin, in one embodiment vitamin D, in particular 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. In one embodiment, the analyte comprises at least 10, in one embodiment at least 15, atoms of the same element, in one embodiment carbon atoms. In one embodiment, the analyte is testosterone and the internal calibrator is 2,3,4-. 13 C 3 -A preparation of testosterone.

[0018] As used herein, the term "sample", also referred to as "test sample", relates in principle to any type of composition of matter, and thus the term may refer to any sample, such as, but not limited to, a biological sample, a chemical sample, an environmental sample, or any other sample that contains or is assumed to contain an analyte of interest. The sample is in one embodiment a sample of the subject, and in one embodiment a medical or diagnostic sample. In one embodiment, the sample is a liquid sample, and in a further embodiment an aqueous sample. In one embodiment, the test sample is selected from the group consisting of physiological fluids, including blood, serum, plasma, saliva, ocular lens fluid, tears, cerebrospinal fluid, sweat, urine, milk, ascites, mucus, synovial fluid, peritoneal fluid, and amniotic fluid, lavage fluids, tissues, cells, and the like. 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, and in a further embodiment a serum or plasma sample. However, the sample may also be a natural or industrial liquid, in particular surface or ground water, sewage, industrial wastewater, process liquids, soil leachates, and the like. In one embodiment, the sample contains or is suspected to contain at least one compound of interest, i.e., a chemical compound to be determined, called "analyte". The sample may contain one or more additional chemical compounds, which are not to be determined, and which are generally called "matrix". Thus, the sample is in one embodiment a complex matrix sample, which in one embodiment contains more than 100, and in a further embodiment more than 1000 chemical compounds. The sample may be used directly as obtained from the respective source, or may be subjected to one or more pretreatment and / or sample preparation step(s). Thus, the sample may be pretreated by physical and / or chemical methods, in one embodiment by centrifugation, filtration, mixing, homogenization, chromatography, precipitation, dilution, concentration, contact with binding agents and / or detection reagents, and / or any other method deemed appropriate by the skilled artisan.

[0019] The term "mass spectrometry", abbreviated as "MS", is understood by those skilled in the art. In one embodiment, the term relates to an analytical technique that involves determining the mass-to-charge ratio of ions generated from an analyte. Thus, an "MS device" in one embodiment includes at least one mass spectrometry unit (MS unit). As used herein, the term "mass spectrometry unit" in one embodiment relates to a mass spectrometry device configured to detect at least one analyte based on the mass-to-charge (m / z) of the analyte or a fragment thereof. In one embodiment, the MS unit is a tandem mass spectrometry (MS / MS) unit, in a further embodiment a triple quadrupole MS (QqQ-MS), in a further embodiment a multiple reaction monitoring (MRM) mode. The MS unit typically comprises at least one detector that records a charge or current induced, in one embodiment proportional to, the amount of ions passing through the mass spectrometry device. The MS device typically further comprises at least one ionization source configured to generate molecular ions and transfer the molecular ions to the gas phase. Ionization methods and suitable ionization units are known in the art and include electron ionization (EI), chemical ionization (CI), electrospray ionization (ESI), atmospheric pressure ionization (APCI), atmospheric pressure photoionization (APPI), and matrix-assisted laser desorption ionization (MALDI), among others.

[0020] In one embodiment, the MS is chromatography MS, in particular gas chromatography MS (GC-MS) or liquid chromatography MS (LC-MS), which are terms understood by the skilled person. Thus, in one embodiment, the MS device is configured to perform a combination of chromatography (e.g., LC or GC) and mass spectrometry (MS). Thus, the device in one embodiment comprises at least one LC and / or GC unit and at least one MS unit, the LC and / or GC unit(s) and the MS unit being coupled via at least one interface. As used herein, the term "liquid chromatography (LC) unit" relates to an analytical module configured in one embodiment to separate one or more analytes of interest of a sample from other components of the sample via liquid chromatography, in an embodiment for detecting one or more analytes using a mass spectrometer. The LC may be based on any separation principle considered appropriate by the skilled person. In one embodiment, the LC is reversed-phase chromatography, hydrophobic interaction chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, or chiral chromatography. In a further embodiment, the LC is reversed-phase chromatography. The LC device may comprise at least one LC column. For example, the 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 transport the analyte(s) of interest. The LC unit may be or may comprise at least one high performance liquid chromatography (HPLC) unit and / or at least one micro liquid chromatography (μLC) device. The term "gas chromatography" is understood by those skilled in the art and in one embodiment, the same separation principles as LC are applicable, but the mobile phase is a gas in GC.

[0021] The terms "mixing" and "admixing" are understood by those skilled in the art and are used interchangeably herein. In one embodiment, the term includes any means deemed appropriate by those skilled in the art to mix, and in one embodiment homogenize, the indicated components and any additional components. Unless otherwise specified, the compounds can be added for mixing in any order. Also, the components may be added in a premixed form.

[0022] The term "isotopologue" is used herein in its conventional sense and refers to a compound or group of compounds that differ from a reference compound only in its or their isotopic composition. Thus, the ions determined for the isotopologue in the method differ in one embodiment only in their isotopic composition, and in a further embodiment, the ions determined for the isotopologue and the analyte differ only in their isotopic composition. As the skilled artisan knows, stable isomers, and thus isotopologues, can exist in a preparation of a chemical compound in a statistical manner, e.g., caused by the natural distribution of isotopes. For example, any carbon atom in a chemical molecule, unless artificially enriched or depleted, 13 C. Thus, and consistent with conventional usage, the term isotopologues also includes populations of molecules that have the same isotopic composition but differ at specific positions in the isotopic label (isotopomers). Thus, for example, 13 C 1 Isotopologues include 1- 13 C 1 -Testosterone, 2- 13 C 1 -Testosterone, 3- 13 C 1 -Testosterone, 4- 13 C 1-testosterone, etc. In view of the above, in one embodiment, an isotopologue is structurally identical to an analyte, but includes at least one isotopic label, i.e., in one embodiment, at least one position where an atom is replaced by its isotope. For the avoidance of doubt, as used herein, the term isotopologue refers to a population of molecules in which the same number of atoms are replaced by the same isotope compared to an analyte, which may be a population of molecules in which the same atom(s) at the same position(s) are replaced, in which case an isotopologue may also be referred to as a "specific isotopologue", whereas an isotopologue may also be a population of molecules in which the same number of the same atoms are replaced by the same number of the same isotopes, in which case an isotopologue may also be referred to as a "mixed isotopologue".

[0023] In one embodiment, the isotope is a stable isotope. In one embodiment, the replaced atom is a carbon atom and the isotope is 13 In a further embodiment, the replaced atom is nitrogen and the isotope is 15 In a further embodiment, the replaced atom is hydrogen and the isotope is 2 In a further embodiment, the replaced atom is oxygen and the isotope is 17 O or 18 O, in one embodiment 18 In a further embodiment, the replaced atom is sulfur and the isotope is 33 S or 34 In one embodiment, at least two, in a further embodiment at least three, in a further embodiment at least four, and in a further embodiment at least five atoms in the analyte structure are replaced by their isotopes, in such a case, in one embodiment, only atoms of the same element are replaced, and the isotopes replacing the atoms are the same isotopes. Thus, in one embodiment, when a carbon atom is replaced, all of the replaced carbon atoms are 13In a further embodiment, at least 10%, in a further embodiment at least 20%, and in a further embodiment at least 30% of the atoms of a particular element are replaced with a particular isotope. As one of skill in the art will appreciate, the above applies mutatis mutandis to fragments of analytes and isotopologues that may be generated and detected during MS.

[0024] As used herein, the term "internal calibrator" refers to a composition of matter that contains a predetermined amount of at least two non-identical isotopologues of an analyte. As will be understood by those skilled in the art in light of this specification, compounds that are non-identical isotopologues of an analyte are, in one embodiment, also isotopologues of each other. Thus, in one embodiment, non-identical isotopologues of each other are not isotopomers and / or are not isotopomers of the analyte.

[0025] In one embodiment, the internal calibrator comprises at least a first and a second isotopologue, and in a further embodiment further comprises at least a third isotopologue, and in a further embodiment at least a fourth isotopologue, of the analyte. The names of the isotopologues are in one embodiment ordered by their relative abundance in the internal calibrator, i.e., in one embodiment, the first isotopologue is the isotopologue present in the highest fraction, the second isotopologue is the isotopologue present in the second highest fraction, and so on. In one embodiment, the internal calibrator comprises at least a first isotopologue of the analyte as the major compound, and at least a second isotopologue in a relative fraction of up to 20%, in one embodiment up to 10%, and in a further embodiment up to 5% of the first isotopologue. In one embodiment, at least two isotopologues or ions derived therefrom have m / z ratios ranging from x to x+(n-1), where x is the m / z ratio of the isotopologue with the lowest m / z ratio and n is the number of isotopologues present in the internal calibrator. Thus, in one embodiment, the internal calibrator provides a series of ions having m / z values ​​that each increase by one mass unit.

[0026] In one embodiment, the first isotopologue in the internal calibrator comprises or is an isotopologue of the analyte that is labeled at a specific position in the molecule, in one embodiment at least two, in a further embodiment at least three, and in a further embodiment at least four positions.Thus, in one embodiment, the first isotopologue is a derivative of two isotopes, in a further embodiment three isotopes, and in a further embodiment four isotopologues of the analyte.Thus, in one embodiment, the first isotopologue is a specific isotopologue as specified herein above.

[0027] In one embodiment, the second isotopologue is present in the preparation of the first isotopologue as a result of natural isotope distribution. In a further embodiment, the second and third isotopologues, and in a further embodiment, the second, third and fourth isotopologues, are present in the preparation of the first isotopologue as a result of natural isotope distribution. Thus, in one embodiment, the second isotopologue and any third and further isotopologue(s) are mixed isotopologues as specified hereinabove. Thus, in one embodiment, all isotopologues of the internal calibrator are included in a single isotopically labeled preparation of the analyte. Since the natural distribution of isotopes of an element is known, it is possible in principle to calculate the fraction of the second isotope and further isotopologues from the probability that an atom is an isotope. In one embodiment, the relative fractions of isotopologues in the internal calibrator are determined, for example, by MS or provided by the manufacturer.

[0028] In an exemplary embodiment, the analyte may be testosterone. In such a case, the first isotopologue may be, for example, 2,3,4- 13 C 3 -testosterone, and thus the first isotopologue has three carbon atoms at positions 2, 3 and 4. 13C isotopologue and therefore has an m / z value increased by +3 compared to testosterone. Furthermore, testosterone has 19 carbon atoms, hence the 2,3,4- 13 C 3 -Each of the 16 formally unlabeled carbon atoms in testosterone is 13 The probability of C is about 1%, so 2,3,4- 13 C 3 -Approximately 16% of testosterone preparations contain one additional 13 C atoms, about 2.4% have two additional 13 Contains C atoms.

[0029] In one embodiment, the second isotopologue comprises or is an isotopologue of the analyte that is labeled at one additional or fewer positions in the molecule compared to the first isotopologue. In one embodiment, the foregoing applies mutatis mutandis to any third and further isotopologues, and thus the second isotope, and any third and further isotope(s) are specific isotopes as specified hereinabove. Thus, when the first isotope is 3,4- 13 C 2 If testosterone, the second isotope may be, for example, 2,3,4- 13 C 3 -testosterone. When the second isotopologue is an isotopologue labeled at one or more specific atoms in the molecule, one skilled in the art will consider that, depending on the natural isotope distribution, a preparation of the first isotopologue may contain a significant amount of the second isotopologue.

[0030] In view of the above, in one embodiment, the relative fraction of the second isotope log is a result of a relative isotope distribution in the manufacturing process of the internal calibrator, which may be a natural relative isotope distribution or an enriched relative isotope distribution.

[0031] As mentioned above, the internal calibrator comprises a predetermined amount of at least two isotopologues of the analyte. As used herein, the term "predetermined amount" relates to an amount that is fixed and recorded at least before the calibration of the method is performed. The predetermined amount of the internal calibrator may relate to any parameter that correlates with the amount of the internal calibrator added to the sample, and in one embodiment is the volume of the internal calibrator stock solution. As the skilled person will appreciate, such a predetermined amount does not necessarily have to be known when the internal calibrator is mixed into the sample, but it must be known in step (c) of the method. Also, as indicated hereinabove, the predetermined amount of isotopologues may in principle be a calculated amount, i.e. the predetermined amount of the internal calibrator may be calculated from the weight of the internal calibrator compound used, the volume in which it is dissolved, and the volume of the resulting solution mixed into the sample. Also, the predetermined amount of isotopologues may be calculated as indicated hereinabove. However, in one embodiment, the predetermined amount of isotopologues in the internal calibrator compound is determined by measurement, for example by determining the MS signal of the internal calibrator. Alternatively, the predetermined amount of an internal standard may be determined by measuring the concentration of the isotopologue in the internal calibrator by standard methods known to those skilled in the art, such as quantitative NMR (Q-NMR), GC, LC and / or spectroscopic or immunological methods.

[0032] The term "MS signal" is used herein in its customary sense as known to those skilled in the art. In one embodiment, the term relates to any intensity parameter or value thereof detected by a detector of an MS instrument for a particular m / z value or range of m / z values. An MS signal determined at an m / z value or range of m / z values ​​of ions generated from an analyte is referred to herein as an "analyte signal" and an MS signal determined at an m / z value or range of m / z values ​​of ions generated from an isotopologue is referred to herein as an "isotopologue signal."

[0033] The term "determining MS signals" is likewise understood by those skilled in the art. Determining MS signals according to the methods specified herein includes determining the MS signals of ions generated from the analyte (analyte signals) and the MS signals of ions generated from said at least two isotopologues (isotopologo signals). The ions generated and detected depend on the analyte, in particular its molecular weight and structure, as well as the ionization method applied and the specific mode of MS detection envisaged, as understood by those skilled in the art. In one embodiment, MS detection includes detection in MS mode, MS / MS mode, or any mode that the skilled artisan deems appropriate, in particular suitable for the MS device specified elsewhere herein. In one embodiment, the term relates to determining a correlation plot between the MS signal and the m / z value or range of m / z values ​​of the ions causing said signal. A graphical representation of the mass spectrum may be provided, for example, as a centroid graph and / or a continuous graph. As mentioned herein, the analyte signal and the isotope signal may be determined as separate signals, whereas the isotopologue signal may be determined as a combined signal. For example, the signal of the first isotopologue (first isotopologue signal) may be determined as part of the determination of the combined analyte signal + first isotopologue signal, the signal of the second isotopologue (first isotopologue signal) may be determined as part of the determination of the combined analyte signal + second isotopologue signal, etc. Thus, the isotopologues do not necessarily have to be determined in an isolated manner. However, the analyte signal is determined in one embodiment as an isolated analyte signal.

[0034] The term "calibration" is used herein in a broad sense consistent with typical usage by those skilled in the art. The term calibration therefore includes the operation of establishing a relationship between the value of a quantity obtained with a measurement standard under specific conditions and the value of the corresponding quantity of a calibrated instrument, i.e., calibration in the narrow sense. However, calibration may also be the verification of a measurement value. The term calibration further includes the means of adjusting or re-adjusting a calibrated instrument or its output so that it matches the value of said quantity obtained with a measurement standard contained in an internal calibrator, i.e., calibration in its usual broader sense. Thus, in the methods specified herein, the term calibration may also relate to providing a correlation between the MS signal determined by the MS instrument and the amount of isotopologues contained in a sample.

[0035] According to step (a) of the method for determining an analyte, a predetermined amount of an internal calibrator is mixed with the sample, the internal calibrator comprising a predetermined amount of at least two non-identical isotopologues of the analyte. Thus, the internal calibrator is mixed with the sample before, during and / or after the sample preparation step. The amount of the internal calibrator is predetermined as described herein above.

[0036] According to step (b) of the method for determining an analyte, the MS signal of an ion generated from the analyte (analyte signal) and the MS signal of an ion generated from at least two isotopologues (isotopologue signal) are determined. In one embodiment, the signal of the analyte is determined at the m / z value of the main ion generated from the analyte, i.e. the ion with the highest abundance, such as the M+H ion. If the signal of the main ion is too high, other ions with lower abundance can be used as well. If the isotopologue in the internal calibrator with the lowest molecular weight is removed by more than one, in one embodiment more than two mass units, the minor ion may be the +1 isotopologue of the analyte as well. In one embodiment, determining the MS signal of an ion in step (b) comprises determining the MS signal of an ion that is a non-identical isotopolog between the analyte and its at least two non-identical isotopologues. In a further embodiment, determining the MS signal of an ion in step (b) comprises determining the MS signal of an ion of said at least two non-identical isotopologues that differ by one mass unit, i.e. one m / z unit. As the skilled person will appreciate, in one embodiment, in each case the corresponding ion is determined, i.e. when the fragment ion of the analyte is determined, the ion of the isotopologue having the same structure is determined. In step (b), the analyte signal and the isotopologue signal may be determined separately, but also the combined analyte + isotopologue signal may be determined, as specified herein above.

[0037] According to step (c) of the method for determining an analyte, a calibration based on the analyte signal and the isotopologue signal determined in step (b) is provided. Thus, the calibration of the method for determining an analyte utilizes the isotopologue signal and the analyte signal. Step (c) may include a substep of calculating the amount of each isotopologue in the sample from a predetermined amount of the internal calibrator added to the sample and a predetermined amount of the isotopologue in the internal calibrator of step (a). In one embodiment, the calibration includes correlating the binding signals of the analyte and the first, second and any further isotopologues, respectively, to a predetermined amount of each isotopologue, i.e., in one embodiment, step (c) includes providing a calibration based on (i) the ratio of the sum of the isotopologue signal and the analyte signal for each of the at least two isotopologues (ii) to the amount for each of the at least two isotopologues. Thus, in one embodiment, the analyte plus a first isotopologue signal correlates with the amount of the first isotopologue, the analyte plus a second isotopologue signal correlates with the amount of the second isotopologue in the sample, etc. As described herein above, said sum may be a calculated sum or may be determined as such in step (b). In one embodiment, said sum is a calculated sum, i.e. calculated as the sum of the analyte signal and each isotopologue signal determined separately in step (b), said analyte signal being linear up to an amount of analyte corresponding to the sum of the amount of analyte and the amount of isotopologue having the highest concentration in the internal calibrator. In one embodiment, said correlation comprises providing a ratio of binding signal to the amount of each isotopologue, said ratio being graphically represented by a graph of the amount of isotopologue versus binding signal. In a further embodiment, the calibration further comprises fitting a regression equation to said ratio. Said regression may be of any type deemed appropriate by the skilled artisan, including linear, polynomial, exponential, or other regression equations. In one embodiment, the regression equation is a linear regression equation.

[0038] According to step (d) of the method for determining an analyte, the analyte is determined based on the calibration provided in step (c). As will be appreciated by those skilled in the art, the calibration of step (c) is, inter alia, already based on the analyte signal, so in one embodiment, this calibration allows the analyte to be determined directly from the calibration. Thus, in particular when the regression equation used for the calibration is linear, the amount of the analyte is determined as the negative value of the solution of the regression equation for a value of the MS signal of the ion being zero. In one embodiment, said amount is the monoisotopic amount of the analyte, i.e. the amount of the analyte represented by its isotopologue with the highest abundance and / or corresponding to a calibrator isotopologue ion with the same m / z value. Thus, in one embodiment, said amount is corrected for the natural abundance of the isotopes in the analyte, the distribution of which is known to those skilled in the art. Thus, in one embodiment, said amount is corrected by adding the amount(s) of the isotopologue(s) of the analyte that are predictably present in the sample.

[0039] Advantageously, in the research underlying the present invention, it has been found that multi-point internal standard regression can be combined with standard addition methods, for example to provide calibration in MS methods. With such a combination, in one embodiment, it is not necessary that the analyte concentration is within the range of known concentrations used for calibration. Thus, the analyte can be determined at any concentration. Furthermore, it has been found that an isotopically labeled preparation of an analyte can contain an amount of isotopomers that can be used to establish a calibration, thus making it possible to provide a calibration curve by adding only one isotopically labeled preparation of the analyte. Furthermore, the relative content of isotopically labeled preparations of an analyte can be established in situ by determining the MS signal of an isotopically labeled preparation of the analyte itself, or from the sample according to step (a).

[0040] The above definitions apply mutatis mutandis below. The following further additional definitions and explanations also apply mutatis mutandis to all embodiments described herein.

[0041] The present invention further relates in one embodiment to a device configured to carry out at least steps (c) and (d) of the method for determining an analyte of the present invention.

[0042] As used herein, the term "apparatus" relates to a collection of means, including at least the indicated means, operatively linked to each other in one embodiment, allowing at least to provide a calibration. Exemplary means for calibration are known to the skilled person and in particular comprise a data processing unit, comprising a data processor, in one embodiment, in which an algorithm for performing the calibration is tangibly embedded. However, the aforementioned computer algorithm may also be included in the memory unit of the apparatus. Thus, the apparatus in one embodiment comprises a microprocessor and a memory unit, and in a further embodiment, the apparatus comprises an algorithm, in one embodiment, in which, tangibly embedded in the memory unit, which, when executed by the microprocessor, causes the apparatus to perform at least step c) of the method for determining an analyte. In one embodiment, the apparatus further comprises at least one data interface for receiving data, such as the value of the MS signal determined in step (b), the predetermined amount of the internal calibrator and / or isotopologue of step (a), and / or for outputting data, such as a regression equation, a calibration graph, and / or an amount of an analyte. The aforementioned output data may also be output via any output unit, such as a screen, a printer, etc. The result may be given as an output of raw data requiring interpretation by a technician. However, in one embodiment, the output of the device is raw data that is processed, i.e. evaluated, and its interpretation does not require a technician. How the means of the device are operationally linked depends on the type of means included in the device. Those skilled in the art will understand how to provide and link suitable means without further ado. In one embodiment, the means are included in a single device.

[0043] The present invention also relates to a system including an apparatus as described hereinabove operatively connected to a mass spectrometer.

[0044] As used herein, the term "system" refers to a system of means comprising at least the indicated devices operatively linked to each other. The system comprises at least one device as specified herein above and at least one MS device as also specified herein above. A person skilled in the art knows how to operatively connect a device to an MS device, and in one embodiment the operative connection comprises at least one common data interface. In one embodiment, the system is adapted to perform the method of determining an analyte as specified herein. In one embodiment, the mass spectrometry unit is adapted to perform at least step (b), in one embodiment at least steps (a) and (b) of the method of determining an analyte, and / or the device is adapted to perform at least step (c), in one embodiment steps (c) and (d) of the method of determining an analyte.

[0045] The present invention further relates in one embodiment to the method for determining an analyte as identified herein above, to the use of a composition comprising at least two non-identical isotopologues of the analyte in a predetermined ratio for the determination of said analyte in a sample.

[0046] The present invention further discloses and proposes a computer program comprising computer executable instructions for carrying out the method according to the present invention in one or more of the embodiments attached hereto when the program is executed on a computer or a computer network. In particular, the computer program may be stored on a computer readable data carrier. Thus, in particular, one, more than one or all of the method steps a) to d) as described above may be carried out using a computer or a computer network, in one embodiment, using a computer program.

[0047] The present invention further discloses and proposes a computer program product having program code means for carrying out the method according to the present invention in one or more embodiments contained herein when the program is executed on a computer or a computer network. In particular, the program code means may be stored on a computer readable data carrier.

[0048] Furthermore, the present invention discloses and proposes a data carrier storing a data structure which, after being loaded into a computer or a computer network, such as the working or main memory of the computer or computer network, is capable of carrying out the method according to one or more of the embodiments disclosed in this specification.

[0049] The present invention further proposes and discloses a computer program product having program code means stored on a machine-readable carrier for performing the method according to one or more of the embodiments disclosed herein when the program is executed on a computer or a computer network. As used herein, a computer program product refers to a program as a tradeable product. The product is generally present in any format, such as a paper format or on a computer-readable data carrier. In particular, the computer program product may be distributed over a data network.

[0050] Finally, the present invention proposes and discloses a modulated data signal containing instructions readable by a computer system or computer network for carrying out the methods according to one or more of the embodiments disclosed herein.

[0051] In one embodiment, referring to computer-implemented aspects of the invention, one or more or all of the method steps of the method according to one or more of the embodiments disclosed herein can be performed by using a computer or a computer network. Thus, in general, any of the method steps involving providing and / or manipulating data can be performed by using a computer or a computer network. In general, these method steps can include any method steps, usually excluding method steps that require manual operations, such as providing a sample and / or certain aspects of performing the actual measurement.

[0052] Specifically, the present invention relates to - a computer or computer network comprising at least one processor, the processor being configured to execute a method according to one of the embodiments described herein; - a computer-loadable data structure configured, when executed on a computer, to carry out a method according to one of the embodiments described herein; a computer program adapted to carry out a method according to one of the embodiments described herein while said program is being run on a computer, a computer program comprising program means for carrying out a method according to one of the embodiments described herein, when said computer program is run on a computer or on a computer network, a computer program comprising program means according to the preceding embodiment stored on a computer readable storage medium; - a storage medium on which a data structure is stored and adapted to carry out a method according to one of the embodiments described herein after the data structure has been loaded into a main and / or working storage of a computer or a computer network, and - a computer program product comprising program code means, the program code means being capable of being stored on a storage medium for carrying out a method according to one of the embodiments described herein when the program code means is executed on a computer or a computer network. is further disclosed.

[0053] In summary of the findings of the present invention, the following embodiments are specifically envisaged.

[0054] Embodiment 1: A method for determining an analyte in a sample by mass spectrometry (MS), comprising: (a) mixing a predetermined amount of an internal calibrator with the sample, the internal calibrator comprising a predetermined amount of at least two non-identical isotopologues of the analyte; (b) determining MS signals of ions generated from the analyte (analyte signal) and MS signals of ions generated from the at least two isotopologues (isotopologue signals); (c) providing a calibration based on the analyte and isotopologue signals determined in step (b); (d) determining the analyte based on the calibration provided in step (c); 1. A method for determining an analyte in a sample by mass spectrometry (MS), comprising:

[0055] Embodiment 2: The method of embodiment 1, wherein the internal calibrator comprises a first isotopologue of the analyte as the main compound and at least a second isotopologue in a relative fraction of up to 20%, in one embodiment up to 10%, and in a further embodiment up to 5% of the first isotopologue.

[0056] Embodiment 3: The method of embodiment 2, wherein the relative fraction of the second isotopologue is a result of a relative isotope distribution in a manufacturing process of the internal calibrator.

[0057] Embodiment 4: The method of embodiment 3, wherein the relative isotopic distribution is a natural relative isotopic distribution or an enriched relative isotopic distribution.

[0058] Embodiment 5: The method of any one of embodiments 1 to 4, wherein the isotopologues are contained in a single isotopically labeled preparation of the analyte.

[0059] Embodiment 6: The method of any one of embodiments 1 to 5, wherein the analyte comprises at least 10, and in one embodiment, at least 15, atoms of the same element, and in one embodiment, carbon atoms.

[0060] Embodiment 7: The method of any one of embodiments 1 to 6, wherein step (c) comprises providing a calibration based on the MS signals and the amounts of the at least two isotopologues determined in step (b).

[0061] Embodiment 8: The method of any one of embodiments 1 to 7, wherein step (c) comprises providing a calibration based on a ratio of (i) the sum of an isotopologue signal and an analyte signal for each of the at least two isotopologues and (ii) an amount for each of the at least two isotopologues.

[0062] Embodiment 9: The method of any one of embodiments 1 to 8, wherein the sum is measured directly in step (b).

[0063] Embodiment 10: The method of any one of embodiments 1 to 8, wherein the sum is a calculated sum, and in one embodiment, the analyte signal is linear up to an amount of analyte that corresponds to the sum of the amount of analyte and the amount of the isotopologue having the highest concentration in the internal calibrator.

[0064] Embodiment 11: The method of any one of embodiments 8 to 10, wherein a regression equation is fitted to the ratio and the amount of analyte is determined as the negative value of the solution of the regression equation for which the MS signal value of the ion is zero.

[0065] Embodiment 12: The method of embodiment 11, wherein the regression equation is a linear regression equation.

[0066] Embodiment 13: The method of any one of embodiments 1 to 12, wherein two of the at least two isotopologues differ in molecular mass by one mass unit.

[0067] Embodiment 14: The method of any one of embodiments 1 to 13, wherein the at least two isotopologues or ions derived therefrom have m / z ratios ranging from x to x+(n-1), where x is the m / z ratio of the isotopologue with the lowest m / z ratio, and n is the number of isotopologues present in the internal calibrator.

[0068] Embodiment 15: The method of any one of embodiments 1 to 14, wherein the isotopologues are not isotopomers of one another.

[0069] Embodiment 16: The method of any one of embodiments 1 to 15, wherein determining the MS signals of ions in step (b) comprises determining the MS signals of ions that are non-identical isotopomers between the analyte and its at least two non-identical isotopologues.

[0070] Embodiment 17: The method of any one of embodiments 1 to 16, wherein determining the MS signals of ions in step (b) comprises determining the MS signals of ions of the at least two non-identical isotopologues that differ by one mass unit.

[0071] Embodiment 18: The method of any one of embodiments 1 to 17, wherein said at least two isotopologues are at least three, and in one embodiment at least four isotopologues.

[0072] Embodiment 19: The method of any one of embodiments 1 to 18, wherein the ions determined for the isotopologues, and in one embodiment for the analyte, are structurally identical.

[0073] Embodiment 20: The method of any one of embodiments 1 to 19, wherein the ions determined for the isotopologues differ only in their isotopic composition.

[0074] Embodiment 21: The method according to any one of embodiments 1 to 20, wherein the isotopologues and the determined ions for the analyte differ only in their isotopic composition.

[0075] Embodiment 22: The method of any one of embodiments 1 to 21, wherein the sample is a complex matrix sample and comprises in one embodiment more than 100, and in a further embodiment more than 1000 chemical compounds.

[0076] Embodiment 23: The method of any one of embodiments 1 to 21, wherein the sample is a subject sample, in one embodiment a medical sample.

[0077] Embodiment 24: The method according to any one of embodiments 1 to 23, wherein the sample is a sample of a body fluid or a tissue sample.

[0078] Embodiment 25: The method of any one of embodiments 1 to 24, wherein the sample is a serum, plasma, or blood sample.

[0079] Embodiment 26: The method of any one of embodiments 1 to 25, wherein the analyte is a low molecular weight compound, in one embodiment, having a molecular mass of up to 2.5 kDa, in one embodiment, up to 1.5 kDa, and in a further embodiment, up to 1 kDa.

[0080] Embodiment 27: The method of any one of embodiments 1 to 26, wherein the analyte is selected from the list consisting of testosterone; amphetamines; cocaine; methadone; ethyl glucuronide; ethyl sulfate; opiates; opioids; clonazepam; methotrexate; voriconazole; mycophenolic acid (total); mycophenolic acid-glucuronide; acetaminophen; salicylic acid; theophylline; digoxin; immunosuppressants; analgesics; antibiotics; antiepileptic drugs; hormones; and vitamins.

[0081] Embodiment 28: The analyte is testosterone and the internal calibrator is 2,3,4- 13 C 3 - The method according to any one of embodiments 1 to 27, wherein the compound is testosterone.

[0082] Embodiment 29: The method of any one of embodiments 1 to 28, wherein at least step (c) of the method, in one embodiment steps (c) and (d), are computer-implemented.

[0083] Embodiment 30: The method of any one of embodiments 1 to 29, wherein the method further comprises outputting the amount or concentration of the analyte in the sample based on the determination in step (d).

[0084] Embodiment 31: An apparatus configured to carry out at least step c) of the method according to any one of embodiments 1 to 30, in one embodiment steps (c) and (d).

[0085] Embodiment 32: The device of embodiment 31, wherein the device comprises a microprocessor and a memory unit.

[0086] Embodiment 33: In one embodiment, the device described in embodiment 32, wherein the device tangibly embedded in the memory unit includes an algorithm that, when executed by a microprocessor, causes the apparatus to perform at least step c) of the method described in any one of embodiments 1 to 28.

[0087] Embodiment 34: A system comprising an apparatus according to any one of embodiments 31 to 33, operably connected to a mass spectrometer.

[0088] Embodiment 35: The system described in embodiment 34, wherein the mass spectrometer is adapted to perform at least step (b), in one embodiment at least steps (a) and (b), of the method described in any one of embodiments 1 to 30.

[0089] Embodiment 36: In one embodiment according to the method according to any one of embodiments 1 to 30, the use of a composition comprising at least two non-identical isotopologues of said analyte in a predetermined ratio for the determination of said analyte in a sample.

[0090] All references cited herein are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification. [Brief description of the drawings]

[0091] [Figure 1] Q-TOF MS spectrum of the artificial sample of Example 1. [Diagram 2] Table 3, Standard Addition Plot of the Data from Example 1. [Diagram 3] Calibration graph for external calibration (Comparative Example 2). [Figure 4] Calibration graph of multipoint internal standard regression (Comparative Example 3). [Diagram 5] Standard addition plot of the data from Example 4.

[0092] The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention in any way.

[0093] Example 1: MS measurement of testosterone 1.1 Chemicals The concentration of testosterone stock solution Cerilliant (1 mg / mL in acetonitrile), lot FE07241802, was determined by Q-PCR to be 1.018 mg / mL ± 2%. 13 C 3 -The concentration of testosterone Cerilliant (0.1 mg / mL in acetonitrile), lot FE06071902, was determined by Q-PCR to be 0.083 mg / mL ± 5-7%.

[0094] Both stock solutions were diluted with acetonitrile to use solutions with a concentration of 10 ng / mL, 255 ng / mL testosterone and 415 ng / mL 2,3,4- 13 C 3 -combined with an artificial sample containing a nominal concentration of testosterone.

[0095] The content of each isotopologue in the artificial samples is shown in Tables 1 and 2. [Table 1] [Table 2]

[0096] The artificial sample was measured using a Q-TOF MS system, and the mass spectrum obtained is shown in Figure 1.

[0097] The area (signal intensity) of each ion was measured and used to calculate the data for the standard addition plot (Table 3), and the standard addition plot was established (Figure 2). [Table 3]

[0098] From the calibration plot in Figure 2 and the corresponding correlation equation y = 574.96x + 117,196, the concentration of the analyte testosterone is determined to be 204 ng / mL, which compares to a nominal concentration of 217 ng / mL, which corresponds to a % drift error of 6.

[0099] The same procedure as above was tested for testosterone concentrations from 100 ng / mL to 1000 ng / mL, and it was found that lower concentrations tended to result in higher % drift error.

[0100] Compared to the comparative examples below, the method of the present invention has a lower % drift error. Furthermore, the method is very flexible in that it compensates for saturation effects and allows for easy addition of additional isotopologues as calibrators.

[0101] Example 2: Comparative Example: External Calibration Various testosterone concentrations were determined with external calibration by Q-TOF MS, the measured signals are shown in Table 4 and the calibration graph is shown in Figure 3. [Table 4]

[0102] The correlation equation obtained was y=283.3x+46,955, and the determined concentration in the test sample was 496 ng / mL, which is compared to the nominal concentration of 415 ng / mL. The drift error was -19.

[0103] Example 3: Comparative Example Multipoint Internal Standard Regression In addition, the 2,3,4- 13 C 3 -Testosterone formulations were used in a multipoint internal standard regression calibration method. The concentrations of the standards and the corresponding signals are shown in Table 5, and the calibration graph is shown in Figure 4. [Table 5]

[0104] The correlation equation obtained was y=512.67x+2118.2, and the determined concentration in the test sample was 372 ng / mL, which is compared to the nominal concentration of 433 ng / mL. The % drift error was 14.

[0105] Example 4: Determination of estradiol The method is essentially the same as in Example 1, using estradiol as the analyte, 13 C 5 A calibration was performed using -estradiol as an internal calibrator on the Q-TOF MS instrument, and the resulting calibration graph is shown in Figure 5.

[0106] The % drift error of the measurements is about 11, which compares to 15 for the external calibration (performed as in Example 2) and 14 for the multipoint internal regression (performed as in Example 3).

[0107] References: - Hoffman et al. (2020), Clin Chem 66(3):474 - International Publication No. 2017 / 178453

Claims

1. A method for determining an analyte in a sample by mass spectrometry (MS), (a) mixing a predetermined amount of an internal calibrator into the sample, wherein the internal calibrator contains at least two non-identical isotopologues of a predetermined amount of the analyte, mixing a predetermined amount of the internal calibrator into the sample; (b) determining the MS signal of the ions generated from the analyte (analyte signal) and the MS signal of the ions generated from the at least two isotopologues (isotopologue signal); (c) providing a calibration based on the analyte signal and the isotopologue signal determined in step (b); (d) determining the analyte based on the calibration provided in step (c). A method comprising the above steps.

2. The method according to claim 1, wherein step (c) comprises providing a calibration based on (i) the sum of the isotopologue signal and the analyte signal for each of the at least two isotopologues and (ii) the ratio of the amount for each of the at least two isotopologues.

3. The method according to claim 1 or 2, wherein a regression equation is fitted to the ratio, and the amount of the analyte is determined as the negative value of the solution of the regression equation when the value of the MS signal of the ions is 0.

4. The method according to claim 3, wherein the regression equation is a linear regression equation.

5. The method according to claim 1 or 2, wherein the internal calibrator contains a first isotopologue of the analyte as the main compound and at least a second isotopologue with a relative fraction of up to 20%, in one embodiment up to 10%, and in a further embodiment up to 5% of the first isotopologue.

6. The method according to claim 5, wherein the relative fraction of the second isotopologue is the result of the relative isotope distribution in the manufacturing process of the internal calibrator.

7. The method according to claim 1 or 2, wherein the isotopologue is included in a single isotope-labeled preparation of the analyte.

8. The method according to claim 1 or 2, wherein the ions determined for the isotopologue and, in one embodiment, for the analyte are structurally identical.

9. The method according to claim 1 or 2, wherein the sample is a sample of interest, in one embodiment a medical or diagnostic sample, and in one embodiment a sample of body fluid or tissue sample.

10. The method according to claim 1 or 2, wherein the analyte is a low molecular weight compound, and in one embodiment a low molecular weight compound having a molecular mass of up to 2.5 kDa.

11. The analyte is testosterone and the internal calibrator is 2,3,4- １３ C ３ -testosterone, the method according to claim 1 or 2.

12. An apparatus configured to perform at least step c) of the method according to claim 1 or 2, and in one embodiment steps (c) and (d).

13. A system comprising the apparatus according to claim 12, operably connected to a mass spectrometer.

14. The system according to claim 13, wherein the mass spectrometer is adapted to perform at least step (b) of the method according to claim 1 or 2, and in one embodiment at least steps (a) and (b).

15. Use of a composition comprising at least two non-identical isotopologues of the analyte in a predetermined ratio for the determination of the analyte in a sample in one embodiment according to the method according to claim 1 or 2.

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