Dual single ion monitoring mass spectrometry

Abstract

The present invention relates to a method for determining an analyte in a mass spectrometry (MS) device comprising a first mass filter and a second mass filter, the method comprising: (i) filtering in the first mass filter for an analyte ion species; (ii) optionally fragmenting at least a portion of the ions obtained by said filtration in step (i) in a collision cell, wherein the collision energy of said fragmentation is selected to be lower than a predetermined collision energy causing fragmentation of said analyte ion species; (iii) filtering in the second mass filter for the analyte ion species filtered in step (i); and (iv) detecting the analyte ion species filtered in step (iii), thereby determining the analyte. Furthermore, the invention relates to devices, systems and uses relating to said method.

Description

[0001] The present invention relates to a method for determining an analyte in a mass spectrometry (MS) device comprising a first mass filter and a second mass filter, the method comprising: (i) filtering for an analyte ion species in the first mass filter; (ii) optionally fragmenting at least a portion of the ions obtained by filtering in step (i) in a collision cell, wherein the collision energy of the fragmentation is chosen to be lower than a predetermined collision energy causing fragmentation of the analyte ion species; (iii) filtering for the analyte ion species filtered in step (i) in the second mass filter; and (iv) detecting the analyte ion species filtered in step (iii), thereby determining the analyte. Furthermore, the invention relates to devices, systems and uses related to the method.

[0002] Mass spectrometry (MS), in particular liquid chromatography tandem mass spectrometry (LC-MS / MS), has become the method of choice for analyte quantification, e.g. in in-vitro diagnostics. Especially in case of small molecules with similar structured metabolites, the specificity and accuracy of MS becomes crucial to produce reliable results. Examples of such small molecules are hormones and immunosuppressive drugs, like cyclosporin A and everolimus.

[0003] MS analysis for in-vitro diagnostic applications is often complicated by low concentrations of the analyte and at the same time by high complexity of the sample matrix, often requiring several analyte enrichment steps to obtain a suitable signal-to-noise ratio in the analysis. Therefore, sample preparation steps are often an important step in MS analysis and other IVD quantification methods / assays, as sample pre-treatment directly influences the accuracy of the analytical method. Precipitation of macromolecules is often used as pre-treatment, however, such methods require removal of the precipitate and can not be compatible with some downstream applications, e.g. bead enrichment. For steroid hormones such as estradiol, various methods have been proposed, such as solid phase (see CN113702558A) or liquid phase (CN111398446A) extraction, followed in each case by multiple reaction monitoring (MRM) mass spectrometry.

[0004] In addition to the analyte enrichment step, MS methods were developed to improve the analyte specific signal. Such methods include for example the use of analyte quantifier / analyte qualifier ion pairs in the MS analysis. A further improvement was the introduction of multiple reaction monitoring (MRM) in which the analyte derived ion is fragmented in a collision cell and the analyte derived product ions are determined. Due to the fact that ionization and fragment generation in both initial ionization and fragmentation are at least partially governed by the chemical structure of the analyte, a double specificity can be achieved. Nonetheless, especially in complex samples such as blood derived samples, the background can be considerable even in MRM. Furthermore, especially in cases where the analyte generates multiple fragments, the signal of the product ions in MRM can be unacceptably low. In addition, various methods were developed that are derived from MRM, although these methods differ greatly, they are all referred to as "pseudo MRM": for analytes that do not generate suitable fragments for MRM, such as menadione, a method was proposed in which a mass filter first quadrupole and a third quadrupole monitor the same molecular ion and are used for quantification (Kamao et al. (2017), Anal Sci 33:863); in this method the target ion is selectively transferred to a third resolving quadrupole mass filter without collision induced dissociation. Kim et al. (2015), Anal Chim Acta 882:38 proposed a Q-TOF instrument based pseudo MRM in which a specific precursor ion is filtered in Q1, the precursor ion is fragmented in Q2, all fragment ions are detected with a TOF detector and the fragments are individually quantified during data analysis. A similar method is proposed in EP 3 557 241 Al. Thus, the above "pseudo MRM" methods in the art differ from the traditional MRM strategy, i.e. filtering a single precursor analyte ion in a first mass filter, fragmenting the precursor analyte ion in a collision cell with a predetermined collision energy that is optimal for a specific fragment ion and detecting this single fragment ion after filtering this single fragment ion in a second mass filter.

[0005] Despite the above improvements, there is still a need in the art for improved tools and methods to determine analytes by MS, avoiding the problems of the prior art. This problem is solved by the tools and methods disclosed herein.

[0006] Thus, the present invention relates to a method of determining an analyte in a mass spectrometry (MS) device comprising a first mass filter and a second mass filter, the method comprising

[0007] (i) filtering in the first mass filter for an analyte ion species;

[0008] (ii) Optionally, at least a portion of the ions obtained by filtration in step (i) are fragmented in a collision cell, wherein the collision energy of the fragmentation is selected to be lower than a predetermined collision energy that causes fragmentation of the analyte ion species.

[0009] (iii) Filtering in a second mass filter for the analyte ions filtered in step (i); and

[0010] (iv) Detect the types of analyte ions filtered in step (iii) to identify the analyte.

[0011] Generally, the terms used herein are given their common and customary meanings to those skilled in the art, and are not limited to specific or customary meanings unless otherwise stated. As used below, the terms “have,” “comprise,” or “include,” or any grammatical variations thereof, 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 this context besides the features introduced by these terms, or to a situation where one or more other features exist. As an example, the expressions “A has B,” “A includes B,” and “A contains 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 those skilled in the art will understand, in one embodiment, the expressions “include one” and “include a” mean “include one or more,” that is, equivalent to “include at least one.” Therefore, unless otherwise stated, a statement involving one of a plurality of items refers to at least one such item in one embodiment and to a plurality of such items in another embodiment; thus, for example, identifying “one cell” refers to identifying at least one cell, and in one embodiment to identifying a plurality of cells. The term “plural” is as understood by those skilled in the art and refers to more than one in one embodiment, i.e., at least two, at least three in another embodiment, at least four in yet another embodiment, and at least five in yet another embodiment; however, a plurality of items may also be at least ten, at least 100, or at least 1000.

[0012] 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 other 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 by using alternative features. Similarly, features introduced by “in one embodiment” or similar expressions are intended to be optional features without limiting other 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.

[0013] In one embodiment, the method specifically described below is an in vitro method. In principle, the method steps can be performed in any order deemed appropriate by those skilled in the art, but in one embodiment they are performed in an indicated order; furthermore, one or more of the steps, in one embodiment all steps, can be assisted or performed by automated equipment. In addition, these methods may also include steps other than those explicitly mentioned above.

[0014] As used herein, unless otherwise specified, the term “about” refers to an indication value having a technical precision generally accepted in the relevant art, in one embodiment involving an indication value ±20%, in another embodiment ±10%, and in yet another embodiment ±5%. Furthermore, the term “substantially” means that there is no deviation affecting the indicated result or use, i.e., a potential deviation that would cause the indicated result to deviate from the specified result by more than ±20%, in another embodiment more than ±10%, and in yet another embodiment more than ±5%. Therefore, “substantially composed 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 effects of the invention. For example, a composition defined using the phrase “substantially composed of…” encompasses any known and acceptable additives, excipients, diluents, carriers, etc. In one embodiment, a composition substantially composed of one set of components will contain less than 5% by weight, less than 3% by weight in another embodiment, less than 1% by weight in another embodiment, and less than 0.1% by weight of (one or more) unspecified components.

[0015] The term "mass spectrometry device," or simply "MS device," is understood by those skilled in the art. In one embodiment, the term refers to a device configured for performing mass spectrometry (MS); thus, an MS device includes at least one mass spectrometry (MS) unit, and the term "MS unit" in one embodiment refers to a mass analyzer operatively connected to a detector configured to detect at least one analyte based on the mass-to-charge (m / z) ratio of the analyte or a fragment thereof. MS detectors are known in the art and particularly include electron multiplier detectors, Faraday cup detectors, photomultiplier detectors, and array detectors. An MS device may further include at least one ionization unit comprising an ionization source configured to generate molecular ions and to transfer the molecular ions into a gas phase for analysis in the MS unit. Ionization methods and suitable ionization units are known in the art and particularly include electrospray ionization (ESI), electron ionization (EI), chemical ionization (CI), atmospheric pressure ionization (APCI), atmospheric pressure photoionization (APPI), and matrix-assisted laser desorption / ionization (MALDI).

[0016] The term "mass filter" is understood by those skilled in the art and, in one embodiment, refers to any unit of an MS device that enables the removal of at least a portion of ions from said ions based on the m / z values ​​of the ions passing through the MS device to the detector. Thus, a mass filter enables the selection of ions within a predetermined range of m / z values. In principle, a mass filter can be configured to allow ions having all m / z values ​​to pass through, for example, for global analysis; however, in one embodiment, the mass filter is configured to select a predetermined range of m / z values. The specific range of m / z values ​​is determined by those skilled in the art based on the particular MS device hardware configuration, requirements arising from the analyte and the sample used, and other parameters known to those skilled in the art. In one embodiment, the mass filter is a quadrupole, as described below.

[0017] The term "collision cell" is also understood by those skilled in the art. Typically, a collision cell is configured to induce collisions of ions that have passed through a first mass analyzer containing an inert gas (such as Ar, He, or N2) to induce (further) fragmentation of said ions. In one embodiment, the collision cell is a quadrupole, as detailed below. The degree of fragmentation and which precursor ions fragment can be adjusted by adjusting the collision energy, which in turn can be adjusted by regulating the potential in the collision cell. Thus, the potential in the collision cell, for example in an MRM, is adjusted such that at least one precursor analyte ion species fragments into product analyte ion species. Appropriate collision energies (typically indicated as the potential applied in the collision chamber, eV, for example) are known in the art and can be determined by those skilled in the art using standard methods. Therefore, the term "collision energy" is used herein in the sense known to those skilled in the art. Specific embodiments of collision energy are described below.

[0018] As is known to those skilled in the art, detecting an analyte based on the mass-to-charge ratio of ions derived from the analyte requires at least one mass filter (such as a quadrupole that applies an electric or magnetic force to ions passing through the mass filter) and at least one detector to determine the ions that have passed through the mass filter. Suitable mass filters and detectors are known in the art. The MS apparatus mentioned herein includes at least a first mass filter and a second mass filter. In one embodiment, the first mass filter is a quadrupole and / or the second mass filter is a quadrupole; thus, in one embodiment, the MS apparatus includes at least two quadrupoles, and in another embodiment at least three quadrupoles. Thus, the MS apparatus can be, for example, a tandem mass spectrometry (MS / MS) apparatus, and in one embodiment, a triple quadrupole MS apparatus. As is known to those skilled in the art, a quadrupole can be used in an MS apparatus as a mass filter (i.e., for selecting ions with a predetermined m / z ratio), as a collision cell, or as a through cell. A quadrupole used as a mass filter can also be referred to as a “mass analyzer”. In the case of a triple quadrupole MS, in one embodiment, the first and third quadrupoles are configured as mass filters, configured in one embodiment to filter for the same m / z ratio, while the second quadrupole is configured as a pass-through pool, or in one embodiment as a collision pool, as described below.

[0019] In one embodiment, the MS device is included in a chromatographic MS system (particularly a gas chromatographic MS (GC-MS) system or a liquid chromatographic MS (LC-MS) system), terms as understood by those skilled in the art. Thus, in one embodiment, the system is configured for a combination of chromatography (e.g., LC or GC) and mass spectrometry (MS). Therefore, the chromatographic MS system may consist of an MS device further comprising at least one chromatographic device, wherein the chromatographic device and the units (particularly ionization units) of the MS device are operatively connected. The chromatographic device may be, in particular, a liquid chromatographic (LC) device or a gas chromatographic (GC) device. As used herein, the term "liquid chromatographic (LC) device" in one embodiment refers to an analytical device configured to separate one or more target analytes from other components of a sample via liquid chromatography, in one embodiment to detect one or more analytes using an MS device. LC can be based on any separation principle deemed suitable 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 apparatus may include at least one LC column. For example, an LC apparatus may be a single-column LC apparatus or a multi-column LC apparatus having multiple LC columns. The LC column may have a stationary phase through which a mobile phase is pumped to separate and / or elute and / or transfer the target analyte. An LC apparatus may be or may include at least one high-performance liquid chromatography (HPLC) unit and / or at least one microfluidic liquid chromatography (µLC) device. The term “gas chromatography” is as understood by a person skilled in the art; in the embodiments, the same separation principle as for LC applies, however, the mobile phase in GC is a gas.

[0020] As used herein, the term "analyte" refers to any chemical compound or group of compounds that should be identified in a sample. In one embodiment, the analyte is a macromolecule, i.e., a compound having a molecular mass 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 fragment thereof. In one embodiment, the analyte is a small molecule chemical compound, i.e., a compound having a molecular mass of at most 1000 u (1 kDa). In one embodiment, the analyte is a compound that produces a product ion from at least one precursor analyte ion in multiple reaction monitoring, in one embodiment producing the product ion with a collision energy of at most 60 Ω. In a further embodiment, the analyte is a compound that is 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. In one embodiment, the analyte comprises an organic at least 3-ring system, in one embodiment an organic at least 4-ring system. Therefore, in one embodiment, the analyte is a hormone, particularly estradiol, cortisol, progesterone, testosterone, 17-hydroxyprogesterone, aldosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), dihydrotestosterone, and / or cortisone; in another embodiment, the analyte is estradiol, and the sample is a serum sample.

[0021] However, the analyte can also be a prohibited drug or its metabolite, such as: amphetamine; cocaine; methadone; ethyl glucoside; ethyl sulfate; opium, especially buprenorphine, 6-monoylmorphine, codeine, dihydrocodeine, morphine, morphine-3-glucuronide and / or tramadol; and / or opioids, especially acetylfentanyl, carfentanil, fentanyl, hydrocodone, norfentanyl, oxycodone and / or hydroxymorphone.

[0022] 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.

[0023] As used herein, the term "subject" refers to a vertebrate, in one embodiment a mammal, particularly livestock, companion animals, or laboratory animals. In another embodiment, the subject is a human.

[0024] The term "analyte ion" is as understood by those skilled in the art. In one embodiment, the term refers to any ion that can be generated from an analyte in the ionization unit of an MS device. As those skilled in the art will understand, in addition to the analyte itself, the specific type(s) of ions(s) generated from the analyte(s) also depends on the specific method used for ionization and the ion mode used for analysis, i.e., whether a negative ion mode or a positive ion mode is used. However, for a given analyte, ionization, and ion mode, the analyte ion and its m / z value are generally known or can be determined by those skilled in the art using standard methods. Thus, in one embodiment, the analyte ion(s) as described herein are known to have a predetermined m / z ratio; thus, in one embodiment, the mass analyzer of the MS device can be configured to select an m / z range including at least one m / z value of at least one analyte ion. As indicated above, a variety of ions(s) with different m / z values ​​can be generated from a given analyte during ionization. As described herein, ions(s) with the same m / z value generated from an analyte are referred to herein as "analyte ion species". When the analyte is a hormone (especially estradiol), in one embodiment the method is carried out in negative ion mode, and in another embodiment ions with a mass of 271.170 or 271.171 are used as the analyte ion species.

[0025] As used herein, the term "sample" refers to any sample that contains or is suspected of containing at least one analyte. In one embodiment, the sample is a biological sample, in one embodiment a sample of body fluids, a sample of isolated cells, a sample from a tissue or organ, or a sample of a wash / rinse solution obtained from an external or internal surface of the body. Samples can be obtained using well-known techniques and include scrapes, swabs, and biopsy samples. Samples can be obtained using a brush, (cotton) swab, applicator, rinse / wash solution, puncture biopsy device, needle puncture of a cavity, or surgical instruments. Tissue or organ samples can be obtained from any tissue or organ, for example, through a biopsy or other surgical procedure. In one embodiment, the sample is a liquid sample, in another embodiment a sample of body fluids (e.g., in one embodiment, blood, plasma, serum, urine, saliva, tears, and fluids obtainable from the mammary gland (e.g., breast milk)). In another embodiment, the sample is a blood, plasma, or serum sample. Blood samples can be obtained by blood collection, for example, by puncturing an artery and / or vein. Plasma and serum samples can be obtained from blood samples using well-known methods. In one embodiment, the sample is a serum sample. In one embodiment, the sample contains or is suspected of containing at least one analyte, as described elsewhere herein. As used herein, the term "sample" refers to a sample such as that obtained from a subject; however, a sample may be pretreated prior to analysis, for example, to release the analyte and / or remove components from the sample matrix. Thus, a sample may be a pretreated sample, which is pretreated by, for example, immunoenrichment, organic solvent treatment, centrifugation, precipitation and / or macromolecular hydrolysis, desalting, addition of excipients, etc. Such pretreated samples shall still be referred to herein as samples.

[0026] As used herein, the term "identifying an analyte" means identifying at least one detectable characteristic of an analyte, which in one embodiment is at least one analyte ion as described above. In one embodiment, identifying an analyte confirms the presence or absence of an analyte in the sample at a concentration higher than the detection limit of the method; that is, in one embodiment, the identification is qualitative. The method for identifying the detection limit is known to those skilled in the art. In another embodiment, identification is a semi-quantitative or quantitative determination of the amount or concentration of the analyte in the sample. For a semi-quantitative determination, the amount may be specified as, for example, two or more predefined categories, such as being specified as above or below a reference value, or as low, medium, or high. For a quantitative determination, the absolute or precise amount of the analyte will be determined, or the relative amount of the analyte will be determined. A relative amount may be determined where the precise amount of the analyte cannot or should not be determined. In such cases, it may be determined whether the amount of the analyte present is increased or decreased relative to a reference sample containing a predetermined amount of the analyte. For quantitative determination, any parameter related to the amount or concentration of the analyte in the sample, or any value derived therefrom through standard mathematical and / or evaluative operations, can be determined and / or output, including multiplication, division, reciprocal formation, scaling, normalization, standardization, error correction, background correction, or mean or median calculations. In one embodiment, an intensity value obtained by or derived from a detector can be determined and / or output. As those skilled in the art will understand, quantitative determination may require the use of internal and / or external calibrations and / or the use of at least one internal and / or external standard.

[0027] Further steps, such as sample pretreatment steps, and particularly enrichment of the analyte beyond other sample components, may precede the determination of the analyte using the methods described herein. Therefore, as indicated above, sample matrix components can be removed by centrifugation, precipitation, desalting, and / or other steps deemed appropriate by a person skilled in the art. The specific steps performed to determine the analyte will also depend on the analyte, its expected concentration, the sample type, and other parameters known to a person skilled in the art. In one embodiment, the analyte is immunoenriched, i.e., in one embodiment, by binding to a binder such as an antibody, via the binder to a solid surface such as beads, and released from the binder after separation of the solid surface and the bound compound from the matrix components. Suitable methods are known to a person skilled in the art. In one embodiment, particularly when the analyte is a hormone, especially estradiol, sample pretreatment includes adding an internal standard to the sample (in one embodiment, a serum sample), treating the sample with an organic solvent (e.g., 30% methanol), adding a bead-bound anti-estradiol antibody, washing the beads, and releasing estradiol from the antibody, followed by application to an LC-MS apparatus; the eluent from the LC unit can be directly transferred to an ionization unit, such as an ESI ionization unit.

[0028] The method includes: step (i) filtering for an analyte ion species in a first mass filter; and step (iii) filtering for the same analyte ion species in a second mass filter. Those skilled in the art will understand the term "filtering" based on the description herein. In one embodiment, filtering is configuring a mass filter to allow ions having an m / z value of the target analyte ion species (i.e., in one embodiment, selected to be most suitable for the analyte ion species determined). Filtering can be a wide-pass filter, i.e., allowing all or most ions generated in the ionization unit to pass through; in one embodiment, filtering is a narrow-pass filter, i.e., configuring a mass filter to allow only ions with m / z values ​​close to the m / z value of the analyte ion species to pass through. As those skilled in the art will understand, the filtering in the first and second mass filters can be identical, i.e., both mass filters can be configured to allow the same range of m / z values ​​to pass through. However, the filtering in the first and second mass filters can also be different; for example, the first mass filter can be configured to perform wide-pass filtering, while the second mass filter can be configured for narrow-pass filtering. In one embodiment, narrow-pass filtering is used to filter ions with m / z values ​​ranging from ±0.5 to ±2 centered on the m / z value of the analyte ion species. In one embodiment, it is used to filter ions with m / z values ​​centered on approximately ±0.8 m / z (i.e., ±0.7% in one embodiment, ±0.3% in another, and ±0.2% in yet another) centered on the m / z value of the analyte ion species. In another embodiment, filtering is used to filter for the m / z value of the analyte ion species with the precision allowed by the instrument used. In any case, in the method described herein, the first mass filter and the second mass filter are for the same analyte ion species, i.e., for the same m / z value or the same range of m / z values.

[0029] The method further includes (iv) detecting the analyte ion species filtered in step (iii) to determine the analyte. The term "detection" is as understood by those skilled in the art and in one embodiment involves measuring and recording a signal generated by the detector of the MS unit for a target m / z value (i.e., typically the m / z value of the analyte ion species). The signal generated by the detector depends on the type of detector used; for example, in an electron multiplier, a voltage pulse is measured. The signal from the detector may be amplified, corrected (e.g., against background), and otherwise further evaluated in a manner deemed appropriate by those skilled in the art before being used to determine the analyte. As understood by those skilled in the art, the signal detected by the detector unit of the MS device can be used to determine the analyte as described above, for example, to determine an absolute or relative concentration. However, depending on the intended use, it may be sufficient to measure and record the signal generated by the detector itself. Therefore, in one embodiment, the determination of the analyte in step (iv) is based on detecting the analyte ion species as the only ion species determined; that is, in one embodiment, the determination does not include fragment ions that determine the analyte ion species.

[0030] In one embodiment, the MS unit of the MS device includes three quadrupoles; that is, in one embodiment, the MS device is a triple quadrupole mass spectrometer (TQMS) device. Such a TQMS device can, for example, be a tandem mass spectrometer consisting of a first quadrupole mass analyzer and a second quadrupole mass analyzer connected in series, with the intermediate pure radio frequency (RF) quadrupole positioned between the first and second mass analyzers; thus, the TQMS device can be configured such that the first quadrupole is the first mass analyzer, the second quadrupole is the intermediate quadrupole, and the third quadrupole is the second mass analyzer. In this case, in one embodiment, the second (intermediate) quadrupole is configured to act as a non-mass-resolved quadrupole, for example, as a through cell or collision cell. Suitable arrangements are known in the art and are used, for example, in multiple reaction measurement (MRM) MS methods.

[0031] In one embodiment, the aforementioned second quadrupole is configured as a collision cell. Therefore, in one embodiment, step (ii) involves fragmenting at least a portion of the ions obtained through filtration in step (i) in the quadrupole, wherein the collision energy for fragmentation is selected to be lower than a predetermined collision energy that would cause fragmentation of the analyte ion species. How to configure a quadrupole as a collision cell is known to those skilled in the art. As illustrated by examples, for instance in estradiol determination, a precursor analyte ion species with a mass of 271.17 can be subjected to a collision energy of 45 to produce a quantitative product analyte ion species with a mass of 145.065. Similarly, in estradiol determination, a precursor analyte ion species with a mass of 271.17 can be subjected to a collision energy of 65 to produce a qualitative product analyte ion species with a mass of 143.050. As understood by those skilled in the art based on the description herein, the ion fragments generated by the aforementioned fragmentation have a lower m / z value compared to their parent ion; therefore, if both the first and second mass filters are used for filtering at the same m / z value, the aforementioned ion fragments will be filtered out in the second mass filter. Thus, in one embodiment, step (ii) may be: (ii) optionally fragmenting at least a portion of the ions obtained by filtration in step (i) in a collision cell, wherein the collision energy of the fragmentation is selected to be lower than a predetermined collision energy that causes fragmentation of a portion of the analyte ion species and filters out that portion. In an optional embodiment, the collision energy remains substantially constant during step (ii) of the method, and in one embodiment, it remains substantially constant during steps (ii) through (iv) of the method, wherein “remaining substantially constant” means keeping the collision energy within ±20% (±10% in one embodiment, ±5% in another embodiment) of a predetermined value.

[0032] However, according to the method described herein, in the embodiments described above, the analyte ion species do not fragment in the collision cell (e.g., the second quadrupole), or the analyte ion species fragment only to a small extent, and the first and second mass filters of the MS unit are configured to filter for the same analyte ion species. For example, up to 30%, up to 20% in one embodiment, and up to 10% in another embodiment, of the analyte ions passing through the first mass filter fragment in the second quadrupole. Therefore, the collision energy in the collision cell is configured to cause fragmentation of the analyte ions passing through the first mass filter to at most the aforementioned extent. Based on the description herein, those skilled in the art will understand that in one embodiment of the method, fragmentation of non-analyte ions is induced, but fragmentation of analyte ions is not induced or is induced only to a low extent; therefore, the signal-to-noise ratio is improved in this method.

[0033] There are various options for determining the collision energy required to achieve the effect of fragmenting matrix-derived ions (but not fragmenting or only fragmenting analyte ions to a low degree through the first mass filter), i.e., for providing an appropriate collision energy according to the method. In one embodiment, a predetermined collision energy is provided (e.g., from the prior art) that provides an optimal signal of product ions from the analyte in the MRM; in another embodiment, the optimal MRM analyte collision energy is predetermined by a standard method for MRM optimization; in yet another embodiment, a predetermined collision energy is determined to cause fragmentation of at least a portion of the analyte ions obtained through filtration in step (i), wherein in one embodiment, the predetermined collision energy is a collision energy that causes fragmentation of at least 90%, at least 75%, and at least 50% of the analyte ions in another embodiment. In all of these cases, the collision energy used in one embodiment is at most 70%, at most 75%, at most 80%, and at most 90% of the aforementioned predetermined collision energy. In another embodiment where a suitable collision energy is determined, the pre-established MRM method is modified by continuously reducing the collision energy in the collision cell until the intensity of the precursor analyte ion species increases by at least 3 times, in one embodiment at least 4 times, and in another embodiment at least 5 times after obtaining the second mass analyzer, and the increased collision energy is used as the collision energy in step (ii). Thus, in one embodiment, the method further includes step (ii), causing at least a portion of the ions obtained through filtration in step (i) to fragment, wherein the collision energy for fragmentation is selected to be lower than a predetermined collision energy that causes fragmentation of the analyte ion species. As those skilled in the art will understand, depending on the specific application, the collision energy may also be significantly lower than the predetermined collision energy, for example, where only the removal of matrix ions fragmented at low collision energies is required. In one embodiment, the collision energy applied in step (ii) is 5% to 90% of the predetermined collision energy, and in another embodiment, it is 10% to 80% of the predetermined collision energy. In one embodiment, the collision energy used in step (ii) is 70% of the aforementioned predetermined collision energy, in another embodiment 75%, in yet another embodiment 80%, and in yet another embodiment 90%.Therefore, in the aforementioned example of estradiol determination, a quantitative precursor analyte ion of mass 271.17 can be subjected to a collision energy of 45 in the MRM to produce a product analyte ion of mass 145.065; thus, according to the method described herein, a quantitative analyte ion of mass 271.17 can be subjected to a collision energy of, for example, 36 (i.e., 80% of 45) to retain an analyte ion of mass 271.17, which is then detected in step (iv).

[0034] Advantageously, in the foundational work of this invention, it was discovered that using an MS device with two mass analyzers to perform double filtration of the same analyte ions can improve analyte detection. Furthermore, it was found that by performing pseudo-MRM, using collision energies low enough to avoid analyte ion fragmentation, analyte detection can be improved by reducing the amount of non-analyte ions, thereby increasing the signal-to-noise ratio in the analysis.

[0035] The definitions made above apply mutatis mutandis to those below. Further additional definitions and explanations below also apply mutatis mutandis to all embodiments described in this specification.

[0036] The present invention further relates to an MS device, the MS device comprising:

[0037] (I) A first quadrupole, which is suitable for use as a first mass filter;

[0038] (II) Second quadrupole;

[0039] (III) A third quadrupole, suitable for use as a second mass filter; and

[0040] (IV) Control unit, which includes a microprocessor,

[0041] The control unit includes tangibly embedded executable code that, when executed on the microcontroller, causes the device to perform the method according to the invention.

[0042] As used herein, the term "device" generally refers to a collection of described tools operatively connected to each other to provide the indicated function. The device may be implemented in a single physical unit or in physically separate units operatively connected to each other. Suitable components and their properties are described above in the context of the method. Therefore, the method of the present invention is implemented by the device described herein. Thus, in one embodiment, the device is configured to perform at least one method as described above. The device may include additional units, particularly input units, data processing units, output units, communication interfaces, and / or any other units deemed appropriate by a person skilled in the art. The device referred to herein is an MS device including the components and properties described above; therefore, an MS device may particularly further include an ionization unit and a detector unit, both as described above.

[0043] In one embodiment, the MS device includes an input unit. As used herein, the term "input unit" refers to any unit configured to transfer information from another entity to the device (particularly its data processing unit or data storage medium), wherein the other entity may be another data processing device or a user. Thus, the input unit may include a user interface; however, the input unit may also be a storage medium comprising a collection of data from which appropriate values ​​can be retrieved. In one embodiment, the input unit is adapted to implement input of at least one method parameter value.

[0044] The term "method parameter" as used herein refers to any parameter that a person skilled in the art would consider relevant to performing the described method. Thus, a parameter can be an analyte identifier, sample type, ion mode, analyte ion species, collision energy, etc. In one embodiment, a method parameter is selected from the m / z value of the analyte ion species, the molecular mass of the analyte ion species, and / or the collision energy. In one embodiment, for example, in a database, at least one method parameter is assigned to an analyte identifier such that, for example, a user or control software can select an appropriate method parameter based on an indication of the analyte to be identified. The database may, for example, be embedded (in one embodiment, tangibly embedded) in a memory cell of an MS device or in a data storage device connected to the MS device via a communication interface. Suitable memory cells are known to a person skilled in the art.

[0045] The term "data processing unit" generally refers to any unit suitable for performing one or more method steps as described above, executed in one embodiment using at least one processor and / or at least one application-specific integrated circuit. Thus, as an example, the at least one data processing unit may include software code stored thereon, comprising a plurality of computer instructions. The data processing unit may provide instructions to one or more hardware elements for performing one or more of the indicated operations, and / or may provide software running thereon to one or more processors for performing one or more of the method steps.

[0046] As used herein, the term "output unit" refers to any arbitrary unit configured to transfer information from a device to another entity, which may be a separate data processing and / or storage device and / or a user. Therefore, an output device may include a user interface (such as a suitably configured display) or may be a printer.

[0047] The term "communication interface" is understood by those skilled in the art to refer to any interface configured for the exchange of information, particularly data. 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, and 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.

[0048] The present invention also relates to an analytical system comprising the MS apparatus and chromatographic apparatus of the present invention (all as described above).

[0049] The present invention also relates to a database preferably tangibly embedded in a data carrier, the database including at least one analyte identifier assigned to at least one method parameter of the present invention.

[0050] As used herein, the term "database" refers to a collection of data that can be physically and / or logically grouped together. Thus, in one embodiment, the database includes assigning at least an analyte identifier to a collision energy value for use in the methods described herein, and in another embodiment, further assigning it to an analyte ion species value, such as its m / z value. Thus, the database implements the assignment of analytes to at least one method parameter. In one embodiment, the database includes additional data (such as upper and / or lower detection limits), data related to authenticity checks, etc. In another embodiment, the database includes data regarding the use of one or more determination methods, batch-specific data (e.g., for internal standards or calibrator samples), etc. In one embodiment, the database may be implemented in a single data storage medium or in physically separate data storage media operatively connected to each other. In one embodiment, the database includes a collection of data on a suitable storage medium, which, in one embodiment, is tangibly embedded therein. Furthermore, in one embodiment, the database further includes a database management system. In one embodiment, the database management system is a web-based, hierarchical, or object-oriented database management system. Additionally, the database may be a federated or integrated database. Also in one embodiment, the database will be implemented as a distributed (federated) system, such as a client-server system.

[0051] The present invention also relates to the use of an MS apparatus according to the invention for determining analytes in a sample.

[0052] The present invention further discloses and proposes a computer program comprising computer-executable instructions for performing the method according to the invention in one or more embodiments appended herein, when executed on a computer or computer network. Specifically, the computer program may be stored on a computer-readable data carrier. Thus, specifically, one embodiment may use a computer or computer network to perform one, more, or even all of the method steps a) to d) as indicated above by using a computer program.

[0053] The present invention further discloses and proposes a computer program product having program code tools so that, when 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.

[0054] Furthermore, the present invention 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.

[0055] 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.

[0056] Finally, the present invention 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.

[0057] 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, in general, any method steps, including providing and / or manipulating 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 work (such as providing samples and / or performing certain aspects of actual measurement).

[0058] Specifically, the present invention further discloses:

[0059] A computer or computer network comprising at least one processor, wherein the processor is adapted to perform a method according to one of the embodiments described herein.

[0060] - A computer-loadable data structure adapted to perform one of the embodiments described in this specification when the data structure is executed on a computer.

[0061] - 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.

[0062] - A computer program, including program means 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.

[0063] - A computer program, comprising program means according to the foregoing embodiments, wherein the program means is stored on a computer-readable storage medium.

[0064] - A storage medium, wherein a data structure is stored on the storage medium and wherein the data structure is adapted to be loaded into the main memory and / or working memory of a computer or computer network, and to be used in a method according to one of the embodiments described in this specification.

[0065] - A computer program product having a program code tool, wherein the program code tool can be stored or stored on a storage medium for performing a method according to one of the embodiments described in this specification if the program code tool is executed on a computer or computer network.

[0066] In summary, the following embodiments are particularly envisioned based on the findings of this invention:

[0067] Example 1: A method for determining an analyte in a mass spectrometry (MS) apparatus including a first mass filter and a second mass filter, the method comprising...

[0068] (i) Filtering is performed in the first mass filter according to the analyte ion species;

[0069] (ii) Optionally, at least a portion of the ions obtained by filtration in step (i) are fragmented in a collision cell, wherein the collision energy of the fragmentation is selected to be lower than a predetermined collision energy that causes fragmentation of the analyte ion species.

[0070] (iii) Filtering in a second mass filter for the analyte ions filtered in step (i); and

[0071] (iv) Detect the types of analyte ions filtered in step (iii) to identify the analyte.

[0072] Example 2: The method according to Example 1, wherein the sample is a biological sample, in one embodiment a sample of the subject's bodily fluids, in another embodiment blood or a blood-derived sample, and in one embodiment a serum sample.

[0073] Example 3: The method according to Example 1 or 2, wherein the analyte is an organic compound, and in one example is a metabolite of the subject.

[0074] Example 4: The method according to any one of Examples 1 to 3, wherein the first mass filter is a quadrupole and / or the second mass filter is a quadrupole.

[0075] Example 5: The method according to any one of Examples 1 to 4, wherein the MS device includes three quadrupoles.

[0076] Example 6: The method according to Example 5, wherein the first quadrupole is used as the first mass filter in step (i), the second quadrupole is optionally used as the collision pool in step (iii), and the third quadrupole is used as the second mass filter in step (ii).

[0077] Example 7: The method according to any one of Examples 1 to 6, wherein the collision pool in step (ii) is a quadrupole.

[0078] Example 8: The method according to any one of Examples 1 to 7, wherein the predetermined collision energy is the collision energy that causes at least 90%, at least 75% in one example, and at least 50% of the analyte ions to fragment, and in one example, the optimal collision energy for the same analyte ion species in a multiple reaction measurement.

[0079] Example 9: The method according to any one of Examples 1 to 8, wherein the collision energy selected in step (ii) is at most 50% of the predetermined collision energy, at most 75% in one embodiment, at most 80% in another embodiment, and at most 90% in yet another embodiment.

[0080] Example 10: The method according to any one of claims 1 to 9, wherein the collision energy selected in step (ii) is 50% to 90% of the predetermined collision energy, and in one embodiment, it is 50% to 80%.

[0081] Example 11: The method according to any one of Examples 1 to 9, wherein the collision energy selected in step (ii) is 70% to 90% of the predetermined collision energy, and in one embodiment it is 70% to 80%.

[0082] Example 12: The method according to any one of Examples 1 to 11, wherein the filtering in step (iii) is a narrow-pass filter.

[0083] Example 13: According to the method of Example 12, the narrow-pass filtering is for filtering ions whose m / z values ​​are within ±2 of the m / z value of the analyte ion species.

[0084] Example 14: The method according to any of Examples 1 to 13, wherein up to 30%, up to 20% in one embodiment, and up to 10% in another embodiment, of the analyte ions passing through the first mass filter are broken up in the second quadrupole.

[0085] Example 15: The method according to any of Examples 1 to 14, wherein the determination in step (iv) is based on detecting the analyte ion species as the only determined ion species.

[0086] Example 16: According to the method of Example 5, wherein the first quadrupole is used as the first mass filter in step (i), the second quadrupole is used to transfer ions from the first quadrupole to the third quadrupole (ii), and the third quadrupole is used as the second mass filter in step (iii).

[0087] Example 17: The method according to any one of Examples 1 to 16, wherein the analyte produces at least one product analyte ion species different from the analyte ion species with a collision energy of up to 60.

[0088] Example 18: The method according to any one of Examples 1 to 17, wherein the analyte comprises an organic 3-ring system, and in one example an organic 4-ring system.

[0089] Example 19: The method according to any one of Examples 1 to 18, wherein the analyte is estradiol.

[0090] Example 20: The method according to any one of Examples 1 to 19, wherein the method further comprises: step (o), ionizing the analyte molecules contained in the sample.

[0091] Example 21: The method according to any one of Examples 1 to 20, wherein the method further includes a sample pretreatment step.

[0092] Example 22: The method according to any one of Examples 1 to 21, wherein the sample pretreatment step includes immunoenrichment and / or chromatography of the sample.

[0093] Example 23: The method according to any one of Examples 1 to 22, wherein the sample pretreatment step includes mixing the internal standard with the sample.

[0094] Example 24: The method according to Example 23, wherein the internal standard is an isotopic configuration of the analyte.

[0095] Example 25: The method according to any one of Examples 1 to 24, wherein the sample is pretreated by immunoenrichment.

[0096] Example 26: The method according to any one of Examples 1 to 25, wherein the sample is pretreated by chromatography.

[0097] Example 27: The method according to any one of Examples 1 to 26, wherein the sample is pretreated by immunoenrichment and by chromatography.

[0098] Example 28: The method according to Example 26 or 27, wherein the chromatography is liquid chromatography (LC).

[0099] Example 29: The method according to Example 28, wherein the LC eluent is used directly for ionization in step (o).

[0100] Example 30: The method according to any one of Examples 1 to 29, wherein in step (iii), specifically, ion species within ±0.7% of the m / z range of the analyte ion species, ±0.3% in one example, and ±0.2% in another example, are detected.

[0101] Example 31: A method for determining an analyte in a mass spectrometry (MS) apparatus comprising a first mass filter, a second mass filter, and a collision cell, the method comprising

[0102] (i) Filtering is performed in the first mass filter according to the analyte ion species;

[0103] (ii) In the collision cell, at least a portion of the ions obtained by filtration in step (i) are fragmented, wherein the collision energy of the fragmentation is selected to be lower than a predetermined collision energy that causes fragmentation of the analyte ion species.

[0104] (iii) Filtering the analyte ions in the second mass filter; and

[0105] (iv) Detect the types of analyte ions filtered in step (iii) to identify the analyte.

[0106] Example 32: The method according to Example 31, further comprising the features described in any one of Examples 1 to 24.

[0107] Example 33: The method according to any one of Examples 1 to 32, wherein the analyte is a small molecule chemical compound with a molecular weight of up to 1000 u (1 kDa).

[0108] Example 34: An MS device comprising

[0109] (I) A first quadrupole, which is suitable for use as a first mass filter;

[0110] (II) Second quadrupole;

[0111] (III) A third quadrupole, suitable for use as a second mass filter; and

[0112] (IV) Control unit, which includes a microprocessor,

[0113] The control unit includes tangibly embedded executable code that, when executed on the microcontroller, causes the device to perform the method described according to any one of Embodiments 1 to 33.

[0114] Example 35: The MS device according to Example 34 further includes an input unit, wherein in one embodiment, the input unit is adapted to implement input of at least one method parameter value.

[0115] Example 36: The MS device according to Example 34 or 35 further includes a memory unit that includes a database that includes at least one analyzer identifier assigned to at least one value of at least one method parameter.

[0116] Example 37: An MS apparatus according to any one of Examples 34 to 36, wherein the at least one method parameter is selected from the m / z value of the analyte ion species, the molecular mass of the analyte ion species, and / or the collision energy.

[0117] Example 38: The MS device according to any one of Examples 34 to 37 further includes an ionization unit and a detector unit.

[0118] Example 39: An analytical system comprising an MS apparatus and a chromatographic apparatus as described in any one of Examples 34 to 38.

[0119] Example 40: The analytical system according to Example 39, wherein the chromatographic device is an LC device.

[0120] Example 41: A database preferably tangibly embedded on a data carrier, the database including at least one analyte identifier assigned at least one method parameter as described in Example 36.

[0121] Example 42: A data carrier comprising a database tangibly embedded according to Example 41.

[0122] Example 43: The MS apparatus according to any one of Examples 34 to 38 is used to determine the analytes in a sample.

[0123] All publicly available information in all references cited in this specification, as well as any publicly available information specifically mentioned herein, is incorporated herein by reference. Attached Figure Description

[0124] Figure 1: Signal intensity of estradiol in LC-MS with retention time. Some measurement results overlap with sample 1 in Table 1; (A) pseudo MRM, signal 271.17 -> 271.17; (B) MRM, signal 271.17 -> 145.066.

[0125] Figure 2: As shown in Figure 2, but using human serum sample W528000 from Table 1. (A) Pseudo MRM, signal 271.17 -> 271.17; (B) MRM, signal 271.17 -> 145.066.

[0126] Figure 3: Area ratio (AR, area analyte / area ISTD) of samples 1 to 5 in Table 1 with increasing spike concentration, measured using (A) pseudo MRM or (B) MRM.

[0127] The following examples are intended to illustrate the invention only. In any case, they should not be construed as limiting the scope of the invention.

[0128] Example 1: Determination of estradiol

[0129] The method described herein is similar to conventional MRM methods, but is essentially a dual single-ion monitoring MS method. Therefore, in accordance with the use of terminology in the prior art, the method of the present invention is referred to as "pseudo-MRM" in the examples provided herein; however, this method should not be confused with methods of the prior art that use the same name thereto and are described above in the introduction.

[0130] 1.1 Sample Preparation

[0131] 150 µL of human serum was mixed with 10 µL of internal standard (10 ng / ml 13C3-estradiol), and vortexed and incubated. 50 µL of 30% MeOH was added, and the sample was incubated after vortexing to dissociate estradiol from binding proteins (e.g., albumin) in human serum. Subsequently, 40 µL of internally prepared magnetic beads (1 mg / ml) coated with E2-MAK-Ab (anti-estradiol antibody) was added and vortexed, followed by incubation to selectively capture estradiol and 13C3-estradiol and immobilize them on the magnetic beads.

[0132] After incubation, the magnetic beads were immobilized using a magnet, the supernatant was removed, and the beads were washed twice with 200 µL of water to minimize matrix components from human serum. Estradiol and 13C3-estradiol were released from the E2-MAK-Ab magnetic beads using 80 µL of 80% MeOH; 50 µL was transferred to a fresh sample container and diluted with 50 µL of water to reduce organic content and thereby improve HPLC performance.

[0133] 1.2 LC Separation

[0134] LC separation of estradiol from other sample components was performed using the following parameters:

[0135] HPLC column: Phenomenex, Kinetex C18; 1.0 mm ID x 50 mm; 2.6 μm

[0136] Column temperature: 55°C

[0137] Flow rate: 440 µL / min

[0138] Eluent A: LC-MS grade water

[0139] Eluent B: LC-MS grade MeOH containing 0.04 mM NH4

[0140] Injection volume: 20 µL (full ring injection)

[0141] Gradient: Maintain equilibration at 39% eluent B for 36 s, LC injection, gradually transition from 39% to 98% eluent B over 36 s (flow rate only switched to MS during this time), maintain rinsing at 98% eluent B for 36 s.

[0142] Total time for LC method: 108 s

[0143] 1.3 Mass Spectrometry

[0144] Use the following parameters to perform MS (CE: collision energy at arbitrary values).

[0145] ESI Temperature: 500℃

[0146] MS polarity: negative mode ionization

[0147] ESI potential: -3.5 kV

[0148] Sprayer gas: 3 L / min

[0149] Assist (heating) gas: 15 L / min

[0150] Air curtain (reverse) gas: 6 L / min

[0151] Collision gas: 0.001 L / min

[0152] Purge gas: 2 L / min

[0153] MS resolution: Resolution per unit at both Q1 and Q3

[0154] MS settings specific to traditional MRM (comparative example)

[0155] 271.170 (Q1) -> 145.065 (Q3) Analyte quantitative ions (67 ms residence time, CE = 45)

[0156] 271.171 (Q1) -> 143.050 (Q3) Qualitative ions of analyte (67 ms residence time, CE = 65)

[0157] 274.180 (Q1) -> 148.076 (Q3) ISTD quantitative ion (10 ms residence time, CE = 45)

[0158] 274.181 (Q1) -> 146.160 (Q3) ISTD qualitative ion (10 ms residence time, CE = 65)

[0159] MS settings specific to pseudo-MRM (based on an example according to the invention)

[0160] Pseudo-MRM: 271.170 (Q1) -> 271.171 (Q3) Analyte quantitative ions (67 ms residence time, CE= 36)

[0161] 274.180 (Q1) -> 274.180 (Q3) ISTD quantitative ion (10 ms residence time, CE = 36)

[0162] 1.4 Optimization of Collision Energy

[0163] The collision energy (CE) in Example 2.3 below was optimized to 45 using the conventional MRM method to obtain the highest signal for the quantitative ions of analytes from 271.170 (Q1) to 145.065 (Q3). Starting from the aforementioned value of 45, the CE was sequentially decreased until the quantitative ion signal for analytes from 271.170 (Q1) to 271.171 (Q3) (pseudo-MRM) increased by 4 to 5 times compared to the quantitative ion signal for analytes from 271.170 (Q1) to 145.065 (Q3) (conventional MRM), which was optimally achieved at 80% of the CE of conventional MRM (i.e., 36 instead of 45).

[0164] 1.5 Results

[0165] The results shown in Table 1 and Figures 1 and 2 below were obtained using the pure diluent (UniDil) and diluents spiked with five different concentrations of estradiol (samples 1 to 5) and human serum samples.

[0166] The area measured against the internal standard in the MRM is 2.8 to 2.9E5, while the area measured against the pseudo-MRM in samples 1 to 5 is 1.4E6. Therefore, the area in the pseudo-MRM according to the invention is increased by approximately 4.5 times. Furthermore, as will be understood from Table 1, the signal-to-noise ratio (S / N) increases by up to 2.5 times when using the pseudo-MRM. The reduction in noise is also shown in Figures 1 and 2.

[0167] As shown in Figure 3, the linearity of the pseudo-MRM measurement results is excellent, with a value of 99.8%, compared to 98.6% obtained using MRM.

[0168] Therefore, pseudo-MRM offers several advantages over traditional MRM, while being implemented on the same device.

[0169] literature

[0170] CN113702558A

[0171] CN111398446A

[0172] EP 3 557 241 A1

[0173] Kamao et al. (2017), Anal Sci 33:863

[0174] Kim et al. (2015), Anal Chim Acta 882:38

[0175]

Claims

1. A method for determining an analyte in a mass spectrometry (MS) apparatus comprising a first mass filter, a second mass filter, and a collision cell, the method comprising (i) Filtering in the first mass filter according to the analyte ion species; (ii) In the collision cell, at least a portion of the ions obtained by the filtration in step (i) are fragmented, wherein the collision energy of the fragmentation is selected to be lower than a predetermined collision energy that would cause fragmentation of the analyte ion species. (iii) Filtering the analyte ions in the second mass filter; as well as (iv) Detect the types of analyte ions filtered in step (iii) to identify the analyte.

2. The method of claim 1, wherein the sample is a biological sample, in one embodiment a sample of bodily fluids of a subject, in another embodiment blood or a blood-derived sample, and in one embodiment a serum sample.

3. The method according to claim 1 or 2, wherein the analyte is an organic compound, in one embodiment a metabolite of the subject, and in another embodiment estradiol.

4. The method according to any one of claims 1 to 3, wherein the MS device comprises three quadrupoles.

5. The method according to claim 4, wherein the first quadrupole is used as the first mass filter in step (i), the second quadrupole is used as the collision pool in step (ii), and the third quadrupole is used as the second mass filter in step (iii).

6. The method according to any one of claims 1 to 5, wherein the predetermined collision energy is a collision energy that causes at least 90% fragmentation of the analyte ions, at least 75% fragmentation in one embodiment, at least 50% fragmentation in another embodiment, and in one embodiment, the optimal collision energy for the same analyte ion species in a multiple reaction measurement.

7. The method according to any one of claims 1 to 6, wherein the collision energy selected in step (ii) is at most 50% of the predetermined collision energy, at most 75% in one embodiment, at most 80% in another embodiment, and at most 90% in yet another embodiment.

8. The method according to any one of claims 1 to 6, wherein the collision energy selected in step (ii) is 50% to 90% of the predetermined collision energy, and in one embodiment, it is 50% to 80% of the predetermined collision energy.

9. The method according to any one of claims 1 to 6, wherein the collision energy selected in step (ii) is 70% to 90% of the predetermined collision energy, and in one embodiment, it is 70% to 80% of the predetermined collision energy.

10. The method according to any one of claims 1 to 9, wherein the filtering in step (iii) is a narrow pass filter.

11. The method of claim 10, wherein the narrow-pass filtering is for filtering ions whose m / z values ​​are within ±2 centered on the m / z value of the analyte ion species.

12. The method according to any one of claims 1 to 11, wherein up to 30%, in one embodiment up to 20%, and in another embodiment up to 10% of the analyte ions passing through the first mass filter are fragmented in the second quadrupole.

13. The method according to any one of claims 1 to 12, wherein the determination in step (iv) is based on detecting the analyte ion species as the only determined ion species.

14. The method according to any one of claims 1 to 13, wherein the method further comprises a sample pretreatment step, wherein in one embodiment, the sample pretreatment step comprises immunoenrichment and / or chromatography of the sample.

15. The method according to any one of claims 1 to 14, wherein the sample pretreatment step comprises mixing an internal standard with the sample, wherein in one embodiment, the internal standard is an isotopic configuration of the analyte.

16. The method according to any one of claims 1 to 15, wherein in step (iv), the ion species are specifically detected within the range of m / z ± 0.7% of the analyte ion species, in one embodiment within the range of m / z ± 0.3% of the analyte ion species, and in another embodiment within the range of m / z ± 0.2% of the analyte ion species.

17. The method according to any one of claims 1 to 16, wherein the analyte is a small molecule chemical compound with a molecular mass of up to 1000 u (1 kDa).

18. An MS device comprising: (I) A first quadrupole, which is suitable for use as a first mass filter; (II) The second quadrupole, which is suitable for use as a collision pool; (III) A third quadrupole, which is suitable for use as a second mass filter; as well as (IV) Control unit, which includes a microprocessor, The control unit includes tangibly embedded executable code that, when executed on the microcontroller, causes the device to perform the method according to any one of claims 1 to 17.

19. The MS device of claim 18, further comprising a memory unit including a database including at least one analyzer identifier assigned to at least one value of at least one method parameter.

20. The MS apparatus according to claim 18 or 19, wherein the at least one method parameter is selected from the m / z value of the analyte ion species, the molecular mass of the analyte ion species, and / or the collision energy.

21. An analytical system comprising an MS apparatus and a chromatographic apparatus according to any one of claims 11 to 13.

22. The use of the MS apparatus according to any one of claims 18 to 21 for determining an analyte in a sample.

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