Derivatization of β-lactam antibiotics for mass spectrometric determination in patient samples

Abstract

The present invention relates to the derivatization of antibiotic analytes and methods for determining the amount or concentration of the derivatized antibiotic analyte in a resulting sample.

Description

[Technical Field]

[0001] The present invention relates to the derivatization of antibiotic analytes and methods for determining the amount or concentration of the derivatized antibiotic analyte in a resulting sample. [Background technology]

[0002] background β-lactam antibiotics are the most commonly prescribed class of antibiotics for patients with bacterial infections, either as specific or broad-spectrum antibiotics. This class of antibiotics acts by interfering with the cross-linking of the peptidoglycan layer, which is most prevalent in Gram-positive bacteria. They exhibit concentration-dependent bactericidal activity. Therefore, it is important to maintain antibiotic concentrations above the MIC. However, higher concentrations can result in adverse effects. Furthermore, the pharmacokinetics of these compounds have been reported to be highly variable and therefore unpredictable (Ronilda D'Cunha et al.; 2018; Antimicrobial Agents and Chemotherapy 62(9)).

[0003] The mechanism of action of these antibiotics is by reacting the four-membered β-lactam ring with D-alanyl-D-alanyl-transpeptidase, thereby inhibiting the formation of cross-links between peptidoglycan polymers of the outer cell wall.

[0004] Thus, the relatively unstable lactam moiety is responsible for the mechanism of action of these antibiotics. However, this instability also leads to partial hydrolysis of these compounds when dissolved in protic solvents. Even more so, hydrolysis is naturally further catalyzed by the presence of acid or base and enhanced at elevated temperatures. Clearly, hydrolyzed antibiotics are no longer active compounds capable of inhibiting bacterial growth.

[0005] Therapeutic drug monitoring (TDM) is a field of medicine that aims to quantify drugs from human sample materials for the purpose of monitoring drug concentrations in the body. Considerable efforts have been made to study and address β-lactam instability in the field of TDM, primarily focusing on storage conditions aimed at preserving compounds in their native (i.e., unhydrolyzed) form (Zander et al.; 2016; Clinical Chemistry and Laboratory Medicine; 54(2)). Obtaining accurate concentrations of natural β-lactam antibiotics in patients is currently very challenging because hydrolysis continues even after patient sampling (e.g., blood draw). Careful monitoring of antibiotic concentrations is crucial, so effective and stable methods for quantifying these compounds from human and animal matrices are needed. Summary of the Invention

[0006] Summary of the Invention In a first aspect, the present invention provides an (automated) method for determining the amount or concentration of one or more derivatized antibiotic analytes in an obtained sample, comprising: a) optionally pre-treating and / or concentrating the sample, in particular using magnetic beads; and b) determining the amount or concentration of one or more antibiotic analytes in the sample; The present invention relates to a method, including:

[0007] In a second aspect, the present invention provides an (automated) method for determining the amount or concentration of one or more antibiotic analytes in an obtained sample, comprising: a) pretreating the sample with a derivatization reagent, wherein the derivatization reagent comprises a nucleophile; b) optionally concentrating the sample obtained after step a), in particular using magnetic beads; and c) determining the amount or concentration of one or more antibiotic analytes in the pretreated sample obtained after step a) or after the optional concentration step b). The present invention relates to a method, including:

[0008] In a third aspect, the present invention relates to an (automated) analytical system (in particular an LC / MS system) adapted to carry out the method of the first or second aspect.

[0009] In a fourth aspect, the present invention relates to a sampling tube for collecting a patient sample that contains a nucleophilic derivatization reagent suitable for stabilizing one or more antibiotic analytes in the sample.

[0010] In a fifth aspect, the present invention relates to the use of a nucleophilic derivatization reagent for determining the amount or concentration of one or more antibiotic analytes in a sample.

[0011] In a sixth aspect, the present invention relates to the use of a nucleophilic derivatization reagent to stabilize an antibiotic analyte in a sample of interest.

[0012] In a seventh aspect, the present invention relates to antibiotic analytes stabilized with a nucleophilic derivatization reagent. [Brief explanation of the drawings]

[0013] Drawing List [Figure 1] Schematic representation of the hydrolysis pathway of piperacillin. [Figure 2A] Measured peak areas of A) native piperacillin (compound 5); B) monohydrolyzed piperacillin (9a or 9b) in water at room temperature over 16 hours. Shown with confidence fit and F-test. A reference line is drawn through the average area values ​​for clarity. [Figure 2B] Measured peak areas of A) native piperacillin (compound 5); B) monohydrolyzed piperacillin (9a or 9b) in water at room temperature over 16 hours. Shown with confidence fit and F-test. A reference line is drawn through the average area values ​​for clarity. [Figure 3] Schematic diagram of the derivatization reaction of meropenem with different reagents: A) propylamine; B) butylamine, C) pentylamine. [Figure 4]Schematic of the derivatization reaction of piperacillin with different reagents: A) propylamine; B) butylamine, C) pentylamine. [Figure 5] Measured peak areas of doubly derivatized piperacillin (compound 7) in water at room temperature over 16 hours for two MRM transitions. Shown with confidence fit and F-test. A reference line has been drawn through the average area values ​​for clarity. [Figure 6] Measured peak areas of meropenem derivatized with the reagents propylamine, butylamine, and pentylamine under different reaction conditions. [Figure 7] Measured peak areas of piperaziline derivatized with the reagents propylamine, butylamine, and pentylamine under different reaction conditions. [Figure 8] A schematic diagram of signal versus concentration is shown, which shows that the spiked concentration is higher than the actual concentration, as shown in Example 4, and the calibration offset arises from the difference between the spiked concentration and the actual concentration. [Figure 9] As shown in Example 4, the area ratio difference between neat and serum-derived samples for four concentrations. [Figure 10] Example 5: Accuracy and precision results. [Figure 11] Correlations were calculated for concentrations from both methods as shown in Example 5. [Figure 12] Correlations were calculated for concentrations from both methods as shown in Example 5. [Figure 13] Difference in accuracy between the two methods per replicate, as shown in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description of the Invention Before describing the present invention in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0015] Several documents are cited herein. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. In the event of a conflict between a definition or teaching of such an incorporated reference and a definition or teaching cited herein, the text of the present specification shall control.

[0016] Each element of the present invention is described below. While these elements are listed with specific embodiments, it is understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application unless the context dictates otherwise.

[0017] definition It will be understood that the word "comprise", and variations such as "comprises" and "comprising", imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0018] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0019] Ratios, concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range." It is understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the numerical values ​​expressly recited as boundaries of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly recited. By way of example, a numerical range of "4% to 20%" should be interpreted not only to include the explicitly recited value 4% to 20%, but also to include each individual value and subrange within the stated range. Thus, this numerical range includes individual values ​​such as 4, 5, 6, 7, 8, 9, 10, ... 18, 19, 20%, etc., and subranges such as 4-10%, 5-15%, 10-20%, etc. This same principle applies to ranges reciting minimum or maximum values. Moreover, such interpretation should apply regardless of the breadth of the range or the described characteristics.

[0020] The term "about," when used in connection with a numerical value, is meant to encompass numerical values ​​within a range having a lower limit of 5% less than the stated numerical value and an upper limit of 5% greater than the stated numerical value.

[0021] The terms "measurement", "determining" or "determining" preferably include qualitative, semi-quantitative or quantitative measurements.

[0022] The term "automated" refers to a method or process that is primarily operated by automatic equipment, i.e., by a machine or computer, to reduce the amount of work done by a human and the time it takes to perform the work. Thus, in an automated method, tasks that were previously performed by a human are now performed by a machine or computer. Typically, a user only needs to configure the tools and define the process. Those skilled in the art are well aware that while some minor aspects may still require manual intervention, the majority of the method is performed automatically.

[0023] In the context of this disclosure, the terms "analyte," "analyte molecule," or "analyte of interest" are used interchangeably to refer to a chemical species being analyzed. A chemical species suitable for analysis, i.e., an analyte, can be any type of molecule present in a living organism, including, but not limited to, nucleic acids, amino acids, peptides, proteins, fatty acids, lipids, carbohydrates, steroids, ketosteroids, and secosteroid molecules. An analyte can also be any substance internalized by an organism, such as, but not limited to, a therapeutic drug, a drug of abuse, a toxin, or a metabolite of such a substance. Therapeutic drugs include antibiotics, i.e., "antibiotic analytes." Antibiotics are substances active against microorganisms. Antibiotics are generally classified based on their mechanism of action, chemical structure, or spectrum of activity. One class of antibiotics is β-lactam antibiotics. β-lactam antibiotics are all antibiotics that contain a β-lactam ring in their molecular structure. These include, but are not limited to, penicillin derivatives (penams), cephalosporins (cephems), monobactams, carbapenems, and carbacephems. Most β-lactam antibiotics act by inhibiting cell wall biosynthesis in bacterial organisms and are the most widely used group of antibiotics. The effectiveness of these antibiotics depends on their ability to reach intact PBPs and bind to penicillin-binding proteins (PBPs).

[0024] The analyte may be present in a sample of interest, e.g., a biological sample or a clinical sample. The terms "sample" or "sample of interest" are used interchangeably herein and refer to a portion or piece of a tissue, organ, or individual, and are usually smaller than such tissue, organ, or individual, which is intended to represent the entire tissue, organ, or individual. Upon analysis, the sample yields information regarding the state of the tissue, or the health or disease state of the organ or individual. Examples of samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, cerebrospinal fluid, urine, saliva, and lymphatic fluid, or solid samples such as dried blood spots and tissue extracts. A further example of a sample is a cell culture or tissue culture.

[0025] In the context of this disclosure, a sample may be derived from an "individual" or "subject." Typically, the subject is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0026] Prior to analysis, samples may be pretreated in a manner specific to the sample and / or analyte. In the context of this disclosure, the term "pretreatment" refers to any measure necessary to enable subsequent analysis of the desired analyte. Pretreatment measures typically include, but are not limited to, elution of solid samples (e.g., elution of dried blood spots), addition of a hemolyzing reagent (HR) to whole blood samples, and addition of an enzyme reagent to urine samples. Similarly, addition of an internal standard (ISTD) is considered sample pretreatment.

[0027] Typically, an internal standard (ISTD) is a known quantity of a substance that exhibits similar properties to the analyte of interest when subjected to a mass spectrometric detection workflow (i.e., including any pretreatment, enrichment, and actual detection steps). The ISTD exhibits similar properties to the analyte of interest, but is clearly distinguishable from it. By way of example, during a chromatographic separation, such as gas or liquid chromatography, the ISTD has approximately the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the mass spectrometer simultaneously. The ISTD, however, exhibits a different molecular weight than the analyte of interest from the sample. This allows ions from the ISTD and the analyte to be distinguished in mass spectrometry using their different mass-to-charge (m / z) ratios. Both are subjected to fragmentation to produce daughter ions. These daughter ions can be distinguished by their m / z ratios and their respective parent ions. As a result, after calibration, the signals from the ISTD and the analyte can be determined and quantified separately. Because the ISTD is added in a known amount, the signal intensity of an analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of the ISTD allows for relative comparison of the amount of analyte detected, allowing for unambiguous identification and quantification of the analyte of interest present in the sample when the analyte(s) reach the mass spectrometer. Typically, although not necessarily, the ISTD is an isotopically labeled variant of the analyte of interest (e.g., 2 H, 13 C, or 15 (including labels such as N).

[0028] The term "immunoglobulin (Ig)," as used herein, refers to immune conferring glycoproteins of the immunoglobulin superfamily. "Surface immunoglobulins" are attached to the membrane of effector cells by their transmembrane regions and include molecules such as, but not limited to, B cell receptors, T cell receptors, class I and II major histocompatibility complex (MHC) proteins, β2-microglobulin (approximately 2M), CD3, CD4, and CDS.

[0029] Typically, the term "antibody," as used herein, refers to a secretory immunoglobulin that lacks a transmembrane region and can therefore be released into the bloodstream and body cavities. Human antibodies are classified into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains, designated by Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the class of antibody (i.e., these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively), and each plays a different role and directs the appropriate immune response to different types of antigens. Different heavy chains vary in size and composition and can contain approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). IgA is found in mucosal areas such as the gastrointestinal, respiratory, and genitourinary tracts, as well as in saliva, tears, and breast milk, where it prevents colonization by pathogens (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389-417). IgD primarily functions as an antigen receptor for unexposed B cells and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437; Chen et al. (2009) Nat. Immunol. 10:889-898). IgE is involved in allergic responses through binding to allergens, which triggers histamine release from mast cells and basophils. IgE is also involved in protection against parasites (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides the majority of antibody-based immunity against invading pathogens and is the only antibody isotype that can cross the placenta and confer passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four distinct IgG subclasses (IgG1, 2, 3, and 4), named in order of abundance in serum, with IgG1 being the most abundant (approximately 66%), followed by IgG2 (approximately 23%), IgG3 (approximately 7%), and IgG4 (approximately 4%).The biological profiles of different IgG classes are determined by the structure of their respective hinge regions. IgM is expressed on the surface of B cells in a monomeric form and in a secreted pentameric form with very high avidity. IgM is involved in the early stage of B cell-mediated (humoral) immunity, eliminating pathogens before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437). Antibodies are not only found as monomers, but are also known to form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM of bony fish), or pentamers of five Ig units (e.g., mammalian IgM). Antibodies are typically made up of four polypeptide chains, including two identical heavy chains and two identical light chains linked via disulfide bonds, resembling a "Y"-shaped macromolecule. Each chain contains several immunoglobulin domains, some of which are constant domains and others are variable domains. Immunoglobulin domains consist of a two-layer sandwich of seven to nine antiparallel chains arranged in two sheets. Typically, an antibody heavy chain contains four Ig domains, three of which are constant (CH domains: CHI, CH2, CH3) domains and one of which is a variable domain (VH). A light chain typically contains one constant Ig domain (CL) and one variable Ig domain (VL). For example, a human IgG heavy chain is composed of four Ig domains linked from N- to C-terminus in the order VwCH1-CH2-CH3 (also referred to as VwCy1-Cy2-Cy3), while a human IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order VL-CL, and is either kappa or lambda type (VK-CK or VA-CA). By way of example, the constant chain of human IgG contains 447 amino acids.Throughout this specification and claims, the numbering of amino acid positions in immunoglobulins is that of the "EU index" as in Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS, and Foeller, C. (1991) Sequences of proteins of immunological interest, 5th ed., US Department of Health and Human Services, National Institutes of Health, Bethesda, MD. "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Thus, the CH domain in the context of IgG is as follows: "CH1" refers to amino acid positions 118-220 according to the EU index as in Kabat; "CH2" refers to amino acid positions 237-340 according to the EU index as in Kabat; and "CH3" refers to amino acid positions 341-447 according to the EU index as in Kabat.

[0030] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment as defined below. These terms specifically refer to an antibody having a heavy chain that includes an Fc region.

[0031] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab fragments" (also called "Fab portions" or "Fab regions"), each containing a single antigen-binding site, and a remaining "Fc fragment" (also called "Fc portion" or "Fc region"), named for its ability to readily crystallize. The crystal structure of the human IgG Fe region has been determined (Deisenhofer (1981) Biochemistry 20:2361-2370). In IgG, IgA, and IgD isotypes, the Fe region consists of two identical protein fragments derived from the CH2 and CH3 domains of the antibody's two heavy chains, while in IgM and IgE isotypes, the Fe region contains three heavy-chain constant domains (CH2-CH4) in each polypeptide chain. Additionally, smaller immunoglobulin molecules exist naturally or have been artificially constructed. The term "Fab' fragment" refers to a Fab fragment that additionally contains the hinge region of an Ig molecule, while a "F(ab')2 fragment" is understood to include two Fab' fragments that are chemically linked or linked via disulfide bonds. While "single-domain antibodies (sdAbs)" (Desmyter et al. (1996) Nat. Structure Biol. 3:803-811) and "nanobodies" contain only a single VH domain, "single-chain Fv (scFv)" fragments contain a heavy-chain variable domain linked to a light-chain variable domain via a short linker peptide (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Bivalent single-chain variable fragments (di-scFv) can be engineered by linking two scFvs (scFvA-scFvB). This can be done by generating a single peptide chain with two VH and two VL regions, resulting in a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is to create an scFv with a linker that is too short to allow the two variable regions to fold together, forcing the scFv to dimerize. Typically, a linker with a length of 5 residues is used to generate these dimers. This type is known as a "diabody."Shorter linkers (one or two amino acids) between the VH and VL domains result in the formation of monospecific trimers, so-called "triabodies" or "tribodies." Bispecific diabodies are formed by expressing chains with the sequences VHA-VLB and VHB-VLA, or VLA-VHB and VLB-VHA, respectively. Single-chain diabodies (scDbs) contain VHA-VLB and VHB-VLA fragments (VHA-VLB-P-VHB-VLA) linked by a linker peptide (P) of 12 to 20 amino acids, preferably 14 amino acids. "Bispecific T cell engagers (BiTEs)" are fusion proteins consisting of two scFvs of different antibodies, one of which binds to T cells via the CD3 receptor and the other to tumor cells via a tumor-specific molecule (Kufer et al. (2004) Trends Biotechnol. 22:238-244). Dual affinity retargeting molecules ("DART" molecules) are diabodies further stabilized by a C-terminal disulfide bridge.

[0032] Thus, the term "antibody fragment" refers to a portion of an intact antibody, preferably comprising its antigen-binding region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab'), Fv fragments; diabodies; sdAbs, nanobodies, scFv, di-scFv, tandem scFv, triabodies, diabodies, scDbs, BiTEs, and DARTs.

[0033] The term "binding affinity" generally refers to the strength of the sum of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including, but not limited to, surface plasmon resonance-based assays (e.g., BIAcore assays, such as those described in PCT Application Publication WO 2005 / 012359); enzyme-linked immunosorbent assays (ELISAs); and competitive assays (e.g., RIAs). Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens rapidly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

[0034] "Sandwich immunoassays" are widely used for detecting an analyte of interest. In such assays, the analyte is "sandwiched" between a first antibody and a second antibody. Typically, sandwich assays require that the capture and detection antibodies bind to different, non-overlapping epitopes on the analyte of interest. The sandwich complex is measured by appropriate means, thereby quantifying the analyte. In a typical sandwich-type assay, a first antibody bound to or capable of binding to a solid phase and a detectably labeled second antibody each bind to the analyte at different, non-overlapping epitopes. A binding agent (e.g., an antibody) specific for the first analyte is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer; the most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support may be a tube, bead, microplate disk, or any other surface suitable for performing immunoassays. The binding process is well known in the art and generally consists of cross-linking, covalent binding, or physical adsorption. The polymer-antibody complex is then washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid-phase complex and incubated for a period of time (e.g., 2-40 minutes, or more conveniently, overnight) sufficient to allow binding between the first or capture antibody and the corresponding antigen, and under appropriate conditions (e.g., room temperature to 40°C, e.g., 25°C to 37°C, inclusive). Following the incubation period, the solid phase containing the first or capture antibody and the antigen bound to the antibody is washed and may be incubated with a second or labeled antibody that binds to a different epitope on the antigen. The second antibody is conjugated to a reporter molecule that is used to indicate binding of the second antibody to the complex of the first antibody and the antigen of interest.

[0035] A highly versatile alternative sandwich assay format involves the use of a solid phase coated with a first partner of a binding pair, e.g., a microparticle coated with paramagnetic streptavidin. Such microparticles are incubated with an analyte-specific binding agent bound to a second partner of the binding pair (e.g., a biotinylated antibody), a sample suspected of containing or containing an analyte whose second partner of the binding pair is bound to the analyte-specific binding agent, and a detectably labeled second analyte-specific binding agent. As will be apparent to those skilled in the art, these components are incubated under appropriate conditions for a period of time sufficient to allow binding of the analyte, the analyte-specific binding agent (bound to) the second partner of the binding pair, and the labeled antibody via the first partner of the binding pair to the solid-phase microparticles. Optionally, such assays may include one or more washing steps.

[0036] The term "detectably labeled" encompasses labels that can be detected either directly or indirectly.

[0037] A directly detectable label either provides a detectable signal, or the label interacts with a second label to modify the detectable signal provided by the first or second label, for example, to provide FRET (fluorescence resonance energy transfer). Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (Briggs et al., "Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058) provide a detectable signal and are generally applicable to labeling. In one embodiment, detectably labeled refers to a label that provides or can be induced to provide a detectable signal, i.e., a fluorescent label, a luminescent label (e.g., a chemiluminescent label or an electrochemiluminescent label), a radioactive label, or a metal chelate-based label, respectively.

[0038] Numerous labels (also referred to as dyes) are available that can be broadly grouped into the following categories, all of which together represent embodiments according to the present disclosure:

[0039] (a) Fluorescent dye Fluorescent dyes are described, for example, by Briggs et al., "Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058.

[0040] Fluorescent labels or fluorophores include rare earth chelates (europium chelates), fluorescein-type labels (including FITC, 5-carboxyfluorescein, and 6-carboxyfluorescein), rhodamine-type labels (including TAMRA), dansyl, Lissamine, cyanine, phycoerythrin, Texas Red, and their analogs. Fluorescent labels can be attached to aldehyde groups contained within target molecules using the techniques disclosed herein. Fluorescent dyes and fluorescent labeling reagents include those commercially available from Invitrogen / Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).

[0041] (b) Luminescent dye Luminescent dyes or labels can be further sub-classified into chemiluminescent dyes and electrochemiluminescent dyes.

[0042] Different classes of chemiluminescent labels include systems based on luminol, acridinium compounds, coelenterazine and analogues, dioxetanes, peroxyoxalic acid and peroxyoxalic acid derivatives. For immunodiagnostic procedures, acridinium-based labels are mainly used (a detailed overview is given in Dodeigne C. et al., Talanta 51 (2000) 415-439).

[0043] The main relevant labels used as electrochemiluminescent labels are ruthenium- and iridium-based electrochemiluminescent complexes, respectively. Electrochemiluminescence (ECL) has proven to be very useful for analytical applications as a highly sensitive and selective method. ECL combines the analytical advantages of chemiluminescence analysis (absence of background light signal) with the ease of reaction control by applying an electrode potential. Generally, ruthenium complexes, especially [Ru(Bpy)3]2+ (emitting photons at approximately 620 nm) regenerated with TPA (tripropylamine) in the liquid phase or at the liquid-solid interface, are used as ECL labels.

[0044] Electrochemiluminescence (ECL) assays provide sensitive and accurate measurements of the presence and concentration of an analyte of interest. Such techniques use a label or other reactant that can be induced to emit light when electrochemically oxidized or reduced in the appropriate chemical environment. Such electrochemiluminescence is triggered by a voltage applied to a working electrode in a specific manner at a specific time. The light produced by the label is measured and indicates the presence or amount of the analyte. For a more complete description of such ECL techniques, see U.S. Pat. No. 5,221,605; U.S. Pat. No. 5,591,581; U.S. Pat. No. 5,597,910; PCT Publication No. WO 90 / 05296; PCT Publication No. WO 92 / 14139; PCT Publication No. WO 90 / 05301; PCT Publication No. WO 96 / 24690; PCT Publication No. US 95 / 03190; PCT Publication No. US 97 / 16942; PCT Publication No. US 96

[0013] Reference is made to PCT Publication Nos. WO 95 / 08644, WO 96 / 06946, WO 96 / 33411, WO 87 / 06706, WO 96 / 39534, WO 96 / 41175, WO 96 / 40978, PCT / US97 / 03653, and U.S. Patent Application No. 08 / 437,348 (U.S. Patent No. 5,679,519). Reference is also made to the 1994 review of analytical applications of ECL by Knight, et al. (Analyst, 1994, 119:879-890), and the references cited therein. In one embodiment, the methods herein are practiced using electrochemiluminescent labels.

[0045] Recently, iridium-based ECL labels have also been described (WO2012107419).

[0046] The radiolabel employs a radioactive isotope (radionuclide), such as H, C, C, F, P, S, Cu, Gn, Y, Zr, TC, In, I, I, I, I, Xe, Lu, At, or Bi.

[0047] (d) Metal chelate complexes suitable as labels for imaging and therapeutic purposes are well known in the art (U.S. Patent Application Publication No. 2010 / 0111861; U.S. Patent No. 5,342,606; U.S. Patent No. 5,428,155; U.S. Patent No. 5,316,757; U.S. Patent No. 5,480,990; U.S. Patent No. 5,462,725; U.S. Patent No. 5,428,139; U.S. Patent No. 5,385,893; U.S. Patent No. 5,739,294; U.S. Patent No. 5,750,660; U.S. Patent No. 5,834,461; Hnatowich et al., J. Immunol. Methods 65 (1983) 147-157; Meares et al., Anal. Biochem. 142 (1984) 68-78; Mirzadeh et al., Bioconjugates Chem.1(1990)59-65;Meares et al,J.Cancer(1990),Suppl.10:21-26;Izard et al,Bioconjugate Chem.3(1992)346-350;Nikula et al,Nucl.Med.Biol.22(1995)387-90;Camera et al. al,Nucl.Med.Biol.20(1993)955-62;Kukis et al,J.Nucl.Med.39(1998)2105-2110;Verel et al.,J.Nucl.Med.44(2003)1663-1670;Camera et al. al,J.Nucl.Med.21(1994)640-646;Ruegg et al,Cancer Res.50(1990)4221-4226;Verel et al,J.Nucl.Med.44(2003)1663-1670;Lee et al,Cancer Res.61(2001)4474-4482;Mitchell,et al,J.Nucl.Med.44(2003)1105-1112;Kobayashi et al Bioconjugate Chem.10(1999)103-111;Miederer et al,J.Nucl.Med.45(2004)129-137;DeNardo et al,Clinical Cancer Research 4(1998)2483-90;Blend et al,Cancer Biotherapy&Radiopharmaceuticals18(2003)355-363;Nikula et al J.Nucl.Med.40(1999)166-76;Kobayashi et al al, J. Nucl. Med. 39 (1998) 829-36; Mardirossian et al, Nucl. Med. Biol. 20 (1993) 65-74; Roselli et al, Cancer Biotherapy & Radiopharmaceuticals, 14 (1999) 209-20). .

[0048] The term "mass spectrometry" ("Mass Spec" or "MS") refers to an analytical technique used to identify compounds by their mass. MS is a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." MS techniques generally involve (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating their mass-to-charge ratio. Compounds can be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. Generally, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrometry instrument, where a combination of magnetic and electric fields causes the ions to follow a path in space depending on their mass ("m") and charge ("z"). The term "ionization" or "ionizing" refers to the process of generating ions of an analyte having a net charge equal to one or more electron units. Anions are those that have a net negative charge of one or more electronic units, while cations are those that have a net positive charge of one or more electronic units. MS methods can be performed in either "negative ion mode," in which anions are generated and detected, or "positive ion mode," in which positive ions are generated and detected.

[0049] "Tandem mass spectrometry" or "MS / MS" involves multiple steps of selective mass analysis, with analyte fragmentation occurring between steps. In a tandem mass spectrometer, ions are generated in an ion source and separated by mass-to-charge ratio in the first stage of mass analysis (MS1). Ions of specific mass-to-charge ratios (precursor ions or parent ions) are selected, and fragment ions (daughter ions) are generated by collision-induced dissociation, ion-molecule reactions, or photodissociation. The resulting ions are then separated and detected in the second stage of mass analysis (MS2). Most sample workflows in MS further include a sample preparation and / or enrichment step, where, for example, the analyte of interest is separated from the matrix using gas or liquid chromatography. Typically, for a mass spectrometry measurement, three steps are performed:

[0050] 1. A sample containing the analyte of interest is ionized, usually by adduct formation with a cation, often by protonation to the cation. Ionization sources include, but are not limited to, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

[0051] 2. The ions are sorted and separated according to their mass and charge. High field asymmetric waveform ion mobility spectrometry (FAIMS) can be used as an ion filter.

[0052] 3. The separated ions are detected, for example, in multiple reaction mode (MRM), and the results are displayed on a chart.

[0053] The term "electrospray ionization" or "ESI" refers to a method in which a solution is passed down a short length of capillary tube and a high positive or negative potential is applied to the end of the capillary tube. The solution that reaches the end of the tube is vaporized (atomized) into a jet or spray of very small droplets in solvent vapor. This mist of droplets passes through an evaporation chamber, which is slightly heated to prevent condensation and evaporate the solvent. As the droplets become smaller, the electrical surface charge density increases until natural repulsion between like charges causes ions and neutral molecules to be released.

[0054] The term "atmospheric pressure chemical ionization" or "APCI" refers to a mass spectrometry method similar to ESI. However, APCI generates ions through ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electrical discharge between the spray capillary and a counter electrode. The ions are then typically extracted into a mass analyzer using a set of differentially pumped skimmer stages. Counterflow drying and preheated N2 gas can be used to improve solvent removal. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar entities.

[0055] "Multiple reaction mode" or "MRM" is a detection mode of an MS instrument in which a precursor ion and one or more fragment ions are selectively detected.

[0056] Mass spectrometers separate and detect ions of slightly different masses, easily distinguishing between different isotopes of a given element. Mass spectrometry is therefore an important method for accurate mass determination and characterization of analytes, including, but not limited to, low molecular weight analytes, peptides, polypeptides, or proteins. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits, and functional interactions, and the total measurement of proteins in proteomics. De novo sequencing of peptides or proteins by mass spectrometry can usually be performed without prior knowledge of the amino acid sequence.

[0057] Mass spectrometric determination may be combined with additional analytical methods, including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and / or ion mobility-based separation techniques.

[0058] The term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of the differential distribution of the chemical components as they flow around or over a stationary liquid or solid phase.

[0059] The terms "liquid chromatography" or "LC" refer to the process of selectively retarding one or more components of a fluid solution as the fluid permeates uniformly through a column or capillary passage of finely divided material. Retardation results from the distribution of mixture components between one or more stationary phase(s) and the bulk fluid (i.e., mobile phase) as the fluid moves relative to the stationary phase(s). Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal phase liquid chromatography (NPLC), while methods in which the stationary phase is less polar than the mobile phase (e.g., a water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) are called reversed phase liquid chromatography (RPLC).

[0060] "High-performance liquid chromatography" or "HPLC" refers to a method of liquid chromatography in which the degree of separation is increased by passing a mobile phase under pressure through a stationary phase, typically a densely packed column. Typically, the column is packed with a stationary phase composed of irregular or spherical particles, a porous monolithic layer, or a porous membrane. HPLC has historically been divided into two distinct subclasses based on the polarity of the mobile and stationary phases. Methods in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) are called normal-phase liquid chromatography (NPLC), while the opposite (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is called reverse-phase liquid chromatography (RPLC). Micro-LC refers to HPLC methods using columns with narrow internal diameters, typically less than 1 mm, e.g., about 0.5 mm. "Ultra-high performance liquid chromatography" or "UHPLC" refers to an HPLC method using a pressure of 120 MPa (17,405 lbf / in2), or approximately 1200 atmospheres. Rapid LC refers to an LC method using a short column with an internal diameter as described above and a length of less than 2 cm, e.g., 1 cm, at the above flow rate and pressure (micro LC, UHPLC). A short rapid LC protocol involves trapping / washing / elution steps using a single analytical column, achieving LC in a very short time of less than 1 minute.

[0061] Additionally, hydrophilic interaction chromatography (HILIC), size-exclusion LC, ion-exchange LC, and affinity LC are well known.

[0062] LC separations may be single-channel LC or multi-channel LC, comprising multiple LC channels arranged in parallel, in which analytes may be separated according to their polarity or log P value, size, or affinity, as is commonly known to those skilled in the art.

[0063] In the context of the present invention, the term "complex" refers to a chemical substance having a specific chemical structure. The complex may contain one or more functional units. Each unit may perform a different function, and two or more functional units may perform the same function.

[0064] In the context of the present invention, the term "nucleophile" refers to a chemical species that donates an electron pair to form a chemical bond. Nucleophiles present in aqueous media include, but are not limited to, -NH2, -OH, -SH, -Se, (R', R'', R''')P, N3-, RCOOH, F-, Cl-, Br-, and I-. In the context of the present invention, the term "nucleophilic derivatization reagent" or "nucleophile derivatization reagent" refers to a reagent that contains such a nucleophile. A nucleophilic derivatization reagent contains a moiety with an orbital that serves as the highest occupied molecular orbital (HOMO) that can attack the lowest unoccupied molecular orbital (LUMO) of a substance of interest, such as an analyte of interest, thereby forming a new molecule composed of the previous nucleophilic unit and the analyte moiety.

[0065] A "kit" is any article of manufacture (e.g., a package or container) containing at least one reagent, such as a drug for treating a disorder or a probe for specifically detecting a biomarker gene or protein of the invention. The kit is preferably promoted, distributed, or sold as a unit for carrying out the method of the invention. Typically, the kit can further include a carrier means compartmentalized to receive in close confinement one or more container means, such as vials, tubes, etc. In particular, each of the container means comprises one of the separate elements used in the method of the first aspect. The kit may further include one or more other containers containing additional materials, including, but not limited to, buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. Labeling may be provided on the container to indicate that the composition is to be used in a particular application and may also indicate instructions for either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or directly to a computer or data processing device. Additionally, the kit may contain standard amounts of biomarkers as described elsewhere herein for calibration purposes.

[0066] "Package insert" is used to refer to instructions customarily included in commercial packaging of a therapeutic or drug product, which contain information about the indications, uses, dosage, administration, contraindications of such therapeutic or drug product, other therapeutic products with which the packaged product may be combined, and / or warnings regarding its use.

[0067] The term "sampling tube" or "sample collection tube" refers to any device having a reservoir suitable for receiving a blood sample to be collected.

[0068] Embodiment Commonly used approaches to measure antibiotics, particularly β-lactam antibiotics, aim to overcome their instability. In contrast, the present invention does not overcome but rather exploits the reactivity of antibiotics by reacting them with a suitable nucleophile, thereby providing an accurate measurement of antibiotics in patient samples.

[0069] In a first aspect, the present invention provides a method for determining the amount or concentration of one or more derivatized antibiotic analytes in a sample obtained, comprising: a) optionally pre-treating and / or concentrating the sample, in particular using magnetic beads; and b) determining the amount or concentration of one or more antibiotic analytes in the sample; The present invention relates to a method, comprising:

[0070] In embodiments, the derivatized antibiotic analyte is an adduct formed between a nucleophilic derivatization reagent and the antibiotic analyte. In certain embodiments, the derivatized antibiotic analyte is a covalent adduct formed between a nucleophilic derivatization reagent and the antibiotic analyte. In embodiments, the derivatized antibiotic analyte exhibits increased stability compared to the same underivatized antibiotic analyte.

[0071] In embodiments, the antibiotic analyte is a lactam antibiotic analyte. In embodiments, the antibiotic analyte is a β-lactam antibiotic analyte. In certain embodiments, the antibiotic analyte is selected from the group consisting of amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, temocillin, phenethicillin, penicillin G, penicillin V, piperacillin, azlocillin, bibampicillin, pivmecillinam, ticarcillin, cephacefetrile, cefadroxil ... Cefadroxil, Cefalexin, Cephalexin, Cephaloglycin, Cephalonium, Cephalonium, Cephaloridine , Cefalotin (cephalothin), Cefapirin (cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (cephazolin), Cephradine (cephradine), Cephradine (cephradine), Cefroxadine, Cefroxadine, Ceftezole, Cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil (cefproxil), cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefdoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine,Cefepime, Cefluprenam, Cefoselis, Cefozopran, Cefpirome, Cefquinome, Ceftobiprole, Ceftaroline, Cefacromezine, Cephaloram, Cefaparol, Cefcanel, Cefedrolol, Cefenpidone, Cefetrizole, Cefibitril, Cefmatilen, Cefmepidium, Cefovecin, Cefoxazole, Cefrotil, Cefsumide, Cefuracetim, The antibiotic analyte is selected from the group consisting of ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreonam, mecillinam, meampicillin, talampicillin, epicillin, sulbenicillin, faropenem, ritipenem, biapenem, pivampicillin, clometocillin, penamecillin, hetacillin, carindacillin, panipenem, tigemonam, carumonam, nocardicin A, penam, sulbactam, tazobactam, clavam, and clavulanic acid. In certain embodiments, the antibiotic analyte is meropenem or piperacillin.

[0072] In embodiments, antibiotic analytes are derivatized with nucleophilic derivatization reagents, particularly reagents containing amine groups, particularly primary or secondary amines, and particularly primary amine groups. Primary amine groups have the advantage of allowing for shorter incubation times compared to secondary amines. In embodiments, antibiotic analytes are derivatized with nucleophilic derivatization reagents containing more than three C atoms, particularly 3 to 20 C atoms, particularly 3 to 10 C atoms, particularly 3 to 5 C atoms, and particularly 4 C atoms. In embodiments, antibiotic analytes are derivatized with linear or branched nucleophilic derivatization reagents, particularly linear amines, particularly linear primary amines, and particularly linear primary amines containing 3 to 5 C atoms. In embodiments, antibiotic analytes are derivatized with nucleophilic derivatization reagents selected from the group consisting of propylamine, butylamine, or pentylamine, particularly primary linear butylamine or primary linear pentylamine. Therefore, MS interference can be reduced or avoided.

[0073] In embodiments, the derivatized antibiotic analyte is derivatized at at least one of its chemical moieties. Those skilled in the art of chemistry will be familiar with chemical moieties that are particularly suitable for derivatization with nucleophilic derivatization reagents. In certain embodiments, the derivatized antibiotic analyte is derivatized at one, two, or three of its chemical moieties.

[0074] In certain embodiments, the antibiotic analyte is meropenem, which is derivatized with a nucleophilic derivatization reagent comprising butylamine. See also Figure 3.

[0075] In certain embodiments, the antibiotic analyte is piperacillin, which is derivatized with a nucleophilic derivatization reagent that includes pentylamine.

[0076] In certain embodiments, where the antibiotic analyte is piperacillin, it is derivatized at two of its chemical moieties, specifically the β-lactam ring and the piperazine ring, with a nucleophilic derivatization reagent that includes pentylamine. See also Figure 4.

[0077] In embodiments, samples containing derivatized antibiotic analytes can be pretreated and / or concentrated by a variety of methods. The pretreatment method depends on the type of sample, such as blood (fresh or dried), plasma, serum, urine, or saliva, while the concentration method depends on the analyte of interest. Those skilled in the art will be familiar with which pretreatment methods are suitable for which sample types. Those skilled in the art will also be familiar with which concentration methods are suitable for which analytes of interest.

[0078] In embodiments where the sample is a whole blood sample, the sample is assigned to one of two predetermined sample preparation (PT) workflows, both of which include the addition of an internal standard (ISTD) and a hemolysis reagent (HR) followed by a predetermined incubation period (Inc), where the difference between the two workflows is the order in which the internal standard (ISTD) and the hemolysis reagent (HR) are added. In embodiments, the ISTD is added to the resulting sample first, followed by the hemolysis reagent. In embodiments, the ISTD is added to the resulting sample after the hemolysis reagent is added. In embodiments, water is added as the hemolysis reagent, particularly in an amount of 0.5:1 to 20:1 mL of water / mL of sample, particularly 1:1 to 10:1 mL of water / mL of sample, particularly 2:1 to 5:1 mL of water / mL of sample.

[0079] In embodiments where the sample is a urine sample, the sample is assigned to one of two other predetermined PT workflows, both of which include the addition of an ISTD and an enzyme reagent followed by a predetermined incubation period, with the difference between the two workflows being the order in which the internal standard and enzyme reagent are added. In embodiments, the ISTD is added to the resulting sample first, followed by the enzyme reagent. In embodiments, the ISTD is added to the resulting sample after the addition of the enzyme reagent. The enzyme reagent is typically a reagent used for glucuronide cleavage or protein cleavage, or any pretreatment of the analyte or matrix. In embodiments, the enzyme reagent is selected from the group consisting of glucuronidase, (partial) exo- or endo-deglycosylating enzyme, or exo- or endo-preotease. In embodiments, the glucuronidase is added in an amount of 0.5 to 10 mg / mL, particularly in an amount of 1 to 8 mg / mL, and particularly in an amount of 2 to 5 mg / mL.

[0080] In an embodiment, the sample is plasma or serum and is assigned to a different predetermined PT workflow that only involves the addition of an internal standard (ISTD) prior to a predetermined incubation time.

[0081] As described herein above or below, the incubation times and temperatures to select for the sample treatment, chemical reaction, or method step under consideration are well known to those skilled in the art. In particular, those skilled in the art know that incubation times and temperatures are interdependent, e.g., higher temperatures typically result in shorter incubation periods, and vice versa.

[0082] The (pre-treated) sample may be further subjected to at least one enrichment workflow. The enrichment workflow may include one or more enrichment methods. Enrichment methods are well known in the art and include, but are not limited to, chemical enrichment methods, including but not limited to chemical precipitation, and enrichment methods using solid phases, including but not limited to solid-phase extraction methods, bead workflows, and chromatographic methods (e.g., gas or liquid chromatography).

[0083] In embodiments, the first enrichment workflow comprises adding a solid phase, in particular solid beads, carrying an analyte-selective group to the (pre-treated) sample. In embodiments, the first enrichment workflow comprises adding magnetic or paramagnetic beads carrying an analyte-selective group to the (pre-treated) sample.

[0084] In embodiments, the magnetic beads comprise a magnetic core coated with a styrene-based polymer that is highly cross-linked by Friedel-Crafts alkylation and further modified with the addition of --OH groups.

[0085] In embodiments, magnetic beads comprise a magnetic core coated with a styrenic polymer that is highly cross-linked via a diamine (e.g., TMEDA) and further modified, where the diamine also serves as a side chain (i.e., in these types of beads, TMEDA provides both quaternary and tertiary amine functional groups). For a complete description of such beads, see WO 2019 / 141779.

[0086] In an embodiment, the addition of magnetic beads involves stirring or mixing. This is followed by a predetermined incubation period to capture the antibiotic analyte(s) of interest on the beads. In an embodiment, the workflow includes a washing step (W1) after incubation with the magnetic beads. Depending on the antibiotic analyte(s), one or more additional washing steps (W2) are performed. One washing step (W1, W2) consists of a series of steps including magnetic bead separation using a magnetic bead manipulation unit including a magnet or electromagnet, liquid aspiration, addition of a washing buffer, resuspension of the magnetic beads, another magnetic bead separation step, and another liquid aspiration. Furthermore, the washing steps may differ in terms of the type of solvent (water / organic / salt / pH) as well as the volume and number or combination of washing cycles. The selection of each parameter is well known to those skilled in the art. The final washing step (W1, W2) is followed by the addition of an elution reagent, followed by resuspension of the magnetic beads, and a predetermined incubation period to release the analyte(s) of interest from the magnetic beads. The unbound magnetic beads are then separated and the supernatant containing the derivatized analyte(s) of interest is captured.

[0087] In embodiments, the first enrichment workflow involves adding magnetic beads with matrix-selective groups to the pretreated sample. In embodiments, adding the magnetic beads involves stirring or mixing. This is followed by a predetermined incubation period to capture the matrix on the beads. Here, the analytes of interest do not bind to the magnetic beads and remain in the supernatant. The magnetic beads are then separated, and the supernatant containing the enriched analyte(s) of interest is collected.

[0088] In an embodiment, the supernatant is subjected to a second enrichment workflow, in particular a chromatographic enrichment workflow. In an embodiment of the present invention, the chromatographic separation is gas or liquid chromatography. Both methods are well known to those skilled in the art. In an embodiment, the liquid chromatography is selected from the group consisting of HPLC, rapid LC, micro LC, flow injection, and trapping and elution. Here, the supernatant is transferred to an LC station or transferred to an LC station after a dilution step by adding a diluent. Different elution procedures / reagents can also be used, such as changing the type of solvent (water / organic / salt / pH) and volume. The various parameters are well known to those skilled in the art and can be easily selected.

[0089] In embodiments, the first enrichment process comprises the use of analyte-selective magnetic beads. In embodiments of the invention, the second enrichment process comprises the use of chromatographic separation, particularly using liquid chromatography. In embodiments, the first enrichment process using analyte-selective magnetic beads is performed before the second enrichment process using liquid chromatography.

[0090] In embodiments, determining the amount or concentration of one or more derivatized antibiotic analytes in the sample is performed in step b). Any suitable method known to those skilled in the art can be used. In certain embodiments, step b) comprises determining the amount or concentration of one or more derivatized antibiotic analytes using immunological methods or mass spectrometry.

[0091] In embodiments where step b) comprises determining the amount or concentration of one or more antibiotic analytes using an immunological method, the method comprises the steps of: i) incubating a patient's (optionally enriched) sample with one or more antibodies that specifically bind to one or more derivatized antibiotic analytes, thereby forming complexes between the antibodies and the one or more derivatized antibiotic analytes; and ii) quantifying the complexes formed in step i), thereby quantifying the amount of one or more derivatized antibiotic analytes in the patient sample.

[0092] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to one or more derivatized antibiotic analytes. As will be apparent to one skilled in the art, the sample can be contacted with the first and second antibodies in any desired order, i.e., the first antibody first, then the second antibody, or the second antibody first, then the first antibody, or simultaneously, for a time and under conditions sufficient to form a first antibody / derivatized antibiotic analyte / second antibody complex. As will be readily apparent to one skilled in the art, it will take no more than routine experimentation to establish appropriate or sufficient times and conditions for the formation of a complex between an antibody and a derivatized antibiotic analyte, or a secondary or sandwich complex comprising the first antibody, the derivatized antibiotic analyte, and the second antibody.

[0093] Detection of the antibody-analyte complex can be carried out by any suitable means. Those skilled in the art are fully familiar with such means / methods. In embodiments, the antibody is detectably labeled, directly or indirectly. In particular embodiments, the antibody is detectably labeled with a luminescent dye, in particular a chemiluminescent dye or an electrochemiluminescent dye.

[0094] In embodiments where step b) comprises determining the amount or concentration of one or more antibiotic-derivatized antibiotic analytes using mass spectrometry, the method comprises the steps of: (i) subjecting ions of the derivatized antibiotic analyte to a first stage of mass spectrometry, whereby the parent ions of the derivatized antibiotic analyte are characterized according to their mass / charge (m / z) ratio; (ii) causing fragmentation of the parent ion of the derivatized antibiotic analyte, thereby producing daughter ions, the daughter ions of the derivatized antibiotic analyte having a different m / z ratio than the parent ion of the derivatized antibiotic analyte; and (iii) subjecting the daughter ions of the derivatized antibiotic analyte to a second stage of mass spectrometry, whereby the daughter ions of the derivatized antibiotic analyte are characterized according to their m / z ratio.

[0095] In an embodiment, the parent ions and / or fragment ions measured are those shown in Table 1. [Table 1]

[0096] In embodiments, derivatized meropenem + H + The parent ion of derivatized piperacillin + H was measured at m / z value 457.164 ± 0.5. + The parent ion of is measured at m / z value 664.235±0.5.

[0097] In embodiments, the fragment ions of the derivatized meropenem are measured at m / z values ​​of 152±0.5 or 173±0.5, and the fragment ions of the derivatized piperacillin are measured at m / z values ​​of 270±0.5 or 464±0.5.

[0098] In embodiments, the method is an automated method. In certain embodiments, the method is performed by an automated system. In certain embodiments, the method does not involve manual intervention.

[0099] In a second aspect, the present invention provides a method for determining the amount or concentration of one or more antibiotic analytes in an obtained sample, comprising: a) pretreating the sample with a derivatization reagent, wherein the derivatization reagent comprises a nucleophile; b) optionally concentrating the sample obtained after step a), in particular using magnetic beads; and c) determining the amount or concentration of one or more antibiotic analytes in the pretreated sample obtained after step a) or after the optional concentration step b). The present invention relates to a method, including:

[0100] In embodiments, the antibiotic analyte is a lactam antibiotic analyte. In embodiments, the antibiotic analyte is a β-lactam antibiotic analyte. In certain embodiments, the antibiotic analyte is selected from the group consisting of amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, temocillin, phenethicillin, penicillin G, penicillin V, piperacillin, azlocillin, bibampicillin, pivmecillinam, ticarcillin, cephacefetrile, cefadroxil ... Cefadroxil, Cefalexin, Cephalexin, Cephaloglycin, Cephalonium, Cephalonium, Cephaloridine , Cefalotin (cephalothin), Cefapirin (cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (cephazolin), Cephradine (cephradine), Cephradine (cephradine), Cefroxadine, Cefroxadine, Ceftezole, Cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil (cefproxil), cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefdoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine,Cefepime, Cefluprenam, Cefoselis, Cefozopran, Cefpirome, Cefquinome, Ceftobiprole, Ceftaroline, Cefacromezine, Cephaloram, Cefaparol, Cefcanel, Cefedrolol, Cefenpidone, Cefetrizole, Cefibitril, Cefmatilen, Cefmepidium, Cefovecin, Cefoxazole, Cefrotil, Cefsumide, Cefuracetim, The antibiotic analyte is selected from the group consisting of ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreonam, mecillinam, meampicillin, talampicillin, epicillin, sulbenicillin, faropenem, ritipenem, biapenem, pivampicillin, clometocillin, penamecillin, hetacillin, carindacillin, panipenem, tigemonam, carumonam, nocardicin A, penam, sulbactam, tazobactam, clavam, and clavulanic acid. In certain embodiments, the antibiotic analyte is meropenem or piperacillin.

[0101] In an embodiment, in step a), the sample is pretreated with a nucleophilic derivatization reagent comprising an amine group, in particular a primary or secondary amine, in particular a primary amine group. In an embodiment, in step a), the sample is pretreated with a nucleophilic derivatization reagent comprising more than 3 C atoms, in particular 3 to 20 C atoms, in particular 3 to 10 C atoms, in particular 3 to 5 C atoms, in particular 4 C atoms. In an embodiment, in step a), the sample is pretreated with a linear or branched nucleophilic derivatization reagent, in particular a linear amine, in particular a linear primary amine, in particular a linear primary amine comprising 3 to 5 C atoms. In an embodiment, in step a), the sample is pretreated with a nucleophilic derivatization reagent selected from the group consisting of propylamine, butylamine, or pentylamine, in particular primary linear butylamine.

[0102] In certain embodiments, in step a), when the analyte is meropenem, the sample is pretreated with a nucleophilic derivatization reagent comprising butylamine.

[0103] In certain embodiments, in step a), when the analyte is piperacillin, the sample is pretreated with a nucleophilic derivatization reagent comprising pentylamine.

[0104] In an embodiment, in step a) the sample is pretreated with a nucleophilic derivatization reagent contained in a solvent, in particular a solvent selected from the group consisting of water, CHCN, THF, dioxane, DMF, DMSO, acetone, t-butyl alcohol, diglyme, DME, MeOH, EtOH, 1-PrOH, 2-PrOH, ethylene glycol, hexamethylphosphoroamide (HMPA), hexamethylphosphorotriamide (HMPT) and glycerin, in particular a solvent selected from the group consisting of water, CHCN, THF, dioxane, DMF, DMSO, acetone, t-butyl alcohol, diglyme and DME.

[0105] In an embodiment, in step a), the sample is pretreated with a nucleophilic derivatization reagent in a solvent further comprising a water-stable and miscible non-nucleophilic base, in particular selected from the group consisting of DBU, TEA, DIPEA, NaPO, NaCO, and CsCO.

[0106] In an embodiment, in step a), the sample is pretreated with a nucleophilic derivatization reagent immediately after obtaining the sample, particularly within less than 10 minutes after obtaining the sample, particularly within less than 5 minutes after obtaining the sample.

[0107] In an embodiment, in step a), the sample is pretreated with the nucleophilic derivatization reagent sample for more than 2 minutes, in particular more than 5 minutes, in particular more than 30 minutes.

[0108] In an embodiment, the sample obtained after step a) comprises a derivatized antibiotic analyte, in particular an antibiotic analyte derivatized with a nucleophilic derivatization reagent.

[0109] In embodiments, the sample obtained after step a) comprises a derivatized β-lactam antibiotic analyte, wherein the β-lactam moiety is destroyed by reaction with a nucleophilic derivatization reagent. In embodiments, the sample obtained after step a) comprises a derivatized β-lactam antibiotic analyte, wherein a covalent adduct of the antibiotic analyte with the nucleophilic derivatization reagent is formed.

[0110] In embodiments, the sample obtained after step a) comprises a derivatized antibiotic analyte that has been derivatized at least one of its chemical moieties. Those skilled in the art of chemistry will be familiar with chemical moieties that are particularly suitable for derivatization with nucleophilic derivatization reagents. In certain embodiments, the sample obtained after step a) comprises a derivatized antibiotic analyte that has been derivatized at one, two, or three of its chemical moieties.

[0111] In certain embodiments where the antibiotic analyte is meropenem, the sample obtained after step a) contains derivatized meropenem, particularly meropenem derivatized with a nucleophilic derivatization reagent that includes butylamine. See also Figure 3.

[0112] In certain embodiments where the antibiotic analyte is piperacillin, the sample obtained after step a) comprises derivatized piperacillin, particularly piperacillin derivatized with a nucleophilic derivatization reagent including butylamine or pentamine. See also Figure 4.

[0113] In certain embodiments where the antibiotic analyte is piperacillin, the sample obtained after step a) comprises derivatized piperacillin, particularly piperacillin derivatized with a nucleophilic derivatization reagent comprising butylamine or pentamine, derivatized at two of its chemical moieties, particularly the β-lactam ring and the piperazine ring. See also Figure 4.

[0114] In embodiments, additional pretreatment methods can be performed in step a). These can be performed before or after pretreatment of the sample with a derivatization reagent. The pretreatment method depends on the type of sample, such as blood (fresh or dried), plasma, serum, urine, or saliva, while the concentration method depends on the analyte of interest. Those skilled in the art will be familiar with which pretreatment methods are suitable for which sample types. Those skilled in the art will also be familiar with which concentration methods are suitable for which analytes of interest.

[0115] In embodiments where the sample is a whole blood sample, the sample is assigned to one of two predetermined sample preparation (PT) workflows, both of which include the addition of an internal standard (ISTD) and a hemolysis reagent (HR) followed by a predetermined incubation period (Inc), where the difference between the two workflows is the order in which the internal standard (ISTD) and the hemolysis reagent (HR) are added. In embodiments, the ISTD is added to the resulting sample first, followed by the hemolysis reagent. In embodiments, the ISTD is added to the resulting sample after the hemolysis reagent is added. In embodiments, water is added as the hemolysis reagent, particularly in an amount of 0.5:1 to 20:1 mL of water / mL of sample, particularly 1:1 to 10:1 mL of water / mL of sample, particularly 2:1 to 5:1 mL of water / mL of sample.

[0116] In embodiments where the sample is a urine sample, the sample is assigned to one of two other predetermined PT workflows, both of which include the addition of an ISTD and an enzyme reagent followed by a predetermined incubation period, with the difference between the two workflows being the order in which the internal standard and enzyme reagent are added. In embodiments, the ISTD is added to the resulting sample first, followed by the enzyme reagent. In embodiments, the ISTD is added to the resulting sample after the addition of the enzyme reagent. The enzyme reagent is typically a reagent used for glucuronide cleavage or protein cleavage, or any pretreatment of the analyte or matrix. In embodiments, the enzyme reagent is selected from the group consisting of glucuronidase, (partial) exo- or endo-deglycosylating enzyme, or exo- or endo-preotease. In embodiments, the glucuronidase is added in an amount of 0.5 to 10 mg / mL, particularly in an amount of 1 to 8 mg / mL, and particularly in an amount of 2 to 5 mg / mL.

[0117] In an embodiment, the sample is plasma or serum and is assigned to a different predetermined PT workflow that only involves the addition of an internal standard (ISTD) prior to a predetermined incubation time.

[0118] As described herein above or below, the incubation times and temperatures to select for the sample treatment, chemical reaction, or method step under consideration are well known to those skilled in the art. In particular, those skilled in the art know that incubation times and temperatures are interdependent, e.g., higher temperatures typically result in shorter incubation periods, and vice versa.

[0119] The pretreated sample may be further subjected to at least one enrichment workflow in step b). The enrichment workflow may include one or more enrichment methods. Enrichment methods are well known in the art and include, but are not limited to, chemical enrichment methods, including but not limited to chemical precipitation, and enrichment methods using solid phases, including but not limited to solid-phase extraction methods, bead workflows, and chromatographic methods (e.g., gas or liquid chromatography).

[0120] In an embodiment, the first enrichment workflow involves adding a solid phase, in particular solid beads, carrying an analyte-selective group to the pre-treated sample.

[0121] In embodiments, the first enrichment workflow involves adding magnetic or paramagnetic beads bearing analyte-selective groups to the pretreated sample. In embodiments, the magnetic beads comprise a magnetic core coated with a styrenic polymer that is crosslinked via Friedel-Crafts alkylation and further modified with the addition of -OH groups. In embodiments, the magnetic beads comprise a magnetic core coated with a styrenic polymer that is crosslinked via a diamine (e.g., tetramethylenediamine (TMEDA)) and further modified, where the diamine also serves as a side chain (i.e., diamine beads bearing TMEDA provide both quaternary and tertiary amine functional groups). For a complete description, see, e.g., WO 2019 / 141779.

[0122] In an embodiment, the enrichment workflow in step b) using magnetic beads includes stirring or mixing. This is followed by a predetermined incubation period to capture the target antibiotic analyte(s) on the beads. In an embodiment, the workflow includes a washing step (W1) after incubation with the magnetic beads. Depending on the antibiotic analyte(s), one or more additional washing steps (W2) are performed. One washing step (W1, W2) consists of a series of steps including magnetic bead separation using a magnetic bead manipulation unit including a magnet or electromagnet, liquid aspiration, addition of a washing buffer, resuspension of the magnetic beads, another magnetic bead separation step, and another liquid aspiration. Furthermore, the washing steps may differ in terms of the type of solvent (water / organic / salt / pH), apart from the volume and number or combination of washing cycles. The method for selecting each parameter is well known to those skilled in the art. The final washing step (W1, W2) is followed by the addition of an elution reagent, followed by resuspension of the magnetic beads, and a predetermined incubation period to release the target analyte(s) from the magnetic beads. The unbound magnetic beads are then separated and the supernatant containing the derivatized analyte(s) of interest is captured.

[0123] In embodiments, the first enrichment workflow involves adding magnetic beads with matrix-selective groups to the pretreated sample. In embodiments, the addition of the magnetic beads involves stirring or mixing. This is followed by a predetermined incubation period to capture the matrix on the beads. Here, the analytes of interest do not bind to the magnetic beads and remain in the supernatant. The magnetic beads are then separated, and the supernatant containing the enriched analyte(s) of interest is collected. In embodiments, the supernatant is subjected to a second enrichment workflow, particularly a chromatographic enrichment workflow. In embodiments, the chromatographic separation is gas or liquid chromatography. Both methods are well known to those skilled in the art. In embodiments, the liquid chromatography is selected from the group consisting of HPLC, rapid LC, micro LC, flow injection, and trapping and elution. Here, the supernatant is transferred to an LC station or transferred to the LC station after a dilution step by adding a diluent. Different elution procedures / reagents, such as varying the type of solvent (water / organic / salt / pH) and volume, can also be used. The various parameters are well known to those skilled in the art and are readily selected.

[0124] In certain embodiments, the first enrichment process comprises the use of analyte-selective magnetic beads. In embodiments of the invention, the second enrichment process comprises the use of chromatographic separation, particularly using liquid chromatography. In embodiments, the first enrichment process using analyte-selective magnetic beads is performed before the second enrichment process using liquid chromatography.

[0125] In embodiments, determining the amount or concentration of one or more derivatized antibiotic analytes in the sample is performed in step c). Any suitable method known to those skilled in the art can be used. In certain embodiments, step c) comprises determining the amount or concentration of one or more derivatized antibiotic analytes using immunological methods or mass spectrometry.

[0126] In embodiments where step c) comprises determining the amount or concentration of one or more antibiotic analytes using an immunological method, the method comprises the steps of: i) incubating a patient sample with one or more antibodies that specifically bind to one or more derivatized antibiotic analytes, thereby forming complexes between the antibodies and the one or more derivatized antibiotic analytes; and ii) quantifying the complexes formed in step i), thereby quantifying the amount of one or more antibiotic analytes in the patient sample.

[0127] In certain embodiments, in step i), the sample is incubated with two antibodies that specifically bind to one or more derivatized antibiotic analytes. As will be apparent to one skilled in the art, the sample can be contacted with the first and second antibodies in any desired order, i.e., the first antibody first, then the second antibody, or the second antibody first, then the first antibody, or simultaneously, for a time and under conditions sufficient to form a first antibody / derivatized antibiotic analyte / second antibody complex. As will be readily apparent to one skilled in the art, it will take no more than routine experimentation to establish appropriate or sufficient times and conditions for the formation of a complex between an antibody and a derivatized antibiotic analyte, or a secondary or sandwich complex comprising the first antibody, the derivatized antibiotic analyte, and the second antibody.

[0128] Detection of the antibody-analyte complex can be carried out by any suitable means. Those skilled in the art are fully familiar with such means / methods. In embodiments, the antibody is detectably labeled, directly or indirectly. In particular embodiments, the antibody is detectably labeled with a luminescent dye, in particular a chemiluminescent dye or an electrochemiluminescent dye.

[0129] In embodiments where step c) comprises determining the amount or concentration of one or more antibiotic-derivatized antibiotic analytes using mass spectrometry, the method comprises the steps of: (i) subjecting ions of the derivatized antibiotic analyte to a first stage of mass spectrometry, whereby the parent ions of the derivatized antibiotic analyte are characterized according to their mass / charge (m / z) ratio; (ii) causing fragmentation of the parent ion of the derivatized antibiotic analyte, thereby producing daughter ions, the daughter ions of the derivatized antibiotic analyte having a different m / z ratio than the parent ion of the derivatized antibiotic analyte; and (iii) subjecting the daughter ions of the derivatized antibiotic analyte to a second stage of mass spectrometry, whereby the daughter ions of the derivatized antibiotic analyte are characterized according to their m / z ratio.

[0130] In an embodiment, the parent ions and / or fragment ions measured are those shown in Table 1.

[0131] In embodiments, derivatized meropenem + H + The parent ion of derivatized piperacillin + H was measured at m / z value 457.164 ± 0.5. + The parent ion of is measured at m / z value 664.235±0.5.

[0132] In embodiments, the fragment ions of the derivatized meropenem are measured at m / z values ​​of 152±0.5 or 173±0.5, and the fragment ions of the derivatized piperacillin are measured at m / z values ​​of 270±0.5 or 464±0.5.

[0133] In embodiments, the method is an automated method. In certain embodiments, the method is performed by an automated system. In certain embodiments, the method does not involve manual intervention.

[0134] In a third aspect, the present invention relates to an analytical system adapted to carry out the method of the first or second aspect.

[0135] In embodiments, the system is a mass spectrometry system, particularly an LC / MS system. In embodiments, the analytical system is an automated analytical system. In certain embodiments, the analytical system does not require manual intervention, i.e., the operation of the system is purely automated. In certain embodiments, the LC / MS system is an automated random access LC / MS system. In embodiments, the MS instrument is a tandem mass spectrometer, particularly a triple quadrupole instrument. In embodiments, the LC is an HPLC, particularly an RP-HPLC, or a fast LC. In embodiments, ion formation is based on electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), particularly positive mode ESI.

[0136] In a fourth aspect, the present invention relates to a sampling tube for collecting a patient sample comprising a nucleophilic derivatization reagent suitable for stabilizing one or more antibiotic analytes in the sample. In an embodiment, the present invention relates to a sampling tube for collecting a patient sample comprising a nucleophilic derivatization reagent that stabilizes one or more antibiotic analytes in the sample.

[0137] Sample collection tubes suitable for use in collecting patient samples are well known in the art and are routinely used by medical practitioners. As those skilled in the art will appreciate, the sampling tube is preferably a tube in nature. In particular, the sampling tube has a size and dimensions adapted to fit the requirements of the sample receiving station of an automated analyzer, such as the Elecsys® analyzer from Roche Diagnostics. The sampling tube may have a conical or, preferably, rounded bottom. In clinical routine, standard tube sizes compatible with commercially available analyzer systems are used. Standard and preferred tubes have, for example, the following dimensions: 13 x 75 mm; 13 x 100 mm, or 16 x 100 mm.

[0138] In an embodiment, the sampling tube according to the present invention is used only once, i.e., a single-use device. In particular, the sampling tube according to the present invention is not only suitable for sample collection, but also adapted to allow further processing of the sample. By collecting the sample in the sampling tube containing the nucleophilic derivatization reagent, the desired result, i.e., derivatization of the antibiotic analyte, is achieved.

[0139] In an embodiment, the nucleophilic derivatization reagent comprises an amine group, particularly a primary or secondary amine, in particular a primary amine group. In an embodiment, the nucleophilic derivatization reagent comprises more than 3 C atoms, in particular 3 to 20 C atoms, in particular 3 to 10 C atoms, in particular 3 to 5 C atoms, in particular 4 C atoms. In an embodiment, the nucleophilic derivatization reagent is a linear or branched chain, in particular a linear amine, in particular a linear primary amine, in particular a linear primary amine comprising 3 to 5 C atoms. In an embodiment, the nucleophilic derivatization reagent is selected from the group consisting of propylamine, butylamine or pentylamine, in particular a primary linear butylamine or a primary linear pentylamine.

[0140] In embodiments, the nucleophilic derivatization reagent derivatizes the antibiotic analyte at at least one of its chemical moieties. Those skilled in the art of chemistry will be familiar with chemical moieties that are particularly suitable for derivatization with a nucleophilic derivatization reagent. In certain embodiments, the nucleophilic derivatization reagent derivatizes the antibiotic analyte at one, two, or three of its chemical moieties.

[0141] In certain embodiments, the nucleophilic derivatization reagent comprises butylamine when the antibiotic analyte is meropenem.

[0142] In certain embodiments, the nucleophilic derivatization reagent comprises pentylamine when the antibiotic analyte is piperacillin.

[0143] In embodiments, the nucleophilic derivatization reagent is included in liquid or lyophilized form. In embodiments, the nucleophilic derivatization reagent further includes a stable, water-miscible, non-nucleophilic base, particularly selected from the group consisting of DBU, TEA, DIPEA, NaPO, NaCO, and CsCO. In embodiments, the nucleophilic derivatization reagent is included in liquid form in a solvent, particularly selected from the group consisting of water, CHCN, THF, dioxane, DMF, DMSO, acetone, t-butyl alcohol, diglyme, DME, MeOH, EtOH, 1-PrOH, 2-PrOH, ethylene glycol, hexamethylphosphoroamide (HMPA), hexamethylphosphorotriamide (HMPT), and glycerin, particularly selected from the group consisting of water, CHCN, THF, dioxane, DMF, DMSO, acetone, tBuOH, diglyme, and DME.

[0144] In a fifth aspect, the present invention relates to the use of a nucleophilic derivatization reagent for determining the amount or concentration of one or more antibiotic analytes in a sample.

[0145] In an embodiment, the nucleophilic derivatization reagent is a reagent comprising an amine group, in particular a primary or secondary amine, in particular a primary amine group. In an embodiment, the nucleophilic derivatization reagent comprises more than 3 C atoms, in particular 3 to 20 C atoms, in particular 3 to 10 C atoms, in particular 3 to 5 C atoms, in particular 4 C atoms. In an embodiment, the nucleophilic derivatization reagent is a linear or branched chain, in particular a linear amine, in particular a linear primary amine, in particular a linear primary amine comprising 3 to 5 C atoms. In an embodiment, the derivatization reagent is selected from the group consisting of propylamine, butylamine, or pentylamine, in particular a primary linear butylamine.

[0146] In embodiments, the antibiotic is a β-lactam antibiotic. In embodiments, the antibiotic is amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, temocillin, phenethicillin, penicillin G, penicillin V, piperacillin, azlocillin, bibampicillin, pivmecillinam, ticarcillin, cephacefetrile, cefadroxil ... xyl), Cephalexin (cephalexin), Cephalexin (cephalexin), Cephaloglycin (cephaloglycin), Cephalonium (cephalonium), Cephaloridine (cephaloradine), Cefalotin (cephalosin) Cephalothin), Cefapirin (Cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (Cefazolin), Cephradine (Cefradine), Cephradine (Cefradine), Cefroxadine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefmetazole, Cefo Nisid, cefotetan, cefoxitin, cefprozil (cefproxil), cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefdoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome,Cefquinome, Ceftobiprole, Ceftaroline, Cefclomedine, Cephaloram, Cefaparol, Cefcanel, Cefedrolol, Cefenpidone, Cefetrizole, Cefibitril, Cefmatilen, Cefmepidium, Cefovecin, Cefoxazole, Cefrotil, Cefsumide, Cefuracetim, Ceftioxide, Ceftolozane, Imiflu The antibiotic analyte is selected from the group consisting of penem, doripenem, ertapenem, meropenem, aztreonam, mecillinam, meampicillin, talampicillin, epicillin, sulbenicillin, faropenem, ritipenem, biapenem, pivampicillin, clometocillin, penamecillin, hetacillin, carindacillin, panipenem, tigemonam, carumonam, nocardicin A, penam, sulbactam, tazobactam, clavam, and clavulanic acid. In certain embodiments, the antibiotic analyte is meropenem or piperacillin.

[0147] In embodiments, the nucleophilic derivatization reagent stabilizes the antibiotic. In embodiments, the nucleophilic derivatization reagent prevents hydrolysis of the antibiotic during determination of the amount or concentration of one or more antibiotic analytes in a sample. In embodiments, the nucleophilic derivatization reagent stabilizes the antibiotic by forming a covalent adduct between the antibiotic analyte and the nucleophilic derivatization reagent.

[0148] In embodiments, the nucleophilic derivatization reagent stabilizes the antibiotic analyte at at least one of its chemical moieties. Those skilled in the art of chemistry will be familiar with chemical moieties that are particularly suitable for derivatization with a nucleophilic derivatization reagent. In certain embodiments, the nucleophilic derivatization reagent derivatizes the antibiotic analyte at one, two, or three of its chemical moieties. In certain embodiments, the nucleophilic derivatization reagent stabilizes the antibiotic analyte by reacting with its β-lactam ring.

[0149] In certain embodiments where the antibiotic analyte is meropenem, a nucleophilic derivatization reagent comprising butylamine is used to stabilize the meropenem. See also Figure 3.

[0150] In certain embodiments where the antibiotic analyte is piperacillin, nucleophilic derivatization reagents including butylamine or pentylamine are used to stabilize the piperacillin. See also Figure 4.

[0151] In certain embodiments where the antibiotic analyte is piperacillin, nucleophilic derivatization reagents including butylamine or pentylamine are used to stabilize piperacillin at two of its chemical moieties, specifically derivatizing it at the β-lactam ring and the piperazine ring. See also Figure 4.

[0152] In embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 2 hours, more than 4 hours, more than 8 hours, more than 12 hours, more than 15 hours, more than 24 hours, more than 48 hours, more than 7 days, more than 2 weeks, more than 4 weeks, more than 2 months, more than 3 months, more than 4 months, more than 5 months, or more than 6 months. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 8 hours, particularly more than 12 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 15 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for at least 16 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for 16 hours.

[0153] In a sixth aspect, the present invention relates to the use of a nucleophilic derivatization reagent to stabilize an antibiotic analyte in a sample of interest.

[0154] In an embodiment, the nucleophilic derivatization reagent is a reagent comprising an amine group, in particular a primary or secondary amine, in particular a primary amine group. In an embodiment, the nucleophilic derivatization reagent comprises more than 3 C atoms, in particular 3 to 20 C atoms, in particular 3 to 10 C atoms, in particular 3 to 5 C atoms, in particular 4 C atoms. In an embodiment, the nucleophilic derivatization reagent is a linear or branched chain, in particular a linear amine, in particular a linear primary amine, in particular a linear primary amine comprising 3 to 5 C atoms. In an embodiment, the derivatization reagent is selected from the group consisting of propylamine, butylamine, or pentylamine, in particular a primary linear butylamine.

[0155] In embodiments, the antibiotic is a β-lactam antibiotic. In embodiments, the antibiotic is amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, temocillin, phenethicillin, penicillin G, penicillin V, piperacillin, azlocillin, bibampicillin, pivmecillinam, ticarcillin, cephacefetrile, cefadroxil ... xyl), Cephalexin (cephalexin), Cephalexin (cephalexin), Cephaloglycin (cephaloglycin), Cephalonium (cephalonium), Cephaloridine (cephaloradine), Cefalotin (cephalosin) Cephalothin), Cefapirin (Cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (Cefazolin), Cephradine (Cefradine), Cephradine (Cefradine), Cefroxadine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefmetazole, Cefo Nisid, cefotetan, cefoxitin, cefprozil (cefproxil), cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefdoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome,Cefquinome, Ceftobiprole, Ceftaroline, Cefclomedine, Cephaloram, Cefaparol, Cefcanel, Cefedrolol, Cefenpidone, Cefetrizole, Cefibitril, Cefmatilen, Cefmepidium, Cefovecin, Cefoxazole, Cefrotil, Cefsumide, Cefuracetim, Ceftioxide, Ceftolozane, Imiflu The antibiotic analyte is selected from the group consisting of penem, doripenem, ertapenem, meropenem, aztreonam, mecillinam, meampicillin, talampicillin, epicillin, sulbenicillin, faropenem, ritipenem, biapenem, pivampicillin, clometocillin, penamecillin, hetacillin, carindacillin, panipenem, tigemonam, carumonam, nocardicin A, penam, sulbactam, tazobactam, clavam, and clavulanic acid. In certain embodiments, the antibiotic analyte is meropenem or piperacillin.

[0156] In embodiments, the nucleophilic derivatization reagent stabilizes the antibiotic. In embodiments, the nucleophilic derivatization reagent prevents hydrolysis of the antibiotic during determination of the amount or concentration of one or more antibiotic analytes in a sample. In embodiments, the nucleophilic derivatization reagent stabilizes the antibiotic by forming a covalent adduct between the antibiotic analyte and the nucleophilic derivatization reagent. In embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 2 hours, more than 4 hours, more than 8 hours, more than 12 hours, more than 15 hours, more than 24 hours, more than 48 hours, more than 7 days, more than 2 weeks, more than 4 weeks, more than 2 months, more than 3 months, more than 4 months, more than 5 months, or more than 6 months. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 8 hours, particularly more than 12 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 15 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for at least 16 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for 16 hours.

[0157] In a seventh aspect, the present invention relates to antibiotic analytes stabilized with a nucleophilic derivatization reagent.

[0158] In embodiments, the nucleophilic derivatization reagent prevents hydrolysis of the antibiotic during determination of the amount or concentration of one or more antibiotic analytes in a sample. In embodiments, the antibiotic is stabilized by the nucleophilic derivatization reagent through the formation of a covalent adduct between the antibiotic analyte and the nucleophilic derivatization reagent. In embodiments, the antibiotic is stabilized by the nucleophilic derivatization reagent for more than 2 hours, more than 4 hours, more than 8 hours, more than 12 hours, more than 15 hours, more than 24 hours, more than 48 hours, more than 7 days, more than 2 weeks, more than 4 weeks, more than 2 months, more than 3 months, more than 4 months, more than 5 months, or more than 6 months. In certain embodiments, the antibiotic is stabilized by the nucleophilic derivatization reagent for more than 8 hours, particularly more than 12 hours. In certain embodiments, the antibiotic is stabilized by the nucleophilic derivatization reagent for more than 15 hours. In certain embodiments, the antibiotic is stabilized by the nucleophilic derivatization reagent for at least 16 hours. In certain embodiments, the antibiotic is stabilized by the nucleophilic derivatization reagent for 16 hours.

[0159] In embodiments, the antibiotic is a β-lactam antibiotic. In embodiments, the antibiotic is amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, temocillin, phenethicillin, penicillin G, penicillin V, piperacillin, azlocillin, bibampicillin, pivmecillinam, ticarcillin, cephacefetrile, cefadroxil ... xyl), Cephalexin (cephalexin), Cephalexin (cephalexin), Cephaloglycin (cephaloglycin), Cephalonium (cephalonium), Cephaloridine (cephaloradine), Cefalotin (cephalosin) Cephalothin), Cefapirin (Cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (Cefazolin), Cephradine (Cefradine), Cephradine (Cefradine), Cefroxadine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefmetazole, Cefo Nisid, cefotetan, cefoxitin, cefprozil (cefproxil), cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefdoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome,Cefquinome, Ceftobiprole, Ceftaroline, Cefclomedine, Cephaloram, Cefaparol, Cefcanel, Cefedrolol, Cefenpidone, Cefetrizole, Cefibitril, Cefmatilen, Cefmepidium, Cefovecin, Cefoxazole, Cefrotil, Cefsumide, Cefuracetim, Ceftioxide, Ceftolozane, Imiflu The antibiotic analyte is selected from the group consisting of penem, doripenem, ertapenem, meropenem, aztreonam, mecillinam, meampicillin, talampicillin, epicillin, sulbenicillin, faropenem, ritipenem, biapenem, pivampicillin, clometocillin, penamecillin, hetacillin, carindacillin, panipenem, tigemonam, carumonam, nocardicin A, penam, sulbactam, tazobactam, clavam, and clavulanic acid. In certain embodiments, the antibiotic analyte is meropenem or piperacillin.

[0160] In an embodiment, the nucleophilic derivatization reagent is a reagent comprising an amine group, in particular a primary or secondary amine, in particular a primary amine group. In an embodiment, the nucleophilic derivatization reagent comprises more than 3 C atoms, in particular 3 to 20 C atoms, in particular 3 to 10 C atoms, in particular 3 to 5 C atoms, in particular 4 C atoms. In an embodiment, the nucleophilic derivatization reagent is a linear or branched chain, in particular a linear amine, in particular a linear primary amine, in particular a linear primary amine comprising 3 to 5 C atoms. In an embodiment, the derivatization reagent is selected from the group consisting of propylamine, butylamine, or pentylamine, in particular a primary linear butylamine.

[0161] In embodiments, the antibiotic analyte is stabilized at at least one of its chemical moieties by a nucleophilic derivatization reagent. Those skilled in the art of chemistry will be familiar with chemical moieties that are particularly suitable for derivatization with a nucleophilic derivatization reagent. In certain embodiments, the antibiotic analyte is derivatized at one, two, or three of its chemical moieties by a nucleophilic derivatization reagent. In certain embodiments, the antibiotic analyte is stabilized by a nucleophilic derivatization reagent by reacting with its β-lactam ring.

[0162] In certain embodiments where the antibiotic analyte is meropenem, a nucleophilic derivatization reagent comprising butylamine is used to stabilize the meropenem. See also Figure 3.

[0163] In certain embodiments where the antibiotic analyte is piperacillin, nucleophilic derivatization reagents including butylamine or pentylamine are used to stabilize the piperacillin. See also Figure 4.

[0164] In certain embodiments where the antibiotic analyte is piperacillin, nucleophilic derivatization reagents including butylamine or pentylamine are used to stabilize piperacillin at two of its chemical moieties, specifically derivatizing it at the β-lactam ring and the piperazine ring. See also Figure 4.

[0165] The present invention further relates to the following items: 1) An (automated) method for determining the amount or concentration of one or more derivatized antibiotic analytes in a sample obtained, comprising: a) optionally pre-treating and / or concentrating said sample, in particular using magnetic beads, and b) determining the amount or concentration of said one or more antibiotic analytes in said sample. A method comprising:

[0166] 2) The method of claim 1, wherein the antibiotic analyte is a lactam antibiotic analyte.

[0167] 3) The method of item 1 or 2, wherein the antibiotic analyte is a β-lactam antibiotic analyte.

[0168] 4) The antibiotic analyte is selected from the group consisting of amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, temocillin, phenethicillin, penicillin G, penicillin V, piperacillin, azlocillin, bibampicillin, pivmecillinam, ticarcillin, cephacefetrile, cefadroxil, cefazolin, cefadroxyl ... Cefalexin (cephalexin), Cefalexin (cephalexin), Cefaloglycin (cephaloglycin), Cefalonium (cephalonium), Cefaloridine (cephaloradine), Cefalotin (cephalothi n)), Cefapirin (Cefapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (Cefazolin), Cephradine (Cefradine), Cephradine (Cefradine), Cefroxadine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefmetazole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil Cefproxil, cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefodoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidin, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomedine,The method of any one of paragraphs 1 to 3, wherein the antibiotic analyte is selected from the group consisting of cephaloram, cefaparole, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, meropenem, aztreonam, mecillinam, meampicillin, talampicillin, epicillin, sulbenicillin, faropenem, ritipenem, biapenem, pivampicillin, clometocillin, penamecillin, hetacillin, carindacillin, panipenem, tigemonam, carumonam, nocardicin A, penam, sulbactam, tazobactam, clavam, and clavulanic acid. In certain embodiments, the antibiotic analyte is meropenem or piperacillin. ,

[0169] 5) The method according to any one of items 1 to 4, wherein the antibiotic analyte is meropenem or piperacillin.

[0170] 6) The method according to any one of items 1 to 5, wherein the antibiotic analyte is derivatized with a nucleophilic derivatization reagent, in particular a reagent comprising an amine group, in particular a primary or secondary amine, in particular a primary amine group.

[0171] 7) The method according to any one of items 1 to 6, wherein the antibiotic analyte is derivatized with a nucleophilic derivatization reagent containing more than 3 C atoms, in particular 3 to 20 C atoms, in particular 3 to 10 C atoms, in particular 3 to 5 C atoms, in particular 4 C atoms.

[0172] 8) The method according to any one of items 1 to 7, wherein the antibiotic analyte is derivatized with a linear or branched nucleophilic derivatization reagent, in particular a linear amine, in particular a linear primary amine, in particular a linear primary amine containing 3 to 5 C atoms.

[0173] 9) The method according to any one of items 1 to 8, wherein the antibiotic analyte is derivatized with a nucleophilic derivatization reagent selected from the group consisting of propylamine, butylamine, or pentylamine, in particular a primary linear butylamine.

[0174] 10) The method according to any one of items 1 to 9, wherein the enrichment step a) comprises at least one enrichment workflow.

[0175] 11) The method according to any one of items 1 to 9, wherein the enrichment step a) comprises using magnetic beads, particularly type A or type B magnetic beads.

[0176] 12) The method according to any one of items 1 to 11, wherein the concentration step a) comprises two concentration steps, in particular a first concentration step comprising the use of magnetic beads and a second concentration step using evaporation.

[0177] 13) The method according to any one of items 1 to 12, wherein in step b), the amount or concentration of the derivatized antibiotic analyte is determined using an immunological assay or LC / MS.

[0178] 14) The method according to any one of items 1 to 13, wherein in step b) the amount or concentration of the derivatized antibiotic analyte is determined using LC / MS, wherein LC is HPLC, in particular RP-HPLC or high performance LC.

[0179] 15) The method according to any one of paragraphs 1 to 14, wherein in step b) the amount or concentration of the derivatized antibiotic analyte is determined using LC / MS, and ion formation is based on electrospray ionization (ESI), in particular positive polarity mode ESI.

[0180] 16) The method according to any one of paragraphs 1 to 15, wherein in step b) the amount or concentration of the derivatized antibiotic analyte is determined using LC / MS, and the MS instrument is a tandem mass spectrometer, particularly a triple quadrupole instrument, in particular an automated random access LC / MS system.

[0181] 17) In step b), the amount or concentration of the derivatized antibiotic analyte is determined using LC / MS, and the amount or concentration of the derivatized meropenem + H + The parent ion of derivatized piperacillin + H was measured at m / z 457.164 ± 0.5. +17. The method according to any one of items 1 to 16, wherein the parent ion of is measured at an m / z value of 664.235±0.5.

[0182] 18) The method of any one of items 1 to 17, wherein in step b), the amount or concentration of the derivatized antibiotic analyte is determined using LC / MS, and the fragment ions of derivatized meropenem are measured at m / z values ​​of 152±0.5 or 173±0.5, and the fragment ions of derivatized piperacillin are measured at m / z values ​​of 270±0.5 or 464±0.5.

[0183] 19) An (automated) method for determining the amount or concentration of one or more antibiotic analytes in an obtained sample, comprising: a) pretreating the sample with a derivatization reagent, wherein the derivatization reagent comprises a nucleophile; b) optionally concentrating said sample obtained after step a), in particular using magnetic beads; and c) determining the amount or concentration of one or more antibiotic analyte(s) in said pretreated sample obtained after step a) or after the optional concentration step b). A method comprising:

[0184] 20) The method of claim 19, wherein the antibiotic analyte is a lactam antibiotic analyte.

[0185] 21) The method of claim 19 or 20, wherein the antibiotic analyte is a β-lactam antibiotic analyte.

[0186] 22) The antibiotic analyte is selected from the group consisting of amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, temocillin, phenethicillin, penicillin G, penicillin V, piperacillin, azlocillin, bibampicillin, pivmecillinam, ticarcillin, cephacefetrile, cefadroxil, cefadroxyl ... Cefalexin (cephalexin), Cephalexin (cephalexin), Cefaloglycin (cephaloglycin), Cephalonium (cephalonium), Cephaloridine (cephaloradine), Cefalotin (cephalothi n)), Cefapirin (Cefapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (Cefazolin), Cephradine (Cefradine), Cephradine (Cefradine), Cefroxadine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefmetazole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil Cefproxil, cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefodoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidin, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomedine,Cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, azt 22. The method of any one of paragraphs 19 to 21, wherein the antibiotic analyte is selected from the group consisting of leonam, mecillinam, meampicillin, talampicillin, epicillin, sulbenicillin, faropenem, ritipenem, biapenem, pivampicillin, clometocillin, penamecillin, hetacillin, carindacillin, panipenem, tigemonam, carumonam, nocardicin A, penam, sulbactam, tazobactam, clavam, and clavulanic acid. In certain embodiments, the antibiotic analyte is meropenem or piperacillin.

[0187] 23) The method according to any one of items 19 to 22, wherein the antibiotic analyte is meropenem or piperacillin.

[0188] 24) The method according to any one of items 19 to 23, wherein in step a) the sample is pretreated with a nucleophilic derivatization reagent containing an amine group, particularly a primary or secondary amine, especially a primary amine group.

[0189] 25) In step a), the sample is pretreated with a nucleophilic derivatization reagent containing more than 3 C atoms, particularly 3 to 20 C atoms, particularly 3 to 10 C atoms, particularly 3 to 5 C atoms, particularly 4 C atoms. The method according to any one of items 19 to 24.

[0190] 26) The method according to any one of items 19 to 25, wherein in step a) the sample is pretreated with a linear or branched nucleophilic derivatization reagent, in particular a linear amine, in particular a linear primary amine, in particular a linear primary amine containing 3 to 5 C atoms.

[0191] 27) The method according to any one of items 19 to 28, wherein in step a), the sample is pretreated with a nucleophilic derivatization reagent selected from the group consisting of propylamine, butylamine, or pentylamine, particularly primary linear butylamine.

[0192] 28) The method according to any one of items 19 to 27, wherein in step a), the sample is pretreated with a nucleophilic derivatization reagent contained in a solvent, particularly a solvent selected from the group consisting of water, CHCN, THF, dioxane, DMF, DMSO, acetone, t-butyl alcohol, diglyme, DME, MeOH, EtOH, 1-PrOH, 2-PrOH, ethylene glycol, hexamethylphosphoroamide (HMPA), hexamethylphosphorotriamide (HMPT) and glycerin, particularly a solvent selected from the group consisting of water, CHCN, THF, dioxane, DMF, DMSO, acetone, tBuOH, diglyme and DME.

[0193] 29) The method according to any one of items 19 to 28, wherein in step a) the sample is pretreated with a nucleophilic derivatization reagent contained in a solvent further comprising a non-nucleophilic base that is stable and miscible with water, in particular selected from the group consisting of DBU, TEA, DIPEA, Na3PO4, Na2CO3, and Cs2CO3.

[0194] 30) The method according to any one of items 19 to 29, wherein in step a) when the analyte is meropenem, the sample is pretreated with a nucleophilic derivatization reagent containing butylamine.

[0195] 31) The method according to any one of items 19 to 30, wherein in step a) when the analyte is piperacillin, the sample is pretreated with a nucleophilic derivatization reagent containing pentylamine.

[0196] 32) The method according to any one of items 19 to 31, wherein in step a), the sample is pretreated with a nucleophilic derivatization reagent immediately after obtaining the sample, particularly within less than 10 minutes after obtaining the sample, particularly within less than 5 minutes after obtaining the sample.

[0197] 33) The method according to any one of items 19 to 31, wherein in step a) the sample is pretreated with a nucleophilic derivatization reagent sample for more than 2 minutes, particularly more than 5 minutes, particularly more than 30 minutes.

[0198] 34) The method according to any one of items 19 to 33, wherein the sample obtained after step a) comprises a derivatized antibiotic analyte, in particular an antibiotic analyte derivatized with a nucleophilic derivatization reagent.

[0199] 35) The method of any one of items 19 to 34, wherein the sample obtained after step a) contains a derivatized β-lactam antibiotic analyte, wherein the β-lactam moiety is destroyed by reaction with a nucleophilic derivatization reagent.

[0200] 36) The method according to any one of items 19 to 35, wherein the concentration step b) comprises at least one concentration workflow.

[0201] 37) The method according to any one of items 19 to 36, wherein the enrichment step b) comprises using magnetic beads, in particular type A or type B magnetic beads.

[0202] 38) The method according to any one of items 19 to 37, wherein the concentration step b) comprises two concentration steps, in particular a first concentration step involving magnetic beads and a second concentration step using evaporation.

[0203] 39) The method according to any one of items 19 to 38, wherein in step c) the amount or concentration of the antibiotic analyte is determined using an immunological assay or LC / MS.

[0204] 40) The method according to any one of items 19 to 39, wherein in step c) the amount or concentration of the antibiotic analyte is determined using LC / MS, wherein LC is HPLC, in particular RP-HPLC or high performance LC.

[0205] 41) A method according to any one of paragraphs 19 to 40, wherein in step c) the amount or concentration of the antibiotic analyte is determined using LC / MS, and ion formation is based on electrospray ionization (ESI), in particular positive polarity mode ESI.

[0206] 42) The method according to any one of paragraphs 19 to 41, wherein in step b) the amount or concentration of the antibiotic analyte is determined using LC / MS, the MS device being a tandem mass spectrometer, in particular a triple quadrupole device, in particular an automated random access LC / MS system.

[0207] 43) In step c), the amount or concentration of the antibiotic analyte is determined using LC / MS and the amount or concentration of the derivatized meropenem + H + The parent ion of derivatized piperacillin + H was measured at m / z 457.164 ± 0.5. + 43. The method according to any one of items 19 to 42, wherein the parent ion of is measured at an m / z value of 664.235±0.5.

[0208] 44) The method of any one of items 19 to 43, wherein in step c) the amount or concentration of the antibiotic analyte is determined using LC / MS, and the fragment ions of derivatized meropenem are measured at m / z values ​​of 152±0.5 or 173±0.5, and the fragment ions of derivatized piperacillin are measured at m / z values ​​of 270±0.5 or 464±0.5.

[0209] 45) An (automated) analytical system (in particular an LC / MS system) adapted to carry out the method according to any one of items 1 to 44.

[0210] 46) A sampling tube for collecting a patient sample containing a nucleophilic derivatization reagent suitable for stabilizing one or more antibiotic analytes in the sample.

[0211] 47) A sampling tube for collecting a patient sample, the sampling tube comprising a device having a reservoir adapted to receive a blood sample to be collected and a nucleophilic derivatization reagent suitable for stabilizing one or more antibiotic analytes in the sample.

[0212] 48) The sampling tube according to item 46 or 47, wherein the nucleophilic derivatization reagent comprises an amine group, particularly a primary or secondary amine, especially a primary amine group.

[0213] 49) The sampling tube according to any one of items 46 to 48, wherein the nucleophilic derivatization reagent contains more than 3 C atoms, particularly 3 to 20 C atoms, particularly 3 to 10 C atoms, particularly 3 to 5 C atoms, and particularly 4 C atoms.

[0214] 50) The sampling tube according to any one of items 46 to 49, wherein the nucleophilic derivatization reagent is a linear or branched chain, particularly a linear amine, particularly a linear primary amine, particularly a linear primary amine containing 3 to 5 carbon atoms.

[0215] 51) The sampling tube according to any one of items 46 to 50, wherein the nucleophilic derivatization reagent is selected from the group consisting of propylamine, butylamine, or pentylamine, particularly primary linear butylamine.

[0216] 52) The sampling tube according to any one of items 46 to 51, wherein the nucleophilic derivatization reagent is contained in a liquid or lyophilized form.

[0217] 53) The sampling tube according to any one of items 46 to 52, wherein the nucleophilic derivatization reagent further comprises a stable, water-miscible non-nucleophilic base, particularly selected from the group consisting of DBU, TEA, DIPEA, Na3PO4, Na2CO3, and Cs2CO3.

[0218] 54) The sampling tube according to any one of items 46 to 53, wherein the nucleophilic derivatization reagent is contained in a liquid form in a solvent, particularly a solvent selected from the group consisting of water, CHCN, THF, dioxane, DMF, DMSO, acetone, tBuOH, diglyme, DME, MeOH, EtOH, 1-PrOH, 2-PrOH, ethylene glycol, hexamethylphosphoroamide (HMPA), hexamethylphosphorotriamide (HMPT), and glycerin, particularly a solvent selected from the group consisting of water, CHCN, THF, dioxane, DMF, DMSO, acetone, tBuOH, diglyme, and DME.

[0219] 55) The sampling tube according to any one of items 46 to 54, wherein when the antibiotic analyte is meropenem, the nucleophilic derivatization reagent comprises butylamine.

[0220] 56) The sampling tube according to any one of items 46 to 55, wherein when the antibiotic analyte is piperacillin, the nucleophilic derivatization reagent comprises pentylamine.

[0221] 57) Use of a nucleophilic derivatization reagent to determine the amount or concentration of one or more antibiotic analytes in a sample.

[0222] 58) The use according to item 57, wherein the nucleophilic derivatization reagent is a reagent containing an amine group, particularly a primary or secondary amine, especially a primary amine group.

[0223] 59) The use according to item 57 or 58, wherein the nucleophilic derivatization reagent contains more than 3 C atoms, particularly 3 to 20 C atoms, particularly 3 to 10 C atoms, particularly 3 to 5 C atoms, particularly 4 C atoms.

[0224] 60) The use according to any one of items 57 to 59, wherein the nucleophilic derivatization reagent is a linear or branched, particularly a linear amine, particularly a linear primary amine, particularly a linear primary amine containing 3 to 5 carbon atoms.

[0225] 61) The use according to any one of items 57 to 60, wherein the derivatization reagent is selected from the group consisting of propylamine, butylamine, or pentylamine, particularly primary linear butylamine.

[0226] 62) The use according to any one of items 57 to 61, wherein the antibiotic is a β-lactam antibiotic.

[0227] 63) The antibiotic is amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, temocillin, phenethicillin, penicillin G, penicillin V, piperacillin, azlocillin, bibampicillin, pivmecillinam, ticarcillin, cephacefetrile, cefadroxil, cefa Cefalexin (cephalexin), Cefalexin (cephalexin), Cefaloglycin (cephaloglycin), Cefalonium (cephalonium), Cefaloridine (cephaloradine), Cefalotin (cephalothin )), Cefapirin (Cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (Cefazolin), Cephradine (Cefradine), Cephradine (Cefradine), Cefroxadine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefmetazole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil (Cefproxil), cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefodoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidin, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomezine,Cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, azt 63. The use of any one of paragraphs 57 to 62, wherein the antibiotic analyte is selected from the group consisting of leonam, mecillinam, meampicillin, talampicillin, epicillin, sulbenicillin, faropenem, ritipenem, biapenem, pivampicillin, clometocillin, penamecillin, hetacillin, carindacillin, panipenem, tigemonam, carumonam, nocardicin A, penam, sulbactam, tazobactam, clavam, and clavulanic acid. In certain embodiments, the antibiotic analyte is meropenem or piperacillin.

[0228] 64) The use according to any one of items 57 to 63, wherein the antibiotic is meropenem or piperacillin.

[0229] 65) The use of any one of items 57 to 64, wherein the nucleophilic derivatization reagent prevents hydrolysis of antibiotics during determination of the amount or concentration of one or more antibiotic analytes in a sample.

[0230] 66) The use according to any one of items 57 to 65, wherein the nucleophilic derivatization reagent stabilizes the antibiotic for more than 7 days, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 2 months, more than 3 months, more than 4 months, more than 4 months, more than 5 months, or more than 6 months.

[0231] 67) Use of nucleophilic derivatization reagents to stabilize antibiotic analytes in samples of interest.

[0232] 68) The use according to item 67, wherein the nucleophilic derivatization reagent is a reagent containing an amine group, particularly a primary or secondary amine, especially a primary amine group.

[0233] 69) The use according to item 67 or 68, wherein the nucleophilic derivatization reagent contains more than 3 C atoms, in particular 3 to 20 C atoms, in particular 3 to 10 C atoms, in particular 3 to 5 C atoms, in particular 4 C atoms.

[0234] 70) Use according to any one of items 67 to 69, wherein the nucleophilic derivatization reagent is a linear or branched, particularly linear amine, particularly a linear primary amine, particularly a linear primary amine containing 3 to 5 carbon atoms.

[0235] 71) The use according to any one of items 6 to 70, wherein the derivatization reagent is selected from the group consisting of propylamine, butylamine, or pentylamine, particularly primary linear butylamine.

[0236] 72) The use according to any one of items 67 to 71, wherein the antibiotic is a β-lactam antibiotic.

[0237] 73) The antibiotic is amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, temocillin, phenethicillin, penicillin G, penicillin V, piperacillin, azlocillin, bibampicillin, pivmecillinam, ticarcillin, cephacefetrile, cefadroxil, cefa Cefalexin (cephalexin), Cefalexin (cephalexin), Cefaloglycin (cephaloglycin), Cefalonium (cephalonium), Cefaloridine (cephaloradine), Cefalotin (cephalothin )), Cefapirin (Cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (Cefazolin), Cephradine (Cefradine), Cephradine (Cefradine), Cefroxadine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefmetazole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil (Cefproxil), cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefodoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidin, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomezine,Cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, azt 73. The use of any one of paragraphs 67 to 72, wherein the antibiotic analyte is selected from the group consisting of leonam, mecillinam, meampicillin, talampicillin, epicillin, sulbenicillin, faropenem, ritipenem, biapenem, pivampicillin, clometocillin, penamecillin, hetacillin, carindacillin, panipenem, tigemonam, carumonam, nocardicin A, penam, sulbactam, tazobactam, clavam, and clavulanic acid. In certain embodiments, the antibiotic analyte is meropenem or piperacillin.

[0238] 74) The use according to any one of items 67 to 73, wherein the antibiotic is meropenem or piperacillin.

[0239] 75) The use according to any one of items 67 to 74, wherein the nucleophilic derivatization reagent prevents hydrolysis of the antibiotic.

[0240] 76) The use of any one of items 67 to 75, wherein the nucleophilic derivatization reagent stabilizes the antibiotic for more than 7 days, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 2 months, more than 3 months, more than 4 months, more than 5 months, or more than 6 months.

[0241] 77) An antibiotic analyte stabilized by said nucleophilic derivatization reagent.

[0242] 78) The antibiotic analyte according to item 77, wherein the nucleophilic derivatization reagent is a reagent comprising an amine group, particularly a primary or secondary amine, especially a primary amine group.

[0243] 79) The antibiotic analyte according to item 77 or 78, wherein the nucleophilic derivatization reagent comprises more than 3 C atoms, particularly 3 to 20 C atoms, particularly 3 to 10 C atoms, particularly 3 to 5 C atoms, particularly 4 C atoms.

[0244] 80) An antibiotic analyte according to any one of items 77 to 79, wherein the nucleophilic derivatization reagent is a linear or branched, particularly a linear amine, particularly a linear primary amine, particularly a linear primary amine containing 3 to 5 C atoms.

[0245] 81) The antibiotic analyte of any one of paragraphs 77 to 80, wherein the derivatization reagent is selected from the group consisting of propylamine, butylamine, or pentylamine, particularly primary linear butylamine.

[0246] 82) The antibiotic analyte of any one of items 77 to 81, wherein the antibiotic is a β-lactam antibiotic.

[0247] 83) The antibiotic is amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, temocillin, phenethicillin, penicillin G, penicillin V, piperacillin, azlocillin, bibampicillin, pivmecillinam, ticarcillin, cephacefetrile, cefadroxil, cefa Cefalexin (cephalexin), Cefalexin (cephalexin), Cefaloglycin (cephaloglycin), Cefalonium (cephalonium), Cefaloridine (cephaloradine), Cefalotin (cephalothin )), Cefapirin (Cephapirin), Cefatrizine, Cefazaflur, Cefazedone, Cefazolin (Cefazolin), Cephradine (Cefradine), Cephradine (Cefradine), Cefroxadine, Cefroxadine, Ceftezole, Cefaclor, Cefamandole, Cefmetazole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil (Cefproxil), cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefodoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidin, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomezine,Cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreona 83. The antibiotic analyte of any one of paragraphs 77 to 82, selected from the group consisting of meropenem, mecillinam, meampicillin, talampicillin, epicillin, sulbenicillin, faropenem, ritipenem, biapenem, pivampicillin, clometocillin, penamecillin, hetacillin, carindacillin, panipenem, tigemonam, carumonam, nocardicin A, penam, sulbactam, tazobactam, clavam, and clavulanic acid. In certain embodiments, the antibiotic analyte is meropenem or piperacillin.

[0248] 84) The antibiotic analyte according to any one of items 77 to 83, wherein the antibiotic is meropenem or piperacillin.

[0249] 85) The antibiotic analyte of any one of paragraphs 77 to 83, wherein the nucleophilic derivatization reagent prevents hydrolysis of the antibiotic during determination of the amount or concentration of one or more antibiotic analytes in a sample.

[0250] 86) The antibiotic analyte of any one of items 77 to 85, which is a derivatized β-lactam antibiotic analyte in which the β-lactam moiety is destroyed by reaction with a nucleophilic derivatization reagent. [Example]

[0251] The following examples are offered to illustrate, but not to limit, the invention claimed herein.

[0252] Example 1: Stability of natural piperacillin The stability of native piperacillin and its hydrolyzed forms was investigated (including compounds 5, 9a, and 9b, respectively. From the hydrolysis pathway of piperacillin (see schematic diagram in Figure 1), it is clear that this compound hydrolyzes at both the piperazine ring and the lactam moiety; only one of the two compounds is monitored to account for the loss of native piperacillin). For this purpose, these compounds were freshly weighed and dissolved in water at a concentration of 1 mg / mL by rotation at room temperature for 15 min. These compounds were then diluted to 5 μg / mL and measured by the appropriate LC-MS / MS method at 0, 2, 4, 6, 8, and 16 h. In this case, a Sunshell C18, 2.6 μm, 2.1 mm × 50 mm column containing solvent A: water with 0.1% HCOOH and solvent B: CH3CN with 0.1% HCOOH was used, and an Agilent Infinity II multisampler / pump system connected to an AB Sciex 6500+MS was used at a flow rate of 0.6 mL per minute. Peaks were integrated using MultiQuant software, and the areas of these peaks are shown in the graphs in Figures 2A and 2B.

[0253] Figures 2A and 2B show the areas of one MRM transition obtained for native piperacillin (compound 5) and its hydrolyzed forms (compounds 9a / 9b), respectively. It is clear that the peak areas obtained change significantly over time (F-test, resulting in a P value of less than 0.0001), with the peak area of ​​the native form decreasing and the peak area of ​​the hydrolyzed forms (compounds 9a / 9b) increasing significantly (F-test, resulting in a P value of less than 0.0001). The reason for this is hydrolysis (schematically shown in Figure 1).

[0254] Example 2: Stability of derivatized piperacillin To assess whether complete β-lactam derivatization could be achieved using simple propylamine, butylamine, or pentylamine, these nucleophiles were added in high excess to solutions of meropenem and piperacillin (1 μg / mL). For schematic diagrams of the chemical reactions, see Figures 3 and 4 for meropenem and piperacillin, respectively.

[0255] The stability of the double butyl amide variant of piperacillin (compound 7, see FIG. 4) was investigated by two MRMs using the same protocol as in Example 1.

[0256] Figure 5 shows the areas obtained for the two MRM transitions of compound 7. It is observed that the peak areas obtained do not change significantly over time, i.e., the derivatized piperacillin does not hydrolyze. This is further supported by an F-test, which gives P values ​​of 0.08 and 0.14.

[0257] Example 3: Stabilization of meropenem and piperacillin in patient samples Derivatization reagent (propylamine, butylamine, or pentylamine) dissolved in water was added to 100 μL of sample (serum spiked with both 1 μg / mL piperacillin and meropenem).

[0258] 100 μL of 1 μg / mL (1.9*10 -9 M) 5*10 for piperacillin 8 , 2.5*10 8 or 2.5*10 6 Equivalent (9.8*10 -5 , 4.8*10 -5 , 1.9*10 -7 Each derivatization reagent (20 μL) was added to the spiked serum. The mixture was then incubated for 3 min, after which a pH-adjusting reagent (40 μL of 1 M HCOOH aqueous solution (pH 2.5) or 500 mM Na3PO4 / Na2HPO4 (pH 12)) was added. Magnetic beads (40 μL, 50 mg / mL) were then added and incubated for 3 min. The supernatant was then removed, and the beads were washed twice with water (150 μL). Next, an elution solution (50 μL of a solution containing 100 mM HCOOH, 100 mM pyrrolidine, or no pH-adjusting reagent in various levels of acetonitrile (10-90%, v / v)) was added. The supernatant (20 μL) was then diluted with water (20 μL).

[0259] To quantify both native (intact) meropenem and piperacillin, as well as their derivatized and hydrolyzed products, an LC-MS / MS method was devised, including tailored MRM transitions for all compounds. Solvent A: water with 0.1% HCOOH, and solvent B: CH3CN with 0.1% HCOOH, was used on a Cortecs C18+C18, 2.6 μm, 2.1 mm x 50 mm column, and an Agilent Infinity II multisampler / pump system connected to an AB Sciex 6500+MS, with a flow rate of 0.6 mL / min. Three MRM transitions were used for each of the derivatized antibiotics (i.e., meropenem (a386) and piperacillin (a0387) derivatized with either propylamine, butylamine, or amylamine). Native meropenem and piperacillin, as well as their hydrolyzed forms (two MRM transitions per analyte), were also included in the analysis. [Table 2]

[0260] For meropenem, the highest peak area results are obtained using pentylamine. Using pentylamine and an optimal workflow, an area of ​​approximately 3E6 should be possible at a concentration of 1 μg / mL of this antibiotic in serum. Using butylamine, an area of ​​1E6 is achievable under optimal conditions. See Figure 6.

[0261] For piperacillin, the highest peak area results are obtained using butylamine. Using butylamine and an optimal workflow (see next section for optimal workflow), an area of ​​2E7 should be possible for this antibiotic at a concentration of 1 μg / mL in serum. See Figure 7.

[0262] Note that when 2.5E8 equivalents of reagent are used, no residual native compound (intact meropenem or piperacillin) is found in the eluate, indicating that the reaction is quantitative in this short time. Furthermore, no increase in the amount of hydrolyzed compound is observed, indicating that the addition of nucleophiles does not catalyze the hydrolysis of the lactam moiety in these compounds, allowing for the differentiation and quantification of intact lactam compounds from hydrolyzed compounds.

[0263] Example 3 shows that the derivatization strategy works for two representative β-lactam antibiotics in combination with three different nucleophilic derivatization reagents, demonstrating the validity and overall robustness of the method. Example 4: Degradation of Piperacillin in Serum

[0264] The main obstacle in quantitating this class of antibiotics is addressed in Figure 8. In general, quantitation, whether by LC-MS / MS, UV, or immunoassay, relies on accurate calibration, and using a reliable calibration method is clearly of paramount importance. However, as can be shown here, β-lactam antibiotics dissolved in serum are highly unstable. This results in spiked concentrations being higher than the actual concentrations, leading to a calibration offset (see Figure 8) and inaccurate results.

[0265] Example 4 shows that when natural β-lactam antibiotics are used for calibration purposes, these compounds decompose faster than the derivatized compounds proposed herein. This means that calibration using natural β-lactam antibiotics will yield inaccurate results. For this reason, the use of stabilized (i.e., derivatized) compounds will yield more accurate results.

[0266] Experimental design We hypothesized that β-lactam antibiotics would be more stable in neat solution (i.e., water containing 50% CH3CN) than in serum-based solutions because they may provide a high concentration of nucleophiles that can hydrolyze or otherwise react with the β-lactam moiety, for example, to give amides or esters. To test this hypothesis, piperacillin was dissolved in a solution of water / CH3CN (1:1, v:v) and used to spike the same solution of serum and water / CH3CN. Although dissolution was performed only once, addition of this stock solution in serum or water / CH3CN was performed three times for four different concentrations of piperacillin.

[0267] Prior to measurement, most methods in routine clinical diagnostics require a purification workflow. Several methods are available, ranging from protein precipitation using organic solvents followed by centrifugation to magnetic bead purification. When quantification is performed by MS / MS, an isotopically labeled internal standard (ISTD) is preferably added at the beginning of this purification workflow to correct for i) analyte loss during this workflow and ii) ion suppression / enhancement, which may differ between calibration samples and patient samples. This can be achieved using an enrichment workflow in which piperacillin is derivatized with butylamine to give a dibutylamide (Figure 4, compound 7, see scheme below). The β-lactam moiety reacts with the butylamide, and the piperazine moiety reacts during this procedure. Preferably, an ISTD is added, which is a stable derivative of piperacillin containing a single butylamide chain and a D5 label on the phenyl moiety. Therefore, this ISTD does not undergo nucleophilic substitution, which would result in the collapse of the β-lactam moiety. However, a second amidation occurs on the piperazine ring during the workflow (see scheme below). Thus, although inherently more stable, ISTD does not degrade as quickly as native piperacillin, and the amidation on the piperazine ring is an in-line control that confirms that amidation with butylamine works. [ka]

[0268] Piperacillin is derivatized using butylamine to give the dibutylamide. [ka]

[0269] Piperacillin-butyramide-D5 is derivatized using butylamine to give piperacillin-dibutylamide-D5.

[0270] Materials and Methods material Piperacillin was obtained from Sigma Aldrich.

[0271] Quality control materials were from Chromsystems and subsequently yielded dissolved concentrations of 19.2 and 97.9 μg / mL.

[0272] method Weighing and Spiking Piperacillin was weighed and dissolved directly in water / CH3CN (1:1, v:v) to obtain a concentration of 1 mg / mL. This stock solution was then used to spike either the serum pool or water / CH3CN (1:1, v:v) to obtain concentrations of 1, 10, 50, and 100 μg / mL. This spiking was repeated three times for each concentration.

[0273] All samples were then homogenized by rolling for 20 minutes. The samples were then placed in the sample preparation module and each sample was processed as described in the following section.

[0274] Sample preparation Preferably, ISTD (piperacillin-butylamide-D5, 20 μg / mL, 20 μL) was added to spiked serum or neat solution (50 μL). To this mixture, n-butylamine (5 M, 50 μL) was added. The mixture was first shaken and incubated at room temperature (rt) for 3 minutes. Next, magnetic beads (bead type B, 50 mg / mL, 40 μL) were added, and the mixture was then shaken again and incubated for approximately 1 minute. Subsequently, the beads were pulled toward the container by applying a magnetic force, and the supernatant was then removed. These beads were washed twice with water (150 μL). Next, acetonitrile containing 0.1% HCOOH (50 μL) was added, and the mixture was then shaken again and allowed to stand for 1 minute. Next, the beads were pulled toward the container, and 20 μL of the supernatant was then removed. The supernatant was then diluted with water (1:1, v:v), and the sample was then measured by LC-MS / MS.

[0275] LC-MS / MS measurement An LC-MS / MS method was developed to quantify the doubly derivatized piperacillin derivatives. The following table shows which fragments were used for this purpose under which settings. [Table 3] [Table 4]

[0276] Solvent A: water with 0.1% HCOOH, and solvent B: CH3CN with 0.1% HCOOH were used on a Kinetex C18, 2.6 μm, 1.0 mm × 50 mm column and an Agilent Infinity II multisampler / pump system connected to an AB Sciex 6500+MS at a flow rate of 0.4 mL per minute, injecting 8 μL per sample.

[0277] result Figure 9 shows the difference in area ratio between the neat and serum-derived samples for four concentrations. For each concentration, this difference is shown to be approximately 30%. The difference in area ratio (for which an internal standard can be used) cannot be attributed to differences in analyte recovery due to different sample preparations for the neat versus serum samples. The internal standard compensates for this effect. Therefore, this difference is most likely due to compound reactivity. Because serum contains many reactive nucleophiles that can react with either lactam or piperazine moieties, spiked concentrations decrease over time in this matrix compared to the same concentrations spiked neat.

[0278] This finding may have implications for the quantification of these antibiotics because the spiked concentrations are higher than the actual concentrations, which are a function of time, temperature, protein concentration, or the concentration of other nucleophiles. Therefore, the use of native piperacillin as a spike material to prepare calibration standards may fail. This also indicates that the quantification of these analytes with the derivatization method described herein will be more accurate.

[0279] Example 5: Comparison of piperacillin derivatization methods with routinely used hospital methods To ensure long-term stability and precise, accurate quantification of β-lactam antibiotics, we envision a strategy that utilizes derivatization of this class of antibiotics. This also involves the use of pre-derivatized calibrators and ISTDs. After development of the in-house assay, experiments were performed using commercially available QC samples routinely used in at least one hospital, e.g., a German hospital. To assess how the derivatization method deviates from the method routinely used in the hospital, 23 patient samples were collected and measured using both methods.

[0280] Example 5 shows that the derivatization method presented here correlates well with routine methods, but an average 20% difference in precision is observed between the two methods. This precision offset is explained in Example 4.

[0281] Materials and Methods material Calibration materials used in derivatization strategies

[0282] Singly derivatized piperacillin (piperacillin-butylamide) i387-2-2 was weighed and spiked directly into serum in powder form and further dilutions were prepared to obtain the calibration series in the table below. [Table 5] [Table 6] [Table 7] [Table 8] [Table 9]

[0283] method Sample preparation for derivatization strategies Preferably, ISTD (piperacillin-butylamide-D5, 20 μg / mL, 20 μL) was added to either a calibration sample, QC sample, or patient sample (50 μL). To this mixture, n-butylamine (5 M, 50 μL) was added. The mixture was first shaken and incubated at room temperature for 3 minutes. Next, magnetic beads (bead type B, 50 mg / mL, 40 μL) were added, and the mixture was then shaken again and incubated for approximately 1 minute. Subsequently, a magnetic force was applied to pull the beads toward the container, and the supernatant was then removed. The beads were washed twice with water (150 μL). Next, acetonitrile (50 μL) containing 0.1% HCOOH was added, and the mixture was then shaken again and allowed to stand for 1 minute. Next, the beads were pulled toward the container, and 20 μL of the supernatant was then removed. The supernatant was then diluted with water (1:1, v:v), and the sample was then measured by LC-MS / MS. All clinical patient samples were processed sequentially in a non-randomized fashion. Thus, there was a time lag of approximately 90 minutes between the processing of sample 1 and sample 23. There was a time lag of approximately 4 minutes between the measurements of the three replicates processed for each sample.

[0284] Sample preparation for hospital methods Preferably, ISTD (piperacillin-D5, 100 μg / mL, 25 μL) was added to either a calibration sample, QC sample, or patient sample (50 μL). This mixture was vortexed briefly and shaken for 5 minutes. MeOH (325 μL) was then added, vortexed briefly, and shaken for 5 minutes. The vials were then centrifuged (14000 rpm at 5°C), and the supernatant (20 μL) was diluted with water (180 μL). These solutions were measured by LC-MS / MS. All clinical patient samples were processed sequentially in a non-randomized manner.

[0285] Derivatization methods for LC-MS / MS measurements An LC-MS / MS method was developed to quantify the doubly derivatized piperacillin derivatives. The following table shows which fragments were used for this purpose under which settings. [Table 10] [Table 11]

[0286] Solvent A: water with 0.1% HCOOH, and solvent B: CH3CN with 0.1% HCOOH were used on a Kinetex C18, 2.6 μm, 1.0 mm × 50 mm column and an Agilent Infinity II multisampler / pump system connected to an AB Sciex 6500+MS at a flow rate of 0.4 mL per minute, injecting 8 μL per sample. [Table 12] [Table 13]

[0287] A Waters XSelect HSS PFP 2.5 μm (2.1 × 100 mm) column with a Waters XSelect HSS PFP Van Guard cartridge (2.1 × 5 mm) was used. Solvent A: water containing 10 mM ammonium formate with 0.2% formic acid, and solvent B: CHCN / MeOH (25:75, v:v) were used with an Agilent Infinity II multisampler / pump system connected to an AB Sciex 6500+MS at a flow rate of 4.5 mL / min, with 2 μL injections per sample.

[0288] result Accuracy and Precision Accuracy can be calculated from the variance of the obtained results, but precision can only be determined if the correct or theoretical concentration is given. As already established in Example 4, the correct concentration is not equal to the spiked concentration, but is a concentration less than this concentration. Nevertheless, to be able to calculate the difference between the derivatization method described here and the reference method, we assume that the spiked concentration is equal to the actual concentration, recognizing that this is not correct. However, as a relative measure of precision, this is still a useful indicator.

[0289] The accuracy in terms of CV is seen to be very low for the quality control samples, which have CVs of less than 4%. The precision for these samples is 86.4 and 80%. Again, this is based on the assumption that the spiked concentrations of the calibrators used in the reference method are equal to the actual concentrations. However, the actual precision is assumed to be closer to 100%. Furthermore, because the relative total error is calculated based on precision, this error is assumed to be closer to 0 than the value calculated in Figure 10.

[0290] Correlation between methods To see how both evaluated methods correlated, Figures 11 and 12 were generated using JMP version 14.3. R2 and F-tests indicating high correlation between the two methods were included in the analysis. Figure 11 shows the correlation concentrations calculated from both methods, with all samples included. Figure 12 shows the correlation concentrations calculated from both methods, with the most highly concentrated sample excluded for clarity. Figure 13 shows the difference in accuracy between the two methods per replicate: (Accuracy Derivatization Method) - (Accuracy Hospital Method).

[0291] All samples processed with the derivatization method were processed three times over a time course of approximately 4 hours between replicates 1 and 3. The samples were placed in the pipetting robot at temperatures between 25 and 30 °C. The significance of this time difference can be clearly seen in Figure 13. Here, we can see that the difference in precision between the two methods is smallest for replicate 1, while replicates 2 and 3 show much larger deviations from the original values. The degradation of this analyte over time is considerable, and therefore the lower precision for replicates 2 and 3 is a consequence of this. This is also much greater than what the method itself is capable of, making any attempt to calculate CV from these values ​​meaningless. Nevertheless, an interesting result from this is that although all replicates of a single sample have the same time lapse between them, the difference in calculated concentration is variable for all samples. For example, clinical sample 42 shows approximately 40% degradation over 4 hours, while clinical sample 137 shows only approximately 10% degradation over the same period. The finding that different clinical samples show differences in the decrease in piperacillin concentration suggests that different clinical samples exhibit different degradation kinetics for piperacillin. This means that it is absolutely crucial to process and measure patient samples as soon as possible after the samples are obtained. More precisely, this also indicates once again that routinely used methods that utilize calibration materials containing spiked piperacillin and ISTD, an isotope-labeled variant of piperacillin, may produce inaccurate results that are an overestimation of the true value.

[0292] This patent application claims priority from European patent application 19209516.4, the contents of which are incorporated herein by reference.

Claims

1. 1. A method for determining the amount or concentration of one or more antibiotic analytes in a sample obtained, comprising: a) pretreating the sample with a derivatization reagent, the derivatization reagent being selected from the group consisting of propylamine, butylamine, or pentylamine; b) concentrating the sample obtained after step a) using magnetic beads; and c) determining the amount or concentration of one or more antibiotic analytes in the pretreated sample obtained after the concentration step b) using LC / MS. Including, the enrichment step b) comprises at least one enrichment workflow, which comprises adding magnetic or paramagnetic beads carrying an analyte-selective group to the pretreated sample of step a); The method wherein the antibiotic analyte is piperacillin or meropenem.

2. 10. The method of claim 1, wherein the derivatization reagent is a primary linear butylamine.

3. 3. The method of claim 1, wherein in step a), the sample is pretreated with a nucleophilic derivatization reagent immediately after the sample is obtained.

4. 4. The method according to claim 1, wherein in step a), the sample is pretreated with a nucleophilic derivatization reagent within less than 10 minutes of obtaining the sample or within less than 5 minutes of obtaining the sample.

5. 5. The method of any one of claims 1 to 4, wherein the sample obtained after step a) comprises an antibiotic analyte derivatized with a nucleophilic derivatization reagent.

6. The method according to any one of claims 1 to 5, wherein in step c) ion formation is based on electrospray ionization (ESI).

7. The method according to any one of claims 1 to 6, wherein in step c) the MS device is a tandem mass spectrometer.

8. The method according to any one of claims 1 to 7, wherein in step c) the MS instrument is a triple quadrupole instrument.

9. The method according to any one of claims 1 to 8, wherein in step c) the MS device is an automated random access LC / MS system.

10. The method of any one of claims 1 to 9, which is automated.

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