Derivatization of β-lactam antibiotics as calibrants / ISTDs in mass spectrometric measurements

Abstract

The present invention relates to a conjugate comprising an antibiotic and a nucleophilic derivatization reagent, a composition comprising the conjugate, a kit comprising the conjugate or composition, and uses of the conjugate or composition.

Description

[Technical Field]

[0001] The present invention relates to conjugates formed between antibiotics and nucleophilic derivatization reagents, compositions comprising the conjugates, kits comprising the conjugates or compositions, and uses of the conjugates or compositions. [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] Therefore, 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 high temperatures. Obviously, hydrolyzed antibiotics are no longer active compounds capable of inhibiting bacterial growth. The terms "hydrolysis" and "hydrolysis" can be used interchangeably in the present disclosure. The term "hydrolysis" is known to those skilled in the art and therefore will not be described in detail.

[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. Obtaining accurate concentrations of natural β-lactam antibiotics is impossible due to continued hydrolysis following patient sampling (e.g., blood draw). Considerable effort has been made to study and address β-lactam instability in the field of therapeutic drug monitoring (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)).

[0006] Because of the importance of carefully monitoring antibiotic concentrations, effective and stable methods for quantifying these compounds from human and animal matrices are needed. Currently, quantification of these antibiotics can be performed by LC-MS / MS. In this method, it is crucial to calibrate the system using the antibiotic of interest as a calibrant. Most commonly, isotope-labeled internal standards (ISTDs) of known concentrations are used to account for analyte losses that occur during compound handling / purification before measurement by MS. ISTDs also correct for ionization variations in different samples. Therefore, a prerequisite for this method is that the concentrations of both the calibrator and the ISTD are known. Because hydrolysis of these compounds occurs under very mild conditions (Wiedemann et al.; 2015; ChemPlusChem, 80), it is difficult to keep these calibrators and ISTDs intact until the time of their use in the measurement. This leads to deviations in the calibrator and ISTD concentrations, making accurate quantification of the analytes of interest in patient samples impossible. Therefore, there is an urgent need for stable calibrators and ISTDs that can measure β-lactam antibiotic levels without the interference with measurement accuracy caused by unstable ISTDs and calibrators.

[0007] Here, for the first time, we are able to show that β-lactam antibiotics can be stabilized to function as stable calibrators and ISTDs to ensure accurate sample measurements. Summary of the Invention

[0008] Summary of the Invention In a first aspect, the present invention relates to a conjugate formed between an antibiotic and a nucleophilic derivatization reagent.

[0009] In a second aspect, the present invention relates to a composition comprising the conjugate of the first aspect.

[0010] In a third aspect, the present invention relates to a kit comprising: a) the conjugate of the first aspect or the composition of the second aspect; and b) a package insert.

[0011] In a fourth aspect, the present invention relates to the use of the complex of the first aspect or the composition of the second aspect for mass spectrometry measurements.

[0012] In a fifth aspect, the present invention relates to the use of a nucleophilic derivatization reagent to stabilize antibiotics, in particular to make them suitable as ISTDs and / or calibrators in mass spectrometric measurements.

[0013] In a sixth aspect, the present invention relates to derivatized antibiotics suitable as ISTDs and / or calibrators in mass spectrometric measurements.

[0014] In a seventh aspect, the present invention relates to the use of derivatized antibiotics as ISTDs and / or calibrants in mass spectrometric measurements.

[0015] In an eighth aspect, the present invention relates to a method for producing a stabilized antibiotic comprising derivatizing an antibiotic with a nucleophilic derivatization reagent. [Brief explanation of the drawings]

[0016] Drawing List [Figure 1]Schematic representation of the hydrolysis pathway of piperacillin (compound 5). [Figure 2A] Measured peak areas of A) native piperacillin (compound 5); and B) singly hydrolyzed piperacillin (compounds 9a or 9b, Figure 1) in water at room temperature over 16 hours. Confidence fit and F-test are shown. A reference line is drawn through the average area values ​​for clarity. [Figure 2B] Measured peak areas of A) native piperacillin (compound 5); and B) singly hydrolyzed piperacillin (compounds 9a or 9b, Figure 1) in water at room temperature over 16 hours. Confidence fit and F-test are shown. A reference line is drawn through the average area values ​​for clarity. [Figure 3] Schematic of the derivatization reaction sequence of meropenem (compound 1) with different reagents: A) propylamine; B) butylamine; C) pentylamine [Figure 4] Schematic of the derivatization reaction sequence of piperacillin (compound 5) 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] Synthetic Routes for Piperacillin-D5 Monohydrate (8) and Piperacillin-n-Butylamide-D5 (9) [Figure 7] Synthetic routes to meropenem-13C2-15N(7) and meropenem-n-butylamide-13C2-15N(8). [Figure 8] Schematic of deuterated piperacillin ISTD (compound 11) [Figure 9] Measured peak areas over 16 hours for singly derivatized piperacillin-D5 (compound 11, FIG. 8) in water at room temperature. A reference line is drawn through the average area values ​​for clarity. Confidence fit and F-test are shown. [Figure 10]Measured peak areas of meropenem (e.g., compounds 2, 3, or 4 shown in Figure 3) derivatized with the reagents propylamine, butylamine, and pentylamine under different reaction conditions. [Figure 11] Measured peak areas of piperacillin (e.g., compounds 6, 7, or 8 shown in Figure 4) derivatized with the reagents propylamine, butylamine, and pentylamine under different reaction conditions. DETAILED DESCRIPTION OF THE INVENTION

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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 characteristics being described.

[0023] 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.

[0024] The terms "measurement", "determining" or "determining" preferably include qualitative, semi-quantitative or quantitative measurements.

[0025] 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.

[0026] 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 with a net negative charge of one or more electron units, while cations are those with a net positive charge of one or more electron units. MS can be performed in either "negative ion mode," where negative ions are generated and detected, or "positive ion mode," where positive ions are generated and detected.

[0027] "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, the following three steps are performed:

[0028] 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).

[0029] 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.

[0030] 3. The separated ions are detected, for example, in multiple reaction mode (MRM), and the results are displayed on a chart.

[0031] 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.

[0032] 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 sustained 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. A counterflow of dry, preheated Ni gas may be used to improve solvent removal. Gas-phase ionization in APCI can be more effective than ESI for analyzing less polar entities.

[0033] "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.

[0034] "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).

[0035] 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 typically be performed without prior knowledge of the amino acid sequence.

[0036] 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.

[0037] 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 bacteria. 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 (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).

[0038] 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.

[0039] 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).

[0040] Prior to analysis by mass spectrometry, 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 by mass spectrometry. 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.

[0041] 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, 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).

[0042] The term "calibrant" typically refers to a complex or composition used as a standard or control to construct a calibration curve that allows for the determination of unknown concentrations or amounts of an analyte of interest in a given sample. Calibrators are run in series over a predetermined linear dynamic range of concentrations. The responses from testing the calibrators are plotted on a signal vs. concentration yx axis graph. Typically, the chemical structure of the calibrator is identical or very similar (exhibits very similar properties) to the chemical structure of the analyte of interest. 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 differential distribution of the chemical components as it flows around or over a stationary liquid or solid phase.

[0043] 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).

[0044] "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.

[0045] Additionally, hydrophilic interaction chromatography (HILIC), size-exclusion LC, ion-exchange LC, and affinity LC are well known.

[0046] 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.

[0047] 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.

[0048] 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 -NH2, -OH, -SH, -Se, (R',R'',R''')P, N3 - , RCOOH, F - , Cl - , Br - , 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 can react with a substance of interest, such as an analyte of interest, thereby forming a new complex that contains a nucleophilic unit and an analyte unit. The terms "nucleophile" and "nucleophilic" can be used interchangeably herein and hereinafter.

[0049] 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 container represents or 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.

[0050] "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.

[0051] Embodiment Commonly used approaches for measuring 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 suitable nucleophiles, thereby providing stable ISTDs and calibrators that ensure accurate measurements of antibiotics in patient samples.

[0052] Thus, in a first aspect, the present invention relates to a conjugate formed between an antibiotic and a nucleophilic derivatization reagent. The conjugate is obtained by reacting the antibiotic with the derivatization reagent to form an amide. In embodiments where the antibiotic is a β-lactam-containing antibiotic, the β-lactam moiety is destroyed upon amide formation. The resulting conjugate comprises an antibiotic unit and a nucleophilic derivatization unit.

[0053] In embodiments, the nucleophilic derivatization reagent is a reagent containing an amine group, particularly a primary or secondary amine. In certain embodiments, the nucleophilic derivatization reagent is a primary amine group. Primary amine groups have the advantage of allowing shorter incubation times compared to secondary amines.

[0054] In embodiments, the nucleophilic derivatization reagent comprises more than 3 C atoms, particularly 3 to 20 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 3 to 10 C atoms, particularly 3 to 5 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 4 C atoms.

[0055] In embodiments, the nucleophilic derivatization reagent is a linear or branched chain, particularly a linear amine, especially a linear primary amine. In certain embodiments, the nucleophilic derivatization reagent is a linear primary amine containing 3 to 5 carbon atoms. Therefore, MS interference can be reduced or avoided.

[0056] In embodiments, the nucleophilic derivatization reagent is selected from the group consisting of propylamine, butylamine, and pentylamine. In certain embodiments, the nucleophilic derivatization reagent is a primary linear butylamine.

[0057] In embodiments, the antibiotic is an antibiotic that includes a β-lactam moiety, i.e., in embodiments, the antibiotic is a β-lactam antibiotic.

[0058] 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 (cephacetrile), cefadroxil (cefadroxyl), cef 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,Selected from the group consisting of cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreonam, and mecillinam.

[0059] In certain embodiments, the antibiotic is meropenem or piperacillin.

[0060] In embodiments, the conjugate comprises one or more isotopic labels. In certain embodiments, the one or more isotopic labels may be included in the nucleophile-derivatized unit and / or the antibiotic unit of the conjugate. In certain embodiments, the one or more isotopic labels are included in the antibiotic unit of the conjugate. In certain embodiments, the one or more isotopic labels are deuterium, 13 C. 15 N and / or 18 In certain embodiments, all isotopic labels are the same label. In alternative embodiments, the isotopic labels may be different, i.e., may include two, three, or four different isotopic labels.

[0061] In certain embodiments, the antibiotic unit of the complex comprises 1 to 10 isotopic labels, particularly all identical. In certain embodiments, the antibiotic unit of the complex comprises 1 to 8 isotopic labels, particularly 1 to 5 isotopic labels. In certain embodiments, the antibiotic unit of the complex comprises 1, 2, 3, 4, or 5 isotopic labels. In certain embodiments, the antibiotic unit of the complex comprises 5 deuterium labels.

[0062] In a second aspect, the present invention relates to a composition comprising a conjugate formed between an antibiotic and a nucleophilic derivatization reagent. In embodiments, the composition further comprises 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, hexamethylphosphoramide (HMPA), hexamethylphosphorus triamide (HMPT), and glycerin. In particular embodiments, the solvent is selected from the group consisting of water, CHCN, THF, dioxane, DMF, DMSO, acetone, t-butyl alcohol, diglyme, and DME.

[0063] In embodiments, the composition further comprises a stable, water-miscible non-nucleophilic base, hi certain embodiments, the non-nucleophilic base is selected from the group consisting of DBU, NaPO, NaCO, and CsCO.

[0064] The conjugate is obtained by reacting the antibiotic with a derivatization reagent to form an amide. In embodiments where the antibiotic is a β-lactam-containing antibiotic, the β-lactam moiety is destroyed upon amide formation. The resulting conjugate comprises an antibiotic unit and a nucleophile-derivatized unit.

[0065] In embodiments, the nucleophilic derivatization reagent is a reagent that includes an amine group, particularly a primary or secondary amine. In certain embodiments, the nucleophilic derivatization reagent is a primary amine group.

[0066] In embodiments, the nucleophilic derivatization reagent comprises more than 3 C atoms, particularly 3 to 20 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 3 to 10 C atoms, particularly 3 to 5 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 4 C atoms.

[0067] In embodiments, the nucleophilic derivatization reagent is a linear or branched chain, particularly a linear amine, especially a linear primary amine. In particular embodiments, the nucleophilic derivatization reagent is a linear primary amine containing 3 to 5 C atoms.

[0068] In embodiments, the nucleophilic derivatization reagent is selected from the group consisting of propylamine, butylamine, and pentylamine. In certain embodiments, the nucleophilic derivatization reagent is a primary linear butylamine.

[0069] In embodiments, the antibiotic is an antibiotic that includes a β-lactam moiety, i.e., in embodiments, the antibiotic is a β-lactam antibiotic.

[0070] 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 (cephacetrile), cefadroxil (cefadroxyl), cef 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,Selected from the group consisting of cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreonam, and mecillinam.

[0071] In certain embodiments, the antibiotic is meropenem or piperacillin.

[0072] In embodiments, the conjugate comprises one or more isotopic labels. In certain embodiments, the one or more isotopic labels may be included in the nucleophile-derivatized unit and / or the antibiotic unit of the conjugate. In certain embodiments, the one or more isotopic labels are included in the antibiotic unit of the conjugate. In certain embodiments, the one or more isotopic labels are deuterium, 13 C. 15 N and / or 18 In certain embodiments, all isotopic labels are the same label. In alternative embodiments, the isotopic labels may be different, i.e., may include two, three, or four different isotopic labels.

[0073] In certain embodiments, the antibiotic unit of the complex comprises 1 to 10 isotopic labels, particularly all identical. In certain embodiments, the antibiotic unit of the complex comprises 1 to 8 isotopic labels, particularly 1 to 5 isotopic labels. In certain embodiments, the antibiotic unit of the complex comprises 1, 2, 3, 4, or 5 isotopic labels. In certain embodiments, the antibiotic unit of the complex comprises 5 deuterium labels.

[0074] In a third aspect, the present invention relates to a kit comprising: a) the conjugate of the first aspect or the composition of the second aspect; and b) a package insert.

[0075] In a fourth aspect, the present invention relates to the use of the complex of the first aspect, or the composition of the second aspect, or the kit of the third aspect, for mass spectrometry measurements.

[0076] In certain embodiments, the complex or composition or kit is used to determine the amount or concentration of an antibiotic analyte in a patient sample by mass spectrometry.

[0077] In certain embodiments, the complex or composition is used to serve as an internal standard (ISTD) in the determination of the amount or concentration of an antibiotic analyte in a patient sample by mass spectrometry. In certain embodiments, the complex of the first aspect or the composition of the second aspect is used as an ISTD by adding a predetermined amount or concentration to a sample of interest. In particular, the complex of the first aspect or the composition of the second aspect is added to the sample prior to measurement of the sample.

[0078] In certain embodiments, the complex or composition is used to serve as a calibrator for the determination of the amount or concentration of an antibiotic analyte in a patient sample by mass spectrometry, hi certain embodiments, the complex of the first aspect or the composition of the second aspect is used.

[0079] In a fifth aspect, the present invention relates to the use of a nucleophilic derivatization reagent for stabilizing an antibiotic analyte in a sample of interest. In particular, the nucleophilic derivatization reagent is used to derivatize and thereby stabilize an antibiotic suitable as an ISTD and / or calibrator in mass spectrometry measurements. In embodiments, the nucleophilic derivatization reagent prevents hydrolysis of the antibiotic. The nucleophilic derivatization reagent prevents hydrolysis by reacting with the antibiotic to form a complex. In embodiments where the antibiotic is a β-lactam-containing antibiotic, complex formation disrupts the β-lactam moiety. The resulting complex comprises an antibiotic unit and a nucleophilic derivatization unit.

[0080] In embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 12 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 12 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 24 hours, particularly more than 48 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 2 weeks. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 2 months. 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.

[0081] In embodiments, a nucleophilic derivatization reagent is used to stabilize the antibiotic, allowing for the use of the derivatized antibiotic as an internal standard (ISTD) mass spectrometric measurement.

[0082] In embodiments, the nucleophilic derivatization reagent is a reagent that includes an amine group, particularly a primary or secondary amine. In certain embodiments, the nucleophilic derivatization reagent is a primary amine group.

[0083] In embodiments, the nucleophilic derivatization reagent comprises more than 3 C atoms, particularly 3 to 20 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 3 to 10 C atoms, particularly 3 to 5 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 4 C atoms.

[0084] In embodiments, the nucleophilic derivatization reagent is a linear or branched chain, particularly a linear amine, especially a linear primary amine. In particular embodiments, the nucleophilic derivatization reagent is a linear primary amine containing 3 to 5 C atoms.

[0085] In embodiments, the nucleophilic derivatization reagent is selected from the group consisting of propylamine, butylamine, and pentylamine. In certain embodiments, the nucleophilic derivatization reagent is a primary linear butylamine.

[0086] In embodiments, the antibiotic is an antibiotic that includes a β-lactam moiety, i.e., the antibiotic is a β-lactam antibiotic.

[0087] 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 (cephacetrile), cefadroxil (cefadroxyl), cef 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,Selected from the group consisting of cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreonam, and mecillinam.

[0088] In certain embodiments, the antibiotic is meropenem or piperacillin.

[0089] In embodiments, the antibiotic and / or the nucleophilic derivatization reagent comprises one or more isotopic labels. In certain embodiments, the one or more isotopic labels may be included in the nucleophilic derivatization unit and / or the antibiotic unit of the conjugate. In certain embodiments, the one or more isotopic labels are included in the antibiotic unit of the conjugate. In certain embodiments, the one or more isotopic labels are deuterium, 13 C. 15 N and / or 18 In certain embodiments, all isotopic labels are the same label. In alternative embodiments, the isotopic labels may be different, i.e., may include two, three, or four different isotopic labels.

[0090] In certain embodiments, the antibiotic unit of the conjugate comprises 1 to 10 isotopic labels. In certain embodiments, the antibiotic unit of the conjugate comprises 1 to 8 isotopic labels, particularly 1 to 5 isotopic labels. In certain embodiments, the antibiotic unit of the conjugate comprises 1, 2, 3, 4 or 5 isotopic labels.

[0091] In certain embodiments, the antibiotic unit of the conjugate comprises five deuterium labels.

[0092] In a sixth aspect, the present invention relates to derivatized antibiotics, in particular derivatized antibiotics suitable for use as ISTDs and / or calibrants in mass spectrometry measurements.

[0093] In embodiments, the antibiotic is derivatized with a nucleophilic derivatization reagent. In embodiments, the nucleophilic derivatization reagent reacts with the antibiotic to form a complex, thereby preventing hydrolysis. In embodiments where the antibiotic is a β-lactam-containing antibiotic, the β-lactam moiety is destroyed upon complex formation. The resulting complex comprises an antibiotic unit and a nucleophilic derivatization unit.

[0094] In embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 12 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 12 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 24 hours, particularly more than 48 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 2 weeks. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 2 months. 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.

[0095] In embodiments, the nucleophilic derivatization reagent is a reagent that includes an amine group, particularly a primary or secondary amine. In certain embodiments, the nucleophilic derivatization reagent is a primary amine group.

[0096] In embodiments, the nucleophilic derivatization reagent comprises more than 3 C atoms, particularly 3 to 20 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 3 to 10 C atoms, particularly 3 to 5 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 4 C atoms.

[0097] In embodiments, the nucleophilic derivatization reagent is a linear or branched chain, particularly a linear amine, especially a linear primary amine. In particular embodiments, the nucleophilic derivatization reagent is a linear primary amine containing 3 to 5 C atoms.

[0098] In embodiments, the nucleophilic derivatization reagent is selected from the group consisting of propylamine, butylamine, and pentylamine. In certain embodiments, the nucleophilic derivatization reagent is a primary linear butylamine.

[0099] In embodiments, the antibiotic is an antibiotic that includes a β-lactam moiety, i.e., the antibiotic is a β-lactam antibiotic.

[0100] 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 (cephacetrile), cefadroxil (cefadroxyl), cef 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,Selected from the group consisting of cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreonam, and mecillinam.

[0101] In certain embodiments, the antibiotic is meropenem or piperacillin.

[0102] In embodiments, the antibiotic and / or the nucleophilic derivatization reagent comprises one or more isotopic labels. In certain embodiments, the one or more isotopic labels may be included in the nucleophilic derivatization unit and / or the antibiotic unit of the derivatized antibiotic. In certain embodiments, the one or more isotopic labels are included in the antibiotic unit of the derivatized antibiotic. In certain embodiments, the one or more isotopic labels are deuterium, 13 C. 15 N and / or 18 In certain embodiments, all isotopic labels are the same label. In alternative embodiments, the isotopic labels may be different, i.e., may include two, three, or four different isotopic labels.

[0103] In certain embodiments, the antibiotic unit of the derivatized antibiotic comprises 1 to 10 isotopic labels. In certain embodiments, the antibiotic unit of the derivatized antibiotic comprises 1 to 8 isotopic labels, particularly 1 to 5 isotopic labels. In certain embodiments, the antibiotic unit of the derivatized antibiotic comprises 1, 2, 3, 4, or 5 isotopic labels.

[0104] In certain embodiments, the antibiotic unit of the derivatized antibiotic comprises five deuterium labels.

[0105] In certain embodiments, when the derivatized antibiotic is intended for use as a calibrant, the derivatized antibiotic does not include an isotopic label.

[0106] In certain embodiments, when the derivatized antibiotic is intended for use as an ISTD, the derivatized antibiotic comprises at least one isotopic label.

[0107] In a seventh aspect, the present invention relates to the use of derivatized antibiotics as ISTDs and / or calibrants in mass spectrometric measurements.

[0108] In embodiments, the antibiotic is derivatized with a nucleophilic derivatization reagent. In embodiments, the nucleophilic derivatization reagent stabilizes the antibiotic. In embodiments, the nucleophilic derivatization reagent reacts with the antibiotic to form a complex, thereby preventing hydrolysis. In embodiments, in which the antibiotic is a β-lactam-containing antibiotic, the β-lactam moiety is destroyed upon complex formation. The resulting complex comprises an antibiotic unit and a nucleophilic derivatization unit.

[0109] In embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 12 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 12 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 24 hours, particularly more than 48 hours. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 2 weeks. In certain embodiments, the nucleophilic derivatization reagent stabilized the antibiotic for more than 2 months. 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.

[0110] In embodiments, the nucleophilic derivatization reagent is a reagent that includes an amine group, particularly a primary or secondary amine. In certain embodiments, the nucleophilic derivatization reagent is a primary amine group.

[0111] In embodiments, the nucleophilic derivatization reagent comprises more than 3 C atoms, particularly 3 to 20 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 3 to 10 C atoms, particularly 3 to 5 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 4 C atoms.

[0112] In embodiments, the nucleophilic derivatization reagent is a linear or branched chain, particularly a linear amine, especially a linear primary amine. In particular embodiments, the nucleophilic derivatization reagent is a linear primary amine containing 3 to 5 C atoms.

[0113] In embodiments, the nucleophilic derivatization reagent is selected from the group consisting of propylamine, butylamine, and pentylamine. In certain embodiments, the nucleophilic derivatization reagent is a primary linear butylamine.

[0114] In embodiments, the antibiotic is an antibiotic that includes a β-lactam moiety, i.e., the antibiotic is a β-lactam antibiotic.

[0115] 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 (cephacetrile), cefadroxil (cefadroxyl), cef 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,Selected from the group consisting of cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreonam, and mecillinam.

[0116] In certain embodiments, the antibiotic is meropenem or piperacillin.

[0117] In embodiments, the antibiotic and / or the nucleophilic derivatization reagent comprises one or more isotopic labels. In certain embodiments, the one or more isotopic labels may be included in the nucleophilic derivatization unit and / or the antibiotic unit of the conjugate. In certain embodiments, the one or more isotopic labels are included in the antibiotic unit of the conjugate. In certain embodiments, the one or more isotopic labels are deuterium, 13 C. 15 N and / or 18 In certain embodiments, all isotopic labels are the same label. In alternative embodiments, the isotopic labels may be different, i.e., may include two, three, or four different isotopic labels.

[0118] In certain embodiments, the antibiotic unit of the conjugate comprises 1 to 10 isotopic labels. In certain embodiments, the antibiotic unit of the conjugate comprises 1 to 8 isotopic labels, particularly 1 to 5 isotopic labels. In certain embodiments, the antibiotic unit of the conjugate comprises 1, 2, 3, 4 or 5 isotopic labels.

[0119] In certain embodiments, the antibiotic unit of the conjugate comprises five deuterium labels.

[0120] In certain embodiments, the derivatized antibiotic is used as a calibrant and does not contain an isotopic label.

[0121] In certain embodiments, derivatized antibiotics are used as ISTDs and include at least one isotopic label.

[0122] In an eighth aspect, the present invention relates to a method for producing a stabilized antibiotic comprising derivatizing an antibiotic with a nucleophilic derivatization reagent.

[0123] In embodiments, the nucleophilic derivatization reagent is provided in an excess of at least 2.5E8 compared to the amount of antibiotic present, particularly an excess of 1E5 to 1E10.

[0124] In embodiments, the nucleophilic derivatization reagent is a reagent that includes an amine group, particularly a primary or secondary amine. In certain embodiments, the nucleophilic derivatization reagent is a primary amine group.

[0125] In embodiments, the nucleophilic derivatization reagent comprises more than 3 C atoms, particularly 3 to 20 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 3 to 10 C atoms, particularly 3 to 5 C atoms. In certain embodiments, the nucleophilic derivatization reagent comprises 4 C atoms.

[0126] In embodiments, the nucleophilic derivatization reagent is a linear or branched chain, particularly a linear amine, especially a linear primary amine. In particular embodiments, the nucleophilic derivatization reagent is a linear primary amine containing 3 to 5 C atoms.

[0127] In embodiments, the nucleophilic derivatization reagent is selected from the group consisting of propylamine, butylamine, and pentylamine. In certain embodiments, the nucleophilic derivatization reagent is a primary linear butylamine.

[0128] In embodiments, the antibiotic is an antibiotic that includes a β-lactam moiety, i.e., the antibiotic is a β-lactam antibiotic.

[0129] 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 (cephacetrile), cefadroxil (cefadroxyl), cef 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,Selected from the group consisting of cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreonam, and mecillinam.

[0130] In certain embodiments, the antibiotic is meropenem or piperacillin.

[0131] In embodiments, the antibiotic and / or the nucleophilic derivatization reagent comprises one or more isotopic labels. In certain embodiments, the one or more isotopic labels may be included in the nucleophilic derivatization unit and / or the antibiotic unit of the conjugate. In certain embodiments, the one or more isotopic labels are included in the antibiotic unit of the conjugate. In certain embodiments, the one or more isotopic labels are deuterium, 13 C. 15 N and / or 18 In certain embodiments, all isotopic labels are the same label. In alternative embodiments, the isotopic labels may be different, i.e., may include two, three, or four different isotopic labels.

[0132] In certain embodiments, the antibiotic unit of the conjugate comprises 1 to 10 isotopic labels. In certain embodiments, the antibiotic unit of the conjugate comprises 1 to 8 isotopic labels, particularly 1 to 5 isotopic labels. In certain embodiments, the antibiotic unit of the conjugate comprises 1, 2, 3, 4 or 5 isotopic labels.

[0133] In certain embodiments, the antibiotic unit of the conjugate comprises five deuterium labels. The present invention further relates to the following items:

[0134] 1) A conjugate formed between an antibiotic and a nucleophilic derivatizing reagent.

[0135] 2) The conjugate according to item 1, wherein the nucleophilic derivatization reagent is a reagent containing an amine group, particularly a primary or secondary amine, particularly a primary amine group.

[0136] 3) The conjugate according to item 1 or 2, 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.

[0137] 4) The conjugate according to any one of items 1 to 3, 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.

[0138] 5) The use according to any one of items 1 to 4, wherein the derivatization reagent is selected from the group consisting of propylamine, butylamine, or pentylamine, particularly primary linear butylamine.

[0139] 6) The use according to any one of items 1 to 5, wherein the antibiotic is a β-lactam antibiotic.

[0140] 7) 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, cephalexin, cephalic acid ... Cefalexin (cephalexin), Cefalexin (cephalexin), Cefaloglycin (cephaloglycin), Cefalonium (cephalonium), Cefaloridine (cephaloradine), Cefalotin (cephalothin), Cefaloglycin Cefapirin (cephapirin), cefatrizine, cefazaflur, cefazedone, cefazolin (cephazolin), cephradine (cephradine), cephradine (cephradine), cefroxadine, cefroxadine, ceftezole, cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil (cefprozil) Xyl), cefuroxime, cefuzonam, cefcapene, ceftaloxime (Cefdaloxime), cefdinir, ceditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefimizole, cefodoxime, ceftalam, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomedine, cephaloram,The conjugate according to any one of Items 1 to 6, wherein the compound is selected from the group consisting of cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreonam, and mecillinam.

[0141] 8) The complex according to any one of items 1 to 6, wherein the antibiotic is meropenem or piperacillin.

[0142] 9) The complex according to any one of items 1 to 8, which comprises an isotope label.

[0143] 10) A composition comprising the complex according to any one of claims 1 to 9.

[0144] 11) A composition comprising the complex according to any one of items 1 to 9, further comprising a solvent, particularly a solvent selected from the group consisting of water, CH3CN, THF, dioxane, DMF, DMSO, acetone, t-butyl alcohol, diglyme, DME, MeOH, EtOH, 1-PrOH, 2-PrOH, ethylene glycol, hexamethylphosphoramide (HMPA), hexamethylphosphorus triamide (HMPT), and glycerin.

[0145] 12) The composition according to item 11, wherein the solvent is selected from the group consisting of water, CH3CN, THF, dioxane, DMF, DMSO, acetone, t-butyl alcohol, diglyme, and DME.

[0146] 13) The composition according to item 11 or 12, further comprising a stable, water-miscible, non-nucleophilic base, particularly selected from the group consisting of DBU, Na3PO4, Na2CO3, and Cs2CO3.

[0147] 14) A kit comprising: a) the compound according to any one of items 1 to 9 or the composition according to any one of items 10 to 13; and b) an accompanying instruction.

[0148] 15) Use of the complex according to any one of Items 1 to 9, the composition according to any one of Items 10 to 13, or the kit according to Item 14 for mass spectrometry.

[0149] 16) Use of the complex according to any one of Items 1 to 9, or the composition according to any one of Items 10 to 13, or the kit according to Item 14 as an internal standard and / or calibration material for mass spectrometry.

[0150] 17) The use of nucleophilic derivatization reagents to stabilize antibiotics, in particular to make them suitable as ISTDs and / or calibrators in mass spectrometric measurements.

[0151] 18) The use according to item 17, wherein the nucleophilic derivatization reagent is a reagent containing an amine group, particularly a primary or secondary amine, especially a primary amine group.

[0152] 19) The use according to item 17 or 18, 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.

[0153] 20) The use according to any one of items 17 to 19, 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.

[0154] 21) The use according to any one of items 17 to 20, wherein the derivatization reagent is selected from the group consisting of propylamine, butylamine, or pentylamine, particularly primary linear butylamine.

[0155] 22) The use according to any one of items 17 to 21, wherein the antibiotic is a β-lactam antibiotic.

[0156] 23) 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,23. The conjugate according to any one of Items 17 to 22, wherein the active ingredient is selected from the group consisting of cephaloram, cefaparol, cefcanel, cefedrolol, cefenpidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxide, ceftolozane, imipenem, doripenem, ertapenem, meropenem, aztreonam, and mecillinam.

[0157] 24) The use according to any one of items 17 to 23, wherein the antibiotic is meropenem or piperacillin.

[0158] 25) The use according to any one of items 17 to 24, wherein the nucleophilic derivatization reagent prevents hydrolysis of the antibiotic.

[0159] 26) The use according to any one of items 17 to 25, wherein the nucleophilic derivatization reagent stabilizes the antibiotic for more than 12 hours.

[0160] 27) Derivatized antibiotics suitable as ISTDs in mass spectrometry measurements.

[0161] 28) Use of derivatized antibiotics as ISTDs and / or calibrants in mass spectrometric measurements.

[0162] 29) A method for producing a stabilized antibiotic, comprising derivatizing the antibiotic with a nucleophilic derivatizing agent.

[0163] 30) The method according to item 29, wherein the nucleophilic derivatization reagent is provided in an excess of at least 2.5E8, in particular an excess of 1E5 to 1E10, compared to the amount of antibiotic present. [Example]

[0164] The following examples are offered to illustrate, but not to limit, the invention claimed herein.

[0165] General Methods: All reactions were magnetically stirred and carried out under a positive pressure of inert gas (N or argon) using standard Schlenk techniques. Glassware was repeatedly dried in vacuo at 620 °C before use. Liquid reagents and solvents were added via syringe or oven-dried stainless steel cannulae through rubber septa. Solids were added under inert gas reflux or dissolved in an appropriate solvent. Low-temperature reactions were carried out in Dewar flasks filled with coolant: acetone / dry ice (-78 °C) or H2O / ice (0 °C). Reaction temperatures above room temperature were carried out in a heated oil bath. When literature procedures were followed, respective references are added in the experimental details. Yields refer to isolated, homogeneous, and spectroscopically pure material unless otherwise indicated.

[0166] Solvents and Reagents: Dry solvents such as dichloromethane (CHCl), tetrahydrofuran (THF), pyridine, N,N-dimethylformamide (DMF), and acetonitrile (MeCN) were purchased from commercial suppliers and used as received. Solvents for extraction and flash column chromatography were purchased at HPLC grade. D-(-)-α-phenylglycine-D5 (1, Figure 6) was purchased from Toronto Research Chemicals. Compound S2 (Figure 7) was purchased from Ambeed Inc. Compound 2 (Figure 6) was synthesized from (+)-6-aminopenicillanic acid (S1, Figure 6) according to De Rosa et al. (Molecules 2015, 20, 22044-22057). All other reagents and solvents were purchased from chemical suppliers (Sigma-Aldrich, Acros Organics, Alfa Aesar, TCI Europe, abcr) and used as received.

[0167] NMR spectroscopy: NMR spectra were measured on an Agilent 400-MR DD2 400 MHz spectrometer equipped with a One NMR Probe operating at 400 MHz for proton nuclei (100 MHz for carbon nuclei). DMSO-d6 and CDCl3 were purchased from Sigma-Aldrich. 1H NMR shifts are reported in ppm relative to the residual shift of TMS. 1 H NMR shifts were calibrated to residual solvent resonances: DMSO-d6 (2.50 ppm) and CDCl3 (7.26 ppm). 13 C NMR shifts were calibrated to the centers of multiple signals of residual solvent resonances: DMSO-d6 (29.84 ppm) and CDCl3 (77.16 ppm). 1 H NMR spectroscopic data are reported as follows: chemical shifts in ppm (multiplicity, coupling constant J, integrated intensity). Multiplicities are s (singlet), br (broad signal), d (doublet), t (triplet), q (quartet), m (multiplet), and m C (centrosymmetric multiplet). In the case of complex multiplicities, the multiplicity with the smaller coupling constant is stated first. Chemical shifts of all signals, except for multiplets, as well as the chemical shifts of centrosymmetric multiplets, are reported as the center of the resonance range. Coupling constants J are reported in Hz. All NMR spectra were analyzed using the program ACD / Spectrus Processor 2015.2.7 from Advanced Chemistry Development, Inc.

[0168] Mass spectrometry: Low-resolution mass spectra (LRMS) were recorded on an HPLC-MS system from Waters GmbH (2695 Separation Module, 996 Photodiode Array Detector, Grace Vydac 218TP C18 5u) computer-controlled via Waters MassLynx V4.1. For each analyte, only the characteristic molecular fragment or molecular ion peak is shown.

[0169] Example 1: Stability of natural piperacillin (compound 5, see Figure 1) The stability of native piperacillin and its hydrolyzed forms was investigated (including compounds 5, 9a, 9b, and 10, respectively. From the hydrolysis pathway of piperacillin (compound 5, see the 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 or degradation of native piperacillin (compound 5). For this purpose, these compounds were freshly weighed and dissolved in water at a concentration of 1 mg / mL by rotation or stirring for 15 min at room temperature. 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, with a flow rate of 0.6 mL per minute, connected to an Agilent Infinity II multisampler / pump system connected to an AB Sciex 6500+MS. Peaks were integrated using MultiQuant software, and the areas of these peaks are shown in the graphs in Figures 2A and 2B.

[0170] 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 the hydrolysis or hydrolysis reaction (schematically shown in Figure 1).

[0171] Example 2: Synthesis of piperacillin-D5 monohydrate (8) and its mono-n-butylamine adduct (9) (see Figure 6 for schematic diagram and compound number). All reactions were magnetically stirred and carried out under a positive pressure of inert gas (N or argon) using standard Schlenk techniques. Glassware was repeatedly dried in vacuo at 620 °C before use. Liquid reagents and solvents were added via syringe or oven-dried stainless steel cannula through a rubber septum. Solids were added under inert gas reflux or dissolved in an appropriate solvent. Low-temperature reactions were carried out in a Dewar vessel filled with the following coolants: acetone / dry ice (-78 °C) or H2O / ice (0 °C). Reaction temperatures above room temperature were carried out in a heated oil bath. When literature procedures were followed, respective references are added in the experimental details. Yields refer to isolated, homogeneous, and spectroscopically pure material unless otherwise indicated.

[0172] Solvents and Reagents: Dry solvents such as dichloromethane (CHCl), tetrahydrofuran (THF), and acetonitrile (MeCN) were purchased from commercial suppliers and used as received. Solvents for extraction and flash column chromatography were purchased at HPLC grade. D-(-)-α-phenylglycine-D5 (1) was purchased from Toronto Research Chemicals. Compound 2 was synthesized from (+)-6-aminopenicillanic acid (S1) according to De Rosa et al. (2015) (Molecules 2015, 20, 22044-22057). All other reagents and solvents were purchased from chemical suppliers (Sigma-Aldrich, Acros Organics, Alfa Aesar, TCI Europe, abcr) and used as received.

[0173] Synthesis procedure [ka]

[0174] Acid (5): 1-Ethylpiperazine-2,3-dione (455 mg, 3.20 mmol, 1.00 equiv.) was dissolved in anhydrous dichloromethane (8.9 mL) under an argon atmosphere at room temperature. After cooling to 0 °C, anhydrous triethylamine (616 μL, 4.42 mmol, 1.38 equiv.) was added, followed by TMSCl (447 μL, 3.53 mmol, 1.10 equiv.), and the resulting mixture was stirred at room temperature for 1 h. The reaction was then cooled to -30 °C, and triphosgene (361 mg, 1.21 mmol, 0.38 equiv.) was added in one batch, and stirring was continued for an additional 1 h. Thorough drying in vacuo afforded intermediate 4 as a moisture-sensitive foam, which was directly redissolved in anhydrous dichloromethane (8.9 mL) for further synthesis. Meanwhile, D-(-)-α-phenylglycine (1, 500 mg, 3.20 mmol, 1.00 equiv.) was dissolved in anhydrous dichloromethane (8.9 mL) at room temperature under an argon atmosphere. Anhydrous triethylamine (693 μL, 6.72 mmol, 2.10 equiv.) and then TMSCl (894 μL, 7.04 mmol, 2.20 equiv.) were added at 0°C, and the resulting mixture was stirred at room temperature for 2 h. A previously prepared solution of intermediate 4 was then added at -40°C, and the reaction was stirred for 30 min, warmed to 0°C, and stirred for an additional 1 h. The reaction was quenched by adding distilled water (approximately 10 mL). After removing the dichloromethane under vacuum, the resulting aqueous suspension was mixed with ethyl acetate (approximately 50 mL). The pH of the aqueous phase was adjusted to 8.0 by adding solid NaHCO3, and the layers were then separated. The aqueous layer was washed with ethyl acetate (20 mL, 3 times). The aqueous phase was then adjusted to pH = 2.0 by adding concentrated aqueous HCl, and then extracted with ethyl acetate (50 mL, 5 times). The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. Acid 5 (1.04 g, 3.04 mmol, 95%) was thus obtained as a colorless foam with a residual content of ethyl acetate (approximately 5 wt%). 1 H NMR (CDCl3,400 MHz): δ=9.88(d,J=6.52 Hz,1H),5.49(d,6.52 Hz,1H),4.15-3.99(m,2H),3.60-3.48(m,4H),1.20(t,J=7.65 Hz,3H)ppm. 13 C NMR (CDCl3,100 MHz): δ=173.2,159.1,155.7,152.2,134.9,128.7-126.5(3C),55.8,43.5,42.6,40.3,12.0 ppm. [Table 1] [ka]

[0175] Ester (6): Acid 5 (932 mg, 2.87 mmol, 1.00 equiv.) and HATU (1.26 g, 3.30 mmol, 1.15 equiv.) were suspended in anhydrous tetrahydrofuran (9.4 mL) at room temperature under an argon atmosphere. After cooling to 0 °C, a solution of anhydrous DIPEA (538 μL, 3.09 mmol, 1.08 equiv.) in anhydrous tetrahydrofuran (3.0 mL) was added, and the resulting mixture was stirred for 5 min. Then, a solution of substance 2 (880 mg, 2.87 mmol, 1.00 equiv.) in anhydrous tetrahydrofuran (3.0 mL) was slowly added, and stirring was continued for 3 h at 0 °C. The reaction was quenched by adding ethyl acetate (100 mL), aqueous phosphate buffer (0.2 m, pH = 7.0, 40 mL), and brine (40 mL) to a stirred biphasic mixture. The layers were separated and the aqueous phase was extracted with ethyl acetate (50 mL, 3 times). The organic layers were combined and dried over NaSO, then filtered and concentrated in vacuo to give crude ester 6 as a yellowish foam. Final purification was achieved by HPLC (C-18 reverse-phase silica, MeCN:HO) to give ester 6 (1.34 g, 2.19 mmol, 76%) as a colorless foam.

[0176] 11H NMR(CDCl3, 400 MHz): δ = 9.93 (d, J = 6.52 Hz, 1H), 7.40 - 7.32 (m, 5H), 6.46 (d, J = 9.16 Hz, 1H), 5.64 (dd, J = 4.02, 9.16 Hz, 1H), 5.45 (d, J = 3.64 Hz, 1H), 5.44 (d, J = 6.52 Hz, 1H), 5.16 (s, 2H), 4.39 (s, 1H), 4.18 - 4.12 (m, 1H), 3.99 - 3.93 (m, 1H), 3.55 (m C , 4H) 1.48 (s, 3H), 1.34 (s, 3H), 1.21 (t, J = 7.63 Hz, 3H) ppm. 13 13C NMR(CDCl3, 100 MHz): δ = 173.3, 168.9, 167.5, 159.4, 155.8, 152.6, 136.2, 134.8, 128.9 - 128.8, 134.9, 127.4 - 126.9 (3C), 70.1, 68.2, 67.7, 64.9, 59.2, 58.9, 43.8, 42.8, 40.8, 31.8, 26.9, 12.3 ppm.

Table 2

Chem.

[0177] Butyramide (7): Ester 6 (100 mg, 0.16 mmol, 1.00 equiv.) was dissolved in anhydrous CHCN (2.5 mL) under an argon atmosphere at room temperature. To this was added a solution of n-butylamine (161 μL, 1.63 mmol, 10.0 equiv.) in anhydrous CHCN (2.5 mL), and the resulting mixture was stirred for 2 h until LCMS control indicated complete consumption of 6. The reaction was quenched by adding it to a stirred biphasic mixture of ethyl acetate (40 mL), aqueous phosphate buffer (0.2 m, pH = 7.0, 10 mL), and brine (10 mL). The layers were separated, and the aqueous phase was extracted with ethyl acetate (30 mL, 4 times). The combined organic layers were dried over NaSO, filtered, and concentrated in vacuo to give crude butyramide 7 as a yellowish foam. Final purification was achieved by HPLC (C-18 reverse-phase silica, MeCN:HO, standard gradient program) to give butyramide 7 (99.2 mg, 0.15 mmol, 89%) as a colorless solid in a 7:7′=95:5 diastereomeric mixture. 1 1 H NMR).

[0178] 1 H NMR (CDCl3,400 MHz): δ=9.88(d,J=7.53 Hz,1H),7.44-7.29(m,5H),6.70-6.55(m,2H),5.33(d,J=5.90 Hz,1H),5.25-5.11(m,3H),4.41(dd,J=4.96,7.53 Hz,1H),4.09(ddd,J=3.64,7.03,13.8 Hz,2 H),3.96(ddd,J=3.89,8.09,13.8 Hz,2 H),3.60-3.45(m,4H),3.42-3.29(m,2H),3.27-3.14(m,2H),1.50-1.42(m,2H),1.38-1.26(m,2H),1.34(s,3H),1.21(t,J=7.22 Hz,3H),1.05(s,3H),0.89(t,J=7.34 Hz,3H)ppm.

[0179] Characteristic signals of diastereomer 17': δ = 6.78 (d, J = 7.15 Hz, 1 H), 6.52-6.46 (m, 1 H), 5.38 (d, J = 6.02 Hz, 1 H), 1.12 (s, 3 H) ppm.

[0180] 13 C NMR (CDCl, 100 MHz): δ = 169.8, 169.2, 168.8, 159.4, 155.8, 152.7, 136.2, 135.1, 128.9, 128.8, 128.8, 72.7, 67.4, 64.9, 59.6, 58.3, 57.4, 43.8, 42.8, 40.7, 39.7, 31.6, 26.4, 26.3, 20.2, 13.9, 12.3 ppm. Note: Deuterium-substituted carbon atoms could not be detected due to line broadening caused by vicinal spin coupling to deuterium. [Table 3] [ka]

[0181] Piperacillin-D5 monohydrate (8): Ester 6 (340 mg, 0.56 mmol, 1.00 equiv.) was dissolved in anhydrous tetrahydrofuran (17 mL) under an argon atmosphere at room temperature. To this was added degassed distilled water (8.5 mL) and palladium on charcoal (10 wt.% Pd, 200 mg, 0.18 mmol, 0.33 equiv. Pd). With vigorous stirring, the resulting suspension was flushed with hydrogen (1 atm, 3 times) and then hydrogenated for 1 h. After filtering through a Celite plug (HO:CHCN = 1:1, ca. 50 mL), the resulting biphasic mixture was concentrated in vacuo (max. 30 °C) to give a colorless foam. The crude material 8 thus obtained was redissolved in a mixture of distilled water (3.4 mL), formic acid (105 μL, 2.78 mmol, 5.00 equiv.), and acetonitrile (1.7 mL) and subjected to purification by HPLC (C-18 reverse-phase silica, MeCN:HO, standard gradient program). After concentration and complete drying in vacuo (maximum 30 °C), semi-pure material 8 (50.0 mg) was dissolved in methanol (1.00 mL). Distilled water (1.85 mL) was added dropwise with stirring until the mixture slowly became cloudy. After cooling to 0 °C for 30 min, another drop of distilled water (1.00 mL) was added dropwise, and the suspension was cooled to 0 °C for another 30 min. The supernatant was then removed from the formed crystals. After washing with ice-cold distilled water (1.00 mL, twice), the remaining solid was thoroughly dried in vacuo (up to 30 °C) to give piperacillin-D5 monohydrate (8,234 mg, 0.43 mmol, 77%) as a colorless solid with a purity of 95.7% (determined by HPLC / UV-Vis).

[0182] 1 H NMR (DMSO-D6,400 MHz): δ=13.35(br,s,1H),9.84(d,J=7.28 Hz,1H),9.31(d,J=7.78 Hz,1H),5.71(d,J=7.28 Hz,1H),5.53(dd,J=5.53,7.78 Hz,1H),5.38(d,J=4.14 Hz,1H),4.19(s,1H),3.89(m C,2 H),3.56(t,J=5.53 Hz,2H),3.39(q,J=7.07 Hz,2H),1.54(s,3H),1.39(s,3H),1.08(t,J=7.07 Hz,3H)ppm.

[0183] 13 C NMR (CDCl, 100 MHz): δ = 173.2, 169.4, 168.9, 159.5, 155.4, 151.9, 137.7, 70.3, 67.0, 63.7, 58.1, 56.5, 42.8, 41.6, 30.2, 26.6, 16.0, 11.9 ppm. Note: Deuterium-substituted carbon atoms could not be detected due to line broadening caused by vicinal spin coupling to deuterium. [Table 4] [ka]

[0184] Piperacillin-n-butylamide-D5(9): Butyramide 7 (95.0 mg, 0.14 mmol, 1.00 equiv.) was dissolved in anhydrous tetrahydrofuran (4.0 mL) under an argon atmosphere at room temperature. To this was added degassed distilled water (2.0 mL) and palladium on charcoal (10 wt.% Pd, 74.2 mg, 0.07 mmol, 0.50 equiv. Pd). With vigorous stirring, the resulting suspension was flushed with hydrogen (1 atm, 3 times) and then hydrogenated for 1 h. After filtering through a Celite plug (HO:CHCN = 1:1, ca. 25 mL), the resulting biphasic mixture was concentrated in vacuo (max. 30 °C) to give a colorless foam. The crude material 9 thus obtained was redissolved in a mixture of distilled water (0.5 mL), formic acid (26.2 μL, 0.70 mmol, 5.00 equiv.), and acetonitrile (1.0 mL) and subjected to purification by HPLC (C-18 reverse-phase silica, MeCN:HO). After concentration and complete drying in vacuo (max. 30 °C), piperacillin-n-butylamide-D5 (9, 52.4 mg, 0.08 mmol, 63%) was obtained as a colorless solid with a purity of 95.8% (determined by HPLC / UV-Vis) in a 9:9′=70:30 diastereomeric mixture (determined by HPLC / UV-Vis).

[0185] 1H NMR (DMSO-D6,400 MHz): δ=12.86(br,s,1H,19+19'),9.84(d,J=7.53 Hz,1H,19'),9.84(d,J=7.53 Hz,1H,19),8.84(d,J=8.78 Hz,1H,19'),8.72(d,J=9.03 Hz,1H,19),8.18(t,J=6.02 Hz,1H,19'),8.02(t,J=5.77 Hz,1H,19),5.72(d,J=7.53 Hz,1H,19+19'),4.84(d,J=6.40 Hz,1H,9),4.71(br,d,J=5.90 Hz,1H,19'),4.63(dd,J=6.40,8.78 Hz,1H,19'),4.34(dd,J=7.03,9.03 Hz,1H,19'),3.92-3.87(m,2H,19+19'),3.57-3.53(m,2H,19+19'),3.44-3.36(m,4H,19+19'),3.16-2.95(m ,2H,19+19'),1.48(s,3H,19'),1.40-1.23(m,4H,19+19'),1.30(s,3H,19),1.10(s,3H,19),1.08(t,J=7.40 Hz,1H,19+19'),0.93(s,3H,19'),0.86(t,J=7.22 Hz,1H,19+19')ppm. [Table 5]

[0186] Example 3: Synthesis of meropenem-13C2-15N1 trihydrate (7), meropenem-13C2-15N-n-butylamide (8) (schematic diagram and compound numbering, see Figure 7) All reactions were magnetically stirred and carried out under a positive pressure of inert gas (N or argon) using standard Schlenk techniques. Glassware was repeatedly dried in vacuo at 620 °C before use. Liquid reagents and solvents were added via syringe or oven-dried stainless steel cannula through a rubber septum. Solids were added under inert gas reflux or dissolved in an appropriate solvent. Low-temperature reactions were carried out in a Dewar filled with coolant: acetone / dry ice (-78 °C) or H2O / ice (0 °C). Reaction temperatures above room temperature were carried out in a heated oil bath. When literature procedures were followed, respective references are added in the experimental details. Yields refer to isolated, homogeneous, and spectroscopically pure material unless otherwise indicated.

[0187] Solvents and Reagents: Dry solvents such as dichloromethane (CHCl), tetrahydrofuran (THF), and acetonitrile (MeCN) were purchased from commercial suppliers and used as received. Solvents for extraction and flash column chromatography were purchased at HPLC grade. All other reagents and solvents were purchased from chemical suppliers (Sigma-Aldrich, Acros Organics, Alfa Aesar, TCI Europe, abcr) and used as received.

[0188] [1]: trans-4-Hydroxy-L-proline (S1, 1.0 g, 7.6 mmol, 1.0 equiv.) was dissolved in aqueous NaOH (0.5 m, 34 mL) and cooled in an ice bath. 4-Nitrobenzoyl chloroformate (1.9 g, 8.6 mmol, 1.1 equiv.) in toluene (25 mL) was added dropwise at 0-5 °C. The biphasic mixture was stirred for 2 h, after which toluene (30 mL) was added and the phases were separated. The aqueous phase was extracted with toluene, and the combined organic phases were extracted with aqueous NaOH (0.5 m). The combined aqueous phases were adjusted to pH = 1 by adding concentrated HCl and extracted with ethyl acetate (3 times). The extract was dried over sodium sulfate, filtered, and concentrated in vacuo. Compound 1 (1.89 g, 6.1 mmol, 80%) was thus obtained as an off-white solid. [Table 6]

[0189] [2]: Substance 1 (1.4 g, 4.5 mmol, 1.0 equiv.) was dissolved in acetonitrile (70 mL) and 1.7 g of HBTU (1.7 g, 4.5 mmol, 1.0 equiv.), and the solution was cooled to room temperature under an argon atmosphere with 416 mg of dimethylamine- 13 C2- 15 N-hydrochloride (0.4 g, 5.1 mmol, 1.1 equiv.) and 2.6 mL of triethylamine (2.6 mL, 19 mmol, 4.2 equiv.) were added sequentially. The mixture was stirred for 90 minutes. Then, acetonitrile was evaporated. The crude product was suspended in ethyl acetate (20 mL) and filtered. The filtrate was washed several times with a saturated solution of bicarbonate. The ethyl acetate was evaporated, and the resulting solid was stored at -20 °C. The product was resuspended in ethyl acetate (20 mL) and filtered. The filtrate was concentrated to a volume of 10 mL, and the solution was cooled to 0 °C. The formed precipitate was filtered off and dried in vacuo to give substance 2 (1.27 g, 3.7 mmol, 83%) as an off-white solid. [Table 7]

[0190] [3]: Substance 2 (1.16 g, 3.41 mmol, 1.00 equiv.) was dried in vacuo and dissolved in anhydrous pyridine (9.0 mL) under an argon atmosphere. Methanesulfonyl chloride (0.53 mL, 6.82 mmol, 2.00 equiv.) was added, and the resulting mixture was stirred at room temperature for 45 minutes. Pyridine was removed in vacuo, and ethyl acetate (200 mL) and saturated aqueous bicarbonate solution (100 mL) were added. The layers were mixed and separated, and the organic phase was extracted with saturated aqueous bicarbonate solution (100 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo to give substance 3 (1.44 g, 3.4 mmol, 99%) as an off-white solid. [Table 8]

[0191] [4]: Material 3 (1.42 g, 3.39 mmol, 1.00 equiv.) was dried in vacuo and transferred to a flame-dried pressure tube with anhydrous DMF (23 mL) under an argon atmosphere. 775 mg of potassium thioacetate (0.78 g, 6.78 mmol, 2.00 equiv.) was added, and the resulting mixture was heated to 70 °C for 1 h. The reaction was monitored by HPLC, and additional potassium thioacetate (0.78 g, 6.78 mmol, 2.00 equiv.) was added, and heating was repeated until >95% of the starting material was consumed. Pyridine was removed in vacuo, and ethyl acetate (200 mL) was added. The resulting suspension was washed with distilled water (100 mL) and brine (100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to give material 4 (1.19 g, 3.0 mmol, 88%) as a colorless solid. [Table 9]

[0192] [5]: Substance 4 (1.12 g, 2.81 mmol, 1.00 equiv.) was dissolved in MeOH (47 mL) and cooled to 0 °C. Aqueous NaOH (1 m, 2.7 mL) was added, and the reaction mixture was stirred for 3 h. The mixture was then acidified to pH = 2 by adding aqueous HCl (0.5 m), and methanol was removed in vacuo. The residue was dissolved in ethyl acetate (350 mL), the layers were separated, and the organic layer was washed with brine (100 mL). The organic phase was dried over sodium sulfate, filtered, and concentrated in vacuo. The crude product was purified by HPLC (C-18 reverse-phase silica, MeCN:HO). Thus, compound 5 (0.45 g, 1.26 mmol, 45%) was isolated as a colorless solid. [Table 10]

[0193] [6]: Hydration buffer for synthesis of substance 7: N-methylmorpholine (720 μL) and acetic acid (360 μL) were added to water (19.0 mL), and the pH was adjusted to 6 by adding acetic acid.

[0194] [7] (Meropenem- 13 C2- 15 N-trihydrate: Substance 5 (0.48 g, 1.36 mmol, 1.00 equiv.) was dissolved in a mixture of ethyl acetate (15 mL) and DMF (3.0 mL). The resulting solution was cooled to below 5 °C. Compound S2 (0.89 g, 1.50 mmol, 1.10 equiv.) and DIPA (0.26 mL, 1.86 mmol, 1.36 equiv.) were then added dropwise. The mixture was then stirred at 0-5 °C for 3.5 h. The reaction was monitored by HPLC. Upon complete conversion of substance 5, hydration buffer (12 mL) was added to the reaction mixture. The mixture was stirred for 3 h and then transferred to a hydration apparatus. Palladium on charcoal (10 wt% Pd, 0.74 g, 0.69 mmol, 0.50 equiv. Pd) was added, and the reaction vessel was purged with hydrogen (4.8 bar). The reaction mixture was hydrogenated for 3 h. The organic layer was then separated and the catalyst was filtered off. The crude product was purified by HPLC (C-18 reverse-phase silica, MeCN:HO). Compound 5 (71.9 mg, 0.16 mmol, 12%) was isolated as a colorless solid. [Table 11]

[0195] [8] Meropenem-butylamide 13 C2- 15 N : Meropenem 13 C2- 15 N-trihydrate (7, 50 mg, 0.13 mmol, 1.0 equiv.) was dissolved in water (0.5 mL) at room temperature under an argon atmosphere. n-Butylamine (4.5 m, 0.12 mL, 0.5 mmol, 3.8 equiv.) was added, and the resulting mixture was stirred for 30 minutes. The reaction was then lyophilized. The residual solid was purified by HPLC (C-18 reverse-phase silica, MeCN:HO). Compound 8 (40 mg, 0.08 mmol, 67%) was thus obtained as an off-white solid. [Table 12]

[0196] [9] Butyramide: Ester 6 (100 mg, 0.29 mmol, 1.00 equiv.) was dissolved in dry CHCN (4.0 mL) at room temperature under an argon atmosphere. To this was added a solution of n-butylamine (282 μL, 2.85 mmol, 10.0 equiv.) in anhydrous CHCN (2.5 mL), and the resulting mixture was stirred for 2 h until LCMS control indicated complete consumption of 6. The reaction was quenched by adding it to a stirred biphasic mixture of ethyl acetate (60 mL), aqueous phosphate buffer (0.2 m, pH = 7.0, 20 mL), and brine (20 mL). The layers were separated, and the aqueous phase was extracted with ethyl acetate (30 mL, 3 times). The organic layers were combined and dried over NaSO, then filtered and concentrated in vacuo to give crude butyramide 9 as a brownish foam. Final purification was achieved by HPLC (C-18 reverse-phase silica, MeCN:H2O) to give butyramide 9 (164 mg, 0.21 mmol, 73%) as a colorless foam in a diastereomeric mixture. [Table 13]

[0197]

[10] Meropene butyramide 13 C2- 15N:Butylamide 9 (100 mg, 0.13 mmol, 1.00 equiv) was dissolved in dry tetrahydrofuran (4.0 mL) under an argon atmosphere at room temperature. To this was added degassed water (2.0 mL) and palladium on charcoal (10 wt% Pd, 68.9 mg, 0.07 mmol, 0.50 equiv Pd). With vigorous stirring, the resulting suspension was flushed with hydrogen (1 atm, 3 times) and then hydrogenated for 3 h. After filtering through a Celite plug (HO:CHCN = 1:1, ca. 25 mL), the resulting biphasic mixture was concentrated in vacuo (max. 30 °C) to give a yellowish liquid. The crude material 8 thus obtained was redissolved in a mixture of distilled water (0.8 mL), formic acid (15.7 μL, 0.42 mmol, 3.00 equiv.), and acetonitrile (0.8 mL) and subjected to purification by HPLC (C-18 reverse-phase silica, MeCN:HO). After concentration and complete drying in vacuo (max. 30 °C), meropenem-butylamide- 13 C2- 15 N (8, 23.3 mg, 0.05 mmol, 39%) was obtained as a slightly yellowish solid in a diastereomeric mixture of excellent purity. [Table 14]

[0198] Example 4: 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 (compound 1, Figure 3) and piperacillin (compounds 5, Figure 1 or 4, 1 μg / mL). For schematic diagrams of the chemical reactions, see Figures 3 and 4 for meropenem and piperacillin, respectively.

[0199] The stability of the double butylamide variant of piperacillin (compound 7, see FIG. 4) was investigated by two MRMs using the same protocol as in Example 1.

[0200] 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.

[0201] Example 5: Stabilization of meropenem (compound 1) and piperacillin (compound 5) in patient samples A derivatization reagent (propylamine, butylamine, or pentylamine) dissolved in water was added to 100 μL of the sample (serum spiked with 1 μg / mL of both piperacillin (compound 5) and meropenem (compound 1)).

[0202] 100 μL of 1 μg / mL (1.9*10 -9 M) 5*10 for piperacillin (compound 5) 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).

[0203] An LC-MS / MS method was devised to quantify native (intact) meropenem (compound 1) and piperacillin (compound 5), as well as both their derivatized and hydrolyzed products, with tailored MRM transitions for all compounds. A Cortecs C18+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, with a flow rate of 0.6 mL / min, connected to an Agilent Infinity II multisampler / pump system connected to an AB Sciex 6500+MS. 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). Natural meropenem (compound 1) and piperacillin (compound 5) and their hydrolyzed forms (two MRM transitions per analyte) were also included in the measurements. [Table 15]

[0204] For meropenem (compound 1), the highest peak area results are obtained using pentylamine. Using pentylamine and an optimal workflow, an area of ​​approximately 3E6 should be possible for this antibiotic at a concentration of 1 μg / mL in serum. Using butylamine, an area of ​​1E6 is achievable under optimal conditions. See Figure 6.

[0205] Piperacillin (compound 5) results in the highest peak area when butylamine is used. 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.

[0206] Note that when 2.5E8 equivalents of reagent are used, no residual native compound (intact meropenem (compound 1) or piperacillin (compound 5)) is found in the eluate, indicating that the reaction is quantitative in this short time. Furthermore, we did not observe an increase in the amount of hydrolyzed compounds, indicating that the addition of nucleophiles does not catalyze the hydrolysis of the lactam moiety in these compounds, allowing us to distinguish and quantify intact lactam compounds from hydrolyzed compounds.

[0207] This patent application claims priority from European patent application 19209520.6, the contents of which are incorporated herein by reference.

Claims

1. A conjugate formed between an antibiotic and a nucleophilic derivatization reagent, the nucleophilic derivatization reagent is a linear primary amine, the linear primary amine comprising a primary amine group and comprising 3 to 10 C atoms; and The antibiotic is meropenem or piperacillin. Complex.

2. The complex of claim 1 , comprising an isotopic label.

3. A composition comprising the complex described in claim 1 or 2, further comprising a solvent.

4. The composition of claim 3, wherein the solvent is selected from the group consisting of water, CH3CN, THF, dioxane, DMF, DMSO, acetone, t-butyl alcohol, diglyme, DME, MeOH, EtOH, 1-PrOH, 2-PrOH, ethylene glycol, hexamethylphosphoramide (HMPA), hexamethylphosphorus triamide (HMPT), and glycerin.

5. The composition described in claim 3 or 4, further comprising a stable, water-miscible non-nucleophilic base.

6. The composition of claim 5, wherein the non-nucleophilic base is selected from the group consisting of DBU, Na 3 PO 4 , Na 2 CO 3 , and Cs 2 CO 3 .

7. A kit comprising: a) the complex according to claim 1 or 2, or the composition according to any one of claims 3 to 6; and b) an attached document.

8. Use of the complex according to claim 1 or 2, or the composition according to any one of claims 3 to 6, or the kit according to claim 7 for mass spectrometry measurement.

9. Use of the conjugate according to claim 1 or 2, or the composition according to any one of claims 3 to 6, or the kit according to claim 7 as an ISTD and / or calibrator for mass spectrometry measurements.

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