The present invention relates to a composition for determining at least one analyte of interest via Mass Spectrometry measurements and uses thereof, a kit and the use thereof, a method of determining the level of at least one analyte of interest in an obtained sample, a sampling tube for collecting a patient sample, an analyzer as well as the use of dialkyl sulfide as an additive in a formulation of P-lactam antibiotic analyte for preventing oxidation.

Background

Beta-lactam antibiotics are a class of antibiotics that are prescribed most commonly as either specific or broad-spectrum antibiotics for patients infected with bacteria. This class of antibiotics works by interfering with the crosslinking of the peptidoglycan layer that is most dominant in Gram-positive bacteria. They exhibit a bacteriocidal effect, which is concentration dependent. Therefore, it is critical to keep the antibiotic concentration above the Minimum Inhibitory Concentration (MIC). However, higher concentrations lead to adverse effects. Moreover, it has been reported that the pharmacokinetics of these compounds is highly variable and therefore unpredictable (Ronilda D'Cunha et al.; 2018; Antimicrobial Agents and Chemotherapy 62 (9)).

Beta-lactam antibiotics often contain a thioether that is sensitive to oxidation to the air, resulting in sulfon and/or sulfoxides. This compromises the stability of these compounds.

A known strategy is to derivatize beta-lactam antibiotics, which enables the use of derivatized internal standards and calibration materials. This method circumvents stability issues with respect to the beta-lactam moiety of this class of antibiotics. However, it does not prevent degradation via oxidation with respect to the thioether. There is no formulation for the compounds having a thioether moiety known preventing degradation via oxidation. A generic formulation that would be suitable to many other compounds would most probably not be applicable in the case of beta-lactam antibiotics, due to reactivity of the thioether bond.

Also, a straightforward strategy like degassing the solution, or storage under N?/Ar would not be suitable if the intention is to use internal standard solutions as part of an assay kit that needs to be filled/stored/shipped and used on analyzers where it is meant to be opened and closed multiple times.

For convenience, environmental reasons and cost-reduction, a formulation that may be used multiple times is naturally better than single-use solutions.

Thus, there is an urgent need in the art to overcome the above mentioned problems. A protective formulation or composition that keeps the analytes having a thioether moiety in their reduced (i.e. intact) form is needed.

For the present invention, a composition for determining at least one analyte of interest via Mass Spectrometry measurements can overcome the above mentioned problems.

It is an object of the present invention to provide a composition for determining at least one analyte of interest via Mass Spectrometry measurements and uses thereof, a kit and the use thereof, a method of determining the level of at least one analyte of interest in an obtained sample, a sampling tube for collecting a patient sample, an analyzer as well as the use of dialkyl sulfide as an additive in a formulation of P- lactam antibiotic analyte for preventing oxidation.

This object is or these objects are solved by the subject matter of the independent claims. Further embodiments are subjected to the dependent claims.

Summary of the invention

In a first aspect, the present invention relates to a composition for determining at least one analyte of interest via at least one Mass Spectrometry measurement or Mass Spectrometry measurements comprising the at least one analyte having a thioether moiety, an additive, wherein the additive is a sulfide or a selenide, preferably wherein the additive is suitable to prevent the oxidation of the thioether moiety.

In a second aspect, the present invention relates to the use of the composition according to any of the aspects for determining the level of at least one analyte of interest via Mass Spectrometry measurements.

In a third aspect, the present invention relates to the use of the composition according to any of the aspects as an ISTD for Mass Spectrometry measurements.

In a fourth aspect, the present invention relates to use of dialkyl sulfide, preferably diethyl sulfide, as an additive in a formulation of P-lactam antibiotic analyte for preventing oxidation.

In a fifth aspect, the present invention relates to a kit comprising the composition according to any of the aspects in a first reservoir, and optionally magnetic beads in a second reservoir.

In a sixth aspect, the present invention relates to the use of the kit according to any of the aspect of the invention for Mass Spectrometry measurements.

In a seventh aspect, the present invention relates to a method of determining the level of at least one analyte of interest in an obtained sample comprising the steps of a) Providing the sample comprising an analyte of interest, wherein the analyte of interest has a thioether moiety, b) Optionally providing a isotopic labeled analyte of interest as in ISTD having a thioether moiety, c) Providing an additive, wherein the additive is a sulfide or a selenide and suitable to prevent the oxidation of the thioether moiety of the analyte of interest and/or the thioether moiety of the isotopic labeled analyte of interest, d) Optionally derivatizing the analyte of interest and/or isotopic labeled analyte of interest with a nucleophilic derivatization reagent, and e) Determining the level of the at least one analyte of interest in the sample, in particular using Mass Spectrometry, preferably LC/MS.

In an eighth aspect, the present invention relates to sampling tube for collecting a patient sample comprising

- an analyte of interest having a thioether moiety, and

- an additive suitable to prevent the oxidation of the thioether moiety of the analyte of interest in a sample, wherein the additive is a sulfide or a selenide.

In a ninth aspect, the present invention relates to an analyzer adapted to perform the method of any of the aspects and/or comprises a composition of any of the aspects.

List of Figures

Figure 1 shows the structures of 1 : Meropenem-butylamide as a deriviatized anaylte of interest, 2: Piperacillin-butylamide as a deriviatized anaylte of interest, 3: diethyl sulfide as an additive, 4: Meropenem-butylamide-sulfoxide as an oxidation product ofMeropenem-butylamide 1, 5: Levetiracetam, which was used to normalization, i.e. compensate for any evaporation effects or variance in injection in LC-MS.

Figure 2 shows the degradation ofMeropenem-butylamide 1 over four weeks in four different formulations with varying Et2S concentrations.

Figure 3 shows the increase of Meropenem-butylamide-sulfoxide 4 over four weeks in four different formulations with varying Et2S concentrations.

Figure 4 shows the degradation of Piperacillin-butylamide 2 over four weeks in four different formulations with varying Et2S concentrations.

Detailed Description of the Invention

Before the present invention is described 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 will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Documents are cited throughout the text of this specification. Each of the documents 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 the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. The term “aspect” and “embodiment” can be used interchangeably.

Definitions

The word "comprise", and variations such as "comprises" and "comprising", will be understood to 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.

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. Percentages, concentrations, amounts, and other numerical data may be expressed or presented herein in a “range” format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of "4% to 20 %" should be interpreted to include not only the explicitly recited values of 4 % to 20 %, but to also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 4, 5, 6, 7, 8, 9, 10, . . . 18, 19, 20 % and sub-ranges such as from 4-10 %, 5-15 %, 10-20%, etc. This same principle applies to ranges reciting minimal or maximal values. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

The term “about” when used in connection with a numerical value is meant to encompass numerical values within a range having a lower limit that is 5% smaller than the indicated numerical value and having an upper limit that is 5% larger than the indicated numerical value.

The term "measurement", "measuring" or "determining" preferably comprises a qualitative, a semi-quanitative or a quantitative measurement.

The term “automated” or “automatic” refers to methods or processes or devices which are operated largely by automatic equipment, i.e. which are operate by machines or computers, in order to reduce the amount of work done by humans and the time taken to do the work. Thus, in an automated method, tasks that were previously performed by humans, are now performed by machines or computers. Typically, the users only need to configure the tool and define the process. The skilled person is well aware that at some minor points manual intervention may still be required, however the large extend of the method is performed automatically.

In the context of the present disclosure, the term “analyte”, “analyte molecule”, or “analyte(s) of interest” are used interchangeably, referring to the chemical species to be analysed. Chemical species suitable to be analysed, i.e. analytes, can be any kind of molecule present in a living organism, include but are not limited to nucleic acid, amino acids, peptides, proteins, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids molecules. Analytes may also be any substance that has been internalized by the organism, such as but not limited to therapeutic drugs, drugs of abuse, toxin, or a metabolite of such a substance. Analytes can be therapeutic drug monitoring (TDM) compounds. Therapeutic drug monitoring compounds include antibiotics, i.e. “antibiotic analytes”. Antibiotics are substance active against microbial organisms. Antibiotics are commonly classified based on their mechanism of action, chemical structure, or spectrum of activity. One class of antibiotics are B- lactam antibiotics. P-lactam antibiotics (beta-lactam antibiotics) are all antibiotic agents that contain a beta-lactam ring in their molecular structures. These include but are not limited to penicillin derivatives (penams), cephalosporins (cephems), monobactams, carbapenems and carbacephems. Most P-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organism and are the most widely used group of antibiotics. The effectiveness of these antibiotics relies on their ability to reach the PBP intact and their ability to bind to the penicillin binding proteins (PBP).

Analytes may be present in a sample of interest, e.g. a biological or clinical sample. The terms "sample" or "sample of interest" or “patient sample” are used interchangeably herein, referring to a part or piece of a tissue, organ or individual, typically being smaller than such tissue, organ or individual, intended to represent the whole of the tissue, organ or individual. Upon analysis, a sample provides information about the tissue status or the health or diseased status of an organ or individual. Examples of samples include but are not limited to fluid samples such as blood, serum, plasma, synovial fluid, spinal fluid, urine, saliva, and lymphatic fluid, or solid samples such as dried blood spots and tissue extracts. Further examples of samples are cell cultures or tissue cultures. Preferabyl, the sample is blood, serum or plasma.

In the context of the present disclosure, the sample may be derived from an “individual” or “subject”. Typically, the subject is a mammal. Mammals include, but are not limited to, domesticated 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).

Before being analysed, a sample may be pre-treated or treated in a sample- and/or analyte specific manner. In the context of the present disclosure, the term “pretreatment” or “treated” refers to any measures required to allow for the subsequent analysis of a desired analyte. (Pre-)treatment measures typically include but are not limited to derivatization of analytes, the elution of solid samples (e.g. elution of dried blood spots), addition of hemolizing reagent (HR) to whole blood samples, and the addition of enzymatic reagents to urine samples. Also the addition of internal standards (ISTD) is considered as (pre-)treatment of the sample.

Typically, an internal standard (ISTD) is a known amount of a substance, which exhibits similar properties as the analyte of interest when subjected to the mass spectrometric detection workflow (i.e. including any pre-treatment, enrichment and actual detection step). Although the ISTD exhibits similar properties as the analyte of interest, it is still clearly distinguishable from the analyte of interest. Exemplified, during chromatographic separation, such as gas or liquid chromatography, the ISTD has about the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the device, e.g. mass spectrometer, at the same time. The ISTD however, exhibits a different molecular mass than the analyte of interest from the sample. This allows a mass spectrometric distinction between ions from the ISTD and ions from the analyte by means of their different mass/charge (m/z) ratios. Both are subject to fragmentation and provide daughter ions. These daughter ions can be distinguished by means of their m/z ratios from each other and from the respective parent ions. Consequently, following calibration, a separate determination and quantification of the signals from the ISTD and the analyte can be performed. Since the ISTD has been added in known amounts, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte. Thus, the addition of an ISTD allows for a relative comparison of the amount of analyte detected, and enables unambiguous identification and quantification of the analyte(s) of interest present in the sample when the analyte(s) reach the mass spectrometer. Typically, but not necessarily, the ISTD is an, preferably stable, isotopically labeled variant (comprising e.g. ² H, ¹³ C, or ¹⁵ N etc. label) of the analyte of interest.

The term “isotopic label” encompasses a label that is part of the original molecular structure of the compound of interest, but which carries preferably heavy atoms such as ¹³ C, ¹⁵ N, ¹⁷ O, ¹⁸ 0, ² H.

The term “Mass Spectrometry” or “Mass Spectrometry measurements” or “Mass Spectrometry measurement” (“Mass Spec” or “MS”) relates to an analytical technology used to identify compounds by their mass. MS is a methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m/z". MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z"). The term "ionization" or "ionizing" refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units. The MS method may be performed either in "negative ion mode", wherein negatively charged ions are generated and detected, or in "positive ion mode" wherein positive ions are generated and detected.

“Tandem mass spectrometry” or “MS/MS” involves multiple steps of mass spectrometry selection, wherein fragmentation of the analyte occurrs in between the stages. In a tandem mass spectrometer, ions are formed in the ion source and separated by mass-to-charge ratio in the first stage of mass spectrometry (MSI). Ions of a particular mass-to-charge ratio (precursor ions or parent ion) are selected and fragment ions (or daughter ions) are created by collision-induced dissociation, ionmolecule reaction, or photodissociation. The resulting ions are then separated and detected in a second stage of mass spectrometry (MS2). Most sample workflows in MS further include sample preparation and/or enrichment steps, wherein e.g. the analyte(s) of interest are separated from the matrix using e.g. gas or liquid chromatography. Typically, for the mass spectrometry measurement, the following three steps are performed:

1. a sample comprising an analyte of interest is ionized, usually by adduct formation with cations, often by protonation forming cations. Ionization source include but are not limited to electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).

2. the ions are sorted and separated according to their mass and charge. High- field asymmetric-waveform ion-mobility spectrometry (FAIMS) may be used as ion filter.

3. the separated ions are then detected, e.g. in multiple reaction mode (MRM), and the results are displayed on a chart.

The term "electrospray ionization" or "ESI," refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

The term "atmospheric pressure chemical ionization" or "APCI," refers to mass spectrometry methods that are similar to ESI; however, APCI produces ions by ionmolecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by an electric discharge between the spray capillary and a counter electrode. Then ions are typically extracted into the mass analyzer by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated N2 gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar entity. "Multiple reaction mode" or "MRM" is a detection mode for a MS instrument in which a precursor ion and one or more fragment ions arc selectively detected.

Since a mass spectrometer separates and detects ions of slightly different masses, it easily distinguishes different isotopes of a given element. Mass spectrometry is thus, an important method for the 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, as well as the global 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.

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.

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 entities as they flow around or over a stationary liquid or solid phase.

The term “liquid chromatography” or "LC" refers to a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this 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, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and methods in which the stationary phase is less polar than the mobile phase (e.g., watermethanol mixture as the mobile phase and Cl 8 (octadecyl silyl) as the stationary phase) is termed reversed phase liquid chromatography (RPLC). "High performance liquid chromatography" or "HPLC" refers to a method of liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column. Typically, the column is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a porous membrane. HPLC is historically divided into two different sub-classes 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, silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and the opposite (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is termed reversed phase liquid chromatography (RPLC). Micro LC refers to a HPLC method using a column having a norrow inner column diameter, typically below 1 mm, e.g. about 0.5 mm. “Ultra high performance liquid chromatography" or “UHPLC” refers to a HPLC method using a pressure of 120 MPa (17,405 lbf/in2), or about 1200 atmospheres. Rapid LC refers to an LC method using a column having an inner diameter as mentioned above, with a short length <2 cm, e.g. 1 cm, applying a flow rate as mentioned above and with a pressure as mentioned above (Micro LC, UHPLC). The short Rapid LC protocol includes a trapping / wash / elution step using a single analytical column and realizes LC in a very short time <1 min.

Further well-known LC modi include Hydrophilic interaction chromatography (HILIC), size-exclusion LC, ion exchange LC, and affinity LC.

LC separation may be single-channel LC or multi-channel LC comprising a plurality of LC channels arranged in parallel. In LC analytes may be separated according to their polarity or log P value, size or affinity, as generally known to the skilled person.

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 that exists in a water medium include but are not limited to -NH2, -OH, -SH, -Se, (R’,R”,R”’)P, N3-, RCOOH, F-, C1-, Br-, I-. In the context of the present invention, the term “nucleophilic derivatization reagent” or “nucleophile derivatization reagent” refers to reagents comprising such nucleophile. A nucleophilic derivatization reagent comprises a moiety, carrying an orbital that serves as the highest occupied molecular orbital (HOMO) that is able to attack the lowest unoccupied molecular orbital (LUMO) of the substance of interest, such as an analyte of interest, thereby forming a new molecule comprised of the formerly nucleophilic unit and the analyte moiety.

The term "sampling tube" or “sample collection tube” refers to any device with a reservoir appropriate for receiving a blood sample to be collected.

In this context "level" or "level value" encompasses the absolute amount, the relative amount or concentration as well as any value or parameter which correlates thereto or can be derived therefrom.

In the context of the present invention, the term “nucleophilic derivatization reagent” or “derivatization reagent” refers to a chemical substance having a specific chemical structure. Said derivatization reagent may comprise one or more reactive groups, which is or are capable of forming a bond, preferably a covalent bond, with the analyte of interest. A derivatized analyte of interest results. Each reactive group may fulfil a different functionality, or two or more reactive groups may fulfil the same funtion. Reactive groups include but are not limited to reactive units, charged units, and neutral loss units. In the context of the present invention, the term “nucleophilic” refers to a chemical species that donates an electron pair to form a chemical bond. Nucleophiles that exists in a water medium include but are not limited to -NH2, -OH, -SH, -Se, (R’,R”,R”’)P, N3-, RCOOH, F-, C1-, Br-, I-. The term “nucleophilic derivatization reagent” can refer to reagents comprising such nucleophile. A nucleophilic derivatization reagent comprises a moiety, carrying an orbital that serves as the highest occupied molecular orbital (HOMO) that is able to attack the lowest unoccupied molecular orbital (LUMO) of the substance of interest, such as an analyte of interest, thereby forming a new molecule comprised of the formerly nucleophilic unit and the analyte moiety.

The term “derivatized analyte of interest” may refer to any molecule that is formed from the original analyte of interest and that is enlarged by a chemical reaction, preferably by reacting with the nucleophilic derivatization reagent. The term "coupling” the analyte of interest and a nucleophilic derivatization reagent via the epoxide moiety as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The term specifically may refer, without limitation, to combining the sample comprising the analyte of interest and the nucleophilic derivatization reagent. Preferably, coupling or combining means to covalently bind the sample, preferably the analyte, and the nucleophilic derivatization reagent via at least one epoxide moiety that can mean that the cyclic structure of the epoxide moiety is broken up.

A "kit" is any manufacture (e.g., a package or container) comprising at least one reagent, e.g., a medicament for treatment of 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 performing the method of the present invention. Typically, a kit may further comprise carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like. In particular, each of the container means comprises one of the separate elements to be used in the method of the first aspect. Kits may further comprise one or more other reagents including but not limited to reaction catalyst. Kits may further comprise one or more other containers comprising further materials including but not limited to buffers, internal standard, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific application, and may also indicate directions for either in vivo or in vitro use. The computer program code may be provided on a data storage medium or device such as a optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device. Moreover, the kit may, comprise standard amounts for the biomarkers as described elsewhere herein for calibration purposes.

Embodiments

In a first aspect, the present invention relates to a composition for determining at least one analyte of interest via at least one Mass Spectrometry measurement or Mass Spectrometry measurements comprising the at least one analyte having a thioether moiety, an additive, wherein the additive is a sulfide or selenide or telluride, preferably wherein the additive is suitable to prevent the oxidation of the thioether moiety.

In embodiments, the additive is an organyl sulfide or an organyl selenide, preferably a diorganyl sulfide or diorganyl selenide. In principle, a telluride as an additive is possible, however not preferred due to their toxicity. In embodiments, the organyl group or organyl groups of the organyl sulfide or the organyl selenide is an organic substituent group(s), regardless of functional type, having one free valence at a carbon atom. Thus, an organyl group can contain organic functional groups and/or atoms other than carbon and hydrogen (i.e. an organic group that can comprise functional groups and/or atoms in addition to carbon and hydrogen). For example, non-limiting examples of atoms other than carbon and hydrogen include halogens, oxygen, nitrogen, and phosphorus, among others. Non-limiting examples of functional groups include ethers, aldehydes, ketones, aldehydes, esters, sulfides, amines, and phosphines, among others. Included in the organyl group can be heteroatom containing rings, heteroatom containing ring systems, heteroaromatic rings, and heteroaromatic ring systems. Preferably, an organyl group is an alkyl group(s) or an aryl group(s).

In embodiments, the additive is a dialkyl sulfide or a diaryl sulfide or an alkyl aryl sulfide.

In embodiments, the additive is a dialkyl selenide or a diaryl selenide or an alkyl aryl selenide. In embodiments, the additive is an alkyl sufide or a thioether with polyethyleneglycol substituents. Preferably, the additive is selected from the group consisting of dimethyl sulfide, diethyl sulfide, dipropyl sulfide, diisopropyl sulfide, dibutyl sulfide, diisobutyl sulfide and ditertbutyl sulfide.

In embodiments, the additive is diethyl sulfide.

In embodiments, the additive is an alkyl selenide or a thioether with polyethyleneglycol substituents. Preferably, the additive is selected from the group consisting of dimethyl selenide, diethyl selenide, dipropyl selenide, diisopropyl selenide, dibutyl selenide, diisobutyl selenide and ditertbutyl selenide.

In embodiments, the additive is diethyl selenide.

In embodiments, the additive is a diethyl sulfide that is dissolved in CH3CN or a mixture of CHsCN/water.

In embodiments, the additive is free of a hydroxyl moiety.

In embodiments, the ratio of the analyte to sulfide is in the range from 1 :0.1 to 1 : 100, preferably 1 : 1 to 1 : 80, more preferabley 1 :25.

In embodiments, the ratio of the analyte to selenide is in the range from 1 :0.1 to 1 : 100, preferably 1 : 1 to 1 : 80, more preferabley 1 :25.

In embodiments, the composition further comprises water and/or CH3CN.

In embodiments, the composition further comprises water and CH3CN, wherein the concentration of the sulfide or selenide is in the range from 0.5 mM to 2 mM.

In embodiments, the content of CH3CN is less than 20%, preferably if the composition further comprises water and CH3CN.

In embodiments, the composition further comprises a solvent, in particular a solvent selected from the group consisting of water, CH3CN, Tetrahydrofuran (THF), Dioxanes, Dimethylformamide (DMF), Dimethyl sulfoxide (DMSO), acetone, t- butyl alcohol, diglyme, dimethyl ether (DME), methanol (MeOH), ethanol (EtOH), 1 -propanol (1-PrOH), 2- propanol (2-PrOH), ethylene glycol, Hexamethylphosphoramiede (HMPA), Hexamethylphosphorous triamide (HMPT), and glycerin.

In embodiments, the composition the thioether moiety is sensitive to oxidation.

In embodiments, the composition the thioether moiety is R1-S-R2, wherein R1 and R2 are each independently selected from alkyl, alkenyl, polyethyleneglycol, a heterocycle, conjugated heterocycle, a (conjugated) aryl or heteroaryl. In embodiments, the composition the thioether moiety is C-S-C.

In embodiments, the composition the additive is a reducing agent.

In embodiments, the additive is suitable to prevent the oxidation of the thioether moiety for at least 15 months.

In embodiments, the composition and/or the formulation is storage stable, preferably for at least 15 months or 16 months or 18 monthes or 20 months.

In embodiments, the at least one analyte is an isotopic labeled Piperacillin or an isotopic labeled Meropenem, wherein at least one analyte is dissolved in the additive, and wherein the additive is a dialkyl sulfide or a diaryl sulfide or an alkyl aryl sulfide.

In embodiments, the composition is a formulation.

In embodiments, the at least one analyte is dissolved in the additive.

In embodiments, the at least one analyte of interest is a P-lactam antibiotic analyte.

In embodiments, the at least one analyte of interest is Piperacillin.

In embodiments, the at least one analyte of interest is Meropenem.

In embodiments, the at least one analyte of interest comprises an isotopic label.

In embodiments, the at least one analyte of interest is used as in ISTD.

In embodiments, the at least one analyte of interest is used as a calibrator.

In embodiments, the at least one analyte is derivatized with a derivatization reagent.

In embodiments, the derivatization reagent is a nucleophilic derivatization reagent.

In embodiments, the analyte of interest is derivatized with a nucleophilic derivatization reagent, wherein the nucleophilic derivatization reagent is a primary amine, secondary amine or thiol, preferably wherein the thiol is RSH, wherein R is selected form the group consisting of alkyl, alkenyl, polyethyleneglycol, a heterocycle, conjugated heterocycle, a conjugated aryl or heteroaryl. In embodiments, the analyte of interest is derivatized with a nucleophilic derivatization reagent, in particular a reagent comprising an amine group, in particular a primary or secondary amine, in particular a primary amine group. A primary amine group has the advantage that the incubation time can be reduced in comparision to a secondary amine. In embodiments, the the analyte of interest is derivatized with a nucleophilic derivatization reagent comprises more than 3 C- atoms, in particular 3 to 20 C-atoms, in particular 3 to 10 C-atoms, in particular 3-5 C-atoms, in particular 4 C-atoms. In embodiments, the analyte of interest is derivatized with a linear or branched nucleophilic derivatization reagent, in particular with a linear amine, in particular with a linear primary amine, in particular with a linear primary amine comprising 3 to 5 C-atoms. In embodiments, the analyte of interest is derivatized with a nucleophilic derivatization reagent selected from the group consisting of propylamine, butylamine, or pentylamine, in particular primary linear butylamine or primary linear pentylamine. Thus, MS interferences can be reduced or avoided.

In embodiments, composition for determining at least one analyte of interest via Mass Spectrometry measurements comprises the at least one analyte having a thioether moiety, an additive, wherein the additive is a sulfide or a selenide, preferably wherein the additive is suitable to prevent the oxidation of the thioether moiety, wherein the composition is free of an oxidized thioether moiety and/or another oxidized moiety.

In embodiments, the thioether moiety is stabilized without an oxidation product.

In embodiments, the additive is an oxygen catcher.

In embodiments, the analyte is a small molecule.

In embodiments, the analyte is not a protein.

In embodiments, the nucleophilic derivatization reagent is a primary amine, secondary amine or thiol, preferably wherein the thiol is RSH, wherein R is selected form the group consisting of alkyl, alkenyl, polyethyleneglycol, a (conjugated) heterocycle, a conjugated aryl or heteroaryl.

In embodiments, the nucleophilic derivatization reagent is substituted or unsubstituted. This can mean in the case of substituted nucleophilic derivatization reagent that the nucleophilic derivatization reagent, e.g. pyrollidine, can comprise other groups, e.g. alkyl or aryl groups that are attached to the nucleophilic derivatization reagent, e.g. pyrollidine. The substituted nucleophilic derivatization reagent can be a derivative of the nucleophilic derivatization reagent. This can mean in case of unsubstituted nucleophilic derivatization reagent that the nucleophilic derivatization reagent, e.g. pyrollidine, does not comprise any other groups, e.g. alkyl or aryl groups that are attached to the nucleophilic derivatization reagent, e.g. pyrollidine. The substituted nucleophilic derivatization reagent is the nucleophilic derivatization reagent without any derivatives.

In embodiments, the at least one analyte is underivatized.

In embodiments, the composition is free of an inert gas, preferably free of argon and/or nitrogen.

In a second aspect, the present invention relates to the use of the composition according to any of the aspects for determining the level of at least one analyte of interest via Mass Spectrometry measurements.

All embodiments mentioned for the first aspect of the invention apply for the second aspect of the invention and vice versa.

In a third aspect, the present invention relates to the use of the composition according to any of the aspects as an ISTD for Mass Spectrometry measurements.

All embodiments mentioned for the first aspect of the invention and/or second aspect of the invention apply for the third aspect of the invention and vice versa.

In a fourth aspect, the present invention relates to use of dialkyl sulfide, preferably diethyl sulfide, as an additive in a formulation of P-lactam antibiotic analyte for preventing oxidation. All embodiments mentioned for the first aspect of the invention and/or second aspect of the invention and/or third aspect of the invention apply for the fourth aspect of the invention and vice versa.

In a fifth aspect, the present invention relates to a kit comprising the composition according to any of the aspects in a first reservoir, and optionally magnetic beads in a second reservoir.

All embodiments mentioned for the first aspect of the invention and/or second aspect of the invention and/or third aspect of the invention and/or fourth aspect of the invention apply for the fifth aspect of the invention and vice versa.

In embodiments, the composition is provided in dissolved form.

In a sixth aspect, the present invention relates to the use of the kit according to any of the aspect of the invention for Mass Spectrometry measurements.

All embodiments mentioned for the first aspect of the invention and/or second aspect of the invention and/or third aspect of the invention and/or fourth aspect of the invention and/or fifth aspect of the invention apply for the sixth aspect of the invention and vice versa.

In a seventh aspect, the present invention relates to a method of determining the level of at least one analyte of interest in an obtained sample comprising the steps of: a) Providing the sample comprising an analyte of interest, wherein the analyte of interest has a thioether moiety, b) Optionally providing an isotopic labeled analyte of interest as in ISTD having a thioether moiety, c) Providing an additive, wherein the additive is a sulfide or a selenide and suitable to prevent the oxidation of the thioether moiety of the analyte of interest and/or the thioether moiety of the isotopic labeled analyte of interest, d) Optionally derivatizing the analyte of interest and/or isotopic labeled analyte of interest with a nucleophilic derivatization reagent, and e) Determining the level of the at least one analyte of interest in the sample, in particular using Mass Spectrometry, preferably LC/MS.

All embodiments mentioned for the first aspect of the invention and/or second aspect of the invention and/or third aspect of the invention and/or fourth aspect of the invention and/or fifth aspect of the invention and/or sixth aspect of the invention apply for the seventh aspect of the invention and vice versa.

In embodiments, the method comprises a further step:

Pre-treating and/or enriching the sample, in particular using magnetic beads.

In embodiments, the method comprises further step:

- Enriching the sample obtained after step b), in particular using magnetic beads.

In embodiments, the enrichment step comprises at least one enrichment workflow.

In embodiments, the samples comprising an analyte may be (pre-)treated and/or enriched by various methods. The (pre-)treatment method is dependent upon the type of sample, such as blood (fresh or dried), plasma, serum, urine, or saliva, whereas the enrichment method is dependent on the analyte of interest. It is well known to the skilled person which (pre-)treatment method is suitable for which sample type. It is also well-known to the skilled person which enrichment method is suitable for which analyte of interest.

In embodiments, wherein the sample is a whole blood sample, it is assigned to one of two pre-defined sample (pre-)treatment (PT) workflows, both comprising the addition of an internal standard (ISTD) and a hemolysis reagent (HR) followed by a pre-defined incubation period (Inc), where the difference between the two workflows is the order in which the internal standard (ISTD) and a hemolysis reagent (HR) are added. In embodiments, the ISTD is added first to the obtained sample followed by the addition of the hemolysis reagent. In embodiments, the ISTD is added to the obtained sample subsequent to the addition of the hemolysis reagents. In embodiments, water is added as a hemolysis reagents, in particular in an amount of 0.5:1 to 20: 1 mL water / mL sample, in particular in an amount of 1 : 1 to 10: 1 mL water / mL sample, in particular in an amount of 2: 1 to 5: 1 mL water / mL sample.

In embodiments, wherein the sample is a urine sample, it is assigned to one of other two pre-defined sample PT workflows, both comprising the addition of an ISTD and an enzymatic reagent followed by a pre-defined incubation period, where the difference between the two workflows is the order in which the internal standard and an enzymatic reagent are added. In embodiments, the ISTD is added first to the obtained sample followed by the addition of the enzymatic reagent. In embodiments, the ISTD is added to the obtained sample subsequent to the addition of the enzymatic reagents. An enzymatic reagent is typically a reagent used for glucuronide cleavage or protein cleavage or any pre-processing of analyte or matrix. In embodiments, the enzymatic reagent in selected from the group consisting of glucuronidase, (partial) exo- or endo- deglycoslation enzymes, or exo- or endo preoteases. In embodiments, glucoronidase is added in amount of 0.5 - 10 mg/ml, in particular in an amount of 1 to 8 mg/ml, in particular in an amount of 2 to 5 mg/ml.

In embodiments, wherein the sample is plasma or serum it is assigned to another predefined PT workflow including only the addition of an internal standard (ISTD) followed by a pre-defined incubation time.

It is well-known to the skilled person which incubation time and temperature to choose for a sample treatment, chemical reaction or method step considered and as named herein above or below. In particular, the skilled person knows that incubation time and temperature depend upon each other, in that e.g. a high temperature typically leads to a shorter incubation period and vice versa.

The sample may be further subjected to at least one enrichment workflow. The enrichment workflow may include one or more enrichment methods. Enrichment methods are well-known in the art and include but are not limited to chemical enrichment methods including but not limited to chemical precipitation, and enrichment methods using solid phases including but not limited to solid phase extraction methods, bead workflows, and chromatographic methods (e.g. gas or liquid chromatography). In embodiments, a first enrichment workflow comprises the addition of a solid phase, in particular of solid beads, carrying analyte-selective groups, to the (pre-treated) sample. In embodiments, a first enrichment workflow comprises the addition of magnetic or paramagnetic beads carrying analyte- selective groups to the pre-treated sample.

In embodiments, the magnetic beads comprise a magnetic core coated with a styrene based polymer that is hypercrosslinked via Friedel-Crafts alkylation and further modified with addition of -OH groups.

In embodiments, the magnetic beads comprise a magnetic core coated with a styrene based polymer that is hypercrosslinked via diamines (e.g. TMEDA) and further modified whereby the diamine also serves as a sidechain (i.e. in these types of beads, TMEDA offers both quaternary and tertiary amine functionalities). For a full description of such beads see: WO 2019/141779

In embodiments, the addition of the magnetic beads comprises agitation or mixing. A pre-defined incubation period for capturing the analyte(s) of interest on the bead follows. In embodiments, the workflow comprises a washing step (Wl) after incubation with the magnetic beads. Depending on the analyte(s) one or more additional washing steps (W2) are performed. One washing step (Wl, W2) comprises a series of steps including magnetic bead separation by a magnetic bead handling unit comprising magnets or electromagnets, aspiration of liquid, addition of a washing buffer, resuspension of the magnetic beads, another magnetic bead separation step and another aspiration of the liquid. Moreover, washing steps may differ in terms of type of solvent (water/organic/salt/pH), aside from volume and number or combination of washing cycles. It is well-known to the skilled person how to choose the respective parameters. The last washing step (Wl, W2) is followed by the addition of an elution reagent followed by resuspension of the magnetic beads and a pre-defined incubation period for releasing the analyte(s) of interest from the magnetic beads. The bound-free magnetic beads are then separated and the supernatant containing derivatized analyte(s) of interest is captured. In embodiments, a first enrichment workflow comprises the addition of magnetic beads carrying matrix-selective groups to the pre-treated sample. In embodiments, the addition of the magnetic beads comprises agitation or mixing. A pre-defined incubation period for capturing the matrix on the bead follows. Here, the analyte of interest does not bind to the magnetic beads but remains in the supernatant. Thereafter, the magnetic beads are separated and the supernatant containing the enriched analyte(s) of interest is collected.

In embodiments, the supernatant is subjected to a second enrichment workflow, in particular to a chromatographic enrichment workflow. In embodiments of the present invention, the chromatographic separation is gas or liquid chromatography. Both methods are well known to the skilled person. In embodiments, the liquid chromatography is selected from the group consisting of HPLC, rapid LC, micro-LC, flow injection, and trap and elute. Here, the supernatant is transferred to the LC station or is transferred to the LC station after a dilution step by addition of a dilution liquid. Different elution procedures/reagents may also be used, by changing e.g. the type of solvents (water/organic/salt/pH) and volume. The various parameters are well-known to the skilled person and easily chosen.

In embodiments, the first enrichment process includes the use of analyte selective magnetic beads. In embodiments, the second enrichment process includes the use of chromatographic separation, in particular using liquid chromatography. In embodiments, the first enrichment process using analyte selective magnetic beads is performed prior to the second enrichment process using liquid chromatography.

In embodiments, wherein step e) comprises determining the level of the at least one analyte of interest in the sample using mass spectrometry, the following steps are comprised:

(i) subjecting an ion of the undervatized or derivatized analyte to a first stage of mass spectrometric analysis, whereby the parent ion of the derivatized analyte is characterised according to its mass/charge (m/z) ratio,

(ii) causing fragmentation of the undervatized or derivatized analyte parent ion, whereby a daughter ion is generated, wherein the daughter ion of the undervatized or derivatized analyte differs in its m/z ratio from the undervatized or derivatized analyte parent ion, and

(iii) subjecting the daughter ion of the undervatized or derivatized analyte to a second stage of mass spectrometric analysis, whereby the daughter ion of the undervatized or derivatized analyte is characterized according to its m/z ratio.

In embodiments, the parent and/or fragment ions measured are those as indicated in Table X.

Table X: MRM transitions of Meropenem and Piperacillin: In embodiments, the parent ion of derivatized Meropenem+HT is measured at a m/z value 457.164±0.5, and the parent ion of derivatized Piperacillin+H ⁺ is measured at a m/z value 664.235±0.5.

In embodiments, the fragment ion of derivatized Meropenem is measured at a m/z value 152±0.5 or 173±0.5, and the fragment ion of derivatized Piperacillin is measured at a m/z value 270±0.5 or 464±0.5. In embodiments, the method is an automated method. In particular embodiments, the method is performed by an automated system. In particular embodiments, the method comprises no manual intervention.

In embodiments, the method is performed in a random-excess mode.

In embodiments, the method is an in-vitro diagnostic method.

In an eighth aspect, the present invention relates to sampling tube for collecting a patient sample comprising

- an analyte of interest having a thioether moiety, and

- an additive suitable to prevent the oxidation of the thioether moiety of the analyte of interest in a sample, wherein the additive is a sulfide or a selenide.

All embodiments mentioned for the first aspect of the invention and/or second aspect of the invention and/or third aspect of the invention and/or fourth aspect of the invention and/or fifth aspect of the invention and/or sixth aspect of the invention and/or seventh aspect of the invention apply for the eighth aspect of the invention and vice versa.

Sample collections tubes suitable to be used for collecting a patient sample are well- known in the art and are used on a routine basis by practioners. As the skilled artisan will appreciate the sampling tube preferably will in fact be a tube. In particular, the sampling tube has a size and dimension adapted to match the requirements of the sample receiving station of an automated analyzer, e.g. an Elecsys® analyzer of Roche Diagnostics. The sampling tube may have a conical or preferably a round bottom. In clinical routine standard tube sizes are used that are compatible with the analyzers systems on the market. Standard and preferred tubes e.g. have the following dimensions: 13x75 mm; 13x100 mm, or 16x100 mm.

In embodiments, the sampling tube according to the present invention is only used once, i.e. it is a single use device. In particular embdiments, the sampling tube according to the present invention is not only appropriate for collection of a sample but it is also adapted to allow for the further processing of the sample. By collecting a sample into a sampling tube containing the nucleophilic derivatization reagent, the desired result, i.e. the derivatization of the analyte, is achieved.

In a ninth aspect, the present invention relates to an analyzer adapted to perform the method of any of the aspects and/or comprises a composition of any of the aspects.

All embodiments mentioned for the first aspect of the invention and/or second aspect of the invention and/or third aspect of the invention and/or fourth aspect of the invention and/or fifth aspect of the invention and/or sixth aspect of the invention and/or seventh aspect of the invention and/or eighth aspect of the invention apply for the ninth aspect of the invention and vice versa.

In embodiments, the analyzer is a mass spectrometry system, in particular an LC/MS system. In embodiments, the analyzer is an automated analytical system. In particular embodiments, the analyzer does not require manual intervention, i.e. the operation of the system is purely automated. In particular embodiments, the LC/MS system is an automated, random-access LC/MS system. In embodiments, the MS device is a tandem mass spectrometer, in particular a triple quadrupole device. In embodiments, the LC is HPLC, in particular is RP-HPLC, or rapid LC. In embodiments, the ion formation is based on electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), in particular positive polarity mode ESI.

In embodiments, the analyser or automatic analyser is a clinical diagnostics system.

A clinical diagnostics system can be a laboratory automated apparatus dedicated to the analysis of samples for in vitro diagnostics. The clinical diagnostics system may have different configurations according to the need and/or according to the desired laboratory workflow. Additional configurations may be obtained by coupling a plurality of apparatuses and/or modules together. A “module” is a work cell, typically smaller in size than the entire clinical diagnostics system, which has a dedicated function. This function can be analytical but can be also pre-analytical or post analytical or it can be an auxiliary function to any of the pre-analytical function, analytical function or post-analytical function. In particular, a module can be configured to cooperate with one or more other modules for carrying out dedicated tasks of a sample processing workflow, e.g. by performing one or more pre- analytical and/or analytical and/or post-analytical steps. In particular, the clinical diagnostics system can comprise one or more analytical apparatuses, designed to execute respective workflows that are optimized for certain types of analysis, e.g. clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separation, mass spectrometry, etc. Thus, the clinical diagnostic system may comprise one analytical apparatus or a combination of any of such analytical apparatuses with respective workflows, where pre-analytical and/or post analytical modules may be coupled to individual analytical apparatuses or be shared by a plurality of analytical apparatuses. In alternative pre-analytical and/or post- analytical functions may be performed by units integrated in an analytical apparatus. The clinical diagnostics system can comprise functional units such as liquid handling units for pipetting and/or pumping and/or mixing of samples and/or reagents and/or system fluids, and also functional units for sorting, storing, transporting, identifying, separating, detecting. The clinical diagnostic system can comprise a sample preparation station for the automated preparation of samples comprising analytes of interest, optionally a liquid chromatography (LC) separation station comprising a plurality of LC channels and/or optionally a sample preparation/LC interface for inputting prepared samples into any one of the LC channels. The clinical diagnostic system can further comprise a controller programmed to assign samples to predefined sample preparation workflows each comprising a pre-defined sequence of sample preparation steps and requiring a pre-defined time for completion depending on the analytes of interest. The clinical diagnostic system can further comprise a mass spectrometer (MS) and an LC/MS interface for connecting the LC separation station to the mass spectrometer.

A sample preparation station can be a pre-analytical module coupled to one or more analytical apparatuses or a unit in an analytical apparatus designed to execute a series of sample processing steps aimed at removing or at least reducing interfering matrix components in a sample and/or enriching analytes of interest in a sample. Such processing steps may include any one or more of the following processing operations carried out on a sample or a plurality of samples, sequentially, in parallel or in a staggered manner: pipetting (aspirating and/or dispensing) fluids, pumping fluids, mixing with reagents, incubating at a certain temperature, heating or cooling, centrifuging, separating, filtering, sieving, drying, washing, resuspending, aliquoting, transferring, storing, etc.).

The clinical diagnostic system, e.g. the sample preparation station, may also comprise a buffer unit for receiving a plurality of samples before a new sample preparation start sequence is initiated, where the samples may be individually randomly accessible and the individual preparation of which may be initiated according to the sample preparation start sequence.

The clinical diagnostic system makes use of mass spectrometry more convenient and more reliable and therefore suitable for clinical diagnostics. In particular, high- throughput, e.g. up to 100 samples/hour or more with random access sample preparation and LC separation can be obtained while enabling online coupling to mass spectrometry. Moreover the process can be fully automated increasing the walk-away time and decreasing the level of skills required.

In further embodiments, the present invention relates to the following aspects:

1. A composition for determining at least one analyte of interest via Mass Spectrometry measurements comprising the at least one analyte having a thioether moiety, an additive, wherein the additive is a sulfide or selenide or telluride, preferably wherein the additive is suitable to prevent the oxidation of the thioether moiety.

2. The composition of aspect 1, wherein the additive is an organyl sulfide or organyl selenide, preferably a diorganyl sulfide or a diorganyl selenide, more preferably a dialkyl sulfide or a diaryl sulfide or an alkyl aryl sulfide.

3. The composition of any of the preceding aspects, wherein the additive is an alkyl sufide, preferably selected from the group consisting of dimethyl sulfide, diethyl sulfide, dipropyl sulfide, diisopropyl sulfide, dibutyl sulfide, diisobutyl sulfide, ditertbutyl sulfide and a thioether with polyethyleneglycol substituents. 4. The composition of any of the preceding aspects, wherein the additive is diethyl sulfide.

5. The composition of any of the preceding aspects, wherein the additive is a dissolved diethyl sulfide.

6. The composition of any of the preceding aspects, wherein the additive is free of a hydroxyl moiety.

7. The composition of any of the preceding aspects, wherein the ratio of the analyte to sulfide or the ratio of the analyte to selenide is in the range from 1 :0.1 to 1 : 100.

8. The composition of any of the preceding aspects, further comprises water and CH ₃ CN.

9. The composition of any of the preceding aspects, further comprises water and CH3CN, wherein the concentration of the sulfide or selenide is in the range from 0.5 mM to 2 mM.

10. The composition of any of the preceding aspects, wherein the content of CH3CN is less than 20%.

11. The composition of any of the preceding aspects, further comprises a solvent, in particular a solvent selected from the group consisting of water, CH3CN, Tetrahydrofuran (THF), Dioxanes, Dimethylformamide (DMF), Dimethyl sulfoxide (DMSO), acetone, t-butyl alcohol, diglyme, dimethyl ether (DME), methanol (MeOH), ethanol (EtOH), 1 -propanol (1-PrOH), 2- propanol (2-PrOH), ethylene glycol, Hexamethylphosphoramiede (EIMP A), Hexamethylphosphorous triamide (HMPT), and glycerin.

12. The composition of any of the preceding aspects, wherein the thioether moiety is sensitive to oxidation.

13. The composition of any of the preceding aspects, wherein the additive is a reducing agent. 14. The composition of any of the preceding aspects, wherein the additive is suitable to prevent the oxidation of the thioether moiety for at least 15 months.

15. The composition of any of the preceding aspects, wherein the composition and/or the formulation is storage stable, preferably for at least 15 months.

16. The composition of any of the preceding aspects, wherein the at least one analyte of interest is a P-lactam antibiotic analyte.

17. The composition of any of the preceding aspects, wherein the at least one analyte of interest is Piperacillin.

18. The composition of any of the preceding aspects, wherein the at least one analyte of interest is Meropenem.

19. The composition of any of the preceding aspects, wherein the at least one analyte is an isotopic labeled Piperacillin or an isotopic labeled Meropenem, wherein at least one analyte is dissolved in the additive, and wherein the additive is a dialkyl sulfide or a diaryl sulfide or an alkyl aryl sulfide.

20. The composition of any of the preceding aspects, wherein the composition is a formulation.

21. The composition of any of the preceding aspects, wherein the at least one analyte is dissolved in the additive.

22. The composition of any of the preceding aspects, wherein the at least one analyte of interest comprises an isotopic label.

23. The composition of any of the preceding aspects, wherein the at least one analyte of interest is used as in ISTD.

24. The composition of any of the preceding aspects, wherein the at least one analyte of interest is used as a calibrator. 25. The composition of any of the preceding aspects, wherein the at least one analyte is derivatized with a derivatization reagent.

26. The composition of any of the preceding aspects, wherein the derivatization reagent is a nucleophilic derivatization reagent.

27. The composition of any of the preceding aspects, wherein the nucleophilic derivatization reagent is a primary amine, secondary amine or thiol, preferably wherein the thiol is RSH, wherein R is selected form the group consisting of alkyl, alkenyl, polyethyleneglycol, a (conjugated) heterocycle, a conjugated aryl or heteroaryl.

28. The composition of any of the preceding aspects, wherein the at least one analyte is underivatized.

29. The composition of any of the preceding aspects, wherein the composition is free of an inert gas, preferably free of argon and/or nitrogen.

30. Use of the composition according to any of the preceding aspects for determining the level of at least one analyte of interest via Mass Spectrometry measurements.

31. Use of the composition according to any of the preceding aspects as an ISTD for Mass Spectrometry measurements.

32. Use of dialkyl sulfide, preferably diethyl sulfide, as an additive in a formulation of P-lactam antibiotic analyte for preventing oxidation.

33. A kit comprising the composition according to any of the preceding aspects in a first reservoir, and optionally magnetic beads in a second reservoir.

34. A kit of the preceding aspect, wherein the composition is provided in dissolved form.

35. Use of the kit of the preceding aspect for Mass Spectrometry measurements. 36. A method of determining the level of at least one analyte of interest in an obtained sample comprising the steps of: a) Providing the sample comprising an analyte of interest, wherein the analyte of interest has a thioether moiety, b) Optionally providing an isotopic labeled analyte of interest as in ISTD having a thioether moiety, c) Providing an additive, wherein the additive is a sulfide or a selenide and suitable to prevent the oxidation of the thioether moiety of the analyte of interest and/or the thioether moiety of the isotopic labeled analyte of interest, d) Optionally derivatizing the analyte of interest and/or isotopic labeled analyte of interest with a nucleophilic derivatization reagent, and e) Determining the level of the at least one analyte of interest in the sample, in particular using Mass Spectrometry, preferably LC/MS.

37. A sampling tube for collecting a patient sample comprising an analyte of interest having a thioether moiety, and an additive suitable to prevent the oxidation of the thioether moiety of the analyte of interest in a sample, wherein the additive is a sulfide or a selenide.

38. An analyzer adapted to perform the method of any of the preceding aspects and/or comprises a composition of any of the preceding aspects.

Examples

The following examples are provided to illustrate, but not to limit the presently claimed invention.

Figure 1 shows the structures of 1 : Meropenem-butylamide as a deriviatized anaylte of interest, 2: Piperacillin-butylamide as a deriviatized anaylte of interest, 3: diethyl sulfide as an additive, 4: Meropenem-butyalmide-sulfoxide as an oxidation product of Meropenem-butylamide 1, 5: Levetiracetam as a labeled anaylte of interest. Using Meropenem-butylamide (1) and Piperacillin-butylamide (2) as model substances for an analyte of interest in a formuation of 20% CH3CN in water with 1 mM diethyl sulfide (3), the inventors show that the stability over time at temperatures between 10 and 35 °C is much better. The example of Meropenem-butylamide- sulfoxide (4) shows that the formation of this compound is reduced in the presence of Et2S (3), thereby further corroborating evidence that oxidation of the thioether is a source of degradation of this compound.

Experimental Design

To assess the protective formulation in terms of stability of the compounds 1 and 2, of which the isotopically labeled forms can to be used as internal standards, a rational approach were taken in which formulations were used in which solubility of all compounds is known and in which the concentration of Et2S is based on the concentrations of compounds 1 and 2. The different formulations were then used in an isochronous stability experiment over four weeks at 35 °C. To control eventual evaporation effects that might lead to concentration of the compounds under evaluation, another compound with known stability (levetiracetam, 5) was spiked into the formulations. All compounds can be measured adequately via LC-MS/MS. Via this approach, analyte areas are measured for each compound and using 5 as an invariable analyte over time, area ratios can be calculated. For this, the area’s of 1, 2 and 4 are divided by the area of 5, thereby correcting for evaporation effects or variability in injection volume.

Materials and Methods

Three concentrations of Et2S in a solution of 20% CH3CN in water (v/v) were prepared with a fourth solution containing no Et2S that served as a negative control. A stock solution of 1 and 2 were prepared by weighing dry powders in a vial. These were dissolved in 20% CEECN in water (v/v), shortly before spiking into the aforementioned four solutions. To this stock solution, also 5 was spiked. The total combined concentration of both compounds 1 and 2 being 210 pM in each formulation and the concentration of 5 being 100 pM. Based on this concentration, a corresponding Et2S concentration of 210 pM or 2 times 210 pM was used. To also test an almost 5 fold concentration, 1 mM was included.

Next, these solutions were aliquoted and stored over 0, 2 and 4 weeks in a temperature controlled stove at 35 °C. Aliquots were either directly, after 2 and after 4 weeks placed in -80 °C freezer. Subsequently, all aliquots were manually diluted a factor 100 with water that was degassed by ultra sonication. Subsequently, all aliquots were measured in one single run via LC-MS/MS, for which we obtained and used MRM transitions for both compounds 1 and 2 as well as compounds 4 and 5. For both of these compounds we used three MRM transitions. In addition, we monitored one of the oxidation (i.e. degradation) products, for which we were able to tune in a MRM transition.

The measurement was performed using a Kinetex Cl 8, 2.6 pm, 1.0 mm x 50 mm column with Solvent A: water with 0.1% HCOOH and Solvent B: CH3CN with 0.1% HCOOH and a flow of 0.4 mL per minute on an Agilent Infinity II multi sampler/pump system connected to an AB Sciex 6500+ MS, injecting 1 pL per sample. For this, the following settings were used:

Table 1. LC Gradient Table 2. MRM transitions and settings

QI: Precursor m/z; Q3: Fragment m/z; DP: Declustering Potential; EP: Entrance

Potential; CE: Collision Enegery; CXP Cell Exit Potential

Results Figure 2 (area ratio as a function of time) shows the degradation of 1. It could be observed a strong Et2S concentration effect with regard to the stability of compound 1. Clearly, without the presence of Et2S, degradation is much faster compared to when 1 mM Et2S is used as an additive. However, also if only 210 pM or 420 pM is present, degradation is slowed down, but to a lesser extent when compared to formulations containing 1 mM Et2S.

Figure 3 (area ratio as a function of time) shows the increase in 4. It is seen that the increase of this compound over time is much less in the presence of 420 pM or 1 mM Et2S. The formation of this compound seems to occur to a higher degree in the presence of only 210 pM compared to a solution without Et2S. However, it needs to be noted that the formation of the sulfoxide is possibly only the first of two oxidation steps, since the sulfoxide might further oxidize to the sulfone. Furthermore, other instabilities might lead to the formation of other products that are not monitored in this experiment. For example, decarboxylation is one of the possible issues that might occur simultaneously. This might explain why the formation of 4 seems to occur at a lower rate in a 210 pM Et2S solution, although other degradation effects might bias this result. Thus, the (lower) increase of 4 might be indicative for the stabilizing effect of Et2S, but is by no means a quantitative metric for this effect. For a full understanding, all degradation products and combinations of decarboxylation and oxidation can be quantified.

Figure 4 (area ratio as a function of time) shows the degradation of 2. Although the presence of Et2S has a positive effect on stabilizing this compound, the stabilization has a less pronounced effect when compared to 1.

This patent application claims the priority of the European patent application 22150342.8, wherein the content of this European patent application is hereby incorporated by references.