Synthesis of stable isotope labeled versions of n-(4-hydroxyphenyl)acetamid

Abstract

The present invention relates to methods to prepare an isotopically labelled N-(4- hydroxyphenyl)acetamid, methods to prepare isotopically labelled 4-aminophenol and isotopically labelled 4-((4-nitrophenyldiazenyl)phenol. The present invention further relates to uses of an isotopically labelled N-(4-hydroxyphenyl)acetamid as an internal standard to determine the presence, amount or concentration of N-(4- hydroxyphenyl)acetamid in a sample and to methods of determining the amount of N-(4-hydroxyphenyl)acetamid in a sample. The present invention also relates to diagnostic compositions comprising an isotopically labelled N-(4- hydroxyphenyl)acetamid and kits for determining the presence, amount or concentration of N-(4-hydroxyphenyl)acetamid in a sample.

Description

[0001] SYNTHESIS OF STABLE ISOTOPE LABELED VERSIONS OF N-(4-HYDROXYPHENYL)ACETAMID Field of the invention The present invention relates to methods to prepare an isotopically labelled N-(4- hydroxyphenyl)acetamid, methods to prepare isotopically labelled 4-aminophenol and isotopically labelled 4-((4-nitrophenyldiazenyl)phenol. The present invention further relates to uses of an isotopically labelled N-(4-hydroxyphenyl)acetamid as an internal standard to determine the presence, amount or concentration of N-(4- hydroxyphenyl)acetamid in a sample and to methods of determining the amount of N-(4-hydroxyphenyl)acetamid in a sample. The present invention also relates to diagnostic compositions comprising an isotopically labelled N-(4- hydroxyphenyl)acetamid and kits for determining the presence, amount or concentration of N-(4-hydroxyphenyl)acetamid in a sample. Background of the invention Paracetamol, also known as acetaminophen or N-(4-hydroxyphenyl)acetamid, is a medication used to treat fever and mild to moderate pain. The combination aspirin / paracetamol / caffeine helps conditions where the pain is mild and is recommended as a first-line treatment for several symptoms and diseases. Paracetamol is effective for post-surgical pain, but it is inferior to ibuprofen. The paracetamol / ibuprofen combination provides further increase in potency and is superior to either drug alone. It is recommended to use paracetamol for short-term and episodic. For people taking paracetamol regularly, regardless the indication, monitoring for liver toxicity is required. Thus, there is a need for accurate methods to determine paracetamol levels in patients, which is capable of high-throughput of samples in humane diagnostics, especially serum analysis. LC-MS-based clinical diagnostics represents one mean to achieve this. This technology employs internal standards to quantify any analyte, such as acetaminophen, in a given sample. Those internal standards are, for several chemical and physical reasons, usually composed of a stable isotope labelled version of the analyte. Syntheses of such stable isotope labelled molecules usually requires different process properties than preparation methods devised for multi kilogram or metric ton scale preparations of other molecules, for example, clinical drugs. The preciousness of isotopically labelled starting materials and the purity requirements in terms of chemical and isotopic purity pose unique challenges to their synthetic manufacturing. Industrial preparation instructions of paracetamol usually refer to the acetylation of 4- aminophenol with acetic anhydride as the last step (J. Chem. Educ.2022, 99(2), 910- 916). They commonly differ in how 4-aminophenol is prepared. Reduction of 4-nitrophenol can yield 4-aminophenol, at which the nitro compounds can be prepared by nitration of phenol. Unfortunately, this reaction is not regioselective, results in tedious separations, low yields and uses conditions capable of hydrogen / deuterium (H / D) exchange in deuterated aromatic ring systems (Synlett 2017, 28, 2153–2158; J. Phys. Org. Chem.2006; 19: 570–578; Tetrahedron Letters, Vo1.29, No.20, pp 2471-2474, 1988). Alternatively, electrolytic reduction of nitrobenzene yields 4-aminophenol in moderate yields, too, and also employs conditions capable of H / D exchange (US2,998,450; J. Label. Compd. Radiopharm. 1988, 12, 1315-1318.). The Celanese synthesis of paracetamol also refers to strongly acidic conditions, rendering the use of fully deuterated solvent mixtures and moisture free reactions necessary in case of the synthesis of deuterated internal standards. (Friderichs E, Christoph T, Buschmann H. "Analgesics and Antipyretics". 2007, Ullmann's Encyclopedia of Industrial Chemistry. Weinheim: Wiley-VCH). Deuterated versions of acetaminophen, or its precursor 4-aminophenol, have also been prepared via H / D exchange starting from the respective unlabelled molecules. However, these methods provide degrees of isotopic labelling per atom site, which are not suitable for quantification in diagnostic tests with low to high µg / ml dynamic concentration range (J. Label. Compd. Radiopharm.1978, 15, 637-643; Chem. Eur. J. 2022, 28, e202201583; Org. Lett. 2021, 23, 9297−9302; J. Label. Compd. Radiopharm. 2000, 43, 817-823; Org. Process Res. Dev. 2019, 23, 648−653; Tetrahedron Letters 2015, 56, 747-749.). Therefore, there remains a need for enhanced methods of preparation of N-(4- hydroxyphenyl)acetamid, which is isotopically labelled, that can provide a higher yield and / or methods, which led to a higher regioselectivity of attaching an amino group at the para-position of phenol to form 4-aminophenol, and / or methods that improve the stability of the isotopic labelling. The problem of low yields, low regioselectivity and / or stability of the isotopic labelling, e.g. reduced H / D exchange, is particularly important when dealing with isotopically labelled molecules, because obtaining compositions comprising molecules with a high degree of isotopic labelling per atom site is decisive for subsequent applications of these molecules, e.g. as an internal standard. Summary of the invention In a first aspect the present invention relates to a method to prepare an isotopically labelled N-(4-hydroxyphenyl)acetamid comprising the following steps i) azo-coupling of an isotopically labelled phenol to4-nitroaniline to form an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol; ii) hydrogenation of the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol according to step i) to form an isotopically labelled 4-aminophenol; and iii) acetylation of the isotopically labelled 4-aminophenol according to step ii) to form the isotopically labelled N-(4-hydroxyphenyl)acetamid. The inventors have surprisingly found that N-(4-hydroxyphenyl)acetamid, in particular its precursor 4-aminophenol, can be obtained with a high regioselectivity of attaching an amino group at the para-position of phenol to form 4-aminophenol and / or high yield of N-(4-hydroxyphenyl)acetamid, in particular its precursor 4- aminophenol. As can be seen in the appended Examples, the inventors have furthermore found that, in case the N-(4-hydroxyphenyl)acetamid shall be formed with deuterium as an isotopic label, despite acidic conditions, the presently claimed invention may lead to a reduction of hydrogen / deuterium (H / D) exchange with the deuterium at the aromatic ring sites, up to the point with a negligible reduction or one below measurement capabilities with mass spectrometry. As can be seen in the appended Example, in particular the NMR data and the lack of detectable ortho product, the present invention leads to a high regioselectivity, in particular in step i) and thus in subsequent steps ii) and iii). The present invention further relates to a second aspect of a method to prepare an isotopically labelled 4-aminophenol comprising the following step ii) hydrogenation of an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol to form the isotopically labelled 4-aminophenol. In a third aspect the present invention relates to a method to prepare an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol comprising the following step i) azo-coupling of an isotopically labelled phenol to 4-nitroaniline to form an isotopically labelled 4-((4-nitrophenyldiazenyl)phenol. In a fourth aspect the present invention relates to a use of an isotopically labelled N- (4-hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable by a method according to the present invention, as an internal standard to determine the presence, amount or concentration of N-(4-hydroxyphenyl)acetamid in a sample. In a fifth aspect the present invention relates to a diagnostic composition comprising an isotopically labelled N-(4-hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4-hydroxyphenyl)acetamid obtained or obtainable by a method according to the present invention, and a suitable excipient. In a sixth aspect the present invention relates to a method of determining the amount of N-(4-hydroxyphenyl)acetamid in a sample, the method comprising the steps of (a) admixing a known amount of an isotopically labelled N-(4- hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable by a method according to the present invention, to the sample, (b) analysing the sample via mass spectrometry, (c) comparing the peak area of N-(4-hydroxyphenyl)acetamid to a standard curve, thereby determining the amount of N-(4-hydroxyphenyl)acetamid in the sample, wherein said standard curve has been created using an isotopically labelled N-(4- hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable by a method according to the present invention; and standards containing N-(4-hydroxyphenyl)acetamid. The isotopically labelled N-(4-hydroxyphenyl)acetamid of the present invention may be particularly useful for determining the presence, amount or concentration of N- (4-hydroxyphenyl)acetamid in a sample, since the isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable may have a higher purity and / or stability of the isotopic labelling. In a seventh aspect the present invention relates to a kit for determining the presence, amount or concentration of N-(4-hydroxyphenyl)acetamid in a sample comprising in a container an isotopically labelled N-(4-hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4-hydroxyphenyl)acetamid obtained or obtainable by a method according to the present invention. List of figures FIG. 1 shows the total ion count evolving with the elution of the sample of Acetaminophen-D4 (SI-5). FIG. 2 shows the total ion count evolving with the elution of the spiked sample of Acetaminophen-D4(SI-5). FIG.3 shows signal / ion count and shape thereof of the mass of isotopically unlabled Acetaminophen in the measured sample of Acetaminophen-D4 (SI-5). FIG. 4 shows signal / ion count and shape thereof of the mass of isotopically unlabelled Acetaminophen in the measured sample of Acetaminophen-D4 (SI-5) that was spiked with isotopically unlabelled Acetaminophen. FIG. 5 shows a simulated signal / ion count of the mass of isotopically unlabelled Acetaminophen. 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 embodiments and examples 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. Several 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 following definitions and embodiments apply to the present disclosure in its entirety, especially to all aspects and embodiments of the invention. 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. 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. The use of the alternative (e.g. “or”) should be understood to mean either one, both or any combination thereof of the alternatives. The term “and / or” should be understood to mean either one, or both of the alternatives. 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. As used herein and unless stated otherwise, it is to be understood that the term “about” is used synonymously with the term “approximately”. Illustratively and unless stated otherwise, the use of the term “about” when used in conjunction with a stated numerical value or range denotes somewhat more or somewhat less than the stated value or range, to within a range of ±15% of that stated, ±10% of that stated, ±5% of that stated, or conveniently ± 2% of that stated. Such values are thus encompassed by the scope of the claims reciting the terms “about” or “approximately”. The term "13C" refers to the stable isotope of carbon with a nucleus containing six protons and seven neutrons. The term “D” refers to deuterium, the stable isotope of hydrogen with a nucleus containing one proton and one neutron. The term "acetylation" refers to an organic esterification reaction with acetic acid. As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to temperatures from at least about 15 °C, e.g. from about 15 °C to about 35 °C, from about 15 °C to about 30 °C, from about 15 °C to about 25 °C or from about 17 °C to about 22 °C. Such temperatures will include 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C and 22 °C. In the context of the present disclosure, the terms “analyte”, “analyte molecule”, or “analyte(s) of interest” are used interchangeably referring to the chemical species to be analysed via mass spectrometry. Chemical species suitable to be analysed via mass spectrometry, i.e. analytes, can be any kind of molecule present in a living organism, include but are not limited to nucleic acid (e.g. DNA, mRNA, miRNA, rRNA etc.), amino acids, peptides, proteins (e.g. cell surface receptor, cytosolic protein etc.), metabolites or hormones (e.g. testosterone, estrogen, estradiol, etc.), fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids, molecules characteristic of a certain modification of another molecule (e.g. sugar moieties or phosphoryl residues on proteins, methyl residues on genomic DNA) or a substance that has been internalized by the organism (e.g. therapeutic drugs, drugs of abuse, toxins, etc.) or a metabolite of such a substance. Such analyte may serve as a biomarker. In the context of present invention, the term “biomarker” refers to a substance within a biological system that is used as an indicator of a biological state of said system. For example, an analyte refers to N-(4- hydroxyphenyl)acetamid. The term "aqueous acid" refers to an acid in an aqueous solution. The term "aqueous hydrochloric acid" refers to an aqueous solution of hydrogen chloride. The term "azo-coupling" refers to an organic reaction that produces an azo compound. The term “azo compound” refers to a derivative of diazene (diimide) HN=NH, in which both hydrogens are substituted for hydrocarbyl groups. It refers to a compound of the general structure R-N=N-R', wherein R and R' may be the same or different and are hydrocarbyl groups. 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. water- methanol mixture as the mobile phase and C18 (octadecylsilyl) 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. 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. The term "comparing" refers to the comparison of a value of interest, in particular an amount of an analyte of interest, with a reference value. In the context of comparing a peak area to a standard curve it may refer to the integral calculated from the peak area, in particular taking into account any internal standard included in the sample, as the value of interest and comparing this value to a previously created standard curve to obtain the real value, e.g. the real amount of analyte of interest in a sample. The comparison may be carried out manually or computer assisted. The value of interest and the reference value can be compared to each other and said comparison can, e.g. be carried out automatically by a computer executing an algorithm for the comparison. In the context of the present invention, the term “compound” refers to a chemical substance having a specific chemical structure. Compounds of the present invention are, for example, N-(4-hydroxyphenyl)acetamid, phenol, 4-aminophenol or 4-((4- nitrophenyl)diazenyl)phenol. The term "composition" may refer to a liquid or solid substance of matter, such as solutions, suspensions or lyophilisates, comprising at least two different substances, for example, solvent and solute. The term "degree of isotopic labelling per atom site" refers to the ratio of the number of molecules with a stable isotope at a specific atom site within the molecular structure compared to the number of molecules with the isotope with the standard atomic weight at the specific atom site within the molecular structure. It usually refers to the molecules present in a composition. For example, phenol according to formula (I) can have a degree of isotopic labelling per atom site at position X2of 98 % when 98 out of 100 molecules in a composition have13C at position X2and 2 out of 100 molecules have12C at position X2. The terms "determine" or “determining” refer to determining the presence or absence of a compound, in particular N-(4-hydroxyphenyl)acetamid. Further, this term refers to determining the amount and / or concentration of a compound, in particular N-(4- hydroxyphenyl)acetamid. Thus, determining as used herein encompasses qualitative as well as quantitative determinations. Quantitative determination includes determining the absolute amount as well as relative amounts, e.g. concentration. The term "diagnostic composition" refers to a composition that can be used for determining the presence, amount or concentration of an analyte of interest, in particular of N-(4-hydroxyphenyl)acetamid, in a sample. The term “fragmentation” refers to the dissociation of a single molecule into two or more separate molecules. As used herein, the term fragmentation refers to a specific fragmentation event, wherein the breaking point in the parent molecule at which the fragmentation event takes place is well defined, and wherein the two or more daughter molecules resulting from the fragmentation event are well characterized. It is well-known to the skilled person how to determine the breaking point of a parent molecule as well as the two or more resulting daughter molecules. The resulting daughter molecules may be stable or may dissociate in subsequent fragmentation events. Exemplified, in case a parent molecule undergoing fragmentation comprises a N-benzylpyridinium unit, the skilled person is able to determine based on the overall structure of the molecule whether the pyridinium unit will fragment to release a benzyl entity or would be released completely from the parent molecule, i.e the resulting daughter molecules would either be a benzyl molecule and a parent molecule lacking of benzyl. Fragmentation may occur via collision-induced dissociation (CID), electron-capture dissociation (ECD), electron-transfer dissociation (ETD), negative electron-transfer dissociation (NETD), electron- detachment dissociation (EDD), photodissociation, particularly infrared multiphoton dissociation (IRMPD) and blackbody infrared radiative dissociation (BIRD), surface-induced dissociation (SID), Higher-energy C-trap dissociation (HCD) or charge remote fragmentation. The term "hydrogenation" refers to a reaction of molecular hydrogen with another compound. For example, with respect to the present invention it refers to a reaction of molecular hydrogen with 4-((4-nitrophenyl)diazenyl)phenol to form 4- aminophenol. The term "internal standard" (ISTD) refers to a known amount of a substance which exhibits similar, preferably identical, 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 upon detection. Exemplified, during chromatographic separation, such as gas or liquid chromatography, the ISTD has about the same elution properties, in particular retention time, as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the 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, 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. Preferably, the internal standard is not naturally occurring in the sample to be analysed. Typically, but not necessarily, the ISTD is a stable isotopically labelled variant (SIL-IS), e.g. comprising2H,13C, or15N etc. label) of the analyte of interest. The term "SIL-IS" refers to stable isotope labelled internal standard. The term "isolated" refers to a characteristic of a molecule or several of the same molecules. The isolated molecule or molecules have been separated from other molecules that differ from the isolated molecule or molecules with respect to their chemical properties or composition, i.e. the atoms in a molecule, in particular a molecule might be isolated, if it has been separated from its structural isomer or stereoisomer, or based on the isotopes present in the molecule. It may refer to a complete or partial separation, i.e. molecules might still be present that differ from the isolated molecule or molecules. The step of “isolating” a molecule refers to the separation of a mixture of molecules, wherein the step is conducted such that the molecule or molecules to be isolated are separated completely or partially. In case a molecule is present in a solution or suspension, it may also include the increase in concentration of the isolated molecule or molecules. Examples for steps of isolating includes crystallization, chromatography, such as size exclusion chromatography, centrifugation, precipitation and filtration. The term "isotopically labelled" refers to a characteristic of a molecule or the same molecules, wherein the molecule or molecules comprise at least one stable isotope. In particular, a molecule is isotopically labelled when one or more atomic sites are enriched with a stable isotope. In particular, it may refer to several molecules, wherein the degree of isotopic labelling per atom site of at least one atom site is higher than 50 %, typically higher than 70 %. 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 methods of the present invention. Typically, a kit may further comprise carrier means being compartmentalized to receive in close confinement one or more container, such as vials, tubes and the like. Kits may further comprise one or more other containers comprising further materials including but not limited to buffers, 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. A 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 on a computer or data processing device. The term “mass Spectrometry” (“Mass Spec” or “MS”) or “mass spectrometric determination“ relates to an analytical technology used to identify and / or quantify compounds by their molecular 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 charged compounds, relating the detected signals to a mass-to-charge ratio and calculating the molecular weight and intensities. 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 negative ions are generated and detected, or in "positive ion mode", wherein positive ions are generated and detected. Determination of an analyte, in particular via mass spectrometry, may include the identification and / or quantification of the analyte. “Tandem mass spectrometry” or “MS / MS” involves multiple steps of mass spectrometry selection, wherein fragmentation of the analyte occurs 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 (MS1). 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, ion- molecule reaction or photodissociation. The resulting ions are then separated and detected in a second stage of mass spectrometry (MS2). 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. 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 electron bombardment or by complex formation with cations / anions, often by protonation to cations / deprotonation to anions. 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. For example, 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 (nebulised) into a jet or spray of very small droplets of solution in solvent vapour. 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 ion- molecule 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 analyser by use of a set of differentially pumped skimmer stages. A counterflow of dry and preheated nitrogen gas may be used to improve removal of solvent. The gas-phase ionization in APCI can be more effective than ESI for analysing less-polar entity. "High-field asymmetric-waveform ion-mobility spectrometry (FAIMS)" is an atmospheric pressure ion mobility technique that separates gas-phase ions by their behaviour in strong and weak electric fields. "Multiple reaction mode" or "MRM" is a detection mode for a MS instrument in which a precursor ion and one or more fragment ions are selectively detected. 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 "regioselectivity" refers to the preference of a chemical reaction for a specific bonding or breaking of a chemical bond in one direction over all other possibilities. For example, within the present invention the term “regioselectivity” may mean that in step i) the azo-coupling leads to the formation of a diazenyl at the para-position of phenol with respect to the hydroxy group, and not to meta or ortho positions. The terms "sample" or "sample of interest" 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. The term "stable isotope" refers to a non-radioactive isotope of an element, in particular an isotope with an atomic mass different from the standard atomic weight, or where applicable the conventional atomic weight, as established by the Commission on Isotopic Abundances and Atomic Weights of the IUPAC (see for example Pure Appl. Chem. 2016, 88, 265; Pure Appl. Chem. 2011, 83, 397). For example,13C is a stable isotope of the element carbon or deuterium is a stable isotope of the element hydrogen. In particular a non-radioactive isotope may refer to an isotope that does not decay into another isotope or element. For example,209Bi was once considered to be the heaviest stable isotope, but has recently been discovered to have a half-life of 1.9 x1019years, while124Xe has the longest so far measured half-life of 1.8x1022years. A stable isotope may thus be considered any isotope with a half-life longer than 1.8x1022years. In particular, a stable isotope may have no or no known half-life at the time of filing. The term "suitable excipient" refers to a chemical substance that is formulated together with a diagnostically relevant substance. A diagnostic relevant substance according to the present invention is a substance that can be used to determine the presence, amount or concentration of an analyte of interest, for example, an isotopically labelled N-(4-hydroxyphenyl)acetamid. Examples of suitable excipients are solvents, such as water, preservatives, such as sodium azide, pH-buffering agents, such as HEPES, or salts, such as sodium chloride. 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. In a first aspect the present invention relates to a method to prepare an isotopically labelled N-(4-hydroxyphenyl)acetamid comprising the following steps i) azo-coupling of an isotopically labelled phenol to4-nitroaniline to form an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol; ii) hydrogenation of the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol according to step i) to form an isotopically labelled 4-aminophenol; and iii) acetylation of the isotopically labelled 4-aminophenol according to step ii) to form the isotopically labelled N-(4-hydroxyphenyl)acetamid. In embodiments of the present invention, the isotopically labelled N-(4- hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with at least one13C, and / or at least one D. Preferably, the isotopically labelled N-(4-hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with at least one13C. The13C labelling has the advantage that an exchange with12C requires breakage of the carbon skeleton structure of the molecule and is therefore more stable compared to labelling with deuterium (D). In case13C is present in the aromatic ring of N-(4- hydroxyphenyl)acetamid the labelling is even more stable compared to labelling with deuterium (D), because the atom bonds within the aromatic ring are more stable. In some embodiments, the isotopically labelled N-(4-hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with at least one D. Deuterium has the advantage that the production of compounds labelled with D is often associated with lower costs. In embodiments of the present invention, the isotopically labelled N-(4- hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three, four, five, six, seven, eight or nine deuterium (D). Preferably, the isotopically labelled N-(4-hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three, four, five or six deuterium (D). Preferably, the isotopically labelled N-(4-hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with three, four, five or six13C. Preferably, the isotopically labelled N-(4-hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with one, two, three or four D. More preferably, the isotopically labelled N-(4-hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with six13C and / or with four D. In embodiments of the present invention, the isotopically labelled N-(4- hydroxyphenyl)acetamid is isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three, four, five, six, seven, eight or nine deuterium (D). Preferably, the isotopically labelled N-(4-hydroxyphenyl)acetamid is isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three or four D. Preferably, the isotopically labelled N-(4-hydroxyphenyl)acetamid is isotopically labelled with six13C and / or with four D. More preferably, the isotopically labelled N-(4-hydroxyphenyl)acetamid is isotopically labelled with six13C. In embodiments of the present invention, the isotopically labelled phenol is isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three, four, five, six, seven, eight or nine deuterium (D). Preferably, the isotopically labelled phenol is isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three, four, five or six D. Preferably, he isotopically labelled phenol is isotopically labelled with six13C, and / or with six D. More preferably, the isotopically labelled phenol is isotopically labelled with six13C. In embodiments of the present invention, the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol is isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three, four, five, six, seven, eight or nine deuterium (D). Preferably, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol is isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three or four D. Preferably, the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol is isotopically labelled with six13C and / or with four D. More preferably, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol is isotopically labelled with six13C. In embodiments of the present invention, the isotopically labelled 4-aminophenol is isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three, four, five, six, seven, eight or nine deuterium (D). Preferably, the isotopically labelled 4-aminophenol is isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three or four D. Preferably, the isotopically labelled 4- aminophenol is isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three or four D. More preferably, the isotopically labelled 4- aminophenol is isotopically labelled with six13C and / or with four D. In embodiments of the present invention, the isotopically labelled N-(4- hydroxyphenyl)acetamid has the formula (IV) the isotopically labelled phenol has the formula (I) the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol has the formula (II) and the isotopically labelled 4-aminophenol has the formula (III) wherein X1, X2, X3, X4, X5and X6are each independently selected from13C and12C; R1, R2, R3, R4, R5and R6are each independently selected from D and H; in each formula (I), (II), (III) and (IV) at least one of X1, X2, X3, X4, X5and X6is13C and / or at least one of R1, R2, R4and R5is D; and in every formula (I), (II), (III) and (IV) each of X1, X2, X3, X4, X5, X6, R1, R2, R4, and R5are selected identically. In embodiments of the present invention, the isotopically labelled N-(4- hydroxyphenyl)acetamid has the formula (IV) the isotopically labelled phenol has the formula (I) the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol has the formula (II) and the isotopically labelled 4-aminophenol has the formula (III) (III); wherein X1, X2, X3, X4, X5and X6are each independently selected from13C and12C; R1, R2, R3, R4, R5and R6are each independently selected from D and H; in each formula (I), (II), (III) and (IV) at least three of X1, X2, X3, X4, X5and X6are13C, and / or at least three of R1, R2, R4and R5are D; and in every formula (I), (II), (III) and (IV) each of X1, X2, X3, X4, X5, X6, R1, R2, R4, and R5are selected identically. In every molecule, N-(4-hydroxyphenyl)acetamid, phenol, 4-((4-nitrophenyl)diazenyl)phenol and 4-aminophenol, the same isotope may be present, because each of phenol, 4-((4-nitrophenyl)diazenyl)phenol and 4- aminophenol may be a precursor molecule of the final product of the presently claimed methods, N-(4-hydroxyphenyl)acetamid. In embodiments of the present invention, X1, X2, X3, X4, X5and X6are13C; and R1, R2, R3, R4, R5and R6are H. In embodiments of the present invention, X1, X2, X3, X4, X5and X6are12C; R1, R2, R3, R4and R5are D; and R6is D or H. In embodiments of the present invention, X1, X2, X3, X4, X5and X6are12C; R1, R2, R3, R4and R5are D; and R6is D. In embodiments of the present invention, the isotopically labelled N-(4- hydroxyphenyl)acetamid has the formula (VIII) In embodiments of the present invention, the isotopically labelled phenol has the formula (V) In embodiments of the present invention, the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol has the formula (VI) In embodiments of the present invention, the isotopically labelled 4-aminophenol has the formula (VII) In embodiments of the present invention, the isotopically labelled N-(4- hydroxyphenyl)acetamid has the formula (XII) In embodiments of the present invention, the isotopically labelled phenol has the formula (IX) wherein each carbon atom represented by * is13C. In embodiments of the present invention, the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol has the formula (X) is13C. In embodiments of the present invention, the isotopically labelled 4-aminophenol has the formula (XI) In embodiments of the present invention, the azo-coupling in step i) is performed at a temperature in the range of -80 °C to 80 °C, -40 °C to 40 °C or -10 °C to 10 °C. Preferably, the azo-coupling in step i) is performed at a temperature in the range of -10 °C to 10 °C. Most preferably, the azo-coupling in step i) is performed at a temperature in the range of -5 °C to 5 °C. In embodiments of the present invention, the azo-coupling in step i) comprises the following steps ia) reacting the 4-nitroaniline with nitrite in the presence of an acid to form a reaction mixture; ib) reacting the reaction mixture according to step ia) with the isotopically labelled phenol to form the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol; and ic) isolating the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol according to step ib). In embodiments of the present invention, the acid in step ia) is an aqueous acid. In embodiments of the present invention, the acid in step ia) is an aqueous hydrochloric acid, tetrafluoroboric acid, perchloric acid, sulphuric acid, methanesulphonic acid or trifluoromethanesulphonic acid. Preferably, the acid in step ia) is aqueous hydrochloric acid. In embodiments of the present invention, the nitrite in step ia) is sodium nitrite. In embodiments of the present invention, the acid is added to a concentration of 0.001 to 10 M in the reaction mixture, preferably the acid is added to a concentration of 0.01 to 2 M in the reaction mixture, most preferably the acid is added to a concentration of 1 to 2 M in the reaction mixture.. In embodiments of the present invention, the pH of the reaction mixture in step ib) is -1 to 4. Preferably, the pH of the reaction mixture in step ib) is 0 to 4. Preferably, the pH of the reaction mixture in step ib) is 0 to 2. In embodiments of the present invention, in step ic) the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol is isolated by filtration. In embodiments of the present invention, the yield of the isolated isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol in step ic) is from 80 to 99 %, preferably 85 to 99 %, more preferably from 95 to 99 %. In embodiments of the present invention, a degree of isotopic labelling per atom site of the isolated isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol in step ic) is the same for X1, X2, X3, X4, X5, X6, R1, R2, R4and R5of the respective isotopically labelled phenol. In embodiments of the present invention, a degree of isotopic labelling per atom site of the isolated isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol in step ic) is from 98 to 100 %. In embodiments of the present invention, in the azo-coupling in step i) 4-nitroaniline couples to the para-position of the isotopically labelled phenol. In embodiments of the present invention, in the azo-coupling in step i) 4-nitroaniline couples to the para-position of the isotopically labelled phenol with a regioselectivity of the coupling to the para-position of 95 to 100 %, preferably 99 to 100 %. In embodiments of the present invention, the hydrogenation in step ii) comprises the following steps iia) reacting the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol according to step i) or step ic) with H2and a Pd / C-based catalyst system in an organic solvent to form the isotopically labelled 4-aminophenol and iib) isolating the isotopically labelled 4-aminophenol according to step iia). In embodiments of the present invention, the organic solvent in step iia) is a linear or cyclic ether, an alcohol, organic acid or ester. Preferably, the organic solvent in step iia) is an alcoholic solvent. In embodiments of the present invention, the organic solvent in step iia) is diethyl ether, tetrahydrofuran, 2-methyl-tetrahydrofuran, ethyl acetate, tertbutyl methyl ether, diisopropyl ether, acetic acid, methanol or mixtures thereof. Preferably, the organic solvent in step iia) is methanol. In embodiments of the present invention, the reacting in step iia) is performed in a continuous flow reactor. Preferably, the system pressure in the continuous flow reactor is 0.5 to 1.5 bar (absolute pressure). In other words the system pressure in the continuous flow reactor is 0.5 × 105to 1.5 × 105Pascal. In embodiments of the present invention, the flow rate is 0.5 to 2.5 mL / min. Preferably, the flow rate is from 1.0 to 1.5 mL / min. In embodiments of the present invention, in step iib) the isotopically labelled 4- aminophenol is isolated by normal phase chromatography. In embodiments of the present invention, the yield of the isolated isotopically labelled 4-aminophenol in step iib) is from 70 to 99 %. Preferably, the yield of the isolated isotopically labelled 4-aminophenol in step iib) is from 74 to 95 %. Most preferably, the yield of the isolated isotopically labelled 4-aminophenol in step iib) is from 74 to 80 %. In embodiments of the present invention, a degree of isotopic labelling per atom site of the isolated isotopically labelled 4-aminophenol in step iib) is the same for X1, X2, X3, X4, X5, X6, R1, R2, R4and R5of the isotopically labelled phenol. In embodiments of the present invention, a degree of isotopic labelling per atom site of the isolated isotopically labelled 4-aminophenol in step iib) is from 98 to 100 %. In embodiments of the present invention, the acetylation in step iii) comprises the following steps iiia) reacting the isotopically labelled 4-aminophenol with acetic acid to form the isotopically labelled N-(4-hydroxyphenyl)acetamid; and iiib) isolating the isotopically labelled N-(4-hydroxyphenyl)acetamid according to step iiia). In embodiments of the present invention, in step iiib) the isotopically labelled N-(4- hydroxyphenyl)acetamid is isolated by normal phase chromatography. In embodiments of the present invention, the yield of the isolated isotopically labelled N-(4-hydroxyphenyl)acetamid in step iiib) is from 70 to 99 %. Preferably, the yield of the isolated isotopically labelled N-(4-hydroxyphenyl)acetamid in step iiib) is from 74 to 80 %. In embodiments of the present invention, a degree of isotopic labelling per atom site of the isolated isotopically labelled N-(4-hydroxyphenyl)acetamid in step iiib) is the same for X1, X2, X3, X4, X5, X6, R1, R2, R4and R5of the isotopically labelled phenol. In embodiments of the present invention, a degree of isotopic labelling per atom site of the isolated isotopically labelled N-(4-hydroxyphenyl)acetamid in step iiib) is from 98 to 100 %. In a second aspect the present invention relates to a method to prepare an isotopically labelled 4-aminophenol comprising the following step ii) hydrogenation of an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol to form the isotopically labelled 4-aminophenol. All embodiments mentioned for the first aspect of the invention apply for the second aspect of the invention and vice versa. In embodiments of the present invention, the method further comprises the following step i) azo-coupling of an isotopically labelled phenol to 4-nitroaniline to form an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol. In a third aspect the present invention relates to a method to prepare an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol comprising the following step i) azo-coupling of an isotopically labelled phenol to 4-nitroaniline to form an isotopically labelled 4-((4-nitrophenyldiazenyl)phenol. All embodiments mentioned for the first 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 a use of an isotopically labelled N- (4-hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable by a method according to the present invention, as an internal standard to determine the presence, amount or concentration of N-(4-hydroxyphenyl)acetamid in a sample. All embodiments mentioned for the first, second and / or third aspect of the invention apply for the fourth aspect of the invention and vice versa. In embodiments of the present invention, the presence, amount or concentration of N-(4-hydroxyphenyl)acetamid in a sample is determined by mass spectrometry. In embodiments of the present invention, the isotopically labelled N-(4- hydroxyphenyl)acetamid has a degree of isotopic labelling per atom site from 98 to 100 %. In embodiments of the present invention, the sample is blood, serum, plasma, synovial fluid, spinal fluid, urine, saliva or lymphatic fluid. In a fifth aspect the present invention relates to a diagnostic composition comprising an isotopically labelled N-(4-hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4-hydroxyphenyl)acetamid obtained or obtainable by a method according to the present invention, and a suitable excipient. All embodiments mentioned for the first, second, third and / or fourth aspect of the invention apply for the fifth aspect of the invention and vice versa. In embodiments of the present invention, the suitable excipient is a solvent, a pH buffering agent, a salt or a combination thereof. In embodiments of the present invention, a degree of isotopic labelling per atom site of the isolated isotopically labelled N-(4-hydroxyphenyl)acetamid is from 98 to 100 %. In a sixth aspect the present invention relates to a method of determining the amount of N-(4-hydroxyphenyl)acetamid in a sample, the method comprising the steps of (a) admixing a known amount of an isotopically labelled N-(4- hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable by a method according to the present invention, to the sample, (b) analysing the sample via mass spectrometry, (c) comparing the peak area of N-(4-hydroxyphenyl)acetamid to a standard curve, thereby determining the amount of N-(4-hydroxyphenyl)acetamid in the sample, wherein said standard curve has been created using an isotopically labelled N-(4- hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable by a method according to the present invention; and standards containing N-(4-hydroxyphenyl)acetamid. All embodiments mentioned for the first, second, third, fourth and / or fifth aspect of the invention apply for the sixth aspect of the invention and vice versa. In embodiments of the present invention, the method of determining the amount of N-(4-hydroxyphenyl)acetamid in a sample is performed in vitro. In embodiments of the present invention, the sample is blood, serum, plasma, synovial fluid, spinal fluid, urine, saliva or lymphatic fluid. In embodiments of the present invention, a degree of isotopic labelling per atom site of the isolated isotopically labelled N-(4-hydroxyphenyl)acetamid is from 98 to 100 %. In embodiments of the present invention the mass spectrometry in step b) is tandem mass spectrometry. In embodiments of the present invention the method further includes a chromatographic step of the sample, preferably a liquid chromatography (LC) step. Preferably the chromatographic step is performed before step b) or after step a) and before step b). In a seventh aspect the present invention relates to a kit for determining the presence, amount or concentration of N-(4-hydroxyphenyl)acetamid in a sample comprising in a container an isotopically labelled N-(4-hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4-hydroxyphenyl)acetamid obtained or obtainable by a method according to the present invention. All embodiments mentioned for the first, second, third, fourth, fifth and / or sixth aspect of the invention apply for the seventh aspect of the invention and vice versa. Suitable container comprising isotopically labelled N-(4-hydroxyphenyl)acetamid, in particular as part of a solid or liquid composition, are known to the skilled person and may be of glass, optionally with a plastic cap, or plastic. In embodiments of the present invention, the kit may comprise several containers comprising isotopically labelled N-(4-hydroxyphenyl)acetamid, in particular at differing amounts and / or concentrations. In embodiments of the present invention, a degree of isotopic labelling per atom site of the isolated isotopically labelled N-(4-hydroxyphenyl)acetamid is from 98 to 100 %. The kit may also comprise further components, such as written instructions or other container comprising different substances or compositions, such as a buffered solution, which can be used to resolubilise a solid composition, such as a lyophilisate. In further embodiments, the present invention relates to the following items: 1. A method to prepare an isotopically labelled N-(4-hydroxyphenyl)acetamid comprising the following steps i) azo-coupling of an isotopically labelled phenol to 4-nitroaniline to form an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol; ii) hydrogenation of the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol according to step i) to form an isotopically labelled 4-aminophenol; and iii) acetylation of the isotopically labelled 4-aminophenol according to step ii) to form the isotopically labelled N-(4-hydroxyphenyl)acetamid. 2. The method according to item 1, wherein the isotopically labelled N-(4- hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with at least one13C and / or at least one D. 3. The method according to item 1 or 2, wherein the isotopically labelled N-(4- hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with one, two, three, four, five or six13C, and / or with one, two, three, four, five, six, seven, eight or nine D. 4. The method according to any one of items 1 to 3, wherein the isotopically labelled N-(4-hydroxyphenyl)acetamid has the formula (IV) the isotopically labelled phenol has the formula (I) the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol has the formula (II) and the isotopically labelled 4-aminophenol has the formula (III) wherein X1, X2, X3, X4, X5and X6are each independently selected from13C and12C; R1, R2, R3, R4, R5and R6are each independently selected from D and H; in each formula (I), (II), (III) and (IV) at least one of X1, X2, X3, X4, X5and X6is13C, and / or at least one of R1, R2, R4and R5is D; and in every formula (I), (II), (III) and (IV) each of X1, X2, X3, X4, X5, X6, R1, R2, R4, and R5are selected identically. according to item 4, wherein X1, X2, X3, X4, X5and X6are13C; and R1, R2, R3, R4, R5and R6are H. according to item 4, wherein X1, X2, X3, X4, X5and X6are12C; R1, R2, R3, R4and R5are D; and R6is D or H. according to item 4, wherein X1, X2, X3, X4, X5and X6are12C; R1, R2, R3, R4and R5are D; and R6is D. 8. The method according to any one of items 1 to 6, wherein the azo-coupling in step i) is performed at a temperature in the range of -80 °C to 80 °C, preferably at a temperature in the range of -10 °C to 10 °C. 9. The method according to any one of items 1 to 8, wherein the azo-coupling in step i) comprises the following steps ia) reacting the 4-nitroaniline with nitrite in the presence of an acid to form a reaction mixture; ib) reacting the reaction mixture according to step ia) with the isotopically labelled phenol to form the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol; and ic) isolating the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol according to step ib). 10. The method according to item 9, wherein the acid is an aqueous hydrochloric acid. 11. The method according to item 9 or 10, wherein the acid is a 0.001 to 10 M aqueous hydrochloric acid. 12. The method according to any one of items 9 to 11, wherein the pH of the reaction mixture in step ib) is -1 to 4, preferably 0 to 2. 13. The method according to any one of items 9 to 12, wherein in step ic) the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol is isolated by filtration. 14. The method according to any one of items 9 to 13, wherein the yield of the isolated isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol in step ic) is from 80 to 99 %, preferably 85 to 99 %, preferably from 95 to 99 %. 15. The method according to any one of items 9 to 14, wherein a degree of isotopic labelling per atom site of the isolated isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol in step ic) is the same for X1, X2, X3, X4, X5, X6, R1, R2, R4and R5of the respective isotopically labelled phenol. 16. The method according to any one of items 9 to 15, wherein a degree of isotopic labelling per atom site of the isolated isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol in step ic) is from 98 to 100 %. 17. The method according to any one of items 1 to 16, wherein in the azo-coupling in step i) 4-nitroaniline couples to the para-position of the isotopically labelled phenol. 18. The method according to item 17, wherein in the azo-coupling in step i) 4- nitroaniline couples to the para-position of the isotopically labelled phenol with a regioselectivity of the coupling to the para-position of 95 to 100 %, preferably 99 to 100 %. 19. The method according to any one of items 1 to 18, wherein the hydrogenation in step ii) comprises the following steps iia) reacting the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol according to step i) or step ic) with H2 and a Pd / C-based catalyst system in an organic solvent to form the isotopically labelled 4-aminophenol and iib) isolating the isotopically labelled 4-aminophenol according to step iia). 20. The method according to item 19, wherein the organic solvent is methanol. 21. The method according to item 19 or 20, wherein the reacting in step iia) is performed in a continuous flow reactor. 22. The method according to item 21, wherein the system pressure in the continuous flow reactor is 0.5 to 1.5 bar (absolute pressure).0.5 to 1.5 bar corresponds to 0.5 × 105to 1.5 × 105Pa. 23. The method according to item 21 or 22, wherein the flow rate is 0.5 to 2.5 mL / min, preferably from 1.0 to 1.5 mL / min. 24. The method according to any one of items 19 to 23, wherein in step iib) the isotopically labelled 4-aminophenol is isolated by normal phase chromatography. 25. The method according to any one of items 19 to 24, wherein the yield of the isolated isotopically labelled 4-aminophenol in step iib) is from 70 to 99 %, preferably 70 to 95 %, most preferably from 74 to 80 %. 26. The method according to any one of items 19 to 25, wherein a degree of isotopic labelling per atom site of the isolated isotopically labelled 4-aminophenol in step iib) is the same for X1, X2, X3, X4, X5, X6, R1, R2, R4and R5of the isotopically labelled phenol. 27. The method according to any one of items 19 to 26, wherein a degree of isotopic labelling per atom site of the isolated isotopically labelled 4-aminophenol in step iib) is from 98 to 100 %. 28. The method according to any one of items 1 to 27, wherein the acetylation in step iii) comprises the following steps iiia) reacting the isotopically labelled 4-aminophenol with acetic acid to form the isotopically labelled N-(4-hydroxyphenyl)acetamid; and iiib) isolating the isotopically labelled N-(4-hydroxyphenyl)acetamid according to step iiia). 29. The method according to item 28, wherein in step iiib) the isotopically labelled N-(4-hydroxyphenyl)acetamid is isolated by normal phase chromatography. 30. The method according to item 28 or 29, wherein the yield of the isolated isotopically labelled N-(4-hydroxyphenyl)acetamid in step iiib) is from 70 to 99 %, preferably from 74 to 80 %. 31. The method according to any one of items 28 to 30, wherein a degree of isotopic labelling per atom site of the isolated isotopically labelled N-(4- hydroxyphenyl)acetamid in step iiib) is the same for X1, X2, X3, X4, X5, X6, R1, R2, R4and R5of the isotopically labelled phenol. 32. The method according to any one of items 28 to 31, wherein a degree of isotopic labelling per atom site of the isolated isotopically labelled N-(4- hydroxyphenyl)acetamid in step iiib) is from 98 to 100 %. 33. A method to prepare an isotopically labelled 4-aminophenol comprising the following step ii) hydrogenation of an isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol to form the isotopically labelled 4- aminophenol. 34. The method according to item 33, wherein the method further comprises the following step i) azo-coupling of an isotopically labelled phenol to 4-nitroaniline to form an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol. 35. A method to prepare an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol comprising the following step i) azo-coupling of an isotopically labelled phenol to 4-nitroaniline to form an isotopically labelled 4-((4-nitrophenyldiazenyl)phenol. 36. Use of an isotopically labelled N-(4-hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4-hydroxyphenyl)acetamid obtained or obtainable by a method according to any one of items 1 to 32, as an internal standard to determine the presence, amount or concentration of N-(4-hydroxyphenyl)acetamid in a sample. 37. The use according to item 36, wherein the presence, amount or concentration of N-(4-hydroxyphenyl)acetamid in a sample is determined by mass spectrometry. 38. The use according to item 36 or 37, wherein the isotopically labelled N-(4- hydroxyphenyl)acetamid has a degree of isotopic labelling per atom site from 98 to 100 %. 39. The use of any one of items 36 to 38 or the method of item 40, wherein the sample is blood, serum, plasma, synovial fluid, spinal fluid, urine, saliva or lymphatic fluid. 40. A diagnostic composition comprising an isotopically labelled N-(4- hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable by a method according to any one of items 1 to 32, and a suitable excipient. 41. The diagnostic composition according to item 40, wherein the isotopically labelled N-(4-hydroxyphenyl)acetamid has a degree of isotopic labelling per atom site from 98 to 100 %. 42. A method of determining the amount of N-(4-hydroxyphenyl)acetamid in a sample, the method comprising the steps of (a) admixing a known amount of an isotopically labelled N-(4- hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable by a method according to any one of items 1 to 32, to the sample, (b) analysing the sample via mass spectrometry, preferably tandem mass spectrometry, (c) comparing the peak area of N-(4-hydroxyphenyl)acetamid to a standard curve, thereby determining the amount of N-(4- hydroxyphenyl)acetamid in the sample, wherein said standard curve has been created using an isotopically labelled N-(4-hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4-hydroxyphenyl)acetamid obtained or obtainable by a method according to any one of items 1 to 32; and standards containing N-(4-hydroxyphenyl)acetamid. 43. The method according to item 42, wherein the isotopically labelled N-(4- hydroxyphenyl)acetamid has a degree of isotopic labelling per atom site from 98 to 100 %. 44. The method according to item 42 or 43, further including a chromatographic step of the sample, preferably a liquid chromatography (LC) step. 45. The method according to item 44, wherein the chromatographic step is performed before step b) or after step a) and before step b). 46. A kit for determining the presence, amount or concentration of N-(4- hydroxyphenyl)acetamid in a sample comprising in a container an isotopically labelled N-(4-hydroxyphenyl)acetamid, in particular an isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable by a method according to any one of items 1 to 32. 47. A kit according to item 46, wherein the isotopically labelled N-(4- hydroxyphenyl)acetamid has a degree of isotopic labelling per atom site from 98 to 100 %.

[0002] Examples The following examples are provided to illustrate, but not to limit the presently claimed invention. All reactions were magnetically stirred and, unless otherwise noted, carried out under a positive pressure of argon utilizing standard Schlenk-techniques. Glassware was dried at 650 °C in vacuo prior to use. Liquid reagents and solvents were added by syringes through rubber septa. Solids were added under inert gas counter flow or were dissolved in appropriate solvents. Low temperature reactions were carried out in a Dewar vessel filled with a cooling agent: acetone / dry ice (−78 °C), NaCl / ice (−20 °C) or H2O / ice (0 °C). Reaction temperatures above room temperature were conducted in a heated oil bath. Yields refer to isolated homogenous and spectroscopically pure materials, if not indicated otherwise. In reaction schemes, a “*” indicates a13C-labelled site. Solvents and Reagents Diethyl ether (Et2O) was distilled under reduced pressure prior to use. Solvents for extraction, crystallization and flash column chromatography were purchased in LiChrosolv®hypergrade from Merck KGaA.13C- and Deuterium-enriched compounds were purchased from Cambridge Isotope Laboratories. All other reagents and solvents were purchased from chemical suppliers (Sigma- Aldrich / Merck KGaA, Acros Organics, Honeywell / Fluka) and were used as received. Chromatography Qualitative thin-layer chromatography (TLC) on silica gel 60 F254 TLC plates from Merck KGaA was used to monitor reactions and preparative chromatography. Analytes on the plates were visualised by irradiation with UV-light (245 nm) and / or staining with an appropriate staining solution. The plate was immersed in the staining solution and then heated with a hot-air gun (350 °C). The following staining solution was applied: (3.0 g KMnO4, 20 g K2CO3, 5.0 mL aqueous 5 % NaOH, 300 mL H2O). Experimental flash column chromatography was performed on Geduran®Si6060 (40 – 63 μm) silica gel from Merck KGaA. All fractions containing a desired substrate were combined and solvents were removed under reduced pressure followed by drying in high vacuo (10– 2mbar) for non volatile substances.10– 2mbar corresponds to 1 Pa. NMR spectroscopy NMR spectra were recorded on an Agilent 400-MR DD2400 MHz spectrometer equipped with an OneNMR Probe operating at 400 MHz for proton nuclei and at 101 MHz for carbon nuclei. The chemical shifts δ of the NMR spectra are reported in ppm relative to the shift of the standard TMS (Tetramethylsilane). NMR shifts are calibrated to the residual solvent resonances of CDCl3(7.26 ppm for1H-NMR and 77.16 ppm for13C-NMR). Spectroscopic data is reported as follows: Chemical shift in ppm (multiplicity, coupling constants J in Hz, integration intensity). In the report of spectroscopic data, the multiplicity of signals is abbreviated with s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and mc (centrosymmetric multiplet). In case of combined multiplicities, the multiplicity with the larger coupling constant is stated first. With exception of the multiplets, the reported chemical shifts of the signal corresponds to the center of the resonance range. The1J13CHcoupling constants exceed any other JHH coupling constants and are therefore reported in the cases of centrosymmetric multiplet. Additionally to1H- and13C-NMR measurements, 2D NMR techniques like homonuclear correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond coherence (HMBC) were used to assign signals. All NMR spectra were analyzed using the program ACD / Spectrus Processor 2015.2.7 from Advanced Chemistry Development, Inc. High Performance Liquid Chromatography (HPLC) HPLC was carried out using HPLC grade solvents and deionized, ultra-filtered water. All separations were conducted at room temperature. Analytical UV-Vis spectra were recorded on a 1260 Infinity HPLC system from Agilent Technologies Inc. that was computer-controlled through ChromeleonTMChromatography Data Systems Software Version 7.2 SR5 Mui (24070) from Thermo Fisher Scientific Inc., using an ACQUITY UPLC, Oligonucleotide 130A, 1.7 μm, BEH C18 column from Waters Corporation, detecting at 265 nm wavelength. LCMS Analysis The determination of the content of non-isotopically labelled acetaminophen (absolute isotopic purity) is verified using high-resolution Liquid Chromatography Mass Spectrometry (LCMS). For this purpose, the sample is dissolved in ethanol, potential interfering impurities are separated via Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC), and detected using high-resolution Mass Spectrometry, ESI-qToF-MS (Resolution >20000) (Waters Synapt-G2Si). The verification of the content of non-isotopically labeled acetaminophen (≤ 0.20 %) is conducted through a threshold test (comparison of sample vs. sample + threshold amount of non-isotopically labelled analyte). Example 1: Synthesis of Acetaminophen-D4Synthesis Azobenzene SI-3 Nitroaniline (SI-1, 2.07 g, 15.0 mmol, 1.0 eq.) was suspended in destilled (dest.) water (15.0 mL) at room temperature, then diluted with concentrated hydrochloric acid (3.70 mL) and subsequently cooled to −5 °C. To this, a second solution, prepared beforehand from sodium nitrite (1.03 g, 15.0 mmol.1.0 eq.) and dest. water (15.0 mL), was added dropwise over the course of 10 minutes. The resulting mixture was stirred for one hour at −5 °C. Then, a solution of Phenol-D6 (SI-2, 1.50 g, 15.0 mmol, 1.0 eq.) and sodium hydroxide (1.20 g, 30.0 mmol, 2.0 eq.) in dest. water (25.0 mL) was added dropwise over the course of 10 minutes, upon which the desired product precipitated and formed a thick, orange suspension. The reaction mixture was brought to pH = 1-2 via the addition of hydrochloric acid (aq., 3 m, 12.5 mL) and left stirring for another hour. Filtration of the precipitate (thorough washings with ice-cold dest. water) and subsequent drying in vacuo yielded azobenzene SI-3 (3.56 g, 14.4 mmol, 96 %) as yellow solid. Rf(n-Hex:Ethyl Acetate= 1:4) = 0.881H NMR (DMSO-D6, 400 MHz): δ = 10.58 (s, br, 1H), 8.39 (d, J = 9.4 Hz, 2H), 7.98 (d, J = 9.4 Hz, 2H) ppm13C NMR (DMSO-D6,100 MHz): δ = 162.2, 155.5, 147.7, 145.3, 125.5 (t, J13CD = 24.4 Hz, 2C), 125.0, 123.0, 115.9 (t, J13CD= 24.4 Hz, 2C) ppm ESI-LRMS for C12H4D4N3O3−calcd. 246.2 [M−H−]: found 246.3 The synthesis of azobenzene SI-3 led to a high yield and high regioselectivity as no by-products, such as 3-((4-nitrophenyl)diazenyl)phenol, were detected via1H and13C NMR. The high regioselectivity at this early step in the synthesis leads to the same regioselectivity in subsequent steps, due to the reactive group (nitrophenyl) introduced at this step. The deuterium of the acidic hydroxyl group of Phenol-D6 was exchanged for a proton during the procedure, which is inconsequential for the isotopic labeling of the arylic positions of SI-3. Phenol-D6was chosen over Phenol- D5as a starting material due to its commercial availability. Synthesis 4-Aminophenol-D4 (SI-4) Azobenzene SI-3 (3.57 g, 14.4 mmol, 1.0 eq.) was dissolved in Methanol (150 mL) under inert gas atmosphere at room temperature. To this, palladium on charcoal (1.54 g, 1.14 mmol Pd, 10 wt-% Pd, 0.1 eq.) was added and the atmosphere was exchanged for hydrogen (1 atm) under vigorous stirring. After three hours control via TLC showed complete consumption of SI-3 and the reaction was subsequently stopped via exchanging back the atmosphere to inert gas. The reaction mixture was filtered over a plug of Celite®(washings with MeOH) and the eluted solution was concentrated in vacuo. Purification via flash column chromatography (n-Hex / EtOAc = 7:3 to 3:7) yielded 4-aminophenol-D4(SI-4, 1.26 g, 11.5 mmol, 80 %) as colourless solid. Rf(n-Hex:Ethyl Acetate= 1:4) = 0.601H NMR (DMSO-D6, 400 MHz): δ = 8.30 (s, 1H), 6.64–6.35 (residual ArH), 4.34 (br, s, 2H) ppm13C NMR (DMSO-D6,100 MHz): δ = 148.1, 140.5, 115.1 (t, J13CD = 23.8 Hz, 2C), 114.8 23.8 Hz, 2C) ppm ESI-LRMS for C6H4D4NO3+calcd. 114.1 [MH+]: found 114.1 Synthesis Acetaminophen-D4(SI-5) 4-Aminophenol-D4 (SI-4, 1.26 g, 11.2 mmol, 1.0 eq.) was dissolved in dry tetrahydrofuran (32.0 mL) under inert gas atmosphere at room temperature. To this, a beforehand prepared solution, made of carbonyldiimidazole (2.05 g, 12.6 mmol, 1.1 eq.) and acetic acid (0.74 mL, 12.9 mmol, 1.2 eq.) in dry tetrahydrofuran (28.0 mL), was added slowly over the course of 15 minutes. The reaction mixture was stirred for five hours until control via TLC indicated full conversion of SI-4. The reaction was quenched via the addition of water (0.50 mL). After 10 minutes the mixture was concentrated in vacuo. The crude solid was redissolved in ethyl acetate (3.0 mL) and purified via flash column chromatography (n-Hex / EtOAc = 7:3 to 0:1). Acetaminophen-D4(SI-5, 1.34 g, 8.96 mmol, 80 %) was obtained as colourless solid after drying in vacuo. Rf(n-Hex:Ethyl Acetate= 1:4) = 0.511H NMR (DMSO-D6, 500 MHz): δ = 9.63 (br, s, 1H), 9.10 (br, s, 1H), 7.32–6.46 (residual ArH), 1.93 (s, 3H) ppm13C NMR (DMSO-D6, 125 MHz): δ = 167.4, 152.9, 130.8, 120.5–120.1 (m, 2C), 114.7–114.3 (m, 2C), 23.6 ppm ESI-LRMS for C8H4D4NO2−calcd. 154.1 [M−H−]: found 154.3 Calculation of absolute isotopic purity according to a threshold in an HR-LCMS experiment, showing the stability of deuterated positions in Acetaminophen-D4(SI- 5). Raw data is shown in Table 1 and FIG.1 to FIG.5. In a first injection, the sample of Acetaminophen-D4 (SI-5) is measured regarding the ion count of the isotopically unlabelled molecule (Table 2, A). In a second measurement, a threshold amount of isotopically unlabelled Acetaminophen is spiked to the first sample and measured again regarding the ion count of the isotopically unlabelled molecule (Table 2, D). Both values are then set in relation so that a value of ≤ 1.0 indicates that the sample of Acetaminophen-D4(SI-5) does not exceed the threshold content of the isotopically unlabelled molecule (here the value lies at ≤ 0.20 mol-%). Table 1 Measured mass intensities of a sample of Acetaminophen-D4 (SI-5) synthesized according to the invention (left row) compared to a spiked sample of isotopically unlabelled Acetaminophen (right row) in HR- LCMS Sample spiked sample Ion Mass Ion count Mass count 152.0033 30 152.0033 70 152.0087 0 152.0087 101 152.0141 0 152.0141 77 152.0195 61 152.0195 15 152.0248 80 152.0248 84 152.0302 389 152.0302 235 152.0356 371 152.0356 331 152.0410 109 152.0410 147 152.0464 55 152.0464 230 152.0517 313 152.0517 752 152.0571 2907 152.0571 11090 152.0625 17070 152.0625 93750 152.0679 43980 152.0679 284300 152.0733 45020 152.0733 320200 152.0786 14350 152.0786 104000 152.0840 1391 152.0840 11430 152.0894 751 152.0894 2182 152.0948 618 152.0948 1271 152.1002 117 152.1002 1060 Table 2 A D Evaluation of isotopic purity ≤ 1.0 Threshold complied with > 1.0 or < 0 Threshold not complied with Ion count of Ion count of isotopically isotopically unlabelled unlabelled A / (D−A) = compound in compound the sample spiked sample 103350 708500 0.2 Example 2: Synthesis of Acetaminophen-13C6 Synthesis Azobenzene SI-7 Nitroaniline (SI-1, 2.07 g, 15.0 mmol, 1.0 eq.) was suspended in dest. water (15.0 mL) at room temperature, then diluted with concentrated hydrochloric acid (3.70 mL) and subsequently cooled to −5 °C. To this, a second solution, prepared beforehand from sodium nitrite (1.03 g, 15.0 mmol. 1.0 eq.) and dest. water (15.0 mL), was added dropwise over the course of 10 minutes. The resulting mixture was stirred for one hour at −5 °C. Then, a solution of Phenol-13C6(SI-6, 1.50 g, 15.0 mmol, 1.0 eq.) and sodium hydroxide (1.20 g, 30.0 mmol, 2.0 eq.) in dest. water (25.0 mL) was added dropwise over the course of 10 minutes, upon which the desired product precipitated and formed a thick, orange suspension. The reaction mixture was now brought to pH = 1-2 via the addition of hydrochloric acid (aq., 3 m, 12.5 mL) and left stirring for another hour. Filtration of the precipitate (thorough washings with ice-cold dest. water) and subsequent drying in vacuo yielded azobenzene SI-7 (3.56 g, 14.4 mmol, 96 %) as yellow solid. Rf(n-Hex:Ethyl Acetate= 1:4) = 0.881H NMR (DMSO-D6, 400 MHz): δ = 10.60 (s, br, 1H), 8.42–8.38 (m, 2H), 8.12– 8.05 (m, 1H), 8.01–7.98 (m, 2H), 7.71–7.65 (m, 1H), 7.21–7.15 (m, 1H), 6.81–6.75 (m, 1H) ppm13C NMR (CDCl3, 100 MHz): δ = 162.3 (dt, J13C13C= 63.83, 8.80 Hz, 1C), 159.5 (d, J13C13C= 14.67 Hz), 147.7 ppm, 145.4 (dt, J13C13C= 64.56, 8.07 Hz, 1C), 126.4–125.1 (m, 2C), 125.0, 123.0, 116.8–115.6 (m, 2C) ppm ESI-LRMS for13C6C6H9N3O3−calcd. 249.1 found 249.1 The synthesis of azobenzene SI-7 led to a high yield and high regioselectivity as no by-products, such as 3-((4-nitrophenyl)diazenyl)phenol, were detected via NMR.

[0003] Synthesis 4-Aminophenol-13C6 (SI-8) Azobenzene SI-7 (3.57 g, 14.4 mmol, 1.0 eq.) was dissolved in Methanol (150 mL) under inert gas atmosphere at room temperature. To this, palladium on charcoal (1.54 g, 1.14 mmol Pd, 10 wt-% Pd, 0.1 eq.) was added and the atmosphere was exchanged for hydrogen (1 atm) under vigorous stirring. After three hours control via TLC showed complete consumption of SI-7 and the reaction was subsequently stopped via exchanging back the atmosphere to inert gas. The reaction mixture was filtered over a plug of Celite®(washings with MeOH) and the eluted solution was concentrated in vacuo. Purification via flash column chromatography (n-Hex / EtOAc = 7:3 to 3:7) yielded 4-aminophenol-13C6 (SI-8, 1.26 g, 11.5 mmol, 80 %) as colourless solid. Rf(n-Hex:Ethyl Acetate= 1:4) = 0.601H NMR (DMSO-D6, 400 MHz): δ = 8.31 (s, br, 1H), 6.72–6.43 (m, 2H), 642–6.11 (m, 2H), 4.35 (s, br, 2H) ppm13C NMR (CDCl3, 100 MHz): δ = 149.4–146.9 (m, 1C), 141.8–139.6 (m, 1C), 116.3–114.3 (m, 4C) ppm ESI-LRMS for13C6H8NO+calcd. 116.1 [MH+]: found 116.3 Synthesis Acetaminophen-13C6 (SI-9) 4-Aminophenol-D4 (SI-8, 1.26 g, 11.2 mmol, 1.0 eq.) was dissolved in dry tetrahydrofuran (32.0 mL) under inert gas atmosphere at room temperature. To this, a beforehand prepared solution, made of carbonyldiimidazole (2.05 g, 12.6 mmol, 1.1 eq.) and acetic acid (0.74 mL, 12.9 mmol, 1.2 eq.) in dry tetrahydrofuran (28.0 mL), was added slowly over the course of 15 minutes. The reaction mixture was stirred for five hours until control via TLC indicated full conversion of SI-8. The reaction was quenched via the addition of water (0.50 mL). After 10 minutes the mixture was concentrated in vacuo. The crude solid was redissolved in ethyl acetate (3.0 mL) and purified via flash column chromatography (n-Hex / EtOAc = 7:3 to 0:1). Acetaminophen-13C6 (SI-9, 1.34 g, 8.96 mmol, 80 %) was obtained as colourless solid after drying in vacuo. Rf(n-Hex:Ethyl Acetate= 1:4) = 0.511H NMR (DMSO-D6, 400 MHz): δ = 9.62 (br, s, 1H), 9.11 (br, s, 1H), 7.33 (dm, J13CH = 163 Hz, 2H), 6.67 (dm, J13CH = 160 Hz, 2H), 1.97 (s, 3H) ppm13C NMR (DMSO-D6, 125 MHz): δ = 167.6, 153.1 (td, J = 66.4, 10.3 Hz, 1C), 131.6–130.5 (m, 1C), 121.4–120.2 (m, 2C), 115.5–114.4 (m, 2C), 23.8 ppm ESI-LRMS for13C6C2H8NO2−calcd. 156.1 [M−H−]: found 156.1

Claims

Claims 1. A method to prepare an isotopically labelled N-(4-hydroxyphenyl)acetamid comprising the following steps i) azo-coupling of an isotopically labelled phenol to 4-nitroaniline to form an isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol; ii) hydrogenation of the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol according to step i) to form an isotopically labelled 4-aminophenol; and iii) acetylation of the isotopically labelled 4-aminophenol according to step ii) to form the isotopically labelled N-(4-hydroxyphenyl)acetamid.

2. The method according to claim 1, wherein the isotopically labelled N-(4- hydroxyphenyl)acetamid, the isotopically labelled phenol, the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol and the isotopically labelled 4-aminophenol are isotopically labelled with at least one13C and / or at least one D.

3. The method according to claim 1 or 2, wherein the isotopically labelled N-(4- hydroxyphenyl)acetamid has the formula (IV)the isotopically labelled phenol has the formula (I)the isotopically labelled 4-((4-nitrophenyl)diazenyl)phenol has the formula (II)and the isotopically labelled 4-aminophenol has the formula (III)wherein X1, X2, X3, X4, X5and X6are each independently selected from13C and12C; R1, R2, R3, R4, R5and R6are each independently selected from D and H; in each formula (I), (II), (III) and (IV) at least one of X1, X2, X3, X4, X5and X6is13C, and / or at least one of R1, R2, R4and R5is D; and in every formula (I), (II), (III) and (IV) each of X1, X2, X3, X4, X5, X6, R1, R2, R4, and R5are selected identically.

4. The method according to any one of claims 1 to 3, wherein the azo-coupling in step i) comprises the following steps ia) reacting the 4-nitroaniline with nitrite in the presence of an acid to form a reaction mixture; ib) reacting the reaction mixture according to step ia) with the isotopically labelled phenol to form the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol; and ic) isolating the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol according to step ib).

5. The method according to claim 4, wherein in the azo-coupling in step i) 4- nitroaniline couples to the para-position of the isotopically labelled phenol with a regioselectivity of the coupling to the para-position of 95 to 100 %.

6. The method according to claim 4 or 5, wherein the acid is an aqueous hydrochloric acid.

7. The method according to any one of claims 1 to 6, wherein the hydrogenation in step ii) comprises the following steps iia) reacting the isotopically labelled 4-((4- nitrophenyl)diazenyl)phenol according to step i) or step ic) with H2 and a Pd / C-based catalyst system in an organic solvent to form the isotopically labelled 4-aminophenol; and iib) isolating the isotopically labelled 4-aminophenol according to step iia).

8. The method according to claim 7, wherein the organic solvent is methanol.

9. The method according to any one of claims 1 to 8, wherein the acetylation in step iii) comprises the following steps iiia) reacting the isotopically labelled 4-aminophenol with acetic acid to form the isotopically labelled N-(4-hydroxyphenyl)acetamid; and iiib) isolating the isotopically labelled N-(4-hydroxyphenyl)acetamid according to step iiia).

10. The method according to claim 9, wherein the yield of the isolated isotopically labelled N-(4-hydroxyphenyl)acetamid in step iiib) is from 70 to 99 %.

11. The method according to claim 9 or 10, wherein a degree of isotopic labelling per atom site of the isolated isotopically labelled N-(4-hydroxyphenyl)acetamid in step iiib) is the same for X1, X2, X3, X4, X5, X6, R1, R2, R4and R5of the isotopically labelled phenol.

12. The method according to any one of claims 9 to 11, wherein a degree of isotopic labelling per atom site of the isolated isotopically labelled N-(4- hydroxyphenyl)acetamid in step iiib) is from 98 to 100 %.

13. Use of an isotopically labelled N-(4-hydroxyphenyl)acetamid obtained or obtainable by a method according to any one of claims 1 to 12 as an internal standard to determine the presence, amount or concentration of N-(4- hydroxyphenyl)acetamid in a sample.

14. The use according to claim 13, wherein the isotopically labelled N-(4- hydroxyphenyl)acetamid has a degree of isotopic labelling per atom site from 98 to 100 %.

15. A method of determining the amount of N-(4-hydroxyphenyl)acetamid in a sample, the method comprising the steps of (a) admixing a known amount of an isotopically labelled N-(4- hydroxyphenyl)acetamid obtained or obtainable by a method according to any one of claims 1 to 12, to the sample, (b) analysing the sample via mass spectrometry, (c) comparing the peak area of N-(4-hydroxyphenyl)acetamid to a standard curve, thereby determining the amount of N-(4- hydroxyphenyl)acetamid in the sample, wherein said standard curve has been created using an isotopically labelled N-(4-hydroxyphenyl)acetamid obtained or obtainable by a method according to any one of claims 1 to 12; and standards containing N-(4-hydroxyphenyl)acetamid.

16. The method according to claim 15, wherein the isotopically labelled N-(4- hydroxyphenyl)acetamid has a degree of isotopic labelling per atom site from 98 to 100 %.

17. A kit for determining the presence, amount or concentration of N-(4- hydroxyphenyl)acetamid in a sample comprising in a container an isotopically labelled N-(4-hydroxyphenyl)acetamid obtained or obtainable by a method according to any one of claims 1 to 12.

18. The kit according to claim 17, wherein the isotopically labelled N-(4- hydroxyphenyl)acetamid has a degree of isotopic labelling per atom site from 98 to 100 %.

Images