WO2023131594A1 - Dérivatisation de composés dans des échantillons de patients pour la pharmacovigilance thérapeutique (tdm) - Google Patents

Dérivatisation de composés dans des échantillons de patients pour la pharmacovigilance thérapeutique (tdm) Download PDF

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Publication number
WO2023131594A1
WO2023131594A1 PCT/EP2023/050039 EP2023050039W WO2023131594A1 WO 2023131594 A1 WO2023131594 A1 WO 2023131594A1 EP 2023050039 W EP2023050039 W EP 2023050039W WO 2023131594 A1 WO2023131594 A1 WO 2023131594A1
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Prior art keywords
analyte
interest
sample
tdm
derivatized
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PCT/EP2023/050039
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English (en)
Inventor
Patrick NEIENS
Tobias Stueckl
Gaston Hubertus Maria VONDENHOFF
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F. Hoffmann-La Roche Ag
Roche Diagnostics Gmbh
Roche Diagnostics Operations, Inc.
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Publication of WO2023131594A1 publication Critical patent/WO2023131594A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9473—Anticonvulsants, e.g. phenobarbitol, phenytoin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry

Definitions

  • TDM therapeutic drug monitoring
  • the present invention relates to a method of determining the level of at least one analyte of interest, sampling tubes for collecting a patient sample, the use of nucleophilic derivatization reagents, an analyzer as well as kits.
  • TDM Therapeutic Drug Monitoring
  • Carbamazepine is an example of the classical antiepileptic drugs, chemically related to the Tricyclic Antidepressants. There are different methods to detect Carbamazepine in a patient sample, e.g. plasma. One possibility is to detect Carbamazepine by Therapeutic Drug monitoring (TDM).
  • TDM Therapeutic Drug monitoring
  • Carbamazepine-10,11- epoxide [I] is an active metabolite of the important antiepileptic drug carbamazepine [II] (see Figure 1). Both analytes and its metabolite should require therapeutic drug monitoring (TDM).
  • TDM Therapeutic Drug monitoring
  • both compounds are quantified mostly by LC-MS/MS, whereby the intact compounds provide the precursor mass, or QI m/z.
  • To quantitate [I] and [II] via LC-MS/MS technology for TDM isotopically labeled internal standards as well as calibration materials that are made up out of these compounds are required.
  • TDM therapeutic drug monitoring
  • the present invention relates to a method of determining the level of at least one analyte of interest in an obtained sample comprising the steps of: a) Providing the sample comprising an analyte of interest, wherein the analyte of interest comprises an epoxide moiety, wherein the analyte of interest is a therapeutic drug monitoring (TDM) compound, b) Derivatization the analyte of interest with a nucleophilic derivatization reagent, preferably by coupling the analyte of interest and a nucleophilic derivatization reagent via the epoxide moiety, and c) Determining the level of the at least one analyte of interest in the sample, in particular using immunological assay or Mass Spectrometry, preferably LC/MS, preferably a method of determining the level of at least one analyte of interest in an obtained sample comprising the steps of: a) Providing the sample comprising TDM (TDM
  • the present invention relates to a sampling tube for collecting a patient sample comprising a nucleophilic derivatization reagent suitable to stabilize one or more therapeutic drug monitoring (TDM) compounds in a sample.
  • a sampling tube for collecting a patient sample comprising a device with a reservoir adapted for receiving a blood sample to be collected, and a nucleophilic derivatization reagent suitable to stabilize one or more therapeutic drug monitoring (TDM) compounds in a sample.
  • the present invention relates to the use of a nucleophilic derivatization reagent for determining the level of at least one analyte of interest, preferably Carbamazepine-10,11 -epoxide, preferably the nucleophilic derivatization reagent is used to stabilize Carbamazepine- 10, 11 -epoxide as an analyte of interest in a sample.
  • a nucleophilic derivatization reagent for determining the level of at least one analyte of interest, preferably Carbamazepine-10,11 -epoxide, preferably the nucleophilic derivatization reagent is used to stabilize Carbamazepine- 10, 11 -epoxide as an analyte of interest in a sample.
  • the present invention relates to the use of a nucleophilic derivatization reagent to stabilize an analyte of interest in a sample, preferably Carbamazepine- 10,11 -epoxide.
  • the present invention relates to the use of one or more derivatized therapeutic drug monitoring (TDM) compounds in a sample as an ISTD and/or calibrator for Mass Spectrometry measurements.
  • TDM derivatized therapeutic drug monitoring
  • the present invention relates to the use of one or more analytes of interest, wherein the analytes of interest are derivatized therapeutic drug monitoring (TDM) compounds in a sample as an ISTD and/or calibrator for Mass Spectrometry measurements.
  • the present invention relates to a kit comprising a derivatized therapeutic drug monitoring (TDM) compound, preferably wherein the derivatized therapeutic drug monitoring (TDM) compound is Carbamazepine- 10, 11 -epoxide.
  • TDM derivatized therapeutic drug monitoring
  • the kit comprising an analyte of interests, wherein the analyte of interests is a derivatized therapeutic drug monitoring (TDM) compound, preferably wherein the derivatized therapeutic drug monitoring (TDM) compound is Carbamazepine- 10,11 -epoxide.
  • the present invention relates to a kit used for an automatic analyzer capable of derivatizing and measuring the at least one analyte of interest comprising a first reservoir having a labeled therapeutic drug monitoring (TDM) compound as an ISTD and/or calibrator, preferably a labeled Carbamazepine-10,11- epoxide, and a second reservoir having a nucleophilic derivatization reagent for derivatization the therapeutic drug monitoring (TDM) compound as an analyte of interest, preferably Carbamazepine- 10, 11 -epoxide, preferably in a eighth aspect, the present invention relates to a kit used for an automatic analyzer capable of derivatizing and measuring the at least one analyte of interest comprising a first reservoir having a labeled analyte of interest, wherein the labeled analyte of interest is a labeled therapeutic drug monitoring (TDM) compound as an ISTD and/or calibrator,
  • TDM therapeutic
  • the present invention relates to an analyzer adapted to perform the method of the first aspect of the invention.
  • Figure 1 shows the structures of Carbamazepine- 10, 11 -epoxide (abbreviated as [I]) and carbamazepine (abbreviated as [II]).
  • Figure 2 shows the derivatization of the analyte of interest with a nucleophilic derivatization reagent.
  • Figures 3 A and 3B show chromatograms of product [III] at 15 seconds, monitored at two different MRM transitions. Chromatograms obtained after derivatization and workup of [I] in serum.
  • Figures 4A and 4B show chromatograms of product [IV] at 23 seconds, monitored at two different MRM transitions. Chromatogram obtained after derivatization and workup of [I] in serum.
  • Figures 5A and 5B show chromatograms of [I] at 48 seconds, monitored at two different MRM transitions. Chromatograms obtained after workup of [I] in serum.
  • Figure 8 shows the Calibration function of [I]
  • Figure 9 shows the comparison of results for three patient samples containing [I], using a state of the art method (“Without”; first three columns on the left side of Figure 9), derivatization with pyrrolidine (second three columns in the middle of Figure 9), or derivatization with n-butylamine (three columns on the right side of Figure 9).
  • A means sample ID U-BS10192.
  • B means sample V-4887785201.
  • C means sample ID W-7050451114.
  • 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, . .
  • measurement preferably comprises a qualitative, a semi-quanitative or a quantitative measurement.
  • automated refers to methods or processes or devices which are operated largely by automatic equipment, i.e. which are operate by machines or computers, in order to reduce the amount of work done by humans and the time taken to do the work.
  • tasks that were previously performed by humans are now performed by machines or computers.
  • the users typically need to configure the tool and define the process.
  • the skilled person is well aware that at some minor points manual intervention may still be required, however the large extend of the method is performed automatically.
  • the term “analyte”, “analyte molecule”, or “analyte(s) of interest” are used interchangeably, referring to the chemical species to be analysed.
  • Chemical species suitable to be analysed can be any kind of molecule present in a living organism, include but are not limited to nucleic acid, amino acids, peptides, proteins, fatty acids, lipids, carbohydrates, steroids, ketosteroids, secosteroids molecules.
  • Analytes may also be any substance that has been internalized by the organism, such as but not limited to therapeutic drugs, drugs of abuse, toxin, or a metabolite of such a substance.
  • Therapeutic drug monitoring (TDM) compound include antiepileptic drugs (AEDs). AEDs provide satisfactory control of seizures for most patients with epilepsy. The drugs have the remarkable ability to protect against seizures while permitting normal functioning of the nervous system.
  • AEDs act on diverse molecular targets to selectively modify the excitability of neurons so that seizure-related firing is blocked without disturbing non-epileptic activity. This occurs largely through effects on voltage-gated sodium and calcium channels, or by promoting inhibition mediated by GABAA (y-aminobutyric acid, type A) receptors.
  • GABAA y-aminobutyric acid, type A
  • AEDs include but are not limited to Acetazolamide, Brivaracetam, Cannabidiol, Carbamazepine, Clobazam, Clonazepam, Eslicarbazepine acetate, Ethosuximide, Everolimus, Fenfluramine, Gabapentin, Lacosamide, Lamotrigine, Levetiracetam, Oxcarbazepine, Perampanel, Phenobarbital, Phenytoin, Piracetam, Pregabalin, Primidone, Rufinamide, Sodium valproate, Stiripentol, Tiagabine, Topiramate, Valproic acid, Vigabatrin and Zonisamide.
  • analyte or Therapeutic drug monitoring (TDM) compound can also include compounds that are selected from the group consisting of Carbamazepine- 10, 11 -epoxide, Chlamy docin and its derivatives, Cytochalasin E17, Epoxy-bicyclic endoperoxides, Ligerinl8, Capsaicin epoxide, Ligerin, Epoxide-containing bicyclic endoperoxide, Trichothecenes, Triptolide, Minnelide, (5R)-5-Hydroxytriptolide (LLDT-28), Cryptophycins derivatives, Beloranib, Sagopilo, Patupilo, Parthenolide and dimethylamino-parthenolid, Withaferin A, Cinobufotalin, Fosfomycin, Carfilzomib, Oprozomib, Eplerenone, Hyoscine/scopolamine, Ixabepilone, May
  • Analytes may be present in a sample of interest, e.g. a biological or clinical sample.
  • sample or “sample of interest” or “patient sample” are used interchangeably herein, referring to a part or piece of a tissue, organ or individual, typically being smaller than such tissue, organ or individual, intended to represent the whole of the tissue, organ or individual.
  • a sample provides information about the tissue status or the health or diseased status of an organ or individual.
  • samples include but are not limited to fluid samples such as blood, serum, plasma, synovial fluid, spinal fluid, urine, saliva, and lymphatic fluid, or solid samples such as dried blood spots and tissue extracts.
  • Further examples of samples are cell cultures or tissue cultures. Preferabyl, the sample is blood, serum or plasma.
  • the sample may be derived from an “individual” or “subject”.
  • the subject is a mammal.
  • Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • a sample Before being analysed, a sample may be pre-treated or treated in a sample- and/or analyte specific manner.
  • pretreatment or “treated” refers to any measures required to allow for the subsequent analysis of a desired analyte.
  • (Pre-)treatment measures typically include but are not limited to derivatization of analytes, the elution of solid samples (e.g. elution of dried blood spots), addition of hemolizing reagent (HR) to whole blood samples, and the addition of enzymatic reagents to urine samples. Also the addition of internal standards (ISTD) is considered as (pre-)treatment of the sample.
  • an internal standard is a known amount of a substance, which exhibits similar properties as the analyte of interest when subjected to the mass spectrometric detection workflow or immunological assay workflow (i.e. including any pre-treatment, enrichment and actual detection step). Although the ISTD exhibits similar properties as the analyte of interest, it is still clearly distinguishable from the analyte of interest. Exemplified, during chromatographic separation, such as gas or liquid chromatography, the ISTD has about the same retention time as the analyte of interest from the sample. Thus, both the analyte and the ISTD enter the device, e.g. mass spectrometer, at the same time.
  • the ISTD however, exhibits a different molecular mass than the analyte of interest from the sample. This allows a mass spectrometric distinction between ions from the ISTD and ions from the analyte by means of their different mass/charge (m/z) ratios. Both are subject to fragmentation and provide daughter ions. These daughter ions can be distinguished by means of their m/z ratios from each other and from the respective parent ions. Consequently, following calibration, a separate determination and quantification of the signals from the ISTD and the analyte can be performed. Since the ISTD has been added in known amounts, the signal intensity of the analyte from the sample can be attributed to a specific quantitative amount of the analyte.
  • 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.
  • the ISTD is an isotopically labeled variant (comprising e.g. 2 H, 13 C, or 15 N etc. label) of the analyte of interest.
  • immunoglobulin refers to immunity conferring glycoproteins of the immunoglobulin superfamily.
  • Surface immunoglobulins are attached to the membrane of effector cells by their transmembrane region and encompass molecules such as but not limited to B-cell receptors, T -cell receptors, class I and II major histocompatibility complex (MHC) proteins, beta-2 microglobulin ( ⁇ 2M), CD3, CD4 and CDS.
  • MHC major histocompatibility complex
  • ⁇ 2M beta-2 microglobulin
  • CD3, CD4 and CDS CDS.
  • antibody refers to secreted immunoglobulins, which lack the transmembrane region and can thus, be released into the bloodstream and body cavities.
  • Human antibodies are grouped into different isotypes based on the heavy chain they possess. There are five types of human Ig heavy chains denoted by the Greek letters: a, y, 5, a, and p. - The type of heavy chain present defines the class of antibody, i.e. these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively, each performing different roles, and directing the appropriate immune response against different types of antigens.
  • Distinct heavy chains differ in size and composition; and may comprise approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science).
  • IgA is found in mucosal areas, such as the gut, respiratory tract and urogenital tract, as well as in saliva, tears, and breast milk and prevents colonization by pathogens (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389-417).
  • IgD mainly functions as an antigen receptor on B cells that have not been exposed to antigens and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118:429-437; Chen et al. (2009) Nat. Immunol.
  • IgE is involved in allergic reactions via its binding to allergens triggering the release of histamine from mast cells and basophils. IgE is also involved in protecting against parasitic worms (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). IgG provides the majority of antibody -based immunity against invading pathogens and is the only antibody isotype capable of crossing the placenta to give passive immunity to fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press).
  • IgGl In humans there are four different IgG subclasses (IgGl, 2, 3, and 4), named in order of their abundance in serum with IgGl being the most abundant (-66%), followed by IgG2 (-23%), IgG3 (-7%) and IgG (-4%).
  • the biological profile of the different IgG classes is determined by the structure of the respective hinge region.
  • IgM is expressed on the surface of B cells in a monomeric form and in a secreted pentameric form with very high avidity. IgM is involved in eliminating pathogens in the early stages of B cell mediated (humoral) immunity before sufficient IgG is produced (Geisberger et al. (2006) Immunology 118:429-437).
  • Antibodies are not only found as monomers but are also known to form dimers of two Ig units (e.g. IgA), tetramers of four Ig units (e.g. IgM of teleost fish), or pentamers of five Ig units (e.g. mammalian IgM).
  • Antibodies are typically made of four polypeptide chains comprising two identical heavy chains and identical two light chains which are connected via disulfide bonds and resemble a "Y"-shaped macro-molecule. Each of the chains comprises a number of immunoglobulin domains out of which some are constant domains and others are variable domains. Immunoglobulin domains consist of a 2-layer sandwich of between 7 and 9 antiparallel —strands arranged in two —sheets.
  • the heavy chain of an antibody comprises four Ig domains with three of them being constant (CH domains: CHI. CH2. CH3) domains and one of the being a variable domain (V H).
  • the light chain typically comprises one constant Ig domain (CL) and one variable Ig domain (V L).
  • the human IgG heavy chain is composed of four Ig domains linked from N- to C-terminus in the order VwCHl-CH2-CH3 (also referred to as VwCyl-Cy2-Cy3), whereas the human IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order VL-CL, being either of the kappa or lambda type (VK-CK or VA.-CA.).
  • the constant chain of human IgG comprises 447 amino acids. Throughout the present specification and claims, the numbering of the amino acid positions in an immunoglobulin are that of the "EU index" as in Kabat, E.
  • CH domains in the context of IgG are as follows: "CHI” refers to amino acid positions 118-220 according to the EU index as in Kabat; "CH2” refers to amino acid positions 237- 340 according to the EU index as in Kabat; and “CEB” refers to amino acid positions 341-44 7 according to the EU index as in Kabat.
  • full-length antibody “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called “Fab fragments” (also referred to as “Fab portion” or “Fab region”) each with a single antigen binding site, and a residual “Fe fragment” (also referred to as “Fe portion” or “Fe region”) whose name reflects its ability to crystallize readily.
  • Fab fragments also referred to as “Fab portion” or “Fab region”
  • Fe portion also referred to as “Fe portion” or “Fe region
  • the Fe region is composed of two identical protein fragments, derived from the CH2 and CH3 domains of the antibody's two heavy chains; in IgM and IgE isotypes, the Fe regions contain three heavy chain constant domains (CH2-4) in each polypeptide chain.
  • CH2-4 heavy chain constant domains
  • smaller immunoglobulin molecules exist naturally or have been constructed artificially.
  • the term "Fab 1 fragment” refers to a Fab fragment additionally comprise the hinge region of an Ig molecule whilst “F(ab')2 fragments” are understood to comprise two Fab' fragments being either chemically linked or connected via a disulfide bond. Whilst “single domain antibodies (sdAb )" (Desmyter et al.
  • scFv single chain Fv
  • di-scFvs Divalent single-chain variable fragments
  • scFvA- scFvB Divalent single-chain variable fragments
  • Bispecific diabodies are formed by expressing to chains with the arrangement VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively.
  • Singlechain diabodies (scDb) comprise a VHA-VLB and a VHB-VLA fragment, which are linked by a linker peptide (P) of 12-20 amino acids, preferably 14 amino acids, (VHA-VLB-P-VHB-VLA).
  • Bi- specific T-cell engagers are fusion proteins consisting of two scFvs of different antibodies wherein one of the scFvs binds to T cells via the CD3 receptor, and the other to a tumor cell via a tumor specific molecule (Kufer et al. (2004) Trends Biotechnol. 22:238-244).
  • Dual affinity retargeting molecules (“DART” molecules) are diabodies additionally stabilized through a C-terminal disulfide bridge.
  • Immunological assay generally refers to a biochemical test that measures the presence or concentration of an analyte of interest in a solution through the use of an antibody or an antigen.
  • the Immunological assay is a Sandwich immunoassay.
  • “Sandwich immunoassays” are broadly used in the detection of an analyte of interest.
  • the analyte is “sandwiched” in between a first antibody and a second antibody.
  • a sandwich assay requires that capture and detection antibody bind to different, non-overlapping epitopes on an analyte of interest. By appropriate means such sandwich complex is measured and the analyte thereby quantified.
  • a first antibody bound to the solid phase or capable of binding thereto and a detectably-labeled second antibody each bind to the analyte at different and non-overlapping epitopes.
  • the first analyte-specific binding agent e.g.
  • an antibody is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g.
  • an extremely versatile alternative sandwich assay format includes the use of a solid phase coated with the first partner of a binding pair, e.g.
  • paramagnetic streptavidin- coated microparticles Such microparticles are mixed and incubated with an analytespecific binding agent bound to the second partner of the binding pair (e.g. a biotinylated antibody), a sample suspected of comprising or comprising the analyte, wherein said second partner of the binding pair is bound to said analyte-specific binding agent, and a second analyte-specific binding agent which is detectably labeled.
  • an analytespecific binding agent bound to the second partner of the binding pair e.g. a biotinylated antibody
  • a sample suspected of comprising or comprising the analyte wherein said second partner of the binding pair is bound to said analyte-specific binding agent
  • a second analyte-specific binding agent which is detectably labeled.
  • these components are incubated under appropriate conditions and for a period of time sufficient for binding the labeled antibody via the analyte, the analyte-specific binding agent (bound to) the second partner of the binding pair and the first partner of the binding pair to the solid phase microparticles.
  • assay may include one or more washing step(s).
  • isotopic label can encompass a label that is part of the original molecular structure of the analyte of interest, but which carries preferably heavy atoms such as 13 C, 15 N, 17 O, 18 O, 2 H.
  • detectably labeled can encompass labels that can be directly or indirectly detected.
  • Directly detectable labels either provide a detectable signal or they interact with a second label to modify the detectable signal provided by the first or second label, e.g. to give FRET (fluorescence resonance energy transfer).
  • Labels such as fluorescent dyes and luminescent (including chemiluminescent and electrochemiluminescent) dyes (Briggs et al "Synthesis of Functionalised Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058) provide a detectable signal and are generally applicable for labeling.
  • detectably labeled refers to a label providing or inducible to provide a detectable signal, i.e. to a fluorescent label, to a luminescent label (e.g. a chemiluminescent label or an electrochemiluminescent label), a radioactive label or a metal-chelate based label, respectively.
  • Fluorescent dyes are e.g. described by Briggs et al "Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids," J. Chem. Soc., Perkin-Trans. 1 (1997) 1051-1058).
  • Fluorescent labels or fluorophores include rare earth chelates (europium chelates), fluorescein type labels including FITC, 5-carboxyfluorescein, 6-carboxy fluorescein; rhodamine type labels including TAMRA; dansyl; Lissamine; cyanines; phycoerythrins; Texas Red; and analogs thereof.
  • the fluorescent labels can be conjugated to an aldehyde group comprised in target molecule using the techniques disclosed herein.
  • Fluorescent dyes and fluorescent label reagents include those which are commercially available from Invitrogen/Molecular Probes (Eugene, Oregon, USA) and Pierce Biotechnology, Inc. (Rockford, Ill.).
  • Luminescent dyes or labels can be further subcategorized into chemiluminescent and electrochemiluminescent dyes.
  • chemiluminogenic labels include luminol, acridinium compounds, coelenterazine and analogues, dioxetanes, systems based on peroxy oxalic acid and their derivatives.
  • acridinium based labels are used (a detailed overview is given in Dodeigne C. et al., Taianta 51 (2000) 415-439).
  • Electrochemiluminescense proved to be very useful in analytical applications as a highly sensitive and selective method. It combines analytical advantages of chemiluminescent analysis (absence of background optical signal) with ease of reaction control by applying electrode potential.
  • Ruthenium complexes especially [Ru (Bpy)3]2+ (which releases a photon at -620 nm) regenerating with TPA (Tripropylamine) in liquid phase or liquid-solid interface are used as ECL- labels.
  • Electrochemiluminescent (ECL) assays provide a sensitive and precise measurement of the presence and concentration of an analyte of interest. Such techniques use labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescense is triggered by a voltage imposed on a working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte.
  • ECL Electrochemiluminescent
  • Radioactive labels make use of radioisotopes (radionuclides), such as 3H, 11C, 14C, 18F, 32P, 35S, 64Cu, 68Gn, 86Y, 89Zr, 99TC, U lin, 1231, 1241, 1251, 1311, 133Xe, 177Lu, 211 At, or 13 IBi.
  • radioisotopes radioisotopes (radionuclides)
  • MS Mass Spectrometry
  • MS is a methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m/z”.
  • MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio.
  • the compounds may be ionized and detected by any suitable means.
  • a "mass spectrometer” generally includes an ionizer and an ion detector.
  • 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.
  • tandem mass spectrometry involves multiple steps of mass spectrometry selection, wherein fragmentation of the analyte occurrs in between the stages.
  • ions are formed in the ion source and separated by mass-to-charge ratio in the first stage of mass spectrometry (MSI). Ions of a particular mass-to-charge ratio (precursor ions or parent ion) are selected and fragment ions (or daughter ions) are created by collision-induced dissociation, ionmolecule reaction, or photodissociation. The resulting ions are then separated and detected in a second stage of mass spectrometry (MS2).
  • aughter ion and “fragment ion” can be used interchangeably.
  • parent ion and “precursor ion” can be used interchangeably.
  • fragment ion can also be used to describe a parent ion.
  • 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.
  • 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.
  • the following three steps are performed: 1. a sample comprising an analyte of interest is ionized, usually by adduct formation with cations, often by protonation to cations.
  • Ionization source include but are not limited to electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).
  • the ions are sorted and separated according to their mass and charge.
  • High- field asymmetric-waveform ion-mobility spectrometry may be used as ion filter.
  • the separated ions are then detected, e.g. in multiple reaction mode (MRM), and the results are displayed on a chart.
  • MRM multiple reaction mode
  • electrospray ionization refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber, which is heated slightly to prevent condensation and to evaporate solvent. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.
  • APCI atmospheric pressure chemical ionization
  • mass spectrometry methods that are similar to ESI; however, APCI produces ions by ionmolecule reactions that occur within a plasma at atmospheric pressure.
  • the plasma is maintained by an electric discharge between the spray capillary and a counter electrode.
  • ions are typically extracted into the mass analyzer by use of a set of differentially pumped skimmer stages.
  • a counterflow of dry and preheated N2 gas may be used to improve removal of solvent.
  • the gas-phase ionization in APCI can be more effective than ESI for analyzing less-polar entity.
  • Multiple reaction mode is a detection mode for a MS instrument in which a precursor ion and one or more fragment ions arc selectively detected.
  • Mass spectrometry is thus, an important method for the accurate mass determination and characterization of analytes, including but not limited to low-molecular weight analytes, peptides, polypeptides or proteins. Its applications include the identification of proteins and their post-translational modifications, the elucidation of protein complexes, their subunits and functional interactions, as well as the global measurement of proteins in proteomics. De novo sequencing of peptides or proteins by mass spectrometry can typically be performed without prior knowledge of the amino acid sequence.
  • Mass spectrometric determination may be combined with additional analytical methods including chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and/or ion mobility-based separation techniques.
  • chromatographic methods such as gas chromatography (GC), liquid chromatography (LC), particularly HPLC, and/or ion mobility-based separation techniques.
  • 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.
  • LC liquid chromatography
  • NPLC normal phase liquid chromatography
  • RPLC reversed phase liquid chromatography
  • 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.
  • NPLC normal phase liquid chromatography
  • RPLC reversed phase liquid chromatography
  • Micro LC refers to a HPLC method using a column having a norrow inner column diameter, typically below 1 mm, e.g. about 0.5 mm.
  • Ultra high performance liquid chromatography or “UHPLC” refers to a HPLC method using a pressure of 120 MPa (17,405 lbf/in2), or about 1200 atmospheres.
  • Rapid LC refers to an LC method using a column having an inner diameter as mentioned above, with a short length ⁇ 2 cm, e.g. 1 cm, applying a flow rate as mentioned above and with a pressure as mentioned above (Micro LC, UHPLC).
  • the short Rapid LC protocol includes a trapping / wash / elution step using a single analytical column and realizes LC in a very short time ⁇ 1 min.
  • LC modi include Hydrophilic interaction chromatography (HILIC), size-exclusion LC, ion exchange LC, and affinity LC.
  • HILIC Hydrophilic interaction chromatography
  • size-exclusion LC size-exclusion LC
  • ion exchange LC ion exchange LC
  • affinity LC affinity LC
  • LC separation may be single-channel LC or multi-channel LC comprising a plurality of LC channels arranged in parallel.
  • LC analytes may be separated according to their polarity or log P value, size or affinity, as generally known to the skilled person.
  • nucleophile refers to a chemical species that donates an electron pair to form a chemical bond. Nucleophiles that exists in a water medium include but are not limited to -NH2, -OH, -SH, -Se, (R’,R”,R”’)P, N3-, RCOOH, F-, C1-, Br-, I-.
  • nucleophilic derivatization reagent or “nucleophile derivatization reagent” refers to reagents comprising such nucleophile.
  • a nucleophilic derivatization reagent comprises a moiety, carrying an orbital that serves as the highest occupied molecular orbital (HOMO) that is able to attack the lowest unoccupied molecular orbital (LUMO) of the substance of interest, such as an analyte of interest, thereby forming a new molecule comprised of the formerly nucleophilic unit and the analyte moiety.
  • HOMO highest occupied molecular orbital
  • LUMO lowest unoccupied molecular orbital
  • sampling tube or “sample collection tube” refers to any device with a reservoir appropriate for receiving a blood sample to be collected.
  • level or “level value” encompasses the absolute amount, the relative amount or concentration as well as any value or parameter that correlates thereto or can be derived therefrom.
  • nucleophilic derivatization reagent or “derivatization reagent” refers to a chemical substance having a specific chemical structure.
  • Said derivatization reagent may comprise one or more reactive groups, which is or are capable of forming a bond, preferably a covalent bond, with the analyte of interest.
  • a derivatized analyte of interest results.
  • Each reactive group may fulfil a different functionality, or two or more reactive groups may fulfil the same funtion.
  • Reactive groups include but are not limited to reactive units, charged units, and neutral loss units.
  • nucleophilic refers to a chemical species that donates an electron pair to form a chemical bond.
  • Nucleophiles that exists in a water medium include but are not limited to -NH2, -OH, -SH, -Se, (R’,R”,R”’)P, N3-, RCOOH, F-, C1-, Br-, I-.
  • the term “nucleophilic derivatization reagent” can refer to reagents comprising such nucleophile.
  • a nucleophilic derivatization reagent comprises a moiety, carrying an orbital that serves as the highest occupied molecular orbital (HOMO) that is able to attack the lowest unoccupied molecular orbital (LUMO) of the substance of interest, such as an analyte of interest, thereby forming a new molecule comprised of the formerly nucleophilic unit and the analyte moiety.
  • HOMO highest occupied molecular orbital
  • LUMO lowest unoccupied molecular orbital
  • the term “derivatized analyte of interest” or “derivatized therapeutic drug monitoring (TDM) compound” may refer to any molecule that is formed from the original analyte of interest or TDM compound and that is enlarged by a chemical reaction, preferably by reacting with the nucleophilic derivatization reagent.
  • the analyte of interest comprises at least one ore more than one epoxide moiety.
  • the epoxide moiety of the anaylte reacts with the nucleophilic derivatization reagent to open the cyclic ring of the epoxide moiety.
  • the term "coupling” the analyte of interest and a nucleophilic derivatization reagent via the epoxide moiety as used herein is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning.
  • the term specifically may refer, without limitation, to combining the sample comprising the analyte of interest and the nucleophilic derivatization reagent.
  • coupling or combining means to covalently bind the sample, preferably the analyte, and the nucleophilic derivatization reagent via at least one epoxide moiety that can mean that the cyclic structure of the epoxide moiety is broken up.
  • analyte of interest and “therapeutic drug monitoring (TDM) compound” can be used interchangeable.
  • analytes of interest and “therapeutic drug monitoring (TDM) compounds” can be used interchangeable.
  • labeled analyte of interest and “labeled therapeutic drug monitoring (TDM) compound” can be used interchangeable.
  • labeled therapeutic drug monitoring (TDM) compound can be used interchangeable.
  • derivatized analyte of interest and “derivatized therapeutic drug monitoring (TDM) compound” can be used interchangeable.
  • kits are any manufacture (e.g., a package or container) comprising at least one reagent, e.g., a medicament for treatment of a disorder, or a probe for specifically detecting a biomarker gene or protein of the invention.
  • the kit is preferably promoted, distributed, or sold as a unit for performing the method of the present invention.
  • a kit may further comprise carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like.
  • each of the container means comprises one of the separate elements to be used in the method of the first aspect.
  • Kits may further comprise one or more other reagents including but not limited to reaction catalyst.
  • Kits may further comprise one or more other containers comprising further materials including but not limited to buffers, internal standard, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a label may be present on the container to indicate that the composition is used for a specific application, and may also indicate directions for either in vivo or in vitro use.
  • the computer program code may be provided on a data storage medium or device such as a optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device.
  • the kit may, comprise standard amounts for the biomarkers as described elsewhere herein for calibration purposes.
  • TDM therapeutic drug monitoring
  • the present invention relates to a method of determining the level of at least one analyte of interest in an obtained sample comprising the steps of: a) Providing the sample comprising an analyte of interest, wherein the analyte of interest comprises an epoxide moiety, wherein the analyte of interest is a therapeutic drug monitoring (TDM) compound, b) Derivatization the analyte of interest with a nucleophilic derivatization reagent, preferably by coupling the analyte of interest and a nucleophilic derivatization reagent via the epoxide moiety, and c) Determining the level of the at least one analyte of interest in the sample, in particular using immunological assay or Mass Spectrometry, preferably LC/MS.
  • TDM therapeutic drug monitoring
  • the method of determining the level of at least one analyte of interest in an obtained sample comprising the steps of: a) Providing the sample comprising an analyte of interest, wherein the analyte of interest comprises an epoxide moiety, wherein the analyte of interest is a therapeutic drug monitoring (TDM) compound, b) Derivatization the analyte of interest with a nucleophilic derivatization reagent to form a derivatized analyte of interest, wherein the derivatized analyte of interest is a derivatized therapeutic drug monitoring (TDM) compound, wherein the derivatized therapeutic drug monitoring (TDM) compound is wherein Rl, R2 are independently selected from the group consisting of H, C1-C20- alkyl, Cl-C20-alkenyl, and -(-O-CH2-CH2-)n-O-CH3 with n being an integer in the range of from 1 to 15 or where
  • the analyte of interest is selected from the group consisting of Carbamazepine- 10, 11 -epoxide, Chlamydocin and its derivatives, Cytochalasin E17, Epoxy -bicyclic endoperoxides, Ligerin 18, Capsaicin epoxide, Ligerin, Epoxide- containing bicyclic endoperoxide, Triterpenoid derivatives, Trichothecenes, Triptolide, Minnelide, (5R)-5-Hydroxytriptolide (LLDT-28), Cryptophycins derivatives, Beloranib, Sagopilo, Patupilo, Parthenolide and dimethylamino- parthenolid, Withaferin A, Cinobufotalin, Fosfomycin, Carfilzomib, Oprozomib, Eplerenone, Hyoscine/scopolamine, Ixabepilone, Maytansine, [N2,-de
  • the analyte of interest is derivatized with a nucleophilic derivatization reagent, wherein the nucleophilic derivatization reagent is a primary amine, secondary amine or thiol, preferably wherein the thiol is RSH, wherein R is selected form the group consisting of alkyl, alkenyl, polyethyleneglycol, a (conjugated) heterocycle, a conjugated aryl and heteroaryl.
  • R is selected form the group consisting of alkyl, alkenyl, polyethyleneglycol, a (conjugated) heterocycle, a conjugated aryl and heteroaryl.
  • the term “(conjugated) heterocycle” can mean that the heterocyle is conjugated or not conjugated.
  • the the analyte of interest is derivatized with a nucleophilic derivatization reagent, in particular a reagent comprising an amine group, in particular a primary or secondary amine, in particular a primary amine group.
  • a primary amine group has the advantage that the incubation time can be reduced in comparision to a secondary amine.
  • the analyte of interest is derivatized with a nucleophilic derivatization reagent comprising more than 3 C- atoms, in particular 3 to 20 C-atoms, in particular 3 to 10 C-atoms, in particular 3-5 C-atoms, in particular 4 C-atoms.
  • the analyte of interest is derivatized with a linear or branched nucleophilic derivatization reagent, in particular with a linear amine, in particular with a linear primary amine, in particular with a linear primary amine comprising 3 to 5 C-atoms.
  • the analyte of interest is derivatized with a nucleophilic derivatization reagent selected from the group consisting of propylamine, butylamine, or pentylamine, in particular primary linear butylamine or primary linear pentylamine.
  • the analyte of interest is derivatized in at least one of its chemical moieties, preferably at least one epoxide moeity.
  • the person skilled in the art of chemistry is well aware of chemical moieties which are suitable to be derivatized, in particular with a nucleophilic derivatization reagent.
  • the derivatized analyte is derivatized in one, two or three of its chemical moieties.
  • the derivatized therapeutic drug monitoring (TDM) compound or the analyte of interest is wherein Rl, R2 are independently selected from the group consisting of H, C1-C20- alkyl, Cl-C20-alkenyl, and -(-O-CH2-CH2-)n-O-CH3 with n being an integer in the range of from 1 to 15 or wherein R1 and/or R2 is a (hetero)cycle that may be conjugated via an alkyl, alkenyl and/or polyethyleneglycol, wherein X represents a sulphur atom, an oxygen atom, or a nitrogen atom, and wherein in case of X is S or O, then R2 is non-existent.
  • Rl, R2 are independently selected from the group consisting of H, C1-C20- alkyl, Cl-C20-alkenyl, and -(-O-CH2-CH2-)n-O-CH3 with n being an integer in the range of from 1 to 15 or
  • this can mean that R1 is selected from the group consisting of H, Cl-C20-alkyl, Cl-C20-alkenyl, and -(-O-CH2-CH2-)n-O-CH3 and R2 is selected from the group consisting of H, Cl-C20-alkyl, Cl-C20-alkenyl, and -(-O-CH2- CH2-)n-O-CH3, or
  • R1 is selected from the group consisting of H, Cl-C20-alkyl, Cl-C20-alkenyl, and - (-O-CH2-CH2-)n-O-CH3 and R2 is a (hetero)cycle that may be conjugated via an alkyl, alkenyl and/or polyethyleneglycol, or
  • R2 is selected from the group consisting of H, Cl-C20-alkyl, Cl-C20-alkenyl, and - (-O-CH2-CH2-)n-O-CH3 and R1 is a (hetero)cycle that may be conjugated via an alkyl, alkenyl and/or polyethyleneglycol, or R1 and R2 are indenpendently from each other a (hetero)cycle that may be conjugated via an alkyl, alkenyl and/or polyethyleneglycol.
  • heterocycle can mean that R1 and/or R2 are a cycle or a heterocycle.
  • the derivatized therapeutic drug monitoring (TDM) compound is: wherein Rl, R2 are independently selected from the group consisting of H, C1-C20- alkyl, Cl-C20-alkenyl and -(-O-CH2-CH2-)n-O-CH3 with n being an integer in the range of from 1 to 15.
  • the derivatized therapeutic drug monitoring (TDM) compound is:
  • the analyte of interest is derivatized with a nucleophilic derivatization reagent, selected from the group consisting of propylamine, butylamine, pentylamine and pyrollidine, in particular primary linear butylamine or pyrollidine.
  • the nucleophilic derivatization reagent is substituted or unsubstituted. This can mean in the case of substituted nucleophilic derivatization reagent that the nucleophilic derivatization reagent, e.g. pyrollidine, can comprise other groups, e.g. alkyl or aryl groups that are attached to the nucleophilic derivatization reagent, e.g.
  • the substituted nucleophilic derivatization reagent can be a derivative of the nucleophilic derivatization reagent. This can mean in the case of unsubstituted nucleophilic derivatization reagent that the nucleophilic derivatization reagent, e.g. pyrollidine, does not comprise any other groups, e.g. alkyl or aryl groups that are attached to the nucleophilic derivatization reagent, e.g. pyrollidine.
  • the substituted nucleophilic derivatization reagent is the nucleophilic derivatization reagent without any derivatives.
  • the method comprises a further step:
  • Pre-treating and/or enriching the sample in particular using magnetic beads.
  • the method comprises further step:
  • the enrichment step comprises at least one enrichment workflow.
  • the samples comprising an analyte may be (pre-)treated and/or enriched by various methods.
  • the (pre-)treatment method is dependent upon the type of sample, such as blood (fresh or dried), plasma, serum, urine, or saliva, whereas the enrichment method is dependent on the analyte of interest. It is well known to the skilled person which (pre-)treatment method is suitable for which sample type. It is also well-known to the skilled person which enrichment method is suitable for which analyte of interest.
  • the sample is a whole blood sample
  • it is assigned to one of two pre-defined sample (pre-)treatment (PT) workflows, both comprising the addition of an internal standard (ISTD) and a hemolysis reagent (HR) followed by a pre-defined incubation period (Inc), where the difference between the two workflows is the order in which the internal standard (ISTD) and a hemolysis reagent (HR) are added.
  • the ISTD is added first to the obtained sample followed by the addition of the hemolysis reagent.
  • the ISTD is added to the obtained sample subsequent to the addition of the hemolysis reagents.
  • water is added as a hemolysis reagents, in particular in an amount of 0.5:1 to 20: 1 mL water / mL sample, in particular in an amount of 1 : 1 to 10: 1 mL water / mL sample, in particular in an amount of 2: 1 to 5: 1 mL water / mL sample.
  • the sample is a urine sample
  • it is assigned to one of other two pre-defined sample PT workflows, both comprising the addition of an ISTD and an enzymatic reagent followed by a pre-defined incubation period, where the difference between the two workflows is the order in which the internal standard and an enzymatic reagent are added.
  • the ISTD is added first to the obtained sample followed by the addition of the enzymatic reagent.
  • the ISTD is added to the obtained sample subsequent to the addition of the enzymatic reagents.
  • An enzymatic reagent is typically a reagent used for glucuronide cleavage or protein cleavage or any pre-processing of analyte or matrix.
  • the enzymatic reagent in selected from the group consisting of glucuronidase, (partial) exo- or endo- deglycoslation enzymes, or exo- or endo schooleases.
  • glucoronidase is added in amount of 0.5 - 10 mg/ml, in particular in an amount of 1 to 8 mg/ml, in particular in an amount of 2 to 5 mg/ml.
  • the sample is plasma or serum it is assigned to another predefined PT workflow including only the addition of an internal standard (ISTD) followed by a pre-defined incubation time.
  • ISD internal standard
  • incubation time and temperature to choose for a sample treatment, chemical reaction or method step considered and as named herein above or below.
  • incubation time and temperature depend upon each other, in that e.g. a high temperature typically leads to a shorter incubation period and vice versa.
  • the sample may be further subjected to at least one enrichment workflow.
  • the enrichment workflow may include one or more enrichment methods.
  • Enrichment methods are well-known in the art and include but are not limited to chemical enrichment methods including but not limited to chemical precipitation, and enrichment methods using solid phases including but not limited to solid phase extraction methods, bead workflows, and chromatographic methods (e.g. gas or liquid chromatography).
  • a first enrichment workflow comprises the addition of a solid phase, in particular of solid beads, carrying analyte-selective groups, to the (pre-treated) sample.
  • a first enrichment workflow comprises the addition of magnetic or paramagnetic beads carrying analyte- selective groups to the pre-treated sample.
  • the magnetic beads comprise a magnetic core coated with a styrene based polymer that is hypercrosslinked via Friedel-Crafts alkylation and further modified with addition of -OH groups.
  • the magnetic beads comprise a magnetic core coated with a styrene based polymer that is hypercrosslinked via diamines (e.g. TMEDA) and further modified whereby the diamine also serves as a sidechain (i.e. in these types of beads, TMEDA offers both quaternary and tertiary amine functionalities).
  • diamines e.g. TMEDA
  • the addition of the magnetic beads comprises agitation or mixing.
  • a pre-defined incubation period for capturing the analyte(s) of interest on the bead follows.
  • the workflow comprises a washing step (Wl) after incubation with the magnetic beads.
  • one or more additional washing steps (W2) are performed.
  • One washing step (Wl, W2) comprises a series of steps including magnetic bead separation by a magnetic bead handling unit comprising magnets or electromagnets, aspiration of liquid, addition of a washing buffer, resuspension of the magnetic beads, another magnetic bead separation step and another aspiration of the liquid.
  • washing steps may differ in terms of type of solvent (water/organic/salt/pH), aside from volume and number or combination of washing cycles. It is well-known to the skilled person how to choose the respective parameters.
  • the last washing step (Wl, W2) is followed by the addition of an elution reagent followed by resuspension of the magnetic beads and a pre-defined incubation period for releasing the analyte(s) of interest from the magnetic beads.
  • the bound-free magnetic beads are then separated and the supernatant containing derivatized analyte(s) of interest is captured.
  • a first enrichment workflow comprises the addition of magnetic beads carrying matrix-selective groups to the pre-treated sample.
  • the addition of the magnetic beads comprises agitation or mixing.
  • a pre-defined incubation period for capturing the matrix on the bead follows.
  • the analyte of interest does not bind to the magnetic beads but remains in the supernatant.
  • the magnetic beads are separated and the supernatant containing the enriched analyte(s) of interest is collected.
  • the supernatant is subjected to a second enrichment workflow, in particular to a chromatographic enrichment workflow.
  • the chromatographic separation is gas or liquid chromatography. Both methods are well known to the skilled person.
  • the liquid chromatography is selected from the group consisting of HPLC, rapid LC, micro-LC, flow injection, and trap and elute.
  • the supernatant is transferred to the LC station or is transferred to the LC station after a dilution step by addition of a dilution liquid. Different elution procedures/reagents may also be used, by changing e.g. the type of solvents (water/organic/salt/pH) and volume. The various parameters are well-known to the skilled person and easily chosen.
  • the first enrichment process includes the use of analyte selective magnetic beads.
  • the second enrichment process includes the use of chromatographic separation, in particular using liquid chromatography.
  • the first enrichment process using analyte selective magnetic beads is performed prior to the second enrichment process using liquid chromatography.
  • step c) comprises determining the amount or concentration of the one or more derivatized analyte using immunological methods or mass spectrometry.
  • step c) comprises determining the level of the at least one analyte of interest in the sample using immunological methods
  • the following steps are comprised: i) Incubating the (optionally enriched) sample of the patient with one or more antibodies specifically binding to the one or more derivatized analyte, thereby generating a complex between the antibody and the one or more derivatized analyte, and ii) Quantifying the complex formed in step i), thereby quantifying the amount of the one or more derivatized analyte in the sample of the patient.
  • step i) the sample is incubated with two antibodies, specifically binding to the one or more derivatized analyte.
  • the sample can be contacted with the first and the second antibody in any desired order, i.e. first antibody first and then the second antibody or second antibody first and then the first antibody, or simultaneously, for a time and under conditions sufficient to form a first antibody/ derivatized analyte /second antibody complex.
  • the antibody/the antibodies is/are directly or indirectly detectably labeled.
  • the antibody is detectably labeled with a luminescent dye, in particular a chemiluminescent dye or an electrochemiluminescent dye.
  • step c) comprises determining the level of the at least one analyte of interest in the sample using mass spectrometry
  • steps are comprised:
  • parent and/or fragment ions measured are those as indicated in Table X.
  • the fragment ion or product ion of derivatized Carbamazepine- 10,11-epoxide +H + is measured at a m/z value 324.055.
  • the fragment ion of derivatized Carbamazepine-10,11-epoxide is measured at a m/z value 236.096.
  • MRM for Pyrrolidine derivate should also be mentioned (QI : 326.055 Q3: 180.093)
  • step b) the sample is treated with a nucleophilic derivatization reagent short after the sample is obtained, in particular within less than 3 hours or 2 hours or 1 hour after the sample was obtained, preferably less than 10 min after the sample was obtained, in particular within less than 5 min after the sample was obtained.
  • the sample obtained after step b) comprises the derivatized analyte of interest.
  • the analyte of interest and/or the derivatized analyte of interest comprises an isotopic label.
  • the derivatized analyte of interest is dissolved or suspended in a solvent, in particular a solvent selected from the group consisting of water, CH3CN, THF, Dioxanes, DMF, DMSO, acetone, t-butyl alcohol, diglyme, DME, MeOH, EtOH, 1-PrOH, 2-PrOH, ethylene glycol, Hexamethylphosphoramiede (EIMP A), Hexamethylphosphorous triamide (HMPT), and glycerin.
  • a solvent selected from the group consisting of water, CH3CN, THF, Dioxanes, DMF, DMSO, acetone, t-butyl alcohol, diglyme, DME, MeOH, EtOH, 1-PrOH, 2-PrOH, ethylene glycol, Hexamethylphosphoramiede (EIMP A), Hexamethylphosphorous triamide (HMPT), and glycerin.
  • the method further comprising a non-nucleophilic base that is stable and miscibile with water, in particular selected from the group consisting of Diazabicyclo(5.4.0)undecene (DBU), triethylamine, diisopropylethylamine, NasPCh, Na2CC>3, and CS2CO3.
  • a non-nucleophilic base that is stable and miscibile with water, in particular selected from the group consisting of Diazabicyclo(5.4.0)undecene (DBU), triethylamine, diisopropylethylamine, NasPCh, Na2CC>3, and CS2CO3.
  • the method is an automated method.
  • the method is performed by an automated system.
  • the method comprises no manual intervention.
  • the method is performed in a random-excess mode.
  • the method is an in-vitro diagnostic method.
  • the present invention relates to a sampling tube for collecting a patient sample comprising a nucleophilic derivatization reagent suitable to stabilize one or more therapeutic drug monitoring (TDM) compounds in a sample.
  • TDM therapeutic drug monitoring
  • the present invention relates to a sampling tube suitable for collecting a patient sample comprising a nucleophilic derivatization reagent suitable for stabilizing one or more therapeutic drug monitoring (TDM) compounds in a sample.
  • TDM therapeutic drug monitoring
  • the present invention relates to a sampling tube suitable for collecting a patient sample comprising a nucleophilic derivatization reagent suitable for stabilizing one or more analyte of interest in a sample, wherein the analytes of interest are therapeutic drug monitoring (TDM) compounds in a sample.
  • TDM therapeutic drug monitoring
  • the present invention relates to a sampling tube for collecting a patient sample
  • a sampling tube for collecting a patient sample comprising a device with a reservoir adapted for receiving a blood sample to be collected, and a nucleophilic derivatization reagent suitable to stabilize one or more therapeutic drug monitoring (TDM) compounds in a sample.
  • TDM therapeutic drug monitoring
  • the present invention relates to a sampling tube suitable for collecting a patient sample comprising a device with a reservoir adapted for receiving a blood sample to be collected, and a nucleophilic derivatization reagent suitable for stabilizing one or more therapeutic drug monitoring (TDM) compounds in a sample.
  • TDM therapeutic drug monitoring
  • the present invention relates to a sampling tube suitable for collecting a patient sample comprising a device with a reservoir adapted for receiving a blood sample to be collected, and a nucleophilic derivatization reagent suitable for stabilizing one or more analyte of interest in a sample, wherein the analytes of interest are therapeutic drug monitoring (TDM) compounds in a sample.
  • TDM therapeutic drug monitoring
  • Sample collections tubes suitable to be used for collecting a patient sample are well- known in the art and are used on a routine basis by practioners.
  • the sampling tube preferably will in fact be a tube.
  • the sampling tube has a size and dimension adapted to match the requirements of the sample receiving station of an automated analyzer, e.g. an Elecsys® analyzer of Roche Diagnostics.
  • the sampling tube may have a conical or preferably a round bottom.
  • Standard and preferred tubes e.g. have the following dimensions: 13x75 mm; 13x100 mm, or 16x100 mm.
  • the sampling tube according to the present invention is only used once, i.e. it is a single use device.
  • the sampling tube according to the present invention is not only appropriate for collection of a sample but it is also adapted to allow for the further processing of the sample.
  • the present invention relates to the use of a nucleophilic derivatization reagent for determining the level of at least one analyte of interest, preferably Carbamazepine- 10, 11 -epoxide.
  • the present invention relates to the use of a nucleophilic derivatization reagent to stabilize an analyte of interest in a sample, preferably Carbamazepine- 10, 11 -epoxide, preferably the use of a nucleophilic derivatization reagent to stabilize Carbamazepine- 10, 11 -epoxide as an analyte of interest in a sample.
  • the present invention relates to the use of one or more derivatized therapeutic drug monitoring (TDM) compounds in a sample as an ISTD and/or calibrator for Mass Spectrometry measurements.
  • TDM derivatized therapeutic drug monitoring
  • the present invention relates to the use of one or more analytes of interest, wherein the analytes of interest are derivatized therapeutic drug monitoring (TDM) compounds in a sample as an ISTD and/or calibrator for Mass Spectrometry measurements.
  • the present invention relates to a kit comprising a derivatized therapeutic drug monitoring (TDM) compound, preferably wherein the derivatized therapeutic drug monitoring (TDM) compound is Carbamazepine- 10, 11 -epoxide.
  • TDM derivatized therapeutic drug monitoring
  • the kit comprising an analyte of interests, wherein the analyte of interests is a derivatized therapeutic drug monitoring (TDM) compound, preferably wherein the derivatized therapeutic drug monitoring (TDM) compound is Carbamazepine- 10,11 -epoxide.
  • the present invention relates to a kit used for an automatic analyzer capable of derivatizing and measuring the at least one analyte of interest comprising a first reservoir having a labeled therapeutic drug monitoring (TDM) compound as an ISTD and/or calibrator, preferably a labeled Carbamazepine-10,11- epoxide, and a second reservoir having a nucleophilic derivatization reagent for derivatization the therapeutic drug monitoring (TDM) compound as an analyte of interest, preferably Carbamazepine- 10, 11 -epoxide.
  • TDM therapeutic drug monitoring
  • the present invention relates to a kit used for an automatic analyzer capable of derivatizing and measuring the at least one analyte of interest comprising a first reservoir having a labeled analyte of interest, wherein the labeled analyte of interest is a labeled therapeutic drug monitoring (TDM) compound as an ISTD and/or calibrator, preferably a labeled Carbamazepine-10,11 -epoxide, and a second reservoir having a nucleophilic derivatization reagent for derivatization the therapeutic drug monitoring (TDM) compound as an analyte of interest, preferably Carbamazepine- 10, 11 -epoxide.
  • TDM therapeutic drug monitoring
  • the present invention relates to an analyzer adapted to perform the method of the first aspect of the invention.
  • the analyzer is a mass spectrometry system, in particular a LC- MS/MS system.
  • the analyzer is an automated analytical system.
  • the analyzer does not require manual intervention, i.e. the operation of the system is purely automated.
  • the LC/MS system is an automated, random-access LC/MS system.
  • the MS device is a tandem mass spectrometer, in particular a triple quadrupole device.
  • the LC is HPLC, in particular is RP-HPLC, or rapid LC.
  • the ion formation is based on electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), in particular positive polarity mode ESI.
  • ESI electrospray ionization
  • APCI atmospheric pressure chemical ionization
  • the analyser or automatic analyser is a clinical diagnostics system.
  • a clinical diagnostics system can be a laboratory automated apparatus dedicated to the analysis of samples for in vitro diagnostics.
  • the clinical diagnostics system may have different configurations according to the need and/or according to the desired laboratory workflow. Additional configurations may be obtained by coupling a plurality of apparatuses and/or modules together.
  • a “module” is a work cell, typically smaller in size than the entire clinical diagnostics system, which has a dedicated function. This function can be analytical but can be also pre-analytical or post analytical or it can be an auxiliary function to any of the pre-analytical function, analytical function or post-analytical function.
  • a module can be configured to cooperate with one or more other modules for carrying out dedicated tasks of a sample processing workflow, e.g.
  • the clinical diagnostics system can comprise one or more analytical apparatuses, designed to execute respective workflows that are optimized for certain types of analysis, e.g. clinical chemistry, immunochemistry, coagulation, hematology, liquid chromatography separation, mass spectrometry, etc.
  • the clinical diagnostic system may comprise one analytical apparatus or a combination of any of such analytical apparatuses with respective workflows, where pre-analytical and/or post analytical modules may be coupled to individual analytical apparatuses or be shared by a plurality of analytical apparatuses.
  • pre-analytical and/or post- analytical functions may be performed by units integrated in an analytical apparatus.
  • the clinical diagnostics system can comprise functional units such as liquid handling units for pipetting and/or pumping and/or mixing of samples and/or reagents and/or system fluids, and also functional units for sorting, storing, transporting, identifying, separating, detecting.
  • the clinical diagnostic system can comprise a sample preparation station for the automated preparation of samples comprising analytes of interest, optionally a liquid chromatography (LC) separation station comprising a plurality of LC channels and/or optionally a sample preparation/LC interface for injecting prepared samples into any one of the LC channels.
  • LC liquid chromatography
  • the clinical diagnostic system can further comprise a controller programmed to assign samples to predefined sample preparation workflows each comprising a pre-defined sequence of sample preparation steps and requiring a pre-defined time for completion depending on the analytes of interest.
  • the clinical diagnostic system can further comprise a mass spectrometer (MS) and an LC/MS interface for connecting the LC separation station to the mass spectrometer.
  • MS mass spectrometer
  • a sample preparation station can be a pre-analytical module coupled to one or more analytical apparatuses or a unit in an analytical apparatus designed to execute a series of sample processing steps aimed at removing or at least reducing interfering matrix components in a sample and/or enriching analytes of interest in a sample.
  • Such processing steps may include any one or more of the following processing operations carried out on a sample or a plurality of samples, sequentially, in parallel or in a staggered manner: pipetting (aspirating and/or dispensing) fluids, pumping fluids, mixing with reagents, incubating at a certain temperature, heating or cooling, centrifuging, separating, filtering, sieving, drying, washing, resuspending, aliquoting, transferring, storing, etc.).
  • pipetting aspirating and/or dispensing
  • pumping fluids mixing with reagents
  • mixing with reagents incubating at a certain temperature, heating or cooling, centrifuging, separating, filtering, sieving, drying, washing, resuspending, aliquoting, transferring, storing, etc.
  • the clinical diagnostic system e.g. the sample preparation station, may also comprise a buffer unit for receiving a plurality of samples before a new sample preparation start sequence is initiated, where the samples may be individually randomly accessible and the individual preparation of which may be initiated according to the sample preparation start sequence.
  • the clinical diagnostic system makes use of mass spectrometry more convenient, more reliable, and therefore suitable for clinical diagnostics.
  • high- throughput e.g. up to 100 samples/hour or more with random access sample preparation and LC separation can be obtained while enabling online coupling to mass spectrometry.
  • the process can be fully automated increasing the walk-away time and decreasing the level of skills required.
  • the present invention relates to the following aspects:
  • a method of determining the level of at least one analyte of interest in an obtained sample comprising the steps of: a) Providing the sample comprising an analyte of interest, wherein the analyte of interest comprises an epoxide moiety, wherein the analyte of interest is a therapeutic drug monitoring (TDM) compound, b) Derivatization the analyte of interest with a nucleophilic derivatization reagent, preferably by coupling the analyte of interest and a nucleophilic derivatization reagent via the epoxide moiety, and c) Determining the level of the at least one analyte of interest in the sample, in particular using immunological assay or Mass Spectrometry, preferably LC/MS, preferably a method of determining the level of at least one analyte of interest in an obtained sample comprising the steps of: a) Providing the sample comprising an analyte of interest, wherein the
  • analyte of interest is selected from the group consisting of Carbamazepine-10,11 -epoxide, Chlamydocin and/or its derivatives, Cytochalasin E17, Epoxy-bicyclic endoperoxides, Ligerinl8, Capsaicin epoxide, Ligerin, Epoxide-containing bicyclic endoperoxide, Triterpenoid derivatives, trichothecenes, Triptolide, Minnelide, (5R)-5-Hydroxytriptolide (LLDT-28), Cryptophycins derivatives, Beloranib, Sagopilo, Patupilo, Parthenolide and/or dimethylamino-parthenolid, Withaferin A, Cinobufotalin, Fosfomycin, Carfilzomib, Oprozomib, Eplerenone, Hyoscine/scopolamine, Ixabepilone, Maytans
  • nucleophilic derivatization reagent is a primary amine, secondary amine, an alcohol, or thiol, preferably wherein the alcohol or thiol is ROH or RSH, wherein R is selected form the group consisting of alkyl, alkenyl, polyethyleneglycol, a heterocycle, a conjugated heterocycle, a conjugated aryl and heteroaryl.
  • Rl, R2 are independently selected from the group consisting of H, C1-C20- alkyl, Cl-C20-alkenyl, and -(-O-CH2-CH2-)n-O-CH3 with n being an integer in the range of from 1 to 15 or wherein Rl and/or R2 is a (hetero)cycle that may be conjugated via an alkyl, alkenyl and/or polyethyleneglycol, wherein X represents a sulphur atom, an oxygen atom or a nitrogen atom, and wherein in case of X is S or O, then R2 is non-existent. 5.
  • the derivatized therapeutic drug monitoring (TDM) compound is: wherein Rl, R2 are independently selected from the group consisting of H, C1-C20- alkyl, Cl-C20-alkenyl and -(-O-CH2-CH2-)n-O-CH3 with n being an integer in the range of from 1 to 15. 6.
  • the derivatized therapeutic drug monitoring (TDM) compound is:
  • nucleophilic derivatization reagent selected from the group consisting of propylamine, butylamine, pentylamine and pyrollidine, in particular primary linear butylamine or pyrollidine.
  • Pre-treating and/or enriching the sample in particular using magnetic beads.
  • step b) the sample is treated with a nucleophilic derivatization reagent short after the sample is obtained, in particular within less than 3 hours or 2 hours or 1 hour after the sample was obtained, preferably less than 10 min after the sample was obtained, in particular within less than 5 min after the sample was obtained.
  • step b) comprises the derivatized analyte of interest.
  • the enrichment step comprises at least one enrichment workflow.
  • a solvent selected from the group consisting of water, CH3CN, Tetrahydrofuran (THF), Dioxanes, Dimethylformamide (DMF), Dimethyl sulfoxide (DMSO), acetone, t-butyl alcohol, diglyme, dimethyl ether (DME), methanol (MeOH), ethanol (EtOH), 1 -propanol (1- PrOH), 2- propanol (2-PrOH), ethylene glycol, Hexamethylphosphoramiede (EIMP A), Hexamethylphosphorous triamide (HMPT), and glycerin.
  • a solvent selected from the group consisting of water, CH3CN, Tetrahydrofuran (THF), Dioxanes, Dimethylformamide (DMF), Dimethyl sulfoxide (DMSO), acetone, t-butyl alcohol, diglyme, dimethyl ether (DME), methanol (MeOH), ethanol (EtOH), 1 -propanol (1- PrOH),
  • a non- nucleophilic base that is stable and miscibile with water, in particular selected from the group consisting of Diazabicyclo(5.4.0)undecene (DBU), tri ethylamine, diisopropylethylamine, NasPCh, Na2COs, and CS2CO3.
  • DBU Diazabicyclo(5.4.0)undecene
  • a sampling tube for collecting a patient sample comprising a nucleophilic derivatization reagent suitable to stabilize one or more therapeutic drug monitoring (TDM) compounds in a sample preferably sampling tube suitable for collecting a patient sample comprising a nucleophilic derivatization reagent suitable for stabilizing one or more therapeutic drug monitoring (TDM) compounds in a sample.
  • a sampling tube for collecting a patient sample comprising a device with a reservoir adapted for receiving a blood sample to be collected, and a nucleophilic derivatization reagent suitable to stabilize one or more therapeutic drug monitoring (TDM) compounds in a sample, preferably a sampling tube suitable for collecting a patient sample comprising a device with a reservoir adapted for receiving a blood sample to be collected, and a nucleophilic derivatization reagent suitable for stabilizing one or more therapeutic drug monitoring (TDM) compounds in a sample.
  • TDM therapeutic drug monitoring
  • nucleophilic derivatization reagent to stabilize an analyte of interest in a sample, preferably Carbamazepine- 10, 11 -epoxide.
  • TDM derivatized therapeutic drug monitoring
  • kits comprising a derivatized therapeutic drug monitoring (TDM) compound, preferably wherein the derivatized therapeutic drug monitoring (TDM) compound is Carbamazepine- 10, 11 -epoxide, in embodiments the kit comprising an analyte of interests, wherein the analyte of interests is a derivatized therapeutic drug monitoring (TDM) compound, preferably wherein the derivatized therapeutic drug monitoring (TDM) compound is Carbamazepine- 10, 11 -epoxide.
  • TDM derivatized therapeutic drug monitoring
  • a kit used for an automatic analyzer capable of derivatizing and measuring the at least one analyte of interest comprising a first reservoir having a labeled therapeutic drug monitoring (TDM) compound as an ISTD and/or calibrator, preferably a labeled Carbamazepine-10,11- epoxide, and a second reservoir having a nucleophilic derivatization reagent for derivatization the therapeutic drug monitoring (TDM) compound as an analyte of interest, preferably Carbamazepine- 10, 11 -epoxide, preferably a kit used for an automatic analyzer capable of derivatizing and measuring the at least one analyte of interest comprising a first reservoir having a labeled analyte of interest, wherein the labeled analyte of interest is a labeled therapeutic drug monitoring (TDM) compound as an ISTD and/or calibrator, preferably a labeled Carbamazepine-10,11 -epoxide,
  • Figure 1 shows the structures of Carbamazepine- 10, 11 -epoxide [I] and carbamazepine [II],
  • FIG. 2 shows the derivatization the analyte of interest with a nucleophilic derivatization reagent.
  • the analyte of interest comprises an epoxide moiety.
  • the analyte of interest is a therapeutic drug monitoring (TDM) compound.
  • the analyte is in this case Carbamazepine- 10, 11 -epoxide [I]
  • the nucleophilic derivatization reagent is pyrrolidineor n-butylamine.
  • the derivatized analyte or derivatized therapeutic drug monitoring (TDM) compound is shown as [III] and [IV],
  • the epoxide may be used in a derivatization reaction with a nucleophile, like an amine, e.g. either a primary or a secondary amine. By doing so, the epoxide is removed from the structure, yielding another amine (reaction with a primary amine yields a secondary amine; reaction with a secondary amine yields a tertiary amine) and a hydroxyl group.
  • a nucleophile like an amine, e.g. either a primary or a secondary amine.
  • Calibrators of [I] were prepared by spiking a stock solution into a buffer mixture with HEPES, BSA, IgG. Three patient samples with concentrations of 3.2, 5.94, and 8.67 pg/mL in serum were used. The concentration of these samples was prior to measurement determined via a reference method.
  • Pyrrolidine and n-butylamine were diluted to a concentration of 5.5 M with water.
  • ISTD solution was prepared by mixing of carbamazepine- 10, 1 l-epoxide- 13 Ce in 20% MeOH.
  • the protocol to measure [III] and [IV] is as follows: To the sample (20 pL), ISTD (i.e. isotopically labeled [I]) (20 pL) was added. To this, derivatization solution (40 pL) was added and the mixture was mixed and incubated. Subsequently, formic acid (2 M, 20 pL) was added, followed by the addition of magnetic beads (50 mg/mL, 20 pL). Next, a magnetic force is applied to the sides of the vessel. Subsequently, the supernatant is removed and the beads are washed twice with water (150 pL).
  • ISTD i.e. isotopically labeled [I]
  • the protocol to measure [I] is as follows: To the sample (20 pL), ISTD (i.e. isotopically labeled [I]) (20 pL) was added. To this, phosphate buffer (250 mM, pH 12, 20 pL) was added, followed by the addition of magnetic beads (50 mg/mL, 20 pL). Next, a magnetic force is applied to the sides of the vessel. Subsequently, the supernatant is removed and the beads are washed twice with water (150 pL). Then, an elution mixture (MeOH/H2O/HCOOH (1 M), 70/20/10, v/v/v) (200 pL) was added.
  • ISTD i.e. isotopically labeled [I]
  • the calibrators were also measured with a standard method that does not comprise derivazation (see Figure 8). Using these calibration functions, the concentration of [I] in the patient samples was obtained. The concentrations obtained via derivatization were then normalized on the concentrations obtained via the state of the art method. The results are summarized Table 2, or for a graphic overview in Figure 9. To conclude, the quantitative results obtained via derivatization are near- to identical to the state of the art method without derivatization. This shows the validity of this method in quantifying carbamazepine- 10, 11 -epoxide in TDM according to this invention. Additionally, as mentioned above the derivatization concept has many advantages in comparision to the state of the art method without derivatization.
  • Figure 9 shows the comparison of results for three patient samples A, B and C containing [I], using a state of the art method (“Without”; first three columns A, B and C on the left side of Figure 9), derivatization with pyrrolidine (second three columns A, B and C in the middle of Figure 9), or derivatization with n-butylamine (three columns A, B and C on the right side of Figure 9).
  • A means sample ID U- BS10192.
  • B means sample V-4887785201.
  • C means sample ID W-7050451114.

Abstract

La présente invention concerne un procédé de détermination du niveau d'au moins un analyte d'intérêt, des tubes d'échantillonnage pour collecter un échantillon de patient, l'utilisation de réactifs de dérivatisation nucléophiles, un analyseur ainsi que des kits.
PCT/EP2023/050039 2022-01-05 2023-01-03 Dérivatisation de composés dans des échantillons de patients pour la pharmacovigilance thérapeutique (tdm) WO2023131594A1 (fr)

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