Method for derivatizing and method for determining an analyte of interest

Abstract

The invention relates in a first aspect to a method for derivatizing an analyte of interest, wherein the method comprises the provision of (a) a liposome particle comprising a derivatization agent for an analyte intended to be analyzed via mass spectrometry, an encapsulating material surrounding the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material and facing towards the outside of the particle; (b) a magnetic particle having on its surface a second member of the binding pair, and optionally a binding agent specific for the analyte of interest; (c) a liquid sample potentially containing an analyte of interest; and (d) optionally a further magnetic particle comprising a binding agent specific for the analyte of interest. The first aspect also relates to a method for determining an analyte of interest, the method further comprising subjecting the derivatized analyte of interest obtained to analysis. A second aspect of the invention is directed to a derivatized analyte of interest, obtained or obtainable from the method of the first aspect of the invention. In a third aspect, the invention is related to a liposome particle comprising a derivatization agent for an analyte intended to be analyzed via mass spectrometry, an encapsulating material surrounding the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material, wherein the encapsulating material comprises one or more lipid(s) or to a liquid mixture comprising said liposome particle in suspended form. A fourth aspect of the invention is related to the use of the liposome particle or the liquid mixture of the third aspect of the invention for derivatization of an analyte of interest or in a method for determining an analyte of interest.

Description

[0001] Roche Diagnostics GmbH October 2, 2025

[0002] RD38809PC

[0003] Method for derivatizing and method for determining an analyte of interest

[0004] Technical Field

[0005] The invention relates in a first aspect to a method for derivatizing an analyte of interest, wherein the method comprises the provision of a particle (a) comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry, an encapsulating material surrounding the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material and facing towards the outside of the particle; (b) a magnetic particle having on its surface a second member of the binding pair, and optionally a binding agent specific for the analyte of interest; (c) a liquid sample potentially containing an analyte of interest; and (d) optionally a further magnetic particle comprising a binding agent specific for the analyte of interest, the method for derivatizing comprising steps (i) to (viii). The first aspect also relates to a method for determining an analyte of interest, the method comprising i) to viii) and further a step (ix) of subjecting the derivatized analyte of interest obtained in viii) to analysis. A second aspect of the invention is directed to a derivatized analyte of interest, obtained or obtainable from the method of the first aspect of the invention. In a third aspect, the invention is related to a particle comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry, an encapsulating material surrounding the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material, wherein the encapsulating material comprises one or more lipid(s), and / or one or more polymer(s) and / or mixed forms thereof (i.e. poly- mersomes); or to a liquid mixture comprising said particle in suspended form. A fourth aspect of the invention is related to the use of the particle or the liquid mixture of the third aspect of the invention for derivatization of an analyte of interest or in a method for determining an analyte of interest.

[0006] Background art

[0007] Mass spectrometry (MS) is a widely used technique for the qualitative and quantitative analysis of chemical substances ranging from small molecules to macromolecules. In general, it is a very sensitive and specific method, allowing even for the analysis of complex biological, environmental or clinical samples. Chemical derivatization of an analyte of interest can be used to enhance the detection sensitivity in MS applications. In most cases, charged (i) or chargeable (ii) compounds are used to produce a permanently charged (i) or chargeable (ii) derivatized analyte in order to improve the mass spec response / sensitivity. As electrospray ionization (ESI) is a commonly used ionization source, most of these reagents aim to enhance the electrospray ionization (ESI) response, but they are not designed to generate a particular product ion by Collision Induced Dissociation (CID) for MS / MS applications. To address this issue, several derivatization reagents were developed carrying a structure suitable for MS / MS detection. “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 (MS 1). 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). Mass spectrometric determination is quite often combined with additional analytical methods including chromatographic methods such as liquid chromatography (LC), particularly HPLC.

[0008] Numerous derivatization reagents are available for liquid chromatography based mass spectrometry (i.e. LC-MS, LC-MS / MS; see reviews: J. Sep. Sci. 2016, 39, 102-114; Biomed. Chromatogr. 2011; 25: 1-10) and laser desorption based applications in combination with a suitable matrix (MALDI, see review: Trends in Analytical Chemistry 143 (2021) 116399; J Mass Spectrom. 2021;56:e4731).

[0009] As outlined above, derivatization is a key tool to achieve ultrahigh sensitivity in LC-MS applications. Theoretically, chemical derivatization can be integrated in a workflow online, offline and pre- or post the chromatographic separation. However, the integration of derivatization into (automated) LC-MS workflows is still difficult due 1) increased number of steps per sample preparation, 2) formation of byproducts after and during derivatization, 3) stability of formed derivatives, 4) loss of analytes during multistep sample preparation, 5) increased time consumption. Therefore, there is an existing need to provide solutions for adopting derivatization into automated sample preparation procedures.

[0010] Especially in view of workflows, which are intended for automation and for enabling a high- throughput, it is of essence that as less as possible steps are performed as fast as possible and that these steps are reliable and reproducible performed at optimal time points of the workflow and using optimal reagents and quantities thereof. Regarding the optimal time point of integrating workflow steps in automated LC-MS( / MS) quantitative assays it is important to note, that available commercialized platforms offer a high degree of flexibility in the early stage of the sample processing due to the possible parallelization of sample-preparation devices. This eases the integration of multiple pipetting steps and long and individual incubation times into the early phase of the workflow. At later stages of such workflows, it is common that the numerous samples prepared during sample preparation are processes through a limited number of integrated workflow units (i.e. for magnetic bead separation or solid phase extraction separation units), a very limited numbers of stationary phases during liquid chromatography and finally a single mass spectrometer. This setup provides a good compromise between throughput, affordable pricing, low maintenance and costs and lab space requirement. However, as outlined above the available degree of parallelization & flexibility of such systems strongly decreases during the time course of the workflow due to fewer numbers of subsequent separation units (i.e. magnetic bead-separation stations or columns for chromatography) and the linear sample processing during mass spectrometry - a general schematic overview is given in Fig. 1. The fluent transition from a parallel to linear sample processing results in continuously shortened available timeframes for each sample at each station of the progressing workflow and thus makes integration of additional or sample-specific pipetting or incubation difficult, if not impossible. Consequently, there is an existing need to perform as much as possible sample-preparation steps in the first steps of automated LC-MS( / MS) quantitative assays. Translating this requirement into the integration of chemical derivatization of an analyte of interest into an automated LC-MS / MS workflow, would favor the so called in-situ derivatization (ISD). In ISD the derivatization reagent is directly added into the sample of interest in the early stage of the workflow, without any previous extraction steps and therefore easy to integrate into automated workflows. However, ISD suffers from several disadvantages, for example, that derivatization of an analyte of interest is carried out at a stage when said analyte is still diluted in it biological matrix , this causes low analyte concentration and the presence of co-reacting matrix components, which are usually present in high concentrations compared to the target analyte and therefore scavenge the derivatization. Consequently, ISD requires very large amounts of derivatization reagent and / or prolonged reaction times to achieve useful yield of the derivatization reaction. This can result in 1) unwanted dilution of the samples, 2) increased background / interferences caused by derivatization reagent, which limits LOQ, 3) formation of by-products, 4) additional clean-up steps prior analysis to remove the excess of derivatization reagent (to avoid ion suppression) and 5) additional pre-concentration steps to the derivatized analyte. [J Sep Sci. 2016 Jan;39(l):102-14], Another drawback of ISD is, that the derivatized analyte is formed in the early stage of the workflow, which makes analyte specific-enrichment by commercially available, highly specific analyte-specific antibodies (usually coupled to a solid phase) impossible. However, such analyte-specific enrichment methods are required to remove interferences to enable high sensitive detection of low abundant analytes (i.e. Annals of Clinical Biochemistry. 2019;56(6):646-653). In summary, ISD would be easy to integrate in high throughput automated workflows due early workflow pipetting, but is not favoured due to the above-mentioned drawbacks. A general schematic overview of an IDS workflow is shown in Fig. 2.

[0011] In contrast to that, ex-situ derivatization (ESD) proceeds via purification / extract! on of the analyte prior derivatization. Compared to ISD this is advantageous because 1) the analytes can be specifically enriched (i.e. using analyte-specific antibody- modified magnetic particles) and separated from isobaric and / or co-eluting interferences, 2) the derivatization reaction proceeds faster (due to pre-concentration of the analyte), which reduces the sample preparation time & increase throughput and 3) requires less derivatization reagent (due to the absence of co-reacting matrix components), which reduces ion suppression & reduces background interferences. A general schematic overview of an ESD workflow is shown in Fig. 3. However, ESD requires dedicated reagent addition of the derivatization reagent after analyte enrichment, which is as outlined above difficult to integrate without lowering the overall throughput and not compatible with existing platforms, i.e. cobas i601 mass spec analyzer. A workflow of a cobas i601 mass spec analyser is generally disclosed in EP 3 391 041 Bl, wherein it can be seen from, for example, Fig. 4, that no addition of derivatization reagent is possible after analyte enrichment - after elution, it is only possible to transfer the probe directly or after dilution to the liquid chromatography analysis.

[0012] In order to allow for, for example, odour masking and stabilisation, WO 2018 / 023005 Al for example thus discloses an encapsulated workflow reagent comprising an encapsulating material and a workflow reagent encapsulated within the encapsulating material, However, said encapsulated workflow reagent is somewhat inflexible as the encapsulated material is pre-installed in a sample preparation device, i.e. the workflow reagent encapsulated within the encapsulating material is static as it is attached to or adsorbed onto the surface of a chromatographic material - which is consequent, as the encapsulated workflow reagent is intended for sample preparation devices such as chromatographic columns. A major drawback of the pre-installed sample preparation device is the uncontrollable release of the workflow reagent into each processed sample once the device is installed. The non-specific release of workflows reagents can cause unwanted side-reactions (i.e. generation of non-ionizable products, isobaric products, co-eluting products) with analytes of interest, which are not intended to react with the workflow reagent. Other problems of this technology are 1) the dilution of the analyte on the stationary column, which will negatively impact the yield of the reaction between the analyte and the workflow reagent, 2) the limitations in terms of incubation time and temperature (vs controlled reactions in solution) and 3) non-constant and non-controllable amount of the release workflow reagent due to aging processes of the stationary device. Such pre-installed sample preparation devices might be beneficial for the overall throughput due to the avoidance of additional pipetting steps and enable some advantages of ESD in an automated manner, but will remain limited and applicable for a few selected & suitable analytes. The possibility to add specific reagents in a sample-or analytespecific manner in precise and reproducible amounts, with full flexibility in terms of reaction times and temperature is clearly preferred to enable a platform for numerous analytes on a single instrument. A general schematic overview of a pre-installed sample preparation device derivatization workflow is shown in Fig. 4.

[0013] In summary, in view of a workflows and reagents for analyte determination via LC-MS including derivatization and analyte-specific enrichment methods, there is still a need for methods, which overcome the above-described disadvantages.

[0014] The objective problem underlying the present invention was thus the provision of a method, which overcome the disadvantages indicated above.

[0015] Summary of the invention

[0016] This problem is addressed by a method for derivatizing an analyte of interest as well as by a method for determining an analyte of interest, a derivatized analyte of interest obtained or obtainable by the method for derivatizing an analyte of interest, a specific particle and its use with the features of the independent claims. Advantageous embodiments which might be realized in an isolated fashion or in any arbitrary combinations are listed in the dependent claims as well as throughout the specification.

[0017] As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements.

[0018] Further, it shall be noted that the terms “at least one”, “one or more” or similar expressions indicating that a feature or element may be present once or more than once, typically will be used only once when introducing the respective feature or element. In the following, in most cases, when referring to the respective feature or element, the expressions “at least one” or “one or more” will not be repeated, notwithstanding the fact that the respective feature or element may be present once or more than once.

[0019] Further, as used in the following, the terms "preferably", "more preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting alternative possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment of the invention" or similar expressions are intended to be optional features, without any restriction regarding alternative embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such a way with other optional or non-optional features of the invention.

[0020] 1staspect - Method for derivatizing an analyte of interest / method for determining an analyte of interest

[0021] This problem is addressed by a method for derivatizing an analyte of interest, the method comprising: i) Providing a) a particle comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry, an encapsulating material surrounding the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material and facing towards the outside of the particle; b) a magnetic particle having on its surface a second member of the binding pair, and optionally a binding agent specific for the analyte of interest; c) a liquid sample potentially containing an analyte of interest; d) optionally a further magnetic particle comprising a binding agent specific for the analyte of interest; ii) Contacting the particle of ia) with the liquid sample of ic) and subsequently with the magnetic particle of ib), and optionally with the further magnetic particle of id), so that the analyte of interest if present is bound to the magnetic particle of ib) and / or optionally to the further magnetic particle of id) via the binding agent; thereby obtaining a liquid remainder of the sample and one or more complexes, wherein at least one complex comprises the particle of ia) bound to the magnetic particle of ib) via the binding pair and optionally the analyte of interest if present bonded via the binding agent to the magnetic particle of ib), and optionally a further complex comprises the analyte of interest if present and the further magnetic particle of id) bonded via the binding agent to each other; iii) Spacely separating liquid remainder of the sample and the complex(es) from each other, thereby obtaining a supernatant comprising the liquid remainder of the sample; iv) Removing the supernatant, thereby obtaining separated complex(es); v) Optionally washing the separated complex(es) at least once, thereby obtaining washed complex(es); vi) Releasing the derivatization agent from the encapsulated system and releasing the analyte from the (optional further) magnetic particle from the complex(es) of iv) or the washed complex(es) of v) by applying release conditions; vii) Optionally separating the remainder(s) of the magnetic particle(s) from the derivatization agent and the analyte if present; viii) Allowing analyte of interest if present and derivatization agent to react, thereby potentially obtaining a derivatized analyte of interest.

[0022] Furthermore, the above-identified problem is addressed by a method for determining an analyte of interest, the method comprising i) to viii) as defined above with respect to the method for derivatizing an analyte of interest and further: ix) Subjecting the derivatized analyte of interest obtained in viii) to analysis, preferably to a mass spectrometric analysis, wherein the mass spectrometric analysis is preferably liquid chromatography-mass spectrometry (LC-MS), more preferably Liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0023] It was found that a particle comprising a derivatization agent encapsulated in an encapsulating material with a first member of a binding pair bonded to at least a part of the encapsulating material (facing to the outer surface of the particle) in combination with a magnetic particle having on its surface a second member of the binding pair, and optionally a binding agent specific for an analyte of interest, allows for a very simplified workflow protocol, wherein concurrent and selective capturing of the analyte and the particle by the magnetic particle, magnetic separation and enrichment, as well as concurrent and selective elution of the analyte and freeing the derivatization agent, followed by instant derivatization in a “one- pot” reaction was achieved in a workflow requiring a minimum of working steps, even in the presence of a (challenging) biological matrix. The method for derivatizing an analyte of interest as well as the method for determining an analyte of interest as described above offer the considerable advantage that the derivatization of analyte of interest occurs after magnetic particle-based enrichment of analyte, which is more efficient due to the absence of interfering matrix components and thus allows to increase the derivatization yield and consequently the sensitivity. The method according to the present invention is advantageous for using with / in a cobas i601 mass spec analyser.

[0024] In the following, when reference is made to “the method”, this means the method for deri- vatizing an analyte of interest as well as the method for determining an analyte of interest except if details regarding step ix) are described, which relates to the method for derivatizing an analyte of interest.

[0025] Derivatization agent

[0026] In some preferred embodiments of the method, the derivatization agent is selected from the group consisting of Cookson-type reagent (4-substituted-l, 2, 4-triazoline-3, 5-dione), Am- plifex Diene ((salt of) 1-propanaminium, 3-[[[l-[4-(3,5-dihydro-3,5-dioxo-4H-l,2,4-tria- zol-4-yl)phenyl]ethylidene]amino]oxy]-N,N,N-trimethyl, CAS 1318851-03-4), Amplifex Keto ((salt of) 3 -(Aminooxy)-N,N,N-trimethyl- 1-propanaminium, CAS 1318790-35-0), Girard T (trimethylacetohydrazide ammonium chloride, CAS 123-46-6), Girard P (l-(2- hydrazinyl-2-oxoethyl)pyridin-l-ium chloride, CAS 1126-58-5), compound having a permanent net charge >1, substituted benzyl compound, and mixture of two or more thereof.

[0027] In some preferred embodiments of the method, the derivatization agent is selected from the group consisting of Cookson-type reagent (4-substituted-l, 2, 4-triazoline-3, 5-dione), Amplifex Diene, Amplifex Keto, Girard T, Girard P, and mixture of two or more thereof.

[0028] In some preferred embodiments of the method, the derivatization agent is a compound having a permanent net charge >1, wherein said compound comprises at least three units Zl, Z2, Q and optional a further unit LI, wherein the units are covalently linked to each other, wherein:

[0029] Q is a reactive unit capable of forming a covalent bond with the analyte,

[0030] Zl is a charged unit comprising at least one permanently charged moiety, in particular a permanently positive charged moiety or a permanently negative charged moiety,

[0031] Z2 is a charged unit comprising at least one permanently charged moiety, in particular a permanently positive charged moiety or a permanently negative charged moiety, and L1 is a substituted or non- substituted linker, preferably a group cleavable via fragmentation, more preferably a fragmentation moiety selected from the group consisting of Me Lafferty fragmentation moiety, Retro Diels Alder fragmentation moiety and aliphatic.

[0032] Preferably, the reactive unit Q is selected from the group consisting of a carbonyl reactive unit, a diene reactive unit, a hydroxyl reactive unit, an amino reactive unit, an imine reactive unit, a thiol reactive unit, a diol reactive unit, a phenol reactive unit, an epoxide reactive unit, a disulfide reactive unit, and an azido reactive unit; and / or, preferably and, Z1 is a charged unit comprising at least one permanently charged moiety, preferably a permanently positive charged moiety or a permanently negative charged moiety, and / or, preferably and, Z2 is a charged unit comprising at least one permanently charged moiety, preferably a permanently positive charged moiety or a permanently negative charged moiety; and / or, preferably and, LI is a substituted linker or non-substituted linker, preferably a cleavable group via fragmentation, more preferably selected from the group consisting of McLafferty fragmentation moiety, Retro Diels Alder fragmentation moiety and aliphatic fragmentation moiety.

[0033] The “net charge” means the total electric charge of the compound. “Permanent” means that said net charge is not readily reversible, for example, via flushing, dilution, filtration, and the like as defined in detail in WO 2021 / 233997 AL Preferably, the compound having a permanent net charge >1 and preferred embodiments thereof are as disclosed in detail in WO 2021 / 233997 AL Said definition and preferred embodiments thereof are incorporated herein by reference. Especially preferred as compound having a permanent net charge >1 are the so called labels 1 to 20, as disclosed in detail on pages 37 to 49 of WO 2021 / 233997 Al; especially these preferred compounds are incorporated herein by reference.

[0034] In some preferred embodiments of the method, the derivatization agent is a compound having a permanent net charge >1, wherein said compound is of formula (A):

[0035] X - LI - Y - L2 - Z (A) wherein

[0036] X is a reactive unit, which is in particular capable of forming a covalent bond with an analyte molecule,

[0037] LI and L2 are independently of each other substituted or non-substituted linker, in particular linear linker,

[0038] Y is a neutral loss unit,

[0039] Z is a charged unit comprising at least one permanently charged moiety, including any salt thereof. Preferably, the reactive unit X is selected from the group consisting of a carbonyl reactive unit, a diene reactive unit, a hydroxyl reactive unit, a diol reactive unit, an amino reactive unit, a thiol reactive unit , a phenol reactive unit, an epoxide reactive unit, a disulfide reactive unit, and an azido reactive unit. The neutral loss unit Y is able to loose a moiety (a neutral entity) having no charge. The neutral loss unit Y is capable of fragmentation, i.e. under conditions of MS, wherein the neutral loss unit Y releases at least one neutral entity upon ionization. The neutral entity is a low molecular weight neutral entity, in particular in a range of 10-100 Da, in particular 20-80 Da, in particular 25-65 Da, wherein more preferably the neutral entity is selected from the group consisting of N2, NO, NO2, S2, SO, SO2, CO, CO2. In particular embodiments, the neutral entity is N2. The charged unit Z is positively or negatively charged, preferably positively charged, wherein the positively charged unit Z, is preferably choosen in a manner that the resulting compound of formula (A) has a pKa of 10 or higher, more particularly has a pKa of 12 or higher. When the charged unit Z is a negatively charged unit, said negatively charged unit Z is preferably chosen in a manner that the resulting compound of formula (A) has a pKb of 10 or higher, more particularly has a pKb of 12 or higher. The linkers LI and L2 are independently of each other linear linkers, wherein the linear linkers L 1 and L2 are independently of each other a single bond between two functional units of the compound of formula (A), or comprise 1 to 10 C-atoms, in particular 1 to 6 C-atoms, in particular 1, 2, or 3 C-atoms. The linear linker LI and L2 comprises independently of each other 1 or more heteroatoms, in particular N, O or S. Preferably, the linkers LI and L2 are independently of each other substituted or unsubstituted, in particular the linker LI and L2 are unsubstituted. Also here, the “net charge” means the total electric charge of the compound. “Permanent” regarding a charged moiety means that said charge is not readily reversible, for example, the charge does not alter based on the surrounding conditions, e.g. the change of the pH value does not lead to a change in the charge of the permanently charged unit as defined in detail in WO 2020 / 020851 AL Preferably, the compound having a permanent net charge >1 and preferred embodiments thereof are as disclosed in detail in WO 2020 / 020851 AL Said definition and preferred embodiments thereof are incorporated herein by reference. Especially preferred as compound having a permanent net charge >1 are the so called labels 1 to 12, as disclosed in detail on pages 30 to 32 of WO 2020 / 020851 Al; especially these preferred compounds are incorporated herein by reference.

[0040] In some preferred embodiments of the method, the derivatization agent is a compound having a permanent net charge >1, which is a compound of general formula (B): X-Ll-Y(-L2-Z)r(B) wherein

[0041] X is a reactive group capable of reacting with an analyte molecule, whereby a covalent bond with the analyte molecule is formed, LI is a bond or a spacer,

[0042] Y is a neutral ion loss unit, L2 is a bond or a spacer,

[0043] Z is a charge unit comprising at least one charged moiety, in particular a permanently charged moiety, r is 0 or 1.

[0044] Preferably, the reactive unit X is selected from the group consisting of a carbonyl-reactive group, a dienophilic group, a carboxylate reactive group, a phenol reactive group, an amino reactive group, a hydroxyl reactive group, a thiol reactive group, an amine reactive group, a dienophilic group, a 1,2 dial reactive group, a 1 -amino 2-hydroxy alkyl reactive group, an 1- amino 2-mercapto reactive group, and a group reacting at the ortho positions of phenols. The neutral ion loss unit Y is a unit, which is able to lose a moiety having no charge. Said moiety includes, but is not limited to ions, atoms and a plurality of atoms. The unit Y is neutral, i.e. it does not carry a positive or negative charge and it is linked to the reactive group X via a linker LI. The neutral unit Y is, under conditions of MS, capable of fragmentation, whereby a neutral species is released. After release of the first neutral species, the remainder of unit

[0045] Y still remains neutral. Typically, but not necessarily, one neutral species is released, i.e. a first neutral species is released. In some embodiments, two neutral species are released. The charge unit Z comprising at least one positively charged moiety. Preferably, the charge unit Z consists of one positively charged moiety and lacks any groups capable to undergo an alternative fragmentation, The charge may be permanent, e.g. when using a quaternary ammonium group, or may be generated by protonation (positive charge) or deprotonation (negative charge), wherein a permanent charge is preferred. Also here, “permanent” regarding a charged moiety means that said charge is not readily reversible, for example, the charge does not alter based on the surrounding conditions, e.g. the change of the pH value does not lead to a change in the charge of the permanently charged unit as defined in detail in WO 2018 / 141821 AL Preferably, the compound having a permanent net charge >1 and preferred embodiments thereof are as disclosed in detail in WO 2018 / 141821 AL Said definition and preferred embodiments thereof are incorporated herein by reference. Especially preferred as compound having a permanent net charge >1 are the compounds (la), (9a), (10a), (4b), (lb), (9b), (10b), as disclosed in detail on pages 60 to 67 of WO 2018 / 141821 Al; especially these preferred compounds are incorporated herein by reference.

[0046] In some preferred embodiments of the method, the derivatization agent is a substituted benzyl derivative, which is preferably a compound of formula (C) wherein one of the substituents B1, B2, B3, B4, B5is a coupling group Q, which is capable of forming a covalent bond with the analyte,

[0047] A1, A2, B1, B2, B3, B4, B5are each independently selected from the group consisting of hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,Ndialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, and isotope thereof,

[0048] A3comprises a group selected from the group consisting of ammonium, pyridinium, and phosphonium, wherein in case of A3being ammonium and B1or B5being the coupling group Q, the coupling group Q comprises a C atom, which is separated by four single or double bonds from the C atom of the CA1A2A3substituent and the coupling group Q comprises a C-atom, which is separated by five single or double bonds from the C atom of the CA1A2A3substituent, preferably A3is ammonium, B1or B5is Q, wherein Q is free of at least one atom, which is selected from the group of oxygen atom, nitrogen atom, sulphur atom and bromine atom.

[0049] Preferably, the compound of formula (C) and preferred embodiments thereof are as disclosed in detail in WO 2021 / 234002 Al - said definition and preferred embodiments thereof are incorporated herein by reference, specially preferred compounds having a permanent net charge >1 are the so called labels 1-25 as disclosed in WO 2021 / 234002 Al in Table 2 on page 39 to page 45; especially these preferred compounds are incorporated herein by reference.

[0050] Encapsulating material

[0051] In some preferred embodiments of the method, the encapsulating material is a material capable of controlled release of the derivatization agent encapsulated therein. In some preferred embodiments of the method, the encapsulating material is a material capable of controlled release of the derivatization agent encapsulated therein upon contact with a compound selected from the group consisting of a polar solvent, preferably a polar protic solvent, an apolar solvent, an organic acid, an organic base, an inorganic acid, an inorganic base, a detergent, an oxidizing reagent, a reducing reagent, and a mixture of two or more thereof; and / or upon application of a treatment selected from the group consisting of heating, light exposure, ultrasound treatment or a combination of two or more of these treatments.

[0052] In some preferred embodiments of the method, the encapsulating material is selected from the group consisting of lipid, detergent, polymer and mixed forms thereof.

[0053] The polymer(s) used as encapsulating material are preferably stimuli-responsive as described by M. A. C. Stuart et al. in Nature Mater 9, 101-113 (2010). Mixed forms of lipid an polymer used as encapsulating material are so called polymersomes, which are as disclosed by T. Anajafi et al. (2015) Polymersome-Based Drug-Delivery Strategies for Cancer Therapeutics, Therapeutic Delivery, 6:4, 521-534; Landfester et al (2018), Liposomes and polymersomes: a comparative review towards cell mimicking, Chem. Soc. Rev., 2018, 47, 8572; Che et al. (2023) Recent advances in permeable polymersomes: fabrication, responsiveness, and applications, Chem. Sci., 2023, 14, 7411-7437; Kumar et al (2024) Polymersomes: Beyond Basics-Synthesis, Stimuli Response and Biomedical Applications, Chemistry Select 2024, 9, e20240204 and Men et al. (2024) Review of Shape Transformation Pathways of Polymersomes: Implications for Nanomotor, Biomedicine, and Artificial Cell Mimics, ACS Appl. Nano Mater. 2024, 7, 13, 14865-14888.

[0054] A detergent is preferably selected from the group consisting of non-ionic detergent, ionic detergent, zwitter ionic detergent and mixtures of non-ionic detergent and ionic detergent. For example, suitable detergent(s) are as disclosed inhttps: / / www.serva.de / enDE / 275_In- formation_Center_Detergents_Classification_of_Detergents.html. For example a non ionic detergent is selected from the group consisting of Polidocanol, Brij 35, Digitonin, Do- decyl-B-D-maltoside, Octyl-B-D-glucopyranodide, Synperonic F68, Synperonic F108, Tergitol 15-S-9, Tween 20, Tween 80, and mixtures of two or more thereof. For example, a ionic detergent is selected from the group consisting of CTAB, Na-cholate, Na-deoxycho- late, Na-N-lauroylsarcosinate, SDS and mixtures of two or more thereof. For example, a zwitterionic detergent is selected from the group consisting of ASB-14, ASB-16, CHAPS, SB 3 - 10, SB 12, and mixtures of two or more thereof. Preferably, the encapsulating material is a lipid. More preferably, the lipid is selected from the group consisting of monoglyceride, diglyceride, triglyceride, wax, sterol, phospholipid and mixtures of two or more thereof.

[0055] In some preferred embodiments of the method, the monoglyceride is an ester of glycerol and a C4 to C28 saturated or unsaturated carboxylic acid; the diglyceride is a diester of glycerol and two C4 to C28 saturated or unsaturated carboxylic acids, which are the same or different; and a triglyceride is a triester of glycerol and three C4 to C28 saturated or unsaturated carboxylic acids, which are the same or different.

[0056] In some preferred embodiments of the method, the sterol is cholesterol.

[0057] In some preferred embodiments of the method, the phospholipid comprises two C4 to C28 saturated or unsaturated carboxylic acids, which are the same or different; wherein the phosphate group is optionally modified with at least one nitrogen containing compound selected from choline, ethanolamine and serine or with glycerol.

[0058] In some preferred embodiments of the method, the encapsulating material comprises a mixture of l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (16:0 PG), 1,2- dipalmitoyl-sn-glycero-3 -phosphocholine (16:0 PC), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(biotinyl) (16:0 Biotinyl PE) and cholesterol.

[0059] In this regard, it is important to note that the herein presented method for derivatizing an analyte of interest preferably uses a lipid as encapsulating material i.e. liposomes for particles comprising a derivatization reagent. As evidenced by Landfester et al (2018), Liposomes and polymersomes: a comparative review towards cell mimicking, Chem. Soc. Rev., 2018, 47, 8572, it is clear to a person skilled in the art that polymersomes can also be used as a particle comprising a derivatization reagent within the herein presented method for derivatizing an analyte of interest.

[0060] Binding pair

[0061] In some preferred embodiments of the method, the binding pair is selected from the group consisting of antigen (including antigen fragments, epitopes and haptens) and corresponding antigen-specific antibody (including antibody fragments); ligand and ligand-specific receptor (both independently selected from a group consisting of peptide, protein, oligonucleotide, polynucleotide and small molecule); complementary nucleic acid (including non-natural nucleic acid like (but not limited to) peptide nucleic acid (PNA), locked nucleic acid (LNA), 2’-0-methyl RNA (2’0Me), phosphorthioate (PS) including L-form thereof,), binder 1 and binder 2 (binder 1 being selected from the group consisting of small molecule, peptide, protein and oligonucleotide and binder 2 being a binder 1 specific aptamer), biotin and avidin; biotin and (poly)streptavidin; biotin and neutravidin; strep-tag and strep-tactin, lectin and carbohydrate; wherein the first member of a binding pair and the respective second member of a binding pair are selected to form one of the aforementioned binding pairs.

[0062] An “oligonucleotide” consists of 2 to 12 nucleotides, a “polynucleotide” has more than 12 nucleotides, both irrespective whether the nucleobases are ribose or desoxyribose. The term “polynucleotide” as well as the term “peptide” includes a respective aptamer, which is a short single-stranded DNA or RNA oligonucleotide having in the range of from 25 to 70 bases or a peptide having in the range of from 25 to 70 amino acids that can bind a specific molecule via their three dimensional structure. A ’’small molecule” is an organic compound that may regulate a biological process, and which has a molecular weight of < 1000 daltons.

[0063] In some preferred embodiments of the method, the binding pair is selected from the group consisting of biotin and avidin; biotin and (poly)streptavidin; and biotin and neutravidin.

[0064] Magnetic particle

[0065] In some preferred embodiments of the method, the magnetic particle of ib) and optionally of id) comprises at least one magnetic core (M), a polymer matrix (P) comprising a, polymer, which is preferably crosslinked and optionally hypercrosslinked, at least partially surrounding the magnetic core (M), wherein the at least one magnetic core (M) comprises a magnetic nanoparticle and optionally a coating (C), and at least one remainder of a functional group (RFG), optionally bonded via a linker (L) to the polymer matrix (P), wherein the RFG is connected to the second member of the binding pair of the magnetic particle of ib) and optionally connected to the binding agent specific for the analyte of interest of ib) or connected to the further magnetic particle to the binding agent specific for the analyte of interest of id). The RFG is connected to the second member of the magnetic particle of ib), optionally to the binding agent specific for the analyte of interest of ib) or connected to the further magnetic particle to the binding agent specific for the analyte of interest of id) by means of covalent or non-covalent bond.

[0066] Magnetic particles are commercially available in a broad variety. For example, suitable magnetic particles are purchasable from Roche, Merck, Thermo Fisher, CD Bioparticles, or Nanocs. A “magnetic particle” may, in principle, display any geometrical form, however, preferably, the particle is substantially spherical. As used herein, the term “substantially spherical” refers to particles with rounded shapes that are preferably non-faceted or substantially free of sharp comers. In certain embodiments, the substantially spherical particles typically have an average aspect ratio of less than 3: 1 or 2: 1, for example, an aspect ratio less than 1.5: 1, or less than 1.2: 1. In a certain embodiment, substantially spherical particles may have an aspect ratio of about 1 : 1. The aspect ratio (AR) is defined as being a function of the largest diameter (dmax) and the smallest diameter (dmin) orthogonal to it (AR = d min / dmax ). The diameters are determined via SEM or light microscope measurements.

[0067] The at least one magnetic core (M) comprises a compound selected from the group consisting of metal, metal carbide, metal nitride, metal sulfide, metal phosphide, metal oxide, metal chelate and a mixture of two or more thereof. In some embodiments, the at least one magnetic core (M) comprises a metal oxide or a metal carbide, more preferably, an iron oxide, in particular an iron oxide selected from the group consisting of FesCU, a-Fe2O3, y- Fe20s, MnFepOq, CoFepOq, NiFepOq, CuFepOq, ZnFepOq„ CdFepOq, BaFepO and SrFepO, wherein p and q vary depending on the method of synthesis, and wherein p is preferably an integer of from 1 to 3, more preferably 2, and wherein q is preferably 3 or 4 most preferably, FesCk Preferably, the at least one magnetic core (M) comprises at least one magnetic nanoparticle, more preferably at least one iron oxide nanoparticle, more preferably a FesCU-nan oparticle.

[0068] The at least one magnetic core (M) comprises, more preferably consists of a magnetic nanoparticle and an optional coating (C).

[0069] The optional coating (C) is present on at least a part of the surface of the magnetic core (M), preferably on at least 90 % of its surface, more preferably on its whole surface. The coating (C) is preferably selected from the group consisting of silica, silicate, silane, phosphate, phosphonate, phosphonic acid, fatty acid, and mixtures of two or more thereof. In some embodiments, the coating (C) is selected from the group consisting of silica, tetraethyl orthosilicate, 3 -(trimethoxy silyl)propyl methacrylate, vinyltrimethoxy silane, vinyltri ethoxysilane, allyltrimethoxysilane, allyltri ethoxy silane, triethoxy vinylsilane, 3 -(trimethoxy si - lyl)propyl acrylate, trimethoxy(7-octen-l-yl)silane, trimethoxymethylsilane, triethoxymethylsilane, ethyltrimethoxysilane, triethoxy(ethyl)silane, trimethoxyphenylsilane, tri- methoxy(2-phenylethyl)silane trimethoxy(propyl)silane, n-propyltriethoxysilane, isobu- tyl(trimethoxy)silane, isobutyltriethoxysilane, vinylphosphonic acid, dimethyl vi- nylphosphonate, diethyl vinylphosphonate, diethyl allylphosphonate, diethyl allyl phosphate, diethyl (2-methylallyl)phosphonate, octylphosphonic acid, butylphosphonic acid, decylphosphonic acid, hexylphosphonic acid, hexadecylphosphonic acid, n-do- decylphosphonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, and arachidic acid,. propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, tridecylic acid, pentadecylic acid, margaric acid, nonadecylic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentaco- sylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid, hexatriac- ontylic acid, heptatriacontanoic acid, octatriacontanoic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, hexadecatrienoic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, heneicosapentaenoic acid, docosapentaenoic acid, clupanodonic acid, docosahexaenoic acid, tetracosapentaenoic acid, tetracosahex- aenoic acid, calendic acid, eicosadienoic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid, tetraco-satetraenoic acid, tetracosapentaenoic acid, 5-dodecenoic acid, 7- tetradecenoic acid, palmitoleic acid, vaccenic acid, paullinic acid, 15-docosenoic acid, 17- tetracosenoic acid, elaidic acid, gondoic acid, mead acid, erucic acid, nervonic acid, rumenic acid, calendic acid, jacaric acid, eleostearic acid, catalpic acid, punicic acid, rumelenic acid, parinaric acid, bosseopentaenoic acid, pinolenic acid, podocarpic acid and mixtures of two or more thereof.

[0070] The magnetic particle has a polymer matrix, wherein the polymer matrix (P) comprises a crosslinked polymer, wherein the crosslinked polymer preferably comprises a co-polymer obtained or obtainable by a method comprising a polymerization of at least two different monomeric building blocks selected from the group consisting of styrene, functionalized styrenes, vinylbenzylchloride, divinylbenzene, vinylacetate, methylmethacrylate and acrylic acid. Preferably, at least one monomeric building block used has one or more functional group(s) reactive towards amine groups or amine groups, wherein the functional group reactive towards amine groups and / or hydroxyl groups is preferably selected from the group of halogenated Cl-C3-alkyl group, halogen atom, epoxy group and activated carboxy group, more preferably the functional group reactive towards amine groups and / or hydroxyl groups is a halogenated Cl-C3-alkyl group, more preferably a -CH2-CI group. Preferably, vinylben- zyl chloride is employed as monomeric building block having functional groups reactive towards amine groups and / or hydroxyl groups. Preferably, at least one further monomeric building block is a crosslinking agent, preferably selected from the group consisting of divinylbenzene, bis(vinylphenyl)ethane, bis(vinylbenzyloxy)hexane, bis(vinylbenzyloxy)do- decane, and mixtures of two or more thereof, wherein more preferably the crosslinking agent comprises at least divinylbenzene. Preferably, the co-polymer is obtained or obtainable by a method comprising a polymerization of at least two different monomeric building blocks selected from the group consisting of the following monomers: with Rv, Rw, Rx, Ryand Rz, are, independently of each other selected from the group consisting of -N3, -NH2, -Br, -I, -F, -NR’R”, -NR’R”R”’, -COOH, -CN, -OH, -OR’, -COOR’, - NO2, -SH2, -SO2, -R’(OH)X, -R’(COOH)X, -R’(COOR”)X, -R’(OR”)X, -R’(NH2)X, - R’(NHR”)X, -R’(NR”R’”)X, -R’(C1)X, -R’(I)X, -R’(Br)x, -R’(F)X, R’(CN)X, -R’(N3)X, - R’(NO2)X, -R’(SH2)X, -R’(SO2)X, alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl and with R’, R” and R’” being, independently of each other, selected from the group consisting of alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, halides, hydrogen, sulfides, nitrates and amines, and wherein x is an integer in the range of from 1 to 3. Preferably, the polymer matrix is obtained or obtainable by a method comprising co-polymerizing at least one monomeric building block, which is a crosslinking agent, and at least one monomeric building block having functional groups reactive towards amine groups and / or hydroxyl groups, wherein the molar ratio of crosslinking agent : monomeric building block having functional groups reactive towards amine groups and / or hydroxyl groups is in the range of from 10: 1 to 1 : 10, preferably in the range of from 5: 1 to 1 :5, more preferably in the range of from 2: 1 to 1 :2, more preferably in the range of from 1.5: 1 to 1 : 1.5, more preferably in the range of from 1.2: 1 to 1 : 1.2.

[0071] A functional group is preferably selected from the group consisting of amino group, azide group, alkyne group, carboxyl group, thiol group, epoxy group, aryl group and alkyl group. “Aryl group” preferably means a phenyl group and “alkyl group” preferably means a C2 to C20 alkyl group. A “remainder of a functional group” (RFG) means the chemical moiety that remains after a bond, preferably an adsorptive bond or a covalent bond, more preferably a covalent bond, has formed between the functional group and the second member of the binding pair of the magnetic particle of ib) and optionally the binding agent specific for the analyte of interest of ib) or the binding agent specific for the analyte of interest of id).

[0072] A linker (L) means a chain of atoms forming a backbone, wherein the backbone has a length in the range of from 2 to 50 atoms, preferably a length in the range of from 2 to 40 atoms, more preferably a length in the range of from 2 to 30 atoms, more preferably a length in the range of from 2 to 20 atoms. All atoms forming the backbone are covalently bondend to each other. In one embodiment, the backbone consists of carbon atoms and one or more heteroa- toms selected from O, N and S, optionally comprising at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (wherein e.g. a phenylene ring accounts for a length of four atoms). Heteroatoms at interior positions are unsubstituted or have one or more substituents selected from the group consisting of hydrogen atom, Cl -CIO alkyl and =0, wherein each Cl -CIO alkyl may be unsubstituted or substituted with a N[(CH2)rCOO(H)]2 group. In some embodiments, the one or more heteroatom(s) are part of a linkage, wherein the linkage is preferably selected from the group consisting of ether linkage, amide linkage, ester linkage, carbamate linkage and urea linkage. Carbon atoms in the chain of atoms of the backbone are substituted with one or more substituents selected from the group consisting of hydrogen atom and Cl to CIO alkyl group, wherein each Cl to CIO alkyl group may be unsubstituted or may have at least one substituent selected from hydroxyl group, amino group or carboxyl goup. The term "alkyl" by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, having the number of carbon atoms designated (i.e. C1-C10 means one to ten carbon atoms). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl.

[0073] Analyte of interest

[0074] The term "Analyte of interest" is used for a compound, which is analyzable or analyzed via mass spectrometry. An analyte of interest can be any kind of molecule present in a living organism, without any limitation as long as it is analyzable via mass spectrometry.

[0075] In some preferred embodiments of the method, the analyte of interest is selected from the group consisting of nucleic acid (e.g. DNA, mRNA, miRNA, rRNA etc.), amino acid, peptide, protein (e.g. cell surface receptor, cytosolic protein), metabolite or hormone (e.g. testosterone, estrogen, estradiol), fatty acid, lipid, carbohydrate, steroid, ketosteroid, secosteroid (e.g. Vitamin D), molecule 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 drug, drug of abuse, toxin.) or a metabolite of such a substance, and mixtures of two or more thereof, preferably the analyte of interest is testosteron.

[0076] In some preferred embodiments of the method, the liquid sample as used herein refers to a biological sample obtained for the purpose of evaluation in vitro. In the methods of the present invention, the liquid sample preferably may comprise any body fluid. Preferred fluid samples are whole blood, serum, plasma, bronchioalveolar lavage (BAL), epithelial lining fluid (ELF), urine or sputum, with plasma or serum being most preferred.

[0077] The term liquid sample includes biological fluid samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides.

[0078] The aforementioned applications for determining analytes in liquid samples may, preferably, be applied or are involved in diagnostic purposes, drug of abuse testing, environmental control, food safety, quality control, purification or manufacturing processes. In diagnostic applications, the qualitative or quantitative determination of an analyte may allow aiding the diagnosis if the analyte is, e.g., a biomarker for a disease or medical condition. Similarly, the qualitative or quantitative assessment of an analyte being an indicator for environmental changes may help to identify pollution or to make assessments of environmental changes. Food safety as well as manufacturing or purification processes may be controlled by qualitative or quantitative determination of indicator analytes. Such indicators may also be determined in connection with general aspects of quality control, e.g., also in storage stability assessments of products and the like.

[0079] The method for derivatizing an analyte of interest as well as the method for determining an analyte of interest are in vitro methods.

[0080] Binding agent specific for the analyte of interest

[0081] In some preferred embodiments of the method, binding agent specific for the analyte of interest is an analyte specific antibody or a fragment thereof, preferably an analyte specific antibody or fragment thereof having sufficient affinity, selectivity, specificity, cross-reactivity, and / or stability as known to the skilled person. Preferably, the analyte specific antibody or a fragment thereof has a dissociation constant (KD) relative to the analyte of interest of at least 1 x 10'6M.

[0082] A fragment of an antibody is preferably selected from the group consisting of F(ab), F(ab‘)2, scFv and dsFv. The binding agent specific for the analyte of interest may be coupled in any suitable way to the (further) magnetic particle as long as the coupling is sufficiently stable to maintain the binding agent connected to the (further) magnetic particle. In some embodiments, the binding agent specific for the analyte of interest may be bound to the (further) magnetic particle also via a binding pair as described in more detail above. In some embodiments, the (further) magnetic particle and the binding agent are bound to each other via a binding pair selected from the group consisting of biotin and avidin; biotin and (poly)strep- tavidin; and biotin and neutravidin.

[0083] Particle - product-by-process

[0084] In some preferred embodiments of the method, a particle comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry, an encapsulating material surrounding the derivatization agent, which comprises one or more lipid(s) and / or one or more polymer(s) and / or mixed forms thereof (polymersomes), wherein at least a part of the lipid(s) and / or of the polymer(s) and / or mixed forms thereof carries the first member of the binding pair and the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material and facing towards the outside of the particle, is obtained or obtainable from sonication of a liquid mixture comprising the lipid(s) and / or of the polymer(s) and / or mixed forms thereof and the derivatization agent.

[0085] Sonication of the liquid mixture comprising the lipid(s) and the derivatization agent is preferably done at elevated temperature (>40°C, more preferably > 50°C, preferably below 100°C) and suitable conditions (i.e. osmality, solvent and ratio of lipids and encapsulant), which are known to the skilled person.

[0086] Contacting step ii)

[0087] In some preferred embodiments of the method, contacting the particle of ia) with the liquid sample of ic) and subsequently with the magnetic particle of ib), and optionally with the further magnetic particle of id) according to ii) is done in liquid phase, preferably in solution, more preferably in aqueous solution, more preferably in buffered aqueous solution; and / or, preferably and, at a temperature in the range of from 0 to 50°C, preferably in the range of from 10 to 30°C; and / or, preferably and, under stirring, preferably under agitation, preferably controlled agitation, wherein“(controlled) agitation” means one or more selected from the group consisting of shaking, rocking, waving rotation, and vortex mixing.

[0088] In some preferred embodiments of the method, ii) comprises ii- 1) Contacting the particle of ia), which carries a first member of a binding pair, with the liquid sample of ic) and subsequently with the magnetic particle of ib), which has on its surface a second member of the binding pair, and a binding agent specific for the analyte of interest, thereby obtaining a liquid remainder of the sample and a complex, comprising the particle of ia) and the analyte of interest if present both bound to the magnetic particle of ib) via the binding pair and the binding agent respectively.

[0089] In some preferred embodiments of the method, ii) comprises ii-2) Contacting the particle of ia), which carries a first member of a binding pair, with the liquid sample of ic)and subsequently with the magnetic particle of ib), which has on its surface a second member of the binding pair, with the further magnetic particle of id) comprising a binding agent specific for the analyte of interest; thereby obtaining at least two complexes, wherein one complex comprises the particle of ia) is bound to the magnetic particle of ib) via the binding pair, and another complex, which comprises the analyte of interest if present is bound to the further magnetic particle of id) via the binding agent, and a liquid remainder of the sample.

[0090] Separation step Hi)

[0091] In some preferred embodiments of the method, spacely separating liquid remainder of the sample and the complex(es) obtained in ii), ii-1) or ii-2) from each other according to iii) is done by application of a magnetic field to enable magnetic bead separation.

[0092] Removing the supernatant, which comprises the liquid remainder of the sample, according to iv) is done by any suitable method known in the art such as, for example, decanting and pipetting. The optional washing step v) is done by any suitable method known in the art such as, for example, by using an aqueous buffer solution having pH in the range of from 5 to 9, preferably in the range of from 6 to 8, more preferably in the range of from 6.5 to 7.5, for example commercially available PreClean M (Phosphate buffer 10 mmol / L; sodium chloride 20 mmol / L; detergent < 0.1 weight-%; preservative; pH 7.0).

[0093] Release step vi)

[0094] As indicated above, releasing the derivatization agent from the encapsulated system according to vi) is done by applying release conditions. Preferably, releasing the derivatization agent from the encapsulated system according to vi) is done upon contact with a compound selected from the group consisting of a polar solvent, preferably a polar protic solvent, an apolar solvent, an organic acid, an organic base, an inorganic acid, an inorganic base, a detergent, an oxidizing reagent, a reducing reagent, and a mixture of two or more thereof; and / or upon application of a treatment selected from the group consisting of heating, light exposure, ultrasound treatment or a combination of two or more of these treatments. In some preferred embodiments of the method, releasing the derivatization agent from the encapsulated system according to vi) is done by contacting the complex(es) with a polar solvent, optionally in combination with an acid.

[0095] In some preferred embodiments of the method, the polar solvent is preferably one or more polar protic solvent(s), more preferably selected from the group of water, Cl to C5 alkyl alcohols and mixtures of two or more thereof.

[0096] In some preferred embodiments of the method, releasing the derivatization agent from the encapsulated system and releasing the analyte from the (optional further) magnetic particle according to vi) is done is done with addition of a mixture containing an acid , preferably an organic acid, more preferably selected from the group consisting of citric acid, formic acid, acetic acid, trifluoracetic acid, difluor acetic acid, trichloroacetic acid, dichloroacetic acid, and mixtures of two or more thereof, more preferably at least with addition of citric acid and / or formic acid. The acid is applied in the form of a mixture for opening the encapsulated system, preferably a mixture of the acid with water and / or alcohol, wherein the alcohol is a Cl to C5 alkyl alcohol, preferably methanol.

[0097] In some preferred embodiments of the method, releasing the derivatization agent from the encapsulated system and releasing the analyte from the (optional further) magnetic particle according to vi) is done in the presence of an additive, preferably selected from the group of lewis bases, more preferably at least m-phenylenediamine.

[0098] Reaction step of viii)

[0099] In some preferred embodiments of the method, at least viii) is done at a temperature in the range of from 0 to 90°C, preferably in the range of from 10 to 90°C, more preferably in the range of from 20 to 85°C, more preferably in the range of from 30 to 80°C, more preferably in the range of from 40 to 75°C, more preferably in the range of from 50 to 70°C.

[0100] Allowance of viii)

[0101] Allowing analyte of interest if present and derivatization agent to react, thereby potentially obtaining a derivatized analyte of interest, of viii) means that the appropriate conditions are applied and sufficient time is given for the reaction to generate a sufficient yield of the derivatized analyte of interest for the intended application (i.e. detection, quantification); the respective conditions and yields are known to the skilled person. Method for determining an analyte of interest - Analysis ix)

[0102] As indicated above, the method for method for determining an analyte of interest comprises steps i) to viii) and further an analysis step ix). The derivatized analyte of interest obtained in viii) is in ix) subjected to analysis, preferably to a mass spectrometric analysis, wherein the mass spectrometric analysis is preferably liquid chromatography-mass spectrometry (LC-MS), more preferably Liquid chromatography-tandem mass spectrometry (LC- MS / MS); the respective conditions are known to the skilled person. The determination is qualitative and / or quantitative. The term “quantitative” as used herein refers to determining the absolute or relative amount of the analyte of interest comprised in a liquid sample, and “qualitative” as used herein refers to the identification of the analyte of interest.

[0103] 2ndaspect - Product-by-process: derivatized analyte of interest

[0104] In a second aspect, the invention relates to a derivatized analyte of interest, obtained or obtainable from the method for derivatizing an analyte of interest as described above in the section related to the first aspect of the invention. All details, embodiments and preferred embodiments described above in the section related to the first aspect of the invention apply also for the second aspect of the invention.

[0105] 3rdaspect - Particle

[0106] In a third aspect, the invention is directed to a particle, preferably a liposome particle and / or a polymer particle, comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry, an encapsulating material surrounding the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material, wherein the encapsulating material comprises, preferably consists of, one or more lipid(s), and / or one or more polymer(s) and / or mixed forms thereof (polymersomes).

[0107] All details, embodiments and preferred embodiments described above in the section related to the first aspect of the invention and in the section related to the second aspect of the invention apply also for the third aspect of the invention.

[0108] According to the third aspect, the invention is also directed to a liquid mixture comprising the particle described above in suspended form. In said liquid mixture, the suspended particles comprise, preferably consists of the components identified above and is neither bonded to nor adsorbed in a solid material, preferably at least only bonded to or absorbed on a solid material up to step ii) as described above. Preferably, neither is the encapsulating material attached to a surface of a scaffolding material, nor is the derivatization agent of the particle attached to or adsorbed onto the surface of a chromatographic material.

[0109] 4thaspect - Use of the particle

[0110] A fourth aspect of the invention is directed to a use of the particle of the third aspect for derivatization of an analyte of interest and / or to a use of the particle of the third aspect in a method for method for determining an analyte of interest.

[0111] All details, embodiments and preferred embodiments described above in the section related to the first aspect of the invention, in the section related to the second aspect of the invention and in the section related to the third aspect of the invention also apply for the fourth aspect of the invention.

[0112] Summarizing and without excluding further possible embodiments, the following embodiments may be envisaged:

[0113] Embodiment 1 : A method for derivatizing an analyte of interest, the method comprising: i) Providing a) a particle comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry, an encapsulating material surrounding the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material and facing towards the outside of the particle; b) a magnetic particle having on its surface a second member of the binding pair, and optionally a binding agent specific for the analyte of interest; c) a liquid sample potentially containing an analyte of interest; d) optionally a further magnetic particle comprising a binding agent specific for the analyte of interest; ii) Contacting the particle of ia) with the liquid sample of ic) and subsequently with the magnetic particle of ib) and, and optionally with the further magnetic particle of id), so that the analyte of interest if present is bound to the magnetic particle of ib) and / or optionally to the further magnetic particle of id) via the binding agent; thereby obtaining a liquid remainder of the sample and one or more complexes, wherein at least one complex comprises the particle of ia) bound to the magnetic particle of ib) via the binding pair and optionally the analyte of interest if present bonded via the binding agent to the magnetic particle of ib), and optionally a further complex comprises the analyte of interest if present and the further magnetic particle of id) bonded via the binding agent to each other; iii) Spacely separating liquid remainder of the sample and the complex(es) from each other, thereby obtaining a supernatant comprising the liquid remainder of the sample; iv) Removing the supernatant, thereby obtaining separated complex(es); v) Optionally washing the separated complex(es) at least once, thereby obtaining washed complex(es); vi) Releasing the derivatization agent from the encapsulated system and releasing the analyte from the (optional further) magnetic particle from the complex(es) of iv) or the washed complex(es) of v) by applying release conditions; vii) Optionally separating the remainder(s) of the magnetic particle(s) from the derivatization agent and the analyte if present; viii) Allowing analyte of interest if present and derivatization agent to react, thereby potentially obtaining a derivatized analyte of interest.

[0114] Embodiment 2: A method for determining an analyte of interest, the method comprising i) to viii) as defined in embodiment 1 and further: ix) Subjecting the derivatized analyte of interest obtained in viii) to analysis, preferably to a mass spectrometric analysis, wherein the mass spectrometric analysis is preferably liquid chromatography-mass spectrometry (LC-MS), more preferably liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0115] Embodiment 3: The method according to embodiment 1 or 2, wherein the derivatization agent is selected from the group consisting of Cookson-type reagent (4-substituted- 1,2,4- triazoline-3, 5-dione), Amplifex Diene ((salt of) 1-propanaminium, 3-[[[l-[4-(3,5-dihydro- 3,5-dioxo-4H-l,2,4-triazol-4-yl)phenyl]ethylidene]amino]oxy]-N,N,N-trimethyl), Amplifex Keto ((salt of) 3 -(Aminooxy)-N,N,N-trimethyl- 1-propanaminium), Girard T (trimethylacetohydrazide ammonium chloride), Girard P (l-(2-hydrazinyl-2-oxoethyl)pyridin-l- ium chloride), compound having a permanent net charge >1, substituted benzyl compound, and mixture of two or more thereof.

[0116] Embodiment 4: The method according to embodiment 3, wherein the derivatization agent is selected from the group consisting of Cookson-type reagent (4-substituted- 1, 2, 4-triazoline- 3,5-dione), Amplifex Diene, Amplifex Keto, Girard T, Girard P , and mixture of two or more thereof.

[0117] Embodiment 5: The method according to embodiment 3, wherein the derivatization agent is a compound having a permanent net charge >1, wherein said compound comprises at least three units Zl, Z2, Q and optional a further unit LI, wherein the units are covalently linked to each other, wherein:

[0118] Q is a reactive unit capable of forming a covalent bond with the analyte,

[0119] Zl is a charged unit comprising at least one permanently charged moiety, in particular a permanently positive charged moiety or a permanently negative charged moiety,

[0120] Z2 is a charged unit comprising at least one permanently charged moiety, in particular a permanently positive charged moiety or a permanently negative charged moiety, and LI is a substituted or non- substituted linker, preferably a group cleavable via fragmentation, more preferably a fragmentation moiety selected from the group consisting of Me Lafferty fragmentation moiety, Retro Diels Alder fragmentation moiety and aliphatic.

[0121] Embodiment 6: The method of embodiment 5, wherein the reactive unit Q is selected from the group consisting of a carbonyl reactive unit, a diene reactive unit, a hydroxyl reactive unit, an amino reactive unit, an imine reactive unit, a thiol reactive unit, a diol reactive unit, a phenol reactive unit, an epoxide reactive unit, a disulfide reactive unit, and an azido reactive unit; and / or, preferably and, Zl is a charged unit comprising at least one permanently charged moiety, preferably a permanently positive charged moiety or a permanently negative charged moiety, and / or, preferably and, Z2 is a charged unit comprising at least one permanently charged moiety, preferably a permanently positive charged moiety or a permanently negative charged moiety; and / or, preferably and, LI is a substituted linker or non- substituted linker, preferably a cleavable group via fragmentation, more preferably selected from the group consisting of McLafferty fragmentation moiety, Retro Diels Alder fragmentation moiety and aliphatic fragmentation moiety.

[0122] Embodiment 7: The method according to embodiment 3, wherein the derivatization agent is a substituted benzyl derivative, which is preferably a compound of formula (C) wherein one of the substituents B1, B2, B3, B4, B5is a coupling group Q, which is capable of forming a covalent bond with the analyte,

[0123] A1, A2, B1, B2, B3, B4, B5are each independently selected from the group consisting of hydrogen, halogen, alkyl, modified alkyl, N-acylamino, N,Ndialkylamino, alkoxy, thioalkoxy, hydroxy, cyano, alkoxycarbonyl, alkoxythiocarbonyl, acyl, nitro, thioacyl, aryloyl, fluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, cyanomethyl, cyanoethyl, hydroxyethyl, methoxyethyl, nitroethyl, acyloxy, aryloyloxy, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, amino, sulfur, and isotope thereof,

[0124] A3comprises a group selected from the group consisting of ammonium, pyridinium, and phosphonium, wherein in case of A3being ammonium and B1or B5being the coupling group Q, the coupling group Q comprises a C atom, which is separated by four single or double bonds from the C atom of the CA1A2A3substituent and the coupling group Q comprises a C-atom, which is separated by five single or double bonds from the C atom of the CA1A2A3substituent, preferably A3is ammonium, B1or B5is Q, wherein Q is free of at least one atom, which is selected from the group of oxygen atom, nitrogen atom, sulphur atom and bromine atom.

[0125] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the encapsulating material is a material capable of controlled release of the derivatization agent encapsulated therein.

[0126] Embodiment 9: The method of any one of embodiments 1 to 8, wherein the encapsulating material is a material capable of controlled release of the derivatization agent encapsulated therein upon contact with a compound selected from the group consisting of a polar solvent, preferably a polar protic solvent, an apolar solvent, an organic acid, an organic base, an inorganic acid, an inorganic base, a detergent, an oxidizing reagent, a reducing reagent, and a mixture of two or more thereof; and / or upon application of a treatment selected from the group consisting of heating, light exposure, ultrasound treatment or a combination of two or more of these treatments.

[0127] Embodiment 10: The method of any one of embodiments 1 to 9, wherein the encapsulating material is a lipid and / or a polymer and / or a mixed form thereof (polysome), preferably the encapsulating material is a lipid, preferably selected from the group consisting of monoglyceride, diglyceride, triglyceride, wax, sterol, phospholipid and mixtures of two or more thereof. Embodiment 11 : The method according to embodiment 10, wherein the monoglyceride is an ester of glycerol and a C4 to C28 saturated or unsaturated carboxylic acid; the diglyceride is a diester of glycerol and two C4 to C28 saturated or unsaturated carboxylic acids, which are the same or different; and a triglyceride is a triester of glycerol and three C4 to C28 saturated or unsaturated carboxylic acids, which are the same or different.

[0128] Embodiment 12: The method according to embodiment 10, wherein the sterol is cholesterol.

[0129] Embodiment 13: The method according to embodiment 10, wherein the phospholipid comprises two C4 to C28 saturated or unsaturated carboxylic acids, which are the same or different; wherein the phosphate group is optionally modified with at least one nitrogen containing compound selected from choline, ethanolamine and serine.

[0130] Embodiment 14: The method according to any one of embodiments 1 to 13, wherein the encapsulating material comprises a mixture of l,2-dipalmitoyl-sn-glycero-3-phospho-(l'- rac-glycerol) (16:0 PG), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (16:0 PC), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (16:0 Biotinyl PE) and cholesterol.

[0131] Embodiment 15: The method according to any one of embodiments 1 to 14, wherein the binding pair is selected from the group consisting of antigen (including antigen fragments, epitopes and haptens) and corresponding antigen-specific antibody (including antibody fragments); ligand and ligand-specific receptor (both independently selected from a group consisting of peptide, protein, oligonucleotide, polynucleotide and small molecule); complementary nucleic acid (including non-natural nucleic acid like (but not limited to) peptide nucleic acid (PNA), locked nucleic acid (LNA), 2’-O-methyl RNA (2’0Me), phosphorthioate (PS) including L-form thereof,), binder 1 and binder 2 (binder 1 being selected from the group consisting of small molecule, peptide, protein and oligonucleotide and binder 2 being a binder 1 specific aptamer), biotin and avidin; biotin and (poly)streptavidin; biotin and neu- travidin; strep-tag and strep-tactin, lectin and carbohydrate; wherein the first member of a binding pair and the respective second member of a binding pair are selected to form one of the aforementioned binding pairs.

[0132] Embodiment 20: The method according to any one of embodiments 1 to 19, wherein a particle comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry, an encapsulating material surrounding the derivatization agent, which comprises one or more lipid(s) and / or one or more polymer(s) and / or mixed forms thereof (polymersomes), wherein at least a part of the lipid(s) carries the first member of the binding pair and the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material and facing towards the outside of the particle, is obtained or obtainable from sonication of a liquid mixture comprising the lipid(s) and the derivatization agent.

[0133] Embodiment 21: The method according to any one of embodiments 1 to 20, wherein contacting the particle of ia) with the liquid sample of ic) and subsequently with the magnetic particle of ib) , and optionally with the further magnetic particle of id) according to ii) is done in liquid phase, preferably in solution, more preferably in aqueous solution, more preferably in buffered aqueous solution; and / or, preferably and, at a temperature in the range of from 0 to 50°C, preferably in the range of from 5 to 50°C , more preferably in the range of from 10 to 30°C; and / or, preferably and, under stirring, preferably under agitation, preferably controlled agitation, wherein “(controlled) agitation” means one or more selected from the group consisting of shaking, rocking, waving rotation, and vortex mixing.

[0134] Embodiment 22: The method according to any one of embodiments 1 to 21, wherein ii) comprises ii- 1) Contacting the particle of ia), which carries a first member of a binding pair, with the liquid sample of ic) and subsequently with the magnetic particle of ib), which has on its surface a second member of the binding pair, and a binding agent specific for the analyte of interest; thereby obtaining a liquid remainder of the sample and a complex, comprising the particle of ia) and the analyte of interest if present both bound to the magnetic particle of ib) via the binding pair and the binding agent respectively.

[0135] Embodiment 23: The method according to any one of embodiments 1 to 21, wherein ii) comprises ii-2) Contacting the particle of ia), which carries a first member of a binding pair, with the liquid sample of ic) and subsequently with the magnetic particle of ib), which has on its surface a second member of the binding pair, with the further magnetic particle of id) comprising a binding agent specific for the analyte of interest; thereby obtaining at least two complexes, wherein one complex comprises the particle of ia) bound to the magnetic particle of ib) via the binding pair, and another complex, which comprises the analyte of interest if present is bound to the further magnetic particle of id) via the binding agent, and a liquid remainder of the sample. Embodiment 24: The method according to any one of embodiments 1 to 23, wherein spacely separating liquid remainder of the sample and the complex(es) obtained in ii), ii-1) or ii-2) from each other according to iii) is done by application of a magnetic field to enable magnetic bead separation.

[0136] Embodiment 25: The method according to any one of embodiments 1 to 24, wherein releasing the derivatization agent from the encapsulated system according to vi) is done from the complex(es) by applying release conditions..

[0137] Embodiment 26: The method according to any one of embodiments 1 to 25, wherein releasing the derivatization agent from the encapsulated system and releasing the analyte from the (optional further) magnetic particle according to vi) is done with addition of a solution containing an acid, preferably an organic acid, more preferably selected from the group consisting of citric acid, formic acid, acetic acid, trifluoracetic acid, difluor acetic acid, trichloroacetic acid, di chloroacetic acid, and mixtures of two or more thereof, more preferably at least with addition of citric acid and / or formic acid.

[0138] Embodiment 27: The method according to any one of embodiments 1 to 26, wherein releasing the derivatization agent from the encapsulated system and releasing the analyte from the (optional further) magnetic particle according to vi) is done in the presence of an additive, preferably selected from the group of lewis bases, more preferably at least m-phenylenedia- mine.

[0139] Embodiment 28: The method according to any one of embodiments 1 to 27, wherein at least viii) is done at a temperature in the range of from 0 to 90°C, preferably in the range of from 10 to 90°C, more preferably in the range of from 20 to 85°C, more preferably in the range of from 30 to 80°C, more preferably in the range of from 40 to 75°C, more preferably in the range of from 50 to 70°C.

[0140] Embodiment 29: A derivatized analyte of interest, obtained or obtainable from the method of any one of embodiments 1 to 28.

[0141] Embodiment 30: A particle, preferably a liposome particle and / or a polymer particle, comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry, an encapsulating material surrounding the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material, wherein the encapsulating material comprises, preferably consists of, one or more lipid(s), and / or one or more polymer(s) and / or mixed forms thereof (i.e. polymersomes).

[0142] Embodiment 31 : A liquid mixture comprising the particle of embodiment 30 in suspended form.

[0143] Embodiment 32: Use of the particle of embodiment 30 or the liquid mixture of embodiment 31 for derivatization of an analyte of interest.

[0144] Embodiment 33: Use of the particle of embodiment 30 or the liquid mixture of embodiment 31 in a method for determining an analyte of interest.

[0145] Examples

[0146] Reference Example 1: Synthesis of liposomes and magnetic particles

[0147] I.1 Liposomes containing derivatization agent and member of a binding pair

[0148] Liposomes, containing Girard's reagent T, with 2 wt.-% 16:0 PE-biotin were prepared according to an established protocol from Edwards et al. (Edwards, K.A., Curtis, K.L., Sailor,

[0149] J.L. et al. Universal liposomes: preparation and usage for the detection of mRNA. Anal Bi- oanal Chem 391, 1689-1702 (2008). https: / / doi.org / 10.1007 / s00216-008-1992-l) with adjustments towards the encapsulant and lipid composition. Shortly, encapsulant was prepared by dissolving 100 mmol L'1Girard's reagent T (Sigma Aldrich as part of Merck Germany) in 4.5 mL 0.2 mmol L1HEPES buffer, pH 7.5 (osmolality 0.475 osmol / kg). 16:0 PC (31.04 mg), 16:0 PG (15.64 mg), cholesterol (19.99 mg) and 16:0 PE-biotin (2.21 mg) were dissolved in 3 mL chloroform and 0.5 mL methanol and thoroughly sonicated in an ultrasonic bath at 60 °C (lipids were purchased from Avanti Polar Lipids, Inc.). Subsequently, 2 mL of preheated (60 °C) encapsulant was added to the lipid solution and emulsified for 4 min at 60 °C, using an ultrasonic bath. After emulsification, the residual solvent was evaporated at 60 °C under reduced pressure. The remaining 2 mL of encapsulant was added after gradual evaporation to 780 mbar and thoroughly vortexed before evaporation was continued to 400 mbar. The remaining solution was extruded at 60 °C successively through 1.0 pm, 0.4 pm, and 0.2 pm membrane using a mini extruder (Avanti Polar Lipids, Inc.) to obtain unilamellar liposomes. Purification was first performed by size-exclusion chromatography with Sephadex® G-50 as stationary phase (column size: 2 cm * 8 cm) and HSS buffer (10 mmol L1HEPES, 200 mmol L1sodium chloride, 200 mmol L1sucrose, 0.01 wt.-% sodium azide), pH 7.5 as mobile phase. Additionally, the liposomes were dialyzed against HSS buffer, before the determination of the hydrodynamic diameter via DLS (156 nm ± 59 nm; Pdl = 0.114) and zeta potential (-19.5 mV) was done. A total lipid concentration of 13.05 mmol L’1was determined through ICP-MS.

[0150] 1.2 Testosterone-specific magnetic beads

[0151] The testosterone-specific magnetic particles were prepared by using commercial streptavidin carrying magnetic particles (Dynabeads™ MyOne™ Streptavidin Cl, Thermo Fisher Scientific Inc.) and a biotinylated testosterone-specific antibody (obtainable from commercial Elecsys® Testosterone II kit). The biotinylated antibody and the streptavidin carrying magnetic particles were incubated overnight at 4 °C for bead functionalization in a coupling ratio of 20 pg antibody per 1 mg bead. Subsequently, the antibody-modified magnetic particles, wherein the testosterone specific antibody was bound to the magnetic particles via the strep- tavidin / biotin binding, were washed three times with PreClean M (Elecsys, cobas e, Roche Diagnostics) for 5 min while shaking and resuspended in an appropriate aqueous storage buffer for storage.

[0152] Reference Example 2: Application of reagents in testosterone-derivatization

[0153] 2.1 Materials, derivatization workflow and general measurement details:

[0154] Stock solutions of testosterone were individually prepared in concentrations of 500 ng / mL (80 volume-% MeOH in EEO) and (4 volume-% MeOH in EEO). HSS buffer (pH=7.5) consists of 10 mM HEPES buffer, 200 mM NaCl, 200 mM Sucrose and 0.01 wt.-% NaNs, supporting the stability of liposomes in solution and during washing steps of the bead workflow procedure. To obtain optimal derivatization reaction conditions, solutions of 4 M Citric acid (MeOH) and 400 mM m-phenylenediamine (MeOH) were prepared correspondingly.

[0155] All described experiments were analyzed by liquid chromatography (LC) and tandem mass spectrometry. As measurement instrument, an ACQUITY-UPLC® (Waters Corp.) coupled to a XEVO® TQ-XS (Waters Corp.) triple quadrupole mass spectrometric instrument was used.

[0156] Data analysis was performed using MassLynx (V4.2, SCN1024, Waters Corp:) instrument software, while integration of resulting peaks was performed using the corresponding Tar- getLynx XS (Waters Corp.) processing software.

[0157] 2.2 Method and acquisition parameters of the respective LC-MS-measurements: Following LC-method was applied to efficiently detect and confirm derivatization reaction products, especially Testosterone-Girard-T [M]+.

[0158] LC-Parameters

[0159] Solvents: A: H2O + 0.1 volume-% Formic Acid (FA), B: MeOH + 0.1 volume-% FA

[0160] Column: ACQUITY UPLC BEH C18, 1.7pm, 2.1x50mm

[0161] Injection volume: 5 pL

[0162] Column temperature: 40°C

[0163] Autosampler: 8°C

[0164] TUV-Detector: Wavelength: 254 nm

[0165] Sample Rate: 20 points / sec

[0166] Details regarding the applied liquid chromatography solvent gradient are listed in Table 1 below, MS Parameters are listed in Table 2:

[0167] Table 1

[0168] Applied liquid chromatography solvent gradient

[0169] Table 2

[0170] VIS-Parameters

[0171] Multiple reaction monitoring (MRM) method settings:

[0172] Testo-GT quan: m / z 402.18 — m / z 343.12 ; CE=30eV ; Dwell 50 ms ; 2.1-2.9 min Testo-GT qual: m / z 402.18 — m / z 162.98 ; CE=30eV ; Dwell 50 ms ; 2.1-2.9 min testosterone: m / z 289.09 — m / z 97.02 ; CE=19eV ; Dwell 50 ms ; 2.8-3.5 min

[0173] Corresponding MRM-transitions were selected based on previous tuning experiments. A daughter scan of pure testosterone-Girard-T [M]+species (see Fig. 5) showed in the total ion chromatogram (TIC) both conformational species - as expected - consisting of cis / trans isomers (not further assigned), while both could be partly separated by liquid chromatography. As the most abundant fragment m / z 343.1 [M-NMes] occurs equally, both isomeric species were integrated for all further MRM-experiments.

[0174] Example 1: Experiment to prove the applicability of liposomes after destruction and release of reagents

[0175] In a first experiment (see Table 3 below, ID #1), the application of the novel liposomes was tested in terms of release of derivatization reagent from the liposomes and subsequent deri- vatization reaction of testosterone.

[0176] Therefore, 20 pL of a liposome stock-suspension of Reference Example 1, 1.1 were treated with 40 pL 80 volume-% MeOH solution containing testosterone (500 ng / mL) as model analyte. This high organic solvent content caused a controlled destruction of said liposomes with subsequent release of the previously incorporated Girard-T reagent. To this reaction mixture, 20 pL of 4 M citric acid (CitAC) in MeOH and 20 pL of 400 mM m-phenylenedi- amine (mPDA) in MeOH were added to obtain optimal derivatization conditions (as previously described in WO 2022 / 084362 Al). Incubation for 30 min at 60 °C with vigorous vor- texing (1400 rpm) initiated a derivatization reaction of testosterone with Girard-T reagent. The reaction mixture was analyzed by LC-MS / MS and product formation of testosterone- Girard-T, containing a single positive charge [M]+was observed, which proves the successful derivatization of testeosterone using the derivatization reagent loaded liposomes. As shown in Fig. 6 the area of the testosterone-Girard-T MRM-quantifier-signal (m / z 402 —> 343) is -73.000 times higher for the above described experiment #1 in comparison to blank experiment #5 (absence of testosterone). The aim of experiment #1 was to prove the applicability of derivatization reagent loaded liposomes as derivatization reagent shuttles. Example 2: Experiment to verify bead capturing and further elution of higher concentrated liposomes

[0177] In a following experiment (see Table 3, ID #2), magnetic bead-based capturing of the liposomes, washing, controlled release of the derivatization reagent from the liposomes and subsequent derivatization of testosterone was tested.

[0178] Therefore, 20 pL of a liposome stock-suspension of Reference Example 1, 1.1 were diluted in 40 pL HSS-Buffer. 10 pL streptavidin magnetic particles were added and the suspension incubated for 20 min at room temperature (RT, in the range of from 20 to 25°C) with gentle vortexing (450 rpm). Magnetic separation, disposal of liquid supernatant and washing with HSS-buffer (3x50 pL) furnished solely liposomes bound to the streptavidin magnetic particles via the streptavidin-biotin interaction. Controlled destruction of the liposomes and thereby release of the Girard-T reagent was triggered by adding 20 pL of 80 volume- % MeOH, together with 20 pL of testosterone solution (80 volume-% MeOH, 500ng / mL) as model analyte. After magnetic bead separation, the Girard-T and testosterone containing supernatant was transferred to another vial, containing 20 pL of 4 M citric acid (MeOH) and 20 pL of 400 mM m-phenylenedi amine (MeOH). The mixture was incubated for 30 min at 60 °C with vigorous vortexing (1400 rpm) to accelerate the derivatization reaction of testosterone by Girard-T. The reaction mixture was analyzed by LC-MS / MS and product formation of testosterone-Girard-T, containing a single positive charge [M]+was observed, which proves 1) selective capturing of the liposomes on magnetic particles and 2) the successful derivatization of testeosterone by the controlled release of the derivatization reagent from the captured liposomes. As shown in Fig. 7 the area of the testosterone-Girard-T MRM- quantifier-signal (m / z 402 —> 343) is -350 times higher for the above described experiment #2 in comparison to blank experiment #5 (absence of testosterone). The aim of experiment #1 was to prove the applicability of derivatization reagent loaded liposomes as derivatization reagent shuttles.

[0179] Example 3: Experiment to verify bead capturing and further elution of lower concentrated liposomes

[0180] In a third experiment (see Table 3, ID #3), magnetic bead-based capturing of the liposomes, washing, controlled release of the derivatization reagent from the liposomes and subsequent derivatization of testosterone was tested using a lower amount / concentration of liposomes. The goal of this experiment was to compare the results of the two different liposome concentrations in order to evaluate the effect of liposome concentration on the derivatization reaction efficiency. Therefore, 5 pL of a liposome stock-suspension of Reference Example 1, 1.1 was diluted in 40 pL HSS-Buffer. 10 pL streptavidin magnetic particles were added and the suspension incubated for 20 min at room temperature (RT, in the range of from 20 to 25°C) with gentle vortexing (450 rpm). Magnetic separation, disposal of liquid supernatant and washing of the magnetic particles with HSS-buffer (3x50 pL) furnished solely liposomes bound to the streptavidin magnetic particles via the streptavidin-biotin interaction. Controlled destruction of the liposomes and thereby release of the Girard-T reagent was triggered by adding 20 pL of 80 volume-% MeOH, together with 20 pL of testosterone solution (80 volume-% MeOH, 500ng / mL) as model analyte. After magnetic bead separation, the Girard-T and testosterone containing supernatant was transferred to another vial, containing 20 pL of 4 M citric acid (MeOH) and 20 pL of 400 mM m-phenylenediamine (MeOH). The mixture was incubated for 30 min at 60 °C with vigorous vortexing (1400 rpm) to accelerate the derivatization reaction of testosterone by Girard-T. The reaction mixture was analyzed by LC-MS / MS and product formation of testosterone-Girard-T, containing a single positive charge [M]+was observed, which proves again 1) selective capturing of the liposomes on magnetic particles and 2) the successful derivatization of testosterone using lower amounts of liposomes by the controlled release of the derivatization reagent from the captured liposomes. As expected, the use of lower amounts of derivatization reagent loaded liposomes in this experiment resulted in a reduced conversion of testosterone into the derivatized testosterone as indicated by the smaller area of the testosterone-Girard-T MRM-quantifier-signal (m / z 402 —> 343) compared to example #2 (see Fig. 7).

[0181] Example 4: Experiment to verify bead capturing with simultaneous immunoprecipitation of testosterone

[0182] In a fourth experiment (see Table 3, ID #4), simultaneous capturing of the liposomes by streptavidin magnetic particles and capturing of testosterone by testosterone-specific magnetic particles, washing, controlled release of the derivatization reagent from the liposomes and testosterone from the antibody-loaded magnetic particles followed by subsequent derivatization of testosterone was tested. This experiment would represent a workflow of the preferred application, by capturing simultaneously the intended analyte and the derivatization reagent containing liposomes, both during a single bead workflow procedure.

[0183] Therefore, 5 pL of a liposome stock-suspension of Reference Example 1, 1.1 were diluted in 20 pL HSS-Buffer and 20 pL of testosterone solution (4 volume-% MeOH, 500ng / mL) as model analyte was added. Next, 5 pL streptavidin magnetic particles and 5 pL of testosterone-specific magnetic particles of Reference Example 1, 1.2 were added. The suspension was incubated for 20 min at room temperature (RT, in the range of from 20 to 25 °C) with gentle vortexing (450 rpm). Magnetic separation, disposal of liquid supernatant and washing with of the magnetic particles with HSS-buffer (2^50 pL) followed the procedure. Controlled release of the derivatization reagent (by destruction of the liposomes attached to the streptavidin magnetic particles) and testosterone (from the antibody-loaded magnetic particles) was triggered by adding 40 pL of 80 volume-% MeOH. After magnetic bead separation, the testosterone and Girard-T containing supernatant was transferred to another vial, containing 20 pL of 4 M citric acid (MeOH) and 20 pL of 400 mM m-phenylenediamine (MeOH). The mixture was incubated for 30 min at 60 °C with vigorous vortexing (1400 rpm) to accelerate the derivatization reaction of testosterone by Girard-T. The reaction mixture was analyzed by LC-MS / MS and product formation of testosterone-Girard-T, containing a single positive charge [M]+was observed, which proves 1) selective capturing of the liposomes on streptavidin magnetic particles 2) selective capturing testosterone by the anti- body-modified magnetic particles and 3) the simultaneous release of derivatization reagent and testosterone followed by successful derivatization of testeo sterone. As shown in Fig. 7 the area of the testosterone-Girard-T MRM-quantifier-signal (m / z 402 —> 343) is -350 times higher for the above described experiment #4 in comparison to blank experiment #5 (see below, absence of testosterone) and similar to the spiking experiment with testosterone (experiment #3).

[0184] Example 5: Control experiment to determine the blank-level of applied workflow reagents in absence of Testosterone

[0185] In a fifth experiment (see Table 3, ID #5), streptavidin magnetic bead-based capturing, washing and controlled release of the derivatization reagent from the liposomes in absence of testosterone was analyzed in order to determine the blank baseline signal of all workflow reagents.

[0186] Therefore, 20 pL of a liposome stock-suspension of Reference Example 1, 1.1 were diluted in 20 pL HSS-Buffer. Next, 10 pL streptavidin magnetic particles and 5 pL of testosteronespecific magnetic particles of Reference Example 1, 1.2 were added. The suspension was incubated for 20 min at room temperature (RT, in the range of from 20 to 25°C) with gentle vortexing (450 rpm). Magnetic separation, disposal of liquid supernatant and washing with of the magnetic particles with HSS-buffer (2x50 pL) followed the procedure. Controlled release of the derivatization reagent (by destruction of the liposomes attached to the streptavidin magnetic particles) was triggered by adding 40 pL of 80 volume-% MeOH. After magnetic bead separation, the Girard-T containing supernatant was transferred to another vial, containing 20 pL of 4 M citric acid (in MeOH) and 20 pL of 400 mM m-phenylenedi- amine (in MeOH). The mixture was incubated for 30 min at 60 °C with vigorous vortexing (1400 rpm) to simulate the derivatization reaction conditions. The resulting blank area of the testosterone-Girard-T MRM-quantifier-signal was analyzed by LC-MS / MS (see Fig. 7).

[0187] In Fig. 7, a logarithmic application of detected Testosterone-Girard-T MRM-Peak area of quantifier-signal m / z 402 —> 343 is shown for the respective experiments #2-#5.

[0188] Experiment #5 displays the blank baseline signal of all derivatization reagents in absence of testosterone (model analyte). Applying these workflow conditions in experiment #2 in presence of spiked testosterone, a significant increase of Testosterone-Girard-T MRM signal is observed. This verifies that the liposomes were captured during the magnetic bead workflow and Girard-T was successfully released during the elution step (controlled destruction of liposomes in 80 volume-% MeOH).

[0189] Another important outcome can be concluded from experiment #3, where lower concentra- tions / amounts of liposomes were used. As expected, lower signals / areas of testosterone- Girard-T were detected under these conditions compared to experiment #2, where higher concentrations of liposomes were used. Applying higher concentrations / excess of derivatization reagent loaded liposomes results in a higher concentration of released derivatiziation reagent, which helps to convert the target analyte into the desired derivatized analyte with higher yield.

[0190] Experiment #4 highlighted another remarkable outcome. In this workflow, testosterone was specifically captured by an anti-testosterone antibody loaded magnetic particle contemporaneous to the liposomes capturing onto the streptavidin-functionalized magnetic particles. After washing, elution and applying suitable derivatization reaction conditions, this experiment resulted in a successful detection of testosterone-Girard-T species. In summary, the combination of both, antibody-based capturing of a target analyte and reagent-containing liposome particles with subsequent derivatization was shown to be effective.

[0191] Example 6: Experiment to verify capturing and concentration of liposomes on the surface of functionalized paramagnetic beads

[0192] In a sixth experiment (see Table 3, ID #6-1 and #6-2), magnetic bead-based liposome capturing, washing, additional bead-transfer and controlled release of the derivatization reagent from modified liposomes was assessed. The aim of this control experiment was to verify, that capturing and concentration of the liposomes merely resulted on the magnetic particle and not by unspecific binding of the liposomes inside the used Eppendorf tubes®.

[0193] Therefore, 20 pL of a liposome stock-suspension of Reference Example 1, 1.1 were diluted in 20 pL HSS-Buffer. 10 pL streptavidin magnetic particle suspension was added and incubated for 20 min at room temperature (RT, in the range of from 20 to 25°C) with gentle vortexing (450 rpm). Magnetic separation, disposal of liquid supernatant and washing with HSS-buffer (3^50 pL) followed the procedure, to generate magnetic-bead bound liposomes through the specific biotin-streptavidin interaction. After said washing step, the liposomebead complex was transferred as thoroughly as possible from previous Eppendorf tube® (experiment ID #6-1) via pipetting into a new, unused Eppendorf tube® (experiment ID #6-2). Both tubes from experiment #6-1 and #6-2 were treated with 20 pL of 80 volume-% MeOH and 20 pL of testosterone solution (80 volume-% MeOH, 500 ng / mL) to cause a possible destruction of liposomes and release of the derivatization reagent. The solution ID #6-1 and the supernatant of ID #6-2 (after magnetic bead separation), were separately transferred to further two vials, containing 20 pL of 4 M citric acid (MeOH) and 20 pL of 400 mM m-phe- nylenediamine (MeOH). The mixtures were incubated for 30 min at 60 °C with vigorous vortexing (1400 rpm) to accelerate the desired derivatization reaction. Both reactions #6-1 and #6-2 were analyzed by LC-MS / MS in terms of testosterone-Girard-T formation to allow direct comparison of both experiments.

[0194] Fig. 8 displays the detected testosterone-Girard-T MRM-peak area of the quantifier-signal for experiments #6-1 and #6-2 in comparison with experiment #2.

[0195] Experiment #6-1 showed only traces of testosterone-Girard-T MRM-peak area, which indicates that unspecific adsorption of the liposomes onto the herein used tubes is very low / neg- ligible and that the liposomes are mainly bound, as anticipated, to the magnetic particles. The residual signal of testosterone-Girard-T in vial #6-1 is likely to be explained by as residual magnetic particles, which remained in vial #6-1 transferring the liposome-bead complex into vial #6-2. In accordance with that observation, the derivatization reaction of vial #6-2 resulted a similar testosterone-Girard-T MRM-peak area compared to experiment #2. This proves again a specific binding of intact, functionalized liposomes on the surface of magnetic beads.

[0196] - 41 -

[0197] Table 3

[0198] Reaction conditions and reagents of experiments #l-#6.

[0199] [Cl]: Extraction of remaining ,empty‘ Eppendorf tube T

[0200] [C2]: Bead suspension transferred via pipette in a new Incubation for Incubation for lOmin at

[0201] Incubation for 30m RT, vortexing at 650rpm Eppendorf tube after 3rd wash step of20min at RT’vor' at 60°C, vortexing texing at 450rpm Magnetic separation; sample #6-1 1400rpm

[0202] Eluate transferred

[0203] Example 7: Experiment to evaluate liposome capturing followed by simultaneous liposome destruction and derivatization reaction in a one-pot reaction

[0204] In a seventh experiment (Table 4, ID #7), a simplified workflow protocol, combining simultaneous immunoextraction of testosterone and liposome capturing onto magnetic particles followed by simultaneous elution of testosterone, liposome destruction, derivatization reagent release and performing derivatization using a LC-MS compatible mixture was tested. Therefore, 10 pL of a liposome stock-suspension of Reference Example 1, 1.1 were diluted in 40 pL HSS-Buffer. Next, 10 pL streptavidin magnetic particles and 10 pL of testosteronespecific magnetic particles of Reference Example 1, 1.2 were added. The suspension was incubated for 20 min at room temperature (RT, in the range of from 20 to 25°C) with gentle vortexing (450 rpm). Magnetic separation, disposal of liquid supernatant and washing with HSS-buffer (3x50 pL) furnished the liposomes being specifically bound to the streptavidin magnetic particles via the biotin handle. Controlled destruction of the liposomes and thereby release of the Girard-T reagent was triggered by adding 20 pL of 80% MeOH + 1%FA (v / v), together with 20 pL of testosterone solution (80 volume-% MeOH + 1 volume-% FA v / v, 500 ng / mL) as a model analyte. The mixture was incubated directly for 30 min at 60 °C with vigorous vortexing (1400rpm) to accelerate the derivatization reaction of testosterone by Girard-T. Magnetic beads were separated, the supernatant reaction mixture was diluted with 40 pL H2O and analyzed by LC-MS / MS. Product formation of testosterone-Girard-T, containing a single positive charge [M]+was observed, which proves again 1) selective capturing of the liposomes on magnetic particles and especially 2) the simultaneous release of derivatization reagent and testosterone followed by successful derivatization of testeosterone in a one-pot reaction using a LC-MS compatible mixture. As shown in Fig. 9, the area of the testosterone-Girard-T MRM-quantifier-signal (m / z 402 —> 343) was ~80 times higher for the above described experiment #7 in comparison to blank experiment #10 (absence of testosterone). This exceeded the aim of providing a simplified workflow protocol, together with more convenient reagents in a less time-consuming manner.

[0205] Example 8: Experiment to evaluate simultaneous immunoextraction and liposome capturing followed by simultaneous elution and derivatization reaction in a one-pot reaction

[0206] In an eighth experiment (Table 4, ID #8), a simplified workflow protocol, combining simultaneous immunoextraction and liposome capturing followed by simultaneous elution of the analyte of interest, liposome destruction and derivatization was tested.

[0207] Therefore, 10 pL of a liposome stock-suspension of Reference Example 1, 1.1 were diluted in 20 pL HSS-Buffer and 20 pL of a testosterone solution (4 volume-% MeOH, 500ng / mL) as model analyte. Next, 10 pL streptavidin magnetic particles and 10 pL of testosteronespecific magnetic particles of Reference Example 1, 1.2 were added. The suspension was incubated for 20 min at room temperature (RT, in the range of from 20 to 25°C) with gentle vortexing (450 rpm). Magnetic separation, disposal of liquid supernatant and washing with HSS-buffer (3^50 pL) furnished the liposomes and testosterone both being specifically bound to the respective magnetic particles , which include but are not limited to: 1) liposomes bound to streptavidin magnetic particle via the biotin handle; 2) liposomes bound to the testosterone-specific antibody-biotin / streptavidin magnetic particle conjugate via the biotin handle and free streptavidin binding sites of the used streptavidin magnetic particle and 3) testosterone bound to the testosterone-specific antibody-biotin / streptavidin magnetic particle conjugate via the specific antigen-antibody interaction.. Elution of testosterone as well as controlled destruction of the liposomes and thereby release of the Girard-T reagent was triggered by adding 40 pL of 80% MeOH + 1%FA (v / v). The mixture was incubated directly for 30 min at 60 °C with vigorous vortexing (1400rpm) to accelerate the derivatization reaction of testosterone by Girard-T. The Magnetic beads were separated, the supernatant of the reaction mixture was diluted with 40 pL H2O and analyzed by LC-MS / MS. Product formation of testosterone-Girard-T, containing a single positive charge [M]+was again observed. This proved: 1) selective capturing of the liposomes on magnetic particles, 2) selective capturing of testosterone by the antibody-modified magnetic particles and especially 3) a simultaneous release of derivatization reagent and testosterone followed by successful derivatization of testosterone within a single step using this simplified protocol and reagents which suitable for LC-MS. As shown in Fig. 9, the area of testosterone-Girard-T MRM- quantifier-signal (m / z 402 —> 343) was ~50 times higher for the above described experiment #8 in comparison to blank experiment #10 (absence of testosterone). This exceeded the aim of providing a simplified workflow protocol for applying immunoprecipitation and derivatization of a molecule of interest, both simultaneously.

[0208] Example 9: Experiment to evaluate simultaneous immunoextraction and liposome capturing followed by simultaneous elution and derivatization reaction in a one-pot reaction in a biological matrix

[0209] In a ninth experiment (Table 4, ID #9), this simplified workflow protocol, combining simultaneous elution, liposome destruction and derivatization step was tested after capturing liposomes with streptavidin magnetic particles and parallelized immunoprecipitation of testosterone in the presence of a biological matrix.

[0210] Therefore, 10 pL of a liposome stock-suspension of Reference Example 1, 1.1 were diluted in previously spiked biological matrix sample, consisting of 20 pL horse serum and 20 pL of testosterone solution (4 volume-% MeOH, 500ng / mL) as model analyte. Next, 10 pL streptavidin magnetic particles and 10 pL of testosterone-specific magnetic particles of Reference Example 1, 1.2 were added. The suspension was incubated for 20 min at room temperature (RT, in the range of from 20 to 25°C) with gentle vortexing (450 rpm). Magnetic separation, disposal of liquid matrix supernatant and washing with HSS-buffer (3 / 50 pL) furnished the liposomes and testosterone both being specifically bound to the respective magnetic particles , which include but are not limited to: 1) liposomes bound to streptavidin magnetic particle via the biotin handle; 2) liposomes bound to the testosterone-specific an- tibody-biotin / streptavidin magnetic particle conjugate via the biotin handle and free streptavidin binding sites of the used streptavidin magnetic particle and 3) testosterone bound to the testosterone-specific antibody-biotin / streptavidin magnetic particle conjugate via the specific antigen-antibody interaction. Elution of testosterone as well as controlled destruction of the liposomes and thereby release of the Girard-T reagent was triggered by adding 40 pL of 80% MeOH + 1%FA (v / v). The mixture was incubated directly for 30 min at 60 °C with vigorous vortexing (1400rpm) to accelerate the derivatization reaction of testosterone by Girard-T. Magnetic beads were separated, the supernatant was diluted with 40 pL H2O and analyzed by LC-MS / MS. Product formation of testosterone-Girard-T, containing a single positive charge [M]+was observed. This further proved 1) selective capturing of the liposomes and testosterone on magnetic particles, even in the presence of a challenging biological matrix, like serum; 2) simultaneous release of derivatization reagent and testosterone followed by successful derivatization of testosterone in a single step using a simplified protocol and reagents which are suitable for LC-MS. As shown in Fig. 9 the area of testosterone- Girard-T MRM-quantifier-signal (m / z 402 —> 343) was ~23 times higher for the above described experiment #9 in comparison to blank experiment #10 (absence of testosterone). This resembled an important outcome and application within, but not limited to, for example diagnostic sample preparation workflows, as it provided a simplified protocol for combining immunoprecipitation and derivatization of a molecule of interest in challenging biological matrices in a fully automatable way.

[0211] Example 10: Control experiment to determine the blank-level of applied workflow reagents in absence of Testosterone within the simplified workflow conditions

[0212] In a tenth experiment (Table 4, ID #10), streptavidin magnetic bead-based capturing of the derivatization reagent loaded liposomes, washing and controlled release of the derivatization reagent from the liposomes in absence of testosterone was analyzed in order to determine the blank baseline signal of all workflow reagents.

[0213] Therefore, 10 pL of a liposome stock-suspension of Reference Example 1, 1.1 were diluted in 40 pL HSS-Buffer. Next, 10 pL streptavidin magnetic particles and 10 pL of testosterone- specific magnetic particles of Reference Example 1, 1.2 were added. The suspension was incubated for 20 min at room temperature (RT, in the range of from 20 to 25°C) with gentle vortexing (450 rpm). Magnetic separation, disposal of liquid supernatant and washing of the magnetic particles with HSS-buffer (2x50 pL) furnished the liposomes being specifically bound to the streptavidin magnetic particles via the biotin handle. Controlled release of the derivatization reagent (by destruction of the liposomes partially attached to the streptavidin magnetic particles) was triggered by adding 40 pL of 80% MeOH + 1%FA (v / v). The mixture was incubated directly for 30 min at 60 °C with vigorous vortexing (1400rpm) to simulate the derivatization reaction conditions. The resulting blank area of the testosterone- Girard-T MRM-quantifier-signal was analyzed by LC-MS / MS after dilution with 40 pL H2O (see Fig. 9, experiment #10).

[0214] Summary Examples #7 to #10

[0215] In Fig. 9, a logarithmic application of detected testosterone-Girard-T MRM-Peak area of quantifier-signal m / z 402 —> 343 is shown for the respective experiments #7-#10. Compared to experiments #l-#6 the workflow protocol was simplified, since it required fewer steps by combining elution of the analyte of interest, liposome destruction and performing derivatization reaction of the analyte of interest in a single step. It is important to note, that the mixture used for this one-pot reaction was especially useful for the subsequent LC-MS, in this example a methanolic solution of formic acid.

[0216] Experiment #10 displayed the blank baseline signal of all reagents in absence of testosterone as a model analyte. Applying the workflow of experiment #7 a significant increase of tes- tosterone-Girard-T MRM signal was observed. This verified that capturing of derivatization reagent-loaded liposomes through their biotin-handle onto streptavidin magnetic beads and subsequent opening of the liposomes could be combined with a derivatization reaction in a single step. Experiment #8 represented another important outcome, as it combined analytespecific immunoprecipitation, liposome-capturing and the above described simplified workflow.

[0217] Experiment #9 represented another important outcome, as it described analyte-specific immunoprecipitation and liposome-capturing a challenging biological sample matrix (in horse serum), followed by the above described simplified workflow. Table 4 leaction conditions and reagents of experiments #7-# 10,

[0218] Incubation for 20 Incubation for 30min at 60°C, min at RT, vortexvortexing at 1400 rpm ing at 450 rpm

[0219] Magnetic separation; Solution transferred

[0220] Short description of the Figures

[0221] Fig- 1 shows a general schematic overview of a workflow intended for automation and for enabling a high-throughput.

[0222] Fig- 2 shows a general schematic overview of an in-situ derivatization (ISD) workflow.

[0223] Fig- 3 shows a general schematic overview of an ex-situ derivatization (ESD) workflow.

[0224] Fig. 4 shows a general schematic overview of a pre-installed sample preparation device derivatization workflow.

[0225] Fig. 5 shows the daughter ion scan (TIC) of pure testosterone-Girard-T (Ipg / mL, 60 vol- ume-% MeOH) with selection of [M]+parent m / z 402.2 and collision energy CE=30eV (top) and the corresponding mass spectrum at 2.50 min retention time (bottom).

[0226] Fig. 6 shows a logarithmic plot of Testosterone-Girard-T quantifier MRM transition peak area resulting after experiment #1 in comparison to blank experiment #5.

[0227] Fig. 7 shows a logarithmic plot of Testosterone-Girard-T quantifier MRM transition peak areas resulting for experiments #2-5.

[0228] Fig. 8 shows a plot of Testosterone-Girard-T quantifier MRM transition peak area of experiments #6-1 and #6-2 in comparison to experiment #2.

[0229] Fig. 9 shows a logarithmic plot of testosterone-Girard-T quantifier MRM transition peak areas resulting for experiments #7-10.

[0230] Cited Literature

[0231] - J. Sep. Sci. 2016, 39, 102-114

[0232] - Annals of Clinical Biochemistry. 2019;56(6):646-653 Biomed. Chromatogr. 2011; 25: 1-10

[0233] Trends in Analytical Chemistry 143 (2021) 116399

[0234] - J Mass Spectrom. 2021;56:e4731

[0235] - EP 3 391 041 Bl - WO 2021 / 233997 Al

[0236] - WO 2021 / 234002 Al

[0237] - Martien A. Cohen Stuart, Wilhelm T. S. Huck, Jan Genzer, Marcus Muller, Christo- pher Ober, Manfred Stamm, Gleb B. Sukhorukov, Igal Szleifer, Vladimir V. Tsukruk, Marek Urban, Frangoise Winnik, Stefan Zauscher, Igor Luzinov, and Sergiy Minko, nature materials, Vol. 9, February 2010, 101-113

[0238] Tayebeh Anajafi, Sanku Mallik (2015) Polymersome-Based Drug-Delivery Strategies for Cancer Therapeutics, Therapeutic Delivery, 6:4, 521-534, DOI: 10.4155 / tde.14.125

Claims

1. Roche Diagnostics GmbH October 2, 2025RD38809PCClaims1. A method for derivatizing an analyte of interest, the method comprising: i) Providing a) a liposome particle comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry, an encapsulating material surrounding the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material and facing towards the outside of the particle; b) a magnetic particle having on its surface a second member of the binding pair, and optionally a binding agent specific for the analyte of interest; c) a liquid sample potentially containing an analyte of interest; d) optionally a further magnetic particle comprising a binding agent specific for the analyte of interest; ii) Contacting the liposome particle of ia) with the liquid sample of ic) and subsequently with the magnetic particle of ib), and optionally with the further magnetic particle of id), so that the analyte of interest if present is bound to the magnetic particle of ib) and / or optionally to the further magnetic particle of id) via the binding agent; thereby obtaining a liquid remainder of the sample and one or more complexes, wherein at least one complex comprises the liposome particle of ia) bound to the magnetic particle of ib) via the binding pair and optionally the analyte of interest if present bonded via the binding agent to the magnetic particle of ib), and optionally a further complex comprises the analyte of interest if present and the further magnetic particle of id) bonded via the binding agent to each other; iii) Spacely separating liquid remainder of the sample and the complex(es) from each other, thereby obtaining a supernatant comprising the liquid remainder of the sample; iv) Removing the supernatant, thereby obtaining separated complex(es); v) Optionally washing the separated complex(es) at least once, thereby obtaining washed complex(es);vi) Releasing the derivatization agent from the encapsulated system and releasing the analyte from the (optional further) magnetic particle from the complex(es) of iv) or the washed complex(es) of v) by applying release conditions; vii) Optionally separating the remainder(s) of the magnetic particle(s) from the derivatization agent and the analyte if present; viii) Allowing analyte of interest if present and derivatization agent to react, thereby potentially obtaining a derivatized analyte of interest.

2. A method for determining an analyte of interest, the method comprising i) to viii) as defined in claim 1 and further: ix) Subjecting the derivatized analyte of interest obtained in viii) to analysis, preferably to a mass spectrometric analysis, wherein the mass spectrometric analysis is preferably liquid chromatography-mass spectrometry (LC-MS), more preferably liquid chromatography-tandem mass spectrometry (LC-MS / MS).

3. The method according to claim 1 or 2, wherein the derivatization agent is selected from the group consisting of Cookson-type reagent (4-substituted-l, 2, 4-triazoline-3, 5-dione), Amplifex Diene ((salt of) 1-propanaminium, 3-[[[l-[4-(3,5-dihydro-3,5-dioxo- 4H-l,2,4-triazol-4-yl)phenyl]ethylidene]amino]oxy]-N,N,N-trimethyl), Amplifex Keto ((salt of) 3 -(Aminooxy)-N,N,N-trimethyl- 1-propanaminium), Girard T (trimethylacetohydrazide ammonium chloride), Girard P ((l-(2-hydrazinyl-2-oxoethyl)pyri- din-l-ium chloride), compound having a permanent net charge >1, substituted benzyl compound, and mixture of two or more thereof; preferably the derivatization agent is selected from the group consisting of Cookson-type reagent (4-substituted-l, 2, 4-tria- zoline-3, 5-dione), Amplifex Diene, Amplifex Keto, Girard T, Girard P, and mixture of two or more thereof; and / or, preferably and, wherein the encapsulating material is a material capable of controlled release of the derivatization agent encapsulated therein; preferably the encapsulating material is a material capable of controlled release of the derivatization agent encapsulated therein upon contact with a compound selected from the group consisting of a polar solvent, preferably a polar protic solvent, an apolar solvent, an organic acid, an organic base, an inorganic acid, an inorganic base, a detergent, an oxidizing reagent, a reducing reagent, and a mixture of two or more thereof; and / or upon application of a treatment selected from the group consisting of heating, light exposure, ultrasound treatment or a combination of two or more of these treatments; and / or, preferably and,wherein the binding pair is selected from the group consisting of antigen (including antigen fragments, epitopes and haptens) and corresponding antigen-specific antibody (including antibody fragments); ligand and ligand-specific receptor (both independently selected from a group consisting of peptide, protein, oligonucleotide, polynucleotide and small molecule); complementary nucleic acid (including non-natural nucleic acid like (but not limited to) peptide nucleic acid (PNA), locked nucleic acid (LNA), 2’-O-methyl RNA (2’0Me), phosphorthioate (PS) including L-form thereof,), binder 1 and binder 2 (binder 1 being selected from the group consisting of small molecule, peptide, protein and oligonucleotide and binder 2 being a binder 1 specific aptamer), biotin and avidin; biotin and (poly)streptavidin; biotin and neutravidin; strep- tag and streptactin, lectin and carbohydrate; wherein the first member of a binding pair and the respective second member of a binding pair are selected to form one of the aforementioned binding pairs; and / or, preferably and, wherein the magnetic particle of ib) and optionally of id) comprises at least one magnetic core (M), a polymer matrix (P) comprising a polymer, which is preferably crosslinked and optionally hypercrosslinked, at least partially surrounding the magnetic core (M), wherein the at least one magnetic core (M) comprises a magnetic nanoparticle and optionally a coating (C), and at least one remainder of a functional group (RFG), optionally' bonded via a linker (L) to the polymer matrix (P), wherein the RFG is connected to the second member of the binding pair of the magnetic particle of ib) and optionally connected to the binding agent specific for the analyte of interest of ib) or connected to the further magnetic particle to the binding agent specific for the analyte of interest of id).

4. The method according to any one of claims 1 to 3, wherein the analyte of interest is selected from the group consisting of nucleic acid, amino acid, peptide, protein, metabolite or hormone, fatty acid, lipid, carbohydrate, steroid, ketosteroid, secosteroid, molecule characteristic of a certain modification of another molecule or a substance that has been internalized by the organism or a metabolite of such a substance, and mixtures of two or more thereof, preferably the analyte of interest is testosterone.

5. The method according to any one of claims 1 to 4, wherein a liposome particle comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry,an encapsulating material surrounding the derivatization agent, which comprises one or more lipid(s) , wherein at least a part of the lipid(s) carries the first member of the binding pair and the derivatization agent, and a first member of a binding pair bonded to at least a part of the encapsulating material and facing towards the outside of the particle, is obtained or obtainable from sonication of a liquid mixture comprising the lipid(s) and the derivatization agent.

6. The method according to any one of claims 1 to 5, wherein contacting the particle of ia) with the liquid sample of ic) and subsequently with the magnetic particle of ib) , and optionally with the further magnetic particle of id) according to ii) is done in liquid phase, preferably in solution, more preferably in aqueous solution, more preferably in buffered aqueous solution; and / or, preferably and, at a temperature in the range of from 0 to 50°C, preferably in the range of from 5 to 50°C , more preferably in the range of from 10 to 30°C; and / or, preferably and, under stirring, preferably under agitation, preferably controlled agitation, wherein“(controlled) agitation” means one or more selected from the group consisting of shaking, rocking, waving rotation, and vortex mixing.

7. The method according to any one of claims 1 to 6, wherein ii) comprises ii-1) Contacting the liposome particle of ia), which carries a first member of a binding pair, with the liquid sample of ic) and subsequently with the magnetic particle of ib), which has on its surface a second member of the binding pair, and a binding agent specific for the analyte of interest; thereby obtaining a liquid remainder of the sample and a complex, comprising the liposome particle of ia) and the analyte of interest if present both bound to the magnetic particle of ib) via the binding pair and the binding agent respectively; and / or, preferably and, wherein ii) comprises ii-2) Contacting the liposome particle of ia), which carries a first member of a binding pair, with the magnetic particle of ib), which has on its surface a second member of the binding pair, with the liquid sample of ic) and subsequently with the further magnetic particle of id) comprising a binding agent specific for the analyte of interest; thereby obtaining at least two complexes, wherein one complex comprises the particle of ia) bound to the magnetic particle of ib) via the binding pair, and another complex, which comprises the analyte of interest if present is bound to the further magnetic particle of id) via the binding agent, and a liquid remainder of the sample.

8. The method according to any one of claims 1 to 7, wherein spacely separating liquid remainder of the sample and the complex(es) obtained in ii), ii-1) or ii-2) from each other according to iii) is done by application of a magnetic field to enable magnetic bead separation.

9. The method according to any one of claims 1 to 8, wherein releasing the derivatization agent from the encapsulated system according to vi) is done by contacting with a compound selected from the group consisting of a polar solvent, preferably a polar protic solvent, an apolar solvent, an organic acid, an organic base, an inorganic acid, an inorganic base, a detergent, an oxidizing reagent, a reducing reagent, and a mixture of two or more thereof; and / or upon application of a treatment selected from the group consisting of heating, light exposure, ultrasound treatment or a combination of two or more of these treatments; and / or wherein releasing the derivatization agent from the encapsulated system and releasing the analyte from the (optional further) magnetic particle according to vi) is done with addition of a mixture containing an acid; and / or wherein releasing the derivatization agent from the encapsulated system and releasing the analyte from the (optional further) magnetic particle according to vi) is done in the presence of an additive, preferably selected from the group of lewis bases, more preferably at least m-phenylenediamine.

10. The method according to any one of claims 1 to 9, wherein at least viii) is done at a temperature in the range of from 0 to 90°C, preferably in the range of from 10 to 90°C, more preferably in the range of from 20 to 85°C, more preferably in the range of from 30 to 80°C, more preferably in the range of from 40 to 75°C, more preferably in the range of from 50 to 70°C.

11. A derivatized analyte of interest, obtained or obtainable from the method of any one of claims 1 to 10.

12. A liposome particle comprising a derivatization agent for an analyte intended to be analysed via mass spectrometry, an encapsulating material surrounding the derivatization agent, anda first member of a binding pair bonded to at least a part of the encapsulating material, wherein the encapsulating material comprises, preferably consists of, one or more li- pid(s).

13. A liquid mixture, comprising the liposome particle of claim 12 in suspended form.

14. Use of the liposome particle of claim 12 of the liquid mixture of claim 13 for derivat- ization of an analyte of interest.

15. Use of the liposome particle of claim 12 or of the liquid mixture of claim 13 in a method for determining an analyte of interest.

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