阅读说明：本技术 过渡金属螯合珠粒 (Transition metal chelate beads ) 是由 G·H·M·冯登霍夫 于 2020-03-27 设计创作，主要内容包括：本发明涉及复合物,其包含：过渡金属阳离子(i)；配体(ii),该配体包含至少一个螯合基团,优选1至4个螯合基团,更优选2或3个螯合基团,其中螯合基团选自异羟肟酸酯基团-N(O～(-))-C(＝O)-R、儿茶酚酯基团、羧酸酯基团、这些螯合基团的部分或完全质子化的形式,以及这些螯合基团和/或它们的部分或完全质子化的形式的混合物,其中R为氢或C1至C5烷基基团；以及磁珠(iii)；其中磁珠(iii)与配体(ii)共价键合。本发明还涉及该复合物用于降低流体样品中的至少一种磷酰氧基物质的含量的用途,该磷酰氧基物质优选地在其结构内包含结构要素-O-P(O～(-))(＝O)-O-,以及涉及用于降低流体样品中的至少一种磷酰氧基物质优选磷脂的含量的方法,该方法包括添加该复合物的步骤。本发明还涉及从该方法获得或能够获得的上清液,以及从该方法获得或能够获得的上清液用于定性和/或定量测定所述上清液中的至少一种分析物的用途。此外,本发明涉及用于定性和/或定量测定流体样品中的至少一种分析物的方法,以及用于测定流体样品中的至少一种磷酰氧基物质的种类和/或量的方法。(The present invention relates to a complex comprising: a transition metal cation (i); a ligand (ii) comprising at least one chelating group, preferably 1 to 4 chelating groups, more preferably 2 or 3 chelating groups, wherein the chelating groups are selected from the group consisting of hydroxamate-groups-N (O) ‑ ) -C (═ O) -R, catechol ester groups, carboxylate ester groups, partially or fully protonated forms of these chelating groups, and mixtures of these chelating groups and/or partially or fully protonated forms of them, wherein R is hydrogen or a C1 to C5 alkyl group; and magnetic beads (iii); wherein the magnetic bead (iii) is covalently bonded to the ligand (ii). Book (I)The invention also relates to the use of the complex for reducing the content of at least one phosphoryloxy species in a fluid sample, the phosphoryloxy species preferably comprising the structural element-O-P (O) within its structure ‑ ) (═ O) -O-, and to a method for reducing the content of at least one phosphoryloxy species, preferably a phospholipid, in a fluid sample, comprising the step of adding the complex. The invention also relates to a supernatant obtained or obtainable from such a method, and to the use of a supernatant obtained or obtainable from such a method for the qualitative and/or quantitative determination of at least one analyte in said supernatant. Furthermore, the present invention relates to a method for the qualitative and/or quantitative determination of at least one analyte in a fluid sample, as well as a method for the determination of the kind and/or amount of at least one phosphoryloxy species in a fluid sample.)

1. A complex comprising

i) A transition metal cation;

ii) a ligand comprising at least one chelating group, preferably 1 to 4 chelating groups, more preferably 2 or 3 chelating groups, wherein the chelating groups are selected from the group consisting of hydroxamate-groups-N (O)^-) -C (═ O) -R, the partially or fully protonated form of the hydroxamate group, and mixtures of hydroxamate groups and their partially or fully protonated form, wherein R is hydrogen or a C1 to C5 alkyl group;

iii) magnetic beads;

wherein the magnetic bead (iii) is covalently bonded to the ligand (ii).

2. The complex of claim 1, wherein the ligand (ii) has the general structure (I)

Wherein:

- -is a bond to the magnetic bead (iii);

a is selected from the group consisting of hydrogen atom, -X-Y- (CH)₂)_m-CH₂-(CHR³)_q-R²-a group and R³A group of (a);

n, m are independently zero or an integer from 1 to 5;

p, q are independently an integer from 1 to 10, preferably an integer from 3 to 5, more preferably 3 or 4;

x is-CH₂-or-NH-;

y is-CH₂-or-C (═ O) -;

R¹、R²independently selected from the group consisting of: hydroxamate group-N (O)^-) -C (═ O) -R, where R is hydrogen or a C1 to C5 alkyl group, and partially or fully protonated forms of the hydroxamate groups;

R³is a hydrogen atom or a-NHZ group, wherein Z is a protecting groupA group, preferably-C (═ O) -O-CH₂-C₆H₅A group (benzyloxycarbonyl group, Cbz) or a tert-butoxycarbonyl group (Boc).

3. The complex of claim 1 or 2, wherein the ligand (ii) has the general structure (Ia):

wherein- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -.

4. The complex of any one of claims 1 to 3, wherein the ligand (ii) has the general structure (Ia1) or (Ia 2):

wherein- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -has the same meaning as in claim 2.

5. The complex according to any one of claims 1 to 4, wherein the transition metal cation (i) is selected from the group of platinum cations, ruthenium cations, iridium cations, scandium cations, titanium cations, vanadium cations, chromium cations, manganese cations, iron cations, cobalt cations, nickel cations, copper cations and zinc cations, more preferably from Fe²⁺、Fe³⁺And Zr⁴⁺And more preferably Zr⁴⁺。

6. The complex according to any of claims 1 to 5, wherein the magnetic bead (iii) comprises a polymer matrix (P), at least one magnetic particle (M) and at least one- (CH) covalently bonded on the surface (S) of the polymer matrix (P)₂)_r-NH- - -group, wherein- - -is a bond to the ligand (ii) and r is zero or an integer in the range of 1 to 10, preferably in the range of 1 to 5An integer within, more preferably an integer in the range of 2 to 4, more preferably 3; wherein the polymer matrix (P) comprises at least one crosslinked (co) polymer.

7. The composite according to claim 6, wherein the polymer matrix (P) comprises a copolymer obtained or obtainable by a process comprising polymerizing at least two different monomer building blocks selected from the group consisting of styrene, functionalized styrene, vinylbenzylchloride, divinylbenzene, vinyl acetate, methyl methacrylate and acrylic acid, preferably at least two different monomer building blocks selected from the group consisting of:

wherein r is zero or an integer in the range of 1 to 10, preferably an integer in the range of 1 to 5, more preferably an integer in the range of 2 to 4, more preferably 3;

R¹、R²、R³、R⁴and R⁵Independently of one another 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’(Cl)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, wherein R ', R "and R'" are independently from each other selected from the group consisting of alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, halide, hydrogen, sulfide, nitrate, and amine; and wherein x is an integer in the range of 1 to 3.

8. The composite according to claim 6 or 7, wherein the (co) polymer of the polymer matrix (P) is cross-linked, wherein the cross-linked (co) polymer of the polymer matrix (P) is obtained or obtainable by copolymerizing at least two different monomeric building blocks according to claim 8 in the presence of at least one monomeric building block being a cross-linking agent, wherein the cross-linking agent is preferably selected from the group consisting of divinylbenzene, bis (vinylphenyl) ethane, bis (vinylbenzyloxy) hexane, bis (vinylbenzyloxy) dodecane and mixtures of two or more of these cross-linking agents, preferably the cross-linking agent comprises at least divinylbenzene.

9. The composite according to any one of claims 6 to 8, wherein the at least one magnetic particle (M) comprises a compound selected from the group consisting of a metal, a metal carbide, a metal nitride, a metal sulfide, a metal phosphide, a metal oxide, a metal carbide, a metal chelate and a mixture of two or more thereof, wherein the at least one magnetic particle (M) preferably comprises a metal oxide or a metal carbide, more preferably comprises an iron oxide, in particular selected from the group consisting of Fe₃O₄、α-Fe₂O₃、γ-Fe₂O₃、MnFe_xO_y、CoFe_xO_y、NiFe_xO_y、CuFe_xO_y、ZnFe_xO_y、CdFe_xO_y、BaFe_xO and SrFe_xO, wherein x and y vary according to the synthesis method, and wherein x is preferably an integer from 1 to 3, more preferably 2, and wherein y is preferably 3 or 4, most preferably comprising Fe₃O₄。

10. Use of a complex according to any one of claims 1 to 9 for reducing the content of at least one phosphoryloxy species in a fluid sample, said phosphoryloxy species preferably comprising the structural element-O-P (O) within its structure^-)(＝O)-O-。

11. A method for reducing the content of at least one phosphoryloxy species, preferably a phospholipid, in a fluid sample, the method comprising the steps of:

a) providing a fluid sample comprising at least one analyte of interest and at least one phosphoryloxy species;

b) optionally adjusting the pH of the fluid sample such that the pH of the fluid sample is in the range of 2.5 to 12, thereby obtaining a pH adjusted fluid sample;

c) adding at least one first complex, preferably in the form of a suspension, more preferably in the form of an aqueous suspension, wherein said complex comprises:

i) a transition metal cation;

ii) a ligand comprising at least one chelating group, preferably 1 to 4 chelating groups, more preferably 2 or 3 chelating groups, wherein the chelating groups are selected from the group consisting of hydroxamate-groups-N (O)^-) -C (═ O) -R, the partially or fully protonated form of the hydroxamate group, and mixtures of hydroxamate groups and their partially or fully protonated form, wherein R is hydrogen or a C1 to C5 alkyl group;

iii) magnetic beads;

wherein the magnetic bead (iii) and the ligand (ii) are covalently bonded; thereby forming a suspension comprising a second complex comprising the first complex and the at least one phosphoryloxy species in complexed form;

d) spatially separating the second complex in the suspension obtained in (c) by applying a magnetic field, thereby obtaining a supernatant substantially free of the second complex;

e) removing the supernatant, thereby obtaining an isolated second complex.

12. A supernatant obtained or obtainable from the method of claim 11.

13. Use of a supernatant obtained or obtainable from the method according to claim 11 for the qualitative and/or quantitative determination of at least one analyte in the supernatant.

14. A method for the qualitative and/or quantitative determination of at least one analyte in a fluid sample, said method comprising the steps of the purification method according to claim 11 and further comprising the step of qualitatively and/or quantitatively determining said at least one analyte in the supernatant obtained from (e) and/or (f) and/or (g).

15. A method for determining the identity and/or amount of at least one phosphoryloxy species in a fluid sample, the method comprising the steps of the purification method according to claim 11 and further comprising:

h) adding an aqueous and/or organic elution solution to the (further) separated second complex obtained according to (e) and/or (f), wherein the aqueous and/or organic elution solution contains a buffering agent and/or a reducing agent, and/or wherein the addition is performed in a reducing atmosphere, thereby separating the at least one phosphoryloxy species from the separated second complex and obtaining a solution comprising the at least one phosphoryloxy species;

j) determining the identity and/or amount of the at least one phosphoryloxy species in the solution obtained according to (h).

Technical Field

The present invention relates to a complex comprising: a transition metal cation (i); a ligand (ii) comprising at least one chelating group, wherein the chelating group is selected from the group consisting of hydroxamate-group-N (O)^-) -C (═ O) -R, catechol ester groups, carboxylate ester groups, partially or fully protonated forms of these chelating groups, and mixtures of these chelating groups and/or partially or fully protonated forms of them, wherein R is hydrogen or a C1 to C5 alkyl group; and magnetic beads (iii); wherein the magnetic bead (iii) is covalently bonded to the ligand (ii).

The invention also relates to the use of the complex for reducing the content of at least one phosphoryl-oxy-substance (phosphorus-oxy-substance) in a fluid sample, the phosphoryl-oxy-substance preferably comprising the structural element-O-P (O-P) within its structure^-) (═ O) -O-, and to a method for reducing the content of at least one phosphoryloxy species, preferably a phospholipid, in a fluid sample, comprising the step of adding the complex. The invention also relates to a supernatant obtained or obtainable from such a method, and to the use of a supernatant obtained or obtainable from such a method for the qualitative and/or quantitative determination of at least one analyte in said supernatant. Furthermore, the invention relates to a method for the qualitative and/or quantitative determination of at least one analyte in a fluid sample, and to a method for the determination of a fluidA method of the type and/or amount of at least one phosphoryloxy species in a sample.

Background

Phospholipids (PPL) are abundant molecules in many human sample materials that may compromise diagnostic assays or have a negative impact on system robustness (see byyda c., Thiele, r., Kobold, u., Volmer, d.a., Analyst, 2014, 139, 2265-. Similarly, many phospho-oxy compounds (e.g. substances with phosphodiesters) such as (oligo) nucleotides or phosphorylated peptides and proteins are common (metabolic) substances which may interfere with analyte quantification.

The increase in S/N (S/N: signal to noise ratio) is a direct indication that there is less interference from substances other than the analyte of interest when measured via LC-MS/MS. Although this technique typically removes most of the interfering matrix via LC and then selects a definite MRM transition, in most cases the accuracy and sensitivity of the quantification is still compromised.

On the other hand, the same substance that is problematic in the quantification of certain analytes may be the analyte of interest itself for further quantification. These highly polar analytes (nucleotides/phosphorylated peptides) or hydrophobic/amphipathic analytes (phospholipids, PPL) are candidates that are difficult to (semi-) selectively isolate and purify from (human) sample material.

Several companies have been working on the removal of phospholipids. Zirconium (Zr) is known for its ability to complex with PPL and, therefore, is also commercially available in different forms. However, in order to remove PPL from the sample, it is not sufficient to add Zr to the sample. The Zr complexed PPL must then be removed from the mixture. There are several commercial methods and materials for removing phospholipids from samples, such as Hybrid SPE^TM (Sigma Aldrich)、Ostro^TM(Waters)、Captiva^TMND (Agilent) and Phere^TM(Phenomenex). However, all commercial materials use a flow-through method whereby a sample is loaded onto the respective material and eluted. Although these materials and methods appear to be effective tools in the decontamination of samples of primarily biological nature, these methods: a) is impractical (i.e., difficult to use with an automated sample preparation tool)A large number of samples (large volumes) are required for process set-up, b) expensive, c) time is required, d) large amounts of waste are generated, e) the removal of phospholipids is only solved and not the purification of the phospholipids themselves, f) the removal of phospholipids is only concerned, and other substances which may cause interference are not concerned.

Magnetic particles are excellent tools for capturing analytes from a sample. The magnetic properties are very important as they allow simple, fast and inexpensive automation on a diagnostic system and also avoid time consuming centrifugation and filtration steps. Superparamagnetic materials gain more attention because they exhibit magnetization only upon application of an external magnetic field. In the absence of an external magnetic field, the magnetization shows zero (no "memory effect"). A wide variety of beads are known and commercially available. However, to date, beads having a transition metal conjugated thereto in the following manner have not been known: that is, each conjugated transition metal atom is still capable of complexing with one or two molecules (such as a phospholipid).

Therefore, the technical problem underlying the present invention is to provide magnetic beads that enable an automated sample preparation workflow and overcome the above mentioned drawbacks.

Disclosure of Invention

This problem is solved by the invention using the features of the independent claims. Advantageous developments of the invention are set forth in the dependent claims and/or in the following description and detailed embodiments, which can be realized individually or in combination.

As used hereinafter, the terms "having," "including," or "containing," or any grammatical variations thereof, are used in a non-exclusive manner. Thus, these terms may refer to the absence of other features in the entity described in this context, in addition to the features introduced by these terms, as well as the presence of one or more other features. As an example, the expressions "a has B", "a includes B" and "a includes B" may refer both to the following cases: the absence of any other element in a than B (i.e., the case where a consists solely and exclusively of B) may also refer to the following case: in addition to B, one or more other elements are present in entity a, such as element C, elements C and D, or even other elements.

Furthermore, it should be noted that the terms "at least one," "one or more," or similar expressions, which indicate that a feature or element may be present one or more than one time, will generally be used only once when introducing the corresponding feature or element. In the following, in most cases, when referring to corresponding features or elements, the expression "at least one" or "one or more" will not be reused, despite the fact that the corresponding features or elements may be present one or more times.

Furthermore, as used below, the terms "preferably," "more preferably," "particularly," "more particularly," "specifically," "more specifically," or similar terms are used in conjunction with the optional features, without limiting the possibilities of substitution. Thus, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As the skilled person will appreciate, the invention may be implemented by using alternative features. Similarly, features introduced by "in embodiments of the invention" or similar expressions are intended to be optional features without any limitation to alternative embodiments of the invention, without any limitation to the scope of the invention, and without any limitation to the possibility of combining features introduced in this way with other optional or non-optional features of the invention.

In a first aspect, the present invention relates to a complex comprising:

i) a transition metal cation;

ii) a ligand comprising at least one chelating group, preferably 1 to 4 chelating groups, more preferably 2 or 3 chelating groups, wherein said chelating groups are selected from the group consisting of hydroxamate-groups-N (O)^-) -C (═ O) -R, catechol ester groups, carboxylate ester groups, partially or fully protonated forms of these chelating groups, and mixtures of these chelating groups and/or partially or fully protonated forms of them, wherein R is hydrogen or a C1 to C5 alkyl group;

iii) magnetic beads;

wherein the magnetic bead (iii) is covalently bonded to the ligand (ii).

It has been found that the complexes of the invention can be used to remove phosphoryloxy species from sample materials, which phosphoryloxy species preferably comprise the structural element-O-P (O) within their structure^-) (═ O) -O-, and its use has a positive effect on S/N, i.e. an increase in S/N of about 2 times compared to the untreated sample. This allows for more sensitive and accurate quantification of the analyte.

The covalent bond between the magnetic bead (iii) and the ligand (ii) may be any kind of suitable bond type, such as an amide bond, an amine bond, an ester bond or a (thio) ether bond. In one embodiment, the magnetic beads (iii) and the ligands (ii) are covalently bonded via amide bonds. Each chelating group (hydroxamate group, catechol group, carboxylate group, and their respective partially or fully protonated forms) has two heteroatoms that are capable of coordinating to a positively charged coordination center (such as a metal cation). That is, each of these chelating groups has two donor atoms.

According to one embodiment of the complex, ligand (ii) has the general structure (I)

Wherein:

- -is a bond to the magnetic bead (iii);

a is selected from the group consisting of hydrogen atom, -X-Y- (CH)₂)_m-CH₂-(CHR³)_q-R²-a group and R³A group of (a);

n, m are independently zero or an integer from 1 to 5;

p, q are independently an integer from 1 to 10, preferably an integer from 3 to 5, more preferably 3 or 4;

x is-CH₂-or-NH-;

y is-CH₂-or-C (═ O) -;

R¹、R²independently selected fromThe group consisting of: hydroxamate group-N (O)^-) -C (═ O) -R, where R is hydrogen or a C1 to C5 alkyl group, a catechol ester group, a carboxylate ester group, and partially or fully protonated forms of these chelating groups;

R³is a hydrogen atom or a-NHZ group, wherein Z is a protecting group, preferably-C (═ O) -O-CH₂-C₆H₅A group (benzyloxycarbonyl group, Cbz) or a tert-butoxycarbonyl group (Boc).

In one embodiment of the complex, ligand (ii) has the general structure (Ia):

wherein- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -has the same meaning as the same as shown as the same as the structure as the same as the.

According to a preferred embodiment of the complex, the ligand (ii) has the general structure (Ia1) or (Ia 2):

wherein- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -has the same meaning as disclosed above for the general structure (I).

In one embodiment of the complex, the transition metal cation (i) is selected from the group of platinum cations, ruthenium cations, iridium cations, scandium cations, titanium cations, vanadium cations, chromium cations, manganese cations, iron cations, cobalt cations, nickel cations, copper cations and zinc cations, more preferably from the group of Fe cations²⁺、Fe³⁺And Zr⁴⁺And more preferably Zr⁴⁺。

A transition metal complex ligand of general structure (I), (Ia1) or (Ia2) is bound to the bead and forms a bidentate, tetradentate or hexadentate complex with the transition metal cation (I). For example, hydroxamates are suitable ligands, and when three hydroxamate groups are in close proximity to a transition metal cation (i) such as Zr⁴⁺Upon standing, a good hexadentate Zr complex was formed. In one embodiment of the complex, the free coordination sites of the transition metal are occupied by solvent molecules (preferably water). Zr⁴⁺An octadentate complex tends to form. (see Guerard F., Lee Y.S., Tripier R., Szajek L.P., Deschammps J.R., Brechbiel M.W., Chem Commun.2013, 49: 1002-. Thus, the presence of 2 or 3 chelating groups (such as hydroxamate groups each having two donor atoms) results in Zr⁴⁺Four or two free coordination sites are created at the cation where the ligand of interest can be bound, i.e. a phosphoryloxy species, which preferably comprises the structural element-O-P (O) within its structure^-) (═ O) — O-, such as PPL, i.e. the exchange of water with a phosphoryloxy species, such as PPL, can occur.

In one embodiment of the complex, the magnetic bead (iii) comprises a polymer matrix (P), at least one magnetic particle (M) and at least one — (CH) covalently bonded to the surface (S) of the polymer matrix (P)₂)_r-NH- - -group, wherein- - -is a bond to the ligand (ii) and r is zero or an integer in the range of 1 to 10, preferably an integer in the range of 1 to 5, more preferably an integer in the range of 2 to 4, more preferably 3; wherein the polymer matrix (P) comprises at least one crosslinked (co) polymer.

Magnetic bead (iii)

The magnetic beads (iii) according to the present invention have a particle size in the range of 1 to 60 micrometer as determined according to ISO 13320.

In principle, the magnetic beads (iii) may exhibit any geometric shape, however, preferably the particles are substantially spherical. As used herein, the term "substantially spherical" refers to particles having a rounded shape that is preferably non-faceted or substantially free of sharp corners. In certain embodiments, the substantially spherical particles generally have an average aspect ratio of less than 3: 1 or 2: 1, such as an aspect ratio of less than 1.5: 1 or less than 1.2: 1. In certain embodiments, a substantially spherical ballThe particles of the shape may have an aspect ratio of about 1: 1. Aspect ratio (A)_R) Is defined as the maximum diameter (d)_max) And a minimum diameter (d) orthogonal thereto_min) Function of (A)_R＝d_min/d_max). The diameter is determined via SEM or optical microscopy measurements.

Polymer matrix (P)

As mentioned above, the magnetic beads (iii) comprise a polymer matrix (P).

In one embodiment of the composite, the polymer matrix (P) comprises a copolymer obtained or obtainable by a process comprising polymerizing at least two different monomer building blocks selected from the group consisting of styrene, functionalized styrene, vinylbenzylchloride, divinylbenzene, vinyl acetate, methyl methacrylate and acrylic acid, preferably at least two different monomer building blocks selected from the group consisting of:

wherein:

r is zero or an integer in the range of 1 to 10, preferably an integer in the range of 1 to 5, more preferably an integer in the range of 2 to 4, more preferably 3;

R¹、R²、R³、R⁴and R⁵Independently of one another 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, wherein R ', R "and R'" are independently selected from the group consisting of alkyl, aryl, cycloalkylHeteroaryl, heterocycloalkyl, halide, hydrogen, sulfide, nitrate, and amine; and wherein x is an integer in the range of 1 to 3.

In one embodiment of the composite, the (co) polymer of the polymer matrix (P) is crosslinked, wherein the crosslinked (co) polymer of the polymer matrix (P) is obtained or obtainable by copolymerizing at least two different monomeric building blocks according to the above embodiments in the presence of at least one monomeric building block which is a crosslinking agent, wherein the crosslinking agent is preferably selected from the group consisting of divinylbenzene, bis (vinylphenyl) ethane, bis (vinylbenzyloxy) hexane, bis (vinylbenzyloxy) dodecane and mixtures of two or more of these crosslinking agents, preferably the crosslinking agent comprises at least divinylbenzene.

Magnetic particle (M)

As mentioned above, the magnetic beads (iii) according to the present invention comprise at least one magnetic particle (M).

In one embodiment of the complex, the at least one magnetic particle (M) comprises a compound selected from the group consisting of: metals, metal carbides, metal nitrides, metal sulfides, metal phosphides, metal oxides, metal chelates, and mixtures of two or more thereof.

It is to be understood that each magnetic particle (M) may comprise a mixture of two or more of the above mentioned groups, i.e. two or more of a metal, a metal carbide, a metal nitride, a metal sulfide, a metal phosphide, a metal oxide, a metal chelate and a mixture of two or more thereof. In addition, mixtures of two or more different metals, two or more different metal oxides, two or more different metal carbides, two or more different metal nitrides, two or more different metal sulfides, two or more different metal phosphides, two or more different metal chelates are contemplated. In addition, it is to be understood that in case the magnetic beads (iii) according to the present invention comprise more than one type of magnetic particles (M), each magnetic particle (M) present in a single magnetic bead (iii) may be the same or may be different from each other. Preferably, all the magnetic particles (M) contained in one magnetic particle are identical. More preferably, the at least one magnetic particle (M) comprises a metal oxide or a metal carbide.

Thus, in one embodiment of the composite, the at least one magnetic particle (M) comprises a metal oxide or metal carbide, more preferably an iron oxide, in particular selected from the group consisting of Fe₃O₄、α-Fe₂O₃、γ-Fe₂O₃、MnFe_xO_y、CoFe_xO_y、NiFe_xO_y、CuFe_xO_y、ZnFe_xO_y、CdFe_xO_y、BaFe_xO and SrFe_xO, wherein x and y vary according to the synthesis method, and wherein x is preferably an integer from 1 to 3, more preferably 2, and wherein y is preferably 3 or 4, most preferably comprising Fe₃O₄。

In a preferred embodiment of the complex, the magnetic particles are superparamagnetic, and thus the magnetic beads (iii) are superparamagnetic as well. The term "superparamagnetic" is known to the person skilled in the art and refers to the magnetic properties encountered especially for particles smaller than a single magnetic monodomain. Such particles are stably oriented upon application of an external magnetic field until a maximum of the global magnetization (called saturation magnetization) is reached. They relax when the magnetic field is removed and are free of hysteresis (no remanence) at room temperature. In the absence of an external magnetic field, superparamagnetic particles exhibit a non-permanent magnetic moment due to thermal fluctuations in the dipole orientation (Neel relaxation) and particle position (Brownian relaxation).

The magnetic particles (M) are present in the center of the magnetic beads, thereby forming one or more so-called "cores", or are homogeneously distributed in the pores throughout the magnetic beads (iii), i.e. substantially homogeneously distributed within the polymer matrix (P).

The magnetic particles (M) preferably comprise, more preferably consist of, nanoparticles. The nanoparticles are preferably the magnetic, preferably superparamagnetic, part of the revealing particles. Nanoparticles are also sometimes referred to herein as "magnetic nanoparticles".

As used herein, the term "nanoparticle" refers to a particle that is less than 100 nanometers in at least one dimension (i.e., has a diameter of less than 100 nm). Preferably, the nanoparticles according to the invention have a diameter in the range of 1nm to 20nm, preferably 4nm to 15nm, as determined according to TEM measurements.

Each nanoparticle has a diameter in the range of 1nm to 20nm, preferably 4nm to 15nm, as determined according to TEM measurements. Preferably, the at least one magnetic nanoparticle is superparamagnetic.

The magnetic particles (M) may comprise only one nanoparticle or more than one nanoparticle. In one embodiment, the magnetic particles comprise 1 to 20 nanoparticles. In another embodiment, the magnetic particle comprises more than 20 nanoparticles, preferably 100 to 150 ten thousand nanoparticles, more preferably 750 to 750,000 nanoparticles, more preferably 1,750 to 320,000 nanoparticles, in particular 90,000 to 320,000 nanoparticles. The nanoparticles may be present as individual (i.e., isolated) particles, for example, the individual nanoparticles may be uniformly distributed throughout the pores of the magnetic beads, or they may form aggregates consisting of several nanoparticles. These aggregates may have different sizes depending on the number of nanoparticles included. In one embodiment, so-called super-particles are formed. According to this embodiment, the magnetic particles (M) comprise more than 20 nanoparticles, and usually more than 100 nanoparticles, wherein these nanoparticles are preferably aggregated with each other to form a super-particle. More preferably, in this case, the magnetic particles (M) comprise nanoparticles consisting of aggregated nanoparticles. Preferably, in this case, the magnetic particles (M) comprise a nanoparticle comprising between 100 and 150 ten thousand nanoparticles, more preferably between 750 and 750,000 nanoparticles, more preferably between 1,750 and 320,000 nanoparticles, in particular between 90,000 and 320,000 nanoparticles. In a preferred embodiment, in the presence of a super-particle, the magnetic particles (M) comprising the super-particle are present in the center of the magnetic bead and form a so-called "core". Preferably, such a core formed by the magnetic particles (M) has a diameter in the range of 80 to 500nm, more preferably 150 to 400nm, and most preferably 200 to 300nm, as determined according to DLS (ISO 22412).

Preferably, the amount of magnetic particles (M) is chosen such that a desired saturation magnetization of the magnetic beads (iii) is achieved. Preferably, the magnetic bead (iii) according to the invention has a molecular weight of at least 1A m²Saturation magnetization of/kg. Preferably, the saturation magnetization is at least 1A m²/kg, more preferably at least 2A m²/kg, more preferably at least 3A m²/kg, more preferably at least 4A m²/kg, more preferably at least 5A m²/kg, more preferably at least 6A m²/kg, more preferably at least 7A m²/kg, more preferably at least 8A m²/kg, more preferably at least 9A m²/kg, and in particular at least 10A m²/kg, such as at 10A m²From/kg to 20A m²In the range of/kg, more preferably in the range of 10A m²From/kg to 30A m²In the range of/kg, as determined according to ASTM A894/A894M.

Use of a complex

The invention also relates to the use of a complex as described above for reducing the content of at least one phosphoryloxy species in a fluid sample, said phosphoryloxy species preferably comprising the structural element-O-P (O) within its structure^-) (═ O) -O-. Preferably, the at least one phosphoryloxy species is selected from the group consisting of: phospholipids, phosphodiesters, oligonucleotides, polynucleotides, phosphorylated peptides and phosphorylated proteins, preferably phospholipids.

Method for reducing the content of at least one phosphoryloxy substance

The invention also relates to a method for reducing the content of at least one phosphoryloxy species, preferably a phospholipid, in a fluid sample, the method comprising the steps of:

a) providing a fluid sample comprising at least one analyte of interest and at least one phosphoryloxy species;

b) optionally adjusting the pH of the fluid sample such that the pH of the fluid sample is in the range of 2.5 to 12, thereby obtaining a pH adjusted fluid sample;

c) adding at least one first complex, preferably in the form of a suspension, more preferably in the form of an aqueous suspension, wherein said complex comprises:

i) a transition metal cation;

ii) a ligand comprising at least one chelating group, preferably 1 to 4 chelating groups, more preferably 2 or 3 chelating groups, wherein said chelating groups are selected from the group consisting of hydroxamate-groups-N (O)^-) -C (═ O) -R, catechol ester groups, carboxylate ester groups, partially or fully protonated forms of these chelating groups, and mixtures of these chelating groups and/or partially or fully protonated forms of them, wherein R is hydrogen or a C1 to C5 alkyl group;

iii) magnetic beads;

wherein the magnetic bead (iii) and the ligand (ii) are covalently bonded; thereby forming a suspension comprising a second complex comprising the first complex and the at least one phosphoryloxy species in complexed form;

d) spatially separating the second complex in the suspension obtained in (c) by applying a magnetic field, thereby obtaining a supernatant substantially free of the second complex;

e) removing the supernatant, thereby obtaining an isolated second complex.

The concentration of the at least one first complex in the suspension, more preferably in the aqueous suspension, is preferably in the range of 10mg/ml to 150mg/ml, more preferably in the range of 25mg/ml to 75mg/ml (50mg/ml +/-25 mg/ml).

In one embodiment, the method for reducing the content of at least one phosphoryloxy species further comprises:

f) applying a wash solution to the separated second complex, followed by spatially separating the second complex by a magnetic field and removing the remaining supernatant, thereby obtaining a further supernatant and a further separated second complex.

In one embodiment, the method for reducing the content of at least one phosphoryloxy species further comprises:

g) fusing the supernatants from (e) and (f).

The present invention also relates to a supernatant obtained or obtainable from the process described above in the section "process for reducing the content of at least one phosphoryloxy species".

The invention also relates to the use of a supernatant obtained or obtainable from a method as described above for the qualitative and/or quantitative determination of at least one analyte in said supernatant.

The present invention also relates to a method for the qualitative and/or quantitative determination of at least one analyte in a fluid sample, comprising the steps of the reduction method as described above and further comprising the step of the qualitative and/or quantitative determination of the at least one analyte in the supernatant obtained from (e) and/or (f) and/or (g).

The invention also relates to a method for determining the identity and/or amount of at least one phosphoryloxy species in a fluid sample, comprising the steps of the reduction method as described above and further comprising:

h) adding an aqueous and/or organic elution solution to the (further) separated second complex obtained according to (e) and/or (f), wherein the aqueous and/or organic elution solution contains a buffer, an acid and/or a reducing agent, and/or wherein the addition is performed in a reducing atmosphere, thereby separating the at least one phosphoryloxy species from the separated second complex and obtaining a solution comprising the at least one phosphoryloxy species;

j) determining the identity and/or amount of the at least one phosphoryloxy species in the solution obtained according to (h).

According to one embodiment, at least one buffer is contained in the aqueous elution solution and/or the organic elution solution, which results in a pH change. A change in pH can cause the phosphoryloxy species to be released from the magnetic beads. The mechanism is simply protonation of the chelating moiety, thereby disrupting the complex, causing release. By buffering agent is meant a system comprising an organic acid and its associated anion, wherein the system is capable of pH buffering. According to another embodiment, the aqueous elution solution and/or the organic elution solution contains at least one acid. There is little restriction on the at least one acid as long as it does not interfere with subsequent measurements. In one embodiment, the at least one acid is selected from the group comprising volatile organic acids, wherein the volatile organic acids comprise all carboxylic acids having a boiling point below 200 ℃, preferably below 150 ℃ at normal pressure (1013 mbar), preferably selected from the group consisting of formic acid, acetic acid and mixtures of formic acid and acetic acid. Non-volatile inorganic acids (such as HCl and phosphoric acid) are excluded. The change in pH causes the phosphoryloxy species to be released from the magnetic beads. The mechanism is simply protonation of the chelating moiety, thereby disrupting the complex, causing release. Another method that may result in the release of the phosphate compound is by reduction of the metal. This also causes destruction of the complex. Thus, according to one embodiment, the aqueous elution solution and/or the organic elution solution contains at least one reducing agent, which causes the release of the phosphate compound by reduction of the metal. This also leads to destruction of the complex. In one embodiment, the at least one reducing agent is selected from the group comprising organic reducing agents, wherein the organic reducing agent is preferably selected from the group consisting of dithiothreitol, dithioerythritol, mercaptoethanol and mixtures of these reducing agents.

According to one embodiment, the addition of the aqueous and/or organic elution solution is performed in a reducing atmosphere, or the aqueous and/or organic elution solution comprising the second complex is exposed to a reducing atmosphere, e.g. a hydrogen atmosphere, whereby also the phosphate compound is released, i.e. the complex is destroyed.

According to one embodiment, the method for disrupting the complex is mixed because: the aqueous elution solution and/or the organic elution solution contains at least one agent selected from the group comprising buffers, acids, reducing agents and mixtures of two or more of these agents and/or wherein the addition of the aqueous elution solution and/or the organic elution solution according to (h) is performed in a reducing atmosphere.

The invention is further illustrated by the following embodiments in combination with the embodiments indicated by the respective dependencies and back references. In particular, it should be noted that in each case where a range of embodiments is mentioned, for example in the context of a term such as "… … according to any one of embodiments 1 to 4", each embodiment within the range is intended to be explicitly disclosed to the skilled person, i.e. the wording of the term should be understood by the skilled person as being synonymous with "described in any one of embodiments 1, 2, 3 and 4.

1. A complex comprising

i) A transition metal cation;

ii) a ligand comprising at least one chelating group, preferably 1 to 4 chelating groups, more preferably 2 or 3 chelating groups, wherein said chelating groups are selected from the group consisting of hydroxamate-groups-N (O)^-) -C (═ O) -R, catechol ester groups, carboxylate ester groups, partially or fully protonated forms of these chelating groups, and mixtures of these chelating groups and/or partially or fully protonated forms of them, wherein R is hydrogen or a C1 to C5 alkyl group;

iii) magnetic beads;

wherein the magnetic bead (iii) is covalently bonded to the ligand (ii).

2. The complex of embodiment 1, wherein the ligand (ii) has the general structure (I)

Wherein:

- -is a bond to the magnetic bead (iii);

a is selected from the group consisting of hydrogen atom, -X-Y- (CH)₂)_m-CH₂-(CHR³)_q-R²-a group and R³A group of (a);

n, m are independently zero or an integer from 1 to 5;

p, q are independently an integer from 1 to 10, preferably an integer from 3 to 5, more preferably 3 or 4;

x is-CH₂-or-NH-;

y is-CH₂-or-C (═ O) -;

R¹、R²independently selected from the group consisting of: hydroxamate group-N (O)^-) -C (═ O) -R, where R is hydrogen or a C1 to C5 alkyl group, catechol ester group, carboxylate ester group, and partially or fully protonated forms of these chelating groups;

R³is a hydrogen atom or a-NHZ group, wherein Z is a protecting group, preferably-C (═ O) -O-CH₂-C₆H₅A group (benzyloxycarbonyl group, Cbz) or a tert-butoxycarbonyl group (Boc).

3. The complex according to embodiment 1 or 2, wherein the ligand (ii) has the general structure (Ia):

wherein- - (O- -X- -O) - -and A have the same meanings as in embodiment 2.

4. The complex according to any one of embodiments 1 to 3, wherein the ligand (ii) has the general structure (Ia1) or (Ia 2):

wherein- - (O- -X- -O) - -and Z have the same meanings as in embodiment 2.

5. The complex according to any one of embodiments 1 to 4, wherein the transition metal cation (i) is selected from the group of platinum cations, ruthenium cations, iridium cations, scandium cations, titanium cations, vanadium cations, chromium cations, manganese cations, iron cations, cobalt cations, nickel cations, copper cations and zinc cations, more preferably from the group of Fe²⁺、Fe³⁺And Zr⁴⁺And more preferably Zr⁴⁺。

6. The complex according to any one of embodiments 1 to 5, wherein the free coordination sites of the transition metal are occupied by solvent molecules (preferably water).

7. The complex according to any of embodiments 1 to 6, wherein the magnetic bead (iii) comprises a polymer matrix (P), at least one magnetic particle (M) and at least one- (CH) covalently bonded on the surface (S) of the polymer matrix (P)₂)_r-an NH — group, wherein — is a bond to the ligand (ii) and r is zero or an integer in the range of 1 to 10, preferably an integer in the range of 1 to 5, more preferably an integer in the range of 2 to 4, more preferably 3; wherein the polymer matrix (P) comprises at least one crosslinked (co) polymer.

8. The composite according to embodiment 7, wherein the polymer matrix (P) comprises a copolymer obtained or obtainable by a process comprising polymerizing at least two different monomer building blocks selected from the group consisting of styrene, functionalized styrene, vinylbenzylchloride, divinylbenzene, vinyl acetate, methyl methacrylate and acrylic acid, preferably at least two different monomer building blocks selected from the group consisting of:

wherein r is zero or an integer in the range of 1 to 10, preferably an integer in the range of 1 to 5, more preferably an integer in the range of 2 to 4, more preferably 3;

R¹、R²、R³、R⁴and R⁵Independently of one another 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’(Cl)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, alkylA group consisting of aryl, cycloalkyl, heteroaryl, heterocycloalkyl, wherein R ', R "and R'" are independently from each other selected from the group consisting of alkyl, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, halide, hydrogen, sulfide, nitrate, and amine; and wherein x is an integer in the range of 1 to 3.

9. The composite according to embodiment 7 or 8, wherein the (co) polymer of the polymer matrix (P) is crosslinked, wherein the crosslinked (co) polymer of the polymer matrix (P) is obtained or obtainable by copolymerizing at least two different monomeric building blocks according to embodiment 8 in the presence of at least one monomeric building block which is a crosslinking agent, wherein the crosslinking agent is preferably selected from the group consisting of divinylbenzene, bis (vinylphenyl) ethane, bis (vinylbenzyloxy) hexane, bis (vinylbenzyloxy) dodecane and mixtures of two or more of these crosslinking agents, preferably the crosslinking agent comprises at least divinylbenzene.

10. The complex according to any one of embodiments 7 to 9, wherein the at least one magnetic particle (M) comprises a compound selected from the group consisting of: metals, metal carbides, metal nitrides, metal sulfides, metal phosphides, metal oxides, metal chelates, and mixtures of two or more thereof.

11. The composite according to any one of embodiments 7 to 10, wherein the at least one magnetic particle (M) comprises a metal oxide or metal carbide, more preferably an iron oxide, in particular selected from the group consisting of Fe₃O₄、α-Fe₂O₃、γ-Fe₂O₃、MnFe_xO_y、CoFe_xO_y、NiFe_xO_y、CuFe_xO_y、ZnFe_xO_y、CdFe_xO_y、BaFe_xO and SrFe_xO, wherein x and y vary according to the synthesis method, and wherein x is preferably an integer from 1 to 3, more preferably 2, and wherein y is preferably 3 or 4, most preferably comprising Fe₃O₄。

12. The complex according to any one of embodiments 1 to 11, wherein the magnetic particle is superparamagnetic.

13. Use of a complex according to any of embodiments 1 to 12 for reducing the content of at least one phosphoryloxy species in a fluid sample, said phosphoryloxy species preferably comprising the structural element-O-P (O) within its structure^-)(＝O)-O-。

14. The use according to embodiment 13, wherein the at least one phosphoryloxy species is selected from the group consisting of: phospholipids, phosphodiesters, oligonucleotides, polynucleotides, phosphorylated peptides and phosphorylated proteins, preferably phospholipids.

14. A method for reducing the content of at least one phosphoryloxy species, preferably a phospholipid, in a fluid sample, the method comprising the steps of:

a) providing a fluid sample comprising at least one analyte of interest and at least one phosphoryloxy species;

b) optionally adjusting the pH of the fluid sample such that the pH of the fluid sample is in the range of 2.5 to 12, thereby obtaining a pH adjusted fluid sample;

c) adding at least one first complex, preferably in the form of a suspension, more preferably in the form of an aqueous suspension, wherein said complex comprises:

i) a transition metal cation;

ii) a ligand comprising at least one chelating group, preferably 1 to 4 chelating groups, more preferably 2 or 3 chelating groups, wherein said chelating groups are selected from the group consisting of hydroxamate-groups-N (O)^-) -C (═ O) -R, catechol ester groups, carboxylate ester groups, partially or fully protonated forms of these chelating groups, and mixtures of these chelating groups and/or partially or fully protonated forms of them, wherein R is hydrogen or a C1 to C5 alkyl group;

iii) magnetic beads;

wherein the magnetic bead (iii) and the ligand (ii) are covalently bonded; thereby forming a suspension comprising a second complex comprising the first complex and the at least one phosphoryloxy species in complexed form;

d) spatially separating the second complex in the suspension obtained in (c) by applying a magnetic field, thereby obtaining a supernatant substantially free of the second complex;

e) removing the supernatant, thereby obtaining an isolated second complex.

16. The method for reducing the content of at least one phosphoryloxy species according to embodiment 15, further comprising:

f) applying a wash solution to the separated second complex, followed by spatially separating the second complex by a magnetic field and removing the remaining supernatant, thereby obtaining a further supernatant and a further separated second complex.

17. The method of embodiment 16, further comprising:

g) fusing the supernatants from (e) and (f).

18. A supernatant obtained or obtainable from the method of any one of embodiments 15 to 17.

19. Use of a supernatant obtained or obtainable from the method of any one of embodiments 15 to 18 for the qualitative and/or quantitative determination of at least one analyte in said supernatant.

20. A method for the qualitative and/or quantitative determination of at least one analyte in a fluid sample, said method comprising the steps of the purification method according to any one of embodiments 15 to 17 and further comprising the step of qualitatively and/or quantitatively determining said at least one analyte in the supernatant obtained from (e) and/or (f) and/or (g).

21. A method for determining the identity and/or amount of at least one phosphoryloxy species in a fluid sample, the method comprising the steps of the purification method according to any one of embodiments 15 to 17 and further comprising:

h) adding an aqueous and/or organic elution solution to the (further) separated second complex obtained according to (e) and/or (f), wherein the aqueous and/or organic elution solution contains an acid, a buffer and/or a reducing agent, and/or wherein the addition is performed in a reducing atmosphere, thereby separating the at least one phosphoryloxy species from the separated second complex and obtaining a solution comprising the at least one phosphoryloxy species;

j) determining the identity and/or amount of the at least one phosphoryloxy species in the solution obtained according to (h).

Drawings

FIG. 1: showing the post-treatment steps for spiked serum prior to final purification with Zr composite beads;

FIG. 2: the S/N ratio of aldosterone samples is shown;

FIG. 3: the S/N ratio of nortriptyline samples is shown;

FIG. 4: the S/N ratio of the benzoylecgonine samples is shown;

FIG. 5: the LPC (18:0) area of samples with or without significant removal of LPCs is shown.

Cited documents

Bylda C.，Thiele，R.，Kobold，U.，Volmer，D.A.，Analyst，2014，139，2265-2276.

Guerard F.，Lee Y.S.，Tripier R.，Szajek L.P.，Deschamps J.R.，Brechbiel M.W.，Chem.Commun.，2013，49：1002-1004.

Guérard，F.，Lee，Y.S.，Brechbiel，M.W.，Chemistry，2014，20(19)：5584-5591.

Examples

1. Chemical product

2. Design of experiments

Zr composite beads were synthesized to remove residual matrix (i.e. impurities) from semi-purified human serum and subsequently evaluated for i) a tendency to increase the signal-to-noise ratio (S/N) of the analyte quantified using LC-MS/MS method and ii) a tendency to remove lysophosphatidylcholine, an important phospholipid.

The increase in S/N is a direct cue that there is less interference from substances other than the analyte of interest when measured via LC-MS/MS. Although this technique typically removes most of the interfering matrix via LC and then selects a definite MRM transition, in most cases the accuracy and sensitivity of the quantification is still compromised. Thus, a cleaner sample allows less ion suppression, thereby allowing for higher sensitivity and higher accuracy. In fact, the impact of the net sample on the robustness of the system is substantial. One directly measurable parameter known to impair the lifetime of an LC-MS/MS system for accurate and sensitive measurements is phospholipids. For this purpose, lysophosphatidylcholine (18:0) (LPC 18:0) was selected as a representative substance and quantified to assess whether the new magnetic beads could remove the substance.

2.1 conjugation of zirconium chelate ligands to magnetic beads and chelation with Zr

Magnetic beads 1 with free amino groups are chosen as suitable magnetic beads. The diacetylated form of ligand 1 (i.e., the dipeptide of O-acetyl hydroxamate-derived ornithine) was coupled to the free amine using standard peptide chemistry. Subsequently, the hydroxyl group was deprotected and the ligand was complexed with Zr (see scheme 2), thereby obtaining Zr composite magnetic beads.

Scheme 1

Conjugating the diacetylated form of ligand 1 to magnetic beads to give hydroxamate beads, deprotecting the hydroxamate group of ligand 1 and complexing with Zr (iv) to give Zr composite magnetic beads.

2.2 evaluation of the ability of Zr composite magnetic beads to remove phospholipids and improve signal-to-noise ratio (S/N) in LC-MS/MS quantification.

The purpose of the Zr composite magnetic beads is to remove as much material as possible from the (semi-clean) biological matrix. The purpose of this experiment was to demonstrate that these beads were able to remove important matrix components, thereby improving: i) quantification of clinically relevant analytes (i.e. S/N) and ii) system robustness (less residual matrix significantly extends the lifetime of the LC-MS/MS system as a whole).

For this purpose, two experiments were performed. The first experiment involved examining S/N from the purified serum and the second involved measuring lysophosphatidylcholine 18:0(LPC 18: 0).

To obtain enough samples containing clinically relevant analytes that were purified from serum using an enrichment workflow that was found to produce high recoveries of these analytes, multiple 100 μ l aliquots of spiked serum were treated 5 times to produce 300 μ l of semi-clean eluate according to the method described below (see also fig. 1). The eluate was then diluted again with water (1: 1). These mixtures were then aliquoted into 9 containers. Containers 1 to 3 were charged with 15. mu.l of water, containers 4 to 6 were charged with 15. mu.l of magnetic beads 1, and containers 7 to 9 were charged with Zr composite magnetic beads. The tubes were then briefly vortexed and allowed to sit for 5 minutes. Subsequently, the supernatant was transferred to HPLC vials. The samples were then measured via LC-MS/MS to determine the S/N of the relevant analytes, and the LPC (18:0) content was compared from these samples.

3. Examples of the embodiments

Reference example 1 Synthesis of ligand 1 and its diacetylated form (O-acetyl hydroxamate-derived bird), respectively Dipeptides of amino acids)

Scheme 2

Synthesis of the diacetylated form of ligand 1

To the carbobenzoxy (Cbz) -protected dipeptide of ornithine is added benzaldehyde to form an imine. The resulting product was then oxidized using mCPBA, followed by hydrolysis and subsequent acetylation. The resulting product (diacetylated form of ligand 1) can be used with standard peptide chemistry, as such via its free carboxylic acid for free amine coupling to appropriate beads.

Example 1 Synthesis of magnet bonded to dipeptide of ornithine derived from O-acetyl hydroxamate via an amide bond Beads (hydroxamate beads)

Approximately 9. mu. mol of ligand (here ligand 1 in diacetylated form) can be coupled to 30mg of magnetic beads 1, assuming a molecular weight of 1 kDa. According to this protocol, DMF (0.25ml) was added to 30mg of magnetic beads 1 and stirred. To this was added a solution of ligand 1 in diacetylated form (20,8mg, 4 equivalents, 36 μmol), HOBt (5mg, 2 equivalents, 18 μmol), DIC (5,6 μ l, 4 equivalents, 36 μmol), N-methylpiperidine (4,3 μ l, 2 equivalents, 18 μmol) in DMF (0,25ml) and the flask was gently mixed by rolling at room temperature for 2 hours.

After the amide conjugation, the reaction mixture was removed from the conjugate beads (magnetic beads 1 coupled to ligands 1 via amide bonds) by applying a magnetic field. The conjugate beads were washed 3 times with MeOH, then 3 times with water, and twice with MeOH. The solvent was then removed and the conjugate beads were resuspended in 6% N-methylpiperidine in MeOH. Again, the conjugate beads were washed 3 times with MeOH, then 3 times with water. Subsequently, the solvent was removed and the conjugate beads were dried under vacuum, yielding 30mg of conjugate beads.

The reaction mixture (0.5ml) used, which was removed from the beads after the reaction, was evaluated for the presence of unreacted dipeptide: a solution of 6% DIPEA in MeOH (1ml) was added and left for 30 minutes. Adding FeCl thereto₃Making it yellow.

The reaction mixture not contacted with the free amine beads (i.e., magnetic beads 1) was used as a negative control. The reaction mixture was subjected to the same treatment (i.e., 6% DIPEA in MeOH was added, the mixture was left for 30 minutes, followed by FeCl addition₃). The color of the mixture was dark brown. This indicates that most, if not all, of the dipeptide reacted with the free amine beads has reacted. That is, any unreacted dipeptide will react with Fe³⁺Complex and produce a brown color, as in the case of the negative control.

Example 2 Synthesis of zirconium composite (Zr composite beads) from the beads of example 1

Hydroxamate beads (30mg) were suspended in water (1ml) and incubated with ZrOCl by rolling for 2 hours at room temperature₂(250. mu.l, 1M aqueous solution) was reacted. The reaction mixture is removed from the conjugate beads by applying a magnetic field. The conjugate beads were then washed 3 times with water. The beads were then resuspended in 1ml of water to give 30mg/ml of Zr composite magnetic beads.

Example 3 post-treatment of spiked serum and Final purification with Zr composite magnetic beads

The standard bead evaluation workflow is as follows (see fig. 1): in the tube, a sample labeled with the analyte of interest (see table 1 for details) is loaded (step 1). The analytes used were aldosterone, benzoylecgonine and nortriptyline. A pH adjusting agent is then added to set the pH of the mixture (step 2). To this was added the bead suspension of Zr composite magnetic beads from example 2, the mixture was shaken and incubated for 5 minutes (step 3). Subsequently, a magnetic field is applied, the magnetic beads are attracted to the side of the vessel (step 4) and the supernatant is removed (step 5). Next, a wash solution is added and the mixture is shaken (step 6), after which the beads are again separated from the supernatant, and the supernatant is then removed again. This process was repeated once more. Subsequently, the elution solution was added (step 7) and the mixture was shaken and incubated for an additional 5 minutes. Next, the beads are separated from the supernatant, which is then transferred to another vial (step 8). To this was added a mixture of internal standards with compounds spiked into the serum sample (step 9). Details are shown in table 1 below. Therefore, enrichment or dilution of the analyte is not achieved using this workflow. For comparison, the same procedure was performed with free 1 and without (control) beads.

TABLE 1

Pooled sera from different donations to which the analyte of interest was added at a ratio of 1: 40 (spiked pool: serum, v/v).

ISTD (internal standard) mixture with isotopically labelled analogues of the added analyte of interest. ISTD allows correction of matrix effects and pipetting inaccuracies. Furthermore, since these concentrations are known, this allows the calculation of analyte recovery.

3.1 bead assessment, reagents and tools

For evaluation of S/N of analytes and comparison of LPC (18:0) content, an Agilent Infinity II multisample sampler and HPLC system was used in combination with an Agilent Poroshell 120 SbAq column (2.1 mm. times.50 mm, 2.7 μm, SEQ ID NO: USFAH01259) or a Thermo Fisher Hypercarb column (2.1 mm. times.50 mm, 3 μm, SEQ ID NO: 10517483). As for the mobile phase, water containing 0.1% formic acid was used as solvent a, and acetonitrile was used as solvent B. For MS/MS, AB-Sciex 6500+ TripleQuad was used, electrospray was used as the ion source. For integration, a MultiQuant software tool is used. The data was then imported into JMP SAS software and analyzed.

3.2S/N comparison of aldosterone, benzoylecgonine, and nortriptyline

One criterion for bead function is a significant improvement in S/N (signal to noise ratio) of clinically relevant analytes. For this purpose, the S/N of aldosterone for different samples was compared. It was shown that treatment with the Zr composite magnetic beads from example 2 resulted in an increase of about 2-fold in S/N compared to the untreated sample. By "free amine beads" is meant magnetic beads 1 with free amino groups for ligand conjugation and Zr complexation. It was observed that the free amine beads also produced samples that allowed better S/N, however the S/N of the Zr composite beads from example 2 outperformed the beads as well. The results are graphically shown in fig. 2. Comparable results were obtained with the other analytes nortriptyline (FIG. 3) and benzoylecgonine (FIG. 4).

3.3 LPC (18:0) comparison

The results of comparing the contents of lysophosphatidylcholine (18:0) (LPC (18:0)) are shown in FIG. 5. It can be seen that the sample treated with the Zr composite magnetic beads from example 2 contained significantly less LPC (18: 0).

It is clear that purging the sample material from which matrix material has been removed has a positive effect on the S/N. This allows for more sensitive and accurate quantification of the analyte. It has been demonstrated that by treating the sample with a new bead type (i.e., Zr composite beads), at least one phospholipid is removed.