WO2011120691A1

WO2011120691A1 - Method for isolating an analyte from a sample fluid with a protein-repellent hydrogel

Info

Publication number: WO2011120691A1
Application number: PCT/EP2011/001611
Authority: WO
Inventors: Hans-Peter Josel; Uwe Kobold; Oswald Prucker; Juergen Ruehe; Hartmut Schetters; Dingding He; Orestes Perivolaris
Original assignee: Roche Diagnostics Gmbh; Albert-Ludwigs-Universität Freiburg; F. Hoffmann-La Roche Ag
Priority date: 2010-04-01
Filing date: 2011-03-30
Publication date: 2011-10-06

Abstract

The present invention relates to a method of the present invention according to which at least one analyte is isolated from a sample fluid containing said at least analyte and at least one protein, comprising the steps of: a) contacting the sample fluid with a protein-repellent hydrogel immobilized on a solid surface to capture said at least one analyte; b) separating the hydrogel obtained in step a); c) optionally washing the hydrogel obtained in step b) with an aqueous solution containing up to ca. 30% by volume of an organic water-miscible solvent; d) contacting the hydrogel obtained in step c) with an aqueous solution containing at least 60% by volume of an organic water-miscible solvent; and e) isolating said at least one analyte obtained in step d); wherein the hydrogel is obtained by co-polymerization of at least one uncharged and polar monomer with at least one hydrophobic monomer and at least one cross-linking monomer.

Description

Method for Isolating an Analyte from a Sample Fluid

with a Protein-Repellent Hydrogel

The present invention is directed to a method for isolating at least one analyte from a sample fluid containing said at least one analyte and at least one protein, comprising the steps of:

a) contacting the sample fluid with a protein-repellent hydrogel immobilized on a solid surface to capture said at least one analyte;

b) separating the hydrogel obtained in step a);

c) optionally washing the hydrogel obtained in step b) with an aqueous solution containing up to ca. 30% by volume of an organic water-miscible solvent;

d) contacting the hydrogel obtained in step c) with an aqueous solution containing at least 60% by volume of an organic water-miscible solvent; and

e) isolating said at least one analyte obtained in step d);

wherein the hydrogel is produced by co-polymerization of at least one uncharged and polar monomer with at least one hydrophobic monomer and at least one cross-linking monomer.

One of the major aspects of modern clinical diagnostics is the analysis and in particular, the quantification of medically relevant substances, for example vitamins and pharmaceuticals, as well as of their corresponding metabolites which often have a low abundance and a low molecular weight. This does not only complicate the enrichment of such substances from often complex samples, such as, for example, human blood but also their detection and quantification in the respective matrix.

However, since the concentration of those compounds in human blood is of high and in case of vitamins or pharmaceutically active ingredients even of essential importance for humans, their quantification necessarily requires a high accuracy. Because of their specific mode of detection conventional methods, such as, for example photometry, high performance liquid chromatography (HPLC) and immunoassays are often quite unspecific and imprecise with respect to medically relevant substances since such samples, for example, blood or serum often represent a very complex biological matrix.

In comparison to said methods mass spectrometry (MS) gained a more and more important status in medicinal analytics for investigation of complex biological matrixes because of its increased measuring sensitivity. In addition, the development of so-called soft ionisation techniques made mass spectrometry applicable for almost all biologically relevant molecules. Further, techniques, such as. for example combination of liquid chromatography with downstream mass spectrometry (LC-tandem MS), and matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) provides high measuring sensitivities and simultaneously low detection limits.

However, a major disadvantage of these analytical systems is the requirement of a relatively sophisticated sample preparation which in most cases includes time consuming, manpower-intensive and hardly automatable working steps, such as, for example protein precipitation followed by ultra centrifugation. Further, trapping systems, mostly in form of preparative trapping columns, are required for separation and enrichment of the analyte from the matrix because of the generally low concentration of the analyte in the sample. In addition, the use of liquid chromatography for sample preparation takes much time when changing from adsorption to desorption conditions in the chromatographic device because of dead volumes in HPLC. Furthermore, the undesirably slow equilibration in the interim phases results in a broadening of the chromatographic peaks which in turn leads to a larger peak volume and a decrease in concentration of the analyte in the eluate. These effects finally result in a less favourable detection limit.

An improvement over the common techniques for preparing samples from complex matrixes is provided by so-called smart hydrogels which allow the adsorption of analytes in the swollen state and their desorption in the collapsed state. In WO 2004/073843 macroporous hydrogels having a large degree of cross-linking of up to 15% are described for separation of substances. The specific kind of cross-linking performed in the synthesis of the hydrogels leads to a high degree of cross-linking. Accordingly, the method of this document leads to the formation of macropores in the resulting hydrogels and moreover, because of the large size of these macropores the hydrogels do not possess the required mechanical stability for use in routine applications. Therefore, the hydrogels of WO 2004/073843 necessarily have to be incorporated into a solid support providing them with the required mechanical stability. Further, the presence of the macropores does not allow drying of such gels because this would result in a loss of the porosity of the hydrogel. Accordingly, macroporous hydrogels are not suitable for disposables used in standard sample preparations for routine medical analytics. A further disadvantage of macroporous hydrogels is the inadequate accessibility of additional capturing groups which are incorporated into the gel during polymerization because these groups can get lost in regions of high monomer concentration during polymerization. Accordingly, such groups may no longer be accessible for the analyte to be captured. Finally, a method and corresponding materials for the enrichment of low abundance and low molecular weight compounds is not disclosed in this document.

WO 2008/1 15653 also describes hydrogels that may be used for capturing analytes from a solution. In this context, the document only focuses on the enrichment of proteins and peptides from biological samples. It is known to the skilled person that these compounds may be present at low concentrations in a biological matrix. However, besides proteins and lipids also hydrophobic substances of medicinal interest, in particular those having low molecular weights, may be present in a biological matrix. Accordingly, isolation and enrichment of the latter compounds from biological matrixes requires much more elaborate methods or selective materials, respectively. Interestingly, WO 2008/1 15653 neither provides any materials nor any methods for the specific isolation and/or enrichment of small molecular weight compounds of medicinal interest from biological samples.

Accordingly, there is a need for a method that provides the selective isolation of medicinally relevant compounds such as vitamins or their metabolites having a low abundance and a low molecular weight from biological samples.

Using a thick layer of hydrogel for capturing at least one analyte from a sample fluid containing at least one analyte and at least one protein does not ensure a successful capturing and enrichment of said at least one analyte. Likewise, a successful isolation and enrichment of analyte(s) from a sample fluid is not provided either by use of hydrogels in a column as used in standard column chromatography because in such a device the hydrogel collapses and seals the column when pressure is applied.

Surprisingly, it has been found that short diffusion path ways for the respective analyte to be captured are preferred in order to achieve a successful isolation of at least one analyte from a sample fluid containing said at least analyte and at least protein. This is not realized by incorporating the hydrogel into a support as described in WO 2004/073843 or by simply using a thick layer of hydrogel. Quite to the contrary, short diffusion pathways for the analyte(s) are preferably realized by thin layers of hydrogel. In order to offer such thin hydrogel layer to the at least one analyte. the hydrogel is preferably immobilized on a solid support.

Therefore, the above-referenced object has been surprisingly solved by the method of the present invention according to which at least one analyte is isolated from a sample fluid containing said at least one analyte and at least one protein, comprising the steps of:

b) separating the hydrogel obtained in step a);

e) isolating said at least one analyte obtained in step d);

The term„isolating" or ..isolation" as well as the verb„to isolate" are used in context of the present invention as known to the person skilled in the art and denote the separation of a substance from a mixture of substances by use of a suitable separation process resulting in the enrichment of the analyte and reduction of sample matrix (e.g. proteins, lipids etc.). Considering the characteristics of the substances in the respective mixture the degree of separation for a particular substance may strongly vary depending on the specific separation process and its underlying separation principle. Accordingly, in order to achieve a degree of separation as high as possible or even an approximately complete separation for the desired compound or group of compounds, respectively, the separation process should be adequately chosen. With regard to isolation and enrichment of medicinally relevant compounds having a low abundance and a low molecular weight from complex biological samples the degree of separation should be as high as possible, in order to provide the desired medicinally relevant compounds in a concentration which is suitable for their exact and routine determination and quantification using standard instrumental analytics.

The term ..sample fluid" is used as known to the skilled person and in context of the present invention relates to any kind of probe containing preferably one or more medicinally relevant substance(s) in a fluid matrix. Further specified examples of such sample fluids are given below. The term ..enrichment^" is used in context of the present invention as known to the person skilled in the art and relates to the increase of concentration of a substance or a group of substances.

The term„analyte" as used in context of the present invention relates to any hydrophobic compound having a molecular weight of preferably below 1.000 Dalton which is typically retained on reverse phase when eluting with hydrophilic eluents, in particular those with a high content of water. In the present context the term ..reverse phase" is understood as defined below. Representative examples of such analyte(s) are mentioned below.

The term hydrophobic" in context of the present invention is used as known to the person skilled in the art and relates to any compounds which are predominantly characterized by van der Waals interactions as major or even solely intermolecular interactions to be considered. The term ..predominantly" in this context indicates that in principle, hydrophilic compounds may be also possible and present but only have a minor importance for the general characterization of the chemical and/or physical properties of the respective analyte. The opposite to the term hydrophobic" in context of the present invention is the term „hydrophilic" which denotes those compounds characterized by hydrogen bonding and which have a strong polar and/or protic character.

The term„protic" in context of the present invention denotes the property of containing or releasing proton(s) and/or of forming hydrogen bond(s), such as, for example water, alcohols, amines etc. The release of protons from a molecule is also known to the skilled person as dissociation. The simplest protic solvent is water, which in a simplified way dissociates into a proton and a hydroxyl ion. Well-known protic solvents are, for example, alcohols in which the release of the proton generally occurs at the hydroxyl group leaving a negatively charged oxygen atom of the former hydroxyl group because the electronegative oxygen atom is able to stabilize the resulting negative charge. Even carbonic acids may be considered as protic solvents, provided that the release of protons from the carboxylic function does not lead to a chemical reaction with a particular substance which for example is to be dissolved in the particular solution. A further group of protic solvents is represented by amines which contain„protons", strictly speaking hydrogen atoms, in their amino group as well as a free electron pair at the corresponding nitrogen atom for forming a hydrogen bond.

As already indicated above, protic solvents may be characterized in that they form hydrogen bonds. In context of the present invention the term ..hydrogen bond" is used as known to the person skilled in the art and denotes the bond being formed between a hydrogen atom which is covalently bound to the atom of an electronegative element (also known as proton donor, X) and another atom of an electronegative element which may be the same or different and which has a free electron pair (also known as proton acceptor, Y). In a simplified form, such a system is accordingly expressed as RX-H^»"Y-R\ wherein R and R' are alkyl residues and the dotted line represents the hydrogen bond. Typical elements for proton acceptor and proton donor are primarily oxygen, nitrogen, sulphur, and also halogens; in some case, for example in the case of HCN, even C (carbon) may act as proton donor. A typical element for a proton acceptor is, in principle, any electronegative element that when being part of an organic molecule has at least one free electron pair, such as, for example oxygen as part of a carbonyl, aldehyde or ether group or of a hydroxyl group, and nitrogen in an amino group. Typical examples for a proton donor are those atoms of electronegative elements which under appropriate conditions can release protons, for example alcohols, thiols or even amino groups. As a representative but not in any way limiting example a hydrogen bond may be formed between the hydrogen atoms of water (proton donor) and the free electron pair of nitrogen atoms in amines (proton acceptor). In a special case of a hydrogen bond specific functional groups may simultaneously act as proton donor and as proton acceptor which is further exemplified by means of hydroxyl groups, carbonic acid groups and most importantly, water.

For example, a compound such as acrylic acid H2C=CH-C(=0)-OH can act as proton acceptor through the free electron pairs of the carbonyl oxygen and as proton donor through the hydrogen atoms of the hydroxyl group. In contrast, a compound such as acrylamide H2C=CH-C(=0)-NH₂ can predominantly act as proton acceptor through the free electron pairs located at the oxygen atom of the carbonyl group and at the nitrogen atom of the amino group. Due to the +1 effect of two methyl groups in N,N-dimethyl acrylamide (DMAA) H₂C=CH-C(=0)-N(CH₃)₂ the proton accepting character is even more pronounced in DMAA than in acrylamide. The respective opposite to protic is„aprotic". The term ..aprotic" in context of the present invention is used as known to the expert skilled in the art and denotes those compounds which, generally, do not have the property of forming hydrogen bonds.

The term„polarity" in context of the present invention is used as known to the expert skilled in the art and denotes the separation of electric charge within a molecule leading to an electric dipole. The phenomenon of polarity in a molecule is based on the fact that electrons of a covalent bond are not always shared equally between two bond atoms, i.e., one atom may exert a stronger attractive force on the electron cloud than the other atom. This ..electron pull^" is attributed to the respective electro-negativity of the involved atoms which represents a measurement for their electron affinity. Accordingly, the unequal sharing of electrons leads to the formation of a dipole. Atoms having a high electronegativity, such as fluorine, oxygen and nitrogen, exert a stronger attractive force on electrons. The characteristic electro-negativity for the atoms of each element has been determined and tabulated as numerical value. For determining the polarity of a covalent bond, the difference between the electro-negativity of the atoms is considered. If the difference is between 0.4 and 1 .7 the bond is generally considered to be a polar covalent bond. In addition, arrangement of the polar bonds in the molecule and impact of non- binding pairs of electrons known as a full molecular orbital should be considered.

Representative polar organic compounds are for example acrylic acid H₂C=CH-C(=0)-OH and its derivatives, such as acrylamide H₂C=CH-C(=0)-NH₂. These compounds have a distinctive electric dipole because of two oxygen atoms or one oxygen atom and one nitrogen atom, respectively, bound to the central carbon atom. In addition, arrangement of the polar bonds in the molecule and impact of non-binding electron pairs also known as free electron pairs should be considered, wherein for this purpose the latter is viewed as a full molecular orbital. In case of acrylic acid the two free electron pairs of each oxygen atom do not point into opposite directions and therefore, contribute vectorially to the distinctive polar character of acrylic acid. Likewise, acrylamide H2C=CH-C(=0)-NH₂ is a polar compound.

The term„organic solvent^" in context of the present invention is used as known to the person skilled in the art and relates to any kind of solvent which primarily consists of carbon and hydrogen atoms. Typically, organic solvents are liquids at room temperature, usually at about 25°C. However, depending on the specific application also organic compounds which are solids at room temperature may be used as organic solvents when they are fluids in the temperature range at which the respective application(s) is/are performed. Typical organic solvents are hydrocarbons.

The term„hydrocarbon" in context of the present invention is used as known to the person skilled in the art and relates to any kind of molecule which primarily comprises carbon and hydrogen atoms. In the simplest case these molecules are linear saturated hydrocarbons (alkanes) entirely composed of carbon-carbon and carbon-hydrogen single bonds and therefore, follow the general formula C_nH_2n+2, wherein n is an integer equal or higher than 1 . Considering that typically organic solvents are fluids at room temperature, only hydrocarbons following the general formula C_nH_2n+2 with n being an integer ranging from 5 (pentane) to approximately 18 (octadecane) are in general considered as organic solvents on hydrocarbon basis. Octadecane having a melting point of from 28-30°C already represents a kind of transition area because depending on the specific application for which it is used it may be still fluid at room temperature or already solid. The hydrocarbons may be substituted with one or more heteroatoms, for example halogens such as fluoro, chloro, bromo or iodo atoms, such as in chloroform CHC1₃ or dichloromethane CH2CI2, primary, secondary or tertiary amino groups, wherein in this context the terms primary, secondary and tertiary are used as common in organic chemistry, or related groups such as amidino or guanidino groups, one or more ether bonds -O- such as in diethylether CH₃-0-CH3, carbonyl groups C(=0) such as an aldehyde group -C(=0)-H, for example in the most simple aldehyde formaldehyde (also known as methanal) H-C(=0)-H, or a ketone group -C(=0)-, for example in the most simple ketone actone CH₃-C(=0)-CH3, ester groups -C(=0)-0- such as ethylacetate (also known as ethyl ethanolate) CH₃-C(=0)-0-CH₂CH3, amide groups -C(=0)-NR'R" such as in acrylamide H2C=CH-C(=0)-NH₂, hydroxyl groups -OH such as in ethanol CH3-OH or in isopropyl CH₃-CH(OH)-CH₃, or nitrile groups -C≡N such as in acetonitrile CH₃-C≡N.

In context of the present invention the term hydrocarbon also relates to hydrocarbons with the same molecular formula following the general formula C_nH_2n+2 but with a different structural formal. Such hydrocarbons are also referred to as branched hydrocarbons in contrast to linear hydrocarbons, for example w-propane (wherein n here stands for normal, i.e., linear) and the branched wo-propane or n-butane and its structural isomers so-butane and /er/-butane. The term hydrocarbon in context of the present invention is not restricted to linear branched or unbranched hydrocarbons (alkanes) but also refers to cyclic hydrocarbons (cycloalkanes) according to the general formula C_nH2„. A typical cycloalkane is cyclohexane having six carbon atoms forming the 6-membered ring of cyclohexane, a further example is cyclopentane having five carbon atoms.

The term cyclic hydrocarbons as used in the context of the present invention also encompass those hydrocarbons having one or more rings built of carbon atoms wherein the rings are connected to each other over one common atom or edge, for example decahydronaphtalene (also known as decalin or as bicycloc[4.4.0]decane), a bicylic organic compound in which two hydrocarbon ring are connected with each other through a common edge. Alternatively, the two rings may be connected through one single bond such as in for example biphenyl (for definition of phenyl see below) in which two phenyl rings are covalently connected through one single carbon-carbon bond (even though biphenyl is a solid at room temperature it is mentioned in the present context for the sake of completeness). The term hydrocarbons as used in the present context also refers to unsaturated hydrocarbons having one or more carbon-carbon multiple bonds, such as double or triple bonds (unsaturated bonds). Unsaturated hydrocarbons having one or more carbon-carbon double bonds are so-called alkenes having the general formula C„H._?„ in case of only one carbon-carbon double bond and those hydrocarbons having one or more carbon-carbon triple bonds are so-called alkynes having the general formula C„H„-2 in case of only one carbon-carbon triple bond. For example, acrylic acid H2C=CH-C(=0)-OH and its derivatives have one carbon-carbon double bond.

Hydrocarbons may also contain so-called conjugated carbon-carbon double bonds of the type -C=C-C=C- such as in cyclic ring molecules. For example, carotenoids and in particular, carotenes have a divalent hydrocarbon chain consisting of 8 conjugated carbon- carbon double bonds which connects the two ionone moieties of a carotenoid.

In case the respective unsaturated cyclic hydrocarbon is ideally planar, i.e., all carbon atoms forming said cyclic hydrocarbon are in the same plane, and follows Hiickel's rule the particular hydrocarbon is aromatic. According to Hiickel's rule a cyclic ring molecule is aromatic when the number of its π-electrons equals 4n+2, wherein n is an integer ranging from 0 to 6, i.e., 0, 1 , 2, 3, 4, 5 or 6. In context of the present invention the term aromatic hydrocarbon relates to from 6- to 14-membered aromatics, for example a 6- membered aromatic having one ring such as benzene, a 10-membered aromatic is a so- called condensed aromatic compound comprised of two rings which are connected through a common edge such naphthalene and a 14-membered aromatic compound having three rings such as anthracene.

Aromatics may also contain heteroatoms, such as, for example nitrogen in pyridine C5H5N or in pyrrole C₄H N, or oxygen in furane QH₄O and benzofurane C₈H₆0, or sulphur in thiophene C₄H₄S. In addition, in aromatic solvents one or more hydrogen atoms may be replaced by hydrocarbon residues, for example in toluene (C₆H₅CH₃) one hydrogen is replaced with a methyl group and in xylene (also known as dimethylbenzen C₆H₄(CH₃)₂) two hydrogens are replaced with methyl groups. Depending, whether the two hydrogen atoms are in position 1 and 2, 1 and 3 or 1 and 4 of the aromatic ring, the xylol is referred to as o-xylol (1 ,2-xylol) , m-xylol (1 ,3-xylol) or p-xylol (1 ,4-xylol).

The term„uncharged^" in context of the present invention is used as known to the person skilled in the art and denotes the opposite of charged, i.e., the fact that a chemical substance, such as an organic compound does not have a charge. The term ,,miscible" in context of the present invention is used as known to the expert skilled in the art and denotes the property of substances such as, for example, solvents to form homogeneous mixtures with one another in any conceivable proportions. The term "homogeneous^" in the present context is understood to mean a uniform system which is characterized in that independently from the number of components the system as a whole has everywhere the same consistency and thus, the same properties. In case of fluids the term homogeneous denotes a system in which there is only one macroscopic phase. In context of the present invention the term ..water-miscible^" is used as known to the expert skilled in the art and denotes the property of a substance, for example, of a solvent to be miscible with water. Suitable solvents having the property of being ..water-miscible" are for example methanol, ethanol, tetrahydrofuran, or dimethylformamide. In order to remove sample matrix still adhered to the hydrogel obtained in step b) said hydrogel is optionally washed with an aqueous solution containing up to ca. 30% by volume of an organic water-miscible solvent, wherein the terms„hydrogel",„organic" and „ water-miscible" are understood as defined above. The term„up to ca. 30%" is used in the present context to mean any conceivable proportion of an organic water-miscible solvent in said aqueous solution in the range of from 0% to ca. 30%, at the most. The term„ca." in context with 30% is used to denote a variation in the range of error of +1-5%, and therefore, the term "up to ca. 30%", is used to mean any upper range of up to +/-30%, i.e., 25%, 30% or 35%. Accordingly, the term "up to ca. 30%", is used to mean any of the following values, such as 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, or 35%.

The term„reverse-phase" in context of the present invention and in particular in context with the meaning of the term„analyte" is used as known to the expert skilled in the art and relates to any chromatographic method involving a non-polar stationary phase, i.e., liquid chromatography as well as gas chromatography. The term ..reverse-phase" is simply derived from the fact liquid chromatography was originally performed on unmodified silica or alumina and thus, on a hydrophilic surface having a distinctive affinity for polar and/or hydrophilic compounds. Accordingly, it was considered as ..normal". The introduction of alkyl chains covalently bound to the support surface changed the polarity of the stationary phase from polar to unpolar and thus, reversed the elution order (see for example I. Molnar, C. Horvarth, Clin. Chem.,„Reverse-phase chromatography of polar biological substances: separation of catechol compounds by high-performance liquid chromatography^", 22: 1497-1502 (1976)): on stationary phase materials having alkyl chains covalently bonded to its surface polar compounds are eluted first while unpolar compounds are retained, and therefore, the respective liquid chromatography technique is called reverse liquid chromatography. All of the known mathematical and experimental considerations used on other chromatographic methods in general also apply to reverse- phase chromatography, such as, for example the preparation resolution in proportion to the column length.

The term stationary phase as used in context with the term reverse-phase denotes any non- polar substance that achieves sufficient packing and that can be used for reverse-phase liquid chromatography. In principle, reverse-phases are obtained by derivatizing the silanol groups (SiOH groups) as main part of the surface of silica by reacting the silica with dialkyl dichlorsilanes, for example. The stable material obtained in that manner can have hydrocarbon chains having a chain length of from 2 to 18 carbon atoms that are referred to as chemically bonded phases. The layer formed on the surface of silica by the alkyl chains is often called brushes. The most popular column for reverse-phase chromatography used today is Ci₈-bonded silica (silica having a bonded hydrocarbon residue with a backbone of 18 carbon atoms). Besides this material also C₈-bonded silica (silica having a bonded hydrocarbon residue with a backbone of 8 carbon atoms), cyano bonded silica and phenyl bonded silica are popular materials for reverse-phase chromatography.

Without exception C|₈, Cg and phenyl bonded silica are reverse-phase stationary materials. However, depending on the analyte and the mobile phase cyano bonded silica can be used in reverse-phase and in normal mode. Surface functionalization of silica can be performed in monomeric or polymeric reaction(s) with different short-chain organosilanes used in a second step to cover remaining silanol groups (end-capping). While the overall retention mechanism remains the same subtle differences in the surface properties will lead to changes in the polarity of the stationary phase. The term ..mobile phase" in the present context is used as known to the expert skilled in the art and relates to any mixtures of water and/or aqueous buffers, and organic solvents being suitable to elute analytes from a chromatography column. The term„to elute^" or „eluting", respectively, in the present context is used as known to the expert skilled in the art and denotes the dissolution, optionally the displacement, of adsorbed substance(s) from solids or adsorbents, which are impregnated with fluids, i.e., the column material to which the substance(s) is/are adsorbed. The term ..adsorption^" in the present context is used as known to the expert skilled in the art and denotes the accumulation of substances from gases or fluids at the boundary phase formed of the gases or fluids with a substance, wherein the latter is able to adsorb the substances at its surface. This adsorption leads to an accumulation of the adsorbed substances at the particular surface. The substance that is able to accumulate substances at its surface is often referred to as adsorbant and the adsorbed material as adsorbate. The term adsorption is usually distinguished from the term ..absorption^" which beyond the accumulation at a surface also refers to the penetration of the accumulated substances into the interior of the adsorbing solid or fluid. In general, adsorption is a physical process in which substances usually molecules adhere to a surface of the adsorbent and thus, are accumulated at the respective surface. The forces being responsible for this adherence are considered to be physical forces rather than chemical bonds and thus, adsorption is also known in the art as physical adsorption or physisorption, which does not necessarily exclude chemical bonding of substances to the surface. The physical forces involved in the adsorption of substances to a surface are in most cases van der Waals-forces, London forces or dipole/dipole interactions, for example hydrogen bonds, or dipole-induced dipole interactions, wherein these terms are used as either explained above or as normally used in context with adsorption. In (column) chromatography usually solvents are used as eluant, i.e., eluting agent in which the substance(s) which are to be eluted are at least sufficiently soluble. Empirically, solvents may be ordered according to their eluting effect in (column) chromatography. In general, the resulting„eluotrop order" is based on a particular adsorbent and goes parallel to the polarity of the solvent.

Solvents suitable for being used as„mobile phase" in reverse-phase chromatography are in general miscible with water. Most commonly acetonitrile, methanol or tetrahydrofuran (THF) are used as solvents in reverse-phased chromatography. Other solvents that may also be used are for example ethanol, 2-propanol (iso-propyl alcohol). Elution can be performed either isocratic or by using a gradient. Isocratic mode of elution means that the proportions in the water/solvent composition do not change while gradient means that the proportions of the water/solvent composition are changed during the separation process.

The term„protein" in context of the present invention is used as known to the expert skilled in the art and denotes all naturally occurring and/or artificial co-polymers which are in general comprised of amino acids as corresponding monomers, wherein the latter term encompasses all 20 naturally occurring amino acids as well as artificial amino acids. In a naturally occurring protein the string of amino acid units and thus, its structure is encoded in the corresponding gene. In a protein the different amino acids are connected to one another through peptide bonds (-NH-CO-) between formed of the carboxy function of a first amino acid and the amino group (-NH?) of a second amino acid. In general, two amino acids can condense to a dipeptide under elimination of water, which reaction is catalyzed by ribosomes during translation. Through repeated condensation tripeptides, oligopeptides, polypeptides, and finally proteins are performed. Besides amino acids proteins may contain at least one ,,ηοη-peptidic" component which may be part of the protein chain and/or of one or more side chain(s). Proteins may be distinguished from the structurally similar polypeptides due to their relatively high molecular weight. From ca. 100 monomer units upwards co-polymers primarily built up of amino acids are considered as proteins. The molecular weight of naturally occurring proteins can be in the range of up to from 10,000 to several million Daltons. Depending on their shape and their behaviour towards water and salts proteins may be groups as follows: globular proteins or spherical proteins, such as for example albumins, globulins, glutelins, histones, prolamines, protamines, as well as scleroproteins or fibrillary, skeletal or fibre proteins (structural proteins), such as, for example keratins, fibroin, elastin, or collagen. In addition, depending on the respective composition of the proteins a further distinction may be made between simple proteins and conjugated proteins. While hydrolysis of simple proteins only gives amino acids, hydrolysis of conjugated proteins furthermore also gives additional components which are considered as essential for the specific properties of conjugated proteins. Those further components are exemplarily represented by nucleoproteins (nucleic acids; chromatin), glycoproteins (carbohydrates; lecithin, immunoglobulins, blood group substances), lipoproteins (lipids), phosphoproteins (phosphoric acid; casein, vitelline), chromoproteins (colourants; hemoglobin, cytochromes, catalase, rhodopsin), metalloproteins (metals; ceruplasmin, plasma transferrins, ferredoxin amongst others, iron proteins) and others.

The term protein in the present context also encompasses proteins in all their different structural appearance forms. Most proteins fold into unique three-dimensional structures, wherein the shape into which a protein generally folds is known as its native conformation.

While many proteins are able to fold unassisted, simply through the chemical properties of their amino acids and/or additional components, other proteins require aid of molecular chaperones to fold into their native states. Accordingly, the term protein in the present context also encompasses the meanings primary structure, secondary structure, tertiary structure and quaternary structure of a proteins, wherein the aforementioned terms are used as known the skilled person. In addition, the term ..protein^" in context of the present invention is also understood to encompass the meaning of single proteins as well as their association to corresponding quaternary structures. Furthermore, the term ..protein" as used in context of the present invention includes enzymes, antibodies, receptors, transport proteins, membrane proteins, cytokines and hormones.

In general, proteins are easily soluble in water. The only important exceptions are membrane proteins. In view of the generally acknowledged solubility characteristics of proteins in water, the term „protein-repellent" in context of the present invention is understood to mean those substances that only interact weekly with proteins, for example in form of temporary dipole/dipole interactions. In this context, the term temporary dipole/dipole interactions is used as known to the expert skilled in the art and denotes that those interactions are only based on the occasional or statistically averaged, respectively, distribution of electron density within a molecule which may be optionally induced through the presence of permanent dipole. On the contrary, so-called„protein-repellent" substances do not form any strong interactions with proteins in form of bonds, such as, for example hydrogen bonds or electrostatic interactions based on charged groups within the respective molecules. The term„repellent" in combination with a specific compound is known in the art for example from the expression ..water repellent" which relates to the phenomenon that water forms droplets on hydrophobic surfaces. This particular phenomenon is attributed to the fact that water can form stronger intramolecular interactions with itself in form of hydrogen bonds than with hydrophobic surface through much weaker van der Waals interactions. Therefore, the approximately spherical form of water droplets is energetically the most favoured. From a macroscopic point of view, water is repelled from the hydrophobic surface.

In context of the present invention the term ..protein-repellent" denotes the property of substances, such as the hydrogel according to the present invention to repel proteins, i.e., not to form any strong intermolecular interactions with proteins. Accordingly, the protein- repellent hydrogel may be compared with the hydrophobic surface from which water drips off. The term„hydrogel" as used in context of the present invention denotes any swellable polymer gels on the basis of polymers which are chemically or physically connected with one another under formation of a three-dimensional network. The chemical connectivity is realized through bond formation, whereas the physical construction of hydrogels may be on the basis of electrostatic, hydrophobic or dipole/dipole interactions between single areas of the respective polymer segments. In context of the present invention the term ..hydrogel" is preferably meant to denote polymer gels in which the three-dimensional network is obtained through chemical bond formation. The network itself can consist of one or more different components. In the presence of a suitable solvent the network swells under simultaneous incorporation of the respective solvent into its three-dimensional network until an equilibrium volume of incorporated volume is reached. In another terminology the swollen state of the network is known as gel and the non-swollen state is known as gelator. In context of the present invention the term hydrogel and/or gel also encompasses the meaning of the term gelator.

Originally, the term ..hydrogel" was meant to denote only those gels constructed of hydrophilic but water-insoluble polymers which swell in the presence of water as solvent. The affinity of hydrogels to water is attributed to salvation and entropic effects of the polymeric network. Beside water also pure hydrophilic organic solvents, such as, for example methanol, ethanol and dimethylformamide as well as their respective aqueous solutions containing the organic solvent in variable amounts effect swelling of hydrogels, wherein the term hydrophilic is understood as explained above. Accordingly, today the term hydrogel is no longer limited to only those gels that swell under incorporation of water into their network but also under incorporation of hydrophilic organic solvents and/or of their respective aqueous solutions and/or mixtures of variable composition.

The term„smart" in context with hydrogels is used as known to the expert skilled in the art and denotes the property of a selected class of hydrogels to selectively react to gradients of surrounding chemical and/or physical parameters with a distinctive change in their volume. Representative chemical parameters are for example concentrations of substances and/or ions, including the charge of the respective ions, polarity of the solvent and pH value of the solutions. Representative physical parameters are for example temperature and pressure. Therefore, smart hydrogels are also considered to have integrated actor-sensor functionalities, i.e., they serve as an automatic controlled system combining sensor(s) and actor(s) in one single element. In the present context the term ..sensor" is used as a chemical equivalent to a technical element and thus, can react either qualitatively or quantitatively in form of a measurable factor to a specific physical and/or chemical parameter, such as, for example temperature, moisture, pressure, radiation or material properties of its surrounding. Accordingly, the term ,, actor" is used as a chemical analogue to a technical converter and therefore, acts as counterpart to the sensor converting a signal such a change of a parameter characterizing the surrounding into a specific action. In context of the present invention the specific action may be understood to be ..mechanical work" in the form of the release of the captured analyte(s) and the accompanied collapsing of the hydrogel effected by the change in the surrounding.

Accordingly, the method of the present invention allows enrichment of low abundance analytes from a sample fluid: if an analyte is present in a sample fluid at low concentration, the method according to the present invention allows capturing of said analyte from the sample and after separation of the hydrogel containing said at least one analyte from the sample fluid, the analyte may be released from that hydrogel by contacting the obtained hydrogel with a second solvent in which the analyte is better soluble than in the first. Because of the better solubility of the respective analyte in the second solvent, a smaller amount of that solvent is required which leads to an increase of concentration for the released analyte in the second solvent. With respect to low abundance analytes which may have concentrations in the original sample below the limits for a reliable and reproducible detection of the analyte and/or its metabolites and/or fragments this means that the method of the present invention allows a concentration of such analytes with a factor of 10 or even up to 100. Such concentrated analytes may subsequently be easily detected and analyzed in standard routine procedures.

In context of the present invention release of the at least one captured analyte is achieved by offering a solvent or a corresponding solution of the solvent in which the analyte(s) is/are better soluble. According to the method of the present invention this is achieved by step (d) in which the hydrogel containing the captured analyte(s) is contacted with an aqueous solution containing at least 60% by volume of an organic water-miscible solvent. Since the release of the analyte(s) is due to their better solubility in the respective solvent or its corresponding solution, a smaller amount of solvent is required for providing sample probes of the desired small molecule compounds. Accordingly, the method of the present invention also provides for a method for enriching a hydrophobic small molecule compounds from a complex biological sample. The term„co-polymerization" in context of the present invention is used as known to the expert skilled in the art and refers to any kind of polymerization in which two or more different monomers are combined to a polymer. Thus, the accumulating co-polymers contain at least two different repeating units within the polymer chain. Depending on the arrangement of the at least two respective monomer units, for example A and B, within the obtained polymer, statistic -ABAABABB-, alternating -ABABABABAB-, block -AAABBBAAA- or graft co-polymers -AA(BBB-)AAA(BBB-)A- are distinguished. The formation of the respective co-polymer may be influenced by the reactivity and/or the structure of the monomers, and the reaction type which is involved in the polymerization involved. Accordingly, the properties of the obtained co-polymers may be selectively influenced and controlled by the type, the proportion and the arrangement of the different monomers within the co-polymer. The term ..polymerization" in context of the present invention is used as known to the expert skilled in the art and refers to any kind of reaction yielding a macromolecule which is comprised of at least one specific kind of repeating unit that is also known as monomer or monomeric unit. The obtained macromolecule may be an oligomer, a polymer or a polymerizate. An oligomer is a molecule which is comprised of several structurally equal or similar units. The exact number of units in an oligomer is unspecific, however in the art this number is mostly in the range of from 10 to 30. Rarely, also molecules having a smaller number of units are considered as oligomers, which is the case for aminoplasts. Often oligomers are associated with a clearly defined number of units, while in contrast polymers almost always have a more or less broad distribution of the molecular weight.

In general, the reaction involved in the production of polymers is a chain-growth polymerization or a step-growth polymerization, such as, for example the addition polymerization or (poly)condensation. Representative examples for polymerization reactions typically used in processes of industrial scale are radical, ionic (anionic or cationic), insertion polymerization, ring opening polymerizations, radiation and photo polymerization. In the present invention radical polymerization is preferred for producing the hydrogel used in the method according to the present invention.

In principle, radicals may be generated by means of the following techniques: (a) homolysis of a bond, (b) reactions with other radicals, and (c) single electron transfer (SET). In context with generating radicals the term„homolysis^" is understood to mean a bond cleavage in a molecule which leads to two fragments both having an unpaired single electron. In general, bond cleavage can be effected either thermally or photolytically and the bond to be cleaved is preferably a labile bond of halogen molecules, azo compounds, peroxides or dialkylmercury compounds. The generation of radicals by reaction of already existing radicals may occur for example through reaction of those radicals with functional groups, such as for example, through radical substitution, or addition. A single electron transfer may be effected for example through anodic oxidation ( olbe synthesis) under evolution of carbondioxide or through reduction of diazonium salts (Sandmeyer reaction).

In most commercial processes radical polymerization are used for producing polymers. Representative examples for polymerization reactions typically used in processes of industrial scale are radical polymerizations. In context of the present invention the performed polymerization reaction is a radical polymerization involving a chain growth. This reaction may be started through action of initiators on monomers.

The term ..radical starter" also known as radical initiator is used as known to the expert skilled in the art and denotes those compounds that produce radicals under mild conditions and thus, promote radical polymerization reactions. Generally, those substances have weak bonds, i.e., bonds with low dissociation energy. Representative examples for initiators are halogen molecules, azo compounds and organic peroxides which form radicals by homolysis.

Like all diatomic homogeneous molecules, halogens can generate two free radicals resulting from the homolysis of the bond, but halogens undergo the homolytic bond cleavage relatively easily. Azo compounds of the type R-N=N-R' can be the precursor of two carbon centred radicals under evolution of nitrogen upon heating or irradiation. For example, azobisisobutyronitrile (AIBN) and l , -azobis(cyclohexanecarbonitrile (ABCN), each yield two isobutyronitrile or cyclohexanecarbontrile radicals, respectively. Organic peroxides, each have a peroxide bond (-0-0-), which can be easily cleaved under formation of two oxygen-centred radicals. Those oxygen-centred radicals also referred to as oxyl radicals are considered as unstable and are believed to fragment into relatively stable carbon centred radicals. For example di-(tertiary)-butylperoxide (tert-Bu-OO-tert- Bu) gives two tert-butanoyl radicals (tert-BuO) that fragment to methyl radical under release of acetone. Another well-known organic peroxide suitable for use as radical initiator is benzoyl peroxide (Ph-CO-OO-CO-Ph) which gives two benzoyloxy radicals (Ph-CO-O) upon heating or irradiation, wherein the latter further fragments to phenyl radicals under evolution of carbon dioxide. Due to their unstable nature azo compounds and organic peroxides often have a high tendency for explosion and therefore, should be handled with care. Accordingly, radical initiators, especially azo compounds and organic peroxides, are often kept cooled or even refrigerated. Usually, radical polymerization is started by action of a radical starter on a monomer, wherein prior to this action the radical starter has fragmented through heat or radiation to at least one radical which reacts with a monomer under formation of a new radical molecule which in turn reacts with another monomer under formation of a larger molecule radical. The continued growth of such molecular is known of chain growth and depending on the reaction conditions and the respective monomers this propagation finally leads to oligomers or polymers, respectively. Chain termination occurs when two radicals react with each other or when a radical molecule fragments under elimination of a small radical molecule. In context of the present invention the term ..monomer^" is used as known to the expert skilled in the art and denotes the low molecular weight molecules which can combine to molecular chains or three-dimensional networks (straight or branched polymers). In context of the present invention the three kinds of polymers are reacted in a radical polymerization yielding a three-dimensional network in form of the hydrogel used in the method according to the present invention.

The term ..cross-linking monomer" in context of the present invention is used as known to the expert skilled in the art and denotes such monomers which react with the formed linear or branched macromolecules or polymer chains which are still soluble under formation of an insoluble network which is no longer soluble, but capable of swelling, i.e., incorporating solvent molecules into its three-dimensional network. In principle, cross-linking may be accomplished by formation of covalent or non-covalent, i.e., coordinative, ionic, physical or salt-like bonds. In a specific form, cross-linking may directly occur simultaneously when building up the polymer, such as, for example in radical polymerization through additional use of monomers having two functionalities suitable for radical polymerization, such as, for example two vinyl functionalities. This particular process is also known to the expert skilled in the art as cross-linking polymerization. Analogous, cross-linking may be achieved in poly additions or poly condensations, respectively, through contribution of monomers having a functionality of equal to or greater than 2.

The degree of cross-linking has considerable effects on the properties of the obtained polymers. Usually, the degree of cross-linking is expressed by means of cross-linking density which denotes the number of cross-linking positions in a given amount of polymer or be the cross-linking index which denotes the number of cross-linked fundamental building blocks per primary molecules. With increasing degree of cross-linking the hardness of the polymers generally increases. Further, the length of the cross-linking bridges or units, respectively, also affects the properties of the obtained polymers.

In context with hydrogels, cross-linking is of major importance because it leads to the formation of the three-dimensional and also to the formation of cavities which allow the swelling behaviour of the hydrogel. Moreover, the degree of cross-linking necessarily affects the size of the pores of the obtained hydrogel and thus, also the size and/or the shape of the analyte(s) that may be captured within the cavities of the hydrogel.

According to the present invention cross-linking in the hydrogel is accomplished during co-polymerization by use of a so-called cross-linking monomer having two functionalities for linking the already formed and still soluble macromolecules or polymer chains, respectively, with one another to give the three-dimensional network which is then capable to swell. The term ..immobilized" in context of the present invention is used as known to the expert skilled in the art and relates to the fixation of the hydrogel on a support. In this context the term support is understood to mean any conceivable macroscopic material which has a surface that can be modified with a linker to covalently attach the hydrogel to the surface of the support. In context of the present invention the support on which the hydrogel is immobilized is a solid surface which facilitates the separation of the hydrogel from the respective sample fluid. In the simplest form the immobilized hydrogel may be separated from the sample fluid by decantation and/or filtration. In such a straight-forward form of separation the support for the immobilized hydrogel may be in principle, any solid stable material.

As already discussed above, thin layers of hydrogel are preferred for successful isolation and enrichment in the method according to the present invention. Therefore, the hydrogel of the present invention is preferably immobilized on a solid surface in such a way that the resulting immobilized hydrogel has a layer thickness in the range of from ca. 1 mm as upper limit to ca. 1 μιη as lower limit. The term„ca." in context of upper and lower limit of the hydrogel layer thickness denotes the range of error which has a value of 5% with regard to the specific layer thickness. In general, the thickness of the layer directly influences the length of adsorption and desorption times. The thicker the layer thickness the longer it takes for the analyte to diffuse into the hydrogel layer. Accordingly, the thinner the hydrogel layer the shorter are the required adsorption/desorption times. Likewise, the larger the surface area of the immobilized hydrogel the better are the adsorption/desorption conditions because a larger contact area is hence provided for said adsorption/desorption processes. Therefore, the combination of a large surface area with a thin layer of the immobilized hydrogel is preferred in context of the method according to the present invention.

Therefore, in a preferred embodiment of the present invention the protein-repellent hydrogel contacted with the sample fluid to capture at least one analyte in step a) of the method of the present invention is a thin layer of a protein-repellent hydrogel immobilized on a solid surface.

The term ,,capturing" as well as the verb ,.to capture" as used in context of the present invention denotes the phenomenon that a compound, i.e. the analyte in context of the present invention, is trapped in the cavities of the hydrogel used in the method according to the present invention. In the swollen state the hydrogel has cavities accessible for the analyte. In order to achieve a successful capturing of the analyte within the cavities of the hydrogel the three-dimensional network of the hydrogel forming the cavities is preferably adequately designed. This means that when the hydrogel swells, i.e., when the hydrogel incorporates solvent molecules into its three-dimensional network the „walls^" of the resulting cavities should fit to the chemical character of the analyte(s) to be captured, such as polarity and/or functional groups. For example, analyte(s) may be adsorbed inside the cavities. In case of hydrophobic compounds adsorption of analyte(s) inside cavities may be realized through hydrophobic interactions for example through hydrophobic components. Respective monomers used in the synthesis of the hydrogel of the present invention and which provide the cavities with a hydrophobic character are for example monomers derived from fatty acids, such as, for example stearyl acrylate or related compounds. The above described effect is attributed to the hydrophobic effect known in the art for the property that non-polar molecules tend to form aggregates of similar molecule and analogous intramolecular interactions in a polar medium. At the macroscopic level, the hydrophobic effect is apparent when oil and water are mixed together and form separate layers or the beading of water on hydrophobic surfaces such as waxy leafs. At the molecular level, the hydrophobic effect is an important driving force for biological structures and is responsible for protein folding, protein-protein interactions, formation of lipid bi-layer membranes, nucleic acid structures, and protein-small molecule interactions.

In thermodynamics, one way to understand the hydrophobic effect is exemplified by a hydrophobic substance on water. While in the absence of any purification pure water adopts a structure which maximizes entropy, a hydrophobic molecule will disrupt this structure and thus, it will lead to a decrease in entropy. This disruption leads to a kind of ..cavity" because the hydrophobic molecule is unable to form bonds with water molecules. In case of several or even many . avities", the surface area of disruptions is high, which in other words means that the surrounding water molecules are limited in their mobility and consequently, the water molecules are higher ordered, i.e., there are fewer free water molecules. However, according to the second law of thermodynamics entropy of a closed system can never decrease and therefore, in order to compensate the loss of entropy the unpolar molecules congregate in a way that the water molecules form a kind of cage structure around them. This cage structure has a smaller surface area than the total sum of the surface areas of the former cavities. Accordingly, the release of water molecules maximizes the amount of free water molecules which in turn increases entropy. In addition, there is a gain in enthalpy because of formation of relatively strong interactions between the released water molecules, in particular dipole-dipole interactions.

In other words, this effect does not involve forces of repulsion between the components such as in case of the above definition of the term protein-repellent. Therefore, capturing of analyte(s) in the cavities formed by the hydrogel should not be confused with the protein- repellent nature of the hydrogel.

Accordingly, in a further embodiment of the present invention, the sample fluid containing at least one analyte and at least one protein is an aqueous sample fluid.

Therefore, in a preferred embodiment the present invention also relates to a method for isolating at least one analyte from an aqueous sample fluid containing said at least one analyte and at least one protein, comprising the steps of:

a) contacting the aqueous sample fluid with a thin layer of a protein-repellent hydrogel immobilized on a solid surface to capture said at least one analyte;

b) separating the hydrogel obtained in step a);

e) isolating said at least one analyte obtained in step d);

Another subject addressed by the present invention is the fact that in complex biological samples low molecular weight molecules are not always present in„free" form but often bound to so-called binding proteins.

In context with the present invention the term„binding protein" is used as known to the expert skilled in the art and denotes the characteristic property of proteins to bind other molecules specifically and tightly. The particular region of the protein which is responsible for binding another molecule is known as the binding site and is often a depression or so- called„pocket" on the molecular surface. This binding ability is mediated through the tertiary structure of the protein, which defines the binding site pocket, in particular through the chemical properties of the side chains of the surrounding amino acids. As representative examples for the extraordinarily tight binding of proteins the binding of ribonuclease inhibitor protein to human angiogenin with a sub-femtomolar dissociation constant (< l O^'13 M) is mentioned. Simultaneously, the binding of proteins is quite specific which is exemplified by the fact that ribonuclease inhibitor protein does not bind at all to its amphibian homolog onconase (> 1 M).

Proteins can bind to other proteins as well as to small molecules, wherein the latter term denotes a small molecular weight organic compound. In general, the term small molecule is by definition not a polymer. Especially within the field of pharmacology, the term small molecule is usually restricted to a molecule that binds with high affinity to a biopolymer such as a protein, nucleic acid, or polysaccharide and in addition, alters the activity or function of the biopolymer.

Further, the term protein binding also referred to as plasma protein binding or plasma albumin binding is also known to denote the reversible binding of substances to plasma proteins, tissue-related proteins and erythrocyte proteins. The occurrence of protein binding is considered to be based on ionic interactions, hydrogen bonds, dipole/dipole interactions as well as hydrophobic interactions. Common proteins that bind drugs and/or small molecules are amongst others human serum, albumin, serum proteins, lipoprotein, glycoprotein, and α, β, and γ globulins.

In principle, all water-miscible organic solvents can be used for this specific separation, wherein the terms water-miscible and organic solvent are to be understood as defined above. Besides pure water-miscible organic solvents also aqueous mixtures of those organic solvents may be alternatively used. For achieving a successful separation of analyte(s) from the binding protein at least a content of up to ca. 50% by volume of an organic water-miscible solvent is required. In this context the term ca. denotes an error range of +/- 5% volume, i.e., 50% +/- 5% by volume. Explicitly, the term ca. 50% by volume denotes any value within the range of from 0% to 55% at the most, such as, for example 0%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, or 55% by volume.

Accordingly, another aspect of the present invention is a method according to the present invention which further comprises prior to step a) the step a') contacting the sample fluid with an organic solvent thereby creating an aqueous mixture containing up to ca. 50% by volume of an organic water-miscible solvent. In a preferred embodiment of said aspect of the present invention the sample fluid of step a^;) is an aqueous sample fluid.

In principle, any conceivable method may be applied for immobilizing the hydrogel. With respect to the surface properties of the solid surface the hydrogel when immobilized on the respective solid surface should be easily accessible for the analyte(s) to be captured. For example immobilizing the hydrogel on a porous solid surface may lead to an immobilized hydrogel which is at least partially ..hidden" in the pores of the support. Therefore, a hydrogel immobilized on a porous solid support may not be easily accessible for the analyte(s) to be captured. In addition, the immobilization of hydrogel within the pores the support may also have a negative impact on the smart behaviour of the hydrogel and on the swelling behaviour of the hydrogel. Accordingly, in context of the present invention, preference is given to a solid support which is non-porous and therefore, the solid support is at best part of a non-porous support material.

Further, in principle any conceivable material may be considered as non-porous support. Since the present invention mainly addresses an analytical field, the method of the present invention is usually performed in a vessel such as vials. With regard to a desired stability of the solid surface towards reaction conditions applied in surface modification and considering that the solid surface should not have any inclination to influence the same probe and the enriched analyte(s), glass showed to be a suitable and therefore, preferred non-porous material. Likewise, a suitable polymer is also preferred as non-porous material. For the person skilled in the art it is obvious that suitable polymers in this context are those which have hydroxyl groups on their surface for forming covalent bonds with the surface modifying agent. Accordingly, polystyrene as used in form of microtiter plates, latex polymers sheets such as hydroxyl latex or carboxyl latex, or nylon sheets may be also used. Alternatively, also pre-activated polymers, such as those having epoxy activated surface hydroxyl groups may be also used.

Accordingly, a further aspect of the present invention is a method according to the present invention, wherein the solid surface is a non-porous material, preferably the non-porous material is glass or a polymer. In case the solid support is not already equipped with or does not have functional groups or moieties for covalently binding the hydrogel in order to accomplish immobilization of the hydrogel the solid support is preferably adequately modified by a surface modifying agent. Since the hydrogel is produced by co-polymerization of at least one uncharged and polar monomer with at least hydrophobic monomer and at least one cross-linking monomer, the solid surface is preferably modified with a group or moiety, respectively, which allows to be involved in the production of the immobilized hydrogel. Moreover, such a surface modifying agent preferably should not disturb production of the hydrogel.

Further, since the hydrogel applied in the method of the present invention is preferably produced by radicalic co-polymerization the surface modifying agent should have a suitable group or moiety which allows for a radicalic addition reaction in order to form a covalent bond to the hydrogel. In this aspect an acrylic acid derivative or methacrylic acid derivative showed to be suitable for this purpose, preferably a methacrylic acid derivative.

Likewise, the surface modifying agent itself should have a suitable group or moiety for forming a covalent bond to the solid surface. Further, the surface modifying agent should preferably form a very strong covalent bond to the solid surface in order to provide a preferably permanent immobilization of the hydrogel on the solid surface. This may be accomplished by using a moiety which includes residues which form easily removable compounds, for example easily removable by distillation due to a low boiling point. For example moieties with alkoxy groups may be applied, which can form the corresponding alcohol molecules which may be distilled from the reaction mixture or may degas from the heated reaction mixture, thereby shifting the reaction equilibrium to the product side, i.e., surface modification of the solid surface. For such a purpose, preferably alkoxy groups, which give alcohols having a relatively low boiling point such as methanol or ethanol, in particular methanol, are preferably used. Moreover the atom or group, which forms the covalent bond to the solids surface should be adequately chosen so that it preferably releases said alkoxy groups easily and itself forms a preferably stable and thus, preferably permanent covalent bond to the surface. In this aspect organosilicon moieties showed to be suitable, which have alkyl or Ci-C₄ alkoxy groups, with the proviso that at least one of the three groups bound to the central silicon atom is a C₁ -C4 alkoxy, wherein C|-C₄ alkoxy is a methoxy, ethoxy, n-propoxy, iso- propoxy, n-butoxy, iso-butoxy or tert-butoxy group, preferably methoxy or ethoxy and more preferably methoxy. In case of glass as solid support the silicon atom of the surface modifying agent may form a chemically stable disiloxan bond (Si-O-Si) under release of the respective equivalents of the corresponding alcohol. In this context an organosilicon methacrylic acid derivative according to Formula I below showed to be a suitable and therefore, preferred surface modifying agent.

In addition, the length of the surface modifying agent should be suitably limited to an upper region in order to avoid that the immobilized hydrogel may be exposed to any shear forces which may have a damaging impact on the hydrogel. In this aspect a linear C|-C|₀ alkylene showed to be suitable. Preferably the linker is a linear Ci-C₈ alkylene, more preferably Ci-C₆ alkylene, particularly preferably C₂-C₄ alkylene and most preferably the linker between the organosilicon moiety and the acrylic acid or methacrylic moiety is C₃ alkylene.

Accordingly, in a further embodiment of the present invention the solid surface is modified by an organosilicon acrylic acid derivative or an organosilicon methacrylic acid derivative of the type given in Formula I

wherein

R⁵ is either H or methyl;

o is an integer from 0 to 9;

A¹, A² and A³ are independently from each other linear or branched Ci-C₄ alkyl or C|-C₄ alkoxy with the proviso that at least one of A¹, A² or A³ is C|-C₄ alkoxy.

In context with the protein-repellent character of the hydrogel of the method according to the present invention derivatives of acrylamide and methacrylamide have shown to be useful for the production of protein-repellent hydrogels. Further, derivatives of acrylamide and methacrylamide are uncharged and polar compounds, which also provide a carbon- carbon double bond which allows their use in polymerization reactions and preferably in radical polymerization reaction, especially in radicalic co-polymerization reactions, which are preferred in context with the production of the hydrogel of the present invention. In this context the at least one uncharged and polar monomer preferably used in context of the present invention is/are acrylamide, N-monoalkylacrylamide. oligoethylene glycol acrylamide, oligoethylene glycol methacrylamide, N,N-dialkylacrylamide and /or an N,N- dialkylmethacrylamide, wherein each alkyl is independently from each other methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, and/or tert-butyl. Preferably, alkyl is methyl, ethyl, n-propyl and/or iso-propyl. In a more preferred embodiment, alkyl of the at least one uncharged and polar monomer is methyl and/or ethyl and in the most preferred embodiment each alkyl group is methyl.

Accordingly, another embodiment of the present invention relates to a method according to the present invention wherein one or more of the uncharged and polar monomer(s) is/are acrylamide, N-monoalkylacrylamide, oligoethylene glycol acrylamide, oligoethylene glycol methacrylamides, Ν,Ν-dialkylacrylamide and /or an N,N-dialkylmethacrylamide, wherein each alkyl group is independently from each other methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso-butyl, and/or tert-butyl. At least one hydrophobic monomer is used in the production of the hydrogel of the present invention. Since the hydrogel according to the present invention is preferably produced in a radicalic co-polymerization reaction derivatives of acrylic acid and/or methacrylic acid have shown to be suitable in this aspect. In addition, the derivatives of acrylic acid and/or methacrylic acid have a hydrophobic moiety, which is preferably connected to the acrylic acid and/or methacrylic moiety through an oxygen atom commonly shared by acid derived moiety(ies) and the hydrophobic moiety. Therefore, the one or more hydrophobic monomer(s) is/are preferably an acrylic acid ester and/or a methacrylic acid ester, with the latter being more preferred. In context of the present invention the hydrophobic moiety is preferably a hydrocarbon chain, which term is used as known to the skilled person and refers to an alkyl, alkenyl or alkynyl residue. As already mentioned above, the hydrogel of the present invention is preferably produced by radicalic co-polymerization. Therefore, if any carbon-carbon multiple bonds are present in the hydrocarbon chain the carbon-carbon multiple bonds are preferably conjugated bonds in order to avoid any interference with the radicalic co- polymerization for production of the hydrogel according to the present invention. In the present context the term conjugated is understood as known to the skilled person and relates to a conjugated system occurring in organic compound in which atoms are covalently bond with alternating single and multiple bonds, e.g. carbon-carbon double bonds, e.g. C=C-C=C-C. The different types of bonds influence each other to produce a region called electron delocalization. In this region electrons do not belong to a single bond or atom, but rather to a group. The formation of a conjugated system results in a general delocalization of electrons across all of the adjacent parallel aligned p-orbitals of the atoms, which increases stability and thereby lowers energy of the molecule. Accordingly, the loss of energy in a conjugated system caused for example through an addition reaction in which a carbon-carbon double bond is involved such as in a radicalic polymerization may not be paid back through the energy of the newly formed bond.

Since hydrocarbon chains are hydrophobic, the incorporation of corresponding monomers is attributed to contribute to the property of the hydrogel of the present invention to isolate one or more hydrophobic analyte(s) from a sample fluid.

Optionally, the hydrocarbon chain may be substituted with one or more phenylen or cyclohexylen groups(s) which may contribute to stiffer hydrocarbon chains. In addition, the presence of optional phenylen group(s) is attributed to provide further possibilities for interaction with the at least one analyte to be captured within the hydrogel of the present invention, for example through π-π stacking.

For the person skilled in the art it is obvious that in order to specifically adjust the hydrogel of the present invention to the particular hydrophobic analytes, the hydrocarbon chain of the hydrophobic monomer may be optionally interrupted by one or more -NH-CO- or -CO-NH- groups, carbonyl groups, -0-, -S- or -NH- linkage or -C(=0)-0- or -0-(0=)C- groups as long as the hydrophobicity of said hydrophobic chain is still maintained.

In context of the present invention the respective hydrophobic moiety is preferably a hydrocarbon chain having from 2 to 20 carbon atoms. Hydrocarbon chains having from 6 to 18 carbon atoms are preferred and even more preferred are those having from 12 to 18 carbon atoms or even from 16 to 18 carbon atoms.

Accordingly, another embodiment of the present invention is a method according to the present invention, wherein one or more of the hydrophobic monomer(s) is/are a methacrylic acid ester and/or an acrylic acid ester, having a hydrocarbon chain comprising from 2 to 20 carbon atoms, wherein the hydrocarbon chain is an alkyl, alkenyl, or alkynyl residue, which may be optionally substituted with one or more phenyl, cyclohexyl, phenylen or cyclohexylen groups with the proviso that carbon-carbon multiple bonds in the hydrocarbon chain if present are conjugated bonds. Preferably the hydrocarbon chain has from 6 to 18. more preferably from 12 to 18 carbon atoms, and also prefered from 16 to 18 carbon atoms.

In general, the cross-linking agent used for producing the hydrogel of the present invention may be represented as a kind of linker having two preferably different functional moieties at its ends. Preferably, the cross-linking agent has two functional moieties which may be subjected to a radical co-polymerization reaction and in this context may be adequately chosen with respect to the at least one uncharged and polar monomer and the at least one hydrophobic monomer. In context of the present invention, acrylate and/or acrylamide moieties of the following type have shown to be suitable moieties of the cross-linking agent which may be subjected to a radicalic co- olymerization:

In these moieties R¹ and R⁴ are independently from each other hydrogen or a C|-C₄ alkyl, i.e., methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl. In context of the present invention methacrylate and/or methacrylamide moieties are preferred, i.e., in the above structures R¹ and R⁴ are independently from each other preferably methyl.

Flexibility of the hydrogel may influence the swelling behaviour of the hydrogel of the present invention. Usually a hydrogel, which is not strictly rigid, is able to expand and to incorporate solvent molecules inside of the expanded„empty room", i.e., the formed cavities. Otherwise, the respective compound may not act as a hydrogel. Accordingly, the linker should provide the final hydrogel with the preferred flexibility and therefore, the linker itself should be flexible. Further, flexible linkers may also provide the final hydrogel with considerable stability in respect to physical influences such as mechanical influences, for example pressure and shear forces. Glycol chains have shown to be suitable linkers in context of the present invention. Suitable glycol chains in context of the present invention may be represented by the alkyleneoxy units of the following type

In case the respective glycol unit is directly bonded to the acrylate moiety the oxygen atom shown in the square brackets is shared together with said acrylate moiety. In the above structures m is an integer from 2 to 10, i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10. Accordingly, depending on the field of application for the hydrogel the length of the straight alkylene in such a glycol chain may range from ethylene to decylene, i.e, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene or decylene. Preferred is a length of the alkylene with m being an integer from 2 to 4, i.e., 2, 3 or 4, in other words the alkylene may be ethylene, propylene or butylenes, and more preferred is ethylene as alkylene, meaning m is 2.

Further, in order to tailor the hydrogel specifically for the analyte(s) to be captured from the sample fluid the alkyleneoxy unit may be substituted with atoms others than the hydrogen typical for an alkyleneoxy unit. Accordingly, R² and R³ in the above structures are independently from each other hydrogen, fluorine, hydroxyl or methyl. Preferred in context of the present invention is that R and R are hydrogen so that the alkylene unit preferably corresponds to an ordinary glycol unit.

The length of the above mentioned linker, i.e., the above-shown structures for the glycol chain may correspondingly vary depending on the respective field of application. In context of the present invention, length of said linker is indicated in the above structures through index n, which is an integer from 1 to 4. Preferred is a length of n = 1 or 2 and more preferred is n with a value of 2.

Further, for specifically tailoring the linker for applications, in which the hydrogel or the cavities formed in the swollen state should be more rigid than provided by ordinary glycol units, one or more of the oxygen-carbon bonds in the glycol chain may be interrupted by a phenylen or cyclohexylen group or an amide group.

Representative but not limiting examples for cross-linking agents based on acrylate are:

with m being an integer from 2 to 10, for example

with n being an integer from 2 to 4;

10

In case of acrylamide moieties as shown above, the linker between said acrylamide moieties may be suitably represented by the following structure,

in which R² and R³ are as defined above. Further, m indicating the length of the straight alkylene unit is an integer from 2 to 10, i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10 in other words the alkylene unit can be ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene or decylene. In a preferred embodiment of the present invention, m is an integer from 2 to 6, i.e., 2, 3, 4, 5 or 6; and in a more preferred embodiment m is 2 and accordingly the alkylene unit preferably is an ethylene.

Further, letter X in the above structure can be a bond or a -CH₂-CH₂-0- or -0-CH₂-CH₂- group. In the latter meaning the respective group forms part of a glycol chain. In context of the present invention the meaning of X being a -CH₂-CH₂-0- or -0-CH₂-CH₂- group is preferred.

Representative but not limiting examples for cross-linking agents based on acrylamide are:

with m from 1 to 6.

Accordingly, a further embodiment of the present invention is a method according to the present invention, wherein the one or more cross-linking agent(s) is/are

i) a glycol acrylate derivative of the Formula II

with

m being an integer from 2 to 10; n being an integer from 1 to 4; being independently from each other H or a C|-C₄ alkyl group; being independently from each other H, F, OH or methyl; wherein one or more of the oxygen-carbon bonds in the glycol chain may interrupted by a phenylen or cyclohexylen group or an amide group; and/or an acrylamide derivative of the Formula III

with

m being an integer from 2 to 10;

X being a bond or a -CH₂-CH₂-0- or -0-CH₂-CH₂- group forming part of a glycol chain; and

R', R², R³ and R⁴ are as defined above. In context of the present invention an ethylene glycol diacrylate derivative and in particular, an ethylene glycol dimethacrylate derivative leads to the best result for synthesis of the hydrogel and therefore, is preferred.

Accordingly, another embodiment of the present invention is a method in which the cross- linking agent is an ethylene glycol diacrylate derivative or an ethylene glycol dimethacrylate derivative.

Examples 9 and 10 further illustrate preferred compositions of the hydrogel of the present invention. In general, successful isolation and enrichment using the method of the present invention can be performed with a hydrogel which is produced by the co-polymerization of from ca. 85 to ca. 98% of at least one uncharged and polar monomer, of from ca. 1 to 10% of at least one hydrophobic monomer and of from ca. 1 to ca. 5% of at least one cross- linking agent. Even better results are obtained for hydrogels produced by co- polymerization of from ca. 90 to ca. 95% of at least one uncharged and polar monomer, from ca. 1 to ca. 5% of the at least one hydrophobic monomer and from ca. 1 to ca. 3% of at least one cross-linking agent which are therefore preferred. More preferred are hydrogels produced by co-polymerization of ca. 94% of at least one uncharged and polar monomer, ca. 5% of at least one hydrophobic monomer and ca. 1% of at least one cross-linking agent. Accordingly, a further embodiment of the present invention is a method according to the present invention, wherein the hydrogel is produced by the co-polymerization of from ca. 85 to ca. 98% of at least one uncharged and polar monomer, of from ca. 1 to 10% of at least one hydrophobic monomer and of from ca. 1 to ca. 5% of at least one cross-linking agent.

As illustrated in Example 1 1 , hydrogels containing Ν,Ν-dialkyl acrylamide showed a protein-repellent character.

Accordingly, another embodiment of the present invention is a method according to the present invention, wherein the uncharged and polar monomer is Ν,Ν-dialkyl acrylamide.

Example 9 shows that stearyl methacrylate is suitable for the production of DMAA- hydrogel for isolating and enriching at least one analyte.

Accordingly, a further embodiment of the present invention is a method according to the present invention, wherein the hydrophobic monomer is stearyl methacrylate. In principle, any commercially available cross-linker which contributes to providing a hydrogel for the method of the present invention may be applied in the synthesis of the hydrogel. As illustrated by Example 10, ethylene glycol dimethacrylamide is a preferred cross-linking agent in context of the present invention. Accordingly, another embodiment of the present invention is a method according to the present invention, wherein the cross-linking agent is ethylene glycol dimethacrylate.

Through the method of the present invention at least one analyte can be isolated from a sample fluid containing additionally at least one protein. Said at least one analyte is in principle any kind of hydrophobic compound or any kind of substance which can be separated on reverse phase chromatography, wherein the term reverse phase chromatography is used as defined above. Preferred analyte(s) is/are hydrophobic synthetic compounds, such as drugs of abuse or immunosuppressiva. Representative but not limiting examples of drugs of abuse are benzoylecgonine, propoxyphen, imipramine, phencyclidine, benzodiazepines, methadone, methaqualone, tetrahydrocannabinol (also known as THC), lysergic acid diethylamide (also known as LSD-25, or simply LSD), amphetamines, opiates and barbiturates. Representative but not limiting examples for immunosuppressiva are tacrolismus, sirolismus, rapamycin, cyclosporine and everolismus. Other preferred analytes are hydrophobic compounds of biological origin, such as vitamins and steroids. Representative but not limiting examples for vitamins are vitamins of the D series, folates, tetrahydrofolates, formyltetrahydrofolate and methyltetra-hydrofolate. Representative but not limiting examples of steroids are testosterone, progesterone, estrogens, corticosteroids, aldosterone and androsterones.

Accordingly, in another embodiment of the method according to the present invention the analyte is a hydrophobic compound.

Since the analytes are either hydrophobic synthetic compounds, such as drugs of abuse or immunosuppressive or hydrophobic compounds of biological origin and furthermore, because the method of the present invention primarily aims at providing sample probes for medicinal investigations the sample fluid to be considered are of human and/or veterinarian origin. Suitable sample fluids in context with the present invention are those which are usually subjects of medical analysis and which are easily obtainable. Therefore, preferred biological fluids are blood, blood components, cerebral spinal fluids, lymph, cell lysates. tissue lysates, stool, urine, ascites, semen, plasma, serum or salivary juice. Preferred samples are whole blood, plasma and serum. In one preferred embodiment the sample will be a serum or a plasma sample.

Accordingly, in a further embodiment of the present invention, the sample fluid for the method according to the present invention is or contains a biological fluid. As known to the skilled person the above-mentioned exemplary sample fluids of human and/or veterinarian original are aqueous or at least essentially aqueous solutions. Therefore, in another embodiment of the present invention the aforementioned sample fluid either being or containing a biological fluid is an aqueous sample fluid. As water-miscible organic solvent in principle any representatives of said class of solvents may be used provided that the desired analyte(s) is/are better soluble in the particular solvent than in water. For example, the skilled person knows that the majority of 250H- vitamin D₃ is not present in free form in human serum but rather it is bound to vitamin D- binding protein. Therefore, it must be released from said protein prior to bringing a sample into contact with the hydrogel of the present invention. Otherwise, in the detection of 250H-vitamin D₃ wrong results would be measured, because a much too low amount of this specific analyte, namely only the free amount of said analyte, can be captured by the hydrogel. Release of said analyte from the binding protein is e.g. affected by adjusting the adsorption solution so that it contains ca. 40% of methanol.

Accordingly, another embodiment is a method of the present invention, wherein the organic water-miscible solvent is acetonitrile, dimethylformamide. ethanol and/or methanol.

The following Figure and Examples are meant to further illustrate the present invention without limiting the scope of the method of the present invention.

Description of the Figure:

Figure 1 shows the swelling behaviour of a hydrogel dependent on the molar content of stearyl methacrylate and the pH value in an aqueous solution of 90% methanol. The upper curve I Fig. 1 shows swelling in acidic environment and the lower curve in basic environment.

Examples:

I. Surface modification of glass

Example 1 : Surface modification of glass with 3-(trimethoxyilyl)propyl methacrylate under reflux heating or using a convection oven i) Preferably, glass vials usually used for gas chromatography (GC) or high performance liquid chromatography (HPLC) were pre-treated with a saturated aqueous solution of NaOH for increasing the number of ..free" hydroxyl groups at the inner glass wall through so-called glass corrosion. The increase in reactivity of the glass surface with 3-(trimefhoxysilyl)propyl-methacrylate is attributed to the enlargement of the number of „free" hydroxyxl groups. For this purpose a saturated aqueous solution of NaOH (15 ml) was transferred into glass vials, sealed and let resting over night at 60°C in a water bath. ii) After discarding the NaOH solution the vials were washed several times with ultrapure water followed by drying at 1 10°C in a convection oven for 1 hour. The modification of the pre-treated glass surface with 3-(trimethoxysilyl)propyl methacrylate was then either performed under reflux heating or using a convection oven. For surface modification at reflux heating, the vials were collected in a 250 ml three- necked flask and covered with toluene. Under slow stirring 2.0 ml 3- (trimethoxysilyl)propyl methacrylate was added first, followed by addition of 7 ml triethylamine. Subsequently, the reaction apparatus was flushed with nitrogen for approximately 10 minutes, and afterwards, the reaction mixture was heated to reflux for 3 hours under a protective gas atmosphere of nitrogen.

For performing the surface modification using a convection oven, each 1 ml of a solution of 3-(trimethoxysilyl)propyl methacrylate (3.0 ml) and triethylamine (1 ml) in toluene (10 ml) was added to vials using an Eppendorf pipette. Subsequently, the vials were first flushed with nitrogen for 10 minutes using needles and then sealed using crimping pliers. Under nitrogen atmosphere the reaction mixture inside the vials was heated at 1 10°C in a convection oven over night.

After completion of the surface modification, the vials were washed each twice with toluene, ethanol, ethanol/water (1 : 1 ; v/v) and diethylether followed by drying in vacuo (approximately at a pressure of 100 mbar). Finally, the vials with modified glass surface were kept under a protective gas atmosphere of nitrogen.

The way surface modification was performed did not show to have any effects on the following experiments. II. DMAA-hydrogel polymerization on modified glass surfaces

In general, polymerization experiments on modified glass surfaces were performed using a so called hydrogel reaction mixture comprising N,N-dimethylacrylamide (DMAA) (mass content of 95%, 5.772 g, 58.227 mmol, 6.000 ml), stearyl methacrylate (mass content of 5%, 1 .038 g, 3.065 mmol, 0.1 16 ml), ethylene glycol dimethacrylate (mass content of 1 %, 0.121 g, 0.613 mmol, 0.1 16 ml) and N.N-dimethylformamide (10.000 ml). For this purpose said monomers, 2,2'-azobis(2-methylpropionitrile) (0.032 g) and the solvent were transferred into a 20 ml GC glass vial, followed by purging with nitrogen for 30 minutes. Then the vial was sealed as quickly as possible, enwrapped in alumina foil and stored in a freezer at -4°C.

Example 2: DMAA-hydrogel polymerization on modified glass surfaces using a convection oven 15 μΐ of the hydrogel reaction mixture was transferred into surface modified GC/HPLC vials using an Eppendorf pipette and subsequently, the vials were sealed with a rubber septum and an alumina cap using crimping pliers. Through two needles the GC/HPLC glass vials were purged with nitrogen for 5 minutes. Then the GC/HPLC vials were placed for 5 minutes inside a convection oven operated at a temperature of 1 10°C. After heating a thin ring of hydrogel formed at the bottom of the vials was observed indicating polymerization and formation of the hydrogel. The GC/HPLC vials were then filled with methanol and let standing over night. After 16 hours (over night) a distinct swelling of the wall layer was observed.

Example 3: DMAA-hydrogel polymerization on modified glass surfaces through

polymerization using centrifugal force i) 50 μΐ of the hydrogel reaction mixture was transferred into surface modified GC/HPLC vials using an Eppendorf pipette and subsequently, the vials were sealed with a rubber septum and an alumina cap using crimping pliers. Through two needles the GC/HPLC glass vials were purged with nitrogen for 5 minutes. Subsequently, the vial was fixed into the thread of a mixing unit operated at 2000 rpm (rounds per minute). The vials were heated using a hot air fan until polymerization started. Alternatively, the hot air fan was replaced with an oil bath operated at a temperature of 200°C. Polymerization under formation of a hydrogel film was observed in form of a thin hydrogel ring at the inner wall of the vials. Afterwards the GC/HPLC vial was filled with methanol and let standing over night. After 16 hours (over night) a distinct swelling of the wall layer was noticed. Through the centrifugal force the reaction solution was distributed in a more uniform way over the wall of the GC/HPLC vials.

Example 4: DMAA-hydrogel polymerization on modified glass surface using a

water bath i) Through a syringe 1 ml of the hydrogel reaction mixture was transferred into surface modified GC/HPLC vials and 2,2'-azobis-(2-methylpropionitrile) AIBN (0.032 g) was added. After complete dissolution of all AIBN the vials were heated for 15 minutes on a water bath operated at a temperature of 73°C. After 15 minutes polymerization was indicated by formation of a thin hydrogel ring at the bottom of the vial. Subsequently, the GC/HPLC vials were filled with methanol and they were let standing over night (16 hours). After 16 hours a distinct swelling of the wall coating was visible.

Summarizing it can be said that, in general, DMAA-hydrogel polymerization on surface modified glass surfaces can be performed by all of the above described methods. Generally, using a smaller amount of hydrogel reaction mixture for production of the hydrogel in the surface modified vials leads to an overreaction in the polymerization reaction which is attributed to the very effective heat transfer through the water bath (75°C). In contrast, synthesis in the convection oven showed to be better controllable.

III. Influence of the geometry of the immobilized hydrogel on

adsorption/desorption characteristics

For determining the effect of the geometry of the immobilized hydrogel on adsorption/desorption characteristics, two different strategies for coating GC/HPLC vials were tested: bottom coating and inner wall coating of GC/HPLC vials.

For this purpose 6 surface modified GC/HPLC vials (modified according to I.) were coated with ca. 10 mg hydrogel using the already described hydrogel reaction mixture. Washing and conditioning were performed as follows: a) washing with 1 ml methanol, b) discarding the washing solution, c) conditioning with 1 ml methanol/water (1/1 ; v/v) and d) discarding the conditioning solution. Afterwards each 1 ml of a standard solution with a concentration of 100 ng/mg 250H vitamin D₃ in methanol/water (1/1 ; v/v) was transferred into the vials which were placed on a pulsating panel operated at 1 ,500 rounds per minute.

Example 5: Preparation of bottom coated GC/HPLC vials i) 100 μΐ of the hydrogel reaction solutionwas transferred into a surface modified GC/HPCL vial and sealed with an alumina cap having a rubber septum. Under nitrogen atmosphere the GC/HPLC vials were placed in a water bath operated at a temperature of 75°C until the polymerization reaction was completed. A hydrogel layer had formed which was firmly connected to the GC/HPLC vialso that it could only be removed from the glass wall of the vials through mechanic impact using a spatula. However, through scratching at the coated inner wall the hydrogel film was partially separated indicating that even after this procedure the hydrogel was firmly connected to the wall. This shows the strong covalent bond between the glass wall and the hydrogel because such a strong connection would not be caused by adhesion forces or van der Waals forces. ii) The hydrogel layer obtained when using 100 μΐ of the hydrogel reaction solution has a cross-section of more than 4 mm in the swollen state. For reducing the layer thickness, the volume of hydrogel reaction mixture was reduced to 50 μΐ. Since performing polymerization reactions with such a small volume of hydrogel reaction mixture is no longer controllable in water bath (conf. overreaction of Example 4) the respective vials were placed in a convection oven. When polymerization reaction is performed in the convection oven at a temperature of 1 10°C almost no overreaction is observed. Moreover, this method even allows reducing the volume of hydrogel reaction mixture to such small volumes as 2 μΐ without substantially any overreaction or any other interferences. As result of a series of tests using 15 μΐ of hydrogel reaction solution showed to be a standard for providing bottom coated vials. Example 6: Preparation of inner wall coated GC/HPCL vials

In order to obtain a complete inner wall coating with the hydrogel, the GC/HPLC vial containing the hydrogel reaction mixture was directly fixed into the screw of a KPG stirrer operated at ca. 2,000 rounds per minute. Due to this setup stirring preferably only happened vertically so that the hydrogel reaction mixture was spun as thin film on the inner wall of the GC/HPLC vial through the resulting rotational forces and thus, covered the complete inside. Afterwards, the vial was heated using a hot air fan at maximum rotational speed of the stirrer until polymerization started. This strategy leads to inner wall coated GC/HPLC vials. As is the case for bottom coated GC/HPLC vials the hydrogel immobilized in this manner also proved to be strongly bound to the wall. Series of tests showed that in the present case use of 200 μΐ of hydrogel reaction mixture leads to a consistently complete inner wall coating of the vials.

Example 7: Washing and conditioning of coated vials i) After synthesis, the hydrogel layer of the coated vials usually still contains residuals of the solvent DMF and of unreacted monomer. Prior to further use, these substances must be removed from the hydrogel in order to avoid undesired interferences when using the method of the present invention for sample preparation. Removal of DMF and of unreacted monomers is performed by addition of methanol. Upon addition of methanol the hydrogel immediately starts to swell which leads to an enlargement of the mesh structure. This enables methanol to penetrate into all areas of the hydrogel and thereby, methanol displaces DMF and unreacted monomers out of the hydrogel. For completion of said displacement this washing was performed several times on a pulsating panel operated at 1 ,500 rounds per minute. ii) Prior to addition of the sample, the hydrogel has to be adjusted to the specific adsorption conditions of said sample. Moreover, the amount of methanol still contained in the hydrogel as a result of the washing procedure must be reduced in order to provide favourable adsorption conditions for the analyte. By this process, also known as conditioning, the hydrogel is adjusted to the proportion of methanol in the later adsorption solution. With regard to the present experiments for adsorption/desorption of 250H- vitamin D₃ which are in general performed in aqueous solution containing varying proportions of methanol, hydrogels are conditioned with the respective water/methanol solutions corresponding to the particular adsorption solution. For completion of conditioning said process is repeated several on a pulsating panel operated at 1 ,500 rounds per minute.

Example 8: Comparative examples for bottom-coated and wall-coated vials in adorption/desorption measurements i) Each 1 ml of a standard solution with a concentration of 100 ng/ml of 250H- vitamin D₃ in methanol/water (1/1 ; v/v) was transferred into the vials washed and conditioned according to Example 7 which were then placed on a pulsating panel operated at 1 ,500 rounds per minute. After end of adsorption time the standard solution was discarded and 500 μΐ of methanol was transferred into the vials for desorption of the analyte. Each 3 vials were subjected to the same adsorption/desorption times of one and four hours. ii) Analysis of the released analyte was performed using an LC-MS system (type Alliance 2695 series of the firm Waters^®). A 150 mm x 2 mm Reprosill OO, ^₈-5μπι column of the firm Dr. Maisch GmbH was used as analytical HPLC column. HPLC module and analytical solumn were connected with a peek capillary of the type 1/16" x 0.1 mm ID of the firm ERC GmbH. The HPLC module worked without a column oven so that chromatography was always performed at room temperature. A photo diode detector of the type 996 of the firm Waters^® was connected through a peek capillary of the type 1/16" x 0.1 mm ID of the firm ERC GmbH to the HPLC module having the analytical column. The MS system itself is a mass spectrometer of the single quadrupole type having an ESI source.

The comparative results for adsorption/desorption measurements for bottom and inner wall coated GC/HPLC vials are summarized below in table 1. adsorption/ remaining in stanafter desorption in remaining in/on retrieval factor desorption time dard solution | ng| standard solution |ng| hydrogel | ng| l% l

1 h/l h bottom 37.3 ± 3.8 28.7 ± 3.5 34.0 ± 3.8 28.7

1 h/1 h wall 1 8.1 ± 2.6 40.4 ± 4.2 41.5 ± 4.6 40.4

4 h/4 h bottom 17.1 ± 2.9 35.3 ± 3.6 47.6 ± 4.7 35.3

4 h/4 h wall 9.3 ± 1 .7 40.7 ± 4.7 50.1 ± 4.2 40.7 Table I : value pairs for adsorption/desorption measurements for different geometries of the bound hydrogel layers. iii) As can be seen from the results, inner wall coated GC/HPLC vials show a better adsorption for shorter adsorption times. In case of inner wall coated vials also the retrieval factors are better; in comparison the differences are less pronounced for longer adsorption times. The better adsorption/desorption behaviour of inner wall coated vials in comparison to bottom coated vials at short adsorption/desorption times is attributed to the larger surface area of the respective hydrogel which is understood to represent a much larger contact area for adsorption. In addition, the distribution of the same mass of hydrogel over a much larger area also results in correspondingly shorter diffusion path ways of the analyte into the hydrogel layer and in short diffusion path ways for the analyte on its way out the hydrogel. Therefore, combination combination of a large surface area with short diffusion path ways results in a better adsorption/desorption behaviour of wall coated vials.

IV. Variation of the composition of the hydrogel

Change in the composition of the hydrogel was made in order to reduce the amount of 250H vitamin D₃ remaining within or on the hydrogel. Studies had shown that hydrogels having a too large proportion of stearyl methacrylate may no longer be able to adsorb 250H-vitamin D₃. This effect is attributed to entropic effects. Further, as can be seen from Figure 1 the swelling behaviour of the hydrogel distinctively decreases with increasing proportion of stearyl methacrylate.

Example 9: Variation of the proportion of stearyl methacrylate in the hydrogel i) A lower proportion of stearyl methacrylate led to longer adsorption times and thus, less favourable adsorption conditions since less C₁₈ chains were available for capturing the analyte. In addition, a reduced proportion of stearyl methacrylate also leads to less 250H- vitamin D₃ adsorbed within or on the hydrogel. Two monomer solutions were prepared; one with the composition 96% N,N-dimethyl acrylamide, 3% stearyl acrylamide and 1 % ethylene glycol dimethacrylate and the other one with the composition 98% N,N-dimethyl acrylamide, 1 % stearyl acrylamide and 1% ethylene glycol dimethacrylate. 2,2'-Azobis(2- methylpropionitrile) was added to both solutions and distributed over 6 surface, modified GC/HPLC glass vials. Then the vials were bottom coated, followed by washing and conditioning as described above. Afterwards each 1 ml of a standard solution of 100 ng/ml 250H vitamin D₃ in methanol/water (1/1 ; v/v) were transferred into the vials and placed at a pulsating panel operated at 1 ,500 rounds per minute. After complete adsorption the standard solution was discarded and 500 μΐ methanol was offered to the hydrogels for desorption. Measurement was performed using mass spectrometry. ii) A better swelling behaviour was observed for hydrogels having a proportion of 1 % stearyl methacrylate. In this case a thicker hydrogel layer was obtained. No significant difference was determined for a hydrogel with 3% stearyl methacrylate (StMA), while a distinctively decreased adsorption was observed for 250H-vitamin D₃. This effect was in particular pronounced for 4 hours of adsorption/desorption. The amount of 250H-vitamin D₃ adsorbed within or on the hydrogel did not show any significant variation, however, a decisively decreased retrieval factor was observed.

Summarizing the above results it can be said that a decrease in the amount of stearyl methacrylate is attributed to lead to a decrease in adsorption/desorption behaviour as shown in Table 2 below.

Table 2: data for adsorption/desorption measurements of varying contents of stearyl methacrylate.

Example 10: Variation of the content of ethylene glycol dimethacrylate in the hydrogel i) For improving the retrieval factor, the structure was further changed. Less cross- linker usually leads to a less compact hydrogel structure. This in turn means a faster fluid exchange and thus, a better diffusion. Accordingly, less cross-linker may lead to faster adsorption/desorption times. On the other hand with less cross-linker the hydrogel structure may get more and more lost. The mesh size or porosity, respectively may get considerably larger which may lead to a decreased swelling behaviour and in particular, separation of the protein may no longer observed. ii) Two monomer solutions were prepared: one with the composition 94.5% N,N- dimethyl acrylamide, 5% stearyl methacrylamide and 0.5 ethyleneglycol dimethacrylate and the other with the composition 94.9% Ν,Ν-dimethyl acrylamide, 5% stearyl methacrylamide and 0.1 ethyleneglycol dimethacrylate. 2,2'-Azobis(2-methyl- propionitrile) was added to both solutions and each solution was distributed over 6 surface modified GC/HPLC glass vials. The vials were bottom coated. Afterwards they were washed and conditioned as described above. Afterwards each 1 ml of a standard solution of 100 ng/ml 250H vitamin D₃ in methanol/water (1/1 : v/v) were transferred into the vials and placed at a pulsating panel operated at 200 rounds per minute. After expiration of the adsorption time the standard solution was discarded and 500 μΐ methanol was offered to the hydrogel for desorption. Each three GC/HPLC vials were subjected to the same adsorption/desorption times for one and four hours. Measurement was performed using mass spectrometry. iii) During the synthesis it was observed that with decreasing amount of cross-linker the hydrogel more and more came closer to a highly viscous fluid. Damages to the hydrogel more frequently appeared during the washing steps. No more swelling was observed and the mass simply lied on the ground as a viscous layer. Data were almost identical for 0.5% ethyleneglycol dimethacrylate and showed no substantial changes. However, for 0.1 % ethylene glycol dimethacrylate a distinctive change was observed. Less favourable adsorption and even less favourable desorption behaviour was observed. Table 3 below presents the corresponding data.

Table 3: data for adsorption/desorption measurements of varying contents of ethylene glycol dimethacrylate. Summarizing the results it can be said that the best results are obtained for a hydrogel composition of 94% Ν,Ν-dimethyl acrylamide (DMAA), 5% stearyl methacrylate (StMA) and 1% ethylene glycol dimethylacrylate (EGDMA).

V. Protein-repellent character of the hydrogel

In case of a protein interaction with the hydrogel, proteins from for example human serum albumin could get into the measuring system and therefore, could lead to a severe interference in the detection of desired unpolar, low-molecular weight compounds. Accordingly, the protein-repellent character of the hydrogel was examined using a solution of bovine serum albumin as test system. Like globulines also albumin belongs to the group of globular proteins which in the human organism provides for maintenance of the colloido osmotic pressure. These substances are proteins of elliptic shape having a molecular weight of approximately 66,000 Daltons, and consist of from 584 to 590 amino acids of which many are sulphur containing.

Example 1 1 : Protein-repellent character of the DMAA-hydrogel i) For testing the protein-repellent character of the hydrogel of the present invention an aqueous solution of bovine serum albumin with a concentration of 5 g/1 was used. A UV/Vis absorption spectrum was measured for this solution showing a maximum absorption at a wave length of 286.9 nm. Each 5 ml of this solution were transferred into two identical measuring cells and a DMAA hydrogel block of 50 mg was added (synthesized and conditioned to a methanol content of 40%prior to addition). Adsorption at a wave length of 286.9 nm was detected for the solutions, wherein the bovine serum albumin solution without hydrogel block served as a reference solution. Afterwards a further measuring was done with bi-distilled water as reference solution. Each 20 test values were recorded and the mean value was formed. Subsequently, the measuring cells were shaken in a rotational panel. After 60, 120, 180 and 240 minutes the same measurement as above was repeated and apart form minor differences the measured values were almost identical. ii) The almost consistent intensity of the measured wave length shows that there is approximately no binding of bovine serum albumin to the hydrogel. In case bovine serum albumin was bound to the surface of the hydrogel its concentration in solution would decrease which would result in a decreasing measured value with ongoing time. The absence of such an effect clearly proves the protein-repellent character of a hydrogel containing Ν,Ν-dimethyl acrylamides an uncharged and polar monomer. iii) Exclusion of larger proteins considerably simplifies sample preparation because protein precipitation using organic solvents and further centrifugation step(s) are no longer required. In addition, the use of protein-repellent hydrogels allows protein purification: small hydrophobic molecules may diffuse from the protein solution into the hydrogel and may be captured there while proteins remain in solution without any interaction with the hydrogel.

VI. Comparative examples

Example 12: Comparing adsoption/desorption behaviour of hydrogel powder and of immobilized hydrogel according to the present invention with Ci₈ SPE material. i) Almost without exception, common analysis methods for 250H-vitamin D₃ based on HPLC or LC/MS, respectively, contain an extraction step: first, for enrichment of the analyte and second, for separation of the analyte from, the complex matrix of human serum to the greatest possible extent. In this aspect, solid-phase extraction methods are based on surface-modified C|₈ material either in form of common SPE cartridges or in form of trapping columns in online processes. Accordingly, such surface-modified Ci₈ material as today's state of the art can be viewed for extraction in the analysis of 250H-vitamin D₃. Therefore, standard C|₈ SPE material is applied for comparative measurements. For comparative measurements the filling of a standard Agilent^® SPE cartridge having a pore size of 5 μιη served as Q material. For comparative measurements each 10 mg of the C]₈ material was transferred in 3 HPLC vial, followed by addition of 1 ml of a methanol/water (1/1 ; v/v) standard solution having a concentration of 100 ng/ml of 250H-vitamin D₃ into each glass vial. Due to the fine-grained character of the SPE material an additional centrifugation for 15 minutes at 15,000 rounds per minute was necessary for collecting the material at the bottom of the vials.

ii) When using 250H-vitamin D₃ as test substance for comparing the method of the present invention or its specific hydrogel, respectively, with common C|₈ SPE material, the amount of organic solvent in the solution may not be reduced in any desired degree. Due to the limited solubility of 250H-vitamin D₃ in water, said proportion of organic solvent in the respective solution may not fall below a certain level, otherwise 250H-vitamin D₃ will inevitably precipitate in the respective solution. Furthermore, the skilled person knows that 250H-vitamin D₃ is not present in free form in human serum but bound to a large extent to vitamin D-binding protein (DBP), also known as gc-globulin (group-specific component). Therefore, prior to the enrichment process itself 250H-vitamin D₃ has to be released from said protein by addition of organic solvent, for this purpose usually acetonitrile is used. Accordingly, if the proportion of organic solvent in the solution is too low, 250H-vitamin D₃ bound to DBP will not be separated from the protein and therefore, it will not be gathered in the measurement; compulsory, this would lead to wrong results in the measurement. iii) For 250H-vitamin D₃ it was found that a solution with a proportion of less than 30% of organic solvent does not allow a transfer of the method of the present invention in the practical working. In this context, a proportion of 40% of methanol in the solution showed to be the optimum. In addition, by varying the length of the adsorption/desorption times in from 1 to 4 hours, it was found that adsorption and desorption times of each 2 hours showed to be the optimum. iv) Under these conditions, i.e., proportion of 40% of methanol and adsorption/desorption times of 4 hours, retrieval factors of 83% were found for the vials coated with the hydrogel (see table 4). Accordingly, under the above-mentioned conditions the retrieval factor measured for glass vials coated with the hydrogel of the present invention is comparable with the respective factor measured for common Ci₈ SPE material. These results are standardized for 10 mg of adsorbing substance.

Table 4: data for hydrogel and C|₈-SPE in comparative measurements for adsorption/desorption times of 2 hours at a content of methanol of 40% (using a solution of 100 ng/ml 250H vitamin D₃ in MeOH/H₂0 (2/3; v/v). VII. Detection of 250H-vitamin D3 from human serum using HPLC glass vials coated with hydrogel

The preceding test measurements using HPLC glass vials coated with hydrogel have illustrated the successful adsorption and following desorption behaviour for 250H-vitamin D₃. The use of standard solutions having a specific concentration of 250H-vitamin D₃ and varying proportions of methanol/water, however, represents ideal conditions for adsorption of 250H-vitamin D₃ and thus, the respective series of tests may only indicate adsorption behaviour of the hydrogel in a sample probe having a complex matrix of human serum. Accordingly, for testing adsorption and desorption behaviour for 250H-vitamin D3 from human serum a corresponding series of tests using serums of a native pool with the known concentration of 22.7 ng/ml was used. Due to the complex of such a matrix whose components may lead to interferences during the detection of 250H-vitamin D3 a system which could provide the required sensitivity was applied, in this case an HPLC triple quadrupole system (type Quantum Ultra of the firm Thermo^®) was used.

Example 13: Determining of 250H- vitamin D₃ from human serum

i) Calibration solutions were prepared starting from a stock solution with a concentration of 100 ng/ml of 250H-vitamin D₃ which was diluted with methanol for obtaining the respective concentrations of the calibrating solutions. For calibration 5 calibration solutions with the concentrations 100 ng/ml, 75 ng/ml, 50 ng/ml, 25 ng/ml and 5 ng/ml were prepared (summarized in table 5 below).

Table 5: preparation of calibration solutions. ii) Preparation of the sample probes of human serums were prepared as follows: a) defrosting the deep frozen serum probes, b) after short mixing, each 8 μΐ of the isotope standard (1ST) D6-250H-vitamin D3 were added to 200 μΐ of the serums using an Ependorf pipette, and c) by adding 200 μΐ of methanol/water mixtures to the serums the final concentrations of 40%, 30% and 20% were adjusted so that the respective methanol/water mixtures had different proportions of methanol. The preparation of the sample probes is summarized in table 6.

Table 6: sample preparation of human serums. iii) The method parameters for measuring human serums using LC/MS are as follows:

extraction:

Waters^® Oasis HLB column (20 x 2.1 ; particle cross-section 25 μηι)

70 μΐ deprotonated probe added on column

mobile phase: water-methanol (95:5; flow rate = 3 ml/min)

wash solution: acetonitrile-MeOH (1 : 1 ; flow rate = 3 ml/min; t = 3.5 min)

column regeneration: water-methanol (95:5) analytics:

LiCropher^® 100 RP18 endcapped column (125 x 4; particle cross-section: 5 μπι) mobile phase: methanol-ammonium acetate_aq (c = 0.5 mmol/1; 90: 10)

flow rate: 0.85 ml/min; t = 3 min; » = 30°C

transfer from extraction column within 2 min.

ESI-MS:

capillary voltage: 3.0 kV cone voltage: 20 V

source temperature: 90°C desolution temperature: 280°C

nitrogen flow: 550 1/h cone gas: 75 1/h

collision energy: 25 V

For performance of the measurements the system setup of the triple quadrupole machine was slightly modified: the preparative column which is normally placed upstream was removed and the eluent flow of the auto sampler was guided directly on the analytical column.

iv) In the series of tests the 250H-vitamin D₃ concentration was detected at proportions of methanol of 40%, 30% and 20% with adsorption/desorption times of 1 , 2 and 3 hours. The tests were performed as described in the above examples. As reference for standardization the enzyme test Elecsys^® of Roche Diagnostics was used.

sample adsorption desorption proportion of mass of hydrogel number time time methanol

1 1 h 1 h 40% (v/v) 10.3 mg

2 2 h 2 h 40% (v/v) 10.7 mg

3 3 h 3 h 40% (v/v) 10.7 mg

4 1 h 1 h 30% (v/v) 10.9 mg 5 2 h 2 h 30% (v/v) 10.3 mg

6 3 h 3 h 30% (v/v) 10.8 mg

7 1 h 1 h 20% (v/v) 9.9 mg

8 2 h 2 h 20% (v/v) 1 1 .0 mg

9 3 h 3 h 20% (v/v) 10.9 mg

Table 7: Overview of the series of tests for detection of 250H-vitamin D3 concentration from human serum

The results of this series of tests are presented in table 8 below.

Table 8: measurement results for detection of the 250H-vitamin D₃ proportions from human serum.

This series of tests shows that the method of the present invention allows sample preparation from human serum for detection of the concentration of 250H-vitamin D₃. Mainly, the calculated concentrations obtained from taking the ratio of area sample with area ISTD come close to the actual sample concentrations, i.e., 22.7 ng/ml of 250H- vitamin D₃. As expected, serum samples with a methanol proportion of 40% in the adsorption solution (sample nos. 2 and 3) gave the best results because the methanol proportion of 40% in the adsorption solution with adsorption/desorption times of 2 hours had already shown the best results for standard solutions of 250H-vitamin D3 (config. Example 12).

VIII. Performance of the method of the present invention

The preparation of thin coatings of hydrogel is preferred for keeping adsorption and desorption times of the analyte(s) as short as possible. In contrast, thick layers of hydrogel lead to longer adsorption and desorption path ways of the analyte(s) into the hydrogel and out of the hydrogel. In addition, thick layers also cause a worse and tedious exchange of fluid between the solvent captured inside the hydrogel and the surrounding medium. Both factors together cause longer adsorption and desorption processes and therefore, thick(er) layers of hydrogel should be avoided.

Example 14: Representative performance of the method of the present invention i) In principle, application of coated GC/HPLC vials is fairly easy. First, a serum probe is transferred into a GC/HPLC vial which has been coated on the inside. The hydrogel interacts with the analyte contained in the serum and adsorbs this analyte. After adsorption of the analyte the serum probe is either removed using a syringe or a pipette or just dumped followed by a short washing step with water for removing remaining residues of the serum matrix. After removal of the washing water the desorption solution is introduced into the vial. The analyte adsorbed inside the hydrogel now desorbs from the hydrogel and can now be applied to analysis. By using a solvent in which the respective analyte is better soluble an enrichment of said analyte is achieved because a smaller volume of solvent is required. ii) In this context it should be considered that the DMAA hydrogel however, starts to swell in the desorption solution and thus, a certain amount of volume is captured within the hydrogel. Therefore, one has to regard that after desorption has started sufficient volume of desorption solvent is present for a subsequent analysis. All of the aforementioned steps can be easily performed by a pipetting robot and therefore, the method of the present invention can be easily made automatable which contributes to the attractiveness of the method of the present invention.

Claims

1. A method for isolating at least one analyte from a sample fluid containing said at least one analyte and at least one protein, comprising the steps of: a) contacting the sample fluid with a protein-repellent hydrogel immobilized on a solid surface to capture said at least one analyte;

b) separating the hydrogel obtained in step a);

c) optionally washing the hydrogel obtained in step b) with an aqueous solution containing up to ca. 30% by volume of an organic water-miscible solvent; d) contacting the hydrogel obtained in step c) with an aqueous solution containing at least 60% by volume of an organic water-miscible solvent;

e) isolating said at least one analyte obtained in step d); wherein the hydrogel is produced by the co-polymerization of at least one uncharged and polar monomer with at least one hydrophobic monomer and at least one cross-linking monomer.

2. The method of claim 1, wherein the protein-repellent hydrogel in step a) is a thin layer of a protein-repellent hydrogel immobilized on a solid surface.

3. The method of claim 1 or 2, further comprising prior to step a) the step of a') contacting the sample fluid with an organic solvent thereby creating an aqueous mixture containing up to ca. 50% by volume of an organic water- miscible solvent.

4. The method of at least one of the claims 1 to 3, wherein the sample fluid is an aqueous sample fluid.

5. The method of at least one of the claims 1 to 4, wherein the solid surface is a non- porous material, preferably the non-porous material is glass or a polymer. The method of at least one of the claims 1 to 5, wherein the solid surface is modified by an organosilicon acrylic acid derivative or organosilicon methacrylic acid derivative of the type given in Formula I

wherein

R⁵ is either H or methyl;

o is an integer from 0 to 9;

A , A and A are independently from each other linear or branched Ci-C₄ alkyl or CrC₄ alkoxy with the proviso that at least one of A¹, A² or A³ is d-C₄ alkoxy.

The method of at least one of the claims 1 to 6, wherein one or more of the uncharged and polar monomer(s) is/are acrylamide, N-monoalkylacrylamide, oligoethylene glycol acrylamide, oligoethylene glycol methacrylamides, N,N- dialkylacrylamide and/or an N,N-dialkylmethacrylamide, wherein each alkyl group is independently from each other methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso- butyl, and/or tert-butyl.

The method of at least one of the claims 1 to 7, wherein one or more of the hydrophobic monomer(s) is/are a methacrylic acid ester and/or an acrylic acid ester, having a hydrocarbon chain comprising from 2 to 20 carbon atoms, wherein the hydrocarbon chain is an alkyl, alkenyl, or alkynyl residue, which may be optionally substituted with one or more phenyl, cyclohexyl, phenylen or cyclohexylen groups with the proviso that carbon-carbon multiple bonds in the hydrocarbon chain if present are conjugated bonds.

The method of at least one of the claims 1 to 8, wherein the one or more cross- linking agent(s) is/are i) a glycol acrylate derivate of the Formula II

with

m being an integer from 2 to 10; n being an integer from 1 to 4;

R¹, R⁴ being independently from each other H or a Ci-C₄ alkyl group;

R², R³ being independently from each other H, F, OH or methyl; wherein one or more of the oxygen-carbon bonds in the glycol chain may be interrupted by a phenylen or cyclohexylen group or an amide group; and/or ii) an acrylamide derivative of the Formula III

with

being an integer from 2 to 10; is a bond or a -CH₂-CH₂-0- or -0-CH₂-CH₂- group forming part of a glycol chain; and

R', R², R³ and R" are as defined above.

10. The method of at least one of the claims 1 to 9, wherein the cross-linking agent is an ethylene glycol diacrylate derivative or ethylene glycol dimethacrylate derivate.

1 1. The method of at least one of the claims 1 to 10, wherein the hydrogel is produced by the co-polymerization of from ca. 85 to ca. 98% of at least one uncharged and polar monomer, of from ca. 1 to 10% of at least one hydrophobic monomer and of from ca. 1 to ca. 5% of at least one cross-linking agent.

12. The method of claim 11 , wherein the uncharged and polar monomer is N,N-dialkyl acrylamide.

13. The method of claim 1 1 or 12, wherein the hydrophobic monomer is stearyl methacrylate.

14. The method of at least one of the claims 1 1 to 13, wherein the cross-linking agent is ethylene glycol dimethacrylate.

15. The method of at least one of the claims 1 to 14, wherein the analyte is a hydrophobic compound.

16. The method of at least one of the claims 1 to 15, wherein the sample fluid is or contains a biological fluid.

17. The method of claim 16, wherein said sample fluid is an aqueous sample fluid.

18. The method of at least one of the claims 1 to 17, wherein the organic water- miscible solvent is acetonitrile, dimethylformamide, ethanol and/or methanol.