DE10155692A1 - Procedure for the detection of endogenous and exogenous substances in the organism - Google Patents

Abstract

The invention relates to a method for simultaneously identifying and/or differentiating endogenic and exogenic substances, in particular substances comprising at least one peptide bond in an organism. According to the invention, endogenic and exogenic substances present in collected endogenic material that is collected in particular from a human or animal organism are accumulated on a support material and said substances are subsequently identified and/or differentiated by means of their differing masses.

Description

The invention relates to a method for detection and / or Differentiation of endogenous and exogenous substances, especially of Substances with at least one peptide bond in an organism.

Exogenous substances, i.e. H. substances created outside of an organism, such as B. recombinant proteins and peptides are used for treatment various diseases, for example the endogenous variants, d. H. those created in the body itself, not by externally supplied or from the special system of the organism resulting substances, insufficiently formed by the body can be. This is already in clinical use Growth hormone for the treatment of growth hormone deficiencies of different origins (Murray, R.D. and Shalet, S.M. (2000). Growth hormone: current and future therapeutic applications. Expert Opin Pharmacother 1, 975-990). Erythropoietin is followed by anemia used for example by tumors (Kasper, C. (2001). Recombinant human erythropoietin in the treatment of anemic patients with hematological malignancies. Ann Hematol 80, 319-329). recombinant Gonadotropins are used for infertility (Garcia-Campayo, V. and Boime, I. (2001). Novel recombinant gonadotropins. trends Endocrinol Metab 12, 72-77). Blood coagulation factors are considered Diseases with increased bleeding tendency used (Monroe, D.M., Hoffman, M., Allen, G.A. and Roberts, H.R. (2000). The factor VII platelet interplay: effectiveness of recombinant factor VIIa in the treatment of bleeding in severe thrombocytopathia. Semin Thromb Hemost, 26, 373-377). Recombinant thyrotropin takes place in patients with a Thyroid cancer application (Baskin, H.J., Atwood, T.M. and Holcomb, L. P. (2000). Recombinant human thyrotropin stimulation of thyroglobulin in the follow-up of patients with stage I or II differentiated thyroid carcinoma. Endocr Pract 6, 430-434).

Most therapeutically important proteins are glycoproteins. The Glycosylation determines stability, folding and biological activity (Lerouge, P., Bardor, M., Pagny, S., Gomord, V. and Faye, L. (2000). N- glycosylation of recombinant pharmaceutical glycoproteins produced in transgenic plants: towards an humanization of plant N-glycans. curr Pharm Biotechnol 1, 347-354). In most cases, the Production of e.g. B. recombinant peptides and proteins Modifications inserted, their functionality in terms of stability and Affect effectiveness (receptor binding). This affects above all Glycoproteins (Garcia-Campayo, V. and Boime, I. (2001). Novel recombinant gonadotropins. Trends Endocrinol Metab 12, 72-77; Lerouge, P., Bardor, M., Pagny, S., Gomord, V. and Faye, L. (2000); Lynch, T.J. (2000). Biotechnology: alternatives to human plasma-derived therapeutic proteins.

Baillieres Best Pract Res Clin Haematol 13, 669-688; Macdougall, I.C. (2001). An overview of the efficacy and safety of novel erythropoiesis stimulating protein (NESP). Nephrol Dial Transplant 16, 14-21). Modifications of the sugar residue are, for example, in the case of erythropoietin carried out to determine the residence time of the recombinant protein in the To extend plasma (Egrie, J.C. and Browne, J.K. (2001). Development and characterization of novel erythropoiesis stimulating protein (NESP). Nephrol Dial Transplant 16, 3-13).

International takes place - with different legislative Framework conditions - use of, for example, growth hormone (also as Growth hormone (GH) or somatotropic hormone (STH)) in animal production. In the case of dairy cows, the optimization of Milk yield in the foreground, for example in the pig higher proportion of lean meat can be achieved (Karg, H., Meyer, H. and Schams, D. (1992). Manipulation of growth and lactation in Cattle. Mh. Vet.-Med. 47, 113-118).

Become both in high-performance sports and popular sports increasingly performance-enhancing preparations in the field of protein and Peptide hormones used (Clarkson, P. M. and Thompson, H. S. (1997). Drugs and sport. Research findings and limitations. Sports Med 24, 366-384; Davey, G. and Nerurkar, R. (1998). Doping in sport. Lancet 352, 1781-1782; Davies, J.S., Morgan, C.L., Currie, C.J. and Green, J. T. (1997). Growth hormone use by body builders. Br J Sports Med 31, 352-353; Haug, E. (1994). [Abuse of doping preparations outside organized sports. A new challenge for health care]. Tidsskr Nor Laegeforen 114, 424-425). This currently affects above all Growth hormone and erythropoietin (Ehrnborg, C., Bengtsson, B.A. and Rosen, T. (2000). Growth hormone abuse. Bailliere's Best Pract Res Clin Endocrinol Metab 14, 71-77; Jelkmann, W. (2000). Use of recombinant human erythropoietin as an antianemic and performance enhancing drug.

Curr Pharm Biotechnol 1, 11-31). Erythropoietin is improving of oxygen transport by increasing the proportion of red Blood cells, the erythrocytes, mainly used in cycling (Audran, M., Gareau, R., Matecki, S., Durand, F., Chenard, C., Sicart, M. T., Marion, B. and Bressolle, F. (1999). Effects of erythropoietin administration in training athletes and possible indirect detection in doping control. Med Sci Sports Exerc 31, 639-645; Breymann, C., Rohling, R., Krafft, A., Huch, A. and Huch, R. (2000). "Blood doping" with recombinant erythropoietin (rhEPO) and assessment of functional iron deficiency in healthy volunteers. Br J Haematol 108, 883-884; Gareau, R., Audran, M., Baynes, R.D., Flowers, C.H., Duvallet, A., Senecal, L. and Brisson, G. R. (1996). Erythropoietin abuse in athletes. Nature 380, 113; Jelkmann, W. (2000). Use of recombinant human erythropoietin as an antianemic and performance enhancing drug. Curr Pharm Biotechnol 1, 11-31). There are also reports of the use of erythropoietin in the Equestrian sport before (Kearns, C.F., Lenhart, J.A. and McKeever, K.H. (2000). Cross reactivity between human erythropoietin antibody and horse erythropoietin. Electrophoresis 21, 1454-1457).

The use of HCG (human chorionic gonadotropin), ACTH (adrenocorticotropic hormone), growth hormone and erythropoietin as well as the substances that regulate their release or their Communicating effect was in 1989 by the Medical Commission of the International Olympic Committee (IOC) banned (Kicman, A. T. and Cowan, D.A. (1992). Peptide hormones and sport: misuse and detection. Br Med Bull 48, 496-517).

Despite these restrictions on use or even prohibition there are problems with humans and animals regarding the abusive Use of these substances (Bidlingmaier, M., Wu, Z. and Strasburger, C. J. (2000). Test method: GH. Bailliere's Best Pract Res Clin Endocrinol Metab 14, 99-109; Breymann, C. (2000). Erythropoietin test methods. Baillieres Best Pract Res Clin Endocrinol Metab 14, 135-145).

The problem of detecting the administration of, for example recombinant growth hormone or erythropoietin in Body fluids are that the endogenous and exogenous, z. B. recombinant form of growth hormone and erythropoietin, if any then differ only to a very small extent (Healy, M. L. and Russell-Jones, D. (1997). Growth hormone and sport: abuse, potential benefits, and difficulties in detection. Br J Sports Med 31, 267-268). The little difference is enough with currently applied analytical Methods are not sufficient to safely endogenously within a sample to distinguish between exogenous forms. Another problem is that Fact that the blood level of natural growth hormone is not is constant, but is subject to rhythmic fluctuations. sudden Increases can therefore only be a spontaneous increase in endogenous Represent growth hormone concentrations. Furthermore, the Release of growth hormone and thus the blood level of quantitative and qualitative changes in the Food composition dependent. Even with strong physical performance there is an increase of growth hormone blood levels (Jenkins, P. J. (1999). Growth hormone and exercise. Clin Endocrinol (Oxf) 50, 683-689.) There is currently no accepted method of detection for doping control in high performance sports and for the control of illegal use in animal production. Therefore, for both Proteohormones indirect detection methods discussed (Bidlingmaier, M., Wu, Z. and Strasburger, C.J. (2000). Test method: GH. Baillieres Best Pract Res Clin Endocrinol Metab 14, 99-109); Birkeland, K.I., Stray- Gundersen, J., Hemmersbach, P., Hallen, J., Haug, E. and Bahr, R. (2000). Effect of rhEPO administration on serum levels of sTfR and cycling performance. Med Sci Sports Exerc 32, 1238-1243; Breymann, C. (2000). Erythropoietin test methods. Bailliere's Best Pract Res Clin Endocrinol Metab 14, 135-145; Choi, D., Kim, M. and Park, J. (1996). Erythropoietin: physico- and biochemical analysis. J Chromatogr B Biomed Appl 687, 189-199). However, indirect detection methods are also included the high risk of a false positive statement. On Increased hematocrit, which leads to exclusion in racing cyclists, z. B. also be physiological or pathophysiological in nature (Brun, J. F., Bouchahda, C., Chaze, D., Benhaddad, A.A., Micallef, J.P. and Mercier, J. (2000). The paradox of hematocrit in exercise physiology: which is the "normal" range from an hemorheologist's viewpoint? Clin Hemorheol Microcirc 22, 287-303; Schmidt, W., Biermann, B., Winchenbach, P., Lison, S. and Boning, D. (2000). How valid is the determination of hematocrit values to detect blood manipulations? Int J Sports Med 21, 133-138; Shaskey, D.J. and Green, G.A. (2000). Sports haematology. Sports Med 29, 27-38). In the foreground is the differentiation based on of the naturally occurring various isoforms of Growth hormone that does not occur with supplementation (Wu, Z., Bidlingmaier, M., Dall, R. and Strasburger, C.J. (1999). Detection of doping with human growth hormones. Lancet 353, 895). The Using the differences in the isotope ratio between endogenous growth hormone and various recombinant preparations is very complex and does not produce uniform results for the individual preparations (Abramson; F.P., Osborn, B.L. and Teffera, Y. (1996). Isotopic differences in human growth hormone preparations. Anal Chem 68, 1971-1972). For erythropoietin either Hematocrit used (Ekblom, B. (1996). Blood doping and erythropoietin. The effects of variation in hemoglobin concentration and other related factors on physical performance. Am J Sports Med 24, S40-42) or other parameters of iron metabolism (Clyne, N., Berglund, B. and Egberg, N. (1995). Treatment with recombinant human erythropoietin induces a moderate rise in hematocrit and thrombin antithrombin in healthy subjects. Thromb Res 79, 125-129; Gareau, R., Brisson, G.R., Chenard, C., Gagnon, M.G. and Audran, M. (1995). Total fibrin and fibrinogen degradation products in urine: a possible probe to detect illicit users of the physical-performance enhancer erythropoietin? Norm Res 44, 189-192). Newer methods allow a certain differentiation based on charge differences in the isoelectric Focusing (Lasne, F. and de Ceaurriz, J. (2000). Recombinant erythropoietin in urine. Nature 405, 635).

What is desired is a test procedure in which an organism endogenous and exogenous substances, in particular peptide compounds, could be demonstrated. It should be a direct and quick proof of this Be possible with a high sensitivity, sensitivity and fabrics Specificity. It is also desirable to distinguish between the various exogenous ones Differentiate substances, especially in the recombinant and genetically engineered substances, these to a certain producer assigned.

The aim of the invention is therefore to provide a method for detection and / or to differentiate between exogenous and endogenous substances to provide what is specific, selective and sensitive enough, by comparison little effort to detect these substances and thus, for example an illegal use of recombinant, genetically engineered Growth hormones to increase performance in humans (doping) and Animal (milk production, fattening).

This object is achieved by the method as set out in claim 1 is defined. Preferred embodiments are in the dependent ones Claims to 2 to 14 set out. The wording of all claims will hereby incorporated by reference into the content of this description.

Surprisingly, it has been found that endogenous and exogenous Fabrics after selective enrichment with the help of a highly sensitive Proof of their different masses can be.

The invention thus relates to a method for detection and / or to differentiate between endogenous and exogenous substances, in particular of substances with at least one peptide bond, where the body material taken in, especially from a the body's own human or animal organism Material, existing endogenous and exogenous substances on one Carrier material are enriched, and then these substances over their different masses demonstrated and / or differentiated become.

According to the invention, they are endogenous and exogenous Substances around peptides, polypeptides, proteins, peptide hormones and / or Proteohormones. With the peptide and / or proteohormones especially STH (somatotropic hormone), erythropoietin, ACTH (adrenocorticotropic hormone), and / or HCG (human Chorionic gonadotropin), but also all other endogenous and exogenous substances, in particular peptide compounds which are prepared with the aid of the invention Proof of procedure can be considered.

Before enrichment and / or detection of endogenous and Exogenous substances can be modified, especially fractionated and / or be marked. So z. B. to differentiate between endogenous and exogenous substances selective digestion or chemical modification of the substances can be used. You can do this on substances bound to a carrier material before mass determination be treated enzymatically or chemically. So it is z. B. possible that by treatment with proteases (e.g. tripsin) peptides and Proteins in, depending on the variant (amino acid sequence), different peptide fragments are cleaved. The individual sections point each have a characteristic molecular weight distribution because the respective enzymes at certain amino acid positions are preferred to cut. The choice of proteases is variant and species specific, because species-specific differences in Amino acid composition of, for example, growth hormones. As The result is one for the respective peptide and protein variant characteristic "fingerprint". This pretreatment of the peptides and Proteins not only improve the distinction less Mass differences, but also allows the differentiation of possible ones Variants with differences in the amino acid sequence.

Other covalent protein modifications such as for example glycosylation or phosphorylation by enzymatic pretreatment of the (enriched and purified) proteins or peptides such as Growth hormone and erythropoietin variants with, for example, glycosidases or detect phosphorylases. Here, the specific on the Carrier-enriched sample according to customary methods spiked with specific enzymes that are complete or selective Allow sugar or phosphate residues to be split off. As enzymes the glycosidases PNGase-F or are suitable, for example Phosphorylases such as alkaline phosphatase and others. Because of the engagement of different glycosidases can be between N- or O- Links between the sugar residues can be distinguished. Another Possibility is the chemical elimination of sugar residues hydrazine is used. The analytical steps take place in Volume, time and temperature adapted to the surface of the substrate, according to known methods (Savage, A. (1997). Glycosylation: A post-translational modification. In Mammalian Cell Biotechnology in Protein Production, (ed. H. Hauser and R. Wagner), pp. 231-276. Berlin, New York: Walter de Gruyter).

Other possibilities of z. B. protein modification, the one Improve the distinction between natural and artificial peptides and proteins among the described enrichment and Measuring conditions can enable, represent various known and future possibilities for the selective labeling of amino acids or covalent modifications that lead to mass changes and so on can be detected by mass spectroscopy.

The body's own material is preferably material that was taken from a living organism, but also that ex mortem removal is provided according to the invention. In a preferred embodiment is the body's own Materials around body fluids, tissues and / or organs. Doing so the body fluids blood, serum, plasma, urine, milk, sweat, Saliva and / or cerebrospinal fluid and all other body fluids. The blood can be punctured by blood vessels (venipuncture) be won. Due to the very small volume that is necessary for the Analysis is needed, blood can also be punctured by Blood capillaries (for example the fingertip) are obtained. The Blood sampling or plasma / serum extraction and extraction other body fluids follow established procedures. they include the puncture of the vessels, separation of the blood components, preferably by centrifugation, and storage, preferably at -80 ° C. In the case In an immediate analysis, the sample is preferably pending analysis Store at 4 ° C. Tissues and organs are established by Biopsy procedure or post mortem and taken accordingly established procedures stored. Before the analysis, the organs and Tissue initially, for example, according to established procedures appropriate buffer can be homogenized. As a buffer, for example Tris-HCl (pH 7.8). The body fluids such as B. serum, plasma, Liquor or urine can be in the original concentration, diluted or concentrated on the carrier material as a defined volume by means of applied with a pipette. To enlarge the Order volume, an adapter can be used. A comparable procedure lends itself to the homogenization of tissues and organs. Dilution steps and sample volume (> 2 µl) depend on the concentration the peptide and protein to be examined in the respective matrix, z. B. solution, depending. A selective enrichment on the Carrier material can be made, for example, by selective fractionation. Methods for tissue homogenates and urine are known to those skilled in the art known.

In a further preferred embodiment, the Support material around a chromatographic support material. This chromatographic support material can be a hydrophobic, hydrophilic, cation-exchanging, anion-exchanging and / or metal ion-binding carrier material, but also all others chromatographic support materials are of the invention Process claimed. The binding takes place during a Incubation period, its parameters (duration, temperature etc.) from the matrix and the concentration of the peptide or protein to be examined are dependent. In a further step (washing step) the selective removal of as many substances as possible, in particular proteins and Peptides, of the matrix or the carrier material, of no interest are. As differentiation criteria between endogenous and exogenous Substances, especially peptide or protein variants, are used for example, special ties to certain chromatographic Surfaces, differences in the binding intensity to these surfaces in Dependence on the stringing of the washing buffer etc. As Carrier material can also be used an immobilized carrier material, which claims in a further embodiment of the method becomes. In a further preferred embodiment, this is immobilized carrier material with antibodies, binding proteins and / or Receptors have been immobilized. The carrier material or the Surface of the carrier material are activated before immobilization. On these activated / inactivated carrier materials is accordingly published standard methods either an antibody (monoclonal / polyclonal), binding proteins and / or receptors immobilized are able to endogenous and exogenous substances, e.g. B. STH or Bind erythropoietin and so together with any existing Fragments or variants from complex matrices, e.g. B. solutions to fish. The coupling of antibodies, binding proteins and / or receptors can be done either directly or via a spacer. Through the Using a spacer can change the orientation of the antibody, Binding protein and / or receptor are controlled. The coupling of antibodies or receptors takes place in the usual way during an incubation period, the parameters (duration, temperature, etc.) of the matrix and the concentration of the substance to be examined, in particular the peptide and / or protein compound. In a further step (washing step) removes all in the Matrix existing substances that are not on the carrier material were bound or are of no interest.

The carrier material can be e.g. B. a chip, preferably a protein chip. On or on this chip the Enrichment, detection and, if necessary, any necessary modification.

In a further preferred embodiment, the endogenous and exogenous substances separated from each other before detection. This separation of the endogenous and exogenous substances can take place before or after enrichment on a suitable carrier material. Furthermore, the separation after modification, in particular fractionation and / or labeling. So z. B. the Modifications of the substances already described above, such as for example glycosylation or phosphorylation, through which enzymatic pretreatment of the on the carrier material (possibly before enriched) purified endogenous and exogenous substances For example, use glycosidases or phosphorylases for the separation.

In a further preferred embodiment, the endogenous and exogenous substances via their different molecular weights demonstrated. So z. B. the so-called SELDI (for surface enhanced laser desorption / ionization) process is a suitable process for the detection of endogenous and exogenous substances using the different molecular weight. An analysis of z. B. time of flight mass spectrometer (TOF [time-of-flight] analyzer) or comparable devices are made, which differentiate on the Basis of molecular weight or for example others Allow selection methods. The substances are added after a energy absorbing matrix ionized with the help of a laser beam and then accelerated in an electric field. The firing frequency of the Laser beam averages 50 to 100 shots. From the Flight time of the substances from the sample carrier to the detector at the end of the Flight tunnels are the molecular mass of the bound particles and their any fragments present are determined. The differentiation between endogenous and exogenous substances usually takes place on the Basis of the difference in the molecular weight of the substances and / or their any fragments present. The accuracy of the system in Relation to the statement about the molecular weight (depending on the molecular size) is approximately 0.1 to 0.01 percent.

In a further preferred embodiment, the endogenous and exogenous substances quantified. This quantification can be done via Standards of endogenous and exogenous substances are made.

Finally, in a particularly preferred embodiment the exogenous substances based on their characteristic modification, especially based on their existing modification, distinguished from each other. So z. B. the differentiation between endogenous and exogenous substances usually based on the Difference in the molecular weight of the substances, in particular peptide and Protein compound, or its fragments. These differences arise for example from one or more additional amino acids. The N-terminal extension of the z. B. recombinant growth hormone leads, for example, to an increase in the molecular weight. The Accuracy of the z. B. TOF analyzer in relation to the statement about the molecular weight (depending on the molecular size is Measuring accuracy 0.1 to 0.01%) therefore not only allows Differentiation between endogenous and exogenous substances, e.g. B. Growth hormone variants, but in the case of differences in the length of the N- terminal extension also indicates the source of the recombinant growth hormone. Also modifications in the area of Sugar residues such as erythropoietin can be so directly or afterwards for example enzymatic modification detected and z. B. for Assignment to a specific producer can be used.

Further details and features of the invention result from the following description of the representation of the invention Method and preferred embodiments in connection with the subclaims. Here, the respective characteristics for themselves be realized alone or in combination with each other.

Experiment 1

Specific monoclonal or polyclonal antibodies against the investigating particles and an IgG control in a concentration between 0.01 and 0.09 mg / ml in a volume of 20 ul the preactivated surfaces of a carrier material are applied. The incubation is then carried out in a steam-saturated one Chamber (incubation chamber) for 1 to 2 hours at room temperature or at 4 ° C overnight (14-16 hours). During this Incubation period, the antibodies are covalently attached to the activated Carrier surfaces bound. The remaining vacancies of the Carrier material are now added by 20 µl Glycerine solution (0.1 M, pH 7.8) blocked. Another incubation for 20 minutes at room temperature in the incubation chamber. The antibody solution is then removed by washing several times. In the first step, the carrier material with 50 mM Tris-HCl (pH 7.8) - Solution (50-100 µl) washed. This is followed by two Wash with 300 µl PBS solution containing 0.5% (v / v) Triton X-100 contains. The incubation period at room temperature is in each case 20 minutes in the incubation chamber. Then two further washing steps with 300 µl PBS (without Triton X-100) for 10 Minutes at room temperature in the incubation chamber.

The sample material is either fed directly onto the Pre-activated and immobilized carrier material with low sample material (Volume <5 µl) or via an adapter (volume> 5 µl). The Incubation takes place for 1-2 hours at room temperature in the Incubation chamber. Then the sample material is pipetted removed, and the loaded carrier material is twice with 300 ul PBS solution containing 0.5% (v / v) Triton X-100 washed. The Incubation time at room temperature is 10 minutes each the incubation chamber. This is followed by two more Wash steps with 300 µl PBS (without Triton X-100) for 10 minutes Room temperature in the incubation chamber. The last two Washing steps are carried out with water (300 µl) for 10 minutes each. The carrier material is then dried. As an energy absorbing Substance becomes, for example, alpha-cyano-4-hydroxy cinnamic acid (2 mg / ml) in 50% (v / v) acetonitrile and 0.2% (v / v) tri-fluoroacetic acid added. Endogenous and exogenous substances are analyzed then using a time-of-flight mass spectrometer.

By this invention, it is possible from z. B. a complex Protein mixture very similar variants of a special peptide or Protein - for example growth hormone and erythropoietin - to enrich and to prove, but also the proof of others endogenous and exogenous substances are possible. The subject of The invention therefore combines, among other things, the high selectivity of Antibodies or other affinity steps with a high accuracy of Determination of the molecular weight of the enriched substances or Molecules. After placing the matrix on the substrate surface can include enrichment, modification (enzymatic Cleavage) and the proof on this. This will also Sources of error and the risk of contamination are reduced. On Analysis run is generally completed within 4 hours. The sample throughput depends on the degree of automation.

Claims (14)

1. A method for the detection and / or differentiation of endogenous and exogenous substances, in particular substances with at least one peptide bond, characterized in that the endogenous and exogenous substances present in the endogenous material removed, in particular endogenous and exogenous substances taken out from a human or animal organism be enriched on a carrier material, and then these substances can be detected and / or differentiated using their different masses. 2. The method according to claim 1, characterized in that it is for endogenous and exogenous substances around peptides, Polypeptides, proteins, peptide hormones and / or proteohormones. 3. The method according to claim 2, characterized in that it is for the peptide and / or proteohormones around STH (somatotropes Hormone), erythropoietin, ACTH (adrenocorticotropic hormone), and / or HCG (human chorionic gonadotropin). 4. The method according to any one of claims 1 to 3, characterized characterized in that before the enrichment and / or the detection of the endogenous and exogenous substances modified them, in particular fractionated and / or marked. 5. The method according to any one of the preceding claims, characterized characterized that it is in the body's own materials body fluids, tissues and / or organs. 6. The method according to claim 5, characterized in that it is in the body fluids around blood, serum, plasma, urine, milk, Sweat, saliva and / or liquor. 7. The method according to any one of the preceding claims, characterized characterized in that the carrier material is a chromatographic support material. 8. The method according to claim 7, characterized in that it is for the chromatographic support material hydrophobic, hydrophilic, cation-exchanging, anion-exchanging and / or metal ion-binding carrier material. 9. The method according to any one of claims 1 to 6, characterized characterized in that the carrier material is a immobilized carrier material. 10. The method according to claim 9, characterized in that it is in the immobilized carrier material by an antibody, Binding proteins and / or receptors immobilized Carrier material is. 11. The method according to any one of the preceding claims, characterized characterized that the endogenous and exogenous substances before the evidence of which is separated from one another. 12. The method according to any one of the preceding claims, characterized characterized in that the endogenous and exogenous substances with the SELDI and / or TOF method can be demonstrated. 13. The method according to any one of the preceding claims, characterized characterized that the endogenous and exogenous substances be quantified. 14. The method according to any one of the preceding claims, characterized characterized in that the exogenous substances based on their characteristic modifications, especially based on their already existing modifications can be distinguished from each other.