WO2018007394A1

WO2018007394A1 - Method for the calibration of a biological sample

Info

Publication number: WO2018007394A1
Application number: PCT/EP2017/066654
Authority: WO
Inventors: Bianca Bethan; Oliver Schmitz; Volker Haake; Oliver Blaesing; Michael Herold; Holger Fahnenstich; Stefan Henkes
Original assignee: Basf Plant Science Company Gmbh
Priority date: 2016-07-08
Filing date: 2017-07-04
Publication date: 2018-01-11

Abstract

The present invention relates to a method for providing a calibrated result of a determination of at least one analyte of interest in a biological sample comprising a) providing a matrix calibration sample, wherein the concentration of at least one reference analyte in said matrix calibration sample was predetermined, b) determining at least one characteristic feature of said at least one analyte of interest in said biological sample, c) determining said at least one characteristic feature of said at least one reference analyte in said matrix calibration sample, and d) based on the results of steps b) and c), providing a calibrated result of the determination of said at least one analyte of interest. Further, the present invention relates to kit, devices, and uses related to said method.

Description

Method for the calibration of a biological sample

Biological samples consist of a broad range of endogenous compounds and may further contain exogenous compounds, like compounds taken up with food, e.g. plant secondary metabolites, or artificial chemical compounds. These compounds reflect a broad range of chemical structures, polarity, volatility and concentrations. The complexity of the compounds present in a biological sample represents a major challenge for the quantification of these compounds, in particular if simultaneous determination of a large number of compounds is attempted.

Conventional quantification methods usually require calibration curves, reagents tagged to the compound of interest and/or use internal standards. These approaches have limitations for the simultaneous quantification of many compounds of various chemical structures and

concentrations. For calibrating results, calibration samples have been widely used, i.e. samples having a defined concentration of an analyte of interest, e.g. reviewed by Bindschedler and Cramer, (201 1 ), Proteomics (1 1 ):756, for plant proteomics. By measuring a characteristic feature of an analyte in a calibration sample in parallel to a sample of interest, the analyte of interest can be quantified. Traditionally, the concentration of an analyte of interest in a calibration sample is defined by adjusting the concentration, e.g. by weighing in a specific amount of an analyte and dissolving the same in a defined amount of solvent. Alternatively, a calibration sample may also be obtained by artificially adding an analyte into an analyte-free sample material at known concentrations or amounts. Further, a series of adding various amounts of analyte of interest into samples containing an unknown amount of the analyte of interest ("spiking") may be used to quantify a result. Also, addition of artificial marker analytes sufficiently similar, but not identical to the analyte of interest, e.g. isotope-labeled compounds in mass spectrometry, was used. Also, combinations of the aforesaid methods have been used (e.g. Leinenbach et al. (2014), Clin Chem 60(7):987). Calibration curves require standards for the compounds of interest. A complex mixture of many different chemical compounds may cause chemical reactions between compounds to occur, leading to changes in the respective concentrations. For quantification of compounds via an internal standard with known concentrations, at least one internal standard for each chemical structure is required, requiring a large number of standards for a broad quantification approach. Addition of an excessive number of compounds to a sample can lead to analytical issues in the quantification process, such as increased ion suppression in mass spectrometry detection. Moreover, appropriate internal standards may not be available for each structural class of compounds.

Further, results of quantitative determinations are frequently validated against reference samples, i.e. samples containing known concentrations of the analyte of interest as reference material (e.g. Phinney et al., (2013) Anal Chem 85:1 1732). As part of a method validation process, it may be evaluated whether the results obtained with a specific quantification method are correct, e.g. are within a given tolerance range, for assessment of trueness and accuracy. Thus, in such validation, a standard reference material is treated as a sample of interest and the result obtained for the analyte of interest is compared to the expected value known for the reference material. Since in this part of validation it is only established whether a given concentration of analyte can be determined correctly, validation results do not generally permit to draw conclusions on concentrations deviating from the validation concentration and, in particular, do not permit calibration.

There is, thus, a need in the art for improved calibration of samples, in particular complex biological samples. The problem is solved by the means and methods provided herein.

Accordingly, the present invention relates to a method for providing a calibrated result of a determination of at least one analyte of interest in a biological sample comprising

a) providing a matrix calibration sample, wherein the concentration of at least one reference analyte in said matrix calibration sample was predetermined, b) determining at least one characteristic feature of said at least one analyte of interest in said biological sample

c) determining said at least one characteristic feature of said at least one reference analyte in said matrix calibration sample, and

d) based on the results of steps b) and c), providing a calibrated result of the

determination of said at least one analyte of interest.

The method for providing a calibrated result of the present invention, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to obtaining a multitude of samples from subjects for step (a), and separating an analyte from further components of a sample before step b).

Moreover, one or more of said steps may be performed by automated equipment.

As used in the following, the terms "have", "comprise" or "include" or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions "A has B", "A comprises B" and "A includes B" may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment of the invention" or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention. Moreover, if not otherwise indicated, the term "about" relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value ± 20%, more preferably ± 10%, most preferably ± 5%.

As used herein, the term "analyte" refers to a molecular species determined according to the invention. Said molecular species can be a metabolite itself which is found in a sample.

Moreover, the analyte may also be a molecular species which is derived from said metabolite. In such a case, the actual metabolite will be chemically modified before and/or during the determination process and, as a result of said modification, a chemically different molecular species, i.e. an analyte, will be the determined molecular species. It is to be understood that in such a case, the analyte represents the actual metabolite. Preferably, the amount of analyte is proportional to the amount of metabolite(s) the analyte is derived from.

Moreover, an analyte according to the present invention is not necessarily corresponding to one molecular species. Rather, the analyte may comprise stereoisomers or enantiomers of a compound. Further, an analyte can also represent the sum of isomers of a biological class of isomeric molecules, or of a subgroup thereof. Said isomers shall exhibit identical analytical characteristics in some cases and are, therefore, not distinguishable or distinguished by various analytical methods including those applied in the accompanying examples described below. However, preferably, the isomers will share at least identical sum formula and, thus, in the case of, e.g., lipids, an identical number of carbon atoms and identical numbers of double bonds in the sum of the fatty acid and other long-chain aliphatic moieties, e.g., sphingobase moieties. Depending on the method for determining at least one characteristic feature of an analyte chosen, an analyte may also represent a sum of a biological class of compounds, such as total sphingomyelin, or a subgroup thereof, e.g. d18:1 -sphingomyelins; an analyte can also comprise isobars, represented by the same nominal mass but different sum formula; and/or an analyte may comprise molecules with a similar sub-structure, in particular in case the method for determining only detects said sub-structure or in case compounds are modified during processing, which may, e.g. include reactions such as losses of chemical groups or a molecular rearrangement occurring when chemical modification is carried out.

The term "analyte of interest", as used herein, relates to an analyte, the concentration or amount of which shall be determined in a biological sample. The term "reference analyte", as used herein, relates to an analyte sufficiently similar to the analyte of interest such that the characteristic feature of the analyte of interest and the characteristic feature of the reference analyte have essentially the same proportionality to the concentration or amount of the respective analyte. Thus, preferably, the characteristic feature of the reference analyte is proportional to the concentration or amount of the reference analyte to the same extent as the analyte of interest is proportional to the concentration or amount of the analyte of interest.

Preferably, the reference analyte and the analyte of interest are members of the same chemical class, e.g. are triglycerides, sphingomyelins, or the like. More preferably, the reference analyte and the analyte of interest are isomers. Most preferably, the reference analyte and the analyte of interest are identical chemical compounds. According to the present invention, the concentration or amount of the reference analyte in said matrix calibration sample was predetermined; i.e. preferably, the concentration or amount of the reference analyte in said matrix calibration sample is known from an earlier quantification. Preferably, the concentration or amount of the reference analyte in said matrix calibration sample is known at the time the method of the present invention is performed. As will be understood, a matrix calibration sample with a known concentration of reference analyte(s) can be used for determining the

concentration of analyte(s) in a further sample, e.g. a further matrix calibration sample, thus providing a further matrix calibration sample with a predetermined concentration of analyte(s). The term "metabolite", as used herein, relates to at least one molecule of a specific metabolite up to a plurality of molecules of the said specific metabolite. It is to be understood further that a group of metabolites means a plurality of chemically different molecules wherein for each metabolite at least one molecule up to a plurality of molecules may be present. A metabolite in accordance with the present invention encompasses all classes of organic or inorganic chemical compounds including those being comprised by biological material such as animals or plants. Preferably, a metabolite has a molecular weight of from 25 Da (Dalton) to 300,000 Da, more preferably of from 30 Da to 30,000 Da, most preferably of from 50 Da to 1500 Da.

Preferably a metabolite has a molecular weight of less than 30,000 Da, less than 20,000 Da, less than 15,000 Da, less than 10,000 Da, less than 8,000 Da, less than 7,000 Da, less than 6,000 Da, less than 5,000 Da, less than 4,000 Da, less than 3,000 Da, less than 2,000 Da, less than 1 ,000 Da, less than 500 Da, less than 300 Da, less than 200 Da, or less than 100 Da. Preferably, a metabolite has, however, a molecular weight of at least 50 Da.

Preferably, the metabolite is a biological macromolecule, e.g. preferably, DNA, RNA, protein, or a fragment thereof, e.g., preferably a fragment produced by processing of sample material. More preferably, in case a plurality of metabolites is envisaged, said plurality of metabolites is representing a metabolome, i.e. the collection of metabolites being comprised by an organism, an organ, a tissue, a body fluid, a cell or a part of a cell at a specific time and under specific conditions.

More preferably, the metabolite in accordance with the present invention is a small molecule compound, such as a substrate for an enzyme of a metabolic pathway, an intermediate of such a pathway or a product obtained by a metabolic pathway. Metabolic pathways are well known in the art and may vary between species. Preferably, said pathways include at least citric acid cycle, respiratory chain, photo respiratory chain, glycolysis (Embden-Meyerhof-Parnas (EMP) pathway), gluconeogenesis, hexose monophosphate pathway, starch metabolism, oxidative and non oxidative pentose phosphate pathway (Calvin-Benson (CB) cycle, glyoxylate metabolism, production and β-oxidation of fatty acids, urea cycle, amino acid biosynthesis pathways, protein degradation pathways such as proteasomal degradation, amino acid degrading pathways, biosynthesis or degradation of lipids, polyketides (including e.g. flavonoids and isoflavonoids), isoprenoids (including e.g. terpenes, sterols, steroids, carotenoids, xanthophylls),

carbohydrates, phenylpropanoids and derivatives, alcaloids, benzenoids, indoles, indole-sulfur compounds, porphyrines, anthocyans, hormones, vitamins, cofactors such as prosthetic groups or electron carriers, lignin, glucosinolates, purines, pyrimidines, nucleosides, nucleotides and related molecules such as tRNAs, microRNAs (miRNA) or mRNAs. Accordingly, small molecule compound metabolites are preferably composed of the following classes of compounds:

alcohols, alkanes, alkenes, alkines, aromatic compounds, ketones, aldehydes, carboxylic acids, esters, amines, imines, amides, cyanides, amino acids, peptides, thiols, thioesters, phosphate esters, sulfate esters, thioethers, sulfoxides, ethers, or combinations or derivatives of the aforementioned compounds. The small molecules among the metabolites may be primary metabolites which are required for normal cellular function, organ function or animal or plant growth, development or health. Moreover, small molecule metabolites further comprise secondary metabolites having essential ecological function, e.g. metabolites which allow an organism to adapt to its environment. Furthermore, metabolites are not limited to said primary and secondary metabolites and further encompass artificial small molecule compounds. Said artificial small molecule compounds are derived from exogenously provided small molecules which are administered or taken up by an organism but are not primary or secondary metabolites as defined above, including, preferably, drugs, herbicides, fungicides, and insecticides. Moreover, artificial small molecule compounds may be metabolic products of compounds taken up, and preferably metabolized, by metabolic pathways of an organism. Moreover, small molecule compounds preferably include compounds produced by organisms living in, on or in close vicinity to an organism, more preferably by an infectious agent as specified elsewhere herein, by a parasitic and/or by a symbiotic organism.

The term "biological sample", as used herein, relates to a sample comprising a biological material, wherein the term "biological material", preferably, includes any substance or mixture of substances produced by a cell, preferably including substances and mixtures of substances produced by such biological material. Preferably, the biological material comprises a multitude of metabolites of a cell. As used herein, the term "multitude of metabolites" preferably relates to at least 50, more preferably at least 100, even more preferably at least 200, most preferably at least 300 metabolites of a cell. Preferably, the biological sample is a sample of a material comprising a non-defined mixture of compounds, such as a cell culture medium comprising serum, a spent cell culture medium, a bodily fluid of an organism, tissue of an organism, and the like. Thus, preferably, the biological sample is a cell culture sample from archaebacterial, bacterial, and/or eukaryotic cells, wherein said cell culture sample preferably comprises cells and/or spent culture medium; preferably, in such case, the biological sample is a sample of cultured bacterial, fungal, plant, such as a dicot or monocot plant, more preferably a crop plant, algae, human or animal cells and/or spent medium of said cells. Most preferably, the biological sample is a sample of and/or spent culture medium from E.coli cells, Bacillus cells, preferably Bacillus acidopullulyticus cells, Bacillus amyloliquefaciens cells, Bacillus lentus cells, Bacillus licheniformis cells, Bacillus subtilis cells, Streptomyces cells, Paenibacillus cells, Basfia succiniciproducens cells, Corynebacterium glutamicum cells, Lactobacillus cells, Schizophyllum commune cells, Aspergillus cells, preferably Aspergillus niger cells, Aspergillus oryzae cells, Chrysosporium cells, preferablyChrysosporium lucknowense cells, Myceliophthora cells, preferably Myceliophthora thermophile cells, Penicillium cells, preferably Penicillium

chrysogenum cells, Penicillium funiculosum cells, Rhizomucor cells, preferably Rhizomucor miehei cells, Trichoderma cells, preferably Trichoderma harzianum cells, Trichoderma longibrachiatum cells, Trichoderma reesei cells, , yeast cells, preferably Candida cells such as Candida rugose cells, Candida lipolytica cells, Candida Antarctica cells, Kluyveromyces cells such as Kluyveromyces lactis cells, Kluyveromyces fragilis cells, Pichia cells such as Pichia pastoris cells, Saccharomyces cells such as Saccharomyces cerevisiae cells,

Schizosaccharomyces pombe cells, CHO cells (Chinese hamster ovary cells), liver cells, hepatocytes, kidney cells, kidney cancer cells, pancreatic cells, pancreatic cancer cells, cardiac cells, cardiac cancer cells, endothelial cells, endothelial cancer cells, fibroblasts, lung cells, lung cancer cells, bladder cells, bladder cancer cells, breast cells, breast cancer cells, colon cells, colon cancer cells, ovarian cells, ovarian cancer cells, duodenum cells, duodenum cancer cells, bile duct cells, bilde duct cancer cells, stem cells or skin cells.

As used herein, the term "plant" relates to a whole plant, a plant part, a plant organ, a plant tissue, or a plant cell. Thus, the term includes, preferably, seeds, shoots, stems, leaves, roots (including tubers), and flowers. Preferably, the term "plant" relates to a member of the clade Archaeplastida. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, preferably Tracheophyta, more preferably Spermatophytina, most preferably monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs; in particular selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana,

Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arabidopsis thaliana, Arachis spp,

Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp.,

Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brachypodium distachyon, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp.,

Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis,

Momordica spp., Morus nigra, Musa spp., Nicotiana spp. (e.g. Nicotiana tabacum), Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp. (e.g. Saccharum officinarum), Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp.,

Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others. Preferably the plant cell, plant or plant part or plant seed is corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), barley (Hordeum vulgare), cotton (Gossypium hirsutum), sunflower (Helianthus annuus), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), soyabean (Glycine max), rape (Brassica napus), sugarcane (Saccharum officinarum), sugarbeet (Beta vulgaris). More preferably the plant cell, plant or plant part is a rice cell, rice plant, rice plant part, or rice seed.

Preferably, the sample is a sample from a multicellular organism. More preferably, the sample comprises a bodily fluid of an organism and/or a tissue of an organism. Preferably, the biological sample is a sample of an animal, preferably a vertebrate, more preferably a mammal. More preferably, the biological sample is a sample of an egg, a, preferably non-human, embryo, or a complete non-human organism, e.g. an insect, a nematode, or a laboratory animal.

Preferably, the biological sample is or comprises a sample of a body fluid, a sample from a tissue or an organ, or a sample of wash/rinse fluid or a swab or smear obtained from an outer or inner body surface. Preferably, samples of stool, urine, saliva, sputum, tears, cerebrospinal fluid, blood, serum, plasma, lymph or lacrimal fluid are encompassed as biological samples by the method of the present invention. In particular in multicellular organisms, biological samples can be obtained by use of brushes, (cotton) swabs, spatula, rinse/wash fluids, punch biopsy devices, puncture of cavities with needles or lancets, or by surgical instrumentation. However, biological samples obtained by well known techniques including, in an embodiment, scrapes, swabs or biopsies are also included as samples of the present invention. Cell-free fluids may be obtained from the body fluids or the tissues or organs by lysing techniques such as

homogenization and/or by separating techniques such as filtration or centrifugation. It is to be understood that a sample may be further processed in order to carry out the method of the present invention. Particularly, cells may be removed from the sample by methods and means known in the art. More preferably, the biological sample is a sample of a body fluid, preferably a blood, plasma, lymph or serum sample. Also more preferably, the biological sample is a tissue sample, preferably a sample of liver tissue, heart tissue, prostate tissue, pancreas tissue, brain tissue, kidney tissue, adipose tissue, gut, skeleton tissue, lung tissue, bladder, breast tissue, cecum and/or skin tissue, such as dermal layer, comprising the epidermis and / or corium and / or subcutis. Also preferably, the biological sample is a sample of an algae or plant, preferably of a monocotyledonous or dicotyledonous plant. More preferably, said biological sample is a tissue sample, preferably leaf tissue, root tissue, shoot tissue, stem tissue, reproductive tissue (such as flower tissue or pollen) and/or seed tissue and/or liquid comprising exudate thereof and/or volatile compounds released thereof.

In the method of the present invention, at least one characteristic feature of an analyte is determined. "Characteristic features", in accordance with the present invention, are features which characterize the physical and/or chemical properties including biochemical properties of an analyte. Such properties include, e.g., molecular weight, viscosity, density, electrical charge, spin, optical activity, colour, fluorescence, chemiluminescence, elementary composition, chemical structure, capability to react with other compounds, capability to elicit a response in a biological read out system (e.g., induction of a reporter gene) and the like. Values for said properties may serve as characteristic features and can be determined by techniques well known in the art. Moreover, the characteristic feature may be any feature which is derived from the values of the physical and/or chemical properties of an analyte by standard operations, e.g., mathematical calculations such as multiplication, division or logarithmic calculus. Preferably, the at least one characteristic feature allows the determination and/or chemical identification of the said at least one analyte and its amount; thus, preferably, the characteristic feature is a quantitative feature. Accordingly, the characteristic value, preferably, also comprises information relating to the abundance of the analyte from which the characteristic value is derived. For example, a characteristic value of an analyte may be a peak in a mass spectrum. Such a peak contains characteristic information of the analyte, i.e. the m/z information, as well as an intensity value being related to the abundance of the said analyte (i.e. its amount) in the sample. Thus, according to the present invention, the analyte is determined quantitatively, i.e. preferably, determination is measuring an absolute amount or a concentration of an analyte. Preferably, the method of the present invention further comprises adding at least one analyte at a predetermined concentration to the matrix calibration sample and/or to the sample of interest. Preferably, the analyte is an analyte not detectable in the matrix calibration sample or a non- biological analyte, the term "non-biological" analyte referring to an analyte not produced by a cell or to an isotopologue produced by a cell at less than 10%, preferably at less than 5% of the major isotopologue. More preferably, a non-biological analyte is a compound not produced by a cell, e.g. an artificial, e.g. synthetic, compound or an isotopologue not produced by a cell.

Preferably, the non-biological analyte is used as an internal standard. More preferably, the non- biological analyte is exclusively used as internal standard.

A sample is, preferably, pre-treated before it is used in the method of the present invention, wherein, as used herein, the term "sample" includes the biological sample and the matrix calibration sample. Preferably, in the method of the present invention, the biological sample and the matrix calibration sample are pre-treated and processed in an identical manner, but separately. An internal standard may be added, before, upon, or after pre-treatment, preferably is added before pretreatment. Preferably, in said internal standard up to four, more preferably up to two standard compounds, most preferably one standard compound are/is comprised. As described in more detail elsewhere herein, the pre-treatment may include treatments required to release or separate the analyte(s) or to remove excessive material or waste. Suitable techniques comprise centrifugation, extraction, fractioning, ultrafiltration, separation (e.g. by binding to paramagnetic beads and applying magnetic force), protein precipitation followed by filtration and purification and/or enrichment of compounds. Moreover, other pre-treatments are preferably carried out in order to provide the analyte(s) in a form or concentration suitable for compound analysis. For example, if gas-chromatography coupled mass spectrometry is used in the method of the present invention, it may be required to derivatize the analyte(s) prior to the said gas chromatography. Suitable and necessary pre-treatments depend on the means used for carrying out the determining at least one characteristic feature and are well known to the person skilled in the art. Pre-treated samples as described before are also comprised by the term "sample" as used in accordance with the present invention.

Preferably, the sample, in particular the biological sample, is a dried sample. Methods for drying samples are known in the art. More preferably, the sample is freeze-dried. Thus, preferably, the biological sample is a sample of freeze-dried fluid or freeze dried tissue material; preferably, the biological sample is a sample of freeze-dried plant material, preferably, freeze-dried leaf material, freeze-dried root material, freeze-dried shoot material, freeze-dried stem material and/or freeze-dried reproductive material, more preferably freeze-dried flower material, freeze- dried tassel, freeze-dried pollen material and/or freeze-dried seed material. The biological sample may also be a sample of homogenized biological material, e.g. ground tissue, preferably ground freeze-dried tissue, lysed cells, and the like. Moreover, the biological sample may also be a fraction from one of the aforesaid biological materials produced by applying a separation method known to the skilled person to said biological material, e.g. sieving, sorting,

centrifugation or extraction. Thus, preferably, the biological sample is an extract of a biological material produced by precipitation, solvent extraction, or the like. Preferably, the pre-treatment of the sample allows for a subsequent separation of analyte(s), in particular of the small molecule analyte(s) as referred to above, comprised by a sample.

Molecules of interest, in particular the analyte(s) as referred to above may be extracted in an extraction step which comprises mixing of the sample with a suitable extraction solvent. The extraction solvent shall preferably be capable of precipitating macromolecules, in particular proteins and/or polynucleotides, in a sample, thereby facilitating the, preferably, centrifugation- based, removal of macromolecule contaminants which otherwise would interfere with the subsequent analysis of the analyte(s) as referred above. Preferably, at least the small molecule analyte(s) as referred to herein are soluble in the extraction solvent. Preferably, the extraction solvent mixture is a phase separating, preferably a two-phase solvent mixture. More preferably, the extraction solvent mixture is a non-phase separating, i.e., a one-phase solvent mixture.

The term "sample source" as used herein, relates to a composition of matter or an organism a sample according to the present invention originates from, i.e. preferably, is withdrawn from. Preferably, a sample source is an archaebacterial, bacterial, or an eukaryotic cell. More preferably, a sample source is a cultured cell, preferably a cultured bacterial, plant, or mammalian cell, or a spent medium thereof. More preferably, the sample source is a subject, preferably a plant or animal subject, more preferably a mammalian subject, most preferably a human subject.

Preferably, the determination of the amount of an analyte as referred to herein is achieved by a compound separation step as specified above and a subsequent mass spectrometry step. Thus, determining as used in the method of the present invention, preferably, includes using a compound separation step prior to the analysis step. Preferably, said compound separation step yields a time resolved separation of analyte(s) comprised by the sample. Suitable techniques for separation to be used preferably in accordance with the present invention, therefore, include all chromatographic and/or electrophoretic separation techniques such as liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra performance liquid

chromatography (UPLC), gas chromatography (GC), thin layer chromatography, size exclusion, affinity chromatography and capillary electrophoresis (CE). Moreover, determination via ion mobility spectrometry, preferably in combination with electrospray/ MS/MS is envisaged. These techniques are well known in the art and can be applied by the person skilled in the art without further ado. Most preferably, GC, LC and/or HPLC are chromatographic techniques to be envisaged by the method of the present invention. Suitable devices for such determination of analyte(s) are well known in the art.

Preferably, mass spectrometry is used, in particular gas chromatography mass spectrometry (GC-MS), liquid chromatography mass spectrometry (LC-MS), direct infusion mass

spectrometry or Fourier transform ion-cyclotrone-resonance mass spectrometry (FT-ICR-MS), capillary electrophoresis mass spectrometry (CE-MS), high-performance liquid chromatography coupled mass spectrometry (HPLC-MS) ultra performance liquid chromatography (UPLC-MS), quadrupole mass spectrometry, ion trap mass spectrometry, including orbitrap mass analyzers, any sequentially coupled mass spectrometry, such as MS-MS or MS-MS-MS, inductively coupled plasma mass spectrometry (ICP-MS), pyrolysis mass spectrometry (Py-MS), ion mobility mass spectrometry or time of flight mass spectrometry (TOF). More preferably, LC-MS, in particular LC-MS/MS are used as described in detail below. The techniques described above are disclosed in, e.g., Nissen 1995, Journal of Chromatography A, 703: 37-57, US 4,540,884 or US 5,397,894, the disclosure content of which is hereby incorporated by reference.

As an alternative or in addition to mass spectrometry techniques, the following techniques may be used for compound determination: nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), Fourier transform infrared analysis (FT-IR), ultraviolet (UV) spectroscopy, refraction index (Rl), fluorescent detection, radiochemical detection, electrochemical detection, light scattering (LS), dispersive Raman spectroscopy, flow injection analysis (FIA), matrix - assisted laser desorption / ionization (MALDI), laser ablation electrospray ionization (LAESI), desorption electrospray ionization (DESI), surface enhanced laser desorption/ionization

(SELDI), charged aerosol detector (CAD), evaporative light scattering detector (ELSD), rapid evaporation ionization, near infrared spectroscopy (NIR), electron capture detector (ECD), nitrogen-phosphorus detector (NPD), thermal conductivity detector (TCD), surface plasmon resonance spectroscopy (SPR) or flame ionization detection (FID). In a preferred embodiment, an analyte may also be determined by its binding to a specific ligand, e.g. to an aptamer, an antibody, and the like. These techniques are well known to the person skilled in the art and can be applied without further ado.

More preferably, said mass spectrometry is coupled with liquid chromatography (LC), such as high performance liquid chromatography (HPLC) MS, in particular HPLC-MS/MS. Liquid chromatography as used herein refers to all techniques which allow for separation of compounds (i.e. metabolites) in liquid or supercritical phase. Liquid chromatography is characterized in that compounds in a mobile phase are passed through the stationary phase. When compounds pass through the stationary phase at different rates they become separated in time since each individual compound has its specific retention time (i.e. the time which is required by the compound to pass through the system). Liquid chromatography as used herein also includes HPLC. Devices for liquid chromatography are commercially available, e.g. from Agilent Technologies, USA. For example, HPLC can be carried out with commercially available reversed phase separation columns with e.g. C8, C18 or C30 stationary phases, normal phase separation or hydrophilic interaction. The person skilled in the art is capable to select suitable solvents for the HPLC or any other chromatography method as described herein. The eluate that emerges from the chromatography device shall comprise the analyte(s) as referred to above.

A suitable solvent for elution for liquid chromatography can be determined by the skilled person. In an embodiment, the solvents for gradient elution in the HPLC separation consist of a polar solvent and a non-polar solvent. Preferably, the polar solvent is a mixture of water and a water miscible solvent with an acid modifier. Examples of suitable organic solvents which are completely miscible with water include the C1 -C3-alkohols, tetrahydrofurane, dioxane, C3-C4- ketones such as acetone and acetonitril and mixtures thereof, with methanol being particularly preferred. Preferably, the non-polar solvent is a mixture of at least one of the above mentioned organic solvents together with hydrophobic solvents from the groups consisting of

dichloromethane (DCM), chloroform, tertiary butyl methyl ether (tBME or MTBE), ethyl ethanoate, and isooctane. Examples of acidic modifiers are formic acid or acidic acid.

Gas chromatography, which may be also applied in accordance with the present invention, in principle, operates comparable to liquid chromatography. However, rather than having the compounds (i.e. metabolites) in a liquid mobile phase which is passed through the stationary phase, the compounds will be present in a gaseous volume. The compounds pass the column which may contain solid support materials as stationary phase or the walls of which may serve as or are coated with the stationary phase. Again, each compound has a specific time that is required for passing through the column. Moreover, in the case of gas chromatography, but also in liquid chromatography, in particular in reverse-phase liquid chromatography, it is preferably envisaged that the compounds are derivatized prior to chromatography. Suitable techniques for derivatization are well known in the art. Preferably, in gas chromatography, derivatization in accordance with the present invention relates to methoxymation and trimethylsilylation of, preferably, polar compounds and transmethylation, methoxymation and trimethylsilylation of, preferably, non-polar (i.e. lipophilic) compounds. More preferably, derivatization comprises, even more preferably consists of, contacting metabolites in a, preferably deproteinized, fraction with a reagent introducing hydrophobic side chains. Preferably, in LC, derivatization in accordance with the present invention relates to introducing hydrophobic side chains, preferably using a reagent derivatizing phenolic hydroxyl groups and amino groups, preferably primary and secondary amino groups. More preferably, said reagent introducing hydrophobic side chains is 5- (dimethylamino)naphthalene-l -sulfonyl chloride (dansylchloride, CAS Registry No: 605-65-2).

For mass spectrometry, the analytes in the sample are ionized in order to generate charged molecules or molecule fragments. Afterwards, the mass-to-charge of the ionized analyte or fragments thereof is measured. Thus, the mass spectrometry step preferably comprises an ionization step in which the analytes to be determined are ionized. Of course, other compounds present in the sample/eluate are ionized as well. Ionization of the analytes can be carried out by any method deemed appropriate, in particular by electron impact ionization, fast atom bombardment, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix assisted laser desorption ionization (MALDI). As set forth above, the mass spectrometry step is carried out after the separation step, in particular the chromatography step. In an embodiment, the eluate that emerges from the chromatography column (e.g. the LC or HPLC column) may be pre-treated prior to subjecting it to the mass spectrometry step.

For example, a blood, serum, or plasma sample (in particular a plasma sample) can be analyzed. The sample may be a fresh sample or a frozen sample. If frozen, the sample may be thawed at suitable temperature for a suitable time. An aliquot of the sample is then transferred to a centrifuge tube and diluted with a suitable diluent. Afterwards an extraction is done (e.g. for 5 minutes using a vortexer). Afterwards, the sample may be centrifuged (e.g. at about 20.000 g). An aliquot of the supernatant (e.g. 200 μΙ) can be used for the quantification of the analyte(s).

The method of the present invention shall be, preferably, assisted by automation. For example, sample processing or pre-treatment can be automated by robotics. Data processing and comparison is, preferably, assisted by suitable computer programs and databases. Automation as described herein before allows using the method of the present invention in high-throughput approaches.

The term "matrix", as used herein, encompasses all constituents of a sample which are not analytes. As will be understood by the skilled person, a biological sample may comprise hundreds or even thousands of species of chemical molecules, of which only a few or even only one may be of interest. Thus, in such case, the compounds which are not compounds of interest will collectively be referred to as matrix. Such further constituents of a sample may have a pronounced impact on the parameters determinable for the analytes of interest, in particular on quantitative parameters, a phenomenon referred to as "matrix effect".

Accordingly, the term "matrix calibration sample", as used herein, relates to a calibration sample comprising the analyte(s) of interest, having essentially the same, preferably having the same, matrix as the biological sample, and comprising the analyte(s) of interest in the concentration present in the biological material used for obtaining the matrix calibration sample. Thus, preferably, the matrix calibration sample, preferably, does not comprise an added analyte, wherein an "added analyte" is an analyte artificially included into a calibration sample. Thus, e.g. in case the biological sample is a serum sample, the matrix calibration sample, preferably, is a blood, plasma, or serum sample, more preferably is a serum sample. Thus, preferably, the matrix calibration sample comprises, preferably consists of, at least one biological sample material. More preferably, the matrix calibration sample comprises, preferably consists of, the same biological sample material as the biological sample.

Preferably, the matrix calibration sample is a sample comprising sample material from at least one sample source. More preferably, the matrix calibration sample comprises sample material from at least five, preferably at least 20, more preferably at least 50 different sample sources, preferably subjects. Preferably, the sample material from said sample sources is mixed intensively, e.g. by stirring or vortexing in the case of liquid samples, by homogenizing in the case of tissue samples, or by mixing in the case of, e.g. seed samples. As used herein, the term " calibrated result" relates to a result of a determination of an analyte being or enabling a quantification of at least one metabolite or group of metabolites; thus, preferably, the calibrated result of the present invention provides sufficient information to allow a metabolite or group of metabolites to be quantified in a biological sample, preferably by standard mathematical operations. Preferably, the calibrated result is a result of a determination of an analyte enabling quantification of a metabolite; thus, preferably, the calibrated result of the present invention provides sufficient information to allow a metabolite to be quantitated in a biological sample, preferably by standard mathematical operations. Preferably, the calibrated result is a value of a quantitative parameter of an analyte, e.g. a concentration, an amount, or a parameter proportional to a concentration and/or an amount of said analyte. Preferably, the calibrated result is a concentration value or an amount. More preferably, the calibrated result is an absolute concentration or an absolute amount of said analyte of interest in said biological sample. Preferably, in case the analyte is identical with a metabolite, the calibrated result may be a concentration value or an amount of a metabolite in a biological sample. Thus, preferably, the method for providing a calibrated result of the present invention is included in a method for quantifying a metabolite in a biological sample comprising the steps of the method for providing a calibrated result, and the further step of providing and/or calculating a quantitative value of said metabolite in a biological sample.

According to the method of the present invention, the calibrated result of the determination of at least one analyte is based on the results of the steps of determining at least one characteristic feature of said at least one analyte of interest and of determining at least one characteristic feature of at least one reference analyte in a matrix calibration sample. Preferably, as used herein, the term "a calibrated result is based on" relates to a calibrated result being calculated by comparing the value of a characteristic feature of an analyte to a value of a characteristic feature of a reference analyte, and calculating the calibrated result by putting the result of said comparison into direct proportionality to the predetermined concentration of the reference analyte in the matrix calibration sample. The skilled person knows that the comparing the value of a characteristic feature of an analyte to a value of a characteristic feature of a reference analyte can be achieved, preferably, via a serial dilution of the matrix calibration sample ("multipoint calibration"), or, more preferably, by directly comparing the value of a characteristic feature of an analyte to the value of a characteristic feature of a reference analyte ("one-point calibration"). Thus, preferably, providing a calculated result comprises a calculation including the formula: calibrated result = (value of the characteristic feature of the analyte) / (value of the characteristic feature of the reference analyte) ^* (concentration or amount of reference analyte in the matrix calibration sample). Preferably, in the method of the present invention, the concentration(s) of the reference analyte(s) in the matrix calibration sample is(are) the only concentration(s) which have been predetermined; i.e., preferably, the predetermined concentration(s) of the reference analyte(s) in the matrix calibration sample is(are) the only quantitative parameter(s) relating to a concentration or an amount used in the method of the present invention. Thus, preferably, the method of the present invention avoids correcting a result of a determination of an analyte by a correction factor. According to the method of the present invention, preferably calibrated results for at least two, more preferably for at least five, even more preferably at least ten, still more preferably at least 50, still more preferably at least 100, most preferably at least 200 analytes of interest are provided. Preferably, such a method comprises providing a matrix calibration sample, wherein the concentration of said at least two, more preferably for at least five, even more preferably at least ten, still more preferably at least 50, still more preferably at least 100, most preferably at least 200 reference analytes in said matrix calibration sample was predetermined. As will be understood from the above, providing calibrated results for at least n analytes does not necessarily mean that concentrations of n reference analytes have to be provided, since a reference analyte may be used for calibrating the concentration or amount of more than one analyte of interest. More preferably, however, a specific reference analyte is provided for each analyte of interest. As also indicated above, an analyte may represent more than one metabolite. Thus, preferably, a calibrated result for at least one analyte of interest is a result representing at least two structurally non-identical metabolites.

Preferably, the at least one analyte of interest comprises analytes selected from at least two, preferably at least five, more preferably at least ten, most preferably all of the following classes of compounds: amino acids, carbohydrates, organic acids, hormones, biogenic amines, acylcarnitines, alcohols, sphingolipids, ceramides, sterols and esters thereof, triacylglycerides, (lyso)phosphatidylcholines, (lyso)phosphatidylethanolamines, fatty acids, carotenoids, hormones, eicosanoids, nucleobases and related, phosphorylated compounds, quinones, quinols, tocopherols, vitamins and secondary metabolites. More preferably, the sample is a plant sample and the at least one analyte comprises analytes selected from at least five, preferably at least ten, more preferably at least 15, most preferably all of the following classes of compounds: amino acids, carbohydrates, organic acids, inorganic acids, hormones, biogenic amines, lipids, acylcarnitines, alcohols, sphingolipids, ceramides, sterols and esters thereof, triacylglycerides, (lyso)phosphatidylcholines, (lyso)phosphatidylethanolamines, fatty acids, eicosanoids, nucleobases and related, phosphorylated compounds, quinones, quinols, tocopherols, alkaloids, glycosides, glucosinolates, carotenoids, terpenes, tannins,

phenylpropanoids, phytohormones, and polyketides.

The calibration according to the method of the present invention may be any calibration method deemed appropriate by the skilled person, including establishing a calibration curve. Preferably, a one-point calibration is used. The skilled person is aware of means and methods of ensuring that a calibration is applied in the linear range of calibration.

Advantageously, it was found in the work underlying the present invention that natural material may be used as a reference sample for calibrating measurement values obtained from a sample having similar or identical composition. In the reference sample, compounds naturally present therein may be quantified by known methods, and the sample used as a calibration sample. Advantageously, such a reference sample compensates in particular the matrix effect which might have an adverse impact on measurement results. Moreover, neither artificial marker compounds nor preparation of calibration solutions, which by itself is error-prone, are required.

The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis. The present invention further relates to a kit for providing a calibrated result of a determination of at least one analyte of interest in a determination method comprising

(i) a matrix calibration sample having a predetermined concentration of at least one reference analyte,

(ii) a data carrier comprising a data collection, said data collection comprising

concentration data and identification data for said at least one reference analyte in said determination method. The term "kit", as used herein, refers to a collection of the aforementioned compounds, means or reagents of the present invention which may or may not be packaged together. The components of the kit may be comprised by separate housings (i.e. as a kit of separate parts) or may be provided in a single housing. Moreover, it is to be understood that the kit of the present invention, preferably, is to be used for practicing the methods referred to herein above. It is, preferably, envisaged that components, in an embodiment all components, are provided in a ready-to-use manner for practicing the methods referred to above. Preferably, all or some of said components are provided in dried, such as in lyophilized form, wherein the component is reconstituted using a liquid such as an aqueous buffered solution. Preferably, all or some of said components are provided in, preferably concentrated, more preferably ready-to-use, liquid form, wherein the concentrated component is diluted using a liquid such as an aqueous buffered solution. Further, the kit, preferably, contains instructions for carrying out said methods and, if applicable, said reconstitution of dried reagents. The instructions can be provided by a user's manual in paper- or electronic form. In addition, the manual may comprise instructions for interpreting the results obtained when carrying out the aforementioned methods using the kit of the present invention, and/or information on the value of the predetermined concentration of at least one analyte in the matrix calibration sample. Preferably, the kit further comprises at least one pre-treatment agent and/or an internal standard. Preferably, the matrix calibration sample has predetermined concentrations of at least ten, preferably at least 50, more preferably at least 100, most preferably at least 200 reference analytes. Preferably, the kit is a kit for use in MS, preferably for use in MS/MS.

As used herein, the term "data carrier comprising a data collection " is used in a wide sense and includes any data carrier physically including the specified data. Preferably, the data carrier comprises concentration data and identification data for at least one reference analyte. As used herein, "identification data" are data allowing a metabolite and/or an analyte to be identified; accordingly, identification data, preferably, are a name, preferably a chemical or lUPAC name of the compound, a chemical formula and/or the like. Accordingly, the data carrier may be a printed information indicating identification data and an indication of the concentration of the identified compound in the matrix calibration sample. More preferably, the data carrier is a machine-readable data carrier, like a CD or DVD, a portable memory device, e.g. comprising a flash memory unit, or the like. Preferably, the data carrier comprises concentration data and identification data for at least ten, preferably at least 50, preferably at least 100, more preferably at least 200 reference analytes. The present invention also relates to a data collection, preferably comprised on a storage medium, said data collection comprising identification data and concentration data for at least one reference analyte comprised in a matrix calibration sample.

Moreover, the present invention relates to a device for determining at least one analyte of interest in a biological sample comprising

an analysis unit adapted for determining at least one characteristic feature of said at least one analyte of interest, and

an evaluation unit having a memory unit comprising identification data and data on the predetermined concentration for at least one reference analyte comprised in a matrix calibration sample.

The term "device", as used herein, relates to a system of means comprising at least the aforementioned means operatively linked to each other as to allow the result of the

determination to be obtained. Preferred means for determining at least one characteristic feature are disclosed above in connection with the methods of the invention. How to link the means in an operating manner will depend on the type of means included into the device. In an embodiment, the means are comprised by a single device.

In an embodiment, the analysis unit comprises a receptacle for a sample. The receptacle may directly contact the sample, or may be a receptacle for a further means receiving the sample, wherein the further means may be e.g. a multi-well plate, to which a sample or a multiplicity of samples may be applied. Moreover, the analysis unit, preferably, comprises a matrix calibration sample, e.g. in a reservoir connected to a dosing means, e.g. a tubing connected to a pump. In an embodiment, the result of the determination is obtained by performing a detection measurement on an appropriate detection unit. Thus, in an embodiment, the analyzing unit of the device of the present invention further comprises a detection unit for at least one characteristic feature of an analyte. Means suitable as a detection unit according to the present invention are known to the skilled person and include, e.g. MS devices. The device of the present invention further comprises a memory unit comprising identification data and data on the predetermined concentration for at least one reference analyte.

In an embodiment, the device of the present invention further comprises an evaluation unit or is part of an analytic system, said analytic system further comprising an evaluation device. As will be understood by the skilled person, the evaluation unit may be comprised in the same housing as the device of the invention as an evaluation unit, or may be a separate device, i.e. an evaluation device. In an embodiment, the evaluation unit or device comprises a microprocessor programmed to receive output data from an output unit of the analyzing unit of the present invention and to perform logical operations providing an evaluation of said output data.

Evaluation of output data may comprise, e.g., correcting data for values measured in one or more control detection reaction, statistical calculations, e.g. calculating means of two or more parallel detection reactions, providing calculated concentration values, e.g. by applying dilution factors, comparing output data to reference values, compiling data in a list, and the like. In an embodiment, the device of the present invention further comprises a data output unit, connected to the detection unit and/or to the evaluation unit or device. The data output unit, in an embodiment, is adapted to output data obtained by the detection unit or generated by the evaluation unit or device. Suitable data output units are known to the skilled person and include simple output units such as an indicator lamp. An output unit may, however, also be an interface to an output device, wherein said interface may be any kind of means of transferring data, including, e.g. cable connections like USB, wireless connections like wireless LAN, bluetooth, and the like, or indirect connections such as data transfer by instant messaging, email, or the like.

In an embodiment, where means for automatically determining at least one characteristic feature are applied, the data obtained by said automatically operating means can be processed by, e.g., a computer program in order to establish or aid in establishing a result. The person skilled in the art will realize how to link the means without further inventive skills. Typical devices are those which can be applied without the particular knowledge of a specialized technician, e.g., electronic devices which merely require loading with a sample. The results may be given as output of raw data, preferably as amounts. Moreover, the present invention relates to a use of a matrix calibration sample having a predetermined concentration of at least one reference analyte for calibrating the determination of an analyte of interest in a sample.

Moreover, the present invention relates to a method for providing a matrix calibration sample, said matrix calibration sample having a predetermined concentration of at least one reference analyte, comprising

a) providing a reference calibration sample essentially consisting of the same

biological sample material as said matrix calibration sample, wherein the concentration of said at least one reference analyte in said reference calibration sample was predetermined,

b) determining at least one characteristic feature of said at least one reference

analyte in said matrix calibration sample,

c) determining said at least one characteristic feature of said at least one reference analyte in said reference calibration sample, and

d) based on the results of steps b) and c), providing a matrix calibration sample having a predetermined concentration of at least one reference analyte.

In view of the above, the following embodiments are preferred: 1 . A method for providing a calibrated result of a determination of at least one analyte of interest in a biological sample comprising

determination of said at least one analyte of interest.

The method for providing a calibrated result of embodiment 1 , wherein said at least one characteristic feature is a quantitative feature.

The method for providing a calibrated result of embodiment 1 or 2, wherein said matrix calibration sample comprises, preferably consists of, at least one biological sample material.

The method for providing a calibrated result of any one of embodiments 1 to 3, wherein said matrix calibration sample comprises, preferably consists of, the same biological sample material as said biological sample.

The method for providing a calibrated result of any one of embodiments 1 to 4, wherein said calibrated result is an absolute concentration or an absolute amount of said analyte of interest in said biological sample.

The method for providing a calibrated result of any one of embodiments 1 to 4, wherein said reference analyte or analytes and said analyte or analytes of interest are identical.

The method for providing a calibrated result of any one of embodiments 1 to 5, wherein said method for providing a calibrated result is a method for providing calibrated results for determinations of at least two, preferably for at least five, more preferably at least ten, even more preferably at least 50, still more preferably at least 100, most preferably at least 200 analytes of interest, and wherein said method comprises

a) providing a matrix calibration sample, wherein the concentration of said at least two, preferably for at least five, more preferably at least ten, even more preferably at least 50, still more preferably at least 100, most preferably at least 200 reference analytes in said matrix calibration sample was predetermined, b) determining at least one characteristic feature of said at least two, preferably for at least five, more preferably at least ten, even more preferably at least 50, still more preferably at least 100, most preferably at least 200 analytes of interest in said sample

c) determining said at least one characteristic feature of said at least two, preferably for at least five, more preferably at least ten, even more preferably at least 50, still more preferably at least 100, most preferably at least 200 reference analytes in said matrix calibration sample, and/or d) based on the results of steps b) and c), providing a calibrated result of the determination of said at least two, preferably for at least five, more preferably at least ten, even more preferably at least 50, still more preferably at least 100, most preferably at least 200 analytes of interest.

The method for providing a calibrated result of any one of embodiments 1 to 6, wherein said at least one analyte of interest comprises analytes selected from at least two, preferably at least five, more preferably at least ten, most preferably all of the following classes of compounds: amino acids, carbohydrates, organic acids, hormones, biogenic amines, acylcarnitines, alcohols, sphingolipids, ceramides, sterols and esters thereof, triacylglycerides, (lyso)phosphatidylcholines, (lyso)phosphatidylethanolamines, fatty acids, carotenoids, hormones, eicosanoids, nucleobases and related, phosphorylated compounds, quinones, quinols, tocopherols, vitamins and secondary metabolites.

The method for providing a calibrated result of any one of embodiments 1 to 8, wherein said matrix calibration sample comprises sample material from at least five, preferably at least 20, more preferably at least 50 different subjects.

The method for providing a calibrated result of any one of embodiments 1 to 9, wherein said biological sample is a cell culture sample from archaebacterial, bacterial, and/or eukaryotic cells.

The method for providing a calibrated result of any one of embodiments 1 to 10, wherein said biological sample is a sample comprising cultured cells, preferably of cultured bacterial, fungal, plant, such as a dicot or monocot plant, more preferably a crop plant, algae, human or animal cells and/or spent medium of said cells.

The method for providing a calibrated result of any one of embodiments 1 to 1 1 , wherein said biological sample is a sample of and/or spent culture medium from E.coli cells, Bacillus cells, Streptomyces cells, Paenibacillus cells, Basfia succiniciproducens cells, Corynebacterium glutamicum cells, Lactobacillus cells, Schizophyllum commune cells, Aspergillus cells, , Chrysosporium cells, Myceliophthora cells, Penicillium cells,

Rhizomucor cells, Trichoderma cells, yeast cells, Candida cells, Kluyveromyces cells, Pichia cells, Saccharomyces cells, CHO cells (Chinese hamster ovary cells), liver cells, hepatocytes, kidney cells, kidney cancer cells, pancreatic cells, pancreatic cancer cells, cardiac cells, cardiac cancer cells, endothelial cells, endothelial cancer cells, fibroblasts, lung cells, lung cancer cells, bladder cells, bladder cancer cells, breast cells, breast cancer cells, colon cells, colon cancer cells, ovarian cells, ovarian cancer cells duodenum cells, duodenum cancer cells, bile duct cells, bile duct cancer cells stem cells or skin cells. The method for providing a calibrated result of any one of embodiments 1 to 9, wherein said biological sample is a sample of an animal, preferably a vertebrate, more preferably a mammal.

The method for providing a calibrated result of any one of embodiments 1 to 13, wherein said biological sample is a sample of an egg, a, preferably non-human, embryo, or a complete non-human organism.

The method for providing a calibrated result of any one of embodiments 1 to 14, wherein said biological sample is a sample of a bodily fluid, preferably a blood, plasma, lymph or serum sample, or is a tissue sample, preferably a sample of liver tissue, heart tissue, prostate tissue, pancreas tissue, brain tissue, kidney tissue, adipose tissue, gut, skeleton tissue, lung tissue, bladder, breast tissue, cecum and/or skin tissue, such as dermal layer, comprising the epidermis and / or corium and / or subcutis.

The method for providing a calibrated result of any one of embodiments 1 to 1 1 , wherein said biological sample is a sample of an algae or plant, preferably of a

monocotyledonous or dicotyledonous plant.

The method for providing a calibrated result of embodiment 16, wherein said biological sample is a tissue sample, preferably leaf tissue, root tissue, shoot tissue, stem tissue, reproductive tissue and/or seed tissue and/or liquid comprising exudate thereof and/or volatile compounds released thereof.

The method for providing a calibrated result of embodiment 16 or 17, wherein said plant includes plants which belong to the superfamily Viridiplantae, preferably Tracheophyta, more preferably Spermatophytina, most preferably monocotyledonous and

dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs; in particular selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arabidopsis thaliana, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brachypodium distachyon, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp.,

Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp. (e.g. Nicotiana tabacum), Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp. (e.g. Saccharum officinarum), Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others. Preferably the plant cell, plant or plant part or plant seed is corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), barley (Hordeum vulgare), cotton (Gossypium hirsutum), sunflower (Helianthus annuus), tobacco

(Nicotiana tabacum), potato (Solanum tuberosum), soyabean (Glycine max), rape (Brassica napus), sugarcane (Saccharum officinarum), sugarbeet (Beta vulgaris). More preferably the plant cell, plant or plant part is a rice cell, rice plant, rice plant part, or rice seed.

The method for providing a calibrated result of embodiment 16 to 18, wherein said biological sample is a sample of freeze-dried plant material, preferably, freeze-dried leaf material, freeze-dried root material, freeze-dried shoot material, freeze-dried stem material and/or freeze-dried reproductive material, preferably freeze-dried flower material, freeze-dried tassel, freeze-dried pollen material and/or freeze-dried seed material.

The method for providing a calibrated result of any one of embodiments 16 to 19, wherein said at least one analyte comprises analytes selected from at least five, preferably at least ten, more preferably at least 15, most preferably all of the following classes of compounds: amino acids, carbohydrates, organic acids, inorganic acids, hormones, biogenic amines, lipids, acylcarnitines, alcohols, sphingolipids, ceramides, sterols and esters thereof, triacylglycerides, (lyso)phosphatidylcholines,

(lyso)phosphatidylethanolamines, fatty acids, eicosanoids, nucleobases and related, phosphorylated compounds, quinones, quinols, tocopherols, alkaloids, glycosides, glucosinolates, carotenoids, terpenes, tannins, phenylpropanoids, phytohormones, and polyketides.

The method for providing a calibrated result of any one of embodiments 1 to 20, wherein compounds comprised in said biological sample and/or matrix calibration sample are separated by chromatography, preferably by liquid chromatography and/or by gas chromatography, before said at least one characteristic feature is determined.

The method for providing a calibrated result of any one of embodiments 1 to 21 , wherein said determining at least one characteristic feature comprises application of mass spectrometry (MS), preferably MS/MS.

The method for providing a calibrated result of embodiment 22, wherein said at least one characteristic feature is a signal at an analyte-specific m/z ratio, preferably a quantitative signal in a detector of an MS device.

The method for providing a calibrated result of embodiment 22 or 23, wherein said at least one characteristic feature is an ion current induced in a detector of an MS device at an analyte-specific m/z ratio.

The method for providing a calibrated result of embodiment 24, wherein said ion current is determined in a total current plot (TIC), preferably mass characteristics in a scan mode, by Selected Ion Monitoring (SIM), by Selected Reaction Monitoring (SRM), and/or by multiple reaction monitoring (MRM).

The method for providing a calibrated result of any one of embodiments 1 to 25, wherein said matrix calibration sample further comprises at least one non-biological compound at a predetermined concentration.

The method for providing a calibrated result of any one of embodiments 1 to 26, wherein said analyte of interest is a compound having a molecular weight of at most 1500 Da.

The method for providing a calibrated result of any one of embodiments 1 to 27, wherein said calibration is a one-point calibration.

A kit for providing a calibrated result of a determination of at least one analyte of interest comprising (i) a matrix calibration sample having a predetermined concentration of at least one reference analyte,

(ii) a data carrier comprising a data collection, said data collection comprising concentration data and identification data for said at least one reference analyte in a determination method.

The kit of embodiment 29, wherein said matrix calibration sample has predetermined concentrations of at least ten, preferably at least 50, more preferably at least 100, most preferably at least 200 reference analytes.

The kit of embodiment 29 or 30, wherein said data carrier comprises concentration data and identification data for analytes selected from at least two, preferably at least five, more preferably at least ten, most preferably all of the following classes of compounds: amino acids, carbohydrates, organic acids, hormones, biogenic amines, acylcarnitines, alcohols, sphingolipids, ceramides, sterols and esters thereof, triacylglycerides,

(lyso)phosphatidylcholines, (lyso)phosphatidylethanolamines, fatty acids, carotenoids, hormones, eicosanoids, nucleobases and related, phosphorylated compounds, quinones, quinols, tocopherols, vitamins and secondary metabolites.

The kit of any one of embodiments 29 to 31 , wherein said determination method comprises MS, preferably MS/MS.

A data collection, preferably comprised on a storage medium, said data collection comprising identification data and concentration data for at least one reference analyte comprised in a matrix calibration sample.

34. A device for determining at least one analyte of interest in a biological sample

comprising

an evaluation unit having a memory unit comprising identification data and data on the predetermined concentration for at least one reference analyte comprised in a matrix calibration sample. 35. The device of embodiment 34, wherein said device further comprises a matrix calibration sample having a predetermined concentration of said at least one reference analyte.

36. Use of a matrix calibration sample having a predetermined concentration of at least one reference analyte for calibrating the determination of an analyte of interest in a sample.

37. The use of embodiment 36, wherein said calibrating is one-point calibrating. A method for providing a matrix calibration sample, said matrix calibration sample having a predetermined concentration of at least one reference analyte, comprising

a) providing a reference calibration sample essentially consisting of the same

analyte in said matrix calibration sample,

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

Figure Legends

Fig. 1 : Quantification of NIST SRM 1950 via MxP Boost approach and comparison to reference values (1 a: high concentrated metabolites; > 500 μΜ), 1 b: low concentrated metabolite; <1 μΜ). A) Determination of MxP Boost accuracy by quantification of NIST SRM 1950 (> 500 μΜ); B) Determination of MxP Boost accuracy by quantification of NIST SRM 1950 (< 1 μΜ)

Fig. 2: Determination of MxP Boost accuracy by quantification of NIST SRM 1950:

Quantification of NIST SRM 1950 via MxP Boost approach and comparison to reference values, metabolites with a concentration between 50 and 500 μΜ.

Fig. 3: Determination of MxP Boost accuracy by quantification of NIST SRM 1950 (1 - 50 μΜ): Quantification of NIST SRM 1950 via MxP Boost approach and comparison to reference values, metabolites with a concentration between 1 and 50 μΜ.

The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

Example 1 : Preparation and quantification of MxP Boost

Human EDTA plasma derived from 20 healthy subjects was pooled, aliquoted into 3.0 ml_ portions, freeze-dried and argonized for long-term storage. The freeze-drying process prevents hydrolysis processes and the argonization inhibits oxidative processes to maximize the long- term stability of the samples. The material was quantified applying several validated quantitative method targeting specific metabolite classes and polarities, respectively yielding into > 700 quantitated metabolites and metabolite class species such as sphingomyelin, total. All methods applied included some metabolites that were also quantified by other methods. Therefore, every method could be cross-validated to some extend showing high accordance with concentrations determined with other methods.

Example 2: Validation of MxP Boost versus NIST SRM 1950 plasma MxPool™ Human plasma was used for one-point calibration of quantifications of metabolites in plasma samples from test subjects. The method was validated against e.g. the reference plasma NIST SRM 1950 provided by the National Institute of Standards and Technology (NIST) of the USA. The experimental design consisted of two sequences (MxP Boost Seq 1 and MxP Boost Seq 2) of four to five aliquots of three different samples, respectively. One sample consisted of commercially available human plasma (four aliquots per sequence) used for normalization of different days (pool normalization). Secondly, five aliquots of MxP Pool were included in each sequence. In addition, five aliquots of NIST SRM1950 plasma were measured in each sequence. Samples within each sequence were measured in an alternating order to exclude systematic effects by order of sample preparation or measurement.

Two sequences of the three different samples were measured on two different timepoints on different LC-MS/MS or GC - MS devices to provide an estimate on possible interday shifts. The results of the accuracy are shown in Figure 1 - Figure 3.

Metabolite concentrations in NIST SRM 1950 plasma as quantified via MxP Boost are given in

Table 1 . Reference values provided by NIST with the associated absolute uncertainty are stated. Moreover, the overall trueness [%] is calculated. Only few metabolites failed to have an overlap with the confidence range given by NIST. However, in these cases, the delta Trueness of the results was lower or at least close to 10 %, which reflects a sufficient accordance of the results. Table 1 Quantification of NIST SRM 1950 Plasma by MxP Boost and comparison to reference value provided by NIST

Metabolite_Name Class Reference absol Mean Delta values ute MxP True¬

NIST SRM uncert Boost ness

1950 ainty Seq1 / [%]

Plasma NIST MxP

SRM Boost

1950 Seq2 Plasm

a

+/-

Alanine Amino acids 300 26.1 293,3 -2.2

Arginine Amino acids 81 ,4 2.3 91 ,9 12.9

Cysteine Amino acids 44,3 6.9 41 ,4 -6.6

Cystine Amino acids 7,8 0.4 7,75 -0.7

Glutamate Amino acids 67 18.0 78,25 9.6

Glycine Amino acids 245 15.9 257 4.9

Histidine Amino acids 72,6 3.6 70,25 -3.2

Isoleucine Amino acids 55,5 3.4 52,05 -6.3

Leucine Amino acids 100,4 6.3 101 ,1 0.7

Lysine Amino acids 140 14.0 150,95 7.8

Methionine Amino acids 22,3 1 .8 21 ,65 -2.9

Phenylalanine Amino acids 51 7.0 49,5 -3.0

Proline Amino acids 177 9.0 180,4 1 .9

Serine Amino acids 95,9 4.3 97,65 1 .8

Threonine Amino acids 1 19,5 6.1 120,4 0.7

Tyrosine Amino acids 57,3 3.0 56,1 -2.1

Valine Amino acids 182,2 10.4 181 ,1 -0.6

Creatinine Amino acids related 60 0.9 57,2 -4.7

Homocysteine Amino acids related 8,5 0.2 8,1 -4.6

Ornithine Amino acids related 52,1 2.8 59,3 13.8

Urea Amino acids related 3900 81.9 4266,85 9.4

Glucose Carbohydrates and 4560 54.7 4347,25 -4.7 related

Arachidonic acid Complex lipids, fatty 984 180.1 980,95 -0.3 (C20:cis[5,8,1 1 ,14]4) acids and related

Behenic acid (C22:0) Complex lipids, fatty 47,8 4.6 44,65 -6.5 acids and related

Cholesterol, total Complex lipids, fatty 3917 86.2 3384,85 -13.6 acids and related

dihomo-gamma-Linolenic Complex lipids, fatty 139 4.0 148,65 6.9 acid (C20:cis[8,1 1 ,14]3) acids and related

Docosahexaenoic acid Complex lipids, fatty 1 18 21.0 1 14,25 -3.2 (C22:cis[4,7,10,13,16,19]6) acids and related

Docosapentaenoic acid Complex lipids, fatty 19,5 0.4 21 ,3 9.2 (C22:cis[4,7,10,13,16]5) acids and related

Docosapentaenoic acid Complex lipids, fatty 38,5 0.7 38,1 -1 .0 (C22:cis[7,10,13,16,19]5) acids and related Docosatetraenoic acid Complex lipids, fatty 25,5 0.6 23,1 -9.4 (C22:cis[7,10,13,16]4) acids and related

Eicoasenoic acid Complex lipids, fatty 1 1 ,5 0.5 10,45 -9.3 (C20:cis[1 1 ]1 ) acids and related

Eicosanoic acid (C20:0) Complex lipids, fatty 18 0.5 20,1 1 1.8 acids and related

Eicosapentaenoic acid Complex lipids, fatty 38,6 0.5 41 ,85 8.4 (C20:cis[5,8,1 1 ,14,17]5) acids and related

Erucic acid (C22:cis[13]1 ) Complex lipids, fatty 3,4 1 .3 3,35 -0.2 acids and related

gamma-Linolenic acid Complex lipids, fatty 39,9 8.5 39,55 -0.8 (C18:cis[6,9,12]3) acids and related

Heptadecanoic acid (C17:0) Complex lipids, fatty 17,6 0.7 15,85 -9.9 acids and related

Laurie acid (C12:0) Complex lipids, fatty 9,47 0.6 8,75 -7.6 acids and related

Lignoceric acid (C24:0) Complex lipids, fatty 46,6 2.6 49,05 5.3 acids and related

Linoleic acid (C18:cis[9,12]2) Complex lipids, fatty 2838 141 .9 2881 ,55 1 .5 acids and related

Linolenic acid Complex lipids, fatty 54,6 3.6 52,1 -4.6 (C18:cis[9,12,15]3) acids and related

Myristic acid (C14:0) Complex lipids, fatty 80,1 17.0 74,05 -7.5 acids and related

Nervonic acid (C24:cis[15]1 ) Complex lipids, fatty 71 ,3 3.2 66,7 -6.4 acids and related

Oleic acid (C18:cis[9]1 ) Complex lipids, fatty 1614 153.3 1521 ,45 -5.7 acids and related

Palmitic acid (C16:0) Complex lipids, fatty 2364 78.0 2217,55 -6.2 acids and related

Palmitoleic acid (C16:cis[9]1 ) Complex lipids, fatty 215 26.0 223,15 3.8 acids and related

Stearic acid (C18:0) Complex lipids, fatty 644 41.2 676,75 5.1 acids and related

Cortisol Hormones, signal 0,23 0.005 0,245 7.6 substances and

Lutein Miscellaneous 0,12 0.040 0,13 8.1

Uric acid Nucleobases and 254 5.1 234,75 -7.6 related alpha-Tocopherol Vitamins, cofactors 19 0.5 20,55 8.2 and related

gamma-Tocopherol Vitamins, cofactors 4, 1 0.4 4,5 9.8 and related

Example 3: Analytical methods Samples were prepared and subjected to LC-MS/MS and GC-MS analysis as described below. Several groups of metabolites were analyzed semi-quantitatively or quantitatively including amino acids, carbohydrates, fatty acids, mono-, di-and triglycerides, other lipids, organic acids, coenzymes, vitamins, secondary metabolites, steroid hormones and catecholamines.

Prospective samples were also analyzed for selected eicosanoids.

Proteins were separated by precipitation from blood plasma. After addition of water and a mixture of ethanol and dichlormethane the remaining sample was fractioned into an aqueous, polar phase and an organic, lipophilic phase.

For the transmethanolysis of the lipid extracts (lipophilic phase) a mixture of 140 μΙ_ of chloroform, 37 μΙ_ of hydrochloric acid (37% by weight HCI in water), 320 μΙ_ of methanol and 20 μΙ_ of toluene was added to the evaporated extract. The vessel was sealed tightly and heated for 2 hours at 100 C, with shaking. The solution was subsequently evaporated to dryness. The residue was dried completely.

The methoximation of the carbonyl groups was carried out by reaction with methoxyamine hydrochloride (20 mg/mL in pyridine, 100 μΙ_ for 1 .5 hours at 60 °C) in a tightly sealed vessel. 20 μΙ_ of a solution of odd-numbered, straight-chain fatty acids (solution of each 0.3 mg/mL of fatty acids from 7 to 25 carbon atoms and each 0.6 mg/mL of fatty acids with 27, 29 and 31 carbon atoms in 3/7 (v/v) pyridine/toluene) were added as time standards. Finally, the derivatization with 100 \ L of N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was carried out for 30 minutes at 60 °C, again in the tightly sealed vessel. The final volume before injection into the GC was 200 μΙ_.

The GC-MS systems consist of an Agilent 6890 GC coupled to an Agilent 5973 MSD (Agilent, Waldbronn, Germany), autosamplers were CompiPal or GCPal from CTC (CTC, Zwingen, Switzerland).

In LC-MS analysis, both fractions were reconstituted in appropriate solvent mixtures. H PLC was performed by gradient elution on reversed phase separation columns. Mass spectrometric detection which allows target and high sensitivity MRM (Multiple Reaction Monitoring) profiling in parallel to a full screen analysis was applied as described in WO2003073464. The H PLC instruments were Agilent 1 100 (Agilent, Waldbronn, Germany), the MS instruments were API4000 from SCI EX (AB SCIEX, Darmstadt, Germany).

Claims

A method for providing a calibrated result of a determination of at least one analyte of interest in a biological sample comprising

a) providing a matrix calibration sample, wherein the concentration of at least one reference analyte in said matrix calibration sample was predetermined, b) determining at least one characteristic feature of said at least one analyte of interest in said biological sample,

determination of said at least one analyte of interest.

The method for providing a calibrated result of claim 1 , wherein said at least one characteristic feature is a quantitative feature.

The method for providing a calibrated result of claim 1 or 2, wherein said matrix calibration sample comprises, preferably consists of, at least one biological sample material.

The method for providing a calibrated result of any one of claims 1 to 3, wherein said matrix calibration sample comprises, preferably consists of, the same biological sample material as said biological sample.

The method for providing a calibrated result of any one of claims 1 to 4, wherein said method for providing a calibrated result is a method for providing calibrated results for determinations of at least ten, preferably at least 50, more preferably at least 100, even more preferably at least 200 analytes of interest, and wherein said method comprises a) providing a matrix calibration sample, wherein the concentration of said at least ten, preferably said at least 50, more preferably said at least 100, even more preferably said at least 200 reference analytes in said matrix calibration sample was predetermined,

b) determining at least one characteristic feature of said at least ten, preferably at least 50, more preferably at least 100, even more preferably at least 200 analytes of interest in said sample

c) determining said at least one characteristic feature of said at least ten, preferably said at least 50, more preferably said at least 100, even more preferably at least 200 reference analytes in said matrix calibration sample, and/or

determination of said at least ten, preferably said at least 50, more preferably said at least 100, even more preferably at least 200 analytes of interest. The method for providing a calibrated result of any one of claims 1 to 5, wherein said at least one analyte of interest comprises analytes selected from at least two, preferably at least five, more preferably at least ten, most preferably all of the following classes of compounds: amino acids, carbohydrates, organic acids, hormones, biogenic amines, acylcarnitines, alcohols, nucleobases and related, phosphorylated compounds, sphingolipids, ceramides, sterols and esters thereof, triacylglycerides,

(lyso)phosphatidylcholines, (lyso)phosphatidylethanolamines, fatty acids, carotenoids, quinones, quinols, tocopherols, vitamins, phytohormones, and secondary metabolites.

The method for providing a calibrated result of any one of claims 1 to 6, wherein said biological sample is a cell culture sample from archaebacterial, bacterial, and/or eukaryotic cells.

The method for providing a calibrated result of any one of claims 1 to 6, wherein said biological sample is a sample of an animal, preferably a vertebrate, more preferably a mammal.

The method for providing a calibrated result of any one of claims 1 to 6, wherein said biological sample is a sample of an algae or plant, preferably of a monocotyledonous or dicotyledonous plant.

The method for providing a calibrated result of any one of claims 1 to 9, wherein compounds comprised in said biological sample and/or matrix calibration sample are separated by chromatography, preferably by liquid chromatography and/or by gas chromatography, before said at least one characteristic feature is determined.

The method for providing a calibrated result of any one of claims 1 to 10, wherein said determining at least one characteristic feature comprises application of mass spectrometry (MS), preferably MS/MS.

A kit for providing a calibrated result of a determination of at least one analyte of interest comprising

The kit of claim 12, wherein said matrix calibration sample has predetermined concentrations of at least 50, preferably of at least 100, more preferably of at least 200 reference analytes. A device for determining at least one analyte of interest in a biological sample comprising

Use of a matrix calibration sample having a predetermined concentration of at least one reference analyte for calibrating the determination of an analyte of interest in a sample.