LU93103B1

LU93103B1 - Quality of samples

Info

Publication number: LU93103B1
Application number: LU93103A
Authority: LU
Inventors: Karsten Hiller; Lisa Krämer; Jean-Pierre Trezzi
Original assignee: Univ Luxembourg
Priority date: 2016-06-10
Filing date: 2016-06-10
Publication date: 2018-01-23

Abstract

The present invention relates to methods for determining the metabolic activity that has taken place in a sample. Furthermore, the present invention relates to uses of a compound (I) for determining the quality of a sample, for determining the metabolic activity of a sample or for reducing false-positive or false-negative results obtained from a sample. Also provided are sample storage devices, filter paper suitable for dried blood spot sampling and vials or containers for sampling comprising compound (I). 93103

Description

SAMPLE QUALITY

FIELD OF THE INVENTION

[1] The present invention relates to methods for determining the metabolic activity that has taken place in a sample. Furthermore, the present invention relates to uses of a compound (I) for determining the quality of a sample, for determining the metabolic activity of a sample or for reducing false-positive or false-negative results obtained from a sample. Also provided are sample storage devices, filter paper suitable for dried blood spot sampling and vials or containers for sampling comprising compound (I).

DESCRIPTION

[2] Metabolomics studies the metabolites, small molecules involved in thousands of enzyme-catalyzed biochemical processes, and is downstream of the central dogma of biology, thereby influenced by genomics, transcriptomics and proteomics. The fast turnover rates and high sensitivity of metabolites to environmental changes, such as the storage time and temperature, metabolomics is the most sensitive approach to evaluate the sample quality. Biospecimen research is studying the impact of environmental or pre-processing conditions on the quality of biological samples.

[3] The quality outcome of a research project or clinical diagnosis is strongly correlated with the quality of the sample it is based on. However, many clinical diagnosis laboratories and research institutes do not know the quality of their samples which leads to wrongly interpreted data or diagnosis.

[4] Up to date, no robust quality control markers predicting/determining the sample quality have been established. Especially in metabolomics studies, pre-analytical markers predicting/determining how long and at what temperature a given sample was stored prior to the sample processing or analysis, would be crucial for subsequent analyses and would enable high quality data e.g. of data collected within a research project. These markers would not only apply on the sample quality for metabolomics studies but also for genomics, transcriptomics and proteomics based applications.

[5] The technical problem can thus be seen in the provision of a method to improve metabolomics studies on samples.

[6] The technical problem is solved by the embodiments reflected in the claims, described in the description, and illustrated in the Examples and Figures. Π The above being said, the present invention relates to a method for determining the metabolic activity that has taken place in a sample, the method comprising a. within 5 minutes or less from the time the sample has been obtained, contacting the sample with a labeled compound (I) that can be metabolized by the sample; and b. determining the presence or absence of one or more labeled metabolites of the labeled compound, wherein the determination of the presence or absence of the one or more metabolites is indicative of the metabolic activity that has taken place in the sample.

[8] The present invention also relates to a method for determining the metabolic activity that has taken place in a sample, the method comprising a. within 5 minutes or less from the time the sample has been obtained, contacting the sample with a labeled compound (I) that can be metabolized by the sample; and b. contacting the sample with a compound (II), which cannot be metabolized by the sample; wherein the ratio between the labeled compound (I) and compound (II) is a predefined ratio; and c. determining the ratio between the labeled compound (I) and compound (II) (labeled compound (l):compound (11)), wherein the determination of the ratio is indicative of the metabolic activity that has taken place in the sample.

[9] In addition, the present invention relates to a use of a labeled compound (I) as defined herein for determining the quality of a sample.

[10] Furthermore, the present invention relates to a use of a labeled compound as defined herein for determining the metabolic activity of a sample.

[11] The present invention also relates to a use of a labeled compound as defined herein for reducing false-positive or false-negative results obtained from a sample. Likewise, the present invention also relates to a use of a labeled compound as defined herein for enhancing the quality of data obtained from a sample.

[12] In addition, the present invention relates to a sample storage device, wherein the sample storage device comprises a labeled compound (I) as defined herein and optionally further comprises a compound (II) as defined herein.

[13] The present invention furthermore relates to a filter paper suitable for dried blood spot sampling, wherein the filter paper comprises a labeled compound (I) as defined herein.

[14] Additionally, the present invention relates to a vial suitable for sampling, wherein the vial comprises a labeled compound (I) as defined herein.

[15] The figures show: [16] Figure 1: Lactic acid M3 enrichment over time. In particular, lactic acid M3 is the 3-fold labeled lactic acid isotopomer. Enrichment is the relative abundance of a specific isotopomer in relation to the sum of all isotopomers for a given compound. In this figure a tube, namely a K2EDTA vacutainer comprising a blood sample a small amount of [U13C]-Glucose has been added to the blood and the sample was then stored at 37°C with slow shaking. At different time points such as 15, 30, 60 and 120 minutes a small amount of blood was taken for metabolite extraction. Additionally, at time point 0, before the labeled glucose has been added, a small amount of blood has been taken for metabolite extraction. Then the labeled lactate has been measured by gas chromatography coupled to mass spectrometry (GC-MS). The enrichment of labeled lactate is depicted for 2 different sample donors. In particular the enrichment is depicted as the fraction of labeled lactate from the total concentration or in other words the total detected amount of lactate (labeled and unlabeled lactate). Over time the labeled compound (I), namely [U13C]-Glucose is gradually metabolized into labeled lactate by biochemical processes. Consequently, the amount of [U13C]-Glucose is decreasing while the amount of labeled lactate is increasing over time. As can be obtained from the graph, the sample is metabolized following a strict time period. Data for both samples (of donor 1 and 2) strongly correlate.

Figure 2 (Table 1): Prediction of pre-processing times from donor 2 based on data from donor 1. To achieve the prediction and/or determination of the time the sample from donor 2 has been metabolically active after sample collection, a linear regression was performed on data obtained from donor 1. The time the sample has been predicted to have been metabolically active is shown in Fig. 2/Table 1 in the column named “predicted time (min)”. This predicted result was then compared to the linear regression generated from data obtained for donor 2. Data for this donor 2 were obtained in the same manner as described in the Example or in the figure legend of figure 1. The so obtained data are shown in column “Time (min)” in Fig. 2/Table 1. Furthermore, the difference between the predicted time of metabolic activity and the measured metabolic activity of the sample obtained from donor 2 is shown in the column named “Difference” in Fig. 2 /Table 1. When looking at the difference it becomes clear that the difference is actually minimal (of about 2 to 3 minutes). Thus, from linear regression the time the sample has been metabolically active after sample collection can be precisely predicted/determined.

[17] Figure 3: Depiction of glycolysis metabolic pathway.

[18] Figure 4: Depiction of glycolysis and gluconeogenesis metabolic pathway.

[19] Figure 5: Depiction of TCA metabolic cycle.

[20] Figure 6: Depiction of urea metabolic cycle.

[21] Figure 7: Depiction of Cori metabolic cycle.

[22] The inventors propose a method for the elucidation of the sample quality by the introduction of a labeled compound directly in the sample. This could be done either by first introducing the labeled compound in the sample collection device prior to sample collection or by adding the labeled compound after the sample has been collected. This labeled compound consists of any compound that is metabolized in the given sample and is different from endogenous compounds (e.g. [U13C]-Glucose instead of [U12C]-Glucose). Notably, [13C]-Glucose can also be present in the sample per se due to the natural abundance of 13C carbon which is about 1.1% of all carbon atoms that exist. However the concentration of such naturally occurring 13C carbon is so low that it can be neglected for the purposes of the present invention. However, the labeled compound must not be biologically different from endogenous compounds. This can for example be the case, when the labeled compound (I), can be differentiated from endogenous compounds e.g. by mass spectrometrial means.

[23] The introduced labeled compound (I) is metabolized directly by the sample, which results in the generation of one or more labeled and also unlabeled metabolites. These metabolites may be e.g. end-products or intermediates of biochemical reactions. The duration and temperature at which a sample has been stored after sample collection is correlating with the amount of the metabolized labeled compound, e.g. correlating with the increase or decrease of labeled metabolism by-products. These labeling patterns can be easily and precisely analyzed by mass spectrometry.

[24] With a regression-based equation, the quality of the sample can be determined by applying the equation to the result obtained by mass spectrometry analysis of labeling patterns.

[25] Therefore, the present invention relates to a method for determining the metabolic activity that has taken place in a sample, the method comprising a. within 5 minutes or less from the time the sample has been obtained, contacting the sample with a labeled compound (I) that can be metabolized by the sample; and b. determining presence or absence of one or more labeled metabolites of the labeled compound, wherein the determination of the presence or absence of the one or more metabolites is indicative of the metabolic activity that has taken place in the sample.

[26] The present invention also relates to a method for determining the metabolic activity that has taken place in a sample, the method comprising a. within 5 minutes or less from the time the sample has been obtained, contacting the sample with a labeled compound (I) that can be metabolized by the sample; and b. contacting the sample with a compound (II), which cannot be metabolized by the sample; wherein the ratio between the labeled compound (I) and compound (II) is a predefined ratio; and c. determining the ratio between the labeled compound (I) and compound (II) (labeled compound (l):compound (11)), wherein the determination of the ratio is indicative of the metabolic activity that has taken place in the sample.

Notably, the methods and also the uses of the present invention are also very robust. This is because the relative abundance/fraction (= enrichment) of the labeled metabolite is comparable between different patients whereas the overall concentrations of the (same) labeled and unlabeled metabolite might be different between patients.

[27] As used herein the term “metabolic activity” means the total of the chemical processes that can occur in the sample. For example such chemical processes are biological reactions that result in growth, production of energy, elimination of waste material, etc. The term metabolic activity is known to the person skilled in the art and, for example, described in Alberts B, Johnson A, Lewis J, et al. New York: Garland Science: 2002. Molecular Biology of the Cell. 4th edition, e.g. in chapters Catalysis and the Use of Energy by Cells and How Cells Obtain Energy from Food or in DeBerardinis and Thompson (2012) “Cellular Metabolism and Disease: What Do Metabolic Outliers Teach Us?” Cell 148; pp. 1132-1144).

[28] In short, metabolic activity can be simplified to those pathways involving abundant nutrients like carbohydrates, fatty acids, and amino acids, essential for energy homeostasis and macromolecular synthesis. Pathways of metabolism can then be separated into three classes: those that synthesize simple molecules or transform them into more complex macromolecules (anabolism); those that degrade molecules to release energy (catabolism); and those that help to eliminate the toxic waste produced by the other classes (waste disposal).

[29] Accordingly, the methods of the present invention can, for example, be directed to the detection of catabolic activity and/or toxic waste production and/or anabolic activity of a sample. Exemplary metabolic activities envisioned by the present invention can include but are not limited to glycolysis, the tricarboxylic acid (TCA) and urea cycle, glycogen catabolism (Cori and Cori), oxidative phosphorylation, cell respiration and the supremacy of ATP in energy transfer reactions (DeBerardinis and Thompson (2012) “Cellular Metabolism and Disease: What Do Metabolic Outliers Teach Us?” Cell 148; pp. 1132-1144). Furthermore, during metabolic activity enzymes can act as catalysts that can e.g. allow the reactions to proceed more rapidly or at lower temperatures.

[30] Notably, the metabolic activity that has been present in a sample after sample collection may be different depending on the type of sample, which is analyzed. Metabolic activity present in a sample can also depend on many factors which are known or unknown to the skilled artesian. For, example it is known that metabolic activity is sensitive to the temperature as e.g. enzymatic activity is optimal at specific temperatures for specific biochemical reactions. For example, usually the metabolic activity is higher at higher temperatures such as temperatures of about 20°C, 25°C, 30°C, 37°C, 40°C, 45°C, 50°C, 60°C, 70°C, 80°C or higher temperatures. The metabolic activity can also be higher in the presence of e.g. more substrate, enzymes etc. On the contrary, metabolic activity can be lower at lower temperatures such as temperatures of about 15°C 10°C, 7°C, 5°C, 4°C, 2°C, 0°C, -5°C, -10°C, -15°C, -20°C, -80°C or fewer degrees.Methods of the present invention can, for example, measure how much and/or for how long metabolic activity has taken place in a sample. These factors also determine the quality of a sample.

[31] In general, the quality of a sample can depend on any factor. The person skilled in the art is also aware of factors that can influence the quality of a sample. For example, the quality of a sample can depend on the temperature at which the sample is stored or on the time period the sample is stored at a specific temperature or on sample storage conditions in general. Storage conditions as referred to herein include storage temperature, pressure, humidity, time, collection devices, as well as the treatment of the stored samples with preserving agents. Sample quality can also depend on the nature of the sample, such as the time that is necessary to obtain a sample. Also the treatment after the sample has been obtained may indirectly influence sample quality. The quality of a sample may also depend on the specific type of sample.

[32] One possibility to determine the quality of a sample is to measure if or for how long a sample has been metabolically active as described herein. One way to avoid that the quality of a sample decreases is fast cooling of the sample to low temperatures directly after the sample has been obtained/collected. Measurement of metabolic activity can then e.g. provide information if the sample has been constantly kept at temperatures e.g. below 4° C or has been stored for a long time. Thus, determination of the metabolic activity that has taken place in the sample can be used to measure the quality of a sample, for example, metabolic activity can be measured by contacting the sample with a labeled compound (I) that can be metabolized by the sample. The rationale behind the use of a labeled compound (I) is that this compound is metabolized within the sample if it is e.g. stored inadequately, such as at high temperatures. Upon biochemical reactions, compound (I) is converted into labeled/unlabeled metabolites. This means the more labeled metabolites are generated the lesser is the amount of compound (I), which is left in the sample. Which concrete metabolites are generated always depends on the compound (I) used. For example, if labeled glucose is used as compound (I) then labeled lactate is a possible labeled metabolite that can be detected. However, if for example compound (I) is citrate, then a labeled metabolite that is measured can be labeled succinate.

[33] The present invention envisions that the labeled compound (I) can be chosen in accordance with the expected metabolic activity e.g. the targeted metabolic pathway present in a sample. The labeled compound can then become metabolized by the sample if the sample is metabolically active. Due to this metabolic activity of the sample one or more labeled metabolites of the labeled compound can be generated. These compounds can then be measured and their presence, absence or abundance indicates if the sample has been metabolically active or not. The present invention also contemplates that the amount of metabolic activity that has taken place in a sample can be determined by the methods of the present invention. This is because the more of the one or more labeled metabolites can be detected the more the sample has been metabolically active and vice versa.

[34] Alternatively or additionally, the sample may be contacted with a compound (II), which cannot be metabolized by the sample. It is further contemplated by the present invention that when the compound (II) is utilized in the methods of the present invention that the ratio between the labeled compound (I) and compound (II) can be a predefined ratio.

[35] In such cases, in principle, any ratio between the labeled compound (I) and the compound (II) can be suitable. Exemplary ratios of the compound (I) divided by the compound (II) (compound (l):compound (II) or compound (l)/compound (11)) are 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.05, 0.01 or less. Yet, the ratio can also be higher than 10, 11, 12, 13, 14, 15, 20 or more. The determination of the ratio is indicative of the metabolic activity that has taken place in the sample. Thus, the more the labeled compound (I) decreased in relation to the compound (II) compared to the predefined ratio the more the sample has been metabolically active and vice versa.

[36] It is further contemplated by the present invention that the sample is of quality when the sample has experienced insignificant metabolic activity or when insignificant metabolic activity has taken place in the sample. Insignificant metabolic activity can mean that the sample has not been metabolically active or metabolic activity was too low to be significant. This can, for example, be the case, when the sample is frozen or freeze-dried directly after it has been obtained. At such low temperatures usually almost any or no metabolic activity is present. Thus, insignificant metabolic activity can comprise the detection of insignificant or no presence of one or more labeled metabolites in the sample.

[37] The exact natures of the one or more metabolites that will be generated depend on the labeled compound that has been utilized in the methods of the present invention. For example, to determine if one or more labeled metabolites have been generated, the fraction of the one or more labeled metabolites from the total concentration or the total measured one or more of the labeled and unlabeled metabolites, respectively, can be determined. The present invention also envisions that in total 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 20, 25, 30 or more metabolites are measured. For example, lactate can be measured by mass spectrometry and the fraction of labeled lactate can be calculated with regard to the total concentration of or the total measured amount of labeled and unlabeled lactate in the sample. Thus, the sample is of quality when the fraction of the one or more labeled metabolites from the total concentration of the one or more of the labeled and unlabeled metabolites can be about 0 %, 0.001%, 0.005%, 0.01%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%4 20% or more. It is further envisioned by the present invention that in a sample of quality the fraction of the one or more labeled metabolites from the total concentration of the one or more of the labeled and unlabeled metabolites is maximally 5%, 7%, 10%, 12%, 15% or 20%. It is also envisioned that the sample is of quality when the fraction of the one or more labeled metabolites from the total concentration of the one or more of the labeled and unlabeled metabolites is maximally 10%.

[38] Alternatively or additionally to measurement of one or more metabolites of compound (I), the methods of the present invention also provide for the measurement of the quality of the sample, with regard to labeled compound (I) itself, for example, labeled compound (I) may be measured in relation to its unlabeled counterpart (unlabeled compound (I)) already present in the sample.

[39] However, labeled compound (I) can also be measured with regard to compound (II) as described herein. Notably, the ratio of the labeled compound (I) and the compound (II) is predefined and therefore known to the person skilled in the art. However, the compound (II) can also be used in order to obtain ratios with regard to e.g. the one or more unlabeled/labeled metabolites to be analyzed. Also in such cases the ratio between the compound (II) and one or more unlabeled metabolites is a predefined ratio.

[40] A predefined ratio as used herein is a ratio, which is known when starting sample analysis. Sample analysis can in general be performed with any technique suitable for this purpose. Exemplary techniques for sample analysis are also described herein. For example, a predefined ratio can be known because the amount/concentration of compound (I) and compound (II) is known when contacting the sample. The present invention also contemplates that the predetermined ratio is determined by analysis of the sample directly after having contacted the sample with the labeled compound (I) and compound (II). “Directly”, when used herein means that contacting of the sample with compound (I) and/or compound (II) is performed within 0, 1,2, 5, 10, 30, 60, 90, 160, 180, 200, or 300 seconds from the time the sample has been obtained. This analysis can be performed by analyzing a small volume of the sample instead of the total volume of the collected sample, which has been contacted with the labeled compound (I) and/or compound (II). Such an analysis can be performed with every technique as described herein or every technique that is able to differentiate the characteristic ions/masses of labeled/unlabeled compounds. For example, the technique can be gas chromatography coupled to mass spectrometry. The analysis result can then be used as the predefined ratio. It is further envisioned by the present invention that the predetermined ratio is determined on the basis of more than 1,2, 3, 4, 5, or more of compound (I) and/or compound (II). As such e.g. two different compounds (I) can be used to calculate a ratio with one compound (II).

[41] It is also contemplated by the present invention that compound (II) serves as an internal standard. An internal standard means that the sample is contacted with a known concentration/amount of compound (II) that is to be analyzed. Thus, the present invention envisions that the sample can be contacted with a known concentration/amount of compound (II) and optionally also with a known concentration/amount of labeled compound (I).

[42] Compound (II) can for example, be such that it is stable during chromatographic measurements. Additionally or alternatively, compound (II) can be such that it does not chemically react with the stationary phase or with any other part of the chromatographic system. Additionally or alternatively, compound (II) can be such that it does not chemically react with any of the analytes. The present invention also contemplates that the compound (II) is soluble in the solvent in which the analyte is dissolved. It is further envisioned that compound (II) behaves similarly to the analyte (like e.g. compound (I) and/or the one or more labeled metabolites) but to provide a signal that can be distinguished from that of the analyte. For example, compound (II) can have similar physico-chemical properties to the analyte such as compound (I) or the one or more labeled metabolites. Such physico-chemical properties can include e.g. boiling point, polarity or functional group type. It is also envisioned by the present invention that any factor that affects the analyte signal will also affect the signal of the compound (II) to the same degree. Thus, compound (II) can be, for example, a compound that is very similar, but not identical to the compound/metabolite of interest in the samples, as the effects of sample preparation should, relative to the amount of each analyte, be the same for the signal from the internal standard as for the signal(s) from the analytes of interest in the ideal case. Therefore, the sample can additionally or alternatively be contacted with an internal standard.

[43] The term “analytes” as used herein can refer to one or more of any of the labeled compound (I), the unlabeled compound (I), the compound (II), one or more labeled metabolites and/or one or more unlabeled metabolites or any other compound as described herein.

[44] The present invention also contemplates that the sample can be contacted with known quantities of analyte(s) of interest (e.g. labeled/unlabeled compound (II), or one or more labeled/unlabeled metabolites). This technique is called standard addition, which is performed to correct for matrix effects. The standard addition method is also known to the skilled person and e.g. described in IOFI Working group on methods of analysis (2011 ) “Guidelines for the quantitative gas chromatography of volatile flavoring substances, from the working group on methods of analysis of the international organization of the flavor industry (IOFI)” Flavor and Fragrance Journal 26, 297-299. Experimentally, 1) The chromatogram of the unknown amount of the analyte is recorded 2) the sample is contacted with a predefined amount of the analyte(s) of interest 3) the sample is analyzed again under the same conditions and the chromatogram is recorded. From the increase in the peak area (or peak height), the original concentration of the analyte of interest can be computed by interpolation. The detector response must be a linear function of analyte concentration and yield no signal (other than background) at zero concentration of the analyte. This ratio for the samples is then used to obtain their analyte concentrations from a calibration curve.

[45] The present invention also contemplates the use of an internal normalization that evaluates the stability of an analyte of interest over the time of measurement. Internal standards can for example be used to normalize for changes due to pipetting errors etc. and correct changes in the signal intensity (decrease of signal over time in a measurement).Such techniques ares also known to the person skilled in the art and, for example, described in IOFI Working group on methods of analysis (2011) “Guidelines for the quantitative gas chromatography of volatile flavouring substances, from the working group on methods of analysis of the international organization of the flavor industry (IOFI)” Flavor and Fragrance Journal 26, 297-299. Therefore the sample can additionally or alternatively be contacted with an external standard.

[46] Additionally or alternatively, also external standards can be used in the present invention. The concept of an external standard is also known to the person skilled in the art and, for example, described in Elcio Cruz de Oliveira Edson I. Muller, Fernanda Abad, Juliana Dallarosa e Cristine Adriano (2010) “Internal standard versus external standard calibration: an uncertainty case study of a liquid chromatography analysis” Quim. Nova, Vol. 33, No. 4, 984-987. For example, academic standards, i.e. standard solutions without matrix, can be used as early markers for the decline of the performance of the analytical system, as metabolites are more prone to adsorb or degrade on the surface of the analytical column in the absence of sample matrix. Another very useful external standard is a pooled sample of all individual samples (pooled QC) measured during a study. A pooled QC can be used to calculate the repeatability and intermediate precision of all detectable metabolites present in the samples and to correct for detector drift and/or variations in mass spectrometry (MS) response between batches. In addition, a pooled QC representative of the samples measured, can be used to correct MS responses of metabolites in individual samples.

[47] As described herein the quality of the sample can also be determined using the ratio of a labeled compound (I) to compound (II) and/or the ratio of compound (II) to one or more labeled metabolites. Accordingly, a sample is of quality when almost no to no metabolic activity has taken place in the sample. This, means that the labeled compound (I) has not or almost not been metabolized. Therefore, the sample is of quality when the ratio of the labeled compound (I) and the compound (II) is approximately the same as the predefined ratio. In this context approximately means that the predefined ratio differs from the determined ratio of about 5, 4, 3, 2, 1, 0.5, 0, 0.05, 0.01 or less. It is also contemplated by the present invention that the sample is of quality when the ratio of the labeled compound (I) and the compound (II) is the same as the predefined ratio.

[481 On the other hand, the sample is of low quality when some to substantive metabolic activity has taken place in a sample, for example, the sample can be of low quality, when the sample has experienced significant metabolic activity after sample collection. Significant metabolic activity can, for example, mean the detection of the significant presence of one or more labeled metabolites in the sample. Significant metabolic activity can also mean the detection of a higher abundance/fraction of one or more labeled metabolites in the sample after storage or after some time after sample collection in comparison of the abundance/fraction of one or more labeled metabolites in the sample measured directly after sample collection. This means that the labeled compound (I) has been metabolized and labeled metabolites of the labeled compound (I) have been generated by biochemical reactions. To measure the metabolic activity that has taken place in the sample, for example, the fraction of the one or more labeled metabolites from the total concentration of the respective one or more of the labeled and unlabeled metabolites can be measured. The fraction of the one or more labeled metabolites from the total concentration of the respective one or more of the labeled and unlabeled metabolites can, for example, be more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or more. The fraction of the one or more labeled metabolites from the total concentration of the respective one or more of the labeled and unlabeled metabolites can also be 15, 20 or 30% or more, of the one or more labeled metabolites from the total concentration of the respective one or more of the labeled and unlabeled metabolites can also be more than 10 %.

[49] Additionally or alternatively, the quality of a sample can be considered low, when only few to none labeled metabolites generated from the labeled compound (I) can be detected in the sample. This means that the ratio of the labeled compound (I) to the compound (II) is decreased compared to the predefined ratio.

[50] Also for determining the presence or absence of the one or more labeled and/or unlabeled metabolites, for example, their level in the sample can be determined. The level can be a measurement of the absolute level. It is also contemplated by the present invention that the level of the one or more labeled metabolites is measured with regard to an external and/or internal standard and/or by using normalization and/or standard addition as described herein. Thus, the determination of the presence or absence can include the determination of the level of the one or more labeled and/or unlabeled metabolites. The determination of the level can also include the determination of the total amount (concentration) of the one or more labeled and/or unlabeled metabolites. The presence or absence/level and/or total amount/concentration of the one or more labeled/unlabeled metabolites can be determined by any technique suitable for this purpose. Exemplary techniques for the purpose are also described herein.

[51] For determining the presence or absence of the compound (I) and/or compound (II), for example, their level in the sample can be determined. The level can be a measurement of the absolute level. Determining the presence or absence of the compound (I) and/or compound (II) can also be done by measuring the relative abundance of compound (I) and/or compound (II) and/or labeled metabolite and/or unlabeled metabolite. The present invention also envisions that the level of the compound (I) and/or compound (II) is measured with regard to an external and/or internal standard and/or by using normalization and/or standard addition as described herein. Thus, the determination of the ratio can include the determination of the level of compound (I) and/or compound (II). The determination of the level can also include the determination of the total amount (concentration) of compound (I) and/or compound (II). The presence or absence/level and/or total amount/concentration of the labeled/unlabeled compound (I) or compound (II) can be determined by any technique suitable for this purpose. Exemplary techniques for this purpose are also described herein.

[52] With the methods, uses and products of the present invention, in principle, any sample can be analyzed, for example, the sample can be a sample, which has been obtained from a subject. The subject can, for example, be an invertebrate, vertebrate or a human being, preferably a human being.

[53] The present invention also contemplates that the sample is a biological sample. It is further envisioned by the present invention that the sample can be used for prognosis and/or diagnosis of disease. For example, a sample obtained from a subject can be used to assign a prognosis or diagnosis for the future occurrence/future non-occurrence or absence/presence of a disease or disorder to that subject. Such prognosis or diagnosis is usually performed by measuring certain biomarkers indicative of a disease or disorder in that sample. Such biomarkers are known to the skilled artesian and for example described in MANOJ KUMAR and SHIV K SARIN “Biomarkers of diseases in medicine” Current Trends in Science, Platinum Jubilee special, p. 403-416 and Ramachandran S. Vasan, MD (2006) “Biomarkers of Cardiovascular Disease" Circulation; 113: 2335-2362.

[54] The sample, can for example be a biological sample. A biological sample can, for example, include a microbial culture, cell culture, a body fluid or a tissue sample. Thus, the sample can be a microbial culture or cell culture or cells. The sample can therefore also be a body fluid sample. Exemplary body fluid samples include samples from blood, cerebrospinal fluid, breath, plasma, feces, serum, urine or saliva. The sample can also be a tissue sample. Exemplary tissue samples include tissue, biopsy or organs. The sample can also be e.g. breath, for the analysis of breath such as volatile, low molecular weight compounds.

[55] Pre-treated samples are also comprised by the term "sample" as used in accordance with the present invention. Suitable and necessary pre-treatments also depend on the technique used for analyzing the sample. The pre-treatment may include treatments required to release or separate the compounds or to remove excessive material or waste. Furthermore, pre-treatments may aim at sterilizing samples and/or removing contaminants such as undesired cells, bacteria or viruses. Suitable pre-treatments comprise centrifugation, extraction, fractioning, ultrafiltration, protein precipitation. Further pre-treatments include filtration and purification and/or enrichment of compounds/metabolites. Moreover, other pre-treatments can be carried out in order to provide the compounds/metabolites in a form or concentration suitable for compound/metabolite analysis.

[56] For example, if gas-chromatography coupled to mass spectrometry is used, it can be required to derivatize the compounds/metabolites prior to the said gas chromatography. Another kind of pretreatment may be the storage of the samples under suitable storage conditions. Storage conditions as referred to herein include storage temperature, pressure, humidity, time as well as the treatment of the stored samples with preserving agents. Suitable and necessary pre-treatments also depend on the techniques used for carrying out sample analysis and are well known to the person skilled in the art. Pre-treated samples as described herein are also comprised by the term "sample" as used in accordance with the present invention.

[57] The present invention contemplates that the sample is contacted with a labeled compound (I) that can be metabolized by the sample and optionally also with a compound (II) which is not metabolized by the sample. The labeled compound (I) may thus be an organic compound or a synthetic compound, which is similar to an organic compound and also becomes metabolized by the sample. The person skilled in the art knows the metabolic pathways that can take place in a sample. Such metabolic pathways are, for example, described by Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002. Molecular Biology of the Cell. 4th edition, e.g. in chapters Catalysis and the Use of Energy by Cells and How Cells Obtain Energy from Food.

[58] Exemplary metabolic pathways include glycolysis (Figures 3 and 4), the tricarboxylic acid (TCA; Figure 5) and urea cycles (Figure 6), glycogen catabolism (Cori and Cori; Figure 7), oxidative phosphorylation, cell respiration and the supremacy of ATP in energy transfer reactions (DeBerardinis and Thompson (2012) “Cellular Metabolism and Disease: What Do Metabolic Outliers Teach Us?” Cell 148; pp. 1132-1144). Therefore, the labeled compound can be a compound that is metabolized in any metabolic pathway. As such the labeled metabolite can, for example, be a metabolic end-product of a metabolic pathway and/or an intermediate present in the metabolic pathway. For example, the labeled compound (I) can be metabolized by glycolysis or gluconeogenesis. In such cases the labeled compound (I) can, for example, be selected from the group consisting of labeled glucose, labeled glucose-6-phosphate, labeled fructose-6-phosphate, labeled fructose-1,6-bisphosphate, labeled glyceraldehyde-3-phosphate, labeled 1,3-bisphosphoglycerate, labeled 3-phosphoglycerate, labeled dihydroxyacetone phosphate, labeled 2-phosphoglycerate, labeled phosphoenolpyruvate, labeled oxaloacetate and/or labeled pyruvate. Accordingly the one or more labeled metabolites can also be selected from the group consisting of labeled glucose-6-phosphate, labeled fructose-6-phosphate, labeled fructose-1,6-bisphosphate, glyceraldehyde-3-phosphate, labeled 1,3-bisphosphoglycerate, labeled 3-phosphoglycerate, labeled dihydroxyacetone phosphate, labeled 2-phosphoglycerate, labeled phosphoenolpyruvate, labeled oxaloacetate and/or labeled pyruvate.

[59] It is also contemplated by the present invention that the labeled compound (I) is metabolized by the TCA cycle. In such cases the labeled compound (I) or the one or more labeled metabolites can, for example, be selected from the group consisting of labeled citrate, labeled cis-aconitate, labeled isocitrate, labeled succinate, labeled alpha-ketoglutarate, labeled succinyl CoA, labeled fumarate, labeled malate and/or labeled oxaloactetate.

[60] It is also contemplated by the present invention that the labeled compound (I) is metabolized by the urea cycle. In such cases the labeled compound (I) or the one or more labeled metabolites can, for example, be selected from the group consisting of labeled glutamate, labeled ammonium, labeled carbamoyl phosphate, labeled citrulline, labeled arginosuccinate, labeled arginine and/or labeled ornithine.

[61] It is also envisioned by the present invention that the labeled compound (I) is metabolized by the Cori cycle. In such cases the labeled compound (I) or the one or more labeled metabolites can, for example, be selected from the group consisting of labeled pyruvate, labeled lactate, labeled lactic acid and/or labeled glucose.The metabolite can also be labeled lactate or labeled lactic acid.

[621 The metabolism of the labeled compound (I) can then be followed because upon biochemical reactions labeled metabolites will be created. The label of the labeled compound (I) can, for example, be an isotopically stable label. Additionally to the labeled compound (I) also the compound (II) as described herein can be labeled, for example, the label of the labeled compound (II) can be an isotopically stable label.

[63] Such isotopically stable labels are known to the person skilled in the art and, for example, described in Gevaert et al. (2008) “Stable isotopic labeling in proteomics” PROTEOMICS Volume 8, Issue 23-24, pages 4873-4885, No. 23-24.

[64] An isotopically stable label is a label that replaces specific atoms by their stable isotope. The shift in the mass in the products due to the stable isotope labeling can then e.g. be measured to determine the sequence the stable isotope followed in the cell's/samples metabolic pathway. It is clear to the person skilled in the art that a labeled compound (I) used in the present invention can be fully labeled, or can be partly labeled. In principle, any isotopically stable label can be used in the present invention. Exemplary isotopic labels include Hydrogen 1H, 2H, Helium 3He, 4He, Lithium 6Li, 7Li, Carbon 12C, 13C, Nitrogen 14N, 15N, Oxygen 16O, 17O, 18O, Fluorine 19F, Sodium 23Na, Magnesium 24Mg, 25Mg, 26Mg, Aluminum 27AI, Phosphorus 31P, Sulfur 32S, 33S, 34S, 36S, Chlorine 35CI, 37CI, Argon 36Ar, 38Ar, 40Ar, Potassium 39K, 41K, Calcium 40Ca, 42Ca, 43Ca, 44Ca, 46Ca, Chromium 50Cr, 52Cr, 53Cr, 54Cr, Iron 54Fe, 56Fe, 57Fe, 58Fe, Cobalt 59Co, Nickel 58Ni, 60Ni, 61Ni, 62Ni, 64Ni, Copper 63Cu, 65Cu, Zinc 64Zn, 66Zn, 67Zn, 68Zn, 70Zn. For example, isotopic labels include one or more of 2H, 13C, and/or 15N, preferably 13C. Furthermore, the labeled compound (I) and/or compound (II) can additionally or alternatively be radioactive (unstable isotope).

However, the labeled compound (I) and/or compound (II) can also be non-radioactive (stable isotope).

[65] In isotopic labeling, there are multiple ways to detect the presence of labeling isotopes; through their mass, vibrational mode, or radioactive decay. Mass spectrometry detects the difference in an isotopomers's mass, while infrared spectroscopy detects the difference in the isotopomers's vibrational modes. Nuclear magnetic resonance detects atoms with different gyromagnetic ratios. The radioactive decay can be detected through an ionization chamber or autoradiographs of gels.

[66] The labeled compound (I) and/or the compound (II) can also be radiolabeled. This means that the nuclides used in isotopic labeling may be stable nuclides or unstable nuclides. A unstable nuclide is an atom that has excess nuclear energy, making it unstable. In principle any unstable isotope can be used in the present invention. Exemplary unstable isotopes include but are not limited to Americium-241, Barium-133, Cadmium-109, Cobalt-57, Cobalt-60, Europium-152, Manganese-54, Sodium-22, Zinc-65, Technetium-99m, Strontium-90, Thallium-204, Carbon-14, Tritium (Hydrogen-3), Polonium-210, Uranium-238, Caesium-137 and/or Gadolinium-153.

[67] The present invention envisions that the labeled compound (I) or the one or more labeled metabolites can be selected from the group consisting of [13C]glucose, [13C]glucose-6-phosphate, [13C]fructose-6-phosphate, [13C]fructose-1,6-bisphosphate, [13C]glyceraldehyde-3-phosphate, [13C]1,3-bisphosphoglycerate, [13C]3- phosphoglycerate, [13C]2-phosphoglycerate, [13C]phosphoenolpyruvate, [13C]oxaloacetate and/or [13C] pyruvate.

[68] In this context it is noted that the person skilled in the art can label basically any compound of interest to provide for a labeled compound (I) and/or labeled compound (II). For example, such a synthesis method is described in Evans (1996) “Synthesis and applications of isotopicaliy labelled compounds 1994” Journal of Labelled Compounds and Radiopharmaceuticals Volume 38, Issue 1, page 103, Evans (1981) “Synthesis of radiolabeled compounds" Journal of Radioanalytical Chemistry, Volume 64, Issue 1-2, pp 9-32 or Dean, et al., ed., John Wiley & Sons, 2004, 522 pp., hard cover, Synthesis and Applications of Isotopicaliy Labelled Compounds volume 8. However, also companies such as SRI International Biosciences provide for any radiolabeled or isotopicaliy stable labeled compound (isotopomer) of interest.

[69] As an alternative to the labeled compound (II), this compound (II) may also be unlabeled. Additionally or alternatively to the labeling or non-labeling, compound (II) can also be a non-naturally occurring compound or a naturally occurring compound. For example, compound (II) can be selected from the group consisting of lipids, amino acids, acylcarnitines or creatinine. Compound (II) may also be a non-endogenous (to the subject or sample) compound.

[70] In principle, the labeled/unlabeled compound (I) as well as the labeled/unlabeled metabolites, but also compound (II) or any other used standard such as e.g. an internal or external standard can all be detected by any means/technique suitable for the detection of such compounds/metabolites. Such means/techniques are well known to the person skilled in the art and, for example, described by Arasaradnam et al. (2014) “Review article: next generation diagnostic modalities in gastroenterology--gas phase volatile compound biomarker detection.” Aliment Pharmacol Ther. 39(8):780-9. Exemplary but non-limiting techniques include chromatographic separation technique, mass spectrometry, nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), Fourier transform infrared analysis (FT-IR), ultraviolet (UV) spectroscopy, light scattering (LS), disperse Raman spectroscopy, flame ionization detection (FID), or a chemical or biological assay.

[71] Mass spectrometry (MS) as used herein encompasses all techniques which allow for the determination of the molecular weight (i.e. the mass) or a mass variable corresponding to a compound/metabolite, to be determined/analyzed in accordance with the present invention. Mass spectrometry can be coupled to different chromatographic techniques. Such chromatographic separation techniques can, for example, be selected from the group consisting of liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography, size exclusion or affinity chromatography, ion exchange chromatography, expanded bed adsorption (EBA) chromatographic separation, reversed-phase chromatography, two-dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography or countercurrent chromatography. The chromatographic separation technique can furthermore be coupled to mass spectrometry.

[72] Also these methods are all known to the person skilled in the art and, for example, described in Gowda and Djukovic (2014) “Overview of Mass Spectrometry-

Based Metabolomics: Opportunities and Challenges” Methods Mol Biol. 1198: 3-12. For example, mass spectrometry may be used in combination of 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 (OEMS), high-performance liquid chromatography coupled mass spectrometry (HPLC-MS), quadrupole mass spectrometry, 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). Thus, mass spectrometry as used herein can relate to GC-MS, LC-MS, direct infusion mass spectrometry, FT-ICR-MS, CE-MS, HPLC-MS, quadrupole mass spectrometry, any sequentially coupled mass spectrometry such as MS-MS or MS-MS-MS, ICP-MS, Py-MS, TOF or any combined approaches using the techniques described herein. Furthermore, for detection of unlabeled/labeled compound (I) and/or compound (II) and/or labeled one or more metabolites LC-MS and/or GC-MS can be used. These techniques are disclosed in, e.g., Nissen 1995, Journal of Chromatography A, 703: 37-57, US 4,540,884 or US 5,397,894.

[73] How to apply these techniques is well known to the person skilled in the art. Moreover, suitable devices are commercially available. Mass spectrometry as used herein can relate to LC-MS and/or GC-MS, i.e. to mass spectrometry being operatively linked to a prior chromatographic separation step. Mass spectrometry as used herein can also encompass quadrupole MS.

[74] Liquid chromatography as described herein refers to all techniques which allow for separation of compounds (i.e. metabolites) in liquid. 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.

[75] Gas chromatography as 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/metabolites will be present in a gaseous volume. The compounds/metabolites 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/metabolite has a specific time which is required for passing through the column.

[76] Moreover, in the case of chromatography such as gas chromatography the compounds/metabolites can be derivatised prior to chromatography. Suitable techniques for derivatisation are well known in the art, for example, derivatisation in accordance with the present invention relates to the substitution of polar functional groups with unpolar or low polarity groups such as methoxyamine and/or trimethylsilylgroups of the target compounds.

[77] 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, fluorescent detection, radiochemical detection, electrochemical detection, light scattering (LS), dispersive Raman spectroscopy or flame ionization detection (FID). These techniques are well known to the person skilled in the art.

[78] The techniques described herein can be assisted by automation, for example, sample processing or pre-treatment can be automated by robotics. Data processing and comparison can be assisted by suitable computer programs and databases. Automation as described herein allows using the method/uses of the present invention in high-throughput approaches.

[79] Moreover, the labeled/unlabeled compound (I) and/or labeled/unlabeled metabolites or compound (II) can also be determined by a specific chemical or biological assay. This assay can comprise means which allow to specifically detect labeled/unlabeled compound (I) and/or labeled/unlabeled metabolites or compound (II) in the sample. Said means can, for example, be capable of specifically recognizing the chemical structure of labeled/unlabeled compound (I) and/or labeled/unlabeled metabolites or compound (II) or are capable of specifically identifying the labeled/unlabeled compound (I) and/or labeled/unlabeled metabolites or compound (II) based on its capability to react with other compounds or its capability to elicit a response in a biological read out system (e.g., induction of a reporter gene). Means which are capable of specifically recognizing the chemical structure of a compound (I) and/or labeled/unlabeled metabolites or compound (II) are, for example, binding proteins such as antibodies or other proteins which specifically interact with chemical structures, such as receptors or enzymes. Specific antibodies, for instance, may be obtained using the biomarker as antigen by methods well known in the art. Suitable antibody and/or enzyme based assays may be RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immune tests, electrochemiluminescence sandwich immunoassays (ECLIA), dissociation-enhanced lanthanide fluoro-immuno assay (DELFIA) or solid phase immune tests.

[80] All of the herein described techniques but also further techniques known to the person skilled in the art may be used to determine the presence or absence and/or the level or amount and/or the concentration of the labeled/unlabeled compound (I) as well as labeled/unlabeled metabolites, but also compound (II) or any other compound. When, for example, analyzing metabolic activity via glycogenesis or glycolysis in a sample, the presence or absence of one or more labeled metabolites can determined by the determination of the presence or absence of one or more of labeled lactate, labeled pyruvate, labeled alanine, labeled glucose-6-phosphate, labeled fructose-6-phosphate, labeled fructose 1,6-bisphosphate, labeled dihydroxyacetone phosphate, labeled glyceraldehyd-3-phosphate, labeled 1,2-bisphosphoglycerate, labeled 3-phosphoglycerate, labeled 2-phosphoglycerate and/or labeled phosphoenolpyruvate, preferably labeled lactate, labeled pyruvate and/or labeled alanine. In such cases labeled compound (I) that has been metabolized can be labeled glucose such as [U13C]-Glucose. However, the labeled compound (I) can also be any other suitable isotopomer, such as 1,2-13C2 Glucose or [U13C]-Ribitol.

[81] Given the techniques that can be used in the present invention the methods and uses of the present invention can further comprise the step of a.1/b.1) extracting the one or more labeled metabolites and/or one or more unlabeled metabolites, extracting labeled compound (I) and/or unlabeled compound (I) and/or extracting compound (II) from the sample.

[82] Additionally or alternatively, the methods/uses of the present invention can further comprise the step a.2/b.2) providing the mass spectrometer with the sample extract.

[83] Additionally or alternatively, the methods/uses of the present invention can further comprise the step a.3/b.3) performing one or more mass spectrometry analysis on the extracted unlabeled and/or labeled compound (I) and/or the extracted one or more labeled and/or unlabeled metabolites and/or extracted compound (II).

[84] Additionally or alternatively, the methods/uses of the present invention can further comprise the step a.4/b.4) performing a chromatographic separation of the extracted unlabeled and/or labeled compound (I) and/or the extracted one or more labeled and/or unlabeled metabolites and/or extracted compound (II).

[85] The chromatographic separation can for example be done on a complex matrix, such as blood in order to separate all compounds with regard to specific physico-chemical characteristics, such as the mass. For a specific compound, the unlabeled and labeled fraction can have the same characteristics.Additionally or alternatively, the methods/uses of the present invention can further comprise the step a.5/b.5) evaluation of results obtained.

[86] When using compound (II) as an internal standard in the methods and uses of the invention for further analysis of the chromatographic and mass spectrometric results, for example, the response factor can be determined. The response factor is known to the person skilled in the art and, for example, described in Jenke and Odufu (2012) “Utilization of Internal Standard Response Factors to Estimate the Concentration of Organic Compounds Leached from Pharmaceutical Packaging Systems and Application of Such Estimated Concentrations to Safety Assessment” J Chromatogr Sci 50 (3): 206-212 and Rome, K. & McIntyre, A. (2012). Intelligent use of Relative Response Factors in Gas Chromatography-Flame Ionisation Detection. Chromatography Today, 52 and IOFI Working group on methods of analysis (2011) “Guidelines for the quantitative gas chromatography of volatile flavouring substances, from the working group on methods of analysis of the international organization of the flavor industry (IOFI)” Flavor and Fragrance Journal 26, 297-299.

[87] In short, the response factor in e.g. chromatography is defined as the ratio between the concentration of a compound/metabolite being analyzed and the response of the detector to that compound/metabolite. A chromatogram will show a response from a detector as a peak. While there are several ways to quantify the peak, one of the most common is peak area, thus:

Response Factor = Peak Area / Concentration.

Ideally, and for easy computation, this ratio is unity (one). To develop calibration curves, one determines the response of analyte compared to that of the internal standard of consistent concentration. Thus, the ratio of the two signals will exhibit less variability than the analyte signal. For example, compound (II) can serve as such internal standard.

[88] Compound (II) as used herein can also be a naturally occurring or non-naturally occurring compound. This compound (II) can be added in a constant or predefined amount to samples.

[89] Compound (II) can, for example, provide a signal that is similar to the analyte signal in most ways but sufficiently different so that the two signals are readily distinguishable by the instrument. In this regard, for example, deuterated compounds (II) can be used in the analysis of volatiles on GC-MS. This is because they are similar to compounds/metabolites to be analyzed but do not occur naturally. Thus, compound (II) can be deuterated. Thus, any suitable deuterated compound can be used as compound (II). Such a deuterated compound can, for example, also be used for the analysis via GC-MS.

[90] As explained herein the compound (I) and optionally compound (II) can be used in the methods and uses of the present invention. It is also envisioned by the present invention that the labeled compound (I) and optionally compound (II) are included in a sample storage device. This means that the labeled compound (I) and optionally the compound (II) are e.g. already comprised in the sample storage device before the sample is obtained, or before the sample is filled in the sample storage device. However, it is also envisioned by the present invention that the sample storage device additionally serves as a device to obtain the probe.

[91] Such a sample storage/sample obtaining device can, for example, be a tube in which the faeces, breath, salvia or urine sample is sampled. It can however also be e.g. a filter paper on which a dried blood spot is obtained, for example, the sample storage device is a container such as a tube. The present invention also contemplates that the sample storage device is a material suitable to obtain dried blood spots such as filter paper, e.g. Whatman filter paper.

[92] Alternatively or additionally, the labeled compound (I) can also be added to the sample, after the sample has been obtained. For example, the labeled compound (I) and optionally the compound (II) can be added to a sample storage device after the sample has been obtained.

[93] The methods of the present invention include that within 5 minutes or less from the time the sample has been obtained, the sample is contacted with the labeled compound (I) that can be metabolized by the sample.

[94] It is also contemplated by the present invention that the sample can be contacted with 1 or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30 or more different labeled compounds (compound (I)) that can be metabolized by the sample). It is further contemplated by the present invention that the sample is first contacted with a first labeled compound (I) and after a period of time with a second labeled compound (I) which is different from the first labeled compound. The present invention also envisions that the labeled compound (I) is added to the sample within less than 240 seconds, 200 seconds, 100 seconds, 90 seconds, 80 seconds, 70 seconds, 60 seconds, 30 seconds, 20 seconds, 10 seconds or less seconds from the time the sample has been obtained. It is further envisioned by the present invention that the compound (compound (I) and/or compound (II)) is applied at the same time to the sample at which the sample is obtained.

[95] It is also envisioned that the sample can additionally be contacted with compound (II). However, the contacting with compound (II) may be performed e.g. shortly before analysis is started. The time point when analysis is performed can however be much more than 5 minutes from the time at which the sample has been obtained. This is because, for example, a sample can be frozen or freeze dried after it has been obtained. Usually such a sample will become defrosted before it will be analyzed. The time period in which the sample has been frozen can be more than 5, 10, 60, 120, 240 minutes and can even be more than 1,2, 3, 4, 5 or more days.

[96] On the other hand, the sample can be contacted simultaneously with compound (II) and the labeled compound (I). It is also envisioned by the present invention that the sample is sequentially contacted by compound (II) and labeled compound (I) or vice versa. Thus, compound (II) can be added to the sample within less than 600 seconds, 500 seconds, 400 seconds, 300 seconds, 240 seconds, 200 seconds, 100 seconds, 90 seconds, 80 seconds, 70 seconds, 60 seconds, 30 seconds, 20 seconds, 10 seconds or less seconds from the time the sample has been obtained. Additionally or alternatively, the sample can be contacted with 1 or more than 1,2,3,4 5,6, 7,8,9, 10, 11, 12, 13, 14, 15, 20, 30 or more compounds (II) that cannot be metabolized by the sample (compound (II)).

[97] The methods of the present invention can furthermore include that the method is a method for determining the time period of metabolic activity of a sample, the method further comprising c. correlating the determined presence of absence of one or more labeled metabolites to a predetermined standard, wherein the correlation determines the time period of metabolic activity of the sample.

[98] The methods of the present invention can furthermore include that the method is a method for determining the time period of metabolic activity of a sample, the method further comprising d. correlating the determined ratio to a predetermined standard, wherein the correlation determines the time period of metabolic activity of the sample.

[99] The determining of the time period of metabolic activity of a sample means, for example, that the metabolic activity can have taken place for a period of time. In principle, the sample can have been metabolically active for any period of time, for example, the period can be a time of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or more minutes. The lesser the period of time the sample has been metabolically active, the higher is the quality of the sample. On the other hand, the higher the period of time the sample has been metabolically active, the lower is the quality of the sample.

[100] The methods/uses of the present invention for determining the time period of metabolic activity of a sample can include obtaining a sample of interest. This sample (of interest) can be a sample in which an unknown period of time of metabolic activity has taken place from the time the sample has been obtained to e.g. the time of analysis. The sample (of interest) can, for example, be analyzed with the techniques as described herein such as GC-MS-or LC-MS Also more than one sample (of interests) can be analyzed at the same time.

[101] The result(s) obtained by the analysis of the sample (of interest) are then correlated to a predetermined standard. This also means that data obtained for the sample (of interest) can be compared to the predetermined standard. Suitable predetermined standards are known to the person skilled in the art. Any suitable predetermined standard can be used in accordance with the present invention.

[102] It is also envisioned by the present invention that the predetermined standard is a calibration such as alinear regression. Such a calibration in form of a linear regression can be determined as described in the Example and figure legends as described herein. For example, a linear regression can be determined by providing a sample which is contacted with labeled compound (I). At different time points such as e.g. 1, 5, 10, 15, 30, 60 and 120 minutes or more minutes a certain amount of the sample can be analyzed. Additionally, before contacting the sample with compound (I) an amount of the sample can be analyzed to provide for a time point 0. It is further envisioned by the present invention that the sample for the (calibration) linear regression is measured with the same technique as the sample (of interest). The different obtained data points which can be at least 3, 4, 5, 6, 7, 8, 9, 10 or more data points or time points after the sample has been contacted with the labeled compound (I) can then be plotted as the enrichment of one or more metabolites and/or labeled compound (I) and/or compound (II) over time. Then a linear regression can be drawn connecting the data points.

[103] This generated (calibration) linear regression can then be used to predict/determine the metabolic activity that has taken place in a sample (of interest). To achieve that the sample (of interest) can be analyzed. The measured result for the sample (of interest) can then be correlated or compared to the (calibration) linear regression as described herein. From this comparison/correlation the time the sample (of interest) has been metabolically active can be predicted/determined. Therefore, the present invention envisions that the predetermined standard is a correlation of the time period of metabolic activity to the presence of one or more labeled metabolites as described herein. The present invention also envisions that the predetermined standard is a correlation of the presence of one or more labeled metabolites to a (calibration) linear regression.

[104] Additionally or alternatively, the present invention contemplates that the predetermined standard is a calibration such as a linear regression, which is generated by the determination of the ratio between compound (I) and compound (II) over time. For example, such a (calibration) linear regression can be determined by providing a sample which is contacted with the labeled compound (I) and a known amount of compound (II). At different time points such as e.g. 1,5, 10, 15, 30, 60 and 120 minutes or more minutes a certain amount of the sample can be analyzed. Additionally, before contacting the sample with labeled compound (I) a certain amount of the sample can be analyzed to provide for a time point 0. The different obtained data points which can be at least 3, 4, 5, 6, 7, 8, 9, 10 or more data points (ratios between compound (I) and compound (II)) can then be plotted as the ratio change of compound (I) and/or compound (II) overtime. Then a linear regression can be drawn connecting the data points in the best way. It is further envisioned by the present invention that the sample for the (calibration) linear regression is measured with the same technique as the sample (of interest).

[105] Also such a generated (calibration) linear regression can then be used to predict/determine the metabolic activity that has taken place in a sample (of interest). To achieve that the sample (of interest) can be analyzed. The measured ratio of labeled compound (I) to compound (II) for the sample (of interest) can then be correlated or compared to data/ratios obtained for the (calibration) linear regression as described herein. From this comparison/correlation the time the sample (of interest) has been metabolically active can be predicted/determined. Therefore, the present invention envisions that the predetermined standard is a correlation of the time period of metabolic activity to the ratio of the labeled compound (I) to the compound (II). The present invention also envisions that the predetermined standard is a correlation of the ratio of the labeled compound (I) to the compound (II) to a (calibration) linear regression.

[106] However, a calibration such as a linear regression as described herein can also be obtained when comparing a predefined concentration/level/amount of the labeled compound (I), with which the sample is contacted, with the concentration/level/amount of the labeled compound (I) after contacting the sample. The comparison/correlation of the analysis result of the sample (of interest) to this (calibration) linear regression can then predict/determine the time the sample (of interest) has been metabolically active. Therefore, the present invention also envisions that the predetermined standard is a correlation of the time period of metabolic activity to the concentration/level/amount the labeled compound (I) measured in the sample to the predefined concentration/level/amount of the labeled compound (I). Therefore, the present invention also envisions that the predetermined standard is a correlation of concentration/level/amount of the labeled compound (I) measured in the sample to the predefined concentration/level/amount of the labeled compound (I), which can e.g. be in the form of a (calibration) linear regression.

[107] It is also envisioned by the present invention that the prediction/determination predicts/determines the time period of metabolic activity with a precision of +/- 20, +/- 15, +/-10 minutes, +/- 8 minutes, +/- 5 minutes, +/- 3 minutes, +/-1 minute.

[108] The present invention also relates to the use of a labeled compound (I), as described herein, for determining the quality of a sample. Notably, the different features as described for the methods of the present invention also apply mutatis mutandis to the uses and products of the present invention.

[109] The quality of the sample may thus be determined by the determination if the sample has been metabolically active. Thus, the present invention also relates to a use of a labeled compound (I) as described herein for determining the metabolic activity of a sample. The quality may also be determined by the determination of the (period of) time the sample has been metabolically active.

[110] The uses of the present invention can further comprise the use of a compound (II) as described herein.

[111] The present invention also provides for a use of a labeled compound (I) as described herein, for reducing false-positive or false-negative results obtained from a sample. This is because the quality outcome of a research project or clinical diagnosis is strongly correlated with the quality of the sample it is based on. However, many clinical diagnosis laboratories and research institutes do not know the quality of their samples which leads to wrongly interpreted data or diagnosis. By determining the quality of a sample, thus the misinterpretation of results obtained from samples can be reduced. Also this use may further comprise the use of a compound (II) as described herein.

[112] In principle there are two ways in which the sample can be contacted by the labeled compound (I) and optionally the compound (II). On the one hand the sample can be contacted with these compounds after the sample has been obtained as described herein. On the other hand the compounds can already be comprised in the sample storage device. In this way the sample is contacted with the compound at the time of sampling. Such a sample storage device is also described elsewhere herein.

[113] Accordingly, the present invention also relates to a sample storage device, wherein the sample storage device comprises a labeled compound (I) as described herein, and optionally further comprises a compound (II) as described herein. Additionally or alternatively, the sample storage device can further comprise a stabilizer, such as e.g. EDTA or Li-heparin. The sample storage device may further be used for determining the quality of a sample. Such sample can for example be used for prognosis and/or diagnosis of disease.

[114] In addition, the present invention also relates to a filter paper suitable for dried blood spot sampling, wherein the filter paper comprises a labeled compound (I) as described herein. The filter paper can further comprise a compound (II) as described herein. The filter paper may additionally or alternatively comprise a stabilizer such as EDTA. The filter paper may further be used for determining the quality of a sample. Such samples can for example be used for prognosis and/or diagnosis of disease.

[115] Furthermore, the present invention relates to a vial or container suitable for sampling, wherein the vial or container comprises a labeled compound (I) as described herein. The vial or container may further comprise a compound (II) as described herein. The vial or container may additionally or alternatively comprise a stabilizer such as EDTA. The vial or container may be used for determining the quality of a sample. Such sample can for example be used for prognosis and/or diagnosis of disease.

[116] The present invention is further characterized by the following items: [117] 1. Method for determining the metabolic activity that has taken place in a sample, the method comprising a. within 5 minutes or less from the time the sample has been obtained, contacting the sample with a labeled compound (I) that can be metabolized by the sample; and b. determining presence or absence of one or more labeled metabolites of the labeled compound, wherein the determination of the presence or absence of the one or more metabolites is indicative of the metabolic activity that has taken place in the sample.

[118] 2. Method for determining the metabolic activity that has taken place in a sample, the method comprising a. within 5 minutes or less from the time the sample has been obtained, contacting the sample with a labeled compound (I) that can be metabolized by the sample; and b. contacting the sample with a compound (II), which cannot be metabolized by the sample; wherein the ratio between the labeled compound (I) and compound (II) is a predefined ratio; and c. determining the ratio between the labeled compound (I) and compound (II) (labeled compound (l):compound (II)), wherein the determination of the ratio is indicative of the metabolic activity that has taken place in the sample.

[119] 3. Method of item 1, wherein the method is a method for determining the time period of metabolic activity of a sample, the method further comprising c. correlating the determined presence of absence of one or more labeled metabolites to a predetermined standard, wherein the correlation predicts and/or determines the time period of metabolic activity of the sample.

[120] 4. Method of item 2, wherein the method is a method for determining the time period of metabolic activity of a sample, the method further comprising d. correlating the determined ratio to a predetermined standard, wherein the correlation predicts and/or determines the time period of metabolic activity of the sample.

[121] 5. Method of item 3 or 4, wherein the metabolic activity has taken place for a period of time.

[122] 6. Method of item 5, wherein the period of time is a time of about 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or more minutes.

[123] 7. Method of any one of claims 1-6, wherein the sample is of quality when the sample has experienced insignificant metabolic activity.

[124] 8. Method of claim 7, wherein the insignificant metabolic activity comprises the detection of insignificant or no presence of one or more labeled metabolites in said sample.

[125] 9. Method of claim 7 or 8, wherein the fraction of the one or more labeled metabolites from the total concentration of one or more of the labeled and unlabeled metabolites is about 0 %, 0,001%, 0.005%, 0.01%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%i19%l20% or more. It is further envisioned that the fraction of the one or more labeled metabolites from the total concentration of the one or more of the labeled and unlabeled metabolites is maximally 5%, 7%, 10%, 12%, 15% or 20%..

[126] 10. Method of item 7, wherein the ratio of the labeled compound (I) and the compound (II) is approximately the same as the predefined ratio.

[127] 11. Method of any one of items 1-6, wherein the sample is of low quality, when the sample has experienced significant metabolic activity.

[128] 12. Method of item 11, wherein the significant metabolic activity comprises the detection of the significant presence of one or more labeled metabolites in said sample.

[129] 13. Method of item 11 or 12, wherein the fraction of the one or more labeled metabolites from the total concentration of one or more of the labeled and unlabeled metabolites is more than 20%, 30%, 40%, 50%, 60%, 70%, 80% or more.

[130] 14. Method of item 11, wherein the ratio of the labeled compound (I) to the compound (II) is decreased compared to the predefined ratio.

[131] 15. Method of any one of items 1-14, wherein the determination of the presence or absence includes the determination of the level of the one or more labeled and/or unlabeled metabolites and/or wherein the determination of the level includes the determination of the total amount of the one or more labeled and/or unlabeled metabolites and/or the total amount of the labeled compound (I) and/or compound (II).

[132] 16. Method of any one of claims 1-15, wherein the sample has been obtained from a subject.

[133] 17. Method of item 16, wherein the subject is an animal, preferably a vertebrate or a human being.

[134] 18. Method of any one of items 1-17, wherein the sample is a microbial culture or cell culture.

[135] 19. Method of any one of items 1-17, wherein the sample is a body fluid sample or a tissue sample.

[136] 20. Method of item 19, wherein the body fluid sample is selected from the group consisting of blood, cerebrospinal fluid, breath, plasma, serum, feces, urine or saliva.

[137] 21. Method of item 19, wherein the tissue sample is selected from the group consisting of tissue, biopsy, cells or organs.

[138] 22. Method of any one of items 1-21, wherein the labeled compound (I) and/or the compound (II) is isotopically stably labeled.

[139] 23. Method of item 22, wherein the stable isotope comprises one or more of 2H, 13C, and/or 15N, preferably 13C.

[140] 24. Method of item 22 or 23, wherein the labeled compound is [13C]glucose, [13C]glucose-6-phosphate, [13C]fructose-6-phosphate, [13C]fructose-1,6-bisphosphate, [13C]glyceraldehyde-3-phosphate, [13C]1,3-bisphosphoglycerate, [13C]3- phosphoglycerate, [13C]2-phosphoglycerate, [13C]phosphoenolpyruvate, [13C]oxaloacetate and/or [13C] pyruvate.

[141] 25. Method of any of items 1-24, wherein the compound (II) is a non-naturally occurring compound or a naturally occurring compound.

[142] 26. Method of item 25, wherein the compound (II) is labeled or non-labeled.

[143] 27. Method of any one of items 1-26, wherein compound (II) is selected from the group consisting of lipids, amino acids, acylcarnitines or creatinine.

[144] 28. Method of any one of items 1-27, wherein determining the presence or absence and/or the determination of the level is performed by a chromatographic separation technique, mass spectrometry, nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), Fourier transform infrared analysis (FT-IR), ultraviolet (UV) spectroscopy, light scattering (LS), disperse Raman spectroscopy, flame ionization detection (FID), a chemical and/or biological assay.

[145] 29. Method of item 28, wherein the chromatographic separation technique is selected from the group consisting of liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography, size exclusion or affinity chromatography, ion exchange chromatography, expanded bed adsorption (EBA) chromatographic separation, reversed-phase chromatography, two-dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography and/or countercurrent chromatography.

[146] 30. Method of item 29, wherein the chromatographic separation technique is coupled to a mass spectrometry analysis.

[147] 31. Method of any one of items 1-30, wherein the presence or absence of one or more labeled metabolites is determined by the determination of the presence or absence of one or more of labeled lactate, labeled pyruvate, labeled alanine, labeled glucose-6-phosphate, labeled fructose-6-phosphate, labeled fructose 1,6-bisphosphate, labeled dihydroxyacetone phosphate, labeled glyceralaldehyd-3-phosphate, labeled 1,2-bisphosphoglycerate, labeled 3-phosphoglycerate, labeled 2-phosphoglycerate and/or labeled phosphoenolpyruvate, preferably labeled lactate, labeled pyruvate and/or labeled alanine.

[148] 32. Method of any one of items 1-31, wherein the labeled compound (I) is included in a sample storage device.

[149] 33. Method of any one of items 1-31, wherein the labeled compound (I) is added to a sample storage device after the sample has been obtained.

[150] 34. Method of item 33, wherein the labeled compound is added to the sample within 200, 100, 90, 80, 70, 60 or less seconds from the time the sample has been obtained.

[151] 35. Method of item 32 or 33, wherein the sample storage device is a material suitable to obtain dried blood spots such as filter paper.

[152] 36. Method of item 32 or 33, wherein the sample storage device is a container such as a tube.

[153] 37. Method of any one of claims 2-36, wherein the predetermined standard is a correlation of the time period of metabolic activity to the presence of one or more labeled metabolites or to the ratio of the labeled compound (I) to the compound (II).

[154] 38. Method of any one of claims 2-37, wherein the prediction/determination predicts/determines the time period of metabolic activity with a precision of +/- 10 minutes, +/- 8 minutes, +/- 5 minutes, +/- 3 minutes, +/-1 minute.

[155] 39. Use of a labeled compound (I), as defined in any one of items 1, 2 or 22-24, for determining the quality of a sample.

[156] 40. Use of item 39, wherein the use further comprises a compound (II) as defined in any one of items 2, 22, 25-27.

[157] 41. Use of a labeled compound as defined in any one of items 1, 2 or 22-24 for determining the metabolic activity of a sample.

[158] 42. Use of item 41, wherein the use further comprises a compound (II) as defined in any one of items 2, 22, 25-27.

[159] 43. Use of a labeled compound as defined in any one of items 1, 2 or 22-24, for reducing false-positive or false-negative results obtained from a sample.

[160] 44. Use of item 43, wherein the use further comprises a compound (II) as defined in any one of items 2, 22, 25-27.

[161] 45. Sample storage device, wherein the sample storage device comprises a labeled compound (I) as defined in any one of items 1, 2 or 22-24, optionally further comprising a compound (II) as defined in any one of items 2, 22, 25-27.

[162] 46. Filter paper suitable for dried blood spot sampling, wherein the filter paper comprises a labeled compound (I) as defined in any one of items 1,2 or 22-24.

[163] 47. Filter paper of item 46, wherein the filter paper further comprises a compound (II) as defined in any one of items 2, 22, 25-27.

[164] 48. Filter paper of item 46 or 47 or sample storage device of item 45 for determining the quality of a sample.

[165] 49. Vial suitable for sampling, wherein the vial comprises a labeled compound (I) as defined in any one of items 1, 2 or 22-24.

[166] 50. Vial of item 49, wherein the vial further comprises a compound (II) as defined in any one of items 2, 22, 25-27.

[167] 51. Vial of item 49 or 50 for determining the quality of a sample.

[168] 52. Method for determining the quality of a sample, the method comprising a. contacting the sample with a labeled compound that can be metabolized by the sample; wherein the contacting is performed in less than 5 minutes from which the sample has been obtained; and b. determining presence or absence of one or more labeled metabolites of the labeled compound, wherein the determination of the presence or absence of the one or more metabolites indicates the quality of the sample.

[169] 53. Method for determining the quality of a sample, the method comprising a. within 5 minutes or less from the time the sample has been obtained contacting the sample with a labeled compound (I) that can be metabolized by the sample; and b. contacting the sample with a compound (II), which cannot be metabolized by the sample; wherein the ratio between compound (I) and compound (II) is a predefined ratio; and c. determining the ratio between compound (I) and compound (II) (labeled compound (l):compound (II)), wherein the determination of ratio indicates the quality of the sample.

[170] 54. Method for determining the time period of metabolic activity of a sample, the method comprising a. contacting the sample with a labeled compound that can be metabolized by the sample; b. determining presence or absence of one or more labeled metabolites of the labeled compound; c. correlating the determined presence of absence of one or more labeled metabolites obtained in step b. to a predetermined standard, wherein the correlation determines the time period of metabolic activity of the sample.

[171] 55. Method for determining the time period of metabolic activity of a sample, the method comprising a. within 5 minutes or less from the time the sample has been obtained contacting the sample with a labeled compound (I) that can be metabolized by the sample; and b. contacting the sample with a compound (II), which cannot be metabolized by the sample; wherein the ratio between compound (I) and compound (II) is a predefined ratio; and c. determining the ratio between compound (I) and compound (II) (labeled compound (l):compound (II)), d. correlating the determined level of the labeled compound obtained in step b. to a predetermined standard, wherein the correlation determines the time period of metabolic activity of the sample.

[172] 56. A method for determining the metabolic activity of cell-containing biological samples obtained from a test subject, the method comprising a. contacting the sample with a labeled compound, that can be metabolized by the sample; and b. determining the metabolic turnover of said labeled compound.

EXAMPLES

[173] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims. EXAMPLE 1 [174] Experimental design

The donors were asked to provide blood via a simple finger prick into a test tube containing EDTA. Directly after the blood collection, metabolites are extracted from a small volume (TO). Subsequently, a small amount of [U13C]Glucose solution is pipetted onto the remaining blood and the blood tube is then incubating at 37°C with slow shaking. At regular time points (15, 30, 60 and 120 min), a small amount of blood is taken out of the tube for metabolite extraction.

The metabolite extracts are measured via gas chromatography coupled to mass spectrometry (GC-MS) and analyzed with the MetaboliteDetector software.

[175] Results

During the incubation, the blood cells (e.g. red blood cells) will produce lactic acid out of glucose via glycolysis. Therefore, the levels of lactic acid are increasing over time. In order to monitor the lactic acid production outside the human body, i.e. due to environmental and pre-analytical conditions, an uniformly labeled glucose tracer ([U13C]Glucose) is added to the blood. The advantage of 13C labeled compounds is that the enzyme activity and the metabolite fluxes will not be altered.

Over time, blood cells will take up the labeled compound and metabolize it to lactic acid which in turn will also be uniformly labeled. On figure 1, the enrichment (fraction of the labeled compound from the total concentration of the compound) of the formed lactic acid is shown over time for 2 different donors.

[176] Both donors showed a similar increase of labeled lactic acid over 2h. Furthermore, based on the pre-processing time and enrichment data from donor 1, the pre-processing time of the blood samples from donor 2 could be predicted (Figure 2/Table 1).

[177] It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[178] All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[179] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[180] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or sometimes when used herein with the term “having”.

[181] When used herein “consisting of excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[182] In each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms.

[183] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[184] When used herein, the term "about" is understood to mean that there can be variation in the respective value or range (such as pH, concentration, percentage, molarity, number of amino acids, time etc.) that can be up to 5%, up to 10%, up to 15% or up to and including 20% of the given value. For example, if a formulation comprises about 5 mg/ml of a compound, this is understood to mean that a formulation can have between 4 and 6 mg/ml, preferably between 4.25 and 5.75 mg/ml, more preferably between 4.5 and 5.5 mg/ml and even more preferably between 4.75 and 5.25 mg/ml, with the most preferred being 5 mg/ml. As used herein, an interval which is defined as “(from) X to Y” equates with an interval which is defined as “between X and Y”. Both intervals specifically include the upper limit and also the lower limit. This means that, for example, an interval of “5 mg/ml to 10 mg/ml” or “between 5 mg/ml and 10 mg/ml” includes a concentration of 5, 6, 7, 8, 9, and 10 mg/ml as well as any given intermediate value.

REFERENCES

Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002. Molecular Biology of the Cell. 4th edition, e.g. in chapters Catalysis and the Use of Energy by Cells and How Cells Obtain Energy from Food.

Arasaradnam RP, Covington JA, Harmston C, Nwokolo CU. Review article: next generation diagnostic modalities in gastroenterology--gas phase volatile compound biomarker detection. Aliment Pharmacol Ther. 2014 Apr;39(8):780-9.

Dean, et al., ed., John Wiley & Sons, 2004, 522 pp., hard cover, Synthesis and Applications of Isotopically Labelled Compounds volume 8.

Dennis Jenke and Alex Odufu (2012) “Utilization of Internal Standard Response Factors to Estimate the Concentration of Organic Compounds Leached from Pharmaceutical Packaging Systems and Application of Such Estimated Concentrations to Safety Assessment” J Chromatogr Sci 50 (3): 206-212

Evans (1996) “Synthesis and application s of isotopically labelled compounds 1994” Journal of Labelled Compounds and Radiopharmaceuticals Volume 38, Issue 1, page 103

Evans (1981) “Synthesis of radiolabelled compounds” Journal of Radioanalytical Chemistry, Volume 64, Issue 1-2, pp 9-32 or

Gowda and Djukovic (2014) “Overview of Mass Spectrometry-Based Metabolomics: Opportunities and Challenges” Methods Mol Biol. 1198: 3-12 IOFI Working group on methods of analysis (2011) “Guidelines for the quantitative gas chromatography of volatile flavouring substances, from the working group on methods of analysis of the international organization of the flavor industry (IOFI)” Flavor and Fragrance Journal 26, 297-299

Kris Gevaert, Francis Impens, Bart Ghesquière, Petra Van Damme, Anja Lambrechts and Joël Vandekerckhove (2008) “Stable isotopic labeling in proteomics” PROTEOMICS Volume 8, Issue 23-24, pages 4873-4885, No. 23-24 MANOJ KUMAR and SHIV K SARIN “Biomarkers of diseases in medicine” Current Trends in Science, Platinum Jubilee special, p. 403-416

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Ralph J. DeBerardinis and Craig B. Thompson (2012) “Cellular Metabolism and Disease: What Do Metabolic Outliers Teach Us?” Cell 148; pp. 1132-1144

Ramachandran S. Vasan, MD (2006) “Biomarkers of Cardiovascular Disease” Circulation; 113: 2335-2362.

Rome, K. & McIntyre, A. (2012). Intelligent use of Relative Response Factors in Gas Chromatography-Flame Ionisation Detection. Chromatography Today, 52

Claims

A method of determining the substance change rate found in a sample. the method comprising a, within 5 minutes each less of the time at which the sample is obtained, confectioning the sample with a labeled compound (i) that can be metabolized to the sample; and b, bestem of presence or absence. of one or more labeled metabolic products of the labeled compound, wherein determining the presence or absence of the one or more labeled storfal products indicates the metabolic activity found in the sample;

2. A method for determining the metabolic activity found in a sample, the method comprising a. within 5 minutes or less from the time the sample is obtained, contacting the sample with a labeled compound (I) that can be metabolized by the sample; and b. Contacting the sample with a compound (II) which can not be metabolized by the sample; wherein the ratio between the labeled compound (I) and the compound (H) is a predefined ratio; and e, determining the ratio between the labeled compound (I) and the compound (II) (labeled compound (1): compound (ii)), wherein the determination of the ratio to the change in activity required in the sample indicates that

3. The method of claim 1, wherein the method is a method for determining the time duration of metabolic activity of a sample, the method comprising c, correcting the determined presence or absence of one or more labeled tissue intermediates having a predetermined level, the correction Period of stoichiographic activity in the sample is predicted and / or determined

4. The method of claim 2, wherein the method is a method of determining the period of time of metabolic activity of a sample, wherein the method comprises temperature d. Kerreiieren the determined ratio with a predetermined standard, the Korreiation predicts and / or determines the period of the Stofiweohaktaktivet in the sample,

5, The method of any of claims 1-4, wherein the sample is of quality if the sample has negligible mass transfer activity. 6. The method of claim 5, wherein the susceptible Stoftwechselaktlvltät the detection of unwesentellcher or kelner presence before? comprising one or more labeled sweetener egg products in the sample,

A method according to claim 5, wherein the ratio of the labeled compound (I) and the compound (U) is about equal to the rate of interpolation.

8. A method according to any one of claims 1-4, wherein the sample is of low quality if the sample has undergone significant metabolic activity.

9. The method of claim 8, wherein the significant metabolic activity comprises detecting the significant presence of one or more labeled suspended tissue products in the sample.

A method according to claim 8, wherein the ratio of the labeled compound (I) to the compound (li) is reduced from the predefined ratio,

A method according to any one of claims 1 to 10, wherein the sample has been obtained from a subject.

12. The method of claim 11, wherein the subject is an animal, preferably a vertebrate or a human,

13. The method according to any one of claims 1-10, wherein the sample is a mikrobakfedelie Kuitur or Zelikuilur.

14. The method according to any one of claims 1-10. wherein the sample is a body fluid sample or a tissue sample.

The method of claim 14, wherein the body fluid sample is selected from the group consisting of slut, cerebrospinal fluid, serum, plasma, serum, stool, urine or saliva.

16. The method according to any one of claims 1-15, wherein the labeled compound (I) and / or the compound (il) is marked isolopically stable,

17. The method of claim 18, wherein the stable isotope comprises one or more of H,! JC and / or preferably S5C

18. The method according to any one of claims 1-17, wherein the compound (II) is a nlcht-naîüdich occurring compound or a natüdich occurring compound.

19. The method according to claim 18, wherein the compound (li) is marked or unmarked 1st

20. The method according to any of claims 1-19, wherein determining the presence or absence and / or the determination of the level with a chromatographic separation technique, mass spectrometry, magnetic nuclear magnetic resonance (NMR), magnetic resonance imaging (MR!) Founer transferm infrared analysis (FT-IR), ultraviolet (UV) spectroscopy, light scattering (LS), disperse rarity spectroscopy, piammionization detection (FfD), a chemical and / or biological examination,

21, A method according to any one of claims 1-20. whereby the labeled compound is sampled within 200, 100, 90: 60, 70, 60 or less after the sample is obtained. The method of claim 3, wherein the predetermined strain is a correlation of the metabolic duration of the metabolic activity to the presence of one or more labeled staging products or the ratio of the labeled compound (I) to the compound (II).

23. Use of a labeled compound (I) as defined in any one of claims 1, 2, 16 or 17 for determining the quality of a sample.

24. Use of a labeled compound (I) as defined in any one of claims 1, 2,16 Eder 17, for determining the metabolism activity of a sample.

Use of a labeled compound (I) as defined in any one of claims 1, 2, 16 or 17 for reducing fungal-positive or false-negative results obtained from a sample.

26. Sample storage device. wherein the sample storage assay comprises a labeled compound (I) as defined in any of claims 1, 2, 10 or 17, optionally further comprising a compound (II) as defined in any one of claims 2, 15-1),

27. A paper towel suitable for dry blood sampling, the filter paper comprising a labeled compound (i) as defined in any of claims 1, 2, 16 or 17, optionally further comprising a compound (II) as in any of claims 2, 16 -19 defined.

A bottle suitable for sampling, the bottle comprising a labeled compound (I) as defined in any one of claims 1, 2, 16 or 17, optionally further comprising a compound (II) as claimed in any one of claims 2 to 10. 19 defined.

29, FSäschchen according to claim 28, filter adapter according to claim 27 or ProbenSagerungsvornchtung according to claim 26 for determining the Quahtät a sample.