of biological samples

The described invention relates to a method to control and validate the quality of quantitative chemical analysis of complex sample mixtures comprising various kinds of analytical instruments by using a ready to use carrier matrix containing a number of analytes embedded in a natural or synthetic polymer.

Moreover, the invention allows the comparison of different experimental batches over time in a close to matrix environment.

Background of the invention

Quality control is important to deliver robust results in analytics. Extraction conditions, sample handling and machine sensitivity can influence the results. The variations make sample to sample and experiment to experiment comparisons difficult.

The state of the art is currently to run a defined sample with each batch

measurement. This sample can contain a number of compounds, which are naturally also occurring in the sample set. This quality control sample is often made by mixing a number of defined chemicals at a given concentrationin a solvent (such as water or methanol) as described in Schiesel et al., Anal BioanalChem (2010) 396, 1655-1679 or Wang et al., Anal BioanalChem (2010) 396, 1513-1538. This way also calibration curves to quantify defined compounds can be generated. However, the sample generation according to this state of the art is quite time consuming for the individual lab. Furthermore, the exact composition of the solution containing the variety of chemicals may change over time, e.g. by way of degradation/decomposition of ingredients, interaction of ingredients or by solvent evaporation.

A common problem in biological sample analysis is the exact determination and quantification of the analyte in question. This is due to a different behavior of the analyte in its natural biological matrix over its synthetically generated matrix as described for the quality control sample. Common problems are shifts in retention time, purity of mass spectra, ion adducts and suppression or compound

concentrations as well as interaction amongst two or more compounds to be analyzed. _ .

Recently, quality control samples from biological extracts are being used in identification of compounds in complex samples. These quality control samples are often extracted from a large amount of biological samples e. g. tomato, ketchup, bacteria, yeast or any other type of biological material. This is produced at one time point and then stored. An aliquot is taken with every new sample set. Often this sample has been homogenized and extracted at a defined time point and thus needs to be injected prior, during or post analysis. This so-called bulk quality control sample has the advantage to resemble the same or close biological material being analyzed. Thus some of the problems described in quantifying and comparing the biological samples can be solved. Nevertheless, the interaction of the quality control sample with the environment, that is solvent evaporation, degradation or reaction with each other cannot be stopped.

The bulk quality control sample has a few drawbacks itself. One of the most important ones is the biological variation of the material. Once it is used up another set has to be prepared. Thus new variation is being introduced and has to be normalized over the experiments between different bulk quality control samples. The storage of bulk quality control samples introduces variation as well, as compounds can be degraded or form by-products. These limitations will not allow doing relative quantifications on the bulk quality control sample or only with labor intensive validation of stability of the sample. Absolute quantification can only be done by using calibration standards with exact concentrations and adding those standard

compounds to the bulk quality control sample or biological sample. Here it needs to be validated whether the concentration of the natural compound in the bulk quality control sample does not vary. Object of the invention

It is therefore an object of the current invention to improve methods of quantitative chemical analysis, especially of complex sample mixtures such as biological entities. It is an object of the invention to overcome the problems of existing methods such as the biological variation of the bulk material, changes due to storage of the materials and the need for calibration standards. Brief description of the invention

The current invention relates to a new quantification process without the problems described above.

It has been found that

a process for parallel quantitative analysis of 10 or more inorganic or organic compounds in a sample, characterized in that,

a carrier matrix comprising

10 or more inorganic or organic compounds to be analysed each compound in a defined amount

embedded in a natural or synthetic polymer

is quantitatively analysed in addition to said sample and the measured results of the sample and the carrier matrix are compared to each other,

is able to overcome the problems described above.

The process is characterized in that a comparison is made between the actual sample which is to be quantitatively analyzed and a carrier matrix containing 10 or more inorganic or organic compounds in a defined amount.

The carrier matrix may essentially be a natural or synthetic polymer, such as starch, gelatin, pectin, polyethylene, polyethyleneglycole, polyorganosiloxane (silicons), polysaccharides, cellulose, DNA, RNA, polyhydroxalkanoate, polypropylene, polyacrylamide or a mixture thereof.

Said carrier matrix may be in the form of a pill, a capsule, a cube, granulate or any other physical form. The term carrier matrix also includes hollow containers filled with the analyte. The carrier matrix may also include material from a biological entity such as a potato, tomato, blood plasma, urine, microbes and so forth. As explained below under certain circumstances it might be helpful to process two or more different carrier matrices which may be of the same or of different form and/or composition. It might e.g. be helpful to use a gelatin cube (125 mm ³ ) together with a hollow body made of polyethylene containing N-Methyl-N-(trimethylsilyl)trifluoroacetamid.

It has to be understood that the carrier matrix according to the present invention is different from the techniques mentioned in certain prior art documents such as Schiesel et al., Anal BioanalChem (2010) 396, 1655-1679 or Wang et al., Anal BioanalChem (2010) 396, 1513-1538. The "matrix -matched calibration standards" . . mentioned in these documents do consist of a simple solution containing a variety of compounds in a defined quantity. Accordingly, the "matrix -matched calibration standards" are not ready to use, not simple to use and in particular not protected from degradation/decomposition of ingredients, interaction of ingredients or by solvent evaporation. In contrast the carrier matrix according to the present invention does consist of a natural or synthetic polymer in which a variety of compounds is embedded. By way of embedding the variety of compounds is protected from degradation/decomposition of ingredients, interaction of ingredients or by solvent evaporation ad can therefore be stored over some time. Moreover, embedding in the sense of this application allows to include poorly soluble or even insoluble analytes. Ebedding also allows to include analytes of high volatility which would easily evaporate from a simple solvent such as water or methanol used in the prior art. Furthermore, the carrier matrix according to the present invention is easy to use and can easily be shipped to various labs to conduct comparison experiments.

The carrier matrix contains a defined amount of a number of inorganic or organic compounds to be analyzed. The number of compounds may be ten or more inorganic or organic compounds. The preferred amount of compounds is 25 or more. It has to be understood that there is no clear upper limit of the number of compounds that are analyzed. In complex situations there may be more than 100 or even several 100 compounds in the carrier matrix. However, it is important that each of the individual compounds contained in the carrier matrix is provided in a defined amount. The compounds may be naturally occurring compounds or they may be chemically synthesized. The compounds may contain stable or radioactive isotopes. The compounds can be in the liquid, gaseous or solid form, and may even change their aggregation state by specific treatment. The compounds may be contained in the natural form or they may be marked with stable or radioactive isotopes. Depending on the number of compounds and their individual physical properties (such as solubility, aggregation form, etc.) the expert skilled in the art is able to select the polymer for the carrier matrix.

It is clear to the expert skilled in the art that the carrier matrix polymer has to be free of the compounds to be measured.

The carrier matrix containing the 10 or more inorganic or organic compounds to be analyzed may be analyzed with a comparable sample of the biological entity. The . _ term biological entity characterizes a sample from a biological product. This may be a plant or part of a plant, such as rice, coffee beans, tomato, wheat, rye, soya or a product from an animal, such as an egg or milk. The sample may also contain animal or human tissue, such as skin, muscle, bone, nerves (incl. spine and/or brain), mucosa, fat, kidney, liver, lung etc.. Said plant, animal or human tissue may be in healthy or pathological form. By way of example: human liver tissue can be in form of healthy liver, in form of liver cirrhosis or in form of cancerous liver. It is furthermore possible to analyze body fluids, such as blood, urine, feces. The biological entity may furthermore be a microbiological entity, such as bacteria, yeast or even a

homogenized animal as a whole (such as a fish, a mouse or a rat). The quantitative detection system may be selected from the usual systems for quantitative analysis such as mass spectroscopy, NMR, FID, PDA, fluorescence, or any other detection system. The detection system may be coupled to separation technologies such as GC, LC, CE or any other separation system.

The carrier matrix comprising the defined amount of the 10 or more inorganic or organic compounds to be analyzed may be used to produce calibration curves by using different quantities or by dissolving the carrier matrix in different volumes to generate different concentrations. The expert skilled in the art is aware of the fact, that the calibration curves may be used to confirm the correct selection of carrier matrix material and compounds to be analyzed.

The carrier matrix comprising the defined amount of the 10 or more inorganic or organic compounds to be analyzed may be manufactured according to methods well known in the art. Generally speaking the natural or synthetic polymer or a mixture of several natural or synthetic polymers is contaminated (e.g. contacted, absorbed to or mixed) with the compounds to be analyzed and divided into small pieces. The compounds to be analyzed may e.g. be solved in an appropriate solvent. The natural or synthetic polymer may be dipped into the solution and the solvent may be removed from the polymer. The resulting carrier matrix comprising the polymer as well as the compounds to be analyzed may then be cut into pieces of (e. g. 100 mg each). It is known to the expert in the art, that it is absolutely essential that a homogeneous distribution of the compounds throughout the carrier matrix material is ensured. . .

Another way to manufacture the matrices according to the present invention is to carefully warm gelatin to more than 50 °C, to add the compounds to be analyzed under stirring and to cool down the gelatin again. The resulting gelatin block may be cut into pieces as described above. It is known to the expert in the art that the polymer used as the carrier matrix material (such as the natural polymer gelatin described above) is free of compounds that are supposed to be analyzed. The very same procedure may be carried out with wax-like compounds or compound mixtures, such as long-chain alkanes or thermoplastic polymers (e.g. polyethylene,

polypropylene).

Details of potential manufacturing processes are known from the production of pharmaceutical or dietetic products and do not need to be further explained here.

A further object of the invention is therefore a carrier matrix for the use in a process for parallel quantitative analysis of 10 or more inorganic or organic compounds in a sample, comprising

10 or more inorganic or organic compounds to be analysed, each of the compounds in a defined amount, embedded in

a natural or synthetic polymer.

In special cases two or more carrier matrices may be used in a process according to the present invention. This may be useful in cases where two or more compounds to be analyzed may react with each other. This is e. g. the case when amongst the compounds to be analyzed an ester is present as well as an enzyme which may decompose the ester, such as an esterase. In another example an organic acid could react with a sugar or sugar alcohol present in the sample. In such cases it may be useful to have one carrier matrix comprising one compound (such as the organic acid) and another carrier matrix comprising the other compound (such as the sugar) which will ultimately be analyzed at the same time in the same analytic process by combining both matrices before or during the analytical process. Another example is an incompatibility of a certain compound with the carrier matrix material. In such cases a different carrier matrix material may be used for this compound. A still further example may be a case in which one or more compounds are highly volatile. In such a case it may be necessary to have such volatile compounds in a separate

compartment, such as a hollow body made of polyethylene which is filled with the volatile compound(s). . .

The expert skilled in the art may be faced with other situations in which two or more different carrier matrices may be useful.

Carrier matrices according to the present invention may also be used to convert certain compounds prior to analysis. It is e. g. possible to combine a carrier matrix comprising a compound containing one or more hydroxyl- or amine-groups with a silylatingcompound (such as trialkylsilylchloride or N-Methyl-N-trimethylsilyltrifluoro- acetamide) which may be present in a second carrier matrix (optionally combined with an auxiliary base.) Upon dissolution of these matrices the compound containing hydroxyl- or amine-groups may react with the silylating agent so that the hydroxyl- or amine-groups are converted into silyl-groups. This is favorable if the analytical method to be used requires evaporation or stabilization of the compound, e. g. in gas chromatography or liquid chromatography.

One of the main advantages of the process described herein is that the carrier matrix containing the compounds to be analyzed may be used in the same sample preparation method as the actual sample being measured. For example if the quantitative analysis of a number of compounds of a certain vegetable requires an extraction of the compounds prior to quantitative analysis the quality control sample may be processed the same way (i. e. extraction) so that alterations of the quantity of compounds by the extraction process may be calibrated.

The expert skilled in the art is aware of the fact that the concentrations of the various compounds may be very different from each other and may even differ in several magnitudes from each other. A specific compound may occur in a given biological entity as several grams per kilogram whereas another compound may occur only in several nanograms per kilogram. It is therefore recommendable to select the defined amount of a compound to be analyzed in the carrier matrix in the same magnitude as it is expected in the analyte.

The present invention is further illustrated in the examples below. Examples

Example 1 - Aroma/Volatile Analysis in Coffee Beans

The analysis of coffee beans on volatile composition using analytical instruments is a time consuming and complex task. Repeated analysis of the same or different samples over a day or after several days is common. A general problem in _ . quantification and normalization of sample data taken over several days exists.

Problems arise through variation in sample preparation and in technical variation caused by the analytical equipment employed. A solution is to use external standards, which can be purchased as pure, isolated compounds. This procedure is time consuming, should contain a large number of compounds interested in and lacks the typical sample carrier matrix. The missing sample carrier matrix is leading to false representation of concentrations or levels of compounds, because interaction between the carrier matrix and several compounds is not possible. Thus, state of the art methods use internal calibration. In this approach a large number of compounds is added (spiked) to the biological sample and compared to the unspiked sample. This is a cumbersome process as many compound solutions have to be prepared and added. Here we describe a novel approach to overcome this hurdle.

A sample carrier matrixcan consist of organic or synthetic polymers. In this example gelatinis used to resemble this sample carrier matrix. The carrier matrix is formed as a gel capsule and encloses for instance a set of at Ieast19 aroma compounds typically present in coffee beans with a defined concentration (Table 1 ).

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Table 1 : List of coffee bean volatiles - -

The capsule is ready to use and does not need cumbersome processing steps prior analysis and application for calibration of compounds. It can be analyzed alone or added to a biological matrix to have a spiked sample in a short time. Furthermore, the so called quality control sample allows the same treatment procedure as every coffee bean sample for volatile analysis. For instance, when the volatiles from the coffee bean are being extracted using heat and a solvent, like water, the same treatment can occur to the quality control sample. This allows the same treatment as compared to biological samples and making comparison of experimental and technical variation possible.

Example 2 - Compound Analysis in Rice

Rice contains several hundred up to thousands of chemical compounds. For the analysis of those compounds analytical instruments are being used. Often only a few, but sometimes a multiple of those compounds shall be investigated at the same time. Common problems in day to day analysis are variations in sample preparation and equipment performance. The comparison of samples run on different days or even on different instruments is nearly impossible with existing methods known in the art. One way to solve this is to run a standardized rice sample along with each analysis. As each biological sample as its own variation a large batch of this standardized sample needs to be prepared and stored. Upon storage degradation, oxidation and other chemical reactions can occur and thus making this sample not as robust as needed. Another way could be the preparation of a chemical standard mix with several folds of the rice containing compounds. This is very time-consuming and expensive and at the same time does not resemble the rice sample, with all its matrix effects, like reactions with the polymeric compounds in the sample, desorption, side reactions, salt ionsand so forth.

A solution to this problem is to produce a standard sample in the form of a pill, which is made out of a rice polymer, for example starch or multicrystalline cellulose, but any other polymer can be used, in which tens to hundreds of compounds with a defined concentration are embedded. A list of such a standard compounds embeddedis shown in table 2, below. This standard sample can be processed in any analytical laboratoryalongside the rice samples under the exact same conditions and thus allows unique comparison of individual samples over several experiments. This - - sample can also serve purpose to establish a calibration curve for the absolute quantification of compounds by adding this standard pill to a biological sample.

Example 3 - Compound analysis in rice using a natural carrier matrix

The described compound analysis in rice in example 2 can also be performed using rice kernel as a carrier matrix and adding typical rice standard compounds in a labeled form, this can be either using stable isotopes, like ¹³ C, ² H (Deuterium) or ¹⁵ N or radioactive labeled molecules, like ¹ C. This sample can then be provided as granulate, a pill, a cube or any other form.

Example 4 - Compound analysis in rice using a stable isotope labeled standard mixture

Given rice example can be standardized using a rice quality batch with a defined quantity of compounds. This quality batch can be grown under controlled ¹³ C atmosphere (containing CO2 marked with ¹³ C, i.e. ¹³ ( >2) allowing full labeling of carbon containing compounds in rice with ¹³ C. This is different to normal atmospheric conditions where a ratio of ¹² C/ ¹³ C is incorporated into organic compounds in planta. The described batch is then extracted and defined quantities of this extract will then be aliquoted into different sizes and provided in the form of granulate, a pill, a cube or any other form in a defined carrier matrix. The composed quality control standard can then be analyzed alongside samples or added to the samples of interest to prepare a spiked biological sample.

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Name CAS Concentration (mg/ml)

Adenosine 58-61-7 2

lnositol-2-phosphate, myo- 7336-80-3 2

Erythritol 149-32-6 2

Homoserine 16504-56-6 2

Glucuronic acid-3,6-lactone 32449-92-6 2

Phosphoric acid 9066-91-5 2

Succinic acid 110-15-6 2

Aspartic acid 56-84-8 2

Malic acid 97-67-6 2

Galactose 59-23-4 2

Octadecatrienoic acid, 6,9,12-(Z,Z,Z)-, n- 86761-55-9 2

Glycerol 56-81-5 2

Pyroglutamic acid 98-79-3 2

Inositol, myo- 87-89-8 2

Glutaric acid, 2-oxo- 328-50-7 2

Tryptophan 73-22-3 2

Shikimic acid 138-59-0 2

Trehalose, alpha.alpha'-, D- 61370-87-2 2

Citric acid 77-92-9 2

Nonanoic acid 112-05-0 2

Glucose, 2-amino-2-deoxy-, D- 3416-24-8 2

Pentacosane, n- 629-99-2 2

Heptacosane, n- 593-49-7 2

Arabinose 5328-37-0 2

Laminaribiose 34980-39-7 2 - -

Galactitol 9001-32-5 2

Octadecanoic acid 57-11-4 2

Tricosane, n- 638-67-5 2

Pyridine, 2-hydroxy- 142-08-5 2

Homoserine lactone 6205-38-0 2

Diethylenglycol 111-46-6 2 myo-lnositol-1 -phosphate 573-35-3 2

Asparagine 70-47-3 2

Phenylalanine 67675-33-6 2

Sorbitol 9001-32-5 2

Butyric acid, 4-amino- 56-12-2 2

Glucose 50-99-7 2

Ornithine-1 ,5-lactam 1892-22-4 2

Gulonic acid, 2-oxo-, DL- 526-98-7 2

Alanine, beta- 107-95-9 2

Lactic acid 9002-97-5 2

Fumaric acid 110-17-8 2

Octadecadienoic acid, n- 61788-89-4 2

Glutamine 5959-95-5 2

Putrescine 333-93-7 2

Hexadecenoic acid, 9-(Z)- 373-49-9 2

Lysine 70-54-2 2

Mannitol 9002-04-4 2

Glucopyranose, D- (=Glucose) see above 2

Ornithine 128551-39-3 2

Aconitic acid, cis- 585-84-2 2 - -

Table 2: List of common compounds in rice - -

Similar results can be achieved with plants grown in an environment containing isotopes from various elements (e.g. H, C, N, O, P, S) in form of isotope labeled compounds such as ¹ NH ₃ , ¹⁵ NH ₄ CI, ( ¹⁵ NH ₄ ) ₂ C0 ₃ , D ₂ 0, H ₂ ¹⁸ 0, K ₃ ³² P0 ₄ , H ₂ ³⁴ S, ¹⁸ 0 ₂ , ³⁴ S0 _2l C ⁸ 0 ₂ or ¹⁴ C0 ₂ . The isotope may be stable or unstable (radioactive). The expert skilled in the art may select the isotope label according tothe analytical task and selected method. The expert skilled in the art will moreover appreciate that the environment does not have to contain 100% of the isotope labeled compound, it may be sufficient to have a higher content of the isotope labeled compound in the environment compared to standard (natural) conditions.