DD204164A5 - TEST SYSTEM AND METHOD FOR DETERMINING CONTENT OF LIQUIDS - Google Patents

Abstract

The invention relates to a test system and method for the determination of ingredients of liquids using pressable matrices impregnated with reagents. The test system is characterized in that it contains at least one imprained matrix in a preferably cylindrical reaction vessel and a device through which the matrix is captured the liquid to be examined is compressed while releasing the reaction product.

Description

Title of the invention;

Test system and method for the determination of ingredients of liquids

Field of application of the invention:

The invention relates to a test system and method for the determination of ingredients of liquids, wherein the test system consists essentially of at least one, arranged in a reaction vessel pressable matrix.

.10 Characteristic of the known technical solutions:

The determination of ingredients of complex systems, in particular, of biological fluids,., Requires a well-defined experimental procedure, the sample with the substance to be determined undergoes various reaction processes. Depending on the method used and the number of samples, the addition and timing of the reagents to be mixed differ. A to be evaluated after the reaction signal to be measured, for the method

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and the substance to be determined is characteristic, finally leads to the concentration of the sought substance. According to the complexity of such processes, a number of different methods have been tested so far; have been designed to provide optimum reliability, simplicity and economy. Especially in clinical chemistry, as a result of the sometimes very high numbers of analyzes, methods have been developed which make it possible to carry out many determinations in the simplest possible and shortest possible time. On the one hand this is achieved by the use of mechanized analysis systems, on the other hand by the provision of the necessary reagents and reaction mixtures in largely prepared form j. In particular, analytical systems are used in which the sample is mixed manually or fully mechanized with reaction solutions from storage containers. However, the provision of the solutions, the possibly necessary preparation of the sample as well as the monitoring and cleaning of the distribution and mixing system require a multitude of work steps and possible sources of error. In particular, the relatively low durability of some analyte substances in solution very often requires an exchange of unused solutions. In addition, the test conditions always require an exactly reproducible addition of different volumes of different solutions, which makes automated systems easy to replace analytical methods impossible.

The difficulties described have led to the development of different test systems. For example, it is known to contact the reagents in solid form either as impregnated support materials, such as impregnated filter papers, or as lyophilizates in corresponding reaction vessels with the sample to be examined; in the-

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Such systems require only pipetting up of solvent and test sample. Attaching different lyophilized reagents at different locations allows the implementation of cascaded reactions. These systems are a simplification for the test procedure, but offer very large problems in the preparation of the corresponding reaction vessels. Above all, these embodiments have the disadvantage common to all aqueous systems of being unable to carry out any separations and separations required for the analysis of body fluids and of having to carry all the reagents added during the course of an analysis.

In the case of the so-called dry analytical methods, on the other hand, some of these problems are eliminated from the outset. In this case, the sample to be examined is applied to a mostly absorbent carrier, which in the simplest case in a single matrix layer contains all necessary reagents in optimum concentration; This eliminates all usual pipetting steps. In addition, the storage of such dry systems in terms of durability is not a serious problem. As a disadvantage of these single-stage systems, however, proves, as well as in the aqueous process, the lack of preparation and preparation possibility and the lack of opportunity to eliminate interfering .Substanzen.

But even in test systems with multi-layered, dry materials, which may contain, for example, layers for the separation of interfering substances, concentration zones and various reaction and blocking areas, the uptake and further transport of the liquid to be examined and the sequence of the individual reaction steps alone from the Diffusion and capillary action dependent and thus virtually neither controllable nor controllable. Thus, it is not possible to kill the reaction at a certain place or the length of stay in one

4, "f W fc I W a

Influence reaction zone. '

From US Pat. No. 3,917,453 there is known a test device which comprises an absorbent material for receiving the sample solution and a compressible layer also consisting of an absorbent material which serves to meter and transport the liquid to be examined. This layer is arranged between two cover layers containing the necessary reagents and can react with the substance to be determined to form a color substance, which is evaluated colorimetrically. However, this device has a number of disadvantages: Quantitative determinations, photometric evaluation and automation are not possible; It is also not possible to carry out reactions which require several successive reaction stages and any necessary separation of undesired substances between two reaction zones.

Aim · · 'de'-r Sjf'in-durig ·: ·

The object of the present invention is to provide devices and methods for the determination of constituents of body fluids which make it possible to avoid the described disadvantages.

According to the invention, this object has been achieved by a test system consisting of a reaction vessel containing at least 'a moldable matrix, whereby a simple, diffusion-independent liquid transport can be carried out, which can be controlled both manually and automatically.

Explanation of the essence of the invention:

The invention relates to a test system for the determination of ingredients of liquids using press-compatible, reagent-impregnated matrices, characterized

characterized in that it comprises at least one impregnated matrix in a preferably cylindrical Reaktricnsgefäß and a device by which the matrix is compressed after receiving the liquid to be examined with delivery of the reaction product.

Furthermore, the invention relates to methods for the determination of ingredients of liquids using pressable, impregnated with reagents matrices, which are dadurc characterized in that applying the liquid to be examined on a arranged in a preferably cylindrical reaction vessel matrix and the reaction product by compressing the matrix of this removed and either directly an evaluation

or only after one or more further passages through impregnated matrices.

Surprisingly, it has been found that the erfindungsge Permitted test system, the application and effectiveness of the analytical methods as well as the reagents in the same way ei allows as a comparable system in aqueous solution, but also additional methods of sample preparation, such as centrifugations, filtrations or chromatographic separations, in unite or replace

The test system for analysis of liquids can be divided into e zelne areas where separate run all necessary for the analysis separation, reaction and determination reaction. This includes, for example, in the analysis of body fluids in addition to the reaction area required for the actual determination of a substance and possibly one

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Display area also preparatory areas, such as for de-whitening or separation of interfering substances, a. The type, number and arrangement of the individual areas are freely combinable. For example, specific provisions may have separate preparation areas, or several different reaction areas may be linked to a common preparation area.

In the test system according to the invention, all are for reaction

, 0 required reagents from the beginning uniform lyophilized in the entire matrix, immobilized or otherwise included included. An application of the to be examined. Liquid leads to a direct mixing of the reactants over the entire matrix area. The individual reaction areas are clearly separated within the reaction system, that is, there is no premature diffusion or mixing. In addition, the device according to the invention offers a number of further advantages over the prior art. Thus, for example, an improvement in mixing and an increase in the reaction rate can be achieved by additional mechanical manipulations, such as compressing the matrix layer during the reaction; the test system allows the use of different or differently prepared matrices for the individual test areas; the evaluation is not only possible visually but also photometrically; the implementation can be done manually and mechanized; The use of the matrix allows for accurate dosing and easy handling of substances and liquids is test system.

The device according to the invention thus has the in the. In addition, the overall process of the reaction is not diffusion-dependent and therefore,

as with the corresponding aqueous systems, influenced and arbitrarily interruptible. A possible modular principle allows an easy replacement of partial areas and overall test systems.

Basis of the test system is a matrix of a flexible, elastic, saügfähigen material that serves as a carrier for reagent and sample and as a reaction space, acts as a separation medium and allows solutions as completely as possible, but at least reproducible, reversible.

release. This matrix is capable of accommodating and storing a certain volume of sample liquid completely and evenly distributed in it, e.g. deliver as completely as possible with an applied slight positive or negative pressure or a corresponding mechanical pressure; this process is repeatable. The matrix must also have sufficient mechanical and chemical stability. Under flexible ur elastic is to be understood in this context a constant under constant external conditions space and form filling of the matrix, without the need for a support vessel is required; it should be able to be changed reversibly by applying pressure. The matrix can consist of one piece, of several segments or of a large number of free or subsequently connected parts, for example, the bonding or crosslinking, of connected parts which satisfy all or most of the corresponding requirements.

Suitable matrix materials are natural and synthetic spongy and foam-like, porous substances, for example open-cell foams and composite foams of various origins and correspondingly elasticized microcapsules, granules or other shaped bodies of absorbent materials. Open celled polyurethane foams, in particular polyester foams and cold foams, are preferred.

In addition, however, special foams are also usable as a matrix, such as foams containing SH groups when using enzymes which require SB groups, or else transparent polymers for visual evaluations.

The compression hardness of these materials should be in the range of.

from about 2 to 10 kPa, those having a compression hardness below 2 kPa show poor mechanical properties, those above 10 kPa require too much pressure in the liquid discharge. The volumetric weight has no significant influence on the usable material.

Some of the commercially available foams have strong enzyme inhibition; for some, severe turbidity may occur during the performance of the preparation process. However, as a rule, the inhibitions can be completely eliminated by the addition of complexing agents. In addition, further improvements can be obtained by appropriate pretreatment. For the kinetic determination of enzymes in liquids, however, enzyra-inhibiting matrices may prove to be particularly advantageous. Thus, it is possible to impregnate the matrix containing the required reagents with the liquid to be examined, the actual determination reaction is not: or runs only slowly. When transferring the solution of gesasite from the matrix into the test area, the undesired inhibitors remain and the kinetic determination is possible at relatively low ¹ initial absorbance.

The introduction of the reagents into the matrix is generally carried out by lyophilizing the reagent solution soaked foam segments. In addition to greater stability of the applied substances, this also has the advantage that only a small amount of reagents is required. Thus it was surprisingly found that, for example, in a separation of protein from a test sample with the aid of a

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Coagulant, such as trichloroacetic acid, an up to ten times lower amount of coagulant is required than in the usual precipitation methods. Thus, by avoiding the otherwise caused by the necessary neutralization high salt loading a series connection of different test areas according to the invention is favored.

In general, all the reagents required for the function of the area in question, separated for each area, are applied to the matrix. In special cases, for example, incompatibility of reagents or simultaneous sequence of multiple reactions with different reagents, it may also be appropriate to give the required reagents separately. This is preferably done by additives amm ensetzen of the entire matrix in this area of individual matrix segments each containing different substances. The matrix segments may be of any desired shape and size, for example they may consist of circular segments, discs or spheres which are independent of one another, or V ^- Or or, after being introduced into the reaction vessel, connected to one another by suitable means such as gluing or mechanical fastening become.

In certain cases it may be necessary, although a complete transfer of the sample solution from one area to the next has to be ensured, but the reagents which interfere in the next area are not to be borne along. This requires the interposition of a correspondingly prepared matrix or immobilization of the reagents, which can be carried out, for example, adsorptively, by encapsulation or by chemical methods. In this way, it is possible to have several consecutively connected regions with very different reaction conditions, for example different pH values or with each other

interfering reagents, to couple. Even in cases where application by soaking and lyophilization is not possible, the substances may be kept in liquid form, for example by inclusion in the matrix.

The most diverse possibilities of immobilizing substances on polymeric support materials by chemical methods are known from the literature. Mention may be made, for example, the binding to carriers with functional groups or crosslinking with glutaric dialdehyde, acrylamide, etc. Also, immobilizations of low molecular weight substances on polyurethane foams, especially on so-called cold foams, by a suitably adapted variation of the so-called triazine method possible (Biochem 109, 137 (1958)). This method makes it possible to bind a multiplicity of compounds with OH, SH or NH groups to the foams with a binding yield of about 1 pmol / ml of foam. In addition to the reagents themselves, it is of course also possible to bind short or long chain, for example bound to polymers such as starch or dextran derivatives or to retain physically or by inclusion in your foam.

The reaction vessel is conveniently in the form of a cylinder with a conical outlet; preferably pipettes, syringes or so-called disposable syringes with a diameter of 5 to 3 0 mm and a length of 10 to 150 mm are used. However, it is also possible to use cuvette-type reaction vessels. These reaction vessels may consist of transparent or partly transparent plastic or solid materials such as glass or metal. Preferred are flexible transparent plastics.

According to a particular embodiment, the test system does not contain just one matrix but a plurality of matrices arranged one above another, which are provided with liquid-permeable matrices.

Orders such as webs, perforated washers or tapers of the reaction vessel are separated. The shape of these arrangements and the size of the holes in the intermediate disks can influence the flow and diffusion processes during the transfer of the liquid to be analyzed between the individual matrix areas. The intermediate discs can be fitted into the reaction vessel so that they are displaceable upon application of a certain pressure.

If the reaction vessel between the individual matrices tapers, they can be closed by laterally applied pressure; If the matrix is likewise pressed together, the liquid exits from the foam in order to be completely absorbed by the foam when it is released. The pressing operation is also feasible by Eerabdrücken a perforated punch.

The figure shows a schematic representation of a device according to the invention. The Ausfühxungsform shown consists of the reaction vessel 1, which terminates in a constriction 2, which is designed as a cuvette. 3 is either the display area of the test system or collection container for the liquid to be dispensed outside the reaction vessel. The reagents impregnated matrices 4 are separated by intermediate plates 5; the upper layer 6 represents a distribution layer, 7 denotes the stamp.

The method is carried out with the aid of the erfindungsge MAESSEN device in such a way that the sample to be examined is applied by a conventional application system on the upper matrix of the reaction vessel; In this case also necessary for dilution solvent is added. It is of course possible by simple

Contact with the sample liquid to suck it by capillary action in the area of this matrix layer. This process can be facilitated by suitable construction of the upper part of the reaction vessel, for example by forming a chamber having a certain volume. It is also possible to install an additional distribution layer over the first matrix layer, which may consist of a filter paper layer, for example. In this case, the solution to be examined can be diluted before or after the application. The process can then either be continued immediately or the reaction vessel can be stored temporarily.

After a fixed reaction time, the first matrix layer is compressed by a suitable device, for example a stamp. The pressure is adjusted so that the matrix is compressed, but the washer maintains its position- This ensures that the liquid contained in the matrix is transferred to the second matrix. The properties of the matrix described make a seal between the individual matrices superfluous.

Various partial reactions can be carried out in this way separately, such as Enteiweißung, separation of the components of the whole blood, separation of isoenzymes, immune reactions, specific precipitation or complex formation, enzymatic or chemical determination reactions. In addition, it is possible / additionally to retain substances by means of appropriate intermediate layers between two areas. These intermediate layers can also

3Q if made of foam, pre-treated foam or filter paper or other absorbent material.

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It is essential that the liquid to be examined be reproducibly transported from one area to another. A quantitative transfer would be preferred, but this can not be achieved due to the matrix materials used. Normally about 2 to 15% of the liquid is retained by the matrix. However, this percentage is reproducible for the matrix material used and enters into the evaluation as a constant factor.

The evaluation is carried out in a conventional manner by all common methods. Preference is given to optical or reflectometric evaluations.

After passage of the (last) matrix layer, the reaction solution either enters a measuring cell within the reaction vessel and is evaluated within the reaction vessel. But it is also possible to transport the reaction solution in a measuring cell located outside of the reaction vessel and to carry out the evaluation therein. But there are also special display reactions feasible, such. As an indication of gas diffusion or an electrochemical determination.

As liquids to be examined mainly body fluids were used, since the inventive method and test system in particular for the determination. the ingredients of 31ut, serum or Haxn is suitable.

The invention is explained in more detail by the following examples:

EMBODIMENTS Example 1

Deoxygenation of a serum sample

Made of a polyurethane cold foam (foam R 6590 the

Company Metzeier, Memmingen), disc-shaped segments (0 15 mm, height 4 mm) were drilled out. These were soaked with 50 μl of a trichloroacetic acid solution (2 mol / l) by application to the middle of the segment and dried until they had a trichloroacetic acid content of 5 × 10 mol. One such prepared matrix segment was inserted into a 10 ml disposable syringe. The center of the segment was in each case mixed with 10 μl of a sample of sera (5.5 mg / dl of protein content) containing various defined amounts of glucose. Subsequently, attached to the punch of the syringe matrix segment of a polyurethane-polyester foam (foam MS 6060 from Metzeier, 0 15 mm, height 16 mm), which was impregnated with 1.1 ml of water, respectively introduced from above together with the punch and pushed through to the stop. In the overall solution expressed, the protein content (biuret method) and the glucose content (glucose dehydrogenase method) were determined according to the following formula:

C = F E

where E is the absorbance measured at 365 nm. The factor F takes into account dilution, blank value and glucose loss in the matrix and was determined by means of test series with standard solutions as 710.

Glucose [mg / 100 ml]

Protein [g / lOC ml]

used found used found 20 19 5.5 0.1 34 32 5.5 0 50 52 5.5 0.2 70 74 5.5 0.1 100 97 5.5 0.1 141 135 5.5 0 167 174 , 5.5 '0.1 250th 245 5.5 0.1 500 478 5.5 0.2

The numerical values show that the deproteinization has succeeded and the glucose values reflect the concentrations used with satisfactory accuracy.

Example 2 Glimmering mood

Cylindrical matrix segments (0 15 mm, height 16 mm) were drilled out and washed out of a polyurethane polyester foam (ME 6060 foam from Metzeier, Memmingen). The dried segments were soaked in 1.1 ml of a solution containing

ο, 2 mol / 1 Phosphorus puifer, pE 7.6, 0 15 mol / 1 Sodium chloride, 5 kU / 1 Glucose dehydrogenase, ο, 11 kU / 1 mutarotase 1 mmol / 1 NAD 'and 25 mmol / 1 Zthylendiamintetraacetat

and then lyophilized. The reaction matrices prepared in this way were mixed in a 10 ml Sinweg syringe by aspiration with 1.1 ml of a deboned serum sample, each containing 10 μ 1 of a defined amount of glucose. After 10 minutes, the total solution was "printed by printing the syringe into a cuvette." At an absorbance of 365 nm (F = 647), the concentration of glucose in the sample was determined. analogous test conditions), the following glucose concentrations [mg / 100 ml] were found which show a good agreement:

manual test 50 80 100 165 200 370 435 500

Matrix systems 50 79 102 161 204 355 427 485

Example 3 Protein determination

From a rigid polyurethane foam (foam R 7295 from Metzeier, Memmingen), cylindrical matrix segments (0 12 mm, height 8 mm) were drilled out, washed out, dried and then impregnated with 0.5 ml of a solution which was

18 mmol / 1 K Na tartrate,

10 mmol / l potassium iodide,

- .. 12 mmol / 1 copper sulfate and

200 mmol / l sodium hydroxide

and then lyophilized. The so-manufactured. The reaction matrices were diluted in a 5 ml disposable syringe each containing a different amount of albumin

Sample solution (10 μΐ sample + 0.5 ml of water) was added by inserting and repeatedly pressing down a perforated punch. Within 10 minutes, the solution in the matrix segment was pressed several times into the space above the matrix and sucked back into the foam ,

During this time, the outlet of the syringe was sealed. Thereafter, the punch was replaced with a non-perforated punch, the whole solution was pressed into a cuvette, and the absorbance of the solution at 546 nm was measured against the blank value of the reagent solution. By conversion, the protein concentration of the original sample can be determined; (F = 15):

, Protein [g / 100 ml]

found used

2.0 2.1

³ '° ³ ' °

^JU 5.0 4.9

6.0 6.2

8.0 7.9

10.0 10.1

12.0 11.8

'/ Γ

L u ü LIQI

Example 4

Determination of Glutamate Oxaloacetate Transaminase (GOT)

From a polyurethane-polyester foam (foam ME 6060 from Metzeier, Memmingen) cylindrical matrix segments (0 20 mm, .Hehe 30 mm) were drilled out and washed out. The dried segments were soaked in half (Segments I) with 3 ml of a solution containing

80 mmol / l phosphate buffer, pH 7.4,

200 mmol / L L aspartate,

0.18 mmol / 1 JiADH,

1 g / l albumin,

έ 1.2 kU / 1 LDH and

^ 0.6 kU / 1 MDE

contained and lyophilized. In a similar manner, the second half of the segments (segments II) were mixed with 3 ml of a solution of

Phosphate buffer, pH 7.4, oxoglutarate and ethylenediaminetetraacetate

spiked and lyophilized. In a 20 ml disposable syringe wx a segment I is overlaid with a perforated polyethylene intermediate disc (0 20 mm, thickness 2 mm) and a segment II. On segment II, 2.5 ml of a standard dilute serum sample (500 μΐ serum with various GOT activities set + 2 ml of physiological saline) was added and after 5 minutes transferred to segment I by squeezing the syringe. Only so much pressure was applied, since the transfer was as complete as possible, but no shifting of the intermediate disc was effected. After a further 2 minutes the total solution was reduced to one by completely depressing the stamper

20 mmol / 1 25 mmol / 1 10 mmol / 1

Cuvette pressed and recorded the extinction curve at 334 nm. From the slope of the curves obtained, the enzyme concentration of the test sample can be calculated (F = 1471):

  9 [u / i] GOT 3 found used 3.2 2, 5.0 S ₁ 14 14 21 23 38 40 58 57 76 74 111 109 140 143

Example 5

Deoxygenation and glucose determination

In a 10 ml disposable syringe, a segment for glucose determination (Example 2) and a segment for deoxygenation (Example 1) were placed one above the other, separated by a multiply perforated polyethylene disc (0 15 μm, thickness 2 mm). In order to examine the glucose content of sera, 10 μl of a serum sample with various defined amounts of glucose (protein content 5.5 mg / dl), as described in Example 1, were added to the upper segment impregnated with trichloroacetic acid, and 1.1 ml Segment pressed for glucose determination. In this case, only so much pressure was applied that as complete as possible transfer of the total solution, but not a displacement of the washer

.30 was effected. After 10 minutes, the entire solution was pressed into a cuvette by pushing the stamp until it stops

pressed. The determination of the glucose concentration was carried out üi a measurement of the absorbance at 365 nm. It is analogous Be game 2 a linear measuring range of 2 0 to> 500 mg / dl glucose.

Example 6

glucose determination

Cylindrical matrix segments (0 15 mm, height 16 mm) were drilled out of a polyurethane-polyester foam (ME 6060 foam from Metzeier Memmingen) and washed out. The dried segments were soaked with 1 ml of a solution containing

20 mmol / l phosphate buffer, pH 7.5,

0.2 kU / 1 diaphorase and

0.6 mol / l 3- (4-iodophenyl) -2- (4-nitrophenyl) -5-

phenyl-2H-tetrazolium chloride

contained and then lyophilized. The reaction matrices thus prepared were overcoated in 10 ml disposable syringes separated by a multi-perforated polyethylene disc with respective segments of reaction matrices for glucose determination (see Example 2). On the latter, 1.1 ml of deproteinized serum sample were applied analogously to Example 2 and the total solution was transferred after 2-3 minutes in the diaphorase / tetrazolium salt segment. After 10 minutes, the evaluation was carried out by means of a visual color comparison of the matrix soaked in the sample with a color comparison table or via an absorbance measurement (400-600 nm) of the solution which had been squeezed out. Analogous results as in Example 2 were obtained.

Example 7

EnteiweiJe and visual glucose determination

In a 10 ml disposable syringe - each of several times perforated polyethylene discs (0 15 mm, thickness 2 mm) separated - from top to bottom, the segments for Enteiweissung (Example 1) and the Glucosebestiinmung (Example 6) stacked. 10 μΐ each of a defined amount of serine sample containing 5.5 mg / dl of protein was applied to the trichloroacetic acid-loaded matrix in the middle. Analogously to Example 5, the deproteinized sample was then transferred together with the rinsing solution into the glucose determination region and after another .2-3 minutes in the diaphorase / tetrazolium salt segment. After a further 10 minutes, the evaluation was carried out visually using a color comparison table or via the extinction (578 nm) of the solution expressed. Analogous results as in Example 5 were obtained.

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Claims (12)

Berlin, 3.11-1982 61 139/18 claim for invention A test system for the determination of ingredients of liquids using pressable, reagent impregnated matrices, characterized in that it contains at least one impregnated matrix in a preferably cylindrical reaction vessel and a device through which the matrix after receiving the liquid to be examined with delivery of the Reaction product is compressed. 2. Test system according to item 1, characterized in that a plurality of matrices are arranged one above the other and separated by liquid-permeable devices. 3.11.1932 61 139/18 3. Test system according to the items 1 or 2, characterized in that the reaction vessel has the shape of a pipette or a syringe, 4. Test system according to items 1 to 3, characterized in that the liquid-permeable arrangements are intermediate disks, webs or tapers of the reaction vessel « 5. Test system according to the items 1 to 4, characterized in that the iJatrices are disc-shaped. 6. Test system according to items 1 to 5, characterized in that a matrix consists of several, with different reagents impregnated, segments » ifi MHV 1Qn η * Γι υ -Z- 3.11.1982 61 139/18 7. (De st syst em nach., The points 1 to 6, characterized in that additional distribution layers, preferably made of! Filter paper, are mounted over at least one matrix. 8. Test system according to items 1 to 7, characterized in that the part of the reaction vessel, which serves to receive the emerging from the (last) matrix liquid, is designed as a display area. 9. A process for the determination of liquid contents using pressable, reagent-impregnated silica, characterized in that the liquid to be tested is applied to a matrix arranged in a preferably cylindrical reaction vessel and the reaction product is compressed by compressing the matrix removed and either directly to an evaluation supplies or only after one or more further passages   by impregnated matrices. 10. The method according to item 9, characterized in that the compression of the matrices is carried out by stamp or brine pressure in a cylindrical reaction vessel · 11.-Verfahren.nach the points 9 and 10, characterized in that a passage successively through a plurality of differently impregnated matrices, wherein each of the Reaktionsproduizt one matrix is converted into the following. -A3 12. The method according to points 8 to 11, characterized in that the determination is carried out by evaluating the liquid contained in the reaction vessel, which is obtained after the passage of the (last) matrix. For this 1 sheet drawing