DE2235152C2 - Diagnostic agent for the detection of blood and other peroxidatically active substances in body fluids - Google Patents

Description

f e d c

(D

NR ₁

in which Rj represents hydrogen or a methyl group, is fused to at least one of the points marked c, d / e, f and g by benzene and / or pyridine rings, two adjacent rings not being allowed to contain one ring nitrogen atom each at the same time.

2. Test strip according to claim 1, characterized in that the quinoline derivative according to formula I _{is substituted at the position denoted by - H and R 1} by lower alkyl groups which can also together form a hydroaromatic ring.

3. Test strip according to one of claims I and 2, characterized in that the carrier additionally contains buffers, wetting agents, thickeners, stabilizers or other auxiliaries.

4. Use of compounds of the general formula I

f e d c

(I)

45

in which Rj represents hydrogen or a methyl group and which is fused to at least one of the points designated by c, d / e, f and g by benzene and / or pyridine rings, two adjacent rings not being allowed to contain one ring nitrogen atom at the same time and where the Compounds I, except in the _{position denoted by —H and R 1} , can be substituted by lower alkyl groups, which together can also form a hydroaromatic ring, for the production of test strips for the detection of peroxidatically active substances.

The detection of small amounts of blood in urine, feces or no longer visible to the naked eye Vomit is very important in diagnosing bleeding in the stomach, intestines, and passages. Such Bleeding is z. B. by tumors, ulcers or inflammation in the corresponding organs caused. In addition, under the influence of certain haemolytic toxins in the urine and plasma free hemoglobin occur. Blood and hemoglobin have a peroxidic effect; that is, they are composed of hydroperoxides Free oxygen and transfer it to certain acceptors. More peroxidatically effective Substances are found in leukocytes and bacteria. Evidence of these substances is important for diagnosing diseases and infections of the kidneys and urinary tract. That is also peroxidic effective myoglobin is found in the urine, for example, after a heart attack. Especially often You may also have blood in your urine if you have bladder stones or kidney stones.

For a sensitive detection of all these substances, their peioxidatic effect is suitable particularly. The oxygen released from a hydroperoxide is transferred to a chromogen, which is oxidized to a dye and so the presence of the peroxidatically active substance indicates. This reaction has been used in medical medicine for a long time, especially for the detection of blood and forensic analytics. It is usually carried out as a test tube or spot reaction carried out, hydrogen peroxide being used as the hydroperoxide in most cases. Come as chromogens especially benzidine, o-tolidine or leuco malachite green are used.

Rapid tests are absorbent carriers, mostly papers, that contain everything necessary for the detection reaction Reagents are impregnated and after simple immersion in the body fluid a color reaction demonstrate. In accordance with the great importance that rapid tests have acquired in recent times, they are already variously developed for the detection of blood in body fluids.

Since the detection of blood depends on the sensitivity of the rapid test and It is also aimed at leukocytes and leukocytes that react less sensitively to peroxidation Attempts have been made on various occasions to detect bacteria, the sensitivity of the known per se Increase detection response through additives. So are for example in the German Auslegeschrift 1 242 905 test papers described the quinoline derivatives as activating additives, preferably Contain quinine. The sensitivity of the test strips is increased by the specified additives, however, according to our own tests, it is practically not possible with the test strips that have been improved in this way Detect leukocytes and bacteria.

The activating effect of the basic body quinoline on blood detection has been around for a long time known (Zeitschrift f. judicial Med. 12, 216 [1928]); however, this is as well as its simple derivatives liquid or volatile and therefore not suitable for use on test strips.

The object was therefore to develop an improved test strip which would be sufficient is sensitive to also detect leukocytes and bacteria.

This object is achieved by the measures specified in the characterizing part of claim 1.

The test strips according to the invention have a previously unattainable sensitivity. Quinoline

Derivatives in which, according to the formula 1 given in claim 1, R _{1 represents} a hydrogen atom are preferred. In the further development according to claim 2, lower alkyl groups are understood to mean alkyl groups with 1 to 4 carbon atoms, 5 and 6 rings being preferred in the event that two alkyl groups together form a hydroaromatic ring.

The following are examples of the most important groups of compounds falling under the general formula 1; the individual connections are all known from the literature.

1. Benzoquinolines

1.1. Benzo [c] quinoline (phenanthridine)

2-methylphenanthridine
6-methylphenanthridine
2-ethylphenantridine

1.2. Benzo [f] quinoline
3-methylbenzo [f] quinine
1,3-dimethylbenzo [f] quinoline 1,2-tetramethylene benzo [f] quinoline 1,2-trimethylene benzo [f] ehinoline

1.3. Benzo [g] quinoline
4-methylbenzo [g] quinoline
2,4-dimethylbenzo [g] quinoline

2. Dibenzoquinolines

2.1. Dibenzofc, fjchinolin (benzo [\] phenanthridine)

2.2. Dibenzo [c, d, e] quinoline (4-azapyren, thebenidine)

3. Pyridoquinolines

3.1.

Pyrido [2,3-f] quinoline (1,7-phenanthroline) 2-methyl-l, 7-phenanthroline 2,8-dimethyl-l, 7-phenanthroline 3.2. Pyrido [3,2-f] quinoline (4,7-phenanthroline) 3-methyl-4,7-phenanthroline 3,8-dimethyl-4,7-phenanthroline 1,3,4,8-tetramethyl-4,7-phenanthroline Pyrido [2,3-g] quinoline (1,6-anthrazoline) 2,7-dimethyl-1,6-anthrazoline

The invention also relates to the use of compounds of those specified in claim 4 Formula I for the production of test strips for the detection of peroxidatically active substances.

It could not be foreseen that the modification according to the invention of the quinoline, which is known per se as an activator, would lead to such an extraordinary increase in effectiveness. Substantially at the ^v orliegenden invention is the fact that an increase in activity can only be achieved by fusion of certain items of Chinolinsystems. So occurs z. B. with annealing in the [b] and [h] position of the quinoline, ie with acridine and benzo- [h] -quinoline, a reduction in activity compared to quino-Hn.

The effectiveness according to the invention can also hardly be explained; if you would, which might be obvious, be attributed to complex formation with the peroxidatically active substance, for example o-phenanthroline, known as a strong complexing agent, has a strong activating effect. However, this is not the case Case, whereas ra- and p-phenanthroline are very effective Represent activators.

Surprisingly, the compounds according to claim 4 increase the sensitivity of psroxidases of vegetable origin, such as B. of horseradish peroxidase, do not, but act specifically on compounds of humans with peroxidic activity or animal organism. This is how it is

ίο possible leukocytes, blood, blood components or Selectively detect bacteria in stool or vomit in addition to plant peroxidases. Here myoglobin is detected with about the same sensitivity as hemoglobin.

t5 The sensitivity of the detection reaction is increased by individual compounds of general formula 1 so that it is possible to detect individual erythrocytes even in urine, which are visible as colored points on the test paper. For example, phenanthridine can be used to produce a test paper with which 5 erythrocytes / mm ³ urine can still be clearly detected. This corresponds to a blood dilution of 1: 1,000,000.

It goes without saying that not all are under

As the general formula I falling compounds in have activating properties to the same extent. So you have it in your hand, the sensitivity for example, to adjust a blood test to the requirements of the practice. One obtains z. B. Test strips increasing activity if one uses the compounds listed below as activators in their order as activators admits: m-phenanthroline ■. p-phenanthroline <Benzo- (f) -quinoline <Phenanthridine.

The sensitivity can be further modified by alkyl substitution; so z. B. by a Methyl substituents in the \ -position to a nuclear nitrogen atom reduce the activation somewhat, while otherwise it usually leads to an increase in activation.

For example, the sensitivity limit of test papers which contain a weak activator, such as 2,8-dimethyl-1,7-phenanthroline, is around 50 erythrocytes / mm ³ urine.

The activating agent according to the invention is used in amounts of about 0.05 to 1.0 ^{a /} _o , preferably 0.2 to 0.5 "" per 100 ml of impregnation solution.

Other components of a test strip for blood are an organic hydroperoxide, an oxidation indicator, a buffer and a surfactant

Means and, if desired, phosphoramides, such as

z. B. phosphoric acid trimorpholide for stabilization, and other auxiliaries.

As hydroperoxides, for. B. cumene hydroperoxide or 2,5-dimethylhexane-2,5-dihydroperoxide Possible indicators are those from the benzidine series, such as o-tolidine, or from the heterocyclic series Azines, such as bis (N-ethyl-quinolone-2) -azine or (N-methylbenzthiazolone - 2) - (1 - ethyl - 3 - phenyl - 5 - methyltriazolone - 2) - azin according to German patent specification 1 648 840 in question.

The indicators are used in amounts of 0.05 to 5 g, preferably 0.2 to 1.0 g per 100 ml of impregnation solution used.

As a buffer z. B. citrate, phosphate, phthalate or succinate buffer in question, with pH and capacity must be chosen so that after immersing the test strip in the body

\ L 2 ⁵ !

liquid on this a pH value of 4 to 7, preferably from 5 to 6 adjusts.

It is also advantageous to add small amounts (about 0.05 to 0.5 g per 100 ml) of a complexing agent to the formulation like adding sodium metaphosphate or ethylenediamine tetraacetic acid, making wrong positive reactions that could be caused by traces of metal can be avoided.

Because the test papers cause bleeding due to the relatively large amounts of water-soluble substances It is convenient to use the formulation of thickeners, such as methylcellulcse and in particular Gelatin, to be added in amounts of about 0.5 to 5 g per 100 ml.

Long-chain organic sulfates or sulfonates, such as z. B. sodium dodecylbenzenesulfonate, dioctyl sodium sulfosuccinate or sodium lauryl sulfate, which are known Stabilize radical cations, such as the oxidized o-tolidine. The wetting agents become the impregnation solution in amounts of 0.5 to 5%, preferably 1 to 3 ° ύ, added.

For the production of the test strips according to the invention absorbent carriers, such as. B. filter paper, cellulose or synthetic fiber fleece, with solutions of the Reagents impregnated in volatile solvents. This is expediently done in two separate ways Steps. First it is impregnated with a solution containing a hydroperoxide, wetting agent, Contains buffers and optionally thickeners. After that, with a solution of an indicator and an activator of the general formula 1 impregnated.

For the detection of peroxidatically active substances in the stool, it is also possible to use the carrier as a to form a waterproof film containing the reagents. This has the advantage that the surface of the Test strip for reading the color reaction can be cleaned by simply wiping it off.

The following examples serve to explain the invention in more detail:

example 1

Filter paper is impregnated one after the other with the following solutions and dried at 40 ° C:

45 Solution 1

1.2 molar citrate buffer from

pH 5.25 35.0 ml

Ethylenediaminetetraacetic acid,

Disodium salt 0.1 g

Dioctyl sodium sulfosuccinate 2.0 g

2,5-dimethylhexane-2,5-dihydro-

peroxide (about 70%) 1.6 g

Phosphoric acid trimorpholide 12.7 g

Ethanol 30.0 ml

Distilled water to 100.0 ml

Solution 2

o-tolidine 0.3 g

Phenanthridine 0.2 g

Toluene to 100.0 ml

A white test paper is obtained which, when exchanged for urine containing blood, after about Turned green for 5 to 20 seconds. If the erythrocytes are intact, the papers are speckled green. 1st Hemolysc has occurred or is located from the front-152

if there is free hemoglobin in the urine, the papers are colored evenly green. The sensitivity is about 5 erythrocytes / mm ³ or the corresponding amount of hemoglobin. A lower number of intact erythrocytes may still cause individual green dots on the test paper. The sensitivity to myoglobin is the same as that of hemoglobin.

White blood cells and bacteria are also detected, intact particles through mottling, lysed by uniform staining.

Example 2

If in solution I of Example 1 instead of 2,5 - dimethylhexane - 2,5 - dihydroperoxide, the equimolar Amount of diisopropylbenzene hydroperoxide is used and the phenanthridine is replaced in solution II the activators listed below give test papers and their sensitivity compared to blood, leukocytes and bacteria is roughly the same order of magnitude as in Example 1:

2-methyl- or 2-ethyl-phenanthridine

Benzo [f] quinoline

1,2-tetramethylene benzo [f) quinoline

Ben7.o [g] quinoline

4-methylbenzo [g] quinoline

Dibenzo [c, d, e] quinoline

Dibcnzo [c, fjquinoline

Pyrido [3,2-f] quinoline

3-methyl-4,7-phenanthroline

Pyrido [2,3-f] quinoline

Example 3

Filter paper is impregnated with the following solutions and dried at 40 C:

Solution 1

1.2 molar citrate buffer from

pH 5.25 40.0 ml

Ethylenediaminetetraacetic acid,

Disodium salt 0.1 g

Dioctyl sodium sulfosuccinate 2.0 g

Gelatin 2.0 g

2,5-dimethylhexane-2,5-dihydro-

peroxide (about 70 ° _o ig) 1.6 g

Ethanol 30.0 ml

Water, dist. To 100.0 ml

Solution 2

o-tolidine 0.3 g

3-methylbenzo [f] quinoline 0.2 g

Toluene to 100.0 ml

The test paper obtained is about a power of ten less sensitive than test papers according to Examples 1 and 2 (about 50 to 100 erythrocytes / mm ³ in 30 to 60 seconds).

Test papers of similar sensitivity are obtained if instead of 3-methylbenzo [f] quinoline the the following activators are used in equimolar amounts.

1,3-dimethylbenzo [f] quinoline

2,4-dimethylbenzo [g] quinoline

6-methylphenanthridine

3,8-dimethyl-4,7-phenanthroline

2,8-dimethyl-1,7-phenanthroline

2,7-Dimethyl-l.fi-anihra7oline

Claims (1)

235 claims: 1. Test strips for the detection of peroxidatically active substances in body fluids, consisting of from a carrier, which with a hydroperoxide, at least one chromogen and is impregnated with an activator consisting of a quinoline derivative, characterized in that that the quinoline derivative according to the general formula I