Compositions and Method for Reducing Turbidity in Samples

The present invention is concerned with a process and a reagent for the removal of turbidities in sample materials, such as biological sample materials.

Reagents to remove turbidity are useful in samples, e.g., serum or plasma-clouded reaction mixtures which are, inter alia, employed in clinical-chemical analysis and foodstuff analysis, as well as in water and effluent analysis. In order to achieve a higher specificity, enzymes are thereby frequently used.

A known problem in photometric analysis is the influence of turbidities of the sample, such as serum or plasma, on the analysis results when the measurement is carried out without sample blank value. The avoidance of the measurement of a sample blank value is desired in order to reduce the reagent consumption, to simplify the measurement and evaluation procedure and to reduce the amount of sample needed. In the case of the use of one-pot reagents, i.e. those containing a mixture of all the reaction components, in principle, the measurement of a sample blank value is even impossible. Such turbidities have various causes, a frequent cause being a high triglyceride content.

Turbidity leads to an increased inherent extinction in a sample-reagent mixture. Since the total extinction is made up of the colored component, which is directly proportional to the concentration of the substrate to be determined, and of a turbidity component, it follows that false results are hereby obtained.

It is known to clear such tubidities by the addition of detergents to such an extent that the turbidity component of the total extinction can be neglected (see German patent specification No. 2,327,894).

An important disadvantage of this process is the fact that very large amounts of detergent are needed. The necessarily high concentrations of detergent are disadvantageous since the activity of the enzymes employed is hereby frequently reduced and, furthermore, such amounts can frequently no longer be introduced into solid material mixtures which are to be dry and sprinklable. This is of particular importance in the case of one-pot reagents.

The present invention overcomes the above-mentioned disadvantages and provides a process and a reagent which permits the above-mentioned turbidities to be removed. By "removal" of turbidity is to be understood, within the scope of the invention, also the prevention or reduction of such turbidities.

The present invention provides a process for the removal of turbidities in reaction mixtures clouded by sample material, especially for photometric measurement by adding to the reaction mixture

(a) at least one low molecular weight organic compound and

(b) at least one detergent which dissolves to give a clear solution and which forms a sparingly soluble complex with the additive (a), in an amount sufficient to bring about the appearance of a precipitation of the complex, and then adding further detergent until the precipitated complex dissolves again.

The present invention is based upon the surprising discovery that the above-mentioned organic compounds, which themselves show, in a clear aqueous solution, no or only a very slight clarifying effect on the turbidities, synergistically extraordinarily increase the already known clarifying action of detergents. The result of this is that, in most cases, not only is a clarification made possible but also the turbidity can be completely removed and, at the same time, the amount of detergent necessary for this is drastically reduced. Examples of compounds (a) which can be used include:

(a) aromatic (condensed and non-condensed) alcohols and amines and preferably those which are strongly electronegatively substituted, preferred electronegative substituents being halogen atoms, for example benzyl alcohol, phenol, mono-, di- and trichlorophenols, di- and tribromophenols, cresols, naphthols, dihydroxynaphthols, aniline, chloroaniline and methylaniline;

(b) esters of aromatic acids and alkanoic acids, for example ethyl p-hydroxybenzoate, ethyl acetate, butyl acetate, ethyl propionate, ethyl trichloroacetate and the like;

(c) straight-chained, cyclic and branched aliphatic alcohols containing more than 3 carbon atoms, for example amyl alcohol, octanol, dodecanol, cyclohexanol and the like;

(d) halogenated, short-chained aliphatic compounds, for example dichloroethane, trichloroethylene, carbon tetrachloride, dichlorobutene, tetrachlorodifluoroethane and the like;

(e) aliphatic ketones, for example butan-2-one, cyclohexanone and the like;

(f) aldehydes, for example cinnamaldehyde and benzaldehyde;

(g) carboxylic acids, for example octanoic acid;

(h) ethers, for example 2-methoxyphenol;

(i) benzene.

The amount of such a compound (hereinafter called an adjuvant) for each particular case can be determined by preliminary experiments. In general, we have found that concentrations of from 0.5 to 300 mM and preferably of from 2 to 10 mM give good results.

The detergents forming sparingly soluble complexes with the above-mentioned adjuvants but which themselves dissolve clearly in the reaction mixture include the non-ionic, ionic and amphoteric detergents, non-ionic polyethylene oxide adducts being preferred. Especially good results are achieved with alkyl, aralkyl and alkylthio ethers, as well as with alkyl and aralkyl esters of polyethylene oxide glycols. In these detergents, the alkyl radicals generally contain 8 to 22 carbon atoms and the aryl radical is preferably a phenyl radical. The glycol residue of these detergents generally contains 3 to 25 ethylene oxide units in condensed form. Typical examples of this especially preferred group of detergents include thesit, Genapol, Tergitol, Triton X100, Lensodel and Brij 35. However, ionic detergents can also be employed, especially alkyl dimethyl benzyl ammonium chlorides, for the alkyl radical of which there applies that which is stated above with regard to the polyethylene oxide adducts. Secondary alkyl sulphates, the alkyl radicals of which also correspond to the above definition, have also proved to be useful. Of the amphoteric detergents, N-lauryldiethanolamide has proved to be useful.

Within the scope of the present invention, as already mentioned, the amount of detergent necessary is very considerably lowered in comparison with the previously known processes. In general, amounts of from 0.05 and 1% suffice, detergent additions of from 0.2 to 0.8% being preferred. Higher concentrations can, of course, also be used but here there is an increased danger of a negative effect on the enzymes. In contradistinction thereto, according to the above-mentioned German patent specification, 10 vol.% of detergent is employed for the clarification.

Especially low detergent concentrations can be used when dissolving of the complex of detergent and adjuvant is carried out by adding a detergent which is different from the detergent contained in the complex. In other words, in the case of this preferred embodiment of the present invention, at least two different detergents are employed. The second detergent used can also be one which is not able to form a sparingly soluble complex with the adjuvant, which can be recognised by a turbidity.

The present invention also provides a clarification agent for reaction mixtures for analysis processes which are clouded by sample material, especially for photometric measurement, with a content of detergent, wherein it consists of or contains

(a) at least one low molecular weight organic compound and

(b) at least one detergent which dissolves to give a clear solution and which forms a less soluble complex with component (a) and optionally

(c) at least one further detergent which is different from (b) and which need not form sparingly soluble complex with (a).

For this clarification agent, there apply the above statements regarding the nature and amount of the detergents and adjuvants.

The process according to the present invention can be used in conjunction with all kinds of measurements in which turbidities can lead to a falsification or disturbance of the measurement and especially in the case of reaction mixtures which are intended for photometric measurement. Such reaction mixtures are well known and are, for example, described for many substances to be determined in "Methoden der enzymatischen Analyse", by H. U. Bergmeyer, pub. Verlag Chemie, Weinheim/Bergstrasse. Therefore, a more detailed explanation of such reaction mixture is here unnecessary.

The following Examples are given for the purpose of illustrating the present invention:

The following combinations according to the present invention gave, within 1 minute, up to a maximum of 2 hours, a removal of the turbidity in the system. 2 ml. test mixture +20 .mu.l drinking milk with 3.5% fat content.

1 cm. optical path length 546 nm; 25.degree. C.

(a) Variation of the adjuvant

In 0.1 M Tris/HCl buffer with 2% MgSO.sub.4.7H.sub.2 O

(b) Variation of the detergent

In 0.1 M Tris/HCl buffer (ph 8.0) with 2% MgSO.sub.4.7H.sub.2 O and 8 mM/l. 2,4-dichlorophenol as additive, good clarification was achieved with the following systems:

Formulation:

0.05 mol/l. K.sub.4 P.sub.2 O.sub.7

10 vol.% ethanol adjusted to pH 8.5 with HCl

1 mg./ml. NADP

1 U/ml. alcohol dehydrogenase EC 1.2.1.5

1000 U/ml. catalase EC 1.11.1.6

0.05 mol/l. disodium oxalate

0.05 mol/l. pyrazole

Because of the given wavelength and the relatively high amount of sample necessary, in the case of lipaemically turbid sample material, the measurement is made difficult or impossible.

For the removal of this turbidity, the following additions were made:

2% butyl acetate

1.5% thesit

0.5% dichloroethane

0.5% hexanol

5%o Genapol X-80 (isotridecanol polyglycol ether)

9%o Genapol X-100 (isotridecanol polyglycol ether)

6%o thesit

Test batch:

2 ml. reagent (with additives (1) or (2))

0.1 ml. sample

start with 0.6 U uricase EC 1.7.3.3.

Measurement at 340 nm and 25.degree. C. to end point.

The effect of the above additives is that the initial extinctions, which are considerably increased by lipaemic, cloudy sample material, are broken down in a very short time.

A turbidity of about 2.0 is reduced within about 10 minutes to a stable level of about extinction 0.2 in comparison with the reagent blank value. It is thereby possible to balance in order to start the reaction.

Formulation:

2.54 g./l. KH.sub.2 PO.sub.4

31.60 g./l. K.sub.2 HPO.sub.4

8 g./l. Na.sub.2 SO.sub.4

200 mg./l. 4-aminoantipyrin

282 mg./l. phenol

pH 7.90

3 U/ml. peroxidase EC 1.11.1.7

0.3 U/ml. cholesterol esterase EC 3.1.1.13

0.3 U/ml. cholesterol oxidase EC 1.1.3.6

The reagent is formulated as a one-pot mixture and must, therefore, be usable without measurement of a blank value. The fulfillment of this requirement is ensured, even in the case of strongly lipaemic, cloudy sample materials, by the following additives: Tubidity-clarifying additives:

815 mg./l. 3,4-dichlorophenol

4 g./l. Genapol Ox-100

3 g./l. sodium desoxycholate

2 g./l. heptanol

8 g./l. thesit

1 g./l. ethyl acetate

7 g./l. thesit

at pH 7.0 and 80 mM K.sup.+ /Na.sup.+ phosphate buffer

1.5 g./l. ethyl p-hydroxybenzoate

4 g./l. thesit

at pH 7.50 in 0.2 M potassium phosphate buffer in addition to the 3 mMol/l phenol needed for colour formation, also

35 mMol/l. phenol

1.5 g./l. Genapol Ox-100

1.5 g./l. Tween 20

1.0 g./l. sodium cholate

Batch: 2 ml. reagent+20 .mu.l. sample, 546 nm, 25.degree. C.

Turbidities of extinction .gtoreq.1.5 caused by the sample material are broken down completely or to a negligible amount within the period of time needed for the reaction.

Formulation:

14.9 g./l. Na.sub.2 HPO.sub.4

13.2 g./l. KH.sub.2 PO.sub.4

8.0 g./l. Na.sub.2 SO.sub.4

30 U/ml. glucose oxidase EC 1.1.3.4

1.8 U/ml. peroxidase EC 1.11.1.7

0.2 U/ml. esterase EC 3.1.1.13

470 mg./l. phenol

156 mg./l. 4-aminoantipyrin

at pH 7.0

Turbidity-clarifying additives:

814 mg./l. 3,4-dichlorophenol

3.6 g./l. Genapol )x-100

3.0 g./l. tauroglycocholic acid

814 mg./l. 3,4-dichlorophenol

2.5 g./l. thesit

3.9 g./l. tauroglycocholic acid

in 0.2 M potassium phosphate buffer, pH 7.0

additionally 1.35 g./l. phenol

489 mg./l. 3,4-dichlorophenol

3.0 g./l. Genapol Ox-100

2.8 g./l. sodium cholate

Measurement here takes place at 578 nm. As already explained above in Example 38, the additives here result in the turbidities in the test batch caused by sample material being broken down in a very short period of time.

The above-given Examples contain those in which are used

(a) one or more detergents which dissolve to give a clear solution and which can form a sparingly soluble complex with the additional material; and those in which are used

(b) additionally to (a), at least one detergent which is not able to form a sparingly soluble complex with the additional material under the conditions in the reaction mixture (designated by *).

It will be understood that the specification and examples are illustrative but not limitative of the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.