Aqueous cholesterol standard solution

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AQUEOUS CHOLESTEROL STANDARD obtained with a content of 8 to 12 vol. percent of an al SOLUTION cohol, for example ethanol, isopropanol or n-butanol, and 0.8 to 1.0 wt. percent sodium chloride. If, instead

The present invention relates to a cholesterol stan of sodium chloride, alkyl dimethyl-benzyl ammonium dard solution, more specifically, an aqueous solution of 5 chloride is used, then the preferred amount is 0.05 to cholesterol with a definite and stable content of choles terol. 0.15 wt. percent.

A number of chemical and enzymatic processes are Another preferred cholesterol standard solution ac known or have been suggested for the quantitative de cording to the present invention comprises 8 to 12 vol. termination of cholesterol in various materials, espe 10 percent of an alcohol, 55 to 60 vol. percent diethylene cially in biological fluids. In the case of these processes, glycol monobutyl ether and 0.05 to 0.15 wt. percent in order to evaluate the measurement results, it is nec alkyl dimethyl-benzyl ammonium chloride. essary to use cholesterol solutions with a definite, Yet another preferred cholesterol standard solution known content of cholesterol as standard. For this pur according to the present invention additionally con pose, cholesterol solutions are used with a definite cho 15 tains a small amount of tetramethylurea. The amount lesterol content which are called cholesterol standards. thereof can be 0.5 to 5 vol. percent and is preferably In the case of the previously used standards, the choles 0.8 to 1.5 vol. percent.

terol is dissolved in glacial acetic acid, chloroform, ab Furthermore, depending upon the expected storage solute alcohol or the like. However, the anhydrous conditions, it can be expedient, for the prevention of standard solution so obtained gives, in the case of a bacterial attack, to add an alkali metal azide. In this cholesterol determination with the Liebermann

Burchardt color reagent, a color intensity which is dif case, the pH value must be adjusted to more than 6.5 ferent from that obtained with serum and can result in by the addition of an appropriate buffer. It is preferred false cholesterol values in the serum (see L. Vass, to add 0.05 to 0.2 percent and more, preferably about Schweiz. med. Wschr., 102914/1972). 25 0.1 percent of an alkali metal azide, for example so This disadvantage can be overcome by the use of an dium azide, in a buffer of pH 6.5 to 8, preferably of 7 aqueous cholesterol standard solution. However, the to 8, for example, a triethanolamine buffer. known aqueous cholesterol standard solutions, which The cholesterol standard solution according to the contain hydroxypolyethoxydodecane as solubilizing present invention can contain up to 400 mg cholesterol agent, in turn suffer from a serious disadvantage insofar 30 in 100 ml of the solvent mixture. The extraordinary as the cholesterol content thereof changes rapidly and storage stability of the solutions necessitates neither the decreases noticeably even in the course of a few days. exclusion of light nor the exclusion of oxygen. This is However, since it is, in the case of a cholesterol stan dard solution, especially important that the cholesterol 35 all the more surprising since, as is known, cholesterol, content thereof remains absolutely constant, it was even in a solid state, must be stored under an inert gas hitherto necessary freshly to prepare the aqueous cho and with the exclusion of light. The stabilizing effect lesterol standard solution before use, which was a con clearly depends upon the interaction of the various siderable disadvantage and, in addition, rendered ques components of the standard solution according to the tionable the dependability of the cholesterol determi 40 present invention since, with the individual compo nation. nents alone, a stabilizing effect cannot be achieved. For The present invention obviates or mitigates these dis example, when a solution which originally contains 400 advantages and provides a stable, aqueous cholesterol mg cholesterol in 100 ml of a mixture of 10 vol. percent standard solution. hydroxy-polyethoxydodecane and 90 vol. percent wa The present invention provides an aqueous choles terol standard solution, comprising, in aqueous solu 45 ter, is stored for 3 weeks at 33°C, only 80 percent of the original cholesterol is still found to be present. In con tion, a known amount of cholesterol, together with 1 to tradistinction thereto, in the case of the standard solu 20 volume percent of a primary or secondary aliphatic alcohol containing up to 4 carbon atoms (for example, tion according to the present invention in its various ethanol, isopropanol or n-butanol); 5 to 20 volume per SO forms, even after storage for three months under the cent of hydroxy-polyethoxydodecane or 40 to 60 vol same conditions, no change of the cholesterol content ume percent diethyleneglycol monobutyl ether; and 0.5 can be ascertained. In the especially preferred embodi to 5 weight percent sodium chloride and/or 0.01 to 0.5 ments of the present invention, complete stability was weight percent alkyl dimethyl-benzyl ammonium chlo still retained after a period of storage of 2 years. ride with 8 to 18 carbon atoms in the alkyl radicals. 55 The following Examples are given for the purpose of The present invention rests on the surprising discov illustrating the present invention:

ery that a solution of cholesterol in dilute aqueous alco hol which, as solubilizing agent, contains the given EXAMPLE 1. amount of hydroxy-polyethoxydodecane or diethylene 40 g recrystallized cholesterol were dissolved in one glycol monobutyl ether, and, as stabilizing agent, so 60 liter hydroxypolyethoxydodecane by heating in a wa dium chloride and/or alkyl dimethylbenzyl ammonium ter-bath at 40°C. This solution was diluted with doubly chloride, is outstandingly stable and, even when stored for a year or more under normal conditions, does not distilled water with a temperature of 40°C, then 100 ml change with regard to the cholesterol content. of a 10 percent aqueous solution of alkyl dimethyl A preferred cholesterol standard solution according benzyl ammonium chloride and 1 liter ethanol were to the present invention contains the dilute alcohol, added thereto and, after cooling to ambient tempera hydroxy-polyethoxydodecane and sodium chloride. In ture, made up to precisely 10 liters with doubly distilled this preferred composition, the best results have been Water.

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EXAMPLE 2 component (d) is alkyl dimethyl benzyl ammonium chloride.

40 g cholesterol were dissolved in 1 liter 7. Standard solution as claimed in claim 1 wherein hydroxy-polyethoxydodecane and 1 liter ethanol and component (d) is a combination of sodium chloride thereafter made up to 10 liters with a 0.9 percent aque and alkyl dimethyl benzyl ammonium chloride. ous solution of sodium chloride. 8. Standard solution as claimed in claim 1 wherein EXAMPLE 3 component (b) is contained in an amount of from 8 to 12 volume percent of said solution.

40 g cholesterol were dissolved in 1 liter 9. Standard solution as claimed in claim 1 wherein hydroxy-polyethoxydodecane, 1 liter n-butanol, 100 ml 10 component of a 10 percent aqueous solution of alkyl dimethyl contained in(c) an is hydroxy-polyethoxydodecane and is amount of from 5 to 20 volume percent benzyl-ammonium chloride and 100 ml tetrame based on the standard solution.

thylurea and then made up to 10 liters with doubly dis 10. Standard solution as claimed in claim 1 wherein tilled water.

component (d) is sodium chloride and is contained in

EXAMPLE 4 an amount of from 0.8 to 1.0 weight percent.

11. Standard solution as claimed in claim 1 wherein 40 g cholesterol were dissolved in 6 liters diethylene component glycol monobutyl ether, 0.5 liters ethanol and 100 ml chloride and(d) is alkyl dimethyl benzyl ammonium is contained in an amount of from 0.05 to of a 10 percent aqueous solution of alkyl dimethyl 0.15 weight percent.

benzyl-ammonium chloride were added thereto and 20 then the mixture was made up to 10 liters with doubly 12. Standard solution as claimed in claim 7 compris distilled water. ing 8 to 12 volume percent of said alcohol as compo It will be understood that the specification and exam nent (b).

ples are illustrative but not limitative of the present in 13. Standard solution as claimed in claim 1 compris vention and that other embodiments within the spirit 25 ing 55 to 60 volume percent of diethylene glycol mono and scope of the invention will suggest themselves to butyl ether as component (c) and 0.05 to 0.15 weight those skilled in the art. percent of alkyl dimethylbenzyl ammonium chloride as What is claimed is: component (d).

1. Cholesterol standard solution comprising in aque 14. Standard solution as claimed in claim 1 addition ous solution 30 ally containing tetramethylurea. a. a defined amount of cholesterol; 15. Standard solution as claimed in claim 14 wherein b. 1 to 20 volume percent, based on the standard so the content of said tetramethylurea is from 0.5 to 5 vol lution, of a primary or secondary aliphatic alcohol ume percent based on said standard solution. of up to 4 carbon atoms; 16. Standard solution as claimed in claim 15 wherein c. 5 to 20 volume percent of hydroxy-polyethoxydo 35 the content of said tetramethylurea is from 0.8 to 1.5 decane or 40 to 60 volume percent of diethylene volume percent based on said standard solution. - glycol monobutyl ether; and 17. Standard solution as claimed in claim 1 also con d. at least one of taining an alkali metal azide and wherein said solution i. 0.5 to 5 weight percent of sodium chloride; and is buffered to a pH of at least 6.5.

ii. 0.01 to 0.5 weight percent of alkyl dimethylben 40 18. Standard solution as claimed in claim 17 wherein zyl ammonium chloride of from 8 to 18 carbon the content of said alkali metal azide is 0.05 to 0.2 per atoms in the alkyl moiety. cent by weight based on said standard solution. 2. Standard solution as claimed in claim 1 wherein 19. Standard solution as claimed in claim 18 wherein said primary or secondary aliphatic alcohol of compo the content of said alkali metal azide is about 0.1 nent (b) is at least one of ethanol, isopropanol or n 45 weight percent.

butanol. 20. Standard solution as claimed in claim 17 wherein 3. Standard solution as claimed in claim 1 wherein said solution is buffered to a pH of 6.5 to 8. component (c) is hydroxy-polyethoxydodecane. 21. Standard solution as claimed in claim 20 wherein 4. Standard solution as claimed in claim 1 wherein said solution is buffered to a pH of 7 to 8. component (c) is diethyleneglycol monobutyl ether. 50 22. Standard solution as claimed in claim 1 wherein 5. Standard solution as claimed in claim 1 wherein the cholesterol content is up to 400 mg. per 100 ml. of component (d) is sodium chloride. said standard solution.

6. Standard solution as claimed in claim 1 wherein se k k