DK142296B - Test strip for detection of ketone substances, especially acetic acetic acid. - Google Patents

Description

(11) PRESENTATION H2296 (m) \ ζ & / DENMARK «'> in. · Ci. · E o 1 n 33/52' (21) Application No 3364/74 (22) Filed on 21 Jun. 1974 (24) Race day 21. June 1974 (44) The application submitted and the notice of publication published on 6 Oct · 1 980

Dl THE RECTORATE FOR

PATENT AND TRADE MARKET (30) Priority requested from it

Nov 8 1973a 125667/73 * JP

(71) jSHIONOGI & CO. LTD., 12, 3-chome, Dosho-machi, Hlgashi-ku, Osaka, JP.

(72) Inventor: Yasunao Ogawa, 5-12-19, Asahigaoka, Ikeda-shi, Osaka Pref., JP: Yukio Yonetani, 3-5-4, Tiyogaoka, Nara-shi, Nara Pref., JP.

(74) Plenipotentiary under consideration:

Th. Ostenfeld Patentbureau A / s._ (54) Test strip for detection of ketone substances, especially acetic acetic acid.

The present invention relates to a test strip for the detection of ketone substances, especially acetacetic acid.

Ketosis is known to occur as a result of disorders of carbohydrate fat metabolism. In patients with ketosis, the content of ketone substances, such as acetic acid, acetic acid, acetone or β-hydroxybutyric acid in the blood, is increased and these substances are excreted mainly as acetic acid in the urine. Ketosis is evidence of the presence of various diseases such as diabetes, digestive disorders, renal insufficiency, uremia, self-poisoning, malignant carcinoma, etc. Thus, the detection of ketones in body fluids such as urine, serum or plasma can be a diagnosis of such diseases.

2 142296

So far, a number of methods for detecting ketone substances have been used in the art. Eg. a sample strip comprising a nitroprusside salt and alkali for detection of ketone substances is known. However, since nitroprusside salts are very unstable against alkali, this method, which is carried out under alkaline conditions, is not suitable for the determination of ketone substances. Furthermore, the test strips used so far in the art suffer from the disadvantages that their production is complicated and that the sensitivity of the strips to ketone substances is not great. Also, from German Patent Specification No. 1,153,920, a diagnostic composition for detecting ketone compounds in body fluids is known which comprises a carrier impregnated with a solution of a nitroprusside, a primary phosphate, a secondary phosphate and an aminoacetic acid, as the amount of the components is adjusted. so that the solution has a pH of 6.6 to 7.5. Nothing is mentioned about heavy metal salts. Surprisingly, it has been found that using a specified nitroprusside salt in combination with a specified heavy metal salt without the addition of a substance to keep the system at an alkaline pH in a simple manner provides a stable test strip with good sensitivity and specific reactivity to ketones. especially acetic acetic acid.

The test strip of the present invention is characterized in that it comprises an absorbent material impregnated with a solution of an inorganic metal salt in the form of nickel chloride, ferric chloride, ferrochloride, ferric ammonium sulfate, ferrous sulfate, cuprochloride, cuprichloride, cobalt dichloride and cobalt dichloride or cobalt dichloride of sodium or potassium nitroprusside, wherein the concentration of metal salt in the solution is from 1 to 20% and the concentration of nitroprusside salt in the solution is from 1 to 20%.

The present test strip can be prepared by dissolving or suspending the nitroprusside salt and the metal salt in a suitable solvent to form a solution. Then, an absorbent material is dipped into the solution and dried in the air or at a temperature of from room temperature to approx. 50 ° C to form a test strip for detection of ketone substances.

The metal salt used is in the form of nickel chloride, ferric chloride, ferrochloride, ferric ammonium sulfate, ferrous sulfate, cuprochloride, cuprichloride, cobalt dichloride or cobalt dichloride. Among the most preferred metal salts are nickel chloride. The metal salts are used at a concentration of from 1 to 20%, e.g. from 5 to 15%.

3 14229G

The nitroprusside salt is used at a concentration of 1 to 20%, e.g. from 5 to 15%.

Examples of solvents for use in the preparation of an impregnation solution containing the metal salt and the nitroprusside salt are water, organic solvents (e.g., dimethylformamide, dimethylsulfoxide, methanol or ethanol) and mixtures thereof.

The absorbent material to be dipped in a solution containing the nitroprusside salt and the metal salt may be a sheet of paper, a piece of cloth or a piece of porous wood. Examples of suitable forms of paper are filter paper, clotting paper, adsorbent paper (e.g. silica gel paper or alumina paper), ion exchange cellulose paper (e.g. phosphonomethylcellulose ethylcellulose cellulose paper, phospho "cellulose paper (anion exchange material made from cellulose, epichlorohydrin and triethanolamine), p-aminobenzyl cellulose paper or polyethyleneimine cellulose paper), ion exchange" Sephadex® "paper and ion exchange resin paper. Among the absorbent materials, ion exchange cellulose paper is preferably used, especially diethylamino cellulose paper.

Usually, the test strip according to the invention is used for dipping into or impregnating with the sample to be tested, thereby producing a detectable color change. Similarly, when plasma or serum is used as the sample to be tested, it is possible to use the test strip according to the invention. The test strip according to the invention can be used, if desired, in more convenient form, such as e.g. adhered to a plastic wrap.

The test strip according to the invention is sensitive to ketone substances, especially uric acid acetic acid. Since a characteristic color change takes place depending on the concentration of the ketone substances to be detected, it is possible to make semi-quantitative determination of such ketone substances. It is possible to detect acetic acetic acid in an amount of more than 1 mg / 100 ml in the urine with the test strip of the invention. In general, the color of the test strip depends on the metal salt used. When nickel or cobalt salts, iron salts and copper salts of the above type are used, the test strip becomes colored purple, orange to brown and blue to purple respectively in the presence of acetic acetic acid. These different tones appear to be due to the formation of particularly colored chelates between the nitroprusside salt, acetic acetic acid and the metal salt. It is believed that this chelate compound contributes greatly to the stability of the nitroprusside salt, thus providing a clear and uniform color with strong intensity. When examining an acidic urine sample, it is preferred to use a test strip which further contains tris (hydroxymethyl) aminomethane which has a buffering effect.

The invention is illustrated in the following by way of example.

Example 1

To a 5% aqueous solution (10 ml) of nickel chloride was added a 5% dimethylformamide solution (3 ml) of sodium nitroprusside in water to give a cloudy, pale yellow solution. One piece (5x6 cm) of diethylaminoethyl cellulose paper (type "DE-81" from Whatman Co.) was dipped in the solution for 1 minute and then the strip was dried at room temperature in a dark place. Then, the strip was cut into smaller pieces (1 x 0.8 cm) and the strips were each adhered to a base, such as a plastic film. The test strips thus obtained were immersed independently in urine samples containing acetic acetic acid at a concentration of 1, 5, 10, 20, 50 and 100 mg / 100 ml, respectively. The test strips became strongly red-violet within 1 minute, depending on the concentration of acetic acid above 1 mg / 100 ml. When the test strip was dipped in a solution which did not contain acetic acid, the strip did not change color and retained its original color (pale yellow).

The test strip did not react with ascorbic acid or thiol compounds, but turned green blue in the presence of pyruvic acid and pale brown in the presence of α-ketoglutaric acid.

Example 2

A 5% dimethylformamide solution (3 ml) of sodium nitroprusside in water was added to a 5% aqueous solution (10 ml) of ferric chloride to form a pale yellow solution. One piece (5x5 cm) of silica gel paper (type "M3F 8860" from Carl Schleicher & Schull Co.) dipped in this solution for 1 minute, dried in the air, cut into pieces (1 x 1 cm) and held to a plastic wrap to form the desired strips.

The sensitivity of the test strips to acetic acid was the same as the sensitivity of the test strips obtained in Example 1.