United States Patent O 3,711,252 COMPOSITION AND METHOD FOR THE DETECTION OF URIC ACID Ashim Chandra Roy, 62 Gloucester Drive, London, England No Drawing. Filed Oct. 13, 1971, Ser. No. 188,942 Claims priority, application Great Britain, Oct. 28, 1970,
51,276/ 70 Int. Cl. C07d 57/34; C09k 3/00; Gln 33/16 US. Cl. 23-253 TP 9 Claims ABSTRACT OF THE DISCLOSURE BACKGROUND OF THE INVENTION It is well known that the amount of uric acid found in the blood or urine is elevated when a person has gout, uremia, leukemia or pneumonia. Uric acid is normally present in blood serum in amounts varying from 0.7 to 6 percent mg. but in the disease conditions stated, values of percent mg. or higher are common.
Medical science has long recognized the usefulness of a test for uric acid in blood serum or urine as an aid in diagnosing abnormal conditions such as gout and distinguishing it from arthritis: in gout the uric acid increases whereas in arthritis no increase occurs. Several methods are known for determining uric acid in body fluids, but no rapid, simple and reliable dip and read test for uric acid has heretofore been reported.
SUMMARY OF THE INVENTION The present invention provides compositions for the detection of uric acid which comprise a ferric salt, a persulfate and either 2,4,6-tri(2-pyridyl)-1,3,5-triazine (hereinafter called TPTZ) or 2,2':6',2"-terpyridyl, in a buffered acidic medium.
DESCRIPTION OF THE PREFERRED EMBODIMENTS Uric acid causes a color change in such above noted compositions which change is directly proportional to the quantity of uric acid present. It is believed that ferric iron forms a complex with either TPTZ or terpyridyl which is reduced by uric acid to a ferrous complex thus causing a color change directly proportional to the concentration of uric acid. The color change reaction may be carried out in aqueous or alcoholic solution or on prepared strips of absorbent material pretreated with the reagent under acid conditions. The addition of the persulfate, e.g. ammonium persulfate, increases the specificity of the reaction, probably by preventing interference by ascorbic acid. It also increases the stability of the composition.
The color change reaction should be effected in an acidic medium of pH 2-6, and preferably about 4. Such a pH can be maintained by the presence of buffering agents such as glycinehydrochloric acid, glycine-acetic acid, sodium acetate-acetic acid, glycine-sulfamic acid or sodium succinate-succinic acid mixtures.
Ferric chloride is the preferred ferric salt, but any other siutable organic or other inorganic ferric salt may be em ice ployed, e.g. ferric sulfate, ferric alum, bromide, fluoride, nitrate, phosphate, acetate, or oxalate. The ferric salt is best employed in a ratio of one part by weight of said salt to 2 parts by weight of TPTZ or terpyridyl.
It is preferable to incorporate the test composition on or with a carrier member and to utilize this combination as a dip-and-read test device. This can be readily achieved, for example, by impregnating a bibulous material with a solution of the test composition and thereafter drying the impregnated test device. Alternatively, a finely divided, dry, intimate mixture of the ingredients can be adhesively attached to the surface of the carrier. Another embodiment comprises incorporating the test composition with a semi-permeable polymeric membrane and using the combination as such or further attaching such membranes to a carrier. The carrier material of the present invention may be filter paper, wood strips, synthetic plastic or non-woven or woven fabric. Filter paper having a thickness of from 0.01 inch to 0.02 inch is preferred as the carrier.
Light interferes with the color change reaction. The compositions of the invention should be stored in the dark, and the color change reaction performed so far as practicable in the dark or at least in the absence of strong light, especially sunlight.
The following examples illustrate the invention.
Example 1 One half m1. of a protein-free serum was mixed with 4 ml. of a 0.1 molar glycine-HCl buflier (pH=3.8), 0.3 ml. of a solution containing 2 mg. of TPTZ per ml. of ethanol, and 0.2 ml. of a solution containing 1.5 mg. of ferric chloride and 10 mg. of ammonium persulfate per ml. of ethanol. The mixture was held in the dark for 15 minutes and the intensity of the blue color which developed was read at 593 millimicrons against a reagent blank in a spectrophotometer. The amount of uric acid present in the serum was obtained by reference to a calibration curve prepared at the same time using ethanol solutions containing known amounts of uric acid.
Example 2 To 300 mg. of TPTZ dissolved in 2 ml. of glacial acetic acid was added ml. of water, 6 grams of glycine, 3 grams of ammonium persulfate and 150 mg. of ferric chloride. The mixture was diluted to ml. with water. Strips of No. Whatman filter paper (8 x 0.5 cm.) were partially dipped in this solution and dried rapidly under a vacuum in the absence of light. The impregnated portion was pale blue-white in color but when it was contacted with a solution of uric acid or a serum or urine containing uric acid, a blue color developed, the intensity of which was dependent upon the concentration of the uric acid present. By comparing the color of the strip with a standard color chart, two minutes after adding the solution, serum or urine, it was possible to estimate the uric acid content.
Example 3 TPTZ (260 mg.) was dissolved in 10 ml. of an aqueous solution containing 18% acetic acid and 60 ml. of an aqueous solution containing 10% glycine were added rapidly. To this mixture was added 15 ml. of an aqueous solution containing mg. of ferric chloride and 3 grams of ammonium persulfate. The resulting mixture was diluted to 100 ml. with water and allowed to stand 2 hours. Strips of absorbent filter paper were impregnated with this reagent solution by dipping and drying said strips in a current of warm air. The strips were used to estimate uric acid as described in the preceding example in sera and urine containing from 0 to 15 percent mg. uric acid.
In any of these examples the TPTZ can be replaced by an equal weight of terpyridyl and essentially the same results obtained.
It has been found that when a persulfate is included in the uric acid, test compositions described herein, there is: no interference due to the presence in the fluid being tested, of; glutathione, L-ergothioneine, cysteine, solicylates, gentisicacid, phenol, acetoacetate, iodide, cupric salts, citrate, lactate, pyruvate and phosphate.
What is claimed is: i
1. A composition for the detection of uric acid which comprises a ferric salt and a substance selected from the group consisting of 2,4,6-tri(2pyridyl)-1,3,5-triazine and 2,2':6',2"-terpyridyl, in a, buffered acidic medium.
2. A composition as claimed in claim 1 in which the ferric salt is ferric chloride. 1
3. A composition as claimed in claim 1 which one part of ferric salt is employedfor every two parts of 2,4,6-tri (2-pyridyl) -1,3,5-triazone or 2,2':6',2"-terpyridyl.
4. A composition as claimed in claim 1 in which the buffer employed is selected from the group consisting of glycine-hydrochloric acid and glycine-acetic acid.
5. A composition as. claimed in claim 1 in which the butter exerts a pH of about 4.
6. A composition as claimed in claim 1 which further includes the use of a persulfate.
7. A composition as claimed in claim 6 in which the persulfate is ammonium persulfate.
8. A test device comprising a carrier member incorporating a composition as claimed in claim 1.
9. A test device as claimed in claim 8 wherein the carrier member is bibulous paper.
References Cited- UNITED STATES PATENTS 1/1967 Bradbury et a1. 260296 D 1/1971 Cottrell 23230 B MORRIS'YO. WOLK, Primary Examiner ROBERT M. REESE, Assistant Examiner