US 3549328 A
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TEST PAPER FOR DETECTOR OF NIACIN Filed Nov. 29, 1967 INVENTOR JAMES 0. KILBURN ATTORNEY! United States Patent 3,549,328 TEST PAPER FOR DETECTOR OF NIACIN James O. Kilburn, Chamblee, Ga., assignor t0 the United States of America as represented by the Secretary of Health, Education, and Welfare Filed Nov. 29, 1967, Ser. No. 686,494 Int. Cl. G01n 31 00, 33/00; C09k 3/00 US. Cl. 23253 3 Claims ABSTRACT OF THE DISCLOSURE This invention relates to a test paper for the detection of niacin.
As is known, the detection of niacin is an important biochemical tool in the identification of Mycobacterium tuberculosis. Several methods of the detection of niacin have been proposed, but they suffer from one or more disadvantages. Among the disadvantages is the requirement for using liquid reagents which are unstable upon exposure to light and air. Accordingly, the reagents must be freshly distilled to avoid colors which mask the color test for niacin. The art has, therefore, long sought a test procedure which could be used for the detection of niacin which avoids the use of liquid reagents.
It is an object of this invention to provide a dry, stable test paper which greatly facilitates the detection of niacin.
Another object is to provide a niacin test paper which yields reliable and reproducible results.
A further object is to provide a method for the detection of niacin which is simple and reliable and avoids the use of liquid reagents.
Other objects and advantages will be apparent to those skilled in the art from the description herein.
The test paper of the invention comprises an inert absorbent substrate, such as filter paper and the like which has been impregnated in separate discrete zones with three reagents. The reagents are solids at ambient temperatures. Solutions of the reagents are employed to impregnate the substrate and the substrate thereafter dried to yield areas of the three reagents on the substrate.
The first reagent is a source of cyanide ion which will react with a halogen to form a cyanogen halide. Suitable solid sources include potassium thiocyanate and potassium cyanide. Aqueous solutions of such reagents may be used to impregnate the substrate.
The second reagent is a source of active free halogen. Suitable solids include chloramine T and alkali metal hypochlorites, which may be used in aqueous solution to impregnate the substrate.
The third reagent is an aromatic amine. Suitable solid amines include p-aminosalicylic acid, benzidine and pmethylaminophenol sulfate. The amine component is preferably dissolved in a non-aqueous solvent for impregnation, since it tends to oxidize in the presence of Water to form colored products.
The accompanying drawing illustrates a test strip prepared in accordance with the invention. The drawing illustrates a strip of filter paper (on an enlarged scale) impregnated with the three reagents at A, B and C. At zone A the paper has been impregnated with the halogen "ice source. At zone B the impregnant is the cyanide ion source. Zone C contains the aromatic amine.
EXAMPLE Filter paper strips, 6 cm. x 8 mm., are impregnated in three zones as shown in the drawing. At zone A,
one drop of 50% aqueous chloramine T was applied. At zone B, a drop of 60% potassium thiocyanate dissolved in 8% aqueous citric acid was applied. At zone C, one drop of a 10% solution of p-aminosalicylic acid in ethanol was applied. The strips were air-dried at room temperature in the dark for 24 hours and thereafter stored at 4 C. in 13 x mm. screw capped tubes. The strips remained stable and useable for more than three months.
In use, 0.6 ml. of an aqueous extract of a culture being tested was added to a 13 x 100 mm. screw capped tube. The test paper strip was dropped into the extract, the p-aminosalicylic acid end first. Diffusion of the ex tract caused mixing of the reagents.
The acidified KCNS mixed with the chloramine T, releasing cyanogen chloride which reacts with niacin and p-aminosalicylic acid to form a yellow color in the liquid. Twelve to fifteen minutes, with occasional shaking, are preferably allowed for best development of the color test.
While it is not intended to be bound by any theory of operation, it is believed that the yellow color is a derivative of glutaconic aldehyde.
In practice, the test strips may bear suita'ble indicia (not shown) to identify one or more reagents on the strip.
While the invention has been described in terms of certain embodiments, they are to be considered illustrative rather than limiting, and it is intended to cover all modifications which fall within the spirit and scope of the appended claims.
1. A dry test strip for the detection of niacin comprising an inert absorbent substrate impregnated in discrete separate zones with (1) a solid stable source of free active halogen ion (2) a solid stable source of cyanide ion, and
(3) a solid stable aromatic amine.
2. The test strip set forth in claim 1 wherein (1) is chloramine T (2) is potassium thiocyanate (3) is p-aminosalicylic acid.
3. A method for the detection of niacin in an aqueous medium comprising adding the test strip of claim 2 to the medium whereby a yellow color develops in said medium if niacin is present.
References Cited FOREIGN PATENTS 3,409,405 11/1968 Mohan 23253 3,411,887 11/1968 Chiu-Choon Kn 23253 3,434,801 3/1969 Scherr 23253' 3,453,180 7/1969 Fraser 23-253 OTHER REFERENCES Goldthorpe et al., Estimation of Nicotinic Acid In Tissues by the Cyanogen Bromide Reaction, Analytical Chemistry, vol. 23, No. 3, March 1951, pages 484- 487.
MORRIS O. W-OLK, Primary Examiner R. E. SERWIN, Assistant Examiner US. Cl. X.R. 23- 230; 252-408