US RE28575 E
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United States Patent [191 Bauer 11 E Re. 28,575
[ Reissued Oct. 21, 1975  INDICATOR FOR DETECTING HYDROGEN PEROXIDE AND PEROXIDATIVE COMPOUNDS CONTAINING ALPHA NAPHTHOFLAVONE  Inventor: Robert Bauer, Bristol, Ind.
 Assignee: Miles Laboratories, Inc., Elkhart,
 Filed: Apr. 8, 1974  Appl. No.'. 459,126
Related US. Patent Documents  Field of Search 252/408; 8/1 R; 23/230 B, 23/253 TP; 195/1035 C  References Cited UNITED STATES PATENTS 2,799,660 7/l957 Nicholls 252/408 3,066,081 11/1962 Rorem 195/1035 X 3,092,465 6/1963 Adams t 23/253 3,183,173 5/1965 Oakes 23/230 3,814,668 6/1974 Blake 23/253 TP X Primary ExaminerBenjamin R. Padgett Assistant Examiner-David Leland Attorney, Agent, or FirmAndrev v L. Klawitter  ABSTRACT Alpha naphthoflavone is an excellent indicator for detecting hydrogen peroxide and peroxidative compounds such as hemoglobin. When formulated with either a peroxidative active compound or a peroxide, this indicator provides a very sensitive chromogenic response to the presence of said constituents in aque- 7 @Claims, No Drawings It/latter enclosed in heavy brackets appears in the original patent but forms no' part of this reissue specification; matt er printed in italics indicates the additions made by reissue.
BACKGROUND OF 'THE INYENTION r The determination of glucose in urine isimportant since thistest is employed to detect'diabetes. Procedures for the detection of sugar in urine are well known in "clinical chemistry. One such procedure utilizes Benedicts copper reduction test, another employs a self heating alkaline copper reduction. testin tablet form, while still'another test depends solely on the action of enzymes. The diagnostic composition in most glucose tests comprises essentially glucose oxidase, peroxidase and an indicator which is oxidizedbyhydr'ogen peroxide and undergoes a color reaction during such oxidation. Typical'indicators employed in' the past include o-tolidine, benzidine dianisidine and 27- diamino'fluorene. I a i [t is well known that glucose oxidase'catalyz'es the aerobic oxidation of glucose to gluconic acid and hydrogen peroxide. However .in the presence .of "iodide, H o oxidizes the iodide to free iodine which produces a color change in the indicator. The color change. pro- SUMMARY OF THE INVENTION This invention is predicated upon the discovery that a-naphthoflavone can be used as an indicator dyestuff in formulations containing either peroxide or a peroxidative active compound when the peroxidative active I compound catalyzes the oxidation of the indicator by the peroxide. Said indicator has the formula and is known chemically as 2-phenyl-4H naphtho l ,2-b]pyran-4-one.
Although the test system may comprise the reagent by dissolving the components in asuitable solvent, impregnating the strips with the resulting solution and drying the impregnated test strips. Details of the compositions contemplated are' set forth in the following examples; I I I I DESCR IPTIONOF THE PREFERRED I EMBODIMENTS.
Example 1 composition was prepared by mixingv the following "components in thegvolumes indicated below: I Glucose oxidase '5 ml. of an aqueoussolution containing 1000 International units per ml. I I Peroxidasel mlfof an aqueous solution containing 2 mg./ml. i i
Example-2' Another composition was prepared. employing no peroxidase as shown below: I t Glucose oxidase3 ml. of an aqueous solution containing 1 0( )0 International units/ml. Potassiumiodi de80 mg. I Ammoniummolyb'datel60 mg. 1 Sodium phosphate buffer3 ml. of a l; M
pH6.. v I v a-Naphthoflavonel ml. of a 0.5% ethanol solution. .Water-S ml. I i i Porous paper strips about A. inch wide long were dipped into the above compositions respectively so that about one-half inch of each strip at one end was completely impregnated. The strips were then dried at 100 C. for 10 minutes. If desired, other porous materials such as wood or plastics can be used as carriers. When contacted with urine containing glucose, such test strips give a positive reaction in 1 minute or less as evidenced by the change in color of the indicator from colorless to blue. The formation of color depends upon the liberation of free iodine from the iodide by the action of hydrogen peroxide. When dipped into urine containing no glucose, the strips show no color change. The intensity of the blue color is enhanced in solution of the presence of polyvinyl alcohol. When molybdates and iodides are substituted for the peroxidase, the a-naphthoflavone minimizes the inhibiting effects of acetoacetate which is also an iodine receptor. It was found that when the strips containing the composition of Example 2 were dipped into a 1% aqueous solution composition in the form of a tablet, powder or solution,
it is preferable to affix said composition on bibulous base materials or carriers such as strips of filter paper of glucose containing 0.1% by weight of acetoacetate they began to change from colorless to blue in 30 seconds and the blue color gradually intensified, whereas strips containing o-tolidine started to show a blue color at 1.5 minutes with very little increase in color intensity even after 5 minutes contact with the same glucose solution.
Example 3 A first solution was prepared containing 1.5 grams of carrageen, 15 grams of polyvinylpyrrolidone, 15 ml. of ethanol and 192 ml. of water.
and 3 inches A second solution was prepared containing 9.24 gramsof citric acid, 40.79 grams of sodium citrate and 124.8 ml. of water. y
A third solution was prepared containing 4.5 grams ofa maleic anhydride methylvinylether copolymer, 1.5 grams of sodium lauroyl sarcosinate and 105 ml. of water.
Still, a fourth solution was prepared containing 0.5 gram of peroxidase and 76 ml. of an aqueous solution of glucose oxidase containing 1,000 international units per ml. of water.
A composition for detecting H and glucose was thereafter prepared containing 9 ml. of a 0.5% ethanol solution of a-naphthoflavone, 0.73 gram of potassium iodide, 9 ml. of ethanol, 5.5 ml. of water, 34.5 ml. of the first solution above, 20.8 ml. of the second solution above, 17.5 ml. of the third solution above and 7.6 ml. of the fourth' solution previously prepared. Bibulous paper strips were dipped in said solution and then dried for 10 minutes at 100 C. These strips readily turned from colorless to blue when contacted with urine containing lucose and the blue color increased with the glucose concentration. Such strips also change from colorless to blue when a drop of bloodcontaining urine and a drop of 3% hydrogen peroxide solution is applied thereto.
in addition to the compositions set forth in the foregoing examples, it was found that the amount of indicator employed could be varied from 0.05% to 0.30% by weight in such compositions whereas the glucose oxidase concentration could vary from 40 to 300 International units per ml. of peroxidase from 0.0 l to 0.05%, the sodium iodide from 0.25% to 1.5%, the ammonium molybdate from 0.1% to 1.5%, and the polyvinyl alcoho] from 0.2% to 2% by weight of ultimate mix at a pH of from 4 to 7.
What is claimed is:
1. In a composition for detecting hydrogen peroxide or peroxidative active compounds utilizing the catalytic oxidation of an indicator system [dyestuff by hydrogen peroxide in the presence of the peroxidative active compound, the improvement which comprises the use of a-naphthoflavone in combination with a water soluble iodide salt as the indicator system [dyestuffi] 2. A composition as claimed in claim 1 in which a-naphthoflavone is present in about 0.05% to 0.30% by weight of said composition.
3. A composition as claimed in claim 1 in which the peroxidative active compound is selected from the group consisting of peroxidase, hemoglobin and molybdate.
4. A composition as claimed in claim 3 in which the molybdate is present in about 0.1% to 1.5% by weight of said composition.
5. A composition as claimed in claim 3 in which the peroxidase is present in about 0.01% to 0.05% by weight of said composition.
[6. A composition as claimed in claim 1 which additionally contains iodide] 7. A composition as claimed in claim I  in which the water soluble iodide salt is present in about 0.25% to 1.5% by weight of said composition.
8. A composition for detecting glucose in aqueous fluids which comprises glucose oxidase, a peroxidative active material, a water soluble iodide salt, and