# US 5,350,694 A · Composition method and device for measuring the divalent cation concentration or specific gravity of a test sample

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- Number: US 5,350,694 A
- Document ID: US5350694A
- Status: Granted
- Kind: A
- Application number: 08/019,668
- Filed: 1993-02-19
- Granted: 1994-09-27
- Earliest claimed: 1993-02-19
- Assignees: Miles Inc.
- Inventors: Chris T. Zimmerle
- Web page: https://patentgrep.com/patent/US5350694A

## CPC

- G01N 33/521: {Single-layer analytical elements}
- G01N 33/52: Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)}
- Y10T 436/108331: Preservative, buffer, anticoagulant or diluent
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

A composition, method and test device for determining the divalent cation concentration or estimating the specific gravity of a test sample are disclosed. The method utilizes a test device comprising a test pad, wherein the test pad includes a carrier matrix incorporating a reagent composition capable of interacting with divalent cations present in the test sample to produce a detectable and measurable response that correlates to the divalent cation concentration or to the specific gravity of the test sample. The reagent composition, comprising a metal-sensitive triphenylmethane dye; a buffer; an optional chelating agent; and suitable carrier, is incorporated into a carrier matrix to provide a test pad of a device useful in a divalent cation assay or a specific gravity assay of a test sample.

## Claims

### Claim 1

A composition capable of exhibiting a detectable and measurable color transition only in response to a total divalent cation concentration of an aqueous test sample, said composition comprising:
(a) about 5 to about 60 millimoles per liter of the composition of a metal-sensitive triphenylmethane dye having the structural formula: ##STR4## wherein X is carbonyl or sulfonyl, n is a number from 0 to about 3, and the R substituents are selected, independently, from the group consisting of hydrogen, hydroxy, an alkyl group including one to about five carbon atoms, bromo, chloro, fluoro, iodo, sulfonate, and phosphate;
(b) a buffer;
(c) a carrier comprising water; and
(d) about 0.5 to about 4 millimoles per liter of the composition of a chelating agent selected from the group consisting of a polyhydroxy compound, a lignosulfonate, a glucoheptonate, a salicyate complex, a dithionate derivative, a polyethyleneamine, a 2,4-pentanedione, a dipyridine, a polypeptide including cysteine, glycine or histidine, a thiocrown ether, a triphenylphosphine, sorbitol, dimethylglyoxime, proline, bissalicylaldehydeethylenediimine, triethyleneamine, triethylenepyridine amine, 1,4,8,11,22,25-octathiacyclooctosane, ethylenediaminetetraacetic acid, N-(hydroxyethyl) ethylenediaminetriacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, aminotris(methylene phosphoric acid), hydroxyethylidene diphosphonic acid, hexamethylenediaminetetra(methylene phosphonate), ethylenediaminediacetic acid, iminodiacetic acid, nitrilopropionic acid, hydroxyethyliminodiacetic acid, pyrophosphoric acid, 1-hydroxyethane-1,1-diphosphonic acid, tripolyphosphoric acid, hexametaphosphoric acid, metaphosphoric acid, and mixtures thereof;
wherein the composition is buffered at a pH of at least about 7.

### Claim 2 (depends on 1)

The composition of claim 1 wherein the metal-sensitive triphenylmethane dye is selected from the group consisting of cresolphthalein complexone, cresolsulfonephthalein complexone, phenolphthalein complexone, phenolsulfonephthalein complexone, bromophthalein complexone, methylthymol blue, xylenol orange, thymolphthalein complexone and mixtures thereof.

### Claim 3 (depends on 1)

The composition of claim 1 wherein the buffer is present in a concentration of about 100 to about 600 millimoles per liter of the composition.

### Claim 4 (depends on 1)

The composition of claim 1 wherein the composition is buffered at a pH of at least about 7 to about 10.

### Claim 5 (depends on 1)

The composition of claim 1 wherein the buffer is selected from the group consisting of acetate; BICINE; phthalate; borate; trichloracetate; sulfosalicylate; phosphate; tartarate; citrate; succinate; maleic acid; 2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol; 3,3-dimethylglutaric acid; 3-N-morpholinopropanesulfonic acid; malonic acid; 1,3-bis[tris(hydroxymethyl)methylamino]propane; tris(hydroxymethyl)aminomethane; tris(hydroxymethyl)aminomethane-maleic acid; tris(hydroxymethyl)aminomethane-malonic acid; 3-N-(trishydroxymethyl)methylamino-2-hydroxypropanesulfonic acid; 2-([tris(hydroxymethyl)methyl]amino)-ethanesulfonic acid; 1,4-piperazinebis(ethanesulfonic acid); 4-morpholinoethanesulfonic acid; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; and mixtures thereof.

### Claim 6 (depends on 1)

The composition of claim 1 wherein the carrier further comprises 0% to about 20% by weight of an organic solvent, based on the weight of the carrier.

### Claim 7

A method of determining a total divalent cation concentration of an aqueous test sample comprising:
(a) contacting the aqueous test sample with a reagent composition capable of exhibiting a color transition only in response to the total divalent cation concentration of the aqueous test sample, said composition comprising:
(i) about 5 to about 60 millimoles per liter of the composition of a metal-sensitive triphenylmethane dye having the structural formula: ##STR5## wherein X is carbonyl or sulfonyl, n is a number from 0 to about 3, and the R substituents are selected, independently, from the group consisting of hydrogen, hydroxy, an alkyl group including one to about five carbon atoms, bromo, chloro, fluoro, iodo, sulfonate, and phosphate;
(ii) a buffer;
(iii) a carrier comprising water; and
(iv) about 0.5 to about 4 millimoles per liter of the composition of a chelating agent selected from the group consisting of a polyhydroxy compound, a lignosulfonate, a glucoheptonate, a salicyate complex, a dithionate derivative, a polyethyleneamine, a 2,4-pentanedione, a dipyridine, a polypeptide including cysteine, glycine or histidine, a thiocrown ether, a triphenylphosphine, sorbitol, dimethylglyoxime, proline, bissalicylaldehydeethylenediimine, triethyleneamine, triethylenepyridine amine, 1,4,8,11,22,25-octathiacyclooctosane, ethylenediaminetetraacetic acid, N-(hydroxyethyl)ethylenediaminetriacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, aminotris(methylene phosphoric acid), hydroxyethylidene diphosphonic acid, hexamethylenediaminetetra(methylene phosphonate), ethylenediaminediacetic acid, iminodiacetic acid, nitrilopropionic acid, hydroxyethyliminodiacetic acid, pyrophosphoric acid, 1-hydroxyethane-1,1-diphosphonic acid, tripolyphosphoric acid, hexametaphosphoric acid, metaphosphoric acid, and mixtures thereof,
wherein the reagent composition is buffered to a pH of about 7 to about 8; and
(b) determining the total divalent cation concentration of the aqueous test sample from the intensity and degree of the color transition of the reagent composition.

### Claim 8 (depends on 7)

The method of claim 7 wherein the aqueous test sample is a biological fluid.

### Claim 9 (depends on 8)

The method of claim 8 wherein the biological fluid is selected from the group consisting of urine, blood plasma, blood serum, and perspiration.

### Claim 10 (depends on 7)

The method of claim 7 wherein the buffer is present in a concentration of about 100 to about 600 millimoles per liter of the composition.

### Claim 11 (depends on 7)

The method of claim 7 wherein the carrier further comprises 0% to about 20% by weight of an organic solvent based on the weight of the carrier.

### Claim 12 (depends on 7)

The method of claim 7 wherein the intensity and degree of the color transition is determined visually or instrumentally.

### Claim 13

A method of semiquantitatively determining a specific gravity of an aqueous solution comprising:
(a) contacting the aqueous solution with a reagent composition capable of measuring the total divalent cation concentration of the aqueous solution, said composition comprising:
(i) about 5 to about 60 millimoles per liter of the composition of a metal-sensitive triphenylmethane dye having the structural formula: ##STR6## wherein X is carbonyl or sulfonyl, n is a number from 0 to about 3, and the R substituents are selected, independently, from the group consisting of hydrogen, hydroxy, an alkyl group including one to about five carbon atoms, bromo, chloro, fluoro, iodo, sulfonate, and phosphate;
(ii) a buffer;
(iii) a carrier comprising water; and
(iv) about 0.5 to about 4 millimoles per liter of the composition of a chelating agent selected from the group consisting of a polyhydroxy compound, a lignosulfonate, a glucoheptonate, a salicyate complex, a dithionate derivative, a polyethyleneamine, a 2,4-pentanedione, a dipyridine, a polypeptide including cysteine, glycine or histidine, a thiocrown ether, a triphenylphosphine, sorbitol, dimethylglyoxime, proline, bissalicylaldehydeethylenediimine, triethyleneamine, triethylenepyridine amine, 1,4,8,11,22,25-octathiacyclooctosane, ethylenediaminetetraacetic acid, N-(hydroxyethyl)ethylenediaminetriacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, aminotris(methylene phosphoric acid), hydroxyethylidene diphosphonic acid, hexamethylenediaminetetra(methylene phosphonate), ethylenediaminediacetic acid, iminodiacetic acid, nitrilopropionic acid, hydroxyethyliminodiacetic acid, pyrophosphoric acid, 1-hydroxyethane-1,1-diphosphonic acid, tripolyphosphoric acid, hexametaphosphoric acid, metaphosphoric acid, and mixtures thereof,
wherein the reagent composition is buffered to a pH of about 7;
(b) determining the total divalent cation concentration of the aqueous solution from the intensity and degree of the color transition of the reagent composition, wherein the color transition is in response only to the total divalent cation concentration of the aqueous solution; and
(c) correlating the total divalent cation concentration of the aqueous solution to the specific gravity of the aqueous solution.

### Claim 14 (depends on 13)

The method of claim 13 wherein the aqueous solution is a biological fluid.

### Claim 15 (depends on 14)

The method of claim 14 wherein the biological fluid is selected from the group consisting of urine, blood plasma, blood serum, and perspiration.

### Claim 16

A method of determining a total divalent cation concentration of an aqueous sample comprising:
(a) contacting the aqueous sample with an analyte detection device comprising a test pad, said test pad having incorporated therein a reagent composition capable of exhibiting a color transition only in response to the total divalent cation concentration of the aqueous sample, said composition comprising:
(i) about 5 to about 60 millimoles per liter of the composition of a metal-sensitive triphenylmethane dye having the structural formula: ##STR7## wherein X is carbonyl or sulfonyl, n is a number from 0 to about 3, and the R substituents are selected, independently, from the group consisting of hydrogen, hydroxy, an alkyl group including one to about five carbon atoms, bromo, chloro, fluoro, iodo, sulfonate, and phosphate;
(ii) a buffer;
(iii) a carrier comprising water; and
(iv) about 0.5 to about 4 millimoles per liter of the composition of a chelating agent selected from the group consisting of a polyhydroxy compound, a lignosulfonate, a glucoheptonate, a salicyate complex, a dithionate derivative, a polyethyleneamine, a 2,4-pentanedione, a dipyridine, a polypeptide including cysteine, glycine or histidine, a thiocrown ether, a triphenylphosphine, sorbitol, dimethylglyoxime, proline, bissalicylaldehydeethylenediimine, triethyleneamine, triethylenepyridine amine, 1,4,8,11,22,25-octathiacyclooctosane, ethylenediaminetetraacetic acid, N-(hydroxyethyl)ethylenediaminetriacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, aminotris(methylene phosphoric acid), hydroxyethylidene diphosphonic acid, hexamethylenediaminetetra(methylene phosphonate), ethylenediaminediacetic acid, iminodiacetic acid, nitrilopropionic acid, hydroxyethyliminodiacetic acid, pyrophosphoric acid, 1-hydroxyethane-1,1-diphosphonic acid, tripolyphosphoric acid, hexametaphosphoric acid, metaphosphoric acid, and mixtures thereof,
wherein the reagent composition is buffered to a pH of about 7, and
(b) determining the total divalent cation concentration of the aqueous sample from the intensity and degree of the color transition of the reagent composition.

### Claim 17

A method of semiquantitatively determining a specific gravity of an aqueous divalent cation-containing sample comprising:
(a) contacting the aqueous sample with an analyte detection device comprising a test pad having incorporated therein a composition capable of undergoing a color transition only in response to the total divalent cation concentration of the aqueous sample, said composition comprising:
(i) about 5 to about 60 millimoles per liter of the composition of a metal-sensitive triphenylmethane dye having the structural formula: ##STR8## wherein X is carbonyl or sulfonyl, n is a number from 0 to about 3, and the R substituents are selected, independently, from the group consisting of hydrogen, hydroxy, an alkyl group including one to about five carbon atoms, bromo, chloro, fluoro, iodo, sulfonate, and phosphate;
(ii) a buffer;
(iii) a carrier comprising water; and
(iv) about 0.5 to about 4 millimoles per liter of the composition of a chelating agent selected from the group consisting of a polyhydroxy compound, a lignosulfonate, a glucoheptonate, a salicyate complex, a dithionate derivative, a polyethyleneamine, a 2,4-pentanedione, a dipyridine, a polypeptide including cysteine, glycine or histidine, a thiocrown ether, a triphenylphosphine, sorbitol, dimethylglyoxime, proline, bissalicylaldehydeethylenediimine, triethyleneamine, triethylenepyridine amine, 1,4,8,11,22,25-octathiacyclooctosane, ethylenediaminetetraacetic acid, N-(hydroxyethyl)ethylenediaminetriacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, aminotris(methylene phosphoric acid), hydroxyethylidene diphosphonic acid, hexamethylenediaminetetra(methylene phosphonate), ethylenediaminediacetic acid, iminodiacetic acid, nitrilopropionic acid, hydroxyethyliminodiacetic acid, pyrophosphoric acid, 1-hydroxyethane-1,1-diphosphonic acid, tripolyphosphoric acid, hexametaphosphoric acid, metaphosphoric acid, and mixtures thereof,
wherein the reagent composition is buffered to a pH of about 7, and
(b) examining the analyte detection device for a color transition in response only to the total divalent cation content of the aqueous sample; and
(c) correlating the color transition to the specific gravity of the aqueous sample.

### Claim 18 (depends on 17)

The method of claim 17 wherein the aqueous sample has a specific gravity of about 1.000 to about 1.015.

### Claim 19

An analyte detection device to determine a total divalent cation concentration of an aqueous test sample comprising:
a support strip;
a test pad; and
a reagent composition incorporated into the test pad, said reagent composition capable of undergoing a color transition only in response to the total divalent cation concentration of the aqueous sample and said reagent composition comprising:
(a) about 5 to about 60 millimoles per liter of the composition of a metal-sensitive triphenylmethane dye having the structural formula: ##STR9## wherein X is carbonyl or sulfonyl, n is a number from 0 to about 3, and the R substituents are selected, independently, from the group consisting of hydrogen, hydroxy, an alkyl group including one to about five carbon atoms, bromo, chloro, fluoro, iodo, sulfonate, and phosphate;
(b) a buffer;
(c) a carrier comprising water; and
(d) about 0.5 to about 4 millimoles per liter of the composition of a chelating agent selected from the group consisting of a polyhydroxy compound, a lignosulfonate, a glucoheptonate, a salicyate complex, a dithionate derivative, a polyethyleneamine, a 2,4-pentanedione, a dipyridine, a polypeptide including cysteine, glycine or histidine, a thiocrown ether, a triphenylphosphine, sorbitol, dimethylglyoxime, proline, bissalicylaldehydeethylenediimine, triethyleneamine, triethylenepyridine amine, 1,4,8,11,22,25-octathiacyclooctosane, ethylenediaminetetraacetic acid, N-hydroxyethyl)ethylenediaminetriacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, aminotris(methylene phosphoric acid), hydroxyethylidene diphosphonic acid, hexamethylenediaminetetra(methylene phosphonate), ethylenediaminediacetic acid, iminodiacetic acid, nitrilopropionic acid, hydroxyethyliminodiacetic acid, pyrophosphoric acid, 1-hydroxyethane-1,1-diphosphonic acid, tripolyphosphoric acid, hexametaphosphoric acid, metaphosphoric acid, and mixtures thereof,
wherein the reagent composition is buffered at a pH of at least about 7.