METHODS AND COMPOSITIONS FOR DETERMINING GLUCOSE IN BLOOD

• US 3,653,841 A
• Filed: 12/19/1969
• Issued: 04/04/1972
• Est. Priority Date: 12/19/1969
• Status: Expired due to Term

• Current Assignee: Bernard Klein
• Original Assignee: Bernard Klein
• Inventors: Klein, Bernard
• Primary Examiner(s): Wolk, Morris O.
• Assistant Examiner(s): Reese, R. M.
• Application Number:
  US04/886,538
• Time in Patent Office: 837 Days
• Field of Search: 23/230,23B 260/239.3D 252/408
• US Class Current: 436/95
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: Y10T 436/118339 with formation of a segmented stream
• CPC Class Codes: Y10T 436/144444 Glucose

Abstract

Colorimetric methods and compositions for quantitatively determining the glucose content of blood plasma or serum by heating a deproteinized sample of blood plasma or serum with an alkaline ferricyanide solution, followed by the addition of ferric ions and a 5-(2-pyridyl)-2H-1,4-benzodiazepine or water soluble salts thereof to produce a brilliant purple colored solution which can be quantitated by standard colorimetric means.

Claims

2. The process in accordance with claim 1 wherein said source of ferric ions consists essentially of an aqueous solution of ferric chloride buffered to a pH of between from about 4.0 to about 5.0.

3. The process in accordance with claim 1 wherein said benzodiazepine compound is added as an aqueous solution buffered to a pH of from about 4.0 to about 5.0.

4. The process in accordance with claim 1 wherein said benzodiazepine compound is selected from the group consisting of 7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1, 4-benzodiazepin-2-one and water soluble acid addition salts thereof.

5. A process for quantitating the glucose content of blood serum or plasma comprising:

a. treating a deproteinized sample of blood serum or plasma with an aqueous alkaline solution containing a source of ferricyanide ions;

b. heating the mixture from step (a) to a temperature of from about 90* C. to about 100*C.;

c. adding to the mixture of step (b) an aqueous solution containing ferric chloride and a buffer;

d. adding to the mixture of step (c) an aqueous solution containing a buffer and a benzodiazepine compound selected from the group consisting of compounds of the formula wherein A is selected from the group consisting of and B is selected from the group consisting of and -CH2-;

R1 is selected from the group consisting of halogen, hydrogen, trifluoromethyl, nitro, and amino;

R2 is selected from the group consisting of , hydrogen, lower alkyl and ;

n is an integer from 2 to 7;

R3 is selected from the group consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and lower alkanoyloxy;

R4 is 2-pyridyl;

R5 is selected from the group consisting of lower alkyl;

hydrogen, and ; and

R5 and R6, where taken together with their attached nitrogen atom, form a radical selected from the group consisting of piperazinyl, lower alkyl substituted piperazinyl, pyrrolidinyl, lower alkyl substituted pyrrolidinyl, piperidinyl, and lower alkyl substituted piperidinyl;

R7 is lower alkyl; and

R8 is selected from the group consisting of lower alkyl and hydrogen and water soluble acid addition salts thereof; and

e. colorimetrically quantitating the glucose present by means of said color.

6. The process in accordance with claim 5 wherein said source of ferricyanide ions is potassium ferricyanide.

7. The process in accordance with claim 5 wherein said buffer present in the aqueous solution containing ferric chloride and the aqueous solution containing the benzodiazepine compound is a buffer pair comprising a water soluble salt of acetic acid and acetic acid.

8. A method for the quantitative analysis of the glucose content of blood plasma or serum consisting essentially of providing in continuous flow, the sequential steps comprising:

a. combining in continuous flow a measured specimen of plasma or serum with an isotonic solution of sodium chloride;

b. passing said mixture through a separating zone, thereby separating by dialysis in said zone from said mixture a clear aqueous solution;

c. mixing sAid clear aqueous solution with a reagent comprising an alkaline aqueous solution of a water soluble ferricyanide salt;

d. passing said aqueous mixture through heating means thereby raising the temperature thereof to from about 95* C. to about 100* C.;

e. mixing said heated aqueous mixture by concurrent flow with a first reagent comprising a buffered aqueous solution of a ferric iron salt and a second reagent comprising a buffered, aqueous solution of a color-forming compound selected from the group consisting of compounds of the formula wherein A is selected from the group consisting of and ;

B is selected from the group consisting of and -CH2-;

R1 is selected from the group consisting of halogen, hydrogen, trifluoromethyl, nitro, and amino;

R2 is selected from the group consisting of , hydrogen, lower alkyl and ;

n is an integer from 2 to 7;

R3 is selected from the group consisting of hydrogen, hydroxy, lower alkyl, lower alkoxy and lower alkanoyloxy;

R4 is 2-pyridyl;

R5 is selected from the group consisting of lower alkyl;

hydrogen, and ; and

R5 and R6, where taken together with their attached nitrogen atom, form a radical selected from the group consisting of piperazinyl, lower alkyl substituted piperazinyl, pyrrolidinyl, lower alkyl substituted pyrrolidinyl, piperidinyl, and lower alkyl substituted piperidinyl;

R7 is lower alkyl; and

R6 is selected from the group consisting of lower alkyl and hydrogen thereof and water soluble acid addition salts thereof thereby forming a colored solution; and

f. flowing said colored solution to an analyzing zone and photometrically determining quantitatively the amount of glucose present during the flow of said colored solution through said analyzing zone.

9. The method in accordance with claim 8 wherein said first reagent and said second reagent are buffered to a pH of from about 4.0 to about 5.0 with a buffer pair comprising a water soluble salt of acetic acid and acetic acid.

10. The method in accordance with claim 8 wherein said colorforming compound is selected from the group consisting of 7-bromo-1,3-dihydro-1-(3-dimethylaminopropyl)-5-(2-pyridyl)-2H-1,4 -benzodiazepin-2-one and water soluble acid addition salts thereof.

11. The method in accordance with claim 8 wherein said water soluble ferricyanide salt is potassium ferricyanide and said ferric iron salt is ferric chloride.