# US 4,399,227 A · Process for determination of glycosylated hemoglobin and reagent therefor

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- Number: US 4,399,227 A
- Document ID: US4399227A
- Status: Granted
- Kind: A
- Application number: 06/350,181
- Filed: 1982-02-19
- Granted: 1983-08-16
- Earliest claimed: 1981-05-13
- Assignees: Panchem Gesellschaft fur chemische Produkte mbH
- Inventors: Cornelia Niederau; Hans Reinauer
- Web page: https://patentgrep.com/patent/US4399227A

## CPC

- G01N 33/723: {Glycosylated haemoglobin}
- Y10T 436/25125: Digestion or removing interfering materials
- 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

An improved process for determining glycosylated hemoglobin wherein the contribution of unstable glucose-aldimine-hemoglobin compounds to the HbA.sub.1 value is substantially eliminated by reacting the hemolysate with a primary amine and a hydrazine compound and thereafter subjecting the hemolysate to chromatography. The process enables reliable evaluation of the HbA.sub.1 value as a long-term parameter in diabetic metabolism control.

## Claims

### Claim 1

In a process for the determination of the glycosylated hemoglobin content of a blood sample comprising the hemolysis of erythrocytes, the chromatographic separation of hemoglobin fractions and their colorimetric measurement, the improvement wherein the contribution of unstable glucose-aldimine-hemoglobin compounds to the HbA.sub.1 value is substantially eliminated by reacting the hemolysate with a primary amine and a hydrazine compound and thereafter subjecting the hemolysate to chromatography.

### Claim 2 (depends on 1)

A process according to claim 1, wherein the hemolysis and the reaction with a primary amine and a hydrazine compound are carried out simultaneously.

### Claim 3 (depends on 1)

A process according to claim 1, or 2, wherein the primary amine comprises hydroxylamine, an alkylamine, a hydroxyalkylamine, an amino acid, aniline or pyridoxamine.

### Claim 4 (depends on 3)

A process according to claim 3 wherein the primary amine comprises hydroxylamine.

### Claim 5 (depends on 3)

A process according to claim 3 wherein the primary amine comprises an alkylamine.

### Claim 6 (depends on 5)

A process according to claim 5 wherein the primary amine comprises ethylamine or propylamine.

### Claim 7 (depends on 3)

A process according to claim 3 wherein the primary amine comprises a hydroxyalkylamine.

### Claim 8 (depends on 7)

A process according to claim 7 wherein the primary amine comprises ethanolamine.

### Claim 9 (depends on 3)

A process according to claim 3 wherein the primary amine comprises an amino acid.

### Claim 10 (depends on 9)

A process according to claim 9 wherein the primary amine comprises lysine.

### Claim 11 (depends on 9)

A process according to claim 9 wherein the primary amine comprises valine.

### Claim 12 (depends on 9)

A process according to claim 9 wherein the primary amine comprises a mixture of lysine and valine.

### Claim 13 (depends on 1, 2)

A process according to claim 1 or 2 wherein the hydrazine compound comprises hydrazine, phenylhydrazine or semicarbazide.

### Claim 14 (depends on 13)

A process according to claim 13 wherein the hydrazine compound comprises hydrazine.

### Claim 15 (depends on 13)

A process according to claim 13 wherein the hydrazine compound comprises phenylhydrazide.

### Claim 16 (depends on 13)

A process according to claim 13 wherein the hydrazine compound comprises semicarbazide.

### Claim 17 (depends on 3)

A process according to claim 3 wherein the hydrazine compound comprises hydrazine, phenylhydrazine or semicarbazide.

### Claim 18 (depends on 9)

A process according to claim 9 wherein the hydrazine compound comprises hydrazine, phenylhydrazine or semicarbazide.

### Claim 19 (depends on 10)

A process according to claim 10 wherein the hydrazine compound comprises hydrazine, phenylhydrazine or semicarbazide.

### Claim 20 (depends on 11)

A process according to claim 11 wherein the hydrazine compound comprises hydrazine, phenylhydrazine or semicarbazide.

### Claim 21 (depends on 12)

A process according to claim 12 wherein the hydrazine compound comprises hydrazine, phenylhydrazine or semicarbazide.

### Claim 22

In a process for the determination of the glycosylated hemoglobin content of a blood sample comprising the hemolysis of erythrocytes, the chromatographic separation of hemoglobin fractions and their colorimetric measurement, the improvement wherein the contribution of unstable glucose-aldimine-hemoglobin compounds to the HbA.sub.1 value is substantially eliminated by reacting the hemolysate with hydroxylamine, an alkylamine, a hydroxyalkylamine or an amino acid and a hydrazine compound and thereafter subjecting the hemolysate to chromatography.

### Claim 23 (depends on 22)

A process according to claim 22 wherein the hydrazine compound is hydrazine.

### Claim 24 (depends on 22)

A process according to claim 22 wherein the hydrazine compound is phenylhydrazine.

### Claim 25 (depends on 22)

A process according to claim 22 wherein the hydrazine compound is semicarbazide.

### Claim 26 (depends on 2)

A reagent for use in the process according to claim 2, said reagent comprising lysine, valine, semicarbazide and digitonin in amounts effective to substantially eliminate the contribution of unstable glucose-aldimine-hemoglobin compounds to the HbA.sub.1 value, together with sufficient phosphate buffer to establish a pH value of approximately 6.6.

### Claim 27 (depends on 22)

A reagent according to claim 22, said reagent containing 20 mM lysine, 20 mM valine, 200 mM semicarbazide hydrochloride, 50 mM sodium phosphate buffer (for pH 6.6) and 0.813 mM digitonin per liter of aqueous solution.