Fructosamine reagent and calibrator system

A method for determining fructosamine in sera which comprises:

• (a) determining a first absorbance of a first color produced by reaction of a first liquid reagent containing a tetrazolium salt with a first amount of sera, said first color produced in consequence of reduction by fructosamine and other interfering substances in said sera of said tetrazolium salt, said first absorbance determined at two predetermined points in time, each within 10 minutes at 37.degree. C.;
• (b) determining a second absorbance of a second color produced by reaction of a second liquid reagent in an amount equal to the amount of said first liquid reagent with sera in an amount equal to said first amount of said sera using a second liquid reagent containing a tetrazolium salt and boric acid which produces a second color change by reaction of the tetrazolium salt with interfering substances in the sera other than fructosamine, said boric acid being in an amount sufficient to inhibit the reaction of fructosamine with the tetrazolium salt, said second absorbance being determined at two predetermined points in time, each within 10 minutes at 37.degree. C.;
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• (c) determining the fructosamine concentration in said sera as a function of the difference in the first and second color change produced using the first liquid reagent which reacts with fructosamine and the second liquid reagent which is inhibited from reacting with fructosamine.

A method of determining the concentrate of fructosamine in sera at 37.degree. C. which comprises:

• (a) adding to a first sample of sera a first liquid reagent which reacts with fructosamine and interfering substances and is an aqueous solution of a carbonate buffer present in a concentration of from about 0.1 to about 0.4 molar, a tetrazolium salt which is reduced by fructosamine and interfering substances in sera present in a concentration of about 0.1 to about 1 molar of a 100% by volume octylphenoxy polyethoxyethanol in a concentration of about 0.1 to about 4% v/v and from 0 to 3% v/v ethoxylated nonylphenol, said solution having a pH of from about 8.5 to about 11.5 and measuring the change in absorbance of the sera added reagent at 500 nm at 2 or 3 minutes of the addition of the reagent and at 2 minutes after the first measurement of absorbance;
• (b) repeating the procedure of step (a) using a second sample of the sera of the same volume as the first sample of sera by addition of a second liquid reagent identical in volume and content of the first reagent and further containing boric acid in a concentration of about 0.05 to about 1 molar to inhibit reaction with fructosamine;
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• (c) determining from the difference in change of absorbance in step (a) and the change of absorbance in step (b) the fructosamine concentration in the sera.

A method of determining the concentrate of fructosamine in sera which comprises:

• (a) adding to a predetermined amount of sera a first liquid reagent containing 0.5 millimolar p-Iodonitrotetrazolium Violet, about 0.1 molar sodium carbonate monohydrate, about 0.1 molar sodium bicarbonate, about 0.1 molar urea, about 1% v/v octylphenoxy polyethoxyethanol, and 1% w/v ethoxylated nonylphenol dissolved in the deionized water and having a pH of about 11 and determining absorbance at 37.degree. C. of 500 nm 2 and 4 minutes or 3 and 5 minutes after addition of the first liquid reagent to the sera;
• (b) repeating step (a) by adding to another sample of the sera, of same proportional amount as the first, a second liquid reagent identical in proportional volume and content of the first reagent but further containing about 0.12 molar boric acid and having a pH of about 11;
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• (c) using the difference in absorbance determined in step (a) and the differential in absorbance determined in step (b) to determine the fructosamine content of the sera.