Two reagent system for the colorimetric determination of chloride ions in body fluids

DETAILED DESCRIPTION OF THE INVENTION The invention is directed to a colorimetric method for measuring chloride ion concentration in a sample of body fluid which method comprises the steps of: (1) Mixing a sample of body fluid with a color reagent; (2) Measuring the absorbance of the mixture of Step 1 at from about 450 to 550 nm; (3) Adding an iron complexing reagent to the mixture of Step 1; and (4) Measuring the absorbance of said mixture of Step 3 at from about 450 to about 550 nm; wherein the difference between the second absorbance measurement and the first absorbance measurement is proportional to the chloride ion concentration in said sample.
The method of the invention provides a procedure whereby a sample blank measurement is made on the same sample and in the same cuvette after the test chloride measurement has been made.
Using Equation 2 for the purposes of illustration, an absorbance measurement is made at about 450 to about 550 nm after the reaction has reached equilibrium.
An iron complexing reagent containing e.
g.
, disodium ethylene diamine-tetraacetic acid (EDTA) is added which chelates the ferric ions to form the essentially colorless Fe(EDTA) complex.
The remaining absorbance of the solution will be due to the reagent absorbance and the sample absorbance.
Thus, the net absorbance of the assay, due to the presence of chloride ion, will be the initial absorbance (before EDTA addition) less the absorbance after EDTA addition and corrections if necessary for volume dilution.
Equation 2 Fe(SCN).
sub.
3 +HgCl.
sub.
2 +NO.
sub.
3.
sup.
- +A.
sub.
1 +A.
sub.
2 +.
.
.
A.
sub.
n +EDTA.
fwdarw.
Fe(EDTA)+2Cl.
sup.
- +NO.
sub.
3.
sup.
- A.
sub.
1 +A.
sub.
2 +.
.
.
A.
sub.
n +3SCN.
sup.
- + 2Hg(EDTA).
As used herein, the term "body fluid" refers to human or animal serum, plasma, sweat, urine and the like.
The method of the invention is particularly suited for determining chloride ion concentration in serum and plasma samples and especially, human serum and plasma samples.
As used herein, the term "color reagent" refers to an aqueous solution of Fe