Reagent and method for direct determination of chloride in serum

A direct, quantitative method is provided for the determination of chloride in blood serum which is based on the reaction between the chloride and ferric ions. A novel reagent is employed in the method which contains no mercury but does contain a surfactant which prevents protein precipitation.

FIELD OF THE INVENTION 
This invention relates to a reagent and method for the direct quantitative 
determination of chloride in blood serum. 
DESCRIPTION OF THE PRIOR ART 
It has long been known in the biological sciences that chloride is the 
major extracellular anion in biological fluids. Movement of the chloride 
into and out of the red cell is essential for transport of bicarbonate 
ions in response to changing amounts of carbon dioxide. Elevated chloride 
values in serum usually indicate dehydration, hyperventillation, 
congestive heart failure, prostatic or other types of urinary obstruction. 
Low serum chloride values are found with extensive burns, excessive 
vomiting, metabolic acidosis, nephritis, intestinal obstruction, 
Addisonian crisis and diarrhea. 
The earliest practical technique for chloride analysis was reported by T. 
Volhard in 1974 (J. Prakt. Chem., 9 217 (1974)). Since that time, dozens 
of direct and indirect methods of analysis have been reported in the 
literature. The most popular methods are the coulometric titration, the 
specific-ion electrode technique and the colorimetric mercuric thiocyanate 
method. 
P. W. West and H. Coll reported in Anal. Chem., 28 (1956) a direct 
spectrophotometric method for chloride in the 0.02-0.2 meq/L range. B. 
Fingerhut later adopted this non-mercurimetric method for serum chloride 
analysis as reported in Clin. Chem. Acta., 41, 247 (1972). Since the 
reagent is in a strong perchloric acid medium, dialysis was employed to 
prevent problems due to protein precipitation. The method did not gain 
wide acceptance in the clinical laboratory, yet the direct colorimetric 
method contains no mercury that causes undesirable disposal problems as in 
other colorimetric methods. It should also be noted that in the original 
West and Coll article, no observable absorbance change was reported below 
an acid concentration of 2.0 N. 
It has now been found that by utilizing a mercury-free reagent which 
contains ferric perchlorate and perchloric acid in dilute quantities and 
preferably, in the presence of a non-ionic surfactant, reliable results 
can be achieved in the direct, quantitative determination of chloride. 
Accordingly, one or more of the following objects can be achieved by the 
practice of this invention. It is an object of this invention to provide a 
novel reagent and method for the direct, quantitative determination of 
chloride in serum. Another object is to provide a reagent for chloride 
determination which utilizes the reaction between the chloride and ferric 
ions. A still further object of this invention is to provide a reagent 
which does not contain mercury. Another object is to provide a method 
which employs a reagent containing perchloric acid at concentrations lower 
than those heretofore used. A still further object of this invention is to 
provide a method for the determination of chloride which has a high 
linearity range for chloride ion determination. Another object of this 
invention is to provide a method for the determination of chloride ion 
which circumvents interference by components present in blood serum. A 
further object of this invention is to provide a bichromatic method which 
facilitates chloride determination on a centrifugal spectrophotometric 
analyzer. These and other objects will readily become apparent to those 
skilled in the art in the light of the teachings herein set forth. 
SUMMARY OF THE INVENTION 
In its broad aspect this invention is directed to a reagent and method for 
the direct, quantitative determination of chloride in serum. The method, 
employing the reagent, is comprised of the steps of: 
(a) contacting a sample of serum with the reagent to form a solution 
wherein the reagent contains: 
(i) from about 0.01 N to about 2.0 N perchloric acid in deionized water, 
(ii) from about 0.01 N to about 1.0 N ferric perchlorate in deionized 
water, and 
(iii) from about 0.1 to about 20 percent by weight of a non-ionic 
surfactant capable of minimizing protein precipitation while not 
interfering with absorbance of the solution. 
(b) measuring the absorbance of the solution after an incubation period, 
(c) comparing the absorbance of the solution with that obtained from 
solutions containing known amounts of chloride, and 
(d) determining the amount of chloride in the serum.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
It has been found that chloride can be determined quantitatively in blood 
serum by a novel method which utilizes the reaction between chloride and 
ferric ions in the presence of a non-ionic surfactant. As hereinbefore 
indicated, the prior art disclosed the use of ferric perchlorate and a 2 
N-8.5 N perchlorate acid in the determination of small amounts of chloride 
in aqueous samples. The yellow chloro complexes of iron (III) exhibit an 
intense absorption band in the vicinity of 340 nm, while the reagent 
containing Fe(III) perchlorate in acidic medium absorbs little light in 
this range, thus lending itself to quantitative measurement. 
Perchloric acid is well known to be a good serum protein precipitant. 
However, in order to determine the chloride concentration in serum, a 
separation process for serum proteins such as the dialysis method reported 
by Fingerhut must be employed before the method can be adapted to an 
automated procedure. Urea has been used to prevent protein precipitation 
in the perchloric acid medium of a different color reaction, but it was 
observed that urea formed a color product with the Fe(III) ions near 340 
nm and was therefore unsuitable for a direct reaction. 
However, in contrast to the prior art methods, it has been found that the 
ferric perchlorate and perchloric acid can be employed in very dilute 
concentrations and the use of a surfactant eliminates a separate protein 
separation step. Hence, through a systematic study of pH dependance of the 
Fe(III)-chloride complexes, it was discovered that ferric chloride species 
formed under well defined conditions of low acidity. For example, it was 
observed that the species absorbs linearly at 340 nm throughout and beyond 
the physiological range of chloride in serum, the standard curve obeying 
Beer's Law. The process also gives sensitivity that is comparable to the 
reagent that contains concentrated perchloric acid. Moreover, it has been 
observed that in the process of this invention, the chloride complex forms 
rapidly in less than 1 minute. 
As previously indicated the perchloric acid and ferric perchlorate are 
employed as dilute solutions. The procedure for preparation of the reagent 
is set forth in Example 1. However, in practice it has been found that the 
composition of the reagent can be varied to obtain good results with 
different sensitivity. Therefore, the reagent of this invention can vary 
in composition as follows: 
1. Perchloric Acid (HClO.sub.4)--About 0.01 N to about 2.0 N. 
2. Ferric Perchlorate, non-yellow [Fe(ClO.sub.4)].sub.3 --About 0.01 N to 
about 1.0 N. 
3. Polyoxyethylene Lauryl Ether (or similar nonionic surfactants)--About 
0.1% to about 20% by weight. 
In actual practice using the particular analyzer hereinafter indicated, the 
reconstituted reagent contained 0.0378 N Fe(ClO.sub.4).sub.3, 9.65 percent 
of polyoxyethylene lauryl ether, and 0.246 N HClO.sub.4. 
As indicated, the process and reagent of this invention utilizes a 
non-ionic surfactant to surpress and/or eliminate blood protein 
precipitation. In contrast to the use of urea, the non-ionic surfactant 
does not form a color product with the Fe(III) ions near 340 nm and hence, 
is ideally suited for this use. 
In practice, the only requirements of the surfactant are that it be 
non-ionic, that it surpresses or eliminates blood protein precipitation, 
and, of course, in contrast to urea, that it does not interfere with the 
absorbance measurement. Preferred surfactants are the family of 
polyoxyethylene ethers having from 2 to 40 ethylene oxide units and 
derived from primary and secondary alcohols having from 8 to 18 carbon 
atoms. A particularly preferred non-ionic surfactant which is well suited 
for use in this invention is polyoxyethylene lauryl ether sold by Atlas 
Chemical Industries under the trademark Brij 35. Other non-ionic 
surfactants can be used as long as they possess the above properties. 
Since the reagent employed in this invention is an oxidizing agent which 
oxidizes bilirubin, it creates a dynamic interference problem for serum 
samples. From a study of the interference, it was found that the general 
strategy of serum blanking, chemical modification, as well as separation 
by extraction were either too complicated or too lengthly. An important 
feature of the bichromatic procedure of this invention was the use of a 
420 nm filter to perform the dynamic blanking procedure. Hence, this 
invention covers the use of filters from 380-450 nm range for bichromatic 
blanking purposes. The 420 nm filter is preferred because of its ability 
to eliminate bilirubin and hemoglobin interferences simultaneously. The 
serum blank changes with time because the bilirubin absorption peak slowly 
shifts from 450 nm to 370 nm. A blank absorbance that matches the one 
measured at 340 nm both for the static hemolysis and the dynamic bilirubin 
interferences can be obtained at 420 nm with proper timing as determined 
experimentally and as depicted in the drawing. This serum blank is then 
stored and subtracted from the final reading taken at 340 nm. This 
procedure is simple, it circumvents the storing of a serum blank and it 
eliminates the bilirubin interference problem. 
While the method of this invention can be employed with any instrument that 
has bichromatic measurement capability, it is particularly useful with the 
CentrifiChem.RTM. analyzer which is marketed by Union Carbide Corporation 
and utilizes a centrifugal field for the mixing and transfer of reagents. 
If an analysis is performed manually with a spectrophotometer thermostated 
at 30.degree. C., a flow-through cell attachment is preferred. With the 
CentrifiChem analyzer, samples and reagents are pipetted into the transfer 
disc with a sample dilution factor of 1:40. The analyzer parameters can be 
dialed in manually or the computer can be programmed to do the automated 
experimental steps. The disc is placed inside the analyzer, parameters are 
loaded and spinning started. After the printout, the analyzer is then 
reset with wavelength changed to produce the final results. 
The following examples illustrate the best mode presently contemplated for 
the practice of this invention: 
EXAMPLE 1 
Reagent Preparation 
A stock solution of ferric perchlorate (0.7 N) was prepared by dissolving 
360 grams of ferric perchlorate, non-yellow crystals into 1 liter with 
deionized water. This solution was then filtered and standardized with 
potassium dichromate. A 5 N stock solution of perchloric acid was prepared 
by diluting 450 milliliters of 70 percent perchloric acid with 1 liter of 
deionized water and standardizing the solution with sodium hydroxide. 
The working solutions were prepared as follows: Solution A was prepared 
from the stock solutions and contained 0.0756 N ferric perchlorate and 
0.492 N perchloric acid. Solution B was prepared as an aqueous solution 
containing 19.3 percent by volume of polyoxyethylene lauryl ether which is 
marketed under the trademark Brij 35 by Atlas Chemical Industries. The 
surfactant is purchased as a 30 weight percent solution and the proper 
dilution made. Solutions A and B are mixed in a 1:1 ratio by volume as the 
working reagent. 
Manual Method 
In practice, 2 milliliters of Solution B are pipetted into a 13.times.100 
mm test tube, then 70 .mu.l sample is pipetted into the tube and the 
contents mixed. A white cloud may appear in serum samples, but will 
disappear after mixing. Thereafter, 2 milliliters of Solution A are added 
and the test tube swirled to insure mixing. 
A reagent blank solution is also prepared by adding to a 13.times.100 mm 
test tube 2 milliliters of Solution B, 70 .mu.l of deionized water, and 2 
milliliters of reagent A, followed by mixing. 
For the serum sample, a serum blank solution is also prepared by pipetting 
4 milliliters of deionized water into a 13.times.100 mm test tube and 
adding 70 .mu.l of serum sample, followed by mixing. 
The test procedure using the manual method is to place all solutions into a 
30.degree. C. water bath or heating block and allow them to incubate for 5 
minutes. Thereafter the solutions are introduced into the 
spectrophotometer and readings taken when the absorbance is stable, 
(approximately 15 seconds). 
Bichromatic Method 
Any instrument that has bichromatic measurement capability such as 
CentrifiChem analyzer can be used with this method. In accordance with the 
procedure for operating the analyzer samples and reagents were pipetted 
into the transfer disc with a sample dilution factor of 1:40. The analyzer 
parameters can be dialed in manually or the computer can be programmed to 
do the automated experimental steps. The disc is placed inside the 
analyzer, parameters are loaded and spinning started. After the printout, 
the analyzer is then reset with wavelength changed to produce the final 
results. 
EXAMPLES 2-9 
Using the reagents prepared in accordance with Example 1, various human 
blood serum samples were analyzed for chloride on the CentrifiChem 
analyzer. The type of serum sample and the results obtained are set forth 
in Table I below: 
TABLE I 
__________________________________________________________________________ 
[Cl.sup.- ] by 
[Cl.sup.- ] 
CORNING 920M 
FOUND 
METHOD 
EXAMPLE 
TYPE OF SAMPLE 
(meq/L) (meq/L) 
USED 
__________________________________________________________________________ 
2 Normal Serum 
102 101 Bichromatic 
3 Iceteric Serum 
97 96 Bichromatic 
4 Hemolyzed Serum 
109 108 Bichromatic 
5 Turbid Serum 
103 104 Bichromatic 
6 Control Serum 
107 106 Bichromatic 
(500 mg/dl hemo- 
globin spike) 
7 Control Serum 
104 104 Bichromatic 
(43.5 mg/dl bili- 
rubin spike) 
8 Deionized Water 
32 1 Bichromatic 
(40 meq/L Br.sup.- spike) 
9 Control Serum 
106 105 Bichromatic 
(very turbid) 
__________________________________________________________________________ 
EXAMPLE 10 
In this example, a comparison was made of the several parameters of the 
invention using the bichromatic procedure and those of the manual method. 
Also the parameters of the hold-blank are set forth. The figures are set 
forth in Table II below: 
TABLE II 
__________________________________________________________________________ 
METHOD 
AMETERS Bichromatic Manual BLANK 
__________________________________________________________________________ 
Linearity 
0-120 meq/L 0-120 meq/L 0-180 meq/L 
Precision 
Within-day 
1.54% 1.7% 1.3% 
Day-to-day 
0.8% 1.4% 1.8% 
Accuracy 
(% Recovery) 95-103% 
Stability 1 year at 25.degree. C. 
Correlation with 
0.9743 0.968 0.9256 
Coulmetric Method 
Interferences 
Br.sup.- none up to 40 meq/L none up to 100 meq/L 
F.sup.- none up to 40 meq/L none up to 40 meq/L 
Bilirubin 
none none up to 5 mg/dl 
none up to 5 mg/dl 
Hemoglobin 
none up to 300 mg/dl 
none up to 300 mg/dl 
none up to 500 mg/dl 
Turbidity 
none up to grossly turbid 
none up to grossly turbid 
none up to grossly 
__________________________________________________________________________ 
turbid 
Although the invention has been illustrated by the preceding examples, it 
is not to be construed as being limited to the materials employed therein, 
but rather the invention encompasses the generic area as hereinbefore 
disclosed. Various modifications and embodiments thereof can be made 
without departing from the spirit and scope thereof.