U.S. patent number 3,816,076 [Application Number 05/261,218] was granted by the patent office on 1974-06-11 for process for the determination of thyroxine.
This patent grant is currently assigned to Merck Patent Gesellschaft mit beschrankter Haftung. Invention is credited to Egbert Tobias Backer.
United States Patent |
3,816,076 |
Backer |
June 11, 1974 |
PROCESS FOR THE DETERMINATION OF THYROXINE
Abstract
Method for the determination of thyroxine in biological fluids
by adsorption on a cation exchanger, subsequent washing out of the
interfering components, eluting the adsorbed thyroxine, liberating
the iodine therefrom, and then carrying out the iodine
determination in a conventional manner, which process is
characterized by the use of an eluent, optionally together with a
basic buffer, which destroys amino groups and liberates iodine from
the thyroxine into the eluate.
Inventors: |
Backer; Egbert Tobias
(Oegstggest, NL) |
Assignee: |
Merck Patent Gesellschaft mit
beschrankter Haftung (Darmstadt, DT)
|
Family
ID: |
5810792 |
Appl.
No.: |
05/261,218 |
Filed: |
June 9, 1972 |
Foreign Application Priority Data
|
|
|
|
|
Jun 15, 1971 [DT] |
|
|
2129553 |
|
Current U.S.
Class: |
436/500; 436/825;
436/531; 436/826 |
Current CPC
Class: |
G01N
33/78 (20130101); Y10S 436/825 (20130101); Y10S
436/826 (20130101) |
Current International
Class: |
G01N
33/74 (20060101); G01N 33/78 (20060101); G01n
033/16 () |
Field of
Search: |
;23/23B |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Sterling et al., Jour. of Clin. Inves., "Measurement of Free
Thyroxine Concentration in Human Serum," Vol. 41, No. 5, 1962 pp.
1031-1032..
|
Primary Examiner: Serwin; R. E.
Attorney, Agent or Firm: Millen, Raptes & White
Claims
What is claimed is:
1. In a process for the determination of thyroxine in biological
fluids which comprises the successive steps of adsorbing the
thyroxine on a cation exchanger, washing the cation exchanger,
eluting the adsorbed thyroxine therefrom and measuring iodine
liberated from the eluted thyroxine, the improvement which
comprises applying a sample of the biological fluid to a column of
the cation exchanger and eluting the adsorbed thyroxine from the
column with an eluent which decomposes the thyroxine adsorbed on
the column and liberates iodine into the eluate.
2. A process according to claim 1 wherein the eluent comprises a
basic buffer.
3. A process according to claim 1 wherein the eluent comprises a
solution of a hypobromite or hypochlorite.
4. A process according to claim 3 wherein the eluent comprises a
basic buffer.
5. A process according to claim 4 wherein the buffer is a borate
buffer.
6. A process according to claim 3 wherein the eluent comprises
sodium hypobromite or sodium hypochlorite.
7. A process according to claim 3 wherein the molarity of the
hypobromite solution is 0.0075 - 0.02M.
8. A process according to claim 7 wherein the molarity is about
0.015M.
9. A process according to claim 1 wherein the eluate is heated.
10. A process according to claim 1 wherein an iodine determination
of the eluate is conducted photometrically, employing the
cerium(IV) arsenite reaction.
Description
BACKGROUND OF THE INVENTION
The present invention relates to a process for the determination of
thyroxine. The exact determination of the thyroxine content is of
importance primarily in biological fluids, e.g., blood serum or
blood plasma, for thyroid diagnostics.
Thyroxine is an iodine-containing hormone which can be synthesized
only by the thyroid gland and is secreted into the blood. This
hormone plays an important part in the regulation of many metabolic
processes. In pathological cases, the thyroxine content in the
blood serum deviates greatly from that which is found under normal
physiological conditions. This is the case, for example, in
Basedow's disease resulting from hyperfunctioning of the thyroid,
resulting in an increased thyroxine content in the blood serum.
Thyroxine normally exits in the blood serum only in a very low
concentration. It is customary to indicate the thyroxine
concentration in .mu.g. of iodine per 100 ml. of serum. The normal
content is approximately 3-7 micrograms of thyroxine iodine per 100
ml. of serum. In pathological cases, the content can be lowered, or
also increased to up to about 20 micrograms of thyroxine iodine per
100 ml. of serum. Thyroxine is bound, in the blood serum, almost
completely to a specific carrier protein, the so-called
"thyroxine-binding globulin." In addition to thyroxine, the serum
also contains very small amounts of other iodine compounds, such
as, for example, iodide, triiodothyronine, thyroglobulin, and
possibly mono- and diiodotyrosine.
A number of processes for the chemical determination of thyroxine
are known.
The method used most in practice is the determination of
protein-bound iodine in the serum, the so-called PBI method. In
this procedure, the organic iodine compounds are precipitated
together with the serum proteins, and the iodine is determined
after preceding alkaline incineration or wet incineration. More
specific is the determination of the iodine which can be extracted
with butanol (BEI method), because in this process only thyroxine
and triiodothyronine, the hormone iodine in the narrower sense, are
picked up. Furthermore, determination methods are known utilizing,
for isolating the hormone iodine, thin-layer chromatography, gel
filtration, or ion exchangers.
However, all conventional methods for thyroxine determination
exhibit considerable disadvantages. A great disadvantage in the PBI
and BEI methods is that the incineration must be conducted in an
expensive incinerator and the average time for the analysis of a
blood sample is very long (approximately 20 hours). A further
essential disadvantage of the PBI and BEI methods is that no
distinction is made between thyroxine and iodine-containing
radiopaque agents or medicines. In patients who were subjected to
radiological examination or therapy with an iodine-containing
agent, the actual concentration of the thyroxine in the blood serum
cannot be determined because these methods in all cases involve the
liberation and determination of total serum iodine. In this
connection, it is to be noted that iodine-containing contrast
agents can reach a concentration in the blood serum which is many
hundred times higher than that of thyroxine. It can take years for
the entire elimination of these contrast agents from the blood
circulation. During this time, when using the PBI method as well as
with the use of the BEI method, one always obtains erroneous,
elevated thyroxine values.
Serum thyroxine determinations involving a thin-layer
chromatography or gel filtration step have the disadvantage that
they are very difficult to process and thus are not feasible as
routine determinations in the clinical laboratory.
A process for the determination of serum thyroxine by means of an
anion exchanger has been described in U.S. Pat. No. 3,471,553 and
in "Clinical Chemistry" 14 (1968) 339. The serum sample is first
brought to a pH of 12-13 by means of a strong base, and introduced
into an anion exchange column. During passage through the column,
the thyroxine is bound to the matrix. The exchange column is then
washed out with several reagents in order to remove the residual
serum components and any iodine-containing contrast agents or
medicines which may be contained in the serum. Thereafter, the
thyroxine is eluted with 50 percent strength acetic acid. The
strong adsorption of thyroxine on the matrix requires the high
acetic acid concentration in the eluent. Either bromine-saturated
water or a solution of KBr and KBrO.sub.3 in water is then added to
the thyroxine-containing eluate, thus producing free bromine in the
acidic eluate. The bromine liberates the iodine from the thyroxine
molecule, which can then be determined in a conventional
manner.
In spite of easy manipulation, this method yet entails grave
disadvantages. The serum specimen cannot be introduced into the
column immediately, but rather must first be adjusted to a specific
pH value. Concentrated acetic acid is necessary for the elution of
the thyroxine, and free bromine is needed for liberating the
iodine, which results in inconvenience in handling due to the
obnoxious odor and possible injury to health. Another disadvantage
is that the iodine is not liberated quantitatively from thyroxine
and consequently the thus-obtained measuring results are not
accurate.
Another method is known for the determination of thyroxine which
utilizes a cation exchanger. See Clin. Chim. Acta 20 (1968), 155.
The serum sample is introduced into a cation exchange column and
then passes through the column, during which step the thyroxine is
bound to the matrix. The column is washed out with various reagents
in order to remove the remaining components of the serum and any
iodine-containing contrast agents or medicines which may be present
in the serum. After elution of the thyroxine with ammonia and
chloric acid incineration, the thus-formed iodine is determined in
the usual manner. Although this method is specific to thyroxine
iodine, it has considerable disadvantages. The 5N ammonia employed
for the elution must again be eliminated from the eluate before it
is possible to effect the liberation of the iodine with chloric
acid under high temperatures. Special apparatus are needed for this
purpose and it is impossible to avoid obnoxious odors and possible
damage to health.
It has now been found that the above-described disadvantages of the
heretofore conventional methods for thyroxine determination can be
avoided by the use of a novel eluent.
SUMMARY OF THE INVENTION
In its process aspect, this invention relates to a process for the
determination of thyroxine in biological fluids in which the
thyroxine is adsorbed on a cation exchanger, interfering components
are washed from and then adsorbed thyroxine is eluted from the
cation exchanger, the iodine is liberated from the eluted thyroxine
and an iodine determination of the liberated iodine is then
conducted, which comprises using eluting the thyroxine with an
eluent, optionally together with a basic buffer, which decomposes
the thyroxine and liberates iodine from the thyroxine into the
eluate.
In its composition aspect, this invention relates to an agent for
the determination of thyroxine in biological fluids, comprising a
thyroxine adsorbing cation exchanger, optionally a salt solution, a
basic buffer, an eluent which destroys amino groups and liberates
iodine from thyroxine, an iodine determination reagent and a
standard solution.
DETAILED DISCUSSION
In the process of this invention, the biological fluid is
introduced, without preceding pH adjustment or treatment with base,
and thus in undiluted form, onto a column of a cation exchanger.
After washing out proteins and any interfering components, such as,
e.g., exogenous iodine-containing compounds, the adsorbed thyroxine
is removed from the exchanger by decomposing the thyroxine by
destroying the amino group, and the iodine is liberated into the
eluate and can be determined directly. Thus, no reagent need be
added to the eluate in order to liberate the iodine.
Thus, for the first time, a simple method is provided for the
routine determination of hormonal iodine (thyroxine and
triiodothyronine), within a relatively short period of time (about
3 hours) and without the use of concentrated bases or specialized
equipment. Accordingly, the novel method requires less than half
the time necessary for the conventional methods. A further
advantage is that there are no obnoxious odors and no
health-impairing reagents involved in the determination.
For the thyroxine determination according to the process of the
present invention, cation exchangers in their H.sup.+ form are
utilized. Suitable are cation exchanger synthetic resins, e.g.,
cross-linked polyvinyl derivatives, for example, polystyrenes,
polyacrylates and polymethacrylates, and polycondensed products,
e.g., phenolic resins having acidic groups, e.g., -SO.sub.3.sup.-,
-COO.sup.-, -O.sup.-, -PO.sub.3.sup.2.sup.-, -PO.sub.3 H.sup.-,
-AsO.sub.3.sup.2.sup.- or -SeO.sub.3.sup.-, and cross-linked
polysaccharides. Preferred are polystyrene cation exchangers,
particularly those having sulfonic acid groups, exhibiting
preferably a particle size of 20-400 mesh, especially 200-400 mesh.
The degree of cross-linking preferably ranges from 2 to 12 percent,
and the exchange capacity is preferably in a range of 0.6 - 2.5
meq./ml., preferably being 1 meq./ml.
When a serum or other biological sample, normally one which has not
been subjected to a pretreatment, is allowed to pass through such
an ion exchange column, the thyroxine contained in the sample is
bound to the column, partially nonionically and partially
ionically, by its amino groups. Most of the serum components,
including any other inorganic iodides which may be present, are not
adsorbed on the column during this step. After the serum is passed
through the column, any residual interfering components are removed
by washing the column, e.g., with a basic buffer, optionally after
a preceding washing step with a salt solution.
The optional washing step with a salt solution is advantageous in
order to remove the proteins contained in the sample and other
components which are not bound to the ion exchanger, and to
neutralize the ion exchange resin. Suitable salt solutions are all
aqueous solutions of salts which cannot be attacked by oxidation
and which do not interfere with the final iodine determination,
such as, for example, sodium chloride, potassium chloride,
potassium dihydrogen phosphate, potassium-sodium tartrate, sodium
acetate, sodium citrate, sodium oxalate, sodium sulfate, calcium
chloride, etc. The concentration of the salt solution is proferably
0.01 to 5M, depending on the solubility of the selected salt.
Particularly advantageous are 0.1-1M sodium chloride solutions.
In the subsequent washing-out step, preferably with a basic buffer,
the last traces of the proteins and any interfering
iodine-containing medicines are removed from the column. It is
advantageous to wash first with a salt solution so that the acidic
ion exchanger is neutralized. In such a case, a small volume and a
low concentration of the basic buffer will suffice without there
being an essential weakening of the buffer capacity thereof.
Suitable basic buffers are those capable of maintaining a pH of
about 7.5 - 9.0, which are inert with respect to oxidizing agents,
and which do not interfere with the iodine determination.
Particularly advantageous are inorganic basic buffer systems, e.g.,
borate and sodium bicarbonate buffer, in a concentration of about
0.05 - 1M, preferably 0.2 - 0.6M, especially a borate buffer of
approximately 0.4M, pH 8.6.
Suitable eluents for the process of this invention are those
comprising an oxidizing agent which is effective in a basic medium
and which is capable of destroying amino groups by oxidation,
thereby liberating iodine from the thyroxine molecule, e.g.,
hypobromite or hypochlorite. Bromine water can also be used as the
eluent. However, agents without troublesome odors, e.g.,
hypobromite or hypochlorite, are preferred. An alkali hypobromite
or alkali hypochlorite solution, e.g., sodium or potassium
hypobromite or hypochlorite, is particularly advantageous.
It is now possible, for the first time, to remove the thyroxine
from the cation exchanger without the use of a concentrated base.
In accordance with the present invention, a solution of
hypobromite, particularly in a weakly basic medium, is the
preferred eluent. This solution is obtained, for example, by mixing
equivalent amounts of sodium hypochlorite in dilute sodium
hydroxide solution and potassium bromide, and diluting the
thus-produced hypobromite solution with the buffer system utilized
for the washing-out step. The concentration of the eluting solution
employed can vary widely but preferably is relatively dilute, e.g.,
less than 0.1M, preferably 0.0075 - 0.02 hypobromite, preferably
about 0.015M.
The thus-obtained, weakly basic eluate is allowed to react until
all of the iodine has been liberated from the thyroxine. To
expedite this reaction, the eluate is preferably heated, e.g., to
about 50-100.degree. C. In order to shorten the reaction time, the
mixture is suitably heated in a water bath, the temperature of the
eluate being maintained at approximately 100.degree. C. When this
is done, the reaction is terminated within a few minutes. At the
same time, any residual organic impurities are also destroyed.
When using hypobromite as the eluent, which is preferred, free
bromine does not evolve at any time during the elution or during
the treatment of the eluate. Also with regard to this factor, the
process of the present invention is superior to the conventional
procedures for thyroxine determination by means of ion exchangers
in which bromine or a mixture of bromide/bromate is added to the
protein-free acid eluate. In addition to the instability of the
aqueous bromine solutions, the extremely poisonous bromine always
escapes into the atmosphere during these conventional
procedures.
The colorimetric determination of the iodine liberated from
thyroxine is effected according to conventional procedures.
Preferably, the conventional cerium(IV) arsenite reaction is
utilized as described, for example, in Clin. Chim. Acta 5 (1960),
301. In general, the eluate, heated for example in a water bath, is
mixed with an acidified arsenite solution and thermostated for
about 10 minutes for temperatur adaption at about 25-40.degree.C.,
preferably at 28.degree. C. Subsequently, an acidic cerium(IV)
ammonium sulfate solution is added thereto and, after a specific
period of time, preferably about 25 minutes, the light absorption
is measured at 420 nm. or 436 nm.
Since, in the process of this invention, the iodine to be
determined is liberated quantitatively from the thyroxine, an
iodate solution can be utilized as the standard. This is a special
advantage, compared to the conventional methods wherein thyroxine
standard solutions are employed. The disadvantages of thyroxine
standard solutions are due to the insolubility of thyroxine in
water, decrease of activity and its undefined iodine content. It is
advantageous in determining the standard to treat the columns with
the eluent.
The agent of this invention for the determination of thyroxine
content of biological fluids, by means of which the above-described
procedure can be carried out, contains the aformentioned reagents.
Preferably, the required reagents are utilized in the form of a
test kit. This novel combination of reagents is characterized in
that it contains
a. a cation exchanger as described above, preferably one or more
units, preferably filled glass or plastic tubes suitable for use as
a column, each in an amount sufficient to adsorb all the thyroxine
in a typical serum sample, e.g., about 1-5, preferably 2 - 3
ml.
b. (optional) an inert (with respect to the thyroxine and eluent)
soluble ionic salt, preferably as an aqueous solution, e.g., about
3 - 10 ml. per unit of cationic exchanger;
c. a basic buffer solution, e.g., about 5 - 15 ml. per unit of
cationic exchanger;
d. eluent solution, e.g., about 5 - 15 ml. per unit of cationic
exchanger;
e. (optional) iodine determination reagent; and
f. (optional) standard iodine solution. In its preferred
embodiment, (b) and more preferably (e) and (f) are present inthe
reagent test kit. However, since some users will have ready access
to (e) and (f) components, they need not be included in the test
kit in order for such users to conduct thyroxine analyses.
A preferred reagent kit contains 1M sodium chloride, e.g., 1M,
solution (optional); borate buffer, e.g., 0.4M, preferably about pH
8.6; potassium bromide solution and a sodium hypochlorite solution,
both preferably about 1M, sodium hydroxide solution, preferably
about 0.1M; and preferably also an indicator reagent for the
measurement of iodine content, e.g., arsenous acid and
cerium-ammonium sulfate and a standard iodine determination
solution, e.g., a potassium iodate solution. The solutions can also
be present in the test kit in the form of concentrates, i.e., to be
diluted prior to use, e.g., 10 to 100 fold.
The thyroxine determination according to the process of the present
invention can also be effected in automatic analyzers. For this
purpose, the thyroxine-containing eluates of various specimens are
fed, via a sample collector, to an analyzing system wherein
arsenite solution and cerium-ammonium sulfate solution are
successively added to the individual samples. After mixing and
elapse of the reaction time, the reaction solution is introduced
into a colorimeter and measured.
The process of this invention can also be utilized in the quality
control of drugs, such as, for example, in the activity
determination of dried and pulverized thyroids. This activity
determination is of great practical importance. Whereas, by means
of the conventional methods, the entire organically bound iodine is
determined during this step, it is possible with the novel process
to determine selectively the biologically active components
(thyroxine and triiodothyronine).
Without further elaboration, it is believed that one skilled in the
art can, using the preceding description, utilize the present
invention to its fullest extent. The following preferred specific
embodiments are, therefore, to be construed as merely illustrative,
and not limitative of the remainder of the disclosure in any way
whatsoever.
EXAMPLE 1
A microcolumn filled with a cation exchanger (polystyrene with
sulfonic acid groups, cross-linked with divinylbenzene, particle
size 200-400 mesh, degree of cross-linking 2 percent) is charged
with 0.5 ml. of serum, and the latter is allowed to pass through.
Then, the column is washed out first with 5 ml. of 1M NaCl solution
and thereafter with a total of 10 ml. of 0.4M borate buffer (pH
8.6). After the washing-out step, two elutions are conducted, with
respectively 5 ml. of a 0.015M solution of hypochlorite/KBr in 0.4M
borate buffer. (The elution can also be effected in one stage with
8 ml. of this eluting solution.) The eluate is heated in a water
bath having a temperature of 100.degree. C. for 8 minutes and is
then cooled to room temperature.
Thereafter, the iodine liberated in the eluate is determined in the
usual manner, for example as follows: The eluate is mixed with 1
ml. of 0.16N arsenite solution and brought to a constant
temperature in a 28.degree. C. water bath for 10 minutes. Then, 1
ml. of 0.017N cerium sulfate solution is added to the thermostated
solution, mixed therewith, and thereupon the mixture is immediately
returned to the water bath. Exactly 25 minutes after adding the
cerium sulfate solution, the absorption of the sample is measured
at 420 nm. against water.
In each determination, in parallel measurements, the blank column
value, the blank reagent value, and two different standard
solutions are measured. The standard reference values are
determined by adding an iodate solution of a known concentration to
the eluate of a blank column value and then conducting the iodine
determination. The columns for obtaining the blank value and the
standard reference values are each treated with the same reagents
as the analysis sample.
In repetitions of the aformentioned determination, the cation
exchangers on polystyrene basis which are commercially obtainable
and are characterized in the following are used in place of the
exchanger utilized above (particle size in mesh, degree of
cross-linking in percent, exchange capacity in meq./ml.):
1. 50-100 mesh; 2%; 0.9 meq./ml.
2. 100-200 mesh; 2%; 0.9 meq./ml.
3. 100-200 mesh; 4%; 1.3 meq./ml.
4. 100-200 mesh; 8%; 1.9 meq./ml.
5. 100-200 mesh; 12%; 2.3 meq./ml.
6. 30-40 mesh; 1.9 meq./ml.
7. 14-52 mesh;
8. 50-140 mesh; 8%.
EXAMPLE 2
A microcolumn filled with cation exchanger (polystyrene basis with
sulfonic acid groups, particle size 50-140 mesh) is charged with
0.5 ml. of plasma, and the latter is allowed to pass therethrough.
Then, the column is washed out first with 5 ml. of 0.5M sodium
acetate solution and thereafter with a total of 10 ml. of 0.1M
sodium bicarbonate solution. After this step, the elution is
effected twice with respectively 5 ml. of a 0.015M solution of
hypochlorite/KBr in 0.1M sodium bicarbonate solution. The eluate is
heated for 5 minutes in a water bath of a temperature of
100.degree. C. and subsequently cooled to room temperature.
The colorimetric determination is thereupon conducted analogously
to Example 1.
In repetitions of the above determination, solutions of the
following salts are utilized in place of the sodium acetate
solution: potassium chloride, potassium dihydrogen phosphate,
potassium-sodium tartrate, sodium citrate, sodium sulfate, sodium
oxalate, and calcium chloride. The washing-out step with the use of
these salt solutions can optionally be omitted, or it can be
effected with 2- or 3-molar solutions.
In place of hypochlorite/KBr as the eluent, bromine water is
utilized in a further modification of the above-described modes of
operation.
EXAMPLE 3
20 mg. of dried and pulverized thyroid is hydrolyzed in a borate
buffer with trypsin and erepsin and, after acidification, an
aliquot is charged into a microcolumn filled with cation exchanger.
After washing out the iodide, monoiodotyrosine, and diiodotyrosine
with borate buffer, the adsorbed triiodothyronine and thyroxine are
eluted with a total of 8 ml. of a 0.015M solution of
hypochlorite/KBr in 0.4M borate buffer. The liberation and
subsequent determination of the iodine are effected analogously to
Example 1.
EXAMPLE 4
A reagent combination for about 50 thyroxine determinations
contains, in addition to an appropriate number of microcolumns
filled with cation exchange resin, the following reagent
solutions:
1. 280 ml. 1M sodium chloride solution
2. 250 ml. 0.05M potassium bromide in 2M borate buffer
3. 10 ml. 1M sodium hypochlorite solution in 0.1M sodium hydroxide
solution
4. 110 ml. 0.16N arsenite solution
5. 110 ml. 0.017N cerium-ammonium sulfate solution
6. 5 ml. standard solution (0.1 mg. KIO.sub.3 /1.)
7. 5 ml. standard solution (0.2 mg. KIO.sub.3 /1.)
Solution 2 is diluted with 1,000 ml. of twice-distilled water.
Shortly before use, 1 ml. of solution 3 is diluted with 100 ml. of
the ready-for-use solution 2. All other solutions are ready for
use.
EXAMPLE 5
A reagent combination for about 25 thyroxine determinations
contains, in addition to a corresponding number of microcolumns
filled with cation exchange resin, the following reagent
solutions:
1. 50 ml. 3M sodium chloride solution
2. 2 .times. 250 ml. 0.4M borate buffer
3. 5 ml. 1M potassium bromide solution
4. 5 ml. 1M sodium hypochlorite solution in 0.1M sodium hydroxide
solution
5. 50 ml. 0.16N arsenite solution
6. 50 ml. 0.017N cerium-ammonium sulfate solution
7. 5 ml. standard solution (0.1 mg. KIO.sub.3 /1.)
8. 5 ml. standard solution (0.2 mg. KIO.sub.3 /1.)
Solution 1 is diluted with 100 ml. of twice-distilled water.
Shortly prior to use, respectively 1 ml. of solutions 3 and 4 are
combined and diluted with 100 ml. of solution 2. All other
solutions are ready for use. Optionally, solution 3 can be omitted
or replaced by a 1M sodium bromide solution.
EXAMPLE 6
2 ml. of 0.15M NaCl solution and 0.5 ml. of serum are successively
introduced into a microcolumn filled with cation exchanger
(polystyrene with sulfonic acid groups cross-linked with
divinylbenzene, particle size 200-400 mesh, degree of crosslinking
2 percent) and allowed to pass therethrough. Then, the column is
washed out with 5 ml. of 0.15M NaCl solution and thereafter with a
total of 10 ml. of 0.4M borate buffer (pH 8.6). After the
washing-out step, the elution is conducted twice with respectively
5 ml. of a 0.015M solution of hypochlorite/KBr in 0.4M borate
buffer. (The elution can also be carried out in one step with 8 ml.
of this eluting solution.) The eluate is heated for 8 minutes in a
water bath of 100.degree. C. and then cooled to room
temperature.
Thereafter, the iodine liberated in the eluate is determined in a
conventional manner, for example as follows: The eluate is mixed
with 1 ml. of 0.22N arsenite solution and brought to a constant
temperature for 10 minutes in a 37.degree. C. water bath. Then, 1
ml. of 0.045N cerium sulfate solution is added to the thermostated
solution, mixed therewith, and the mixture immediately returned
into the water bath. Exactly 25 minutes after adding the cerium
sulfate solution, the absorption of the sample is measured at 436
nm. against water.
EXAMPLE 7
A reagent combination for about 20 thyroxine determinations
contains, in addition to a corresponding number of microcolumns
filled with cation exchange resin, the following reagent solutions,
ready for use:
1. 250 ml. 0.15M sodium chloride solution
2. 250 ml. 0.4M borate buffer
3. 250 ml. 0.015M potassium bromide solution in 0.4M borate
buffer
4. 3.75 ml. 1M sodium hypochlorite solution in sodium hydroxide
solution
5. 25 ml. 0.22N arsenite solution
6. 25 ml. 0.045N cerium-ammonium sulfate solution
7. 5 ml. standard solution (0.845 mg. KIO.sub.3 /1.)
8. 5 ml. standard solution (1.69 mg. KIO.sub.3 /1.)
The preceding examples can be repeated with similar success by
substituting the generically or specifically described reactants
and/or operating conditions of this invention for those used in the
preceding examples.
From the foregoing description, one skilled in the art can easily
ascertain the essential characteristics of this invention, and
without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions.
* * * * *