U.S. patent number 3,615,228 [Application Number 04/878,573] was granted by the patent office on 1971-10-26 for glucose determination method employing orthotoluidine.
This patent grant is currently assigned to The Dow Chemical Company. Invention is credited to John Di Giorgio, Robert F. Mack, Bernard J. Thiegs.
United States Patent |
3,615,228 |
Thiegs , et al. |
October 26, 1971 |
**Please see images for:
( Certificate of Correction ) ** |
GLUCOSE DETERMINATION METHOD EMPLOYING ORTHOTOLUIDINE
Abstract
An improved orthotoluidine reagent composition for use in
determination of glucose in biological fluids effective sufficient
hydrazine to provide a uniform intensity of color when the
composition is reacted with a predetermined amount of glucose, such
as a standard solution.
Inventors: |
Thiegs; Bernard J. (N/A),
Mack; Robert F. (N/A, IN), Giorgio; John Di (N/A,
CA) |
Assignee: |
Company; The Dow Chemical
(MI)
|
Family
ID: |
25372309 |
Appl.
No.: |
04/878,573 |
Filed: |
November 20, 1969 |
Current U.S.
Class: |
436/95;
436/166 |
Current CPC
Class: |
G01N
33/66 (20130101); Y10T 436/144444 (20150115) |
Current International
Class: |
G01N
33/66 (20060101); G01N 021/22 (); G01N 031/22 ();
G01N 033/16 () |
Field of
Search: |
;23/230,23B,253TP
;252/408 ;195/103.5 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
3Analytical Abstracts, 7 5353 (1960), 8 2074, 3412 (1961), 9 4350
(1962), 10 4306 (1963), 13 787, 4295 (1966), 14 7648
(1967).
|
Primary Examiner: Wolk; Morris O.
Assistant Examiner: Katz; Elliot A.
Claims
We claim:
1. In a method for determination of glucose comprising the steps of
mixing together predetermined quantities of a color reagent
composition comprising orthotoluidine as the color forming reagent
and a sample composition comprising glucose, mixing together an
additional quantity of said color reagent composition and a
standard composition comprising glucose at a predetermined
concentration in said standard composition, and developing color in
the mixtures resulting from both said mixing steps under
substantially identical conditions whereby the intensity of said
developed color in each such mixture is proportional to the
concentration of glucose therein;
the improvement which comprises the step of adding hydrazine to the
color reagent composition prior to the step of developing color,
the hydrazine being added in an amount sufficient to provide a
maximum intensity of said developed color equivalent to an
absorbance of about 0.28 to about 0.35 for light of a wavelength
between 590 and 625 millimicrons when the concentration of glucose
in the ultimate mixture in which color is developed is from about
1.5 to about 2.5 milligrams of glucose per 100 milliliters of
ultimate mixture.
2. The method of claim 1 wherein the hydrazine is added in an
amount sufficient to provide a hydrazine concentration of from
about 60 to about 200 micromoles of hydrazine per 100 milliliters
of reagent composition.
3. The method of claim 1 wherein the hydrazine is added in the form
of hydrazine hydrate.
4. The method of claim 1 wherein the reagent composition is
prepared by mixing together orthotoluidine, thiourea and glacial
acetic acid in predetermined amounts, and wherein the hydrazine is
mixed with the orthotoluidine prior to preparation of the reagent
composition.
5. The method of claim 4 wherein the hydrazine is employed in an
amount sufficient to provide from about 9 to about 20 micromoles of
hydrazine per gram of orthotoluidine.
6. In a reagent composition useful for determination of glucose by
measuring the intensity of color produced by the reaction of
glucose with orthotoluidine comprising a solution of orthotoluidine
in glacial acetic acid, the concentration of orthotoluidine being
from about 5 to about 10 grams of orthotoluidine per 100
milliliters of said solution; the improvement wherein the reagent
composition further comprises from about 9 to about 20 micromoles
of hydrazine per gram of orthotoluidine, said hydrazine being
sufficient to decrease the intensity of the color produced when
said composition is reacted with glucose.
7. The composition of claim 6 further comprising from about 0.1 to
about 1 gram of thiourea.
8. The composition of claim 6 wherein the composition consists
essentially of about 9 grams of orthotoluidine and about 0.5 gram
of thiourea per 100 milliliters of composition and contains from
about 80 to about 180 micromoles of hydrazine per 100
milliliters.
9. The composition of claim 8 further comprising from about 1.8 to
about 2.1 milligrams of glucose per 100 milliliters, and wherein
said hydrazine is present in an amount sufficient to decrease the
intensity of color obtained by reaction of said glucose with said
orthotoluidine to provide an intensity of said color equivalent to
an absorbance of from about 0.30 to about 0.34 for light having a
wavelength of from 590 to 620 millimicrons.
Description
BACKGROUND OF THE INVENTION
The determination of glucose in biological fluids using an
orthotoluidine reagent composition is an accepted method in many
hospital, industrial, reference and commercial clinical
laboratories. The method is based on the color formed when glucose
reacts with orthotoluidine in glacial acetic acid during a short
heating period. The intensity of the resulting blue-green color is
measured photometrically.
Conventional orthotoluidine reagents generally contain from about 5
to about 10 grams per 100 milliliters of orthotoluidine in glacial
acetic acid and often contain a small amount of thiourea usually in
a concentration of from about 0.1 to 1 gram per 100 milliliters, to
prevent subsequent discoloration of the reagent.
The glucose determination is usually performed on a biological
fluid such as serum or plasma; however, whole blood, cerebrospinal
fluid, urine, and other biological fluids may be analyzed. A
predetermined volume of sample fluid, generally 0.100 milliliter,
is mixed with a predetermined volume of orthotoluidine reagent
composition, generally from about 3 to about 5 milliliters in a
suitable container such as a colorimeter cuvette or test tube.
A predetermined amount of a glucose standard is added to a second
container containing the same volume of reagent as used for the
sample. The glucose standard will often contain 100 milligrams of
glucose per 100 milliliters. Both containers are capped, mixed, and
heated to develop color, typically in a heating block at
100.degree. C. or boiling water bath for a predetermined period of
time, usually from 7 to 10 minutes. The tubes are simultaneously
removed at the end of the heating period and cooled simultaneously,
typically in a water bath at a temperature below 25.degree. C. for
3 minutes. The intensity of blue-green color in the glucose
standard and serum sample is measured photometrically in a
spectrophotometer or suitable filter colorimeter at a wavelength
from 590 to 625 millimicrons or nanometers. The instrument employed
will have been previously adjusted to indicate zero absorbance
using an appropriate blank composition, e.g. reagent without
glucose. Such procedure prevents measurement of intensity of color
due to the reagent along, so the measurements are made only on the
color produced by the glucose-orthotoluidine reaction. In the
present specification and claims, it is understood that the
absorbance of the reagent composition alone is accounted for by
setting the measuring instrument to read zero absorbance with
reagent blank containing no glucose, and terms such as
"absorbance," "final absorbance" and the like are employed to refer
solely to the absorbance or color intensity produced by the
reaction of the reagent composition with glucose. The concentration
of glucose in the serum sample is calculated by multiplying the
ratio of the final absorbances obtained with the serum sample to
that obtained with standard solution by the concentration of
glucose in the standard. The final absorbance obtained in a given
case depends on such factors as the exact reagent and standard used
and the heating time employed for color development.
As a practical matter, prepared is desirable that different batches
of an orthotoluidine reagent composition give nearly the same
intensity of color when reacted with the same amount of glucose
standard in identical determination procedures. So long as each
batch of reagent composition is prepared from identical homogeneous
lots of ingredients, substantially uniform final absorbances can
generally be obtained. However, in practice, different batches of
orthotoluidine reagent compositions prepared with ingredients from
different lots have been found to give wide variations in
absorbance values when reacted with an identical glucose standard
under identical conditions. Moreover, even batches of reagent
composition prepared from the same homogeneous lot of
orthotoluidine may differ in final absorbance if the lot of
orthotoluidine is stored for extended periods between formulation
of the different batches. Furthermore, photometric error is
increased with color intensities of high absorbance. Thus, it is
undesirable that a reagent give excessive color intensity with a
sample containing glucose at a concentration within the normal
physiological range. For example, if a reagent gives a high
absorbance value with a glucose standard containing 100 milligrams
glucose per 100 milliliters, then sera from untreated diabetic
patients will develop even more color, thereby requiring the sample
to be read in the extreme upper end of the colorimeter absorbance
scale--the region of maximum photometric error. Thus, it is
important to be able to control the formulation of a reagent so
that final absorbances will be both predictable and optimum over a
reasonable range of sample glucose concentrations. A maximum
absorbance of from about 0.28 to 0.35 for a standard containing 100
milligrams of glucose per 100 milliliters is desirable.
In a typical glucose determination mixture containing about 50
parts by volume of reagent composition and one part by volume of a
typical standard solution or control serum, the glucose
concentration is about 15 to about 18 to about 21 to 25 micrograms
glucose per milliliter most generally about 19.6 micrograms per
milliliter and a final absorbance of 0.28 to 0.35 is desirable a
absorbance of 0.30 to 0.34 being considered preferable. Optimum
final absorbances could be obtained by changing the amount of
orthotoluidine employed in formulating various batches of reagent
composition, or by altering the determination procedure in the
relative proportions of reagent composition and standard or sample
employed; however, such modifications of formulation and procedure
or both are not desirable.
There is a need for a simple improvement in orthotoluidine reagents
which provides for regulation of the color developed with
predetermined amounts of glucose to obtain uniformly optimum
absorbance from batch to batch without necessitating changes in
analytical procedure or extensive reformulation in each batch of
reagent composition.
BRIEF SUMMARY OF THE INVENTION
This invention relates to an improvement in orthotoluidine reagent
compositions in biological fluids. The invention gives a uniform
intensity of color when reacted with a specified quantity of
glucose. The intensity of color developed by the reagent with such
quantity of glucose can be predetermined, adjusted or regulated
whenever a new batch of reagent is produced from new or different
lots of ingredients to achieve optimum final absorbances in each
lot.
It has now been found that the intensity of color developed in
glucose determinations carried out with a conventional
orthotoluidine color reagent composition can be regulated by the
addition of a minor amount of hydrazine to the reagent composition
prior to mixing the reagent composition with glucose. More
particularly it has been discovered that the addition of small
amounts of hydrazine to an orthotoluidine reagent composition, in
an amount sufficient to lower the final absorbance in glucose
determinations, provides for control of the final color intensity
within the optimum range for colorimetric determinations, and
additionally increases the storage stability of the reagent
composition thus treated.
In formulating and using orthotoluidine reagents according to the
invention, the reagent composition can be formulated with the same
orthotoluidine and glacial acetic acid and thiourea (if desired) in
the same proportions as one typically employed, and the glucose
determination can be carried out in the identical procedures, with
the same proportions of reagent composition and glucose standard or
sample as have been heretofore employed, with the sole modification
being the addition to the reagent composition of sufficient
hydrazine to provide a final absorbance after maximum color
development of about 0.28-0.35 for from about 15 to about 25
micrograms glucose per milliliter of the ultimate mixture of
sample, control serum or standard with the reagent composition. The
hydrazine can be added to the reagent composition at any time prior
to use, and is preferably added thereto by mixing the required
amount of hydrazine with the orthotoluidine prior to formulating
the reagent composition, generally in amounts of from about 9 to
about 15 to about 20 micromoles of hydrazine per gram of
orthotoluidine.
The exact amount of hydrazine to be added to any particular batch
of orthotoluidine reagent composition can vary depending upon the
proportion of ingredients employed in formulating the desired
composition and the exact procedure to be employed in using the
same, as well as depending upon the extent to which the final
absorbance obtained with a particular batch varies from the optimum
range. In practice the relative proportions of ingredients in the
reagents composition, and the parameters of the analytical
procedure such as proportions of reagent composition and standard
or sample, heating time and temperature for color development,
wavelength of light at which color intensity is measured, etc. are
predetermined. Consequently, the exact amount of hydrazine to be
added to control color intensity for a particular batch of a
predetermined composition in a predetermined procedure is dependent
upon the color intensity obtained with the unmodified batch.
In a convenient procedure, a series of aliquots of the reagent
composition batch are prepared containing varying concentrations of
added hydrazine, and the aliquot compositions are employed in the
test procedure to measure the final absorbances obtained with a
standard glucose composition containing 100 milligrams of glucose
per 100 milliliters, and to ascertain the concentration of
hydrazine required to provide the desired final absorbance. The
hydrazine concentration required to provide a particular absorbance
in successive batches of reagent composition remains the same, so
long as the orthotoluidine and glacial acetic acid ingredients are
from identical homogeneous lots of such ingredients. Once the
hydrazine concentration required to control absorbance is
determined for one batch prepared from particular lots of
ingredients, additional batches of reagent composition formulated
from the same ingredients lots can be treated to contain the same
concentration of hydraine to standardize the final absorbances
obtained with any such batch. In a preferred embodiment, the
required hydrazine concentration is determined for a given set of
ingredient lots, and the amount of hydrazine required to provide
the same concentration in subsequent batches is added to the
orthotoluidine. This procedure eliminates the need for measuring
additional quantities of hydrazine each time a new batch of reagent
composition is prepared from the same lots of orthotoluidine and
glacial acetic acid, and the addition of the hydrazine to the
orthotoluidine also enhances the stability of the orthotoluidine
during storage.
The hydrazine can be conveniently employed in the present invention
as liquid hydrazine or as hydrazine hydrate, and a concentrated
aqueous solution of hydrazine hydrate is particularly convenient.
The orthotoluidine reagent composition and glucose determination
procedure are conventional in determination of glucose by reaction
with orthotoluidine as the essential color forming reactant.
Preferably the reagent composition contains from about 5 to about
10 grams of orthotoluidine per 100 milliliters of ultimate
composition and from about 0.1 to about 1 gram of thiourea per 100
milliliters, the remainder of the composition being glacial acetic
acid. A particularly preferred composition is prepared by
dissolving 9 grams orthotoluidine and 0.5 gram thiourea in glacial
acetic acid to obtain a final volume of 100 milliliters. Such
reagent composition is preferably employed in glucose determination
my mixing one part by volume of a biological fluid sample or a
glucose standard solution with about 30 to 50 parts by volume of
reagent composition, heating the mixture in a tube in a heating
block or water bath at a temperature of about 100.degree. C. for
about 7 minutes, then cooling in a cold water bath (temperature of
water bath below 25.degree. C.) for about 3 minutes before
measuring color intensity with light having a wavelength of 590 to
625 millimicrons (nanometers). With such a reagent composition and
method, excellent control of final absorbance is obtained with
concentrations of hydrazine equaivalent to from about 3 to about 10
milligrams of hydrazine hydrate per 100 milliliters of reagent 180,
to about 200 micromoles of hydrazine per 100 milliliters of
composition. Compositions so prepared are stable for over 6 months
at room temperature, producing substantially uniform optimum final
absorbances of about 0.28 to 0.35 when color is developed in the
ultimate mixture of reagent and sample or standard containing 1.5
to 2.5 milligrams glucose per 100 milliliters. The reagent
compositions give excellent results in glucose determination on
biological fluids, including sera from either normal or diabetic
subjects.
DESCRIPTION OF PREFERRED EMBODIMENTS
The following examples are illustrative and representative of the
invention.
EXAMPLE 1
A series of orthotoluidine reagent compositions is prepared by
mixing together the following ingredients in the following
proportions:
orthotoluidine 9.0 grams Thiourea 0.5 grams Glacial acetic acid to
a final volume of 100 milliliters
Each such composition is prepared in an identical procedure, with
the sole variation being the use of orthotoluidine from one of two
homogeneous lots, A and B, and the use of glacial acetic acid from
one of three lots, 1, 2 and 3.
Each composition is employed in a glucose determination procedure
by mixing 5 milliliters of reagent composition with 0.1 milliliter
of an aqueous glucose standard solution containing 100 milligrams
glucose per 100 milliliters of standard solution, heating the
mixtures for 7 minutes in a tube heating block set at 100.degree.
C., cooling the mixtures for 2 to 3 minutes in a cold water bath,
and determining the absorbance of light by the blue-green reaction
product in a colorimeter with light having a wavelength of 595
millimicrons. The colorimeter is adjusted to read zero absorbance
with a reagent blank, that is, the reagent composition to which no
glucose has been added, prior to measuring absorbance of the
corresponding mixture of standard solution and reagent composition.
The final absorbances obtained with the compositions reacted with
the glucose standard are found to vary from 0.34 to 0.408.
Each reagent composition is then divided into aliquots and varying
amounts of hydrazine are added to each aliquot, the hydrazine being
added as an 85 percent aqueous solution of hydrazine hydrate. The
aliquot compositions are then reacted with glucose and the final
absorbances of the products are determined in duplicate operations
carried out as described above. In a representative operation
carried out with aliquots of the reagent composition prepared from
Lot A orthotoluidine and Lot 3 glacial acetic acid, the following
final absorbances are found with aliquots having the following
amounts of added hydrazine
---------------------------------------------------------------------------
Added Hydrazine (grams of hydrazine hydrate per 100 milliliters)
Final Absorbance Run I Run II Average
__________________________________________________________________________
0.0 0.410 0.405 0.408 0.004 (80)* 0.362 0.360 0.361 0.005 (100)
0.330 0.330 0.330 0.006 (120) 0.325 0.325 0.325 0.008 (160) 0.310
0.315 0.313
__________________________________________________________________________
EXAMPLE 2
The compositions of example 1 containing various amounts of added
hydrazine sufficient to lower the final absorbances to a desired
value are prepared and compared with the compositions without added
hydrazine by the procedure described in example 1, Duplicate runs
are carried out in each case. 0.1 milliliter of an identical
glucose standard solution containing 100 milligrams glucose per 100
milliliters of glucose standard is employed in each determination.
The results are set forth in following table. ##SPC1##
EXAMPLE 3
A series of seven orthotoluidine reagent compositions is prepared
using the same lot of orthotoluidine in each case and one of seven
lots of glacial acetic acid (a)-(g) in each case. The final
absorbances with and without added hydrazine are determined as set
out above in example 1, The average results obtained in duplicate
determinations are set out in the following table. ##SPC2##
EXAMPLE 4
The reagent compositions of examples 1-3 containing from about 80
to about 160 micromoles of hydrazine per 100 milliliters and giving
final absorbances between 0.28 and 0.35 when 5 milliliters of the
glucose standard solution (equivalent to glucose concentration of
1.96 milligrams of glucose per 100 milliliters of ultimate mixture)
are employed in various operations to determine glucose in
biological fluids according to the procedure described above.
Excellent results are obtained in determination of glucose
concentrations in 0.1 milliliter samples of serum containing known
glucose concentrations ranging from 60 to over 190 milligrams per
100 milliliters of serum, with excellent precision between and
within runs.
* * * * *