U.S. patent number 3,795,484 [Application Number 05/211,075] was granted by the patent office on 1974-03-05 for automated direct method for the determination of inorganic phosphate in serum.
This patent grant is currently assigned to Union Carbide Corporation. Invention is credited to John A. Daly, Gerhard Ertingshausen.
United States Patent |
3,795,484 |
Daly , et al. |
March 5, 1974 |
AUTOMATED DIRECT METHOD FOR THE DETERMINATION OF INORGANIC
PHOSPHATE IN SERUM
Abstract
A direct method is provided for the spectrophotometric
determination of inorganic phosphate in fluids, particularly body
fluids, such as blood serum. The process requires only a single
reagent addition and comprises reacting the phosphate-containing
fluid with an ammonium molybdate solution and thereafter measuring
the absorbance within a specified time interval before the reaction
has measurably proceeded and at the end of the reaction by means of
a centrifugal analytical photometer. Inasmuch as a linear
relationship exists between the phosphate concentration and the
change of absorbance, the concentration is an unknown sample can be
conveniently calculated by comparison with the results obtained
from the simultaneous measurement of a sample of known
concentration.
Inventors: |
Daly; John A. (Valley Cottage,
NY), Ertingshausen; Gerhard (Riverdale, NY) |
Assignee: |
Union Carbide Corporation (New
York, NY)
|
Family
ID: |
22785506 |
Appl.
No.: |
05/211,075 |
Filed: |
December 22, 1971 |
Current U.S.
Class: |
436/46; 356/433;
436/164; 356/427; 436/105 |
Current CPC
Class: |
G01N
33/84 (20130101); Y10T 436/166666 (20150115); Y10T
436/112499 (20150115) |
Current International
Class: |
G01N
33/84 (20060101); G01n 021/22 (); G01n
033/16 () |
Field of
Search: |
;23/23B,23R,253R,259
;250/218 ;356/197,246,196,204 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
3547586 |
December 1970 |
Denney et al. |
|
Other References
Coleman et al., Chem. Abs., Vol. 75, No. 6, p. 291 (July 26,
1971)..
|
Primary Examiner: Scovronek; Joseph
Attorney, Agent or Firm: Moran; William R.
Claims
1. A process for the determination of inorganic phosphate in a
phosphate-containing fluid which comprises the steps of:
a. forming a mixture of said phosphate containing fluid and an
ammonium molybdate solution;
b. measuring by means of a centrifugal analytical photometer a
first absorbance reading at 340 nanometers within two seconds after
said mixture is formed,
c. measuring a second absorbance reading at 340 nanometers within
10 minutes after said mixture is formed,
d. comparing the absorbance differential with at least one other
differential obtained simultaneously under the same conditions from
a fluid containing a known concentration of inorganic phosphate,
and
e. determining the amount of inorganic phosphate in said
4. The process of claim 1 wherein said mixture contains a surface
active
5. The process of claim 4 wherein said surface active agent is a
sorbitan monooleate.
Description
This invention relates in general to a process for the
determination of inorganic phosphate in fluids, particularly body
fluids. In one aspect, the invention relates to a process for the
determination of inorganic phosphate in blood serum. In a further
aspect the invention relates to a process for the determination of
phosphate using a centrifugal analytical photometer.
In recent years the need for more sophisticated quantitative
analytical methods has increased markedly due to the numerous
microanalytical studies in biochemical research, routine clinical
testing for physicians and hospitals, and the like. In addition to
the increased demand for new methods of analysis, in certain fields
it is often highly desirable that the method be simple to perform,
be rapid and yet provide consistently reliable results. This is
particularly important for clinical testing of body fluids where a
proper diagnosis of treatment often depends upon the information
provided by analyses. However, few methods are available which can
rapidly and accurately handle the increasing number and varied test
desired by clinicians.
For example, the determination of inorganic phosphate in body
fluids, such as blood serum, is assuming a steadily growing share
of the clinical laboratory's work load. The determination of
phosphate in serum is important in several diseases in particular
uremia and chronic renal diseases were phosphate retention occurs.
Only the so-called inorganic phosphate is estimated since the
significance of changes in phospholipids, phosphate esters and
nucleotide phosphate is not easily related to clinical problems.
However, in spite of the advent of many new chemical methods, the
photometric determination of inorganic phosphate in biological
samples is conventionally performed by the use of the molybdenum
blue reaction (I.M. Kolthoff and P.D. Elving, Eds., Part II, Vol. 5
pages 317-402, 1961).
It has now been found that inorganic phosphate can be determined
conveniently and accurately by a method which utilizes a
centrifugal automatic analyzer. Analytical photometers which
utilize a centrifugal field have recently become available for the
rapid microanalysis of a wide variety of liquids such as body
fluids, e.g., blood serum, food products, and the like. Since
numerous analyses can be performed rapidly and simultaneously these
devices are of particular interest wherein a large number of
samples is involved or a variety of tests on one sample is desired.
Moreover, since these devices allow the use of relatively small
volumes of reagents, i.e., in the microliter range, the use of
expensive reagents can be minimized.
One such device which utilizes a centrifugal field in
microanalytical studies is described in U.S. Pat. No. 3,555,284.
This device employs the principle of double-beam spectrophotometry
wherein absorbances of a liquid sample and a reference solution are
intercompared. The system is basically a series of cuvets arranged
around the periphery of a rotor so that when it is spun,
centrifugal force transfers reagents and samples to the cuvets
where the concentration is measured spectrophotometrically. A
sample loading disk is provided which consists of rows of cavities
arranged concentrically. Reagents are placed in the inner-most
cavity and serum samples in the center cavity of the sample loading
disk which is then indexed and positioned in the rotor with each
reagent and serum sample having its respective cuvet. As the rotor
is accelerated, centrifugal force moves the reagents and sample to
the outer-most cavity where they are transferred through a small
channel to the cuvet. During the transfer, the reagent and sample
mix. The filled cuvets rapidly spin past the fixed light beam and
the transmission of light is measured.
As previously indicated, most of the approaches for the
determination of inorganic phosphate involve the use of the
molybdenum blue reaction. This reaction involves the formation of a
phosphate molybdate complex which is subsequently reduced by means
of stannous chloride, phenylhydrazine, ascorbic acid, amino
naphtholsulfonic acid or other reducing agents. A blue colored
complex of the reduce heteropolyacid is formed and the absorbance
of the complex measured at around 700nm (nanometers). The
preparation of a protein free serum sample is required for this
test, which makes the test cumbersome to perform. Additionally, the
sensitivity of the test is low. Moreover, in order to perform the
test properly, at least two sequential additions of reagents are
required.
Attempts to measure inorganic phosphate by means of the yellow
molybdovanadophosphate heteropolyacid have been suggested but have
failed to date to become accepted as a routine procedure. Even less
effort has been made to quantitate the unreduced phosphomolybdate
complex prior to its reduction. The absorbance maximum of this
heteropolyacid complex lies in the ultraviolet and high sample and
reagent blanks have to be eliminated.
It is therefore an object of this invention to provide a process
wherein many of the disadvantages indicated above are eliminated or
minimized. A further object is to provide a process for the
determination of inorganic phosphate which is accurate and requires
only a single reagent addition. Another object of the invention is
to provide a process for the determination of inorganic phosphate
in which the background absorbances of sample and reagent are
automatically eliminated. A further object is to provide a process
which utilizes a centrifugal analytical photometer. These and other
objects will readily become apparent to those skilled in the art in
the light of the teachings herein set forth.
In its broad aspect the invention is directed to a process for the
determination of inorganic phosphate in body fluids. The process
comprises the steps of:
a. forming a mixture of the phosphate containing fluid and an
ammonium molybdate solution;
b. measuring by means of a centrifugal analytical photometer a
first absorbance reading at 340nm within two seconds after said
mixture is formed,
c. measuring a second absorbance reading at 340nm within ten
minutes after said mixture is formed
d. comparing the absorbance differential with at least one other
differential obtained simultaneously under the same conditions from
a fluid containing a known concentration of inorganic phosphate,
and
e. determining the amount of inorganic phosphate in the
phosphate-containing fluid.
The process of this invention provides an accurate and rapid method
for the quantitative determination of inorganic phosphate in body
fluids. In view of the fact that a linear relationship exists
between the change of absorbance and the phosphate concentration,
up to at least 10 milligram per 100 milliliters phosphate can be
measured. Results of precision studies and correlation with a known
method indicated that this method was of equal accuracy.
Moreover, the process simplifies the diagnostic reagent by
measuring the color formed in the first reaction alone. The
absorption of the unreduced phosphomolybdate complex occurs in the
ultraviolet and can be quantitatively measured at 340nm. In
addition protein does not have to be removed from the sample and is
kept in solution.
As previously indicated the process comprises the determination of
inorganic phosphate by measurement of changes in absorbance due to
the interaction of inorganic phosphate and ammonium molybdate.
For the analysis of inorganic phosphate by the process of this
invention a centrifugal rotary photometer supplied by Union Carbide
Corporation under the trademark "CentrifiChem" was utilized. In
this instrument, a Teflon disk containing samples and reagents is
inserted into a rotor with 30 radially arranged cuvets. When the
rotor starts spinning, the reagent rises from every individual
reagent well up to individual sample cavities, and the sample
reagent mixture is transferred into the single cuvets within 1.5
seconds. One cuvet containing water is used as a reference when the
cuvets spin past the stationary light beam of a spectrophotometer
which measures the absorbance and displays it on an oscilloscope.
Two sets of digitized absorbance readings of each cuvet can be
stored simultaneously and the difference between them processed in
a computer. A reading is taken of the first absorbance after start
and the time interval after which the second measurement occurs
also noted. A number of readings can be taken after the initial
one. In the rate mode the absorbance change per interval is
expressed in .DELTA.A/min. In the end point mode the absorbance
differences between initial and every individual subsequent set of
readings is measured. This approach allows compensation for cuvet
to cuvet variations and for serum and reagent blanks if readings
are taken before the reaction has started to a measurable degree. A
blank reading recorded in a preceding run can be used as initial
reading in a subsequent run. After a selected time interval has
elapsed, the readings are printed out. The last set of data stored
in the memory can be multiplied by means of the computer which
permits direct print-out in concentration units. The rotor is
thermostated within .+-.0.1.degree.C. in an air bath.
A particular advantage of the process of this invention is that
removal of protein which normally would interfere with the reaction
is avoided. It was observed that serum protein could be kept in
solution and turbidity minimized by the addition of a small amount
of a surface active agent. Although a wide variety of surface
active agents can be employed, it has been found that the non-ionic
surface active agents are preferred. Illustrative agents include
among others, the sorbitan monooleates, sold by Atlas Powder
Company under the trademark "tween". "Tween-80" is particularly
effective in eliminating protein interference. The amount of
surface active agent employed need only be such as will prevent
precipitation and turbidity. In practice it has been found that if
the molybdate solution contains from about 0.1 to about 1.0 per
cent of the surface active agent that protein interference is
suppressed.
The single active reagent employed in the process of this invention
is an ammonium molybdate solution. The solution is prepared by
dissolving 2.0 grams of ammonium molybdate ( (NH.sub.4).sub.6
Mo.sub.7 O.sub.24.sup.. 4H.sub.2 O) in one liter of 1.2N H.sub.2
SO.sub.4. The solution is stable indefinitely. The surface active
agent can be added to the molybdate solution. For example, 0.9
milliliters of a solution containing 1 part of the surface active
agent to two parts of water can be added to each 100 milliliter of
the molybdate solution.
A standard phosphorous reference solution can be prepared by
dissolving 439 milligrams of KH.sub.2 PO.sub.4 in 100 milliliters
of water. A few drops of chloroform can be added as a preservative.
5.0 milliliters of this stock solution is then diluted to 100
milliliters with water.
In practice, 400 microliters of the molybdate reagent are pipetted
into the innermost reagent cavity of the sample holder disk and 10
microliters of serum sample is pipetted into the sample cavity.
With the analyzer in operation an initial absorbance reading is
taken at 2.0 seconds and a final reading at 10 minutes. The
instrument is equipped with a filter permitting readings to be
taken at 340 nanometers.
The optical system of the analyzer permits the measurement of
absorbance in a linear dynamic range from 0 to 2.5 at 340 nm. Since
the reaction is linear to above 10 milligram per 100 milliliter
phosphate, and the unreduced form of the phosphomolybdate complex
can be employed, the inorganic phosphate can be determined rapidly
and accurately. By measuring the difference in the two absorbance
readings and comparing it with a solution containing a known
quantity of phosphorous, the concentration can easily be
determined. Comparison of the method of this invention with
conventional literature methods confirmed its accuracy. The
following example is illustrative:
EXAMPLE I
The majority of the sample cavities of a Teflon disk sample holder
of a CentrifiChem Automatic Analyzer were loaded with 10
microliters of human blood sera. The reference position contained
400 microliters of distilled water in the innermost cavity. The
remaining cavities were loaded with 10 microliter aliquots of
various phosphorous standards ranging from 2 to 10 milligrams per
hundred milliliters. An ammonium molybdate solution was prepared
containing 2 grams of (NH.sub.4).sub.6 MO.sub.7 O.sub.24 --4H.sub.2
O and 3 milliliters of a non-ionic surface active agent, sold by
Atlas Powder Company under the trademark "tween-80". In one liter
of 1.2N H.sub.2 SO.sub.4.sup.. 400 microliters of this solution was
then placed in the innermost reagent cavity of the sample
holder.
The initial reading of all 30 cuvettes was taken two seconds after
starting the rotor spin. The second reading was taken after ten
minutes. The absorbance change was used to determine phosphorus
concentrations based upon the standards. The test was run with a
340nm interference filter.
According to Beer's Law, the absorbance of a solution is
proportional to the concentration of the solution's chromophore as
long as the concentration is low. Accordingly, the concentration of
phosphomolybdate formed in the reaction is at any time proportional
to the absorbance it generates.
Absorbance X F = Concentration
In order to convert absorbance into concentration, the absorbance
has to be multiplied with a factor F. In the analyzer employed this
is achieved by setting the appropriate factor on a digital switch
which automatically multiplies the absorbance whereupon
concentration units will be printed out directly.
During the analytical run, a standard of known phosphorus
concentration is always run with the samples. If the results for
the standard show slight variations from the correct
concentrations, a setting of a digital switch is changed until the
correct value is printed out. The same correction is then
automatically applied to the results from the unknown sample.
Although the invention has been illustrated by the preceding
disclosure, it is not to be construed as being limited to the
particular embodiments or materials disclosed therein. Rather, the
invention encompasses the generic area hereinbefore disclosed.
Various modifications and embodiments thereof can be made without
departing from the spirit and scope thereof.
* * * * *