Photometric method for the determination of inorganic phosphate in liquid samples

An improved photometric method for the determination of inorganic phosphate in fluids is disclosed. The method makes use of two reagents, an acid reagent and an ammonium molybdate reagent. Utilizing these reagents, the method comprises the following steps: PA0 (a) forming a first mixture consisting of a sample fluid and acid reagent; PA0 (b) measuring the light absorbance of said first mixture; PA0 (c) forming a second mixture consisting of said first mixture and ammonium molybdate reagent; PA0 (d) measuring the light absorbance of said second mixture; PA0 (e) calculating the difference in light absorbance (.DELTA.A sample) between the light absorbances measured for said first and second mixtures; and, PA0 (f) determining the concentration of inorganic phosphate in said sample fluid by comparing the difference in light absorbance (.DELTA.A sample) for said sample fluid to the difference in light absorbance (.DELTA.A standard) for a standard fluid having a known concentration of inorganic phosphate, .DELTA.A standard being determinable in a like manner to that set forth in steps (a)-(e).

FIELD OF THE INVENTION 
This invention relates generally to a photometric method for the 
determination of inorganic phosphate in fluids, especially body fluids, as 
well as to reagents useful in the practice of this method. The method 
disclosed is particularly adapted for use with certain centrifugal 
photometric analyzers of known design. 
BACKGROUND OF THE INVENTION 
The majority of phosphorus (80-85%) present in the body is found in the 
bones as hydroxyapatite. The remainder is present as inorganic phosphate 
and phosphate esters. A determination of the concentration of inorganic 
phosphate in various body fluids can be highly useful in the diagnosis of 
various disease states. For example, increased serum phosphorus can be 
indicative of hypervitaminosis D. hypoparathyroidism, and renal failure. 
Similarly, reduced serum phosphorus levels are seen in rickets (Vitamin D 
deficiency), hyperparathyroidism, and Fancony's syndrome. 
The inorganic phosphate content of body fluids has typically been 
determined using one of two known methods, or variations thereof. In the 
first method, that of Fiske and Subbarow [Biol. Chem. 66, 375 (1925)], a 
heteropoly compound formed between ammonium molybdate and inorganic 
phosphate is reduced under mild conditions to form the phosphomolybdenum 
complex, which is measured photometrically. In the second more widely used 
method, that of Daly and Ertingshausen [Clin. Chem. 18, 263 (1972)], 
inorganic phosphate is reacted with ammonium molybdate in sulfuric acid 
solution to form the unreduced phosphomolybdate complex, which is measured 
photometrically at about 340 nm. 
Due to the immense volume of testing done by modern clinical laboratories, 
as well as the need for consistently accurate test results, inorganic 
phosphate determinations are typically performed using high speed, 
automatic instrumentation. In particular, the use of so-called centrifugal 
analyzers for the determination of inorganic phosphate has become 
widespread. An exemplary device of this type is described in U.S. Pat. No. 
3,555,284. 
Generally speaking, centrifugal photometric analyzers comprise an array of 
transparent reaction chambers or cuvettes arranged around the periphery of 
a centrifuge rotor. One or more solution receiving chambers are located 
radially inwardly from each cuvette and passageways are provided 
communicating between each cuvette and its associated solution receiving 
chambers. The solution receiving chambers are shaped to retain liquid when 
the rotor is at rest and to release liquids to the cuvettes when the rotor 
is spun. Thus, spinning of the rotor causes the transfer of sample, 
reagent or both to the cuvettes, where the samples and reagents react. As 
the rotor continues to spin, each cuvette passes, in turn, between a light 
source and a photodetector where the phototransmittance of the contents of 
each cuvette is read. These readings are then used to determine the 
concentration of a given species in each of the samples. 
A typical prior art approach for the determination of inorganic phosphate 
utilizing a centrifugal analyzer is described in U.S. Pat. No. 3,795,484. 
In this method, which makes use of the unreduced phosphomolybdate complex 
chemistry described previously, samples under study are reacted in their 
respective cuvettes with a single reagent, which comprises ammonium 
molybdate, sulfuric acid and surfactant. Approximately two seconds after 
the rotor has begun to spin and, presumably, before the reaction has 
proceeded to a significant degree, a first or "blank" absorbance reading 
is taken for each cuvette. The purpose of this reading is to determine the 
inherent absorbance of the cuvette and unreacted sample and reagent. A 
second reading is taken after 10 minutes, the time required for the 
adequate reaction of sample and reagent. The change in absorbance between 
the first and second readings is compared to the change in absorbance 
obtained in a like manner for a standard having a known concentration of 
inorganic phosphate, in order to determine the phosphate content of each 
sample. 
In an improvement of the above method, described in U.S. Pat. No. 
4,220,451, a reduction of the time required for adequate reaction of 
sample and reagent--from 10 to about 2 to 4 minutes--is accomplished by 
optimizing the surfactant concentration of the reagent. 
Although the above mentioned analytical methods certainly represent 
significant advances in the field of clinical chemistry, it will be 
appreciated that they nevertheless possess certain deficiencies. For 
example, in these prior methods the initial absorbance readings are not 
true blank readings, in that they are made after the reaction has already 
proceeded to an extent. Further, in view of the heavy workload of many 
clinical laboratories, the time required in these methods for reaction to 
proceed to an adequate extent and for a final reading to be taken, even at 
2 to 4 minutes, is too long. 
Accordingly, it is the object of this invention to provide an improved 
method for the determination of inorganic phosphate in fluids. 
More particular objects are to provide an improved method for determining 
inorganic phosphate concentration which can be conveniently carried out 
using a centrifugal photometric analyzer, which allows for true sample 
blanking, and which is significantly more rapid than known methods. 
SUMMARY OF THE INVENTION 
The above and other objects are satisfied by the present invention which 
provides a method and reagents for the determination of inorganic 
phosphate utilizing a modification of the unreduced phosphomolybdate 
chemistry of Daly and Ertingshausen previously discussed. Although the 
method provided is particularly suitable for use with centrifugal 
analyzers, it should be recognized that it may also be practiced manually 
or adapted for use with other types of instrumentation. 
In contrast to prior methods, the present method makes use of two reagents, 
an acid reagent and an ammonium molybdate reagent. Utilizing these 
reagents, the method comprises the following steps: 
(a) forming a first mixture consisting of a sample fluid and acid reagent; 
(b) measuring the light absorbance of said first mixture; 
(c) forming a second mixture consisting of said first mixture and ammonium 
molybdate reagent; 
(d) measuring the light absorbance of said second mixture; 
(e) calculating the difference in light absorbance (.DELTA. A sample) 
between the light absorbances measured for said first and second mixtures; 
and, 
(f) determining the concentration of inorganic phosphate in said sample 
fluid by comparing the difference in light absorbance (.DELTA. A sample) 
for said sample fluid to the difference in light absorbance (.DELTA. A 
standard) for a standard fluid having a known concentration of inorganic 
phosphate, .DELTA. A standard being determinable in a like manner to that 
set forth in steps (a)-(e).

DETAILED DESCRIPTION OF THE INVENTION 
As previously indicated, although the method provided by the invention can 
be carried out manually or using other instrumentation, it is intended 
primarily for use with centrifugal photometric analyzers of the type which 
are capable of adding at least two different reagents to a sample being 
tested. An exemplar of this type of machine is the COBAS BIO.RTM. 
automated chemistry analyzer, manufactured by Hoffmann-La Roche Inc. 
Accordingly, the invention is described herein in terms of how it can be 
practiced in an exemplary fashion utilizing a centrifugal analyzer such as 
the COBAS BIO.RTM.. Those skilled in the art will, of course, comprehend 
the manner in which the invention could be practiced manually or with 
other instrumentation. 
As previously mentioned, two reagents are required for the practice of the 
invention, an acid reagent and an ammonium molybdate reagent. 
While prior art techniques make use of a single reagent comprising ammonium 
molybdate, sulfuric acid and surfactant, it has been found that, in the 
practice of the present method, in order for a proper sample blank reading 
to be obtained, the pH during the sample blank reading should be about the 
same as that during the final transmittance reading. Accordingly, before 
taking the sample blank reading the sample is first diluted with the 
acidic reagent. Later, when molybdate reagent is added to this acid 
reagent/sample mixture, the acid takes part in the formation of the 
phospho-molybdate complex. It has been found possible to either place the 
full amount of acid required in the so-called acid reagent or to split the 
acid between both the acid and molybdate reagents. 
In order to prevent precipitation where proteinaceous samples are used. The 
final mixture of both reagents and sample optimally contains a minor 
amount of a non-ionic surface-active agent such as Brij 35, Triton X-100, 
Triton 405, Tween 80 or Tween 20. This surfactant may be placed in either 
reagent or divided between the two. 
An appropriate acid reagent would comprise an aqueous solution containing 
sulfuric acid, at a concentration ranging between about 0.05 and 0.34 
moles per liter, and surfactant, at a concentration ranging between about 
0 and 1.2 percent by volume. 
An exemplary acid reagent contains about 0.255 moles per liter of sulfuric 
acid and about 0.8 percent by volume of Brij 35 surfactant. 
An appropriate ammonium molybdate reagent would comprise an aqueous 
solution containing ammonium molybdate, at a concentration of about 0.0054 
moles per liter, sulfuric acid, at a concentration ranging between about 
0.93 and 0 moles per liter, and surfactant, at a concentration ranging 
between about 0 and 1.2 percent by volume. 
An exemplary ammonium molybdate reagent contains about 0.0054 moles per 
liter of ammonium molybdate, and about 0.255 moles per liter of sulfuric 
acid. 
In order to calculate the phosphate concentration of a sample, readings 
taken for the sample must be compared to those taken for one or more 
standards having known concentrations of phosphate. Readings taken for 
standards also serve as controls, verifying proper instrument operation. 
An advantageous aspect of the invention is that suitable inorganic 
phosphate standards can be either aqueous or protein based, regardless of 
whether the sample is aqueous or proteinaceous. It is preferred to utilize 
three separate standards. Suitable standards are available commercially or 
can be readily prepared by those skilled in the art using known per se 
techniques. 
In the practice of the method provided by the invention, as it is performed 
using the COBAS BIO.RTM. instrument, 5 .mu.l aliquots of each of a number 
of sample fluids, as well as three standard phosphate solutions, are 
automatically drawn up, diluted with 50 .mu.l portions of water, and 
pipetted into respective sample receiving chambers of a rotor. In order to 
provide a reagent blank reading, a 55 .mu.l portion of water only is 
charged into one sample receiving chamber. Similarly, 150 .mu.l portions 
of acid reagent are deposited in the reagent receiving chambers of the 
rotor. The rotor is then rapidly spun, centrifugally driving both the 
sample (or standard fluids) and the acid reagent into the peripherally 
diposed cuvettes, where the two combine to form a first mixture. In order 
to compensate for any endogenous sample interference, an initial 
absorbance reading, termed an "acid sample blank," is taken at this point 
as each cuvette passes through the machine's optical path. This reading 
can be taken in the range between about 320 and 380 nm., with 340 nm. 
being preferred. Rapid rotation of the rotor is then halted while the 
instrument automatically pipettes 50 .mu.l portions of ammonium molybdate 
reagent, followed by 20 .mu.l of water, into each sample receiving chamber 
of the rotor. Rapid rotation of rotor is again commenced, centrifugally 
driving the molybdate reagent into the cuvettes, where a second mixture 
consisting of sample or standard fluid and acid reagent (i.e. the 
previously formed first mixture) and molybdate reagent is formed. At this 
point, the following reaction takes place: 
##STR1## 
The reaction yielding the phosphomolybdate complex is conveniently carried 
at a temperature ranging between about 24.degree. and 40.degree. C., and 
preferrably is carried out at 37.degree. C. The time required for the 
substantial completion of the reaction will, depending upon reaction 
temperature, range between about 10 and 50 seconds, with the time required 
at 37.degree. C. being about 15 seconds. 
At the completion of the complex forming reaction, a final absorption 
reading is taken for the second mixture in each cuvette. 
Upon completion of the final absorption reading, the instrument calculates 
a factor using the following formula: 
##EQU1## 
then: 
Concentration of the sample (mg/dl)=(.DELTA.A 
sample-.DELTA.A.sub.RB).times.F where: 
conc=Concentration in mg/dl 
St.sub.1 =Standard One 
St.sub.2 =Standard Two 
St.sub.3 =Standard Three 
.DELTA.A=Change in Absorbance 
RB=Reagent Blank (Cuvette containing only 55 .mu.l of water and reagent). 
FNT Note: .DELTA.A.sub.RB is normally a value close to zero. 
It is to be understood that the foregoing description of the invention is 
by way of illustration and not of limitation and that persons of ordinary 
skill in the art may employ other embodiments without departing materially 
from the spirit or scope of the invention.