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C LIN IC AL C HE MIS TR Y, V ol. 18 , N o. 3 , 1 972 2 63
Direc t Method for Determining Inorgan icPhospha te in Serum with the Cen trifiChemJ ohn A . D aly an d G erh ard E rting sh aus en
A direct m ethod was developed for determ in inginorganic phosphate in serum , which requiresonly a sing le reagent addition. The m ethod quanti-ta tes the unreduced phosphom olybdate hetero-po lyacid at 340 nm and is linear to at least 10 m g ofphosphate per 100 m l. Only 10 M l o f serum is re-quired. The unique blanking capabilities of cen-trifuga l ana lyzers perm it the on run e lim inationof serum and reagent background absorbances,w hich are autom atically subtracted. D ata on pre-cis ion, corre la tion, and recovery are presented.K in etics o f th e re actio n w ere stu die d, an d th eo reti-ca l lim its of automatic blanking when applied to afirs t-o rd er re ac tio n a re d is cu ss ed .Add it ional Keyph rases direct uv methodreagent {149} a uto m atic b la nk in g {149} kinetics single
Inorganic phosphate in b iological samples isconventionally determ ined photometrically by useof the molybdenum blue reaction 1 . Variousreducing agents such as FeSO4, SnC12, ascorb icacid, and am inonaphtho lsulfon ic acid have beenused . These vary w ith respect to the stability andcolor intensity of the reduced phosphomolybdateheteropolyacid they all p roduce 2 , 3 . Measure-m ent of inorganic phosphate via the yellow molyb-dovanadophosphate heteropolyacid has been sug-gested but has not yet been accepted as a routineprocedure 4).Even less effort has been made to quantitate the
phosphomolybdate complex before it is reduced.This heteropolyacid complex absorbs maximally inthe ultrav io let, and high sample and reagent blankshave to be elim inated.From the Union Carbide Research Institu te, Tarry town, N . Y .
10591.Received Sept. 7 , 1971; accep ted Dec. 9, 1971.
The optical system of the CentrifiChem ,which perm its absorbance to be measured in alinear range from 0 to 2.5 , enabled us to use theunreduced form of the phosphomolybdate complexto determ ine inorganic phosphate. In addition,the unique blanking capabilites of centrifugalanalyzers provide blank measurements in less than3 s after sample and reagent are m ixed. In-div idual blank measurements for all cuvets areautomatically subtracted from the final absorbancereading , thus elim inating reagent and sampleblanks sim ultaneously. If small amounts ofTween-80, a polyoxycthylene sorbitan mono-oleate, are added to the m olybdate reagent, pro-tein need not be removed from serum samples, anda single-step reagent for the determ ination wasdeveloped. The reaction is linear to at least 10 m gof inorganic phosphate per 100 m l, and is completew ith in 12 mm . Only 10 of serum is required pertest.Ma te ria ls a nd Meth od sReagents
H 2S04, 0.60 m ol/liter. Pipet 33 m l of reagent-grade H2S04 into 500 m l of distilled water. Cool toroom temperature and dilute to 1 liter.
M olybd ate solution. D issolve 2 .0 g of ammo-nium molybdate [ N114)sM o7Ose4 1120; Mathe-son, Coleman and Bell, Norwood, Ohio 45212] in 1liter of H2S04, 0.60 m ol/liter. S table indefinitely.
Tween 80. Mix one volume of Tween 80 FisherScientific Co., Springfield, N . J. 07081) w ith twovolumes of water.
W orking reagen t. Mix 100 m l of molybdate so lu-tion w ith 0.9 m l of the diluted Tween 80. Useafter 30 mm .
Stock phosphate standard . 100 mg/i 00 m l. Dis-solve 439 mg of KH2PO4 in water and dilute to 100
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CENTRIF ICHEM m g/ 1Q O nI P HO SP HA TEFig. 1. C orrela tion between Technicon SM A 12/60molyb-denum blue m ethod a nd C en trifiC he m p ho sp ho mo ly b-date method n = 95
264 C LIN ICAL CH EM ISTRY, Vol. 18, N o. 3, 1972
m l. Add a few drops of chloroform as a preserva-tive.
W orking phosphate standard. Dilute 5.0 m l of thestock standard to 100 m l w ith water.Instrumentat ionA CentrifiChem Union Carbide Corp., Tarry-
town, N . Y . 10591) as previously described [Daly,J. A ., Fabiny, D . L ., and E rtingshausen , G .,CentrifiChem , a high speed analyzer for the clinicallaboratory, CLIN CHEM . 16, 530 1970), abstract],is used as supplied by the manufacturer. A lO -M lserum sample is pipetted into the CentrifiChemtransfer disk, while 400 M1 of work ing reagent ispipetted into the reagent cavity. An initial readingT0) is taken at 2.0 s and a final reading 12 mm laterat 340 nm . Standards are included in assigned posi-tions to determ ine the appropriate factor for relat-ing change in absorbance to concentration of in-o rg an ic p ho sp ha te .Resu lts and D is cu ss io nP re cis io n a nd A cc ura cyW ithin-run precision , as determ ined by assaying
27 aliquots of a serum pool on a single rotor w as1 .5 cv) at a concentration of 3.0 m g/100 m l.D ay-to-day precision was studied for 10 days;
the coefficient of variation was 2.7 for a commer-cial control contain ing 8.2 mg of phosphate per 100m l. Results of our method were compared tothose obtained w ith the molybdenum blue methodused w ith the SMA 12/60 Technicon Corp .,Tarrytown, N . Y . 10591). N inety-five sampleswere analyzed, w ith the results shown in Figure 1.Recovery experim ents Table 1) showed theaccuracy of the method.KineticsThe kinetics of the overall molybdenum blue
reaction were studied by Crouch and M alm stadt5). The reaction rate w ith respect to phosphatewas first order. They also found that the velocity ishigh ly sensitive to changes in pH and molybdateconcentration. The relationsh ips depended verym uch on the kind of acid used and on the abso lu teranges of pH and molybdate concentration in thereaction m ixture. It w as, therefore, im portant toinvestigate the reaction rate w ith respect to allthose variables in the new reagent.The possibility of an initial rate reaction was
elim inated when it w as discovered that serumpro tein had an accelerating effect on the reaction.This phenomenon varied from serum to serumwithout affecting the final absorbance differencebetw een the blank and the reaction product.
T ab le 1 . P erc en ta ge R ec ov ery o f P ho sp ha teA dded to Tw o S eraIn s erum Added Expected Found
mg p hosphate/100 m l Recovery6.5 2 .4 8.9 8.8
4 0 10 5 10 42.4 1.0 3.4 3.42.4 4.8 4.8
The dependence of the reaction rate on the pHwas studied at three different sulfuric acid concen-trations and it w as confirm ed that the speed ofreaction was inversely proportional to the 9thpower of the H + concentration. This relationshipwas determ ined by plo tting in itial reaction rate vs.the. H concentration on log-log paper and measur-ing the slope of the curve Figure 2).The test is also high ly sensitive to molybdate
concentration Figure 3). The plots of the in itialrate of the reaction vs. the m olybdate concentra-tion on log-log paper yielded a positive slope of 5 ,indicating the reaction velocity to fo llow Equation1:
a[PM I [P043j[M o}5 1at
M is p ho sp ho mo ly bd ate )This expression is very sim ilar to that found byM alm stadt and Crouch.The high dependence of the reaction rate on the
pH provided a convenien t parameter to use in de-sign ing a test such that, at the tim e of the blankreadings at 2 s, the reaction was less than 2 com -plete.The overall reaction when applied to th is test
w as assumed to be first o rder because of the high
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C LIN ICAL C HEM ISTR Y, Vol. 18, No. 3, 1972 26 5
F ig . 2 . Dependen ce o f re ac tio n ra te o f p ho sphom oly b-da te me thod on ac id ity , a s measu red a t th ree phospha tec on ce ntr atio ns a nd th re e a cid itie s:- - - 0.50; 0.55;and- . - 0 .6mo la r H2S04
F ig . 3 . D ep en de nc e o f re actio n ra te o f phosphomolyb.date m ethod on m olybdate concentration, as m eas-ured at tw o phosphate concentrations and three m olyb-da te concen trat ions : ... 2.0; - 1 .6 ; a nd - -, 1. 2mmol/ l iter
and therefore practically constan t) concentrationof H and M o6. The reaction is described by theformula
In a = k t2 t = t2 - Iia-x2 to,.
in w hich a is the initial phosphate concentration,x1 and x2 are the concentrations of phosphomolyb-date at ti an d t2 , an d t11, is the half-life tim e ofthe reaction. If t1 is 2 s and t2 equals t112, the f or-mula can be arranged to
ln_2_ t1) ________=ln 2 a - x, )a - X2
which means that a reaction w ith a half-life tim e of68.6 s is 2 complete 2 s after its start. Reagentparameters w ere according ly adjusted such thatsera reacted w ith a half-life tim e longer than 70 s.Under these conditions the slower-reacting water-
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F ig . 4 . Illu s tr ation o f au toma tic e lim i na tion o f d iffe ren tserum blanks for samples w ith identica l phosphate con-centration 3.8 m g/1 00 m l p ho sp ha te
based standards have half-life tim es of about lOOs,and have reacted to 99 completion after 12 mm .
Othe r Ana ly tic a l Conside ra tionsThe lim it of the molybdate concentration was
determ ined by the absorbance of the reagent blankat 340 nm , which is considerable, but combinedreagent and sample blanks having absorbancevalues near 0.8 could be tolerated because theoptical system of the instrument is linear to wellbeyond an absorbance of 2.0.M any organic polymers were checked as to their
properties to keep serum protein in solution :Tween 80 most effectively prevented proteinprecipitation and turb idity.F igure 4 illustrates the blanking capabilities of
the CentrifiChem . Two sera w ith equal phosphateconcentration but different serum blanks were runsimultaneously. Readings of blanks, which ord inar-ily are subtracted from the final absorbance, wereretrieved and the absolute absorbance was mea-sured every 2 mm throughout the test. F igure 4makes it obvious that the two absorbance changes
) are the same, even though the initial b lank valuesd if fe r c on si de ra bl y.References1. R ieman, W ., and Beukenkam p, J., In Treatise on Anal yt ic alChem istry, 5, Part II. I. M . Kolthoff and P . D . E lv ing, Eds.W iley , N ew York, N . Y ., 1961, pp 317-402.2. Sumner , J. B ., A method for the colorimetric determ ination of3) phosphorus. Science 100, 4 13 1 944 ).3 . Fiske, C . H ., and SubbaRow , Y ., The colorim etric determ ina-tion o f p h os ph o ru s . J. B iol. Chein. 66, 37 5 1 92 5).4. Sim onsen , D . G ., W ertman, M ., W estover, L . M ., and M ehI,0 . W ., The determ ination of serum phosphate by the molyb-d ivan ad ate m eth od . J. Biol. Chein. 166, 747 1946).5 . Crouch, S. R ., and Ialm stadt, H . v., A m echan istic in vesti-gation of molybdenum blue m ethod for the determ ination ofphosphate. A na l. C hern . 39, 1084 1967).
