A spectrophotometric micromethod for calcium determination in blood serum

703

SHORT COMMUNICATIONS

A spectrophotometric

micromethod

for calcium determination

in

blood serum The observation that chloranilic acid reacts with the diazonium salt of sulfanilic acid giving rise to a yellow colour, with an absorption maximum at 355 rnp (Fig. I), an E&z0 value of 1.550 and a linear titration curve (Fig. 2), induced us to find out the best conditions for the application of this reaction to serum calcium determination. It is known, in fact, that chloranilic acid reacts with Ca++ at neutral or slightly acid pH giving a precipitate of calcium chloranilate, that can be redissolved by complexing with EDTAl-3. 1.4r

6.6 ,F *@.. 0.5 -

h

d

‘\

3 si 0.4. 5

+., I\

0 6 0.3-

‘i \ .\

’

,i

330

,

,

,

350

370

390

Wavelength

“‘iyb,.y

410

2 4 Cont. of chloranilic

430

(p/ml

(mp)

Fig. I. Absorption spectra of the colour produced salt of sulfanilic acid. Fig. 2. Calibration

curve of the chloranilic

by reaction of cbloranilic

6

8 acid

1

acid with diazonium

acid reaction with cliazonium salt of sulfanilic acid

Reagents (I) Chloranilic acid solutiolz: I g of chloranilic acid (B.D.H.) is dissolved in 80 ml of H,O by addition of 7 ml of N NaOH; the pH is adjusted between 3.5 and 7.0 by further addition of NaOH or of chloranilic acid. The solution is taken to IOO ml with water, kept in refrigerator overnight and then filtered through a Whatman No. I filter paper; it is stored in refrigerator and eventual additional precipitates filtered off, when used. (21 Iso~7o~a~o~~ate7 (50 : 50 v/v). (3) 5% solution of the tetrasodium salt of ethylendiaminotetracetic acid (B.D.H.). (4) 0.4% sulfanilic acid (C. Erba) solution in 0.x N HCI. IO ml of this solution are mixed with 0.3 ml of a freshly prepared 2 y(, NaNO, solution, immediately before use. (5) N HCI. (6) Standard calcium solution: 0.2497 g of CaCO, are dissolved in IO ml of

704

SHORT COMMUNICATIONS

N HCl and taken to 1000 ml with glass-distilled Ca++ for IOO ml is thus obtained.

water. A concentration

Procedure To 0.1 ml of serum in a conical test tube, 0.1 ml of chloranilic

of IO mg of

acid solution

is

added with mixing. After 30 min at room temperature, the precipitate is centrifuged down for IO min at 3000 rev./min in a radial centrifuge and the supernate cautiously poured. The precipitate is resuspended with reagent (2) and washed once or twice with 0.5-1 ml of this reagent. The washed precipitate is dissolved with 0.1 ml of reagent (3), and 0.9 ml of H,O is added. To 0.3 ml of this solution, 3.7 ml of diazotized sulfanilic acid, and after 30 min I ml of N HCl are added. Readings are taken at 355 rnp against a blank prepared by mixing 0.3 ml of H,O with 3.7 ml of reagent (4) and I ml of reagent A parallel determination is run on 0.1 ml of the standard calcium solution. Calculation:

O.D. sample _~~----. -~~~~~~~ X I0 = mg Ca++ per 100 ml of serum. O.D. standard

Study of the conditions fey the reactiolz In order to select the optimal following variables were investigated. (I)

then more the blank (2) centration

(5).

conditions

for the colour

development,

the

Concentration of sulfanilic acid. The colour increases rapidly up to 0.4%, slowly until 1% concentration; however, a slight colour appears also in at the higher concentrations. HCl concentration of the diazonium salt soZution. Decreasing the HCl confrom I to 0.1 N a sharp colour increase is observed. At lower concentrations

somewhat more colour develops, however some colour appears also in the blank. (3) Colour development in time. Colour intensity increases logarithmically with time. 30 min after the reaction, as much as 907; of the colour developed at 2 h is already present. Addition of I ml of N HCl stops the colour increase at the selected time, usually 30 min. After acidification the colour remains stable for at least 4 h. (4) Reproducibility. A standard deviation of &30/ was found on repeated tests on various sera. A comparison with Ferro’s method20n 20 different sera gave a correlation factor of 1.01 f 0.03. Institute Brescia

of Pathology,

Spedali

L. SPANDRIO

Civili,

(Italy)

REFERENCES I P. V. FERRO AND A. B. HAM, Am. J. Clin. Pathol., 2 P. V. FERRO AND A. B. HAM, Am. J.Clin. Pathol., 3 L. SPANDRIO, Clin.Chim. Ada, IO (1964) 376. Received Clin. Chim.

July Ada,

rzth,

1965

12 (1965) 703- 704

28 (1957) z8(1957)

346. 689.