ANALYST, FEBRUARY 1988, VOL. 113 251

Fluorimetric Determination of Calcium in Serum with Calcein Blue

Konstantinos A. Matsoukas Industrial Chemistry Laboratory, Department of Chemistry, University of loannina, loannina 45332, Greece and Mavroudis A. Demertzis" Analytical Chemistry laboratory, Department of Chemistry, University of loannina, loannina 45332, Greece

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Calcium was determined in serum by fluorimetry with calcein blue (CB) in a strongly alkaline solution (0.1 M KOH). Triethanolamine and N-(2-hydroxyethyl)iminodiacetic acid were used to mask iron, copper, zinc and magnesium. The relative standard deviation of the slope of a typical working curve was 0.45%. This value corresponds to confidence limits of 3.53 k 0.02 at the 95% level. The correlation coefficient was 0.9999. The mean value of calcium in a 204 sample of Quality Control Serum was within the appropriate limits of the reference sample. The changes in the structure of CB on standing at high pH are discussed. Keywords: Serum calcium determination; masking of iron, copper, zinc and magnesium; fluorimetry

Calcein blue1 or umbellikomplexon2 are alternative names for the compound prepared by condensation of 4-methylumbelli- ferone (MU), formaldehyde and iminodiacetic acid.

The analytical applications of CB are based on its fluores- cence and absorption maxima which depend on the pH and cations added. 1.2 Direct and indirect complexometric determi- nations of metal ions, alone or in mixtures, with or without prior separation, using CB as a fluorescent indicator, have been reported.1-3,4 A direct titration of silver ions with iodide, using CB as a fluorescent absorption indicator because of its large polarisability , has been reported.5 The residual fluores- cence of CB at the end-point of the titration of calcium was obtained by adding a few drops of rhodamine B solution.6

The properties of CB and the complexation of CB with various metal ions have been reported.1.2 In addition, information on purity, composition, fluorescence as a function of pH and the properties of CB as an acid have been described.' The structures of CB and of its calcium derivative were also investigated by Huitink et al. ,7 but their results were in disagreement with those reported previously.8~9

Fluorimetric determinations of zirconium, fluorides and sulphates have been developed using CB as a reagent.l*J' Recently, calcium was determined fluorimetrically in strongly alkaline solutions,l2 although CB was not previously con- sidered to be suitable for this purpose.7 Iron, copper, zinc and to a certain extent magnesium are masked with triethanol- amine (TEA). The practice of inclusion of a known amount of magnesium in the blank was applied.

In this paper, a further investigation of the properties of CB and its analytical potential is reported. Variations in the fluorescence of CB on standing depend on the concentration of hydroxide, potassium and calcium ions. A calibration graph can be linearly extended to 0-40 p . ~ of calcium in 0.1 M KOH. The upper limit of the linear part of the graph depends on the concentration of CB.

Experimental Reagents All solutions were stored in polyethylene bottles and were prepared using distilled water obtained from a borosilicate autostill (Jencons Ltd.).

A stock calcium solution of 10-3 M was prepared from reference calcium carbonate (Thorn-Smith) dissolved in a

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* To whom correspondence should be addressed.

minimum amount of 0.1 M HCl and diluted to volume with water. This stock solution was titrated against a standard solution of ethylenediaminetetraacetic acid disodium salt (EDTA) (obtained from BDH).

Standard copper (CuCl,) and zinc (ZnSO,) solutions, iron(II1) nitrate [Fe(N03)3.9H20] and magnesium sulphate (MgS04.7H20) of analytical-reagent grade, all purchased from Merck, were used to prepare stock solutions of 10-3 M.

Other stock solutions used were standard potassium hydroxide (1 M), TEA (1 M) and tris(hydroxymethy1)amino- methane (Tris) (0.1 M), all obtained from Merck. Tetramethyl- ammonium hydroxide pentahydrate (TMAH) (1 M), N-(2- hydroxyethy1)iminodiacetic acid (HEIDA) (0.01 M), MU (10-3 M) and CB (10-3 M in Tris at pH 7.00)13 were obtained from Fluka. The stock solution of CB was stored in a refrigerator at 5 "C and was stable for at least 6 months. A Quality Control Serum (BC40, lot K8618, from Wellcome) was used.

Apparatus Fluorescence spectra were obtained using a Perkin-Elmer LS 3 spectrofluorimeter. Quantitative fluorescence measure- ments were obtained using a Turner Associates (Palo Alto, CA) Model 110 filter fluorimeter. A band-pass filter with maximum transmittance at 405 nm and a sharp-cut filter with 37% transmittance at 455 nm were used as primary (excita- tion) and secondary (emission) filters, respectively. Neutral density filters or black pieces of cardboard with suitable openings were used to adjust the instrument sensitivities. Round borosilicate cuvettes were used. All vessels, pipettes and cuvettes were frequently washed in detergent or in a sodium dichromate - sulphuric acid mixture and were rinsed thoroughly with tap water, then with distilled water and finally dried in an electric oven.

Procedure Standard solutions are directly prepared in a set of five cuvettes, in which volumes of 15, 30, 45, 60 and 75 pl of the 10-3 M calcium solution are placed in turn. A 20-p.1 volume of serum is placed in a sixth cuvette. Into each cuvette 0.5 ml of 1 M KOH, then 1 ml of 1 M TEA, followed by 50 pl of 10-2 M HEIDA are transferred and the vofume is made up to 4.8 ml with distilled water. Finally, 200 p1 of 10-3 M CB are added at 1-min intervals and the contents thoroughly mixed. After 20 min the fluorescence is measured at 1-win intervals. The

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252 ANALYST, FEBRUARY 1988, VOL. 113

amount of calcium in the serum is determined from a calibration graph of fluorescence vs. calcium concentration.

The final concentration of the sample is 4 1.11 of serum per ml of solution and the five standard solutions are from 3 to 15 VM in calcium. Moreover, each solution is 0.1 M in KOH, 0.2 M in TEA, 10-4 M in HEIDA and 40 PM in CB.

Results and Discussion Stability of CB in Alkaline Solutions The change in fluorescence of CB on standing in highly alkaline solutions is shown in Fig. 1 and depends on the hydroxide concentration or the degree of complexation of potassium and calcium. The decrease in fluorescence was attributed to hydrolysis of the coumarin ring of CB giving fluorescent hydroxycoumarinic acid.’

A change in the structure of CB must be assumed to explain the increase in fluorescence at lower KOH [Fig. 1 (a)] , TMAH [Fig. 1 (b)] and calcium [Fig. 1 (c)] concentrations. A four-month-old solution of CB in 0.01 M KOH (“old CB”) did not react with calcium. This is presumed to be due to the absence of the imino group which is responsible for metal complexation. The product formed by CB without the imino group, similar to MU, would be structurally less flexible and should fluoresce more intensely. This assumption is further supported by the synchronous scanning fluorescence spectra of MU, CB and “old CB” shown in Fig. 2. The spectra of the MU and “old CB” are comparable, whereas that of CB is different. The intensity of the “old CB” spectrum is less than that of the MU spectrum because the hydroxycoumarinic acid present gives no spectrum under the particular experimental conditions used.

The complexation of calcium with CB [Fig. 1 (c)] restricts the rotation of the imino group and favours the hydrolysis of the coumarin ring. The rate of hydrolysis with respect to hydroxides can be seen [Fig. 1 (d)] when CB is completely complexed with an excess of calcium. The same result is obtained if KOH is used instead of TMAH. With TMAH [Fig. 1 (b)] , where no complexation is expected, the removal of the imino group is favoured, and is easier at elevated tempera- tures. The complexation of potassium14 with CB can be seen by comparing Fig. 1 (a) with 1 (b) and 1 (c). The effects seen in Fig. 1 are unchanged in the presence of the masking agents TEA and HEIDA.

100

80

E al

60 iz al .-

40 - al a

20

I l l I l l

0 15 30 45 0 15 30 45 0 15 30 45 0 15 30 45 60

Fig. 1. Variation of the fluorescence of CB solutions with time. (a) CB 20 p ~ ; KOH 0.01-0.6 M. (b) CB 40 VM; TMAH 0.01-0.6 M. (c) CB 30 p ~ ; KOH 0.1 M; calcium &30 p ~ . ( d ) CB 20 VM; TMAH 0.01-0.6 M; calcium 40 p~

Umin

100 I A

500 450 400 350 Excitation wavelengthhm

Fig. 2. Synchronous scanning fluorescence spectra of (A) CB, (B) MU and (C) “old CB.” In all instances pH = 10.88 universal buffeP), concentration of each substance = 20 VM an 6 far UV cuvettes and the same instrumental conditions were used Wavelength difference between excitation and emission monochromators was 15 nm

Table 1. Errors of calcium recovered in duplicate from solutions of known concentration under various experimental conditions. Measurements were made at room temperature. In all instances KOH was 0.1 M and CB was 40 p~

Concentration Error, YO

Solution TEA/ HEIDA/ Ca/ Fe/ CU/ Znf Mg/ 10min* 15min* 20min* 25min* No. M PM PM PM PM PM PM

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16

0.01 0.08 0.2 0.2 0.2 0.01 0.08 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

40 100

- 40

100 100 100 100 100 100 100

10 10 10 10 10 10 10 10 10 10 5

10 15 5

10 15

10 10 10 10 10 10 10 10 10 10 0.5 1 1 0.5 1 1

10 10 10 10 10 10 10 10 10 10 0 5 1 1 0.5 1 1

10 10 10 10 10 10 10 10 10 10 0.5 1 1 0.5 1 1

- 10 10 10 10 10 -

- 2 3 3

0.0 0.0 -0.5 +0.5 -0.7 +0.5 +1.4 +2.8 +4.2 +2.1 +2.1 +0.7 +4.9 +1.4 +1.4

+30.0 +30.0 +24.0 +9.0 +10.4 +9.7 +5.6 -1-4.2 t-5.6 +5.6 +2.8 0.0 +2.1 +0.7 -0.7 +4.2 +4.2 +3.5 +3.5 +3.5 +3.5 +3.5 +2.1 +4.9 +1.4 0.0 -0.7 +1.4 +0.7 -0.7 +1.4 +0.7 -0.7

+2.1 +0.7

-0.7 -2.8 +2.8 +3.5 +2.8 -2.8 -0.7 -2.8

* Time between the addition of CB and the measurement of the fluorescence.

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ANALYST, FEBRUARY 1988, VOL. 113 253

Table 2. Accuracy in fluorimetric determination of calcium in a solution 0.1 M in KOH, 0.2 M in TEA, 10-4 M in HEIDA and 4 x 10-5 M in CB

Calcium concentration/yM Deviation, %

i* i-t [ 1 O O ( i -i)/i] 0.0 0.03 co 3.0 3.00 0.00 6.0 5.97 -0.50 9.0 8.93 -0.78

12.0 12.04 +0.33 15.0 15.01 +0.07

* Prepared values. t Mean values of triplicate solutions calculated from the fluores-

cence using equation (1).

Accuracy and Precision The percentage errors of calcium determined in duplicate in prepared solutions under various conditions are shown in Table 1. The errors are related to the time interval between the addition of CB and the recording of the fluorescence measurements. In the absence of magnesium a small interfer- ence occurs from iron, copper and zinc at lower TEA concentrations (solutions 1 and 2). At higher TEA concentra- tions and with a combination of TEA plus HEIDA, the errors increase (solutions 3-5 and 11-13). In the presence of magnesium TEA is an insufficient masking agent and the large positive errors decrease as the TEA concentration increases (solutions 6-8). The recovery of calcium in the presence of magnesium is satisfactory if both TEA and HEIDA are present as masking agents (solutions 9, 10 and 14-16). In this instance, a fluctuation in the concentration of all the metal ions considered does not essentially influence the recovery of calcium. It is important to note that the errors change from positive to negative on standing. Hence, the choice of the measurement time can affect the recovery. Although an explanation is difficult, the decrease in the fluorescence cannot merely be related to changes in the structure of the CB molecule. Other combinations of TEA and HEIDA which secure good results can also be used.

Deviations in the calibration graph for the determination of calcium are shown in Table 2. The relative standard deviation of the curve was 0.45%, corresponding to confidence limits of 3.53 A 0.02 at the 95% level. The correlation coefficient was 0.9999. The values were calculated by the method of least squares using equation (l), with CY = 9.88. The linear part of the calibration graph is larger at lower KOH concentrations. However, such conditions are not recommended because the masking of iron, copper and zinc with TEA is incomplete.

y = a r + b x . . . . . . * ‘ (1) Good agreement of the proposed method with established

methodsls+fc, was found. A standard reference serum (Well- come Quality Control BC40) €or clinical chemistry was used to

check the accuracy and precision of this method. The reference serum calcium, repeatedly determined between the first and fourth day after reconstitution, was found to lie within the appropriate limits (10.2-10.6 mg of calcium per 100 ml of serum). Five solutions of the same concentration in serum (4 pl of serum per ml of final solution) were prepared and their calcium content was calculated from equation (1). The relative standard deviation of the calcium content was 1.14%, corresponding to confidence limits of 10.50 f 0.15 at the 95% level; CY and b for each individual working curve were 8.70 and 3.40, respectively.

Conclusion Calcium at a limiting concentration of 10-5 M can be determined fluorimetrically with CB in the presence of iron, copper, zinc and magnesium. The concentration of magne- sium can be at least equal to that of calcium. TEA is a perfect masking agent for iron, copper and zinc in 0.1 M KOH. If magnesium is also present then HEIDA must be added to mask it, because TEA alone is an inappropriate reagent for this purpose. However, a combination of TEA and HEIDA causes positive deviations for the calcium values when magnesium is absent and iron, copper and zinc are present. Serum calcium determination is not affected by proteins, lipids and bile pigments.

1. 2. 3. 4. 5. 6.

7.

8. 9.

10.

11. 12.

13.

14. 15. 16.

17.

References Wilkins, D. H., Talanta, 1960, 4, 182. Eggers, J. H., Talanta, 1960, 4, 38. Escarrilla, A. M., Talanta, 1966, 13, 363. Elsheimer, H. N., Talanta, 1967,14,97. Escarrilla, A. M., Anal. Chim. Acta, 1968,43,353. Kirkbright, G. F., and Stephen, W. I., Anal. Chim. Acta, 1962, 27, 294. Huitink, G. M., Poe, D. P., and Diehl, H., Talanta, 1974, 21, 1221. Willuns, D. H., Talanta, 1959,2,277; 1960,4, 80. Korbl, J., and Svoboda, V., Talanta, 1960,3,370. Hems, R. V., Kirkbright, G. F., and West, T. S., Anal. Chem., 1970, 42, 784. Tan, L. H., and West, T. S., Analyst, 1971,96,281. Matsoukas, K. A., and Demertzis, M. A., “Proceedings of the 1 l th Greek National Chemistry Conference, Athens, Decem- ber 2-5, 1986,” Chimika Cheonika (New Series), Association of Greek Chemists, Athens, 1986. Bates, R. G., “Determination of pH, Theory and Practice,” Wiley, New York, 1964, p. 160. Matsoukas, K. A., and Demertzis, M. A., unpublished results. Kessler, G., and Wolfman, M., Clin. Chern., 1964, 10,686. Bachra, B. N., Daver, A., and Sobel, A. E., Clin. Chem., 1958,4, 107. Dean, J. A., “Lange’s Handbook of Chemistry,” McGraw- Hill, New York, 1979, pp. 5-81.

Paper A71222 Received June 2nd, 1987

Accepted August l l th, 1987

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