Kinetic spectrophotometric method for the determination of urinary cobalt based on its catalytic effect on the oxidation of l -adrenaline hydrochloride by hydrogen peroxide

ANALYTICA CHIMICA

AClit ELSEVIER

Analytica Chimica Acta 290 (1994) 363-369

Kinetic spectrophotometric method for ,the determination of urinary cobalt based on its catalytic effect on the oxidation of L-adrenaline hydrochloride by hydrogen peroxide Maria G. Angelova *, Alexander A. Alexiev Department of Chemhy

and Biochemistry, Medical University,5&00Pkuen, Bulgaria

(Received 17th August 1993; revised manuscript received 2nd December 1993)

Abstract A catalytic reaction for determination of nanomblar concentrations of Co(II), i.e., oxidation of L-adrenaline hydrochloride with H,O, in alkaline medium, is proposed. The reaction gives a low limit of detection of 2.5 X low9 M cd111 in the reaction mixture, good reproducibility with a relative standard deviation (R.S.D.) of 4-5% in the Co(I1) concentration range 8.0 X 10P9-8.0 x 10-s M and good selectivity. On the basis of this indicator reaction, a catalytic-spectrophotometric method for the determination of cobalt in small urine samples (5.00 ml) was elaborated. The analysis of 17 urine samples, taken from healthy persons of different ages, gave cobalt concentrations in the range 0.20-1.50 pm01 1-l. The R.S.D. for ten replicate analyses of a urine sample with an average cobalt content of 0.63 pm01 I-’ was 5.6%. The reliability of the method was verified by a comparative photometric method (r = 0.9755) and by a determination based on known additions of cobalt (r = 0.9894). Z&y words: Catalytic methods; Kinetic methods; UV-Visible

spectrophotometry;

1. Introduction

this work was the study of the oxidation reaction of adrenaline salts with H,O, to establish the optimum conditions for performing the reaction and to develop a method for the determination of cobalt in biological materials.

In the last few years, several catalytic-spectrophotometric methods for the determination of cobalt have been described [l-lo], but there are few data concerning their practical application to the determination of cobalt in blood serum, urine and other biological samples [ll-141. The determination of traces of cobalt by the catalytic reaction of the o-dihydroxy derivatives with H,O, oxidation has a low limit of detection [1,3,15]. Adrenaline is an o-dihydroxy derivative with a lateral substituent in the benzene nucleus that has not been studied previously. The aim of

Cobalt; Urine

2. Experimental 2.1. Reagents All solutions were prepared from analytical-reagent grade substances and doubly distilled wa-

0003-2670/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0003-2670(93)E0702-9

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ter. Laboratory glassware was immersed for 12 h in aqua regia, washed ten times with distilled water and five times with doubly distilled water and dried at 140°C. The glassware was kept in hermetically closed vessels before and after the taking samples. A 0.01 M solution of Co(H) was prepared by dissolving 0.2379 g of CoCl, - 6H,O (Chemapol, Czechoslovakia) in 100 ml of 0.01 M HCl (Merck), the exact concentration being controlled complexometrically. The initial 1.6 M solution of H,O, was prepared from 30% H,O, (Merck) whose exact concentration was controlled permanganometrically. Because of their limited stability, solutions with concentrations of 0.16 M H,O, and 1 x lo-’ M Co@11 were prepared every week by successive tenfold dilutions of the initial solutions with doubly distilled water. Working solutions of Co(B) (1 X 10e6 and 1 X lo-’ M) were prepared by dilution daily starting from a 1 x 10m5 M solution of CoCl, * 6H,O. A 0.1 M solution of the substrate L-adrenaline hydrochloride (AD - HCl) was prepared by dissolving 0.1838 g of L-adrenaline (Fluka) in 100 ml of 0.1 M HCl. This solution could be used for 1 week if kept in dark glass vessels at 5-10°C. The pH was maintained between 9.23 to 11.02 with the aid of borate buffer [16]. Buffer solutions were prepared by mixing 0.1 M NaOH (solution A) and 0.5 M sodium tetraborate [12.367 g of H,BO, (Fluka) + 100 ml of 1 M NaOH solution, diluted to 1 1 with doubly distilled water] (solution B) according to 1161.For a buffer solution of pH 10.75, solution B was added to 48.85 ml of solution A to a final volume of 100 ml. The initial concentrations of the reagents in the reaction mixture and the experimental conditions are described in the text where necessary.

2.2. Apparatus A Specol 11 spectrophotometer with EK-5 temperature-controlled cells and a Specord UVvisible recording spectrophotometer were used. A PHM 64 research pH meter was used for measuring the pH of the solutions.

2.3. Initial rate method

The catalytic procedure for the determination of Co@) in solutions by the initial rate method is as follows. In a three-compartment reaction vessel, 0.40 ml of AD * HCl (0.10 M) was placed in the first compartment, 0.40 ml of H,O, solution (0.16 M) in the second compartment and O.OO0.40 ml of Co(B) solution (1 X low6 M) in the third compartment. Volumes of 4.20-3.80 ml of borate buffer (pH 10.75) were added to each compartment in approximately equal portions, so that the total volume of the reaction mixture was the same in each experiment (5.00 ml). After the components had been brought to the reaction temperature (50°C) and mixed by vigorous shaking, the reaction mixture was transferred into a l-cm temperature-controlled cell in the Specol spectrophotometer. The change in the absorbance (A) at 360 nm as a function of time (7) was evaluated. The measure of the initial rate of the indicator reaction, tan (Y= AA/Ar, was determined from the initial straight-line portions of the kinetic curves of A vs. 7. A calibration graph of initial rate vs. co(B) concentration was constructed. 2.4. Fixed-time method In addition to the initial rate method to obtain a calibration graph for the determination of Co(H) in the same concentration interval, it is possible to apply the technically simpler fixed-time method. Instead of the three-compartment reaction vessel a test-tube can be used. The conditions for the determination, i.e. pH, temperature concentrations of oxidant, substrate and catalyst, volumes of the reagents, tota volume of the reaction mixture and wavelength for absorption measurement, were the same as for the initial rate method. The calibration graph in the fiiedtime method was prepared as follows: to a cobalt solution H,O, and buffer solutions were successively added, the solution was kept at 50°C for 15 min, the substrate solution was added and the start of reaction was registered. After 3 min from the start of the reaction the reaction mixture was placed back in the thermostat and the absorption was measured exactly.

M.G. Angelova, Ad. Akriev/Analyrica

2.5. Catalytic method for the determination of cobalt in urine A 5.00~ml urine sample is placed in a porcelain crucible and evaporated to dryness in a drying oven at 140°C for 4 h, then the residue is heated on a hot-plate for 4 h. In order to carbonize the residue, it is heated in a muffle furnace at 450°C for 2 h and finally at 600°C for 6 h. These conditions ensure complete mineralization without loss of cobalt. To the crucible containing the mineralized sample, 2.40 ml of 4 M HCl are added and the sample is heated again until complete dissolution of the residue. The turbid solution is left to cool and then filtered with the help of a fine-pore filteo To 0.60 ml of the clear filtrate 0.25 ml of 10% sodium citrate solution is added (to mask the iron) together with of 0.40 ml of 9.85 M NaOH, 0.10 ml of anhydrous acetic acid and 0.15 ml of doubly distilled water. Thus the total volume of the neutral solution was 1.50 ml. To three dry tubes containing aliquots of 0.15 ml of neutral solution, 0, 0.10 and 0.30 ml of 1 X 10m6 M Co(H) solution are added, followed by 4.05, 3.95 and 3.75 ml of borate buffer (pH 10.751, respectively, and 0.40 ml of 0.16 M H,O,. The samples are placed in a thermostat at 50°C

Chimica Acta 290 (1994) 363-369

36.5

for 15 min and then 0.40 ml of 0.10 M adrenaline hydrochloride initial reagent is added. In this way the total volume of the solution in each tube is 5.00 ml. The reaction mixture is shaken and placed in the thermostat again at the same temperature. After 3 min from the start of the reaction the absorption is measured at 360 nm in a l-cm cell against doubly distilled water and a plot of absorbance (A) vs. added C&II) concentration is constructed. Blank determinations are run in the same way with 0.15-ml portions of doubly distilled water and a plot of absorbance vs. concentration constructed. The cobalt content is determined from the distance between the intercepts of the calibration straight lines with the abscissa. A comparative method, a variant of the spectrophotometric method with 1-nitroso-Znaphthol [17], adapted as described [12], was used.

3. Results and Discussion 3.1. Study of optimum reaction conditions In a study of the optimum conditions for performing the indicator reaction, its velocity was followed spectrophotometrically according to the

Wavelength nm Fig. 1. Absorption spectra of the reaction product: (a) in the absence of catalyst; (b) in the presence of catalyst. Conditions: 1.60 X lo-* M AD * HCI; 1.28 X lo-* M H,O,; 2.00 X lo-’ M Cd&; pH 10.50; 40°C after 2.2 min of reaction.

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initial rate method. The factors studied were the absorption spectra of the reaction products and the influence of the acidity of the medium, the temperature and the concentrations of the reagents and the catalyst on the reaction velocity. In Fig. 1 the absorption spectra of the reaction products of the catalysed and uncatalysed reactions are shown. In the range 340-360 nm there is well formed shoulder for the absorption curve of the catalysed reaction and an optimum difference between the absorption of the catalysed and uncatalysed reactions can be observed. The wavelength of 360 mn, selected for further studies, gives the possibility of following the reaction rate with the spectrophotometer in a reproducible way. In Figs. 2b and 3b, the effects of the concentrations of the oxidant and substrate on the cobah(catalysed reaction are shown. The maximum reaction rate is observed within a narrow concentration intervals for the two reagents. On the other hand, it can be seen (Figs. 2a and 3a) that a change in the concentration of the reagents hardly affects the rate of the uncatalysed reaction

a

0

q

1

2

3

4

I H2021 x lo2 M

Fig. 2. Dependence of the initial reaction peroxide concentration: (a) in the absence the presence of catalyst. Conditions: 8.00~ 2.00x lo-’ M &Cl,; pH 10.50; 40°C; 360

rate on hydrogen of catalyst; (b) in 1O-3 M AD.HCI; nm.

620

1

2

3

4

5

6

7

0,

IAD.HCLIK~~~ M

Fig. 3. Dependence of the initial reaction rate on AD .HCl de concentration: (a) in the absence of catalyst; (b) in the presence of catalyst. Conditions: 1.28 X lo-’ M H,02; 2.00 x lo-’ M CoCl,; pH 10.50; 40°C; 360 nm.

under the studied conditions. The selected concentrations of the oxidant and substrate for the determination of Co(H) were 1.3 x 10e2 M H,O, and 8.0 X 10m3 M AD - HCl in the reaction mixture. The effect of the acidity on the rates of the catalysed and uncatalysed reactions is presented in Fig. 4. The studies showed that for buffering of the reaction mixture, the borate buffer (Na,B,O,-NaOH) is the most appropriate, because it has no side-effects. With a decrease in the acidity of the medium, the rates of the catalysed and uncatalysed reaction increase quickly, whereas at pH 10.50-11.02 there is no change in the reaction rates. At the same time in this pH interval, the difference in the reaction rate between the catalysed and uncatalysed reactions is maximum. This is why a pH of 10.75 was selected as the optimum acidity for the remainder of the studies. The influence of temperature on the reaction rates was studied according to the conditions showed in Fig. 5. With increase in temperature the rates of the catalysed and uncatalysed reactions increase and the difference in the reaction

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rates becomes larger. For practical purposes [low reaction background, high slope of the calibration graph and good reproducibility of the results in the determination of Co(II)], a temperature of 50°C was selected. 3.2. Calibration For the selected optimum conditions for the reaction, in which a minimum limit of detection and a minimum background were found, the calibration graph exhibits a clear linear range from 8.0 x 1O-9 to 8.0 x 1O-8 M. The regression equations of the linear calibration plots obtained by the initial rate and fmedtime methods were tan (Y= O.O2NKl+ 0.01875 Cco(,~) and A = 0.1833 + 0.00484 Cco(I,j, respectively, with correlation coefficients of r = 0.9973 and 0.99028, respectively. The standard deviations of the intercept (s,), the slope (sJ, tan (r (So,,,) and A (sA) [18] and the number of data points W for the calibration lines obtained by the initial rate method were S, = 0.001990, sI, =

TEMPERATURE,"C

Fig. 5. Dependence of the initial reaction rate on temperature: (a) in the absence of catalyst; (b) in the presence of catalyst. Conditions: 1.28X 10d2 M H,O,; 8.00~ lo-’ AD. HCI; pH 10.75; 360 nm. Table 1 Effect of other ions on the reaction rate Ion

P"

Fig. 4. Dependence of the initial reaction rate on acidity: (a) in the absence of catalyst; (b) in the presence of catalyst. Conditions: 1.28x10v2 M H,O,; 8.00~10-~ M AD.HCI; 6.00x 10-s M CoCl,; 40°C; 360 nm.

M&I) NKII) CuUI) Fe(III) MdVI) Fe(I1) Crw) Cd(II) PMII) Zn(I1) Ag(I) Cat II) Mg(I1) Oxalate IBrCitrate Borate Phosphate Tartrate

Limiting concentration @I) 2.00x

10-7

4.00x10-7 2.00x 10-6 4.00 x 10-6 4.00x 10-6

6.00~10-~ 6.00x lo+ 6.00x lo+ 6.00x 1O-6 6.00x lo+ 1.00x10-’ 2.00x 10-4 4.00x 10-4 4.00x 10-6 1.00x 10-S 1.00x 10-5

Limiting mole ratio,

lIonl/lCo0II1 5 10 50 100 100 150 150 150 150 150 250 5000 10000 100 1000 loo0

Conditions: 1.28x1O-2 M H202; 8.OOx1O-3 M AD.HCI; 4.00~ lo-* M CoCI,; 5o”C, pH 10.75; 360 nm.

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MC. Angelova, A.A. Alexiev /Analytica Chimica Acta 290 (1994) 363-369

oooo4069 Stana=

0.001870 and N = 5 and those obtained by the fiied-time method were s, = 0.002070, s,, = 0.0000276, sA = 0.014298 and N = 10. Under these conditions the detection limit of the method was 2.5 x 10e9 M CoGI) in the reaction mixture [18]. In the concentration range 8.0 x 10e9-8.0 x 10m8 M Co(R), the relative standard deviation (R.S.D.) varied from 4 to 5%.

3.3. Selectivity Solutions containing 4.0 x 10-s M CoW and different concentrations of other ions in the range 1.0 X lo-‘-1.0 X 10m9 M were examined according to the catalytic initial rate method for the determination of cobalt. In the same concentration range the influence of other ions on the

Table 2 Determination of cobalt in urine Catalytic method Co in urine (firno 1-l)

Added (wmoll-l)

Found wmol I-’

(o/o)

0.20

0.44 1.24 2.04 0.44 1.24 1.24 2.04 0.28 0.44 0.44

0.40 1.20 2.08 0.48 1.24 1.24 1.96 0.28 0.40 0.44

90.91 96.77 101.96 109.08 100.00 100.00 96.07 100.00 90.91 100.00

0.12 0.28 1.64 1.24 2.04 0.28 0.84 1.64 0.28 0.84 1.64 0.44 1.24 2.04 0.28 0.84 1.64 0.44 1.24 2.04 0.28 0.84 1.64 0.84 1.24 0.28 0.48 1.64

0.12 0.28 1.64 1.32 2.00 0.28 0.84 1.68 0.40 0.84 1.64 0.48 1.44 2.04 0.26 0.84 1.52 0.44 1.24 1.88 0.30 0.80 1.56 0.96 1.24 0.28 0.88 1.71

100.00 100.00 100.00 106.45 98.04 100.00 100.00 102.44 142.85 lOOSKI 100.00 109.08 116.13 100.00 92.85 100.00 92.68 100.00 100.00 92.16 107.14 95.24 95.12 114.28 100.00 100.00 104.76 104.87

0.28 0.44 0.48 0.52 0.60 0.64 0.68

0.68 0.84

0.84

1.00

1.20

1.20

1.24

1.36 1.50

Recovery

Spectrophotometric method: Co in urine (pm01 I-‘) 0.22

0.29 0.41 0.42 0.49 0.64 0.64 0.64

0.70 0.81

0.76

0.85

1.17

1.14

1.25

1.29 1.38

M.G. Angdoua, AA ALsieu/Ana&ica

background reaction was studied. The results, presented as concentrations and the mole ratios at which the other ions have no influence on the background and catalytic reactions, are summarized on Table 1. The most disturbing effects on the reaction are exerted by manganese(R), nickel011 and copper(R) when they are present in concentrations 5, 10 and 50 times higher than those of Co(H), respectively. At these concentrations the three ions increase the rates of the background and catalytic reactions. Increases in the velocities of the catalysed and uncatalysed reactions are also induced by other metal ions and by oxalate, bromide and iodide anions (Table 1). Interfering effects occur at molar ratios higher than 100. Citrate, borate, phosphate and tartrate ions have no effects on the catalysed and uncatalysed reactions in the concentration interval studied. The results in Table 1 show that the reaction exhibits relatively good selectivity. 3.4. Analysis of urine samples The complex composition of biological materials is responsible for the significant and unpredictable influence of the matrix on the determination of nanomolar cobalt concentrations. For this reason urine samples had to be pretreated. Dry mineralization of the samples was applied. After dissolving the dry residue, the solution was neutralized and diluted to a definite volume. The good selectivity and the low detection limit of the proposed reaction permitted the direct determination of cobalt in neutral solution, without extraction and re-extraction. The method of the standard additions helps to eliminate the effects of the non-organic matrix and the background of the catalytic reaction. The results for seventeen urine samples (Table 2), taken from healthy persons of different ages, show cobalt concentrations in the range 0.2-1.5 pmol/l. The R.S.D. for ten replicate analyses of a urine sample with an average content of cobalt of 0.63 pmol/l was 5.6%.

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369

The repeatability of the method was verified by comparison with a spectrophotometric method (Table 2). The correlation coefficient, calculated on the basis of the results obtained by the two methods, was r = 0.9755. The reliability of the method was confirmed by the results for the determination of known cobalt additions (Table 2). The correlation coefficient between the added and found concentrations of cobalt in urine was r = 0.9894.

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