Determination of copper, zinc, and selenium in human plasma and urine samples by potentiometric stripping analysis and constant current stripping analysis

Clinica Chimica Acta 285 (1999) 53–68

Determination of copper, zinc, and selenium in human plasma and urine samples by potentiometric stripping analysis and constant current stripping analysis ` Maria Luisa Gozzo*, Luigi Colacicco, Cinzia Calla, Giuliano Barbaresi, Raffaella Parroni, Bruno Giardina, Silvio Lippa Istituto di Chimica e Chimica Clinica, Universita` Cattolica del S. Cuore, Roma, Italy Received 26 October 1998; received in revised form 26 February 1999; accepted 22 April 1999

Abstract Potentiometric stripping analysis and constant current stripping analysis are proposed as routine methods for analysis of copper, zinc and selenium in plasma and urine samples. The analytical performance of these methods is comparable with that reported for atomic absorption spectrometry. However the low cost, greater simplicity of the apparatus, and the facility of execution make this methodology a valid candidate for routine application in Clinical Chemistry laboratories.  1999 Elsevier Science B.V. All rights reserved. Keywords: Copper; Zinc; Selenium; Potentiometric stripping analysis; Constant current stripping analysis

1. Introduction Clinical Chemistry laboratories are concerned with the analytical techniques useful for routine determination of copper, zinc and selenium in plasma and *Corresponding author. Corresponding address: Laboratorio di Chimica Clinica, Universita` Cattolica del S. Cuore, Facolta` di Medicina e Chirurgia ‘A. Gemelli’ Largo Francesco Vito 1, 00168 Roma, Italy. Tel.: 139-6-3015-4222; fax: 139-6-3550-1918. E-mail address: [email protected] (M.L. Gozzo) 0009-8981 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 99 )00085-6

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urine samples, because of the well known biological roles and clinical interest of these trace elements [1–3]. At present, the analytical technique most frequently employed is atomic absorption spectroscopy (AAS) both as flame atomic absorption spectrometry (FAAS) for zinc and copper and electrothermal atomic absorption spectrometry (EAAS) for selenium. Inductively coupled plasma emission spectrometry (ICPES) or mass spectrometry methods provide the capability of multielement analysis but offer no real advantage for routine analysis [4]. Moreover all these methodologies require a complex and expensive apparatus and this hinders their use for routine in Clinical Chemistry laboratories. Stripping potentiometry is an electroanalytical technique characterized by a remarkable analytical specificity and sensitivity that, up to now, has been used to measure trace elements in water, air and foods whereas only a few applications to biological fluids have been reported [5–7]. Stripping potentiometry includes two methodologies: potentiometric stripping analysis (PSA) and constant current stripping analysis (CCSA). Both these procedures consist of two steps. The first one is an electrolysis during which the metal ions are reduced to metal by an applied potential and dissolved into a thin mercury film plated on a working glassy-carbon electrode. During the second step, the applied potential is removed and the stripping of amalgamated metals can occur either by their chemical oxidation (PSA), or by a constant current applied to the working electrode (CCSA). The change of potential at the working electrode with time is then registered and the area of the peak obtained is directly proportional to the amount of the metal in the sample. PSA is used for determination of copper and zinc while CCSA is employed for selenium analysis. This paper describes the application of stripping potentiometry for routine determination of copper, zinc and selenium in digested plasma and urine samples. The methods supplied by Radiometer for water, air and foods [7,8] were modified in order to make these suitable for biological samples.

2. Materials and methods

2.1. Instrumentation The ‘TracelabE’ (Radiometer A / S Copenhagen Denmark) consisting of the potentiometric stripping unit ‘PSU 20’ and the sample station ‘SAM20’, connected to a PC Olivetti M240 with TAP2 software package (Radiometer), is used to carry out the analysis. The ‘PSU 20’ unit was equipped with three electrodes: the working glassy-carbon electrode (F3600), the calomel (K436) electrode as reference electrode and the platinum (P 136) electrode as counter

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electrode to provide or consume electrons for the electrolytic reaction which takes place at the working electrode. This latter electrode, in the case of selenium determination, has a 0.1 mol / l HCl salt bridge to avoid the deleterious effects of chlorine evolution (P736). The microwave system MLS-1200 Mega (Milestone s.r.l. Bergamo, Italy), equipped with the microwave digestion rotor (MDR 10) or with evaporation rotor (ECR 6), is used for sample digestion and concentration respectively. The wattages and times of the digestion program were: 250 (2 min), 0 (2 min), 250 (10 min), 400 (5 min), 500 (5 min), 600 (5 min), 0 (2 min) and 650 (10 min). The wattages and times of the concentration program were: 900 (10 min), 0 (2 min), 1000 (10 min), 0 (2 min), and 1000 (7 min) followed by ventilation for 10 min. The A.S. 5000 atomic absorption spectrophotometer (Perkin-Elmer) was employed for atomic absorption spectrometry. All glassware, pipettes and vessels were cleaned with 1 mol / l HNO 3 and rinsed several times with Millipore-Q water. A special rinsing program is available from Milestone for tetrafluoromethoxyl (TFM) vessels used for digestion.

2.2. Chemicals Mercury plating solution (S2201) and electrode test solution (S2202) were supplied by Radiometer. Hydrogen peroxide solution 30%, Selectipur was from Merck (Darmstadt, Germany). 37% Hydrochloric acid, 65% nitric acid and 70% perchloric acid, ultrapure for trace metal analysis, were from Carlo Erba (Milano, Italy). 96% acetic acid and sodium acetate anhydrous were suprapur from Merck (Darmstadt, Germany). Gallium (III)–nitrate-nonahydrate was from Merck (Darmstadt, Germany). The suprapur copper, zinc, and selenium standard solutions, certified for atomic absorption spectrometry, were from Merck (Darmstadt, Germany). HgCl 2 p.A. grade, was from Merck (Darmstadt, Germany).

2.3. Samples In order to evaluate the reliability of the procedure we used: • 2 Li-heparinized plasma pools obtained from patient’s samples; • 24 h urine patient’s samples; • SeronormE Trace Elements Control Serum from Nycomed Pharma (Oslo, Norway) batch no. 311089;

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• Randox assayed bovine sera normal (batch no. 082SN) and elevated (batch no. 107SE) from Randox laboratories LTD (UK); • Lyphochek Urine Metals Control Levels I (lot no. 44501 and 69011) and II (lot no. 44502 and 69012) from BioRad (ECS division California USA).

2.4. Preparation of the Tracelab Every morning the glassy-carbon electrode was polished with aluminum oxide (Radiometer) and then rinsed with Millipore-Q water. The mercury plating of the working electrode was then executed, according to Radiometer procedure, in order to cover the electrode surface with an homogeneous mercury film. The quality of the mercury film was evaluated by performing the electrode test program according to the Radiometer specifications.

2.5. Samples pretreatment 2.5 ml of Li-heparinized plasma or 6.5 ml of 24 h urine collection were added to 3 ml of 30% H 2 O 2 , 3 ml of 70% HClO 4 and 0.3 ml of 65% HNO 3 in TFM vessels of the microwave digestion system MLS-1200 Mega and the digestion program was executed. The vessels were then transferred to the evaporation rotor MCR6 and the concentration program was executed. The residue of the pretreated sample was diluted with milli-Q water to a final volume of 10 ml. Aliquots of this digested sample were used for determination of all trace elements. The smallest amount of plasma sample that can be digested is 0.250 ml for copper or zinc and 1 ml for selenium. In these cases the whole residue of the pretreated sample is used for the determinations.

2.6. Copper determination The test mixture included: 1 ml of water diluted pretreated Li-heparinized plasma or 3 ml of water diluted pretreated urine, 0.5 ml of oxidant solution (800 mg Hg 11 / ml in 1.3 mol / l HCl), 0.5 ml of HCl 37% and milli-Q water to a final volume of 25 ml. We used an electrolysis potential of 2 900 mV for 120 s with stirring and 30 s without stirring for plasma samples and for 300 s with stirring and 30 s without stirring for urine samples. During the electrolysis step the copper is firstly reduced and then amalgamated at the working electrode (Cu 11 1 2e 2 ⇒ CuHg). During the following stripping phase the amalgamated copper is chemically oxidized and stripped back into the solution in ionic form (CuHg 1 Hg 11 ⇒ Cu 11 1 Hg). Mercury (II) ions are used as oxidant. The stripping potential as a function of time was then registered and the copper

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concentration was then calculated from the peak area using a preliminary calibration line. This latter was done by three sequential additions to the reagent blank, of a working standard solution (0.5 ml of a 1000 mg / l or 100 mg / l standard solution for plasma or urine samples respectively) at the beginning of the routine work and remained valid for all the following ten samples.

2.7. Zinc determination The test mixture included 1 ml of water diluted pretreated plasma or 1 ml of water-diluted pretreated urine, 6 ml of Na acetate buffer 2 mol / l pH 4.7, 2 ml of oxidant solution (1600 mg Hg 11 / ml in 0.06 mol / l HCl), 0.5 ml of 10 mg / l gallium solution and milli-Q water to a final volume of 25 ml. Gallium (III) is added to remove the copper interference [8]. We used an electrolysis potential of 2 1400 mV for 120 s with stirring and 30 s without stirring. During the electrolysis step the zinc is firstly reduced and then amalgamated at the working electrode (Zn 11 1 2e 2 ⇒ ZnHg). During the stripping phase the amalgamated zinc is chemically oxidized and stripped back into the solution in ionic form (ZnHg 1 Hg 11 ⇒ Zn 11 1 Hg). Mercury (II) ions are used as oxidant. The stripping potential as a function of time was then registered and the zinc concentration was then calculated from the peak area using a preliminary calibration line. This latter was done by three sequential additions of 0.2 ml of the working standard solution (1000 mg / l) to the reagent blank for both serum and urine samples at the beginning of the routine work, and remained valid for all the following ten samples.

2.8. Selenium determination The test mixture included: 4.0 ml of water diluted pretreated Li-heparinized plasma or 3.0 ml of water diluted pretreated urine, 9 ml of 6 mol / l HCl, 0.5 ml of oxidant solution (800 mg Hg 11 / ml in 1.3 mol / l HCl) and deionized water to a final volume of 20 ml. During the electrolysis step Se 16 is firstly reduced to Se 14 in a strong acidic medium containing a high concentration of chloride ions, with a consequent evolution of chlorine at the platinum electrode. The Se 14 is then reduced to Se 22 according to the following reactions: H 2 SeO 3 1 6e 2 1 6H 1 ⇒ H 2 Se 1 3H 2 O H 2 Se 1 Hg

11

⇒ HgSe 1 2H

1

In the stripping step the Hg bound as HgSe is reduced to metallic mercury: HgSe 1 2e 2 1 2H 1 ⇒ Hg 1 H 2 Se.

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In this way Selenium is determined indirectly by measuring the signal coming from the HgSe derivative. We used the followings CCSA operating conditions: • Electrolysis potential: 100 mV for 500 s, 2150 mV for 50 s • Electrolysis time: 550 s with stirring and 30 s without stirring • Constant current: 210 mA The stripping potential as a function of time was then registered and the selenium concentration was calculated from the peak area using a calibration line. This was constructed at the beginning of the routine work by three sequential additions of 0.5 ml of a standard solution (100 mg / l) to the reagent blank for both plasma and urine samples, and remained valid for all the following ten samples. Zinc and copper were determined with FAAS according to the method reported by Alcock [9] and Chou [10] respectively whereas selenium was measured with EAAS according to Morisi [11].

3. Results and discussion The potentiometric methods supplied by Radiometer for trace elements determination in water were optimized for use in plasma and urine samples.The modified parameters were: the electrolysis times, the reaction volumes, and, in selenium determination, the intensity of the applied constant current. Moreover a suitable program of plasma and urine sample digestion was introduced. A single digestion procedure can be used for the determination of all the trace elements in the same sample. The linearity of the proposed methods was determined by analyzing, in triplicate, scalar dilutions of convenient standard solutions. The results obtained are showed in Figs. 1–4. In our analytical conditions all the linearity ranges are sufficiently wide to include the concentrations usually found in normal and pathological plasma and urine samples. In fact they were: for copper 1.6–1000 mg / dl in plasma and 0.5–50 mg / l in urine, for zinc 1.5–300 mg / dl both in plasma and urine and for selenium 0.5 –400 mg / l also for both plasma and urine. The lower values of the reported linearity ranges were the detection limits determined as mean62.6 SD of ten determinations of the reagent blanks. Imprecision studies for copper, zinc and selenium are reported in Tables 1–3, respectively. Both within-run and between-days imprecisions were evaluated from replicate determinations on the same digested samples preserved at 48C for between days studies. Total imprecision includes the variability due to different plating and calibration procedures as well as that due to the digestion process; in

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Fig. 1. Linearity of plasma copper method determined by analyzing scalar dilutions of a working standard solutions of 1000 mg / dl. Data reported are the mean of three different determinations, and are expressed both in mg / dl (A) and mmol (B).

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Fig. 2. Linearity of urine copper method determined by analyzing scalar dilutions of a working standard solutions of 50 mg / l. Data reported are the mean of three different determinations, and are expressed both in mg / dl (A) and Gmmol / l (B).

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Fig. 3. Linearity of zinc method determined by analyzing scalar dilutions of a working standard solution of 300 mg / dl. Data reported are the mean of three different determinations, and are expressed both in mg / dl (A) and mmol / l (B).

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Fig. 4. Linearity of selenium method determined by analyzing scalar dilutions of a working standard solution of 400 mg / l. Data reported are the mean of three different determinations, and are expressed both in mg / dl (A) and mmol / l (B).

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Table 1 Imprecision of copper determination by PSA method Target value

Within run a

Between run b

Total imprecision c

(mg/dl)

(mg/dl)

(mg/dl)

(mg/dl)

Mean

SD

CV%

Mean

SD

CV%

Mean

SD

CV%

Plasma pool

–

151

1.8

1.2

154

2.1

1.3

155

4.0

2.6

Urine pool 1

–

43

0.8

2.0

45

1.1

2.5

45

3.2

7.1

Urine pool 2

–

–

–

–

–

–

–

95

3.2

3.4

Randox N

118–145

–

–

–

–

–

–

133

4.2

3.1

Randox E

195–243

–

–

–

–

–

–

232

8.1

3.5

(69011) Level 1

38–57

–

–

–

–

–

–

48

3.5

7.3

(69012) Level 2

53–80

–

–

–

–

–

–

67

4.0

5.9

Lyphocheck urine control

a

Within run imprecision was calculated from 20 consecutive determinations on the same digested sample. b Between days imprecision was evaluated from the data of the same digested sample but determined on 20 different days. c Total imprecision was calculated from the data of the same sample but from 20 independent digestions, each on a different day.

fact it was calculated from results of replicate determinations in different days of the same samples from independent digestion procedures. The imprecision of the proposed methods is satisfactory for clinical purposes and agrees with that reported for AAS methods [9,10,12]. The inaccuracy was evaluated from both recovery experiments and comparing the performance of the candidate methods with that of reference methods using a split sample study. The recovery experiments were performed by analyzing six patients plasma and urine samples before and after the addition of various amounts of each oligoelement. The results of recovery experiments, reported in Table 4, clearly confirm the elevated specificity of the potentiometric technique and demonstrate that in our conditions the contamination or memory effects of TFM vessels used in the digestion procedure are quite absent, and in particular, that selenium is not lost by formation of volatile acids. The split sample studies between the reference methods and the candidate ones were performed on fresh patient plasma samples with values distributed in a wide concentration range assayed over a period of several days. The linear regression plots for copper, zinc and selenium are shown in Figs. 5–7 respectively. The linear regression equations for all oligoelements clearly demonstrate the agreement with the reference methods and the smaller incidence of matrix

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Table 2 Imprecision of zinc determination by PSA method Target value (mg/dl)

Plasma pool no. 1 pool no. 2 Urine pool Seonorm (311089) Randox N (082N) Randox E (107SE) Lyphocheck urine control (44501) Level 1 (44502) Level 2

Within run a (mg/dl)

Between run b (mg/dl)

Total imprecision c (mg/dl)

Mean

SD

CV%

Mean

SD

CV%

Mean

SD

CV%

150

77 180 – 147

1.9 4.0 – 2.6

2.5 2.2 – 1.8

76 182 – 150

2.3 8.9 – 5.5

3.0 4.9 – 3.7

78 185 65 154

4.8 12.3 3.4 8.0

6.1 6.6 5.2 5.2

130–187

182

4.9

2.7

180

7.8

4.3

177

11.4

6.4

163–234

237

6.8

2.9

240

10.3

4.3

241

15.2

6.3

57–86 87–131

56 113

1.5 2.4

2.7 2.1

60 110

2.4 3.8

4.0 3.5

58 109

3.1 5.1

5.3 4.7

a

Within run imprecision was calculated from 20 consecutive determinations on the same digested sample. b Between days imprecision was evaluated from the data of the same digested sample but determined on 20 different days. c Total imprecision was calculated from the data of the same sample but from 20 independent digestions, each on a different day.

effects in our specimens compared with non digested samples which were used in AAS. Furthermore a satisfactory agreement was achieved, for all sera and urine controls employed, between the obtained and the expected values assigned by AAS methods. The matrix effects which are present in the AAS methods, since the sample pretreatment is omitted, could explain the minor deviations found. Copper and selenium concentrations determined in homologous serum and Li-heparinized plasma samples of ten subjects, did not show significant differences as was evident from the Student’s t test for paired data (P.0.3). Preliminary digestion procedure of the samples which requires about 40 min for 10 samples and can be unique for all trace elements determinations, makes possible the elimination of both the matrix effects and the interfering substances which could damage the mercury film, thus avoiding a frequent plating of the working electrode and new calibration. In our conditions 10 samples can be analyzed without a new plating of the working electrode and with the same calibration. The smallest amount of plasma sample that can be digested is 0.25 ml for copper or zinc and 1 ml for selenium. In these cases the whole residue of

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Table 3 Imprecision of selenium determination by CCSA method Within run a (mg / dl)

Target value (mg / dl)

Plasma pool no. 1 pool no. 2 Urine pool 1 Seronorm Lyphocheck urine control Level 1 Level 2

Between run b (mg / dl)

Total imprecision c (mg / dl)

Mean

SD

CV%

Mean

SD

CV%

Mean

SD

CV%

– – – 86

105 – – 86

1.8 – – 1.9

1.7 – – 2.1

104 – – 86

4.5 – – 3.6

4.3 – – 4.1

106 167 86 86

4.1 4.6 5.4 4.3

3.9 2.7 6.2 5.0

55.4–83.2 158–238

88 202

2.0 1.0

2.2 0.5

88 202

3.8 3.5

4.3 1.7

88 203

4.8 4.7

5.5 2.3

a

Within run imprecision was calculated from 20 consecutive determinations on the same digested sample. b Between days imprecision was evaluated from the data of the same digested sample but determined on 20 different days. c Total imprecision was calculated from the data of the same sample but from 20 independent digestions, each on a different day.

the pretreated sample is used for the determinations. Furthermore the sample pretreatment procedure makes the copper, zinc and selenium determinations possible also in cells and tissue homogenates (data not reported). Finally the practicability of the proposed procedure allows the maintenance of Table 4 Results of the recovery experiments a Oligoelement

Copper

Zinc

Selenium

Plasma

Urine

Amount

Amount

Recovery%

Amount

Amount

Recovery%

added

recovered

6SD

added

recovered

6SD

(mg)

(mg)

(mg)

(mg)

50

45

9064.0

50

46

9263.7

100

97

9763.2

100

98

9862.6

200

170

8563.9

200

168

8463.8

50

43

8663.7

50

42

8463.2

100

85

8563.5

100

92

9263.0

200

198

9962.0

200

202

10162.0

50

48

9663.1

50

48

9662.2

100

92

9263.8

100

94

9463.0

200

178

8964.1

200

198

9963.6

a The recovery experiments were performed by analyzing 6 different plasma and urine samples both before and after the addition of the reported amounts of each oligoelement.

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Fig. 5. Correlation between plasma levels of copper obtained with flame atomic absorption spectrometry (reference method) versus potentiometric stripping analysis (candidate method).

a good performance with a short training of technical staff for a proper handling of the instrumentation, samples and reagents. In conclusion, we are confident that the reliability and accuracy of the

Fig. 6. Correlation between plasma levels of zinc obtained with flame atomic absorption spectrometry (reference method) versus potentiometric stripping analysis (candidate method).

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Fig. 7. Correlation between plasma levels of selenium obtained with electrothermal atomic absorption spectrometry (reference method) versus constant current stripping analysis (candidate method).

proposed method, resulting from the reported data, and the low cost of the equipment, could make it suitable for routine purposes in Clinical Chemistry laboratories

4. Abbreviations AAS FAAS EAAS ICP-ES PSA CCSA TFM

Atomic Absorption Spectrometry Flame Atomic Absorption Spectrometry Electrothermal Atomic Absorption Spectrometry Inductively Coupled Plasma Emission Spectroscopy Potentiometric Stripping Analysis Constant Current Stripping Analysis Tetrafluoromethoxyl

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[3] Vallee BL, Falchuk KH. The biochemical basis of zinc physiology. Physiol Rev 1993;73:79– 118. [4] Savory J, Wills MR. Trace metals: essential nutrients or toxins. Clin Chem 1992;38(8):1565– 73. [5] Ostapczuk P. Direct determination of Cadmium and lead in whole blood by potentiometric stripping analysis. Clin Chem 1992;38:1995–2001. [6] Jagner D, Josefson M, Westerlund S, Aren K. Simultaneous determination of cadmium and lead in whole blood and in serum by computerized potentiometric stripping analysis. Anal Chem 1981;53:1406–10. [7] Danielssons L, Jagner D, Josefson M, Westerlund S. Computerized potentiometric stripping of cadmium, lead, copper and zinc in biological materials. Anal Chim Acta 1981;127:147– 56. [8] Hua C, Jagner D, Renman L. Determination of selenium by means of computerized flow constant current stripping at carbon fiber electrodes. Anal Chim Acta 1987;197:257–64, analysis for the determination. [9] Chou PP. Zinc. In: Pesce AJ, Kaplan LA, editors, Methods in clinical chemistry, St. Louis, Washington, DC, Toronto: C.V. Mosby, 1987, pp. 596–602. [10] Alcock NW. Copper. In: Pesce AJ, Kaplan LA, editors, Methods in clinical chemistry, St. Louis, Washington, DC, Toronto: C.V. Mosby, 1987, pp. 527–38. [11] Morisi G, Patriarca M, Menotti A. Improved determination of Selenium in serum by Zeeman atomic absorption spectrometry. Clin Chem 1988;34:127. [12] Shamberger RJ. Selenium. In: Pesce AJ, Kaplan LA, editors, Methods in clinical chemistry, St. Louis, Washington, DC, Toronto: C.V. Mosby, 1987, pp. 543–50.