1080 Journal of the Chinese Chemical Society, 2008, 55, 1080-1085 Simultaneous Determination of Cd(II), Cu(II), Pb(II) and Zn(II) in Human Plasma by Potentiometric Stripping Analysis Shu-Jie Wang a ( ), Hao Zheng b ( ) and Bao-Xian Ye a, *( ) a Department of Chemistry, Zhengzhou University, Zhengzhou 450052, P. R. China b Zhengzhou Environmental Monitoring Centre, Zhengzhou 450007, P. R. China In this paper, Potentiometric Stripping Analysis (PSA) was simultaneously used to determine the concentrations of trace metals (Zn, Cd, Pb and Cu) in human plasma. The metal ions were concentrated as their amalgams on the glassy carbon surface of a working electrode that was previously coated with a thin mercury film and then stripped by a suitable oxidant. The selection of the experimental conditions was made by using the experimental-designed methodology. The optimum conditions of the method includes a 0.2 M HAc-NaAc buffer mixture (ph 4.5) as supporting electrolyte, and an electrolysis potential of -1220 mv. The limits of detection (LOD) were obtained 1 gl -1 for Zn(II) and Pb(II), 0.5 gl -1 for Cu(II) and 2 g L -1 for Cd(II) in the studied medium. The good recoveries were obtained for the analysis in human plasma. The method was applied to blood samples, using the method of standard additions and the results were compared with Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) as reference method. Furthermore, a simple digestion protocol of samples is investigated compared to the conventional digestion method. Keywords: Potentiometric stripping analysis; Inductively Coupled Plasma-Atomic Emission Spectrometry; Sample preparation. INTRODUCTION For metal ion analysis, electrochemical techniques are potentially the cheapest and quickest methods for carrying out the determination, that is, when they are compared with other instrument techniques such as atomic absorption spectroscopy and inductively coupled plasma, etc. 1 In recent years, this method has been used in a variety of matrices such as tap water, sediment sludge, etc. The instrumentation of Potentiometric Stripping Analysis (PSA) is small and does not need any servicings or special installation requirements. PSA is similar to anodic stripping voltammetry (ASV) in the first step, but differs from ASV in the stripping step, because the deposited metal ions are chemically oxidized and the potential vs. time behaviour at the working electrode is measured. In this paper, derivative potentiometric stripping analysis (dpsa) was utilized for simultaneous determination of Zn(II), Cd(II), Pb(II) and Cu(II) in human blood sample. In the dpsa, potential and time data are digitally converted into dt/de and E is plotted against dt/de, thus both the sensitivity of the method and resolution of the analysis are increased. 2-4 Electroanalytical determination of trace and ultratrace concentrations of Zn(II), Cu(II), Pb(II) and Cd(II) have traditionally been performed by stripping techiques on mercury or other solid electrodes. 5-8 Of all the trace element, lead and cadmium are important to consider because of their toxicity. 9 Lead and cadmium cause both acute and chronic poisoning, adverse effects on kidney, liver, heart, vascular and immune system. Moreover, lead and cadmium exposure cause chromosome aberration, cancer and birth defects. 10,11 However the micronutrients as copper and zinc, are essential for human nutrition at low doses but may also be toxic for humans at high doses. 12 As bioessential elements, 8,13,14 they participates in many biological functions such as components of enzymatic and redox systems induces both lipid peroxidation of lipid membrane and the peroxidationoffattyacidinoils,leadingtoarapidformation of undesirable products that negatively influence the quality of oil, particularly its organoleptic properties. It is known that a deviation of copper or zinc level from normal values in the human body causes serious effects on health. Therefore, the simultaneous determination of these toxic * Corresponding author. Fax: +86-03-71-67763220; E-mail: yebx@zzu.edu.cn
Simultaneous Determination of Cd(II), Cu(II), Pb(II) and Zn(II) J. Chin. Chem. Soc., Vol. 55, No. 5, 2008 1081 (cadmium, lead) and available essential (zinc, copper) elements in human plasma samples has important significance in clinical analysis. Sample preparation is a critical step in the whole analytical procedure for the determination of heavy metals. In reported literatures, the human plasma sample preparation must be done by the conventional digestion method, which involves in tedious and cumbersome procedures by strong mixed acids under high temperature. 15,16 In our study, we have found a pretreating human plasma sample method by simply using mixed diluent acid under normal temperature. This digestion method is feasible and convenient compared to the conventional digestion method and is very suitable for the simultaneous determination of trace Zn, Cu, Cd, and Pb, by potentiometric stripping analysis. EXPERIMENTAL SECTION Apparatus and Instrumentation Potentiometric analyses were carried out by using a home-made IVY200 potentiometric stripping analyzer, which is connected to a PHILIPS compatible personal computer. A 10 ml cell was used with a three-electrode system: the working electrode is a glassy carbon disk electrode (GCE) coated with thin mercury film; the reference electrode is a saturated calomel electrode (SCE), and a platinum wire electrode is used as counter electrode. The ph of the solution was measured with a PHS-2C Digital ph meter (Shanghai Lida, China) equipped with a combined glass electrode, which was calibrated regularly with standard buffer solutions (ph 4.00 and 6.86) at 25 0.1 C. Reagents and Solutions Mercury plating solution (3 mm Hg 2+ ) was prepared from 0.01 g ultra-pure Hg(NO 3 ) 0.5H 2 O (Shanghai Reagent Corporation, China), 2.53 g KNO 3 and 0.063 ml 65% HNO 3 (Luoyang Reagent Corporation, China) dissolving to final volume of 100 ml with redistilled water. Zn(II), Pb(II) and Cd(II) standard stock solutions (1 g L -1 ) was prepared by dissolving zinc granule, lead powder and cadmium powder (Beijing Chemical Plant, China) in 100 ml of dilute acid. Cu(II) standard stock solution (1 g L -1 ) was prepared by dissolving an appropriate weight of CuSO 4 5H 2 O (Beijing Chemical Plant, China) in 100 ml of redistilled water, and working solutions at desired concentration was prepared by diluting the stock solution with doubly-distilled water. Other commercial reagents (65% HNO 3, HAc and NaAc) used were of the ultra-pure grade and the doublydistilled water was used for the preparation for solutions. Preparation of Pre-Plating Mercury Film Prior to each new experiment, a fresh mercury film was plated onto the glassy carbon surface of the working electrode. The rotating glassy-carbon electrode was polished with 0.05 m alumina oxide on polishing pad in routine experiments and then rinsed with redistilled water. Subsequently, in a 10 ml cell, 5 ml mercury plating solution was injected into and then the three-electrode were inserted. The mercury plating of the working electrode was then executed. The optimal plating mercury conditions were investigated. For used pre-plating mercury film in this experiments, pre-plating four times with 60s every time at -1.0 V were the best conditions. Under this condition, a satisfactory and homogeneous mercury film was plated on the surface of the working electrode. Analytical Procedure Pre-treating of samples Plasma samples obtained from healthy volunteers were stored under 5 C.1.0mLofplasmawasaddedto5 ml mixed diluent acid digestive solution (DADS, selfpreparation) in a 10 ml test tube and then wobbled about 1 min. The vessel was placed in ultrasonic for 5 min, and then hold centrifugal separation for 1 min. The upper clear solution can be used for direct determination of zinc, copper, lead and cadmium. Derivative potentiometric stripping analysis Before each analysis, the plating of the working electrode was executed as procedure that preparation of preplating mercury film. The concentrations of Cd(II), Cu(II), Pb(II), and Zn(II) were simultaneously determined in 5 ml 0.2 M HAc-NaAc buffer mixture (ph 4.5) containing 0.25 ml of 10 g ml -1 Ga(III), which was used to prevent Cu- Zn complex formation on the mercury film. 8 The stirred electrolysis was executed at -1220 mv for 120s. After the rest time of 20s, the stripping step was performance by the dissolved oxygen as oxidant to dissolve metals deposed on Hg electrode. 17 The Zn(II), Cd(II), Pb(II) and Cu(II) stripping peaks were respectively registered at -990 mv, -640 mv, -480 mv, -120 mv. The quantitative analysis was executed by the multiple point standard additions method. RESULTS AND DISCUSSION Optimization of experimental conditions Supporting electrolytes Various supportive electrolytes, such as NH 4 Cl-HCl,
1082 J. Chin. Chem. Soc., Vol. 55, No. 5, 2008 Wang et al. NH 2 CH 2 CH 2 NH 2 -HCl, HCl, PBS, H 3 PO 4, HAc-NaAc were tested. The results show that the best stripping peak response of Zn(II), Cd(II), Pb(II) and Cu(II) is in HAc-NaAc solution. When the measurements were performed in this buffer solution, the largest stripping peaks and the good reproducibility were obtained. The concentration of electrolyte has no obvious effect on the stripping peak and shape by testing electrolyte concentration in range of 0.1 M ~ 0.4 M. Hence, we selected 0.2 M HAc-NaAc (ph = 4.5) as the supporting electrolyte for the bigger buffer capability and little blank. The effect of solution ph In this paper, we must consider that the stripping behavior of each element is determined simultaneously in the same supporting electrolyte. In our experiments, we found that the lower the ph, the better for determining of Cd(II), Pb(II) and Cu(II), but the stripping peak of Zn(II) disappeared at ph 2.0. Furthermore, considering the change of the solution ph after adding the pretreated samples, large range of ph was tested. For selecting the solution ph, the stripping sensitivity of each element and the buffer capability were in consideration. Then, we found that the best results were obtained in the solution of ph 4.5. Suitable oxidant The stripping step is controlled by oxidant in the potentiometric stripping analysis, and so the selection of suitable oxidant looks very important. There are some requirements for an oxidant: (i) the medium oxidation competence; (ii) no interference between the reduced matters; (iii) the lesser background; (iv) the less mucosity. 18 In this works, with respect to the convenience without deoxygenize, the dissolved oxygen was elected as the oxidant in the stripping step. The stir of solution In the electrodeposit process, stirring the solution can improve the accumulation efficiency consumedly. For the sake of no-impurity were introduced into the solution, and good reproducibility were obtained, rotating electrode instead of stirring with a muddler was utilized. The rotary speed of the working electrode is controlled by inner software and fixed at 2000 rpm. This speed is the condign velocity in potentiometric stripping analysis for getting higher repeatability. 18 Mechanism of the electrode reaction The above information, especially the well-defined peaks, suggests that the surface process at the mercury film electrode could be exploited systematically by derivative potentiometric stripping analysis with some advantages in sensitivity. Accordingly, the connected experiment was performed and the derivative potentiometric stripping plot isshowninfig.1. The electrode mechanism reaction of the method probably involves two steps. In the first step, the M 2+ is reduced to M 0 at closed circuit, forming metal amalgam on the electrode surface by the application of a sufficiently negative potential. In the second step, the reduced M 0 is oxidized to M 2+ by dissolved oxygen. The recognition mechanism of metal ions can be described as following: Step 1: M 2+ +ne+hg M(Hg) Step 2: M(Hg) + 4 mh + +mo 2 M 2+ +Hg+2mH 2 O Electrochemical parameters The optimized electrochemical parameters ensure the maximum analytical sensitivity for simultaneous determination of Zn(II), Cd(II), Pb(II), and Cu(II) in human plasma are given in Table 1. In this condition, the peak potentials of 10 gl -1 Zn(II), Cd(II), Pb(II) and Cu(II), were observed at 990, 640, 480 and 120 mv (Fig. 1) respectively. Calibration equations and detection limits In optimal condition, a good linearity was obtained in the ranges of 2~650 g L -1 for Zn(II), 8~500 g L -1 for Cd(II), 4~750 g L -1 for Pb(II), and 4~220 g L -1 for Cu(II), respectively. The calibration equations and linear relation coefficients are: Fig. 1. Simultaneous determination of 10 gl -1 Zn(II), Cd(II), Pb(II), and Cu(II) in 0.2 M HAc-NaAc (ph = 4.5) using dpsa. The electrochemical conditions are given in Table 1.
Simultaneous Determination of Cd(II), Cu(II), Pb(II) and Zn(II) J. Chin. Chem. Soc., Vol. 55, No. 5, 2008 1083 Table 1. Optimised analytical conditions for the simultaneous determination of Zn(II), Cd(II), Pb(II), and Cu(II) in human plasma Parameters Zn Cd Pb Cu Deposition potential (mv) -1220-1220 -1220-1220 Stripping Potential range (mv) -1220~0-1220~0-1220~0-1220~0 Deposition time (s) 120 120 120 120 Peak potential (mv) 990 640 480 120 Cleanout potential (mv) -100-100 -100-100 Cleanout time (s) 30 30 30 30 dt de -1 = 1.535C Zn(II) + 53.13 (2~650 gl -1 ) R = 0.999 dt de -1 = 1.796C Cd(II) + 218.3 (8~500 gl -1 ) R = 0.998 dt de -1 = 1.510C Pb(II) + 352.1 (4~750 gl -1 ) R = 0.997 dt de -1 = 62.75C Cu(II) + 1540 (4~220 gl -1 ) R = 0.998 A key feature of any analytical method is its detection limit. For the method described here, the detection limits have been calculated by dividing three times the standard deviation of the peak value of the blank by the slope of the analytical curve. 19 From these values, detection limit of 1 g L -1 for Zn(II) and 0.5 g L -1 for Cu(II), 2 g L -1 for Cd(II) and 1 g L -1 Pb(II) were found. The relative standard deviations were 2.3% for zinc, 2.9% for cadmium, 0.5% for lead, and 3.1% for copper (five repetition), respectively. Application to real samples In order to validate the feasibility of the proposed method in practical analytical applications, three healthy volunteers plasma samples were used for simultaneous determination of Zn(II), Cd(II), Pb(II) and Cu(II). The recovery experiments were performed by analyzing each plasma sample before and after the addition of relevant metal standard solutions. Then the two shares samples were pretreated simultaneously as mentioned in procedure of pre-treating of samples. Only 20 L pretreated samples were required by this analysis and were intruded into 5 ml electrolytes. The determination results are listed in Table 2. In order to assess the accuracy of the method described and ensure that analytes loss or sample contamination did not occur during the chronopotentiometric analysis, recovery tests were performed. The recovery data generated from the samples was summarized in Table 3. Furthermore, the results obtained by proposed method were compared with that by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) to validate the accuracy of this method. The Table 2. Concentrations of Cu(II), Pb(II), Cd(II), and Zn(II) found in human plasma samples by dpsa and ICP-AES Samples Zn Cd Pb Cu 1 Detected ( g ml -1 ) 5.42 ND 0.21 0.91 ICP-AES ( g ml -1 ) 5.20 0.23 0.88 2 Detected ( g ml -1 ) 7.10 ND 0.14 0.72 ICP-AES ( g ml -1 ) 6.97 0.15 0.70 3 Detected ( g ml -1 ) 6.23 ND 0.30 1.31 ICP-AES ( g ml -1 ) 6.41 0.28 1.34 4 Detected ( g ml -1 ) 10.020 ND 0.17 2.01 ICP-AES ( g ml -1 ) 10.330 0.18 1.95 ND: none detected. Each value was the mean of five determinations. Table 3. Recovery data of Cu(II), Pb(II), Cd(II), and Zn(II) in human plasma samples Samples Zn Cd Pb Cu 1 Expected ( g ml -1 ) 10.42 5.00 0.71 1.41 Found ( g ml -1 ) 9.56 5.21 0.69 1.49 Recovery (%) 92 104 97 105 2 Expected ( g ml -1 ) 12.10 5.00 0.64 1.22 Found ( g ml -1 ) 12.65 5.36 0.66 1.31 Recovery (%) 104 107 103 107 3 Expected ( g ml -1 ) 11.23 5.00 0.80 1.81 Found ( g ml -1 ) 10.96 5.12 0.85 1.90 Recovery (%) 98 102 106 105 4 Expected ( g ml -1 ) 15.02 5.00 0.67 2.51 Found ( g ml -1 ) 15.52 5.09 0.71 2.44 Recovery (%) 103 102 105 97 comparison is illustrated in Table 2 as well. The excellent average recoveries and the good matching with the results of ICP-AES suggest that the proposed method holds the promise for human plasma samples. CONCLUSIONS Derivative stripping potentiometry is an attractive alternative to atomic absorption spectroscopy and atomic
1084 J. Chin. Chem. Soc., Vol. 55, No. 5, 2008 Wang et al. Fig. 2. (a) Calibration curve of zinc in ph 4.5 HAc-NaAc buffer in the concentration range of 2~650 gl -1. (b) Calibration curve of cadmium in ph 4.5 HAc-NaAc buffer in the concentration range of 8~500 g L -1. (c) Calibration curve of lead in ph 4.5 HAc-NaAc buffer in the concentration range of 4~750 gl -1. (d) Calibration curve of copper in ph 4.5 HAc-NaAc buffer in the concentration range of 4~220 gl -1. emission spectroscopy for the detection of Zn(II), Cd(II), Pb(II) and Cu(II) concentrations in biological samples. The proposed method based on derivative potentiometric stripping analysis provides a rapid, sensitive and reliable procedure to simultaneously detect the trace levels of Zn, Cd, Pb, and Cu in human plasma samples. Sample preparation played an important role in ensuring the accuracy of determination because trace metals were often present in low concentrations in a complex matrix. Herein, we have found a simple and feasible pretreatment of human plasma samples compared to regular digestion of blood samples. The results of Zn, Cd, Pb, and Cu determination using the pro-
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