Determination of Zinc levels in Healthy Adults from the West of Algeria by Differential Pulse Anodic Stripping Voltammetry

a Laboratoire de Chimie Analytique et d’Electrochimie, Département de Chimie, Faculté des Sciences, Université Abou Bekr Belkaid, 13000, Tlemcen, Algeria. b Laboratoire Antibiotiques, Antifongiques, Physico-chimie, Synthèse et Activité biologique, Département de Biologie, Université Abou Bekr Belkaid, Tlemcen 13000, Algeria. * Corresponding author: E-mail address: t_attar@mail.univ-tlemcen.dz att.tarik@gmail.com ABSTRACT


INTRODUCTION
Environmental pollution has given rise to concern about the accumulation of heavy metal in human body [1]. In order to trace the exposure and absorption of such element, biological indicators such as blood, hair, and urine are often analyzed [2]. Zinc is one of the important trace elements related to health and disease. It plays an important role in human growth; it has a recognized action on more than 300 enzymes, by participating in their structure or in their catalytic and regulatory actions [3,4]. It is a structural ion of biological membranes and it's closely related to protein synthesis [5,6]. Zinc deficiency has long been recognized as a role-playing in a number of physiological disorders, including dermatologic conditions such as eczema, acne, and psoriasis [7][8][9][10][11]; poor wound healing [12][13][14]; growth retardation [15][16][17]; delayed sexual maturity [18]; hypogeusia and chronic immunodeficiency [19]. In most cases, abnormally low levels of zinc appear to involve a malfunction of the metal-binding protein metallothionein [20,21]. The choice of a method will depend in particular on nature of the studied medium, amongst elements to be proportioned and their concentrations, amongst analyses to be carried out, of the speed required of the analysis, the precision imposed on proportioning and finally of the equipment which one lays out or which one can lay out. The electrochemical methods have many advantages compared with the spectrometric methods, the equipment being less expensive and measurements being able to be realized in difficult mediums of access [22]. These methods ensure the transformation of a concentration of species into solution into an electric quantity measured by means of simple mathematical relations. Among the electrochemical methods most recent, the stripping voltammetry is a precise, significant and selective method electrochemical [23]. The objective of this work is to study the influence of the principal experimental parameters on the current of stripping, namely the effect of electrolyte support, potential of accumulation, the time of deposit and stirring rate of the solution in order to determine the concentration of the zinc dissolved in whole human blood.

Apparatus
Anodic stripping voltammetry measurements were performed using a commercially available TraceLab 50 trace analysis system. It consists of POL150 polarographic analyzer, MDE150 polarographic stand and TraceMaster 5 software (Radiometer Analytical S.A, France). The three electrodes system consists of a working hanging mercury dropping electrode (HMDE) that was renewed prior to each measurement, a platinum auxiliary electrode and Ag|AgCl saturated with KCl as reference electrode.

Reagents
All chemicals used were of analytical-reagent grade or the highest purity available. Aqueous solutions were prepared by dissolving a certain amount of chemicals into high-purity deionized (DI) water (MilliQ water system). Acids used for the analysis, the nitric acid (69.5%, Fluka) and the perchloric acid (70-72%, Merck). Stock solutions of zinc (1000 ppm, atomic adsorption standard, Aldrich), were diluted daily as required.

Sample digestion
A 1.0 ml of whole blood in a long-necked 50 ml flask, 4.0 ml of acid mixture (3 ml HNO3 and 1 ml HClO4) was added in flask. The temperature is maintained with 150°C during 4 hours and then the temperature is fixed at 200°C until quasi total evaporation of the contents [24]. After cooling, we add the same mixture of acids to the residue, then we let evaporate until obtaining a dry residue [25]. This residue is taken again by 5 ml of nitric acid with 0.25% and preserved in polyethylene tubes [26,27]. In this case, the mixtures of strong acids used allow, in combination with high temperatures, to destroy the organic matter and to transform it into by-products of gas reaction, which are then eliminated under the hood. It is in fact necessary, to reduce the contaminations, to use quantities as weak as possible reagents.

Voltammetric Measurement
A 10 mL sample solution, containing of perchloric acid was transferred to the voltammetric cell and purged with nitrogen for 5 min. The accumulation potential (-1150 mV) was applied to a fresh mercury drop for 60 s while the solution was stirred. Following the accumulation period, the stirring was stopped, and after 20 s the second-order derivative linear scan voltammogram was recorded by applying a positive-going potential scan at 20 mV s -1 . The concentration of zinc in the samples was determined using standard addition method. Reagent blanks were taken along with each batch of samples and the metal concentration observed in these blank samples were subtracted from the corresponding batch of field samples.

Effect of concentration of electrolyte support
In a first step, the effect of concentration supporting electrolyte was investigated. Fig. 1 shows the influence of the concentration HClO4. The effect of electrolyte support on peak current was studied with concentration in the range from 0.01 to 0.1M. A decrease in the current is observed when the concentration electrolyte support is higher than 0.02M.

Effect of deposition potential
In the stripping procedure, the determination of the elements is based on the use of a deposition potential. The optimum deposition potential must be obtained. For this purpose, the deposition was carried out on the HMDE for 60 s at several selected deposition potentials. The relation between the peak current and the deposition potential is shown in Fig.  2. The effect of the accumulation potential on the peak current was examined over the range from -1200 to -1050 mV. The peak current increased gradually with changing potential from -1200 to -1150 mV, reached its maximum at -1150 mV and decreased at more positive potentials. Therefore, an accumulation potential of -1150 mV was used in all experiments.

Effect of accumulation time
The accumulation time is always important factor in stripping voltammetric analysis because of its prevailing influence on sensitivity and detection limit of the method [28]. The effect of accumulation time on the zinc peak current was studied in the 0 -75 s range in solutions containing 70 µg L -1 of Zn, as illustrated in Fig. 3. It is seen that the peak current of the zinc increases linearly as accumulation time increases. At longer times the peak current decreased notoriously became almost constant, probably due to saturation in the surface of the mercury electrode.

Effect of stirring rate
Stirring intensity is one of the important parameters that affect the accumulation efficiency. Agitation of the sample solution facilitated the mass transfer process. In present work, the samples were agitated at various stirring rates (150, 225, 300, 400 and 525 rpm). The result showed that the current intensity efficiency was first improved with increased agitation rate before 400 rpm and then decreases starting after this agitation rate (Fig.4). Hence, a stirring rate of 400 rpm was chosen for further studies.

Calibration data
For the evaluation of the analytical parameters (the recovery percentage, standard deviation, accuracy and precision), a study of the influence of the concentration of the zinc was made in aqueous solution under the optimal conditions mentioned above. It is observed that the precision is 3.96%. This is strong evidence that this method is precise and reproducible. Furthermore, the percentage recovery of the zinc determination was tested by measurement of a standard reference material, and by comparison of results obtained by ASV with those obtained by inductively coupled plasma sector field mass spectrometer (ICP-SFMS) for the same set of samples, seronorm (level 3,O512627) was used as the standard whole blood certified reference materials with the recommended concentration of zinc is 97.75% which is an indication that this method is accurate for zinc is 97.42%. A linear response over the concentration range of 2.87 10 -3 to 1625 µg L -1 zinc was observed under optimum conditions, with correlation coefficient of 0.9999. The detection limit for zinc was found to be 0.86 µg L -1 estimated from 10 replicate determination of blank solution. This limit is better than coupling ICP-MS [29].  (Table 1). No significant differences were observed P < 0.001 in blood zinc concentrations among women and men after applying to them the Student's t-test. D e c e m b e r 1 5 , 2 0 1 3 The level concentration of zinc that we found in this study (mean 6700.313 µg L -1 ) that we found is very much in line with the values for an Italian population 6597 µg L -1 [30], Czech Republic population 6842 µg L -1 [31] and Chinese population 6399 µg L -1 [32]. As an application the figure below represents a voltammograms relating to the proportioning of zinc in the blood starting from the conditions which were optimized previously (Fig.5). The zinc concentration of this sample is equal 7169.263 µg L -1 .

Conclusion
The optimized method has been successfully applied to the determination of zinc in whole blood with good accuracy and precision. The proposed method is inexpensive and fast. The detection limit of 0.86 µg L -1 can be lowered further by increasing accumulation time. Acceptable agreement was found between the results obtained and the values of certified reference material. Moreover, and in the lack of bench-mark data regarding the standard rates of the blood of the trace elements in a healthy Maghrebian population, this work constitutes a first contribution to the establishment of a specific database to the town of Tlemcen. D e c e m b e r 1 5 , 2 0 1 3