- Email: [email protected]
temperature-controlled microwave mineralization
ANALYTIcA CHIMICA
ACIYA
ELSEVIER
Analytica
Chimica Acta 317 (1995) 311-318
Anodic stripping voltammetric determination of total lead in anencephalic fetuses after pressure/temperature-controlled microwave mineralization Jorge E. Tahb, Lorenia Marcano I, Romer A. Romero Laboratorio
*
de Instrumentacich Analitica, Departamento de Quimica, Facultad Experimental de Ciencias, La Uniuersidad de1 Zulia, Apartado Postal 15202, Las Delicias, Maracaibo 4003-A, Venezuela Received 8 May 1995; revised 13 July 1995; accepted
13 August 1995
Abstract The development of a closed-vessel mineralization method for the decomposition of brain, liver, kidney and lung specimens of anencephalic (A) fetuses and controls (C) from the eastern coast of lake Maracaibo, Venezuela, is presented. Digestion was done in a laboratory microwave oven provided with pressure sensing tube and fiberoptic temperature probe to monitor and control pressure and temperature conditions inside the lined digestion vessels. Total lead was subsequently determined by differential pulse anodic stripping voltammetry (DPASV) with a hanging mercury drop electrode. The optimized conditions for maximal pressure and temperature set up were 1260 Wa and 190°C. Three samples and one blank were routinely prepared for simultaneous digestion. After sample mineralization, the lead oxidation peak appeared at a potential of -0.45 V vs. Ag/AgCl, pH 4.70. Lead concentrations obtained by DPASV analysis of the mineralized biological materials were compared with those provided by electrothermal atomization atomic absorption spectrometry (ETA-AAS) on the same digestion samples. The correlation between the two methods was excellent: y = 1.142x - 0.0035, r = 0.9999, n = 40, p < 0.001, where y and x were the lead concentrations determined by DPASV and ETA-AAS, respectively. For the DPASV determination of total lead, precision (R.S.D.) was better than 3.8%, for within- and between-run analyses. The detection limit of the electrochemical method, defined as three times the standard deviation of a blank solution, was 0.03 pg Pb g-’ (in solid sample), equivalent to 0.1 pg Pb 1-r in the diluted test portions. The dry-weight metal concentrations (k 1 SD., Kg g-‘) found in brain, liver, kidney and lung were as follows: (brain, undetectable in A and in C; liver, 2.1 + 1.1 in A, 0.5 f 0.2 in C; right lung, 1.1 ? 0.8 in A, 0.6 + 0.1 in C; left lung, 0.6 _t 0.2 in A, 0.7 + 0.1 in C; right kidney, 1.4 + 0.7 in A, 1.5 f 0.03 in C and left kidney, 1.7 + 0.9 in A, 0.7 +_0.2 in C. The proposed DPASV method constitutes an analytical alternative, as reliable as ETA-AAS, for the voltammetric determination of total lead in solid samples. Keywords:
Total lead; Stripping
* Corresponding author. ’ Present address: Departamento
voltammetry;
Anencephaly;
de Anatomia
Patokjgica,
Pressure/temperature-controlled
Hospital
microwave
mineralization
‘Pedro Garcia Clara’, Ciudad Ojeda, Venezuela.
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOOO3-2670(95)00422-X
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J.E. Taha’n et al./Analytica
1. Introduction Anencephaly is a life-incompatible congenital malformation characterized by deficient development of the brain organ in the fetus. Epidemiological studies have indicated that Jamaica has the lowest rate of anencephaly with an incidence of 0.26 per 1000 total births 111 while the highest rate of 8.6/1000 has been found in Belfast [2]. In Venezuela, a relatively high anencephaly rate of 5.1 was reported in 1994 for the eastern coast of lake Maracaibo, area located in the north-western part of the country [3]. This situation has been associated with the presence of lead (Pb) in the environment [4]. Lead-induced teratogenicity [5] and neurotoxicity [6] have been experimentally demonstrated. Automobile combustion of tetraethyl lead-containing gasolines still represents the main source of lead pollution in Venezuela (ca. > 90%) [7]. Our studies have shown that the lead concentration in the Venezuelan gasolines ranges between 700 and 800 mg 1-l (depending on the octane number) [8]. These values are one order of magnitude higher than the lead levels allowed in most European countries and in the United States. To assess the extent of the possible harmful effect played by lead in anencephaly, data are required on the total metal concentrations in body tissues and organs (e.g., brain, liver, kidney and lung). The role of lead in anencephaly is worth studying using reliable analytical methods of measurement. Differential-pulse anodic stripping voltammetry (DPASV) has been recently used for the determination of lead in environmental [9,10], geological [ll] and food samples [12]. The extremely high sensitivity of this technique is due to its effective in situ preconcentration step [ 121. However, the direct DPASV determination of total metal in biological materials is interfered with by the presence of organic matter [13,14]; this may bind lead, modifying the metal speciation and, therefore, its availability. Hence, the stripping procedures always require a preliminary destruction of organic matter to free the analyte metal from organic ligands and to produce a single labile metal species that can subsequently be determined by DPASV. With this approach, the lead determined by DPASV in mineralized brain, liver, kidney and/or lung, or any other metal electrochem-
Chimica Acta 317 (1995) 311-318
ically measured in these materials or in any sample matrix regardless of its complexity, will produce quantitative data statistically similar to those obtained by using techniques such as electrothermal atomization atomic absorption spectrometry (ETAAAS), frequently employed for the purpose of total metal determination. Otherwise, serious underestimations of the metal will be obtained [13-151. In previous papers, Tahan and Romero [14] reported the DPASV determination of total soluble copper in haemodialysis water with a high organic content [15] and in blood plasma after high-pressure mineralization. The same group [13] also described the determination of total soluble aluminum in haemodialysis water with a high organic matter-content, decomposed by microwave heating and posteriorly analysed by differential-pulse polarography. In this paper we present the development of a closed-vessel mineralization method for the decomposition of brain, liver, kidney and lung specimens from anencephalic fetuses and controls; pressure and temperature inside the digestion vessels were controlled and monitored. This procedure was tested for its suitability to permit the posterior determination of total lead by DPASV with the hanging mercury drop electrode (HMDE). Lead was also determined by an ETA-AAS-based method [16] for accuracy evaluation purposes.
2. Experimental 2.1. Apparatus The DPASV measurements were done using a Model 273A potentiostat/galvanostat system (Princeton Applied Research (PAR)) in conjunction with a PAR Model 303A SMDE electrode assembly and a Hewlett-Packard ColorPro Graphics Plotter, being the whole system controlled from a computer based on the Intel 80386 microprocessor running the 5.0 MS-DOS operating system (IBM Personal System/2 Model 40 SX> and the PAR Head Start Creative Electrochemistry Software. A large drop size was used with the static mercury drop electrode device throughout this work. During the deposition step, samples were stirred by means of a PAR Model
J.E. Tahrin et al. /Analytica
305 stirrer. DPASV determinations were done at 20°C under oxygen-free nitrogen. A Perkin-Elmer Model 2380 atomic absorption spectrometer with a Perkin-Elmer Model HGA-500 graphite furnace atomizer and a deuterium arc background corrector were used for the ETA-AAS determination of total lead in the mineralized samples, according to the method reported by Granadillo and Romero [16]. Biological materials were freeze-dried in a Labconco Model 6 liter lyophilizer, kept at -52°C for 48 h. Mineralizations were performed in a CEM Model MDS-2100 laboratory microwave oven (950 W for 100% power) provided with pressure sensing tube and fiberoptic temperature probe to monitor and control pressure and temperature conditions inside the lined digestion vessels. This type of vessel consists of a lOO-ml PTFE PFA (a tetrafluoroethylene with a fully fluorinated alkoxy side chain) inner linner and a PTFE PFA cover; the casing and cap of the lined digestion vessel are made from microwave-transparent polymer (polytherimide). The collection vessel placed in the center of the turntable collects, condenses and vents vapors released when the rupture membrane breaks down. In this system, pressure tubing attached to a sample vessel (control vessel) is routed outside the microwave cavity through one of the inlet/outlet ports; pressure is sensed by a transducer and displayed graphically and digitally on the display screen of the oven. The microwave transparent fiberoptic temperature probe is inserted into the thermowell of the control vessel and exits the microwave cavity via a bulkhead connector and leads to a special temperature control PC board mounted on the system CPU board; the temperature sensor is a phosphor located at the tip of the probe. 2.2. Reagents and samples All chemicals used were of analytical reagent grade. The lead stock solution (ca. 1000 mg 1-l > was prepared from a Titrisol concentrate (Merck). The lead working standard solutions were freshly prepared by serial dilution of the stock with grade I (as established by the American Society for Testing and Materials, ASTM, ca. electrical resistivity > 16.6 MR cm-’ at 25°C) [17] triply-distilled and deionized water; nitric acid was purchased from Riedel-de
Chimica Acta 317 (1995) 311-318
313
I-&en, and had a metal content below the ETA-AAS detection limit (0.1 pg Pb 1-l) 1161. The supporting electrolyte was 6 M acetate buffer (pH 4.701, prepared by mixing acetic acid (Riedel-de Hfien) and sodium hydroxide (Merck). Concentrated perchloric acid (Merck) was used for the microwave mineralization. For accuracy evaluation, the following standard reference material was used: Sargasso (NIES no. 9) from the National Institute for Environmental Studies (Ibaraki, Japan). This material was mineralized as stated below to provide aqueous solutions whose final concentration was within the range of the lead concentrations expected in the biological materials analysed. Complete organs (brain or brain-like tissue in anencephalics, kidney, liver and lung) were obtained by autopsy from both anencephalic and control fetuses; it should be pointed out that anencephalic brain consisted of a cerebrovascular mass covered by a translucent membrane. The autopsies were performed at the Department of Pathological Anatomy of ‘Pedro Garcia Clara’ Hospital by qualified pathologists who were told about the risk of metal contamination from dust, surgical glove powder, surgical instruments, etc. Organs were excised from every subject with plastic knifes, trimmed of fat, gristle, sheath and connective tissue, rinsed free of blood with grade I ASTM triply distilled and deionized water, blotted with pieces of deionized filter paper and placed in metal-free polyethylene bags as soon as possible and then quickly frozen. Once in the laboratory, whole organs were thawed, blended, lyophilized, ground, mixed and kept in polyethylene bags at 4°C until analysis. Special care was exercised to avoid contamination during the handling of samples for preparation for analysis. In this sense, sample manipulation, solution preparation and instrumental measurements were conducted in a clean room with Class-100 work area. The clinical protocol was approved by an ad hoc medical committee of the hospital. 2.3. Closed-Llessel microwave logical samples
mineralization
of bio-
Approximately 150 mg of lyophilized material (e.g., brain, liver, kidney, lung, standard reference material) and 8 ml of concentrated perchloric acid
314
J.E. Tahrin et al. /Analytica
were placed into the vessel liner and capped with a cover that employs pressure assisted type seal for leak-free operation (e.g., as internal vapor pressure rises from microwave heating of the liquid, the seal is energized, forming a progressively tighter seal with increasing pressure) [181. The capped liner was put into the microwave-transparent body of the lined digestion vessel and closed by tightening the cap. Three samples and one blank were routinely prepared for simultaneous digestion. The four vessels (including the control vessel which always contained the largest lyophilized test portion to mineralize) were placed on the turntable, inserted in the oven, and irradiated for 16 min (at 100% power, equivalent to 950 W and 2450 MHz); the optimized conditions for maximal pressure and temperature were 1260 kPa and 190°C. The turntable was rotated continuously. After cooling to ambient temperature, the caps were removed and contents were transferred quantitatively into 50-ml polypropylene calibrated flasks and diluted to volume with 6 M acetate buffer to reach pH 4.70. Blanks were prepared with the same reagent, without the samples, undergoing a similar high-pressure closed-vessel decomposition treatment by microwave heating. Aliquots (ca. 8-10 ml) were transferred into the SMDE sample cells kept at 20°C degassed with oxygen-free nitrogen for 2 min and analysed voltammetrically. 2.4. Statistical analysis Statistical comparisons of data obtained sequentially by DPASV and ETA-AAS were done by repeated measurements of analysis of variance [19]; significant F values were tested with Tukey test [20] with the help of a commercial statistical package (Statgraphics, STSC, Rockville, MD); values of p < 0.05 were considered significant.
3. Results and discussion Initially, the deposition potential, deposition time, potential scan rate, drop size and nitrogen purge time were optimized with synthetic lead samples with the following results: -0.70 V vs. Ag/AgCl, 2 min with stirring (plus 40 s quiescent), 5 mV s-l, large (equivalent to ca. 2.5 mm*) and 2 min with stirring,
Chimica Acta 317 (1995) 311-318
respectively. These conditions were applied in the DPASV analysis of real samples. In the determination of total metals in biological materials, the transformation of the solid sample into solution needs careful consideration in order to avoid serious underestimation of the analyte [7,21]. In this sense, the presence of organic matter has been reported to be a source of interference for the quantitative determination of metal elements by voltammetric means with the SMDE electrode assembly [13151. Therefore, samples were pre-treated by using microwave-assisted pressurized acid mineralization, carried out in closed vessels whose inside pressure and temperature conditions were controlled and monitored from the oven microcomputer board. The aim of the digestion procedure was to remove concomitant substances and produce digestion solutions suitable for the subsequent evaluation of total lead by DPASV. It should be pointed out that interference studies on common ions (sodium, potassium, calcium, magnesium, iron, copper, zinc, cadmium, chloride, acetate, sulphate, etc.) did not have any effect on the electrochemical determination of lead. The results obtained electrochemically for total lead in mineralized samples were verified by ETAAAS evaluation of the same digestion samples. Initial microwave mineralizations were done with the addition of 15 ml of concentrated nitric acid to different test portion weights (ca. 100, 150, 200 and 300 mg), using several mineralization times (ca. 5, 10, 20, 40 and 60 min); maximal pressure and temperature inside the digestion vessels were set up at 1260 kPa and 190°C as reported already [18]. In pressure/temperature-controlled microwave mineralization experiments, optimum mineralization conditions are obtained once pressure and temperature reach maximal values; in general, the faster in reaching maximal values the better the oxidizing agent [18]. For nitric acid mineralization, one control vessel, three sample vessels and two blank vessels were employed for simultaneous digestion. The initial results were unsatisfactory; not more than 50% of the total lead content could be quantified by DPASV, at an oxidation potential of - 0.45 V vs. Ag/AgCl, pH 4.70, and the rest was interfered by the presence of a more cathodic peak signal appearing at - 0.53 V vs. Ag/AgCl (Fig. la). It was demonstrated that this latter voltammetric peak was not the result of the
J.E. Tahbn et al./Analytica
Potential (V vs Ag/AgCl) (al
I I 4.7
d.e
4.6
d..
*.,
Potential (V vs Ag/AgCl) (bl Fig. 1. (a) DPASV voltammogram for liver lead after mineralization by microwave irradiation using 15 ml of concentrated nitric acid, 150 mg of test portion weight, 16 min of oven time and six vessels (including blank). (b) Triplicate voltammograms for liver lead after mineralization by microwave irradiation using 8 ml of concentrated perchloric acid, 150 mg of test portion weight, 16 min of oven time and four vessels (including blank). In both, the pressure and temperature inside the control vessels were set up at 1260 kPa and 190°C; deposition potential, -0.70 V; deposition time, 2 min with stirring (plus 40 s quiescent); potential scan rate, 5 mV s-‘.
catalytic reduction of hydrogen or dissolved oxygen; it may be due to a dissolved electroactive species formed by the decomposition of nitric acid (e.g., NO, NO,). This was an unfortunate situation because nitric acid proved to be an adequate oxidizing agent for the biological materials undergoing microwave decomposition, as seen in Fig. 2a. This figure shows the monitoring of the pressure and temperature inside the sample vessel and makes perceptible that the optimum mineralization conditions were obtained in a relatively short period of time (ca. < 2 min); after this time, samples were left at an additional period of time in the oven to ensure the quantitative destruction of organic concomitants. The selection of the total oven time was established by visual inspection
Chimica Acta 317 (1995) 311-318
315
of digestion solutions; digestion solutions freed of particles and colourless were obtained after 16-min oven time. The addition of perchloric acid to the biological material undergoing microwave heating favoured the electrochemical evaluation of total lead because the interference produced by the cathodic peak disappeared. Consequently, 12 ml of concentrated perchloric acid, added to 250 mg of biological material (e.g., brain, liver, kidney, etc.) test portion, were used in a first digestion trial intended to mineralize the largest fraction of the organics. Fig. 2b shows the typical traces for pressure and temperature obtained under the experimental conditions mentioned above; however, quantitative determination by DPASV was not possible because mineralization was incompletely achieved (recovered lead was 60-800/c lower than total lead and the reproducibility was 7.5% (R.S.D.)); this latter situation is depicted in Fig. 2b, in which one sees that the highest pressure reached (ca. < 700 kPa) was much lower than the pressure set up of 1260 kPa. As expected, pressure inside the digestion vessel increased when the sample weight and perchloric acid volume added decreased, because of the faster rate of microwave power absorption by the sample/acid system. The best results were obtained using less sample weight and less perchloric acid volume under the following conditions: addition of 8 ml of concentrated perchloric acid to 150 mg of test portion weight, using four digestion vessels (one control vessel, two sample vessels and one blank vessel) irradiated for 16 min (Fig. 2~). Fig. 2c shows that the optimum mineralization conditions were reached in less than 9.5 min; after this time, lined digestion vessels containing the test portions spent about 6.5 min more in the microwave oven to complete mineralization for a total oven time of 16 min, as was the case for nitric acid mineralization (Fig. 2a). Under these conditions, the DPASV lead concentrations correlated statistically with the results obtained by ETA-AAS. Hydrochloric acid and hydrogen peroxide were tried, but unsuccessfully. After the microwave mineralization, the digestion solutions were buffered at pH 4.70; no further sample treatment was required and the lead oxidation peak appeared at a potential of - 0.45 V vs. Ag/AgCl (Fig. lb). Monitoring and controlling pressure and tempera-
316
J.E. Tatin et al./Analytica
ture inside the digestion vessels guaranteed a much safer mineralization than those reported previously [22], avoiding chances for explosion of the reactor system and potential injuries to the analyst. This is of particular interest when perchloric acid is used in closed digestion systems; thus, no threatening situa-
Chimica Acta 317 (1995) 311-318
tions did exist during these experiments. The risks of lead losses by volatilization and of contamination were reduced because pressurized vessels were employed. Furthermore, lined digestion vessels are able to deal with a large organic sample size (ca. < 0.5 g), which favoured the adjustment of the detection limit
P
Time (min> (b) Fig. 2. Monitoring pressure (P) and temperature (T) inside the lined digestion vessel heated by microwave irradiation. (a) Sample 150 mg of anencephalic liver; nitric acid volume, 15 ml. (b) Sample weight, 250 mg of anencephalic liver; perchloric acid volume, (cl Sample weight, 150 mg of anencephalic liver; perchloric acid volume, 8 ml. In all cases, pressure and temperature were set up kPa and 190°C. For case (a), one control vessel, three sample vessels and two blank vessels employed. For cases (b) and (cl, one vessel, two sample vessels and one blank vessel employed.
weight, 12 ml. at 1260 control
J.E. TahCn et al. /Analytica Table 1 Within- and between-run Sample h
precision
study for the DPASV determination
Mean Pb concentration
1 2
1.5 1.3
from anencephalic
fetuses a
Between-run
S.D. ( pg Pb g-‘)
R.S.D. (o/o)
S.D. ( /qg Pb g- ‘)
R.S.D. (o/c)
0.01 0.03
0.6 2.3
0.04 0.05
2.7 3.8
a Five replicates of each sample were done (six runs each). b Randomly collected from anencephalic fetuses at the Department coast of lake Maracaibo, Venezuela).
of the DPASV analysis. In general, pressure/temperature-controlled microwave mineralization seems to be a reliable alternative for sample decomposition. To the best of our knowledge, no other sample microwave mineralization work has reported on the use of this type of digestion system that deserves to be further considered for application development. Linear calibration graphs were obtained up to 100 pg Pb 1-r. The biological material test portions analysed were within this linear range. The current, Z ( PA), was found to be linearly related to the concentration, C ( pg Pb l-l>, by the equation Z = 0.002X + 0.0037 (the standard deviation of the slope and intercept were 0.0002 and 0.0022, respectively, for eight six-point calibration graphs; correlation coefficient = 0.971). The standard addition method was also employed for quantification purposes and produced graphs with slopes statistically indistinguishable from those of the aqueous standard calibration graphs. This implied the absence of interferences in the DPASV determination of lead in the biological materials after adequate microwave mineralization as proposed, and the alternative possibilities of employing either the calibration graphs or the standard addition method for quantification. Lead concentrations obtained by DPASV analysis
Table 2 Dry-weight 20 controls
of total lead in two kidney samples
Within-run
(PgPbg-‘)
317
Chimica Acta 317 (1995) 311-318
of Pathological
Anatomy
of ‘Pedro Garcia
Clara’ Hospital
of the mineralized biological materials from anencephalic fetuses and controls were compared with those obtained by ETA-AAS on the same digestion samples. The correlation between the two methods was excellent: y = 1.142~ - 0.0035, r = 0.9999, n = 40, p < 0.001, where y and x were the lead concentrations determined by DPASV and ETAAAS, respectively. The standard error of the estimate (S,,,) is 0.397 pg Pb 1-l and the standard deviations of the intercept and slope are 0.383 Fg Pb 1. ’ and 0.00069, respectively. The accuracy of the proposed DPASV-based method was also tested by analysing the standard reference material Sargasso (NIES no. 9) supplied by the National Institute for Environmental Studies (Japan), with a target lead concentration of 1.35 +_ 0.05 pg Pb g-l; the concentration found for lead using the proposed method was 1.30 f 0.02 pg Pb did not differ signifig _ ‘. This mean concentration cantly (p > 0.001) from the certified value. The reliability of the method was further assessed through a recovery study. This was done by five replicate DPASV determinations of lead in two real samples of brain, liver, kidney and of lung (one from an anencephalic fetus and one from a control fetus). The average recovery was 99.8% (range 96-105%).
lead concentrations (mean f 1 S.D., pg g-’ ) found in brain, liver, kidney and lung samples from 20 anencephalic evaluated by DPASV
Groups
Brain
Liver
Right lung
Left lung
Right kidney
Left kidney
Anencephaly Control
UD UD
2.1 f 1.1 * 0.5 * 0.2
1.1 f0.8 * 0.6 + 0.1
0.6 f 0.2 0.7 + 0.1
1.4 + 0.7 1.5 + 0.03
1.7 + 0.9 0.7 * 0.2
UD = Undetectable by DPASV. Statistical differences with respect to controls
as follows:
(eastern
* p < 0.001; * * p < 0.005.
fetuses and
*-
318
J.E. Tahrin et al./Analytica
The within- and between-run precisions for the electrochemical determination of lead in two kidney samples are shown in Table 1. Analysis of five replicates of each sample were done (six runs each) using the proposed microwave mineralization procedure. The standard deviation ranges were 0.01-0.03 pg Pb g-’ (within-run) and 0.1-0.4 pg Pb g-l (between-runs). These results can be considered adequate for this type of analysis and show that the proposed mineralization method is highly reproducible. The detection limit of the electrochemical method, defined as three times the standard deviation of a blank solution, was 0.03 pg Pb g-’ (in solid sample), equivalent to 0.1 pug Pb 1-l in the diluted biological material test portions, for a sample weight of 150 mg; this limit can be adjusted by varying the sample size. This value is similar to that of the ETA-AAS method 1161. The proposed method has been used in the laboratory to establish the mean concentration (k 1 S.D., pg g-t) of lead in brain, liver, kidney and lung samples from anencephalic fetuses and controls from the Department of Pathological Anatomy of ‘Pedro Garcia Clara’ Hospital, eastern coast of lake Maracaibo (Venezuela). Results of a survey of twenty anencephalic fetuses and twenty control fetuses are shown in Table 2. Liver (p < 0.0011, right lung (p < 0.005) and left kidney (p < 0.005) lead was significantly increased in anencephaly. These results indicate that lead should be seriously considered for cause/effect studies when the high rate of anencephaly incidence in Venezuela is considered [3,4]. In conclusion, the proposed DPASV method was sensitive, accurate and precise, giving an important analytical alternative, as reliable as ETA-AAS, for the customary voltammetric determination of total lead in a wide range of concentrations in solid samples.
Acknowledgements This research was partially supported by Consejo de Desarrollo Cientifico y Humanistico (CONDES) from La Universidad de1 Zulia and Consejo National
Chimica Acta 317 (1995) 311-318
de Investigaciones ICIT).
Cientificas
y Tecnologicas
(CON-
References [l] K. Coard, C. Escoffery, J. Golding and D. Ashley, Teratology, 41(1990) 173. [2] J.H. Elwood and J.M. Elwood, Int. J. Epidemiol., 13 (1984) 45. [3] L.Ch. Barrios, J.E. Tahan, L. Marcano, V.A. Granadillo, H.S. Cubillan, J.M. Sanchez, M.C. Rodriguez, F.G. de Salazar, 0. Salgado and R.A. Romero, Ciencia, 3 (1995) 49. [4] J.E. Tahan, L.Ch. Barrios, L. Marcano, V.A. Granadillo, H.S. Cubillln, J.M. Sanchez, M.C. Rodriguez, F.G. de Salazar, 0. Salgado and R.A. Romero, Trace Elements Electrolytes (in press). [5] L. Friberg, G.F. Nordberg and V.B. Vouk, Handbook of the Toxicology of Metals, Elsevier, Amsterdam, 1986, p. 404. [6] M. Newland, S. Yezhon, B. Logdberg and M. Berlin, Toxicol. Appl. Pharm., 126 (1994) 6. [7] J.E. Tahan, J.M. Sanchez, V.A. Granadillo, H.S. Cubillan and R.A. Romero, J. Agric. Food Chem., 43 (1995) 910. [8] V.A. Granadillo, J.E. Tahan, 0. Salgado, L.E. Elejalde, B. Rodriguez-Iturbe, G.B. Romero and R.A. Romero, Clin. Chim. Acta, 233 (1995147. [9] D.Q. Nam, F. Skkel and P. Buryan, Sci. Total Environ., 144 (19941 87. [IO] J.D. Lee and J.M. Lo, Anal. Chim. Acta, 287 (19941259. [ll] T. Nedeltcheva, L. Costadinnova and M. Athanassova, Anal. Chim. Acta, 291 (1994) 75. [12] S. Mannino and J. Wang, Electroanalysis, 4 (1992) 835. [13] R.A. Romero, J.E. Tahan and A.J. Moronta, Anal. Chim. Acta, 257 (1992) 147. [14] J.E. Tahln and R.A. Romero, Anal. Chim. Acta, 273 (1993) 53. [15] J.E. Tahan, A.J. Moronta and R.A. Romero, Anal. Chim. Acta, 2 (1990) 449. [16] V.A. Granadillo and R.A. Romero, J. Anal. At. Spectrom., 8 (1993) 615. [17] Standard Specification for Water, American Society for Testing and Materials ASTM, Philadelphia, 1977, Document 1193-77. [18] M.C. Rodriguez, J.M. Sanchez, H.S. Cubilhin and R.A. Romero, Ciencia, 2 (1994) 99. [19] S. Wallenstein, CL. Zucher and J.L. Fleiss, Circ. Res., 47 (1980) 1. [20] B. Ostle, Estadistica Aplicada, Limusa, 4th edn., Mexico, 1974, pp. 373-396. [21] V.A. Granadillo, H.S. Cubillan, J.M. Sanchez, J.E. Tah&n, E. Marquez and R.A. Romero, Anal. Chim. Acta, 306 (1995) 139-147. [22] C. Cabrera, M.L. Lorenzo, C. Gallego, M.C. Lopez and E. Lillo, J. Agric. Food Chem., 42 (1994) 126-128.
ACIYA
ELSEVIER
Analytica
Chimica Acta 317 (1995) 311-318
Anodic stripping voltammetric determination of total lead in anencephalic fetuses after pressure/temperature-controlled microwave mineralization Jorge E. Tahb, Lorenia Marcano I, Romer A. Romero Laboratorio
*
de Instrumentacich Analitica, Departamento de Quimica, Facultad Experimental de Ciencias, La Uniuersidad de1 Zulia, Apartado Postal 15202, Las Delicias, Maracaibo 4003-A, Venezuela Received 8 May 1995; revised 13 July 1995; accepted
13 August 1995
Abstract The development of a closed-vessel mineralization method for the decomposition of brain, liver, kidney and lung specimens of anencephalic (A) fetuses and controls (C) from the eastern coast of lake Maracaibo, Venezuela, is presented. Digestion was done in a laboratory microwave oven provided with pressure sensing tube and fiberoptic temperature probe to monitor and control pressure and temperature conditions inside the lined digestion vessels. Total lead was subsequently determined by differential pulse anodic stripping voltammetry (DPASV) with a hanging mercury drop electrode. The optimized conditions for maximal pressure and temperature set up were 1260 Wa and 190°C. Three samples and one blank were routinely prepared for simultaneous digestion. After sample mineralization, the lead oxidation peak appeared at a potential of -0.45 V vs. Ag/AgCl, pH 4.70. Lead concentrations obtained by DPASV analysis of the mineralized biological materials were compared with those provided by electrothermal atomization atomic absorption spectrometry (ETA-AAS) on the same digestion samples. The correlation between the two methods was excellent: y = 1.142x - 0.0035, r = 0.9999, n = 40, p < 0.001, where y and x were the lead concentrations determined by DPASV and ETA-AAS, respectively. For the DPASV determination of total lead, precision (R.S.D.) was better than 3.8%, for within- and between-run analyses. The detection limit of the electrochemical method, defined as three times the standard deviation of a blank solution, was 0.03 pg Pb g-’ (in solid sample), equivalent to 0.1 pg Pb 1-r in the diluted test portions. The dry-weight metal concentrations (k 1 SD., Kg g-‘) found in brain, liver, kidney and lung were as follows: (brain, undetectable in A and in C; liver, 2.1 + 1.1 in A, 0.5 f 0.2 in C; right lung, 1.1 ? 0.8 in A, 0.6 + 0.1 in C; left lung, 0.6 _t 0.2 in A, 0.7 + 0.1 in C; right kidney, 1.4 + 0.7 in A, 1.5 f 0.03 in C and left kidney, 1.7 + 0.9 in A, 0.7 +_0.2 in C. The proposed DPASV method constitutes an analytical alternative, as reliable as ETA-AAS, for the voltammetric determination of total lead in solid samples. Keywords:
Total lead; Stripping
* Corresponding author. ’ Present address: Departamento
voltammetry;
Anencephaly;
de Anatomia
Patokjgica,
Pressure/temperature-controlled
Hospital
microwave
mineralization
‘Pedro Garcia Clara’, Ciudad Ojeda, Venezuela.
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOOO3-2670(95)00422-X
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1. Introduction Anencephaly is a life-incompatible congenital malformation characterized by deficient development of the brain organ in the fetus. Epidemiological studies have indicated that Jamaica has the lowest rate of anencephaly with an incidence of 0.26 per 1000 total births 111 while the highest rate of 8.6/1000 has been found in Belfast [2]. In Venezuela, a relatively high anencephaly rate of 5.1 was reported in 1994 for the eastern coast of lake Maracaibo, area located in the north-western part of the country [3]. This situation has been associated with the presence of lead (Pb) in the environment [4]. Lead-induced teratogenicity [5] and neurotoxicity [6] have been experimentally demonstrated. Automobile combustion of tetraethyl lead-containing gasolines still represents the main source of lead pollution in Venezuela (ca. > 90%) [7]. Our studies have shown that the lead concentration in the Venezuelan gasolines ranges between 700 and 800 mg 1-l (depending on the octane number) [8]. These values are one order of magnitude higher than the lead levels allowed in most European countries and in the United States. To assess the extent of the possible harmful effect played by lead in anencephaly, data are required on the total metal concentrations in body tissues and organs (e.g., brain, liver, kidney and lung). The role of lead in anencephaly is worth studying using reliable analytical methods of measurement. Differential-pulse anodic stripping voltammetry (DPASV) has been recently used for the determination of lead in environmental [9,10], geological [ll] and food samples [12]. The extremely high sensitivity of this technique is due to its effective in situ preconcentration step [ 121. However, the direct DPASV determination of total metal in biological materials is interfered with by the presence of organic matter [13,14]; this may bind lead, modifying the metal speciation and, therefore, its availability. Hence, the stripping procedures always require a preliminary destruction of organic matter to free the analyte metal from organic ligands and to produce a single labile metal species that can subsequently be determined by DPASV. With this approach, the lead determined by DPASV in mineralized brain, liver, kidney and/or lung, or any other metal electrochem-
Chimica Acta 317 (1995) 311-318
ically measured in these materials or in any sample matrix regardless of its complexity, will produce quantitative data statistically similar to those obtained by using techniques such as electrothermal atomization atomic absorption spectrometry (ETAAAS), frequently employed for the purpose of total metal determination. Otherwise, serious underestimations of the metal will be obtained [13-151. In previous papers, Tahan and Romero [14] reported the DPASV determination of total soluble copper in haemodialysis water with a high organic content [15] and in blood plasma after high-pressure mineralization. The same group [13] also described the determination of total soluble aluminum in haemodialysis water with a high organic matter-content, decomposed by microwave heating and posteriorly analysed by differential-pulse polarography. In this paper we present the development of a closed-vessel mineralization method for the decomposition of brain, liver, kidney and lung specimens from anencephalic fetuses and controls; pressure and temperature inside the digestion vessels were controlled and monitored. This procedure was tested for its suitability to permit the posterior determination of total lead by DPASV with the hanging mercury drop electrode (HMDE). Lead was also determined by an ETA-AAS-based method [16] for accuracy evaluation purposes.
2. Experimental 2.1. Apparatus The DPASV measurements were done using a Model 273A potentiostat/galvanostat system (Princeton Applied Research (PAR)) in conjunction with a PAR Model 303A SMDE electrode assembly and a Hewlett-Packard ColorPro Graphics Plotter, being the whole system controlled from a computer based on the Intel 80386 microprocessor running the 5.0 MS-DOS operating system (IBM Personal System/2 Model 40 SX> and the PAR Head Start Creative Electrochemistry Software. A large drop size was used with the static mercury drop electrode device throughout this work. During the deposition step, samples were stirred by means of a PAR Model
J.E. Tahrin et al. /Analytica
305 stirrer. DPASV determinations were done at 20°C under oxygen-free nitrogen. A Perkin-Elmer Model 2380 atomic absorption spectrometer with a Perkin-Elmer Model HGA-500 graphite furnace atomizer and a deuterium arc background corrector were used for the ETA-AAS determination of total lead in the mineralized samples, according to the method reported by Granadillo and Romero [16]. Biological materials were freeze-dried in a Labconco Model 6 liter lyophilizer, kept at -52°C for 48 h. Mineralizations were performed in a CEM Model MDS-2100 laboratory microwave oven (950 W for 100% power) provided with pressure sensing tube and fiberoptic temperature probe to monitor and control pressure and temperature conditions inside the lined digestion vessels. This type of vessel consists of a lOO-ml PTFE PFA (a tetrafluoroethylene with a fully fluorinated alkoxy side chain) inner linner and a PTFE PFA cover; the casing and cap of the lined digestion vessel are made from microwave-transparent polymer (polytherimide). The collection vessel placed in the center of the turntable collects, condenses and vents vapors released when the rupture membrane breaks down. In this system, pressure tubing attached to a sample vessel (control vessel) is routed outside the microwave cavity through one of the inlet/outlet ports; pressure is sensed by a transducer and displayed graphically and digitally on the display screen of the oven. The microwave transparent fiberoptic temperature probe is inserted into the thermowell of the control vessel and exits the microwave cavity via a bulkhead connector and leads to a special temperature control PC board mounted on the system CPU board; the temperature sensor is a phosphor located at the tip of the probe. 2.2. Reagents and samples All chemicals used were of analytical reagent grade. The lead stock solution (ca. 1000 mg 1-l > was prepared from a Titrisol concentrate (Merck). The lead working standard solutions were freshly prepared by serial dilution of the stock with grade I (as established by the American Society for Testing and Materials, ASTM, ca. electrical resistivity > 16.6 MR cm-’ at 25°C) [17] triply-distilled and deionized water; nitric acid was purchased from Riedel-de
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313
I-&en, and had a metal content below the ETA-AAS detection limit (0.1 pg Pb 1-l) 1161. The supporting electrolyte was 6 M acetate buffer (pH 4.701, prepared by mixing acetic acid (Riedel-de Hfien) and sodium hydroxide (Merck). Concentrated perchloric acid (Merck) was used for the microwave mineralization. For accuracy evaluation, the following standard reference material was used: Sargasso (NIES no. 9) from the National Institute for Environmental Studies (Ibaraki, Japan). This material was mineralized as stated below to provide aqueous solutions whose final concentration was within the range of the lead concentrations expected in the biological materials analysed. Complete organs (brain or brain-like tissue in anencephalics, kidney, liver and lung) were obtained by autopsy from both anencephalic and control fetuses; it should be pointed out that anencephalic brain consisted of a cerebrovascular mass covered by a translucent membrane. The autopsies were performed at the Department of Pathological Anatomy of ‘Pedro Garcia Clara’ Hospital by qualified pathologists who were told about the risk of metal contamination from dust, surgical glove powder, surgical instruments, etc. Organs were excised from every subject with plastic knifes, trimmed of fat, gristle, sheath and connective tissue, rinsed free of blood with grade I ASTM triply distilled and deionized water, blotted with pieces of deionized filter paper and placed in metal-free polyethylene bags as soon as possible and then quickly frozen. Once in the laboratory, whole organs were thawed, blended, lyophilized, ground, mixed and kept in polyethylene bags at 4°C until analysis. Special care was exercised to avoid contamination during the handling of samples for preparation for analysis. In this sense, sample manipulation, solution preparation and instrumental measurements were conducted in a clean room with Class-100 work area. The clinical protocol was approved by an ad hoc medical committee of the hospital. 2.3. Closed-Llessel microwave logical samples
mineralization
of bio-
Approximately 150 mg of lyophilized material (e.g., brain, liver, kidney, lung, standard reference material) and 8 ml of concentrated perchloric acid
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were placed into the vessel liner and capped with a cover that employs pressure assisted type seal for leak-free operation (e.g., as internal vapor pressure rises from microwave heating of the liquid, the seal is energized, forming a progressively tighter seal with increasing pressure) [181. The capped liner was put into the microwave-transparent body of the lined digestion vessel and closed by tightening the cap. Three samples and one blank were routinely prepared for simultaneous digestion. The four vessels (including the control vessel which always contained the largest lyophilized test portion to mineralize) were placed on the turntable, inserted in the oven, and irradiated for 16 min (at 100% power, equivalent to 950 W and 2450 MHz); the optimized conditions for maximal pressure and temperature were 1260 kPa and 190°C. The turntable was rotated continuously. After cooling to ambient temperature, the caps were removed and contents were transferred quantitatively into 50-ml polypropylene calibrated flasks and diluted to volume with 6 M acetate buffer to reach pH 4.70. Blanks were prepared with the same reagent, without the samples, undergoing a similar high-pressure closed-vessel decomposition treatment by microwave heating. Aliquots (ca. 8-10 ml) were transferred into the SMDE sample cells kept at 20°C degassed with oxygen-free nitrogen for 2 min and analysed voltammetrically. 2.4. Statistical analysis Statistical comparisons of data obtained sequentially by DPASV and ETA-AAS were done by repeated measurements of analysis of variance [19]; significant F values were tested with Tukey test [20] with the help of a commercial statistical package (Statgraphics, STSC, Rockville, MD); values of p < 0.05 were considered significant.
3. Results and discussion Initially, the deposition potential, deposition time, potential scan rate, drop size and nitrogen purge time were optimized with synthetic lead samples with the following results: -0.70 V vs. Ag/AgCl, 2 min with stirring (plus 40 s quiescent), 5 mV s-l, large (equivalent to ca. 2.5 mm*) and 2 min with stirring,
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respectively. These conditions were applied in the DPASV analysis of real samples. In the determination of total metals in biological materials, the transformation of the solid sample into solution needs careful consideration in order to avoid serious underestimation of the analyte [7,21]. In this sense, the presence of organic matter has been reported to be a source of interference for the quantitative determination of metal elements by voltammetric means with the SMDE electrode assembly [13151. Therefore, samples were pre-treated by using microwave-assisted pressurized acid mineralization, carried out in closed vessels whose inside pressure and temperature conditions were controlled and monitored from the oven microcomputer board. The aim of the digestion procedure was to remove concomitant substances and produce digestion solutions suitable for the subsequent evaluation of total lead by DPASV. It should be pointed out that interference studies on common ions (sodium, potassium, calcium, magnesium, iron, copper, zinc, cadmium, chloride, acetate, sulphate, etc.) did not have any effect on the electrochemical determination of lead. The results obtained electrochemically for total lead in mineralized samples were verified by ETAAAS evaluation of the same digestion samples. Initial microwave mineralizations were done with the addition of 15 ml of concentrated nitric acid to different test portion weights (ca. 100, 150, 200 and 300 mg), using several mineralization times (ca. 5, 10, 20, 40 and 60 min); maximal pressure and temperature inside the digestion vessels were set up at 1260 kPa and 190°C as reported already [18]. In pressure/temperature-controlled microwave mineralization experiments, optimum mineralization conditions are obtained once pressure and temperature reach maximal values; in general, the faster in reaching maximal values the better the oxidizing agent [18]. For nitric acid mineralization, one control vessel, three sample vessels and two blank vessels were employed for simultaneous digestion. The initial results were unsatisfactory; not more than 50% of the total lead content could be quantified by DPASV, at an oxidation potential of - 0.45 V vs. Ag/AgCl, pH 4.70, and the rest was interfered by the presence of a more cathodic peak signal appearing at - 0.53 V vs. Ag/AgCl (Fig. la). It was demonstrated that this latter voltammetric peak was not the result of the
J.E. Tahbn et al./Analytica
Potential (V vs Ag/AgCl) (al
I I 4.7
d.e
4.6
d..
*.,
Potential (V vs Ag/AgCl) (bl Fig. 1. (a) DPASV voltammogram for liver lead after mineralization by microwave irradiation using 15 ml of concentrated nitric acid, 150 mg of test portion weight, 16 min of oven time and six vessels (including blank). (b) Triplicate voltammograms for liver lead after mineralization by microwave irradiation using 8 ml of concentrated perchloric acid, 150 mg of test portion weight, 16 min of oven time and four vessels (including blank). In both, the pressure and temperature inside the control vessels were set up at 1260 kPa and 190°C; deposition potential, -0.70 V; deposition time, 2 min with stirring (plus 40 s quiescent); potential scan rate, 5 mV s-‘.
catalytic reduction of hydrogen or dissolved oxygen; it may be due to a dissolved electroactive species formed by the decomposition of nitric acid (e.g., NO, NO,). This was an unfortunate situation because nitric acid proved to be an adequate oxidizing agent for the biological materials undergoing microwave decomposition, as seen in Fig. 2a. This figure shows the monitoring of the pressure and temperature inside the sample vessel and makes perceptible that the optimum mineralization conditions were obtained in a relatively short period of time (ca. < 2 min); after this time, samples were left at an additional period of time in the oven to ensure the quantitative destruction of organic concomitants. The selection of the total oven time was established by visual inspection
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315
of digestion solutions; digestion solutions freed of particles and colourless were obtained after 16-min oven time. The addition of perchloric acid to the biological material undergoing microwave heating favoured the electrochemical evaluation of total lead because the interference produced by the cathodic peak disappeared. Consequently, 12 ml of concentrated perchloric acid, added to 250 mg of biological material (e.g., brain, liver, kidney, etc.) test portion, were used in a first digestion trial intended to mineralize the largest fraction of the organics. Fig. 2b shows the typical traces for pressure and temperature obtained under the experimental conditions mentioned above; however, quantitative determination by DPASV was not possible because mineralization was incompletely achieved (recovered lead was 60-800/c lower than total lead and the reproducibility was 7.5% (R.S.D.)); this latter situation is depicted in Fig. 2b, in which one sees that the highest pressure reached (ca. < 700 kPa) was much lower than the pressure set up of 1260 kPa. As expected, pressure inside the digestion vessel increased when the sample weight and perchloric acid volume added decreased, because of the faster rate of microwave power absorption by the sample/acid system. The best results were obtained using less sample weight and less perchloric acid volume under the following conditions: addition of 8 ml of concentrated perchloric acid to 150 mg of test portion weight, using four digestion vessels (one control vessel, two sample vessels and one blank vessel) irradiated for 16 min (Fig. 2~). Fig. 2c shows that the optimum mineralization conditions were reached in less than 9.5 min; after this time, lined digestion vessels containing the test portions spent about 6.5 min more in the microwave oven to complete mineralization for a total oven time of 16 min, as was the case for nitric acid mineralization (Fig. 2a). Under these conditions, the DPASV lead concentrations correlated statistically with the results obtained by ETA-AAS. Hydrochloric acid and hydrogen peroxide were tried, but unsuccessfully. After the microwave mineralization, the digestion solutions were buffered at pH 4.70; no further sample treatment was required and the lead oxidation peak appeared at a potential of - 0.45 V vs. Ag/AgCl (Fig. lb). Monitoring and controlling pressure and tempera-
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ture inside the digestion vessels guaranteed a much safer mineralization than those reported previously [22], avoiding chances for explosion of the reactor system and potential injuries to the analyst. This is of particular interest when perchloric acid is used in closed digestion systems; thus, no threatening situa-
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tions did exist during these experiments. The risks of lead losses by volatilization and of contamination were reduced because pressurized vessels were employed. Furthermore, lined digestion vessels are able to deal with a large organic sample size (ca. < 0.5 g), which favoured the adjustment of the detection limit
P
Time (min> (b) Fig. 2. Monitoring pressure (P) and temperature (T) inside the lined digestion vessel heated by microwave irradiation. (a) Sample 150 mg of anencephalic liver; nitric acid volume, 15 ml. (b) Sample weight, 250 mg of anencephalic liver; perchloric acid volume, (cl Sample weight, 150 mg of anencephalic liver; perchloric acid volume, 8 ml. In all cases, pressure and temperature were set up kPa and 190°C. For case (a), one control vessel, three sample vessels and two blank vessels employed. For cases (b) and (cl, one vessel, two sample vessels and one blank vessel employed.
weight, 12 ml. at 1260 control
J.E. TahCn et al. /Analytica Table 1 Within- and between-run Sample h
precision
study for the DPASV determination
Mean Pb concentration
1 2
1.5 1.3
from anencephalic
fetuses a
Between-run
S.D. ( pg Pb g-‘)
R.S.D. (o/o)
S.D. ( /qg Pb g- ‘)
R.S.D. (o/c)
0.01 0.03
0.6 2.3
0.04 0.05
2.7 3.8
a Five replicates of each sample were done (six runs each). b Randomly collected from anencephalic fetuses at the Department coast of lake Maracaibo, Venezuela).
of the DPASV analysis. In general, pressure/temperature-controlled microwave mineralization seems to be a reliable alternative for sample decomposition. To the best of our knowledge, no other sample microwave mineralization work has reported on the use of this type of digestion system that deserves to be further considered for application development. Linear calibration graphs were obtained up to 100 pg Pb 1-r. The biological material test portions analysed were within this linear range. The current, Z ( PA), was found to be linearly related to the concentration, C ( pg Pb l-l>, by the equation Z = 0.002X + 0.0037 (the standard deviation of the slope and intercept were 0.0002 and 0.0022, respectively, for eight six-point calibration graphs; correlation coefficient = 0.971). The standard addition method was also employed for quantification purposes and produced graphs with slopes statistically indistinguishable from those of the aqueous standard calibration graphs. This implied the absence of interferences in the DPASV determination of lead in the biological materials after adequate microwave mineralization as proposed, and the alternative possibilities of employing either the calibration graphs or the standard addition method for quantification. Lead concentrations obtained by DPASV analysis
Table 2 Dry-weight 20 controls
of total lead in two kidney samples
Within-run
(PgPbg-‘)
317
Chimica Acta 317 (1995) 311-318
of Pathological
Anatomy
of ‘Pedro Garcia
Clara’ Hospital
of the mineralized biological materials from anencephalic fetuses and controls were compared with those obtained by ETA-AAS on the same digestion samples. The correlation between the two methods was excellent: y = 1.142~ - 0.0035, r = 0.9999, n = 40, p < 0.001, where y and x were the lead concentrations determined by DPASV and ETAAAS, respectively. The standard error of the estimate (S,,,) is 0.397 pg Pb 1-l and the standard deviations of the intercept and slope are 0.383 Fg Pb 1. ’ and 0.00069, respectively. The accuracy of the proposed DPASV-based method was also tested by analysing the standard reference material Sargasso (NIES no. 9) supplied by the National Institute for Environmental Studies (Japan), with a target lead concentration of 1.35 +_ 0.05 pg Pb g-l; the concentration found for lead using the proposed method was 1.30 f 0.02 pg Pb did not differ signifig _ ‘. This mean concentration cantly (p > 0.001) from the certified value. The reliability of the method was further assessed through a recovery study. This was done by five replicate DPASV determinations of lead in two real samples of brain, liver, kidney and of lung (one from an anencephalic fetus and one from a control fetus). The average recovery was 99.8% (range 96-105%).
lead concentrations (mean f 1 S.D., pg g-’ ) found in brain, liver, kidney and lung samples from 20 anencephalic evaluated by DPASV
Groups
Brain
Liver
Right lung
Left lung
Right kidney
Left kidney
Anencephaly Control
UD UD
2.1 f 1.1 * 0.5 * 0.2
1.1 f0.8 * 0.6 + 0.1
0.6 f 0.2 0.7 + 0.1
1.4 + 0.7 1.5 + 0.03
1.7 + 0.9 0.7 * 0.2
UD = Undetectable by DPASV. Statistical differences with respect to controls
as follows:
(eastern
* p < 0.001; * * p < 0.005.
fetuses and
*-
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The within- and between-run precisions for the electrochemical determination of lead in two kidney samples are shown in Table 1. Analysis of five replicates of each sample were done (six runs each) using the proposed microwave mineralization procedure. The standard deviation ranges were 0.01-0.03 pg Pb g-’ (within-run) and 0.1-0.4 pg Pb g-l (between-runs). These results can be considered adequate for this type of analysis and show that the proposed mineralization method is highly reproducible. The detection limit of the electrochemical method, defined as three times the standard deviation of a blank solution, was 0.03 pg Pb g-’ (in solid sample), equivalent to 0.1 pug Pb 1-l in the diluted biological material test portions, for a sample weight of 150 mg; this limit can be adjusted by varying the sample size. This value is similar to that of the ETA-AAS method 1161. The proposed method has been used in the laboratory to establish the mean concentration (k 1 S.D., pg g-t) of lead in brain, liver, kidney and lung samples from anencephalic fetuses and controls from the Department of Pathological Anatomy of ‘Pedro Garcia Clara’ Hospital, eastern coast of lake Maracaibo (Venezuela). Results of a survey of twenty anencephalic fetuses and twenty control fetuses are shown in Table 2. Liver (p < 0.0011, right lung (p < 0.005) and left kidney (p < 0.005) lead was significantly increased in anencephaly. These results indicate that lead should be seriously considered for cause/effect studies when the high rate of anencephaly incidence in Venezuela is considered [3,4]. In conclusion, the proposed DPASV method was sensitive, accurate and precise, giving an important analytical alternative, as reliable as ETA-AAS, for the customary voltammetric determination of total lead in a wide range of concentrations in solid samples.
Acknowledgements This research was partially supported by Consejo de Desarrollo Cientifico y Humanistico (CONDES) from La Universidad de1 Zulia and Consejo National
Chimica Acta 317 (1995) 311-318
de Investigaciones ICIT).
Cientificas
y Tecnologicas
(CON-
References [l] K. Coard, C. Escoffery, J. Golding and D. Ashley, Teratology, 41(1990) 173. [2] J.H. Elwood and J.M. Elwood, Int. J. Epidemiol., 13 (1984) 45. [3] L.Ch. Barrios, J.E. Tahan, L. Marcano, V.A. Granadillo, H.S. Cubillan, J.M. Sanchez, M.C. Rodriguez, F.G. de Salazar, 0. Salgado and R.A. Romero, Ciencia, 3 (1995) 49. [4] J.E. Tahan, L.Ch. Barrios, L. Marcano, V.A. Granadillo, H.S. Cubillln, J.M. Sanchez, M.C. Rodriguez, F.G. de Salazar, 0. Salgado and R.A. Romero, Trace Elements Electrolytes (in press). [5] L. Friberg, G.F. Nordberg and V.B. Vouk, Handbook of the Toxicology of Metals, Elsevier, Amsterdam, 1986, p. 404. [6] M. Newland, S. Yezhon, B. Logdberg and M. Berlin, Toxicol. Appl. Pharm., 126 (1994) 6. [7] J.E. Tahan, J.M. Sanchez, V.A. Granadillo, H.S. Cubillan and R.A. Romero, J. Agric. Food Chem., 43 (1995) 910. [8] V.A. Granadillo, J.E. Tahan, 0. Salgado, L.E. Elejalde, B. Rodriguez-Iturbe, G.B. Romero and R.A. Romero, Clin. Chim. Acta, 233 (1995147. [9] D.Q. Nam, F. Skkel and P. Buryan, Sci. Total Environ., 144 (19941 87. [IO] J.D. Lee and J.M. Lo, Anal. Chim. Acta, 287 (19941259. [ll] T. Nedeltcheva, L. Costadinnova and M. Athanassova, Anal. Chim. Acta, 291 (1994) 75. [12] S. Mannino and J. Wang, Electroanalysis, 4 (1992) 835. [13] R.A. Romero, J.E. Tahan and A.J. Moronta, Anal. Chim. Acta, 257 (1992) 147. [14] J.E. Tahln and R.A. Romero, Anal. Chim. Acta, 273 (1993) 53. [15] J.E. Tahan, A.J. Moronta and R.A. Romero, Anal. Chim. Acta, 2 (1990) 449. [16] V.A. Granadillo and R.A. Romero, J. Anal. At. Spectrom., 8 (1993) 615. [17] Standard Specification for Water, American Society for Testing and Materials ASTM, Philadelphia, 1977, Document 1193-77. [18] M.C. Rodriguez, J.M. Sanchez, H.S. Cubilhin and R.A. Romero, Ciencia, 2 (1994) 99. [19] S. Wallenstein, CL. Zucher and J.L. Fleiss, Circ. Res., 47 (1980) 1. [20] B. Ostle, Estadistica Aplicada, Limusa, 4th edn., Mexico, 1974, pp. 373-396. [21] V.A. Granadillo, H.S. Cubillan, J.M. Sanchez, J.E. Tah&n, E. Marquez and R.A. Romero, Anal. Chim. Acta, 306 (1995) 139-147. [22] C. Cabrera, M.L. Lorenzo, C. Gallego, M.C. Lopez and E. Lillo, J. Agric. Food Chem., 42 (1994) 126-128.