Comparison of flameless atomic absorption spectrophotometry and anodic stripping voltammetry for the determination of blood lead

MICROCHEMICAL

35, 70-82 (1987)

JOURNAL

Comparison of Flameless Atomic Absorption Spectrophotometry and Anodic Stripping Voltammetry Determination of Blood Lead S. COSTANTINI,* Laboratory

of Applied

R. GIORDANO, Toxicology,

Istituto

for the

AND M. RUBBIANI Superiore

di Sanitci,

Rome,

Italy

Received July 8, 1986; accepted September 17, 1986

INTRODUCTION

In relation to the known lead toxicity and function of blood lead concentration as exposure indicator, numerous methodologies (1, 2, 4-11) for its analysis have been developed. This work was addressed to a critical evaluation of analytical possibilities of the techniques currently most used for this analysis, such as anodic stripping voltammetry (ASV) and electrothermal atomic absorption spectrometry (ETA-AAS). Relating to the electrochemical technique, the possibility to use two ASV instrumentations, equipped with different kinds of graphite electrodes and based on different reading systems, allowed comparison between a digestion procedure and a direct method using a prepackaged decontaminated reagent kit. As far as atomic absorption spectrometric analysis is concerned, the use of stabilized temperature platform furnace, coupled with a suitable matrix modifier experimentally chosen among several combinations of reagents, allowed direct calibration with lead standard solutions which represented in the past a very critical point of this determination. The high accuracy of the methods proposed was demonstrated using reference standard materials by the agreement of results with the certified values. MATERIALS

AND METHODS

Reagents All reagents used were of guaranteed-reagent grade as follows: From Merck Suprapur: nitric acid 65% m/m, perchloric acid 70% m/m, sulfuric acid 96% m/m, hydrochloric acid 30% m/m, acetate buffer (sodium acetate 1 M + sodium chloride 0.2 M), magnesium nitrate. From Merck pro analysis: Tiiton X-100, ammonium phosphate monobasic, mercuric(I1) chloride. From ESA (Environmental Sciences Associated, Burlington, MA): Metexchange M Reagent (chromium chloride 1.07 wt%, calcium acetate 1.43 * To whom correspondence

should be addressed. 70

0026-265x/87 $ I .50 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

Pb DETERMINATION

wt%, mercuric 2.9-ml volume.

71

IN BLOOD

ion 0.0028 wt%) certified to contain less than 2.0 ng lead per Standard

Solutions

Lead nitrate standards prepared from a certified Chemicals. Lead standard Metexchange M Kit from ESA.

solution

1 g 1 -i Pb from BDH

Apparatus

ESA Model 2014 anodic stripping voltammetry analyzer equipped with composite graphite mercury electrodes (CGME) and an A-25 Varian recorder. ESA Model 3010 microprocessor stripping voltammeter equipped with a CGME electrode (high working speed) and a Watanabe X-Y recorder. Perkin Elmer Model 430 atomic absorption spectrometer equipped with a deuterium background system, a lead electrode-discharge lamp, and a Perkin Elmer HGA 500 graphite furnace equipped with an AS-40 autosampler. The signals from the spectrophotometer were registered on a Perkin Elmer Model 56 recorder. A digestion system including 20-mm digestion cells, digestion racks, and a thermoregulated hot-plate. Gas Supplies

High-purity

argon and nitrogen

were used as purge gases.

Cleaning

of Equipment

Glassware and plasticware were soaked for 24 hr in HNO, 10% v/v, repeatedly rinsed with doubly distilled water and then filtered-air dried. The digestion cells were cleaned by carrying out the typical digestion procedure adopted for the samples, using 2 ml of acid mixture. CGME

Coating

Procedure

As far as the ESA 2014 Model is concerned, the electrode surface preparation was performed according with Constructor’s Instructions. Mercury coating was carried out by soaking the electrode in 5 ml of 3.2 x lop3 M mercury plating solution and setting the plating potential at - 500 mV. Nitrogen was initially bubbled through for 2 min and the stirring was then stopped. The electrode was allowed to plate from a quiet solution for 3 hr, then nitrogen stirring was continued for 30 min to complete the mercury coating. This combination of plating solution concentration/plating time was chosen because it had previously given the best results (3) for sensitivity, reproducibility, and lifetime of the coating. The ESA 3010 electrode mercury coating was performed using a plating solution prepared in Metexchange reagent from ESA according with Constructor’s Instructions. Preparation

of Blood

Samples

Anodic Stripping Voltammetry Depending on the apparatus used, the ASV analysis for lead determination

in

72

COSTANTINI,

GIORDANO,

AND

RUBBIANI

blood can be performed in two different ways: (a) acid digestion of samples and determination in buffered solution (ESA 2014); (2) direct analysis using Metexchange M reagent without sample pretreatment (ESA 3010). Analysis after acid digestion. Blood samples were shaken thoroughly and the foam was allowed to settle. A 200~~1 aliquot of venous blood containing anticoagulant was pipetted into a digestion cell using a Gilson pipet and 500 ~1 of concentrate HNO,-HClO,-H,SO, (24 + 24 + 1) was added. After expelling the blood from the tip of the pipet (with the plunger still depressed) doubly distilled water was drawn up into the tip and this rinse solution was expelled into the sample analysis cell. Duplicate aliquots of the blood samples were usually taken. The sample was then placed in a digestion rack on a hot plate at IOO- 120°C and evaporated to about half its volume in lo-15 min. The temperature of the hot plate was then increased up to 220-250°C. After 20-30 min most of the acid had evaporated and approximately 100-200 ~1 remained in the bottom of the cell. At this point the digestion was stopped to prevent evaporation to dryness. After cooling, the sample was reconstituted with 4.5 ml of acetate buffer and then analyzed after the addition of 50 ~1 of concentrated perchloric acid. The pH value was about 4.9. Direct analysis. A lOO+l aliquot of venous blood was pipetted into a plastic vial containing 2.9 ml of Metexchange reagent using a Gilson pipet and, after 10 min, directly analyzed on the ESA 3010 voltammeter. Atomic Absorption

Spectrometry

Blood samples were diluted 1 + 9 in a solution containing NH,H,PO, and Mg(NO,), 0.05% w/v and then analyzed into the furnace. RESULTS Anodic

Stripping

The analysis was performed

AND DISCUSSION

Voltammetry-Analysis

after Digestion

under the following

Initial potential Plating potential Auto sweep hold Sweep rate Plating time Current range

0.1% w/v

-900 -900 -800 + 60 30 0.2

instrumental

conditions:

mV mV vs Ag/AgCl mV mV s-l min mA full-scale

Oxygen-free nitrogen was bubbled through the cell during the entire plating and stopped just before the stripping step. Calibration

Graph

In consideration of the lead concentrations expected in blood, the calibration graph was constructed by running different lead standards (0,20, 40, 60, 100, and 150 ng) prepared in acetate buffer at pH 4.9 under the same conditions used for the samples analysis. At these concentrations the contribution from Pb zero standard was negligible and each single point was taken to describe the calibration graph. Samples were run in duplicate interspersed with blanks and controls at the level of one blank and one standard for each ten analyses. Under the working

Pb DETERMINATION

IN BLOOD

73

conditions adopted, utilizing 200 ~.LAfull-scale, we found 1 PA = 0.98 ng of lead. The equation calculated by the linear regression method was Y = 1.027X - 0.034

Y = 0.9996.

The validity of this calibration method was verified by running some blood samples from unexposed subjects, spiked with different amounts of lead before the digestion procedure. In this way, the calibration straight line of peak intensity versus amount of lead, corrected for the blank (reagent lead content + endogenous lead), was coincident with that previously constructed by directly running lead standards. To verify the range of linearity for higher concentration of lead, a second calibration graph was constructed up to 500 ng using a suitable full-scale (1 mA) on the instrument. The response was still found linear and therefore the concentration range of standards could be increased, if necessary, in relation to a larger amount of metal in the samples. Interference Study

The effect of thallium as an interfering ion on the ASV determination of lead was studied under the chosen instrumental condition. Thallium forms a shoulder on the cathodic edge of the Pb peak. In samples where thallium could co-occur, i.e., in workers of certain ore smelting or roasting industries, the Pb peak must be observed for possible broadening. Interfering effects were evaluated by analyzing a 50-ng Pb standard spiked with different amount of interfering element. The results are given in Table 1. It can be seen that the effect of Tl on the peak of Pb was negligible until the concentration became nearly equal. From this point, the interference gradually raised with increasing amount added. The effect observed in this study was overcame without difficulty re-running the sample after the addition of 50 ~1 of I M EDTA to estimate the thallium. Anodic Stripping

Voltammetry-Direct

Analysis

The direct method without sample treatment required the use of a trace metal analyzer equipped with an electrode of different shape and speed response in comparison with that mounted on the instrument used in the method previously TABLE 1 Effect of Thallium on the ASV Determination Lead (50 ng Pb Standard) Thallium added (ng)

Lead found (m)

10 20 30 50 75 100

49.4 50.5 49.4 51.4 68.4 75.6

of

Error

% - 1.2 + 1.0 - 1.2 +2.8 +36.8 +51.2

(2All measurements were made in triplicate and results averaged.

74

COSTANTINI,

GIORDANO,

AND RUBBIANI

described (Fig. 1). .This instrument, equipped with a microprocessor, was calibrated by means of human blood lead standard solutions (low level 2 + 2 ng, high level 96 + 4 ng) from ESA prepared in Metexchange reagent, using the following formula: Optimum setting of calibration dial =

Assigned Hi level Calibration dial control value X setting used Digital reading during the run.

The response, given in pg/lOO ml, was read directly on the digital readout. Since the measure is based on the peak area obtained by electronically integrating the stripping current, the integration set point value (ISP, volts) was found to be a critical parameter for a correct measure of the metal. In fact, the optimum value of the ISP can differ for the same metal in two different matrices and may even differ for the same metal and matrix from day to day, depending on the surface electrode coating and maintenance. For this reason, after each electrode coating, an accurate “integration set point study” was done using the digital readout on the instrument to determine the correct ISP value. During the experiment this value was checked every day. An example of this study (ISP versus digital response) is shown in Fig. 2. The instrument conditions usually adopted were the following: Initial potential Final potential Sweep rate Recorder set point Integration set point Analysis time Scale expansion

s==

- 1.085 v -0.130 v 14 mV/step - 0.700 v -0.465 V 1 min Xl

Nitrogen

1 Test electrode 2 Counter electrode 3-Reference

electrode

4- Ce// head 5 Counter electrode compartment 6. Reference compartment

electrode

1 -Counter Z-Reference 6

7

electrode electrode

3-Stirring I.- Cell 5-Counter

bar head electrode

6-Reference electrode ‘I-Test electrode B-Stirring

wire wire

(platinum) (Ag/AgCi)

propeller

0. Bubbler

FIG. 1. Electrodes for ASV determination. Model Analyzer (direct method).

(A) 2014 Model Analyzer (digestion method). (B) 3010

Pb DETERMINATION

IN BLOOD optimum

isp

75 setting

100~

0 z : 2 L

50-

-G .. 9 a

i / (+)

O-

C-1

: /

-0.465

-

Integration set point ,vo/ts, FIG. 2. Integration

set point study in ASV determination

(direct method).

X-Y recorder tracings obtained for low and high level blood standards are shown in Fig. 3. Interference Study

The application heparin-stabilized

of the direct method, using the Metexchange reagent, requires blood. It is known that chelating agents in blood compete with

FIG. 3. ASV determination (direct method): X-Y recorder tracing for low- and high-level blood lead standards. Low level: 2 k 2 ng; high level: 96 2 4 ng.

76

COSTANTINI,

GIORDANO,

AND RUBBIANI

binding sites in the red blood cells for lead and establish an equilibrium with a portion of lead bound by the chelate. Although Metexchange reagent was designed to quickly displace lead from the associations commonly formed with the red blood cells, the exchange rate with EDTA is very slow. To evaluate this effect, a blood sample having an endogenous lead content of 0.08 mg liter-r (determined by ETA-AAS) was used to prepare heparin-stabilized samples and EDTA samples, respectively, spiked with 50 ng lead standard. The EDTA sample was analyzed just after dilution in Metexchange reagent, after 10, 20, 30, 40, 50, 60 min and also after the addition of 100 ~1 of 1O-2 g ml-’ of nickel prepared in 0.04 M HCl. In the last case, the same addition of Ni was done to the low- and highlevel controls too. Results are reported in Table 2. It can be seen that results for heparin-blood and EDTA-blood become equal after 50 min. Alternatively, the addition of Ni was found to be effective in accelerating the lead/Metexchange ion exchanges. Graphite Furnace Atomic Absorption

Spectrometry

In recent years a great number of reports (1,2,5-9) have appeared in literature describing the determination of blood lead by ETA-AAS with and without sample treatment. Although the high sensitivity of graphite furnace atomization often allows the determination of trace elements in biological samples without sample treatment and by simple dilution with a suitable diluent or matrix modifier, the determination of blood lead shows some problems due to the blood matrix complexity and sample contamination. Usually, for an accurate calibration, aqueous standards cannot be used and the standard addition method is commonly em-

TABLE 2 Effect of EDTA on the ASV Determination of Blood Lead (Endogenous Lead Content: 8 ng; Added Lead Standard: 50 ng) Direct Method” Sample Heparin-stabilized blood EDTA-stabilized blood Soon after dilution in Metexchange reagent After 10 min 20 min 30 min 40 min 50 min 60 min Soon after the addition of 100 ~1 10e2 g ml-’ Ni in 0.04 M HCl

Lead found hit)

Error %

57

- 1.72

39 40 43 49 53 58 57

- 32.7 -31.0 -25.8 - 15.5 -8.6 0.0 - 1.72

59

+ 1.72

n Results were made in triplicate and averaged.

Pb DETERMINATION

ployed. In our work, blood was directly diluted experimentally chosen among the following: (1) (2) (3) (4) (5) (6)

Triton-X Triton-X Triton-X Triton-X Triton-X Mg(NO,),

77

IN BLOOD

(1 + 9) with a matrix

100 (from 0.01 to 1% v/v) 100 0.01% + NH,H,PO, 0.35% 100 0.1% + H,PO, 0.2% 100 0.1% + HNO, 0.5% 100 0.1% + Mg(NO,), 0.025% + NH,H,PO, 0.05% + NH,H,P04 0.1%

modifier

0.1%

The best results, as far as sensitivity and peak shape are concerned, were obtained with the last combination. The dilution ratio (1 + 9) represented the best compromise between sensitivity of analysis and the need to avoid a build-up of deposit on the platform. To set up the best analytical conditions for the drying and ashing steps, some preliminary measurements were made. Temperature and duration of the drying period were found to be crucial factors to avoid splattering. Visual inspection during the drying stage was advisable to check sample behavior. Figure 4 shows the absorbance as a function of the charring and atomization temperatures for a blood sample containing 50 kg liter-’ lead. For a lo-p,l sample injection the optimum charring temperature was 650°C which represented the maximum still utilizable without loss of analyte and still able to prevent deposit formation. The atomization temperature was fixed at 2200°C. However, in the range 2000-22Oo”C, sensitivity was found to be independent on the temperature of atomization. The instrumental settings for subsequent analyses were the following: Wavelength (nm) Spectral bandwidth (nm) EDL lamp source (watt) Background correction Sample aliquot (~1) Purge gas Signal mode Graphite tube The furnace program was established Step Step Step Step Step Step Step

1 2 3 4 5 6 7

Drying I Drying II Ashing I Ashing II Atomization Cleaning Cooling

283.3 0.7 10 deuterium lamp 10 argon UPP absorbance, peak height pyrocoated with 1’Vov platform as follows: 90°C 130°C 650°C 650°C 2200°C 2650°C 20°C

10 s ramp 30 s 30 s 1s 0s 1s 1s

4 s hold 10 s 50 s 11 s 8s 6s 20 s

The argon flow used to purge the tube during a run (flow 300 ml min-l) was stopped in the second ashing step and during the atomization stage which was performed in the “maximum power” mode. As expected, calibration with

78

COSTANTINI,

GIORDANO,

AND RUBBIANI 0

A

0.2 i

0.1

:T

8"8-k

I,,

, Loo

, , 600

, , , $ , , , , 800 lwo 16w l&m Temperature

, zoo0

,

, 2200

,

, ZLCO

OC

FIG. 4. Flameless atomic absorption determination: The absorbance as function of the charring (A) and atomization (B) temperatures. Blood sample containing 50 pg liter-’ lead.

aqueous standard solutions was found not to be adequate, as shown in Fig. 5. On the contrary, a calibration graph constructed by running lead standards (0, 10, 20, 30, 50, 70 ng liter-‘) prepared in Mg(NO,), 0.05% + NH,H,PO, 0.1% was coincident with that obtained analyzing a blood sample diluted I + 9 in the same matrix modifier and spiked with the same amounts of lead. Each value, taken to describe the calibration graph, was always corrected for the blank (Pb zero standard) .

FIG. 5. Flameless atomic absorption determination: (A) and blood matrix (B).

Calibration curves for lead in aqueous matrix

411

784

416

772 Theoretical value I-% I-’ 194 310 650

190 321 660

132

126

Found (I% I-‘)

5.9 4.7 4.1

4.2

5.0

5.4

C.V. (%)

97.9 103.5 101.5

101.5

98.7

104.7

198 322 638

624

346

76

Found (I.% I-9

6.8 5.9 4.9

5.9

7.4

11.2

C.V. (%)

102.1 103.8 98.1

80.8

83.1

60.3

Recovery (%o)

ASV direct method

was made in triplicate and averaged

Recovery (%)

ASV digestion method

a Each value represents the mean of five analyses; each determination

I 2 3

Bovine blood matrix BCR Reference Material n. I94 BCR Reference Material n. 195 BCR Reference Material n. 196 Human blood matrix

Sample

Certified lead value (I# I-‘)

TABLE 3 Accuracy and Precision0

190 314 661

763

409

128

Found b% I-‘)

4.7 4.0 3.9

3.8

3.7

4.1

C.V. (%)

ETA-AAS

97.9 101.2 101.7

98.8

98.3

101.5

Recovery (%)

F

2 5 gj z ?

E $

2 g -1

COSTANTINI,

80 The equation

calculated

GIORDANO,

AND RUBBIANI

from the calibration

curve was

Y = 3.24 x 1O-3X - 5.71 x 1O-4 Performance

r = 0.9993.

Tests

To evaluate accuracy and precision (Table 3) all of the methods described were applied to certified samples of bovine blood from BCR (Bureau Central Reference, CEE) and a number of human blood samples spiked with suitable amounts of lead. In particular, the preparation of spiked human blood samples was necessary for the evaluation of the ASV direct method because the calibration was made, as required, by lead human blood standards from ESA. In fact, as it is possible to see, the values obtained for bovine blood matrix were of poor accuracy whereas good results were found for human blood matrix. For the ASV digestion method and ETA-AAS one, simple linear regression equations were calculated between theoretical values (independent variable, X value) and found values (dependent variable, Y value). The results were as follows: ASV digestion method:

Y = 1.0145x - 0.9735

ETA-AAS:

Y = 0.9977x

+ 0.4591

P r P r

< = < =

0.001 0.9997 0.001 0.9996.

A correlation plot of values for 31 samples from exposed subjects, by ETA-AAS method versus ASV direct method is reported in Fig. 6 Detection limits were calculated as the concentration that gives a value equal to three times the value of

ETA

FIG.

6. Correlation

plot by ETA-AAS

-AAS

lead

pg/dL

versus ASV direct method. Samples from exposed people.

Pb DETERMINATION

IN BLOOD

81

TABLE 4 Detection Limits Method

Detection limit (+g I-’ lead)

ASV digestion method ASV direct method ETA-AAS

15 21 7

the background standard deviation (based on ten determinations) obtained by running the blank reagents. The values, referred to original samples before treatment or dilution, are given in Table 4. CONCLUSIONS

Although general results indicate that all techniques used are suitable for the determination of lead in blood, some considerations can be done. Atomic absorption technique is characterized by better detection limit and precision. However, the largest difference in precision occurs mainly at low nontoxic lead levels and the detection limit values of the ASV techniques are not critical parameters for a good determination also at low levels, such as those expected in unexposed people. In any case, atomic absorption technique might be preferred for infant blood analysis. As far as the comparison between the ASV methods is concerned, accuracy for human blood is comparable, whereas the digestion procedure seems to show a better precision. However, a rapid analysis and minimum sample contamination risk are practical advantages making the direct method particularly suitable when a large number of analyses are required. SUMMARY

Three different procedures, based on electrothermal atomic absorption spectrometry (ETA-AAS) and anodic stripping voltammetry (ASV) methods for blood lead are presented. Atomic absorption procedure is simple and quite rapid using a dilution with a matrix modifier as the only sample preparation. An easy digestion method of samples and a quicker direct one are adopted in the electrochemical determinations. Precision and accuracy are evaluated by blood samples from reference standard materials. Detection limit values demonstrate the applicability of the methods to lead blood determinations. REFERENCES 1. Brodie, K. G., and Routh, M. W., Trace analysis of lead in blood, aluminium and manganese in serum and chromium in urine by graphite furnace atomic absorption spectrometry. C/in. Biochern. 17, 19-26 (1984). 2. Corradetti, E., Mannozzi, A., and Marinelli G., Dosaggio di piombo e cadmio nel sangue per spettrofotometria di assorbimento atomic0 con fornetto di grafite:metodo diretto. B&l. Chim. Igien. 35, 395-403 (1984). 3. Costantini, S., Giordano, R., Rizzica, M., and Benedetti, F., Applicability of anodic stripping

82

4. 5. 6. 7. 8. 9. 10. Il.

COSTANTINI,

GIORDANO,

AND RUBBIANI

voltammetry and graphite furnace atomic absorption spectrometry to the determination of antimony in biological matrices. A comparative study. Analysr 110, 1355- 1359 (1985). Khandekar, R. N., and Mishra, U. C., Determination of lead, cadmium, copper and zinc in human tissues by differential pulse anodic stripping voltammetry. Fresenius Z. Anal. Chem. 319, 577-580 (1984). Lagesson, V., and Andrasko, L., Direct determination of lead and cadmium in blood and urine by flameless atomic absorption spectrophotometry. C/in. Chem. 25/11, 1948-1953 (1979). MacLeod, K. E., and Lee, R. E., Selected trace metal determination of spot tape samples by anodic stripping voltammetry. Anal. Chem. 45(14), 2380-2383 (1973). Nise, Cl., and Vesterberg, O., Blood lead determination by flameless atomic absorption spectroscopy. Clin. Chim. Acta 84, 129-136 (1978). Paschal, D. C., and Bell, C. J., Improved accuracy in the determination of blood lead by electrothermal atomic absorption spectrometry. At. Spectrosc. 2(5), 146-150 (1981). Pruszkowska, E., Carnrich, G. R., and Slavin, W., Blood lead determination with the platform furnace technique. A?. Spectrosc. 4(2), 59-61 (1983). Searle, B., Chan W., and Davidow, B., Determination of lead in blood and urine by anodic stripping voltammetry. Clin. Chem. 19(l), 76-80 (1973). Wang, J., Stripping analysis of trace metals in human body fluids. J. Electroanal. Chem. 139, 225-232 (1982).