Determination of Lead in Blood and Urine by Anodic
Stripping Voltammetry
Bernard Searle, Wing Chan, and Bernard Davidow
An anodic stripping procedure for determining lead
in blood and urine was evaluated, and methods of
calibration were developed. The technique requires
as little as 50 tl of blood or 0.5 ml of urine.Comparisons were made between results obtained by anodic
stripping voltammetry, for finger-puncture
blood, and
by atomic absorption spectrophotometry,
for venous
blood from 200 children. The two methods were also
compared with respect to lead in urines from patients hospitalized for suspected lead poisoning and
in urines from normal healthy volunteers. Major advantages of the anodic stripping method are its increased sensitivity and lower cost as compared to
many current methods for detecting and measuring
lead.
Additional Keyphrases: environmental hazards
lead
poisoning
atomic absorption spectrophotometry
compared
Pb in capillary and venous blood
ultramicromethods for trace metals
.
-
The analysis for lead in biological tissues was always considered
one of the more difficult clinical
laboratory
procedures,
which require large amounts
of blood or urine. The dithizone method developed in
1942 by Bamback and Berkey (1) was intended as a
micro-scale
method, but required as much as 10 ml
of blood. The polarographic
procedure of Nylander
and Holmquist
(2) required only 5 ml of blood, but
its precision was rather low and the technique
was
lengthy and complex.
In i968, Hessel (3) developed an atomic absorption
method that required only i-S ml of blood. The procedure is relatively simple and is very precise. More
recently Kahn et al. (4) developed an atomic absorption “Sampling
Boat Technique,”
in which whole
blood could be examined without pretreatment.
Fernandez
and Kahn (5) improved
upon the latter
method with the development
of the “Delves Sampling Cup” technique,
which requires only 10 tl of
blood. In this procedure the cup has to be replaced
after analysis of about 35 samples.
More recently, very sensitive flameless atomic-absorption techniques
(6, 7) have been developed. They
involve electrothermal
heating of the sample to produce atomic lead vapor. Although initial results appear promising, the method requires further study.
Anodic stripping voltammetry
has been recognized
From The New York City Department of Health,
Ave.,New York,N.Y. 10016.
ReceivedAug. 16,1972;accepted Oct.30,1972.
76
CLINICAL CHEMISTRY, Vol. 19, No. 1, 1973
455 First
for many years as one of the more sensitive techniques for metal analysis. It depends on concentrating metal ions on a carbon electrode by electrodeposition from solution. It is not necessary to carry the
plating step to completion,
because a reproducible
portion of the sample can be deposited
in a fixed
time interval. Reversal of the current causes the element to be stripped from the electrode, which gives
rise to a peak current measurable
on a recorder. Nanogram quantities
of material can be analyzed rapidly with relatively
good precision. The use of the
method for analysis of lead in blood was proposed by
Matson (8) in 1970. We therefore undertook a study
to determine
its application
to the ultramicroscale
analysis of lead in blood and urine.
Materials and Method
Equipment
Anodic Stripping Voltammeter,
Model 2014 (Environmental
Science
Associates,
Burlington,
Mass.
01803. Heating blocks (Environmental
Science Associates). Strip Chart Recorder, Model G2500 (Varian
Aerograph,
Walnut Creek, Calif. 94598). Eppendorf
pipet (0.05 ml) (Brinkmann
Instruments
Inc., Westbury, N.Y. 11590). Atomic Absorption
Spectrophotometer, Model 403 (Perkin Elmer Corp., Norwalk,
Conn. 06856).
Reagents
Perchloric
acid, 70%, doubly vacuum-distilled,
lead free (G. Fredrick Smith Chemical Co., Columbus, Ohio 43223). All-glass doubly-distilled
water.
“Prepurified”
grade nitrogen, in tank equipped with
two-stage
regulator
(Matheson
Co., East Rutherford, N.J. 07073). Lead nitrate, Certified ACS (Fisher Scientific Co., Springfield, N.J. 07081).
Anodic Stripping Method of Analysis
Calibration for Blood
and
Blood specimens, obtained by finger puncture with
a sterile lancet, were aspirated into a 0.05-ml Eppendorf pipet, and immediately
expelled into the 20-ml
cells supplied with the Model 2014 Anodic Stripping
Voltammeter.
The cells were covered with Parafilm,
and could be shipped to the laboratory in this condition without fear of spilling the sample. After the
specimens
reached the laboratory,
0.2 ml. of 70%
perchloric
acid was added to the cells, which were
then heated on a hot plate in metal heating blocks
for 30 mm. The temperature
of the hot plate was ad-
justed so that condensate
did not extend more than
half way up the wall of the test tube. At the end of
this period the heating blocks were removed from the
hot plate, the cells allowed to cool for several minutes, and 4 ml of distilled water was added. The cells
were then placed on the plating module. A negative
potential,
-800 mV, was applied for a period of 20
mm, to concentrate
the lead ions on the electrode.
After the plating period, the metal was “stripped”
off the electrode. The stripping gives a sharp current
peak, proportional
to the concentration
of the metal
in solution.
Oxygen-free
nitrogen
was bubbled
through the cells during the entire plating and stripping period at a pressure of 41.4 kPa (6 lb/in.2).
The instrument
was calibrated
by analyzing four
aqueous standards
containing 0.05 ml of lead nitrate
(100 pg of lead per 100 ml of water), 0.2 ml of 70%
perchloric acid, and 4 ml of water. After plating for
20 mm and then stripping,
the four peak heights
were averaged and used in formula 1 (below) to obtain the blood factor required in subsequent
calculations. A reagent blank containing
water instead of
the lead solution was treated in the same way, and
was also run in quadruplicate.
The average of the
four blanks was subtracted
from the average of the
four standards
in formula i to obtain the blood factor.
Blood factor = 100/(Av peak height of stds.
Av peak height of reagent blank) (1)
-
Formula 2 was used to calculate pg of lead
ml of blood. The product of the peak height
sample and the blood factor equals the lead
pg/100 ml of blood, after correcting for the
blank.
pg lead/100 ml
per 100
of each
value in
reagent
blood factor X
(Peak height of sample
Av peak height of reagent blank)
blood
L
Blood
(Samples
L......Reagent
Blanks
Fig. 1. Recorder
tracings
for anodic stripping analysis of
Quadruplicate runs are shown for the aqueous lead standard (0.5 ml
lead. 100 pg lead/100 ml water), reagent blank. and blood sample. The
first peak is lead, measured in microamperes. The second and third
peaks are copper and mercury, respectively,neither of which were measured
standards
containing
0.5 ml of lead nitrate (20 pg
lead/iOO ml water) plus 0.4 ml of perchloric
acid.
Four reagent-blanks
were also run, consmstmng of 0.5
ml of water plus 0.4 ml of perchloric acid. The four
peak heights of the reagent blanks were averaged,
and this value was subtracted
from the average of
the four standards.
The urine factor was calculated
as shown in formula3.
Urine factor = 20/(Av peak height of stds.
Av peak height of reagent blanks)
The pg lead/i00
(2)
in formula
and Calibration
Standards
I
-
Figure 1, a copy of one of the recorder tracings,
shows in quadruplicate
the peak heights,
in microamperes, of a standard,
a reagent-blank,
and a
sample. There are three peaks for most of the tracings. The first peak is due to lead, the next to copper, and the third to mercury on the electrode; the
latter peak is an inherent function of the electrode,
and will vary widely in height, depending
on the
exact potential of the reference electrode in the cells.
We measured
only the height of the lead peak, in
microamperes,
by counting
the number of spaces
that the pen traversed on the cross-hatched
recorder
paper.
of Analysis
L......Aqueous
lead
=
-
Method
..__J
for Urine
As in the procedure
for lead in blood, the 20-ml
cells were placed on the hot plate in heating blocks
after 0.5 ml of urine and 0.4 ml of 70% perchloric
acid had been added. After a 30-mm digestion, the
tubes were cooled and 4 ml of distilled water was
added. The plating time was also 20 mm.
The instrument
was calibrated
by analyzing four
pg
lead/100
ml urine was calculated
(3)
as shown
4.
ml
= urine factor X
(peak height of sample
Av peak height of reagent blanks)
urine
-
(4)
Results
Lead nitrate solutions in concentrations
of 30 to
800 pg of lead per 100 ml of water were analyzed by
anodic stripping;
results were linear over this wide
concentration
range (Figure 2).
We also compared the variation in values for blood
lead as obtained
by atomic absorption
(3) and by
anodic stripping voltammetry.
In this study we used
four different blood samples that had been shown by
atomic absorption
to contain 23, 33, 53, and 83 pg of
lead per 100 ml of blood. For atomic absorption
analyses 3.5 ml of blood was used, whereas for anodic stripping 50 and 100 pl of blood was used.
Table
1 summarizes
the results,
showing
the
amounts of lead recovered and the standard
deviation of the replicate samples. It also shows that the
coefficient of variation was largest for the low, nontoxic concentrations
of blood lead.
CLINICAL CHEMISTRY,
Vol.19, No. 1, 1973
77
Table 1. Comparison of Results for Blood Lead,
as Determined by Atomic Absorption
and by Anodic Stripping
No
samples
Total
Pb re-
Pb
present
covered,
av
%
Vol
of
blood,
ml
5.4
6.0
3.5
3.5
CV,
SD
pg/laoml
Atomic absorption
12
12
23
33
17
35
0.91
2.09
12
12
53
83
53
0.91
1.7
3.5
85
2.24
2.6
3.5
23
23
33
33
29
30
34
32
2.26
4.22
1.13
2.40
7.8
14.1
3.3
7.5
0.10
0.05
0.10
0.05
53
57
2.27
4.0
0.10
53
83
58
85
3.16
4.10
5.4
4.8
0.05
0.10
83
83
2.49
3.0
0.05
Anodic stripping
12
12
12
12
12
12
12
12
Lied.
jig/tOOmI of Wat.r
Fig. 2. Demonstration of the linearityof the anodic stripping method for lead analysis over a wide concentration
range
Table 2. Comparison of Results by Anodic
Stripping (ASV) and Atomic Absorption (AA)
Techniques for Measuring Lead in Venous
Blood Specimens’
ASV
AA
pg
Deviation
ASV
Pb/100 ml
56
54
48
65
43
43
19
12
29
28
52
41
20
17
37
17
26
29
57
45
20
16
20
13
AA
Deviation
pg Pb/i 00 ml
+8
67
76
39
42
0
+7
+3
23
29
20
53
29
22
54
37
-5
15
71
41
16
69
37
26
28
65
55
77
58
-11
-4
0
+1
+17
+4
Av. deviation (ASV-AA)
=
-0.9
an = 24 (different specimens).
-9
-3
-6
-2
-8
+2
+4
-2
-12
-3
pg Pb/100 ml blood
_______
___________
In Table 2, results for atomic absorption
and anodic stripping voltammetry
are compared for 24 different venous-blood
samples. The average deviation
between the two different types of analysis is 0.9
pg/100 ml blood. A paired t-test (9) showed that the
results were not significantly different (P <.05).
We studied the blood of 200 children. Both capillary (finger-puncture)
and venous blood was sampled
from each child, and analyzed, respectively,
by anodic stripping and atomic absorption
analyses. The
volume of capillary blood used for anodic stripping
analysis was 50 p1, and of venous blood for atomic
absorption analysis, 3.5 ml.
Figure 3 is a scatter diagram comparing the results
of the two methods, and Table 3 gives a frequency
distribution
of the absolute differences. The average
deviation between the two methods is 1.1 pg of lead
per 100 ml of blood. The data for the 200 children by
anodic stripping
were 2.1 pg of lead per 100 ml of
blood lower than the atomic absorption
results. A
78 CLINICAL CHEMISTRY. Vol.
19, No. 1, 1973
40
of Blood
Spectrophotometry(AAS)
Lead,pg/IOOml
Atomic
Absorption
Fig. 3. Scatter diagram comparing the results of anodic
strippingand atomic absorption analysis
paired t-test showed that this difference was insignificant (P <.05). The correlation
coefficient
(10) is
high (r = 0.87), which indicates the close relationship between results obtained by the two methods.
Results for lead in urine were also compared,
by
anodic stripping
and atomic
absorption.
In the
atomic absorption
procedure,
100 ml of urine was
adjusted
to pH 2.5 with glacial acetic acid. Five
milliliters of ammonium
pyrrolidmne dithiocarbamate
(APDC) solution (2 g/100 ml of water) and 20 ml of
methyl isobutyl ketone (MIBK) were added. The
mixture was throughly shaken in a 250-ml separatory
funnel, allowed to stand for 10 mm, and the lower
layer discarded.
After centrifugation
for 5 mm at
2500 rpm, the organic solvent was aspirated into the
atomic absorption spectrophotometer.
Standards
consisted of 0.00, 0.03, 0.06, and 0.09
mg of lead per 100 ml of water. These were similarly
extracted at pH 2.5 and measured in the atomic ab-
Table 3. Frequency Distribution: Differences
between Results of Anodic Stripping and
Atomic Absorption Methods for
Determining Blood Leada
Deviation
between two
methods,
pg Pb/l00 ml
0
+1
Frequency
15
9
11
+2
-2
+3
-3
9
12
8
10
9
3
+4
-4
+5
7
-5
10
+6
-6
7
9
3
8
+7
-7
+8
5
-8
+9
-9
11
2
6
3
+10
-10
5
+11
-11
6
2
3
4
+12
-12
+13
-13
+14
-14
Frequency
In percent
7.5
Deviation
between two
methods,
pg Pb/i 00 ml
4.5
5.5
4.5
6.0
4.0
5.0
4.5
1.5
3.5
5.0
3.5
4.5
1.5
4.0
2.5
5.5
+15
-15
+16
-16
+17
-17
+18
-18
+19
-19
+20
-20
+21
-21
+22
-22
1
4
1
2
0
0.0
1.0
0.0
1.0
0.5
0.0
0.5
0.5
0.5
0.0
0.0
0.0
Frequency
2
0
2
1
0
1
1
1
0
0
0
1.0
+23
0
3.0
1.5
2.5
-23
+24
0
0
-24
0.5
3.0
1.0
1.5
2.0
0.5
0.5
0.5
+25
-25
+26
-26
+27
0
2
0
0
0
0
0
1
0
-27
+28
-28
Frequency
In percent
of the
anodic
ASV
AA
pg/lOO ml
0.5
0.4
1
2.0
0.6
0.5
I.0
1
1
1.0
0.0
0.0
0.0
0.0
1.0
0.0
0.0
0.0
0.0
0.0
0.5
0.0
Discussion
advantage
Leadadded
1
1
sorption spectrophotometer
to provide a calibration
curve.
Table 4 shows a comparison
of anodic stripping
and atomic absorption results for urine to which lead
was added in amounts covering the normal and toxicologic range. Except for the low, nontoxic lead concentration,
recoveries for the anodic stripping
and
atomic absorption methods correlated well.
Table 5 shows a comparison
of results by anodic
stripping
and atomic absorption,
for urine of patients hospitalized
for suspected lead poisoning. The
concentration
of lead found was about the same by
both methods.
In Table 5 are compared
the results of anodic
stripping
and atomic absorption,
for urine from 10
normal, healthy laboratory
personnel. In this study,
10 ml of MIBK was used instead of 20 ml, because of
the low concentration
of lead present. The smaller
volume of solvent resulted in greater precision, and
thus in better correlation
between the methods at
the low normal concentrations.
major
Lead recovered
0.4
0.6
#{176}In
200 children.
The
Table 4. Comparison of Results by Anodic
Stripping (ASV) and Atomic Absorption (AA),
for Urine with Added Lead
stripping
6
6
6
6
6
6
6
6
6
5
6
6
6
6
6
12
11
11
12
10
12
10
11
11
12
10
11
12
12
11
25
25
25
26
28
25
26
26
26
25
25
25
25
50
26
51
25
50
50
50
50
48
49
51
49
50
49
50
92
92
93
102
101
101
101
101
50
50
100
100
100
100
100
95
102
Table 5. Comparison of Anodic Stripping
Voltammetry and Atomic Absorption Analysis
for Lead In Urine
Anodic stripping
Atomic
pg Pb/l00
absorption
ml
Patients hospitalized with suspected lead poisoning
16.0
16.0
19.0
20.0
23.0
23.0
2.6
5.0
6.8
8.0
23.0
13.0
19.0
12.0
Healthy volunteers
2.3
2.4
3.6
0.4
4.7
0.7
1.1
1.1
0.7
1.3
1.1
1.1
2.2
2.1
1.2
1.4
0.7
1.7
1.4
CLINICALCHEMISTRY,
1.1
Vol.19. No. 1. 1973
79
method is its increased sensitivity as compared to
the dithizone procedure of Bamback and Berkey (1)
and the ammonium
pyrrolidine-methyl
isobutyl ketone procedure
of Hessel (3). In addition,
repeated
analyses are facilitated,
because lead is not lost during processing.
The decreased
cost of instrumentation and specimen collection adds a financial benefit
(e.g., the anodic stripping device costs about half as
much as the double-beam
atomic absorption
instrument we used).
In the replicate studies performed with blood, the
relative standard deviation was somewhat higher for
the anodic stripping method than for the atomic absorption technique
of Hessel. However, the largest
difference in precision occurred mainly at low, nontoxic lead concentrations.
Comparison
between the methods on 24 different
venous blood samples showed the results were not
significantly, different. Similarly,
the work with 200
children,
in which fmger-puncture
blood was analyzed by anodic stripping voltammetry
and venous
blood by atomic absorption,
showed no statistically
significant difference.
Studies on urine showed good correlations between
the two methods in both recovery experiments,
and
in comparative
analyses on urine containing
lead in
either normal or toxicologic amounts.
Although the anodic stripping techniqt,ie requires a
larger volume of blood than the Delves atomic absorption technique (5) (50 pl for anodic stripping vs.
10 pl for the Delves) the collection of 50 pl of blood
by finger puncture
does not present
any greater
problem than the collection of 10 pl when microcapilary tubes are used for this purpose. It is expected
that the precision would be greater in collecting the
larger volume of sample.
The skill required of the technicians
is about the
same for the anodic stripping
voltammetry
method
and the ultramicroscale
absorption
techniques.
The
increased
sensitivity
and lower cost of the anodic
stripping
voltammetry
instrument
as compared
to
the atomic absorption
spectrophotometer
used in
this study makes the anodic stripping voltammetry
method
appropriate
for laboratories
that receive
80
CLINICAL CHEMISTRY,
Vol.19,No. 1,1973
fewer than 25 specimens
per day. The somewhat
greater error of replication
of the anodic stripping
voltammetry
can be compensated
by analyzing duplicate specimens.
For laboratories
that receive fewer than 100 specimens per day, the ultramicroscale
atomic absorption
techniques
appear suitable.
To process more than
100 specimens
per day, we think the macroscale
atomic absorption technique of Hessel is most convenient. We think that all of these techniques
will
eventually undergo further modifications
to improve
sample handling and to decrease analysis time.
We are indebted
to Miss Chi-Fang
Shih, for statistical
evaluation of results.
The work on which this publication
is based was performed
pursuant to Contract No. 68-03-0004, Environmental Health Service, Environmental
Control Administration,
USPHS, Dept. of
HEW.
References
1. Bamback,
K., and Berkey, R. E., Microdetermination
of lead
bydithizone. md. Eng. Chem. 14, 904(1943).
2. Nylander,A.,and Holmquist,C.,Polarographicdetermination
of lead in blood.A.M.A.
Arch. md. Hyg. Occup.
Med. 10, 183
(1954).
3. Hessel,D. W., A simple and rapid determination of lead in
blood. At. Absorption
Newslett.
7,55 (1968).
Kahn, H. L., Peterson, G. E., and Shallis, J. E., Atomic absorption microsampling with the “sampling boat” technique. At.
Absorption
Newslett.
7, 35 (1968).
4.
5. Fernandez, F. J., and Kahn, H. L., Determination of lead in
whole blood by atomic absorption with the “Delves sampling
cup” technique.At. Absorption Newslett.
10,1(1971).
6. Amos, M. D., Bennett, P. A., Brodie, K. G., Lung, P. W. Y,
and Matousek, J. P., Carbon rod atomizer in atomic absorption
and fluorescence
spectrometry
and its clinical
application.
Anal.
Chem. 43, 211 (1971).
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L., Microdetermination
of lead in blood by flameless atomic absorption spectrometry. Anal. Chem. 43, 1319(1971).
8. Matson, W. R., Griffin, R. M., and Schreiber, G. B., Rapid
subnanogram simultaneous analysis of Zn, Cd, Pb, Cu, Bi. In
Trace Substances
in Environmental
Health,
4, D. Hemphill,
Ed.
University of Missouri, Columbia, Mo., 1971, pp 396-406.
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Chemistry:
Principles
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Iowa State University Press, Ames, Iowa, 1967, p 172.