89

S
A
Atomic Spectroscopy
Vol. 19(3), May/June 1998
INTRODUCTION
Large quantities of toxic
elements such as cadmium, lead,
nickel, and chromium have been
released to the environment in past
decades and exposure to these
elements may pose a health risk to
populations living in or near indus-
trially polluted areas. Long-term
studies on changes in health status
and the quality of the environment
cannot be successfully conducted,
however, unless reliable and com-
prehensive reference data on envi-
ronmental trace element levels are
established (1). Unfortunately, valid
reference values for many toxic and
carcinogenic metals are lacking or
inadequate. In this respect, the
EUROTERVIHT project (Trace Ele-
ment Reference Values in Human
Tissues) aims to establish and com-
pare trace element reference values
(baseline values) in biological fluids
and tissues from unexposed popula-
tions within different regions of the
European Union (2). The reference
values have a key role in the verifi-
cation or reconsideration of legal
limits of exposure to protect the
general population and in the estab-
lishment of appropriate monitoring
strategies for occupationally
exposed groups (3).
For acquired data to be consid-
ered acceptable for establishing
reference values, rigorous control
of pre-analytical and analytical fac-
tors must be applied (4,5). This
entails the use of well-characterized
materials for sample collection
and analysis (6), validated and docu-
mented sampling procedures and
validated analytical methods with
comprehensive quality assurance
procedures. Electrothermal atomic
absorption spectrometry (ETAAS)
has long been the preferred analyti-
cal method for the routine determi-
nation of trace metals in biological
fluids owing to its reliability, sensi-
tivity, and relatively low cost (7).
A major limitation with current
instrumentation, however, is the
single-element capability. Multi-
element studies become costly in
terms of analyst and instrument
time, while the risk of contamina-
tion is greatly magnified by repeated
sample manipulation.The recent
introduction of simultaneous multi-
elemental electrothermal atomic
absorption instrumentation offers
the opportunity to overcome these
limitations (8).
In this paper, methods are
described for the simultaneous
determination of chromium and
nickel in serum, and the simultane-
ous determination of cadmium and
lead in both whole blood and urine
matrices. With the selection of
compatible elements and the
careful optimization of instrument
conditions, these elements can be
determined with minimum sample
pre-treatment and with sufficient
sensitivity to establish reference
values for healthy unexposed
populations.
EXPERIMENTAL
Instrumentation
All analyses were performed
using a Perkin-Elmer SIMAA

6000
atomic absorption spectrometer
(Perkin-Elmer, Norwalk, CT USA)
with transversely heated graphite
furnace and longitudinal Zeeman-
effect background correction. All
automated dilutions and injections
were made with a Perkin-Elmer
AS-72 autosampler, fitted with an
80-position tray and microdispenser
capable of delivering 0.1-mL
volumes. The spectrometer and
autosampler were controlled
by AA Winlab

software,
(Perkin-Elmer, Norwalk, CT USA).
*Corresponding author.
Simultaneous Multielement AAS Determination of
Trace Elements in Human Body Fluids to Establish
Reference Values for European Populations
*M.A. White
1,2
and A.Panayi
1
1
Life Sciences Unit, Environment Institute, European Commission Joint Research Centre
Ispra, I-21020 Italy
2
Health and Safety Laboratory, Broad Lane, Sheffield, S3 7HQ UK
ABSTRACT
Sensitive and rapid techniques
are described for the simultane-
ous determination of chromium
and nickel in serum and lead and
cadmium in both blood and urine
matrices using a multielemental
electrothermal atomic absorption
spectrophotometer.
Sample pretreatments were
kept to a minimum to avoid
unnecessary risk of contamina-
tion. Serum and urine samples
were simply diluted 1+1 (v/v)
with 0.1% HNO
3
/0.1% Triton

X-100 and 0.2% HNO

3
/0.05%
Triton X-100, respectively. Whole
blood samples were diluted 1+3
(v/v) with a diluent containing
NH
3
/Na EDTA/NH
4
H
2
PO
4
. Addi-
tionally, a 1% NH
4
H
2
PO
4
chemical
modifier was used to stabilize Cd
at elevated ashing temperatures.
Limits of detection for the ele-
ments were 0.05 g/L for Cr,
0.2 g/L for Ni, 0.06 g/L for Cd
in urine, 0.2 g/L for Cd in blood,
2.6 g/L for Pb in urine and 4.5
g/L for Pb in blood.
The accuracy of the methods
was evaluated by analysis of certi-
fied reference materials and sam-
ples from international Quality
Assurance programs.
AA-1244
Return to Document Menu
90
Hollow cathode lamps (Perkin-
Elmer) were used for chromium,
nickel, and lead measurements, and
an electrodeless discharge lamp for
cadmium determinations. A pyrolyt-
ically coated tube with integral plat-
form (Perkin-Elmer No. B050-4033)
was used for all determinations.
The instrument was located in
a specialized laboratory, which was
supplied with filtered air. All metal
surfaces were sealed with a resin-
based paint and all work surfaces
and cabinets were manufactured
from high-density plastic (Tema Srl.,
Faenza, Italy).
Calibration standard solutions
and manual dilution of samples
were made with calibrated
automatic pipettes (Eppendorf,
Germany). Polypropylene sample
cups and pipette tips were acid-
washed in 10% nitric acid, rinsed
twice in Milli-Q

ultrapure water
(Millipore, Molsheim, France) and
air-dried in an ultraclean room
before use.
Reagents and Standard
Solutions
Working calibration standards
were prepared from 1.00 g/L certi-
fied atomic absorption standards
(Spectrosol grade, BDH, UK). Supra-
pure double sub-boiling distilled
HNO
3
and NH
3
were from Romil
Chemicals Ltd, Loughborough, UK.
Ammonium dihydrogen orthophos-
phate, sodium EDTA, and Triton

X-100 diluent were from Merck,

Darmstadt, Germany, and Chelex

-
100 ion exchange resin from Bio-
Rad, Richmond, USA.
The following reference materi-
als were used: National Institute of
Standards and Technology (NIST)
Standard Reference Materials (SRM)
2670 Human Urine, 1598 Bovine
Serum, 1643c Trace Elements in
Water, Seronorm trace elements in
human whole blood, serum and
urine (Nycomed Pharma, Norway).
Ultrapure Milli-Q water was used
as diluent throughout.
Sample Preparation
All sample preparations were
performed under clean room
conditions.
For the determination of
chromium and nickel in serum,
samples were diluted 1+1 (v/v)
with 0.1% HNO
3
/0.1% Triton X-100
diluent. A bovine serum was used
as the calibration sample for
method of additions calibration.
Spike additions of 2.67 g/L, 5.33
g/L, and 10.67 g/L Cr and Ni
were prepared automatically from
a 20-g/L aqueous solution. Thirty
microliter volumes were injected
into the furnace. Urine samples
were also diluted 1+1 (v/v) with
a 0.2% HNO
3
/0.05% Triton X-100
diluent. The method of additions
calibration used a fresh urine sam-
ple with spike additions of 1.0 g/L,
2.0 g/L, 4.0 g/L Cd, and 10.0
g/L, 20.0 g/L, and 40 g/L Pb pre-
pared automatically from an aque-
ous stock solution. Ten microliter
volumes were injected into the fur-
nace together with 5 mL of a 1%
NH
4
H
2
PO
4
solution, as a chemical
modifier, to stabilize Cd. For the
determination of Pb and Cd in
whole blood, samples were diluted
1+3 (v/v) with a diluent containing
0.14 M NH
3
, 0.03 M NH
4
H
2
PO
4
,
0.003 M NaEDTA and 0.5% Triton
X-100 diluent. A human whole
blood sample was used for method
of additions calibration. Spike addi-
tions of 1.33 g/L, 4.0 g/L, 8.0
g/L, 12.0 g/L Pb, and 0.53 g/L,
1.6 g/L, 3.2 g/L, and 4.8 g/L Cd
were prepared from an aqueous
stock standard. Fifteen microliter
volumes were injected into the fur-
nace together with 5 mL of 1%
NH
4
H
2
PO
4
as a chemical modifier.
In all cases, the sample diluent
was used as an analytical blank.
RESULTS AND DISCUSSION
Initial studies were undertaken
to optimize instrument parameters
and furnace conditions for the ana-
lytical procedures (9). Selection of
element combinations was made
based on the manufacturers recom-
mended instrument settings (10).
These settings were then used as
the starting conditions for method
optimization. The furnace programs
shown in Tables IIII gave optimum
absorbance intensities for the
selected element combinations in
the three biological media studied.
Attempts to determine three ele-
ments simultaneously resulted in a
marked deterioration in analytical
performance as optimum instrument
conditions were compromised.
Drying times for serum and whole
blood samples could not be short-
ened as this resulted in excessive
bubbling of the samples and
a marked loss of reproducibility.
An ashing temperature of 1200
o
C
and an atomization temperature of
2200
o
C were selected for Cr and
Ni determinations. With these tem-
peratures, the analyte peak shapes
for both elements were well
formed and clearly resolved from
background signals. The atomiza-
tion peak and background peak
profiles for Ni and Cr in bovine
serum spiked at 2.0 g/L are shown
in Figure 1. A graphite tube lifetime
of several hundred firings with no
evidence of degraded response was
achieved with this furnace program
and sample treatment.
91
A
tomic
S
pectroscopy
Vol. 19(3), May/June 1998
TABLE I
Instrumental Conditions and Funace Program
Chromium and Nickel in Serum
Instrument SIMAA 6000 AA
Wavelength Cr 357.9 nm, Ni 232.0 nm
(2-lamp mode)
Signal Zeeman AA-BG
Measurement Peak area
Sample volume 25 uL
Matrix modifier None
Calibration Method of additions
Furnace Program
Step Temp Ramp Hold Gas flow Read
1 130 40 20 250
2 550 30 20 250
3 1200 20 15 250
4 20 2 3 250
5 2200 0 5 0 X
6 2450 1 3 250
TABLE II
Instrumental Conditions and Furnace Program
Lead and Cadmium in Whole Blood
Instrument SIMAA 6000 AA
Wavelength Pb 283.3 nm, Cd 228.8 nm
(2-lamp mode)
Signal Zeeman AA-BG
Measurement: Peak area
Sample volume 15 L
Matrix modifier 5-L 1% ammonium
dihydrogen phosphate
Calibration Method of additions
Furnace Program
Step Temp Ramp Hold Gas flow Read
1 110 60 40 250
2 550 40 20 250
3 1800 0 5 0 X
4 2200 1 2 250
TABLE III
Instrumental Conditions and Furnace Program
Lead and Cadmium in Urine
Instrument SIMAA 6000 AA
Wavelength Pb 283.3 nm, Cd 228.8 nm
(2-lamp mode)
Signal Zeeman AA-BG
Measurement Peak area
Sample volume 10 L
Matrix modifier: 5-L 1% ammonium
dihydrogen phosphate
Calibration Method of additions
Furnace Program
Step Temp Ramp Hold Gas flow Read
1 100 5 10 250
2 150 15 30 250
3 300 1 5 250
4 750 1 10 250
5 20 1 5 250
6 1800 0 5 0 X
7 2450 1 4 250
Fig. 1. Absorbance peak and background peak profiles for Cr
and Ni in bovine serum spiked with 2.0 g/L of the elements.
92
For the determination of Cd
and Pb in both blood and urine
matrices, a NH
4
H
2
PO
4
chemical
modifier was employed to increase
the thermal stability of Cd during
the ashing stage of the furnace pro-
gram (11). In the urine matrix, opti-
mal analyte signal resolution for the
two elements was achieved with an
ashing temperature of 750
o
C and
atomization temperature of 1800
o
C
(Figure 2). Although a cool-down
step is not generally used with the
new transversely heated graphite
tube, the addition of this step in the
furnace program for the determina-
tion of lead and cadmium in urine
was found to give better signal
peaks and temporal separation of
the cadmium and background sig-
nals. Temporal resolution of back-
ground and analyte signals for Cd
overcame the problem of variable
urine matrix composition which
may affect the determination of this
element (12). Satisfactory analyte
signals for Cd and Pb in a whole
blood matrix were obtained with
a lower ashing temperature of
550
o
C (Figure 3). Again, analyte
and background signals for Cd
were temporarily resolved.
In all three sample types, matrix
interference effects were fully cor-
rected for by using Zeeman effect
background correction, matrix-
matched calibration standards,
and integrated absorbance measure-
ments. No significant build-up of
carbonaceous residue was observed
with this sample treatment and fur-
nace program.
Calibration and Calibration
Blank
The use of ultrapure reagents
and clean room conditions for
sample preparation was essential
for obtaining analytical blanks with
negligible absorption signals for the
analytes of interest. In particular,
random, spuriously high blank read-
ings for Ni and Cr were observed
if clean room conditions were not
applied. To obtain a satisfactory
analytical blank reading for the
determination of Pb and Cd in
blood and urine, a further clean-up
procedure with Chelex-100 ion
exchange resin was necessary to
remove Pb and Cd contaminants
from the blood diluent and chemi-
cal modifier solutions (13).
Fig. 2. Absorbance peak and background peak profiles for
lead and cadmium in human whole blood spiked with
4 mg/L of lead and 1.6 mg/L of cadmium.
Fig. 3. Absorbance peak and background peak profiles for
lead and cadmium in human urine spiked with 25 mg/L
of lead and 1.4 mg/L of cadmium.
93
A
tomic
S
pectroscopy
Vol. 19(3), May/June 1998
Calibration curves were linear
up to 10 g/L for Cr and Ni, 6 g/L
for Cd in both blood and urine
matrices, 15 g/L for Pb in blood,
and 40 g/L for Pb in urine. The
calibration slopes for aqueous stan-
dards and matrix-matched standards
were not parallel and, therefore,
matrix-matched clibration standards
were used for all determinations.
The use of matrix-matched standards
also ensured that standards and
unknown samples had similar vis-
cosities and pipetting characteristics.
Analytical Sensitivity and
Precision
Characteristic masses for the
individual elements, defined as the
amount of analyte giving an inte-
grated absorbance signal of 0.0044 s,
were as follows: 1.4 pg for Cd,
26 pg for Pb, 6.2 pg for Cr, and
25 pg for Ni. These values fell
within 20% of the recommended
characteristic mass values for the
analytes using single-element analyt-
ical conditions.
Detection limits were established
by analysis of samples with a low
endogenous content of the
elements to be determined. The
limit of detection (LOD 3 , n=10)
was 0.05 g/L for Cr, 0.2 g/L for
Ni, 0.05 g/L for Cd in both matri-
ces, 1.2 g/L and 2.6 mg/L for Pb
in blood and urine, respectively.,
These detection limits are sufficiently
sensitive for the accurate determi-
nation of values in the general pop-
ulation, with the possible exception
of Ni, for which the reported range
of reference values (0.161.2 g/L)
(14) spans the LOD. The LOD
for Ni and Cr, however, could be
reduced further with repeated
sample injection and pyrolysis
prior to atomization (15).
Analytical recovery experiments
were performed by spiking previ-
ously analyzed serum, blood, and
urine samples with known amounts
of NIST CRM 1643 Trace Elements
in Water. The recovery results are
shown in Table IV, together with
the analytical precision figures for
the methods. Recoveries of all ele-
ments are considered satisfactory,
relative to the small amounts of
analyte spikes.
Analytical Accuracy
The accuracy of the methods
was assessed by analyzing certified
reference materials and commer-
cially available quality control mate-
rials of serum, blood, and urine.
All reference materials were diluted
appropriately to fall within the cali-
bration ranges of the respective
methods and treated as normal
samples. The results of analyses are
shown in Table V. The method for
Pb and Cd in whole blood was fur-
ther evaluated by analyszing sam-
ples from the U.K. external quality
assurance scheme for blood lead
and cadmium (UKEQAS). The corre-
lation between values obtained
with the described method and
the consensus mean values are dis-
played in Figures 4 a and b. The
excellent coefficients of correlation
for both elements (r
2
=0.99 for Cd
and Pb) show that the method is
accurate over a range of Cd and Pb
concentrations, which include the
levels expected in a non-occupa-
tionally exposed population.
TABLE IV
Analytical Recovery and Precision for Methods Described
Element and matrix Amount Analytical Analytical
of spike recovery precision
(% RSD)
Chromium in serum 2.5 g/L 102 2% (n=10) 8.0
Nickel in serum 2.5 g/L 99 6% (n=10) 8.0
Lead in blood 4 g/L 104 8% (n=6) 4.2
Cadmium in blood 1.6 g/L 98 6% (n=6) 8.5
Lead in urine 9 g/L 111 5% (n=6) 4.8
Cadmium in urine 3.3 g/L 108 5% (n=6) 8.5
TABLE V
Analysis of CRMs and Commercial QA Materials
Using Methods Described
Element and matrix Reference material Range of Reference
values found value
(g/L) (g/L)
Lead in blood Seronorm 30.5 33.8 31 41
whole blood (level 1)
Cadmium in blood Seronorm 0.8 1.08 0.8 1.0
whole blood (level 1)
Chromium in serum NIST 1598 0.1 0.16 0.06 0.22
Nickel in serum Seronorm serum 2.2 3.3 2.5
Lead in urine Seronorm urine 100 104 85 97
(100 added)
Lead in urine NIST 2670 102, 103 109
Cadmium in urine Seronorm urine 5.9 6.7 5.5
94
CONCLUSION
These studies demonstrate
that concentrations of trace
elements can be accurately deter-
mined simultaneously in different
human body fluid matrices. Opti-
mum analytical performance was
achieved with combinations of two
elements having compatible condi-
tions for their determination by
ETAAS. As previously observed, the
transversely heated graphite
furnace with longitudinal Zeeman
effect background correction gives
significantly improved signal-to-
noise ratios due to its simpler opti-
cal system (15). This enables
improved detection limits to be
achieved for many elements in
these complex matrices. The
improved detection limits enable
the realistic determination of
reference values for unexposed
populations with minimal sample
manipulation. With this improved
analytical sensitivity, however,
control of contamination risks is
critical, and the use of ultrapure
reagents and ultra-clean room con-
ditions are essential in achieving
reliable quantitative data.
Received January 6, 1998.
REFERENCES
1. E.I. Hamilton, E. Sabbioni, and
M.T. Van der Venne, Sci. Total
Environ. 158, 165 (1994).
2. C. Minoia, E. Sabbioni , P. Apostoli,
R. Pietra, L. Pozzoli, M. Gallorini,
G. Nicolau, L. Alessio, and E.
Capodaglio, Sci. Total Environ.
95, 89 (1990).
3. L. Alessio, Sci. Total Environ. 120,
1 (1992).
4. J. Verseick, R. Barbier, R. Cornelis,
and J. Hoske, Talanta 29, 973
(1982).
5. C. Minoia, R. Pietra, E. Sabbioni,
A. Ronchi, A. Gatti, A. Cavallieri,
and L.Manzo , Sci. Total Environ.
120, 63 (1992).
6. S. Bro, P.J. Jorgensen, J.M. Christensen,
and M.J.Horder, Trace Elem. Elec-
trolytes Health Dis. 2, 31 (1988).
7. D.L. Tsalev and Z.K. Zaprianov
(Eds.), Atomic absorption spec-
trometry in occupational health
and environmental health prac-
tice, CRC Press, Boca Raton, FL
USA, Volume 1 (1983).
8. B. Radziuk, G. Rodel, H. Stenz,
H. Becker -Ross and S.J. Florek,
Anal. At. Spectrom. 10, 127 (1995).
9. B.S. Iversen, A. Panayi, J.P. Camblor,
and E. Sabbioni, J. Anal At. Spec-
trom. 11, 591 (1996).
10. Perkin-Elmer Corporation, The
THGA graphite furnace,
techniques and recommended
conditions, Publication B3210,
(1992).
11. K.S. Subramanian, Prog. Anal.
Spectrosc. 9, 237 (1986).
12. A. Taylor, S. Branch, H.M. Crews,
D.J. Halls, and M.A. White, J. Anal.
At. Spectrom. 10, 1R (1994)
13. M. Agarwal, R. Bennet, I.G. Stump,
and J.M. DAvia, Anal. Chem. 47,
924 (1975)
14. E. Neiboer, W.E. Sanford, and
B.C. Stace, in Nickel and human
health, E. Neiboer and J.O. Nriagu
(Eds.), J Wiley, New York, Ch. 4,
(1992)
15. I.L. Shuttler, At.. Spectrosc 13, 174
(1992).
Fig. 4. Analytical performance of the method for lead and cadmium in blood assessed by analysis of:
(a) lead and (b) cadmium in samples from UK External Quality Assurance scheme (UKEQAS).
(a)
(b)