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Atomic Spectroscopy

Vol. 19(3), May/June 1998

INTRODUCTION

Large quantities of toxicelements such as cadmium, lead,nickel, and chromium have beenreleased to the environment in pastdecades and exposure to theseelements may pose a health risk topopulations living in or near indus-trially polluted areas. Long-termstudies on changes in health statusand the quality of the environmentcannot be successfully conducted,however, unless reliable and com-prehensive reference data on envi-ronmental trace element levels areestablished (1). Unfortunately, validreference values for many toxic andcarcinogenic metals are lacking orinadequate. In this respect, theEUROTERVIHT project (Trace Ele-ment Reference Values in HumanTissues) aims to establish and com-pare trace element reference values(baseline values) in biological fluidsand tissues from unexposed popula-tions within different regions of theEuropean Union (2). The reference

values have a key role in the verifi-cation or reconsideration of legallimits of exposure to protect thegeneral population and in the estab-lishment of appropriate monitoringstrategies for occupationallyexposed groups (3).

For acquired data to be consid-

ered acceptable for establishingreference values, rigorous controlof pre-analytical and analytical fac-tors must be applied (4,5). Thisentails the use of well-characterizedmaterials for sample collectionand analysis (6), validated and docu-

mented sampling procedures and

validated analytical methods withcomprehensive quality assuranceprocedures. Electrothermal atomicabsorption spectrometry (ETAAS)has long been the preferred analyti-cal method for the routine determi-nation of trace metals in biologicalfluids owing to its reliability, sensi-tivity, and relatively low cost (7).

A major limitation with currentinstrumentation, however, is the

single-element capability. Multi-element studies become costly interms of analyst and instrumenttime, while the risk of contamina-tion is greatly magnified by repeatedsample manipulation.The recentintroduction of simultaneous multi-elemental electrothermal atomic

absorption instrumentation offersthe opportunity to overcome theselimitations (8).

In this paper, methods aredescribed for the simultaneousdetermination of chromium andnickel in serum, and the simultane-ous determination of cadmium andlead in both whole blood and urinematrices. With the selection ofcompatible elements and thecareful optimization of instrumentconditions, these elements can bedetermined with minimum sample

pre-treatment and with sufficientsensitivity to establish reference

values for healthy unexposedpopulations.

EXPERIMENTAL

Instrumentation

All analyses were performedusing a Perkin-Elmer SIMAA 6000atomic absorption spectrometer(Perkin-Elmer, Norwalk, CT USA)

with transversely heated graphite

furnace and longitudinal Zeeman-effect background correction. Allautomated dilutions and injections

were made with a Perkin-ElmerAS-72 autosampler, fitted with an80-position tray and microdispensercapable of delivering 0.1-mL

volumes. The spectrometer andautosampler were controlledby AA Winlab software,(Perkin-Elmer, Norwalk, CT USA).

*Corresponding author.

Simultaneous Multielement AAS Determination ofTrace Elements in Human Body Fluids to Establish

Reference Values for European Populations*M.A. White1,2 and A.Panayi1

1 Life Sciences Unit, Environment Institute, European Commission Joint Research CentreIspra, I-21020 Italy

2 Health and Safety Laboratory, Broad Lane, Sheffield, S3 7HQ UK

ABSTRACT

Sensitive and rapid techniquesare described for the simultane-ous determination of chromiumand nickel in serum and lead andcadmium in both blood and urinematrices using a multielemental

electrothermal atomic absorptionspectrophotometer.Sample pretreatments were

kept to a minimum to avoidunnecessary risk of contamina-tion. Serum and urine samples

were simply diluted 1+1 (v/v)with 0.1% HNO3/0.1% Triton

X-100 and 0.2% HNO3/0.05%Triton X-100, respectively. Wholeblood samples were diluted 1+3(v/v) with a diluent containingNH3/Na EDTA/NH4H2PO4. Addi-tionally, a 1% NH4H2PO4 chemicalmodifier was used to stabilize Cdat 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 Cdin urine, 0.2 g/L for Cd in blood,2.6 g/L for Pb in urine and 4.5g/L for Pb in blood.

The accuracy of the methodswas evaluated by analysis of certi-fied reference materials and sam-ples from international Quality

Assurance programs.

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Hollow cathode lamps (Perkin-Elmer) were used for chromium,nickel, and lead measurements, andan 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 ina specialized laboratory, which wassupplied with filtered air. All metalsurfaces were sealed with a resin-based paint and all work surfacesand cabinets were manufacturedfrom high-density plastic (Tema Srl.,Faenza, Italy).

Calibration standard solutionsand manual dilution of samples

were made with calibratedautomatic pipettes (Eppendorf,Germany). Polypropylene samplecups and pipette tips were acid-

washed in 10% nitric acid, rinsedtwice in Milli-Q ultrapure water(Millipore, Molsheim, France) andair-dried in an ultraclean roombefore use.

Reagents and StandardSolutions

Working calibration standardswere prepared from 1.00 g/L certi-fied atomic absorption standards(Spectrosol grade, BDH, UK). Supra-pure double sub-boiling distilledHNO3 and NH3were from RomilChemicals 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 ofStandards and Technology (NIST)Standard Reference Materials (SRM)2670 Human Urine, 1598 BovineSerum, 1643c Trace Elements in

Water, Seronorm trace elements inhuman whole blood, serum andurine (Nycomed Pharma, Norway).

Ultrapure Milli-Q water was usedas diluent throughout.

Sample Preparation

All sample preparations wereperformed under clean roomconditions.

For the determination ofchromium and nickel in serum,samples were diluted 1+1 (v/v)

with 0.1% HNO3/0.1% Triton X-100diluent. A bovine serum was usedas the calibration sample formethod of additions calibration.Spike additions of 2.67 g/L, 5.33g/L, and 10.67 g/L Cr and Ni

were prepared automatically from

a 20-g/L aqueous solution. Thirtymicroliter volumes were injectedinto the furnace. Urine samples

were also diluted 1+1 (v/v) witha 0.2% HNO3/0.05% Triton X-100diluent. The method of additionscalibration used a fresh urine sam-ple with spike additions of 1.0 g/L,2.0 g/L, 4.0 g/L Cd, and 10.0g/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%

NH4H2PO4 solution, as a chemicalmodifier, to stabilize Cd. For thedetermination of Pb and Cd in

whole blood, samples were diluted1+3 (v/v) with a diluent containing0.14 M NH3, 0.03 M NH4H2PO4,0.003 M NaEDTA and 0.5% Triton

X-100 diluent. A human wholeblood sample was used for methodof additions calibration. Spike addi-tions of 1.33 g/L, 4.0 g/L, 8.0g/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 aqueousstock standard. Fifteen microliter

volumes were injected into the fur-nace together with 5 mL of 1%NH4H2PO4 as a chemical modifier.

In all cases, the sample diluentwas used as an analytical blank.

RESULTS AND DISCUSSION

Initial studies were undertakento optimize instrument parameters

and furnace conditions for the ana-lytical procedures (9). Selection ofelement combinations was madebased on the manufacturers recom-mended instrument settings (10).These settings were then used asthe starting conditions for methodoptimization. The furnace programsshown in Tables IIII gave optimumabsorbance intensities for theselected element combinations inthe three biological media studied.

Attempts to determine three ele-ments simultaneously resulted in amarked deterioration in analyticalperformance as optimum instrumentconditions were compromised.Drying times for serum and wholeblood samples could not be short-ened as this resulted in excessivebubbling of the samples anda marked loss of reproducibility.

An ashing temperature of 1200oCand an atomization temperature of2200oC were selected for Cr andNi determinations. With these tem-peratures, the analyte peak shapes

for both elements were wellformed and clearly resolved frombackground signals. The atomiza-tion peak and background peakprofiles for Ni and Cr in bovineserum spiked at 2.0 g/L are shownin Figure 1. A graphite tube lifetimeof several hundred firings with noevidence of degraded response wasachieved with this furnace programand sample treatment.

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TABLE IInstrumental 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 2504 20 2 3 250

5 2200 0 5 0 X

6 2450 1 3 250

TABLE IIInstrumental 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% ammoniumdihydrogen phosphate

Calibration Method of additions

Furnace Program

Step Temp Ramp Hold Gas flow Read

1 110 60 40 250

2 550 40 20 2503 1800 0 5 0 X

4 2200 1 2 250

TABLE IIIInstrumental 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% ammoniumdihydrogen phosphate

Calibration Method of additions

Furnace Program

Step Temp Ramp Hold Gas flow Read

1 100 5 10 250

2 150 15 30 2503 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 Crand Ni in bovine serum spiked with 2.0 g/L of the elements.

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For the determination of Cdand Pb in both blood and urinematrices, a NH4H2PO4 chemicalmodifier was employed to increase

the thermal stability of Cd duringthe ashing stage of the furnace pro-gram (11). In the urine matrix, opti-mal analyte signal resolution for thetwo elements was achieved with anashing temperature of 750oC andatomization temperature of 1800oC(Figure 2). Although a cool-downstep is not generally used with thenew transversely heated graphitetube, the addition of this step in thefurnace program for the determina-tion of lead and cadmium in urine

was found to give better signalpeaks and temporal separation ofthe cadmium and background sig-nals. Temporal resolution of back-ground and analyte signals for Cd

overcame the problem of variableurine matrix composition whichmay affect the determination of thiselement (12). Satisfactory analyte

signals for Cd and Pb in a wholeblood matrix were obtained witha lower ashing temperature of550oC (Figure 3). Again, analyteand background signals for Cd

were temporarily resolved.

In all three sample types, matrixinterference effects were fully cor-rected for by using Zeeman effectbackground correction, matrix-matched calibration standards,and integrated absorbance measure-ments. No significant build-up of

carbonaceous residue was observedwith this sample treatment and fur-nace program.

Calibration and CalibrationBlank

The use of ultrapure reagents

and clean room conditions forsample preparation was essentialfor obtaining analytical blanks withnegligible absorption signals for theanalytes of interest. In particular,random, spuriously high blank read-ings for Ni and Cr were observedif clean room conditions were notapplied. To obtain a satisfactoryanalytical blank reading for thedetermination of Pb and Cd inblood and urine, a further clean-upprocedure with Chelex-100 ionexchange resin was necessary to

remove Pb and Cd contaminantsfrom the blood diluent and chemi-cal modifier solutions (13).

Fig. 2. Absorbance peak and background peak profiles forlead and cadmium in human whole blood spiked with4 mg/L of lead and 1.6 mg/L of cadmium.

Fig. 3. Absorbance peak and background peak profiles forlead and cadmium in human urine spiked with 25 mg/Lof lead and 1.4 mg/L of cadmium.

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Calibration curves were linearup to 10 g/L for Cr and Ni, 6 g/Lfor Cd in both blood and urinematrices, 15 g/L for Pb in blood,

and 40 g/L for Pb in urine. Thecalibration 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 standardsalso ensured that standards andunknown samples had similar vis-cosities and pipetting characteristics.

Analytical Sensitivity andPrecision

Characteristic masses for theindividual elements, defined as theamount 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, and25 pg for Ni. These values fell

within 20% of the recommendedcharacteristic mass values for theanalytes using single-element analyt-ical conditions.

Detection limits were establishedby analysis of samples with a lowendogenous content of theelements to be determined. Thelimit of detection (LOD 3 , n=10)

was 0.05 g/L for Cr, 0.2 g/L forNi, 0.05 g/L for Cd in both matri-ces, 1.2 g/L and 2.6 mg/L for Pbin blood and urine, respectively.,

These detection limits are sufficientlysensitive for the accurate determi-

nation of values in the general pop-ulation, with the possible exceptionof Ni, for which the reported rangeof reference values (0.161.2 g/L)

(14) spans the LOD. The LODfor Ni and Cr, however, could bereduced further with repeatedsample injection and pyrolysisprior to atomization (15).

Analytical recovery experimentswere performed by spiking previ-ously analyzed serum, blood, andurine samples with known amountsof NIST CRM 1643 Trace Elementsin Water. The recovery results areshown in Table IV, together withthe analytical precision figures for

the methods. Recoveries of all ele-ments are considered satisfactory,relative to the small amounts ofanalyte spikes.

Analytical Accuracy

The accuracy of the methodswas assessed by analyzing certifiedreference materials and commer-cially available quality control mate-rials of serum, blood, and urine.

All reference materials were dilutedappropriately to fall within the cali-bration ranges of the respective

methods and treated as normalsamples. The results of analyses areshown in Table V. The method forPb and Cd in whole blood was fur-ther evaluated by analyszing sam-ples from the U.K. external qualityassurance scheme for blood leadand cadmium (UKEQAS). The corre-lation between values obtained

with the described method andthe consensus mean values are dis-played in Figures 4 a and b. Theexcellent coefficients of correlationfor both elements (r2=0.99 for Cdand Pb) show that the method isaccurate over a range of Cd and Pbconcentrations, which include thelevels expected in a non-occupa-tionally exposed population.

TABLE IVAnalytical Recovery and Precision for Methods Described

Element and matrix Amount Analytical Analyticalof 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 VAnalysis of CRMs and Commercial QA Materials

Using Methods Described

Element and matrix Reference material Range of Referencevalues found value

(g/L) (g/L)

Lead in blood Seronorm 30.5 33.8 31 41whole blood (level 1)

Cadmium in blood Seronorm 0.8 1.08 0.8 1.0whole 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

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CONCLUSION

These studies demonstratethat concentrations of traceelements can be accurately deter-mined simultaneously in differenthuman body fluid matrices. Opti-mum analytical performance was

achieved with combinations of twoelements having compatible condi-tions for their determination byETAAS. As previously observed, thetransversely heated graphitefurnace with longitudinal Zeemaneffect background correction givessignificantly improved signal-to-noise ratios due to its simpler opti-cal system (15). This enablesimproved detection limits to beachieved for many elements inthese complex matrices. Theimproved detection limits enable

the realistic determination ofreference values for unexposedpopulations with minimal samplemanipulation. With this improvedanalytical sensitivity, however,control of contamination risks iscritical, and the use of ultrapurereagents and ultra-clean room con-ditions are essential in achievingreliable quantitative data.

Received January 6, 1998.

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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)