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in workers in a lead mill (The Black Angel, Greenex A/S,. Marmorilik, Greenland) wehave compared analytical results from five laboratories that examined ...
GUN. CHEM. 24/10, 1797-1800 (1978)

InterlaboratoryComparison of Lead and Cadmium in Blood, Urine, and Aqueous Solutions Poul-Erik Paulev,’ Poul Solgaard,2 and Jens Christian Tje113

Analysis for lead and cadmium in biological liquids (blood and urine)isdifficult. Resultsof such analyses from five laboratories are compared forsamples withknown additionsof lead and cadmium. The data, evaluated in terms of inter- and intralaboratory reproducibility and accuracy,

suggest that laboratories should voluntarily participate in quality control programs. Users ofroutinelaboratories advised to use theirown qualitycontrolprogram. Additional

Keyphrases: trace elements

Reliability of the atomic method for lead in biological

are

quality control

absorption spectrophotometric liquids such as blood and urine

is important. A high precision (i.e., reproducibility) in a single laboratory may coexist with low accuracy (i.e., a large difference between the measured and the true value). Few interlaboratory calibration programs are reported in the literature. The results of Keppler et al. (5) showed a poor quality of lead analyses in blood, even for samples with high lead concentrations. A 1975 review (6) showed that the findings of 30% of the laboratories engaged in routine analysis for lead in blood

differed

by more

than

15% from

the true

value. In relation to a study of possible subclinical effects of lead in workers in a lead mill (The Black Angel, Greenex A/S, Marmorilik, Greenland) we have compared analytical results from five laboratories that examined aliquots of urine, blood, and salt solutions to which known amounts of lead and cadmium were added. The object of the present work was to estimate the precision and accuracy in measurement of the concentrations of these two metals in these fluids. This interlaboratory comparison program covered three laboratories in Scandinavia, one in England, and one in Canada.

Materials and Methods Samples: A. Blood (swine, heparmnized) containing a low concentration of added lead and cadmium (1 g Na2SO3 added per liter to preserve the sample). B. Blood (swine, heparinized) containing a high concentration of lead and cadmium (1 g Na2SO3 added per liter). C. Urine (human) with a “normal” concentration of lead and cadmium (10 ml of concentrated HC1 added per liter). 1 Institute of Medical Physiology B, University of Copenhagen, Raadmandsgade 71, DK-2200 Copenhagen N, Denmark. 2Research Establishment, Ris#{216}, DK-4000 Roskilde, Denmark. 3Department of Sanitary Engineering, Technical University of Denmark, Building 115, DK-2800 Lyngby, Denmark. Received May 16, 1978; accepted July 17, 1978.

D. Urine (human) with a high concentr8n of lead and cadmium (10 ml of concentrated HC1 added liter)’. E. Sodium chloride solution (10 g/liter) witha low concentration of lead and cadmium (2 ml of concentrated HC1 added per liter). F. Sodium chloride solution (10 g/liter) with a high concentration of lead and cadmium (2 ml of concentrated HC1 added per liter). G. Water (2 ml of concentrated HC1 added per liter). The difference in the two metals between the low and the high concentration was known. The concentrations of lead and cadmium were within the range that may be encountered in practice. A clean, lead-free bottle of each of the above samples (A-G) was marked with the letter and forwarded from the University of Copenhagen to five different laboratories (The National Occupational Hygiene Service Ltd., Manchester; Cominco Analytical Services Laboratories, Canada; Medicinsk Laboratorium A/S, Copenhagen; Ris#{216} Research Establishment, Roskilde; and the Department of Sanitary Engineering, The Technical University of Denmark, Lyngby. The coordinating department (Dr. Paulev, University of Copenhagen) did not participate in the analytical work. The individuallaboratories are anonymous, and the numbers (lab. 1-5) bear no relation to any system of names or sequence of addresses. Three other laboratories did not want to participate in the interlaboratory calibration project. The reason given was that these laboratories were satisfied with their own research results-although previous experience had shown that their results were far from correct. Procedures The methods and analysis were those ordinarily used in the laboratories to determine total concentrations of lead and cadmium. Laboratory 1: Urine (3 ml) or blood (1 ml) is wet-ashed in a glass-stoppered test tube with 1 ml of HNO3 plus 0.1 ml HC1O4 (Merck, Suprapur), slowly heated until nearly dry, and the residue dissolved in about 5 ml of 0.2 mol/liter HNO3. Lead is extracted into 2 ml of xylene containing, per liter, 2 g of diethylammonium diethyldithiocarbamic acid after adding 2 ml of sodium citrate (1 mol/liter, pH 4.5), to separate lead from other sample constituents. The measurement is done on the xylene phase by atomic absorption, with a Model 300 instrument (Perkin-Elmer Corp., Norwalk, Conn. 06856), with graphite furnace HGA-72 and with use of background compensation with a deuterium lamp. Laboratory 2: Photometry of lead in urine, involving wetashing of the urine to destroy organic matter, followed by extraction of the lead by a citrate-cyanide medium with a CLINICAL CHEMISTRY,

Vol. 24, No. 10, 1978

1797

Table 1. Results of an interiaboratory Comparison of Lead Determination Mean for

LaboratorIes Lead add.d

Sample

2

3

_________________ all 5 4 5 CV, % labs.

Pb, mg/lIter

A: Blood B: Blood 01ff. (B C: Urine

X1 + 0.2 X1 + 0.67 -

A):0.47 X2

D:Urine X2+ 0.124 Dlff.(D-C):0.124 E:Saltsolutlon X3+0.124 F: Salt solution X3 + 0.380 Diff. (F E): 0.256 G: Water 0.273 -

0.19 0.59 0.40 0.01 0.14

0.22 0.63 0.41 0.02 0.17

0.19 0.60 0.41 0.01 0.14

0.24 0.71 0.47 0.01 0.13

0.18 0.43 0.25 0.01 0.12

0.12

0.15

0.13

0.12

0.11

0.12 0.36 0.24 0.25

0.16 0.44 0.28 0.30

0.16 0.43 0.27 0.27

0.13 0.43 0.31 0.26

0.13 0.22 0.09 0.25

0.20 0.59

12.3 17.2

0.14

13.4

0.14 0.41

13.4 24.7

X1, X2, andX3 aretheunknownconcentrations of lead present In the samples before further addItIon of lead.

carbon tetrachloride solution of dithizone. Excess dithizone was removed from the extract by treatment with a second cyanide solution at a slightly higher pH. The dithizone-CC14 layer is drained into a 2-cm test tube supplied with the Evelyn Colorimeter and measured at 515 nm, with the colorimeter set to 100% transmittance with a reference solution. The concentration of cadmium in blood was also determined colorimetrically. Dr. Craw, Cominco, has developed this unpublished method. The coefficient of variation was 5% for a mean lead concentration of 0.4 mg/liter and the SD was ±0.02 mg/liter. As the cadmium analyses are at or near the limit of detection, the coefficient of variation is meaningless. Laboratory 3: Lead: Blood (1 ml) was diluted and hemolyzed with 4 ml of a 10 ml/liter solution of the surfactant Triton X-100. Urine (4 ml) was treated with 1 ml of a 250 g/kg solution of CC13COOH in concentrated HC1O4. To 10 parts of the supernate was added one part of Triton X-100. Lead was measured directly in the salt solutions and compared to an array of standards of a similar composition as the test samples. Cadmium: HNO3/H20

Blood

was diluted

and precipitated

(5/95 by vol). Urine and aqueous solutions

with were

solvent extracted with a solution of sodium diethyldithiocarbamate in methylisobutyl ketone. Apparatus: All measurements were performed with a Perkin-Elmer atomic absorption spectrophotometer, Model 400 SG, with a graphite furnace HGA 74 (controlled by a HGA controller 2100). The samples were applied with a PerkinElmer Autosampler AS 1. All measurements were corrected for background absorption with a deuterium lamp. The CV was 5% for lead in blood, 8% for lead in salt solutions, 13% for cadmium in blood, and 15% for lead and cadmium in urine and for cadmium in salt solutions. Laboratory 4: Lead and cadmium were determined in blood by the punched paper disc method (4-mm disc) with use of graphite oven (Varian CRA 90) on a Varian AA6 atomic absorption spectrophotometer. Cadmium in urine, aqueous solutions, and water was measured with the carbon cup furnace, ramp mode (10-id additions). Lead in salt solutions and water was measured with monocolor dithizone, and, after wet ashing, also in urine. Laboratory 5: Blood (1 ml) was diluted and hemolyzed with 1 ml of water, lead was dissociated by adding 1 g of Hg2 per liter, and complexed as nitrate by adding 2 ml of 6 mol/liter HNO3. Lead was separated from organic matter and the majority of inorganic ions by absorption on strongly basic anion 1798 CLINICALCHEMISTRY,Vol. 24, No. 10, 1978

exchanger and finally determined by atomic absorption spectrophotometry of a 1 mol/liter HNO3 eluate (PerkinElmer Model 305 B with HGA 72 carbon furnace).

Results

Lead The methods used differed in details and in principle in the five laboratories, and still the mean concentration found by all five laboratories was equal to the added lead, so the concentrations of lead found in the samples before addition of lead to the blood (X1) must have approached zero. In the urine (X2) the lead concentration before addition of lead was about 0.01 mg/liter (or less) and in the aqueous solution (X3) about 0.02-0.03 mg/liter (Table 1). Therefore the added lead can be accepted as the true known value. The degree of association between the absolute values for lead concentrations was good for all five laboratories, except for sample F in one laboratory (Table 1). The differences (B-A), (D-C), and (F-E) also agreed well with the true difference (Table 1). The reproducibility is measured quantitatively as the CV in % (coefficient of variation). The interlaboratory CV varied between 12.3 and 24.7% (Table 1). Within a given laboratory the coefficient of variation in this study was 5%, which reflects an acceptable (interlaboratory) reproducibility (or precision) of the lead measurements. These relationships show an acceptable accuracy of the methods in all laboratories. Cadmium Two of the laboratories (1 and 5) did not measure cadmium concentrations. Laboratory 2 uses the spectrophotometric method and their results are expressed to two decimal places, as compared to three in the data from Laboratories 3 and 4-both of which used atomic absorption methods. Laboratories 3 and 4 seem to have a rather high degree of association, until the differences (B-A), (D-C), and (F-E) are considered (Table 2). The cadmium analyses are not reliable. Although cadmium concentrations in physiological liquids are about 10-fold lower than the lead concentrations, and the analytical sensitivity of the methods employed is 10-fold greater for cadmium analysis than for lead, it is evident that the analytical performance for cadmium is lagging behind. lntraiaboratory

Comparison

In one laboratory (Laboratory 2) four lead analyses were performed on each sample (Table 3). A high degree of asso-

Table 2. Results of an Interlaboratory Comparison of Cadmium Determination Laboratories Cadmium added

Sample

A: Blood B: Blood 01ff. (B C: Urine D: Urine Duff. (0

Y1 + 0.020 Y1 + 0.067 -

A): 0.047

-

C): 0.0 12

Y2 + 0.012

2 Cd, mg/lIter

3

4

0.01 0.04 0.03 0.Ola 0.01 0

0.018 0.062 0.044 0.001 0.010 0.009

0.021 0.062 0.041 0.001 0.023 0.022

E:Saltsolution

Y3 + 0.0124

0.02

0.012

0.015

F: Salt solution Duff. (F E): 0.026 G: Water

Y3 + 0.0380

0.03 0.01 0.03

0.027 0.015 0.022

0.030 0.015 0.027

-

0.0273

V1. V2. V3 indicate the unknown concentrations in thesamples before cadmium was added. 8 LimIt of method.

Table 3. Results of Intralaboratory Comparison (Laboratory No. 2) of Lead Determination Sample

A: Blood B: Blood 01ff. (B C: Urine 0: Urine

1stanal.

-

A)

Diff.(D-C)

0.21 0.64 0.43 0.01 0.15 0.14

3rd anal. Pb, mg/liter

4th anal.

0.23 0.62 0.39 0.02 0.18 0.16

E: Aqueous solution

0.17

0.15

0.17

F: Aqueous solution Diff. (F E) G: Water

0.44 0.27 0.30

0.44 0.29 0.29

0.44 0.27 0.30

-

CV

0.22 0.64 0.42 0.02 0.17 0.15

2nd anal.

-

0.65

-

0.45 -

0.30

Mean

0.22 0.63 0.41 0.02 0.17 0.15

0.16 0.44

0.28 0.30

5% at mean concentration of 0.4 mg/liter. SO = ±0.02 mg/liter.

was found from analysis to analysis (Table 3). Both and accuracy of the spectrophotometric method applied is extremely high (Table 3), and the atomic absorption methods had the same reliability.

ciation

precision

Sensitivity The sensitivity of the graphite furnace method was in the order of 4-5 ig of lead per liter of fluid. The spectrophotometric method needed higher minimum concentrations-up to two orders of magnitude.

Discussion Lead This study was initiated on the basis of a workshop report showing that nine chemical laboratories of superior status using atomic absorption or anodic stripping voltammetry techniques could not demonstrate a reliable capability in analysis for lead in sea water (2). Three other laboratories disagreed in 28 cases out of 35 when any of them reported a blood value above the accepted international safety limit. They concluded that diagnostic or change-of-work decisions should not be made on the basis of blood lead determinations alone (3). The European comparison study showed a poor reproducibility, when multiple aliquots of a single sample were for-

warded to laboratories without their knowledge (2). A much better reproducibility was obtained when replicate aliquots were analyzed within each laboratory. Thus the intralaboratory reproducibility (performed by persons knowing the previous results from a particular aliquot) was better than the results from analyses performed on aliquots that were not known to originate from the same sample. Other interlaboratory calibration programs also showed a rather poor reliability for lead analyses (1, 5). A recent report does not recommend the extraction/tantalum boat-atomic absorption method for lead in blood, because of a poor precision, as judged from a coefficient of variation of 23% (4). Since the state of the art was evaluated in an excellent review in 1975 (6), many methodological improvements have been introduced. The data on lead in our material shows that it is possible to obtain a high precision and accuracy in lead analyses, even without any attempt to standardize the methods and principles used. Today the atomic absorption methods can be just as reliable as the classical spectrophotometric dithizone method. Although we may now measure blood and urine lead concentrations precisely and even accurately, we are still faced with the same basic man-machine interface problems as before. A high degree of precision in a single laboratory (Table 3) may lead the scientist to believe that his method is more reliable than it actually is, in terms of accuracy (Table 1). CLINICAL CHEMISTRY,

Vol. 24, No. 10, 1978

1799

Cadmium Acceptable results were not obtained for cadmium. Accurate and precise results have recently been obtained for the determination of cadmium on filters by the use of external calibration curves (4).

Conclusions Concerning Lead 1. The methods studied all have an acceptable reproducibility and accuracy. The same precision and accuracy is obtained by use of the classical dithizone method as by graphite furnace atomic absorption spectrophotometry. Neither is clearly superior. 2. Reproducibility at an individual laboratory is generally much better than itsaccuracy. 3. Assuming the validity of the present material, the results suggest that laboratories should participate voluntarily in quality control programs. The control program must include the following factors: reproducibility (precision) as evaluated by replicate analyses in each laboratory, and accuracy, by analytical recovery of known additions of lead to blood and urine, including differences between two additions. 4. Customers dependent upon blood lead analyses are advised to use their own quality control program.

Conclusions Concerning Cadmium

Reliablecadmium analyses were not available. Methodological improvements

and quality

control

1800 CLINICALCHEMISTRY, Vol. 24, No. 10, 1978

programs are

necessary before cadmium analyses in fluids such as blood and urine are reliable. We are grateful to Chief Chemist D. A. Craw, Analytical Services Laboratories, Cominco Ltd., Canada, and to Paul Persson,Medicinsk Laboratorium A/S, Copenhagen, for help with theanalyses. We thank Greenex A/S for financial support of the project, and Lene Eriksen and Margit Hartyani for their excellent technical assistance.

References 1. Berlin, A., Lauwerys, R., Buchet, J. P., et al., Intercomparison program of lead, mercury, and cadmium analysis in blood, urine, and aqueous solutions. Clin. Chem. 21, 551 (1975). 2. Brewer, P., et al. (Workshop participants and advisors), Interlaboratory lead analyses of standardized samples of sea water.Marine Chem. 2,69 (1974). 3. Browne, R. C., Ellis, R. W., and Weightman, D., Interlaboratory variation in measurement of blood lead levels. Lancet ii, 1112 (1974). 4. Eller, P. M., and Haartz, J. C., A study of methods for the determination of lead and cadmium. Am. md. Hyg. Assoc. J. 38, 116 (1977). 5. Keppler, J. F., Maxfield, M. E., Moss,W. D., et al., Interlaboratory evaluation of the reliability of blood lead analyses. Am. md. Hyg. Assoc. J. 31,412 (1970). 6. Pierce, J. 0., Koirtyohann, S. R., Clevenger, T. E., and Lichte, F.

E., The determination of lead in blood.A review and critique of the state of the art,1975. Internat. Lead Zinc Res.Org., Inc., New York, N.Y., 1976.