sulfate should be a constant factor, provided the sample volume and the pH during measurement are kept constant. A brief study of the solubility of barium sulfate in solutions of varying acidity was made to determine its solubility in the 0.005N hydrochloric acid used in this analysis. The results are given in Table V. The solubility was found to be about 600 pg barium sulfate in 100 ml of 0.005N hydrochloric acid. In the sulfation plate analysis, the 20-ml aliquot may contain up to 120 pg dissolved barium sulfate (50 pg sulfate). Thus, in every analysis, 50 pg sulfate may be lost due to barium sulfate dissolving in the solution. If the standards are run a t the same pH as the samples, the dissolved barium sulfate will be compensated for and the observed results will be correct. This contrasts with the standard method ( 3 ) where there may be a discrepancy of up to 30 kg sulfate between samples and standards, since only 20 pg sulfate are soluble in 20 ml deionized water at pH 6 ( 6 ) . Any changes in the results of the analysis of a specific sample due to standing for a period of time were studied using a standard solution of sodium sulfate (25 pg/ml) in 0.005N hydrochloric acid. Twenty-ml samples of the stock solution were read a t 15-minute intervals for 3 hours. The results indicated a loss of 5-770 of measurable sulfate. Samples should, therefore, be analyzed on the same day as acidification is made.
(6) "Handbook of Chemistry and Physics." Chemical Rubber Publishing Co.. New York, N.Y., 1970, p B-91
Table V. Solubility of Barium Sulfate in Hydrochloric Acid Solutions of Varying Concentration Normality of hydrochloric acid solution
0.000 0.001 0.004 0.005 0.008 0.010 0.050 0.100 a
Solubility of barium sulfate (I.rg BaSOti100 ml)
90 360 580 6004 720 820 1810 2510
This value determined by extrapolation.
In conclusion, we have shown that careful pH control of samples and standards is required for the accurate determination of sulfation rates. Further, we recommend that the standard 3-hour heating step be eliminated. Our modifications have been in successful routine use in our laboratory for over one year.
ACKNOWLEDGMENT The authors wish to thank D. P. Horning and N. McQuaker for their assistance in preparing this manuscript.
RECEIVEDfor review July 18, 1973. Accepted March 22, 1974.
Determination of Cadmium, Lead, Thallium, and Nickel in Blood by Atomic Absorption Spectrometry Francis Amore Division of Laboratories, Illinois Department of Public Health, Springfield, Ill. 6270 1
Presently there is great concern about possible poisoning from industrial exposure or environmental contamination by heavy metals (1-3). Often requests for analysis of suspected heavy metal intoxication are based on general symptoms so that a possible causative agent is not known. From a toxicological point of view, it is desirable to be able to rapidly screen for more than a single suspected element for two reasons: first, it decreases the possibility of missing the toxic metal and, second, it gives a clue to the possible source. In most instances, the sample is limited so that as much information as possible must be obtained from the amount available. Atomic absorption spectrophotometry was chosen as the preferred method because of its more general availability ( 4 ) compared to other methods (5-7) and its speed and sensitivity. (1) (2) (3) (4) (5) (6) (7)
N. A. Schroeder and A. P. Nason, Clin. Chem., 17, 461 (1971). J. McCaull, fnvlron. Quart.. 13, 16 (1971).
Chem. Eng. News. 49, (29), 29 (1971). E. Berman. Progr. Chem. Toxicol., 4, 155 (1969). J. G. Osteryoung and R. A. Osteryoung, Amer. Lab., 4 (7), 18 (1972).
K. M. Harnbidge, Anal. Chem.. 43, 102 (1971). M F. Lubozynski, R. J. Baglan, G. R. Dyer, and A. B. Brill, J. Appl. Radiat. lsotopes, 23, 487 (1972).
Many procedures for the analysis of blood for individual metals have been reported (8-12); however, few procedures have been reported for the simultaneous extraction of multiple elements (23).The elements considered in this paper were chosen because of several requests recently received in this laboratory. Our laboratory is routinely using a procedure developed by Hessel (14) for the determination of lead in blood. Since this procedure was the most rapid and precise extraction method tested by us, it was decided to apply the method to cadmium, thallium, and nickel. Although slightly different p H values are recommended for the extraction of different elements (9, 15),all the elements are extractable a t the p H of blood with either ammonium pyrrolidine dithiocarbamate (APDC) or sodium diethyldi(8) S.Selander and K. Crarner, Brit d lnd. Med., 25, 209 (1968). (9) E. Berman, At. Absorption Newsleft.. 6, 57 (1967). (10) F. W. Sunderman, Amer. J. Clin. Pathol., 44, 182 (1965). (11) W. Slavin, S. Sprague, F. Rieders, and V. Cordova, At. Absorption News- left.,3, 7 (1964). (12) M. D. McNeely, F. W. Sunderman. M. W. Nechay, and H. Levine. Clin. Chem., 17, 602 (1971). (13) S. L. Sachdev and P. W. West, Anal. Chem. Acta, 44, 301 (1969). (14) D. W. Hessel, At. AbsorptionNewsleft., 7, 55 (1968). (15) E. Berman, At. Absorption Newsleft., 3, 11 (1964).
A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 1 1 , SEPTEMBER 1 9 7 4
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Table IV. Analytical Results for TI
Table I. Analytical Results for Cd
Absorbance
Absorbance
Concn, rg/ml
Aqueous
0.00 0.02 0.04 0.06 0.08 0.10
0.0000 0.0276 0.0528 0.0800 0.1048 0.1280
Blood
Concn, sg/ml
Aqueous
Blood
0.0000 0.0265 0.0505 0.0762 0.1021 0.1240
0 .o 0.2 0.4 0.6 0 .a 1.o
0.0000 0.0117 0.0253 0,0400 0.0542 0,0684
0.0000 0.0114 0,0255 0.0397 0,0545 0.0689
Table V. Comparison of Single Element and Multielement Spikes
Table 11. Analytical Results for Pb Concn, a/ml
0 .o 0.2 0.4 0.6 0.8 1.o
Absorbance Aqueous
Absorbance Blood
Element
Single
Multi
Cd Pb Ni TI
0,1096 0.0640 0 ,0449 0.0501
0,1129 0.0645 0.0403 0.0467
0 .oooo 0.0106 0.0218 0,0345 0.0451 0.0556
0.0000 0 ,0095 0.0204 0.0312 0.0412 0.0514
Table VI. Standard Deviation for Nickel Concn,
Table 111. Analytical Results for Ni Absorbance
Concn,
0 .o 0.2 0.4 0.6 0.8
1.o
Aqueous
Blood
0.0000 0,0157 0.0311 0.0424 0.0530 0.0724
0.0000 0 .0144 0,0289 0.0437 0.0595 0.0742
thiocarbamate (SDDC) since the extractions are reported to be efficient over a broad pH range (16, 17) for most metals. Whole blood was chosen for testing because two of the elements (Le., P b and Cd) are reported to be present mainly in the erythrocytes, while the other two elements ( i e . , T1 and Ni) are about evenly distributed between the plasma and red blood cells. EXPERIMENTAL
Apparatus. A Perkin-Elmer Model 303 atomic absorption spectrophotometer with a recorder readout module and a PerkinElmer 56 strip chart recorder was used for all measurements. The instrument was equipped with a three-slot Boling burner. A vortex mixer was used for all stirring. Samples were centrifuged in an International Centrifuge. Reagents. Reagent grade chemicals were used throughout. Stock metal solutions were prepared by determinate weighing of salts to give a concentration of 1000 ppm of the elements. All solutions were made 0.1% in nitric acid (Ultrex-J. T. Baker). Working standards were prepared by appropriate dilutions of the stock solutions. The methyl isobutyl ketone (MIBK) was used without further purification. The Triton X-100 was from Baker. The sodiumN,N-diethyl dithiocarbamate was a 25% aqueous solution obtained from K & K Laboratories, Inc. Procedure. Five milliliters of whole blood treated with citrate or heparin as anticoagulant are pipetted into a 100-mm X 10-mm test tube. One milliliter of a solution containing 2% SDDC and 5% Triton X-100 is pipetted into the blood. The solution is mixed on a vortex mixer for 10 seconds, then left standing for ten minutes for complete hemolysis of the blood to occur. Three milliliters of MIBK are pipetted into the tube which is then stoppered with a polyethylene stopper and shaken gently. The solution is centrifuged a t approximately 1200 rpm for five minutes. The MIBK layer is aspirated directly into an air-acetylene flame and the % (16)E.Montford, Can. Spectrosc., 13, 2 (1968). (17)C.E. Mulford, At. Absorption Nswsletf.,5,88 (1966). 1598
rdml
Av absorbance
0.2 0.4 0.6 0.8 1.o
0,0144 0.0289 0.0437 0.0595 0 ,0742
Std dev
+O ,00029 i-0.00104 1 0 ,00110 +~0,00087 10.00000
absorption recorded. Water saturated MIBK is used to set 0% absorption. Measurements are made at 2833 A, 2288 A, 2767 A, and 2322 A for lead, cadmium, thallium, and nickel respectively. Standards are prepared by spiking pooled human blood or cows blood to contain 0,0.2,0.4,0.6,0.8,1.0 pg/ml of lead, thallium, and nickel and 0.02,0.04,0.06,0.08, 0.1 oglml of cadmium.
RESULTS AND DISCUSSION
The applicability of the procedure to the determination of each element was investigated by comparison of results obtained using water spiked with the metal to results obtained using spiked blood. Although metals which exist in physiological fluids as a result of ingestion and metabolism might behave differently than metals added directly to the fluid, this approach was necessitated because of the lack of specimens with known levels of these elements as determined by a digestion procedure. Tables I-IV give the results in absorbance obtained for water and blood spiked with Cd, Pb, Ni, and T1, respectively. In all cases, there is excellent agreement between the aqueous standards and the blood. Differences observed are within the 3-5% relative per cent standard deviation found for the procedure. The data show that none of the metals are more strongly complexed by blood components when compared to the dithiocarbamate complexing agent used. The data presented are for SDDC; however, the same results were obtained with APDC. The above results were obtained for solutions of the individual elements. A similar study was carried out using multiple metal spikes to determine if any metal exerted an effect on the extraction or atomic absorption behavior of another metal. No interactions were observed. The same signals, within experimental error, were obtained for all elements when extracted from multielement spikes as from single element spikes. This is shown in Table V in absorbance for all metals at a concentration of 0.6 ,ug/ml, except cadmium which was 0.06 Qg/ml.
ANALYTICAL CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
Table VI contains some data on precision for nickel. Data for all the other metals fall within the same range so they are not included. The relative per cent standard deviations are from 2 to 5% for all elements a t the lowest concentration. The results are based on triplicate samples of spiked blood taken through the entire procedure. The effect of EDTA on the procedure was investigated since EDTA is widely used in the treatment of metal intoxication. EDTA is also used as an anticoagulant in blood samples. The EDTA was added as 1 ml of a 0.01M solution to aqueous and blood standards. The level added corresponds to 2.5 mg/ml in blood. When used as an anticoagulant, EDTA levels of 1.5 mg/ml are expected. The levels expected during EDTA therapy are variable due to the varied treatment schemes in use. The results are shown in Table VII. The data illustrate that only Ni is significantly affected by the presence of EDTA. A decrease of approximately y3 in the readout signals is observed with Ni. This decrease is attributed to the complexing of Ni by EDTA preventing its complexation and extraction as the SDDC complex. Despite this interference, the method is still applicable to Ni since a measurable amount (Le., approximately f/3) of the Ni expected in cases involving nickel intoxication would be detected. Another method of analysis is required to quantitate the amount of nickel present. Although the method was not tried with other metals, it is expected that it will be applicable to a large number of metals ( i e . , Bi, Cu, Co, etc.) which are extractable as the SDDC complexes into MIBK. Under the conditions used, it is possible to analyze for only four elements at a time because of the limited volume of MIBK available for aspiration into the flame. The number of metals measured in a single 5-ml sample can be increased by decreasing the aspiration rate and/or increasing the amount of MIBK used for extraction. Although these steps decrease the sensitivity of the method, sufficient sensitivity is available for the determination of these elements. It is also possible to use the new micro nonflame systems to greatly increase the number of elements determined without increasing the sample size.
Table VII. Effect of EDTA Concn,
Absorbance
Element
i.lg/ml
No E D T A
With E D T A
Cd Cd Ni Ni Pb Pb TI TI
0.02 0.10 0.2 1.o 0.2 1.o 0.2 1.o
0.0429 0.1765 0.0915 0.3675 0,0246 0.0985 0.0165 0.0820
0.0391 0.1752 0.0292 0,1454 0.0250 0.0946 0.0168 0,0757
The sensitivity of the method for P b and T1 is 0.08 pg/ ml. This is far below the level expected in blood for lead poisoning (>0.4 pg/ml) or thallium poisoning (>0.1 pglml). The sensitivity of the method for Ni is 0.06 pg/ml. Normal Ni values are
