Determination of Lead in Biological and Related Material by Atomic Absorption Spectrophotometry David W. Yeager, Jacob Cholakl, and Ethel W. Henderson Kettering Laboratory, Dept. of Environmental Health, University of Cincinnati, Cincinnati, Ohio 45219 ~
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A simple single extraction procedure applicable to the atomic absorption determination of trace quantities of lead in all types of biological and related material has been developed. The method involves the chelation of lead with ammonium pyrrolidine dithiocarbamate (APDC)a t p H 8.5 and extraction of the chelate into a small volume of methyl isobutyl ketone f o r aspiration into the burner of the instrument. Potassium cyanide is used to mask the effects of iron, zinc, and copper, Only bismuth and cadmium of the metal ions that react with APDC at p H 8.5 have been encountered as interferences. Procedures are suggested for analyzing materials containing large quantities of bismuth and cadmium. The method is particularly useful for the determination of lead in the range of 0.05 to 20 pg. It may be equally adaptable to the determination of bismuth, cadmium, manganese, antimony, selenium, tellurium, and thallium.
A
tomic absorption spectrophotometry (AAS) is widely used for the rapid determination of lead in urine and blood to evaluate the hazard to workmen of their exposure to the metal. AAS may be applied directly to urine and to solutions of ashed samples of blood, but to improve reproducibility and to provide greater sensitivity, it is generally combined with a chelation and extraction technique in which lead is complexed with ammonium pyrrolidine dithiocarbamate (APDC) and is then extracted into methyl isobutyl ketone (MIBK)for aspiration into the burner of the instrument (Allan, 1961; Berman, 1964; Hessel, 1968; Perkin-Elmer Corp., 1968; Pierce and Cholak, 1966). The AAS procedure most widely used for determining the lead content of blood employs heparinized samples and involves precipitating the proteins, adding APDC,and extracting the resulting complex into MIBK (Berman, 1964; Perkin-Elmer Corp., 1968 ; Pierce and Cholak, 1966). In the case of urine, the chelation and extraction is simply done on the acidified fresh sample (Berman, 1964; Pierce and Cholak, 1966). In attempting to apply the chelation and extraction technique at p H 2-3 to solutions of ashed biological material (clotted blood, particularly), recoveries of lead were inconsistent, often appreciably below known added amounts o r those reported by the colorimetric procedure used in this laboratory (Cholak et al., 1948). It was determined that iron was the main interference when present in milligram quantities (as in blood) since it prevented the complete chelation and extraction of the lead. Farrelly and Pybus (1969) noted this interference in working with digested red cells and employed a double extraction procedure involving an initial extraction with diethyl ether followed by the chelation with APDC at p H 2-3 and final extraction into MIBK. In investigating other methods for eliminating the interference by iron, the observation by Malissa and Schoffmann (1955) that lead reacts To whom correspondence should be addressed. 1020 Environmental Science & Technology
with APDC at p H 7-8 and that such metal ions as iron, copper, and zinc d o not react in the presence of potassium cyanide offered the possibility for the development of a simple single extraction procedure. This paper, therefore, describes such a n AAS procedure especially applicable to the accurate quantitative determination of the lead content of body tissues and fluids as well as of related material. While most useful for the determination of lead in the range of 0.05-20 pg of lead per sample, the method has been used with. equal confidence for the analysis of samples containing up to 100 pg of lead.
Experimental Special Reagents. All reagents used are analytical grade throughout and when used in the amounts specified yield a blank of 0.1 pg of lead per sample. AMMONIUM CITRATE BUFFERp H 8.5. Dissolve 400 grams of citric acid in 100 ml of distilled water and 400 ml of reagent ammonium hydroxide. Allow to cool and adjust to p H 8.5 with ammonium hydroxide with phenol red indicator (0.1 aqueous, n o preservative added). Make up to 1 liter with distilled water. POTASSIUM CYANIDE SOLUTION10% wjv. Dissolve 10 grams of potassium cyanide in a small amount of distilled water. Add sufficient distilled water to make 100 mi of solution. AMMONIUM PYRROLIDINE DITHIOCARBAMATE (APDC)2 % wjv. Dissolve 2 grams APDC in a small amount of distilled water and add sufficient distilled water to make 100 ml of solution. This solution is prepared daily, The APDC used in this work was prepared by the method described by Malissa and Schoffmann (1955). A product of suitable purity may also be obtained from Fisher Scientific. 4 - METHYL - 2 - PENTANONE[METHYL ISOBUTYLKETONE (MIBK)].The MIBK is stored in a glass-stoppered bottle containing sufficient water to saturate the MIBK. LEADSTANDARDS. Stock: Dissolve 0.5 gram of lead metal (Merck, 99.95% lead) in approximately 100 ml of 50% v/v distilled nitric acid and make up to 1 liter with distilled water. 1 ml = 500 pg lead. Other standard solutions are prepared as needed by diluting appropriate aliquots of the stock s o h tion with 1% vjv nitric acid. ASH AID SOLUTION. Dissolve 25 grams of potassium sulfate in sufficient distilled concentrated nitric acid to make 100 ml. Instrumentation. The Perkin-Elmer Model 303 atomic absorption spectrophotometer equipped with a three-slot Boling burner was used for this work. The analyses were performed at the 283.3-nm resonance line at the standard conditions for lead as given in the supplier's analytical methods manual (Perkin-Elmer Corp., 1968). Absorption was recorded on a strip chart. Although the 217-nm line may also be used, the 283.3-nm line was preferred because of the better signal-to-noise ratio. Procedures Sample Preparation. Samples may be prepared by employing wet or dry ashing procedures. I n this work, samples were prepared essentially by the methods used for many years
in this laboratory and which were described by Bambach and Burkey (1942). Sizes of samples varied from 5 t o 20 grams of blood, 1 t o 10 grams of tissue, 50 to 100 m l of urine, 2 t o 15 grams of dried food material, and 5 to 10 grams of feces. Glazed silica dishes (Vitreosil) are recommended for the ashing a t 500" C. A n exception to the earlier procedure for ashing blood was the substitution of 2 m l of ash aid solution for the 10 ml of nitric acid. The ash aid served to reduce the possibility of flashing and of loss of lead (Keenan et al., 1963). The entire prepared sample is then used, o r the volume of the prepared solution is adjusted to 25 ml in glassstoppered borosilicate cylinders for storage o r for aliquoting. Atomic Absorption Determination. Transfer the entire prepared sample o r a n appropriate aliquot containing not more than 100 pg of lead to a 50-ml volumetric glass-stoppered flask. Add 2 drops of phenol red indicator, 5 m l o f ammonium citrate buffer, and ammonium hydroxide dropwise until a red color is obtained (approximate p H = 8.5). If a precipitate forms, the solution must be discarded and a smaller sample o r aliquot taken. (Note: F o r bone and kidney, the aliquots taken should contain not more than 1 gram of fresh tissue.) Add 1 ml of 10% w/v potassium cyanide solution, 1 m l of the APDC reagent, followed by an accurately measured 4 ml of MIBK. Stopper the flask and shake vigorously for 30 sec. Add distilled water to bring the MIBK layer up into the neck of the flask. Invert the flask several times for mixing and let it stand for a few minutes. Aspirate the aqueous saturated MIBK (to establish the base line) and then the MIBK layer of the sample into the atomic absorption spectrophotometer and record the absorptions o n the strip chart. Repeat the procedure for each sample. The absorptions are then converted to lead values by carrying known amounts of lead through the entire procedure. Since the reagents may contain traces of lead, a reagent blank is carried through the procedure and its absorption is subtracted from the absorptions of the samples and standards.
Table I. Recoveries of Known Amounts of Lead Added to Blood and Urine Lead added (mg/100 grams) to 5 grams of blood 0.060 0.120 0.180 0.300 Lead recov (mg/100 grams) 0.056 0.118 0.182 0.302 0,068 0.120 0.180 0.302 0.056 0.118 0.180 0.296 0.070 0.116 0.178 0.264 Mean 0.063 0.118 0.180 0.291 %Recov 105.0 98.3 100.0 97.3 Lead added (mg/liter) to 50 ml of urine 0.060
0.180
0.300
Lead recov (mg/liter) 0.052 0.164 0.298 0.064 0.166 0.314 0.056 0.198 0,292 0.058 0.186 0,296 Mean
% Recov
0,058
0.179
96.8
99.4
0.300 100.0
Table 11. Analysis of Replicate Samples of Blood and Urine (5 grams blood-50 ml urine) Blood, mg lead/100 grams Urine, mg lead/liter 0.027 0.020 0.027 0.018 0.026 0.014 0.025 0,012 0.026 0,020 0.027 0.018 0.025 0.014 0.028 0.020 0.025 0.016 0.023 0.016 Mean 0.026
Mean 0.017
0.001
S D ~0,003
SDa a
Standard deviation.
Results and Discussions Table I shows the recoveries obtained on the analysis of quadruplicate samples of blood and urine to which known amounts of lead had been added. Mean lead recoveries ranged from 97.3 to 105 % for the various quantities added to the blood, and 96.8 to 100% for the quantities added to the urine. The precision of the method was tested by 10 replicate determinations o n one sample of blood and o n one sample of urine. The results are listed in Table 11. Standard deviations from the mean concentrations of 0.026 mg/100 grams of heparinized whole blood and 0.017 mg/liter of urine were 0.001 mg/100 grams and 0.003 mg/liter, respectively. The results obtained o n analyzing different biological materials by this procedure are plotted in Figure 1 in relation to the results obtained o n the same samples by the colorimetric dithizone procedure used for many years in this laboratory (Cholak et al., 1948). The calculated regression line for the 213 pairs of results had a slope of 1.007 with a standard deviation of 0.022 at the 95 % confidence interval, indicating that, at this interval, the results obtained with the respective methods differ by approximately 2.0%. The slopes of the regression lines and the intercepts for the different types of material are presented in Table 111. The values indicate that, except for the tissues, essentially the same results were obtained by the two methods. I n comparing the results by the two methods for the tissues, a n approximate 10 % difference was obtained. Some of this difference may be explained by the fact that to obtain a more accurate analysis by what is generally considered the standard method for determining biological lead, the aliquots selected for the colorimetric method were generally larger than those used for the A A S procedure. The need for this adjustment was due to the small quantity of the original sample available for analysis and the small quantity of lead often present. In agreement with the observation by Chan and Lum-ShueChan (1969), loss of lead chelate to the aqueous phase as occurs with some metal chelates did not result when distilled water was used to bring the MIBK layers into the neck of the 50-ml volumetric flask. Although the procedure has been described for the use of 4 ml of MIBK in a 50-ml volumetric flask, increased sensitivity can be obtained by using a 25-ml flask and reducing each reagent addition by 50z. F o r larger Volume 5, Number 10, October 1971
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Table 111. Comparison of Results Obtained by the Dithizone and AAS Methods for Various Biological Materials Type of No. of pairs Slope of material of analyses Range, pg Pb/gram regression linea Intercept Blood 13 0.14-0,70 1.010 =k 0.163 0.0013 Urine 49 0,008-0,046 0.895 f 0.120 0.0015 Tissue 85 0.03-2.36 1.103 f 0.046 0 0017 Feces 32 0.30-5.10 1.015 =t 0.049 - 0,0466 Food 16 0.62-4.10 0.949 =t 0.093 0 0047 Bone 18 0.19-17.6 1.039 + 0.097 -0.3347 All samples 213 0.008-17.6 1.007 i 0.022 - 0,0004 a
Range indicates 95
confidence intervals.
samples, the extraction procedure should be carried out in 100-ml flasks with a doubling of all reagent additions. Although iron was the principal interference eliminated by chelating and extracting lead at p H 8.5 from solutions of ashed material in the presence of ammonium citrate and potassium cyanide, the extraction of copper and zinc is also prevented. With the procedure described in this paper, extraction of 10 pg of lead was complete in the presence of 10 mg of iron, 10 mg of zinc, or 5 mg of copper. When the extractions were repeated at p H 2.5 and in the absence of potassium cyanide, recoveries of the lead were erratic with precipitates being present in the MIBK in each case. The most serious interference with the poorest recovery was observed to occur when 5 mg of copper was present. The ammonium citrate is added to prevent the precipitation of phosphate and iron which otherwise would occur at p H 8.5.
50 0
Bismuth and cadmium are not masked by cyanide when is used for chelation purposes at p H 8.5. Bismuth is not normally present in biological material and has not been detected in urine, blood, or tissues, even after a bismuth derivative has been used as a gastrointestinal antiseptic. However, on the ingestion of such a derivative, the feces will contain large amounts of bismuth which is easily recognized by the yellow precipitate in the MIBK. Because of the large quantity of bismuth that may be present, it is impractical to aliquot or adjust the size of the sample to eliminate the interference so that when such samples are encountered, the procedure is to forego the chelation and extraction procedure and to determine the lead directly on the acid solution of the ashed feces, with an appropriate background correction for flame scatter (Pierce and Cholak, 1966). The interference due to cadmium has been observed only in the case of kidney in which it may be present in milligram quantities. In the presence of KCN, cadmium reacts with APDC at p H 8.5, giving a white precipitate in the MIBK which interferes with the lead extraction. The interference in this case may be eliminated by limiting the amount of kidney in the aliquot to not more than 1 gram of wet tissue. APDC
Literature Cited
Allan, J. D., Spectrochim. Acta 17, 467 (1961). Bambach, K., Burkey, R. E., Anal. Chem. 14, 904 (1942). Berman, E., A t . Absorption Newslett. 3, 111 (1964). Chan, Y . K., Lum-Shue-Chan, K., Anal. Chim. Acta 48, 434 (1969). Cholak. J.. Hubbard. D. M.. Burkev. R. E.. Anal. Chem. 20. 671 (1948). Farrelly, R. O., Pybus, J., Clin. Ckem. 15, 566 (1969). Hessel. D. W.. At. Absorotion Newslett. 7. 55 (1968). Keenan, R. G:, Byers, D.. H., Saltzman, B. E.; Hyslop, F. L., Amer. Ind. Hyg. Ass. J . 24, 481 (1963). Malissa, H., Schoffmann, E., Mikrochim. Acta 1 (Ger), 187 (1 95 5). Perkin-Elmer Corp., “Analytical Methods for Atomic Absorption Spectrophotometry,” revised, Norwalk, Conn., 1968. Pierce, J. O., Cholak, J., Arch. Enciron. Health 13, 208 (1966). _
DITHIZONE W g m
Figure 1. Plot comparing results by zone colorimetric procedure
AAS
with those obtained by the dithi-
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Receiced for reciew December 3, 1970. Accepted April 2, 1971. This research was sponsored in part by the Center for the Study of the Human Encironment under grant no. USPHS ES-00159,and in part under contracts with the National Air Pollution Control Administration ( C P A - 7 0 - 1 4 ) , International Lead Zinc Research Organization, and the American Petroleum Institute.
