Atomic Absorption Determination of Cadmium and Lead in Whole Blood by a Reagent-Free Method Thomas R. Hauser, Thomas A. Hinners, and Jane L. Kent Environmental Protection Agency, National Environmental Research Center, Division of Health Effects Research, Bio-Environmental Laboratory Branch, Research Triangle Park, N . C . 27711
A method is presented whereby blood can be analyzed for cadmium and lead with relative detection limits of 0.2 ng/ml and 2 ng/ml, respectively. Whole blood was added to a flame-purged tantalum sampling boat, dried in an oven, and ashed in a low temperature asher. Analysis was achieved by directly flaming the ash in the atomic absorption apparatus. Calibration was accomplished by flaming aliquots of metal standard solution in the same sampling boat used for the blood analysis. Recovery of cadmium and lead from spiked blood samples was complete. The analysis of twelve 0.5-ml replicates of one blood sample for cadmium gave a mean of 3.5 ng/ml and a standard deviation of 0.9 ng/ml; for lead, a mean of 213 ng/ml and a standard deviation of 37 ng/ml were obtained. The method offers a 25-fold improvement in detection for the analysis of blood cadmium in comparison to extraction procedures.
THEPRESENCE of cadmium and lead in trace quantities in the environment has caused great concern about the effects of these metals on man. The biological influences of cadmium and lead have been reviewed recently by Underwood ( I ) . Brain damage can be caused by lead poisoning, and mice treated with lead have an increased susceptibility to infection (2). Additionally, atmospheric lead fallout has been implicated by Bazell (3) as a cause of some sickness observed in zoo animals in New YorkCity. Cadmium has been associated with arterial hypertension in man ( 4 ) ; when administered to rats, cadmium has produced hypertension (5) and increased mortality (6). Environmental cadmium poisoning has been established as the cause for an estimated 100 human deaths in Japan (7). To determine whether exposure of humans to low environmental levels of these metals results in increased blood levels, it is necessary to have a very sensitive method of analysis for both cadmium and lead. Extraction procedures coupled with atomic absorption analysis have been published (8, 9) that give a blood cadmium detection limit of 5 ng/ml. Kubota et al. ( 9 ) reported that 5 5 of 243 blood specimens contained cadmium below this detection limit. Existing analytical methods with sufficient sensitivity are available for blood lead analysis, but the accuracy of these techniques in routine usage
(1) E. J. Underwood, “Trace Elements in Human and Animal Nutrition,” 3rd ed., Academic Press, New York, N.Y., 1971. (2) F. E. Hemphill, M. L. Kaeberle, and W. B. Buck, Science, 172, 1031 (1971). (3) R. J. Bazell, ibid., 173, 130 (1971). (4) H. A. Schroeder, Arch. Emiron. Health, 21, 798 (1970). (5) H. A. Schroeder and W. H. Vinton, Jr., Amer. J . Physiol., 202, 515 (1962). (6) H. A. Schroeder, W. H. Vinton, Jr., and J. J. Balassa, J . Nutr., 80, 48 (1963). (7) N. Yamagata and I. Shigematsu, Bull. Inst. Pub. Health, Tokyo, 19, l(1970). (8) E. Berman, At. Absorption Newslett., 6 , 57 (1967). (9) J. Kubota, V. A. Lazar, and F. Losee, Arch. Enciron. Health, 16,788 (1968).
is questionable. Keppler et a / . (IO) conducted a collaborative test for blood lead analysis involving a variety of methods and laboratories, and reported that only one laboratory out of 43 obtained acceptable blood lead values for the complete set of 12 blood specimens in the study. While the speed of the Delves’ method for blood lead determination (11) is attractive, its dependence on the method of additions for calibration and on partial oxidation is unattractive. Use of the method of additions implies that recovery is incomplete, variable, or both. Partial oxidations are susceptible to variability. Application of the Delves’ method to blood cadmium analysis has not been reported. Electrically-heated carbon atomizers offer impressive detection limits for standards, but lead and cadmium in blood may be volatilized during the ashing phase required before atomization. The investigation described in this report was carried out to develop a method demonstrating an increased sensitivity for blood cadmium while, at the same time, offering simplicity and accuracy in the analysis for both lead and cadmium in blood. The method presented appears to be suitable for the analysis of other biological and environmental samples for cadmium, lead, and other elements with volatile salts (excluding any liberated during ashing). EXPERIMENTAL
Apparatus. A Perkin-Elmer Model 403 Atomic Absorption Spectrophotometer equipped with a 3-slot burner, tantalum sampling boats and holder, a Deuterium Background Corrector, cadmium and lead hollow cathode lamps, a 10-mV chart recorder, commercial grade acetylene, and compressed air was used for all analyses. A 5-chamber Trapelo Model 505 Low Temperature Dry Asher was employed for ashing operations. A vacuum oven (National Appliance Co. Model 5830) was used to dry the samples prior to ashing. Pipets and containers were acid cleaned with 1 :3 nitric acid and rinsed three times with deionized water. Reagents. Harleco standard solutions (1000 ppm) of cadmium chloride and lead nitrate were obtained from HartmanLeddon Company, Philadelphia, Pa. 19143. As a precautionary measure, 1 nitric acid was used a as diluent for the standard solutions to prevent the metals from binding to the container walls. The nitric acid used was “Baker Analyzed” Reagent; and water was deionized by passage through an IonXchanger, research model, deionizing column from Illinois Water Treatment Company, Rockford, Ill. 61105. The nitric acid and deionized water were determined to be free of detectable cadmium and lead by the boat technique. Procedure. Operating parameters of the atomic absorption instrument were established as follows. An air flow of 22 liters/minute and an acetylene flow of 7 liters/minute were maintained throughout all analyses, and the instrument was operated in the concentration mode using wavelength and slit settings as recommended by the instrument manufacturer. (10) J. F. Keppler, M. E. Maxfield, W. D. Moss, G. Tietjen, and A. L. Linch, Amer. Ind. Hyg. Ass. J . , 31,412 (1970). (11) H. Delves, Analyst (London),95,431 (1970).
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Table I. Response to Standards with and without Ashing Element Cadmium 1 ni2 5 ng Lead 100 ng 200 ng
Average signal area (mm2) of triplicates With LTA Without LTA treatment treatment 18
21 104
112
84 176
88 179
five microliters of standard solution (50 ng/ml cadmium or 2000 ng/ml lead) was added to the boat, and the liquid evaporated with the boat a/s inch from the flame. The boat was reflamed while the recorder pen was tracing to obtain the calibration response. Frequently, the calibration procedure was repeated with larger volumes of standard solution to confirm linearity of response. A plot of the peak area VS. quantity of metal is shown in Figure 1.
RESULTS AND DISCUSSION
Figure 1. Calibration curves The optic chamber of the atomic absorption spectrophotometer was purged with air when the Deuterium Background Corrector was used. The recorder response control was set at 1 (1/4-secondtime constant), and the chart speed was 6 inches/ minute; 0.25 and 1.O absorbance scale settings were employed for cadmium and lead, respectively. The burner height was adjusted to give maximum response (a setting of 5.0 for cadmium and 6.4 for lead), and water was continuously aspirated at approximately 4 ml/minute to cool the burner head. The recorder response was modified by setting the concentration dial at 10 for cadmium and 50 for lead (where 1000 is full scale). Each new sampling boat was mounted in the boat holder, leveled, and positioned 1/4 inch above the burner head. Sampling boats were not readjusted on subsequent use because the arms of the boats become brittle after flaming. With the appropriate lamp in place and the flame burning, the boat was repeatedly flamed until no absorption response was indicated on the recorder chart. With tweezers and forceps, the boat was removed from the holder and placed in a glass ashing dish of the low temperature asher (LTA). Each dish is capable of holding four sampling boats (without stacking), which permits 20 boats per ashing cycle with a 5-chamber asher. Whole blood (0.5 ml) was then added to the flame-purged boat. The dish containing the boat(s) was then placed in a vacuum oven where the blood was dried at 60-62 "C and 0.5 atmosphere of pressure for 1 hour. The dish was then transferred to the LTA. Ashing was completed in 16 hours (overnight) at a R F forward power setting of 140 watts, an oxygen flow rate of 50 cc/min, and a pressure of 1 mm. Once the instrument parameters were established, and the instrument was determined to be operating properly, the sampling boat containing the ashed blood was again positioned in the boat holder by means of forceps and tweezers. While the recorder pen was tracing, the boat was pushed into the flame and kept there until the recorder response returned to base line. An empty boat should be carried through the full procedure for a blank. Calibration. The metal under study was quantitated by comparing the peak area observed for the sample with the response obtained with standards. Peak area was evaluated by multiplying the peak height by the width at half peak height. After the blood ash was flamed and the recorder pen returned to base line, the boat was withdrawn from the flame and reinserted to verify that the boat did not alter the base-line tracing. The boat was withdrawn from the flame again and allowed to cool while still attached to the boat holder. Twenty1820
Procedure. Tantalum boats as received from the supplier must be cleaned prior to analytical usage. Intense absorption was observed at wavelengths for both cadmium and lead when new boats were flamed for the first time. Application of the Deuterium Background Corrector did not diminish this intense absorption; the tantalum boats were cleaned by repeated purging in the spectrophotometer flame until no absorption response was noted on the recorder. It is speculated that this boat contamination may result from oil used in the manufacture of the boats. After the boats were cleaned, all subsequent manipulations of the boats were managed with tweezers and forceps. The response obtained from empty boats carried through the entire procedure can be attributed to dust. During the initial phase of this research, the dishes containing blood specimens in boats were placed in the LTA without prior drying, and, as the air was pumped from the LTA to establish the necessary vacuum for operation,the blood foamed and frothed. The rate of frothing of blood under sufficiently reduced pressure can cause sample loss from shallow containers. Therefore, blood samples were dried in a vacuum oven at 60-62 "C under 0.5 atmosphere of pressure for one hour. After the blood was dried, ashing was accomplished in the LTA using the parameters previously described. Complete ashing was obtained in 16 hours. The same analytical results were obtained with a 42-hour ashing as with the 16hour ashing. It is possible that less ashing time is needed; the 16-hour period was chosen so that samples could be conveniently ashed overnight (4 p m . to 8 a.m.). However, maximizing for the shortest ashing time could result in sufficient heating to volatilize some cadmium and lead compounds. For our ashing conditions, no loss of cadmium or lead was evidenced for standards alone or for standards added to blood samples (Tables I and 11). When the ash product has retained color or formed a lump, wetting and drying the ash has been of assistance in obtaining a completely ashed and dispersed specimen. High values were obtained when ashing was excluded. Previous experimenters (11, 12) have shown that an interfering absorption signal is obtained when blood ash is flamed in an atomic absorption apparatus in the analysis for lead. Kahn (12) used a Deuterium Background Corrector to free (12) H. L. Kahn, Ai. Ahsorpriau Newsleri., 7,40 (1968).
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the blood lead signal from this interfering signal; both Kahn (12) and Delves (11) used a fast recorder chart speed to isolate the lead signal. Interfering absorption was observed also at the wavelength used for cadmium analysis. In this study the Deuterium Background Corrector (12) was used and eliminated interference from nonspecific wavelength background absorption. For cadmium analysis, the Deuterium Background Corrector (12) was essential. A fast recorder chart speed did not permit cadmium analysis because the typical cadmium content of blood is so low that the cadmium signal could not be fully distinguished in the presence of the massive interfering absorption. For cadmium, the recorder scale was adjusted to obtain the optimum signal-to-noise ratio while preserving the shortest time constant in order to maximize detection of the shortlived signal. For lead, the recorder scale was adjusted to encompass the “normal” range of concentrations expected for blood lead, 100 to 400 ng/ml. For routine analysis, 0.5 ml of whole blood was added to the flame-purged boats. However, blood samples with high metal content require a volume of less than 0.5 ml to keep the instrument response on scale. Calibration and Recovery. The first calibration curves used were prepared by plotting peak height (adsorbance) us. quantity of metal standard loaded in the boat. For new boats this method appeared to be quite adequate, but with repeated usage the peak height response from boats decreased and became variable. This is probably caused by an uneven volatilization of the metal from the boat as crust accumulates in the boat. Peak area and not peak height was a more reproducible measure of instrument response; therefore, the calibration curves were prepared by plotting peak area us. quantity of metal analyzed. Replicate analyses for blood lead with one boat indicated that boats could be reused at least eight times with no loss in precision. To determine whether flame-dried standards (as described in the Experimental Procedure) were equivalent to standards carried through the entire analytical procedure, equal amounts of standard solutions were either flame-dried or given the oven drying-LTA treatment (Table I). The calibration response by both procedures was essentially the same, and calibration could be more conveniently achieved with flame-dried standards. Tests revealed that one standard solution could be used to establish a calibration curve by altering the volume analyzed instead of using a constant volume of many standard solutions of different concentrations. Although fresh dilutions of standards were prepared frequently, occasional tests of standard solutions (prepared with 1% nitric acid and stored in plastic bottles) showed no change in concentration after several weeks. Triplicate analyses demonstrated that recovery was complete when 0.5-ml aliquots of one blood specimen were fortified with 5 ng of cadmium and 200 ng of lead (Table 11). The relative detection limits of 0.2 ng/ml for cadmium and 2 ng/ml for lead are based on 0.5-ml aliquots of blood. The absolute detection limits are 0.1 ng cadmium and 1 ng lead. The term absolute detection limit as used here is defined as the quantity of metal needed to give an instrument response twice the size of the background variations. If the instrument detects 0.1 ng of cadmium, blood must contain 0.2 ng/ml of cadmium (relative detection limit) in order that 0.5 ml will provide the 0.1 ng cadmium needed for detection. In this case the relative detection limit is relative to the volume analyzed. The relative detection limits could be lowered by in-
Table 11. Recovery of Cadmium and Lead Added to Blood (Averages of 3) Blood lead, ng/ml Blood cadmium, ng/ml 8.2 226 610 (400 ng/ml added) 19.7 (10 ng/ml added) Table 111. Twelve Replicate Analyses for Blood Cadmium and Lead Blood lead, ng/ml Blood cadmium, ng/ml 4.8 24 1 4.4 276 4.4 184 3.8 238 3.8 282 4.5 154 5.0 246 1.9 225 2.3 163 2.8 169 2.1 153 2.3 237 X = 213 ng/ml 2 = 3.5 ng/ml Std dev = 37 ng/ml Std dev = 0.9 ng/ml
creasing the volume analyzed. However, no blood specimens tested to date have had cadmium (or lead) contents below detection when using 0.5-ml aliquots of blood. When one blood specimen was analyzed with 12 replicates for both cadmium and lead, the means and standard deviations obtained were 3.5 f 0.9 ng/ml and 213 f 37 ng/ml, respectively (Table 111). Comparison of this method with an existing extraction method (8) for blood lead gave excellent agreement. For a blood specimen found to contain 213 i 37 ng/ml lead (mean f. standard deviation) by the boat technique, the extraction method gave 180 st 20 ng/ml. Twelve replicate analyses were performed by both methods. In this study the extraction method appeared to offer better precision for blood lead than the boat method; however, other tests with the extraction method did not always show full recovery in spite of meticulous pH control. This same comparison could not be made for cadmium since the cadmium content of the blood was below the detection limit of the extraction procedures ; however, the majority of blood cadmium values measured to date by this method has been below 10 ng/ml, which is consistent with the report by Kubota et al. (9) for “normal” blood using a different method. Additional confirmation of the boat method was obtained when a correlation between exposure and blood lead values was revealed after 129 specimens were analyzed with knowledge of the exposure unknown to the analyst. An increased analysis rate might be achieved by substituting the smaller Delves’ cups (11) for the tantalum boats in this method by allowing more samples per ashing. The boat technique described offers a method to analyze cadmium and lead in blood without prior separation or extraction. For cadmium, the method offers a 25-fold improvement in detection over published extraction methods; for both cadmium and lead, the method offers increased simplicity and ease of analysis. RECEIVED for review February 28, 1972. Accepted May 18, 1972. Reference to brand names of equipment or chemical sources does not constitute endorsement by the U.S. Government.
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