Microdetermination of Cadmium and Lead in Whole Blood by Flameless Atomic Absorption Spectrometry Using Carbon-Tube and Carbon-Cup as Sample Cell and Comparison with Flame Studies F. D. Posma and Johannes Balke Laboratory for Analytical Chemistry, University of Amsterdam, The Netherlands
R. F. M. Herber and E. J. Stuik’ Coronel Laboratory, Medical Faculty, University of Amsterdam, The Netherlands
Determination of cadmlum and lead in whole blood at a concentration level of i ppb and 100 ppb, respectlvely, was performed by flameless and flame atomic absorption spectrophotometry. For the flame, a routine analysis procedure was used. In the case of flameless atomlc absorptlon, carbon-tube and carbon-cup were used as sample cell for lead and cadmium, respectively. A method of direct analysls, requiring a total sample volume of 50 pl or less, was developed. Regression analysis has been applied to the calibration curves and confidence intervals were calculated, providing a constant index of precision. The analytical results obtalned with both flameless and flame atomic absorption were compared and evaluated.
A need exists for a direct and rapid method of determining cadmium and lead in whole blood that can be used for screening populations. Flame atomic absorption ( I ) has been shown to possess the required sensitivity in case of lead; however, for cadmium the detection limit turns out to be too high in most cases. Besides the sample volume, 10 ml and 15 ml blood for duplicate lead and cadmium determinations, respectively, is too large. Recent developments of flameless atomic absorption offer the opportunity for the direct determination of trace metals in biological materials requiring microliter sample volumes. Among the non-flame atomization methods proposed for lead determinations on 100 ~1 or less are atomic absorption by graphite sample cells e.g., carbon rod, graphite tube (2-8), by the tantalum strip ( 9 ) ,and the flame atomization techniques using a sampling cup (10) or sampling boat ( 1 1 ) .For cadmium determination, the r.f. carbon bed atomizer (12),and the sampling cup technique (13, 14) serve the required sample volume. However, a number of these methods cannot be considered as direct methods since there is need for external ashing of the sample or for solvent extraction (8-10, 13). Besides, in most cases the use of a deuterium background corrector is recommended or necessary (2-7,9,11,14). In this study, the possibilities for the direct analysis of lead and cadmium in whole blood using the carbon-tube and the carbon-cup as sample cells and without the need of a background corrector are experimentally investigated. The problem appeared to be the separation of the atomic absorption signal from all of the background signal because both cadmium and lead are relatively volatile elements and Present address, Laboratory of Molecular Biology, Agriculture University, Wageningen, The Netherlands. 834
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
their analytical signals are usually superimposed on the background signal caused by combustion products from the blood matrix.
EXPERIMENTAL Apparatus. The carbon rod atomizer Model 63 (Varian Techtron) was used in conjunction with a Varian Techtron A.A.5 spectrophotometer. Two different graphite sample cells coated with pyrolytic graphite were used, viz. the carbon-tube and the carboncup; the dimensions of these cells were as described by Matousek and Brodie (15). The sample cells were sheathed with nitrogen a t a flow rate of 4.5 l./min to prevent entry of atmospheric oxygen. Signals were registered with a fast response system consisting of a P.A.R. Model 126 lock-in amplifier, a transient recorder (Biomation Model 8021, and a Varian Strip Chart recorder Model A-25, as described previously by one of the authors (16). Sampling was accomplished with a 5-pl “Excalibur Autopette” fitted with disposable tips. The Perkin-Elmer Model 303 atomic absorption spectrophotometer with an air-acetylene flame was used for comparative study. Conventional hollow cathod lamps were used (Varian Techtron and Perkin-Elmer) as light sources, operated at the recommended current. The resonance lines utilized were: Pb, 217.0 nm; and Cd, 228.8 nm. Reagents and Standards. Analytical grade cadmium sulfate and lead nitrate were dissolved in a 1% “ 0 3 solution to produce 1 g/l. stock solutions. Standards were freshly prepared by serial additions of different quantities of the stock solutions to blood samples. To prevent coagulation, all standards and samples were treated with heparin. Nitric acid 65% and hydrogen fluoride 38-40%, both “Merck Suprapur”, were used for oxidation and destruction of the blood matrix. Procedure. For all lead analyses, the carbon-tube was used as sample cell and for all cadmium analyses the carbon-cup was used. T h e reason for this will be explained in the section on optimization of analytical results. Five-pl samples were deposited into tube or cup followed by the introduction of 10 ~1 of nitric acid in case of lead analyses and 15 p1 of hydrogen fluoride for cadmium analyses. Next, the mixture was subjected to three successive phases: the dry phase, the ash phase, and the atomization phase. The carbon rod power supply (Varian Techtron) automatically performs the sequential drying, ashing, and atomization phases according to preset conditions (2). The dry phase turns out to be very critical, especially for cadmium analyses; therefore, drying has to be accomplished by gently boiling the mixture in the carbon-cup. In case of cadmium analyses, the gas flow was stopped during the ash phase. This appeared to be necessary to obtain a sufficient separation of atomic absorption signal from the background signal. The time constant of the fast response amplifier-recorder system used for cadmium and lead analyses was 0.01 sec. The final experimental conditions used were as listed in Table I. Comparative flame studies were done using a routine analysis procedure similar to that used by U. Westerlund-Helmerson ( I ) . This method involves extraction of lead or cadmium from hemolyzed blood by APDC into MIBK and measurement vs. aqueous standards. However, for comparison of the flameless technique with
Table I. Experimental Conditions for the Analysis of Cadmium a n d Lead i n Whole Blooda Ash
Dly
Atomization
Element
settings
Temperature
Settings
T emperatwe
Rate
Cut-off voltage
Temperature
Cd Pb
0.5 V, 20 sec 0.5 V, 20 sec
150 "C 150 "C
2.3 V, 30 sec 2.3 V, 30 sec
530 "C 530 "C
0.4 V/sec 0.2 V/sec
4.8 V 4.2 V
1340 "C 1250 "C
Dry and ash phase are accomplished by applying a selected definite voltage (V)for a preset period of time (sec). The denoted voltage2 pertain to rms voltages. C The temperatures denote the finally reached temperature at each phase. During the atomization phase, the voltage is linearly increased a t a preselected rate (V/sec) to a chosen cut-off voltage.
;r
100-
80
-
60
-
-
3
40-
z
P
=
&
51
z
20-
L 6
0-
0 __t
TimelSecl
-
,L~ 0
a
b
,
,
3
6
\
i
9
*
Timelsecl
Figure 2. Recorder traces for cadmium in whole blood at 228.8 nm with the cadmium lamp (a) and the deuterium lamp (b)measured during the atomization phase
9
Figure 1. Recorder traces for lead in whole blood at 217.0 nm with the lead lamp (a) and the deuterium lamp (b); measured during the atomization phase Zero of time axis is arbitrary chosen
the flame method, blood standards were used throughout the present study.
RESULTS AND DISCUSSION Optimization of Analytical Signals. The problem, caused by superposition of analytical signals of both cadmium and lead on the background signal can be dealt with in several ways. However, the possibilities are limited since simultaneous background correction was not feasible with our equipment. Preliminary investigations demonstrated that the addition of acids as nitric acid, hydrogen fluoride, and perchloric acid not only destroyed the organic matrix upon heating during the dry and ash phase but also favored the resolution of the atomic absorption signal from the nonatomic absorption signal during the atomization phase. Besides, the separation of peak signals could be enhanced upon using a fast response amplifier-recorder system as was clearly demonstrated previously (16). Further experimental optimization proved that the addition of nitric acid, in the case of lead analyses, immediately after sample deposition provided excellent destruction and oxidation of the organic materials during the dry and ash phase. While during the atomization phase, the nonatomic peak, caused by residual inorganic material, split into two signals with the atomic absorption signal for lead in between. The optimum resolution of the lead signal from the nonatomic signal is shown in Figure l a together with the signal, from the same sample, using a deuterium continuum lamp Figure llr. I t is clear that the first and the last peak in Figure l a are nonatomic, caused by volatilization of the matrix. Further increase of the ash voltage, above that mentioned in 'Table I, reduced the peak separation with a consequent hampering of analytical interpretation.
A gas stop was applied during the preceding ash phase. Zero of time axis is arbitrary chosen
In case of cadmium analyses, the analytical signal remained superimposed on the nonatomic signal. However, by stopping the gas flow during the ash phase, the entry of atmospheric oxygen caused effective oxidation of both matrix and cadmium, assuming cadmium to be converted into cadmium oxide, and resulted in the shift of the atomic absorption peak with a consequent increase in resolution. Besides, the difference in volatility (Cd, bp 765 "C and CdO, bp 1599 "C) allowed the ash voltage to be increased without loss of cadmium signal as was experimantally confirmed, and ashing could be completed before the atomization phase. The addition of hydrogen fluoride for destruction of the blood matrix gave optimum results in this case. However, frothing of the sample during the dry and ash phase resulted in loss of sample and the remaining inorganic material was left in the form of a small plug or web that shielded off some of the radiation from the hollow cathode lamp. This led to the use of the carbon-cup. Frothing could be better handled by easier visual inspection and the inorganic material remained on the bottom of the cup. Although the sensitivity decreased with respect to the carbon-tube, the final analytical results were much better in the case of the carbon-cup. The optimum resolution of cadmium from the background signal is demonstrated in Figure 2a together with the signal using a deuterium continuum lamp in Figure 2b. Also, it is clear that the first and the last peak in Figure 2a are nonatomic. However, it must be mentioned that in the experimental conditions used to obtain the signals shown in Figures 1 and 2, the ash voltage is decreased with respect to those summarized in Table I to demonstrate more clearly the peak separation, but this does not affect the final analytical results. The use of a fast amplifier-recorder system that enhanced the separation of the atomic absorption signal from the background signal for both cadmium and lead matched the transient nature of the signals obtained in ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
835
015
01c
0 w
U
mu
0.05
0
100
300
200 P O D PO-
P P b Cd
Figure 3. method of
__c
Calibration curves for cadmium in whole blood by the standard additions and drawn as least-squares lines
Concentration in ppb Cd denotes the added concentration to whole blood. The horizontal bars represent 9 5 % confidence intervals (cf. Table II), 1 ppb = 0.1 fig/IOO ml. (0) Flameless atomic absorption method using a sample volume of 5 fit whole blood. (El) Flame atomization method; the absorbance values have been multiplied by 10 to avoid the large difference in slope between the curves
flameless atomic absorption and turned out to be an indispensable part of the analysis equipment. With a conventional amplifier-recorder system, the signal separation appeared to be always insufficient, a t least in the present case. Analytical Results. Calibration curves for cadmium and lead in whole blood based on the method of standard additions and drawn as least squares lines are presented in Figures 3 and 4, using the same set of standards as references for both flameless atomic absorption and flame atomic absorption. For the flameless atomization method, triplicate measurements of each standard and sample were performed while in the case of flame atomic absorption, the sample size (30 ml or less) permitted only duplicate measurements of standards and samples. Comparison of the analytical lines, obtained with both analysis methods, was done by statistical analysis of the data. The homogeneity of the variances of the standards as well as the homogeneity of the variances of samples and standards was verified according to Cochcranes criterion and F distribution ( 17). For a confidence level of 95%, the hypothesis of homogeneity could be accepted in all cases. Also the hypothesis of linearity (17) has been verified by comparing the ratio of variances, due to reproducibility and to the dispersion of the mean values with respect to the calibration line, with the tabulated values of the F distribution for the 5% level of significance. All analytical lines proved to be linear. Using the calibration line, an approxi836
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
Calibration curves for lead in whole blood by the method of standard additions and drawn as least-squares lines Figure 4.
Concentration in ppb Pb denotes the added concentration to whole blood. Symbols as defined in Figure 3
mate standard deviation for the estimate of a concentration can be derived by the law of propagation of error but this does not take into account the relationship between slope and intercept of the analytical line (18, 19). In this case, as was done previously (20), a better approach is to calculate the confidence interval for the estimate of the concentration of an unknown sample. For comparison of the analytical lines obtained with both flameless atomization and flame atomization, confidence intervals for concentrations corresponding to the mean absorbance of the standards were calculated and inserted in Figures 3 and 4 as horizontal bars. Table I1 summarizes the numerical values of the confidence intervals. The variation of the intervals with the concentration appears to be small and intervals for intermediate concentrations can easily be estimated. Furthermore, the results in Table I1 show that the 95% confidence intervals provide a constant index of precision for all calibration lines: f 3 8 ppb and f 3 3 ppb for lead analyses with flameless and flame atomic absorption, respectively; and f 0 . 9 ppb and f 0 . 3 ppb for cadmium analyses with flameless and flame atomic absorption, respectively. In the present cases, the confidence intervals for the calibration lines obtained with flame atomic absorption turn out to be smaller than those obtained with flameless atomic absorption, especially for cadmium, resulting in less precise results for the flameless technique. Also, the accuracy has been examined. First, the methods were investigated in more detail by taking the flame method as reference method and comparing it with the flameless method by calculating regression lines. The equation of the regression line was y = rnx + b where x is the estimate of a known concentration determined by the flame method and y the estimate of
Table 11.95% Confidence Intervalsa for Concentrations Corresponding to the Mean Absorbance of the Standards for the Cadmium a n d Lead Calibration Curves Mead
Calibration curve Element
Method
Cadmium
Flameless
Lead
Slope X
lo4
152
*confidence Concenba-
absorbance
Intercept
X
246
lo4
X
lo4
Relestd dev, %
tion, ppbf
intewal, *PPb#
223 605 818 1469
20.0 11.0 4.9 10.3
-0.2 2.4 3.8 8.0
io.9 0.9 0.8 1 .o
Flame
9.6
16
15 38 54 92 172
8.5 3.4 1.7 0 0.9
-0.1 2.2 4.0 7.9 16.1
0.3 0.3 0.3 0.3 0.4
Flameless
7.2
850
880 1490 2357 2990
4.0 4.0 6.2 10.9
4.0 89.0 210.0 297.0
36.0 36.0 40.0
30 59 90 108
15.6 4.2 1.7 0
-8.0 103.0 217.0 288.0
36.0 30.0 32.0 35.0
Flame
0.28
31
40.0
a 95% confidence interval: the proportion of cases in which the confidence interval fails to include the true value of unknown sample is 570. Curves were obtained with both flameless and flame atomic absorption. The calibration curves are given as least-squares lines (cf. Figures B; intercept B in absorbance and slope M in absorbance per ppb; Mean absorbance values relate 3 and 4 ) . Calibration curve Y = M X to triplicate and duplicate measurements for flameless and flame atomic method. respectively. e Relative standard deviation of the absorbance measurements. f Estimate of the concentration corresponding to the mean absorbance value and calculated by means of the calibration curve. 8 Confidence interval expressed as precision index (ppb).However, the confidence intervals are not symmetrical around the estimated concentration but deviations of the precision index values from the unsymmetrical confidence intervals are neglectable.
+
Table 111. Comparison of Results for Flameless a n d Flame Atomic Absorption Regression linea
Analytical
Correlation Element
Method
coefficient
Slope
Intercept
Detection
Characteristic
rate
limit, ppbb
concentration, ppbc
debnnlday d
Flameless 0.9986 1.015 0.1 0.2 0.3 40 Flame 4 .O 5 .O 20 Pb F lamele s s 0.9974 1.045 11 .o 15.0 5.0 40 Pb Flame 40.0 160.0 20 a The regression line v . = mx + b was calculated where x is the result of the flame method and the result of the flameless method; interCd Cd
cept b in ppb. Detection limit calculated for a signa1:noise ratio = 2 in ppb (1 ppb = 0.1 pg/lOO ml) using a sample volume of 5 p1 in case of the flameless method. Sensitivity as the concentration in ppb producing a signal of 1%absorption. Analytical rate denotes the number of complete analyses that can be performed per day.
the same known concentration determined by the flameless method. The results together with other data relating to the analytical usefulness of the methods are given in Table 111. If there were no differences between the results from the two methods, the regression line should have a slope of 1.00 and an intercept of zero. A deviation from slope 1.00 implies a difference in response between the methods. Deviations from intercept values of zero indicate a constant difference between the methods. The correlation coefficients together with slope and intercept values of the regression lines (Table 111) show good agreement between the methods. This comparison agrees with or compares favorably to the comparison of flameless and flame technique made by others ( 3 , 6 , 1 4 , 2 1 ) . The analysis results for lead and cadmium of 21 blood samples, obtained by venipuncture, are given in Table IV. The blood samples represent an arbitrary collection of samples from female volunteers including both smokers and non-smokers. The individual cadmium results cannot be compared since, in most cases, the concentration levels found were lower than the detection limit of the flame atomization method. However, in the case of samples 14, 18, 19, and 20, the flame method gives significantly higher re-
sults than the flameless atomization method. The mean blood cadmium of the 21 female volunteers, as given in Table IV, i.e., 1.3 ppb, closely agrees with the mean values of 1.3 ppb and 1.1ppb as reported by Ulander and Axelson ( 2 2 )and by Nygaard et al. ( 2 3 ) ,respectively. The mean blood lead values of 111 ppb and 93 ppb show no significant difference as revealed by Student’s test. The individual results show in 18 cases no significant difference between the methods; Le., no judgment can be pronounced on whether a significant difference is apparent on the basis of the confidence intervals. In three cases, samples 9, 11, and 12, the existence of a significant difference between the methods has been proved. The sensitivity in analytical chemistry is defined as the slope of the calibration curve and in atomic absorption spectrometry always given as the characteristic concentration corresponding to a signal of 1%absorption. The data in Table I11 show that the characteristic concentration, for 1%absorption, of the flameless atomization method is superior by an order of magnitude or more to the conventional flame method. Also the detection limits, calculated as the concentration producing a signal equal to two standard deviations of the noise and summarized in Table 111, demANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
837
Table IV. Comparison of Blood Cadmium and Blood Lead Concentrations as Determined by Flameless and Flame Atomic Absorption Cadmium, ppb" Sample no.
Flameless
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Mean result Std dev
0.6 1.4 2.5 1.6 1.6 0.4
Flame
Lead, ppb Flameless
Flame
N R ~
80.0 50.0 130.0 130.0 110.0 140.0 110.0 120.0 70.0 110.0 100.0 140.0 NR 70.0 90.0 100.0 60.0 0.9 1.7 210.0 120.0 120.0 80.0 1.3 2.2 140.0 60.0 50.0 0.7 130.0 2.0 100.0 130.0 0.8 4.3 90.0 100.0 NR 0.9 90.0 80.0 NR NR 80.0 120.0 NR 50.0 80.0 2.4 1.5 80.0 4 .O 100.0 NR 180.0 13.4 160.0 12.9 150.0 4:4 120.0 0.9 NR 70.0 60.0 1.3 111.0 93 .O ... 36.0 1.04 ... 33.0 a Results in ppb; 1 ppb = 0.1 fig/lOO ml. NR (no result) denotes NR NR NR NR NR NR NR NR NR NR NR NR
recorded. In case of the flameless atomization method, triplicate measurements of standards and samples were performed while, for the flame atomization method, duplicate measurements were executed. The flameless atomic absorption method appears to be about twice as fast as the flame method, however, with less precise results. This means that the number of standards can be increased especially near the ends of the calibration line with a corresponding reduction in the error of the calibration line to increase the precision of the results obtained with the flameless method without appreciable loss of time.
ACKNOWLEDGMENT The authors are greatly indebted to M. Hinlopen and D. Couzy-Greidanus for their technical assistance.
LITERATURE CITED
a concentration lower than the detection limit.
onstrate the capability of the flameless atomization method for the detection of very low cadmium and lead concentrations in micro samples. However, for lead, the gain in sensitivity is partly offset by the noise resulting in a reduced gain in detection limit. Table I11 also shows the number of complete analyses that can be performed per day. For the calculation of these rates, standard preparation, time for working curves, and calculation of each result have been included. One set of standards is used for the whole batch of samples and for both methods; every 10 samples, a working curve has been
838
ANALYTICAL CHEMISTRY, VOL. 47, NO. 6, MAY 1975
(20) (21) (22) (23)
U. Westerlund-Helmerson, At. Absorpt. Newsl, 9, 133 (1970). J. P. Matousek and B. J. Stevens, Clin. Chem., 17, 363 (1971). N. P. Kubasik, M. T. Volosin, and M. H. Murray, Clin. Chem., 18, 410 (1972). N. P. Kubasik and M. T. Volosin, Clin. Chem., 16, 1415 (1972). N. P. Kubasik. M. T. Volosin, and M. H. Murray, Clin. Biochem., 5, 266 (1972). J. F. Rosen, J. Lab. Clin. Med., 80, 567 (1972). B. Welz and E. Wiedeking, Fresenius' 2.Anal. Chem., 252, 111 (1970). E. Norval and L. R . P. Butler, Anal. Chim. Acta, 56, 47 (1972). J. Y. Hwang, P. A. Ulluci, S. B. Smith Jr., and A. L. Malenfant, Anal. Chem., 43, 1319 (1971). H. T. Delves. Ana/yst(London), 95, 431 (1970). H. L. Kahn and J. S.Sebestyen, At. Absorpt. Newsl., 9, 33 (1970). J. W. Robinson, D. K. Wolcott, P. J. Slevin. and G. D. Hindman, Anal. Chm. Acta, 66, 13 (1973). A. A. Cernik, At. Absorpt. Newsl., 12, 163 (1973). J. D. Bogden and M. M. Joselow, Am. ind. Hyg. Assoc. J., 35, 88 (1974). J. P. Matousek and K. G. Brodie. Anal. Chem., 45, 1606 (1973). F. J. M. J. Maessen and F. D. Posma, Anal. Chem., 46, 1439 (1974). N. N. Nalimov, "The Application of Mathematical Statistics To Chemical Analysis", Pergamon Press, Elmsford, NY, 1963. J. Mandel and F. J. Linnig, Anal. Chem., 29, 743 (1957). J. Mandel, "The Statistical Analysis of Experimental Data", Interscience, New York. NY. 1964. F. J. M. J. Maessen, F. D. Posma, and J. Balke. Anal. Chem., 46, 1445 (1974). W. N. Anderson, P. M. G. Broughton, J. B. Dawson, and G. W. Fisher, Clin. Chim. Acta, 50, 129 (1974). A. Uiander and 0. Axelson, Lancet, April 13, 682 (1974). S. P. Nygaard, G. J. Bonde, and J. C. Hansen. Nord. Hyg. Tidskr., 54, 153 (1973).
RECEIVEDfor review July 12,1974. Accepted December 23, 1974.
