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H. Fore and R. A. Morton, Biochem. J . , 51. 594-598 (1952). M. Suzuki and W. E. C. Wacker, Anal. Biochem., 57, 605-613 (1974). D. J. D'Amico and H. L. Klawans, Anal. Chem., 48, 1469-1472 (1976). F. Bek, J. Janouskova, and 8. Moldan, At. Absorp. Newsl., 13, 47-48 (1974). (8) S. B. Gross and E. S. Parkinson, At. Absorp. Newsl., 13, 107-108 (1974). (9) J. Smeyers-Verbeke, C. May, P. Drochmans, and D.L. Massart, Anal. Biochem., 83, 746-753 (1977). (4) (5) (6) (7)
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J. Smeyers-Verbeke, Y. Michotte, P. Van den Winkel, and D. L. Anal. Chem., 48, 125-130 (1976).
RECEIVED for review April 13, 1979. Accepted July 25, 1979. D.I.P. was the holder of an Australian Wool Corporation Postgraduate Scholarship.
Determination of Aluminum in Blood, Urine, and Water by Inductively Coupled Plasma Emission Spectrometry Pierre Allain" and Yves Mauras Laboratoire de Pharrnacologie, C.H.U., 49036 Angers Cedex, France
A method is described for the determination of aluminum in water, urine, and blood by inductively coupled plasma using a concentric pneumatic nebulizer. Optimum working conditions are determined. Interferences are systematically studied using different metals and metalloids and especially those commonly found in biological samples. Some metals, particularly Ca, Ll, Sr, Na, Fe increase background Intenshy and alkali metals and alkaline earth metals Increase the net signal intensity of AI. The limits of detection are: 0.4 pg/L in water, 1 pg/L In urine, and 4 pg/L in blood. Sampling preparation for blood and urine Is reduced to a simple dilution with demineralized water. Aluminum assays on 14 healthy subjects gave the following results: blood 12.5 f 4 (std dev) yg/L, urine 4.7 f 2.5 (std dev) pg/L.
Aluminum assays in body fluids and water have taken on considerable importance over the past few years, ever since the metal was first suspected of being involved in cases of encephalopathy in patients with renal insufficiency under dialysis. Recent reports ( I - 1 1 ) show aluminum is commonly determined by graphite furnace atomic absorption spectrometry. Our experience with this technique often produced manifestly erratic results so that reliable assays could be obtained only by frequently repeated measurements. These difficulties have led us t o carry out aluminum assays by inductively coupled plasma emission spectrometry as described below.
EXPERIMENTAL Apparatus. Plasma emission spectrometry was carried out using a Jobin Yvon Elemental Analyzer J Y 38 P, consisting of a Plasmatherm source inductively coupled to a high frequency (27.12 MHz) magnetic field operating a t 1.5 kW, a thermoregulated monochromator H-R 1000, and an electronic readout console. The monochromator in Czerny-Turner configuration includes a holographic grating with 2400 grooves/mm. The focal length is 1 m, wavelength range 190-700 nm, dispersion 0.4 nm/mm. The gas used as coolant and carrier was argon and the samples were introduced into the plasma by means of a concentric pneumatic nebulizer. Atomic absorption spectrometry was carried out using a Perkin-Elmer HGA 2100 graphite furnace mounted on an Instrumentation Laboratory IL 151 spectrophotometer with correction for nonspecific absorption. Reagents. The calibration for aluminum and the evaluation of spectral interference were based on standard Merck Titrisol metal solutions of 1 g/L. All solutions were prepared in plastic 0003-2700/79/0351-2089$01.00/0
laboratory ware with water demineralized after reverse osmosis. Working Conditions. The influence of wavelength, excitation level, nebulization, and height above load coil on the signal intensity and the background intensity was studied using a 1 mg/L solution of aluminum in water, in order to determine optimum working conditions for the best signal/background ratio. Evaluation of Interference. Spectral interference was studied by nebulizing 1 g/L solutions of different metals and recording the spectra for wavelength sweeps about 394.40 and 396.15 nm. The effect on the signal intensity and on the background intensity was studied by adding increasing concentrations of different metals to a 1 mg/L solution of aluminum and measuring the signal strength at the wavelength of aluminum, 396.15 nm, and the background level a t a lower value, 396.09 nm. The effect of anions was studied by comparing the results obtained with 1 mg/L solutions of aluminum in sodium chloride, nitrate, sulfate, phosphate, and EDTA solutions at appropriate concentrations so that each sample contained 0.5 g/L of sodium. Procedure. Water, urine, and blood aluminum assays were carried out at 396.15 nm. Urine samples were diluted to 1/4, blood samples to l / i o while water samples were examined pure or diluted in the case of concentrated solutions such as used in dialysis. The calibration was carried out using an additive technique: each sample was successively added to using standard solutions in appropriate concentrations so as to obtain standard additions of 31.25, 125, and 500 pg/L. After centrifugation, each tube was measured by taking five readings of 5 s each. The background intensity, measured after a wavelength displacement of 0.06 nm, was subtracted from each measurement. The standard addition lines were calculated by the method of least squares, and the concentration of the samples was determined by extrapolation.
RESULTS O p t i m u m Working Conditions. Among the various known lines of aluminum, only two, 394.40 and 396.15 nm, produce a signal strong enough to allow aluminum determinations a t concentrations of less than 1 mg/L. For each of these lines, the best working conditions were obtained at 1 kW, at a height of 25 m m above the load coil, with nebulization at a pressure of 30 psi and argon flow at 1 L/min corresponding t o a nebulization speed of about 6 mL/min. The coolant gas (argon) flow was adjusted to 13 L/min. Under these conditions, a 1 mg/L solution of aluminum gave a n overall signal-background ratio of 40 at 396.15 n m and 20 at 394.40 nm. Interference. Interference was evaluated using different metals and metalloids, especially those commonly found in biological samples. T h e interaction of the matrix elements on aluminum determination can be classified under three headings: spectral interference, modification of background 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979 ca
300
I
tI
i /
/
1
/
/
2,
--
11
Figure 2. Spectra obtained for wavelength sweeps from 395.90 to 396.35 nm with pure water (l),pure water containing 250 pg/L AI (2), 250 mg/L Ca (3), 250 pg/L AI + 250 mg/L Ca (4)
INTERFERING
METALS
g/L
Flgure 1. Action of different metals on the background measured at
the peak base of aluminum line: 396.09 nm level, and modification of signal strength. Spectral Interference. No spectral interference was observed with Na, K, Mg, Sr, Li, and T1. With Ca, Fe, Pb, Zn, Hg, Cu, Bi, La, and Rb, signals of identical relative intensity obtained a t exactly the wavelength of aluminum 394.40 and 396.15 nm, were attributed to aluminum impurities in concentrations less than 20 pg/L. The only element which gave several peaks around 394.40 and 396.15 nm was boron at 1 g/L and may therefore possibly introduce errors in aluminum determination. However, these peaks disappear at boron concentrations of less than 1 mg/L. Calcium produced an emission at 396.8 nm, but this could easily be separated from the aluminum line by the high resolution of the monochromator. Modification of Background Leuel. Some metals in solution can increase the background level of demineralized water. In Figure 1, signal counts are plotted against concentrations of interfering metals, with 100 counts as the base value of demineralized water for background correction at 396.09 nm under the working conditions described. Among the metals, Ca, Li, Sr, Na, Fe, K, Cu, and Mg, calcium provoked the greatest modification of background level, a fourfold increase at a concentration o f 1 g/L. Figure 2 shows that, as mentioned above, the interference is not due to the presence of a calcium line but to an increase in background around 396 nm. At biological concentrations, and taking into account the dilutions necessary for blood and urine assays, Na, K, Ca, and Fe are the only metals likely to lead to increase in background. When several of these metals are involved, the increase in background is roughly equal to the sum of the component increases. The metals, Pb, Bi, Zn, and Rb, and the anions, chloride, sulfate, nitrate, phosphate, and EDTA, showed no action on background intensity. Similar results were obtained for aluminum determination at 394.40 nm.
A2DED
Figure 3.
METAL
9-,
Action of different metals on 1 mg/L AI signal intensity
Modification of Signal Strength. Figure 3 shows signal counts, with background intensity subtracted, plotted against concentrations of added metals, with 100 counts as the base level for a 1 mg/L aluminum solution in demineralized water. It can be seen that the aluminum line intensity at 396.15 nm is modified by the presence of other metals in solution. Alkali metals and alkaline earth metals increase the signal strength by 20-30% at a concentration of 0.01 g / L and by almost 100% at 1 g/L. Copper is the only element tested which reduced the signal intensity. When several metal ions are present, the increase in signal strength is roughly equal to that of the element producing the highest increase on its own. The anions, chloride, sulfate, nitrate, phosphate, and EDTA, showed no action on signal strength. Similar results were obtained for aluminum determination at 394.40 nm. Blood, Urine, and Water Aluminum Determinations. The calibration graphs for aluminum in blood, urine, and water are remarkably linear. Over a wide range of concen-
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
Table I . Reproducibility of 20 Day-to-Dag Replicate Determinations mean X standard coefficient PgIL deviation of variation water urine blood
12.6
1.1
8.7
4.4
394
16.3 1.5 12.4
40
2.7
3.1 6.8
483
23.5
4.9
374 21
7.1
trations (0-2000 pg/L) linear regression coefficients are 0.9999 in blood, 0,9999 in urine, and 0.9998 in water. The detection limit was calculated as the concentration corresponding to twice the standard deviation of background noise (12, 13). With the dilutions used, we find 0.4 pg/L for water, 1 pg/L for urine, and 4 pg/L for blood. Table I indicates the reproducibility of the method used. The same measurements were repeated each day for 20 days. The coefficient of variation is 6-9% at low concentrations and 3-5% a t high concentrations. Aluminum assays on 14 healthy subjects of both sexes gave the following results: blood, 12.5 A 4.0 (std dev) pg/L; urine, 4.7 f 2.5 (std dev) pg/L. Aluminum assays on some patients under dialysis sometimes indicate concentrations as high as 500 pg/L or even higher. Ordinary tap water from our town supply contains less than 30 pg/L of aluminum. The results of 16 blood samples routinely assayed using plasma spectrometry and graphite furnace atomic absorption techniques show a good correlation between the two methods ( r = 0.991). DISCUSSION The study of spectral interference showed that only the presence of boron at concentrations higher than 1mg/L could introduce an error in aluminum assays. Boron, a t such concentrations, is very rare in biological samples and could be easily dealt with by identification at 249.6 nm, a wavelength a t which there is no interference with aluminum. The signal count a t 396.15 nm could then be corrected for the concentration of boron in the sample. Aluminum assays could also be carried out a t 394.40 nm, but this wavelength has the disadvantage of lower sensitivity although the interference and the interaction due to other metals in the matrix remain unchanged. Besides, it should be noted that the interaction on the background and on the signal intensity vary with working conditions: height above load coil, nebulization and plasma torch characteristics. Thus the values indicated in Figures 1 and 3 are mean values obtained under the defined conditions. The matrix of the individual samples, especially in the case of urine and water, is unknown and may vary considerably causing changes in signal intensity and in background level. This is why it is better to calibrate for each sample by using the additive technique and measuring the background a t peak base by wavelength shifts. For blood samples, however, we observed almost no differences in background intensity, which may be explained by their relatively constant viscosity and their similar Na, K, Ca, and Fe content. During the nebulization of blood, the main problem encountered was due to the presence of high concentrations of organic compounds which burn in the neighborhood of the induction coils and clog the central orifice of the plasma torch. Increasing the dilution of the samples to 1/15 or 1/20 produced only slight improvement while the sensibility fell
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sharply. T o overcome this difficulty, we had new torches constructed, with a larger diameter for the upper orifice of the central tube, which allow several hundreds of blood samples, diluted to 1/10, to be assayed for aluminum without clogging and without loss of sensitivity compared to standard equipment. The nebulization of undiluted urine raised no special problems, although it was found preferable t o dilute the samples so as to dissolve minerals precipitated during storage a t 4 "C as well as to decrease background level. One of the main advantages of the plasma torch is to allow blood and urine aluminum assays without modification of samples, thus decreasing the risk of loss or contamination which easily occurs with techniques based on mineralization, precipitation, or extraction. Centrifugation after dilution, destined to prevent clogging of the nebulizer, does not alter the signal strength. The only disadvantage of the present technique is that it requires fairly large test samples of a t least 2 mL but we have not yet had any experiences with the ultrasonic nebulizer. We are currently experimenting with the introduction of microsamples using a graphite furnace. The sensitivity of aluminum assays in water samples corresponds to that indicated by Boumans and Barnes ( 1 4 ) . The linearity of the calibration graphs extends to very high aluminum concentrations so that practically all blood and urine samples can be directly assayed. For the same reason, in routine assays on large numbers of biological samples, it is possible to use a single addition for calibration. Our aluminum assays on healthy subjects are in good agreement with those obtained by other authors (1, 11,15) using atomic absorption spectrometry. However, the reproducibility of results is far more satisfactory with the plasma torch method. Our experience with graphite furnace absorption spectrometry in routine aluminum assays showed the appearance of frequent erratic peaks in spite of all the modifications we attempted in the graphite tube as well as in the techniques of sample preparation. The poor consistency of the method sometimes obliged us to inject each sample, three or even five times over as did Crapper (16). Having encountered no difficulties of this kind during hundreds of blood, urine, and water assays with the plasma torch, we have finally decided to drop graphite furnace atomic absorption spectrometry in favor of inductively coupled plasma spectrometry for aluminum determination. LITERATURE CITED J. P. Clavel, M. C. Jatidon, and A. Galli, Ann. Bioi. Clin., (Paris), 36, 33 (1978). C. Fuchs, M. Brashe, K. Pashen, H. Nordbeck, and E. Quellhorst, Ciin. Chim. Acta, 52,71 (1974). K.Garrnestani, A. J. Bbtcky, and E. P. Rack, AMI. Chem., 50,144 (1978). J. E. Gorsky and A. A. Dietz, Clin. Chem. ( Winston-Salem, N.C), 24, 1485 (1978). K. Julsharnn, K. J. Andersen, Y. Willassen, and 0. R. Braekkan, Anal. Biochem., 88,552 (1978). M. T. Kovalchik, W. D. Kaehny, A. P. Hegg, J. T. Jackson, and A. C. Alfrey, J . Lab. Ciin. Med., 92, 712 (1978). F. J. Langrnyhr, and D. L. Tsalev, Anal. Chim. Acta 92, 79 (1977). G. R. Legendre and A. C. Alfrey, Clin. Chem., ( Winston-Salem, N . C . ) . 22, 53 (1976). Y. Pegon, Anal. Chim. Acta. 101, 385 (1978). J. A. Persson, W. Frech, and A. Cedergren, Anal. Chim. Acta, 92, 95 (1977). S. Ranisteanc-Eavdon, F. Prouikt, and R. Bowdon, Ann. @hi.CMn. (Pad), 36, 39 (1978). P. W. J. H. Bournans, F. J. De Boer, Spectrochim. Acta, Part B , 32, 365 (1977). S. Greenfield, ICP Inform. Newsi., 4, 199 (1978). P. W. J. M. Bcumans and R. M. Barnes, ICPInfcnm. News/., 3,445 (1978). W. D. Kaehny, A. C. Alfrey, R. E. Holrnan, and W. J. Shorr, Kidney Int., 12, 361 (1977). D. R. Crapper, S. S. Krishnan, and S. Quittkat, Brain, 99, 67 (1976).
RECEIVED for review April 2, 1979. .4ccepted July 25, 1979.
