2086
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
Microdetermination of Manganese in Animal Tissues by Flameless Atomic Absorption Spectrometry David I. Paynter' Department of Animal Science and Production, University of Western Australia, Nedlands, Western Australia 6009
A method is described for the microdetermination of manganese In animal soft tissues. Plasma and homogenates of tissues were acidified with HCI, followed by heating at 60 OC and Centrifugation. This treatment effectively liberates the manganese Into the supernatant fraction where its concentration was determined using flameless atomic absorption spectrometric methods. Optimal instrument operating parameters are discussed for both the Varlan model 63 and model 90 carbon rod atomizers. Matrix Interferences were not detected, and the use of background correction and manganese standard additions was found to be unnecessary. Udng a number of liver samples, good agreement was obtained between the proposed flameless method and results obtained using complete wet digestion followed by conventional flame atomic absorption analysis. Relative standard deviation for a sample of plasma extract, containing 2.07 ng Mn/mL, was 3.5 %
.
Manganese is a n essential trace element in animals. In tissues, it is involved in a number of enzyme reactions as an activator, and in a limited number of enzymes as an integral bound metal ion ( 1 , Z ) . T h e low concentrations of manganese present in plasma and most soft tissues have in the past presented problems in analysis. Neutron activation methods, while offering the required sensitivity ( 3 ) ,are not generally applicable t o routine use. Other methods previously used including colorimetric ( 4 ) and conventional flame atomic absorption spectrometry ( 5 ) , although relatively free from interference, lack the sensitivity required for many tissues, and entail relatively time-consuming sample preparation. The development of flameless (furnace) atomic absorption spectrometry has considerably lowered detection limits for manganese, and methods have now been reported for the determination of this element in biological material such as serum (6, 7) cerebrospinal fluid ( 6 ) ,and tissue fractions (8, 9). Matrix interferences have been found to occur in the flameless determination of manganese (10). In methods involving biological samples, these interferences have been at least partially compensated for by using standard additions of manganese and/or background correction (6-9). T h e small final sample size actually used in the determination in flameless methods, and the relative heterogeneity of some tissues, necessitates some form of tissue digestion or homogenization. In the present study, a method involving tissue homogenization, followed by acid treatment, permitted the determination of manganese in a variety of animal tissues by flameless atomic absorption spectrometry. Matrix interferences were not encountered in the present method and neither standard additions or background correction were necessary. Present address: Attwood Veterinary Research Laboratories, Mickleham Road, Westmeadows, Victoria, Australia 3047. 0003-2700/79/0351-2086$01 .OO/O
EXPERIMENTAL Apparatus. An AA-3'75 double beam atomic absorption spectrophotometer with simultaneous deuterium arc background correction facilities, equipped with either a CRA-63 or CRA-90 carbon rod atomizer, an ASD-53 autosampler, chart recorder, and digital printout recorder (all Varian Techtron products) was used for these studies. The manganese hollow-cathode lamp was operated at 5 mA, with the AA-375 set at 279.8 nm, using CRA slit and peak height modes. For 10-pL sample sizes, internally threaded graphite rods were used; for smaller sample sizes ( 2 and 5 pL), the normal nonthreaded rods were used. All rods were pyrolytically coated, and during use were purged with nitrogen at a flow rate of 6 L/min. The work head was equipped with a single beam mask with a 3-mm aperture. Sample Preparation. Plasma was obtained from rat heparinized whole blood. Tissues from the same animals were washed in cold 0.9% (w/v) NaCl after removal, then stored at 4 "C or -20 "C prior to analysis. Tissues were homogenized with an aqueous 0.2 70(v/v) Triton X-100 solution, using all-glass tissue homogenizers (Kontes). Homogenates of 10% (w/v) were prepared, using 0.5 or 1.0 g (wet weight) samples of tissue. To aliquots of these homogenates (in 3-mL snap cap tubes) HCl appropriately diluted with water was added, to give the required final concentration of manganese, in 2 M HC1. The acid treated samples were then heated at 60 "C for 1 h in a water bath, cooled, and then centrifuged. The clear supernatants, without further treatment, were used for injection into the carbon rod atomizer. With plasma, HC1 was added directly to the sample, followed by heating and centrifugation as described for tissue homogenates. All samples were diluted with HC1 such that total manganese in the 2-pL sample (or 10 FL in the case of plasma) applied to the carbon rod atomizer was in the range of 5-60 pg. For flape atomic absorption spectrophotometric analysis of livers, 1.0-g samples were wet ashed with 10 mL of a 9:l mixture (by volume) of nitric and perchloric acids until the digestion had been a t white fumes for 30 min. Digest volumes were then adjusted to 10 mL with water and analyzed for manganese using an oxidizing air-acetylene flame. Chemicals, A manganese standard solution, containing loo0 pg Mn/mL (as the chloride) was obtained from BDH. Dilutions in water or HC1 from a 10-pg Mn/mL stock to working concentrations were prepared on the day of use. Xitric acid was distilled before use; all other chemicals were of analytical reagent grade. RESULTS AND DISCUSSION P r e l i m i n a r y E x p e r i m e n t s . Attempts to determine manganese in untreated tissue homogenates by the flameless atomization method were unsuccessful. A considerable proportion of manganese in these samples was associated with the particulate fraction, and settling of the fraction occurred with standing of samples in the autosampler. This and the small sample size applied to the rod (2 pL) contributed to the poor reproducibility encountered. Difficulties were also found with untreated plasma samples. While a previous report has indicated t h e successful determination of manganese in untreated serum samples using a flameless atomization method (6), we encountered difficulties with spluttering and foaming of plasma samples during the drying stage, even with a 2-pL sample size. Addition of Triton X-100 (0.1 to 1.0% (v/v) final concentration) alleviated this problem, but created '01979 American
Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
Final
HCI
2087
(MI
Figure 2. Effect of HCI concentration on recoveries of manganese
I
from heart homogenate. Recovery from homogenates without manganese standard additions, 0 ; and with standard additions, A. Values for the 5 M HCI treatment value were used as the 100% recovery value
Htln
Mn
added
(ng/rnl)
Figure 1. Effect of acid treatments on recoveries of manganese from heart homogenates. Standards in H,O only, 0;standards in homogenate with final acid concentrations of 10% (v/v), 0 ; 25% (v/v), A;50% (v/v)
others. Creepage on the rod occurred, with the degree of creepage being related to the previous history of the rod being used. Standard additions were required to obtain accurate results. To overcome these matrix effects and to ensure homogenous samples, further sample treatment was desirable. Proposed Method. Several acids, including trichloroacetic, hydrochloric, nitric, perchloric, and sulfuric, and one alkali (KOH) were added a t several concentrations to samples of tissue homogenates, along with standard additions. After heating a t 60 "C for 1 h, the samples were centrifuged, then assayed for manganese. The curves for homogenate with standard additions were then compared to curves of standards in water only. The results for three of these acids (HC1, "OB, and HCLOJ are shown in Figure 1. Only HC1 treatment gave samples free of matrix interferences, as indicated by the similar slopes for standard additions of manganese t o water only and to tissue homogenates treated with HC1. Similar results were obtained for both liver and heart homogenates. Other treatments led to either excessive manganese contamination (e.g., trichloroacetic acid, KOH) and/or matrix interferences, as occurred with nitric and perchloric acids. In a previous study, significant matrix interferences by the nitrates and chlorides of both calcium and magnesium have been reported in the determination of manganese by flameless methods (10). In the present study, interferences due to chlorides of these elements, have not been observed with either tissue homogenates or plasma samples when the acid used is HC1. Significant suppression of the manganese signal was found to occur when HC1 was replaced by HNOBfor sample acid treatment. The differences in matrix effects observed between these studies relate to a number of factors, including
the types of carbon rod used, the sample sizes applied to these rods, and the relative concentrations of calcium and magnesium in the final sample. As additions of HC1 to homogenates and plasma did not completely digest these samples, the effect of HC1 concentration on solubilizing the manganese fraction was investigated. At each of several acid concentrations (0 to 5 M HCl), recoveries of manganese in heart homogenate supernatants was determined after manganese standard additions to these samples of 0 and 13.5 ng Mn/mL, followed by heating (60 "C for 1 h) and centrifugation. The results are shown in Figure 2. Without acid treatment, only 37% of the manganese inherent in the sample, and 63% of the added manganese, was recovered in the supernatant fraction following centrifugation. In contrast, addition of HC1 a t concentrations of 0.1 M and greater gave near 100% recoveries in this fraction. The low concentration required to liberate manganese from the homogenate particulate fraction, suggests that manganese is released from this fraction by a pH rather than hydrolysis-type action. Effect of Furnace Conditions on Results. In drying the sample on the rod, no problems were encountered with creepage or spluttering of the sample. In general the drying phase was very similar to that of aqueous standards, presumably owing to the removal of much of the matrix from these samples by treatment with HC1. For most samples a 2-pL sample size was used t o minimize the drying time required, and avoid unnecessary sample dilution. For plasma, however, a 2-pL sample gave insufficient sensitivity. This was overcome by the use of internally threaded graphite tubes, on which the sensitivity was increased by using a 10-pL sample size. Drying parameters for both these sample sizes are shown in Table I. In the ash cycle following drying, liver, heart, and kidney samples gave a single sharp ash peak which volatilized a t C600 "C. Above this temperature no ot,her background peaks were apparent and ash temperatures of up to 1500 OC for 10 s could be used before Mn volatilization became apparent (Figure 3), giving a relatively large range of satisfactory ashing temperatures for which background correction was unnecessary. For plasma derived samples, a second non-atomic ash fraction was present volatilizing a t approximately 1100 "C. Failure
2088
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
Table I. Operating Parameters for the Determination of Manganese, Using the Varian Carbon Rod Atomizers, and HCl Treated Samples CRA-90
carbon rod atomizer furnace type sample volume ( p L ) dry ash plasma homogenates
plain 2
5
: 2c3
-
3 350
-
3 'c;
-
10 105 'C, 60 s 1300 'C, 1 0 s
100 'C, 25 s
900 " C , 10 s
atomize
CRA-63 threaded
plain
threaded 10 5, 60 s 6.75, 20s
2
5.75, 25
s
6.5, 1 5 s
ramp rate 700 " C i s final temp. 2500 " C hold at final temp. 0.5 s
7,2 s
(step mode)
c
c5G
\
t
i
\I;
Terrpercture
I'c)
Flgure 3. Effect of ashing temperatures on the manganese signal obtained in the atomize phase from HCI treated plasma and heart
203
!'C/iecl
Rc*e
Figure 4. Effect of temperature ramp rate in atomize phase, on peak height and peak area of HCI treated samples. Peak height mode with plasma (10-pL sample size), U; Peak height mode, 0 ;and peak area mode, A ; for heart (2-pL sample size)
homogenate samples. Heart homogenate without background correction, 0 ; Plasma with background correction, A ; and without background correction,
.
..
0 .
to remove this peak resulted in considerable signal interference unless background correction was used (Figure 3). T h e ash parameters listed in Table I are for measurement of plasma samples without the use of background correction. In practice, we have observed slightly better precision when this non-atomic peak in plasma is removed prior to atomization, rather than relying on background correction for its removal. I n the atomize phase, increasing the ramp rate ("C rise in temperature/s) considerably increased the peak height values for homogenate and plasma extracts without affecting the peak area (Figure 4). Atomize settings shown in Table I enable maximum peak heights and sensitivities to be obtained. The high ramp rate used with the CRA-90, is similar to t h a t obtained using step-mode atomization available with the CRA-63 (approximately 800 "C/s); both atomizers gave similar final results a t the settings shown in Table I. Only one peak (corresponding to the element peak) occurs in the atomize phase, using the ash parameters shown in Table I. At these atomize settings, rod life usually exceeds 100 firings. Accuracy and Precision. Accuracy of the method was investigated by comparing results obtained by the proposed flameless method and by conventional flame atomic absorption spectrometry. In the latter, wet digestion of samples was performed in duplicate (see Experimental section). In the former method, a single homogenate and acid extract was prepared for each sample, with duplicate firings on the carbon rod. A total of 27 livers, obtained from rats fed diets containing 0.2 t o 30 pg Mn/g diet, were assayed. Manganese content of these livers was distributed through the range of 0.25 to 2.5 pg Mn/g wet weight. No attempt was made to re-assay any sample, and the individual results are shown in Figure 5. The Mn content of these livers, determined by the flame method (x) and the proposed flameless method (y) were
832
6cIC
LOC
Romp
. *
0 5
.
*
*
I O Mn-
I 5 Flame
2 0
2 5
! 1418 I
Figure 5. Comparison of liver manganese concentratlons determined by the proposed fhmeless method and by the conventional flame method
related according to the equation y = 1 . 0 3 ~- 0.004 pg/g wet weight, with a coefficient of determination of 0.96, showing good agreement between the two methods. Relative standard deviations for 10 replicate determinations of manganese in liver and plasma extracts were 0.5% and 3.5%, respectively. For the liver extract, the final concentration of manganese after acid treatment was 26 ng/mL and 2 pL samples were applied to the rod. The plasma extract contained 2.07 ng M n / m L final concentration and 10-pL samples were applied.
ACKNOWLEDGMENT The author is indebted to B. S. Fleming for his technical assistance and to R. J. Moir for his discussions. LITERATURE CITED (1) M. C. Scrutton, Biochemistry, 10, 3897-3904 (1971). (2) E. J. Underwood, "Trace Elements in Human and Animal Nutrition", 4th ed., Academic Press, New York, London, 1977, pp 170-195. (3) P. S.Papavasiliou and G. C. Cotzias, J . Biol. Chem., 236, 2365-2369 (196 1).
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
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)
2089
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 W o r k i n g 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
