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Fast furnace determinations of aluminum and iron in bone and soft tissues
S ecfrochwrca Acta Vol 47B. No 2. pp 239-244. 1992 Pprmted m Great Brttam
osM-8547/92 $5 cm + al 0 1992 Pergamon Press plc
Fast furnace determinations of aluminum and iron in bone and soft tissues* LIANGIIAN? Brooks Rand Ltd, 3950 Stxth Avenue Northwest, Seattle, WA 98107 U S A (Recerved 25 March 1991, accepred 3 Jufy 1991) Abstract-A fast method for Zeeman graphtte furnace AAS was developed for the determmatton of Al and Fe m bone and soft ttssues No pyrolysts step and no modtfier was used The analyttcal time was reduced to less than 1 min per determmatton wrth no loss of analyttcal precision and accuracy A simple HNO, dtgestton resulted m clear soluttons which could be quantified wtth pure aqueous standards The analyttcal performance of the Al determmation m bone and soft tissues was considerably improved by using the STPF technique and a residual problem was solved by simply pretreating a L’vov platform wtth a bone dtgestate For the determmatton of Fe, the use of HNO, was required to ensure highest accuracy
INTRODUCTION MANY publications have documented that Al and Fe accumulate in dialysis patients with chronic renal failure and subsequently induce associated diseases [l-3]. Consequently, serum or plasma Al and the plasma concentration of ferritin, are routinely monitored [2] and many analytical methodologies have been developed based on the informatton obtained from such monitoring. However, the concentration of Al in serum or plasma does not represent the true body store of this element. Rather, bone and tissues have been suggested as preferable specimens to use m the evaluation of this element [4]. In the case of Fe, an increased plasma ferritm concentration usually reflects an increase m total body Fe. But tt does not distinguish between parenchymal and reticuloendothelial Fe and thus is not specific for determining Fe-toxicity [5]. Therefore, the determination of Al and Fe in bone and various soft tissues, as well as the development of analytical methods, are necessary. Because of its high sensttivity, the ability to use small sample sizes and its relatively low cost, graphite furnace atomic absorption spectrometry (GFAAS) is now the most widely used technique for trace element determination in biological materials [6-g]. With the use of STPF techniques, modern GFAAS has become an accurate method. But compared to flame AAS and ICP, it is slow, requiring 2-4 min per determination. Experience has demonstrated that this time can be reduced, however, by eliminating the pyrolysis step (and usually the matrix modifier). These fast methods have been used for the determination of other metals in a wade variety of materials [9-141 and excellent agreement with certified values has been obtained In general, since elimmating the pyrolysis step may increase the background, Zeeman background correction is particularly important. As some modifiers provide significant background themselves, avoiding the use of a matrix modifier will often decrease the background. Compared to serum or plasma, concentrations of Al and Fe in bone and soft tissues are quite different between tissue types and individuals This makes estimating adequate dilution difficult, especially m samples from dialysis patients in which Al and Fe accumulate and from animals loaded with the two elements for pharmacokinetics tests. In these cases, the analyst must pay careful attention to the data from measurement of digests and to dilute those sample (specimens) which are too high. Thus, the
* This work was presented by the author, as a part of her Ph D. Thests, at the Department of Nephrology-Hypertension of Antwerp University (Belgium) and revtsed at Brooks Rand Ltd. t Permanant address Guangxi Anti-Eprdemic and Hygiene Statton, Nannmg, Guangxt, People’s Repubhc of Chma 239
240
LIANG
LIAN
Table 1 Furnace program for the pretreatment Step Temperature (“C) Ramp time (s) Hold time (s) Gas flow rate (ml mm-l)
of the L’vov platform
1
2
3
4
loo 10 90 300
200 5 20 300
2400 1 4 300
2600 1 3 300
analytical time becomes particularly important. This fast furnace method was developed during a study of biliary and urinal excretion of aluminoxamine and fe~oxamine in dogs. It has been used in other projects and for routine analysis.
EXPERIMENTAL
A Perkm-Elmer Zeeman 3030 AAS equipped with an HGA-600 graphite furnace, an AS-60 autosampler and an Anadex Silent Scnbe printer, was used. Pyrolytically coated graphite tubes were preferred, although uncoated tubes were also used. In some of the expenments mentioned below, the uncoated tubes provided no special problems for this analysts. Also, tt was judged that coated tubes were too expensive to use for routine application of the methods developed m this paper. ~rol~l~ly coated L’vov platforms were used for Fe determina~on whtle, for Al, platforms were pretreated by sequentially mJectmg 50 p.1of a bone digest and then atomrzing, as presented m Table 1 A total of 10 ahquots of drgestate were required to stabthze the output of the pretreated platform. The bone digest used for pretreatment was obtained by digestmg 4 g of bone, then dtlutmg to 50 ml wrth water, thus yteldmg a liquid containing 80 mg/ml bone matnx Reagents A stock Al standard soiution (1.00 g/l) (Phtlhpsburg, NJ, U S A ); 1.00 g/I stock Fe standard solution (Sigma, St LOUIS, MO, U S A ), Suprapure concentrated HNOs (Cat. No. 441) Sample dlgestton Bone and soft tissues were collected using the method of D’HAESE et al. [15]. Special care was taken to avoid contamination, from the moment of samphng unttl analysis. Wet samples weighing 0 l-O.5 g were stored m stoppered polystyrene tubes at -20°C unttl analysts. For the GFAAS analysis, samples were transferred quantttatrvely to 10 ml PTFE tubes and 1 ml of concentrated HN03 was added Each tube was stoppered, placed m an oven and drgested at 90°C for 3-4 h unttl a clear digest was obtained. This was then transferred to a polyethylene volumetnc flask and adlusted to 50 ml with doubly Qsnlled water Depending upon the expected Al or Fe ~ncentra~on, this solutton was, for Al, Qluted with water, or, for Fe, wtth 2% (v/v) HNOJ pnor to GFAAS analysts. For bone analysis, the matnx concentration should not be higher than 20 mg/ml. Working standards Standards containing 0, 25, 50, 100 &l of Al or Fe were prepared by diluting solutions with water for Al, or with 2% (v/v) HN03 for Fe, respecuvely.
the stock
RESULTS AND DISCUSSION
For the determination of Al or Fe m bone and soft trssue lgestion liqurds, the results obtained were identical with or without the pyrolysis step. Table 2 presents an example Absorbance in the peak area mode was identical. We noted that, without pyrolysis, the peak height/peak area ratios (PH/PA) were greater than with pyrolysis for each type of tissue. Moreover, the relative standard deviations of PHYPA between
Determmatton of ahumnum and non m bone
241
Table 2 Companson of the absorbance for the determmatton of Al (800 pg) m organs wtth and wtthout the pyrolysts step Samples are from a dog free of metal supplements Absorbance
Kidney Liver Heart
Peak Height (PH) Wtth Wtthout
Peak Area (PA) With Without
0 348 0 378 0 354
0.243 0 243 0 239
0 413 0408 0 414
Wtth
0244 0.244 0244 RSD%
PH/PA Wtthout
1 43 1 56 161 59
169 1 67 1 70 12
organs, using pyrolysis, were greater than without, indicating the analysis to be more free from matrix interferences when pyrolysis was not used. In addition, in two pharmacokinetics studies, concentrations in dog and rat samples were determined using both conventional analysis (with the pyrolysis step) and fast analysis (without the pyrolysis step) and the correlations of results are listed in Table 3. Except for Al in heart and muscle (where concentrations were close to the detection limits), the slopes of the regression equation and the correlation coefficients were close to 1, while the intercept of the regression equation was close to zero. These data indicated that the pyrolysis step was not necessary for these situations. With respect to the drying temperature, different temperatures (10°C) combined with relative ramp and hold times were examined. The best analytical precision was obtained by the fast program, as can be seen in Table 4. It is expected that a higher temperature (400°C) could be used in the presence of some Triton X-100, as in the slurry work by MILLER-IHLI [16], resulting in a further shortening of drying time, with no loss of precision. Matrix inter$erences and calibration Aluminum. The GFAAS determination of Al was highly susceptible to many interferences and was consequently reviewed by SLAVIN [17,181. However, when a Table 3 Correlation of results obtamed by conventtonal and fast furnace analysts. Because of the great &fference of the analyte concentratton between samples, no mean *SD was calculated Regression equation Type
n*
Element
Bone
22
Al
Bram
18
Heart
22
Liver
22
Lung
16
Ktdney
20
Muscle
20
Spleen
20
Fe Al Fe Al Fe Al Fe Al Fe Al Fe Al Fe Al Fe
Analyte cone range (pdg)
slope
mtercept
13-106 13-312 0 1-3.6 15-95 0.2-l 1 45-474 0 4-348 96-5239 1 l-4 8 57-1875 0 2-329 54-1875 0 l-l 6 13-200 2 2-226 231-5758
0 98 1 03 0 95 0 98 0.85 102 0 95 099 0 93 0% 0.98 0.95 0.84 0% 102 097
01 -1 00 -1 +o 1 -3 4 +10 +10 +o 1 +6 +08 +7 +o 1 +2 -1.2 +13
* Includmg dogs and rats loaded or unloaded wrth Al and Fe
Correlation cocffictent
0 9975 0 9971 09943 0 9981 0 9500 0 9988 0.9925 0 9954 0 9914 09996 09991 0.9994 0 9781 0.9991 0.9982 0.9952
LIANGLIAN
242
Table 4 Furnace condlttons Conventlonal Step Temperature
(“C)
Ramp time (s) Hold time (s) Glas Flow Rate (ml mu-l) Sample volume (~1) Wavelength (nm) Sht width (nm)
Fast
1
2
3
4
1
2
3
4
100
2400
2700
100
200
2400
2700
30
1450 (Al) 1100 (Fe) 5 35
1 3
5 20
1 5
300
300
0 3 (Al) 5 (Fe) 0
300
300
300
Al 10 309 3 07
Fe 10 248 3 02
5
3 PAI) 5 (Fe) 0
1 3 300
Al/Fe Slgnal mode peak area Zeeman background correction HCL current 25 mA
modern GFAAS technique with STPF was used, this susceptibility was no longer observed and the analytical performance of the Al determination was considerably improved (In this work, it was found that there remained some problems of matrix interference.) Compared to the sensitivity of the Al determination m water, soft tissues were found to enhance the sensitivity, resulting m calibration problems, implying that the matrixfree aqueous standards could not be used. This enhancement effect was more pronounced in the bone matrix. Moreover, it was found that, when the bone matrix concentration was above 20 mg/ml, the sensitrvtty decreased remarkably with increasing bone matrrx concentration. However, it was found that when a graphite tube with a L’vov platform became aged (typically after about 100 firings for biological materials) this enhancement effect was no longer observed. At that point, the Al determination was found to be free of matrix interferences for liver matrix concentrations rangmg between O-50 mg/ml and for bone matrix concentrations, ranging from O-20 mg/ml. It was noted that no sigmficant differences existed in matrix interference patterns m a variety of soft tissues. Apparently, aqueous standards free of matrix can be used for calibration when an aged tube is used The above finding was attributed to the formatron of refractory carbides due to the reaction between some refractory elements present in the matrix and the carbon from the graphite tube, which resulted m carbide coating of the platform. This encouraged consideration of whether coating the L’vov platform with ztrcomum (Zr) before analysis would provide a similar beneficial effect [19]. Here, it was found that a different interference pattern resulted-the sensitivity decreased remarkably with increasing matrix concentration, m the presence of a Zr-coated platform. However, it was found that when a L’vov platform was pretreated with bone digestion liquid, interferences were no longer observed, just as when using an aged tube Since the procedure of pretreating a platform is easier, it is suggested that pretreated platforms for Al determination m bone and soft trssues are used. Using a pretreated platform, matrix interferences were further examined by measuring 800 pg of Al m a variety of specimens (25-50 kg of brain, kidney, liver, heart, muscle, lung, spleen and bone) from a dog which was free of metal supplements. After subtracting Al blanks, absorbances in the peak area mode were identical between specrmen types (0.238 2 0 001 As) and with those found m water (0.238 As), indicating that matrix interferences were completely eliminated. Therefore, measurements can be quantified using an aqueous calibration curve. It was noted that although peak area absorbance was identtcal between bone and soft tissues, the PH/PA ratio differed significantly (for bone, the ratio was 2.3, while for soft trssues other than bone, it was 1.69 & 0.04) indicating that the matrix constituents of bone differed, while it was similar between various soft tissues. Earlier, using a less sophisticated AAS instrument,
Determmation 03
of aluminum and Iron m bone
243
IL ,r, r
*-*
*H
*
2
02 01
/
0
(
05
HNO,
1
15
2
concentration(%l
Fig 1 Influence of HNO, on the Fe GFAAS determmatlon 6 t.q of dry muscle matnx
The sample was 800 pg of Fe m
significant matrix-induced differences between bone and soft tissues were found [20]. To eliminate these, two different procedures were developed, one to adapt the determination of bone and one for soft tissues. It has now been shown, however, that Al in bone and soft tissue can be measured by one procedure and only aqueous standards are required when modern instrumentation is used with a pretreated platform. Iron. It was found that the integrated absorbance strongly depended on the HN03 concentration. Using a muscle reference sample (BCR CRM, No. 278) the relationship between the absorbance and HN03 concentration 1s illustrated m Fig. 1. As have others 1211, it was found that the absorbance increases with increasing HN03 concentration up to 1.5% HN03. When the concentration was equal to or greater than lS%, the results were found to be close to the certified values using aqueous standards, indicating that the analysis was free from matrix interferences. In order to avoid rapid damage to the graphite tubes, an HN03 concentration of 2% (v/v) was used throughout the study. The slopes of matrix matched curves for bone and a variety of soft tissues were identical to the slope of the aqueous standard curve. These data indicate that aqueous standards containing 2% HN03 can be used for the Fe determmation in bone and soft tissues. For both Al and Fe determmation it was noted that the mterferences stated above exhibited similar patterns using pyrolytically coated and uncoated tubes, but the sensitivity was higher and the accuracy and precision was better when a coated tube was used. The use of a L’vov platform was found to be essential. Analytical preckon and accuracy Aluminum. The precision was evaluated by repeatedly determining (10 times) 5 mg/ml liver digestion liquids from an unsupplemented rat m which 10, 20, 40, 60 and 80 l&l Al were added, respectively The relative standard deviations (RSD) were found to be 2.5-3%, except for the lowest concentration where it was 5%. Since no suitable reference materials with certified values for Al in bone or tissue were used, the accuracy was evaluated by recoveries of added Al into liver and bone digestates. A total of 35 hver digest soluttons containing lo-80 l.~g/lAl and l-50 mg/ml matrix were analyzed. The mean recovery was 101 + 2%. For 20 bone digest solutions containing lo-80 &I of Al in a l-20 mg/ml matrix, 102 k 3% was found. Iron. The analytical performance was evaluated by repeated analysis (10 times) of a reference muscle sample at different dilutions. The results are listed in Table 5. Good agreement between observed and certified values was obtained, indicating the method to be reliable, and the RSDs showed the excellent precision of the method. Acknowledgements-4 would hke to thank MS SHARON GOLDBLATT, of Brooks Rand, Ltd, for her conslderable edltonal assistance m the preparation of this manuscnpt
LIANGLIAN
244
Table 5 Determmatton of Fe m reference muscle ttssue (BCR CRM, No 278) The matnx weight was m 10 ul of digest The certified value IS 133 2 4 ug/g Matrtx Weight (dry, cLg) 1 18 2 36 4 72 9 44
Fe contents @g/g) found (mean ?S D ) 132 * 129 ” 130 * 130 f
3 3 2 3
RSD (%) 23 23 15 23
REFERENCES [l] M E De Broe, P C D’Haese, F L Lamberts, F L Van de Vyver and W J Vtsser, Alumrnrum Toxrnty m Duzlysrs Patrents Guldefrnes nnd Prmctples Lovens kemtske Fabrtk (LEO), Brussels (1988) [2] P C D’Haese, Ph.D Thesis Umversrty of AmsterdamlAntwerp (1988) [3] F L Van Vyver, A Vanheule, A H Verbueken, P C D’Haese, W J Vtsser, A B Bekaert, N Buyssens, W De Keersmaeker, W Vanden Bogaert and M E De Broe, Contr Nephrology 38, 153 (1984) [4] J Savory and M R Wills, Analytical techniques for the analysts of alummum, m Alununum and Health, a Crltrcal Review Ed, H J Gttelman New York and Base1 (1989) [5] C A Finch, New Engl J Med 1702 (1982) [6] W Slavm, Scr Total Envu-on. 71, 17 (1988) [7] D C. Paschal, Specrrochmr. Actu 44B, 1229 (1989) [8] N J Miller-Ihh, Spectrochrm Actu 44B, 1221 (1989) [9] W Slavm, N J Miller-Ihh and G R Carnnck, Fasr Furnace Anafysrs and Sfurry Sumphng Amencan Laboratory (1990) [lo] T Hadetsht and R D McLaughlin, Analyt Chem 48, 1009 (1976) [ll] W. Slavm, D C. Manmng and G R Camnck, Talanta 36, 171 (1989) [12] W. Slavm, D C. Manning and G R Camnck, Spectrochlm Acta 44B, 1237 (1989) [13] D Bradshaw and W Slavm, Spectrochrm Acta 44B, 1245 (1989) [14] C Bendtcho and M T C De Loos-Vollebregt, Spectrochrm Acta 45B, 679 (1990) [15] P C. D’Haese, F L Van de Vyver, F A de Wolff and M E De Broe, Chn Chem 1, 24 (1985) [16] N. J Miller-Ihh, J Anulyt Atom Specfrom 3, 73 (1988) [17] W Slavm, J Analyt Atom Specfrom 1, 281 (1986) [18] W Slavm, Graphrte Furnace AAS, A Source Book, part no 0993-8139 Perkm-Elmer Corporatton, Norwalk U S A (1984) [19] S Costantmt, R Gtordano and I Vemtllo, Mlcrochem J 30, 425 (1984) [20] L Ltang, P C D’Haese, F L Lamberts and M E De Broe, Clan Chem 37(3), 461 (1991) [21] R Karwowska, E Bulska and A Hulamckt, Talanta 27, 387 (1980)
osM-8547/92 $5 cm + al 0 1992 Pergamon Press plc
Fast furnace determinations of aluminum and iron in bone and soft tissues* LIANGIIAN? Brooks Rand Ltd, 3950 Stxth Avenue Northwest, Seattle, WA 98107 U S A (Recerved 25 March 1991, accepred 3 Jufy 1991) Abstract-A fast method for Zeeman graphtte furnace AAS was developed for the determmatton of Al and Fe m bone and soft ttssues No pyrolysts step and no modtfier was used The analyttcal time was reduced to less than 1 min per determmatton wrth no loss of analyttcal precision and accuracy A simple HNO, dtgestton resulted m clear soluttons which could be quantified wtth pure aqueous standards The analyttcal performance of the Al determmation m bone and soft tissues was considerably improved by using the STPF technique and a residual problem was solved by simply pretreating a L’vov platform wtth a bone dtgestate For the determmatton of Fe, the use of HNO, was required to ensure highest accuracy
INTRODUCTION MANY publications have documented that Al and Fe accumulate in dialysis patients with chronic renal failure and subsequently induce associated diseases [l-3]. Consequently, serum or plasma Al and the plasma concentration of ferritin, are routinely monitored [2] and many analytical methodologies have been developed based on the informatton obtained from such monitoring. However, the concentration of Al in serum or plasma does not represent the true body store of this element. Rather, bone and tissues have been suggested as preferable specimens to use m the evaluation of this element [4]. In the case of Fe, an increased plasma ferritm concentration usually reflects an increase m total body Fe. But tt does not distinguish between parenchymal and reticuloendothelial Fe and thus is not specific for determining Fe-toxicity [5]. Therefore, the determination of Al and Fe in bone and various soft tissues, as well as the development of analytical methods, are necessary. Because of its high sensttivity, the ability to use small sample sizes and its relatively low cost, graphite furnace atomic absorption spectrometry (GFAAS) is now the most widely used technique for trace element determination in biological materials [6-g]. With the use of STPF techniques, modern GFAAS has become an accurate method. But compared to flame AAS and ICP, it is slow, requiring 2-4 min per determination. Experience has demonstrated that this time can be reduced, however, by eliminating the pyrolysis step (and usually the matrix modifier). These fast methods have been used for the determination of other metals in a wade variety of materials [9-141 and excellent agreement with certified values has been obtained In general, since elimmating the pyrolysis step may increase the background, Zeeman background correction is particularly important. As some modifiers provide significant background themselves, avoiding the use of a matrix modifier will often decrease the background. Compared to serum or plasma, concentrations of Al and Fe in bone and soft tissues are quite different between tissue types and individuals This makes estimating adequate dilution difficult, especially m samples from dialysis patients in which Al and Fe accumulate and from animals loaded with the two elements for pharmacokinetics tests. In these cases, the analyst must pay careful attention to the data from measurement of digests and to dilute those sample (specimens) which are too high. Thus, the
* This work was presented by the author, as a part of her Ph D. Thests, at the Department of Nephrology-Hypertension of Antwerp University (Belgium) and revtsed at Brooks Rand Ltd. t Permanant address Guangxi Anti-Eprdemic and Hygiene Statton, Nannmg, Guangxt, People’s Repubhc of Chma 239
240
LIANG
LIAN
Table 1 Furnace program for the pretreatment Step Temperature (“C) Ramp time (s) Hold time (s) Gas flow rate (ml mm-l)
of the L’vov platform
1
2
3
4
loo 10 90 300
200 5 20 300
2400 1 4 300
2600 1 3 300
analytical time becomes particularly important. This fast furnace method was developed during a study of biliary and urinal excretion of aluminoxamine and fe~oxamine in dogs. It has been used in other projects and for routine analysis.
EXPERIMENTAL
A Perkm-Elmer Zeeman 3030 AAS equipped with an HGA-600 graphite furnace, an AS-60 autosampler and an Anadex Silent Scnbe printer, was used. Pyrolytically coated graphite tubes were preferred, although uncoated tubes were also used. In some of the expenments mentioned below, the uncoated tubes provided no special problems for this analysts. Also, tt was judged that coated tubes were too expensive to use for routine application of the methods developed m this paper. ~rol~l~ly coated L’vov platforms were used for Fe determina~on whtle, for Al, platforms were pretreated by sequentially mJectmg 50 p.1of a bone digest and then atomrzing, as presented m Table 1 A total of 10 ahquots of drgestate were required to stabthze the output of the pretreated platform. The bone digest used for pretreatment was obtained by digestmg 4 g of bone, then dtlutmg to 50 ml wrth water, thus yteldmg a liquid containing 80 mg/ml bone matnx Reagents A stock Al standard soiution (1.00 g/l) (Phtlhpsburg, NJ, U S A ); 1.00 g/I stock Fe standard solution (Sigma, St LOUIS, MO, U S A ), Suprapure concentrated HNOs (Cat. No. 441) Sample dlgestton Bone and soft tissues were collected using the method of D’HAESE et al. [15]. Special care was taken to avoid contamination, from the moment of samphng unttl analysis. Wet samples weighing 0 l-O.5 g were stored m stoppered polystyrene tubes at -20°C unttl analysts. For the GFAAS analysis, samples were transferred quantttatrvely to 10 ml PTFE tubes and 1 ml of concentrated HN03 was added Each tube was stoppered, placed m an oven and drgested at 90°C for 3-4 h unttl a clear digest was obtained. This was then transferred to a polyethylene volumetnc flask and adlusted to 50 ml with doubly Qsnlled water Depending upon the expected Al or Fe ~ncentra~on, this solutton was, for Al, Qluted with water, or, for Fe, wtth 2% (v/v) HNOJ pnor to GFAAS analysts. For bone analysis, the matnx concentration should not be higher than 20 mg/ml. Working standards Standards containing 0, 25, 50, 100 &l of Al or Fe were prepared by diluting solutions with water for Al, or with 2% (v/v) HN03 for Fe, respecuvely.
the stock
RESULTS AND DISCUSSION
For the determination of Al or Fe m bone and soft trssue lgestion liqurds, the results obtained were identical with or without the pyrolysis step. Table 2 presents an example Absorbance in the peak area mode was identical. We noted that, without pyrolysis, the peak height/peak area ratios (PH/PA) were greater than with pyrolysis for each type of tissue. Moreover, the relative standard deviations of PHYPA between
Determmatton of ahumnum and non m bone
241
Table 2 Companson of the absorbance for the determmatton of Al (800 pg) m organs wtth and wtthout the pyrolysts step Samples are from a dog free of metal supplements Absorbance
Kidney Liver Heart
Peak Height (PH) Wtth Wtthout
Peak Area (PA) With Without
0 348 0 378 0 354
0.243 0 243 0 239
0 413 0408 0 414
Wtth
0244 0.244 0244 RSD%
PH/PA Wtthout
1 43 1 56 161 59
169 1 67 1 70 12
organs, using pyrolysis, were greater than without, indicating the analysis to be more free from matrix interferences when pyrolysis was not used. In addition, in two pharmacokinetics studies, concentrations in dog and rat samples were determined using both conventional analysis (with the pyrolysis step) and fast analysis (without the pyrolysis step) and the correlations of results are listed in Table 3. Except for Al in heart and muscle (where concentrations were close to the detection limits), the slopes of the regression equation and the correlation coefficients were close to 1, while the intercept of the regression equation was close to zero. These data indicated that the pyrolysis step was not necessary for these situations. With respect to the drying temperature, different temperatures (10°C) combined with relative ramp and hold times were examined. The best analytical precision was obtained by the fast program, as can be seen in Table 4. It is expected that a higher temperature (400°C) could be used in the presence of some Triton X-100, as in the slurry work by MILLER-IHLI [16], resulting in a further shortening of drying time, with no loss of precision. Matrix inter$erences and calibration Aluminum. The GFAAS determination of Al was highly susceptible to many interferences and was consequently reviewed by SLAVIN [17,181. However, when a Table 3 Correlation of results obtamed by conventtonal and fast furnace analysts. Because of the great &fference of the analyte concentratton between samples, no mean *SD was calculated Regression equation Type
n*
Element
Bone
22
Al
Bram
18
Heart
22
Liver
22
Lung
16
Ktdney
20
Muscle
20
Spleen
20
Fe Al Fe Al Fe Al Fe Al Fe Al Fe Al Fe Al Fe
Analyte cone range (pdg)
slope
mtercept
13-106 13-312 0 1-3.6 15-95 0.2-l 1 45-474 0 4-348 96-5239 1 l-4 8 57-1875 0 2-329 54-1875 0 l-l 6 13-200 2 2-226 231-5758
0 98 1 03 0 95 0 98 0.85 102 0 95 099 0 93 0% 0.98 0.95 0.84 0% 102 097
01 -1 00 -1 +o 1 -3 4 +10 +10 +o 1 +6 +08 +7 +o 1 +2 -1.2 +13
* Includmg dogs and rats loaded or unloaded wrth Al and Fe
Correlation cocffictent
0 9975 0 9971 09943 0 9981 0 9500 0 9988 0.9925 0 9954 0 9914 09996 09991 0.9994 0 9781 0.9991 0.9982 0.9952
LIANGLIAN
242
Table 4 Furnace condlttons Conventlonal Step Temperature
(“C)
Ramp time (s) Hold time (s) Glas Flow Rate (ml mu-l) Sample volume (~1) Wavelength (nm) Sht width (nm)
Fast
1
2
3
4
1
2
3
4
100
2400
2700
100
200
2400
2700
30
1450 (Al) 1100 (Fe) 5 35
1 3
5 20
1 5
300
300
0 3 (Al) 5 (Fe) 0
300
300
300
Al 10 309 3 07
Fe 10 248 3 02
5
3 PAI) 5 (Fe) 0
1 3 300
Al/Fe Slgnal mode peak area Zeeman background correction HCL current 25 mA
modern GFAAS technique with STPF was used, this susceptibility was no longer observed and the analytical performance of the Al determination was considerably improved (In this work, it was found that there remained some problems of matrix interference.) Compared to the sensitivity of the Al determination m water, soft tissues were found to enhance the sensitivity, resulting m calibration problems, implying that the matrixfree aqueous standards could not be used. This enhancement effect was more pronounced in the bone matrix. Moreover, it was found that, when the bone matrix concentration was above 20 mg/ml, the sensitrvtty decreased remarkably with increasing bone matrrx concentration. However, it was found that when a graphite tube with a L’vov platform became aged (typically after about 100 firings for biological materials) this enhancement effect was no longer observed. At that point, the Al determination was found to be free of matrix interferences for liver matrix concentrations rangmg between O-50 mg/ml and for bone matrix concentrations, ranging from O-20 mg/ml. It was noted that no sigmficant differences existed in matrix interference patterns m a variety of soft tissues. Apparently, aqueous standards free of matrix can be used for calibration when an aged tube is used The above finding was attributed to the formatron of refractory carbides due to the reaction between some refractory elements present in the matrix and the carbon from the graphite tube, which resulted m carbide coating of the platform. This encouraged consideration of whether coating the L’vov platform with ztrcomum (Zr) before analysis would provide a similar beneficial effect [19]. Here, it was found that a different interference pattern resulted-the sensitivity decreased remarkably with increasing matrix concentration, m the presence of a Zr-coated platform. However, it was found that when a L’vov platform was pretreated with bone digestion liquid, interferences were no longer observed, just as when using an aged tube Since the procedure of pretreating a platform is easier, it is suggested that pretreated platforms for Al determination m bone and soft trssues are used. Using a pretreated platform, matrix interferences were further examined by measuring 800 pg of Al m a variety of specimens (25-50 kg of brain, kidney, liver, heart, muscle, lung, spleen and bone) from a dog which was free of metal supplements. After subtracting Al blanks, absorbances in the peak area mode were identical between specrmen types (0.238 2 0 001 As) and with those found m water (0.238 As), indicating that matrix interferences were completely eliminated. Therefore, measurements can be quantified using an aqueous calibration curve. It was noted that although peak area absorbance was identtcal between bone and soft tissues, the PH/PA ratio differed significantly (for bone, the ratio was 2.3, while for soft trssues other than bone, it was 1.69 & 0.04) indicating that the matrix constituents of bone differed, while it was similar between various soft tissues. Earlier, using a less sophisticated AAS instrument,
Determmation 03
of aluminum and Iron m bone
243
IL ,r, r
*-*
*H
*
2
02 01
/
0
(
05
HNO,
1
15
2
concentration(%l
Fig 1 Influence of HNO, on the Fe GFAAS determmatlon 6 t.q of dry muscle matnx
The sample was 800 pg of Fe m
significant matrix-induced differences between bone and soft tissues were found [20]. To eliminate these, two different procedures were developed, one to adapt the determination of bone and one for soft tissues. It has now been shown, however, that Al in bone and soft tissue can be measured by one procedure and only aqueous standards are required when modern instrumentation is used with a pretreated platform. Iron. It was found that the integrated absorbance strongly depended on the HN03 concentration. Using a muscle reference sample (BCR CRM, No. 278) the relationship between the absorbance and HN03 concentration 1s illustrated m Fig. 1. As have others 1211, it was found that the absorbance increases with increasing HN03 concentration up to 1.5% HN03. When the concentration was equal to or greater than lS%, the results were found to be close to the certified values using aqueous standards, indicating that the analysis was free from matrix interferences. In order to avoid rapid damage to the graphite tubes, an HN03 concentration of 2% (v/v) was used throughout the study. The slopes of matrix matched curves for bone and a variety of soft tissues were identical to the slope of the aqueous standard curve. These data indicate that aqueous standards containing 2% HN03 can be used for the Fe determmation in bone and soft tissues. For both Al and Fe determmation it was noted that the mterferences stated above exhibited similar patterns using pyrolytically coated and uncoated tubes, but the sensitivity was higher and the accuracy and precision was better when a coated tube was used. The use of a L’vov platform was found to be essential. Analytical preckon and accuracy Aluminum. The precision was evaluated by repeatedly determining (10 times) 5 mg/ml liver digestion liquids from an unsupplemented rat m which 10, 20, 40, 60 and 80 l&l Al were added, respectively The relative standard deviations (RSD) were found to be 2.5-3%, except for the lowest concentration where it was 5%. Since no suitable reference materials with certified values for Al in bone or tissue were used, the accuracy was evaluated by recoveries of added Al into liver and bone digestates. A total of 35 hver digest soluttons containing lo-80 l.~g/lAl and l-50 mg/ml matrix were analyzed. The mean recovery was 101 + 2%. For 20 bone digest solutions containing lo-80 &I of Al in a l-20 mg/ml matrix, 102 k 3% was found. Iron. The analytical performance was evaluated by repeated analysis (10 times) of a reference muscle sample at different dilutions. The results are listed in Table 5. Good agreement between observed and certified values was obtained, indicating the method to be reliable, and the RSDs showed the excellent precision of the method. Acknowledgements-4 would hke to thank MS SHARON GOLDBLATT, of Brooks Rand, Ltd, for her conslderable edltonal assistance m the preparation of this manuscnpt
LIANGLIAN
244
Table 5 Determmatton of Fe m reference muscle ttssue (BCR CRM, No 278) The matnx weight was m 10 ul of digest The certified value IS 133 2 4 ug/g Matrtx Weight (dry, cLg) 1 18 2 36 4 72 9 44
Fe contents @g/g) found (mean ?S D ) 132 * 129 ” 130 * 130 f
3 3 2 3
RSD (%) 23 23 15 23
REFERENCES [l] M E De Broe, P C D’Haese, F L Lamberts, F L Van de Vyver and W J Vtsser, Alumrnrum Toxrnty m Duzlysrs Patrents Guldefrnes nnd Prmctples Lovens kemtske Fabrtk (LEO), Brussels (1988) [2] P C D’Haese, Ph.D Thesis Umversrty of AmsterdamlAntwerp (1988) [3] F L Van Vyver, A Vanheule, A H Verbueken, P C D’Haese, W J Vtsser, A B Bekaert, N Buyssens, W De Keersmaeker, W Vanden Bogaert and M E De Broe, Contr Nephrology 38, 153 (1984) [4] J Savory and M R Wills, Analytical techniques for the analysts of alummum, m Alununum and Health, a Crltrcal Review Ed, H J Gttelman New York and Base1 (1989) [5] C A Finch, New Engl J Med 1702 (1982) [6] W Slavm, Scr Total Envu-on. 71, 17 (1988) [7] D C. Paschal, Specrrochmr. Actu 44B, 1229 (1989) [8] N J Miller-Ihh, Spectrochrm Actu 44B, 1221 (1989) [9] W Slavm, N J Miller-Ihh and G R Carnnck, Fasr Furnace Anafysrs and Sfurry Sumphng Amencan Laboratory (1990) [lo] T Hadetsht and R D McLaughlin, Analyt Chem 48, 1009 (1976) [ll] W. Slavm, D C. Manmng and G R Camnck, Talanta 36, 171 (1989) [12] W. Slavm, D C. Manning and G R Camnck, Spectrochlm Acta 44B, 1237 (1989) [13] D Bradshaw and W Slavm, Spectrochrm Acta 44B, 1245 (1989) [14] C Bendtcho and M T C De Loos-Vollebregt, Spectrochrm Acta 45B, 679 (1990) [15] P C. D’Haese, F L Van de Vyver, F A de Wolff and M E De Broe, Chn Chem 1, 24 (1985) [16] N. J Miller-Ihh, J Anulyt Atom Specfrom 3, 73 (1988) [17] W Slavm, J Analyt Atom Specfrom 1, 281 (1986) [18] W Slavm, Graphrte Furnace AAS, A Source Book, part no 0993-8139 Perkm-Elmer Corporatton, Norwalk U S A (1984) [19] S Costantmt, R Gtordano and I Vemtllo, Mlcrochem J 30, 425 (1984) [20] L Ltang, P C D’Haese, F L Lamberts and M E De Broe, Clan Chem 37(3), 461 (1991) [21] R Karwowska, E Bulska and A Hulamckt, Talanta 27, 387 (1980)