Anal. Chem. 1993, 65,2174-2176
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Determination of Parts-per-Billion Concentrations of Indium in Biological Materials by Electrothermal Atomic Absorption Spectrometry Following Ion Pair Extraction Wei Zheng, I. Glenn Sipes, and Dean E. Carter' Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721
INTRODUCTION Indium is a group 111-Bheavy metal distributed in a minute quantity, about 0.1 pg/g of earth's crust, in nature.' In modern industry, indium compounds are mainly used in decorative coatings, bearings, low-melting alloys, glass-sealing alloys, brazings, semiconductor research, and nuclear reactor control rods.' In clinical medicine, radioactive 111In-labeled antibodies, proteins, and cells have been widely used in diagnosis of a variety of malignant tumors in liver, lung, ovary, and breast2-4 and of certain inflammatory diseases such as rheumatoid arthritis, myocyte necrosis, and prosthesis infections."' With its increasing use as an important metal in both industry and clinical medicine, the potential toxicities caused by indium have become a serious concern during the past decade. The limited information available suggests that indium may cause damage to liver, kidney, and bone marrow upon its entering the systemic circulation.a10 Although a number of studies indicate that indium may accumulate in liver following administration of l"In-labeled antibodies,11J2 little is known about its absorption, distribution, and elimination in experimental animals and in humans after exposure to different indium compounds. Lack of sensitive methods for the determination of indium in biological matrices has hampered such studies. Electrothermal atomic absorption spectrophotometry (ETAAS) has been used to determine indium in geological samples.13-16 However, this method is not applicable to quantification of indium in biological samples, because of a much lower concentration of indium in biological tissues and a severe interference from certain biologically abundant ions such as iron(II1) and c ~ p p e r ( I I ) . ~ J ~ J ~
* Author to whom correspondence should be addressed.
(1)Trace metals in the environment; Smith, I. C., Carson, B. L., Hoffmeieter, F., Eds.; Ann Arbor Science: Ann Arbor, MI, 1978 Vol. 5, pp 1-42. (2)Divni. C. R.: McDermott. K.: Johnson. D. K.: Schnobrich. K. E.: Finn; R. S,;Cohen, A. M.; Larson, S . M. Znt.'J. Radiat. Appl. Instrum: 1991,18,705-710. (3)Greager, J. A,; Chao, T. C.; Blend, M. J.; Atcher, R. W.; Gansow, 0. A,; Brechbiel, M. W.; DasGupta, T.K. J. Nucl. Med. 1990,31,13781383. (4)Kosmas, C.; Kalofonos, H. P.; Epenentos, A. A. Deu. Biol. Stand. 1990,71,93-102. (5)Oyen, W. J.;van Horn, J. R.; Claessens, R. A.; Slooff, T. J.; van der Meer. J. W. D.: Corstens. F. H. Radiolom 1992. 182. 195-199. (6jRydgren,' L.; Wolloher, P.; HultquTit, R.; Guskson, T.Scand. J. Rheumatol. 1991,20,319-325. (7)Bhattacharya, S.;Lahiri, A. Eur. J. Nucl. Med. 1991,18,889-895. (8) Toxicology and biological monitoringofmetak in humans.;Carson, B. L., Ellis, H. V., McCann, J. L. Eds.; Lewis: Chelsea, MI, 1986; pp 115-120. (9)Woods, J. S.;Carver, G. T.; Fowler, B. A. Toxicol. Appl. Pharmacol. 1979,49,455-461. (10)Castronovo, F. P.;Wagner, H. N. Br. J. Exp. Pathol. 1971,52, 543-559. (11)Fritzberg, A. R.; Beaumier, P. L. J.Nucl. Med. 1992,33,394-397. (12)Jones, P.L.; Brown, B. A.; Sands, H. Cancer Res. 1990,50,852s856s. (13)Zhou, L.; Chao, T. T.; Meier, A. L. Anal. Chim. Acta 1984,161, 369-373. (14)Shan, X.Q.;Ni, Z. M.; Yuan, Z. N. Anal. Chim. Acta 1985,171, 269-277. (15)Hayashibe, Y.;Kurosaki,M.;Takekawa,F.; Kuroda,R.Mikrochim. Acta 1989,2,1023-1027. (16)Kuroda, R.; Wada, T.; Boma, T.; Rsobo, N.; Ogaha, K. Analyst 1990,115,1535-1537. (17)Clark, J. R. J. Anal. A t . Spectrom. 1986,I, 301-308. 0003-2700/93/0365-2 174$04.00/0
The objective of this study was to develop a sensitive ETAAS technique in order to determine parts-per-billion levels of indium in biological matrices. Our results indicated that following a selective extraction of indium ions by methyltricaprylammonium ion (Aliquat 336) into a hexane-methyl isobutyl ketone solution,the principal interferencesfrom iron and copper could be excluded with the use of masking agents. The method allows determination of 0.5-1.0 ng/mL concentrations of indium in rat tissue matrices following a minimum sample dilution.
EXPERIMENTAL SECTION Apparatus. A Thermo Jarrell Ash Smith-Hieftje 12 atomic absorption spectrophotometer (AAS)equipped with a CTF 188 graphite furnace and an automated sampling device were used for determination of indium. Absorbance at 303.9 nm was corrected for background using the Smith-Hieftje technique. The hollow cathode lamps were supplied by the Thermo Jarrell Ash Co. (Franklin, MA). Lamp current was 2.0 mA with a bandwidth of 0.5 nm. Reagents. Chemicals were obtained from the following sources: certified atomic absorption standard containing 990 mg/L In(II1) and methyltricaprylammonium chloride (Aliquat 336) from Aldrich Chemical Co., Milwaukee, WI; sulfuric acid, hydrogen peroxide, glacial acetic acid, and hexane from Fisher Scientific,Pittsburgh, PA; sodiumthiosulfate and hydroxylamine hydrochloride from Mallinckrodt Chemical Works, Paris, KT; methyl isobutyl ketone (MIBK) and ultrapure nitric acid from Baxter Healthcare, McGaw Park, IL; palladium chloride from Johnson Matthey Electronics, Ward Hill, MA. All reagents used in this experiment were of analytical grade, HPLC grade, or the best availablepharmaceuticalgrade. Solutionswere freshlymade prior to the experiment. Procedure. Tissues were weighed (0.5 g) or pipeted (0.5mL) in Pyrex glass tubes. Aliquots (0,5,10,25, and 50 pL)of standard indium solution (1000 ng/mL) were added to the above tissue samples to produce final concentrations of 0, 1, 2 , 5 , and 10 ng of In/mL in a total volume of 5 mL. In the recovery study, the indium was not added to the tissue preparations until after they were digested,extracted, and ready for ET-AASanalysis. Tissues were digested with 0.5 mL of concentrated HzSOl by gradually raising the temperature to 210-220 "C and maintaining it for 15 min. A total volume of 3 mL of HzOz (30%)was added dropwise to the digest, and the solution was heated for another 5 min until no bubbles were generated. The final digestion solution was clear and lightly yellow. The digestion solution was mixed with 2 mL of a 27% (w/v) aqueous solution of NaCl and vortexed for 10 8. Aliquots of 0.2 mL of 2.5% Na&03 and 0.2 mL of 2.5% NHZOH-HCl were added; the mixture was vortexed and allowed to stand for 10 min. An organic phase consisting of Aliquat 336-hexane-MIBK (5:5:90,v:v:v) was used to extract ion pairs formed between InCLand R4N+(Aliquat ion). Following addition of 3 mL of organic phase, the mixture was stirred vigorously in a vortex mixer for 30 s. The organic and aqueous phases were then separated by centrifugation at 2000 rpm for 2 min. The upper organic phase was transferred to another set of test tubes. The remaining aqueous phase was extracted once again with an additional 3 mL of hexane-MIBK mixture (5:95, v:v). A pool of the organic phase (approximately 6 mL) was then back-extracted twice with 2 mL of an aqueoussolution containing 5 % concentrated HNOs and 5 % glacial acetic acid. The mixture was thoroughly vortexed for 30 s followed by centrifugation at 2000 rpm for 2 min to allow the separation of two phases. The aqueous phase was used for the determination of indium. 0 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 85, NO. 15, AUGUST 1, 1993
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Table I. CTF 188 Graphite Furnace Program Settings. DVrOlYSiS mode dry 1 2 atomization cleanup temp(OC) ramp (8) hold (a) purge (8)
150 2 0 1
450 5 14 2
795 5 14 2
2000 0 5 0
44
I
3000
2200 3 3
A single beam mode and a built-in automatic background compensation program were used. Lamp current was 2.0 mA with a bandwidth of 0.5 nm.
0
Prior to ET-AASanalysis,the aqueous solution (approximately 3.8 mL) was mixed with an aliquot of 1%PdClz to a final concentration of 50 pg/mL PdC12. The sampleswere then diluted to 5 mL with distilled-deionized water. The assay solution was dispersed into a pyrolytically coated atomization graphitecuvette for 40 s at a flow rate of 1.5 mL/min by a Model-K FASTAC aerosol deposition module. The settings for AAS are listed in Table I. The peak height mode was used for the quantitation of assay solution. Detection Limit and Precision Study. The detectionlimit was computed as follows:18 3SD(blank)X conc(std) X vol(std) ABS of std where SD(blank)is the standard deviation of the blank-corrected signal, conc(std) is the concentration of the low-level standard used (ng/mL), vol(std) is the volume of the standard solution injected into the graphite tube (mL),and ABS of std is the mean absorbance of the blank-corrected low-level standard. The precision study was conducted at a final concentrationof 5 ng/mL. For intraday precision, the digestion and extraction procedures were repeatedly operated for each individual tissue (n = 4-5). The interday precision study was performed on the blank samples only. The same procedure was used at 6 different days within 1 month. detection limit =
RESULTS AND DISCUSSION Thismethod was established for determination of low levels of indium in biological tissues. It coupled ion pair extraction with ET-AAS analysis. Following digestion, the standard indium ions preadded to various tissues were effectively extracted by a two-step extraction procedure. Typical ETAAS traces of indium standard in liver and blood are illustrated in Figure 1. The indium recovered from the digestion and extraction procedure was about 97 7% in blank samples, which had no tissues present (Table 11). The recoveriesof indium added to samples containing tissue (such as blood, bone, intestine, kidney, liver, lung, and urine) ranged from 88 to 110% a t a concentration of 5 ng/mL (Table 11). The calibration curves established for blood, kidney, liver, and urine showed a linearity (r2)of 0.990-0.998 (Figure 2). The best linearity range, in general, was obtained between 1 to 10 ng/mL. The absorbance displayed a small deviation from linearity at indium concentrations exceeding 50 ng/mL. The intraday precision measured as a coefficient of variation (CV) at 5 ng/mL was less than 13.9% for those samples containing tissues (Table 11). The higher variation observed in large intestine samples (15.7 % could have resulted from the partial loss of sample during the digestion process. The day-to-day precision determined for blank samples was 8.6%. The method permits measurement of 5-10 ng of indium per gram of tissue. A number of methods using AAS technique to determine indium in geological samples have been reported in the 1iterature.lS-16 However, a suitable AAS technique that could quantify indium in biological matrices was not available.
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Figure 1. Typlcal ET-AAS traces of lndlum standard In llver (A) and blood (B) samples: dashed line, lndlum slgnal; dotted line, background slgnal; solM line, temperature profile. Standard indlum (final concentration of 5 ng/mL) was added to the liver and whole blood prbr to digestion and extraction. Table 11. Intraday Precision and Recovery of ET-AAS Determination of Indium in Biological Matrices CV (%) at CV ( % ) at tissues" 5ng/mU % rec tissues" 5nglmLb % rec blanke blood bone kidney
11.5 13.9 8.8 9.4
liver lung urine large intestine
96.7 97.5 110 97.0
8.3 13.5 2.4 15.7
87.5 93.2 104 106
aAll samples contained about 0.5 g of tissues except for bone samples which had 0.25 g of tissue (n = 4 for liver; n = 5 for blank and all other tissues). CV, coefficient of variation. No tissue present. ".
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In (ppb) Flgure 2. Callbratlon curves of lndlum extracted from rat tissues. Indium standards were added to 0.5 g or 0.5 mL of tissue samples prior to digestion and extraction. Data represent lndlum flnal concentratlons(ppb or ng/mL) In assay solutbn after extraction.Values In parentheses represent correlation coefficients (P).
Unlike geological samples, the indium ions in the biological samples are typically below 5 ng/mL levels after a minimum dilution for sample preparation. Such a low indium level renders the assay susceptible to interference by the more biologically abundant ions such as iron(II1) and ~ o p p e r ( I I ) . ~ ~ J ~
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 15, AUGUST 1, 1993
It has been reported that the high molecular weight quaternary amines can form a relatively stable ion pair with a number of metals. The formed ion pairs can be easily extracted from aqueous solution into certain organic solvents such as methyl isobutyl ketone (MIBK).17.19~2~ Among the quaternary amines, Aliquat 336 is strongly recommended in that it is capable of extracting metal ions from both acidic and alkaline solutions. Extraction using Aliquat 336 has been successfully applied in determination of tissue concentrations of gallium,m an element in the same group as indium in the periodic table. In this study, the ion pairs formed between InCb- and Aliquat 336 are of the type
+
In3+ 4C1InC1;
-
+ R3N+CH3
InC1; R3N(CH3).InC1,
of the aqueous phase, but also generated a great number of air bubbles in the aspiration system. This produced an inaccurate aspiration flow. In a parallel study, we found that elimination of HzOz from the back-extraction solution did not change the recovery of indium from the organic phase to any significant extent. Hence, we utilized an aqueous solution consisting of 5 % HN03 and 5 % CH3COOH in the ET-AAS determination of indium. The low sensitivity of indium determination by ET-AAS is sometimes associated with the vaporization of molecular species of indium during the preatomization stages. In the current study, the direct application of the aqueous extract to ET-AAS, without any matrix modifier, resulted in signal peaks appearing in the low temperature region, typically 11200 OC. Occasionally,an “M”-typepeak could be observed. It has been reported that the presence of microgram amounts of palladium allows a great increase in the tolerable ashing temperature for indium in aqueous solution, thereby improving the sensitivityby a factor of 3 as compared to samples without Pd addition.14 Addition of Pd is thought to stabilize indium by forming more thermostable complexes or alloys and to decrease the formation of volatile species such as InO. Although we did not investigate the mechanism by which Pd increases the sensitivity of the indium analysis, we did find that the presence of Pd in aqueous solution (final concentration of 50pg/mL) shifted the indium signal peak to a higher temperature range and modifiedthe peak to a more symmetric shape. Consequently, the absorbance readout as peak area was increased.
where RsN+CH3 is Aliquat 336. However, the direct application of a metal-containing organic phase into ET-AAS results in the volatilization of samples at low temperatures prior to the atomization. The matrix of the organic extract also produces background interferences for most metals when determinations are made by ET-AAS.17 Therefore,in addition to extracting InCb-&N+ ion pairs from the digests into the MIBK phase, the backextraction method of Clark and Viets21was adapted to extract indium ions into a dilute acid solution. This procedure avoids the problemsassociatedwith organicsolvents. Data presented in Table I1 and Figures 1 and 2 indicate that this two-step extraction procedure is of practical use for determination of indium in animal tissues. CONCLUSIONS Fe(II1) and Cu(I1)can be extracted concomitantly into the MIBK organic phase. Fe causes a significant suppression of This technical note reports for the first time an extraction indium absorbance,13J5and Cu produces a severe interference method followed by ET-AAS analysis to determine the ppb with indium signals.17 Since Fez+ is unlikely to be extracted concentrations of indium in tissue matrices. The method to any appreciable extent into the MIBK phase, reduction of excludes the major interferences from some biologically Fe3+ to Fez+ can effectively diminsh the Fe i n t e r f e r e n ~ e . ~ ~ . ~abundant ~ ions such as iron and copper. The method has the One of the commonly used reducing agents in this type of detection limit of 0.5-1.0 ng/mL indium in rat tissue matrices study is ascorbic acid.13 However, the reaction catalyzed by after a minimum dilution and permits measurement of 5-10 ascorbic acid requires a relatively long time to reach comng of indium from per gram of tissue. This procedure should pletion,a and the ascorbic acid per se also impairs the precision be useful in better understanding the relationship between of the indium determination.15 Thus, we chose hydroxylamine the disposition and toxicity of indium compounds. hydrochloride to reduce Fe3+ to Fez+. In order to minimize the possible interference from Cu, sodium thiosulfate was ACKNOWLEDGMENT used to mask Cu by forming a stable complex. The authors gratefully acknowledge the technical assistance A mixture of aqueous solution containing HN03, CH3of Michael J. Penessa, Jonathan P. Monteleone, and Darren COOH, and HzOz has been suggested to strip the MIBK Wong. This research was supported by the National Toxorganic phase to back-extract a number of metal ions.’7921 icology Program under Contract N01-ES-85230. However, the presence of HzOz not only increased the viscosity (19) McDonald, C. W.; Rhodes,T.Anal. Chem. 1974,46, 300-301. (20) Scott, N.;Carter, D. E. Anal. Chem. 1987,59,888-890. (21) Clark, J. R.;Vieta, J. G. Anal. Chem. 1981,53,65-70.
RECEIVEDfor review February 12, 1993. Accepted May 9, 1993.
