Anal. Chem. 1989,61 1235-1238
other measurement methods such as CVAA will be equally applicable. ACKNOWLEDGMENT We thank John J. McNerney and Douglas M. Dowis (Arizona Instruments, Jerome, AZ) for much valuable discussion and for their help in many other ways. Registry No. Hg, 7439-97-6; HzO, 7732-18-5.
LITERATURE CITED Airey, D. Sci. TotalEnviron. 1982, 25, 19-40. Taylor, D. Mercury as an Environmental Pollutant-A Bibliography; Kynock Press: Birmingham, UK, 1980. Eaton, A. J.-Water Pollut. Control Fed. 1988, 6 0 , 752-772. Brandenberger, H.; Bader, H. At. Absorpt. Newsl. 1967, 6 , 101. Hatch, W. R.; Ott,W. L. Anal. Chem. 1988, 4 0 , 2085-2087. Annual Book of ASTM Standards; Volume 11.01, “Water”, Standard D 3223-80, American Society of Testing and Materials: Philadelphia. PA, 1984. StandardMethods for the Examination of Water and Wastewater, 16th ed.; American Public Health Association: Washington, DC, 1985; Method 320A, p 232. United Kingdom Department of the Environment Methods Exam. Waters . .-.. . Assoc. . ... .Mater. ... 1007. - - - - ,39. Bureau International Technique Du Chlore, Anal. Chim. Acta 1979, 709. 209-228. United States Environmental Protection Agency, Method 245.1, €PA Methods for Chemical Analysis of Water and Wastes, Publication No. 60014-79-020; 1983. U S . Department of Heaith, Education and Welfare, National Institute for Occupational Safety and Health. NIOSH Manual for Analytical Methods, 2nd ed., Clncinnati, OH, 1977; Method P & CAM 165. Streufert, D. Z . Chem. 1987, 2 7 , 209-211. Omang, S.H. Anal. Chim. Acta 1971, 5 3 , 415-420. Rains, T. C.; Menis, 0. J. Assoc. Off. Anal. Chem. 1972, 55. 1339- 1344.
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(15) Fitzgerald, W. F.; Lyons, W. B. Mfure(London) 1973, 242, 452-453. (16) Ahmed, R.; May, K.; Stoeppler, M. Fresenius’ 2.Anal. Chem. 1987, 326, 510-516. (17) Kopp, J. F.; Longbottom, M. C.; Lobring, L. B. J.-Am. Water Works Assn. 1972, 6 4 , 20-25. (16) Adeioju, S . B.; Mann, T. F. Anal. Lett. 1987, 2 0 , 985-1000. (19) Uchino, E.; Kosuga, T.; Konishi, S.; Nishimura, M. Environ. Sci. Techno/. 1987. 2 7 , 920-922. (20) Magos, L. Analyst(London) 1971, 9 6 , 847-2353. (21) Magos, L.; Clarkson, T. W. J . Assoc. Off. Agric. Chem. 1972, 55, 961-971. (22) Toffaleti, T.; Savory, J. Anal. Chem. 1975, 4 7 , 2091-2095. (23) Margel, S.;Hirsh, J. Clin. Chem. 1984, 30, 243-245. (24) Anderson, D. H.; Evans, J. H.; Murphy, J. J.; White, W. W. Anal. Chem. 1971, 4 3 , 1511-1512. (25) McNerney, J. J.; Busek, P. R.; Hanson, R. C. Science 1972, 778, 61 1-612. (26) McNerney, J. J. United States Patent 3,714,562, Jan 30, 1973. (27) McNerney, J. J. Sensors 1986, 3(2), 39-41. (28) Murphy, P. J. Anal. Chem. 1979, 57. 1599-1600. (29) Mudroch, A.; Kokotich, E. Analyst (London) 1987, 772, 709-710. (30) Fenton, H. J. H. J. Chem. SOC. 1894, 65,899. (31) Walling, C. Chem. Rev. 1975, 8 , 125-131. (32) Sansoni, B.; Kracke, W. Z . Anal. Chem. 1968, 243, 209-241. (33) Barbeni, M.; Minero, C.; Peiizzetti, E.; Borgareilo. E.; Serpone, N. Chemosphere 1987, 76,2225-2237. (34) Puppo, A.; Halliwell, B. Biochem. J. 1988, 249, 185-190. (35) Thurman, E. M.; Malcolm, R. L. Environ. Sci. Techno/. 1981, 15, 463-466. (36) Carron, J.; Agemian, H. Anal. Chim. Acta 1977, 9 2 , 61-70. (37) Kunai. A.; Hata, S.;Ito, S.;Sasaki, K. J. Am. Chem. SOC. 1986, 708, 6012-6016. (38) American Society Committee on Environmental Improvement Anal. Chem. 1980, 5 2 , 2242-2249.
RECEIVED for review December 5, 1988. Accepted March 9, 1989.
Graphite Furnace Atomic Absorption Spectrometric Determination of Chromium, Nickel, Cobalt, Molybdenum, and Manganese in Tissues Containing Particles of a Cobalt-Chrome Alloy Foster Betts* and Alicia Yau
The Hospital for Special Surgery, 535 East 70th Street, New York. New York 10021 An Investigation of wear debrls generation by cobatt-chrome alloy Joint prostheses required analysis of human tissue contalning metal In lonlred or partlculate form, at levels from less than 1 to over 3000 pg/g of dry tlssue. Wet ashlng by a number of published methods failed to dlssolve high levels of the alloy particles. Other methods Inadequately digested tlssue llplds or were unsuitable for samples wlth very low metal content. A procedure using wet ashlng was developed that has broad appllcabillty to direct analysis, by flameless atomlc absorptlon wlth Zeeman background correction, of tissues contalnlng nonvolatlle metals In lonlc or partlculate form. Direct analysis of a standard reference material containing from 0.4 to 2.4 pg/g of the elements of Interest yielded average values wHhln 5 % or less of the certlfled content for all five metals, wlth a coefficient of variation less than 5 % . When the standard materlal was spiked wlth powdered alloy at 3000 pg/g, recovery values for all metals ranged from 97 YO to 100% wlth Individual coefflclent of varlatlon values of 4 % or less. The detectlon limlt was below 0.1 pg/g of dry tlssue for Cr and NI and below 0.03 pg/g for Co, Mn, and Mo.
INTRODUCTION The expanded use of metal alloy bioprosthetic materials
has necessitated the development of methodology to monitor the accumulation of metal debris (in particulate and ionic form) in surrounding and peripheral tissues. The constituent elements (usually nonvolatile metals such as Cr, Co, Ni, Mo) are readily analyzed in wet ashed tissues by flameless atomic absorption spectrophotometry when present in ionized form. A large body of literature exists describing tissue digestion in various concentrated acids, and recent papers have compared recoveries of the relevant metals using different acid combinations (1-3). However, the cobalt-chrome surgical alloys are extremely corrosion resistant, and accurate determination of these elements in soft tissues that may contain significant amounts of alloy particles is more complicated. For direct analysis in the graphite furnace a procedure was sought that would completely digest tissues with appreciable lipid content, be usable a t metal levels less than 1kg/g dry weight, and dissolve alloy particles a t more than 3000 pg/g. Digestion by refluxing in concentrated nitric acid gave excellent analytical results on a standard reference material having certified values for the metals of interest, but failed to dissolve particles of a cobalt-chrome prosthesis alloy when they were added to the standard material. Refluxing in mixtures of H N 0 3 and HC1 also failed to dissolve the particles. Mixtures of HN03 and H2S04dissolved the metal particles,
0003-2700/89/0361-1235$01.50/00 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 11, JUNE 1, 1989
but the sulfuric acid is difficult to evaporate. As a result, for tissue digests with low metal content that must be analyzed with minimum dilution, the high concentrations of sulfuric acid introduced into the furnace greatly reduced graphite tube life and may produce analytical interferences for some elements. Hydrogen peroxide has been used t o digest tissues containing titanium alloy particles ( 4 ) ,but produced incomplete digestion of lipids in tissues with substantial fat content, resulting in very low metal recovery, and incompletely dissolved cobalt alloy particles. This paper describes procedures for tissue digestion and direct analysis by flameless atomic absorption with Zeeman background correction, which yield accurate results for cobalt (Co), chromium (Cr), nickel (Ni), molybdenum (Mo), and manganese (Mn) in tissues containing these metals a t trace levels in ionic form or as alloy particles. This method has applicability to the determination of nonvolatile metal debris in a broad range of body tissues.
EXPERIMENTAL SECTION Materials. Water for making solutions, dilution, and rinsing of labware was distilled and deionized with a Millipore Corp. Milli-Q deionizer. The 25-mL volumetric flasks used for tissue digestion were soaked overnight in concentrated HNO,, triplerinsed, dried, and stored until use in sealed plastic bags. All other labware, including autosampler cups, was soaked overnight in 5% HNO, and triple-rinsed. Acids used for preparation of standards and for tissue digestion were of J. T. Baker Ultrex grade. Calibration standards were prepared from Aldrich atomic absorption standard solutions. The magnesium nitrate used as a matrix modifier was of J. T. Baker Ultrex grade. A standard reference material TORT-1, lobster hepatopancreas, having certified values for the elements of interest, was obtained from the National Research Council of Canada, Marine Analytical Chemistry Standards Program, Ottawa, Canada. Tissue Preparation and Digestion. Precautions to avoid metal contamination were taken at all steps of tissue preparation and analysis. Handling of tissues and digests for weighing, dilution, etc., was carried out in a laminar flow hood under class 100 conditions. All human tissues and the TORT-1 material were dried to constant weight at 100 "C. Weighed tissue samples of approximately 300 mg were placed in 25-mL volumetric flasks, 5 mL of concentrated HNO, was added, and the flasks, up to 16 at a time, were placed on a hot plate in the laboratory fume hood and covered with a glass plate. The temperature setting of the hot plate was raised slowly over a period of hours to avoid foaming, until refluxing of the HNO, was seen in the necks of the flasks (approximately 140 "C). Efficient refluxing was provided by the necks of the flasks, eliminating the need for separate reflux columns. When the flasks were nearly dry, an additional 5 mL of concentrated HNO, was added, and refluxing continued just to dryness but without charring. Nearly complete digestion of organic material was achieved, leaving any alloy particles in the tissue free but not completely dissolved. Then 5 mL of 12 N HCl was added, and the mixture was boiled gently to dissolve alloy particles. It was found that if any significant amount of H N 0 3 was allowed to remain in the flasks for this step, the cobalt-chrome alloy particles could not be dissolved, even with additional HCl and prolonged heating. When most of the HC1 was evaporated, a final 5 mL of concentrated HNO, was added to complete digestion of organic material, and the flasks were refluxed until nearly dry. Since HC1 has a lower boiling point than "OB, these steps eliminate excess HCl, which could cause analytical interferences. After cooling, the flasks were brought to volume with 1% HNO,, yielding a colorless solution with no visible precipitate. Control blanks, containing only the acids used for digestion, were carried through all steps. The procedure described yielded quite low sample contamination, as the results from the TORT-1 standard material demonstrate. These metals can be measured accurately in biological tissues at levels below 1 pg/g of dry tissue. However, during digestion in the fume hood, dust-laden air is drawn around the flasks, and significant contamination of samples with very low metal content may occur, especially for Cr and Ni. Potential
contamination has been reduced by performing digestions with the hot plate placed in a small class 100 laminar flow hood, which is vented into the laboratory fume hood. For tissue samples with very low metal content, Ni and Cr contamination from the commercial acids used for digestion may be appreciable, although the other metals were undetectable in the control blanks, making it desirable to reduce the volume of acid necessary. Acid evaporation during sample digestion was reduced by careful temperature control, keeping the reflux line during the HNO, steps just into the neck of the volumetric flask. With the reduction in the rate a t which HNO, boiled away, it was possible to eliminate the second addition of nitric acid, while still obtaining adequate digestion. In the HCl step, maintaining the flasks overnight a t a temperature just below the boiling point yielded complete dissolution of metal particles with 2 mL of 1 2 N HC1 rather than 5 mL. Bringing the digests to a final volume of 10 mL, rather than 25 mL, reduced the dilution by a factor of 2.5, giving better overall accuracy and lower detection limits for metal in the tissue. Graphite Furnace Analysis. Metal analyses were performed on a Perkin-Elmer 5100/Zeeman atomic absorption spectrophotometer with an AS-60 autosampler, using Zeeman background correction. All dilutions of the digests were performed with 1% HNO,, as recommended by Perkin-Elmer (5) to eliminate possible interference from small amounts of chlorides present. Standards were also prepared in 1% HNO,, although measured values were independent of nitric acid concentration from 0.2% to 2% in the standards. Pyrolytic graphite tubes with L'vov platforms were used for Cr, Co, Ni, and Mn, with magnesium nitrate as a matrix modifier for all elements except Ni. Mo was atomized from the wall with no matrix modifier. Analyses for each element were carried out under stabilized temperature platform furnace (STPF) conditions by using furnace programs and instrument settings recommended by Perkin-Elmer ( 5 ) . A drying step of 40 s at 140 "C was used. Background absorbances, even for undiluted digests, were comparable to those for the standards, indicating low organic content of the digests. No analytical interferences were detected, although periodic checks (especially on digests analyzed with no dilution) were performed by spike recovery measurements, which can be done automatically by the autosampler in the 5100/Zeeman instrument. Automatic recalibration of the instrument was performed after every six to seven samples, and each measurement was performed with three replications. Metal carry-over in the furnace from sample to sample, especially for Mo and Ni, is a major source of error when one is measuring high- and low-concentration samples intermixed on the autosampler tray. Also, high-concentration samples read immediately before the automatic recalibration produce carry-over into the standard blank, causing too large an automatic zero correction and erroneous recalibration. To eliminate these problems, low-concentration samples should be measured together and a standard blank or empty cup read just before recalibration. Precision is increased for samples near the detection limit by using multiple injections of sample into the graphite furnace, increasing the number of replicate measurements, and by reading blanks immediately preceding and following these low-concentration samples.
RESULTS AND DISCUSSION Metal Recovery in Standard Reference Material. The ability of these procedures to give reliable results on samples containing trace levels of metal, as well as those containing considerable amounts of alloy particles, was verified by analyzing the TORT-l standard reference material and samples of this material spiked with known amounts, approximately 1000 pg, of cobalt-chrome alloy filings (No. 30 mesh). This is a more severe test than a n equal weight of prosthesis wear particles, which are known from microscopic examination to be 3-5 pm in average size. Digestion was performed with a total acid volume (HC1 plus "OB) of 20 m L and a final dilution volume of 25 mL. T h e average values for all metal levels in TORT-1 samples (n = 4) agreed with the certified values to within 3% (except for Ni), with a coefficient of variation (cv) of 6% or better (Table I). T h e slightly higher
ANALYTICAL CHEMISTRY, VOL. 61, NO. 1 1 , JUNE 1 , 1989
pg/g for Cr, and significantly less for the other elements. For tissue digestion with reduced volumes of acid as described above, the average weight of metal in blanks was below 0.02 pg for Ni and below 0.01 pg for Cr. For 0.3-g tissue samples and a final dilution volume of 10 mL, the corresponding contamination levels would be below 0.06 pg/g for Ni and below 0.03 pg/g for Cr. In practice, concentrations in the digests below 1pg/L are difficult to measure reliably by the graphite furnace method even with multiple injections of the sample into the furnace. This corresponds to a practical detection limit for these methods of the order of 0.03 pg/g of dry tissue for Cr, Co, Ni, Mo, and Mn. Analysis of Tissues. More than 150 tissue samples obtained a t surgery for replacement of cobalt-chrome alloy prostheses have been analyzed for Cr, Co, Ni, and Mo, and a few for Mn. Most samples had more than 2-3 pg/g of each metal, yielding within-run cv's for tissue metal levels generally better than 5%, similar to the accuracy achieved for the TORT-1 material. Repeat measurements generally agreed to within 7 % . Table I11 presents representative data for Cr, Co, Ni, and Mo on samples of tissue from joint replacement surgery. The first 11samples are listed in order of decreasing levels of cobalt (the major cobalt-chrome alloy constituent), and the proportions of the metals relative to cobalt are shown for each sample. For the two samples in Table I11 that contained the largest amounts of metal, the relative proportions of the metals agreed quite well with the composition of the alloy sample used in the spike recovery measurements, typical of samples with similar to higher metal levels. The data on tissue sample A demonstrate that very high levels of these metals (over 2500 pg/g) can be digested. The metal was found, by X-ray examination of undigested remnants of tissue, to have come from pieces of stainless steel surgical wire used when the prosthesis was implanted, and the metal proportions are in good agreement with the composition of surgical stainless steel. As the level of cobalt in the tissues decreased, the levels of Cr and Ni tended to approach a constant value in the range of 0.4-0.7 pg/g, even in some tissue samples in which Co, M, and Mn were below the detection limit, indicating that metal accumulated in the tissue in forms other than as alloy particles. The low-metal samples yielded concentrations in the digests of 1or 2 pg/L or less, which are difficult to measure accurately, especially for Ni and Mo for which the sensitivity is poorer, and within-run cv's were often greater than 15%. Carry-over errors sometimes caused day-to-day repeat measurements on low-concentration samples to differ by 50-100'70 for Ni, Mo, and Mn, which are minor alloy constituents. These errors occurred for some samples in Table I11 that were analyzed
Table I. Analysis of TORT-1 Samples sample
Cr 2.41" 2.38 2.50 2.23
1
2 3 4 mean
2.38 4.6% NRC' 2.40 (0.6) recover9 99.3 CVb
co
Ni
Mo
Mn
0.43" 0.38 0.43 0.40
2.53" 2.37 2.41 2.41
1.42" 1.70 1.59 1.54
24.6" 23.4 24.4 23.6
0.41 5.9% 0.42 (0.05) 97.6
2.43 2.9% 2.40 (0.3) 105.6
1.56 7.4% 1.5 (0.3) 104.0
24.0 2.6% 23.4 (1.0) 101.7'70
" Micrograms of element per gram of dry sample. *Coefficient of variation in percent. National Research Council of Canada certified value for TORT-1. Values in parentheses are 95% confidence limits. dMean sample value as a percentage of certified value. Table 11. Analysis of TORT-1 Samples Spiked with Powdered Alloy sample 1
2 3 4 mean CVb
alloy recoveryd
Cr
Co
Ni
Mo
0.288" 0.286 0.289 0.275
0.616" 0.639 0.634 0.620
0.007 15" 0.0661" 0.008 52" 0.00734 0.0658 0.00834 0.00727 0.0645 0.00840 0.00700 0.0670 0.00776
0.285 2.3% 0.291 97.9%
0.627 1.8% 0.646 97.1%
0.007 19 2.1% 0.00720 99.9%
0.0659 1.6% 0.0680 96.9%
1237
Mn
0.008 26
4.1% 0.00850 97.2%
"Grams of element measured in digest per gram of alloy in spiked sample. *Coefficient of variation in percent. Grams of element per gram of alloy dissolved in HCl. dMean sample value as a percentage of the alloy value. value for Ni was found t o result from Ni in the acids used. T o estimate recovery in the digests of the spiked samples, measured values of metal in the digests, corrected for tissue metal content, were compared to results obtained on 100-mg samples of the alloy dissolved in HC1. Recovery was between 97 and loo%, and the cv was 4% or less for all elements (Table
11). Control blanks yielded concentrations below 1.5 pg/L for Cr and 1-3 pg/L for Ni, corresponding to metal levels of 0.1-0.2 pg/g for 0.3-g tissue samples. Control concentration readings for the other elements were considerably below 1 pg/L. For tissue samples weighing 0.3 g and with correction for average metal levels in the control blanks, this is equivalent to contamination levels well below 0.15 pg/g for Ni, below 0.1
Table 111. Analysis of Tissues from Joint Replacement Surgery sample 1
2 3 4
5 6 7 8 9 10 11
Ad
Cr
co
Ni
Mo
Cr
51.8" 23.5 8.2 5.2 0.99 1.03 1.07 0.70 0.62 0.64 0.54 1460.60
97.2' 53.1 13.6 13.1 1.92 1.90 1.87 0.57 0.42 0.34 0.17 1.29
2.08" 0.75 0.78 0.94 0.60 0.47 0.46 0.61 0.38 0.51 0.62 1065.98
11.68" 7.55 0.99 1.99
0.53 0.44 0.63 0.40 0.52 0.54 0.57 1.22 1.48 1.88 3.17 1.37
b
0.099 0.125 b b b b 148.1
ratio of metal proportions co Ni 1 1 1 1 1 1 1 1 1 1 1 0.001
Mo
0.021 0.014 0.057 0.07 0.31 0.24 0.24 1.07 0.90 1.50 3.65
0.12' 0.14 0.07 0.15
1
0.14O
b
0.05 0.07
b b b b
" Micrograms of element per gram of dry sample. Data unreliable due to sample weight
