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ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
Determination of Chromium in Biological Materials by Stable Isotope Dilution Claude Veillon," Wayne R. Wolf, and Barbara E. Guthrie' Nutrition Institute, Human Nutrition Center, USDA, Beltsville Agricultural Research Center, Beltsville, Maryland 20705
Lyophilized samples are ashed in an oxygen plasma discharge and the Cr is extracted as the trifluoroaceiylacetone chelate. Samples are spiked with "Cr and the isotope ratio is measured by combined gas chromatography-mass spectrometry using dual ion monitoring. Precise determinations at sub-ppb Cr levels are obtained, and the accuracy of the method is verified by independent means. Urinary excretion levels of this nutritionally essential element are shown to be about an order of magnitude lower than previously believed.
Sixteen trace elements are known t o be essential for t h e maintenance of life in man and t h e higher animals. These include Co, Cr, Cu, F, Fe, I, Mn, Mo, Se, Sn, and Zn, as well as As, Cd, Ni, Si, a n d V. T h e first eleven are known t o be essential in humans, some knowledge of their function exists, a n d dietary deficiencies have been identified. T h e latter five have only thus far been shown to be essential to higher animals in controlled environment/purified diet studies. Their essentiality t o humans is suspected, but has not been demonstrated. Chromium was first shown t o be essential in animals about 20 years ago ( I ) . Subsequently, it has been found t o be a n essential micronutrient for man, and is involved in glucose metabolism and t h e mechanism of action of insulin. Dietary deficiencies have been identified, and supplementation was shown to be effective in some types of impaired glucose tolerance ( 2 ) . Current theories on Cr metabolism suggest that t h e Cr nutritional status of a n individual is reflected in the urinary Cr excretion (31, and balance studies have necessitated t h e postulation of a highly-available form of dietary Cr. However, a recent study ( 4 ) suggests t h a t human urinary Cr excretion may be an order of magnitude lower than previously reported. Apparently, early data were affected by the extreme difficulty of determining Cr a t sub-ppb levels in a urine matrix. Serious questions were raised regarding the ability of present d a y commercial furnace atomic absorption spectrometers employing deuterium background correction t o measure Cr in ashed urine ( 4 ) . Using a modification of the sample preparation procedures described earlier (51, a volatile, thermally-stable trifluoroacetylacetone (tfa) chelate of Cr is isolated and measured by combined gas chromatography-mass spectrometry (GC/MS). Stable isotope dilution techniques permit a n accurate, sensitive, and precise measurement of urinary Cr levels with no observed matrix effects. Accuracy of t h e method is verified with standard reference materials (SRM) of known Cr content, and with parallel measurements on a novel continuum source, echelle monochromator, wavelength-modulated atomic absorption spectrometer (CEWM/AA) (6).
EXPERIMENTAL Instrumentation. The combined GC/MS system consisted of a quadrupole mass spectrometer (Model 3200, Finnigan Corp., 'Present address: Nutrition Department, University of Otago, Dunedin, New Zealand.
Sunnyvale, Calif.) coupled to a gas chromatograph (Model 9500, Finnigan Corp.). The MS was equipped with an electron impact source (70 eV), electron multiplier detector (-2800 V), 4-channel multiple ion monitor for specific peak monitoring, and a 4-channel, strip-chart recorder. For this investigation, only 2 channels were used. The GC was equipped with a 5 ft X 1/4 in. 0.d. (150 cm x 6.4 mm) glass column packed with 1% SE-30 on Chromasorb-W (Pierce Chemical Co., Rockford, Ill.) and operated at 125 "C. The GC/MS interface is all glass and equipped with a jet separator to reduce the amount of He carrier gas introduced into the ion source. A manually operated by-pass valve was provided to divert the column effluent until the hexane solvent peak had eluted. The CEWM/AA system used in the verification studies has been described (6). Reagents. n-Hexane specified as >99% pure was used for extractions without further treatment. The tfa (Pierce Chemical Co.) used for chelation was stored at 4 " C and aliquots were double-distilled prior to use. These aliquots were stored in the dark a t 4 "C for a maximum of 2 weeks before redistillation. Stabilize1 H202(50%, Fisher Scientific Co., Fairlawn, N.J.), used as an ashing aid in part of the procedure (vide infra), was stored at 4 "C. In order to maintain acceptably low blank levels, it was found necessary to purchase numerous containers of H 2 0 2and analyze them for Cr with the graphite furnace CEWM/AA, retaining only those having less than about 0.3 ppb Cr (good ones have contained less than 0.1 ppb; some are in excess of 1 ppb). The chelation/extraction was carried out in a 0.1 M sodium acetate bliffer (ultrapure grade, Alfa/Ventron, Danvers, Mass.), adjusted to pH 4.7 with HC1 prepared by isothermal distillation (7). This buffer was found to be Cr-free by graphite-furnace CEWM/AA. The 1 M NaOH used to remove excess ligand following chelation was prepared from reagent grade material. Spike solutions for the stable isotope dilution method were prepared from a stock solution of 50Cr containing 101.1 ppm Cr and 96.79 at. '% 50Cr. The enriched 50Cr203was obtained from Oak Ridge National Laboratory (Union Carbide, Oak Ridge, Tenn.), oxidized with HCIO,, reduced back to Cr(II1) and made 1 M in HN03. The isotopic composition was verified by thermal ionization mass spectrometry at the National Bureau of Standards (8) and the values (Table I) agreed with those specified by the supplier. Standard solutions of Cr for the graphite furnace CEWM/AA measurements were prepared by dilution of a 1000-ppm stock solution of (NH,)2Cr0, (Alfa/Ventron). Water used in the preparation of solutions and cleaning of apparatus was of 18-MQresistivity and was obtained from the purification system described elsewhere (7). Apparatus. In the initial phases of this study, a wet ashing sample digestion procedure was used This all-glass reflux system and procedure have been described in detail elsewhere ( 5 ) . Chelation of the Cr was carried out in 10 X 100 mm reaction tubes (No. 29560, Pierce Chemical Co.) fitted with Teflon valves. Small samples (1 mL) were dried and oxygen-plasma ashed in Coors porcelain crucibles ( 4 ) ,while large samples (10 mL) were dried and oxygen-plasma ashed in the 35mL round bottom flasks from the reflux digestion system. All glassware and containers were cleaned by heating to -90 "C for 1 h in a 2% solution of detergent/decontaminant ("Isoclean", Isolab, Inc., Akron, Ohio) followed by sonication for 15 min. The apparatus was then thoroughly rinsed with 18-MQ water, dried in a 60 "C vacuum oven, and stored in plastic containers. All sample handling and solution preparations were carried out in a Class-100 laminar flow work station (Model TT-4830, Environmental Air Control, Hagerstown, Md). Liquid samples were dried either in a vacuum oven or by lyophilization. The vacuum
This article not subject to U.S. Copyright. Published 1979 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
oven (Model 29, Thelco, Chicago, Ill.) has an all-aluminum interior, and was connected to an oil-less vacuum pump (Model 1VBF18-M100X, Gast Mfg. Corp., Benton Harbor, Mich.) capable of 27-in. Hg vacuum (-75 Torr). Oven temperature was maintained a t 60 "C and air filtered through a 0.2-pm membrane filter (No. 12106, Gelman Instrument Co., Ann Arbor, Mich.) was bled into the furnace at a rate sufficient to maintain the vacuum at 25-in. Hg (-125 Torr). The lyophilizer was a corrosion-resistant unit (Model FD-6-84-VP, FTS Systems, Stone Ridge, N.Y.) having a polypropylene condensation chamber and a titanium refrigeration coil operated at -100 "C. A shop-fabricated base plate of methylmethacrylate (35 X 35 X 2.5 cm) was connected directly to the condensation chamber with a short piece of 50-mm methylmethacrylate tubing solvent-welded to the plate, and covered with a 1.5-mm sheet of neoprene rubber. Thus, inverting a polycarbonate bell jar (No. 5300-1212, Nalgene, Rochester, N.Y.) over the base plate formed a large, metal-free, sample freeze-drying chamber. Air used to vent the system to atmospheric pressure following lyophilization was filtered through a 0.2-pm membrane filter (No. 12106, Gelman). Oxygen-plasma ashing of the dried samples was performed in a 5-chamber unit (LTA-505,LFE Corp., Waltham, Mass.) operated at 1 Torr O2 pressure, 400-W RF forward power, -5-51.' RF reflected power, and with a 4 0 "C cold trap between the unit and the vacuum pump. Samples. A large urine pool sample was prepared from 24-h urine collections from seven healthy adult males aged 2G40 years. The urines were mixed thoroughly in a single polypropylene container, divided into aliquots while continuously mixing, and frozen and stored at -20 "C. Aliquots were thawed immediately prior to sample preparation. For method verification, samples of spray-dried brewer's yeast (SRM 1569, National Bureau of Standards, Washington, D.C.) certified to contain 2.12 f 0.05 pg Cr/g, were also carried through the sample preparation procedure. Procedure. In the initial stages of this investigation, the reflux/digestion procedure described in detail earlier (5)was used, both for the urine and brewer's yeast samples. While completely satisfactory for the brewer's yeast, this procedure gave unacceptably large blanks for the urine samples, primarily due to the extra NaOH and relatively large amounts of HzOZused in this method (5). Subsequently, the following procedure was developed which minimizes the quantity and types of reagents necessary. The 35-mL round-bottom flasks used in the reflux system were cleaned by refluxing 5 mL of buffer and 0.2 mL of tfa for 1 h, followed by 3 hexane rinses and several water rinses. Ten mL of urine and 12.5 ng of 50Cr spike were then added to the flasks and the samples shell frozen in a dry ice-isopropanol bath. Blanks consisted of 10 mL of H 2 0 plus 5 ng of the 50Crspike. The frozen samples were then placed in the lyophilizer chamber and freeze-dried overnight. The chamber was vented to atmospheric pressure and the samples were immediately placed in the oxygen plasma asher to prevent moisture pickup. After overnight ashing, the samples were removed, allowed to cool to room temperature, and 1 mL of 50% H z 0 2was added. They were then placed in the 60 "C vacuum oven and allowed to stand at this temperature for 3 h before evacuating the oven. After drying overnight, the white ash was dissolved in 2.5 mL of buffer with vigorous swirling. Though rarely necessary, any ashed samples that were not white a t this point were treated with an additional 0.5 mL of HzOzand re-dried. Two 1-mL aliquots from each flask were transferred to 2 separate hydrolysis tubes, to each of which 0.15 mL of double distilled tfa was added. The tubes were closed and placed in a 100 "C oven for 2 h. Since the Cr(tfa), chelate does not form to any appreciable extent a t room temperature, the procedure from this point on is simplified with respect to contamination control. The hydrolysis tubes were opened and the chelate and excess ligand extracted with two 0.5-mL aliquots of hexane which were combined in 5-mL disposable glass culture tubes. The hexane extract was then washed with 0.5 mL of 1 M NaOH for 1 min to remove excess ligand. The excess ligand is extracted rapidly into the aqueous layer as the Na-salt, while the Cr(tfa), chelate is removed quite slowly at a linear rate of 2.2% per min. Alternate methods of removing the excess ligand have been described (9). The base-washed hexane layer was transferred to a glass "Hypo-Vial'' (No. 12901, Pierce Chemical Co.) and allowed to evaporate at room temperature (the Cr(tfa)achelate is not volatile
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Table I. Relative Abundance of Cr Isotopes in Nature and in Spike Solution natural W r spike isotope abundance, % ( 8 ) , 9i "Cr 4.31 96.79 s2Cr 83.76 2.98 53Cr 9.55 0.18 54Cr 2.38 0.05 at room temperature). To each vial, 0.15 mL of hexane was added and the vials were sealed with Teflon-faced silicone rubber septa (No. 12813) crimped on with aluminum seals (No. 13213) using a hand crimper (No. 13211, Pierce Chemical Co.). The now completed samples can then be measured at any later time with the GC/MS.
RESULTS AND DISCUSSION Naturally-occurring Cr consists of four isotopes, as shown in Table I. Also shown are the data for the 50Cr spike solution. T h e Cr(tfa)3 chelate exists in cis and trans isomeric forms, with about 84% in the trans form (10). T h e mass spectrum of this compound exhibits several groups of peaks corresponding primarily to Cr(tfa)+, Cr(tfa)F+, Cr(tfa)2+, and Cr(trfaI3+ (11, 12). T h e most intense group is t h a t of t h e Cr(tfa)2+ion at about 358 amu, and was used throughout this study. This ion group consists of four peaks, a t 356.18, 358.18, 359.18, and 360.18 amu, corresponding, respectively, t o the mass 50, 52, 53, and 54 Cr isotopes in Cr(tfa)2+. I n addition, the 356-360 amu mass region is virtually free of interferences caused by other organic species. For all determinations, the 50Cr/52Crisotope ratio was measured a t the 356 and 358 amu Cr(tfa)2+ion peaks and the total Cr originally present in the samples calculated. Because of occurrence of isotopes like 13C,l80etc. , in the Cr(tfa),+ ion, the calculation must take these into account. For example, the occurrence of an l80or 2 13C atoms in the ion contributes to the 358 (52Cr)peak, and reduces the 356 (52Cr)peak. Since we are using the 50Cr/52Cr isotope pair, the corrections are smaller than for pairs separated by 1 amu. Frew e t al. (12) have discussed these corrections in detail. From the data in Table I, it can be shown that: ()'
= "bP'
0.8568 - 0.0381R 0.7430R - 0.0381
where Cr(,,) is the total ng of Cr present in the original urine sample, Cr(sp)is t h e ng of 50Cr spike added, and R is the observed ratio of the 356/358 amu peaks. T h e numerical values of the constants in Equation 1 are valid only for t h e "Cr spike given in Table I. Not included in this derivation are the 2H, " 0 , and 14C isotopic contributions; their low abundance renders their contribution negligible. The error in the total Cr determination by isotope dilution is a minimum when the amount of spike added is approximately equal to the amount of Cr present in the sample (12), Le., for observed isotope ratios near unity. For t h e determinations reported herein, the isotope ratios were all within a range of about 0.2-2.0, where t h e error due to a n error in the measured ratio is still quite small (12). T h e accuracy of the method was verified using the NBS SRM 1569 brewer's yeast sample. T h e results of three determinations are shown in Table 11, and excellent agreement with the certified value is obtained. Illustrated in Table I11 are the results obtained for the urine pool sample, both by the stable isotope dilution GC/MS method described herein, and using t h e graphite furnace CEWM/AA ( 4 , 6). A description of the latter system and procedure is in preparation for later publication. T h e results agree within experimental error. T h e precision for the GC/MS method is considerably better t h a n for the
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 7, JUNE 1979
Table 11. Analysis of NBS SRM 1569 Brewer's Yeasta "Cr spike, sample, g 1.577 1.570 1.474
pg
Certified Value:
observed ratio,
5oCr/52Cr
p g Cr/g
2
0.710
2.15
2 2
0.770
1.97
0.763 2.12 mean: 2.08 i 0.09
2.12 z 0.05 p g Cr/g.
Table 111. Chromium Content of Urine Pool Sample G C . l I S , n g Cr m L
0.37
0.31
0.17
0.30
0.26 0.27 -
0.32
0.32
I
0.02
ACKNOWLEDGMENT T h e authors gratefully acknowledge Vincent P. Flanagan and Aldo Ferretti of t h e Lipid Nutrition Laboratory for the use of their G C / M S instrumentation. We thank Laura P. Dunstan and E. L. Garner of NBS for their analysis of t h e 50Cr spike solution.
CEU'MiAA, ng CrimL
0.30
-
Guthrie e t al. ( 4 ) t h a t a deuterium background corrector is inadequate for its purpose in the determination of Cr in urine. More importantly, t h e method and procedures described herein constitute a very powerful analytical method for t h e determination of Cr a t sub-ppb levels in difficult matrices with good precision and accuracy.
0.34
I
0.1
CE\I'\I/.A.4 method. This is due primarily to the following procedural differences: ( a ) the Cr content of this pool sample is quite near the detection limit for the CELVR.1, AA system (0.05 ppb); ( b ) the blank was relatively large (10-20% of the sample readings); (c, contamination problems were greater due to the fact that the CE\VM 'AA instrument area was not a controlled environment space; (d, smaller samples were used (1 mL vs. 10 mL); and (e) considerable tube-to-tube variation was found for the graphite furnace (1). T h e results of this study are significant in three important aspects. Nutritionally, the fact that urinary Cr excretion is an order of magnitude lower than previously believed removes the necessity of postulating a form of Cr in foods 10-fold more available biologically than inorganic Cr to account for differences in food content, excretion, and measured availability. Analytically, the results are significant in two aspects. Considerable support is lent to the earlier conclusion of
LITERATURE CITED (1) K. Schwarz and W. Mertz, Arch. Biochem. Biophys., 85, 292 (1959). (2) K. N. Jeejeebhoy, R. C. Chu, E. B. Marliss, G. R. Greenberg, and A. Bruce-Robertson, Am. J . Clln. Nutr., 30, 531 (1977). (3) W. Mertz, Physiol. Rev., 4S, 163 (1969). (4) 6. E. Guthrie, W. R. Wolf, and C. Veillon, Anal. Chem., 50, 1900 (1978). ( 5 ) W. R. Wolf, J . Chromatogr.. 134, 159 (1977). (6) A . T. Zander, T. C. O'Haver and P. N. Keliher. Anal. Chem., 49, 838 (1977). (7) C. Veillon and B. L. Vallee, in "Methods in Enzymology", Voi. 54, Academic Press, New York, Chapter 25, pp 446-484 (in press). (8) L. P. Dunstan and E. L. Garner, National Bureau of Standards, Washington, D.C., personal communication, 1976. (9) E. L. Arnold and B. L. Dold, Anal. Chem., 50, 1708 (1978). (IO) R. C. Fay and T. S. Piper, J . Am. Chem. Soc., 85, 500 (1963). (1 1) W. R. Wolf, M. L. Taylor, B. M. Hughes, T. 0. Tlernan, and R. E. Severs, Anal. Chem., 44, 616 (1972). (12) N. M. Frew, J. J. Leafy,and T. L. Isenhour, Anal. Chem., 44 665 (1972).
RECEIVED for review January 18, 1979. Accepted March 12, 1979. B.E.G. was supported in part by a U S . Public Health Service International Research Fellowship (No. 1F05 T W 02529-01). Presented in part a t the Federation of Analytical Chemistry and Spectroscopy Societies 5th Annual Meeting, Boston, Mass., November 1978. Specific manufacturer's products are mentioned herein solely t o reflect t h e personal experiences of the authors and do not constitute their endorsement nor t h a t of the Department of Agriculture.
Propagation of Random Errors in Estimating the Levels of Trace Organics in Environmental Sources Konanur G. Janardan Math Systems, Sangamon Sta te University, Springfi8ld, Illinois 62708
David J. Schaeffer" Illinois Environmental Protection Agency, 2200 Churchill Road, Springfield, Illinois 62706
Equations for the propagation of random errors (sampling through analysis) for a trace organlc pollutant are developed. The relationship of random errors to the response factor Is described.
In collecting material for trace organic analysis, three key steps can be distinguished: capture (collection and storage), recovery (extraction and concentration), and quantitation (isolation, identification, and quantitation) ( I ) . For example, capture on macroreticular resins (Z), granulated activated carbon ( 2 ) ,or foam plugs ( 3 ) ;recovery by solvent extraction; and quantitation by gas chromatography (GC)/mass spec0003-2700/79/035 1-1024$01 .OO/O
trometry (MS) is a widely employed sequence. Where liquid-liquid solvent extraction is used, capture and recovery are combined. Because these processes affect t h e accuracy of t h e final estimate, usually to an unknown degree, the propagation of random and systematic errors through the sequence should play a significant role in the analyst's choice of methods. General equations for the propagation of random errors are developed below. T h e effects of systematic (determinate) errors are not considered here.
RESULTS AND DISCUSSION T h e propagation of random errors is a well known problem in analytical chemistry ( 4 ) . Solution of this problem using
0 1979 American Chemical
Society
