Anal. Chem. 1990, 62, 111-115
the ion source housing so as to give the same length (area) and position. Since these emitter conditions were carefully controlled, it is certain that the observed large difference in i,(T) behavior is due to the differences in the properties of the emitter materials. It was found that the Ir emitter gave comparable results. The i vs T characteristics were essentially the same. The ion signal increased with heating current and leveled off at saturation values. Gaseous Environment around the Emitter. It could be concluded from the previous study that an oxidized emitter should be used in order to improve sensitivity and stability. Such an oxidized emitter can be obtained if air (oxygen) is added to the emitter surface from another gas line. The addition of 10 mL of air to helium as a carrier gas had effects on the signals observed by the APIMS(S1D). This effect was an increase in total ion intensity with a slight corresponding change in product ion distribution. This suggests that air serves the purpose of modifying the surface leading to a change in the work function as well as the chemical property of the surface, which is responsible for the difference in response.
CONCLUSION This study demonstrated the existence and theoretical basis of the SID mechanism; positive surface ionization is the mechanism of ion formation in SID. Thus, the probability of ion formation is related to the IE of the adsorbed species. On the hot surface, some of the compounds decompose through a series of unimolecular reactions. At each step of the decomposition newly formed species may be ionized. If the species has a low IE, its positive ion is formed. The charge carriers collected at the conventional SID collector may not be those originally formed. The ion of GC(S1D) moving through a gas in a longer electric field than the present experimental setup of the API(S1D)MS may ex-
111
perience charge stripping, charge transfer, ion/molecule reaction, etc. However, the SID response depends only on the total intensity of ion species initially formed. The incorporation of the SID into the ion source of an APIMS is very easily done. Since a heat Pt emitter reacts under atmospheric pressure condition as SID does to ionize compounds, the APIMS(S1D) may provide a powerful combination of functions which should be useful for trace organic analysis of specific substances. This is particularly the case in the parallel use of an ion counting system.
ACKNOWLEDGMENT The authors are grateful to M. L. Messersmith at Yokota Air Base for manuscript preparation. H.J. is a graduate student from Meisei University. Registry No. Pt, 7440-06-4; Ir, 7439-88-5.
LITERATURE CITED Fujii, T.; Arimoto, H. Ana/. Chem. 1985, 57, 2625. Fujii, T.; Arimoto, H. J . Chromatogr. 1988, 355, 365. Zandberg, E. Ya.; Ionov, N. I . Swface Ionization; Israel Program for Scientific Translations: Jerusalem, 197 1. McKeown, M.; Siegel, M. W. Am. Lab. 1975, 89. Horning, E. C.; Carroll, D. I.; Dzidic, I.; Lin. S. N.; Stiilwell, R. N.; Thenot, J. P. J . Chromatogr. 1977, 142, 481. Grimsrud, E. P.; Kim, S. H.; Gobby, P. L. Anal. Chem. 1979, 5 1 , 223. Fujii, T.; Jimba, H.; Ogura, M.; Arimoto, H.; Ozaki, K. Analyst 1988, 1134, 789. Fujii, T.; Kitai, T. h i . J . Mass Spectrom. Ion Processes 1986, 7 1 , 129. Fujii, T.; Suzuki, H Obuchi, M. J . Rtys. Chem. 1985. 89, 4687. Fujii, T.; Ogura, M.; Jimba, H. Anal. Chem. 1989, 6 1 , 1026. Fujii, T. Int. J . Mass Spectrom. Ion Processes 1984, 57, 63.
RECEIVED for review May 16, 1989. Revised manuscript received October 6, 1989. Accepted October 18,1989. Work was supported in part by the Ministry of Education, Science, and Culture of Japan; Grand-in-Aid for General Scientific Research (No. 63540452).
Determination of Chromium in Urine by Stable Isotope Dilution Gas Chromatography/Mass Spectrometry Using Lithium Bis(trifluoroethy1)dithiocarbamate as a Chelating Agent Suresh K. Aggarwal, Michael Kinter, Michael R. Wills, John Savory, and David A. Herold*
Departments of Pathology, Biochemistry, and Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
An Isotope dllutlon gas chromatography/mass spectrometry method uslng llthlum bls(trlfiuoroethyi)dlthiocarbamateas a chelating agent Is descrlbed for the determlnatlon of chromium In urine. A wet digestion procedure wlth "0,-H,O, Is used for oxldizlng the organic matter associated with urine samples. The Isotope ratios are measured by selected ion monitoring In a general-purpose mass spectrometer using a 10-m fused silica capliiary column. Memory effect, in sequential analyses of samples wlth different Isotope ratios, was evaluated by preparing a series of synthetic mixtures and was found to be negligible. The accuracy of the method was verlfied by quantitatlon of chromlum in the NIST freeze-dried urine reference material, SRM-2670, wlth a recommended chromlum concentration of 13 pg/L In the normal level and certlfled chromlum concentratlon of 85 f 6 pg/L In the elevated level. 0003-2700/90/0362-0111$02.50/0
INTRODUCTION Chromium (Cr) has been recognized as an essential micronutrient for humans that is involved in important biochemical processes such as glucose metabolism and the action of insulin (I). Current knowledge about the role of Cr in human nutrition has been reviewed in a recent article by Offenbacher and Pi-Sunyer (2). Nutrient Cr, Cr(III), is present in food in the trivalent form, while hexavalent chromium, Cr(VI), is considered to be an occupational hazard because of its allergenic and carcinogenic activities. Since the major pathway of elimination of absorbed Cr is excretion in urine, urinary Cr levels have been suggested as an indicator of total body burden and recent uptake (3). Blood Cr levels have been suggested to reflect long-term exposure to Cr (4). As with most other metals, Cr in biological materials is generally determined by electrothermal atomic absorption 0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 2, JANUARY 15, 1990
spectrometry (EAAS) because of the speed, minimum need for sample preparation, possibility of automation, and good sensitivity of this technique (5). However, EAAS measurement of Cr is very susceptible to matrix effects and, therefore, requires standards similar to the samples. Urinary Cr concentrations in normal subjects have been found to be below 1 pg/L with a range of 0.2-1 pg/L (6-9). In serum, Cr values ranging from C0.05 to 0.29 pg/L (mean = 0.11 f 0.07 pg/L; n = 15) have been reported recently by EAAS (10). In a recent report on the determination of Cr in biological materials by members of the International Union of Pure and Applied Chemistry (IUPAC), it has been emphasized that analytical chemists should provide methods that are sufficiently sensitive to give accurate results at levels of less than 10 pg/L Cr (11). Stable isotope dilution mass spectrometry, which has long been used by nuclear and geological scientists, is a well recognized technique for trace metal determinations in complex matrices. This technique offers the advantage of freedom from matrix effects and the constraints of quantitative sample preparation. Since the method employs an ideal internal standard, i.e. an enriched isotope of the same element, a high degree of accuracy can be readily achieved in concentration determinations. Stable isotope dilution mass spectrometry would supplement EAAS methods, providing an alternative phvsicochemical principle for the quantitation of chromium. The use of general-purpose mass spectrometers for trace metal determinations is attractive because these instruments are widely available, which would allow many clinical laboratories to carry out trace metal determinations without additional specialized and expensive instrumentation. Further, combined gas chromatography/mass spectrometry (GC/MS) also offers the advantage of shorter analysis times and the ability to separate different metal chelates. The potential of general-purpose mass spectrometers for trace metal determinations has been demonstrated in a few published studies (12-15). However, one of the fundamental problems preventing the widespread application of GC/MS methods has been the observation of carryover, or memory, in the sequential analysis of samples with different isotope ratios (13). This can be a serious problem and must be evaluated for the metal and the chelating agent under investigation. We have recently developed an isotope dilution GC/MS method using lithium bis(trifluoroethyl)dithiocarbamate, Li(FDEDTC), as a chelating agent for the quantitation of nickel (16,17). In this paper, we continue this line of research by demonstrating the suitability of Li(FDEDTC) as a chelating agent for the determination of Cr in biological materials by isotope dilution GC/MS; we validate the method by using NIST reference materials. The method offers high sensitivity with detection limits down to the sub-part-per-billion level (pg/L) with the inherent advantages of stable isotope dilution, most notably that the accuracy and precision of the analyses are not affected by incomplete recovery. EXPERIMENTAL SECTION Instrumentation. The GC/MS system consisted of a double-focusing, reverse geometry mass spectrometer (Model 8230, Finnigan MAT, San Jose, CA) coupled to a gas chromatograph (Varian 3700). The mass spectrometer, equipped with a SpectroSystem 300 data system for on-line data acquisition and processing, was operated as previously described ( I 7). Reagents. The "Cr-enriched CrZO, (>96 atom % "Cr) used as a spike for isotope dilution was obtained from Oak Ridge National Laboratory (Oak Ridge, TN). Certified Atomic Absorption Standard (potassium dichromate) was purchased from Fisher Scientific (Fairlawn, NJ) and used as the primary standard for spike calibration. Double sub-boiling quartz distilled HNO,
and HzS04 in Teflon bottles were obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD). Ultrex grade ammonium hydroxide solution (30%)was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ), and stabilized hydrogen peroxide (50%) was obtained from Fisher Scientific. Potassium permanganate was obtained from Mallinckrodt, Inc. (Paris, KY). The standard reference material, freeze-dried urine SRM 2670 (normal and elevated levels of toxic metals), was purchased from the NIST and prepared according to their directions. Lithium bis(trifluoroethyl)dithiocarbamate, Li(FDEDTC), was synthesized by using bis(trifluoroethy1)amine from PCR Research Chemicals (Gainesville, FL), and n-butyllithium and carbon disulfide from Aldrich Chemical Co. (Milwaukee, WI) in an inert atmosphere at -70 "C (18). Several precautions were necessary to minimize the potential for Cr contamination from the apparatus, reagents, personnel, and the laboratory environment, as previously reported (17). Since the overall blank defines the detection limit of the method and limits the applicability of this technique at extremely low levels, it was necessary to identify a lot of HzOz(Lot No. 791310) containing minimium amounts of Cr. The levels of Cr, determined by EAAS, present in various reagents used were as follows: chelating agent Li(FDEDTC), 0.06 rg/L; 4% solution of ammonium hydroxide, 0.016 rg/L; hydrogen peroxide, 0.4 g / L ; 0.5% (w/v) solution of KMnO,, 1.5 pg/L; pH 3 acetate buffer, 0.8 pg/L. An overall blank of less than 1ng was present due to the volumes of these different reagents used in the procedure for digestion and chelate formation. Preparation and Standardization of Spike Solution. A 50Crspike solution was prepared by dissolving the Tkz03in a minimum amount of Ultrex HCIO, with heating in a Teflon beaker. The solution was brought to volume with 0.5 M "OB. Diluted spike solutions were prepared from this stock solution, on a weight basis, for isotope dilution experiments. The isotopic composition of Cr in the spike was determined experimentally by preparing Cr(FDEDTC), chelate. The spike solution was calibrated as previously reported by reverse-isotope dilution GC/MS using the natural Cr primary standard (17, 19). Digestion and Chelate Formation. A known volume (1 mL) of the reconstituted urine reference material was mixed with a weighed amount of "Cr spike solution in a Teflon beaker. The amount of Cr in the spike solution added to the urine sample was optimized to obtain an isotope ratio in the mixture, corresponding to the geometric mean of isotope ratio m/z 562/564 in the sample and the spike. This is not an essential requirement of the method, but it is advisable for obtaining the best results. The spiked mixture was treated with 1 mL of concentrated HNO, and was allowed to stand overnight to allow the partial digestion of the organic matter and reduce foaming during subsequent heating. The partially digested solution was heated gently on a hot plate at 50 "C to reduce the volume to about 100 rL, and then 100 pL of 50% HzOzwas added. The solution was again heated gently and inspected periodically. The contents were mixed and the beaker was tapped gently to disperse frothing. The digestion with H20zwas performed four or five times until a white residue remained on complete evaporation of the solution. The procedure required about 1 mL of HzOzand took 3-4 h. The dried residue was dissolved in 1 mL of deionized water (DW), and the solution was again heated until completely dry. The residue was redissolved in 2 mL of DW, and the solution was transferred to a polypropylene (PP) tube. To the solution in the PP tube, 50 pL of 0.5% (w/v) KMnO, solution followed by 100 pL of 1M HzS04 was added to oxidize Cr(II1) to Cr(V1). The solution in the PP tube was heated in a boiling water bath for about 30 min to complete the oxidation of Cr. The solution was allowed to cool to room temperature and adjusted t o pH 3 by using 50-100 pL of a 4% solution of ammonium hydroxide. Subsequently, 500 pL of pH 3 acetate buffer was added and the Cr chelate formed by adding 200 pL of 20 mM solution of the chelating agent Li(FDEDTC). The Cr chelate was extracted with two 500-pL aliquots of CH2Cl2.The organic extract containing the Cr chelate was allowed to evaporate to dryness at room temperature in the laminar flow hood and reconstituted in 20 pL of CHZClzprior to GC/MS analysis. Samples containing 10 r g of Cr(V1) were used for optimizing the conditions of chelate formation. The chelate formation, at this high concentration, was immediately evident
ANALYTICAL CHEMISTRY, VOL. 62, NO. 2, JANUARY
15, 1990
113
Table I. Theoretically Expected and Measured Abundances of Different Ion Peaks in Natural Chromium atomic mass
atom % abund'
ion m / z
"Cr
4.345 83.789 9.501 2.365
818 820 821 822
S3Cr MCr
'Taken from ref
20.
molecular ion peak (M'+) calcd % measdb % 2.95 57.69 19.62 19.75
3.08 57.66 19.38 19.88
ion m / z
fragment ion peak (M - L)+ calcd % measdb %
562 564 565 566
3.32 64.58 17.09 15.01
3.36 64.11 17.18 15.35
Iniectine 5 ne of Cr on column.
Table 11. Theoretically Expected and Measured Abundances of Different Ion Peaks in Chromium Spike atomic mass
atom % abund"
ion mlz
"Cr
96.40 3.33 0.22 0.05
818 820 821 822
Wr 53Cr "Cr
molecular ion peak (Me+) calcd 90 measdb % 69.27 22.59 4.89 3.25
68.44 22.92 5.11 3.53
ion m / z
fragment ion peak (M - L)+ calcd % measdb %
562 564 565 566
78.28 17.46 2.62 1.64
78.04 17.68 2.61 1.67
'Values given by the supplier (Oak Ridge National Laboratory) of enriched isotope. Injecting 5 ng of Cr on column. from a change in the color of the solution as soon as chelating agent was added. Gas Chromatography/Mass Spectrometry. Prior to Cr isotope ratio measurements, the focusing conditions of the mass spectrometer were optimized and mass calibration was established by using perfluorokerosene. The Cr isotope ratios were measured in duplicate by injecting 1 KLof the chelate solution and monitoring the group of peaks corresponding to the fragment ion (M - L)+, formed by the loss of one ligand molecule. Data were obtained in a selected ion monitoring (SIM) experiment by integrating the chromatographic peak areas as previously described (17).
R E S U L T S AND DISCUSSION Precision a n d Accuracy in Isotope Ratio Measurements. The mass spectrum exhibits several isotopic groups of peaks corresponding to Cr(FDEDTQ3'+, Cr(FDEDTQ2+, and Cr(FDEDTC)+ designated by Me+,(M - L)+, and (M 2L)+, respectively (16). Among the various ion peaks containing the metal atom, the fragment ion Cr(FDEDTC)2+ formed by the loss of one ligand is of maximum intensity. The most abundant isotopic group of Cr(FDEDTC)2+ions nominally a t mlz 562 was used throughout the experiments to achieve high sensitivity. This ion group consists of four peaks a t mlz 561.88, 563.88, 564.88, and 565.88, corresponding respectively to W r , 52Cr, 53Cr, and 54Cr isotopes in Cr(FDEDTC)2+.Further, the presence of a molecular ion as well as the observation of symmetrical and sharp chromatographic peaks indicates the thermal stability of the Cr chelate a t nanogram levels under the experimental conditions used. Tables I and I1 present the results obtained for isotope ratio measurements of natural Cr and Wr-enriched spike, respectively. For these measurements, 1 KL of the chelate solution containing 5 ng of Cr was injected. The atom percent abundances of different isotopes in natural Cr (Table I) are the recommended values based on the values measured experimentally by various laboratories (20). The atom percent abundances of different Cr isotopes in "Cr spike (Table 11) are the values provided by the supplier of the enriched isotope (Oak Ridge National Laboratory). The theoretically calculated values are also given in these tables for the abundances of various Cr-containing peaks in the molecular ion, Cr(FDEDTC)3*+,and in the fragment ion, Cr(FDEDTC),+. These values have been obtained by calculating the contributions of the isotopes of chromium, carbon, nitrogen, and sulfur in these ions (21, 22). As can be seen from the data in Tables I and 11, there is excellent agreement among the calculated and measured abundances of the various ions in the natural Cr as well as in the "Cr spike. This shows that,
Table 111. Precision in Determining Isotope Ratios of Natural Cr by Using Cr(FDEDTC)3 5621564
mean of means within-run precision, % between-run precision, '70 overall precision: %
0.0543 1.9 6.1 6.4
isotope ratio" 5651564 5661564
0.2728 1.2 3.6 3.8
0.2432 1.3 3.0 3.2
'Injecting 5 ng of Cr on column. Overall precision S, was calculated by combining the within-run precision (Si)and betweenrun precision (&), using the formula S, = (St + S2)1/2, within the experimental uncertainties, there was no mass discrimination due to the use of different accelerating voltages when isotope ratios were determined by voltage peak switching. This lack of bias is likely due to the small relative mass difference at the high m / z values of the ions. In the presence of mass discrimination, a general trend toward decreasing abundances with increasing mass of Cr isotope would have been observed. Since this was not observed, no correction was applied to the experimentally determined isotope ratios over the 4-amu mass range measured a t either the molecular ion (mlz 818-822) or the fragment ion (mlz 562-566). Moreover, any mass discrimination factor that might be observed is canceled in isotope dilution experiments as the spike calibration is performed by reverse-isotope dilution using a primary standard in the same experiment (19). Precision in the determination of various isotope ratios was evaluated by performing measurements of chelated natural Cr on three different days. Five to 10 replicate injections of 5 ng of Cr were made on each of the three days. Mean values and standard deviations were calculated from the data obtained on each day. These mean values were used to calculate the mean of means and its standard deviation, referred to as between-run precision and given in Table 111. The within-run precision was calculated by considering the standard deviation values obtained on different days. Overall precision was calculated by combining the within-run precision and between-run precision values. This was done to evaluate the effects of any variations in the mass spectrometer operating parameters that may occur from one day to another. Overall precision values of 3-6% were obtained at the 5-ng level. As expected, the precision is better when the ratios to be measured are closer to unity (for example, mlz 5651564 and 566/564 versus 5621564 in Table III), and this can be achieved by optimum spiking in the isotope dilution step.
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 2, JANUARY 15, 1990
Table IV. Determination of Chromium in Spike Solution Using Five Synthetic Mixtures with Altered Isotope Ratios Ranging from 0.2 to 2
4,301
ion used
4.20
Cr(FDEDTC),'+ Cr(FDEDTC)2C
0.056
Natural
Natural
-I
isotope ratio
mean concn,O pg of Cr/g of soln
std dev, rglg
range, ccg/g
8181820 8181821 8181822 562/564 562/565 5621566
2.31 2.29 2.33 2.22 2.31 2.35
0.04 0.03 0.04 0.06 0.06 0.05
2.26-2.34 2.24-2.33 2.28-2.34 2.15-2.29 2.20-2.37 2.27-2.41
Injecting 5 ng of Cr on column, mean from five synthetic mixtures.
Table V. Standardization of 'Wr Spike Solution by Reverse Isotope Dilution
0.054
isotope ratio
mean concn," pg of Cr/g of soln
std dev, alg
range, ccglg
818/820 818/821 8181822 5621564 5621565 5621566
2.31 2.31 2.31 2.26 2.31 2.30
0.02 0.03 0.05 0.04 0.02 0.02
2.30-2.36 2.28-2.35 2.27-2.38 2.21-2.32 2.28-2.33 2.28-2.33
0.052 0.050
n A R -.-~ .-
,
ion used Cr(FDEDTQ3*+ I
2
3
4
5
6 7 8 9 101112131415
Injection Number Figure 1. Evaluation of cross-contamination between samples of very
Cr(FDEDTCI2+
different isotopic compositions, in consecutive analyses. Injections 1-5 and 11-15 are from a natural Cr sample and injections 6-10 are from a 50Cr spike.
"Injecting 5 ng of Cr on column, mean from five independent samples.
Memory Effect. One problem with the GC/MS analyses of metal chelates for isotope ratio measurements is the cross-contamination in the GC/MS system between sequential analyses of samples with widely different isotope ratios. This can be the limiting factor for some metals and chelating agents. This phenomenon, referred to as the memory effect, can adversely affect the accuracy of the measurement of altered (nonnatural) isotope ratios. Memory effect has been an analytical problem due, in part, to the nonavailability of a suitable chelating agent. The Li(FDEDTC) chelating agent used in this work offers advantages in comparison to trifluoroacetylacetone (TFA) used earlier for Cr determination in biological materials (22), geological materials (23),and seawater (24). The advantages include the quick preparation of chelates a t room temperature, negligible memory effect, and the measurement of isotope ratios over a limited range (2-4 amu) a t high mass (mlz 560 or 830),thereby reducing the mass discrimination effects and chemical interferences in the mass spectrometer. Also, the use of Li(FDEDTC) as a chelating agent is promising because of its applicability to several elements which can be readily separated on a capillary GC column (25). In the present studies of Li(FDEDTC) as a chelating agent, the memory effect was evaluated by using two different approaches. The first method involved the sequential analysis of a solution of natural Cr and a solution of the 50Cr spike measuring the mlz 5621564 isotope ratio. The analyses were carried out in the following sequence: five injections of natural Cr, five injections of W r spike, five injections of natural Cr. The results obtained are shown in Figure 1. For these data, no appreciable memory or carryover is observed when these samples with isotope ratios differing by a factor of about 90 are analyzed sequentially. The second approach used to investigate the suitability of GC/MS using Cr(FDEDTC)3chelate was an evaluation of the accuracy of determining isotope ratios different from those of natural samples. For this experiment, five synthetic mixtures differing in the mlz 5621564 ratio by a factor of 10 (range
0.2-2) were prepared by mixing weighed aliquots of the primary standard solution and the 50Cr spike solution. The mixtures were prepared to contain almost equal amounts of total Cr. Three injections, each with 5 ng of Cr, were made from each of the mixtures, and the isotope ratios mlz 5621564, 5621565, and 5621566, corresponding to the different Cr isotopes in the fragment ion Cr(FDEDTC)2+,were determined. On another day, the isotope ratios mlz 818/820,818/821, and 8181822, corresponding to different Cr isotopes in the molecular ion Cr(FDEDTC)3*+,were also determined by using these synthetic mixtures. The mixtures were analyzed in the sequence of increasing isotope ratios. The isotope ratios determined from these mixtures were used to calculate the Cr concentration in the spike solution by using reverse-isotope dilution methodology, and the results are given in Table IV. The consistency in the concentration of Cr in the spike solution, shown by low standard deviations and narrow ranges of the calculated concentration, shows that the isotope ratios were measured accurately. Further, the constancy in the isotope ratios determined by replicate injections from each of the synthetic mixtures also demonstrated no appreciable memory effect. Isotope Dilution Results. The 50Crspike solution was calibrated by reverse isotope dilution using a primary standard of natural Cr. For this standardization, five samples were prepared by mixing weighed amounts of primary standard and 50Crspike solutions to achieve an optimum isotope ratio mlz 5621564 in the spiked mixtures. The results obtained for Cr concentration in the spike solution are given in Table V. The concentration values calculated from the different isotope ratios are all in good agreement. The concentration values are given in units of pg/g since the aliquots were taken on a weight basis to eliminate pipetting errors. The standard deviation observed for the concentration values in Table V is better compared to that in Table IV. This is because the concentration values in Table V were derived from samples with optimum isotope ratios (i.e. mlz 5621564 0.5) whereas
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 2, JANUARY 15, 1990
Table VI. Determination of Cr in SRM-2670Urine by Isotope Dilution GC/MS mean concn: p g
NIST value, pg/L
85 f 6 13 f ?
562/564 562/565 562/566 562/564 562/565 562/566
s t d dev,
soln
rg/L
range, r g / L
89 (n = 11) (n = 11) (n = 11) (n = 5) (n = 5) (n = 5)
6 5
76-99 89-101 73-101 11-15 11-17 9-17
Injecting 2 n g
94 92 13 13 12
Patrick K. Anonick for assistance in synthesis. Registry No. Li(FDEDTC), 74613-66-4; Cr, 7440-47-3.
LITERATURE CITED
of C r / L of isotope r a t i o
115
9
2 3 3
of Cr o n column.
the values in Table 1V were from samples with a range of isotope ratios (Le. m / z 562/564 from 0.2 to 2). The calibrated 50Crspike solution was then used to quantitate Cr in the NIST freeze-dried urine reference material SRM 2670. This reference material consists of two different urine samples containing 13 pg of Cr/L (recommended value) and 85 f 6 pg of Cr/L (certified value). The signal-to-noise ratio (>lo0 to 1 for 13 pg of Cr/L) observed in the GC peak indicates potential limits of detection down to sub-partsper-billion levels. In principle, the detection limit of the method (2-3 ng/L) limits the quantitative measurement of Cr; however, contamination from reagents, laboratory, and analyst makes the practical limit 0.1-1 pg/L with reasonable precautions. All the isotope ratios corresponding to different Cr isotopes in the fragment ion Cr(FDEDTC)2+were recorded and used for calculating the Cr concentration in the urine samples. The results obtained are shown in Table VI and have been corrected for Cr blank. Since the Cr concentration values in the urine reference material are provided in units of pg/L by NIST, the urine sample aliquots were taken on a volume basis instead of weight basis so that the Cr concentration values in urine are given in pg/L. The concentration values calculated by using the different isotope ratios are in good agreement with one another as well as with the NIST values in the reference material SRM 2670.
CONCLUSION The results of this work demonstrate that isotope dilution GC/MS using Li(FDEDTC) as a chelating agent can be used for determining Cr in urine. Results obtained are shown to be accurate and precise. No significant memory effect is seen in the determination of altered isotope ratios. This absence of memory effect not only allows accurate quantitation but also shows that the technique can be used for isotope ratio measurements in metabolic and bioavailability studies (26).
ACKNOWLEDGMENT The authors thank James Nicholson for providing the electrothermal atomic absorption spectrometric results and
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RECEIVED for review March 27, 1989. Revised manuscript received October 10, 1989. Accepted October 18, 1989. Funding for the purchase of the high-resolution mass spectrometer was obtained from the National Institutes of Health, Division of Research Resources Shared Instrumentation Grant Program, Grant Number 1-S10-RRO-2418-01. Additional funding support from the John Lee Pratt Fund of the University of Virginia and Grant ESO 4464 of the National Institute of Environmental Health Sciences is also gratefully acknowledged. S.K.A. Thanks the Division of Experimental Pathology, Department of Pathology, University of Virginia Health Sciences Center, for a postdoctoral fellowship and the authorities at Bhabha Atomic Research Center, Trombay, Bombay 400 085, India, for granting leave.
