ANALYTICAL
BIOCHEMISTRY
194,
140-145
(1991)
Determination of Copper in Urine and Serum by Gas Chromatography-Mass Spectrometry Suresh
K. Aggarwal,
Department
Received
July
of Pathology,
Michael
Kinter,
University
of Virginia
and David Health
A. Heroldl
Sciences Center, Charlottesville,
Academic
22908
31, 1990
A stable isotope dilution gas chromatography-mass spectrometry method using enriched “Cu as an internal standard is described for the determination of Cu in urine and serum. Chelating agents N,N’-ethylenebis(trifluoroacetylacetoneimine) [H,(enTFA,)] and lithium bis(trifluoroethyl)dithiocarbamate [Li(FDEDTC)] were used and evaluated for memory effect. H,(enTFA,) did not show any appreciable memory effect, whereas Li(FDEDTC) was found to have a strong memory effect. Overall precision of 1.6% was obtained for determining Cu isotope ratios at a lo-ng level using H,(enTFA,). Cu concentrations in the National Institute of Standards and Technology (NIST) reference materials, freeze-dried urine SRM 2670, and human serum SRM 909 determined using the H,(enTFA,) chelating agent were in good agreement with the NIST-certified values. Isotope ratios determined by gas chromatography-mass spectrometry on samples with altered isotopic composition were in good agreement with the inductively coupled plasma-mass spectrometry data. 0 1991
Virginia
Prese,
Inc.
Copper (Cu) is an essential trace element for several enzyme systems. It is also a very toxic metal. Consequently, a complex transport system is required to deliver Cu to the sites of utilization. Defects in these systems can result in deficiency or excess body burden of copper. Two rare but well-characterized Cu-related diseases are Menke’s (steely-hair) syndrome, a fatal Cu deficiency caused by defective intestinal absorption, and Wilson’s disease, a treatable Cu excess caused by defective bilary excretion and impaired incorporation of Cu in ceruloplasmin. The diverse physiological consequences of Cu in enzymatic reactions and the effect of ’ To whom correspondence should be addressed at University of Virginia Health Sciences Center, Department of Pathology, Box 214, Charlottesville, VA 22908. Fax: (604) 9246060.
excess or deficient Cu on the central nervous system emphasize the importance of understanding Cu absorption, metabolism, and elimination (1,2). These studies demand the development of precise and accurate methods for Cu determination in biological samples. A few attempts have been reported for Cu determination in biological samples using mass spectrometry with volatile chelates. Terlouw et al. (3,4) used acetylacetone as a chelating agent and fully deuterated copper acetylacetonate and “5Cu-labeled chelate as the internal standards. Hui et al. (5) used tetraphenylporphine (H,TPP)2 for Cu derivatization, and deuterated TPP chelate and 65Cu as the internal standards. Johnson (6) also used Cu-TPP chelate for investigating Cu absorption studies in human subjects. Buckley et al. (7) performed a detailed investigation for Cu isotope ratio measurements using acetylacetone, trifluoroacetylacetone, heptafluorodimethyloctanedione, and 8-hydroxyquinoline. They reported a significant memory effect for the sequential analyses of two Cu samples with 65Cuabundances of 30.83 and 39.98% using these chelating agents. H,TPP was the only chelating agent found suitable for a limited range of isotope ratio measurements. All these studies used a direct probe for sample introduction, which necessitates the prior separation of other isobaric, interfering metals. The use of a gas chromatographic column for sample introduction is attractive since this provides a separation step for different metal chelates. Moreover, the availability of autosamplers in the commercial gas chromatography-mass spectrometry (GC-MS) systems would provide a high throughput of samples. An extensive literature survey showed that there have only been two reports on the GC-MS determination of Cu using H,(enTFA,) and Na(FDEDTC)
2 Abbreviations used: TPP, tetraphenylporphine; enTFA,, N,N’ethylenebis(trifluoroacetylacetoneimine); FDEDTC, bis(trifluoroethyl)dithiocarbamate; NIST, National Institute of Standards and Technology; SIM, selection ion monitoring.
140 All
Copyright 0 1991 rights of reproduction
ooo3-2697191$3.00 by Academic Press, Inc. in any form reserved.
CHROMATOGRAPHY-SPECTROMETRY
chelating agents (8,9). Hachey et al. (8) investigated H,(enTFA,) for Cu isotope ratio measurements using chemical ionization; however, the multiplicity of ion molecule products contributing to the mass peaks of interest limited the accuracy and precision. For Na(FDEDTC), the authors report no data (9) for either precision and accuracy of natural and altered Cu isotope ratios or memory effect. These are the problems which have limited the development of GC-MS methods for trace metals determination and must be investigated. Growing interest in the use of stable enriched isotopes for studying the bioavailability of various trace metals and the easy accessibility of organic mass spectrometers to clinical and biomedical laboratories encouragedus to develop GC-MS methods for the determination of trace metals in biological samples (10). We have previously synthesized lithium bis(trifluoroethyl)dithiocarbamate [Li(FDEDTC)] and used it as a chelating agent for the determination of Ni, Cr, Co, and Pt at the nanogram levels in urine with excellent accuracy and precision and without any appreciable memory effect (11-14). In this paper, Li(FDEDTC) and H,(enTFA,) chelating agents were investigated for Cu analysis in biological samples. Precision and accuracy in determining Cu isotope ratios at the nanogram levels were evaluated and memory effect in measuring altered isotope ratios were studied. Finally, stable isotope dilution GC-MS analysis using enriched 65Cu was validated for the quantitation of Cu in the National Institute of Standards and Technology (NIST) freeze-dried urine reference material SRM 2670 and human serum SRM 909.
EXPERIMENTAL
Instrumentation The mass spectrometer was a double-focusing, reverse-geometry instrument (Model 8230, FinniganMAT, San Jose, CA). The instrument is equipped with a SpectroSystem 300 data acquisition and processing system and a Varian 3700 gas chromatograph with on-column injection. Details of the GC-MS system have been previously described (10). For the present studies, a 10 m X 0.32 mm, nonpolar fused-silica capillary column, DB-1 (dimethylpolysiloxane) (J. W. Scientific, Ranch0 Cordova, CA), with a 0.25pm film thickness was used. Samples were injected at an oven temperature of 100°C followed by a 25”C/min ramp to 300°C. The ion source was heated to a temperature of 200°C and the GC-MS interface was at 280°C. The secondary electron multiplier was at 2 kV. GC-MS data were acquired at 2 Hz, yielding approximately 20 data points across the 10-s wide GC peak. A mass resolution of 1000 was used throughout. The isotope ratios were calculated by integrating the ion current for the various chromatographic
DETERMINATION
OF
COPPER
IN
141
URINE
peaks. Details of the measurement been reported previously (10).
methodology
have
Reagents The 65Cu-enriched copper oxide (99.61 atom % of 65Cu) used as an internal standard for isotope dilution was obtained from Oak Ridge National Laboratory (Oak Ridge, TN). Certified Atomic Absorption Standard (copper nitrate in 2% nitric acid) purchased from Fisher Scientific (Fairlawn, NJ) was used as the primary standard for calibration. Double sub-boiling quartz-distilled HNO, in Teflon bottles was obtained from NIST (Gaithersburg, MD). The standard reference materials, freeze-dried urine SRM 2670 and human serum SRM 909, were also purchased from NIST and prepared according to their directions. 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. Deionized water was used throughout these experiments. Although high-quality clean room facilities are not necessary for Cu analysis in biological samples precautions must be taken to prevent adventitious contamination of samples with Cu from the apparatus, reagents, personnel, and the laboratory environment as previously reported (11,12). The levels of Cu, determined by electrothermal atomic absorption spectrometry, in various reagents used were as follows: hydrogen peroxide, 0.5 pg/liter; 4% solution of ammonium hydroxide, 0.03 pg/liter; pH 3 acetate buffer, 0.5 pg/liter. No detectable amounts of Cu were observed in methylene chloride and the chelating agents. An overall background of about 1 rig/sample was present due to the volumes of these different reagents used for digestion and chelate formation.
Preparation
of Chelating
Agents
Chelating agents were prepared using the published procedures. Li(FDEDTC) was synthesized by reacting bis(trifluoroethyl)amine (PCR Inc., (Gainesville, FL) and n-butyllithium (Aldrich Chemical Co., Milwaukee, WI) in an inert atmosphere at -70°C followed by the addition of carbon disulfide (Aldrich) (10,15). H,(enTFA,) was synthesized by mixing 10 ml of trifluoroacetylacetone (Aldrich) and 40 ml of ethanol (Aldrich) and heating to 70°C under argon (16). Then 3 ml of ethylenediamine (Aldrich) was added slowly and the heating continued for 30 min. The solution was cooled and filtered under suction to remove the precipitated white crystalline solid. This was washed with ethanol and recrystallized from ethanol.
142 Preparation
AGGARWAL,
and Standardization
KINTER,
AND
of Spike Solution
A 65Cu solution was prepared by dissolving 65Cu0 in 0.5 M nitric acid with heating. Diluted solutions were prepared on a weight basis from this stock solution for isotope dilution experiments. Isotopic composition of Cu in this solution was determined experimentally by GC-MS analyses of Cu(enTFA,) chelate. The spike solution was calibrated, as reported previously (11,12), by reverse isotope dilution GC-MS using the Cu primary standard and preparing the Cu-chelate. Concentration of Cu in the spike solution was calculated from the mass spectrometrically determined isotope ratios and the weights of the standard and spike solutions taken for mixing (17).
f ;(j&
50
Digestion of Urine and Serum Samples and Chelate Formation A known volume (1 ml) of the reconstituted urine or serum reference material was mixed with a weighed amount of =Cu solution in a Teflon beaker. The spiked urine samples were digested using nitric acid and hydrogen peroxide as previously reported (11,12). The serum samples were first deproteinized using 100 ~1 of concentrated nitric acid added dropwise with continuous vortexing of the solution. Following this addition, the samples were heated on a water bath at 50°C for about 30 min and the precipitated proteins pelleted by centrifugation (18). The supernate was taken for digestion with nitric acid and hydrogen peroxide, as for the urine samples (llJ2). Cu(FDEDTC), was prepared at a pH of 3, using 1 ml of acetic acid and sodium acetate buffer and 100 ~1 of a 20 mM solution of Li(FDEDTC) in deionized water. Cu(enTFA,) was prepared using a 50 mM solution of H,(enTFA,) in methylene chloride by the procedure of Belcher et al. (16). For this procedure, the digested biological extracts were dissolved in 500 ~1 of deionized water, and 2 ml of ethanol was added. The pH of the solution was adjusted to be greater than 11.5 by addition of about 1 ml of ammonia solution. A loo-~1 sample of the chelating agent was added and the solution vortexed and allowed to stand for 5 min. The reaction mixture was then extracted by addition of 1 ml of methylene chloride followed by 2 ml of deionized water to induce phase separation. The organic extract containing Cu(enTFA,) was allowed to evaporate to dryness at room temperature in the laminar flow hood and reconstituted in 20 ~1 of methylene chloride for GC-MS analysis. Spectrometry
The Cu isotope ratios were measured in duplicate by injecting 1~1 of the chelate solution and monitoring the group of peaks corresponding to the molecular ion. Data
,,,,~,,~!,:r ,,,,1;,,,)
100
150
200
250
,,,,,I[
300
350
400
m/z FIG.
Gas Chromatography-Mass
HEROLD
1.
Electron-ionization
mass
spectrum
of Cu(enTFA,).
were acquired in a selected ion monitoring (SIM) experiment, without using a lock mass, and employing voltage peak switching (10). RESULTS
AND
DISCUSSION
The 70-eV EI mass spectra of Cu(FDEDTC), and Cu(enTFA,) exhibit two groups of ions containing Cu isotopic information. These ions are the molecular ion CM)+’ and a fragment ion resulting from the loss of one ligand. The mass spectrum of Cu(FDEDTC), showed an intense peak at m/z 224 corresponding to (CF,CH,),NCS, but it is not useful for Cu isotopic analysis (10). The mass spectrum of Cu(enTFA,) is shown in Fig. 1. The molecular ions, Cu(enTFA),+*, and Cu(FDEDTC)i’ with nominal m/z 393 and 575, respectively, were used for Cu isotope ratio measurements throughout these experiments. The high-intensity fragment ion at nominal m/z 228 in Cu(enTFA,) may be used with this chelate to increase sensitivity in samples containing extremely low levels of Cu. Evaluation of Memory Effect Memory effect in GC-MS analyses of metal chelates refers to the cross-contamination during sequential analyses of samples with varying isotope ratios. In the present studies, memory effect was investigated using four synthetic mixtures prepared by mixing weighed aliquots of the primary standard solution and the %Cu solution in differing proportions but containing almost equal amounts of Cu. These mixtures covered a range from 1.4 to 4.2 of “5Cu/63Cu isotope ratios. Chelates were prepared from these mixtures and replicate analyses (3 or 4), each with 10 ng of Cu, were made for each mixture in the sequence of increasing isotope ratios and then in the reverse sequence. Figures 2 and 3 present the results obtained for m/z 3951393 and m/z 577/575 isotope ratios determined in the mixtures using, respectively,
CHROMATOGRAPHY-SPECTROMETRY
DETERMINATION
OF
COPPER
IN
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143
Spike Mixture 4
32.0 30.0 28.0
-CT 8
3.0
2 ’
v) i?
Mixture 3
2.5 2.0
Analysis Number FIG. 4. Evaluation of the cross-contamination between samples of widely varying isotopic compositions in consecutive analyses, using H,(enTFA,).
18 19 2021 2223242525272829
Analysis Number FIG. 2. Evaluation yses of samples with
of the cross-contamination altered isotope ratios,
using
in consecutive H,(enTFA,).
anal-
Cu(enTFA,) and Cu(FDEDTC), chelates. As can be seen, no appreciable memory effect is observed for Cu(enTFA,), whereas there is a strong memory effect encountered in using Cu(FDEDTC),. The memory is seen in two ways. First, within a set of analyses (i.e., analyses 14 to 18 of mixture 3), there is a trend toward increasing ratios in the set. This is a result of memory from the analysis of mixture 2. The reverse is seen for analyses 27 to 32, in which the memory of the analysis of mixture 3 is affecting the ratios obtained for the analysis of mixture 2. Memory can also be seen in the average ratios obtained on the ascending vs descending portions
Mixture
4
Analysis Number FIG. 3. Evaluation yses of samples with
of the cross-contamination altered isotope ratios,
using
in consecutive Li(FDEDTC).
anal-
of the figure; the values obtained are not equal. For example, the average for mixture 3 obtained in analyses 14 to 18 is 2.12 + 0.08 (after analysis of mixture 2) but in analyses 23 to 26, the average is 2.53 f 0.03 (after analysis of mixture 4). This is an excellent illustration of the deleterious effects which memory can have on measurements requiring isotope ratios analyses. It is interesting to note that this GC-MS system has already been characterized for the fluorinated diethyldithiocarbamates of Ni (ll), Cr (12), Co (13), and Pt (14) and no appreciable memory was observed. This along with the observation that no appreciable memory is observed for Cu(enTFA,) illustrates the importance of the choice of chelating agent. It is our belief that the availability of suitable chelating agents is the major determinant for the presence or the absence of memory in the GC-MS analyses of metals. In turn, we also believe that memory is the key limitation to the use of these methods. A more rigorous approach was used for H,(enTFA,) chelating agent to determine if memory effect could be observed. This involved the sequential analyses of a solution of natural Cu and enriched 65Cu measuring the ml.2 3951393 isotope ratio. The results are shown in Fig. 4. In these two samples with ratio m/z 395/393 differing by a factor of 60, no appreciable memory effect was seen. It is important to make this evaluation of possible memory effect by comparing the value of the natural Cu ratios before and after analysis of the enriched 65Cu. The ratio of the natural Cu is near the measurement optimim, while the enriched sample, being far from optimum, shows a lack of precision. This rigorous evaluation enhances the confidence in using H,(enTFA,) as a chelating agent for Cu determination by GC-MS in unknown samples. Precision and Accuracy in Isotope Ratio Measurements Table 1 shows the precision and accuracy achieved for isotope ratio measurements of natural Cu using
144
AGGARWAL, TABLE
Precision
and Accuracy of Natural
KINTER,
AND HEROLD
1
TABLE
in Isotope Ratio Determination Cu as Cu(enTFA,)
Determination
of Cu in Urine
Day 1 Day 2 Day 3 Day 4 Mean of means Within-run precision (%) Between-run precision (%) Overall precision ’ ( W) Calculated isotope ratio Difference * (%)
0.4895 0.4928 0.5038 0.4911
k + -+ +
+ SD)
0.0068 0.0058 0.0065 0.0150 0.4943 0.9 1.3 1.6 0.4594 +7.6
(n (n (rz (n
= = = =
5) 5) 4) 5)
a Overall precision S, was calculated by combining the within-run precision (SJ and between-run precision (S,) according to the formula S, = (Sf + S:)“‘. * Difference of the mean of means with respect to the calculated isotope ratio.
Cu(enTFA,) at a lo-ng level. Overall precision in determining isotope ratios was evaluated by performing measurements of chelated natural Cu on different days, with replicate analyses on each day. The mean of means included in Table 1 was calculated by using the mean values obtained on different days. The mean value for each day was used to calculate the standard deviation on the mean of means, referred to as between-run precision in Table 1. The within-run precision was calculated by using
the standard
deviation
values
obtained
on the indi-
vidual days. Overall precision was arrived at by combining the within-run and between-run precision values. This was done to include the effects of any variations in the mass spectrometric operating parameters that may affect the quality of isotope ratio data from one day to another. Overall precision of 1.6% is obtained using Cu(enTFA,). The calculated isotope ratio for this chelate, given in Table 1, was obtained by including the contributions of Cu, C, N, and 0 isotopes (19). A difference of 7.6% in the ratio for Cu(enTFA,) is obtained for the mean of means with respect to the calculated value. It should be noted that this represents a statistically significant bias. We have not been able to explain the source of this bias, so no correction is applied to the measured isotope ratios. However, this bias would be canceled in any isotope dilution experiment in which the internal standard solution is calibrated in the same experiment. The method, therefore, remains a fundamentally sound means of performing isotope dilution.
Results on Urine and Serum Samples The calibrated 66Cu spike was used to quantify Cu in the NIST reference materials, freeze-dried urine SRM
NIST value (fig/liter)
Sample Urine SRM-2670 Human serum SRM-909
370+
and Serum
Concentration of solution (@&/liter) mean + SD (n = 4)
Isotope ratio 395/393 (mean
2
Range &g/liter)
30
410 t 30
370 to 430
1100 + 100
1000 + 30
970 to 1040
2670 and human serum SRM 909. Since the certified concentrations in these samples are provided in the units of microgram/liter by NIST, the samples were taken on a volume basis instead of weight basis. The results are shown in Table 2. The Cu concentrations determined by using H,(enTFA,) are in good agreement with the NIST certified values in the urine and serum samples.
Comparison of Isotope Ratios Determined and ICP-MS
by GC-MS
This GC-MS method was also validated by comparison of the results obtained by ICP-MS. For this purpose, four synthetic mixtures were prepared by mixing primary standard solution and enriched ‘Yu solution as previously described. A comparison of the results for the m/z 3951393 isotope ratios calculated from the YZu/ 63Cu ratios obtained by ICP-MS and those determined by GC-MS is shown in Table 3. The calculated ratios for the m/z 3951393 in Cu(enTFA,) were obtained by using the experimentally determined Y!u/~CU ratios by ICP-MS and including the contributions of C, N, and 0 isotopes. As is seen, a mean value of 1.006 f 0.005 for the measured/calculated ratio is obtained. A regression analysis of 65Cu/63Curatios (x) by ICP-MS (column 2) and m/z 3951393 ratios (y) by GC-MS (column 4) gave an equation y = A + Bx with A = 0.015, B = 1.0025, and a
TABLE A Comparison
of Cu Isotope
by GC-MS
3
Ratios in Synthetic and ICP-MS
Samples
m/z 3951393 Synthetic mixture SM-11 SM-12 SM-13 SM-14 Mean
65cu/~cu by ICP-MS 1.3835 1.8691 2.6115 4.2308
Calculated 1.397 1.883 2.625 4.245
Measured/ calculated
Measured GC-MS 1.407 1.901 2.648 4.241
+ -t k f
0.003 0.020 0.010 0.050
1.007 1.009 1.009 0.999 1.006
+ 0.005
CHROMATOGRAPHY-SPECTROMETRY
correlation coefficient of 1.000. However, a slope of 0.89 was reported by Hachey et al. (8) for Cu(enTFA,) chelate during linear regression analysis of the measured isotope ratios by chemical ionization in the chelate and 65Cu/63Cu ratios. A small constant bias in using CuTPP chelate, with no appreciable memory effect in the limited isotope ratio range, was reported by Buckley et al. (7). They suggested that this may be due to the exchange of Cu in CuTPP with Cu-containing parts of the mass spectrometer. The absence of bias in the present studies (except for the natural Cu experiment) and good agreement in the isotope ratios obtained by GC-MS and ICP-MS enhances the confidence in using H,(enTFA,) chelating agent for Cu. CONCLUSIONS
The results of the present work show that H,(enTFA,) is a better chelating agent compared to Li(FDEDTC) for Cu determination by GC-MS. Results obtained on concentration in the NIST reference materials, freeze-dried urine and human serum, are shown to be accurate and precise. ACKNOWLEDGMENTS The authors thank P. J. Paulsen, J. D. Fassett, and J. R. Moody of NIST (Gaithersburg, MD) for the ICP-MS results; Kathy Winborne for providing the electrothermal atomic absorption spectrometric results; and Patrick K. Anonick and Frank Gordon for assistance in the syntheses of the chelating agents. The authors also thank Professors Savory and Wills for their interest in the present work and allowing use of the facilities available in the trace metals laboratory. Funding for the purchase of the high-resolution mass spectrometer was obtained from the National Institute of Health, Division of Research Resources Shared Instrumentation Grant Program (Grant l-SlORRO-241801). Additional funding from the John Lee Pratt Fund of the University of Virginia is gratefully acknowledged. S.K.A. thanks the Division of Experimental Pathology, Department of Pathology,
DETERMINATION
OF
COPPER
IN
145
URINE
University of Virginia Health Sciences Center, for a post-doctoral fellowship and the authorities at Bhabha Atomic Research Center (Trombay, Bombay, India) for granting leave from July 1987 to July 1989 and deputation from June 1990 to August 1990.
REFERENCES 1. Baselt, R. C., and Carvey, R. H. (1989) in Disposition of Toxic Drugs and Chemical in Man (Baselt, R. C., and Carvey, R. H., Eds.), pp. 220-223, Yearbook Medical Publishers, Chicago. 2. Danks,
D. M.
(1988)
Annu.
Reu. Nutr.
8,235-257.
3. Terlouw, J. K., DeRidder, J. J., Heerma, W., and Dijkstra, G. (1970) 2. Anal. Chem. 249,296-301. 4. Terlouw, J. K., and DeRidder, J. J. (1970) 2. Anal. Chem. 260, 166-168. 5. Hui, K. S., Davis, B. A., and Boulton, A. A. (1977) Neurochem. Res. 2,495-506. 6. Johnson, P. E. (1982) J. Nutr. 112, 1414-1424. 7. Buckley, W. T., Huckin, S. N., Budac, J. J., and Elgendorf, G. K. (1982) Anal. Chem. 54, 504-510. a. Hachey, D. L., Blais, J. C., and Klein, P. D. (1980) Anal. Chem. 52, 1131-1135. 9. Soltani-Neshan, A., Dorner, K., and Schaub, J. (1988) Fresenius’ 2. Anal. Chem. 331,202-204. 10. Aggarwal, D. A. (1989) 11. Aggarwal, D. A. (1989) 12. Aggarwal, D. A. (1990) 13. Aggarwal, 14. Aggarwal,
S. K., Kinter, Anal. Chim. S. K., Kinter, Anal. Chem. S. K., Kinter, Anal. Chem. S. K., Kinter,
M., Wills, M. R., Acta 224,83-95. M., Wills, M. R., 61,1099-1103. M., Wills, M. R., 62,111-115. M., and Herold,
S. K., Kinter, M., and Herold, 15. Sucre, L., and Jennings, W. (1980) Anal. 16. Belcher, R., Martin, R. J., Stephen, W. I., malizad, A., and Uden, P. C. (1973) Anal.
Savory,
J., and Herold,
Savory,
J., and Herold,
Savory,
J., and Herold,
D. A., communicated. D. A., communicated. Lett. 13(A6), 497-501. Henderson, D. E., KaChem. 45,1197-1203.
17. Aggarwal, S. K., Duggal, R. K., Rao, R., and Jain, H. C. (1986) Int. J. Mass Spectrom. Ion Processes 71, 221-231. 18. Brown, S., Bertholf, R. L., Wills, M. R., and Savory, J. (1984) Clin. Chem. 30,1216-1218. 19. DeBievre, P., and Barnes, I. L. (1985) Int. J. Mass Spectrom. Ion Processes 65, 211-230.
BIOCHEMISTRY
194,
140-145
(1991)
Determination of Copper in Urine and Serum by Gas Chromatography-Mass Spectrometry Suresh
K. Aggarwal,
Department
Received
July
of Pathology,
Michael
Kinter,
University
of Virginia
and David Health
A. Heroldl
Sciences Center, Charlottesville,
Academic
22908
31, 1990
A stable isotope dilution gas chromatography-mass spectrometry method using enriched “Cu as an internal standard is described for the determination of Cu in urine and serum. Chelating agents N,N’-ethylenebis(trifluoroacetylacetoneimine) [H,(enTFA,)] and lithium bis(trifluoroethyl)dithiocarbamate [Li(FDEDTC)] were used and evaluated for memory effect. H,(enTFA,) did not show any appreciable memory effect, whereas Li(FDEDTC) was found to have a strong memory effect. Overall precision of 1.6% was obtained for determining Cu isotope ratios at a lo-ng level using H,(enTFA,). Cu concentrations in the National Institute of Standards and Technology (NIST) reference materials, freeze-dried urine SRM 2670, and human serum SRM 909 determined using the H,(enTFA,) chelating agent were in good agreement with the NIST-certified values. Isotope ratios determined by gas chromatography-mass spectrometry on samples with altered isotopic composition were in good agreement with the inductively coupled plasma-mass spectrometry data. 0 1991
Virginia
Prese,
Inc.
Copper (Cu) is an essential trace element for several enzyme systems. It is also a very toxic metal. Consequently, a complex transport system is required to deliver Cu to the sites of utilization. Defects in these systems can result in deficiency or excess body burden of copper. Two rare but well-characterized Cu-related diseases are Menke’s (steely-hair) syndrome, a fatal Cu deficiency caused by defective intestinal absorption, and Wilson’s disease, a treatable Cu excess caused by defective bilary excretion and impaired incorporation of Cu in ceruloplasmin. The diverse physiological consequences of Cu in enzymatic reactions and the effect of ’ To whom correspondence should be addressed at University of Virginia Health Sciences Center, Department of Pathology, Box 214, Charlottesville, VA 22908. Fax: (604) 9246060.
excess or deficient Cu on the central nervous system emphasize the importance of understanding Cu absorption, metabolism, and elimination (1,2). These studies demand the development of precise and accurate methods for Cu determination in biological samples. A few attempts have been reported for Cu determination in biological samples using mass spectrometry with volatile chelates. Terlouw et al. (3,4) used acetylacetone as a chelating agent and fully deuterated copper acetylacetonate and “5Cu-labeled chelate as the internal standards. Hui et al. (5) used tetraphenylporphine (H,TPP)2 for Cu derivatization, and deuterated TPP chelate and 65Cu as the internal standards. Johnson (6) also used Cu-TPP chelate for investigating Cu absorption studies in human subjects. Buckley et al. (7) performed a detailed investigation for Cu isotope ratio measurements using acetylacetone, trifluoroacetylacetone, heptafluorodimethyloctanedione, and 8-hydroxyquinoline. They reported a significant memory effect for the sequential analyses of two Cu samples with 65Cuabundances of 30.83 and 39.98% using these chelating agents. H,TPP was the only chelating agent found suitable for a limited range of isotope ratio measurements. All these studies used a direct probe for sample introduction, which necessitates the prior separation of other isobaric, interfering metals. The use of a gas chromatographic column for sample introduction is attractive since this provides a separation step for different metal chelates. Moreover, the availability of autosamplers in the commercial gas chromatography-mass spectrometry (GC-MS) systems would provide a high throughput of samples. An extensive literature survey showed that there have only been two reports on the GC-MS determination of Cu using H,(enTFA,) and Na(FDEDTC)
2 Abbreviations used: TPP, tetraphenylporphine; enTFA,, N,N’ethylenebis(trifluoroacetylacetoneimine); FDEDTC, bis(trifluoroethyl)dithiocarbamate; NIST, National Institute of Standards and Technology; SIM, selection ion monitoring.
140 All
Copyright 0 1991 rights of reproduction
ooo3-2697191$3.00 by Academic Press, Inc. in any form reserved.
CHROMATOGRAPHY-SPECTROMETRY
chelating agents (8,9). Hachey et al. (8) investigated H,(enTFA,) for Cu isotope ratio measurements using chemical ionization; however, the multiplicity of ion molecule products contributing to the mass peaks of interest limited the accuracy and precision. For Na(FDEDTC), the authors report no data (9) for either precision and accuracy of natural and altered Cu isotope ratios or memory effect. These are the problems which have limited the development of GC-MS methods for trace metals determination and must be investigated. Growing interest in the use of stable enriched isotopes for studying the bioavailability of various trace metals and the easy accessibility of organic mass spectrometers to clinical and biomedical laboratories encouragedus to develop GC-MS methods for the determination of trace metals in biological samples (10). We have previously synthesized lithium bis(trifluoroethyl)dithiocarbamate [Li(FDEDTC)] and used it as a chelating agent for the determination of Ni, Cr, Co, and Pt at the nanogram levels in urine with excellent accuracy and precision and without any appreciable memory effect (11-14). In this paper, Li(FDEDTC) and H,(enTFA,) chelating agents were investigated for Cu analysis in biological samples. Precision and accuracy in determining Cu isotope ratios at the nanogram levels were evaluated and memory effect in measuring altered isotope ratios were studied. Finally, stable isotope dilution GC-MS analysis using enriched 65Cu was validated for the quantitation of Cu in the National Institute of Standards and Technology (NIST) freeze-dried urine reference material SRM 2670 and human serum SRM 909.
EXPERIMENTAL
Instrumentation The mass spectrometer was a double-focusing, reverse-geometry instrument (Model 8230, FinniganMAT, San Jose, CA). The instrument is equipped with a SpectroSystem 300 data acquisition and processing system and a Varian 3700 gas chromatograph with on-column injection. Details of the GC-MS system have been previously described (10). For the present studies, a 10 m X 0.32 mm, nonpolar fused-silica capillary column, DB-1 (dimethylpolysiloxane) (J. W. Scientific, Ranch0 Cordova, CA), with a 0.25pm film thickness was used. Samples were injected at an oven temperature of 100°C followed by a 25”C/min ramp to 300°C. The ion source was heated to a temperature of 200°C and the GC-MS interface was at 280°C. The secondary electron multiplier was at 2 kV. GC-MS data were acquired at 2 Hz, yielding approximately 20 data points across the 10-s wide GC peak. A mass resolution of 1000 was used throughout. The isotope ratios were calculated by integrating the ion current for the various chromatographic
DETERMINATION
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peaks. Details of the measurement been reported previously (10).
methodology
have
Reagents The 65Cu-enriched copper oxide (99.61 atom % of 65Cu) used as an internal standard for isotope dilution was obtained from Oak Ridge National Laboratory (Oak Ridge, TN). Certified Atomic Absorption Standard (copper nitrate in 2% nitric acid) purchased from Fisher Scientific (Fairlawn, NJ) was used as the primary standard for calibration. Double sub-boiling quartz-distilled HNO, in Teflon bottles was obtained from NIST (Gaithersburg, MD). The standard reference materials, freeze-dried urine SRM 2670 and human serum SRM 909, were also purchased from NIST and prepared according to their directions. 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. Deionized water was used throughout these experiments. Although high-quality clean room facilities are not necessary for Cu analysis in biological samples precautions must be taken to prevent adventitious contamination of samples with Cu from the apparatus, reagents, personnel, and the laboratory environment as previously reported (11,12). The levels of Cu, determined by electrothermal atomic absorption spectrometry, in various reagents used were as follows: hydrogen peroxide, 0.5 pg/liter; 4% solution of ammonium hydroxide, 0.03 pg/liter; pH 3 acetate buffer, 0.5 pg/liter. No detectable amounts of Cu were observed in methylene chloride and the chelating agents. An overall background of about 1 rig/sample was present due to the volumes of these different reagents used for digestion and chelate formation.
Preparation
of Chelating
Agents
Chelating agents were prepared using the published procedures. Li(FDEDTC) was synthesized by reacting bis(trifluoroethyl)amine (PCR Inc., (Gainesville, FL) and n-butyllithium (Aldrich Chemical Co., Milwaukee, WI) in an inert atmosphere at -70°C followed by the addition of carbon disulfide (Aldrich) (10,15). H,(enTFA,) was synthesized by mixing 10 ml of trifluoroacetylacetone (Aldrich) and 40 ml of ethanol (Aldrich) and heating to 70°C under argon (16). Then 3 ml of ethylenediamine (Aldrich) was added slowly and the heating continued for 30 min. The solution was cooled and filtered under suction to remove the precipitated white crystalline solid. This was washed with ethanol and recrystallized from ethanol.
142 Preparation
AGGARWAL,
and Standardization
KINTER,
AND
of Spike Solution
A 65Cu solution was prepared by dissolving 65Cu0 in 0.5 M nitric acid with heating. Diluted solutions were prepared on a weight basis from this stock solution for isotope dilution experiments. Isotopic composition of Cu in this solution was determined experimentally by GC-MS analyses of Cu(enTFA,) chelate. The spike solution was calibrated, as reported previously (11,12), by reverse isotope dilution GC-MS using the Cu primary standard and preparing the Cu-chelate. Concentration of Cu in the spike solution was calculated from the mass spectrometrically determined isotope ratios and the weights of the standard and spike solutions taken for mixing (17).
f ;(j&
50
Digestion of Urine and Serum Samples and Chelate Formation A known volume (1 ml) of the reconstituted urine or serum reference material was mixed with a weighed amount of =Cu solution in a Teflon beaker. The spiked urine samples were digested using nitric acid and hydrogen peroxide as previously reported (11,12). The serum samples were first deproteinized using 100 ~1 of concentrated nitric acid added dropwise with continuous vortexing of the solution. Following this addition, the samples were heated on a water bath at 50°C for about 30 min and the precipitated proteins pelleted by centrifugation (18). The supernate was taken for digestion with nitric acid and hydrogen peroxide, as for the urine samples (llJ2). Cu(FDEDTC), was prepared at a pH of 3, using 1 ml of acetic acid and sodium acetate buffer and 100 ~1 of a 20 mM solution of Li(FDEDTC) in deionized water. Cu(enTFA,) was prepared using a 50 mM solution of H,(enTFA,) in methylene chloride by the procedure of Belcher et al. (16). For this procedure, the digested biological extracts were dissolved in 500 ~1 of deionized water, and 2 ml of ethanol was added. The pH of the solution was adjusted to be greater than 11.5 by addition of about 1 ml of ammonia solution. A loo-~1 sample of the chelating agent was added and the solution vortexed and allowed to stand for 5 min. The reaction mixture was then extracted by addition of 1 ml of methylene chloride followed by 2 ml of deionized water to induce phase separation. The organic extract containing Cu(enTFA,) was allowed to evaporate to dryness at room temperature in the laminar flow hood and reconstituted in 20 ~1 of methylene chloride for GC-MS analysis. Spectrometry
The Cu isotope ratios were measured in duplicate by injecting 1~1 of the chelate solution and monitoring the group of peaks corresponding to the molecular ion. Data
,,,,~,,~!,:r ,,,,1;,,,)
100
150
200
250
,,,,,I[
300
350
400
m/z FIG.
Gas Chromatography-Mass
HEROLD
1.
Electron-ionization
mass
spectrum
of Cu(enTFA,).
were acquired in a selected ion monitoring (SIM) experiment, without using a lock mass, and employing voltage peak switching (10). RESULTS
AND
DISCUSSION
The 70-eV EI mass spectra of Cu(FDEDTC), and Cu(enTFA,) exhibit two groups of ions containing Cu isotopic information. These ions are the molecular ion CM)+’ and a fragment ion resulting from the loss of one ligand. The mass spectrum of Cu(FDEDTC), showed an intense peak at m/z 224 corresponding to (CF,CH,),NCS, but it is not useful for Cu isotopic analysis (10). The mass spectrum of Cu(enTFA,) is shown in Fig. 1. The molecular ions, Cu(enTFA),+*, and Cu(FDEDTC)i’ with nominal m/z 393 and 575, respectively, were used for Cu isotope ratio measurements throughout these experiments. The high-intensity fragment ion at nominal m/z 228 in Cu(enTFA,) may be used with this chelate to increase sensitivity in samples containing extremely low levels of Cu. Evaluation of Memory Effect Memory effect in GC-MS analyses of metal chelates refers to the cross-contamination during sequential analyses of samples with varying isotope ratios. In the present studies, memory effect was investigated using four synthetic mixtures prepared by mixing weighed aliquots of the primary standard solution and the %Cu solution in differing proportions but containing almost equal amounts of Cu. These mixtures covered a range from 1.4 to 4.2 of “5Cu/63Cu isotope ratios. Chelates were prepared from these mixtures and replicate analyses (3 or 4), each with 10 ng of Cu, were made for each mixture in the sequence of increasing isotope ratios and then in the reverse sequence. Figures 2 and 3 present the results obtained for m/z 3951393 and m/z 577/575 isotope ratios determined in the mixtures using, respectively,
CHROMATOGRAPHY-SPECTROMETRY
DETERMINATION
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143
Spike Mixture 4
32.0 30.0 28.0
-CT 8
3.0
2 ’
v) i?
Mixture 3
2.5 2.0
Analysis Number FIG. 4. Evaluation of the cross-contamination between samples of widely varying isotopic compositions in consecutive analyses, using H,(enTFA,).
18 19 2021 2223242525272829
Analysis Number FIG. 2. Evaluation yses of samples with
of the cross-contamination altered isotope ratios,
using
in consecutive H,(enTFA,).
anal-
Cu(enTFA,) and Cu(FDEDTC), chelates. As can be seen, no appreciable memory effect is observed for Cu(enTFA,), whereas there is a strong memory effect encountered in using Cu(FDEDTC),. The memory is seen in two ways. First, within a set of analyses (i.e., analyses 14 to 18 of mixture 3), there is a trend toward increasing ratios in the set. This is a result of memory from the analysis of mixture 2. The reverse is seen for analyses 27 to 32, in which the memory of the analysis of mixture 3 is affecting the ratios obtained for the analysis of mixture 2. Memory can also be seen in the average ratios obtained on the ascending vs descending portions
Mixture
4
Analysis Number FIG. 3. Evaluation yses of samples with
of the cross-contamination altered isotope ratios,
using
in consecutive Li(FDEDTC).
anal-
of the figure; the values obtained are not equal. For example, the average for mixture 3 obtained in analyses 14 to 18 is 2.12 + 0.08 (after analysis of mixture 2) but in analyses 23 to 26, the average is 2.53 f 0.03 (after analysis of mixture 4). This is an excellent illustration of the deleterious effects which memory can have on measurements requiring isotope ratios analyses. It is interesting to note that this GC-MS system has already been characterized for the fluorinated diethyldithiocarbamates of Ni (ll), Cr (12), Co (13), and Pt (14) and no appreciable memory was observed. This along with the observation that no appreciable memory is observed for Cu(enTFA,) illustrates the importance of the choice of chelating agent. It is our belief that the availability of suitable chelating agents is the major determinant for the presence or the absence of memory in the GC-MS analyses of metals. In turn, we also believe that memory is the key limitation to the use of these methods. A more rigorous approach was used for H,(enTFA,) chelating agent to determine if memory effect could be observed. This involved the sequential analyses of a solution of natural Cu and enriched 65Cu measuring the ml.2 3951393 isotope ratio. The results are shown in Fig. 4. In these two samples with ratio m/z 395/393 differing by a factor of 60, no appreciable memory effect was seen. It is important to make this evaluation of possible memory effect by comparing the value of the natural Cu ratios before and after analysis of the enriched 65Cu. The ratio of the natural Cu is near the measurement optimim, while the enriched sample, being far from optimum, shows a lack of precision. This rigorous evaluation enhances the confidence in using H,(enTFA,) as a chelating agent for Cu determination by GC-MS in unknown samples. Precision and Accuracy in Isotope Ratio Measurements Table 1 shows the precision and accuracy achieved for isotope ratio measurements of natural Cu using
144
AGGARWAL, TABLE
Precision
and Accuracy of Natural
KINTER,
AND HEROLD
1
TABLE
in Isotope Ratio Determination Cu as Cu(enTFA,)
Determination
of Cu in Urine
Day 1 Day 2 Day 3 Day 4 Mean of means Within-run precision (%) Between-run precision (%) Overall precision ’ ( W) Calculated isotope ratio Difference * (%)
0.4895 0.4928 0.5038 0.4911
k + -+ +
+ SD)
0.0068 0.0058 0.0065 0.0150 0.4943 0.9 1.3 1.6 0.4594 +7.6
(n (n (rz (n
= = = =
5) 5) 4) 5)
a Overall precision S, was calculated by combining the within-run precision (SJ and between-run precision (S,) according to the formula S, = (Sf + S:)“‘. * Difference of the mean of means with respect to the calculated isotope ratio.
Cu(enTFA,) at a lo-ng level. Overall precision in determining isotope ratios was evaluated by performing measurements of chelated natural Cu on different days, with replicate analyses on each day. The mean of means included in Table 1 was calculated by using the mean values obtained on different days. The mean value for each day was used to calculate the standard deviation on the mean of means, referred to as between-run precision in Table 1. The within-run precision was calculated by using
the standard
deviation
values
obtained
on the indi-
vidual days. Overall precision was arrived at by combining the within-run and between-run precision values. This was done to include the effects of any variations in the mass spectrometric operating parameters that may affect the quality of isotope ratio data from one day to another. Overall precision of 1.6% is obtained using Cu(enTFA,). The calculated isotope ratio for this chelate, given in Table 1, was obtained by including the contributions of Cu, C, N, and 0 isotopes (19). A difference of 7.6% in the ratio for Cu(enTFA,) is obtained for the mean of means with respect to the calculated value. It should be noted that this represents a statistically significant bias. We have not been able to explain the source of this bias, so no correction is applied to the measured isotope ratios. However, this bias would be canceled in any isotope dilution experiment in which the internal standard solution is calibrated in the same experiment. The method, therefore, remains a fundamentally sound means of performing isotope dilution.
Results on Urine and Serum Samples The calibrated 66Cu spike was used to quantify Cu in the NIST reference materials, freeze-dried urine SRM
NIST value (fig/liter)
Sample Urine SRM-2670 Human serum SRM-909
370+
and Serum
Concentration of solution (@&/liter) mean + SD (n = 4)
Isotope ratio 395/393 (mean
2
Range &g/liter)
30
410 t 30
370 to 430
1100 + 100
1000 + 30
970 to 1040
2670 and human serum SRM 909. Since the certified concentrations in these samples are provided in the units of microgram/liter by NIST, the samples were taken on a volume basis instead of weight basis. The results are shown in Table 2. The Cu concentrations determined by using H,(enTFA,) are in good agreement with the NIST certified values in the urine and serum samples.
Comparison of Isotope Ratios Determined and ICP-MS
by GC-MS
This GC-MS method was also validated by comparison of the results obtained by ICP-MS. For this purpose, four synthetic mixtures were prepared by mixing primary standard solution and enriched ‘Yu solution as previously described. A comparison of the results for the m/z 3951393 isotope ratios calculated from the YZu/ 63Cu ratios obtained by ICP-MS and those determined by GC-MS is shown in Table 3. The calculated ratios for the m/z 3951393 in Cu(enTFA,) were obtained by using the experimentally determined Y!u/~CU ratios by ICP-MS and including the contributions of C, N, and 0 isotopes. As is seen, a mean value of 1.006 f 0.005 for the measured/calculated ratio is obtained. A regression analysis of 65Cu/63Curatios (x) by ICP-MS (column 2) and m/z 3951393 ratios (y) by GC-MS (column 4) gave an equation y = A + Bx with A = 0.015, B = 1.0025, and a
TABLE A Comparison
of Cu Isotope
by GC-MS
3
Ratios in Synthetic and ICP-MS
Samples
m/z 3951393 Synthetic mixture SM-11 SM-12 SM-13 SM-14 Mean
65cu/~cu by ICP-MS 1.3835 1.8691 2.6115 4.2308
Calculated 1.397 1.883 2.625 4.245
Measured/ calculated
Measured GC-MS 1.407 1.901 2.648 4.241
+ -t k f
0.003 0.020 0.010 0.050
1.007 1.009 1.009 0.999 1.006
+ 0.005
CHROMATOGRAPHY-SPECTROMETRY
correlation coefficient of 1.000. However, a slope of 0.89 was reported by Hachey et al. (8) for Cu(enTFA,) chelate during linear regression analysis of the measured isotope ratios by chemical ionization in the chelate and 65Cu/63Cu ratios. A small constant bias in using CuTPP chelate, with no appreciable memory effect in the limited isotope ratio range, was reported by Buckley et al. (7). They suggested that this may be due to the exchange of Cu in CuTPP with Cu-containing parts of the mass spectrometer. The absence of bias in the present studies (except for the natural Cu experiment) and good agreement in the isotope ratios obtained by GC-MS and ICP-MS enhances the confidence in using H,(enTFA,) chelating agent for Cu. CONCLUSIONS
The results of the present work show that H,(enTFA,) is a better chelating agent compared to Li(FDEDTC) for Cu determination by GC-MS. Results obtained on concentration in the NIST reference materials, freeze-dried urine and human serum, are shown to be accurate and precise. ACKNOWLEDGMENTS The authors thank P. J. Paulsen, J. D. Fassett, and J. R. Moody of NIST (Gaithersburg, MD) for the ICP-MS results; Kathy Winborne for providing the electrothermal atomic absorption spectrometric results; and Patrick K. Anonick and Frank Gordon for assistance in the syntheses of the chelating agents. The authors also thank Professors Savory and Wills for their interest in the present work and allowing use of the facilities available in the trace metals laboratory. Funding for the purchase of the high-resolution mass spectrometer was obtained from the National Institute of Health, Division of Research Resources Shared Instrumentation Grant Program (Grant l-SlORRO-241801). Additional funding from the John Lee Pratt Fund of the University of Virginia is gratefully acknowledged. S.K.A. thanks the Division of Experimental Pathology, Department of Pathology,
DETERMINATION
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IN
145
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University of Virginia Health Sciences Center, for a post-doctoral fellowship and the authorities at Bhabha Atomic Research Center (Trombay, Bombay, India) for granting leave from July 1987 to July 1989 and deputation from June 1990 to August 1990.
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D. M.
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