Anal. Chem. 1994,66, 856-860
Determination of Natural and Isotopically Enriched Chromium in Urine by Isotope Dilution Gas Chromatography/Mass Spectrometry Claude Veiiion' and Kristine Y. Patterson Beltsville Human Nutrition Research Center, Vitamin and Mineral Nutrition Laboratory, Unlted States Department of Agriculture, 1 17 Building 307, Beltsvllle, Maryhnd 20705 Michelle A. Rubin and Phyiis B. Moser-Veiiion Department of Human Nutrition and Food Systems, University of Maryland, College Park, Maryland 20742
A method is described for the determination of chromium and its enriched stable isotopes in human urine by isotope dilution mass spectrometry. A volatile chelate is formed with trifluoroacetylacetone (TFA) and the fragment ions corresponding to Cr(TFA)*+in the 356-360 m/zregion are monitored. The chelate is thermally stable and exhibits no memory effects when isotope ratios change. The detection limit for the method is 0.03 ng of Cr/g, and the accuracy is verified by certified reference materials and by an independent method. The method is highly specific for chromium, due to the combined properties of the chelating agent, chromatographic column, and massspecific detector. In additionto total chromiumdeterminations, the method can also be used to quantitate enriched stable isotopes of chromium used as metabolic tags in tracer experiments in human nutrition studies.
Chromium is recognized as an essential trace element in the human diet, and its primary function appears to be associated with glucose metabolism and/or insulin response. The dietary/metabolic history of chromium dates back to 1959, when Schwarz and Mertz proposed "glucose tolerance factor" (GTF) as the active form of chromium in foods' and Wacker and Vallee noted a high concentration of chromium in isolated nucleic acids2 Interestingly, in the intervening 33 years, GTF has not yet been isolated and characterized (although one can purchase it in health food stores!). Perhaps the best first evidence of the essentiality of chromium is the study of Jeejeebhoy et al., who successfully reversed insulinrefractory diabetes in a patient on total parenteral nutrition (TPN) with chromium ~upplementation.~ The analytical chemistry of chromium during this period is a good example of why biological reference materials with certified trace element content are essential to this and related fields. The levels of chromium in readily accessible human samples, such as serum, plasma, and urine, are usually well below 1 ng/g, and often much closer to 0.1 ng/g. At these concentration levels, perhaps only three analytical methods have sufficiently low quantitation limits for the determinations: neutron activation analysis (NAA), mass spectrometry (1) Schwarz, K.; Mertz, W. Arch. Biochem. Biophys. 1959. 85, 292-295. (2) Wacker, W. E. C.; Vallee, B. L. J. Biol. Chem. 1959, 234, 3257-3262. (3) Jeejeebhoy, K. N.; Chu, R. C.; Marliss, E. B.; Greenberg, G. R.; BruceRobertson, A. Am. J. Clin. Nutr. 1977, 30, 531-538.
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(MS), and graphite furnace atomic absorption spectrometry (GFAAS). The first two are not widely available, and the latter is the most susceptible to matrix interference effects. By 1978, it had become evident that the determination of chromium in biological samples had serious problems. Most of the literature values were obtained by GFAAS, and values reported for the same or similar samples differed by several orders of magnitude. This was especially disconcerting for urinary chromium values, since urinary chromium excretion had been proposed by Mertz in 196g4 as an indicator of chromium nutritional status. Most of the reported values for urinary chromium were in the 2-20 ng/g range, which was at odds with the -0.5% dietary chromium absorption figure found by Doisy5 in radiotracer experiments. Daily urinary chromium excretions of, say, 10 pg (e.g., 10 ng/g urinary chromium concentration X 1000 g of urine per day) meant that the dietary intake had to be 2 mg of chromium (at an absorption of 0.5%) and yet no reasonab1eU.S. diet can supply much more than 100 pg/day. In 1978, Guthrie et a1.6 demonstrated that the GFAASs in use at the time and employing deuterium lamp background correction were only measuring background absorption, due to the low lamp intensity at the chromium wavelength. This cast serious doubt on the validity of all previous urinary chromium values reported by GFAAS. At about the same time, several developments in instrumentation began to show that urinary chromium levels were in fact well below 1 ng/g. This was accomplished with wavelength mod~lation,~ highintensity background correction lamps? and modified furnace conditions to reduce background a b s ~ r p t i o n .These ~ new GFAAS values were confirmed by an independent method, namely, isotope dilution mass spectrometry.1° The use of a volatile chelate for measuring chromium by combined gas chromatography/mass spectrometry (GC/MS) was pioneered (4) Mertz, W.Phys. Rev. 1969, 49, 163-203. (5) Doisy, R. J.; Streeten, D. H. P.;Souma, M. L.; Kalafer, M. E.; Reliant, S. I.; Dalakos, T.G. In Newer Trace Elements in Nutrition; Metrz, W., Cornatzer, W. E., Eds.; Marcel Dekkcr: New York, 1971; pp 155-168. (6) Guthrie, B. E.; Wolf, W. R.; Veillon, C. Anal. Chem. 1978, 50. 19OC-1902. (7) Hardy, J. M.; OHaver, T. C. Anal. Chem. 1977, 49, 2187-2193. (8) Kayne, F. J.; Komar, G.; Laboda, H.; Vanderlindc, R. E. Clin. Chem. 1978, 24, 2151-2154. (9) Routh, M. W. Anal. Chem. 1980, 52, 182-185. (IO) Veillon, C.; Wolf, W. R.; Guthrie, B. E. Anal. Chem. 1979, 51, 1022-1024.
0003-2700/94/0366-0856$04.50/0
0 1994 American Chemical Society
earlier by Wolf et ala," who also reported vapor pressure measurements and thermodynamics of various metal 6-diketonates.12 Wolf also investigated combined GC/AAS for chromium determinations with volatile ~helates.13-1~ Isotope dilution mass spectrometry (IDMS) can also be used to measure enriched stable isotopes of chromium, which can be used as tracers in human metabolic studies. We describe herein a method for quantitating these enriched isotopes in human urine samples, which has the additional advantage of reducing errors caused by sample contamination with unenriched (natural) chromium. EXPERIMENTAL SECTION Overall Procedure. Samples are spiked with a known quantity of enriched stable isotope (W2r) and then acid digested, and the chromium is chelated as the trifluoroacetylacetone (TFA) derivative. The Cr(TFA)3chelate is extracted into hexane for introduction into the mass spectrometer via a gas chromatograph, and the ions corresponding to Cr(TFA)2+ (the most intense fragment) are monitored. From the isotope ratios, one calculates the amount of natural (unenriched) chromium in the sample, as well as the amounts of any enriched chromium isotopes used as tracers and present in the sample. Total chromium in the sample is the sum of these and was verified both by analyzing certified Standard Reference Materials (SRMs) and by an independent method (GFAAS with Zeeman-effect background correction). Instrumentation. The GC/MS system employed a quadrupole mass spectrometer (Model 4000, Finnigan MAT, San Jose, CA) coupled to a gas chromatograph (Model 9610, Finnigan) and equipped with a programmable multiple ion monitor (PROMIM) so that the desired number of isotopes could be monitored simultaneously. The GC used a 3-m silanized glass column (2-mm i.d.) packed with 1% SP-2401 on loo/ 120-mesh Chromasorb 750 (Supelco, Inc., Bellefonte, PA) interfaced to the electron impact (EI) ion source through a glass jet separator. Operating conditions were as follows: injector 165 OC; column 130 OC; separator and transfer line 175 OC; helium carrier gas flow rate 20 mL/min. Reagents. Enriched stable isotopes of chromium were obtained from Oak Ridge National Laboratory (Oak Ridge, TN) as Cr2O3 and dissolved as previously described.10 Standard solutions of chromium were obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD) as SRM 3 112 and diluted as required. These were used to calibrate the enriched stable isotope solutions by reverse isotope di1uti0n.l~ Deionized reverseosmosis water (18 MQ resistivity) was used for all dilutions and rinsing. TFA (Eastman Kodak Co., Rochester, NY) was purified by double low-temperature, low-pressure distillation16 and stored at -20 OC in amber glass bottles that the reagent was originally supplied in. HPLC-grade hexane (Fisher (11) Wolf, W. R.; Taylor, M.L.; Hughes, B. M.;Tiernan, T. 0.;Sievers, R. E. Anal. Chrm. 1972, 44,616-618. (12) Wolf, W. R.;Sievers,R. E.;Brown,G.H. Inorg. Chem. 1972,11,1995-2002. (13) Wolf, W. R. Anal. Chem. 1976,48, 1717-1720. (14) Wolf, W. R. J . Chromarogr. 1977, 134, 159-165. (1 5 ) Patterson, K. Y.;Veillon, C.; Moser-Veillon, P. B.; Wallacc,G. F. And. Chim. Acta 1992, 258, 317-324. (16) Veillon, C.; Patterson, K. Y.;Bryden, N. A. Anal. Chim. Acta 1984, 164, 67-76.
Scientific, Pittsburgh, PA) was used without further purification, Hydrochloric, nitric, and acetic acids were subboiling distilled (Seastar Chemical, Seattle, WA), and ammonium hydroxide was prepared by isothermal di~ti1lation.l~ The pH 5.7 ammonium acetate buffer used for the chelation was made from the ammonium hydroxide and acetic acid, and the pH adjusted with either as needed. For use in sample digestion, several lots of hydrogen peroxide (30%) from several sources were tested by GFAAS in order to find one with the lowest amount of chromium contamination (this varies with manufacturer and lot number, so no specific recommendations can be made). For removal of excess chelating agent after chelation, reagent-grade 0.1 M NaOH was used; the purity of this reagent is not critical since TFA reacts very slowly with chromium at room temperature. Magnesium nitrate was used as an ashing aid, taken from a solution of 0.186 g of Mg(NO3)~6H20(Puratronic grade, Johnson Matthey Chemicals, Ltd.) dissolved in 1 mL of water Using these reagents and exercising strict contamination control measures, it was possible to keep the overall chromium blank for the procedure below 0.2 ng. This blank was almost entirely "reagent" blank, and as such was quite reproducible for a given set of reagents. Digestion and Chelation. All sample preparation procedures, including digestion, were carried out in class-100clean areas. Urine samples, usually 3 g, were accurately weighed into acid-cleaned, silanized quartz tubes.16 To this was added an accurately weighed amount of the enriched 50Cr internal standard (spike) and 50 pL of the magnesium nitrate solution. Tubes were placed in multihole aluminum heating blocks (Multi-Blok Heater, Lab-Line Instruments, Inc., Melrose Park, IL) maintained at 90 OC and heated to dryness (usually overnight). When dry, samples were allowed to cool, 100 pL of concentrated nitricacid was added, and the resultant mixture was returned to the heating block (swirl gently until any foaming subsides). When nearly dry, 50-pL aliquots of hydrogen peroxide were added at 2-h intervals until the residue was white (occasionally additional nitric acid was necessary). When white and dry, sample residues were dissolved in 50 pL of 6 M HCl and taken to dryness again. Cooled sample residues were dissolved in 50 pL of 1 M HCl, and 1 mL of 1 M ammonium acetate buffer (pH 5.7) and 100 pL of purified TFA were added. Sample tubes were then tightly capped with polyethylene stoppers and heated at 70 OC for 1.5 h. After cooling, the Cr-TFA chelate was extracted with three 0.5-mL aliquots of hexane, which were combined, and the excess TFA extracted from this hexane with 1.5 mL of 0.1 M NaOH for 40 s with vortexing. The hexane layer was quickly separated from the base wash solution, placed in a small screw-cap vial, and allowed to evaporate. The residue was redissolved in 25 pL of hexane and the vial capped for subsequent analysis. Vial caps sealed with unpunctured, Teflon-faced rubber septa prevent evaporation of the hexane before samples are analyzed and are convenient to open and close. Analysis. In the mass spectrum, ion peaks are seen for the parent ion, as well as various fragment ions. The most intense ion cluster is that of Cr(TFA)2+, where one TFA ligand has
.
(17) Veillon, C.; Reamer, D. C. Anal. Chcm. 1981,53, 549-550.
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been lost. This cluster appears at m/z 356,358,359, and 360 amu's, corresponding to 5oCr(TFA)2+,52Cr(TFA)2+,W r (TFA)2+, and 54Cr(TFA)2+,respectively. Four of the GC/ MS PROMIM channels were tuned to these masses by use of unenriched (natural) Cr-TFA chelate dissolved in hexane. The maximum availablesampling (dwell) time of 100 ms was used for each channel, since the peaks are several seconds wide under the operating conditions used. The nonsymmetry of the TFA ligand means that Cr(TFA)2+ occurs in both cis and trans forms, and one indeed observes twochromatographic peaks. These are well separated on the SP-2401 column, and the more abundant trans form emerges first, followed by the much smaller cis peak. All measurements of ion intensities and experimental isotope ratios were made using the trans peak. The "response" of the quadrupole MS is not perfectly linear over even this narrow mass range, due to slight mass dependence of quadrupole transmission, detector ion conversion, etc., so small correctionsneed to be applied to the observed isotope ratios to reconcile them with the expected or theoretical isotope ratios. These corrections are small in comparison to the larger, but completely predictable, ones necessary for the isotopic contributionsfrom carbon, hydrogen, and oxygen in the TFA in arriving at expected isotope ratios. The latter corrections are covered in detail in refs 10 and 18. RESULTS AND DISCUSSION
Table 1. Atom Parcent " d a m a s of Natural Cr and Erwkhod I s o t o p s of Cr
isotope
natural CP
enriched Wrb
enriched Wrb
50 52 53 54
4.3452 83.7895 9.5006 2.3647
96.79 2.98 0.18 0.05
0.027 1.665 98.230 0.077
a
*
From ref 19. Oak Ridge National Laboratory.
Table 2. Theodcal Parcent Aknrdamas of tho Isotopm of Cr Adjwted for the Cr(TFA)*+ Chdate
ion mass (m/z)
natural Cr
enriched Wr
enriched Wr
356 358 359 360
3.8472 74.23810 16.774 4.0856
85.76 3.81
0.024 1.474 87.138 9.891
0.46 0.10
16 15 14 13 12 11
in
Injection #
'9
8 7
5 4 3 2 1
Isotope Dilution Analysis. A primary assumption in isotope dilution analyses is that, at some point and before any analyte element is lost or gained, the spike (internal standard) chemically equilibrates with the analyte. Once this occurs, quantitative recovery is no longer required for an accurate determination. The best way of assuring this equilibration of spike and analyte is to "destroy the chemical history" of the sample by, for example, complete digestion to an inorganic solution. This is especially true for biological samples, because the analyte is often bound to or part of some organic species, which may behave quite differently from an added inorganic spike in subsequent sample processing. More importantly, this method allows one to quantitatively measure other enriched isotopes present in the sample, providing one with nonradioactive tracers that can be used, for example, in metabolic studies involving children, pregnant women, and lactatingmothers, to name a few groups for which radiotracer studies are now essentially impossible. The spike constitutes an ideal internal standard, since it is chemically identical to the analyte. Isotope dilution analysis is an "absolute" method, in that one does not have to calibrate instrument response against standards but merely measure isotope ratios. On the other hand, the accuracy of the method depends very heavily on the accuracy of the single spike addition, but since this can be calibrated by reverse isotope dilution, many of the uncertaintiesare canceled out. Perhaps the greatest strength of IDMS methods is that essentially all of the sources of error are predictable and measurable. Shown in Table 1 are the atom percent abundances for natural chromium (from ref 19 ) and for an individual lot (18) Frew, N.M.; k r y , J. J.; Isenhour, T.L. Ancrl. Chem. 1972,44,665-671. (19) Holden, N. E.; Martin, R. L.; Barnes, I. L. Pure Appl. Chem. 1983,55,11191136.
0
0.2
0.4
.
0.6
.
.
0.8
-
1
1.0
-
-
'
1.2
m/z 3561358 Ratio (50152) Flgure 1. Measured isotope raw of successive injectkms of natural chromium(1-5and 12-16)andnaturalchromiumspiked withenriched 60Cr (7-1 1).
each of 50Cr and W r obtained from Oak Ridge National Laboratory, indicating a high degree of enrichment above natural levels. In Table 2, the theoretical or expected abundances are shown, now taking into account the contributions from isotopes of carbon, hydrogen, and oxygen in the Cr(TFA)2+ molecular ion. Substantial changes are noted, especially for "M + 1" ions, which is not too surprising when one considers that this ion contains several carbon atoms, of which over 1% of each is I3C. As mentioned earlier, these corrections are entirely predictable. Stability of Cr-TFA Chelate. When volatile chelates are used to introduce metals into mass spectrometers via gas chromatographs, it is important that the chelate be stable.20 It should not decompose significantly,although this requirement is not essential in isotope dilution measurements. More importantly, it must not exchange its analyte element for another atom of the same element. To do so would alter the ratios of enriched isotopes, and this is easily verified. Likewise, the system should be free of "memory" effects, where the isotope ratio is affected by previous samples with different ratios. The trivalent chromium-TFA chelate is very well behaved in these regards. This is demonstrated in Figure 1, where five successive injections of natural chromium (50/52 =0.05) are followed by five injectionsof a sample enriched in (20) Aggamal, S. K.; Kinter, M.;Wills, M.R.;Savory, J.; Herold, D. A. Anal. Chem. 1990,62, 1 1 1-1 15.
1.5
T1#. 9. Comparbon d hAytkal Values to Cu#fkatr V . l w r for NIST 8RY 2670 Toxk M o t h In F r w z r 9 r k d Urine Cr concn (ng/mL)
normal elevated
n
experimental
6
11 0.1” 84*2a
certificate
*
3
86
* 6c
Mean & standard deviation. b Information value (not yet certified). c Certified value uncertainty.
*
a
EE
1-
8
w
0.5
Tabh 4. Comparison d Ullnary CI Values by QWMS and Zooman Qraphko Fumace Atomk AbrorpHon 8p.ctromotry
urine sample
GC/MS (ng/g)
A B
0.30 0.23 0.30 0.20
C
D
Cr concn Zeeman GFAAS (ng/mL) 0.30 0.22 0.29 0.23
50Cr(50/52 = 1,2),followed again by five injections of natural Cr. Analytical Results. Results for urinary chromium were verified by two methods: analysis of certified reference urine and analysis of human urine samples by an additional, independent method (Zeeman GFAAS). SRM-2670, Toxic Metals in Freeze-Dried Urine (NIST, Gaithersburg, MD), was analyzed for chromium, and the results are shown in Table 3. The “normal” sample has an “information” value that is not yet certified. The term normal for this reference material is misleading in that the value of 13 ng of Cr/mL is -50 times higher than the concentrations found in normal human urine.21 To successfully quantify chromium in human urine, detection limits of C0.05 ng of Cr/mL are needed. The detection limits calculated for this method, defined as three times the standard deviation of the blank, and for 3-mL urine samples, are 0.03 ng of natural Cr/g (n = 10 blanks) and 0.02 ng of 53Cr/g (n = 23 blanks). Four 24-h urine samples from apparently healthy subjects were collected and analyzed by both GC/MS and Zeeman GFAAS. Results are presented in Table 4, and these values are typical of North Americans not taking chromium supplements.22 The Zeeman GFAAS method23employsstandard additions with no pretreatment of the urine samples; thus, the two methods are independent. It should be pointed out that samples (as well as reagents and standards) are normally aliquoted by weight, due to increased speed, accuracy, and precision obtainable with modern top-loading balances (perhaps the more correct term now would be scales). Earlier references often list concentrations on a volume basis, but since the specific gravity of urine is only about 1-2% greater than water, it is not unreasonable to compare values expressed on either a volume or weight basis. Measurement of Enriched Stable Isotopesin Urine. In order to estimate how long an oral dose of an enriched stable isotope of chromium could be followed in human urine, a volunteer (21) Dub, P.AM/ySt 1988, 113,917-921. (22) Andemn, R.A.; Polansky, M.M.;Brydcn, N. A,; Patterson, K.Y.;Veillon, C.; Glinsmann, W.H.J. Nurr. 1983, 113, 276-281. (23) Veillon, C.;Patterson,K.Y.;Bryden, N. A. Clin. Chcm. 1982,28,2309-2311.
1
--
I
I
I
72
gS
z
0
I
0
24
I
48
120
4
I 144
Hours after 53Cr Dose Flgurr 2. Urinary excretionof enriched %r ( 2 4 4 collections)following an oral dose of 200 pg of %r as the chlorkle in water.
was given an oral dose of 200 pg of enriched 53Cr (as CrCl3) in water, and 24-h urine collections were made for 10 days following the ingestion of the tracer. The urine samples were analyzed for both natural chromium and the enriched 53Cr metabolic tag. The natural chromium was essentiallyconstant, at a level below 0.3 ng of Cr/g for all samples. The enriched 53Crtracer was highest in the first 24-h collection (- 1.3 pg/ day), rapidly dropping to a level of 0.1 pg/day. Beyond day 6, the level fell below the quantitation level of the method (Figure 2). The cumulative excretion of the tracer during the first 6 days is only -2 pg, or 1% of the oral dose. This is in keeping with the well-known poor absorption of this essential trace element.5
-
CONCLUSIONS Isotope dilution analysis has some significant advantages for accurately measuring trace elements in general and enriched stable isotopes in particular. The methodology used here has been shown to be quite robust over the years, and one might be tempted to compare it to newer techniques such as inductively coupled plasma mass spectrometry (ICP-MS), The latter may someday have an advantage of speed over this method, when the isobaric interferences at some of the chromium isotopes are overcome. With the TFA chelate, this method operates in the m/z region near 360 amu, where background and interferences are virtually nonexistent. Other volatile metal chelate methods, such as GC-AA, have never proved very successful,because of the necessity of quantitative recovery throughout the various steps, which an absolute method with an ideal internal standard like isotope dilution does not require. Errors in the method are predictable and measurable, and the spike is calibrated by reverse isotope dilution on the same instrument used to measure the analyte, resulting in a high level of accuracy. Specificity of the method is alsovery high, due to thecombined propertiesof thechelating agent, gas chromatograph, and mass spectrometer. For metabolic studies of trace elements in humans using enriched stable isotopes, the method has additional advantages. The primary advantage is nonradioactivity of the tracer, and the fact that the enriched tracer can still be quantitated in the Analytlcel Chemkrv, Vol. 66, No. 6, Mer& 15, 1994
819
presence of varying levels of sample contamination with natural, unenriched analyte. For urinary chromium measurements, the method described here is shown to be capable of measuring urinary chromium at normal concentrations, as well as enriched isotope tracers inurine. The Cr(TFA)s chelateisquitestableandanexcellent choice for GC/MS analysis for chromium. The accuracy of the technique has been established with both SRMs and by a second, independent method, namely, Zeeman GFAAS.
ACKNOWLEDGMENT Specific manufacturer's products are mentioned herein solely to reflect the personal experiences of the authors and do not constitute their endorsement nor that of the Department of Agriculture. Received for revlew September 8, 19Q3. Accepted December 17, 1993.' Abstract published in Aduuncr ACS Absfrucrs, February 1, 1994.
