Anal. Chem. 1980, 52, 1131-1135
1131
High Precision Isotopic Ratio Analysis of Volatile Metal Chelates David L. Hachey,
' Jean-Claude
Blais,2 and Peter D. Klein'
Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439
together with chelate formation, have been used to quantitate nanogram amounts of copper in rat brain (9). Iron kinetics have been studied in animals using "Fe ( I O ) . Hileman has described the use of 65Cufor kinetic studies in animals (11). Chromium levels in urine have been quantitated by IDMS techniques (12). Although GC analysis of volatile metal chelates has been studied extensively and has been reviewed comprehensively ( 1 3 ) ,only a few reports have appeared describing GC/MS studies of volatile chelates.
High precision Isotope ratio measurements have been made for a series of volatile alkaline earth and transition metal chelates using conventional GC/MS instrumentation. Electron ionization was used for alkaline earth chelates, whereas isobutane chemical ionization was used for transition metal studies. Natural isotopic abundances were determined for a series of Mg, Ca, Cr, Fe, NI, Cu, Cd, and Zn chelates. Absolute accuracy ranged between 0.01 and 1.19 at. %. Absolute precision ranged between fO.O1-0.27 at. YO (RSD f 0.07-10.26%) for elements that contained as many as eight natural isotopes. Calibration curves were prepared using natural abundance metals and their enriched 50Cr,"Ni, and 65Cuisotopes covering the range 0.1-10.7 at. % excess. A separate multiple Isotope callbration curve was similarly prepared using enriched 'ONi (0.02-2.15 at. YO excess) and 62Ni (0.23-18.5 at. % excess). The samples were analyzed by GC/CI/MS. Human plasma, containing enriched 26Mg and 44Ca, was analyzed by EI/MS.
EXPERIMENTAL Instrumentation. Isobutane CI mass spectra were recorded at 1 Torr ion source pressure using a Chemetron Medical Products BIOSPECT mass spectrometer. Electron ionization spectra (70 eV) were recorded using a Perkin-Elmer model 270 GC/MS. Quantitative isotope ratio measurements were made using custom-built isotope ratiometers (14) or a similar, commercially available microprocessor based instrument (Chemetron Medical Products, St. Louis, Mo.). The mass spectrometers were operated at an ion source temperature of 13e-180"C, depending on sample volatility. GC/MS analyses were performed using glass columns (1.8 m X 1 mm id.) packed with either 1% SE-30 or 6% SE-30 on Gas Chrom Q (100/120) and extensively silanized. Reagents. l,l,l-Trifluoro-2,4-pentariedione (TFA), 1,1,1,2,2pentafluoro-6,6-dimethylheptane-3,5-dione (PPM), 1,1,1,2,2,3,3heptafluoro-7,7-dimethyloctane-4,6-dione (FOD), and sodium diethyldithiocarbamate (DDC) were obtained from commercial sources. Most chelating agents were used without further purification. Sodium-DDC was recrystallized twice from ethanolethyl ether (1:2). N,N'-Ethylene-bis(trifluoroacety1acetoneimine) (enTFA2)was prepared by established procedures (15). Metal isotopes were obtained from Oak Ridge National Laboratory as 50Cr203(96.80 at. %), 60Ni(99.62 at. YO),62Ni(96.45 at. YO),and "CuO (99.69 at. 701. Stock solutions of @"i, 6'Ni, and W u were prepared by dissolving the enriched materials in nitric acid. The refractory oxide %Cr203was oxidized by sodium peroxide fusion to dichromate, then reduced to Cr3+ with a small volume of sulfurous acid. High purity, redistilled nitric acid was used for preparation of reagent stock solutions and for digestion of biological samples. Preparation of Metal Chelates. Magnesium and calcium chelates (PPM, FOD, respectively) were prepared by refluxing an ethanolic solution of the metal oxide or hydroxide with a threefold excess of chelating agent. Alternatively, these chelates could be prepared by solvent extraction from a strongly basic ammonia solution using 0.1 M chelating agent in toluene or ethyl acetate. Copper and zinc DDC chelates were prepared by ethylacetate extraction of aqueous metal stock solutions treated with 1 mL of 0.006 M NaDDC. Fe(TFA),, Cr(TFAI3, Cu(enTFA2), and Ni(enTFA,) were all prepared by established procedures
T h e essential roles that trace elements play in maintaining good nutrition in man have been extensively documented. Numerous disease states have been identified that result from trace element deficiencies, genetic defects in mineral metabolism, or specific toxic effects of trace elements ( I ) . There are important areas of clinical nutrition that remain largely unexplored, either because ethical constraints dictate against using radioisotopes in certain clinical populations or because inadequacies of existing technology prohibit certain clinical studies. Stable isotopes of metallic elements, although readily available, have not achieved wide use as isotopic tracers in human studies. The reason for their limited use is that their analysis by thermal ionization- mass spectrometry (TI/MS) is tedious, requires extensive sample preparation, and demands expensive, dedicated instruments. Moore and Rosman have reported kinetic studies of calcium metabolism in infants using "%a and *Ca by T I / M S ( 2 ) . Rabinowitz and co-workers have performed elegant studies of lead metabolism in adult males using '04Pb and 207Pb (3). Gastrointestinal absorption of magnesium has been studied using stable =Mg and radioactive 28Mg ( 4 ) . Spark source mass spectrometry has been used to quantitate up to 20 trace elements simultaneously using stable isotope dilution techniques ( 5 ) . Schulten et al. have used field desorption mass spectrometry to quantitate picogram quantities of thallium and lithium in biological samples (6). Isotope dilution mass spectrometry (IDMS) has been used to quantitate chromium and zirconium, as volatile fluorinated P-diketones, in lunar and geologic samples using the direct inlet port of the mass spectrometer ( 7 , 8 ) . IDMS techniques,
(I0-1 2 ) . Digestion of Biological Samples. Human plasma, 2.0 g, was treated with 26Mgand 44Ca and transferred to a clean Teflon beaker. The plasma was dried under an infrared heat lamp, treated with 10 mL of nitric acid, and evaporated under the heat lamp almost to dryness. The process was repeated 4 or 5 times until a colorless or pale yellow solution was obtained. The ash products were dissolved in 2 mL of water. The aqueous solution was saturated with ammonia gas, then extracted with 50 pL PPM in 2 mL of redistilled ethyl acetate. Isotope Ratio Measurements. Natural abundance isotope ratio measurements were made using monomeric ions that were
'Present address: Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas 77030. On leave from the Fondation Curie, Institut du Radium, Paris, France. 0003-2700/80/0352-1131$01 0010
c
1980 American Chemical Society
1132
ANALYTICAL CHEMISTRY, VOL. 52, NO. 7, JUNE 1980
either the parent ion or the base peak in the mass spectrum. Ions used for isotope ratio measurements were Mg(PPM)*,m / z 514 [ML,+];Mg(FOD),, m / z 557 [ML2- 57'1; Ca(PPM)2,m / z 530 [ML,+]; Cr(TFAI3,m / z 512 [MLB+ H+];Fe(TFA),, m / z 516 [ML3 + H+];Cu(enTFA,), m / z 394 [ML + H+] and m / z 411 [ML H2 + F+];Ni(enTFAz),m / z 389 [ML + H+] and m / z 406 [ML - Hz+ F+];Ni(DDCIz, m / z 355 [ML, + H']; and Zn(DDC)*,m / z 361 [MLz + H+]. Isotope dilution curves were prepared for Cr(TFA), using enriched "Cr and natural abundance 52Crcovering the range 0.12-8.5 at. 70excess. Similar curves were prepared using enriched 65Cu(0.20-9.35 at. % excess) and enriched fiONi (0.12-10.7 at. % excess). A separate multiple isotope calibration curve was prepared using @"i (0.024-2.51 at. 70excess) and fi2Ni (0.23-18.5 at. % excess). For natural abundance measurements, all isotopic abundances of a given element were quantitated simultaneously. Three to six measurements were made for each sample for statistical calculation. For calibration curves, only the enriched isotopes and the most abundant natural isotope were quantitated. Sample sizes were 5C500 ng, calculated on the basis of the free metal content. N a t u r a l Abundance Calculations. Fractional isotopic abundance values are calculated from experimentally measured isotopic ratios by solving a set of simultaneous linear equations. Each equation quantitatively describes all isotopic contributors to a given ion ( 1 6 ) and has the general form:
Table I. Isobutane Chemical Ionization Mass Spectra of 2 p g Cu(enTFA,) and Ni(enTFA,)
~
I, =
E
x=i+j
AiX,
(1)
where I , is the relative ion abundance for the xth ion. The unknown fractional isotopic abundance is X,. The relative abundance ( A , ) of the ligand isotopic contributors may be calculated or may be obtained from published mass spectral isotopic abundance tables ( 17). Ligand isotopic contributions are calculated only for elemental formulas corresponding t o the four or five most intense ions. Relative contributions below 0.02% were considered insignificant. The set of simultaneous equations can be expressed in matrix notation as:
I = Ax
(2)
The least squares solution of X can be obtained by simple matrix algebra. The value of X (equation 3) is the product of the inverse of A transpose A(ATA)-', A transpose (AT) and I .
X
(3)
= (ATA)-'ATI
As a practical example, the specific matrix for Ni(enTFA2) is shown (Matrix 1). The ligand isotopic contributions were based Matrix I
'
X 5Y 100 1.312 0.043 0 0
X,, 0 100
14.016 1.312 0
X,., 0 0 100
14.016 0.013
XA,
x,,
0
0
0 0 100
0 0 0 100
1.312
on the elemental formula C12H13N202Ffi, for ulhich the relative isotope contributions calculated were 100% (MI, 14.01670 (M + l), 1.312% (M + 2), and 0.43% (M + 3). Each column in the matrix represents the relative contributions to the ion abundance (Ix)due to the various isotopic species present in the chelating agent for a specific metal isotope (X,).
RESULTS A N D D I S C U S S I O N Mass S p e c t r o m e t r y . E1 mass spectra of alkaline earth P-diketonates reportedly contain polymeric ions in the gas phase (18). Our results support these observations. T h e Ca(FOD), spectrum contains intense ions m / z 1319 [Ca3L4F+], m / z 925 [Ca,L3], and m / z 689 [Ca2L2F+].T h e presence of polymeric ions in the E1 mass spectrum of calcium and magnesium chelates depends strongly on the preparative methods chosen. Preparation of chelates in anhydrous ethanol results in a predominance of polymeric species, because hydration of the cation from metal oxides or hydroxides is
fragment [ML + HI [ M L + H + 171' [ML + H + 381' [(ML), + HIC [(ML), + H + 171" +
Cu(en TFA,) intensity miza c/ob'
Ni(enTFA,) intensity, mlza c/oh
394 41 1 432 787
389 406 427 777 794
20.1 1.2 2.4 100
0.3
804
100 18.5
3.4 7.2
2.1
a miz reported only for the most abundant isotopes, 63Cuand "Ni. Ion intensity reported for the spectrum at the apex of the chromatographic peak. Abundance of dimeric ions is concentration dependent.
minimal. Extraction of the chelate from aqueous solutions a t high p H gives predominantly a monomeric molecular ion a t m / z 530 in the spectrum of Ca(PPM),. T h e presence of polymeric ions in the spectrum of Mg(PPM), is less dependent on preparation methods than in the case of the calcium chelates. The two most intense ions in the spectrum of Mg(PPM), occur a t m / z 457 [ML2- 57'1 and a t m / z 514 [MLz+]. For isotope ratio studies, the most suitable ions were m / z 530 [Ca(PPM),], m / z 557 [Mg(FOD),], and m / z 457 [Mg(PPM),]; the latter two ions represent [ML, - C4H9]species. Under isobutane CI conditions, only ions corresponding to the free ligand were observed in several calcium chelates. Apparently, proton transfer competes effectively for the coordination site of the ligand. The CI spectrum of Mg(PPM), exhibited several intense ions due t o polymeric species and to fluorine attachment ions. Important ions in the isobutane spectrum were m / z 515 [ML2H+],m / z 534 [ML,FH+], m / z 760 [M2L3H+l,m / z 779 [M2L3FH+], m / z 760 [M2L3H+], m/z 779 [M2L3FH+],and m / z 1029 [M2L4H+]. Whether these polymeric ions form in the gas phase or occur in solution is uncertain, but we suspect that gas phase polymerization prodominates. Thus chemical ionization is of limited use for isotopic tracer studies involving alkaline earth chelates. EI/MS studies of transition metal TFA, DDC, and enTFAz chelates have been reviewed (19). The isobutane and methane CI mass spectra for a series of transition metal 2,4-pentanedionates and TFA chelates have been published (20,21). Our data generally support the published spectra, with the only notable exception occurring in the isobutane CI spectra of fluorinated chelates, where an ion 17 amu higher than the protonated molecular ion is frequently observed. This ion probably results from attachment of fluorine to the molecular ion, followed by loss of hydrogen. Isobutane CI spectra of Cu(enTFA,) and Ni(enTFA,) obtained by GC/CI/MS are simple, as shown in Table I. T h e spectrum of Cu(enTFA,) has both monomeric ions, a t m / z 394 [ML + H+] and m / z 411 [ M L + H + 17'1, as well as dimeric species a t m / z 787 [M2L2 H+] and m / z 804 [M,L2 + H + 17'1. T h e spectrum of the nickel chelate is similar. Attempts t o prepare Zn(enTFA,) were unsuccessful. T h e mechanism for addition of 17 amu to give ions a t m / z 411 and m / z 804 is unknown, but their addition may represent attachment of fluorine and loss of hydrogen. Formation of dimeric ions in the gas phase is more pronounced for Cu(enTFA,) than for the nickel chelate. T h e enTFA2 chelates of Cu and Ni and Cr(TFA)3show no tendency to exchange ligands and thus they are well suited for isotopic trace studies. N a t u r a l Isotopic A b u n d a n c e Measurements. Natural abundance isotope ratio measurements were undertaken to study the precision and accuracy of the analytical methodology. Volatile metal chelates used for isotopic tracer studies show increased complexity of the natural metal isotope pattern
+
ANALYTICAL CHEMISTRY, VOL 52, NO. 7, JUNE 1980
Table 11. Isotopic Abundances Obtained from a Series of Alkaline Earth p-Diketonates '3 abundancea
chelate
isotope
Mg(PPM),
24Mg 25Mg 26Mg 24Mg
Mg(FOD),
Ca(PPM),'
found
78.44 t 10.92 t 10.64 t 78.71 r 25Mg 1 0 . 6 0 t 26Mg 10.69 40Ca 96.87 + ",Ca 0.96 i"Ca 2.18 f
difference'
0.06 ( n = 4 ) 0.09 0.05
0.27 ( n = 5 ) 0.19 0.15 0.07 ( n = 5 ) 0.06 0.04
0.26 -0.79 0.53 -0.01
-0.41 0.48 0.42 -0.32 -0.11
Corrections for ligand isotope contributions were based on the elemental formulas Mg(PPM), = C,4Hl,F,o04, Mg(FoD)2 = C 1 6 H l i F 1 4 0 4 3 = C18H~OF1004 (25)' Difference between the established natural abundance and the experimentally determined values. N o attempt was made to quantitate minor isotopes (
