1334
Anal. Chem. 1986, 58,7334-1340
Inductively Coupled Plasma Mass Spectrometry Applied to Isotopic Analysis of Iron in Human Fecal Matter Bill T. G. Ting and Morteza Janghorbani* Department of Pathology (Nutrition), Boston University School of Medicine, M1008, Boston, Massachusetts 02118
Inductively coupled plasma mass spectrometry combined with stable isotope dilution Is applied to accurate Isotopic analysis of human fecal matter for 54Feand "Fe. Argon plasma generated Interferences are of minor concern. The interference from 54Cr can be corrected instrumentally, whereas '*Ni must be removed chemlcaily. The ratio of the stable isotopes of interest can be measured routinely with a relative standard deviation of about 1%. The overall accuracy of the method for quantitative Isotopic analyses is evaluated in Standard Reference Material (SRM) 1577a (Bovine Liver), fecal homogenate subsampies, and synthetic solutions of iron. For SRM 1577a, the respectlve comparisons are (pg/g) 192.2 f 2.2 (present method) vs. 194 f 20 (certifled value). For the fecal matrix, the present method yields (pg/mL) 15.14 f 0.36 vs. 15.82 f 0.48 based on atomlc absorption spectrophotometry. For an iron solution (250 ppm), repilcate analyses yleld the value of 245.4 f 1.5 PPm*
Studies of iron bioavailability in infants have not been carried out extensively because such studies require application of isotopic techniques (1). The accepted isotopic methods, widely employed in studies with adults, involve application of radioiron (56Fe/69Fe)(2). Application of radioisotopes in studies with infants is, at present, not permitted in many institutions and, in general, poses serious ethical issues. For these reasons, and in spite of the importance of these issues to nitritional well-being of infants, little accurate experimental data are available to serve as the basis for judicious judgement on iron requirements of infants and supplementation of infant foods (3). Much effort has been expended in the recent past to develop suitable tracer methods based on the nonradioactive stable isotopes of iron (1,4-8). These methods have, in general, not been successful (1). The reasons for the difficulty relate to lack of availability of suitable isotope measurement methodology and have been discussed in detail previously (1, 9). Recently, the method of inductively couplied plasma mass spectrometry (ICP/MS) has been applied to this problem (9). It has been established that the most critical measurement requirement, viz., achievement of sufficient measurement precision in the determination of isotopic ratio for 5sFe/57Fe in infant's blood following oral administration of a physiologically acceptable dose of 5sFe (-1 mg), can be met with this new technique. Thus, the broad area of iron nutriture of infants can now be explored with a safe, nonradioactive isotopic method. Application of the hemoglobin incorporation method to studies of iron absorption requires knowledge of the extent of incorporation of the absorbed isotopic label into hemoglobin (10). In the healthy adult with normal iron stores, several groups have shown that this is an acceptably constant value (11,12); 8045% is now taken as the accepted factor. In the normal adults without iron stores, but normal erythropoietic apparatus, incorporation is complete (11,12). On the other 0003-2700/88/0358-1334$0 1.5010
hand, many disease states result in dramatic reduction of this value (11,12). Therefore, application of the hemoglobin incorporation method to iron absorption studies in such physiologic states is obviously not a suitable approach. Little is known about the quantitative aspects of hemoglobin incorporation in infants. The two studies available, which are based on radioiron, indicate a wide range (10,13). Therefore, it is not clear at this time whether and over what age range the hemoglobin incorporation method can serve as a sound methodologic base for the measurement of iron availability in infants. This important problem can be resolved readily by the combination of isotope balance (1) and hemoglobin incorporation methods, if the dose of labeled iron is absorbed sufficiently (>lo%) to permit reasonably accurate estimation of its absorption from fecal isotope excretion data (I). This can be done by administration of the label in the ferrous (Fe2+) form (2, 9). However, in order to carry out such a study successfdy, it must first be shown that quantitatively accurate measurements of absolute amounts of the relevant stable isotopes of iron can be made in infants' stools. This has not been shown previously. We have addressed this issue, viz., accurate analysis of selected stable isotopes of iron in fecal matter, employing the new method of ICP/MS and isotope dilution analysis (IDA). Our results are reported in this paper.
EXPERIMENTAL SECTION 1. Instrumentation. The ICP/MS employed in these studies was an Elan Model 250 system (SCIEX, Thornhill, Ontario, Canada). It was operated in the isotope ratio mode. The nebulizer employed here was Meinhard concentric glass type, TR-30C (Meinhard Associates, CA). Sample solution was aspirated into the argon plasma via a peristaltic pump (Rabbit, Rainin Instrument Co., Inc., Woburn, MA). The flow rate was in the range 1.5-1.8 mL/min. All isotropic analyses were carried out in the peak switching mode, with three sequential ion collections for each mass spectral peak. The dwell time for each ion collection was set at 3 s. Typically, on any one solution, 10 sequential isotope ratio measurements were made and, the mean It l a was recorded as a single data point. These could be repeated as many times as was desired. The instrument settings used in this study were as follows: rf power, 850 W; nebulizer pressure, 37 psi; lens settings (ring/ B/A/C), 15/51/95/50. The instrument was operated in the high-resolution mode. The distance from load coil to the sampler was 27 mm. Prior to each experiment, we typically adjust these instrument settings to first provide maximum intensity for 56Feemploying a 1 ppm iron solution and repetitive scanning over the 56 mass. Subsequently, we readjust these parameters to obtain as reproducible a mass peak as judged necessary. This often involves a compromise between maximum ion beam intensity and mass peak reproducibility. The instrument employed for atomic absorption measurements was a Perkin-Elmer Model 5000 system (Perkin-Elmer Corporation, Norwalk, CT). All measurements were carried out with the standard flame operating conditions as recommended by the manufacturer. 2. Chemistry. All reagents used were analytical grade purchased from commercial vendors and used without further pu0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
J
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rification. Enriched stable isotopes of iron were purchased from Oak Ridge National Laboratory, Isotope Division, Oak Ridge, TN. These were either as Fe203or especially prepared elemental iron shipped under inert atmosphere. Ferric oxide preparations were dissolved in aqua regia and diluted to provide working stock solutions. 51Fe-spiked calibration solutions were prepared by appropriate addition of the 57Fe-enrichedstock solution to iron standard solutions (Stock Standard Solution, 1000 ppm Fe, EM Science, Cherry Hill, NJ). The resultant mass isotope ratios ( M R ) were calculated based on the calculated concentrations of the stock solutions, the known natural abundances of stable isotopes (14) converted to weight basis, and the certified isotopic composition of the enriched isotopes supplied by the vendor and converted to weight basis. All wet oxidations were carried out according to our usual practices employing the method of HN03/H202(15). Storage and sampling of the SRM 1577a was carried out as per recommendations contained in the certification sheet (16). Fecal homogenate5 were prepared by blending appropriate portions of fresh feces with deionized water in a modified home blender equipped with a side stopcock to permit continuous sampling while blending. Because of the expected major isobaric interferences due to 68Ni,several variants of a separation scheme were tested as given in Scheme I. Following these evaluations, the complete analytical scheme given in Scheme I1 was devised.
RESULTS AND DISCUSSION 1. Choice of Isotopes. There are four stable isotopes of iron (9). Of these, 5sFe (natural abundance 0.322 wt %) is most suitable as the in vivo spike (9). 56Feis not a suitable reference isotope because at concentrations required to achieve satisfactory ion intensities for minor abundant isotopes, 56Fe intensities will drive the system into nonlinearity. Iron-54 (natural abundance 5.605 wt %) serves well for this purpose. Iron-57 (natural abundance 2.183 wt %) then serves as the in vitro spike. An available 57Fe-enrichedpreparation (Oak Ridge National Laboratory, Isotope Division, Oak Ridge, TN) possesses the following isotopic composition (all in wt %): "Fe 0.304, =Fe 13.32, 57Fe86.28, and W e 0.102. The atomic weight of iron in this preparation and its calculated gravimetric factor (preparation is as Fe203)are 56.792 and 0.7029, respectively. Isotope abundance is usually expressed as atom %. In contrast, all investigators dealing with studies of iron in man universally employ the mass scale. This is also the case for studied of mineral metabolism for other minerals (1). Thus, measured isotope ratios must be eventually converted to isotope excess expressed on the mass scale (e.g., micrograms of 58Fe excess recovered in feces). While the conversion to
the mass scale could be performed after determination of atom 570 excess, manipulations on the atom % scale are awkward for researchers interested in these applications. Thus, we have chosen to express all isotope ratios on the mass scale. Therefore, for any isotope pair we define a ratio (mass isotope ratio, MIR) as the weight ratio of the two isotopes. If this ratio refers to unenriched material, it is designated as MIR". We do realize that this notation may not appeal to others, especially those used to the traditional approach, but it is our judgement that this is most consistent with the needs of the field of mineral metabolism. We have employed this notation previously (1). The absolute level of the in vitro spike must be optimized in relation to the expected isotopic content of the sample. In the classical application of isotope dilution analysis, the isotopic ratios in the prespiked sample are known (natural ratios). The applicable equation is given below (eq l),where MIR57/54
+
57FeU 57Fes MIR57/54
=
+
54FeU 54Fes
is the ratio of 57Fe/54Fein the spiked unknown sample, expressed as ratio of weights (denoted as mass isotope ratio), 54FeUand 57Feuare the absolute amounts of the two isotopes in the sample before spiking, 54Fe9and 57Fesare the absolute amounts of the two isotopes in the spike, and
MIR057/54= 57Fe,/54Fe,
(2)
where MIR057/54is the mass isotope ratio for the sample before spiking. It is clear that the only two unknowns are 54FeUand 57Feu. Solving these two equations for 54Feugives (1
54FeU=
- 0.003523MIR57/54)57Fe8 MIR57/54 - 0.3895
(3)
The two factors 0.003 523 and 0.3895 are related to the isotopic ratio of 57Fe/"Fe in the spike and the unknown, respectively. It is clear that the larger the level of 57Festhe larger the magnitude of MIR57/54and the larger the denominator of eq 3. The uncertainty in the estimated value of 54FeUwill be an inverse function of the level of the spike. The magnitude of
1338
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
this uncertainty is given by eq 4.
For convenience, the
0.003523~MIR 054,u/54Feu =
1 - 0.003523MIR
t
,0200
2 \
subscript for MIR57/54 has been deleted from eq 4. The quantitative behavior of the error function ( u ~ ~ , , / ~ ~ F as~a, , ) function of level of spike is given in Figure 1. The calculated curve clearly shows that for the measurement precision of 1%, the overall analytical uncertainty (U ~ , , / ~ ~ Franges ~ , , ) between 2.2% (for 57Fes= 57Fe,) and 1.5% (for 57Fe, = 10 X 57Fe,). The curve is initially steep. There is little improvement for spike levels higher than 5 X 57Fe,,. For the case of in vivo labeled feces (in vivo label: 58Fe), the appropriate set of equations is (MIRo57154)54Fe, + 57Fes =
+ 58FeU+ 58Fes MIR58/54 = 54FeU+ 54Fes
MIR57/54
54Feu 54Fes
(5)
In deriving these equations, we have assumed that the ratio of 57Feto 54Fein the feces prior to in vitro spiking with 57Fe is equal to the natural ratio. This assumption is not exactly correct as the in vivo spike will invariably also introduce some 57Fe. In a typical experiment, a 1-mg dose of enriched 58Fe preparation is administered to infants whose daily iron intake might be in the range 3-10 mg. A typical 1-mg 58Fepreparation introduces 0.0107 and 0.01845 mg of j4Fe and 57Fe, respectively (9). If absorption of this label is l o % , and if the unabsorbed label is excreted effectively over a 72-h period, the true value of MIR57/54(value before in vitro spiking) will be in the range 0.3971-0.4145 (cf. 0.3895 as natural ratio). Thus, the resultant error will be relatively small. The error can, of course, be eliminated altogether if the base-line ratio for 57Fe/54Feis measured experimentally, but this will require twice the number of measurements. The increase in the required number of isotopic analyses may not be justifiable in light of the small magnitude of this error. 2. Potential Spectral Interferences. Potentially, a number of mechanisms could lead to mass spectral interferences for stable isotopes of iron. These could be a serious source of error in isotopic analysis of fecal matter. Potential interferences are generated via the argon plasma itself, chemical reactions between the argon plasma and reagents employed in the processing of fecal samples, and isobaric interferences resulting from selected other mineral components of the fecal matter. (a) Argon Plasma Background. When aspirating aqueous solutions into the argon plasma, mass peaks at 54,56,57, and 58 are observed (Figure 2). We have assigned the following species to these mass positions: 54, 40Ar14N+; 56, 40Ar160+; 57, 40Ar160H+;58, 40Ar180+.We have confirmed the nature of the species at mass numbers 56 and 58 by observing the expected changes in peak intensity upon substitution of Hz"O for H20 (Figure 2). In isotopic analysis of fecal matter, the final solution used for the measurement of mass isotope ratios will have an iron concentration around 20 ppm. We have shown previously that this concentration provides the optimum ion intensities for the least abundant isotope, 58Fe(9). Compared to ion intensities of iron isotopes from such a SOlution, the background mass spectral ion intensities constitute a 0 4 . 4 % contribution (Table I). Therefore, the background peaks at mass numbers 54 and 57 are not of any practical concern.
J
,0180 ,0160
t
a"
,0140
,0120
'oloo
2
4
6
8
10
57Fe, ; x 5'Fe, Flgure 1. Error function for isotope dilution analysis showing the relationship between the expected precision of the determinations and the level of spiking: 57Fe8,amount of 57Feadded to the sample as spike; 67Feu,amount of 57Fepresent in the sample prior to spiking.
8000
1
I 1
2ooo~
,
,I,I
,
,
,
lbi
I$'
'
0 53.0
54.0 5 5 . 0
56.0
57.0 58.0 59.0 60.0 61.0
Mass
Figure 2. Mass spectrum of the reglon of interest to isotopic analysis for Iron: (a) distilled deionized water and (b) H,"0 (15 % "0). ~~~
Table I. Comparison of Peak Intensities for Deionized/Distilled Water (DI/dist. HzO)and DI/dist. HzO Containing 20 ppm Fe
mass no. 54 56 57 58
obsd peak intensity, ions/s DI/dist. H20 20 ppm Fe 540 f 20 8200 f 140
131 120 f 1090
60 f 7
47080 f 390 6685 f 102
ND"
Not detectable.
(b) Reagent Mass Spectra. As processing of fecal matter to yield suitable solutions for introduction into the argon plasma requires the use of strong inorganic acids, we have inves$igated any potential background peaks due to such reagents. Of the ashing procedures commonly employed, wet oxidation with HNO3/HzO2is particularly convenient for oxidation or organic matter in feces (15). In Figure 3, we have presented the spectral region of interest for solutions of dilute HN03 (Figure 3a, top) and dilute HC1 (Figure 3b, bottom). The concentration of acids in these solutions corresponds to approximately 5%. It is clear that introduction of HN03 at such concentrations does not introduce additional mass spectral peaks of concern (cf. Figure 2). Introduction of HN03 may increase the background contribution of mass 54 somewhat (cf. Figure 3a and Figure 2), but in quantitative relation
ANALYTICAL CHEMISTRY, VOL. 58,NO. 7, JUNE 1986
i337
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1400
-
1000
1600 1400-
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nn
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52 0
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56 0
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Mass
Figure 4. Mass spectrum of synthetic mixture: 5 ppm Fe, 0.5 ppm Ni, 0.25 ppm Cr in dilute "0,.
12001000-
soo-
one application. The general nature of this potentially important problem has not yet received adequate attention. (c)Isobaric Interferences. In contrast to the blood matrix (9),isobaric interferences are potentially serious for stable isotopes of iron in fecal matter. These interferences are summarized in Table 11. While fecal content of Fe, Ni, and Cr may vary over a wide range due to variations in intake, the values given in Table 11 for 54Cr/54Feand 58Ni/58Feare the expected upper limits. It is clear that while "Cr may not pose a major problem for 54Fe,58Niclearly constitutes a potentially serious interference. A typical mass spectrum obtained from a synthetic mixture of Fe (5 ppm), Ni (0.5 ppm), and Cr (0.25 ppm) is shown in Figure 4. The measured relevant ion intensities for individual isotopic components of this mixture are as follows (ions/s, mean f lu): 54Fe40050 f 468, 54Cr1069 f 37, 58Fe1984 f 27, and 58Ni32210 f 305. The measured isotope ratios (54Fe/57Feand 58Fe/57Fe)for a solution containing 5 ppm Fe alone as well as for the synthetic mixture are given in Table 111. The mass spectrometer provides both uncorrected ion intensities as well as ion intensities corrected for known isobaric interferences. Both sets of data are included in Table I11 for 58Fe/57Fe.It is clear that the corrected ratio for "Fe/57Fe is acceptably close to the ratio obtained from the solution containing only Fe (2.728 f 0.039 vs. 2.731 f 0.018). Therefore, as expected, 54Crinterference is not a major issue. Based on these observations, we believe that the necessary isobaric correction for 54Crcan be carried out by the instrument algorithm under the conditions prevalent for fecal matter. In stark contrast, both uncorrected and corrected isotope ratios for SsFe/57Fereported for the synthetic mixture bear no resemblance to the interference-freeobserved ratio of 0.1353 f 0.0021. Therefore, since the major potential isobaric interference is due to SsNicontent of fecal matter, the chemical separation procedure must focus specifically on this issue. Nickel forms a stable complex with ",OH, Ni(NH3)2+,while Fe3+does not (17).A separation procedure was devised based on this difference (Scheme I). Variations of this general procedure were tested on aliquots from a fecal homogenate (samples 0-8,
Mass
Flgure 3. Mass spectral region of interest to isotopic analysis of Iron showing the effect of inorganic acids: top, spectrum of 5 % HN03, and bottom, spectrum of 5 % HCI.
Table 11. Potential Isobaric Interferences for Stable Isotopes of Iron in Fecal Matter isotope of interest
potential isobaric interference
expected ion peak ratio for isobaric interference: isotope of interestn
54Fe 57Fe 58Fe
54Cr none 58Ni
54Cr/54Fe,0.020 58Ni/5sFe,20
Calculated for typical Cr/Fe and Ni/Fe content of human feces.
to the peak intensity of "Fe arising from a 20 ppm Fe solution (Table I) this is clearly insignificant. Additionally, the concentration (5%) of HN03 used here is at the higher end of the expected acid concentrations usually encountered in any actual analysis. Similarly, while introducing HC1 also does not alter the background peak intensities at mass numbers 54,57, and 58, two major peaks at mass numbers 51 (35C1160+) and 53 (37C1160+) are present. The contribution of HC1 to the peak at mass number 54 (37C1170+) is negligibly small. A small peak does appear at mass number 55 (37Cli80+). Despite the lack of quantitatively significant interferences from these two acids in specific reference to the stable isotopes of iron, these data, as well as our experience with other elements (unpublished), clearly indicate that reagent-generated mass spectra could present a real complicating problem for certain applications of ICP/MS. Such problems should be assessed individually and carefully in specific relation to any
Table 111. Measured Ratios (MR) for S4Fe/S'Feand S8Fe/S7Fefor Sample Aliquots of Scheme I sample designation 5 ppm Fe 5 ppm Fe 0 1
6 7
+ 0.5 ppm Ni + 0.25 ppm Cr
uncorrected MR58/57 0.1424 f 0.0024 2.430 f 0.014 0.5188 f 0.0033 0.1425 f 0.0009 0.1490 f 0.0010 0.1447 f 0.0012
corrected MR54/.57
2.731 2.728 2.713 2.712 2.710 2.717
f
0.018
f 0.039 f 0.021 f 0.018 f 0.012
f 0.024
MR58/57
0.1353 0.0165 0.1142 0.1336 0.1346 0.1324
ion intensities, ions/s 67Fe 6oNi 52Cr
f 0.0021
f 0.0284 f 0.0038 f 0.0010 f 0.0012
f 0.0014
13300 f 12470 f 11780 f 12350 f
152 260 120 175
2177 44 64 59
f 29 f2 f2
f3
268 468 483 420
f 16 f 12 f 10
f 13
1338
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
Table IV. Reproducibility of Nickel Separation Scheme" 60Ni intensity,
sample
* * 49.0
6 2 0 54.0 6 6 . 0 66.0 80.0 82 0
Mass
66.0 49.0
62 0. 54 0 56.0 88.0 60.0 82 0
no.
MR54/mb
MR58/57*
ions/s
1 2 3 4 5 6 7 8 5 ppm Fe
2.712 f 0.018 2.762 f 0.021 2.754 f 0.011 2.690 f 0.031 2.695 f 0.013 2.710 f 0.012 2.717 f 0.024 2.708 f 0.007 2.731 f 0.018
0.1336 f 0,0010 0.1352 f 0.0010 0.1341 f 0.0025 0.1325 f 0.0013 0.1338 f 0.0022 0.1346 f 0.0012 0.1324 f 0.0014 0.1325 f 0.0011 0.1353 f 0.0021
44 f 2 40 f 3 34 f 2 48 f 3 51 A 1 64 f 2 59 f 3 59 f 3
" See Scheme I for sample identification. Instrument-corrected ratios; data are as mean f l a for 10 sequential measurements.
68.0
Mass
Flgure 5. Mass spectra of various stages in the separation of stable isotopes of iron from fecal matter (Scheme I,Table 111). For identification of spectra see Scheme I.
Scheme I). The results of this experiment have also been summarized in Table 111. The resultant mass spectra are also shown in Figure 5. In Table 111, we have presented the information in three categories, first ion intensities for 57Fe, 60Ni,and 52Cr. These data are presented to show the extent of recoveries for Fe, Ni, and Cr. The selected isotopes are expected to be interference free. Second, uncorrected isotope ratios are summarized for 5sFe/57Fe. This ratio should indicate the extent of 58Niinterference to 58Feion peak. Thirdly, the instrument-corrected isotope ratios for 5sFe/57Feand 5LFe/57Fe are also summarized. The last category represents the isotope ratio data that are normally employed. The ion intensity data clearly show that in either variation of the method (cf. samples 1,6, and 7) 89-94% of iron is recovered in the final solution. Similarly, chromium is also not lost, However, the corrected ratio for 54Fe/57Fedoes not appear to be significantly different from the ratio obtained from a 5 ppm solution of Fe or the synthetic mixture. Therefore, even for the fecal sample, the extent of 54Crinterference is not a matter of concern. The uncorrected ratio of 58Feto 57Fein the fecal sample prior to nickel removal step is 0.5188 f 0.0033 (cf. similar ratio for 5 ppm Fe solution of 0.1424 f 0.0024) reflecting the contribution made by 58Ni. This is reduced to the range 0.1425-0.1490 (samples 1,6, and 7) after the nickel separation procedure. The resultant ratio is slightly larger than that from the 5 ppm Fe solution reflecting the small amount of residual Ni (see column with the heading 60Ni). The instrument corrected ratio for 58Fe/57Fein the same sample (sample 0 before Ni separation, Table 111) is 0.1142 f 0.0038, also significantly different from the interference-free value of 0.1353 f 0.0021 (Table 111) in the iron-only solution. This ratio becomes quantitatively the same as the ratio for the 5 ppm Fe solution in all samples (1,6, and 7) undergoing the nickel separation procedure. Therefore, in spite of the very small residual Ni present in samples 1, 6, and 7 (2-3% of original nickel content of the sample; see 6oNi peak intensities of Table 111),the instrument-corrected 58Fe/57Feratios are not different from the ratio observed in the 5 ppm Fe solution. The validity of instrument correction algorithms depends strongly on the actual magnitude of the isobaric interferences, as clearly seen from the data for "Cr in comparison with 58Ni. Therefore, instrument correction should not be dependent upon if the magnitude of the isobaric interference is large (e.g., 58Ni),but for smaller contributions (54Crin these samples) the correction appears satisfactory. In general, the correct approach to the elimination of isobaric interferences is, of course, by means of selective chemical separation schemes. Reproducibility of the measurements is shown in Table IV. These data indicate that the variations of the separation
Table V. Data for 57Fe-SpikedStandard Solutions solution
MR57/54"
no.
MIR67/54
7/19/85
7/29/85
0 1 2 3 4 5 6 7
0.3895 0.4073 0.4255 0.4609 0.5679 0.7460 1.102 2.163 3.217 3.915
0.4010 f 0.0053 0.4188 f 0.0040 0.4422 f 0.0051 0.4731 f 0.0035 0.5765 f 0.0041 0.7563 f 0.0032 1.083 f 0.0115 2.172 f 0.0122 3.188 f 0.0206 3.881 f 0.0171
0.4012 f 0.0077 0.4284 f 0.0036 0.4426 f 0.0021 0.4733 f 0.0075 0.5867 f 0.0043 0.7631 f 0.0029 1.127 f 0.0076 2.191 f 0.0183 3.235 f 0.0437 3.952 f 0.0419
a
9
Linear regression equations: 7/19/85, MR57/54= 0.01654 0.9875(MIR57/54), r2 = 0,9999; 7/29/85, MR57,54 = 0.01496 1.0042(MIR57/54), r2 = 0.9999.
+ +
scheme are not substantially consequential to the accuracy of the procedure. Furthermore, the resultant isotope ratio measurements are not materially different from the results obtained from the 5 ppm Fe solution. 3. Analytical Scheme. The complete analytical scheme for isotopic analysis of in vivo spiked 58Fefecal matter is given in Scheme 11. The separation scheme is self-explanatory, Quantitative recovery of iron is, of course, not necessary because of the application of IDA. The achievable precision of the isotope ratio measurements depends on the number of mass spectra collected for each solution and could approach the value of 0.1% (9). This high degree of precision is not necessary for fecal samples (1). For fecal samples in the present application of the method, overall precision and accuracy of 1% in the absolute recovery of excreted isotopes are satisfactory. ( a ) 57Fe-SpikedCalibration Plots. In the measurement of absolute isotope content, the measured isotope ratios for 57Fe/54Feand 58Fe/54Femust be converted to true mass isotope ratios (eq 5 and 6). This requires construction of appropriate calibration curves relating the measured ratio (MR) to the true ratio (MIR). Previous experience with isotopic measurements for iron in blood (9) indicates that the linear regression parameters relating these two variables are strongly matrix dependent, Additionally, they also vary for different instrument operating parameters (9). Because the accuracy of the estimated values of MIR (from the measured value of MR) has such an important impact on the overall accuracy of isotopic analyses, this issue deserves careful scrutiny. We have previously attempted to prepare fecal matrix spiked with variable but known amounts of the appropriate stable isotope so as to prepare a set of spiked matrix-matched standards whose MIR covers the range of interest. We have encountered much difficulty as preparation of such standards requires accurate analysis of elemental composition of a large fecal
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
1330
Table VI. IDA and Atomic Absorption Measurements of Iron" Fe content determined sample matrix
IDA
atomic absorption
iron solution NBS SRM 1577a fecal homogenate
245.4 f 1.5 pg (5) 192.2 f 2.2 pg/g (3) 15.14 f 0.36 pg/mL (4)
192.3 f 3.6 kg/g (3) 15.82 0.48 ,ug/mL (3)
comments
*
known content = 250 pg certified value: 194 i 20 pg/g
"Value in parentheses refers to the number of replications. Data correspond to mean f la. composite for the particular element. To circumvent these difficulties in the present work, we decided to start with a standard solution of iron (1000 ppm atomic absorption standard) to which then is added known but variable quantities of 57Feas an in vitro spike to provide the desired range of MIRs. Using these spiked standards and neglecting any potential matrix effects, we have then analyzed subsamples of both National Bureau of Standards Standard Reference Material Bovine Liver (NBS SRM 1577a) and a fecal homogenate pool, both with IDA employing 57Feas the in vitro spike and atomic absorption spectrophotometry employing the iron standard solution (1000 ppm) as the atomic absorption standard. Our reasoning has been that if the final analyses of iron in fecal homogenate pool and the Bovine Liver agree, within the acceptable limits, between our IDA and the atomic absorption methods or the certified value for the SRM 1577a, then matrix matching is not necessary in the preparation of spiked standards. Aliquots each consisting of 500 p L of lo00 ppm Fe solution were added to 5 mL of concentrated HC1 to which had been added a precisely calculated amount of 57Fe-enrichedsolution; final volume of the resultant spiked standards was adjusted to 100 mL with deionized water. Ten such solutions were prepared whose values of MIR for 57Fe/64Fecovered the range from 0.3895 (natural) to 3.915. These 10 standards have been run on two occasions, each with either 57Fe-spiked Bovine Liver test samples or 57Fe-spikedfecal homogenate test subsamples, all prepared according to Scheme 11. The calibration data for both occasions are given in Table V. The linear regression parameters are also given in the table. It is clear that for both occasions, an excellent linear relation was obtained for the two variables (R2 = 0.9999). However, the slope and intercepts appear to be slightly different for the two occasions. As the operating instrument parameters could vary substantially on different occasions (more than for the data presented here), it becomes clear that construction of calibration plots is necessary for any set of analyses. ( b ) Accuracy of Isotopic Analyses. Three sets of samples have been analyzed for their iron content using the 57Fe-spiked IDA method in comparison with atomic absorption spectrophotometry: iron solutions derived from atomic absorption standard, NBS SRM 1577a (Bovine Liver), and aliquots from a fecal homogenate. In every case, the 67Fe spike added corresponded to about 4-5 times the estimated value of 57Fe initially present in the sample (Figure 1). The results of these analyses are given in Table VI. The iron solution employed as unknown was actually derived from the atomic absorption standard (1000 ppm) containing 250 pg of Fe/sample. The calculations were carried out according to eq 1and the results of 54Fethen expressed as natural iron. Therefore, these results actually reflect the accuracy of "Fe isotopic analyses. It is clear that in every case the IDA method agrees closely with other estimates of iron content. For the NBS SRM 1577a, the certified value (16) is 194 f 20 rg/g. We have obtained values of 192.2 f 2.2 and 192.3 f 3.6 with the IDA and atomic absorption methods, respectively (Table VI). For the fecal homogenate, the IDA method gives 15.14 i 0.36 rg/mL as the iron concentration, while the measurement with the atomic absorption yields the
value of 15.82 f 0.48. Thus, these data clearly establish the accuracy of the IDA method for isotopic ("Fe) analysis of iron. We have not explicitly established the absolute accuracies of 58Fe analyses (similar to data reported in Table VI). However, the isotope ratio data given in Table I11 clearly demonstrate the accuracy of the measurement in the ratio 5sFe/57Fe. Calculations involved to estimate either iron or 58Fefrom 57Fe/5sFedata are similar to those carried out for 57Fe/54Fe.Therefore, we see no reason for repetition of these calculations to establish accuracy of these analyses based on 58Fe. We fully expect the overall performance of these analyses to be similar to those reported in Table VI based on calculations using "Fe. Therefore, we conclude that the proposed IDA method provides accurate data on absolute contents of MFeand 58Fein fecal matter derived from in vivo %Fe-labeled studies in humans.
CONCLUSION We have developed an accurate method, based on the application of isotope dilution analysis and ICP/MS, for isotopic analysis of iron in human fecal matter. We have established the accuracy of isotopic analyses in relation to the studies of iron utilization in infants and have shown this to be sufficient for the analytical requirements of such studies. Combination of this method with our previously reported measurement of 58Fe enrichment in infant's blood (9) now permits the investigation of quantitative aspects of iron utilization in the early period of infancy, an important problem in the nutritional management of infants with respect to iron. The method reported herein is the first application of ICP/MS to accurate isotopic analysis of iron in fecal matter. Its combination with measurements in blood (9) provides, for the first time, a unified method for application to the problem of iron utilization in infants. The method of ICP/MS appears to provide a major instrumental breakthrough for metabolic studies of this type for a number of minerals (elements) of importance to human health and disease. However, due to the many potential interference problems associated with the nature of ion generation employed in this method and the very complex nature of fecal matter, the necessary analytical chemistry methodology should be developed carefully. The capabilities and limitations should be evaluated for each specific set of isotopes and in relationship to the accuracy requirements of the intended application. The magnitude of interferences should be assessed in relation to the overall scheme of analysis. Registry No. 54Fe,13982-24-6;58Fe,13968-47-3;54Cr,1430497-3; 5sNi, 13981-79-8; 57Fe,14762-69-7;Fe, 7439-89-6; "OB, 7697-37-2; HCl, 7647-01-0. LITERATURE CITED Janghorbanl, M.; Young, V. R.; Ehrenkranz, R. A. Trace Nemenb in Nutrition of Children; Raven: New York, 1985; pp 63-85. Bothwell, T. H.; Charlton, R. W.; Cook, J. D.;Flnch, C. A. Iron Metabolism in Man ; Blackwell Scientific Publications: London, 1979. Stekel, A., Ed. Iron Nutrition in Infancy and Childhood; Nestle's Nutrition Workshop Serbs; Raven: 1984; Vol. 4. Janghorbanl, M.; Ting, B. T. G.; Young, V. R. J . Nutr. 1980, 770, 2190. Werner, E.; Hansen, C.; Wlttmaack, K.; Roth, P.; Kaltwasser, J. P. INSERM Svmr, Ser. Paris) 1983. 773. 201. King, J. C.iRaynolds,'W. L., Margen, S . ' A m . J . Clin. Nutr. 1978, 3 7 , 1198.
1340
Anal. Chem. 1988, 58, 1340-1344
(7) Miiier, D. D.;Van Campen, D. M . J. Clin. Nutr. 1979, 32, 2354. (8) Carni, J. J.; James, W. D.;Koirtyohann, S. R.; Morrix, E. R. Anal. Chem. 1960, 52, 216. (9) Janghorbanl, M.; Ting, B. T. G.; Fomon, S. J. Am. J . Hematol. 1986, 21, 277. (IO) Gorten, M. K.; Hepner. R.; Workman, J. 8 . J . Pediatr. (St. Louls) 1963, 83, 1063. (11) Dubach, R.; Moore, C. V.; Minnich, V. J . Lab. Clin. Med. 1946, 31, 1201. (12) Finch, C. A.; Gibson, J. G.; Peacock, W. C.; Fiuharty, R. G. Blood 1949, 4, 905. (13) Gorky, L.; Sjoiin, S. Acta Paediatr. 48 Scand. Suppl. 1959, 177, 24.
(14) Chart of the Nuclides; General Electric, Nuclear Energy Division: San Jose, CA, 1972. (15) Janghorbani, M.; Ting, B. T. G.; Young, V. R. Clin. Chim. Acta 1980, 108, 9. (16) National Bureau of Standards, Certificate of Analysis, Standard Reference Material 1577a,Washington, DC, June 15, 1982. (17) Meites, L., Ed. Handbook of Analytical Chemistry, 1st ed.; McGrawHili: New York, 1963;Table 1-17, pp 1-39.
RECEIVED for review October 10, 1985. Accepted January 9, 1986.
High-Performance Liquid Chromatographic Separation of Biologically Important Arsenic Species Utilizing On-Line Inductively Coupled Argon Plasma Atomic Emission Spectrometric Detection W. D. Spall,* J. G. Lynn,J. L. Andersen, J. G. Valdez, and L. R. Gurley Toxicology Group, Life Science Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
An anlon exchange, high-performance liquid chromatography technlque uslng a l5mIn linear gradient from water to 0.5 M ammonium carbonate to separate arsenlte, arsenate, methylarsonlc acid, and dimethylarsinic acld from neutral arsenlc containlng compounds was developed for application to a study of arsenlc metabolism In cultured cell suspensions. Arsenlc detection was accomplished by the direct coupilng of the column effluent to an inductively coupled argon plasma atomic emisslon spectrometer (ICAP-AES) set to monltor the arsenic emisslon line at 197.19 nm. The analysts requlres 20 min and is sensltlve to as low as 60 ng of arsenlc Injected to the column.
Arsenic, an important environmental toxicant, enters the environment from a variety of sources, including nonferrous smelting operations, coal-fired power plants, and agricultural organomenical herbicide applications ( I , 2). Arsenic residues are generally reported as total arsenic with no designation of the molecular form of the arsenic. From a biological and toxicological perspective, it is necessary to know the chemical species distribution of the arsenic, since the toxicological properties of these compounds vary widely (3-5). It is well-established that many organisms, including man, chemically transform arsenic ( I ) . Inorganic arsenite and arsenate ions, methylarsonic acid (MAA), dimethylarsinic acid (DMA, cacodylic acid), phenylarsonic acid, several organic esters of arsenic oxyacids, and volatile alkyl arsines are among the known products of these biological transformations (6, 7). It is obvious that an accurate assessment of the toxicological behavior of arsenic is not possible without knowledge of the concentrations, chemical forms, and interactions of the different molecular species comprising the total arsenic load in the environment. Although the toxicology of arsenic is reasonably well-understood, the biochemical aspects of arsenic transformation by mammalian cells has not been determined. We are undertaking an investigation of the fate of arsenic when mammalian cell cultures are exposed to arsenic in different chemical forms. This is a logical extension of our ongoing study of the effects of heavy metals on the cell cycle of mammalian cells in culture (8-11).
Sensitive techniques for the determination of some arsenic species exist and are generally based on the differential generation of arsine (12) or on the column chromatographic separation of arsenic species. The most sensitive of these techniques uses graphite furnace atomic absorption (GFAA) spectroscopy for the determination of arsenic in the column effluents (13-16). The disadvantage of GFAA is that the arsenic measurement is discontinuous. Because GFAA is not a flow method, its use requires that small portions of the column effluent be collected periodically and individually analyzed. Therefore, the arsenic concentration determined for each portion of the effluent is essentially an instantpeous arsenic concentration in the column effluent at the time of sampling. The problems of discontinuous analysis are not readily appreciated until one considers the details of the analysis procedure. The volume collected for the analysis is usually small, on the order of 50-100 pL. The time required for the actual analysis is limited by the recycle rate of the graphite furnace and varies from 30 to 60 s/analysis, depending on the furnace programming sequence selected by the analyst. The column effluent that elutes between samples (e.g., during the time required for the analysis) is generally not analyzed, but instead is sent to waste. The concentration of the eluting arsenic compound may be determined as either the area under the peak generated from the discontinuous plot of arsenic concentration at each sample point vs. elution time or as the summation of the histogram of the individual arsenic concentration determinations. In either case, the sampling frequency must be high enough to ensure that the estimation technique produces as small an error as possible. The number of samples required is then determined by the degree of accuracy desired and the recycle rate of the graphite furnace. If the recycle rate is assumed to be 30 s, and at least five samples are deemed necessary for accurate concentration determination, then a minimum peak base width of 2.5 min will be required. In practice, it is desirble to use 10 or more samples to determine peak areas, and recycle times are more commonly 45 s, so the eluting peaks should have at least a 5-min base width. It must be emphasized that this technique does not cause chromatographic peak broadening, but rather, requires wider peaks for accurate analysis, a condition not
0003-2700/86/0358-1340$01.50/00 1986 American Chemical Society
