Anal. Chem. 1980, 52, 1811-1814
abundances) can be used directly to obtain reasonably accurate relative molar concentrations of the oligomers. T h e FDMS method is particularly appealing since it provides a means for direct determination of Mnand Mw It is to be expected that other "good desorbing" polymer systems can also be characterized in this manner. Polymers containing highly polar groups would likely yield less accurate results (due to poor desorption characteristics). T h e results for polystyrene are quite encouraging and serve to demonstrate the potential of higher mass field desorption analysis of polymers.
ACKNOWLEDGMENT T h e support a n d encouragement of J. B. Pausch (The BFGoodrich Co.) and C. C. Fenselau and R. J. Cotter (The J o h n s Hopkins University) are greatly appreciated. J. D. Wenrick (The BFGoodrich Co.) gave helpful suggestions regarding the computer programs. R. E. Harris ( T h e BFGoodrich Co.) assisted with the mass spectroscopy (Varian M A T 311A). LITERATURE CITED (1) Beckey, H. D. "Principles of Field Ionization and Field Desorption Mass Spectrometry"; Pergamon Press: New York. 1977. (2) Schulten. H.-R. Adv. Mass Spectrom. 1977, 7 , 83-96.
181 1
(3) Schulten, Methods Biochem. Anal. 1977, 2 4 , 313-448. (4) Mead, W. L. Anal. Chem. 1968, 40, 743-747. (5) Wiley, R. H.; Cook, J. C., Jr. J . Macrornol. Sci., Chem. 1976, A10, 8 11-814. (6) Wiley, R. H . Macromol. Rev. 1979, 14, 379-417. (7) Lattimer, R. P.; Welch, K. R.; Pausch, J. 13.; Rapp, U. Varian MAT 31 1A Application Note No. 27; Varian MAT Mass Spectrometry, Florham Park, NJ, 1978. (8) Lattimer, R. P.; Harmon, D. J.; Welch, K. R. Anal. Chem. 1979, 51, 1293-1296. (9) Matsuo, T.; Matsuda, H.; Katakuse, 1. Anal. Chem. 1979, 57, 1329- 133 1, (10) Lattimer, R. P.; Welch. K. R. Rubber Chem. Technol. 1978. 51, 925-939. (11) Lattimer, R. P.; Welch, K. R. Rubber Chem. Technol. 1980, 53. 151-159. (12) Altares, T., Jr.; Wymon, D. P.; Alien, V. R . J . Polym. Sci., PartA 1964, 2 . 4533-4544. (13) Flory, P J. "Principles of Polymer Chemistry"; Cornell University Press: Ithaca. NY: ChaDter 7. (14) Matsuo, T.; Matsuda, H.: Katakuse, I. A17al. Chem. 1979, 51, 69-72. (15) Daly. N. R.; McCormick, A,; Powell, R. E. Rev. Sci. Instrum. 1966, 39, 1163-1167. (16) Daly, N. R.; McCormick, A.; Powell, R. E.; Hayes, R. Int. J . Mass Spectrom. Ion Phys. 1973, 11, 255-276.
RECEIVED for review April 8, 1980. Accepted July 7 , 1980. Appreciation is expressed to The BFGoodrich Co. and to the National Science Foundation for support of this work.
Isotope Ratio Measurements of Urinary Calcium with a Thermal Ionization Probe in a Quadrupole Mass Spectrometer Alfred L. Yergey,' Nancy E. Vieira, and James W. Hansen Intramural Research Program and Neonatal and Pediatric Medicine Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205
A thermal ionization mass spectrometric technique using standard quadrupole instrwnentation and a special solids probe has been developed for the measurement of calcium isotope ratios in biological samples. Calcium is coated onto disposable rhenium filaments from an acidic solution after being isolated from urine by precipitation with basic oxalate. Isotope ratios of natural abundance standards were measured with an average accuracy of about 1 % ; urinary calcium concentrations were determined using an isotope dilution technique which gave results that differed by less than 10 % from values determined by two independent methods; overall reproducibility of isotope ratio measurements was about 1 % standard deviation. The method uses standard quadrupole instrumentation.
T h e rates of calcium flux across gastrointestinal and renal barriers along with interchange between soft tissue and skeletal compartments axe of great importance in the study of calcium homeostasis and skeletal development. Such rates can be readily determined in vivo by measuring the time dependent changes in the specific activity of radioactive tracers ( I , 2 ) . When newborns and children are the subjects of such investigations, however, it is both desirable and necessary to use nonradioactive tracers. Recent work with stable calcium tracers in newborns has shown ( 3 )that, measuring the atom percent enrichment of two isotopes increases the precision of determining kinetic parameters compared to the use of a single tracer. These determinations of specific activity have been
made by measuring isotope ratios using thermal ionization mass spectrometry. The thermal ionization technique has been used to measure calcium isotope ratios with relative accuracies of better than 0.05% with relative precisions of less than 0.01% standard deviation ( 4 , 5 ) . T h e mass spectrometers for such measurements are specially designed for the task. Typical measurements involve elapsed times of several hours per sample and generally require breaking vacuum between samples to change filaments. Isotope ratio measurements of the high quality provided by such thermal ionization mass spectrometry tend to be more accurate than necessary for biological samples. In this paper we report the development of a satisfactorily accurate, rapid method of measuring calcium isotope ratios using a thermal ionization method in a standard quadrupole mass spectrometer with a specially designed solids probe. We report very reproducible measurements that are of acceptable accuracy for the systems studied, but which require less time per sample and use more generally available instrumentation t h a n do typical thermal ionization methods.
EXPERIMENTAL Mass Spectrometer. A Finnigan Mdel4OOO quadrupole was used as supplied by the manufacturer with one exception. For convenience the multiple-turn diaphragm type solids inlet valve was replaced with a CON-VAC quarter-turn butterfly type valve manufactured by Consolidated Instruments, 7541 Belair Road, Baltimore, Md. 21236. Solids Probe and Disposable Filaments. The solids probe designed for this work is shown schematically in Figure 1. The
This article not subject to U.S. Copyright. Published 1980 by the American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
I
I 40
I
I
I
42
1 44
m z-
Figure 2.
Figure 1. Thermal ionization solids inlet probe and disposable filament. Note two different scales
probe tip serves as a receptacle for the disposable filament assemblies from which the samples are ionized. The vacuum seal of the probe is effected a t its base using a polyester material that flows at 200 "C. Filaments are heated using a commercial 0-16 VDC, Cb8 A power supply (Epsco EFB Filtered DC Power Supply). Positive biasing of the filament relative to ground is obtained with a battery and potentiometer; 60 Hz pickup is reduced with a 1-pF capacitor to ground from one side of the power supply output. The filament assemblies shown in Figure 1 are made from two 2.5-cm lengths of $16 ga nickel wire sealed together with a bead of Corning G-12 glass. The separation of the leads is 0.64 cm at the base. A rhenium ribbon filament (0.002 x 0.05 X 0.6 cm) is spot welded across the tips of the supports. The filament supports are cleaned in an ultrasonic bath for 45 min using a 3% "OB solution followed with a 15-min ultrasonic rinse using deionized water both before and after spot welding. The assembled filaments are dried and then degassed for 1 h a t lo4 Torr and white incandescence. Degassed filaments are stored in a clean container until use. Materials and Reagents. @CaC03was obtained from the Oak Ridge National Laboratory. H N 0 3 used for washing filaments and plasticware was Baker Reagent Grade; H N 0 3 used for preparing solutions was Baker Ultrex Grade. Deionized water was used for preparing solutions and for rinsing. Standards were prepared and reagents were stored in polypropylene vessels. Biological samples were prepared in 12-mL glass centrifuge tubes. The storage vessels, glassware, and disposable pipet tips were soaked overnight in 10% HN03, rinsed in deionized water, and air dried under cover. Sample Preparation. Natural abundance and spiked samples for isotope recovery studies were prepared by placing 15 gL of approximately 0.02 M CaC03in 3% "OB on an inverted filament and dried using a n infrared lamp for 1 h. Spiked urine samples were prepared by adding 150 gL of a 34.7 gg/mL 44Casolution (82.9% %a in 3% "0,) to 5 mL of urine. Calcium was removed from the urine using a modified version of published calcium extraction procedures (6, 7). Five milliliters of a saturated solution of ammonium oxalate made to pH 10 by the addition of concentrated ammonium hydroxide was added to 5 mL of urine. A granular CaC204precipitate was prepared by heating the solution for a few minutes in steam, cooling in an ice bath, and then concentrating it a t the base of the tube by centrifugation. The precipitate was washed in the basic ammonium oxalate and centrifuged again. The supernatant was then decanted and the precipitate dried a t 100 "C; the precipitate was heated in a flame to convert the oxalate into the carbonate and/or oxide and the residue dissolved in about 200 gL of 3% HN03. When a discolored residue was present after heating, a drop or two of concentrated H N 0 3 was added to the residue and the drying and heating repeated. A 15 pL aliquot of this solution was applied to a sample filament and dried. Total calcium recoveries from urine were greater than 98%. Data Acquisition. Data were acquired by using the NIH Isotope Ratio Calculator, described elsewhere in detail (8). Briefly. this is a microprocessor controlled, specific ion monitoring device that is capable of accumulating intensities for up to six different ions per scan cycle. The basic scan speed is 250 ms per ion; scan width is variable, but the same for all channels followed; scan starting points are separately adjustable for each channel. The
Partial mass spectrum of K+ and Ca'
desired wide dynamic range of intensities that the device follows is accomplished by employing a 10-bit A/D converter that uses only the eight most significant bits. The A/D signal is input from a program selectable eight-position variable gain amplifier. This combination of A/D converter and amplifier results in an overall dSnamic range of 214. The device is capable of accumulating both signal and base line/background data separately. At the end of data and base-line acquisition, the device prints (1)the number of base-line and signal scans acquired, (2) the actual, net (pro rata subtraction of base line) and gain adjusted counts acquired for the signal, and (3) the number of base-line counts. The ratio of the gain adjusted counts in appropriate channel pairs is calculated and printed. Operating Conditions. Dried sample filaments were placed in the probe tip, the probe was inserted into the mass spectrometer, and the filament was heated slowly. After a conditioning period of several minutes a t about 1.8-2 A, filament current was increased to a level where W a c was just observed. Over a period of a few minutes, the intensity of the Ca+ signal increased; when the signal had stabilized, the filament current was increased so that the @2a+ signal was in the range of 0.2-1.0 X lo4 A, typically requiring the application of 3-4 A. Electron multiplier high voltage for this signal was about 1950 V with a probe bias voltage of 5
v.
The mass spectrometer was placed under control of the isotope ratio calculator and 480 scans of each of the desired masses were made. Other ions could be monitored as required. A t the end of the data acquisition, the filament power was turned off, and 25 scans of the base line for each channel were acquired. The background levels of calcium were determined by heating clean, degassed, unloaded filaments in the same manner as sample filaments. In general, after observing an initial surge of calcium ions lasting a few seconds at intensities of about 1% of the final intensities of sample filaments, the Ca+ diminished to much lower levels or disappeared entirely.
RESULTS AND DISCUSSION Measurements on Natural Abundance Calcium. Figure 2 shows a thermal ionization spectrum obtained prior t o the burnoff of potassium from a sample filament operating at about 3 A. Note that at these masses, t h e resolution of t h e mass filter is sufficient t o permit separations of t h e ions a t t h e base line. Note also that at this current level, t h e potassium ions appear to be somewhat broader with noiser tops than d o the calcium ions. At lower filament currents, t h e K+ signals are shaped similarly t o the CaC signals, b u t to burn off t h e potassium and t o produce t h e calcium ions, t h e filament current, and thus filament temperatures, are elevated considerably over those required for production of potassium ions. Although filament temperatures cannot be measured in the present apparatus, it is apparent that, above minimum emission temperatures, ions will be released from the filament surface with broadened energy distributions compared to that which they have at temperatures just sufficient to release them from the surface. Since the temperature required for calcium t o be ionized is much greater t h a n for potassium ( 9 ) , t h e potassium ion signal, at temperatures where calcium ions are observed, will have considerable excess energy compared t o t h e calcium ions. Under these circumstances, some peak
ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
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--+2%
Q
0 X +
_______________-__
0
.28$-
/L---L--L' 0
1
-2 Yo
3
2
REPLICATE 0
Figure 4.
4
2
filaments
Time, min
Figure 3.
Evolution of Ca+ from thermal ionization filament
Table I. Calcium Isotope Ratios of Natural Abundance Material Relative to m / z 4 2 mt z
40 42 43 44 46 48
observed ratio 154
2
3
--N/A 3.19 i 0.010 N/A 0.289 = 0.005
Replicate measurements of (4eCa/44Ca) using two different
Table 11. Isotope Dilution Curve for 44Ca in Natural Abundance Calcium 44Ca added
%
ratio from best measured values ( 10 ) 151.04
___ i
0.2078 i 3.203 = 0.00487 i 0.2842 T
7.38 16.2 39.1
0.01
0.0001
broadening accompanied by noise on the peak tops seems quite likely. Figure 3 shows the intensity of the 4oCa+ion as a function of time. This signal intensity is achieved only by use of the probe bias circuitry described. Use of the instrument's quadrupole bias voltage results in greatly broadened peaks for intensities of the same level produced by using the probe bias. These intensity differences undoubtedly are due to differences between the sites of ion formation a t the probe tip a n d the normal ion formation point of the source. Note the initial rise, during which time the final K+ burnoff occurs. and t h e plateau in the intensity that persists for 12-20 min in a useable filament. Isotope ratio measurements are made in this plateau region of the Ca+ signal. At this point in the development of the methodology, the success rate for the production of coated sample filaments to give the response shown in Figure 3 is greater t h a n 90%. Measurements of isotope ratios of natural abundance (n.a.) calcium are given in Table I along with ratios calculated from the best measured values ( I O ) for comparison. A single replicate of each measurement required slightly more than 6 min to obtain, including time for base-line data acquisition and printing the results. T h e ratio 46Ca/42Cacannot be measured in t h e present system because ions m / z 46 are of too low an intensity to be observed in the system as it is configured. The ratio of 43Ca/42Cais not reported because there are background ions present at m / z 43 that interfere with the measurements. These background ions are seen in Figure 3 and are deduced to originate from hydrocarbon sources. The intensity of these interfering ions is highly variable but diminishes with time. They are present despite extensive degassing of the sample filaments prior to loading. Their presence is great enough during the Ca+ signal measurements to prevent measurement of the 43Ca/42Caratio. T h e reproducibility and the degree of isotope fractionation of successive measurements from two filaments are shown in Figure 4. I t is seen in this figure that there are no time dependent trends in the two series of three measurements on the same filaments a n d that the various replicates are scattered about the overall grand average value for this mea-
7.4
?
0.1
0.3 0.9 8 2 r 1.6
:15 36
81.1
0,0001 0.001
0.00003
observed atom 70 enrichment of "Ca i i
Table 111. Adequacy of Precipitation Purification Cat 42 Ca 4'Ca/42 precipi- cation precipication tation exchange tation exchange 44
sample A2 52 MU6 MU16
3.70 4.44 3.70 3.88
3.71 4.37 3.75 3.92
0.283 0.292 0.406 0.341
0.283 0.288 0.425 0.346
surement. T h e lack of an apparent time dependent change in the isotope ratios supports the conclusion that if there is isotope fractionation occurring, the systematic error from it is no greater t h a n random errors of measurement or errors from background sources. Isotope D i l u t i o n s a n d Biological Samples. For the planned clinical studies, the range over which the ratio measurements for a particular pair of isotopes is required to be linear is from about 10 to 400 atom 70excess of the isotope of interest. An isotope dilution study was performed to test the utility of our methodology a t the lower end of this range of enrichments. T h e results of this study are shown in the second column of Table I1 along with the reproducibility of the measurements given as standard deviation of the mean. The measurements were carried out using the 44Ca/42Caratio to determine the enrichment but with a simultaneous measurement of the 48Ca/42Caratio serving as an internal check for the stability of the instrumentation. T h e regression line through the points of Table I1 is y =: 1 . 0 1 -~ 1.5. The slope and intercept do not differ significantly from the ideals of 1.0 a n d 0, respectively. We tested the acceptability of precipitation alone, without use of an ion exchange extraction as used in previously reported methods, as the means of purifying and concentrating calcium in urine. Two normal urines to which an aliquot of 44Cawas added and two urines from a subject in a calcium isotope tracer study in which 44Caand 48Cawere enriched in vivo were each treated in two ways. First, the precipitation method was used as described, and second, the precipitation method was preceded by a cation exchange extraction ( 4 ) (neutral urine passed over Bio-Rad AG-SOW X8 a n d eluted with 6 N HC1). T h e results of this experiment are given in
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
Table IV. Label Recovery sample A1 J1 N1 El
A2 52
44Ca/42Ca, obsd 3.63 + 0.08 4.29 ?I 0.07 4.58 i 0.05 3.87 i 0.02 3.70 * 0.04 4.44 i 0.00
e?Ca/42Ca, recovery, calcd % 3.70 4.42 4.59 4.02 3.71 4.43
98.1 97.0 99.9 96.3 99.7 100.2
Table 111with the values for the two normal urines listed first. T h e average difference between the ratios of the two sets of samples is 0.73% indicating t h a t there is no significant systematic difference between them (two-sided t test, P > 0.05) showing t h a t use of a cation exchange extraction offers no advantage over direct purification and concentration by precipitation. T h e ability of our method to measure calcium isotope ratios accurately in real biological samples was determined by a label recovery experiment. An aliquot of 82.9 atom % 44Cawas added to six normal urine samples, the calcium extracted by oxalate precipitation, and the 44Ca/42Caratio measured. These observations were compared to the ratios expected from calculations based on independently determined calcium concentrations, i.e., atomic absorption spectrometry and an EGTA titrametric method with a fluorescein end point (Calcette). T h e results are given in Table IV. The mass spectrometric measurements are given in the second column and expected ratios calculated from the average of atomic absorption and Calcette measurements are given in the third column (estimated standard deviation of 3%). It is seen that the measured ratios agree quite well with the calculated values; t h e average difference between the observed and calculated values is 1.3 7’0, a difference t h a t is not significant (two-sided t test, P > 0.05). Comparison of Results with Other Thermal Ionization Methods. Before the development of this thermal ionization method was begun, we attempted to use various calcium pdiketonates as the basis of the isotope ratio measurements. We were unable to find any chelating agent, including the use of the intramolecular P-diketonates of Alberts and Cram ( I I ) , t h a t gave satisfactory results (12). Our results show an average relative accuracy of about 1% for natural abundance isotope ratio measurements, and an ability to determine urinary calcium isotope ratios that differ by about 1%from expected results. T h e overall reproducibility of all our isotope ratio measurements is about 1%. Although these measurements are less accurate and reproducible than those typically obtained in thermal ionization studies ( 4 , 5 ) , they are acceptable for biological studies and
the overall ease of making the measurements, the short times involved per measurement, and the wide availability of the quadrupole instrumentation more than compensate for decreased accuracy. The present determinations were made in approximately 10% of the time required for those of the typical measurement ( 4 ) . In addition, the present measurements did not require the use of a specially designed mass spectrometer. A single other report of a quadrupole mass spectrometer being used for thermal ionization studies has appeared recently (13) but has not been applied to the measurement of calcium isotopes. The methodology used by these workers for the measurement of uranium isotope ratios and trace metal studies involves a special thermal ionization source in conjunction with the quadrupole filter. High ion transmissions are observed, but, as with most magnetic thermal ionization mass spectrometers, one is faced with breaking vacuum betwen sample filaments when using such a system.
CONCLUSIONS T h e data reported show t h a t our method for measuring calcium isotope ratios provides results t h a t are sufficiently accurate and reproducible to be used with confidence in the analysis of calcium isotopes in urine. The method is one that is likely to be easily applicable to a wide variety of mass spectrometer types since the major instrumental modification is to the solids probe. In addition, the simplicity and ease of sample preparation should make it an attractive method for analysis of calcium isotopes from other sources such as feces and milk that can be made to yield ionic calcium t h a t is relatively free of organic contamination. LITERATURE CITED Aubert, J. P.; Milbaud, G. Biochim. Biophys. Acta 1960, 3 9 , 122-139. Harris, W. H.; Heaney, R. P. N. Engl. J . Med. 1969, 280, 193-202. Moore, L. J.; Lim, M. 0.; Machhn, L. A,; Hansen, J. W. Unpublished wcfk, Neonatal & Pediatric Research Branch, NIH, 1978. Moore, L. J.; Machlan, L. A. Anal. Chem. 1972, 4 4 , 2291-2296. Stahl, W. R o c . Int. Conf. Mass Spectfosc. Kyoto, 1969, p 707. Kolthoff, I . M.; Sandell, E. B. “Textbook of Quantitative Inorganic Analysis”. 3rd 4.;The Macmillan Co.: New York, 1952; Chapter XXI. Hansen, J. W.; Gordan, G. S.;Prussin, S. G. J. Clin. Invest. 1973, 52, 304-315. Yergey. A. L.; Clem, T. Unpublished work, IRP. NICHD, NIH, 1979. Inghram, M. C.; Chupka, W. A. Rev. Sci. Instrum. 1953, 24, 518-520. Russell, W. A.; Papanastassiou. D. A.; Tombrello, T. A. Geochim. Cosmochim. Acta, 1978, 42. 1075-1090. Alberts, A. H.; Cram, D. J. J. Am. Chem. SOC.1977,99, 3880-3882. Yergey, A. L. Unpublished work, IRP, NICHD. NIH, 1979. Huber, W. K.; Rettinghaus, G. 8th International Mass Spectrometry Conf., 1979, Oslo, Norway, Aug. 12-18.
RECEIVED for review March 18,1980. Accepted June 16,1980. A preliminary report of this work was presented a t the 27th Annual Conference on Mass Spectrometry and Allied Topics, Seattle, Wash., June 3-8, 1979.
