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(5) Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa, S.; Singh, R. M. Biochemisfry~W66,5 , 467-477. (6) Edsall, J. T.; Wyman, J. Biophysical Chemlstry”; Academic Press: New York, 1958. (7) Bates, R. G. “Determination of pH, Theory and Practice”; Wiley: New York, 1964. (8) Perrin. D. D.; Dempsey, B. “Buffers for pH and Metal Ion Control”; Chapman and Hall: New York, 1974. (9) Smith, P. K.; Smith, E. R. B. J . Biol. Chem. 1042, 744, 737-745. (IO) Meyerhof, 0.;Lohmann, K. Biochem. Z . 1027, 785,113-119. (11) Grunwald, E.; Berkowitz, B. J. J . Am. Chem. SOC. 1051, 7 3 , 4939-4944.
(12) Barcarella, A. L.; Grunwaid, E.; Marshall, H. P.; Purlee, E. L. J . Org. Chem. 1055, 2 0 , 747-762. (13) Born, M. Z.fhys. 1020, 7 , 45-49. (14) Smith, L. A,; Caprioli, R. M. Biomed. Mass Spectfom. 1083, 70,
98-102.
M.;Smith, L. A. 31st Annual Conference on Mass Spectrometry and Allied Topics, Boston, MA, May 1983.
(15) Caprioli, R.
for review June
279
1983* Accepted September
1983.
Determination of Stable Isotopes of Calcium in Biological Fluids by Fast Atom Bombardment Mass Spectrometry David L. Smith Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112
Hlgh-resolutlon fast atom bombardment mass spectrometry was used to measure the Isotopic abundances of calclum In plasma and urine. Since the method Is very sensitive (>5 X 10‘’ lons/pg of calclum), only 2 to 5 pL of sample Is requlred. RepetRlve measurementsof the 42Ca/44Caratlo In plasma and urine require about 3 mln and typically have a relatlve standard devlatlon of 0.2-0.5 %. No sample preparation is necessary and measurements are made by using Instrumentatlon that Is often avallable In larger research laboratories.
As many as 20 different metals perform functions necessary for life, growth, and reproduction in mammals. Substantial information about the levels of both essential and nonessential elements in tissues of normal and diseased individuals has been obtained through use of atomic absorption, neutron activation analysis, and X-ray fluorescence spectrometry. In addition, radioisotopes have been used as tracers to elucidate processes by which metals are absorbed, distributed, stored, and excreted by the body. Our understanding of these processes is, however, particularly deficient in humans because ethical constraints severely restrict the use of radioisotopes. In general, radioisotopes may be ingested by children and child-bearing adults only in very special cases. Although stable isotopes of most of the essential metals are available highly enriched, they have been used as tracers for bioiogical studies in only a few instances. This is primarily because a satisfactory method of analysis of stable isotopes in biological fluids has not been available. Although the isotopic abundances of calcium may be measured by GC/MS analysis of volatile chelates (1, 2), thermal ionization (3),field desorption ( 4 ) ,and inductively coupled plasma (5,6) mass spectrometry, there are no reports in the literature indicating the clinical or routine use of these methods to follow the biological pathways of calcium. This is presumably because none of these methods satisfactorily fulfii all of the rigorous requirements of sensitivity, precision, and simplicity necessary for such studies. The results to be presented here show that high-resolution fast atom bombardment (FAB) mass spectrometry can be used to measure the isotopic abundances of calcium and may be well suited for tracer studies.
Fast atom bombardment mass spectrometry (7,B) has been used to desorb a variety of high mass organic ions which are initially dissolved in glycerol. The process by which ions are extracted from the surface into the gas phase is presumed similar, if not identical, to that found in secondary ion mass spectrometry (SIMS). However, use of a beam of neutral projectiles to desorb ions of interest makes the technique readily adaptable to high resolution mass spectrometers and reduces the probability of electrostatically charging the sample target, We report here the use of high-resolution FAB mass spectrometry to measure the relative abundances of %a, 42Ca, and 44Cain human plasma and urine.
EXPERIMENTAL SECTION The analyses were made with a Finnigan MAT 731 high-resolution mass spectrometer which was equipped with an FAJ3 gun (Ion Tech). A beam of xenon atoms with an energy of approximately 7 keV was used to desorb ions from the surface of the sample. The desorbed or sputtered ions were accelerated to 8 keV and mass analyzed in the usual manner. Ions present in mass windows, which had been selected so that only ions having the same exact mass as the calcium isotopes, were detected by use of a 16-stage electron multiplier and pulse amplifier/discriminator (GalileoPAD-401). The peak match unit was used to accurately and repetitively switch between the exact masses of the isotopes of interest. A signal averager (Nicolet 1170) was used to integrate ion intensities over time and to switch the peak match unit between the masses of interest. Since the intensity of the *Oca and 42Ca beams normally differ by approximately a factor of 100, it is important to spend a proportionately longer time monitoring the minor beam. This was achieved by using single channel dwell times of 10 ms and 0.10 ms for the 42Caand 40Casignals, respectively. A typical measurement was made by integrating the signals for 64 repetitive scans, and this required about 3 min. The sample tip used for these measurements,which was similar to one described by Martin et al. (9)was made from copper. The copper peaks at mlz 63 and 65 were used to tune the ion source, set the resolution, and calibrate the peak match unit. To make isotopic ratio measurements, the resolution was first set at 10000. The collector slit was then opened to give a flat top peak and a final resolution of approximately 5000. The blood was drawn from a chemically normal human adult in hepranized vacuum tubes and centrifuged to separate the erythrocytes from the plasma. From 2 to 5 pL of plasma or urine was placed on the probe tipe, dried in a stream of nitrogen, and
0003-2700/83/0355-2391$01.50/0 0 1983 American Chemical Society
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TIME ( M I N )
Flgure 2. The time dependence for the %a signal for 5 pL of plasma: m l A m = 5000, FAB gun power 8 W. Figure 1. Integrated ion intensity for mass intervals centered around the exact mass values of 40Ca, 42Ca, and 44Cafor a 2-pL sample of plasma. Peaks vertically aligned wlth the arrow are due to isotopes of calcium while other peaks are unidentified.
analyzed directly. A sample dispersing medium such as glycerol was not used since the analyte was monatomic. A reference sample of calcium was made by dissolving reagent grade CaClzin distilled water. Enriched W a (93.77%)was purchased from the Stable Isotopes Division of Oak Ridge National Laboratory.
RESULTS AND DISCUSSION Species composed of only a few atoms may be dislodged or sputtered from a surface which is bombarded by a beam of high-energy atoms. Emitted particles having an imbalance in charge, negative or positive, may be detected, identified, and quantitied by mass spectrometry. This observation may be used to measure the relative abundance of stable isotopes of calcium in biological fluids. Since the sample target is electrically conductive, high intensity, continuous beams of either neutral or charged projectiles may, in principle, be used. A beam of neutral xenon atoms was used for the present measurements. An important goal of the present method is to quantify the isotopes of calcium, which are present as only a minor constituent in a complex biological matrix, without the need for sample preparation such as ashing, extraction, or chelation. The technique of sputtering is used to nonspecifically remove ions from the sample. High-resolution mass spectrometry is used to isolate and quantify the ions of interest. Since calcium is very mass deficient in comparison to other ions present in the mass spectrum, a modest increase in mass resolving power is sufficient to separate calcium ions from other ions which have the same nominal mass. The necessity for using high resolution for the measurements is illustrated in Figure 1,which shows the ion intensity in mass intervals around the exact masses of 40Ca,42Ca,and W a present in a 2-pL sample of plasma. These mass spectra are the summations of 512 scans and were obtained by using a resolving power of 1OOOO. It is evident from these data that, while 40Cais the major ion a t m/z 40, the less abundant isotopes of calcium may be only minor contributors to the total ion beam at their respective nominal masses. Precise measurement of the abundances of calcium isotopes by FAB mass spectrometry can therefore be expected only if a high resolving power is used. Some ions may be present which cannot be resolved from the calcium ions with the resolving power available to most commercial high-resolution mass spectrometers. For example, there is a minor isotope (0.012%) of potassium at m / z 40 which can be resolved from @Caonly if a resolution of 28000 is used. The contribution of 40K to the 40Casignal was calculated by measuring the 41Ksignal d was negligible in the present measurements. Such interferences are regarded as
atypical since a resolving power of 3500 is sufficient to separate calcium ions from all other ions likely to be present in these samples. Thus, a resolving power of 5000 probably represents a good compromise between selectivity and sensitivity. The sensitivity of FAB mass spectrometry for calcium was determined by recording the @Casignal as a function of time for a 200-ng sample of calcium (555 ng of CaClZ).The normal analogue detection system was used for these measurements because the ion signal for pure salts was too high for the pulse counting system. The 40Casignal was integrated over time until the signal had decreased to about 30% of the peak value. For a resolving power of 5000, a sensitivity of 1 X C/pg was determined for calcium. Since more than 5 X 1O1O ions/pg are detected, FAB mass spectrometry may be used to quantify calcium in small amounts of sample. This feature is particularly important in studies of infants where only microliter quantities of plasma are available. To be clinically useful, the analytical method must be able to measure relative isotopic abundances with a standard deviation of less than 1% and preferably less than 0.1%. If ion statistics determine the precision of the measurement, lo6ions must be recorded for a standard deviation of 0.1%. The present sensitivity measurements show that the abundance of minor isotopes present at the 1%level may be precisely determined for subnanogram quantities of calcium. The ability of FAB mass spectrometry to detect calcium, which is present only as a minor constituent in a complex biological matrix, is illustrated in Figure 2. The calcium signal from a 5-pL sample of plasma was monitored by using the same instrumental conditions described above for the sensitivity measurements. Since the calcium concentration in plasma is normally about 90 ng/pL, this sample contained a total of approximately 450 ng of calcium. After evaporation of the water, calcium was present in the remaining matrix of proteins, lipids, salts, etc. at a level of approximately500 ppm. The results in Figure 2 show that the 40Casignal rises to a broad maximum of about 2 x IO5ions/s and persists above lo5 ions/s for over 20 min. Only a few seconds are required to record a statistically acceptablenumber of ions of the major isotope while a proportionately longer time is needed for minor isotopes. Other measurements show that the signal level may be increased by setting the FAB gun for higher power. These results were obtained by using an FAB gun power of 8 W. The data in Figure 2 were recorded with a 1-s time constant and show that the short term stability of the signal is good. A high rate of switching between the isotopes, or even simultaneous recording of the isotopes, is, therefore, not required for high precision measurements. A curve similar to the one in Figure 2 was also obtained for urine. The linearity of response was tested by spiking four aliquots of plasma with enriched 42Ca(93.77%). The quantity of spike was selected so that the 42Calevel increased from 5 to 35% above that due to endogenous calcium. The 42Ca/40Caratio
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
for the best thermal ionization measurements (IO),it is sufficient for most calcium tracer studies. This is illustrated in Figure 3 which is a plot of the normalized 42Ca/40Caratio vs. time from ingestion for urine samples from a 77-kg male who had ingested 4 mg of enriched 42Cadissolved in 250 mL of orange juice. The standard deviation for three replicate measurements of each sample is indicated by the error bars in Figure 3. It is evident from these results that only a relatively small quantity of enriched calcium is required for FAB mass spectrometry to follow absorption and subsequent excretion of calcium by an adult. In the case of infants, even smaller amounts of enriched calcium are required for clinically useful measurements.
1.140 1.1 20 1.1 0 0
I .08C
RI R’
1.060 1.040 1.02c
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ACKNOWLEDGMENT I
2
I
4
6
Q
TIME ( h r ) Flgure 3. Variation in the 42Ca/40Caratio in urine samples from a 77-kg male who had ingested 4 mg of 93.77% enriched 42Caat 0 h.
was measured for the four spiked samples and was normalized to the ratio of a fifth, unspiked, sample. When the measured ratios were plotted against the known percentage of spike as calculated from the gravametric preparation of the samples, a straight line with a correlation coefficient of 0.999 97 was found. Each of these measurements used 5 p L of plasma. The natural abundance 42Ca/40Caratio for a 5-pL sample of urine was measured 10 times to illustrate the typical precision of the present method. Each measurement was the s u m of 32 scans and required a total of 3 min. The mean value was 0.006 20 and the relative standard deviation was 0.3%. This is equal to the theoretical relative standard deviation, which is based on the number of ions recorded in each measurement. The observed precision for these measurements is therefore apparently limited by ion statistics and higher precision could, in principle, be achieved by counting for longer times. Although the precision of isotopic ratio measurements using the present instrumentation is not as high as that reported
The author thanks C. L. Atkin for helpful discussions and J. A. McCloskey for making the intrumentation available for this study. Registry No. Calcium-42,14333-05-2;calcium-44,14255-03-9; calcium-40, 14092-94-5.
LITERATURE CITED (1) Hachey, D. L.; Blals, J. C.; Klein, P. D. Anal. Chem. 1980, 52, 1131-1135. (2) Kowantzkl, R.; Peters, F.; Rell, G. H.; Haass, G. Biomed. Mass Spectrom. 1980, 7 , 540-543. (3) Yergey, A. L.; Vlelra, N. E.; Hansen, J. W. Anal. Chem. 1980, 52, 1811-1814. (4) Lehmann, W. D.; Kessler, H. “Stable Isotopes”; Elsevier: Amsterdam, 1982; pp 649-654. (5) Houk, R. S.; Thompson, J. J. Blomed. Mass Specfrom. 1983, 70, 107-1 12. (6) Douglas, D. J.; Quan, E. S. K.; Smith, R. G. Specfrochlm. Acta, Pari 8 1983, 388, 39-48. (7) Barber, M.; Bordoll, R. S.; Sedgewick, R. D.; Tyler, A. N. J. Chem. Soc., Chem. Common. 1981, 7 , 325-327. (8) Surman, D. J.; Vlckerman, J. C. J. Chem. SOC.,Chem. Commun. 1981, 7, 324-325. (9) Martin, S. A.; Costello, C. E.; Blemann, K. Anal. Chem. 1982, 54, 2362-2368. (10) Russell, W. A.; Papanastasslou, D. A,; Tombrello, T. A. Geochlm. Cosmochlm. Acta 1978, 42, 1075-1090.
RECEIVED for review June 20,1983. Accepted August 15,1983. Partial support for this work was from NIH Grant GM 21584.
