Anal. Chem. 1987, 59, 2570-2574
2570
Quantitation of Stable Isotopic Tracers of Calcium by Fast Atom Bombardment Mass Spectrometry Xiangyu Jiang and David L. Smith* Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907
Instrumentation and methodology developed for quantltation of stable isotopic traces in urine are described. Calcium is isolated from urine as the insoluble oxalate salt which is subsequently dissolved in hydrochloric acid. The Isotopic content of the acid solution is determined by use of a conventional mass spectrometer equipped with a fast atom bombardment ion source. Calcium ions are desorbed from the sample surface by a beam of high-energy xenon atoms and detected with a high-resolution mass spectrometer. A data acquidtlon system has been developed to control the mass spectrometer and record the ion signals. Detailed analysis of potential sources of error indkates that the precidon of the method is presently i M e d primarily by an mope effect that occurs during ion desorption. Results presented here demonstrate that the relative abundances of calcium isotopes In urine can be determined with high precision (coefficient of variation < 0.2%) and that the method Is a viable attemative to conventional thermal ionization mass spectrometry. The method is especiaHy attractive because it uses a conventional high-resolutlon mass Spectrometer which is routinely used for analysis of organic substances.
Stable isotopic traces of calcium could play a major role in extending our understanding of calcium absorption and metabolism in humans. Information derived from tracer studies will be particularly important for studies of calcium utilization in children because of their high rates of bone development. For example, absorption of calcium when ingested with specific foods can be determined by feeding human test subjects these foods plus a tracer isotope ( I ) . In addition, calcium absorption from specific foods, such as human milk, has been determined by intrinsically labeling the food (2). The tendency of highfiber diets to reduce calcium absorption in humans may also be determined directly by using isotopic tracers. Decay cuwes of tracers administered intravenously may be used to determine the turnover rate of the exchangeable calcium pool. This information has been used to quantitatively determine the rates at which calcium moves to and from the bone in infants (3) and adults (4). Despite the enormous potential for using stable isotopic tracers to study calcium nutrition and bone development in children, they have rarely been used because they are difficult to quantify. Although several methods have been used to determine isotopic abundances of calcium, each is fraught with significant obstacles. Thermal ionization mass spectrometry is the standard method for determining isotopic abundances of metals and has been used to establish accepted values for the natural abundances of the calcium isotopes. If the samples are vigorously purified, very high precision can be achieved with thermal ionization mass spectrometry (5). The isotopic abundance of calcium in samples which have been only partially purified have also been determined by thermal ionization, but with a concomitant reduction in precision (3, 6). Isotopic abundances of a variety of metals have also been determined by neutron activation analysis and inductively
coupled plasma discharge mass spectrometry (1, 7). Fast atom bombardment mass spectrometry (FABMS), a form of secondary ion mass spectrometry (SIMS), or particle desorption mass spectrometry (PDMS) provides the basis for an alternative method for determining the isotopic abundances of metals (8-11). This method uses a high-energy (8 keV) beam of xenon atoms to desorb ions from the surface of the sample. These ions are accelerated to high energy and analyzed by a high-resolution mass spectrometer. Because the sample has not been highly purified, high-resolution mass analysis is required to separate the calcium ions from other ions having the same nominal mass which are also sputtered from the sample surface. Although FABMS may not replace thermal ionizatior mass spectrometry as a reference method for determining isotopic abundances, it does provide a favorable compromise between sample preparation time and the precision with which isotopic abundances can be determined. In addition, the analysis is performed with a conventional high-resolution mass spectrometer which is routinely used for analysis of organic substances. Such instruments are generally more available than conventional isotope ratio mass spectrometers, and may, therefore, be the method of choice in some instances. The potential of FABMS for detecting isotopic tracers of calcium was demonstrated previously (8,9). The purpose of this report is to describe recent improvements in methodology with special emphasis on identifying those parameters which limit the accuracy with which tracers may be determined. EXPERIMENTAL SECTION All mass spectral analyses were performed on a Kratos MS-50 high-resolution mass spectrometer equipped with a high field magnet, a fast atom gun (Ion Tech, Model llNF), and a postacceleration detector which includes a 16-stageelectron multiplier. The FAB gun was normally operated with xenon and an anode current and voltage of 1mA and 6 kV, respectively. Similar results were also obtained with argon. Because of collision-induced broadening of the peak, the 110 L/s turbomolecular pump normally supplied on the analyzer was replaced with a 170 L/s pump. This modification was necessary to achieve a flat-top peak at a mass resolving power of 3500. Since the ion beam typically drifted less than 1 ppm/min, and since the flat portion of the peak was about 100 ppm wide, the reference beam remained centered in the collector slit for the duration of the analysis. The voltage on the conversion dynode was 4 kV. A relatively simple data system has been developed to select, deflect, and record ions. This system consists of an IBM personal computer with 512K memory, a Data Translation interface card (Model DT2801) which has a 12-bit analog-to-digital converter (ADC) and a 16-bit digital port. Specific bits in the digital port were set high or low, as required to control microrelays which selected the desired ion beam, or deflected all of the ions so that an electrometer zero reading could be taken. As many as four isotopes could be recorded automatically. Software has been written in BASIC especially for this application and was normally run in the compiled form for higher speed. Urine samples were prepared for isotopic analysis by adding two volumes of saturated ammonium oxalate solution (pH >9) to one volume of urine, allowing the mixture to stand for 30 min, and briefly centrifuging it. After the supernate was discarded, the calcium oxalate was dissolved in reagent grade hydrochloric
0003-2700/87/0359-2570$01,50/0C 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 21, NOVEMBER 1, 1987
acid (10%). Although 1 mL of urine was normally used in the preparation, only 1% of the sample was actually analyzed by FABMS. Much smaller quantities of urine would most likely give similar results. Isotopic analysis was performed by placing 1 pL of the solution on a stainless steel probe tip which had an area of approximately 4.5 mm2. The probe was subjected to the low vacuum system for approximately 1min to remove water before insertion to the high vacuum chamber where the sample was immediately bombarded by the neutral beam. The system was normally allowed to stabilize for 2 min before data acquisition was started. After each use, the probe tip was washed in deionized water, electrocleaned, washed in deionized water, and rinsed with acetone or methanol. The electrocleaning conditions (concentrated phosphoric acid, 0.1 A current, and 20 s) effectively removed calcium from previous samples. After extensive use, the probe tip became pitted and was polished with 600-grit paper. Total calcium content of urine and standards was determined by atomic absorption spectrophotometry. A calibration w e was made from solutions of calcium carbonate (NJ3S SR 915) dissolved in 0.5% lanthanum chloride.
RESULTS AND DISCUSSION Fast atom bombardment mass spectrometry is a potentially useful alternative to traditional mass spectrometric methods for determining isotopic abundances of metals present in biological materials. This method is attractive because it does not require a mass spectrometer specifically designed for isotope ratio measurements, nor does it require extensive sample preparation. This latter feature is especially important for studies of mineral nutrition and metabolism which often require analyzing many samples. Since the very isotopes used as tracers are normally present at their natural abundance levels, it is the change in isotopic abundance that must be determined to quantify stable isotopic tracers. Because of the desirability of using only tracer quantities of an enriched isotope and because enriched isotopes contribute significantly to the cost of a study, experiments are usually designed to keep the change in isotopic abundance small, typically less than 50%. It follows that, to be useful for quantitative isotopic tracer studies, the method must be able to determine ion abundances with high precision (preferably with coefficient of variation
