2474
Anal. Chem. 1905, 57,2474-2470
Determination of Nanogram Quantities of Vanadium in Biological Material by Isotope Dilution Thermal Ionization Mass Spectrometry with Ion Counting Detection J. D. Fassett* and H. M. Kingston Center for Analytical Chemistry, National Measurement Laboratory, National Bureau of Standards, Gaithersburg, Maryland 20899
A procedure has been developed for the determlnatlon of nanogram quantities of vanadlum In biological matrices by Isotope dllutlon mass spectrometry that uses a 50V spike enriched to 64 atom %. The V Is chemlcally purifled by a Chelex-100 separation and loaded onto a carburlred Re fllament. Stable signals of 10'-10~ Ions per second are malntalned for greater than 30 mln with 1 pg V samples. Corrections for 50Cr and "TI are made to the 50V signal by measurement of 52Crand 4% The chemlcal blank has been reduced to 150 pg. The V, Cr, and TI chemical blanks are the limltlng sources of error at the nanogram level. The procedure has been applied to the determlnatlon of V In varlous NBS Standard Reference Materials: Oyster Tissue, SRM 1566; Citrus Leaves, SRM 1572; Bovlne LIver, SRM 1577a; and Human Serum, SRM 909. The concentratlon levels in these materlals range from 2.6 ng/g In the Human Serum SRM to 2.3 pg/g in the Oyster Tissue SRM wlth measurement preclslons ranglng from 11 % to 0.28 % relatlve standard devlatlon, respectlvely. The certified concentratlon of V In the Human Serum SRM Is the lowest of any Standard Reference Material.
Data on the V content of biological materials are both sparse and highly conflicting, presumably because of the analytical difficulties attendant in measuring natural concentrations. It is now accepted that V is broadly distributed at low concentrations and is not concentrated in any particular organ or tissue in higher animals ( I ) . The V content of human serum has been reported a t 0.03 ng/g (2) and in the range of 10-400 ng/g in various human tissues ( I ) . The V content of foods and feeds is similarly very low, from less than 0.1 ng/g in vegetable and fruits to 2-10 ng/g in liver, fish, and meat. The daily dietary intake of V is on the order of g, and can be expected to vary widely (3). Vanadium is a recently recognized essential trace element (3, although a specific physiological role for V has yet to be established. Studies have demonstrated that V deficiency is manifested in impaired growth and reproduction in both chicks and rats (2). Knowledge is clearly incomplete on the conditions that produce V deficiency, as well as on the dietary components that affect V metabolism. As with most essential elements, V is toxic at excessive concentrations, as demonstrated on animal models. Vanadium poisoning in man has not been documented. The ability to measure V at low levels with high precision and accuracy is essential for establishing the natural levels and defining the role of V in nutrition and health. High sensitivity measurements of V are made by neutron activation analysis (NAA). A multielement NAA method has been reported with detection limits of 0.8 ng/g (4).The first reliable measurements of V in human serum, as stated above (2), were made by NAA which demonstrated a relatively small This article not subject to
Table I. Sources of Vanadium Blank for Human Serum (SRM 909) Analysis
reagents HNO, HClO, CH,COONH, H2O ",OH
vanadium amt vanadium concn, used, total, Pglg g Pg 1.8 13 24 7 14 2.0 1.0 22 22 5 0.5 10 0.7 13 9
loading blank: range 2-41 pg, average 19 pg total of known sources of blank:
93 pg
containers resins environmental contamination total of unknown sources of blank: measured blank: range 100-170 pg, average
5 1 pg
150 pg
range of V in women (standard deviation, 0.010 ng/mL, N = 36), a larger range in men (0.024-0.939 ng/mL, N = 36). One ICP-AES limit of quantitative determination for V has been stated as 0.7 ng/g (5), although demonstration of this limit in the analysis of biological materials a t the nanogram per gram level was not made. Mianzhi and Barnes state a limit of quantitation for V in serum at 1.9 ng/g by ICP-AES;bovine serum and human serum were analyzed and levels of 82 and 98 ng/g of V, respectively, were determined (6). A major hindrance in the development of sensitive and accurate methods of analysis has been the lack of reference materials that reflect natural levels of V. Measurements of V in geochemical and cosmochemical materials have been made by gas source mass spectrometry using VOFB (7) and by double filament thermal ionization mass spectrometry (8). Although the primary purpose of these studies was the observation of variations in the isotopic composition of V, isotope dilution measurements were also made by Balsiger et al., whose procedure for the determination of V showed a reagent blank of 150 ng and chemical yields that were on the order of 30% (9). The mass spectrometric procedure described here has picogram sensitivity using single-filament thermal ionization with pulse counting detection. The use of high sensitivity mass spectrometry is promoted by the development of clean and efficient dissolution and separation procedures which reduce the uncertainty from blank variability to a relatively small level.
EXPERIMENTAL SECTION Reagents and Laboratory Ware. High-purity reagents, produced by subboiling distillation at NBS (10) and stored in Teflon (FEP) bottles, were used in this study. All reagents were analyzed by isotope dilution mass spectrometry prior to use in sample digestion. Specific reagents were purified to reduce the
US. Copyright. Publlshed 1985 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
vanadium blank levels. Ammonium acetate was prepared from high-purity ammonium hydroxide and acetic acid and purified by passing a 1-L quantity through a Chelex-100 resin. The concentrationsof V in reagents used are included in Table I, which is further discussed below. The Chelex-100 resin (Bio-Rad Laboratories, 200-400 mesh) was poured as a slurry into acid cleaned 5-mL polypropylene columns. Each column contained 3.2-3.4 mL of hydrated resin. The resin was cleaned in situ with four column volumes of 2.5 mol/L "OB, rinsed with water, and reactivated with three column volumes of 2.0 mol/L ammonium hydroxide. The resin was rinsed with water prior to use and conditioned with 3 mL of 1.0 mol/L ammonium acetate previously adjusted to pH 6.2 (11). The cation exchange resin (Bio-RadLaboratories, AG50X8, 100-200 mesh) was placed in acid-cleaned 5-mL polypropylene columns. Each column of 4 mL of hydrated resin was prepared by washing with 4 column volumes of 6 mol/L HC1 and 5 volumes of 0.05 mol/L HC1 and 1% H202 All sample handling was performed in a clean room equipped with laminar flow hoods which met Class 100 specifications (12). The enriched WV spike was purchased from Oak Ridge National Laboratory (ORNL). Spike solutions were prepared in dilute "OB and were stored in Teflon bottles. The isotopic composition of the spike was determined in the course of the measurement program and was 36.089% 50V and 63.911% 51V (60V/51V= 0.56468, standard deviation of a single measurement, s = 0.001 13, number of determinations, N = 15) whereas natural V is 0.25% 50V and 99.75% 51V (13). The V concentrations of the spike solutions were determined mass spedrometrically compared to gravimetrically prepared solutions of natural V. Long-term stability of the V spike solutions has been verified by calibrations spanning 2 years. The Re V-shaped filaments were filled with a slurry of spectroscopically pure graphite in acetone. These filaments were outgassed at 1750 OC for 30 min in vacuo under a potential field. Chemical Preparation and Separation Procedures. The chemical procedure consisted of spiking and dissolution of the sample followed by high yield separations of the V. The measurement procedure centered on a column chelation separation of v. Six samples, one each from six different bottles of oyster tissue, were placed in weighing bottles and dried in a vacuum chamber for 24 h at a pressure of 30 Pa using a cold trap as specified in the SRM certificate. Digestion of the spiked, weighed samples was accomplished in a mixture of HN03, HF, and HClO, in open Teflon (FEP) beakers. The resulting solutions were taken to dryness; the residues were redissolved in 4 mL of 0.1 mol/L HC1 and 1% H2OP Each solution was heated for 10 min and then added to a column containing 5 mL of 100-200 mesh cation exchange resin (Bio-Rad AG 50x8). The column was washed with five 2-mL volumes of 0.05 mol/L HC1 and 1% H202. Both the sample solution and wash solutions were collected and taken to dryness. The isotopically equilibrated V fractions were redissolved in 5 mL of 1 mol/L ammonium acetate previously adjusted to pH 6.2. The samples were added to a 3.5-mL column of Chelex-100 chelating resin. Five 3-mL aliquots of the ammonium acetate solution were applied to the column and the eluate was discarded. A 2-mL portion of water was applied to the column followed by four 3-mL aliquots of 2x)mol/L ammonium hydroxide (11-14). The ammonium hydroxide fraction containing the V was collected in a Teflon beaker. The sample solutions were taken to dryness several times with 1-2 drops of dilute HCl. The sample was redissolved in a single drop of 5 mol/L HC1, resulting in vanadium oxychloride. Six samples, one each from six different bottles of citrus leaves, were dried at 85 "C for 2 h. Digestion and separation procedures were essentially the same as for the oyster tissue. Six samples, one each from six different bottles of bovine liver, were dried for 24 h at 30 Pa. Samples ranging from 2 to 3 g were weighed into Teflon (PFA) open beakers and digested using a combination of high purity, concentrated HN03 and HCIO1. The digested samples were dried and then redissolved in 5 g of 1mol/L ammonium acetate (pH 6.2). The pH of the samples was monitored and, if necessary, adjusted to between 5.3 and 5.6 by the addition of dilute ammonium hydroxide. The V was then separated using Chelex-100. After separation, the samples were
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Table 11. Chemical Blanks, ng determination
oyster tissuen
citrus leavesu
bovine liver
human serum
2.35 1.30 0.34
2.32 3.07 4.09
0.39 0.54 0.30
0.17
1.33
3.16
0.41
1 2
3 4 5
0.10 0.14 0.16 0.16
mean
0.15
a Determined with more concentrated spike and overspiked ratio measurement.
"
52
50
51
49
Mass
Figure 1. Mass scan of loading blank containing 7 pg of natural V and 1 ng of V spike. The V slgnal was slowly decaying. Time of scan was 500 s. further treated with H2S04and HCIOl to destroy residual organic material. Seven human serum samples, one each from seven containers, were sampled according to the protocol suggested for this SRM. The samples were weighed by difference and were transferred using all-polyethylenesyringes and Teflon needles. The samples were digested using HN03 and HCIOl in closed Teflon (PFA) vessels. Blank Measurements. Mass spectrometric loading blanks and chemical blanks were measured in this study. The mass spectrometric loading blank was determined by directly loading known amounts of the spike solution on the Re filaments in the same manner as the samples. The measured loading blanks averaged 19 pg with a range from 41 to less than 2 pg (n = 8). Chemical blanks were measured by using 9 to spike quantities of the reagents equal to those used to dissolve the samples, evaporating this material to dryness, separating the V in the same manner as a sample, and determining the V by isotope dilution. The blanks determined for the four materials analyzed are summarized in Table 11. The blank level progressively decreased in this measurement program. Mass Spectrometry. A single magnetic sector thermal ionization mass spectrometer of basic NBS design, which possessed a high-sensitivity ion counting detector system, was used. This instrument has been described previously (15). The chemically separated V sample was dried in the bottom of a Teflon beaker, taken up in a drop of dilute HC1, and dried on the surface of the carburized Re filament. After the sample dried, additional graphite was added to the V filament by adding a drop of the slurry of graphite in acetone. This slurry was again dried under a heat lamp. The mass spectrometric procedure consisted of gradual heating of the filament in a controlled and reproducible fashion to 1400 "C. Mass scans were made prior to data acquisition which occurred in a 45-min window of the procedure. Figure 1 illustrates the mass scan for a loading blank in which 1ng of spike was loaded directly on the Re fiiament. The data acquisition procedure began with integration of the base line for 25 s at the low mass side of mass positions 52, 51, 50, and 49 before and after each 5oV/51V ratio set. The signals at masses 52 and 49 were integrated for 25 s both before and after the acquisition of the s'V/51V ratio set. A summary of the measurement sequence is illustrated in Table 111. Four ratio sets were taken and the ratios were corrected for
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985
Table 111. Measurement Sequence step
mass positionu
time on mass, s
1 2 3 4 5 6 7-15
52 49 48.5 51.5 50.5 49.5 50, 51, 50,
25 25 25 25 25 25 10 s each 25 25 25 25 25 25
etc. 16 17 18 19 20 21
52 49 48.5 51.5 50.5 49.5
purpose Cr interference Ti interference base line
ratio: 50/51 Cr interference Ti interference base line
" A 5-9 delay follows each shift in magnet position.
the contribution of 50Tiand 50Crto the 50V signal.
RESULTS AND DISCUSSION Chemical Separation. The primary goal in the chemical processing of samples is the efficient separation of V from the matrix in which it is contained. The process not only must be efficient but must be accomplished with a minimum amount of both reagents and manipulation to minimize contamination by V and subsequent changing of the V isotopic ratio established by mixing and equilibrating the sample and the spike. Because of the isobaric interferences caused by both Cr and Ti in the mass spectrometric measurement, it is critical that the process specifically eliminate these two elements. Equilibration of the natural and spike V is required for accurate measurement and must be assured in the digestion of a sample prior to any chemical manipulation. Vanadium has several oxidation states which can be present in acid solutions; the two primary forms are V(1V) and V(V). During the dissolution process the spiked sample was heated for several hours to fumes of concentrated HC104. This step left the V uniformly in the V(V) oxidation state. A procedure to separate V from many other metal ions using cation exchange was used as a preseparation step in the processing of the oyster tissue and citrus leaves. It relies on the affinity of the resin for most metal ions in very dilute acid and on the elimination of this affiiity in the case of V through the formation of peroxide complex. Under these conditions, V is oxidized to its +5 oxidation state and passes through the column quantitatively (16). The cation resin also allows the anions and many of the digestion products, such as phosphate from the organic matrix, to pass through the column with the V. Therefore, this separation could not be used alone to purify the V. The efficiency of the separation was checked using both 48Vand 51Crradiotracers. The V passed through the column quantitatively; several percent of the Cr also passed. The Chelex-100 separation uses a similar design to that used for the separation of trace elements from seawater (11). The effective removal of V from the chelator using ammonium hydroxide has been previously discussed (14). The V is chelated with other transition metals from the 1mol/L ammonium acetate buffer while the majority of the digestion products pass through the column and are eliminated. The V is chelated and eluted with greater than 98% efficiency under these conditions. The alkali and alkaline-earth elements are removed with the ammonium acetate washings. The Cr remaining in the V fraction is less than 2% of its original concentration. The retention of other metal ions was evaluated by use of 54Mn,one of the most weakly held transition metals, and "Wd. These metals were quantitatively retained by the resin and were not eluted with the V. They may be
eluted after the V using an acid solution (11). Although the hydrogen peroxide was not used in the Chelex-100 resin separation, it was tested to determine its affect on the V-Chelex complex. The chelation of the V by the resin was prevented by a 1%HzOzconcentration in the sample. This observation would indicate that once formed, the vanadium peroxide competes strongly with the Chelex-100 resin. This effect is relevant since these two separations are used sequentially. Complete destruction of the hydrogen peroxide in the sample is necessary between the ion exhchange and chelating separations to prevent interference with the formation of the V-Chelex 100 complex. This combination of separations, the cation procedure followed by chelation chromatography, gave the best separation of V from Cr and Ti. However, this combination of separations also limits the analysis to samples with higher levels of V, such as the oyster tissue and citrus leaves, because of the relatively high blank caused by the increase in reagents and laboratory manipulation when two distinct separation steps are used. The blank levels associated with the oyster tissue and citrus leaves precluded the use of the above separation procedures for the determination of the V in bovine liver and human serum samples. Reduction of the blank was achieved for these materials by eliminating the cation separation, improving reagent purity, reducing the quantities of reagents, and optimizing and improving experimental conditions and sample handling methods under clean laboratory conditions. When the chelation procedure is used, complete destruction of the protein in the human serum and bovine tissue samples is essential. Chelex-100 concentrates undigested amino acids and will release them in the ammonium hydroxide fraction with the V. For the bovine liver samples, open Teflon (PFA) beakers were used in the digestion. In contrast, sealed Teflon (PFA) digestion vessels were used in the digestion of the human serum samples. These sealed digestion vessels enabled the use of higher temperatures and higher pressures. As a result, the complete decomposition of the organic matrix was achieved in much less time than the conventional procedure using the open beakers. Perhaps more importantly, the sealed vessels prevented the loss of acid, which reduced the amounts of reagents required in the dissolution. The contribution of V from the reagents to the blank in the serum analysis is summarized in Table I. It is seen that the major source of blank can be attributed to the reagents used in dissolution and separation. Thus, these closed digestion vessels were a major factor in producing the smallest blank of the four materials analyzed, as indicated by Table 11. The sealed digestion procedure, coupled with the use of chelation separation, reduced the blank by an order of magnitude. A significant increase in the relatiue level of interference from the isobars of Cr and Ti for both the bovine liver and human serum samples was also observed. Further, specific chemical separation of V from the residual Cr and Ti is feasible. However, further separative procedures will inevitably increase the V blank. Clearly, there exists a trade-off between sample blank corrections and measurement interference corrections which must be balanced in the separation chemistry. Both the accuracy and precision of the ultimate measurement must be considered. Mass Spectrometry. The success of these experiments was contingent upon the development of a single-filament thermal ionization procedure with relatively high ionization efficiencies and low backgrounds of V, Cr, and Ti. Initial experiments in which V salts were dried directly upon Re filaments, or dried directly followed by H2 reduction (171, resulted in erratic and inefficient mass spectrometric behavior. It was observed that VO was preferentially evaporated and
ANALYTICAL CHEMISTRY, VOL. 57, NO. 13, NOVEMBER 1985 ~~~
Table IV. S°Crand sOTi Corrections to the Measurement, '70 chromium range median
material oyster tissue citrus leaves bovine liver human serum
0.33 0.22 2.61 8.45
0.00-0.99 0.05-1.44 2.08-17.7 2.16-19.34
'OV
titanium range median 0.04-2.40 0.84-5.42 4.10-9.23 4.13-21.2
0.18 2.23 6.02 10.14
ionized at relatively low temperature (1200 "C). Since this mass spectrometric behavior is analogous to U, a single-filament procedure developed for the measurement of U isotopes was attempted. This procedure consists of mixing the V sample (dissolved in HC1) with graphite and drying, which results in the in situ reduction of V prior to vaporization and ionization in the mass spectrometer. The procedure resulted in a controlled emission of V+ ions in the temperature range of 1350-1450 "C (measured pyrometrically without correction). An estimate of the ionization and mass spectrometric transmission efficiency for V was made by integrating the ions detected for a known number of atoms loaded upon a filament. A sample containing 1 ng, or 1.2 x 1013atoms, of V resulted in detection of between lo7 and los ions for a measurement efficiency of 104-10-6. Although this efficiency does not match the 2 X efficiency reported for U (18),the efficiency of both elements is within an order of magnitude of ionization efficiencies calculated from the Saha-Langmuir equation using the experimental temperatures, a work function of 5.25 eV for carburized Re, and ionization potentials of 6.0 eV for U and 6.7 eV for V. The mass spectrometric loading procedure was demonstrably low in contamination by V, Cr, and Ti, as illustrated by the loading blank of Figure 1. The correction to the measured signal at mass 50 for @Crranged from 3.12 to 5.80% or 18-33 pg equiv of the correction to the measured signal at mass 50 for 5oTi ranged from 0.36 to 1.59% or 2-9 pg equiv of "V. The corrected 50V/51Vratio showed a -1.1% deviation from the spike ratio, which represents a loading blank of 7 pg of natural V. Contamination by Cr and Ti has the potential for severely comprising the quality of V mass spectrometric measurements. The observed mass spectrometric behavior of these two elements is considerably different. The optimum temperature for Cr emission is at a lower temperature than for V, whereas the optimum temperature for emission of Ti is at a higher temperature than V. A Cr signal, if present, tends to decrease in time relative to V, while a Ti signal, if present, tends to increase in time relative to V. Corrections for 50Tiand 50Crinterferences were made by measuring 49Ti and 52Crand using the known natural isotopic abundances of these elements, 9 i / 4 g I= 0.954 and mCr/52Cr = 0.051858 (13),to determine the interferences. Although @Ti, which is 13.6 times as intense as 4?l?i,could have provided a more precise measurement of the 50Tiinterference, it was not measured because of the potential interference of this isotope by 48Ca. A significant Ca signal was observed in the serum samples. Table IV summarizes the Cr and Ti corrections made in the materials analyzed here. No other inter-
ferences were observed in the mass spectrometry of V; the mass region from m / e 49 to 52 is free of thermally ionized hydrocarbon species. Measurement Uncertainties. The sources of uncertainty in this procedure are the spike calibration, isotopic ratio measurement, blank correction, and fractionation correction. Two isotopic spikes were used in this study: a more concentrated spike was used in the isotope dilution of the sample materials and a less concentrated spike was used in the determination of blanks. The concentrated spike was 48.68 X mol/g and calibrated with gravimetrically prepared natural V solutions. The uncertainty in this spike calibration was 0.15% (Is, N = 4). The uncertainty in measurement of the mass 50/mass 51 ratio is dependent upon the absolute signal levels, and thus, the amount of V present on the filament. The uncertainty of the calculated 5oV/51Vratio includes the uncertainty due to the Cr and Ti corrections. The uncertainty in the calculated ratio will increase as the magnitude of these corrections increases. The uncertainty in the 50V/51Vratio is magnified in the calculation of concentration by isotope dilution (19). This magnification factor, defined as the ratio of relative uncertainty in concentration to the relative uncertainty in the measured isotope ratio, is dependent upon the ratio of spike to sample and the isotopic purity of the spike and sample. The effect of the ratio measurement uncertainty on the calculated concentration is highest for the material with the lowest level of V, which is the human serum, because of all the above mentioned factors. Murphy (20) and others have noted that the analytical blank is often the limiting factor in ultratrace determinations and thus must be extensively characterized and controlled. The three major sources of blank result from the sampling and storage process, contamination from the environment during chemical processing, and contamination from the reagents and materials used in chemical processing. Environmental contamination is controlled in clean areas during sample handling and chemical separations (12). The uncertainty in the blank correction must be included in any statement of uncertainty for the calculated concentration in isotope dilution. The absolute blank correction for each material is summarized in Table 11. In high-precision isotopic ratio measurement procedures, the reproducibility of isotopic fractionation is often the limiting source of measurement uncertainty. In isotope dilution analysis, fractionation is not a systematic error if it is the same in the isotopic and concentration calibration of the spike and in the isotopic and concentration determination in the sample. To guarantee this assumption, it is important to carefully reproduce all aspects of the mass spectrometric procedure to minimize possible systematic changes in isotopic fractionation. The relative contributions of the sources of uncertainty are listed ih Table V for the materials analyzed here. The typical uncertainty in measured ratio, column 1,is the mean standard deviation for the four ratio sets measured per sample. The error magnification factor, as discussed above, typical for each material is listed in column 2. The product of the error magnification factor and the uncertainty in the ratio directly reflects the uncertainty in the calculated concentration caused
Table V. Measurement Uncertainties, %
material
ratio
oyster tissue citrus leaves bovine liver human serum
0.22 0.20 0.36 0.53
ratio measurement magnification actual 1.25X 1.8X 2.8X 4.2X
0.28 0.36 1.01 2.23
2477
blank correction
uncertainty
spike uncertainty
0.07 1.3 0.16 0.53
0.05 0.36
0.15
0.05 0.10
0.15 0.15
0.15
std error 0.16 0.28 0.51 1.12
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Anal. Chem. 1985, 57. 2478-2481
Table VI. V Concentrations, bg/g
material
determn
oyster tissue citrus leaves bovine liver human serum
6 6
5 7
concn 2.316 0.245 0.0987 0.00263
RSD, % 0.28 2.0 1.6 11.6
by the ratio measurement. The relative magnitude and uncertainty of the blank correction are listed in columns 4 and 5, and the relative uncertainty of the spike concentration is listed in column 6. The calculated standard error which combines the sources of uncertainty (21) is listed in column 7 of Table V ( N = 4 for ratio measurement; N = 3-5 for blank measurement; N = 4 for spike calibration). Vanadium Content. The V contents of the materials analyzed here are summarized in Table VI. The measurement uncertainty, as expressed by the standard deviation of the six or seven samples analyzed, includes the above-mentioned uncertainties for a single measurement and any uncertainty caused by sample inhomogeneity. The reproducibility of the mass spectrometric measurement procedure was not explicitly checked by replicate determinations of V in subsamples from a single dissolved sample. However, the measured uncertainty for replicate determinations from different bottles of each SRM can be compared with the uncertainties associated with single measurements for each SRM (column 7, Table V). It is possible that the significantly larger measurement uncertainty for the replicate determinations of V in citrus leaves and human serum is caused by the variability of V in these materials. Nonetheless, such inhomogeneity is minor at the ultralow V levels of these materials, which can now be used for the validation of V ultratrace measurement in biological matrices. Registry No. V, 7440-62-2.
LITERATURE CITED (1) Underwood, E. J. “Trace Elements in Human and Animal Nutrition“; Academic Press: New York, 1977; pp 388-397.
(2) Cornelis, R.; Versieck, J.; Mees, L.; Hoste, J., Barbier, F. 8iol. Trace Elem. Res. 1981, 3 , 257-263. (3) Nielson, F. H. I n “Inorganic Chemistry in Biology and Medicine”; Martell, A. E., Ed.; American Chemical Society: Washington, DC, 1980; pp 32-35. (4) Ward, N.; Bryce-Smlth, D.; Minskl, M.; Zaaljman, J.; Pim, B. I n “Trace Element-AnalyticalChemistry in Medicine and Biology, Vol. 2”; Blatter, P., Schramel, P., Eds.; Waiter de Gruyter: New York, 1983; pp 483-498. (5) Haas, W. J.; Fassel, V. A.; Grabau, F.; Kniseley, R. N.; Sutherland, W. L. I n “Ultratrace Metal Analysis in Biological Sciences and Environment”; Amerlcan Chemical Society: Washington, DC, 1979; p 93. (6) Mianzhi, 2.; Barnes, R. M. Appl. Spectrosc. 1984, 38, 635. (7) Flesch, G. D.; Capellen, J.; Svec, H. J. Adv. Mass Spectrom. 1966, 3 , 571. (8) Balsiger, H.; Mendia, M. D.; Peily, 1. 2 . ; Llpschutz, M. E. Earth Planet. Scl. Lett. 1976, 28, 379-384. (9) Peily, I.2.; Llpschutz, M. E.; Balsiger, H. Geochim. Cosmochim, Acta 1970, 34, 1033-1036. (10) Kuehner, E. C.; Alvarez, R.; Pauisen, P. J.; Murphy, T. J. Anal. Chem. 1972, 4 4 , 2050-2056. (11) Kingston, H. M.; Barnes, I.L.; Brady, T. V.; Rains, T. C.; Champ, M. A. Anal. Chem. 1978, 50, 2064. (12) Moody, J. R. Phiios. Trans. R . SOC.London, A 1962, 305, 669-680. (13) Peiser, H. S.;Holden, N. E.; De Bievre, P.; Barnes, I. L.; Hagemann, R.; DeLaeter, J. R.; Murphy, T. J.; Roth, E.; Shima, M.; Thode, H. G. Pure Appl. Chem. 1984, 56, 695. (14) Riley, J. P.; Taylor, D. Anal. Chim. Acta 1968, 4 1 , 175-178. (15) Shields, W. R., Ed. ”Analytical Mass Spectrometry Section: Summary of Activities;” National Bureau of Standards: washington, DC, 1967; NBS Tech. Note (U.S.) 426. (16) Fritz, J. S.; Abbink, J. E. Anal. Chem. 1982, 3 4 , 1080. (17) Moore, L. J.; Machlan, L. A.; Shields, W. R.; Garner, E. L. Anal. Chem. 1974, 46, 1082. (18) Chen, J.; Wasserburg, G. J. Anal. Chem. 1981, 53, 2060-2067. (19) Jamleson, R. T.; Schreiner, G. D. L. I n “Electromagnetically Enriched Isotopes and Mass Spectrometry”; Smith, M. L.; Ed.; Butterworths Sclentiflc Pubiicatlons: London, 1956; pp 169-176. (20) Murphy, T. J. I n “Accuracy in Trace Analysis: Sampling, Sample Handling, and Analysis”; Natlonal Bureau of Standards: Washington, DC, 1976; NBS Spec. Publ. 422, pp 509-539. (21) Eisenhart, C. Science 1969, 160, 1201-1204.
RECEIVED for review May 20, 1985. Accepted July 1, 1985. Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.
Graphite Furnace Atomic Absorption Spectrometry with Nitric Acid Deproteinization for Determination of Manganese in Human Plasma Kunnath S . Subramanian* and Jean-Charles Meranger Environmental Health Directorate, Health and Welfare Canada, Tunney’s Pasture, Ottawa, Ontario K I A OLZ, Canada A nltrlc acid deprotelnlzatlon-graphlte furnace atomlc absorptlon spectrophotometric method has been developed for determlnlng nanogram per mllllllter levels of manganese In human plasma samples. Values for manganese In the sample are obtained by the use of matrlxmatched callbratlon graphs. The detection llmft (three standard devlatlons of blank) for manganese Is 0.1 ng/mL, and Is sufficiently low for base llne studies. Data are presented on the degree of accuracy and preclslon of the method. At least 15 samples can be analyzed per hour. The sensitlvlty and slmpllclty of the procedure make It attractlve for routine environmental surveillance Involvlng large throughput of samples.
The biological role of manganese has not yet been com-
pletely elucidated because of the difficulty of measuring this metal in biological materials (1). Manganese occurs at the sub-nanogram-per-milliliterlevels in human plasma or serum (2) necessitating the use of very sensitive analytical methods. Among the various techniques used for measuring manganese in plasma/serum, electrothermal atomization atomic absorption spectrometry (ETAAS) is gaining increasing attention because of its excellent sensitivity and selectivity for this metal. Several ETAAS methods have been published for manganese in plasma/serum (3-6). Sample treatments vary widely and include none (7-11), dilution with water (3, 12, 13), Triton X-100 (4, 14), and ethylene glycol (5), and matrix-modification with ammonium oxalate (6). In our hands all these methods resulted in the deposition of a carbonaceous crust in the graphite tube partially obstructing the light path
0003-2700/85/0357-2478$01.50/0 0 1985 American Chemical Society
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