Anal. Chem. 1995,67,1568-1574
isotope Dilution Analysis of Selenium in Biological Materials by Nitrogen Microwave-InducedPlasma Mass Spectrometry Jun Yorhinaga,*st Toshihiro Shimsaki,* Konosuke Oishi,' and Masatoshi Morttat National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, lbaraki 305, Japan, and Hitachi Instmments Engineethg Co. Ltd. and Hitachi Ltd., 882 Ichige, Katsura, lbaraki 312, Japan
Nitrogen microwave-inducedplasma mass spectrometry was applied to isotope dilution analysis of selenium in biological matrices. The precision of isotope ratio (80Se/ '%e) measurements of a 100 ng/mL standard solution was 0.5%under practical analysis conditions and could be further lowered by increasing the integration time. Spectroscopic interference at m/z = 80 arising from SO3+ was observed, but the extent was not so much as to cause a serious problem at the sulfur level normally found in biological materials. Isotopic ratios in digested clinical specimens agreedwell witb those in the standard solution, indicating no other spectroscopic interferences on selenium isotopes in real samples. "he proposed method was succesdidly applied to the analysis of selenium in biological reference materials. "he results showed good agreement with the c e d e d values. Isotope dilution analysis is considered to be a defmitive method because it provides the most accurate values. In inorganic elemental analysis, thermal ionization mass spectrometry (TIMS) has been the most commonly used method for isotope dilution analysis. The TIMS analysis provides high sensitivity and good accuracy and precision, but at the same time the method requires tedious sample pretreatment and skillful operation. The use of inductively coupled plasma mass spectrometry (ICPMS) for isotope dilution analysis has been gaining wider acceptance because ICPMS is highly sensitive and requires minimum sample treatment, although analytical precision (0.11%)is not as good as that obtained by TIMS. A number of reports have been published on isotope dilution analysis of elements in a variety of matrices by ICPMS.1-9 Although ICP is an excellent ionization source, formation of molecular ions from plasma gas (Ar) and/or other matrix elements hampers sensitive analysis of ' National Institute for Environmental Studies. Hitachi Instruments Engineering Co. Lid. Hitachi Ltd. (1) McLaren, J. W.; Beauchemin, D.; Berman, S. S. Anal. Chem. 1 9 8 7 , 5 9 , 6 1 0 (2) Beauchemin, D.; McLaren, J. W.; Mykytiuk, A P.; Berman, S. S. Anal. Chem. 1987, 59, 778. (3) Garbarino, J. R;Taylor, H. E. Anal. Chem. 1987, 59, 1568. (4) Beary, E. S.; Brletic, IC A; Paulsen, P. J.: Moody, J. R Analyst 1987, 112, 441. (5) Beauchemin, D.; McLaren, J. W.; Berman, S. S. Anal. Chem. 1988, 60, t 5
687. (6) Paulsen, P.J.; Beary, E. S.; Bushee, D. S.; Moody, J. R Anal. Chem. 1988, 60, 971. (7) Wang, X.;Vczian, M.; LasZtity,A; Barnes, R M. J. Anal. At. Spectrom. 1988, 3, 821. (8) Klinkhammer, G. P.; Chan, L. H. Anal. Chim. Acta 1990,232,323. (9) Okamoto, K. Spectrochim. Acta 1991, 46B, 1615.
1568 Analytical Chemistry, Vol. 67,No. 9, May 1, 1995
some elements and isotopes. Selenium is one such example: major isotopes of selenium, Le., mlz = 76 (relative intensity 9.0), 78 (23.6), and 80 (49.7), are subject to severe interference from An+. Other isotopes of lower abundance are also subject to molecular interferences from halogens. Selenium is an important element from both nutritional and toxicological viewpoints. An association between marginal deficiency and occurrences of cancer or cardiovascular diseases has been proposed.1° Various analytical techniques have been estab lished for the determination of selenium in biological matrices. However, the demand still exists for a precise and accurate definitive analytical method. Besides TIMS, isotope dilution analysis of selenium has been performed using gas chromatograph mass spectrometry.ll ICPMS is also used for this purpose after separation of selenium from interfering molecules based on the hydride generation technique.12-14In general, however, these methods require intensive sample pretreatment, i.e., digestion, reduction to S e o ,and derivatization. In addition, the hydride generation technique suffers from reagent impurity and memory effect as well as from interference in the hydride-generating step. Shen et al.15J6demonstrated the possibility of using nitrogen microwave-induced plasma mass spectrometry (MIPMS) for selenium isotopic analysis because they found no spectroscopic interferences in the mlz region of selenium. They employed a moderate power (500W) nitrogen MIP therefore, a critical deficit of the system was lower sensitivity compared with that of ICPMS. Recently, Oishi et a1.l' and Okamotols reported operating nitrogen MIPMS at higher power (1.3 kW). This system could achieve sensitive detection, comparable to that of ICPMS, greater for many elements than that obtainable by the earlier low-power MIPMS system by a magnitude of 2-3. In the present paper we report isotope dilution analysis of selenium using MS with a 1.3 kW nitrogen MIP ion source by means of direct solution nebulization. (10) Levander, 0. A In Trace Elements in Human and Animal Nutrition; Meltz, W., Ed.; Academic Press: Orlando, FL, 1986. (11) Reamer, D. C.; VeiUon, C. Anal. Chem. 1981.53, 2166. (12) T i g , B. T. G.; Mooers, C. S.; Janghorbani, M. Analyst 1989, 114, 667. (13) Buckley, W. T.; Budac, J. J.; Godfrey, D. V.; Koenig, K. M. Anal. Chem. 1992, 64, 724. (14)Tao, H.: Lam, J. W. H.; McLaren, J. W. J.Anal. At. Spectrom. 1993,8,1067. (15) Shen, W.-L.; Davidson, T. M.; Creed, J. T.; Caruso, J. A Appl. Spectrosc. 1990,44,1003. (16) Shen, W.-L.; Davidson, T. M.; Creed, J. T.; C m s o , J. A Appl. Spectrosc. 1990,44,1011. (17) Oishi, K.; Okumoto, T.; Shirasaki, T.; Iino, T.; Koga, M.; Furuta, N. Spectrochim. Acta 1994, 49B, 901. (18) Okamoto, Y. J. Anal. At. Spectrom. 1994, 9, 745. 0003-2700/95/0367-1568$9.00/0 Q 1995 American Chemical Society
This technique enables precise, accurate, and rapid determination of selenium in biological samples.
%
EXPERIMENTAL SECTION Instrumentation. The instrument used was a nitrogen MIP mass spectrometer (Hitachi P-7000). A detailed description of the instrument is published elsewhere.17 Typical operating conditions for MIPMS are as follow: incident power, 1.3 kW; reflected power, e10 W plasma gas (nitrogen) flow, 13 Wmin; nebulizer gas (nitrogen) flow, 1.3 L/min; nebulizer, concentric type; temperature of spray chamber, 5 "C; sampling cone, 0.8 mm odice (Pt); skimmer cone, 0.4 mm orifice (Cu); sample uptake rate, 0.25 mL/ min. Reagents. All of the reagents used were of the purest quality available. Water used was Milli-Q purified water @W, 18 MP). Ultrapure nitric acid and hydrofluoric acid were purchased from Kanto Chemic4 Co. Ltd. (Tokyo, Japan). Selenium-78 spike (metal powder form) was purchased from Oak Ridge National Laboratory (Oak Ridge, TN). A 25 mg portion of it was dissolved with concentrated nitric acid and then diluted to 500 pglg with DW. Accurate selenium concentration in this solution was determined against standard selenium solution prepared from high-purity selenium metal (99.999%,Wako Pure Chemical Co., Osaka, Japan) by a reverse isotope dilution technique. Selenium standard solution prepared from a commericall000 mg/L standard for atomic absorption (Kanto Chemicals Co. Ltd., Tokyo, Japan) was also used for the optimization of the mass scanning condition. There were no detectable differences between selenium isotope ratios in standard solutions prepared from pure metal and those in commercial standard solution. Suprapur grade sodium nitrate, potassium nitrate, and calcium nitrate (Merck, Germany) and 99.999%ammonium dihydrogen phosphate (Aldrich Chemical Co. Inc., Milwaukee, WI) were dissolved in water to make 10 000 pg/mL solutions. An appropriate amount of each solution was added to selenium standard solution to test for matrix effect. A 10 000 pg/mL sulfur standard solution for ICP/DCP (Aldrich) was used for quantitative estimation of the S03+ interference. Sample Preparation. Approximately 100 mg of each biological reference materials (NIST 1577a bovine liver; BCR no. 186 pig kidney; BCR no. 397 human hair) was accurately weighed into a PFA vial. One milliliter of NIST 1598 bovine serum or reconstituted NIST 2670 freeze-dried urine was also accurately weighed. An appropriate amount of the selenium-78spike solution was then added to make the soSe/78Seratio in the spiked sample in the range of 0.5-1. For the natural selenium isotopic ratio analysis, no spike solution was added before acid digestion. The sample was digested with 2 mL of concentrated nitric acid at 140 "C for 4 h by the double digestion vessel method.19 After the sample was cooled, 100 pL of hydrofluoric acid was added, and the mixture was heated at 140 "C on a hot plate to dryness. Another 100 pL of nitric acid was then added, and the mixture was evaporated to dryness to remove residual hydrofluoric acid. The residue was dissolved with 100 pL of nitric acid, made up to 10 g with DW (ca. 0.14 M HNOJ, and filtered with a 0.45 pm membrane filter. A candidate certifed reference material (NIES no. 13 human hair, National Institute for Environmental Studies, (19) Okamoto, IC; Fuwa, K. A d Chem. 1984,56,1758. (20) IUPAC. Pure Appl. Chem. 1991,63,975.
Ibaraki, Japan) was also prepared as described above. Analytical blank was prepared in the same manner. All procedures were carried out in a class 1000 clean room. Mass scanning Condition. Mass scanning conditions to give optimum reproducibility of the isotope ratio measurement were examined using a 100 ng/mL selenium standard solution. Monitored isotope ions of selenium were m/z = 76, 77, 78, 80, and 82. The ion of m/z = 74 was not monitored because of its low abundance and isobaric interference from 74Ge. Relative standard deviations (RSDs) of five 80Se/78Seratio measurements were calculated and used as a measure of precision. In the first experiment, dwell time was fixed at 2 ms, and the dependence of RSD on the number of sweeps was examined. Correspondingly, the dependence of RSD on integration time was examined. Integration time per mass (dwell time x number of sweeps) tested was from 0.5 to 10 s. In the second experiment, integration time was fixed at 3 s/mass, and the dependence of RSD on the dwell time was examined. Dwell time was tested from 0.2 to 100 ms (corresponding to 30- 15 000 sweeps). Data acquisition was 1 point/mass, and mass scanning was done by peak jumping mode: it was fixed throughout the experiments. SpectroscopicInterference. In the present MIPMS system, virtually no interfering molecular ions have been reported in the m/z = 74-82 region, at least when water and dilute nitric and hydrochloric acids were introduced to plasma. Dilute sulfuric acid gave a minor peak at m / z = 80, which was assigned to SO3+.I7 However, this does not exclude the possibility that unpredictable spectroscopic interference exists in a real sample with a complex matrix. We therefore recommended measuring natural (unspiked) isotopic ratios in the sample matrix and confirming whether any deviation from the theoretical value exists, which would indicate spectroscopic interference. The unspiked, digested reference materials were measured for natural isotopic ratios of selenium (76/78,77/78,80/78, and 82/78), and the values were compared with those obtained for standard solution and reported values. Analysis of Standard Reference Materials. The spiked, digested reference materials were measured for 80Se/78Se.Three aliquots were digested and measured for each reference material. A 0.14 M nitric acid was introduced for washing (-3 min) between samples, and then ion counts at m/z = 78 and 80 of analytical blank were measured. 80Se/7sSein a sample was obtained after subtracting ion counts of the analytical blank obtained just before the sample measurement. Selenium concentration in the reference materials was calculated from the following equation:
C = W,M,@,- RBJ/w,W(RB, - A,) where C is the concentration of selenium in the sample (in ng/ g), w, is the atomic weight of natural selenium (78.96),20w, is the atomic weight of spike selenium (77.93), M, is the amount of added spike selenium (in ng) ,A, is the abundance of %e in the spike (0.01), B, is the abundance of 78Sein the spike (0.9858), A, is the abundance of %e in natural selenium (0.4961),21 B, is the abundance of 78Sein natural selenium (0.2378),2l and W is the weight of the sample for digestion (in g). (21) IUPAC. Pure ApPL Chem. 1991,63,991.
Analytical Chemistry, Vol. 67, No. 9,May 1, 1995
1569
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Figure 2. Dependence of the RSD (“h)of the 80Se178Seisotopic ratio measurements on dwell time. The RSD is derived from 5 measurements/ sample. Integration time was fixed at 3 s/mass. Dwell time is inversely related to the number of sweeps. The results of two independent measurements are shown.
RESULTS AND DISCUSSION Optimum Mass Scanning Condition. The precision of the
isotopic ratio measurement is one of the major determinants of the overall precision of the isotope dilution analysis. In order to obtain optimum condition for precise measurement of selenium isotopic ratios, two parameters, i.e., integration time and dwell time, were examined using 100 ng/mL selenium standard solution. Figure 1 shows the dependence of the RSD (%)of the %e/ 78Se ratio measurement on the number of sweeps. Since dwell time was fixed at 2 ms, the number of sweeps corresponds to the integration time. In this figure, theoretical RSD is also presented. These values were calculated from the following equationz2based on the ion count at each number of sweeps, on the assumption that statistical error of ion counting was independent between the two masses (mlz = 78 and 80): VAR (c80/c,$ =
(Cao/C78)2[vm(C,d/ (C8d2 + vm(c78)/(c78)21 where VAR is the variance, and C78 and Ca are the ion counts at m / z = 78 and 80. The measured RSD decreases from -2% at 250 sweeps (0.5 s integration/mass; ion count at m / z = 80, 1.8 x 104) to 0.3%at 3000 sweeps (6 s integration/mass; ion count at m / z = 80,2.3 x lo5),and it becomes constant thereafter. Although the measured RSD exceeds the theoretical when the number of sweeps is less than 3000, the measured coincides with the theoretical RSD (22) Mood, A M.; Graybill, F. A; Boes, D. C . Introduction to the Theory of Statistics; McGraw-Hill-Kogakusha: Tokyo, 1974.
1570 Analytical Chemistry, Vol. 67,No. 9,May 1, 1995
thereafter. This indicates that a factor or factors other than statistical ion counting error, which worsens the RSD, are present when the integration time is shorter. The most probable factor may be that ion counting at two masses is not done simultaneously but rather sequentially in a quadrupole mass spectrometer. When the integration time is shorter, cyclic fluctuation of the ion transmission to the detector due to, for instance, shrink-expand cycle of plasma at several hundred hertz,23 may influence the precision, but when the integration time is longer, this influence may be averaged out. Figure 2 shows the dependence of the RSD of the *Se/78Se ratio measurement on dwell time when the integration time per mass is set at 3 s. Two independent measurements were carried out for each dwell time. From this figure, it can be concluded that RSD has little dependence on dwell time. Although the difference is small, a 1ms dewll time seems to give the best result (0.2-0.3%). The optimum dwell time may come from two consistent requirements: a short dwell time is good for simultaneous counting of ions at two m/z values, but too short a dwell time may result in deteriorated precision, presumably due to instability of electronics due to a too rapid quadrupole scan. From the above two experiments,the optimum mass scanning conditions for selenium isotope ratio measurement were chosen as follow: integration time, 3 s/mass; dwell time, 1 ms; number of sweeps, 3000; number of measurement/sample,5; total analysis time/sample, 7.5 min. Although it was shown that longer integration time/mass provided a better E D in the data presented (23) Furuta, N. /.Anal. At. Spectmm. 1991, 6, 199.
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Se Concentrat ion
(ng/mL) Figure 3. Dependence of the RSD (%) of the 80Se/78Seisotopic ratio measurements on selenium concentration. The RSD is derived from 5 measurements/sample. Dwell time, 1 ms/mass; integration time, 3 s/mass; number of sweeps, 3000. 2.2 2.18 2.16 e,
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Se Concentrat ion (ng/mL) Figure 4. 80SeP8Seas a function of selenium concentration. The horizontal line indicates the true value (80Se/78= 2.086).*O Other details are as in the footnote of Figure 3. Table 1. Deviatlon of Measured Isotope Ratios of Selenium from Natural Values'
%e/78Se measd value 1 measd/true measd value 2 measd/true IUPAC value
77%/78Se
%e/%e
Table 2. WithinmDay and Between-Day Variation in Selenium Isotopic Ratio Measured by MIPMS'
82Se/78Se
0.387 0.98
0.321 1.00
2.121 1.02
0.380 1.04
0.383 0.97
0.319 0.99
2.144 1.03
0.389 1.06
0.394
0.321
2.086
0.367
Measured values 1 and 2 were isotope ratios in 100 ng/mL standard solution measured on separated days. IUPAC value is from ref 21. in Figure 1,total analysis time in these cases would be > 10 min/ sample. In addition, introduction of dilute nitric acid for 3 min was necessary to lower the signals to the background level even when 3 s integration was chosen; this would require a longer time when the integration time is longer. This will further reduce sample throughput. For this practical reason, 3 s integration/ mass was chosen for routine analysis. If better precision is needed, it can be achieved by sacrificing sample throughput. Effect of Selenium Concentration on Precision of Selenium Isotopic Ratio Measurement. Figure 3 demonstrates the dependence of the RSD on selenium concentration under the proposed optimum mass scanning conditions. Three individual measurements were made for each concentration. The RSD is expected to improve as selenium concentration increases. It was approximately 2%when the selenium concentration was 10 ng/ mL (ion count at m / z = 80,104) and < 1%when the concentration exceeded 50 ng/mL. This must also be a consequence of decreased statistical error with increasing ion count.
day 1 2 3 4
80Se/78Se mean f SD RSD (%)
+
2.117 0.009 2.144 f 0.008 2.144 f 0.007 2.128 f 0.006
0.44 0.37 0.32 0.30
bias from true value (%) +1.5 f2.8 +2.8 +2.0
Mean and standard deviation of 80Se/78Sein standard solution (100 ng/mL) measured several times a day (typically three measurements, i.e., at the beginnin , at the middle, and at the end of each working day). True value, &e/78Se
= 2.086.21
Accuracy of Selenium Isotopic Ratio Measurements. None of the parameters examined (number of sweeps, dwell time, and selenium concentration) had any influence on the measured 80Se/78Sevalue. An example is shown in Figure 4. This indicates that these parameters did not affect the relative sensitivity to the m/z values monitored. However, it was noticed that measured ratios deviated from the values which have been shown as natural abundance.21 As shown in Table 1, the extent of the deviation was proportionalto the mass difference,indicating that this is from mass discrimination. The mass discrimination is constant within any particular working day (RSD < 0.4%),but it fluctuated from day to day (Table 2). This indicates that instrumental mass discrimination can vary on a daily basis. This probably results from differences in the settings of several parameters, e.g., ion lens voltage, which were set daily to provide optimum signal response. It is clear from this table that the standard solution must be measured daily for its selenium isotopic ratios and that Analytical Chemistry, Vol. 67, No. 9,May 1, 1995
1571
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Mat r i x Concent r a t ion (ug/mL) Figure 6. 80Se/78Seas a function of matrix element concentration. Vertical axis denotes 80Se/78Serelative to that in standard solution (100 p@L) without matrix elements. Analytical conditions are as in the footnote of Figure 3.
measured values in real samples must be corrected by an appropriate factor, obtained daily. Effect of Matrix Elements. Figures 5 and 6 show the effects of increasing matrix element concentration on ion count and measured @W7*Se,respectively. Matrix elements tested included sodium, potassium, calcium, and phosphorus. The vertical axes of Figures 5 and 6 denote ion count and YW7%e,respectively, relative to those measured in standard solution without matrix elements. Although the ion count rate is suppressed by the presence of some of the matrix elements F i e 5), the measured isotope ratios are within 0.996-1.008, thus indicating no effect on isotope ratio measurement by the coexisting matrix element at the level examined (< 200 pg/mL) . This indicates that matrixinduced mass discrimination does not occur when selenium isotopes are measured by the present instrument when biological matrix elements coexist at this level. This trpe of discrimination is reported when the isotope ratio of a lighter element (e.g., boron) is measured in the presence of a heavier matrix element (e.g., sodium, >3000 pg/mL) by ICPMSF4 Table 3 shows the RSD of the %e/7*Se measurement of the 100 ng/mL standard solution as a function of matrix element level. The RSD of the %e/'%e measurement seems to deteriorate to the extent of -1% when sodium or phosphorus is present; however, this is not the case for the other two matrix elements (potassium and calcium) examined. Moreover, there is no linear relationship between the RSD and the matrix element level. Both signal suppression in the presence of a matrix element and salt buildup in the interface might cause this result in a complex manner. (24) Gregoire, D. C. Anal. Chem. 1987, 59, 2479.
1572 Analytical Chemistry, Vol. 67, No. 9,May 1, 1995
Table 3. Precision of Sqhnium Isotope Ratio Measurement and Matrix Element Level.
matrix element concn @g/mL)
Na
P
K
Ca
0 50 100 200
0.19 0.68 0.43 0.92
0.21
0.55 0.46 0.45 0.73
0.67 0.48 0.69 0.73
0.51 0.42
Figures in the table are the RSD (%) of the 80Se/78Seratio measurement. Selenium concentration was 100 ng/mL.
Calibration Curve and Detection Iimit. Figure 7 shows calibration curves for selenium isotopes over 0-100 ng/mL, which were linear in this concentration range. The detection limit of %e was calculated from the slope of the calibration curve and the standard deviation of the background counts. It was 35 pg/ mL when 3a definition was used. The background counts in the mlz = 75-82 region were approximately 20-30 counts/s earlier in our experiment. The background count increased to 140 counts/s after the original photon stopper was replaced with that of smaller diameter (1.04) to increase ion transmission efficiency. The ion count of the 100 pg/L standard solution at m / z = 80 was 3.5 x 104 and 8 x 104 counts/s before and after the replacement, respectively. Replacement of the photon stopper does not substantially affect the detection limit. Spectroscopic Interferences. An interference at m / z = 80 from SO3+ is expected when the sample contains sulfur at higher concentration^.^^ Figure 8 shows the background-subtractedmass spectrum over m / z = 73-83 of the 1000 pg/mL sulfur standard solution containing no selenium. A peak was observed at m / z =
35000 30000 n
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0
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82Se
5000 0
0
IO
20
30
40
50
70
60
80
100
90
Se concentrat ion (ng/mL) Figure 7. Calibration curves of selenium isotopes. Measurement conditions are as in the footnote of Figure 3 Detection limit of 80Se( 3 4 was 35 pg/mL. Table 4. Selenium Isotopic Ratios in Unspiked Reference Materials and Standard Solution'
bovine liver (NIST) human urine (NIST) pig kidney (BCR) human hair (BCR) standard solution IPACZ1
76Se/78Se
77Se/78Se
YW718Se
0.353 f 0.016 0.379 f 0.008 0.377 f 0.003 0.408 f 0.014 0.385 f 0.003 0.394
0.378 f 0.022 0.309 f 0.009 0.317 f 0.008 0.311 f 0.004 0.321 f 0.003 0.321
2.085 f 0.062 2.136 f 0.043 2.153 i 0.030 2.169 f 0.041 2.152 f 0.014 2.086
approx Se concn (ppb)
82Se/78Se
0.396 f 0.023 0.384 f 0.004 0.395 f 0.009 0.399 f 0.011 0.389 f 0.003 0.367
9 50 110 40 100
Figures in this table are mean and la of five measurements.
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0 73
Table 5. Selenium Concentratlons In Cllnical Reference Materials Determined by Isotope Dilution MIPmMS'
74
75 76 7 7
78 79 80 81 82 8 3 m 0h
Figure 8. Background-subtractedmass spectrum over mlz = 7383 when 1000 pg/mL sulfur standard solution was introduced to plasma. The peak at mlz = 80 can be assigned to S03+. The ion count at the mlz was 110 countsls, corresponding to 0.2 ng/mL selenium.
80 which can be assigned as SO3+. The ion count was 110 counts/ s, which corresponds to 0.2 ng/mL selenium. Among the reference materials examined in the present study, hair has the greatest sulfur:selenium ratio (approximately 50 0OO:l). Even in this case, however, SO3+contributed less than 1%of the selenium ion count at m/z = 80. The interferencefrom SO3+ can be ignored in the case of biological materials. When this interference is a problem, subtraction of counts of m/z = 80 derived from SO3+or use of 77Seor as reference mass is recommended. Table 4 shows selenium isotopic ratios of the unspiked reference materials along with that of standard solution (100 ng/ mL) and a recent IUPAC value. For the measured ratio in standard solution was 2-3% higher than the IUPAC value.21 As has been mentioned, this probably arose from an instrumental mass discrimination effect, the extent of which changed on a daily basis. With regard to unspiked reference materials, the imprecision values of the ratios are larger than those of the standard solution. This is primarily due to lower selenium concentrations in the sample solutions prepared from these reference materials, which
NIST 1598 bovine serum NIST 2670 human urine (elevated level) NIST 1577a bovine liver BCR pig kidney BCR human hair NIES CRM 13bhuman hair
ng/g
pg/mL pg/g
pg/g pg/g pg/g
found
certified
45.2 f 1.4 0.461 f 0.002
42.4 f 3.5 0.46 f 0.03
0.709 f 0.025 10.5 f 0.06 2.02 f 0.02 1.83 f 0.01
0.71 f 0.07 10.3 f 0.5 2.00 f 0.08 1.70 f 0.11
a Determined after spiking 78Seto make in the range of 0.5-1.0, nitric acid digestion, and dilution. Dilution factor was -10 for serum and urine and 100for the others. Candidate NIES certified
reference material. Not certified. Mean of acceptable values of neutron activation analysis from cooperating laboratories is shown.
are shown in the last columns of Table 2. The presence of matrix elements may also contributed to variability. The measured ratios are in agreement with those in standard solution when analytical error is taken into consideration. This holds true in the case of hair reference material as we have evaluated it. Deviations of the isotopic ratios were evident in the case of NIST bovine liver, but they could be attributed to poorer precision of the isotopic ratio measurement due to low selenium concentration in measured sample solution (-9 ng/mL). No systematic deviation in isotopic ratios from standard solution indicates that interferences in this m/z region are, if present at all, not problematic, at least in terms of practical viewpoints. Janghorbani and co-workers reported selenium isotope ratio analysis of various biological materials by the hydride generation ICPMS t e c h n i q ~ e . ~They ~ . ~ ~reported 1%overall RSD in 74Se/ 77Seand 82Se/77Semeasurements.12 They also showed 0.2-1.3% (25) Janghorbani, M.; Ting, B. T. G. Anal. Chem. 1989,61, 701.
Analytical Chemistv, Vol. 67, No. 9,May 1, 1995
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RSD in the same ratios in biological materials determined by the same system.25 The precision obtainable by their system may be comparable to, or even better than, that by our system when taking into consideration the fact that they used less abundant isotopes to avoid Arz+ interferences. This might be due to the higher ion count associated with higher analyte transportation efficiency in a gas introduction system than in solution nebulization. However, as with other studies in which the hydride generation technique is used,13J4control of memory effect was important in this technique. Analysis of Reference Materials. Table 5 shows the results of selenium isotope dilution analyses of clinical reference materials by the present method. 78Sewas spiked, and the 80Se/78Seratio was measured. Instrumental mass discrimination was corrected according to the IUPAC value (80Se/78Se= 2.086).21 Excellent agreements with respective certified values were obtained for all of the reference materials analyzed. Moreover, precision of analysis, as represented by the RSD of the three analyses, is typically < 1%,except for the cases where the selenium level was low. The result for the NIES candidate reference material agree favorably with that obtained by neutron activation analysis. The present result will be used for the certification of this reference material. CONCLUSION A nitrogen MIPMS was examined for its applicability to isotope dilution analysis of selenium. The isotopic ratio could be
1574 Analytical Chemistty, Vol. 67, No. 9, May 1, 1995
measured with good precision (RSD 0.5%)under routine analytical conditions. No detectable interference in the m / z region of selenium arising from sample matrix was noticed, though SO3+ may be a minor interferant at mlz = 80. The nitrogen MIPMS system enables precise and accurate determination of selenium in biological materials by the isotope dilution technique if instrumental mass discrimination is properly corrected. This was v e ~ e dby the analyses of several clinical reference materials, which showed excellent agreement with certified values with high precision. This method will be of great value in cases where an accurate value is required, e.g., certdication of selenium in candidate reference materials. ACKNOWLEDGMENT
The authors appreciate Drs. H. Nitta and k Tanaka, National Institute for Environmental Studies,Japan, for valuable discussion on the manuscript.
Received for review October 26, 1994. Accepted February 16, 1995.@ AC941050N @Abstractpublished in Advance ACS Abstracts, March 15, 1995.
