Optimum Procedure for the Determination of Selenium in Biological Specimens Using 77mSe Neutron Activation A. J. Blotcky, L. J. Arsenault, and E. P. Rack’ Medica/ Research, V.A. Hospitai, Omaha, Neb. 68105
An optimum procedure is outlined for the rapid quantitative determination of trace ( 2 0 . 0 6 pg) selenium in serum and tissue using 77mSeneutron activation. Sample preparation, irradiation procedures, types of detector assemblies, and spectral analyses and data reduction are discussed. The optimum procedure utilizing a low-power nuclear reactor ( - 3 X 10” n/cm2 sec) consisted of irradiating lyophilized tissue samples or dialyzed-lyophilized serum samples for 20 sec, with a 20-sec decay time prior to radioassay. The most suitable radioassay assembly was the 5-mm X 3-in. N a l (TI) detector system. Spectral analyses and data reduction consisted of eliminating the interferences of isotopes of relatively long half-lives by spectrum subtraction and correcting for the 23Ne[‘3Na ( n , ~ ) ’ ~ N e and ] contributions in the region of the 77n’Se photopeak. Comparisons between the results obtained by the discussed 77n’Se procedure and values quoted for an NBS Bovine Livei Standard were excellent.
Because of the importance that trace quantities of selenium play in the physiology of living species, various analytical procedures have been developed for analyses of microquantities of selenium iri serum and tissue. Some of the most popular techniques involve neutron activation analysis of the longer-lived selenium radioisotopes 75Se ( T1l2= 120.4 days) ( 1 , 2) and slSe ( T ~ , = z 18 min) ( 3 ) . One of the most potentially useful isotopes, 77mSe,has been used only in a few reported studies (4-7), mainly because its short half-life (17.6 & 0.15 sec) (8) restricts postirradiation chemistry of the biological sample. An advantage of utilizing 77mSe is that a large number of samples can be analyzed routinely and can be available for additional analyses such as for total protein, nitrogen, etc., if a nondestructive technique is employed. In our attempt to do nondestructive 77mSe analysis using the previously reported techniques (4-7), we encountered experimental problems not previously reported. In order to develop an optimum routine procedure, employing a low-power nuclear reactor, utilizing the 77mSe isotope, we found it of importance to understand the effects of various factors on the method. These included sample preparation prior to irradiation, irradiation
*
Department N e b . 68508.
of
C h e m i s t r y , U n i v e r s i t y of Nebraska, L i n c o l n ,
( 1 ) H . L. Rook,Ana/. Chem.. 44, 1276 (1972).
(2) K. Samsahl, Anal. Chem. 39, 1480 (1967). (3) H . J. M. Bowen and P. A. Cawse. Anaiyst i i o n d o n i . 88, 721 (1963) (4) K . P. McConnell, Proc. int. Coni.. Mod. Trends Act. Anal. 137 (1961) ( 5 ) M . Okada, Nature (London). 187, 594 (1960). ( 6 ) R. H . Filbey and W. L. Yakeley, Radiochem. Radioana/. L e t t , 2, 307 ( 1969) (71 R. C. Dickson and R. H . Tomlinson. Ciin. Chim. Acta. 16, 311 (1967) ( 8 ) W. T. K . Johnson, Doctoral Thesis, American University, Washington, D.C.. 1970. Unlversity Microfilms Order No. 70-24,279.
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A N A L Y T I C A L CHEMISTRY, V O L . 45, N O . 7, JUNE 1973
procedure including activation and counting time, type of detector assembly, and spectral analysis and data reduction.
INSTRUMENTATION Neutron Irradiation. All samples were irradiated in the Omaha V. A. Hospital TRIGA reactor, operating with a thermal neutron flux of 3.1 x 10’1 n/cm2 sec. A “shuttle rabbit” system similar to the one described by Anders (9) was used for all irradiations. Radioassay Equipment. Two different y-ray detector systems were used in conjunction with a Nuclear-Chicago 400-channel analyzer: (1)two each 3- x 3-in. NaI (Tl) detectors in parallel; (2) two each 5 mm X 3-in. NaI (Tl) detectors in parallel. In addition, one Harshaw Chemical 12.2% efficient Ge(Li) detector (FWHM 2.2 keV) was used with a Nuclear-Data N.D. 2400 1024-channel analyzer. The shield for the NaI (Tl) detectors was of a design similar to that described by Heath (10) allowing 32 in. from the detector to the shield wall. EFFECTS OF EXPERIMENTAL FACTORS ON THE ANALYSIS Since most of the activated trace elements in biological specimens (tissue and serum) have radioactive half-lives longer than that of 7TmSe, short irradiation times minimize their contribution to the y-ray spectrum of the neutron-irradiated sample. However, all biological samples contain appreciable quantities of oxygen, sodium, and chlorine which, upon irradiation, form radionuclides. Previously reported techniques utilizing the longer-lived isotopes (for which a much longer neutron irradiation time is required) require postirradiation chemistry for the removal of these radioactive contaminants. We irradiated 1.5 grams of wet rat liver and 2.0 ml of human serum for 20 sec in the nuclear reactor and, with only a 20-sec delay, analyzed the radioisotopes present using the 3- x 3-in. NaI (Tl) detectors. Depicted in Figure l a is the gross y-ray spectrum of the wet tissue in the range of 0 to 4 MeV. As can be seen, in addition to the 77mSe photopeak, other activities such as 1 9 0 , 56Mn, *7Mg, 24Na, and 38Cl are present. We also found a photopeak a t 0.44 MeV which has an experimentally determined half-life of 38.7 sec. This photopeak’s origin is from the 23Na(n,p)23Ne reaction, unreported in previous 77mSe analyses. Analysis of a pure neon spectrum showed that the Compton continuum from 23Ne contributed to the activity under the 77mSe photopeak. Presented in Figure 2a is the y-ray spectrum of 2 ml of untreated human serum. The gross spectrum is similar to that of wet tissue. It is important to observe that sodium, chlorine, and neon photopeaks all have Compton contributions that add to the activity under the 77mSe photopeak. It is obvious from inspecting Figures l a and 2a that irradiating the wet liver (9) 0. Anders, Anal. Chem.. 33, 1709 (1961). (10) R . L. Heath, “Scintillation Spectrometry,” 2nd ed., Vol. 1. Phillips Petroleum Company. Idaho Falls, Idaho, 1964, p 14.
3000 2000
IO00
3000
g 2000 r
:
IO00 3000
2000
+
s=
IOOC
z
t
t
e
U t
MeV
Figure 2. The y-ray spectra of neutron-irradiated human serum ( a ) Raw serum with 3- X 3-in. Nal (TI); ( b ) lyophilized serum with 3- X 3-in. Nal (Ti); ( c ) lyophilized serum with 5-mm X 3-in. Nal (TI) 40C
300 200
688 30C ~
+ ‘>e.
200
Figure 1. The y-ray spectra of neutron irradiated rat liver ( a ) Wet liver with 3- X 3-in. Nal (TI); ( b ) lyophilized liver with 3- X 3-in. N a i (TI), ( c ) lyophilized liver with 5-mrn X 3-in. Nal ( T I ) ; ( d ) lyophillzed liver with Ge(Li)
I0C t3
zI rc)
and raw serum and using a 3- X 3-in. NaI (Tl) crystal assembly is not suitable for performing an accurate nondestructive analysis for selenium. Sample Preparation. Both wet tissue and raw serum contain appreciable quantities of water which add to the I9O content of the irradiated samples. For example, normal human serum contains approximately 945 grams of water per liter of serum and wet human liver contains approximately 711 grams of water per kilogram of tissue. Lyophilizing both tissue and serum samples greatly reduces the oxygen content as can be seen in Figures l b and 2b. An important question to consider is whether any biologically bound selenium is lost during a lyophilization step. Routine comparisons of the selenium content of both lyophilized and nonlyophilized serum samples showed no appreciable difference within experimental error. Therefore, we would not expect to lose any biologically-bound selenium in h lyophilization step. In order to optimize the radioassay procedure, it is necessary to minimize the contributions due to 23Ne, 24h’a, and 3 W l . Since we were interested in a nondestructive technique we substituted a 5-mm x 3-in. NaI (Tl) assembly for the 3- x 3-in. system. These thin crystals greatly reduce the sodium and chlorine radioactive contributions. Figures IC and 2c show the results of using the 5-mm x 3-in. NaI (Tl) crystal assembly.
0 [L
W
o. v)
t-
5 80C
0 60C
Z
50C 40C
30C 20c
2 I0C
__j
MeV
Figure 3. The -,-ray spectra of dialyzed-lyophilized human
serum (a) 3- X 3-ln Nal (TI), ( b ) 5-mm X 3-in Nal (TI). ( c ) Ge(Li)
ANALYTICAL C H E M I S T R Y , VOL. 45, NO. 7, JUNE 1973
1057
600 500 400 300
W
N
00 Z - 80 z 60 m 0 50
t-
3
Z 200
E
IO0
1
40
W a 30
80 60 50 40 30
m I-
Z
3 0 0
Z >-
20
I'
5 -4 .-
-
3 --
IO
5 -
o a
3
I-
6 --
20
8 6 5
I0
Se
4
2
2 '-
0
0.25
0
c
__j MeV Figure 4. The y-ray spectrum of N B S Bovine Liver Standard
Sample
Solid liver Hydro I yzed Iive r Solid liver i- 0.4 p g Se Hydrolyzed liver i- 0.4 p g Se added before hydrolysis Hydrolyzed liver 0.4 p g Se added after hydrolysis
+
aN
Itissue ;dJe?:
Counts per pg Se added
Relative std dev, %
4677
14.1 6.9
4898
3.8
4700
5.1
6.5
0.7316 0.6836
= 5, all samples from same liver and lyophilized prior to irradiation.
As can be seen in Figure IC, no further treatment is necessary for tissue samples prior to data reduction for selenium content. Since 2 ml of normal human serum contains about 4.5 times the sodium content as 1 gram of liver, resulting in a much greater neon contribution from the 23Na(n,p)23Ne reaction, additional procedures other than a choice of crystals were required €or the serum. Most of our research samples receive postirradiation analysis for nitrogen or protein. Consequently, we chose to use 1058
05
Figure 5. The ',-ray spectrum of dialyzed-lyophilized human
serum; decay spectrum subtracted
Reference Material (SRM 1577);decay spectrum subtracted Table I. Sell-Absorption S t u d p
0.2 4 MeV
a dialysis method rather than a chemical one to remove the sodium and chloride ions from the serum. Using 2 ml of the same serum whose y-ray spectrum has been shown in Figure 2a, we dialyzed and lyophilized the sample. Presented in Figure 3a is the y-ray spectrum employing a 3x 3-in. NaI crystal system. Presented in Figure 36 is the y-ray spectrum using the 5-mm x 3-in. NaI (Tl) system, It is interesting to observe the results of dialysis as shown in Figures 3a and 3 b and compare them to the nondialyzed-lyophilized samples presented in Figures 2b and 2c. McConnell (11-13) has shown that dialysis of rat plasma protein did not alter the 75Se protein fraction appreciably within a p H range of 2.2 and 8.4. In order t o determine whether our serum samples had a loss of selenium in dialysis, a comparison was made between the 77mSe activity in dialyzed-lyophilized and nondialyzed-lyophilized pooled human serum. All samples contained pooled human serum and were lyophilized prior to irradiation. Dialysis was performed against deionized water. The
ANALYTICAL CHEMISTRY, VOL. 45, NO. 7 , JUNE 1973
(11) K . P. McConnell, 0 . M Roth, and H . Hoch, rex. Rep. Biol. Med., 26, 5 3 5 (1968). (12) K. P. McConnelt and D. M . Roth, Arch. Biochem. Biophys.. 124, 29 (1968), (13) K . P. McConnell, C. H . Wabnitz, and D. M. Roth, rex. Rep. Bioi. Med.. 18, 438 (1960).
Table II. Cornpallison of 5-mm X 3-in. Nal(TI) System with 12% Efficient Ge(Li) Detectora Detector
5 mm
Sample no.
Net counts perb g liver
m Se per g liver
Std dev
NBS wg Se per g liver
Nal(T1). %
1891 2220 2000 2200 2213 2148 2087 1907 1816 2054
1.02 1.20 1.08 1.19 1.20 1.16 1.13 1.03 0.98 1.11
0.08
1.1 f 0.1c
100.0
824 786 880 727 668 745 861 743 865 789
1.24 1.20 1.33 1.10 1.01 1.13 1.30 1.13 1.31 1.19
0.11
1.1 f 0.1c
38.4
Nal
(TI) 1 2 3 4 5 6 7 8 9
Mean Ge(Li) 1 2 3 4 5 6 7 8 9
Mean
All samples contained 0.4898 to 0.7600 g of NBS Standard Reference Material Bovine Liver No. 1577. *Corrected for neon and oxygen contribution. Two times the standard deviation.
mean “microgram per gram of sample” value for six nondialyzed samples was 1.38 with a 35% relative standard deviation. The mean “microgram per gram of sample” value for six dialyzed samples was 1.51 with a 10% relative standard deviation. Since there appears to be no significant difference in the 77mSe content due to dialysis, we do not believe that any biologically-bound selenium is lost in the dialysis. Solid biological tissues may have different thicknesses. If self-absorption of the iimSe is a factor, apparent differences in selenium concentration may result. We compared the selenium activity of liver samples which were lyophilized with those which were digested and lyophilized. Table I shows that no significant difference in selenium content was found in the processed liver samples having different thicknesses.
IRRADIATION PROCEDURE Irradiation Time. Because 23Ne, I9O, 24Na, and other elements in biological specimens (which have been activated by neutron irradiation) have half-lives longer than that of 7imSe, short irradiation times are necessary. For example, when comparing an irradiation time of 20 sec us. 70 sec, the per cent increase in the 77mSe was 173 while in the 1 9 0 and 24Na, it was 205 and 259, respectively. For this reason an irradiation time of 20 sec with a 20-sec decay time before radioassay was used. Decay Correction. As it is not possible to measure the counting rate a t an instant in time but only over a period of time, it is the usual convention t o consider the decay time of a radioisotope to extend from the time the sample was removed from the reactor to the midtime of the counting period. The error resulting from this method has been shown to be 2% for a counting period equal to the half-life and only 0.6% for one-half of a half-life (13). Since some samples may have a longer analyzer counting time than others, due to an increase in the activity of the sample, it is necessary to incorporate a counting time correction into the solution. A mathematical correction (14) can be formulated in the following manner
where A = the observed mean activity over the duration of the measurement, Ac = the corrected activity a t onehalf of the elapsed counting time, t 1 / 2 = the half-life of the nuclide, and S t = the counting interval. In analyzing a typical set of tissues, the counting interval A t varied from 0.30 to 0.37 min giving a resultant error of 2.1 to 3.2%. Therefore, the counting interval At was measured for each sample and after determining the counts under the selenium photopeak a t time zero, the counting time correction was applied. This correction has also been incorporated into a computer program which will be discussed in the data reduction section. Types of Detectors Employed. Using a 1.5-gram lyophilized rat liver sample, we compared the y-ray spectra using all three detector assemblies as shown in Figures I b , IC,and Id. A similar comparison was made for a 2-ml dialyzed-lyophilized sample of human serum as shown in Figures 3a, 3b, and 3c. It is obvious from inspecting these figures that the most suitable radioassay assemblies are the 5-mm x 3-in. NaI (Tl) and the Ge(Li) detector system. The 5-mm x 3-in. XaI (Tl) assembly is only 76% as efficient as a 3- x 3-in. NaI (Tl) system for selenium while the Ge(Li) detector used is 29% as efficient as the 3x 3-in. assembly.
SPECTRAL ANALYSIS AND DATA REDUCTION As can be seen by looking at Figures IC and 3b, the sodium and chlorine still contribute to the activity under the selenium photopeak even though the 5-mm x 3-in. NaI (Tl) assembly is used. The activity from these contaminants contributes to the dead time of the analyzer which in turn contributes to an increased counting time of the 17.6-sec 77mSe.Therefore, the upper level discriminator of the analyzer must be set to cut off the spectrum above 0.5 MeV. (14) G . B. Cook and J F. Duncan. “Modern Radiochemical Practice.” Clarendon Press. Oxford. 1952. p 57
ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, JUNE 1973
1059
Elimination of the interferences from isotopes of relatively long half-life such as sodium and chlorine was accomplished by subtracting an 80-sec decay spectrum from a 20-sec decay spectrum. The elimination of the interferences far outweighed the possible loss of activity. A subtracted spectrum of NBS Bovine Liver No. 1577 is shown in Figure 4 and the subtracted spectrum of the same serum sample used in Figures 2 and 3 is shown in Figure 5. It is important to note that the oxygen and neon are still present; and, consequently their contributions must be subtracted from the area under the selenium peak. By utilizing y-ray spectra obtained from pure neon and water, the 23Ne and 1 9 0 contributions in the region of the 77mSe photopeak were obtained as a function of the counts in the respective photopeak of the contaminant. A Fortran computer program was then written for an IBM 1620 so that merely by feeding the 20- and 80-sec spectra (obtained on paper tape from the analyzer) into the computer, the channel sums are taken, contributions subtracted, and corrections made for the inherent error existing when counting short-lived activities in a period comparable to the radioisotope half-life. The selenium standards were prepared from dissolved and lyophilized Johnson Matthey spectrographically standardized selenium shot containing 0.03-ppm sodium and no reportable oxygen. Interfering ion corrections were not applied to the selenium standard.
niques. The National Bureau of Standards Standard Reference Material No. 1577 Bovine Liver had a reported selenium content of 1.1 f 0.1 pg/gram of sample (95% confidence limit). The reported selenium content was determined by neutron activation analysis employing the 75Se isotope ( 2 ) and isotope dilution mass spectrometry. Presented in Table I1 is a comparison between our analyses of the NBS Bovine Liver Standard employing both the Ge(Li) detector system and the 5-mm X 3-in. NaI (TI) crystal assembly. We can readily see that the results compare quite well to the NBS reported values. It should be pointed out that the Bovine Liver Standard contained quantities of selenium a factor of 10 higher than those found in typical serum samples. At high selenium concentrations, there is no apparent difference using a Ge(Li) detector or a 5-mm x 3-in. NaI (Tl) detector assembly. However, for typical biological systems using a low-power nuclear reactor, the 5-mm X 3-in. NaI (Tl) is preferred. As can be seen from a comparison of the actual values of selenium content in identical samples presented in Table 11, the relative standard deviation is less than 10%. The sensitivity of our described procedure is L0.6 pg of selenium, using a reactor power level of -3 x 10’1 n/cm2 sec. During a normal working day, the number of selenium analyses that can be run-including spectral analyses and data reduction-approaches 150 samples.
APPLICATION OF PROCEDURE It was of importance to compare analytical results using our procedure with a sample analyzed by different tech-
Received for review October 12, 1972. Accepted January 8, 1973.
Nuclear Magnetic Resonance Determination of Water and an Oxygen Titration for Nitric Oxide in Liquid Nitrogen Tetroxide Stephen P. Vango and Stanley L. Manatt Space Sciences Division and Propulsion Dwision. Jet Propulsion Laboratory. California lnstitute of Technology. Pasadena, Calif. 97 703
An NMR procedure is described for the rapid quantitative analysis of water in nitrogen tetroxide oxidizer. This technique is capable of detecting as little as 0,001 wt YO of H20 (10 p g of water per gram) and gives results in the concentration range of 0.01 to 0.02 wt YO with a precision of about f0.002 wt YO.Because many samples of oxidizer contain NO, a procedure for 02 titration of NO prior to N M R analysis for H 2 0 is described which gives the NO concentration accurate to f0.05 wt YO.
The quantity of water present in nitrogen tetroxide profoundly affects its rate of attack on metals. A few tenths of one per cent of water in nitrogen tetroxide to be utilized as an oxidizer in a propulsion system cannot be tolerated. As part of a quality control program of propellantgrade nitrogen tetroxide, we have had to consider carefully the merits of the various methods for detection and quantitation of water in the latter (1-4). Very recently, it has been stated that an NMR method for this analysis 1060
ANALYTICAL CHEMISTRY, VOL. 45, NO. 7, JUNE 1973
“remains unattractive as a quality control procedure because of the high cost of instrumentation and the intricacy of sample preparation” (4). We feel this statement is not warranted at the present time, as NMR instrumentation is very wide-spread in control laboratories and the sample preparation requires no more effort than that involved in sampling for the analyses of other components of a Nz04 oxidizer ( i e . , for total N204, NOC1, and ash). Thus, we describe here the details of our NMR water procedure for nitrogen tetroxide oxidizer which has been in common usage in our laboratories for some years. It is believed that a mixture of a small amount of water in nitrogen tetroxide a t ambient temperature is converted (1) Military Specification; Propellant, Nitrogen Tetroxide, Mil-P-26539
USAF, July 18, 1960. (2) G. C. Whitnack and C.J. Holford, Anal. Chem., 21, 801 (1949). ( 3 ) N. V. Sutton, H. E. Dubb, R . E. Bell. I . Lysyj, and B. C. Neale, “Advanced Propellant Chemistry,” Advan. Chem. Serios, 54, 231 (1966). (4) R. F. Mufaca, E. Willis, C. H. Martin, and C. A . Crutchfield, Ana/. Chem.. 41, 295 (1969).
