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ANALYSIS BY STABLE-ISOTOPE-DILUTION MS
Measuring Selenium in Environmental Samples
by F. MacLeod and B. A. McGaw
School of Applied Sciences,
The Robert Gordon University, Aberdeen (U.K.); C. A. Shand,
The Macaulay Land Use Research Institute, Aberdeen (U.K.)
Selenium (Se) is an element which has aroused much recent
interest. It is an essential trace metalloid for animals and
is known to be a cofactor in two enzyme systems. The first,
glutathione, acts as an antioxidant by destroying peroxides
which attack cellular membranes. The second, iodothyronine
5'-deiodinase, converts thyroxine (T4) to triiodothyronine
(T3) with the release of iodine.
Selenium in the diet is obtained from plants and animal
products. The Se level in plants is dependent largely on the
selenium content and the chemical species in the soils.
Figure 1. Formation of 5'-nitropiazselenol (Se-NPD).
Analytical Options
The analysis of selenium is difficult, because it is
potentially volatile and present in very low levels in most
soils (<2 µg/g) and plants (<100 ng/g). Current methods
include fluorimetry, neutron activation analysis (NAA), and
atomic absorption spectrometry (AAS) techniques. Fluorimetry
is an established low-cost method, but it involves a
digestion step, and care has to be taken to avoid
interferences. NAA, although accurate, is not widely
available. Flame AAS has high detection limits and lacks
precision. Hydride generation AAS is prone to chemical
interferences from copper and arsenic. Graphite-furnace AAS
has good sensitivity, but suffers from interferences that
need to be overcome by using matrix modifiers and background
correction.
Figure 2. Mass spectrum scan of selenium standard
as SE-NPD showing peaks at m/z 223-231.
GC/MS for Plants and Soils
Using isotope-dilution mass spectrometry (ID-MS) by GC/MS as
an alternative method for selenium analysis has the advantage
of offering accurate and precise Se measurements without
requiring quantitative recovery. The method was first
employed by Reamer and Veillon1 for the analysis of
biological samples and has since been extended by Ducros and
Favier2 to human body fluids.
Our article describes the first application of this method
for the quantitation of selenium in plants and soils and
evaluates the analytical performance of a standard benchtop
GC/MS. More comprehensive details are published elsewhere3.
Figure 3. Mass spectrum scan of 76Se isotope solution
as Se-NPD showing peaks at m/z 223-231.
Preparing Isotope Solution
Elemental 76Se (Europa Scientific, England) was dissolved in
concentrated nitric acid and then diluted with water. Using
reverse-isotope-dilution mass spectrometry with natural
abundance AAS Se standard, the concentration was accurately
determined as 184.77 µg/g. This solution was diluted to 18.61
µg/g for the spiking purposes.
Digestion and Derivatization
We used plant and soil certified reference materials (CRM)
from the Laboratory of the Government Chemist (C85-04 and
C74-05). Prior to digestion, plant material (5 gram) or soil
(0.5 gram) was spiked with 100 µL of the 76Se isotope
solution.
Plant material was digested on a block (150°C) with a
mixture of 18 M HNO3 and 30 volume H2O2, until a clear
digest was obtained. Soils were heated with 18 M HNO3 in
Teflon bombs (Savillex, U.S.A.) in a microwave oven, with
occasional venting to eliminate organic material. After the
addition of 29 M HF, the samples were digested on a hotplate
(180°C) for 6 hours before evaporation of the acids (120°C).
The digest was transferred to a Pyrex tube, 200 µL of 6 M HCl
was added, and the tube was heated in a water bath (85°C) for
5 minutes. This step was repeated until 1 cm3 of HCl had been
added. The tube was cooled, and 2 cm3 of an indicator
mixture (20 mM EDTA, 10 mg/L bromocresol purple and 7 M
ammonium hydroxide) were added. After heating for 1.5 hours,
the tube was cooled, 5 cm3 0.1 M HCl added, and left
overnight in darkness. The derivatizing agent
4-nitro-1,2-phenylenediamine was added (0.5 cm3 of 100 mg
dissolved in 25 cm3 0.1 M HCl).
The tube was heated in a water bath (40°C) for 30 min. After
cooling, 5 cm3 of chloroform was added to extract the
5'-nitro piazselenol (Se-NPD), and the tube shaken. The
aqueous layer was discarded. The chloroform was evaporated
using a rotary film evaporator. Prior to the analysis, the
Se-NPD was redissolved in 20 µL of chloroform.
Obtaining Relative Abundance
Derivatized Se standards and the 76Se isotope solution were
injected into the GC/MS and a scan of each obtained (Figures
2 and 3) to identify the Se-NPD and show the isotopes
present. The relative abundances of the selenium isotopes as
Se-NPD were obtained in the SIM mode (Table 1). This process
was repeated 10 times.
Quantitation of Selenium Levels
The selenium levels were calculated using the Pickup and
McPherson equation2
x=y(RQl-Qk)/(Pk-RPl)
where x=moles of Se in sample, y=moles of Se in spike, R=m/z
229/225, Pk and Pl are the relative abundances of m/z 229 and
225, respectively, in the standard, and Qk and Ql are the
relative abundances of m/z 229 and 225 of the spike solution.
Therefore,
x=y(91.35R-0.11)/(46.56-8.45R)
The results for Se levels in the plant and soil CRMs are
shown in Table 2. For both CRM materials, the random errors
for our measurements are lower than the uncertainty values
given for the certified materials, and the mean selenium
concentrations lie within their 95% confidence levels. Our
conclusion: isotope-dilution MS offers accurate results with
high reproducibility.
A practical detection limit of 10 ng per 2 µL injection was
obtained by diluting a standard until m/z 229/225 became
significantly different (p>0.1).
References
- Reamer, D. C. and Veillon, C. Anal. Chem 53, 2166 (1981).
- Ducros, V. and Favier, A. J. Chromatogr. 583, 35 (1992).
- MacLeod, F., McGaw, B. A., and Shand, C. A. Talanta (in press).
| Instrumentation |
|---|
| Gas Chromatograph: | HP 5890
| | Mass Spectrometer: | HP 5791A (Quadrupole)
| | Capillary Column: | DB-1; 25 m x 0.2 mm i.d. x 0.33 µm film
| | Data Collection: | HP MS ChemStation
| | Experimental Conditions |
|---|
| Carrier Gas: | Helium, 50 cm3/min
| | Injection: | Splitless mode
Injector line temp. 250°C
Transfer line temp. 280°C
Purge gas flow rate 20 cm3/min
| | Oven: | 120°C (0 min), to 200°C at 5°C/min
| Mass Spectrom.
Program: | Electron impact ionization; 70 eV
Selected ion monitoring of peaks at m/z 225 and 229, which correspond to the 5'-nitropiazselenol derivatives of 76Se and 80Se, respectively.
Scanning time for each selected peak, 420 ms
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Table 1.
Isotopic Composition of Natural Abundance of
Selenium (Se-NPD) and Enriched 76Se (76Se-NPD)
| Se Isotope
| Se-NPD Ion (m/z)
| Measured % Abundance Se-NPD (Natural Se) n=10*
| C.V.(%)
| Theoretical % Abundance
(Natural Se)**
| Measured % Abundance Se-NPD (76Se Solutn) n=10*
| 74 | 223 | 0.77 | 7.79 | 0.80 | 1.07
| 76 | 225 | 8.45 | 0.24 | 8.29 | 91.35
| 77 | 226 | 7.54 | 0.26 | 7.61 | 6.84
| 78 | 227 | 22.47 | 0.4 | 22.22 | 0.63
| 80 | 229 | 46.56 | 0.19 | 45.93 | 0.11
| 82 | 231 | 8.35 | 0.6 | 8.85 | 0.0
* n = Number of replicates
** Calculation based on the known isotopic composition of C, H, N, O, Se
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Table 2.
Selenium Levels in CRM Cabbage Leaves and Soil
| Sample (Reference No.) | Certified Se (µg/g) | Measured Se (µg/g)
|
|---|
| Cabbage Leaves (C85-04) | 0.083 ± 0.008 | 0.091 ± 0.007
| | Chinese Soil (C74-05) | 1.56 ± 0.12 | 1.67 ± 0.04
| Values are means derived from triplicate analysis of three individual
samples. Errors are standard deviations
of the means (n=3).
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How Important is Selenium?
Selenium plays a major role in agriculture. Its presence in
the soil of grazing land and in the feedstock has important
implications in animal husbandry and the dairy and beef
industry.
Low concentrations of Se in the diet of farm animals can lead
to impaired locomotion, low conception rates, high incidence
of abortion, and anemia. In horses, difficulty in swallowing
and respiratory distress has been observed. Cattle and sheep
have been diagnosed with infertility and poor growth as a
result of selenium-deficient feed. Acute lack of the trace
mineral has caused excessive deaths in farm pig populations.
In agricultural areas of known Se deficiency, farmers often
add small dosages of the mineral to the feed of their
livestock.
Grossly excessive selenium content in the soil and in streams,
on the other hand, has led to major birth defects in water
fowl and migratory birds visiting runoff-polluted geographies.
High dietary Se levels have caused cancer in laboratory
animals.
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