Fresenius' Joumal of
Fresenius J Anal Chem (1994) 348:792-805
Analytical methods for the speciation of selenium compounds: a review
X. Dauchy 1, M. Potin-Gautier 2, A. Astruc 2, and M. Astruc 2
BRGM, Drpartement Analyse, BP 6009, F-45060 Orleans Cedex 2, France
2 Laboratoire de Chimie Analytique, Facult6 des Sciences et Techniques, Avenue de l'Universit6, F-64000 Pau, France
Received: 7 July 1993
Abstract. Selenium, like sulphur, exists in the environment
in several oxidation states and as a variety of inorganic and
organic compounds. Dissolved inorganic selenium can be
found in natural waters as selenide Se ( - I I ) , as colloidal
elemental selenium Se (0), as selenite anions HSeO3 and
SeO 2- i.e. Se (+IV) and as the selenate anion (SeO4z ) i.e.
Se (+VI). Organic forms of selenium that may be found in
organisms, air or in the aqueous environment, are volatile
(methylselenides) or non volatile (trimethylselenonium ion,
selenoamino acids and their derivatives). Knowledge of the
different chemical forms and their environmental and bio-
medical distribution is important because of the dependence
of bioavailability and toxicity on speciation. This paper re-
views the different analytical methods used for the specia-
tion of selenium compounds, with special attention to inor-
ganic selenium and organoselenium species.
1 Introduction
Selenium is now well known as an essential element for
biological systems both as a nutrient and as a potential toxi-
cant, the difference between the necessary daily intake and
the toxical value being narrow.
A number of analytical procedures exists for the deter-
mination of selenium in samples from different compart-
ments of the environment. However only total selenium is
reached with these methods whereas it is recognised that
the toxicity and the bioavailability of selenium depend on
its chemical form. Thus the development of reliable tech-
niques to study the speciation of selenium in waters, soils,
vegetable and other biological materials has been a neces-
sary step to understand the biogeochemical cycle, mobility,
transfer and uptake of selenium, as well as its toxicity.
In this review, we distinguish the papers related to the
speciation of inorganic selenium from those concerning
specific organoselenium compounds. In 1982, Robberecht
and Van Grieken [1] have published a review on selenium
in waters. Their data are not included in this paper.
Correspondence to : M. Potin-Gautier
Springer-Verlag 1994
2 Speciation of inorganic selenium
Selenium has four formal oxidation states:
The oxidation state ( - I I ) selenium exists as hydrogen
selenide (HSe-) at natural pH-values, and as a number of
metallic selenides. H2Se is a toxic gas at room temperature
and is thermodynamically unstable in aqueous solutions.
Heavy metal selenides may be one of the insoluble forms
in soils.
Elemental selenium [Se(0)] exists in several allotropic
forms. It is very stable and highly insoluble, but may have
colloidal forms.
Selenium in the (+IV) state occurs as selenite (HSeO~
or SeO~-) in natural media.
Selenium in the (+VI) state or selenate (SeO]-) is
stable under alkaline and oxidizing conditions.
The relative importance of these various oxidation
states is different in natural conditions and in the compart-
ments of the environment.
a) Speciation by separation
and direct species determination
This method is only used to separate selenite and selenate
ions. Some authors suggest to use ion-pairing HPLC [2, 3],
but the most commonly used separation method is ionic
chromatography [4-20]. On the other hand, the detection
methods are very variable: conductimetry [4, 5, 15], U. V.
[12, 14], atomic absorption [16], HG-AAS [17], neutron
activation [6, 7], fluorimetry [9], ICP-AES [20] or ICP-MS
[2].
Table 1 summarises the different experimental condi-
tions proposed for this analytical procedure.
b) Speciation by specific oxido-reduction reactions
This method is more often used than the first one because
it allows the specific determination of Se (+IV), Se (+VI),
Se (0) and Se (-II).
1 Determination of selenite ion Se (+IV). Selenium (+IV)
is directly determined in water samples either by GC-ECD
[21-28], GC-MS [29], GC-IDMS [301, fluorimetry [ 3 1 -
35] after formation of volatile piazselenol complex with an
appropriate diamine reagent or by HG-AAS [36-42], HG-
AAS with radiotracer techniques [43-45], HG-DCP-AES
 793

Table 1. Speciation of inorganic selenium by direct determination

Species Sample Procedure Detector Detection Ref.

type limit

Se (+IV) Soil Vydac 302 IC 4.6 Conductimetry 90 ng. m1-1 [5]

Phtalic acid 1.5 mM 9 ng
Se (+IV) Fresh Dionex 30589 ETAAS 20 ng - rnl-' [16]
Se (+VI) water Na2CO3 8 mM 20 ng
Se (+IV) Urine BioRad X AG.2. X 8 10 rig. ml I [6]
TMSe LiOH 0.5 M NAA 10 ng
Se (+IV) Water Whatman Partisil 5ODS-3 ICP-AES 40 to 70 [19]
Se (+VI) Water-CH3OH 90/10 ng. ml -~
(Bu4N)3PO4 5 mM
Se (+ IV) Soil Dionex Omnipac Conductimetry 30 ng- ml - ~ [13]
Se (+VI) PAX-500 AC
Na2CO3 2 mM-NaHCO3
1.7 mM
Se (+IV) Soil Wescan 269-029 Conductimetry 110 ng. ml -~ [11]
Se (+VI) p-hydroxybenzoic acid 60 ng • m1-1
2.0-6.0 mM
Se (+IV) Water Dionex AS4A U.V. 100 ng. rnl-~ [12]
Se (+VI) Na2CO3 2.25 m M +
NaHCO3 0.75 mM
Se (+VI) Water Dionex ItPIC-AS5 Conductimetry 4.8 ng. rnl-~ [10]
Na2CO3 2.5 m M +
NaOH 1.0 mM

[46], HG-ICP-AES [47], HG-ICP-MS [48], or again by
electrochemical methods such as differential-pulse polaro-
graphy, square-wave voltammetry, differential-pulse cath-
odic stripping or adsorptive stripping voltammetries [ 4 9 -
6O].
For more complex samples such as animal tissues or
geological materials, the determination of selenite requires
a pretreatment (ultrasonic waves, centrifugation, removal of
organic species by XAD resin [61]).
The procedures using acid leaching [6•] may be uncer-
tain because the oxidation state of selenium is controlled
by the experimental redox treatment.
2 Determination of total selenium Se~. The most commonly
used techniques (voltammetry, GC, HG-AAS, HG-
ETAAS), depend on the formation of Se (+IV). The
Se ( - I I ) and Se (0) species must be oxidized and Se (+VI)
reduced at the same time.
For aqueous samples, these conversions may be realised
in different ways: bromine-bromide redox buffer solution
[26], effect of a concentrated sulphuric acid and titanium
trichloride solution by heating the mixture before addition
of the bromine-bromide redox buffer solution and heating
again[21, 24], effect of hydrobromic acid and bromine solu-
tion by boiling [25], addition of a potassium persulphate
solution in hot hydrochloric acid solution [38-41, 63, 64]
or in oxalic acid solution [39], with a mixture of nitric and
perchloric acids followed by a reduction with hydrochloric
acid [65].
Animal tissues and fluids [22, 26, 44, 61, 66, 67], soils
[35, 67-73], plants [26, 31, 35, 68, 74], foods [75, 76],
sediments [41, 61, 69, 77, 78], and incinerator ashes [62]
require a digestion with boiling nitric acid [22, 26, 50, 62]
or a nitric and perchloric acids mixture with hydrogen per-
oxide [6•, 68] and sometimes with the addition of hydrox-
ylamine [79] or a mixture of nitric, perchloric and hydroflu-
oric acids for geological materials [69]. Simple microwave
digestion procedures have been developed [66, 67]. Sele-
nate is then reduced to selenite with boiling hydrochloric
acid. Kuldvere [78] converted selenium to the (+IV) oxida-
tion state with aqua regia and reversed aqua regia. Selenium
(+V1) is reduced to selenium (+IV) and simultaneously
the lower oxidation states are oxidized to selenium (+IV).
Potin-Gautier et al. have compared the total selenium
concentrations determined with different pretreatments and
analytical techniques in water and soil samples [73] and in
a vegetable [74].
3 Determination of selenate ion Se (+V I). The selenium
(+VI) determination is never realised directly but necessi-
tates two treatments of the sample and two measurements.
Two procedures are used:
a) the difference between the total selenium (SET)
content and the sum SeR = [Se ( - I f ) , (0), (+IV)] obtained
by mild oxidation of selenide and elemental selenium mix-
ture to selenite, represents the Se (+VI) fraction;
b) Se (+VI) level is the difference between the direct
Se (+IV) determination and the Seo = [Se (+VI) + Se
(+IV)] analysis obtained by mild reduction of selenate
ions.
For aqueous samples, the oxidation of selenide and ele-
mental selenium to selenite is realised by a bromine solu-
tion [26] or a bromine solution in concentrated hydrochloric
acid [25], whereas selenate is reduced to selenite in a boil-
ing hydrochloric acid solution [33, 36, 49, 50], a mixture
of titanium trichloride/hydrogen peroxide/hydrochloric acid
[37] or by U. V. irradiation [51].
794
For animal materials [23, 61], vegetable [26], biological
samples [22, 26] and sediments [61], a wet digestion is
required by a single acid such as nitric acid [22, 23, 26] or
a mixture of acids [61]; then Se (+VI) is reduced to Se
(+IV) by classical hydrochloric acid treatment or again in
an hydrochloric persulphate solution [61].
4 Determination of the sum of selenide Se (-II) and ele-
mental selenium Se (0). Two schemes are used for this de-
termination:
a) the sum Se (-II) + Se (0) is the difference between
the SeR data and the Se (+IV)-value obtained prior to oxi-
dation (two measurements);
b) the sum Se (-II) + Se (0) is the difference between
SeT and Seo (two measurements).
The quality of the results obtained by these methods of
speciation is dependent on the selectivity of the redox reac-
tions used. Several critical studies [50, 70, 78-83] have
been realised to compare the different procedures.
The results of various studies are presented in Table 2.
Some authors have studied inorganic selenium with total
organic selenium [50] or particulate selenium [38, 50] at
the same time.
3 Speciation of organic selenium
a) Identification and determination
of volatile alkylselenium compounds
Concentrations of atmospheric selenium in remote areas of
the earth are far in excess of predictions from anthropo-
genic or known natural sources [84]. A large fraction of
atmospheric selenium appears to exist in an unidentified
vapor phase [84, 85], which probably plays an important
role in the global atmospheric selenium budget [86]. A po-
tentially important source for the atmospheric enrichment
of selenium is natural biomethylation [84].
The methylation of selenium has been known for some
time [87]. It has been well documented that volatile al-
kylselenium compounds are produced from inorganic sele-
nium salts through a biomethylation process by various or-
ganisms. Challenger [88] first pointed out that two different
strains of fungus produced dimethyl selenide (DMSe) from
sodium selenate or selenite. Rats fed with selenite and sele-
nate exhaled a volatile selenium compound identified as
DMSe [89-92], methylation probably taking place in the
liver [93-95]. The release of selenium in volatile form
from plants has been suggested [96, 97] and confirmed [98,
99]. In other studies, dimethyl selenide and/or dimethyl dis-
elenide were identified in vapors given off by plants [100-
103].
A variety of microorganisms such as fungi, molds and
bacteria are capable of producing volatile selenium com-
pounds (DMSe, DMDSe, (CH3)z SeO2) with and without
added inorganic or organic selenium species [104-106].
Methylated selenium compounds have been observed ema-
nating from soils [107-113], biologically active lake sedi-
ments [114], and sewage sludge [109, 115-117]. Alkylsel-
enides have also been detected in the atmosphere just above
freshwater lakes [117, 118], in natural aqueous systems
[119] and in air particulates [120].
It is generally believed that the formation of these meth-
ylated compounds constitutes a major mechanism of sele-
nium detoxification for microorganisms [109], animals [89,
95, 121] and probably man, since dimethyl selenide is con-
sidered to be about 500 times less toxic than selenite [95,
122]. Several cycles have been proposed for the methyl-
ation of selenium [88, 101, 109, 119, 123, 124].
Data on the determination of these volatile alkylsele-
nides are scarce because efforts to speciate these gases have
been hampered by difficulties of collection and lack of suf-
ficiently sensitive Se-selective detectors.
1 Collection of volatile alkylselenium compounds. Since the
alkylselenide species levels in air are very low, an efficient
sampling technique for preconcentrating the analyte is nec-
essary. The common air sampling procedure is based on
cryogenic trapping. The volatile selenides are removed
from the sample by sucking with a pump (from air) or by
gas stripping (from water, soil, sewage sludge and sedi-
ment), and swept into the cold trap. A variety of liquid
trapping media (hexane, aqueous solution of EDTA, etc.)
contained in tubes was investigated [98, 110]. None of the
aqueous media described by Lewis et al. [98] has been
proven to be effective in retaining organoselenide com-
pounds. Various kinds of solid adsorbent such as activated
carbon [98, 100, 113, 125, 126], glass wool [36, 117, 118],
or gas chromatographic stationary phases [102, 105, 106,
109, 119, 127, 128] were found to be successful for trap-
ping volatile Se species. Immersion of the charcoal tubes
in liquid nitrogen did not increase the efficiency of adsorp-
tion of alkylselenide compounds on charcoal [98]. How-
ever, quantitative collection can be achieved with other ad-
sorbents at liquid nitrogen temperature [119, 120], at ca.
-130°C [117, 118], - 8 0 ° C [127] or - 2 0 ° C [106]. Effects
of cryogenic trap temperature and of several adsorbents on
the collection efficiency have been investigated by Jiang et
al. [117, 129].
The volatile selenium compounds are then thermally
desorbed [102, 103, 106, 118-120, 127] or extracted from
the adsorbent with various solvents [98, 100, 109, 113, 126]
prior to gas chromatographic analysis. The losses for the
least volatile species may be due to incomplete recoveries
from the cryogenic trap. The thermal desorption procedure
must be done very carefully [120]. Reported recoveries
were 95-98% for DMSe, 84-98% for DMDSe and 8 9 -
100% for DEtSe [117, 120, 127, 129].
The precolumn adsorption technique is important. This
step allows an accumulation of the volatile selenides to lev-
els suitable for detection. Collecting sample vapors directly
in the analytical column [102] resulted in severe peak
broadening.
2 Analytical methods for speciation of volatile alkylsele-
nides. Evans et al. [100, 130] developed separation pro-
cedures using gas chromatography. However, the chromato-
grams obtained were relatively difficult to interpret. The
largest response to the monoselenides was obtained with an
FID, whereas diselenides and ethyl selenocyanate gave the
largest response with an ECD [130]. Differences in solubil-
ity and in chromatographic behaviour were used to separate
the components [100]. Gas chromatography with FID was
also used by Fleming et al. [115] for the identification of
microbial metabolites. In all these methods, the detection
timits were in the microgram range.
 795

Table 2. Speciation of inorganic selenium by specific oxidoreduction reactions

Determined Calculated Sample type Procedure Detection Ref.

species species limit

Se (+IV) Se (+VI) Water GC-ECD 2 ng. 1-1 [26]

SeR Se ( - I I ) + Se (0)
Set
SeR Se (+VI) Plant GC-ECD 5 ng • g 1 [26]
SeT Biological
material
Se (+IV) Water GC-ECD 2 ng • 1 1 [24]
Sex
SeR Se (+VI) Bovine liver GC-ECD 1 ng • m1-1 [23]
SeT
Se (+IV) Se (+VI) Water GC-ECD 2 ng• 1-~ [25]
Ser~ Se ( - I I ) + Se (0)
Sex
Se (+IV) Se (+VI) Water HG-ETAAS 5 ng • 1 1 [36]
Seo 0.5 ng
Se (+IV) Se (+VI) Water HG-AAS 20 ng• 1-1 [17]
Seo 1 ng
Se (+IV) Se (+VI) Sea water HG-AAS 0.01 nmol- kg -1 [38]
Seo Se ( - I I ) + Se (0)
Sex
Sep~ti~
Se (+IV) Se (+VI) Sediment DPP or DPCSV 10 ng • 1-~ [61]
Se0 Se ( - I I ) + Se (0) Animal tissue 2 ng
SeT
Se (+IV) Se (+VI) Natural water DPP 76 nM [50]
Seo Seorga 0.51 nM
Seorga + Se
(+IV)
Sep~ti~
Se (+IV) Se (+VI) Agricultural HG-AAS 1 ng• ml -~ [39]
Seo Seo~g~+ Se (0) drainage
Sex and soil
Se (+IV) Se (+VI) Water DPP [49]
Seo
Se (+IV) Se (+VI) Garbage HG-AAS [62]
Seo Se (0) incineration XRFS
Sex
Sex Se (+VI) Water IDMS 12 pg- g ~ [65]
Seo Se ( - I I ) + Se (0)
Se (+IV)
Seo Se (+VI) Sea water Fluorimetry [34]
Se (+IV)
Se (+IV) Se (+VI) Human blood GC-ECD 2 ng - ml 1 [22]
Seo or plasma
Se (+IV) Se (+VI) Ground water Zirconium 5 ng- 1-1 [33]
Sex precipitation
Fluorimetry
Se (+IV) Se (+VI) Natural water Fluorimetry 50 ng • 1 i [321
Seo Seo~go
Sex

Successful speciation assemblies usually involve gas
chromatography with either atomic absorption spectrometry
[102, 103,114, 1 1 8 - 1 2 0 , 127] or microwave plasma detec-
tion [109, 116]. The detection limits approach 0.1 ng Se for
AAS [36, 114, 1 1 8 - 1 2 0 , 127] and 0.02 ng for MPD [109,
116]. SCD [106] and NDAFS [131] have proven to be ex-
cellent detectors for the speciation of alkylselenides. A very
sensitive method for the determination of volatile selenium
species has been developed by Jiang et al. [110]. These
compounds were separated by GC followed by sequential
796

Table 3. Speciation of volatile alkylselenides

Species Sample type Column Detector Detection limit Ref.

DMSe, DMDSe, DEtSe, Pure compound 5 ft. X 1/8 in. Hydrogen FID - [130]
DEtDSe, DPrSe, 20% polymetaphenylether on or ECD
DPrDSe and ethyl 60/80 chromosorb W coated
selenocyanate with hexamethyldisilizane
Four compounds Volatile selenium com- 5 ft.×l/8 in. Hydrogen FID - [100]
One of them is DMDSe pound released from 20% polymetaphenylether on or ECD
plants 60/80 chromosorb W coated
with hexarnethyldisilizane
DMSe, DMDSe and one Synthetic air samples 1.8 m X 6 rnm AAS with 0.1 ng Se [127]
unknown volatile sele- and headspace gases of 3% OV-1 on chromosorb W, silica furnace [114]
nium compounda culture flasks (soil, lake, 80-100 mesh
sediment)
DMSe, DMDSe Cultures of a strain of Chromosorb 101 (60/ FID - [115]
penicillium isolated 80 mesh) and 5% FFAP
from raw sewage on chromosorb G (100/
120 mesh)
DMSe, DMDSe, DEtSe, Vapors given off by 122 cm X 3 mm 20% poly- AAS with a - [102]
and 3 peaks, uniden- plants metaphenylether on 60/ quartz "T" [103]
tiffed 80 mesh chromosorb W tube furnace
DMSe, DMDSe and Volatile selenium me- 1.2 m X 2 mm 3% OV - MPD 20 pg of DMSe [109]
(CH3)2 SeO2 tabolites evolved by mi- 101 on 100/120 mesh chro- [116]
croorganisms in sewage mosorb 750
sludge and soil
DMSe, DMDSe, DEtSe, Air samples collected at U-tube filled with 0.1 g of GFAAS DMSe = 0.1 ng [120]
Dimethyl Selenone various aquatic environ- 10% polymetaphenylether on DMDSe = 0.2 ng [118]
ments 80-100 mesh chromosorb DEtSe = 0.1 ng
W
DMSe, DMDSe Pure organic chemicals 10 m × 0.53 mm HP-1 wide NDAFS 10 pg Se [131]
bore column coated with a
2.65 gm thick film of
methylsilicone gum
DMSe, DMDSe Surface water and oxic 25 cm glass column 5% AAS 0.1 ng Se [119]
groundwater OV-3 on chromosorb WHP
(80-100 mesh)
DMSe, DMDSe, Di- Headspace gas samples 30 m x 0.25 mm DB-5 with SCD DMSe = 22 pg Se [106]
methyl selenyl sulfide from biological cultures 1 ~tm film of methyl phenyl DMDSe -- 8 pg Se
(5%) silicone
DMSe, DMDSe, DEtSe Gaseous selenium me- U-shaped glass column 1.5% GFAAS DMSe = 5 pg Se [110]
tabolites evolved from OV-1 coated on shimalite W DEtSe = 5 pg Se
soil DMDSe = 20 pg Se
DMSe, DMDSe Natural waters 6 m × 4 . 8 mm stainless steel AAS with a 0.1 ng Se [36]
column packed with 16.5% quartz
silicone oil DC-550 on chro- furnace
mosorb WAW DMCS ( 8 0 -
100 mesh)
DMSe, DMDSe Gaseous evolved from 25 m CPS.2 capillary column MS - [1131
soil
DMSe Breath air 100 × 0.3 cm VZ-7, 6 0 - Hydrogen 1 gg/ml [126]
of mice 80 mesh (gasukurokogyo FID
Co.)
DMSe, DMDSe Gas evolved from 1.8 mX 3 mm chromosorb MS - [105]
amended soil 101 (60/80 mesh)

According to Reamer et al. [109], the unknown species is probably (CH3)z SeO2.

introduction of the separated species into a graphite fur- ing their chromatographic behaviour with those of pure ref-
nace. erence compounds [100, 102, 103, 127].
The identification of the organoselenide species was Selected gas-chromatographic selenium speciation ap-
confirmed by GC-MS [106, 109, 1 1 3 - 1 1 5 ] or by compar- plications are listed in Table 3.

b) Identification and determination
of selenoamino acids
In this chapter, we will confine ourselves to mentioning
those studies which have established the occurrence of sele-
noamino acids, as well as the analytical methods used.
Other aspects of selenoamino acid biochemistry (incorpora-
tion into the proteins, metabolic actions, biosynthesis etc.)
have been reviewed elsewhere [124, 132-137[.
Several investigators have demonstrated that in some
biological systems selenium compounds primarily occur as
selenium analogs of sulphamino acids. Selenocystine
[138-140], selenomethionine [97, 137, 138, 141-145],
Se-methylselenocysteine [97, 140, 146-148], selenocys-
tathionine [140, 149], Se-methylselenomethionine [97, 124,
139, 150] and selenohomocystine [151] have all been re-
ported as present in certain biological tissues or protein
fractions (leaves or seeds of plants, cultures of bacteria or
yeast, organs of animals). The selenoamino acids isolated
probably originate from several enzymatic systems.
Indeed, it was established that several enzyme-cata-
lyzed reactions required the participation of a selenium-
containing protein [152-154]. The chemical form of sele-
nium in clostridil glycine reductase [155, 156], glutathione
peroxidase [157-159], formate dehydrogenase [160] and
hydrogenase [161] has been shown to be selenocysteine
[162]. On the other hand, selenomethionine has been iden-
tified as a constituent of clostridial thiolase [I63-165]. In
addition, proteins of unknown catalytic function have been
reported to contain selenocysteine [166, 167], or uniden-
tified forms of selenocompounds have been found in sele-
noproteins [148].
Selenoamino acids have been hypothetized as metabo-
lites in the biological pathways of selenium excretion by
living organisms [168, 169]. Their total determination in
urine has been achieved by Blotcky et al. [7].
The identity of organoselenium compounds in the
aquatic environment has not been fully established. Several
of the possible species are selenium-containing amino
acids, primarily in the form of dissolved peptides [38, 170,
17l]. According to Cooke et al. [119], Se-methyl-
selenomethionine released from plant detritus or microbial
biosynthesis is a likely candidate. In marine bacteria and
plankton, selenium is predominantly found as selenoamino
acids in proteins [172-174]. Selenomethionine has been
isolated from the hydrophilic fulvate fraction of soil [175],
and from proteins of marine algae [176].
Two separation methods have been suggested in the lit-
erature: they involve ion exchange or gas chromatography.
For gas chromatography, it is necessary to convert sele-
noamino acids to highly volatile derivatives. Various rea-
gents have been studied, such as bis (trimethylsilyl)
acetamide [142, 177], N-methyl-N(tertbutyldimethylsilyl)
-trifluoroacetamide [175] and cyanogen bromide [141,178,
179]. Identification of the chemical form of selenium was
generally achieved by mass spectrometry [142, 175] or by
co-chromatography with derivatives of standard seleno-
amino acids [141, 177-180].
The classical method of separation of selenoamino
acids is ion-exchange chromatography. It has permitted the
separate elution of selenocystine, selenocysteine and sele-
nomethionine from other amino acids [140, 181-184].
This procedure has been applied to crude animal or plant
extracts. However, attempts to identify the chemical struc-
797
tures of organoselenium in proteins were unsuccessful until
1976. When the native proteins are subjected to the pro-
cedures commonly employed to cleave them into their con-
stituent amino acids, selenocysteine decomposes due to its
acid lability [185[. The need to protect selenocysteine resi-
dues prior to hydrolysis and isolation has been commented
by several authors [142, 155-157, 160, 162]. In most
cases, it has been achieved after reduction of the protein
and treatment with alkylating agents. Selenocysteine is
identified as its carboxymethyl-, aminoethyl-, carboxy-
ethyl- and 2,4 dinitrophenyl derivative from digests of the
pure protein [155-158, 160-162, 166, 167, 186]. The rou-
tine method for determining the aminoacids can only be
used for the detection of selenomethionine [141]. Indeed,
its presence did not depend on prior alkylation of the pro-
tein [163, 164]. However, Ouyang et al. [141], who have
compared the results of gas chromatography with those ob-
tained by acid hydrolysis/ion-exchange chromatography,
found that the latter gave much lower values. They have
suggested that this may be due to the oxidation of Se-Met
during acid hydrolysis.
According to Stadtman [133, 162], claims in the litera-
ture prior to 1975 that selenocysteine (or selenocystine) was
identified in acid hydrolyzates of native proteins, are espe-
cially suspect, and probably incorrect for the most part.
As for gas chromatography, the primary means of
identification of selenoamino acids are chromatographic
comparison with known standards [157, 161] or mass spec-
trometry [158, 186]. However, paper chromatography, thin-
layer and gel permeation chromatography have also been
proven to be useful tools to demonstrate the existence of
selenoamino acids [143, 144, 157, 158, 160, 162, 187].
Selenocysteine residues in glutathione peroxidase, total
selenoarnino acids in urine and selenomethionine metabo-
lites in cell cultures have been determined by X-ray crystal-
lographic analysis [159], NAA [7] and HPLC [137] respec-
tively. To our knowledge, only Cooke et al. [119] have
established an analytical method for the measurement of
selenium-containing free amino acids (and/or colloidal ele-
mentary selenium) in aquatic samples. They have calcu-
lated these data by difference between the sum [Se (-II)
+ Se (0)[ and the Cls solid phase extraction fraction which
appeared to isolate dissolved organic selenides (associated
with soluble peptides, proteins and higher molecular weight
organic compounds) from the free selenoamino acids.
Selected analytical procedures for the speciation of sel-
enoamino acids are listed in Table 4.
c) Identification and determination
of trimethylselenonium ion
Trimethylselenonium (TMSe +) has been reported to be pre-
sent in the urine of rats fed or injected with relatively high
levels of selenite [191-193[ and other forms of selenium
[136, 193-195]. It has also been identified in human urine
[6, 7, 196-201] and serum samples [7].
The level of TMSe + in urine of normal subjects is quite
low (0.2 < [TMSe +] < 60 ng Se/ml) [6, 7, 199, 200]. In
pooled serum samples, TMSe + level was in the range 3 6 -
132 ng Se/ml [7]. Foster et al. [136] have published a study
on the distribution of TMSe + in rat tissues. There is evi-
dence that the entry rate of the dose as well as the chemical
form of ingested selenium influence the ratio of TMSe + to
total urinary Se [6, 136, 192, 193, 198, 200]. These obser-
798

Table 4. Speciation of selenoamino acids

Species Sample type Procedure Detector Detection limit Ref.

Se-Met and its sulfur Serum, liver or muscle Paper chromatography (buta- NAA 0.1 g mol m1-1 [1871
analog nol-pyridine-water solvent)
Se-Met, selenocystine Pure compounds Gas-liquid chromatography of Hydrogen - [177]
and their sulfur analogs silylated derivatives of seleno- FID
amino acids
Selenocystine, Se-Met, Seeds of plants Ion-exchange chromatography Spectro- - [140]
Se-Cysta, Se-Cys, (gradient elution with buffers photo-
selenohomocystine and prepared from sodium citrate metry
their sulfur analogs and hydrochloric acid)
Se-Cys Clostridial glycine 7sSe-labeled protein reduced Gamma - [155]
reductase and formate and treated with various counter [160]
dehydrogenase alkylating agents prior to [162]
hydrolysis and separation on the
column of the amino-acid ana-
lyzer
Se-Cys Rat liver glutathione Carboxymethylation and Gamma - [157]
peroxidase aminoethylation of 75Se- counter
labeled protein prior to hy-
drolysis and separation on the
columns of an amino acid ana-
lyzer
Se-Met Clostridial thiolase Cochromatography of native Gamma - [163]
7sSe-thiolase with authentic Se- counter [164]
Met on an amino acid analyzer
Se-Cys Pure ovine erythrocyte Derivatization of the reduced MS [158]
glutathione peroxidase enzyme with 1-fluoro-2,4-dini- [1861
trobenzene. Separation by gel
filtration and reversed phase
HPLC. Transformation to Se-
Methyl-N (2,4-dinitrophenyl)
selenocysteine
Se-Met, Se-Cys Standard amino acid Ion-exchange chromato- Spectro- [181]
mixture graphy photo-
metry
Se-Met metabolites and Cell culture Single HPLC run with a HP Spectro- 20 pmol [1371
their sulfur analogs Hypersil column photo- per compound
(2.1 × 100 ram) metry
Se-Met Pure compound Determination of Se-Met by re- FID 8 ng [1781
and yeast action with cyanogen bromide [1411
and gas chromatography on a [1791
column (2 m × 2.5 ram) packed
with 10%. Ethylene glycol suc-
cinate on chromosorb W-AW
DMCS ( 6 0 - 80 mesh)
Se-Met Pure compound Se-Met reacts with CNBr to ECD [180]
form methylselenocyanate,
which is extracted and
digested to give SeO~-.
This is then determined as
piazselenole by GC
Se-Met Soybean protein Proteolytic digestion. Subffac- MS [1421
tionation on a gel permeation
colunm. Se-contalning fraction
was chromatographed on cellu-
lose thin-layer plate and identi-
fied by GC-MS
Se-Met Raw materials HPLC [sperisorb 005-2 (5 gin) Spectro- 5 gg • m1-1 [1881
and tablets 12 cm × 4.6 mm] with a mobile photometry
phase of acetonitrile and sodium
acetate buffer
 799

Table 4 (continued)

Species Sample type Procedure Detector Detection limit Ref.

Se-Met Soil extract Fractionation using Amberlite MS - [175]

XAD-8 resin with a pH gradi-
ent. Derivatization and analysis
by GC-MS
Total selenoamino Urine Derivatization with o-phthalal- NAA 40 ng• ml i [7]
acids dehyde and 2-mercaptoethanol.
Trapping of the amino acids on
an anion exchange resin
Selenocysteine Tissue and protein Carboxymethylation of 758e. Gamma - [166]
samples Labeled crude homogenates or counter
protein fractions prior to hy-
drolysis and separation on an
amino acid analyzer
Se-Met, Se-Cys, Health food HPLC (Nucleosil 5NO2) using AAS Low ng [189]
selenocystine supplements a methanolic mobile phase con- level [190]
taining triethylamine and acetic
acid. Thermochemical hydride
generation integrated in a silica
interface

vations seem to support the hypothesis that this seleno-me-
tabolite does not constitute a quantitatively significant uri-
nary excretory product under low dosing conditions. How-
ever, it is generally believed that TMSe + assumes a signifi-
cant role in the detoxification of excess selenium intakes
[191-193, 196, 198]. Many questions such as the kinetics
of its synthesis, organ specificity and details on metabolic
processes remain to be elucidated. In addition, the chemical
forms of selenium in urine have not yet been completely
characterized. Other forms such as selenoniocholine [20]
and selenoamino acids [7] may constitute a significant por-
tion of Set in urine.
Recently, two studies have demonstrated the occurrence
of TMSe + in lake water samples with a mean concentration
of 12 ng/1 [65, 202].
Early techniques to measure TMSe + levels in urine in-
volved the use of the radiotracer 75Se with y-radiation
counting or autoradiography after ion-exchange purification
[136, 191, 196] or paper chromatography [192-194]. Its
use is limited because of the relatively long biological half-
life of this radiotracer and the associated issues of radiation
exposure.
Most of the methods used recently are based on ion-
exchange chromatography and fractional determination.
TMSe + is concentrated and isolated from other forms of
selenium on the ion-exchange resin [6, 7, 65, 193, 198,
202]. Single-column procedures were developed for water
and urine samples [136, 193, 195]. Indeed, one paper [198]
showed that this method may underestimate the TMSe +
level in urine samples. Only 10% of the labeled 75Se
TMSe + was recovered in the eluate. A modified dual-col-
umn procedure was reported [198] in which the urine was
first passed through an anion-exchange resin, connected in
series to a cation-exchanger. This method has also been
used by Blotcky et al. [199] and gave a good recovery for
TMSe + in urine (92%).
Alternatively, the ion-exchange column is used to trap
selectively TMSe + (the resin containing the selenium me-
tabolite is analyzed for 77~nSeby NAA) [199] or to convert
the TMSe-reineckate to TMSe C1 [200].
High-performance liquid chromatography appeared to
be clearly superior to earlier methods. The high resolution
obtained by HPLC permits a clear separation of the TMSe +
ion from other organoselenium compounds [195, 20l]. The
TMSe + level was also calculated indirectly by substracting
non-TMSe + selenium from total urine selenium [198], or
by forming the volatile compound DMSe after thermal de-
composition of TMSe + in highly alkaline solutions [65,
200]. This alkyl selenide is collected in HNO3 and analyzed
by ICP/MS [200] or IDMS [65].
All these techniques are rather time consuming due to
the number of chemical manipulations that are required and
employ high-cost detectors such as NAA [6, 7, 198, 199]
and ICP/MS [200].
Selected analytical techniques for separating TMSe +
from other organoselenium compounds are listed in
Table 5.
d) Identification and determination
of other organic selenium compounds
Some authors have already demonstrated the occurrence of
organic selenium compounds other than alkylselenides,
TMSe +, and selenoamino acids. Previous investigations
dealing with the total organoselenium determination will
not be described in this article.
Tanzer et al. [65] have developed a selective determina-
tion of acidic, neutral and basic organoselenium compounds
in water samples. The speciation analysis was carried out
by adsorbing these compounds on a XAD-2 column at dif-
ferent pH (pH 8 for the neutral and basic forms, and pH 3
for acidic forms). The acidic organoselenium forms (94 and
28 pg/g) were higher than the basic and neutral ones (49
and 18 pg/g).
The determination of non-trimethylselenonium sele-
nium in urine [198] was achieved by a selective wet oxida-
tion. Except for TMSe +, all urine selenium is oxidized to a
common pool of selenite. Kraus et al. [195] have published
a rapid and simple method for the separation of acetic acid-
(2-dimethylselenonium) bromide and acetic acid-(2-di-
 80O

Table 5. Speciation of trimethylselenonium ion

Species Sample Column Solvent Detector Detection Ref.

type system limit

TMSe +, Human 1.5 X 10 cm glass 100 ml NAA - [198]

total selenium, urine column packed with AG 6 tool/1 C1
non TMSe + 2 × 10 (200-400 mesh)
selenium connected in series
to 1.5 × 10 cm glass
column packed with
AG 50 W × 8
( 2 0 - 5 0 mesh)
TMSe +, Rat urine 4 X 200 mm Gradient Gamma - [195]
selenomethionine, (75Se-labeled Macherey-Nagel elution counter
Se-methylseleno- compounds) Nucleosil 0.003-0.33 mol/1
cysteine, dimethyl 5 grrdSA (NH4)2
selenocysteine HPO4
selenonium, (pH 4)
Se-methyl seleno- 0.33-0.5 mol/1
methionine, and two (NH4)2
acetic acid (2-Se HPO4
dimethylselenonium) (pH 4)
TMSe +, Human 0.7 × 4 cm polystyrene 97 ml of NAA - [199]
total selenium urine column packed with 0.5 tool/1 LiNO3
AG 2 × 8 resin (200-
400 mesh) and
0.7 × 10 cm borosilicate
glass column packed
with AG 50 W × 8
(200- 400 mesh)
TMSe +, SeO 2 , Human urine 0.7 × I0 cm borosilicate 0.5 mol/1 LiOH NAA 10 ng of [6]
total selenium, and serum column packed with Se/ml [7]
total selenoamino AG 2 X 8 resin
acids (200- 400 mesh)
TMSe +, Human urine 2.8 × 14 cm packed Distilled ICP/MS 40 ng Se [200]
total selenium with Dowex water
1 X 8 (100-200 mesh)
• TMSe +, Spiked 0.46 × 150 mm cyano- 70% AAS TMSe + = [201]
selenoniocholine human urine propyl bonded phase methanol (quartz 44 ng
(5 gm silica support) 29% diethyl T-tube) selenonio-
ether choline =
1% acetic 31 ng
acid 0.01%
triethylamine
0.2 mg/ml
trimethyl-
sulphonium
iodide
TMSe +, Water 1 × 10 cm column 25 ml of GFAAS 10 ng/2 1 [136]
selenite, samples containing DOWEX 4 tool/1 HC1
selenate 50 W × 8 (H +) resin
TMSe +, selenite, Water 0.8 × 25 cm pyrex glass 5 mol/1 HC1 IDMS 10 pg. g-1 [65]
selenate, total samples column containing
selenium, acidic DOWEX 50 W × 8
organoselenium, in the H + form
neutral and basic
organoselenium

methylselenonium) bromide ethyl ester in standard com-
pound mixtures (see Table 5). Selenoniocholine, a potential
selenonium metabolite, has been analyzed b y H P L C - A A S
in urine samples which have been spiked [201] (see
Table 5). Cooke et al. [119] have examined dimethyl-
selenonium ions in surface waters. The procedure devel-
oped minimizes loss and conversion of D M S e + - R to
DMSe, and is presented in Table 6. D M S e + - R comprised
1 - 2 1 % o f the total selenium.
A rapid and sensitive method for the resolution of some
selenium oxide enantiomers has been reported b y Gargaro
et al. [203]. Such a technique is directly applied to several
 801

Table 6. Speciation of other organic selenium compounds

Species Sample Column Solvent Detector Detection Ref.

type system limit

Basic and neutral Lake 0.8 × 25 cm pyrex glass 25 ml CH3OH IDMS 10 pg/g [65]
organoselenium water column containing 25 ml NH3
compounds, acidic samples XAD - 2 resin (pH 10)
organoselenium
compounds
DMSe+-R, Surface DOWEX AG 50 cation - AAS 0.01 mmol/1 [119]
DMSeO2, water samples exchange resin
DMSeO
Selenoxides
enantiomers
O Pure 150 × 4 mm IPA/CH2Clfl U.V. - [203]
II compound stainless- hexane
Se mixtures steel packed with 20/20/60 or
X-CH2 / \Ar Lichrosorb 6/14/80
X=C6H5 Si 100 DACH-
Ar= C6H5 DNB, 5 pm
X = C (C6Hs)OCH~
Ar=C6H5
X=C(C6Hs)OCH3
Ar = CH3OCeH4
Selenourea Mixtures 20 × 20 cm frosted glass 1,1' oxybis-ethane/ Spot - [204]
Selenoxanthene a of O, S and Se sheets (TLC). The ad- ethanol (85/15) coloration
Selenoxanthone analogs sorbent was "Kieselgel CH2C12/tri- observed
G Merck nach Stahl" chloroethane/ in normal
cyclohexane and U.V.
(40/20/30) light
Selenochrom-4-one, Pure 20 × 30 cm frosted trichloroethene/ Spot [205]
Selenochroman-4-ol, compound glass sheets (TLC). trichloromethane coloration
2-nitrobenzyl phenyl mixtures The adsorbent was (70/30) observed
selenide, "Kieselgel G. Merck in normal
2-aminobenzyl nach Stahl" and U.V.
phenyl light
selenide,
6H-dibenzo [b, d]
selenopyran
Selenols Deproteinized 3.2 × 100 mm 20 mg/1 sodium Electro- 5 • 10 -6 mol/1 [207]
diselenides and plasma spiked Biophase octyl sulphate, chemical 1 " 10 -7 tool/1
selenenyl with those ODS column, 5% CH3CN, detector l • 10 -7 tool/1
sulfides compounds 3 gm 0.05 mol/1 Nail2
PO4, H3PO4 to
give pH 2.9
GS Se SG, Pure his 3.2× 100 mm 80 rag/1 sodium Electro- <1 • 107 molB [208]
PS Se SR (alkylthio) biophase ODS octyl sulfate, chemical
PS Se SG, selenides column, 3 gm 0.1 mol/l NaH2PO4 detector
(P = penicillamine mixtures H 3 P O 4 to give
G = glutathione) pH 2.5,
gradient elution
CH3CN 4%--~50%

a Xanthenes are not easily separated even by solvent mixtures.

areas of chemical, pharmacological, medical and biochemi-
cal research (Table 6). The thin-layer chromatographic be-
haviour on silica gel (mono- and two-dimensional tech-
niques) of some organic selenium compounds and their sul-
phur and oxygen analogs has been studied by Bottura et al.
[204, 205]. Their results are also summarized in Table 6.
Rabenstein et al. [206] have developed a method for the
determination of the selenol group of selenocysteamine by
titration with methylmercuric hydroxide with end-point de-
tection by NMR. Selenols, diselenides, bi(alkylthio)sele-
nides and selenenyl sulphides can all be determined by
liquid chromatography with electrochemical detection
[207, 208] (Table 6).
4 Conclusion
In recent years, the chemistry and biology of selenium spe-
cies have been the object of increasing attention, due to the
importance of this element as both an essential and toxic
802

substance. However, the accurate speciation of selenium is MES: Microwave emission spectrometry
still a major challenge for analytical chemists, and the Methane selenol: C H 3 - S e - H
knowledge of its pathways in the environment and living Methyl methylselenite: CH3-Se-OCH3
organisms is still limited. II
While a majority of methods for the determination of O
selenium in environmental and biological matrices give the MPD: Microwave plasma detector
total selenium content, only a few procedures are capable MS: Mass spectrometry
of distinguishing the different chemical forms of selenium- NAA: Neutron activation analysis
NDAFS: Non-dispersive atomic fluorescence spectrometry
containing compounds. The development of selective ana- NMR: Nuclear magnetic resonance
lytical techniques for this element is limited by three diffi- SCD: Sulphur chemiluminescence detector
culties: Selenenyl sulfides: R - S e - S - R
- the low concentration of organoselenium species in Se-Cysta: selenocystathionine: CH2-Se-CH2
the samples, I I
- the insufficient knowledge on the chemical structure CH2 HCNHz
of organic selenium compounds present in the samples, I I
- the relatively low sensitivity of usual detectors for HCNH2 COOH
this element. I
COOH
Several investigations reported in this article show that
one cannot ignore the importance of organoselenium com- Se-Met: Selomethionine: CH3- S e - (CH2)2- C H - COOH
pounds in biological and environmental systems. However, I
NH2
further improvements are necessary in the area of analytical
chemistry to obtain more information about the speciation Selenols: R - S e H
of selenium. Medical and environmental research are di- Selenoniobetaine: (CH3)z Se+-CH2-COOH
rectly interested in the development of such methods. Selenoniocholine: (CH3)~ Se+-CH2-CH2OH
Selenourea: Se = C (NH~)~
9H-Selenoxanthene:
Abbreviations used

AAS: Atomic absorption spectrophotometry

Acetic acid-(2-dimethylselenonium) bromide:
(CH3)~ Se+-CH2-COOH
Acetic acid-(2-dimethylselenonium) bromide ethylester: Selenoxanthone: O
(CH3)2- Se+ - CH2- CO2- C2H5
Bis (alkylthio) selenide: R - S - S e - S - R
DCP-AES: Direct current plasma - atomic emission spec-
trometry
DEtDSe: Diethyl diselenide: CzHs- Se - Se - C2H5
DEtSe: Diethyl selenide: CzHs-Se-C~H5
DMDSe: Dimethyl diselenide: CH3-Se-Se-CH3
DMSe: Dimethyl selenide: CH3-Se-CH3 Se-Cys: S e - methylselenocysteine:
Dimethyl selenenyl sulfide: CH3-Se-S-CH3 CH3-Se-CH2-CH-COOH
Dimethyl selenocysteine selenonium: I
(CH3)2 Se+-CH2-CH-COOH NH2
I Se-methyl selenomethionine selenonium:
NH2 (CH3)2 Se+-(CH2)2-CH-COOH
DMSe+-R: Dimethylselenonium ion I
NH2
/O TLC: Thin-layer chromatography
Dimethyl selenone: (CH3)2 Se TMSe+: Trimethyl selenonium ion: (CH3)3 Se +
\O XRFS: X-Ray fluorescence spectrometry
DMSeO: Dimethyl selenoxide
DPrDSe: Di-n-propyl diselenide
DPrSe: Di-n-propyl selenide
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