Analyst, August 1996, Vol. 121 (77R-83R) 77R Speciation Analysis of Some Organic Selenium Compounds A Review Krystyna Pyrzynska Department of Chemistry, University of Warsaw, Pasteura I , 02-093 Warsaw, Poland Summary of Contents Introduction Sampling and Storage Sample Preparation Analytical Methods for Selenium Determination Total Selenium Selenium Speciation Chromatographic Separation and Determination of Organoselenium Species Gas Chromatography Liquid chromatography Electrophoretic techniques Conclusion References Keywords: Speciation analysis; organoselenium compounds; methylselenides; selenoamino acids; review species into volatile compounds such as dimethylselenide (DMSe) and dimethyldiselenide (DMDSe), which are exhaled and excreted through the skin. The trimethylselenonium ion (TMSe+), the major product of selenium metabolism, leaves the body of humans in urine.The biomethylation processes are considered to be detoxification steps, because DMSe and TMSe+ are less toxic than other selenium compounds. Several schematic biogeochemical cycles which link selenate and selenite with simple methylated and more complex organic selenium compounds have been proposed.17-20 Compared with the extensive investigations on total selenium or selenite and selenate determination, very little work has been carried out on organic selenium compounds. The organically bound selenium is an essential part of the natural selenium cycle and seems to be the most readily available for humans; hence, the study of organoselenium species is important.Introduction Selenium has been recognized as an essential nutrient for humans, based on its presence at the active sites of glutathione peroxidase. This enzyme protects membranes from damage caused by the peroxidation of lipids. 1 Selenium compounds also catalyse the reactions of intermediate metabolism and inhibit the toxic effects of heavy metals such as arsenic, cadmium and mercury.24 For human health, selenium is an essential trace elements at concentrations ranging from 0.8 to 1.7 pmol l-1. At higher concentrations, selenium becomes toxic for man, animals and marine organisms. The toxic dose of selenium is very much dependent on its chemical form, with different toxicity for organic and inorganic compounds.6~7 In environmental and biological samples, selenium can exist in inorganic forms (as elemental selenium, metal selenides, and selenite and selenate ions) and as organic species with direct Se-C bonds (methylated compounds, selenoamino acids, sele- noproteins and their derivatives).Selenate (Se042-) and selenite (Se032-) appear to be the predominant species in natural waters. Hydrogen selenide is easily oxidized to non- toxic elemental red selenium, which is insoluble in water. Over the years, several organic selenium compounds have been identified in biological samples (Table 1). Selenoamino acids are the principal dietary forms of selenium; selenome- thionine is derived from plants8 and selenocysteine from animals.' Dietary selenium in the form of its amino acids is absorbed to a greater extent than inorganic species.However, selenium from selenite may be more rapidly incorporated into the enzyme glutathione peroxidase.10.11 In the aquatic envi- ronment, such as in marine bacteria and plankton, selenium is predominantly found in proteins as selenoamino acids. l2-I4 Selenomethionine has been isolated from the hydrophilic fulvate fraction of soil15 and from proteins of marine algae.16 A number of bacteria are able to transform inorganic selenium Sampling and Storage Various factors affect the losses of selenium or interconversion of one species into another during sampling and sample storage. The collection of samples for the determination of the alkylselenium species, such as DMSe and DMDSe, is a significant problem because of the volatility of these com- pounds. The common sampling procedure is based on scryo- genic trapping.The volatile selenides are removed from the sample by sucking with a pump (from air) or by helium gas stripping (water, soil and sediment) and swept into a cold trap. Various types of solid adsorbents such as activated carbon,21-23 glass W O O I ~ ~ , ~ ~ and GC stationary phases19,21,22,26,27 were found to be successful for trapping selenium species. The pre-column adsorption technique allows an accumulation of the selenides to levels suitable for detection. Selenium species are then thermally desorbedl9.27 or extracted from the adsorbent with Table 1 Organoselenium compounds found in living organisms Compound Formula Selenoc y steine Se-methylselenoc y steine Selenomethionine Se-methyiselenomethionine Selenocy stine Selenourea Selenoniocholine Selenobetaine Dimeth ylselenide Dimethyldiselenide Trimethylselenonium HSe-CH2CH(NH2)-COOH CH3Se-CH2CH(NH2)-COOH CH3Se-CH2CH2CH(NH2)-COOH ( CH2)2Se+-CH2-CH2-CH(NH2)- HOOC-CH(NH2)CH2-Se-Se-CH2- Se=C(NH2)2 (CH3)2Se+CH2CH2-COOH (CH3)2Se+CH2-COOH CH3-Se-CH3 CH3-Se-Se-CH3 (CH3)3Se+ COOH CH(NH2)XOOH78R Analyst, August 1996, Vol.121 organic solvents23 prior to analysis by GC or AAS. However, when a high volume of air sample is used some losses of selenium may occur.28 C~tter2~ stated that the volatile methyl- ated selenium compounds should be frozen (with liquid nitrogen) because even in airtight containers they are com- pletely lost within a day. Several papers concerning the stability of inorganic selenium species have been published.During the storage of selenium solutions, adsorption, desorption, volatilization processes, etc., may occur, altering the original concentration. These processes depend on pH, storage medium, temperature, selenium concen- tration, container material and the ratio of surface area per unit v0Iume.~,~Y-3~ Significant SeIV losses were observed at pH 6 in PTFE containers.34 Selenium(v1) is more stable than Serv in aqueous solutions and less dependent on the acidic conditions of the sample. The optimum temperature at which there is no significant risk of inorganic selenium losses at 10 and 50 pg 1-1 concentration levels over the 12 months tested was -20 "C.34 Samples stored at this temperature need not be acidified, which is an advantage. Acidification can cause changes in selenium speciation, as well as causing hydride generation difficulties during analysis.The literature indicated that only a few storage experiments had been conducted with selenoamino acids.35>36 Campanella et al.35 investigated selenium losses from samples containing 100 vg 1-1 of selenourea stored in PTFE containers at 4 "C and at room temperature. In both instances, no loss of selenium was observed. There was also no significant influence of tem- perature, selenium concentration and container material on the stability of selenomethionine in a high ionic strength matrix over a 120 d period.36 A significant loss of this compound was found in low concentration (10 pg 1-1) solutions stored in borosilicate glass and polyethylene containers in a low ionic strength matrix.Sample Preparation Total selenium determination requires that its organic forms must be transformed into inorganic selenium. The decomposi- tion will also destroy organic matter that may interfere with the detection method. The most popular methods involve digestion with strong acids (HN03, HC104, H2S04)37-40 or UV irradiation after addition of hydrogen per0xide.3~7~~ Concentrated HC1 is avoided, since selenium forms volatile chloride adducts. The main difficulty with wet oxidation is preventing selenium from being volatilized. Lowering the digestion temperature is effective; however, it prolongs the time of digestion. Micro- wave heating was more effective for the drying and wet ashing (with HN03) of fish tissues when applied to selenium determination.42 Compared with thermal heating in an open vessel, much shorter digestion times and smaller amounts of chemicals were used.A comparison between six decomposition methods for various organoselenium compounds followed by their determination by HGAAS was presented by Ornemark et al.43 They found that the use of peroxodisulfate at pH 2 2 was the most efficient method. It decomposed all the species tested, including the very resistant TMSe+. This substance was not decomposed by permanganate or by oxidative UV irradiation. Selenomethionine from biological samples can only be con- verted into inorganic selenium after digestion with an HN03- H2S04-HC104 mixture at 310 0C.44 The efficiency of the mineralization of selenourea solutions was higher using UV photodegradation than with wet acid digestion.35 Moreover, the former method requires shorter operating times, viz., 2 h against However, the best digestion method for a particular purpose is not easy to define and no single procedure is suitable in all instances.The choice of acids and other reagents used for 8-9 h. oxidation should also take into consideration the instrumental technique used for detection. Some of the methods for the determination of selenium require the conversion of its species into SeIV because Sevl does not react with most of the complex-forming agents used for spectrophotometric detection. Moreover, the hydride generation process is most efficient when selenium occurs as SeIv. Hence, after decomposition of organoselenium species, HCI reduction to SeIV is mostly applied.It is recommended that this process be performed at high temperature (90-100 "C).24,45,46 However, if the sample is boiled for too long, reduction to elemental selenium may occur24 or selenium may be lost owing to the formation of volatile compounds.3 Recently, an on-line reduc- tion in a closed system at 140 OC47 and a microwave energy source with continuous sampling flow48 have been proposed. Automation of these systems has resulted in a shortening of the analysis time and has minimized the risk of losses. Despite the very sensitive analytical methods available for selenium, it is seldom possible to perform direct determinations at the concentration levels present in natural samples. The total selenium content in environmental samples ranges from 0.1 to 400 pg 1-1 in waters to about 1 ng 1-1 in atmospheric aerosols and 1-80 pg g-1 in soils, but, depending on geological factors, groundwater may contain much higher concentrations up to 6 mg 1-1.33334749 This means that the analyte has to be pre- concentrated.Moreover, this could also serve as a convenient method for the separation of selenium from the matrix. Several preconcentration-separation procedures for selenium have been described in the literature, including coprecipitation,5&52 derivatization followed by extraction into an organic sol- vent53-55 or application of solid sorbents.3.56-60 Supercritical fluid extraction (SFE) is fast becoming an alternative technique to conventional extraction methods for solid samples.This technique is more effective, removing the need for toxic solvents and is very suitable for on-line automation.61 Although the growing list of SFE applications to environmental samples has been discussed,61,62 this technique does not yet appear to have been fully used for the speciation of selenium in soil and sediment samples.63 Hydride generation, coupled to AAS and ICP detectors, is the technique most commonly used for the separation of selenium from non-volatile matrix components. The enrichment of the hydrogen selenide generated can be achieved using a liquid nitrogen cold trap.24,44,64,65 Also, commercial graphite furnaces (in AAS methods) can be used as both the trapping medium and as the atomization cell.66367 Ethylation, using NaBEt, as reductant, is an alternative to the hydride generation method.68 This reaction does not allow the speciation of inorganic selenium species because a single derivative (Et2Se) is generated from both SeIv and Sevl.It has not yet been explored for organic selenium compounds. Chromatographic and electrophoretic techniques coupled with different detection systems have been extensively used for the separation and determination of all selenium species. The application of these methods to the speciation of organoselen- ium compounds and the sample characteristics required for a given separation technique will be discussed later. Analytical Methods for Selenium Determination Total Selenium Several analytical techniques have been applied to the determi- nation of total selenium content at trace levels in environmental and biological samples (Table 2).The determination is usually carried out after mineralization of the organic matrix and an appropriate derivatization process fhydride generation, ethyla- tion, complexation with an aromatic o-diamine). Methods for selenium determination have recently been reviewed. *8,33,79,80 The literature data also reviewed the quantification of seleniumAnalyst, August 1996, Vol. 121 79R in different matrices: ~ater,~~,"t81 urine,*9,82 blood,83,84 and environmenta16x.85 and biological ~ a m p l e s . 2 ~ ~ ~ " * ~ Selenium Speciation A sequential extraction procedure termed 'phase speciation'87 only provides information about the partitioning of selenium between exchangeable, carbonate, iron and manganese oxide, organic and resistant mineral fractions from the suspended particles and bottom sediments of natural waters.This informa- tion helps in the understanding of the processes of removal of selenium from the dissolved to the particulate state and the potential for its bioavailability. However, the problem with all sequential extraction procedures is that they cannot preserve the chemical forms of selenium because of the reagents and conditions employed.88 The identification and determination of definite selenium compounds requires more refined separation techniques. Most procedures involving selenium speciation distinguish between two main categories of species: (1) non-volatile, which includes inorganic species such as SelV and Sevl, elemental selenium and matrix-bound organic compounds; and (2) volatile organic selenides such as DMSe and DMDSe.Volatile selenium species are purged from the sample matrix and trapped either on a solid adsorbent or in a liquid nitrogen trap, followed by thermal desorption prior to GC analysis. 19,24,56,89 Non-volatile selenium species in four oxidation states (-11, 0, IV and VI) have been determined in natural ~ a t e r ~ ~ ~ , ~ ~ , ~ ~ 9 ~ by analysing three separate sample aliquots: (1) with no further chemical treatment-determination of Se" using fluorimetry, HGAAS, HG-ICP or electrochemical methods; (2) after oxidation by UV irradiation or wet acid digestion-the sum of Se-I1 + SeO + Seiv; and ( 3 ) after reduction to selenite with hydrochloric or hydrobromic acid-all selenium species.The difference between total selenium and the sum of SelV and Sevl is attributed to organic selenium compounds and elemental selenium. The error for the species determined by difference is always much higher than for the species determined directly. Usually, a preconcentration step (solvent extraction, coprecipi- tation, sorption on solid sorbents) is required to achieve a sufficient concentration level for detection. The analytical methods proposed differ mainly in the way in which inorganic selenium species are converted into Se'" and in the methods used for preconcentration of selenium. Some workers have also studied a colloidal selenium fraction.35-88,92,96 Selenium speciation in plant and biological samples has focused on two fractions: (1) organic selenium and Se-'I; and (2) inorganic species, including Se'" and Sevl.The scheme determines the sum of the Se-I' and SeIV content after wet oxidation of the matrix. Total selenium is determined after Table 2 Analytical methods for the determination of selenium Technique Spectroscupy- UV-visible Fluorimetry X-ray fluorescence HGAAS ETAAS Mass spectrometry ICP-AES Detection Matrix limit Ref. Plants 80 mg I-' 69 Sediment 0.28 pg 1-1 70 Waters 60 ng 1-1 71 Tissues 20 ng 1-1 72 Urine 20ng I-' 73 Sediment 0.40 pg I-' 70 Waters 10 ng 1- 1 74 Electrochemical- Anodic stripping voltammetry Waters 80 mg 1-' 75 Cathodic stripping voltammetry Waters 2 ng 1-1 76 Neutron activation analysis Human diets 0.5 pg g- 1 1 78 Differential-pulse polarography Fish 5 ng 77 boiling the digested samples with concentrated HC1.Sel- enium(1v) is found by difference. This procedure has been used, with minor modifications to the oxidation step, for the speciation analysis of selenium in milk products,y7 body fluids98 and fish.">l00 The separation and determination of the dissolved organic selenide fraction can be achieved by using LC methods at atmospheric pressure.56J01-105 Organoselenium species (other than TMSe+) were separated from natural water samples by adsorption on Amberlite XAD-2 resin at pH 8 and 3, respectively.56 The species adsorbed at pH 8 were classified as neutral and basic forms, while those adsorbed at pH 3 as acidic. As the acidic forms dominated in lake water samples, it was concluded that most of these compounds were selenium- containing humic substances.Blocky et ul. developed a procedure for the determination of total selenoamino acids in urine. These compounds were derivatized with o-phthalal- dehyde and 2-mercaptoethanol, followed by their retention on an anion-exchange resin, Bio-Rad AG2-X8. Lithium hydroxide solution was used as the eluent, which, in addition to its elution characteristics, facilitated the continuous denaturating of urine. Dissolved organoselenium species (mainly the selenoamino acid fraction) were separated from inorganic selenium spe- cies103,1*4 or from their sulfur analogue105 using Amberlite XAD-8 resin. Sep-Pak C 18 cartridgesi9 and copper-treated Chelex-100 resinI01 were also applied for this purpose. Chromatographic Separation and Determination of Organoselenium Species Chromatographic methods are based on dynamic partitioning of analytes carried by a mobile phase through a stationary phase between these two phases.These techniques are at present virtually the only ones used in speciation analysis. The elution mode, apart from producing good separations, leaves the column in its original condition, ready for another sample. Gus chromatography Gas chromatography is the most frequently used technique for the determination of volatile selenium species, such as DMSe and DMDSe. These naturally occurring compounds are suffi- ciently volatile to be separated by packed'9~2-?~")6~107 or ~ a p i l l a r y ~ 7 , 1 0 ~ ~ ~ 0 " ~ ~ ~ columns. The packed columns can be conveniently cleaned and have a large sample capacity. In recent years there has been a trend towards capillary or open- tubular megabore columns with thin polymethoxysilane coat- ings, which provide higher resolution and sharper bands.However, their limited capacity is a disadvantage. The chromatographic columns are usually coupled to AAS detection methods which use flame atomization, a quartz tube (QT) or a graphite furance (GF). The sensitivity of GC-AAS is affected by the temperatures of the injector, detector and chromatograph. At low temperatures, the species tend to adsorb onto the injector. On the other hand, excessive temperatures lead to thermal decomposition of the compounds. Temperatures of 100 and I60 "C for the injector and detector, respectively, are satisfactory.1'0 The detection limits of GC-AAS approach 0.1 ng of selenium using a QT24 and 5 pg of selenium using a GF.I07 The successful speciation of volatile alkylselenides has also been carried out with other detection techniques, such as microwave-induced plasma atomic emission spectrometry (MIP-AES),I I I atomic fluorescence spectrometry (AFS), l 2 and MSI"6 and with a sulfur chemiluminescence detector (SCD)? Selected GC applications for selenium speciation are presented in Table 3.Gas chromatography has also been applied to study the transformation of inorganic selenium species into methylated80R Analyst, August 1996, Vol. 121 compounds in laboratory experiments with animals.23.82 After administration of selenite and selenocystine in the drinking water of mice, DMSe was exhaled as the predominant species.When selenomethionine was administered, both DMSe and DMDSe were detected.' 10 These results represent a step forward in understanding the metabolism of these compounds. Similar experiments were performed to study the biomethyla- tion of inorganic selenium administered to fungal cultures.27 Both DMSe and DMDSe were separated and detected in the low picogram range by GC-SCD. Liquid chromatography The separation of selenium species by LC offers a number of potential benefits. These include minimal preparation of liquid samples and separation at ambient temperature, thus avoiding the risk of thermal decomposition of unstable compounds. Another advantage is that both the stationary and mobile phases can be varied simultaneously to achieve better separation.'8 This technique is mainly applied as HPLC, in which the size of the particles used for the stationary phase is very small to ensure good separation.HPLC provides, depending on the type of detector, low detection limits (in the pg 1-1 range) with fairly short chromatographic run-times. Current HPLC methodology for selenium employs conduc- tometric,79,' 13-1 14 UV"5 and fluorimetric22 detection. Nume- rous interferences, mainly ionic, from the sample and reagents constitute the major drawback of these detectors. The applica- tion of selenium-specific detectors, such as ETAAS,' '6 ICP- AES117 or ICP-MS,56,118 is very helpful for elimination of these interferences. ~~~ Table 3 Speciation analysis of volatile alkylselenides Species* Matrix Detectiont Ref. DMSe, DMDSe Surface water, groundwater AAS 19 DMSe Air exhaled by mice FID 23 DMSe, DMDSe Gases from biological samples SCD 27 DMSe, DMDSe Gas evolved from soil cultures MS 106 DMSe, DMDSe, DESe Gas evolved from soil ETAAS 107 DMSe, DMDSe Gas evolved from sediments FID 108 DMSe, DMDSe, Gases evolved from DESe sewage sludge and soils MIP 111 * DMSe = Dimethylselenide; DMDSe = dimethyldiselenide; DESe = diethylselenide.t FID = Flame ionization detector; SCD = sulfur chemiluminescence detector; MIP = microwave-induced plasma. A major concern in the development of HPLC coupled with spectroscopic detection has been the interface. Organic solvents can impair the performance because of the instability of the plasma to organic vapours and deposition of carbon on the sampling cone and torch.Improvements to the HPLC-ICP interface have involved a direct injection nebulizer118 and a thermospray vaporizer,'I7 which allow the use of a higher concentration of organic modifiers in the mobile phase. The addition of oxygen to the nebulizer gas flowt19 or nebulization of 2% nitric acid between chromatographic runs'20 can reduce some of these problems. Hill et a1.121 have reviewed the coupling of various chromatographic methods to both ICP-AES and ICP-MS. Blais et al. 122 developed a post-column thermochemical hydride generator (THG) as an interface for HPLC-AAS and optimized the determination of low nanogram amounts of selenoniocholine and TMSe+ in urine. The THG interface was also used for the determination of selenomethionine in complex matrices such as nutritional supplements and mixtures of free amino acids.123 Selenomethionine was first derivatized with 1 -fluoro-2,4-dinitrobenzene and, after acidification, was ex- tracted with diethyl ether. The coupling of HPLC with ETAAS detection has the disadvantage of using discrete sample volumes and does not allow the continuous monitoring of the effluent from the chromatograph. A computerized system for the collection and treatment of data improves the quantification of the analy- tes.*z4 Several types of chromatographic mode, including normal- and reversed-phase partitioning, ion pairing and ion exchange, have been applied to the separation and determination of selenium species. The stationary phase is usually silica, bonded or coated. The mobile phase is aqueous, but contains a polar organic solvent (usually methanol or acetonitrile) in various proportions to overcome the instability of the analyte complexes in water.Trimethylselenonium, as a major metabolite of selenium, is mainly determined in urine and water samples (Table 4). Its concentration level in the urine of normal subjects is in the range 10-60 ng ml-1.102 Two studies have demonstrated the occur- rence of TMSe+ in lake water samples with a mean concentra- tion of 12 ng 1-l.53956 The information concerning the TMSe+ content is useful in predicting the intake of excess of selenium and the detoxification mechanism in living organisms. 130 However, the chemical forms of selenium in urine have m f yet been completely characterized. Other potential metabolites, such as selenoniocholine, may constitute a significant portion of total selenium.29~102~122 The high resolution obtained by HPLC permits a clear separation of selenoamino acids from other selenium species -- Table 4 Speciation of trimethylselenonium ion (TMSe+) Species Matrix Column and mobile phase Detection* TMSe+, Se'", Sev' Water Dowex 50W-X8; 4 moll-' HC1 TMSe+, Se'", SevL, total organic selenium Dowex 50W-X8; 5 mol 1-I HC1 TMSe+, Serv, total selenoamino acids Bio-Rad AG2-X8; 0.5 mol 1-1 LiOH Water Urine, serum ETAAS IDMS NAA TMSe+, Se'", Sev' Water Waters IC-PAK; ammonium citrate, pH 3.3 ICP-AES TMSef, selenoniocholine Urine Cyanopropyl-bonded silica; methanol with TMSe+, selenocysteine, selenomethionine Urine Nucleosil; gradient, 0.003-0.5 mol 1-1 TMSef, Se'", Sevr Urine Nucleosil 100-SB; ammonium citrate, pH 3 and 7 ETAAS TMSe+, Serv, Sevl Urine Hamilton PRP-1; methanol with TMSe+, Se'", SevL Water Hamilton PRP-X-100; phosphate buffer, pH 6.8 HGAAS * IDMS = Isotope dilution mass spectrometry; THGAAS = thermochemical hydride generation atomic absorption spectrometry.CH,COOH and triethylamine THGAAS (NH4)2P04 Radiometric tetrabut y lammonium ICP-MS Ref. 53 56 102 127 117 121 125 126 128 129Analyst, August 1996, Vol. 121 81R Table 5 Speciation of selenoamino acids Species Matrix Column and mobile phase Detection* Ref, Selenomethionine Food supplements Selenoc ysteine Water Selenomethionine Soil extract Selenocystine, selenomethionine Extract of white Selenocysteine, selenomethionine Extract from the clover liver of marine mammals Selenocy steine Spiked plasma Nucleosil; aqueous methanol with trietylamine THGAAS 123 VBondapak CI8; methanol-H20 (30 + 70) uv 134 Amberlite XAD-2; pH gradient MS 135 Hamilton PRP-1; aqueous acetonitrile with Et4NBr ETAAS 136 AminoPac PA1; gradient elution with NaOH, sodium borate and sodium acetate IPAD 137 CIS; methanol-H20 (20 + 80) Fluorimetry 138 * THGAAS = Thermochemical hydride generation atomic absorption spectrometry; IPAD = integrated pulsed amperometric detection.and from other amino acids.102,121,12’,1”-13~ Several seleno- amino acids and related selenium compounds have been identified in biological tissues or protein fractions. The selenoamino acids isolated probably originate from several enzymic systems which require the participation of selenium- containing proteins.Selected analytical procedures for the determination of selenoamino acids are presented in Table 5. Several other organic selenium compounds, such as seleno- carbohydrates, I39 ~elenoproteins*~0,~~1 and selenonucleo- sides142 have been separated using LC. They were further characterized by gel chromatography or electrophoresis. Electrophoretic techniques These techniques are based on differences in the electrophoretic mobilities of ions and are realized in three basic modes: zone, isotachophoresis and isoelectric focusing. The electrophoretic mobility of an ion is largely determined by its mass-to-charge ratio, physical dimensions and interactions with buffer compo- nents. CZE is a relatively new technique that is potentially applicable to the separation of a range of species from small molecules and ions to large biomolecules.The wider use of this technique is hampered by the lack of a sensitive and specific detection system in the on-line mode. Work on interfacing CZE and ICP-MS is in progress,143 but this coupling has not yet been applied in selenium speciation analysis. Usually, UV detection has been used.5J4 CZE was applied to the separation of inorganic selenium forms from selenomethionine and selenocystines and dialkyl- selenium compounds, * 44 but applications to real samples were not reported. The electrokinetic potential was modified by the addition of a cationic surfactant. The identification of the analyte peak when complex matrices are analysed plays a major role, because the migration times of molecules present at low concentrations are influenced by closely migrating substances present at high concentrations.145 As a consequence, the migration time for a specific analyte in a real sample is different from that in a model or standard solution. Michalke 146 proposed a method for the identification and quantification of selenocystine, selenocystamine and sele- nomethionine in human milk samples despite the shift of migration times caused by the different ionic composition. Conclusion Selenium speciation studies have become increasingly impor- tant in recent years as more information is sought in order to gain a better understanding of the role of selenium in the environment and human health. The measurement of total selenium content at trace levels in water and biological samples can be reliably performed by several established techniques.The accurate determination of different selenium species, particularly organic compounds, is still a major challenge for the analyst. Many of the speciation procedures rely on the separation technique and there are still limitations with sample preparation. Various factors affect the sorption or loss of selenium, and the interconversion of one species into another during sampling, sample storage and analysis. The most efficient way for the quality control of analytical results is through the analysis of certified reference materials. Although several certified reference materials having different matrices are available,33 they are only certified for total selenium content.The methods developed for selenium specia- tion should be validated by using spiked samples. 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