Separation and on-line preconcentration of nonsteroidal anti-inflammatory drugs by microemulsion electrokinetic chromatography.

The MEEKC separation and on-line preconcentration of nonsteroidal anti-inflammatory drugs (NSAIDs) were investigated. In this work, two microemulsion (ME) systems were employed. Complete separation could be achieved at a pH of 5.5 with octane as the oil phase (ME I). The LODs for the determination of flurbiprofen, fenoprofen, naproxen, ketoprofen, suprofen, and indoprofen were 0.45, 0.32, 0.10, 0.48, 0.77, and 0.29 mg/L, respectively. To enhance the sensitivity, anion selective exhaustive injection-sweeping (ASEI-sweeping) and stacking with reverse migrating pseudostationary phase (SRMP) were compared. For having a simple process without polarity switching and shorter analysis time, a suppressed EOF and di-n-butyl-L-tartrate as the oil phase (ME II) were used for the simultaneous determination of NSAIDs. The sensitivity for ASEI-sweeping was enhanced approximately 280-fold. However, there was much background noise, and some of the analytes were co-eluted. When SRMP was used, all of the analytes could be detected simultaneously, and baseline separation could be achieved. The sensitivities of all of the analytes, except for indoprofen, were enhanced approximately 170-fold compared with the normal MEEKC, and the LODs were 2-45 µg/L. Herein, we propose a simple method for the direct determination of NSAIDs at therapeutic and toxic levels in urine throughout a wide concentration range.

An investigation into the stability of microemulsions in electrophoresis.

A typical oil-in-water microemulsion (ME) was applied for the capillary electrophoretic separation of nonsteroidal anti-inflammatory drugs (NSAIDs). As the high-pH ME was introduced into the capillary and a voltage was applied, we observed an unusual phenomenon during the preconditioning process: a sharp inflection point occurred at 56.2 min ( ± 4.8%) (N = 15). Both before (region A) and after (region B) that point, a steady state was observed. Highly reproducible results were obtained for this event. Two different absorbance patterns were observed for the hydrostatic injection of either freshly prepared ME or ME collected from the outlet of the capillary column during the steady state of region B. The latter had an inflection point at approximately 9 min, whereas the former had a constant absorbance over the entire range. To further characterize this property, regions A and B were used for the separation of NSAIDs. The results showed that both the detection limit and the reproducibility of the separation were superior in region B. For deep insight into the stability of the ME in an electric field, the effects of the oil, cosurfactant, pH, and voltage were systematically investigated in the neat ME. From our findings, it can be concluded that the inflection time might be the migration time of the ME in an electric field, and it might actually be the result of equilibration. Moreover, the existence of many unexpected phenomena seems to be the result of a change in the properties of ME droplet in an electric field.

Stationary phases for the enrichment of glycoproteins and glycopeptides.

The analysis of protein glycosylation is important for biomedical and biopharmaceutical research. Recent advances in LC-MS analysis have enabled the identification of glycosylation sites, the characterisation of glycan structures and the identification and quantification of glycoproteins and glycopeptides. However, this type of analysis remains challenging due to the low abundance of glycopeptides in complex protein digests, the microheterogeneity at glycosylation sites, ion suppression effects and the competition for ionisation by co-eluting peptides. Specific sample preparation is necessary for comprehensive and site-specific glycosylation analyses using MS. Therefore, researchers continue to pursue new columns to broaden their applications. The current manuscript covers recent literature published from 2008 to 2013. The stationary phases containing various chemical bonding methods or ligands immobilisation strategies on solid supports that selectively enrich N-linked or sialylated N-glycopeptides are categorised with either physical or chemical modes of binding. These categories include lectin affinity, hydrophilic interactions, boronate affinity, titanium dioxide affinity, hydrazide chemistry and other separation techniques. This review should aid in better understanding the syntheses and physicochemical properties of each type of stationary phases for enriching glycoproteins and glycopeptides.

An insight into the mechanism of CEC separation of template analogues on a norepinephrine-imprinted monolith.

A monolith molecularly imprinted polymer (MIP) column was prepared from template (-)-norepinephrine, functional monomer (itaconic acid), and a cross-linker (either ethylene glycol dimethacrylate or divinylbenzene) in porogen N,N-dimethylformamide. Understanding the molecular recognition of a template using an MIP seems feasible. However, it is hard to explain the recognition properties of their analogues on an MIP. The separation mechanism was investigated with the addition of charged surfactants, native and derivatised ß-cyclodextrin (ß-CD), achiral crown ether, etc. to determine the retention behaviour of the template analogues. The addition of organic modifiers and the adjustment of separation conditions were used to manipulate the selectivity. No chiral recognition was observed under most of the test conditions except the experiment with the charged ß-CD on the divinylbenzene-MIP column. The different experimental conditions led to differences in the mobilities of the analytes and resulted in remarkable enantiomeric separation of the template. We confirmed the presence of mixed-mode selectivity of the stationary phase based on hydrogen bonding, hydroelectric and hydrophobic interactions, and the electrophoretic mode.

Zirconia nanoparticles-coated column for the capillary electrochromatographic separation of iron-binding- and phosphorylated-proteins.

A ZrO(2) nanoparticles (ZrO(2)NPs)-coated column was prepared through a sol-gel process using zirconium(iv) oxychloride, which reacted with silanol groups of the fused-silica capillary. The condensation reaction was carried out at 350 °C for 8 h. Electroosmotic flow (EOF) measurements and scanning electron microscopy (SEM) images were used to characterize the ZrO(2)NPs fabricated on the inner wall of the capillary. Below the pI value (pH 5-6), cathodic EOF elucidated that the phosphate buffer adsorbs tightly on the zirconia surface, resulting in a negatively charged surface. In this work, iron-binding proteins, phosphorylated proteins and glycoproteins were selected as the model compounds. The effects of pH, concentration, buffer type and the organic modifier were studied to optimize the separation efficiency. Iron-binding proteins exhibited a retention time for myoglobin (Mb) < hemoglobin (Hb), which corresponded to the binding constants for ZrO(2)NPs. The α- and ß-subunit of Hb could be separated in borate buffer (20 mM, pH 9.0) with MeOH (20%, v/v). Greater affinity of α-casein and bovine serum albumin (BSA) for the stationary phase as the pH decreased was found by comparison with that of conalbumin (ConA) and transferrin (Tf). Interestingly, 14 peaks for glycoisoforms of ovalbumin (OVA) were observed using borate buffer (40 mM, pH 9.0). The established method was also applied to the determination of analytes in the egg whites of chicken and duck eggs.

Preparation and evaluation of a monolithic molecularly imprinted polymer for the chiral separation of neurotransmitters and their analogues by capillary electrochromatography.

A monolithic molecularly imprinted polymer (MIP) column was prepared as the stationary phase for the capillary electrochromatographic (CEC) separation of a group of structurally related compounds including dopamine (DA), (±)-epinephrine (EP), (-)-isoproterenol (ISO), (±)-norepinephrine (NE), (±)-octopamine (OCT), and (±)-synephrine (SYN). Here, (-)-NE was used as the template. Either methacrylic acid (MAA) or itaconic acid (IA) together with a mixture of ethylene glycol dimethacrylate (EDMA) and α,α'-azobis(isobutyronitrile) (AIBN) in N,N-dimethylformamide (DMF) was introduced into a pre-treated, silanised, fused-silica capillary by a thermal non-covalent polymerisation procedure. Optimised conditions for the polymerisation reaction were assessed by the separation efficiency of the template. Both the template/monomer/cross linker molar ratio and the compositions of the functional monomer, cross-linker, and porogen affected polymerisation. The optimum in situ polymerisation reaction was performed at 65 °C for 17 min. By varying CEC parameters like eluent composition and pH, we observed that the addition of SDS to the eluent clearly improved the CEC separations. With a mobile phase of citrate buffer (10 mM, pH 3)/SDS (40 mM)/acetonitrile (2/2/1, v/v/v) solution and an applied voltage of 10 kV, the six related structures of the template and their enantiomeric mixtures were satisfactorily separated at 30 °C.

Bioconjugation of 32-macrocyclic polyammonium cations-functionalized gold nanoparticles with BSA.

Water-dispersed, spherical, underivatized Au-NPs with particle size less than 5 nm were synthesized from an aqueous solution of 32-macrocyclic polyammonium chloride, [32]ane-(NH(2)(+))(8).8Cl(-) (32-MCPAC) using sodium borohydride (NaBH(4)) as the reducing agent. The bioconjugation of the synthesized Au-NPs at different pHs (3.6-5.6) with bovine serum albumin (BSA) protein was studied using UV-Vis, fluorescence, and Raman spectroscopy. These studies support that the Au-NPs were incorporated into the protein moiety and bound to it chemically. The binding constants (K(b)) and stoichiometries (n) (i.e., the number of Au-NPs bound by the proteins) of BSA protein to the Au-NPs at different pHs were determined by measuring the quenching of the fluorescence intensity of the tryptophan residues of the protein molecules after conjugation. The values for K(b) (n) were found to be 1.05 x 10(10) M(-1) (1.66), 2.09 x 10(10) M(-1) (2.30), and 1.86 x 10(10) M(-1) (1.75) at pH 3.60, 4.60, and 5.60 for BSA-Au-NPs conjugations, respectively. The results show that BSA binds to the Au-NPs strongly at pH 4.60, which is equivalent to its isoelectric point (pI 4.6).

NaDDBS as a dispersion agent for multiwalled carbon nanotubes in capillary EKC separation of nucleotides.

A novel pseudostationary phase (PSP) of multiwalled carbon nanotubes (MWCNTs) dispersed with sodium dodecylbenzenesulfonate (NaDDBS) was used for the EKC separation of nucleotides. NaDDBS has a long hydrophobic chain and a benzylsulfonate group. It suspends more MWCNTs (about 100-fold) than SDS, and the π-π interaction between the benzene ring of NaDDBS and MWCNTs prolongs the slurry suspension time. Using NaDDBS as a surfactant can reduce the required amount of MWCNTs and decrease the baseline noise. To produce a stable suspension, the optimum ratio (w/w) of MWCNTs to NaDDBS was investigated with turbidimetry. In this context, several parameters affecting EKC separation were studied, including buffer pH, composition, concentration, and the organic modifier. Use of NaDDBS (8 mg/L)/MWCNTs (0.8 mg/L) as the PSP in a phosphate buffer (30 mM, pH 8) yielded complete resolution of seven geometric isomers of a nucleoside monophosphate. In stacking mode, with 10% MeOH in the sample plug, the mixture of nucleoside mono-, di-, and tri-phosphates was satisfactorily separated in phosphate buffer (50 mM, pH 9). The results indicate that nucleotides with bases containing more electron-withdrawing groups interact more strongly with MWCNTs. The system has been used to separate oligonucleotides, and to analyze nucleotides in a complex matrix sample.

Functionalized carbon nanotubes as the pseudostationary phase for capillary EKC separation of non-steroidal anti-inflammatory drugs.

Functionalized multiwalled carbon nanotubes (f-MWCNTs) can serve as the pseudostationary phase (PSP) for the capillary EKC separation of non-steroidal anti-inflammatory drugs (NSAIDs). To increase their hydrophilicity, we treated MWCNTs, with a sonochemical process in a concentrated nitric/sulfuric acid mixture. The oxidized MWCNTs were then characterized by FT-IR, transmission electron microscopy, and X-ray photoelectron spectroscopy. We evaluated the potential of the PSP and the effects of buffer composition, pH, addition of organic modifier, and injection temperature on the NSAID separation. The PSP created a network structure of pi-pi interactions, hydrophobic forces, hydrogen bonding, and electrostatic interactions to separate NSAIDs, providing a different separation mode from SDS micelles. We achieved complete separation of six NSAIDs using a mixture of a borate buffer (75 mM, pH 10) with methanol (5%, v/v) containing 0.02 mg/mL f-MWCNTs, an applied voltage of +12 kV and detection at 214 nm. Better precision was obtained with a low injection temperature. The method was also satisfactorily applied to the analysis of NSAIDs spiked into a urine sample.

Preparation and characterization of monoliths covalently bonded chelating groups for capillary electrochromatographic separation of metal ions.

In this work, a novel polymer-based monolithic column was prepared using an o-phthalaldehyde-l-phenylalanine Schiff base complex as the reactive center and a mixture of methanol and n-propanol as the porogen. The monolithic column was employed for the separation of a metal ion mixture including Pb(II), Mn(II), Cu(II), Ni(II), Cr(III), Fe(III) and Cr(VI). Tetrabutylammonium bromide (TBAB) was used as a mobile phase additive to enhance the separation efficiency of metal ions by EDTA precomplexation. Using a phosphate buffer (20 mM, pH 3.0), TBAB (10 mM), MeOH (15%, v/v), an applied voltage of -15 kV, and detection at 220 nm, the metal ion mixture was satisfactorily resolved. The average theoretical plate number was 17,900 plates/m. The separation was also carried out in the absence of TBAB, leading to dissimilar elution order and shorter retention time. The separation behavior of the monolithic column was also compared with that of the blank polymer. The unique properties of the monolithic column might be mediated by a combination of electrophoretic behavior and chromatographic retention involving hydrophobic and hydrophilic interactions, as well as ligand exchange.

Preparation and application of ionic liquid-coated fused-silica capillary fibers for solid-phase microextraction.

A simple and cost effective solid-phase microextraction device has been developed. Fused-silica capillaries were etched with ammonium hydrogen difluoride prior to coating with an ionic liquid. For comparison, both a bare fused-silica capillary and one pretreated with a Nafion membrane were coated with the ionic liquid. All three coated capillaries were employed for the head space microextraction of polycyclic aromatic hydrocarbons (PAHs) which were then separated with an established GC system. Efforts to optimize the extraction process indicated that the etched fiber displayed the most efficient extraction, giving not only highly reproducible extraction results but also greater extraction efficiency. The Nafion membrane-supported fiber was inferior to the etched fiber, while the untreated fused-silica had the lowest extraction efficiency. The Nafion membrane contains negatively charged sulfonate groups, and the increase in ionic liquid binding was due to electrostatic attractive forces. However, due to the hydrophobic interactions of the PAHs with the polymer matrix in the Nafion membrane, a more complex adsorption/desorption mechanism might reduce the efficiency. The established method was successfully applied for the analysis of PAHs released from burning of mosquito coil incense.

Novel stationary phase for complexation gas chromatography originating from ionic liquid and metallomesogen.

A metallomesogen of a polycatenar oxazoline copper(II) complex, [Cu(S-C(12))(2)], that exhibited a columnar mesophase with a helical organization was prepared and employed as the stationary phase for the GC separation with polycyclic aromatic hydrocarbons (PAHs) as model compounds. For introducing the mesogen into the capillary column, an ionic liquid (BeMIM-TfO) was used as the vehicle. The results of thermal analyses and UV-vis spectroscopy indicated that some beneficial interactions occurred between the metallomesogen and the ionic liquid. Various parameters affecting the separation efficiency were studied. Different ratios of BeMIM-TfO and Cu(S-C(12))(2) (1:0, 1:1, 1:2 and 1:3 (w/w)) were tested for the separation of the PAHs. As the amount of Cu(S-C(12))(2) was increased, complete separation could be achieved. The stationary phase with the ratio of 1:1 provided the most satisfactory result having average theoretical plate number of 5.2 x 10(3)plates/m. With an optimized temperature program, 11 PAH mixtures were completely separated within 27 min. The interaction between PAH and these fascinating and interesting stationary phases was discussed.

Synthesis of 28-membered macrocyclic polyammonium cations functionalized gold nanoparticles and their potential for sensing nucleotides.

A new synthesis of underivatized gold nanoparticles (Au-NPs) in water stabilized by the highly water soluble 28-membered macrocyclic polyammonium chloride, [28]ane-(NH(2)(+))(6)O(2)6Cl(-) (28-MCPAC) is reported. In addition to providing stability, 28-MCPAC with its cationic form functionalizes the Au-NPs for sensing anions in water. The 28-MCPAC-Au-NPs show a surface plasmon band in the visible region (>520 nm). By tuning the 28-MCPAC:HAuCl(4) ratio, Au-NPs with different core diameters ranging from 4 nm to 6 nm, as determined by TEM analysis, can be obtained. Particles are spherical, discrete, and appeared to have narrow size distributions. Raman spectroscopy confirms that the physisorption is responsible for the interaction between Au-NP surface and 28-MCPAC. The potential of the as-synthesized particles for sensing monophosphorylated nucleosides (nucleotides): 5-adenosine monophosphate (5-AMP), 5-cytosine monophosphate (5-CMP), 5-guanine monophosphate (5-GMP), and 5-uridine monophosphate (5-UMP) is investigated spectroscopically. Nucleotides-assisted agglomerations of 28-MCPAC-Au-NPs follow the order: 5-UMP>5-GMP>5-CMP>5-AMP. An attempt is taken to prepare Au-NPs in water at pH 4.55 without an added stabilizer. Particles without an added stabilizer are short lived, and the TEM image shows that the particles aggregate following a quasi-two-dimensional self-assembly array.

Capillary electrochromatographic separation of peptides using a macrocyclic polyamine for molecular recognition.

A macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N(8)), in the bonded phase was employed as a molecular receptor for CEC separation of oligopeptides. Parameters affecting the performance of the separations were considered. Baseline separation for the mixture of angiotensin I, angiotensin II, [Sar(1), Thr(8)]-angiotensin II, beta-casomorphin bovine, beta-casomorphin human, oxytocin acetate, tocinoic acid, vasopressin, and FMRF amide could be achieved using phosphate buffer (30 mM, pH 7) as the mobile phase. Column efficiency with average theoretical plate numbers of 69 000 plates/m and RSDs of <1% (n = 6) was achieved. [Met(5)]-enkephalin and [Leu(5)]-enkephalin, which have identical pI values and similar masses could be completely separated using acetate buffer (30 mM) with pH gradient (pH 3 inlet side and pH 4 outlet side). The results suggest that the mechanism for the peptide separation was mediated by a combination of electrophoretic migration and chromatographic retention involving anion coordination and anion exchange. After long-term use, the deviation of the EOF of the column after more than 600 injections was still within 6.0% of that for a freshly prepared column.

Separation of corticosteroids by microemulsion EKC with diethyl L-tartrate as the oil phase.

A novel microemulsion based on a mixture of diethyl L-tartrate (DET) and SDS was developed for the microemulsion EKC (MEEKC) determination of structurally related steroids. The system consisted of 0.5% w/w DET, 1.7% w/w SDS, 1.2% w/w 1-butanol, 89.6% w/w phosphate buffer (40 mM, pH 7.0), and 7% w/w ACN. With an applied voltage of +10 kV, a baseline separation of aldosterone (A), cortisone acetate (CA), dexamethasone (D), hydrocortisone (H), hydrocortisone acetate (HA), prednisolone (P), prednisolone acetate (PA), prednisone (Ps), triamcinolone (T), and triamcinolone acetonide (TA) could be achieved. Under the optimized conditions, the reproducibility of the retention time (n = 4) for most of the compounds was less than +/-0.8% with the exception of A, Ps, and T. The average number of theoretical plates was 18 800 plates/m. The results were compared with those achieved by the modified micellar EKC (MEKC). MEEKC showed obvious advantages over MEKC for the separation of highly hydrophobic substances. To further evaluate the system, we tested the MEEKC method by analyzing corticosteroids in a spiked urine sample.

Separation of dialkyl sulfides by metallo-mesogenic stationary phases for complexation gas chromatography.

A copper mesogenic side-chain polymer (P-C(15)CuC(18)) was cross-linked onto the capillary wall as a stationary film for gas chromatography (GC) separation of alkyl sulfides. These organic sulfides are of interest for their large health impact because of their wide range of volatiles and high reactivities toward metals. Different GC parameters for optimal separation efficiency are discussed for use with a mesogenic polymer column along with flame photometric detection (FPD). Both the carrier gas flow-rate and column temperature were studied to determine the relationship of plate height to the chemical structure of the solutes, as well as to determine the morphology of the mesogenic polymer. Van 't Hoff plots show phase transitions of the stationary mesophase as the column temperature was varied. The results reveal that the separation mechanism might be based on ligand exchange and polarity interaction between the analytes and the stationary phase, with the vapor pressure of the analytes also being important. The former interaction dominates in the lamellar crystalline phase and the latter interaction dominates in the hexagonal columnar-discotic phase. With high reproducibility for retention time (RSD< or =0.37%) and for peak area (RSD< or =5.16%), the GC-FPD system produced linear calibration graphs (r> or =0.9918) for the determination of 13 sulfides with a detection limit below 2.5 ng.

A macrocyclic polyamine as an anion receptor in the capillary electrochromatographic separation of carbohydrates.

An analytical approach of the 32-membered macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N8) was described for the capillary electrochromatographic (CEC) separation of derivatized mono- and disaccharides. The column displayed reversal electroosmotic flow (EOF) at pH below 7.0, while a cathodic EOF was shown at pH above 7.0. The reductive amination of saccharides was carried out with p-aminobenzoic acid. Some parameters that affect the CEC separations were investigated. Several competitive ligands, such as Tris, EDTA and phosphate were also examined for the effect on the performance. We achieved a complete separation of all compounds as well as the excess derivatizing agent by using borate buffer (pH 9.0) in a mode of concentration gradient (60 mM inlet side and 70 mM outlet side). The relative standard deviation of the retention time measured for each sample was less than 4% in six continuous runs, suggesting that the bonded phase along with the gradient formed inside the column was quite stable. With the mixing modes of anion coordination, anion exchange, and shape discrimination, the interaction adequately accomplishes the separation of carbohydrates which are epimers or have different glycosidic linkage, although the electrophoretic migration is also involved in the separation mechanism.

Synthesis, isolation, and redispersion of resorcinarene-capped anatase TiO2 nanoparticles in nonaqueous solvents.

C-undodecylcalix[4]-resorcinarene (C(11)-resorcinarene)-capped anatase TiO(2) nanoparticles have been synthesized and could be isolated and redispersed in different nonaqueous solvents. The adsorption of C(11)-resorcinarene onto the surface of TiO(2) nanoparticles led the shifting of the onset wavelength of the optical absorption in the visible range along with a broad band centered at 422 nm corresponding to ligand-to-metal charge transfer transition within the surface titanium(IV)-C(11)-resorcinarene complex. The interaction of TiO(2) nanoparticle with C(11)-resorcinarenes was investigated by photoluminescence (PL). Proton nuclear magnetic resonance ((1)H NMR) spectroscopy study revealed that the C(11)-resorcenarene molecules adsorbed chemically onto the surfaces of TiO(2) nanoparticles. The average particle diameter of bare anatase TiO(2) and C(11)-resorcinarene-capped TiO(2) was determined using transmission electron microscopy (TEM) and was found to be equal to ca. 5 nm.

Capillary electrochromatographic separation of proteins on a column coated with titanium dioxide nanoparticles.

A TiO2 nanoparticle (TiO2 NP)-coated open-tubular column for the capillary electrochromatographic separation of proteins is described. The surface chemistry of the TiO2 NPs on the inner wall of the fused silica was significantly affected by the running buffer. By varying of the phosphate buffer pH, only cathodic EOF was indicated. The results showed that TiO2 NPs are existed as a complexed form with the buffer ligand. Good separation of conalbumin (ConA), apo-transferrin (apoTf), ovalbumin (OVA), and BSA could be achieved with phosphate buffer (40 mM, pH 8.0) and an applied voltage of 15 kV. Five peaks of glycoisoforms of OVA were observed under these conditions. In comparison with the retention behavior of the analytes on the bare fused-silica column, the new column's high resolving power seems to be predominantly derived from the ligand exchange of the analytes with the phosphate adsorbed onto the TiO2 NPs. The method was also used to separate egg-white proteins. Both acidic and basic proteins in egg white were separated in a single run. The microheterogeneities of OVA could also be found in it. The separation efficiency for the main peak of OVA in egg white was around 10,000 plates/m.

Speciation of selenium compounds by open tubular capillary electrochromatography-inductively coupled plasma mass spectrometry.

We introduce a T-type interface and a crossflow nebulizer to find ways to combine CEC with inductively coupled plasma MS (ICP-MS) detection for selenium speciation. For CEC separation, we employed a macrocyclic polyamine-bonded phase capillary as the separation column and a bare fused-silica capillary filled with the make-up liquid (0.05 M HNO3). The effect of nebulizer gas flow rate, make-up liquid flow, type, concentration and pH of the mobile phase on the separation have been studied. Tris buffer of 50 mM at pH 8.50 gave the best performance for selenium speciation. The reproducibility of the retention time indicated that sample injection by electrokinetic and nebulizer gas flow was better than that by self-aspiration alone. The detection limits for selenate, selenite, selenocystine and selenomethionine were found to be 2.40, 3.53, 12.86 and 11.25 ng/mL, respectively. Due to the high sensitivity and element-specific detection, as well as the high selectivity of the bonded phase, quantitative analysis of selenium speciation in urine was also achieved.