Improving sensitivity by large-volume sample stacking combined with sweeping without polarity switching by capillary electrophoresis coupled to photodiode array ultraviolet detection

An easy, simple, and highly efficient on-line preconcentration method for polyphenolic compounds in CE was developed. It combined two on-line concentration techniques, large-volume sample stacking (LVSS) and sweeping. The analytes preconcentration technique was carried out by pressure injection of large-volume sample followed by the EOF as a pump pushing the bulk of low-conductivity sample matrix out of the outlet of the capillary without the electrode polarity switching technique using five polyphenols as the model analytes. Identification and quantification of the analytes were performed by photodiode array UV (PDA) detection. The optimal BGE used for separation and preconcentration was a solution composed of 10 mM borate-90 mM sodium cholate (SC)-40% v/v ethylene glycol, without pH adjustment, the applied voltage was 27.5 kV. Under optimal preconcentration conditions (sample injection 99 s at 0.5 psi), the enhancement in the detection sensitivities of the peak height and peak area of the analytes using the on-line concentration technique was in the range of 18-26- and 23-44-fold comparing with the conventional injection mode (3 s). The detection limits for (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (C), (-)-epigallocatechin gallate (EGCG), and (-)-epicatechin gallate (ECG) were 4.3, 2.4, 2.2, 2.0, and 1.6 ng/mL, respectively. The five analytes were baseline-separated under the optimum conditions and the experimental results showed that preconcentration was well achieved.     

Optimization of sample stacking for the simultaneous determination of nonsteroidal anti-inflammatory drugs with a wall-coated histidine capillary column

A wall-coated histidine capillary column was developed for the on-line preconcentration of nonsteroidal anti-inflammatory drugs (NSAIDs) in capillary electrochromatography (CEC). A wide variety of experimental parameters, such as the sample buffer, background electrolyte (BGE) composition, concentration, sample plug lengths, water plug, and the effect of organic modifiers were studied. The relationship between peak height and injection times for the NSAIDs by variation of sample and BGE buffer concentration was investigated. On addition of sodium chloride (0.3-0.6%) to the sample zone, the stacking efficiency was increased. With acetate buffer (100 mM, pH 5.0)/ethanol (20% v/v) as BGE and sample solution in acetate buffer (0.2 mM, pH 5.0)/ethanol (20% v/v)/NaCl (0.3% w/v), NSAIDs could be determined at low microM levels without sample matrix removal. The detection limit was 0.096 microM for indoprofen, 0.110 microM for ketoprofen, 0.012 microM for naproxen, 0.023 microM for ibuprofen, 0.110 microM for fenoprofen, 0.140 microM for flurbiprofen, and 0.120 microM for suprofen. The method could be successfully applied to the simultaneous determination of NSAIDs in urine. The recoveries were better than 82% for all the analytes. The present method enables simple manipulation with UV detection for the determination of NSAIDs at low concentration levels in complex matrix samples.     

Online preconcentration by electrokinetic supercharging for separation of endocrine disrupting chemical and phenolic pollutants in water samples

Online preconcentration using electrokinetic supercharging (EKS) was proposed to enhance the sensitivity of separation for endocrine disrupting chemical (methylparaben (MP)) and phenolic pollutants (2‐nitrophenol (NP) and 4‐chlorophenol (CP)) in water sample. Important EKS and separation conditions such as the concentration of BGE; the choice of terminating electrolyte (TE); and the injection time of leading electrolyte (LE), sample, and TE were optimized. The optimum EKS‐CE conditions were as follows: BGE comprising of 12 mM sodium tetraborate pH 10.1, 100 mM sodium chloride as LE hydrodynamically injected at 50 mbar for 30 s, electrokinetic injection (EKI) of sample at –3 kV for 200 s, and 100 mM CHES as TE hydrodynamically injected at 50 mbar for 40 s. The separation was conducted at negative polarity mode and UV detection at 214 nm. Under these conditions, the sensitivity of analytes was enhanced from 100‐ to 737‐fold as compared to normal CZE with hydrodynamic injection, giving LOD of 4.89, 5.29, and 53 μg/L for MP, NP and CP, respectively. The LODs were adequate for the analysis of NP and CP in environmental water sample having concentration at or lower than their maximum admissible concentration limit (240 and 2000 μg/L for NP and CP). The LOD of MP can be suitable for the analysis of MP exists at mid‐microgram per liter level, even though the LOD was slightly higher than the concentration usually found in water samples (from ng/L to 1 μg/L). The method repeatabilities (%RSD) were in the range of 1.07–2.39% (migration time) and 8.28–14.0% (peak area).     

Characterization of protein hydrolysates of cosmetic use by CE-MS

Protein hydrolysates have been used as active principles in cosmetic products conferring different properties to the final formulations, which are mostly controlled by the peptide size and its amino acid sequence. In this work, capillary electrophoresis coupled to mass spectrometry analyses were carried out in order to investigate such characteristics of protein hydrolysates. Samples of different origins (milk, soy and rice) were obtained from a local company, and were analyzed without a previous preparation step. The background electrolyte (BGE) and sheath liquid compositions were optimized for each sample. The best BGE composition (860 mmol/L formic acid--pH 1.8--in 70:30 v/v water/methanol hydro-organic solvent) was chosen based on the overall peak resolution whereas the best sheath liquid was selected based on increased sensitivity and presented different compositions to each sample (10.9-217 mmol/L formic acid in 75:25-25:75 v/v water/methanol hydro-organic solvent). Most of the putative peptides in the hydrolysate samples under investigation presented molecular masses of 1000 Da or less. De novo sequencing was carried out for some of the analytes, revealing the hydrophobicity/polarity of the peptides. Hence, the technique has proved to be an advantageous tool for the quality control of industrial protein hydrolysates.     

On-line preconcentration of perfluorooctanoic acid and perfluorooctanesulfonic acid by nonaqueous capillary electrophoresis

Separation of major environmental pollutants as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) by capillary electrophoresis is reported for the first time. It is not possible to resolve the solutes in an aqueous media. However, the use of methanol and acetonitrile as the background electrolyte (BGE) solvents allowed their rapid separation in an uncoated capillary. A major effort was put into BGE optimization in respect to both separation efficiency and detection for further on‐line preconcentration. 5 mmol.L^?1 naphthalene‐1‐sulfonic acid and 10 mmol.L^?1 triethylamine dissolved in ACN/MeOH (50:50 v/v) provided best separation and detection conditions. Next, the large‐volume sample stacking and the field‐amplified sample injection were applied and compared. Large‐volume sample stacking improved limits of detection (LODs) with regard to the standard injection by 69 times for PFOA and 143 times for PFOS with LODs of 280 and 230 nmol.L^?1, respectively. Field‐amplified sample injection improved LODs 624 times for PFOAand 806 times for PFOS with LODs 31 and 40 nmol.L^?1, respectively. Both preconcentration methods showed repeatabilities of migration times less than 1.2% RSD intraday and 6.6% RSD interday. The method was applied on PFOA and PFOS analysis in a sample of river water treated with solid‐phase extraction, which further improved LOD toward 5.6 × 10^?10 mol.L^?1 for PFOS and 6.4 × 10^?10 mol.L^?1 for PFOA and allows the method to be used for river water contamination screening or decomposition studies.     

三角形法和四面体法优化选择毛细管区带电泳背景电解质

建立了两种高效、快速的毛细管区带电泳背景电解质(BGE)的优化方法三角形优化法和四面体优化法。以色谱指纹图谱指数F和色谱指纹图谱相对指数Fr作为评价毛细管电泳分析系统的目标函数,以雪莲药材水提取液为样品,考察一定浓度的硼砂、硼酸、磷酸氢二钠和磷酸二氢钠溶液按三角形优化法和四面体优化法构成背景电解质时对样品的分离情况,通过添加有机改性剂和调节pH进行再优化。用三角形法优化出以50 mmol/L硼砂-含3%乙腈的150 mmol/L磷酸二氢钠(体积比为1∶1)作为BGE时分离效果最佳,用四面体法优化出以50 mmol/L硼砂-150 mmol/L磷酸二氢钠-200 mmol/L硼酸(体积比为1∶1∶2,用0.1 mol/L氢氧化钠调pH 8.55)作为BGE时分离效果最佳,分别获得28个和25个电泳峰。所建立的方法操作简捷,适用于中药材水提取液或醇提取液的毛细管区带电泳BGE的选择。     

Tungstate as complex-forming reagent facilitating separation of selected polyphenols by capillary electrophoresis and its comparison with borate

A novel electrophoretic BGE containing tungstate as complex-forming reagent is suitable for the separation of polyphenols. Similar to molybdate-containing BGE reported earlier (cf. M. Polásek, et al.., Talanta 2006, 69, 192) addition of tungstate to BGE affects significantly migration of compounds/ligands with vicinal -OH groups due to the formation of negatively charged complexes involving W(VI) as central ion. Baseline separation of mixtures of flavonoids (apigenin, luteolin, hyperoside, quercetin, and rutin) and phenolic acids (chlorogenic and p-coumaric acid) was achieved within 15 min with optimized BGE of pH 7.4 containing 50 mM N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES), 2.2 mM tungstate, and 25% v/v of methanol. The separation was performed in a 75 cm (effective length 42 cm)x 75 microm id uncoated fused-silica capillary at 30 kV with spectrophotometric detection at 275 nm. The calibration curves were rectilinear for 25-175 microg/mL of all analytes (cinnamic acid as the internal standard). The LODs ranged from 1.8 to 6 microg/mL for all analytes except for chlorogenic acid. Intraday precision (n = 6) of migration times (RSD < or = 1.2%) and peak areas (RSD < or = 5.6%) was evaluated. The tungstate-based BGEs can be alternatively utilized for the analysis of polyphenols at considerably lower pH than with conventional alkaline borate-based BGEs.     

On‐line preconcentration strategy for the simultaneous quantification of three local anesthetics in human urine using CZE

The simultaneous determination of usually employed anesthetics (procaine, lidocaine, and bupivacaine) has been developed and validated using CE with ultraviolet detection at 212 nm. The separation of these three drugs has been achieved in less than 7 min, using a temperature of 25ºC and 25 kV, with a 150 mM citrate buffer (pH 2.5) as BGE. Field‐amplified sample injection (FASI) has been used for on‐line sample preconcentration. Ultrapure water and ACN 50/50 (v/v) mixture gave the greatest enhancement factor when it was employed as an injection solvent. Injection voltage and time were optimized, being 13 kV and 13 s, the optimum values, respectively. To avoid the possible irreproducibility associated with the electrokinetic injection, an internal standard such as tetracaine, was employed. The instrumental detection limits (LOD S/N = 3) for the compounds ranged between 2.6 and 7.0 μg L⁻¹ and the quantitation limits (LOQ S/N = 10) between 37.8 and 55.9 μg L⁻¹. The detection limits obtained in real human urine samples ranged between 55.2 and 83.6 μg L⁻¹ and the quantitation limits between 196.0 and 276.0 μg L⁻¹. The proposed method has demonstrated its applicability to the analysis of these local anesthetics in urine samples without any pretreatment, allowing the rapid determination of these target analytes.     

CE‐ESI‐MS coupled with dynamic pH junction online concentration for analysis of peptides in human urine samples

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE‐ESI‐MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol–water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L ‐Ala‐L ‐Ala (pI=5.57), L ‐Leu‐D ‐Leu (pI=5.52), Gly‐D ‐Phe (pI=5.52) and Gly‐Gly‐L ‐Leu (pI=5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2–2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45–3.57 and 4.93–6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.     

Analysis of neutral nitromusks in incenses by capillary electrophoresis in organic solvents and gas chromatography-mass spectrometry

Nitromusks used as fragrances in a variety of personal-care products, cleansers, and domestic deodorants, including incense sticks, are neutral nitro aromatic compounds; some of these have been reported as photosensitizers. In this work, their analysis was performed by capillary electrophoresis (CE) in a methanol-based background electrolyte (BGE). In particular, a 10 mM solution of citric acid in methanol was used; under these conditions the strong suppression of the electroosmotic flow favored the use of negatively charged surfactants as additives for the anodic migration of the studied analytes. To this end, sodium taurodeoxycholate (TDC) was supplemented at high concentration (190 mM) to the organic background electrolyte (BGE), showing strong indication of the ability to give micelle-like aggregates. Since nitromusks are characterized by the presence of a nitroaromatic ring with low charge density, their association with TDC aggregates can be ascribed to donor-acceptor interactions. Separation of musk xylene, musk ketone, and the banned musk moskene and musk ambrette was obtained under full nonaqueous BGE; the addition of relatively small water percentages (15% v/v) was found to be useful in improving the separation of pairs of adjacent peaks. Under optimized conditions (190 mM sodium TDC in methanol-water, 85-15 v/v containing citric acid 10 mM) the system was applied to the analysis of nitromusks in incense sticks extracted with methanol. The results were compared with those obtained by the analysis of the same samples using gas chromatography with mass detector. The expected different selectivity of separation obtained using the two techniques can be useful in the unambiguous determination of the analytes; furthermore, a substantial accord of the preliminary quantitative results achieved with the two methods was assumed as the confirmation of the potential reliability of CE performed with high percentage of organic solvent.     

Quantification of buserelin in a pharmaceutical product by multiple-injection CZE

An efficient and rapid separation method based on reversed-polarity multiple-injection CZE (MICZE), has been developed for the quantification of buserelin in a pharmaceutical product. The determinations were performed by serially injecting five standard solutions of buserelin (50-300 microg/mL) and one reference analyte into a Polybrene-coated capillary. All the samples contained goserelin, an analog peptide to buserelin, as internal standard (IS). Immediately after pressure injection, the applied sample plugs were subjected to electrophoresis for 2 min at -25 kV. Consequently, each sample plug became isolated from its neighboring plugs by the BGE, composed of 100 mM phosphate-triethanolamine buffer at pH 3.0 containing 10% v/v ACN. During separation the individual sample components migrated at similar velocities and as distinct zones through the capillary giving 24 peaks, 12 from the analyte and the IS and 12 from the sample matrix. The buserelin content of the pharmaceutical product was determined to be 0.94 +/- 0.05 mg/mL, which is only a slight deviation from the declared concentration (1 mg/mL).     

Simultaneous separation of 17 anions by capillary electrophoresis with the addition of an organic solvent

Seventeen inorganic and organic anions, that normally are insufficiently separated via ion chromatography, were completely separated by the addition of an organic solvent to a solution of BGE combined with an adjustment of the apparent pH via CE in combination with indirect UV absorbance detection. Methanol, ethanol, and acetonitrile were examined for their utility in manipulating the selective separation of anions. Methanol and acetonitrile were better modifiers than ethanol at enhancing the resolution of anions comigrating in an aqueous solution of BGE. Methanol was selected as the modifier that provided the largest separation window that could achieve a complete separation of the target analytes. Via the use of methanol, manipulation of the selectivity between inorganic anions and that between inorganic and organic anions was enhanced, but the separation between organic anions remained difficult when only methanol was used. By varying the apparent pH of the BGE in the presence of 10% v/v methanol, however, the separation selectivity between organic anions was substantially improved. Eventually, 7 inorganic and 10 organic anions were simultaneously separated using BGE at a pH of 6.3 in the presence of 10% v/v methanol.     

Enhanced sensitivity and resolution for the analysis of paralytic shellfish poisoning toxins in water using capillary electrophoresis with amperometric detection and field‐amplified sample injection

The profiling of the most lethal paralytic shellfish poisoning toxins (PSTs) in freshwater has increased the need to establish an alternative analytical method with high sensitivity and resolution. In this paper, a coupling technique of field‐amplified sample injection (FASI) and CE with end‐column amperometric detection (CE‐AD) was developed to improve the detection sensitivity and separation of PSTs by electrokinetically injecting a water plug of analytes to the capillary filled with a high‐conductivity BGE. Parameters affecting FASI and CE process were carefully adjusted to achieve the highest response and resolution. Separation selectivity for PSTs, especially for the analogues and epimers, was greatly enhanced by using 40 mM Britton–Robinson buffer (pH 9.5) as BGE, which altered the EOF and mobility of the analytes that interacted with polyborate ions. Satisfactory linear relationship between peak current and concentration of toxins were gained over a wide range of 1.95–254 μg/L. The detection limits (S/N = 3) for five PSTs ranged from 0.63 to 3.11 μg/L, which are below the health alert level in drinking water. In comparison with the up‐to‐date reporting chromatographic methods, the FASI‐CE‐AD method was simple, low‐cost, selective, and sensitive enough for direct quantification of PSTs at very low levels, implying a potential for screening and monitoring of PSTs in surface waters.     

MEKC–MS/MS method using a volatile surfactant for the simultaneous determination of 12 synthetic cannabinoids

This study describes a method for the simultaneous determination of 12 synthetic cannabinoids by MEKC–MS/MS using a volatile surfactant (ammonium perfluorooctanoate) as a constituent of the micellar pseudostationary phase. Although most synthetic cannabinoids comigrated by a CZE method, sufficient separation could be achieved by the proposed method. The best separation was made possible by 50 mM ammonium perfluorooctanoate in 20% v/v acetonitrile/water (apparent pH* 9.0) as the BGE, followed by MS detection using a sheath liquid composed of 5 mM ammonium formate in 50% v/v methanol/water mixed hydro‐organic solvent. The standard calibration curve for all analytes showed good linearity (r > 0.99). Satisfactory recoveries, ranging from 89.5 to 101.7%, were obtained. The LODs were 6.5–76.5 μg/g for the target analytes. This method appears to be a useful tool for the identification of synthetic cannabinoids in illegal herbal incense blends.     

Determination of malachite green in fish water samples by cloud‐point extraction coupled to cation‐selective exhaustive injection and sweeping‐MEKC

We have employed a high‐sensitivity off‐line coupled with on‐line preconcentration method, cloud‐point extraction (CPE)/cation‐selective exhaustive injection (CSEI) and sweeping‐MEKC, for the analysis of malachite green. The variables that affect CPE were investigated. The optimal conditions were 250 g/L of Triton X‐100, 10% of Na₂SO₄ (w/v), heat‐assisted at 60°C for 20 min. We monitored the effects of several of the CSEI‐sweeping‐MEKC parameters – including the type of BGE, the concentrations of SDS, the injection length of the high‐conductivity buffer, and the injection time of the sample – to optimize the separation process. The optimal BGE was 50 mM citric acid (pH 2.2) containing 100 mM SDS. In addition, electrokinetic injection of the sample at 15 kV for 800 s provided both high separation efficiency and enhanced sweeping sensitivity. The sensitivity enhancement for malachite green was 1.9×10⁴ relative to CZE; the coefficients of determination exceeded 0.9928. The LOD, based on an S/N of 3:1, of CSEI‐sweeping‐MEKC was 0.87 ng/mL; in contrast, when using off‐line CPE/CSEI‐sweeping‐MEKC the sensitivity increased to 69.6 pg/mL. This proposed method was successfully applied to determine trace amounts of malachite green in fish water samples.     

Separation and determination of psoralen and isopsoralen by microemulsion electrokinetic chromatography

The use of microemulsion electrokinetic chromatography was proposed to separate psoralen and isopsoralen in Psoralea corylitolia L. and its preparations. After conducting a series of optimizations, baseline separation was obtained for the analytes under the optimum conditions [sodium dodecyl sulfate 1.05% (m/v), ethyl acetate 0.96% (v/v), butan-1-ol 0.24% (v/v), 25 mm borate, pH 8.5, applied voltage 17.5 kV and detection at 254 nm]. Regression equations revealed linear relationships (correlation coefficients 0.9997 for psoralen and 0.9999 for isopsoralen) between the peak area of each analyte and the concentration. The limits of detection (defined as a signal-to-noise ratio of about 3) were 0.42 microg/mL for psoralen and 0.32 microg/mL for isopsoralen, respectively. The analytes were successfully determined with recoveries ranging from 95.50 to 102.03%. The method has been successfully applied for the analysis of psoralen and isopsoralen in medical samples. Furthermore, a simple and effective extraction method, with methanol in an ultrasonic water bath for 20 min three times, was used for sample preparation.     

Development of an ultrasensitive stacking technique for 5‐nitroimidazole determination in untreated biological fluids by micellar electrokinetic chromatography

Cation‐selective exhaustive injection and sweeping followed by a MEKC separation is evaluated for the sensitive analysis of 5‐nitroimidazoles in untreated human serum and urine. Deproteinized serum and urine samples were diluted 76 and 143 times, respectively, in a low‐conductivity solvent (5.00 mM orthophosphoric acid containing 5.0% v/v methanol). Samples were electrokinetically injected at 9.8 kV for 632 s in a previously conditioned fused‐silica capillary (65.0 cm × 50 μm id). Separation was performed at –30 kV and 20°C using 44 mM phosphate buffer (pH 2.5), 123 mM SDS, and 8% v/v tetrahydrofurane as BGE. Signals were monitored at 276 nm and peak area was selected as analytical response. Good linearity (R² ≥ 0.988) and LODs lower than 1.5 and 1.8 μg/mL were achieved in serum and urine, respectively.     

Determination of nitrate in seawater by capillary zone electrophoresis with chloride-induced sample self-stacking

A capillary zone electrophoresis (CZE) method was established to determine low concentration nitrate which was online preconcentrated with chloride-induced leading-type sample self-stacking for seawater samples. The sample self-stacking was based on transient isotachophoresis in which chloride served as leading ion, and dihydrogenphosphate in the background electrolyte (0.1 M phosphate) as the terminating one. Due to the small mobility difference between nitrate and chloride, the isotachophoresis time was so long that nitrate could not separate from the rear sharp boundary between chloride and the background electrolyte (BGE) when it migrated to the detection window. A zwitterionic surfactant, 3-(N,N-dimethyldodecylammonio)propane sulfonate was added to the BGE to enlarge the mobility difference for its selective interaction with anions. Thus, a highly conductive sample could be injected in a large volume with about fourfold sensitivity enhancement compared to that of field amplification sample stacking in which nitrate was dissolved in pure water. The relative standard deviations (n=5) of migration time, peak area, peak height were 0.1, 3.0, 1.5%, respectively. The limit of detection (S/N=3) for nitrate was 35 microg/l in seawater samples with relatively low concentration BGE (0.1 M sodium phosphate, pH 6.2). The overall procedure consisting of online preconcentration and separation was as simple as routine CZE except for a slightly longer sample injection time (3-4 min).     

Capillary electrophoretic separation of tricyclic antidepressants using a polymer-coated capillary and beta-cyclodextrin as an electrolyte additive

A new method for the CE separation of nine tricyclic antidepressants (TCAs), viz. amitriptyline, clomipramine, desipramine, doxepin, fluphenazine, imipramine, nortriptyline, promazine, and thioridazine, is described. The capillary was statically coated with a layer of poly(N,N-dimethylacrylamide) (PDMA) to suppress the EOF, and beta-CD was used as an additive in the BGE solution. The optimal resolution of nine TCAs was obtained by using a 1% v/v PDMA-coated capillary and a BGE solution of 50 mM sodium phosphate buffer (pH 3.0) containing 0.5 mM beta-CD. Efficiencies were typically >10(5 )plates/m. Complete separation of nine TCAs could be achieved in about 28 min; the two diastereomers of doxepin and the two enantiomers of thioridazine could also be separated. The RSD values of migration time and peak area of the TCAs were in the ranges 0.5-0.8 and 3.3-4.9% (n = 10), respectively. In combination with a suitable sample clean-up technique, such as hollow fiber-based liquid phase microextraction (HF-LPME), the polymer-coated capillary can be employed for the CE-UV analysis of TCAs in human plasma.     

Determination of ammonia, creatinine and inorganic cations in urine using CE with contactless conductivity detection

CE with capacitively coupled contactless conductivity detection (C(4)D) was used to determine waste products of the nitrogen metabolism (ammonia and creatinine) and of biogenic inorganic cations in samples of human urine. The CE separation was performed in two BGEs, consisting of 2 M acetic acid + 1.5 mM crown ether 18-crown-6 (BGE I) and 2 M acetic acid + 2% w/v PEG (BGE II). Only BGE II permitted complete separation of all the analytes in a model sample and in real urine samples. The LOD values for the optimized procedure ranged from 0.8 microM for Ca(2+) and Mg(2+) to 2.9 microM for NH(4)(+) (in terms of mass concentration units, from 7 microg/L for Li(+) to 102 microg/L for creatinine). These values are adequate for determination of NH(4)(+), creatinine, Na(+), K(+), Ca(2+) and Mg(2+) in real urine samples.