Determination of flavonoids in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. by capillary electrophoresis with electrochemical detection

Four flavonoids (rutin, hyperoside, quercitrin and quercetin) in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. were determined by capillary electrophoresis with wall-jet amperometric detection. The working electrode was a 500 μm diameter carbon disc electrode and the detection potential was +0.95 V (versus Ag/AgCl). Effects of several important factors, such as the running buffer and its corresponding pH and concentration, separation voltage, injection time were investigated to acquire the optimum conditions for separation of these four flavonoids. Baseline separation for the four flavonoids was obtained within 21 min in a 60 cm length capillary at a separation voltage of 15 kV with a 60 mmoL/L Na₂B₄O₇-120 mmoL/L NaH₂PO₄ buffer (pH 8.8) as running buffer. The relationship between peak currents and analyte concentrations was linear over about two orders of magnitude with detection limits (defined as S/N = 3) ranging from 0.02 to 0.05 μg/mL for all analytes. This method was applied for the determination of the above four flavonoids in H. cordata Thunb. and S. chinensis (Lour.) Bail. with simple extraction procedures, and the assay results were satisfactory.     

Determination of mannitol and three sugars in Ligustrum lucidum Ait. by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection has been developed for the separation and determination of mannitol, sucrose, glucose, and fructose in Ligustrum lucidum Ait. for the first time. Effects of several important factors such as the concentration of NaOH, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions. The detection electrode was a 300 μm diameter copper disc electrode at a working potential of +0.65 V (versus saturated calomel electrode (SCE)). The four analytes can be well separated within 13 min in a 40 cm length fused-silica capillary at a separation voltage of 12 kV in a 75 mM NaOH aqueous solution. The relation between peak current and analyte concentration was linear over about three orders of magnitude with detection limits (S/N = 3) ranging from 1 to 2 μM for all analytes. The proposed method has been successfully applied to monitor the mannitol and sugar contents in the plant samples at different growth stages with satisfactory assay results.     

Miniaturized capillary electrophoresis system with a carbon nanotube microelectrode for rapid separation and detection of thiols

Multi-walled carbon nanotube (CNT) was mixed with epoxy to fabricate microdisc electrode used as a detector for a specially designed miniaturized capillary electrophoresis (CE)-amperometric detection system for the separation and detection of several bioactive thiols. The end-channel CNT amperometric detector offers favourable signal-to-noise characteristics at a relatively low potential (0.8 V) for detecting thiol compounds. Factors influencing the separation and detection processes were examined and optimized. Four thiols (homocysteine, cysteine, glutathione, and N-acetylcysteine) have been separated within 130 s at a separation voltage of 2000 V using a 20 mM phosphate running buffer (pH 7.8). Highly linear response is obtained for homocysteine, cysteine, glutathione, and N-acetylcysteine over the range of 5-50 μM with detection limits of 0.75, 0.8, 2.9, and 3.3 μM, respectively. Good stability and reproducibility (R.S.D. < 5%) are obtained reflecting the minimal adsorption of thiols at the CNT electrode surface. The new microchip protocol should find a wide range of bioanalytical applications involving assays of thiol compounds.     

Determination of ethylenediaminetetraacetic acid, ethylenediaminedisuccinic acid and iminodisuccinic acid in cosmetic products by capillary electrophoresis and high performance liquid chromatography

A capillary electrophoresis (CE) and a high performance liquid chromatography (HPLC) method are described for the simultaneous determination of ethylenediaminetetraacetic acid (EDTA), S,S′-ethylenediaminedisuccinic acid (EDDS) and R,S-iminodisuccinic acid (IDS) complexing agents as their Fe(III) complexes in cosmetics like shower cream and foam bath. The non-biodegradable EDTA is used in combination with biodegradable analogues like EDDS and IDS in many commercial products. The HPLC method involves separation by reversed-phase ion pair chromatography on a C₁₈ column using methanol-formate buffer (20 mM tetrabutylammonium hydrogen sulfate, 15 mM sodium formate adjusted to pH 4.0 with formic acid) (10:90, v/v) as mobile solvent at a flow rate of 0.8 mL min⁻¹ at 24 °C using UV detection at 240 nm. The CE separation was performed in a fused silica capillary of 50 μm i.d. with the total length of 50 cm with a 10 mM MES and MOPSO (pH 5.5) at an applied voltage of −25 kV. The samples were introduced by applying a 50 mbar pressure for 2 s. Absorbances at 215 and 225 nm were monitored for the detection of the complexes. The methodology performance of the two methods was evaluated in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ) and reproducibility. The LOD values obtained from HPLC are low when compared with CE. The applicability of both the methods was demonstrated for the analysis of cosmetic products such as shower cream and foam bath. The results obtained by both CE and HPLC were found to be comparable and in good agreement.     

Separation and determination of acrylamide in potato chips by micellar electrokinetic capillary chromatography

A simple and rapid method using micellar electrokinetic capillary chromatography (MEKC) was developed for the separation and determination of acrylamide in potato chips at low levels for the first time. The experimental conditions for the separation and quantification of acrylamide were optimized at first. The optimized conditions were: 50 mmol/L Na₂B₄O₇ and 40 mmol/L SDS at pH 10.0, 12 kV applied voltage, 76 cm total length (67 cm effective length) and 75 μm i.d. capillary, 198 nm wavelength, 15 cm high 25 s hydrodynamics sample injection, 20 °C air-cooling. The linear response of acrylamide concentration ranges from 0.5 to 100 μg/mL with high correlation coefficient (r = 0.9986, n = 9). The LOD and LOQ were estimated to be 0.1 and 0.33 μg/mL based on S/N = 3 and 10. The precision values (expressed as R.S.D.) of intra- and inter-day were 0.86-4.35% and 2.61-9.65%, respectively. Recoveries spiked at levels 2, 20, 60 μg/mL ranged between 90.86% and 99.6% with R.S.D. less than 6.5%. Finally, the developed method has been applied to the analysis of real samples and has achieved satisfactory results. All of these indicated that it was a reliable method for the quantification of acrylamide in potato chips.     

Determination of nickel in natural waters by FAAS after sorption on octadecyl silica membrane disks modified with a recently synthesized Schiff's base

A simple, highly sensitive, accurate and selective method for the determination of trace amounts of Ni²⁺ ions in water samples is proposed. The method is based on the separation and preconcentration of Ni²⁺ on an octadecyl-bonded silica (ODBS) membrane disk modified by a recently synthesized Schiff’s base N,N′-bis (3-methylsalicylidene) ortho phenylene diamine (MSOPD) at pH 7. The synthesis of this extractant ligand is also described. The retained nickel on the membrane was eluted with 2×5 ml 0.5 M HNO₃ and measured by flame atomic absorption spectrometry (FAAS) at 232.0 nm. The extraction efficiency and the influence of the type and least amount of eluent for the stripping of Ni²⁺ from the disks, pH, flow rates of sample solution and eluent, amount of MSOPD, effect of other ions, and breakthrough volume were evaluated. The maximum capacity of the membrane disks modified by 3 mg of MSOPD was found to be 146±4 μg Ni²⁺. The 3σ limit of detection of the method was 30 ng per 1000 ml and also an enrichment factor of 250 was obtained. The proposed method has been applied to the determination of nickel in several water samples with satisfactory results.     

Identification and quantitation of iodotyrosines and iodothyronines in hydrolysate of iodinated casein by capillary electrophoresis

In this paper, a new method for separation, identification and quantitation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine (MIT), 3,5-diiodo-L-tyrosine (DIT), L-thyronine (T₀), 3,5-diiodo-L-thyronine (T₂), 3,5,3′-triiodo-L-thyronine (T₃) and 3,3′,5,5′-tetraiodo-L-thyronine (T₄)] was described by using capillary electrophoresis with photodiode-array ultraviolet-visible detection (CE-UV). The certain influence factors were systematically investigated, including the type, concentration and pH of buffer, and additive. We found that 10 mM sodium borate running buffer (pH 8.5) containing 0.10 mM β-CD as additive reagent allowed the best instrumental conditions for the optimum separation of the iodotyrosines and iodothyronines. Under optimized conditions, the analytical time was within 6 min, using an uncoated fused-silica capillary of 75 μm inner diameter with an effective length of 30 cm. The reproducibility of the migration time and peak area was less than 0.6% and 6.8%, respectively. A linear range from 10-1000 μg/mL and low limits of detection from 1.3-3.4 μg/mL were obtained at the detection wavelength of 280 nm. Our preliminary results show that the method is well suitable for determination of the hydrolysate of iodinated casein.     

Separation and determination of alpinetin and cardamonin in Alpinia katsumadai Hayata by flow injection-micellar electrokinetic chromatography

A simple, rapid, and accurate method for the separation and determination of alpinetin and cardamonin in Alpinia katsumadai Hayata was developed by combination of flow injection (FI)-micellar electrokinetic chromatography (MEKC) for the first time. The analysis was carried out using an unmodified fused-silica capillary (50 μm i.d.; total length 13.6 cm; effective length 10.3 cm) and direct ultraviolet (UV) detection at 214 nm. The sample throughput was 11-24 samples per hour using the background electrolyte (BGE) containing 4 mM sodium borate-8 mM NaH₂PO₄ (pH 8.1)-8 mM sodium dodecyl sulfate (SDS)-19% (v/v) ethanol. The repeatabilities (n = 4) reached relative standard deviation values (R.S.D.) of 3.0% and 2.5% for the peak areas and 2.5% and 3.1% for peak heights of alpinetin and cardamonin, respectively. Regression equations revealed linear relationships (r²: 0.9993-0.9994) between the peak area of each analyte and the concentration. Recoveries were in the range 90-92% and 99-105% for alpinetin and cardamonin, respectively.     

Separation and determination of nitroaniline isomers by capillary zone electrophoresis with amperometric detection

In this paper, capillary zone electrophoresis with amperometric detection (CZE-AD) was firstly applied to the simultaneous separation and determination of nitroaniline positional isomers. The three analytes could be perfectly analyzed by using the buffer of extreme pH. The effects of several important factors were investigated to find optimum conditions. A carbon-disk electrode was used as working electrode. The optimal conditions were 40 mmol/L tartaric acid-sodium tartrate (pH 1.2) as running buffer, 17 kV as separation voltage and 1.10 V (versus saturated calomel reference electrode, SCE) as detection potential. Under the optimum conditions, o-, m- and p-nitroaniline were separated successfully and good linearity, reproducibility and recovery results were obtained. The detection limit for m-nitroaniline was as low as at 9.06 × 10⁻⁹ mol/L. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 1.8% for migration time and 1.1% for peak areas. The utility of this method was demonstrated by monitoring dyestuff wastewater and the assay results were satisfactory.     

Quantitative analysis of flavonoids and phenolic acids in Arnica montana L. by micellar electrokinetic capillary chromatography

Arnica montana preparations have been used in Europe for centuries to treat skin disorders. Among the biologically active ingredients in the flower heads of the plant are sequiterpenes, flavonoids and phenolic acids. For the simultaneous determination of compounds belonging to the latter two groups a micellar electrokinetic capillary chromatography (MEKC) method was developed and validated. By using an electrolyte solution containing 50 mM borax, 25 mM sodium dodecyl sulfate and 30% of acetonitrile the separation of seven flavonoids and four caffeic acid derivatives was feasible in less than 20 min. The optimized system was validated for repeatability (σ_rel ≤ 4.4%), precision (inter-day σ_rel ≤ 8.13%, intra-day σ_rel ≤ 4.32%), accuracy (recovery rates from 96.8 to 102.4%), sensitivity (limit of detection (LOD) ≤ 4.5 μg mL⁻¹) and linearity (R² ≥ 0.9996), and then successfully applied to assay several plant samples. In all of them the most dominant flavonoid was found to be quercetin 3-O-glucuronic acid, whereas 3,5-dicaffeoylquinic acid was the major phenolic acid; the total content of flavonoids and phenolic acids varied in the samples from 0.60 to 1.70%, and 1.03 to 2.24%, respectively.     

Trace determination of short-chain aliphatic amines in biological samples by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

In this study, a new capillary electrophoresis (CE) method is described originally for the sensitive and selective determination of short-chain aliphatic amines in biological samples. These amines were converted into their N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA) derivatives and measured by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection. The derivatization conditions and separation parameters for the aliphatic amines were optimized in detail. The SIFA-labeled amines were fully separated within 8.5 min using 25 mM pH 9.6 boric acid electrolyte containing 60 mM sodium dodecyl sulfate (SDS). The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.02 to 0.1 nM, which was the lowest value reported by CE methods. The developed method was successfully employed to monitor aliphatic amines in serum and cells samples. After comparison of other CE methods using different fluorescent probes, the present method represents a powerful tool for the trace determination of aliphatic amines in complex biological samples.     

Catalytic adsorptive stripping voltammetry versus electrothermal atomic absorption spectrometry in the determination of trace cobalt and chromium in human urine

Two methods of the determination of cobalt and chromium in human urine of non-occupationally exposed populations—highly sensitive catalytic adsorptive stripping voltammetry (CAdSV) and electrothermal atomic absorption spectrometry (ET-AAS)—are evaluated and compared. The CAdSV methods are based on adsorptive accumulation of a cobalt-nioxime (1,2-cyclohexanedione dioxime) or a chromium-DTPA (diethylenetriammine-N,N,N′,N″,N″-pentaacetic acid) complexes on a hanging mercury drop electrode, followed by a stripping voltammetric measurement of the catalytic reduction current of the adsorbed complex in the presence of sodium nitrite in case of cobalt or in the presence of sodium nitrate in case of chromium determination. In the CAdSV procedure UV-photolysis was used for the sample pre-treatment; the ET-AAS determination did not require any separate preliminary decomposition of the analyte urine samples. The accuracy of the procedures was checked by the analysis of commercially available quality control urine samples. The detection limits (3σ) were 0.13 μg l⁻¹ for Co and 0.18 μg l⁻¹ for Cr in ET-AAS determination and 0.007 μg l⁻¹ for Co and 0.002 μg l⁻¹ for Cr in CAdSV measurements. Precision (R.S.D.) was less than 5% for both methods. The study has shown that the CAdSV is a more reliable and sensitive technique for the determination of very low cobalt and chromium contents in urine, the detection of which is not possible when using the AAS technique.     

Determination of methylamines and trimethylamine-N-oxide in particulate matter by non-suppressed ion chromatography

An ion chromatography method with non-suppressed conductivity detection was developed for the simultaneous determination of methylamines (methylamine, dimethylamine, trimethylamine) and trimethylamine-N-oxide in particulate matter air samples. The analytes were well separated by means of cation-exchange chromatography using a 3 mM nitric acid/3.5% acetonitrile (v/v) eluent solution and a Metrosep C 2 250 (250 mm × 4 mm i.d.) separation column. The effects of the different chromatographic parameters on the separation were also investigated. Detection limits of methylamine, dimethylamine, trimethylamine, and trimethylamine-N-oxide were 43, 46, 76 and 72 μg/L, respectively. The relative standard deviations of the retention times were between 0.42% and 1.14% while the recoveries were between 78.8% and 88.3%. The method is suitable for determining if methylamines and trimethylamine-N-oxide are a significant component of organic nitrogen aerosol in areas with high concentration of these species.     

Separation of chelating agents as copper complexes by capillary zone electrophoresis using quaternary ammonium bromides as additives in N-methylformamide

This study presents the use of quaternary ammonium bromides as additives in N-methylformamide (NMF) for the separation and quantification of chelating agents as copper complexes by capillary zone electrophoresis (CZE). The new quaternary ammonium bromides were synthesized in our laboratory and used for the first time for CZE applications performed in NMF media. The methods were developed and optimized for determination of six chelating agents (trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid (HEDTA), nitrilotriacetic acid (NTA) and triethylenetetraaminehexaacetic acid (TTHA)) as copper complexes. Among the tested electrolyte additives in NMF media (pH_app 10.2) dimethyldioctylammonium bromide (DMDOAB), dimethyldinonylammonium bromide (DMDNAB) and dimethyldidecylammonium bromide (DMDDAB), at a concentration of 20 mmol L⁻¹ improved the separation of the copper complexes. The optimized methods require only 12 min for one analysis, and the detection limits for copper complexes of DMDNAB, the best-performing additive, were ≤24 μmol L⁻¹. Relative standard deviations (R.S.D.) for migration times were ≤2.5, ≤2.1, ≤3.1% and for peak areas, ≤3.1, ≤3.0, ≤3.2% for DMDOAB, DMDNAB and DMDDAB used as additives, respectively. All three methods were successfully applied to the analysis of natural and wastewater samples. No matrix effects from these samples were observed. The interaction between quaternary ammonium bromides and copper complexes is discussed.     

Highly sensitive method for determination of N-nitrosamines using high-performance liquid chromatography with online UV irradiation and luminol chemiluminescence detection

A new method for the measurement of N-nitrosamines in part-per-trillion concentrations from water samples without preconcentration steps has been developed. This method is based on online UV irradiation after high-performance liquid chromatographic separation and subsequent luminol chemiluminescence detection without addition of an oxidant. It was confirmed that N-nitrosamines in basic aqueous solution were transformed to peroxynitrite by UV irradiation. The detection limits for this method were 1.5 ng/L, 2.9 ng/L, 3.0 ng/L, and 2.7 ng/L for N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine, respectively, at a signal-to-noise ratio of 3. The calibration graphs were linear in the range of 5–1000 ng/L for these N-nitrosamines. This method was used for the determination of N-nitrosamines in tap water, river water, and industrial plant effluent samples. The recoveries of N-nitrosodimethylamine, N-nitrosomorpholine, N-nitrosomethylethylamine, and N-nitrosopyrrolidine present in tap water sample at a concentration of 10 ng/L (mean ± standard deviation, n = 4) were (94.8 ± 2.7)%, (102.0 ± 6.9)%, (99.3 ± 3.9)%, and (102.8 ± 2.5)%, respectively. These results indicate that our proposed method can be applied satisfactorily to the determination of N-nitrosamines in water samples.     

Rapid on-line preconcentration and suppressed micro-bore ion chromatography of part per trillion levels of perchlorate in rainwater samples

The development of a rapid method for the determination of perchlorate in rain and drinking waters is presented. In the optimised method, an on-line preconcentration technique was employed utilising a 10 mm × 4.6 mm Phenomenex Onyx monolithic guard cartridge coated with (N-dodecyl-N,N-dimethylammonio)undecanoate for selective preconcentration, with subsequent elution into a fixed volume injection loop (‘heart-cut’ of the concentrator column eluate) and separation using an IonPac AS16 (250 mm × 2 mm) anion exchange column and a potassium hydroxide concentration gradient. Off-line optimisation studies showed that the coated monolith displayed near quantitative recovery up to 50 μg/L perchlorate level from standards prepared in reagent water. On-line preconcentration of perchlorate obtained detection limits down to 56 ng/L in reagent water, between 70 and 80 ng/L in rainwater samples and 2.5 μg/L in non-pretreated drinking water. After an additional sample sulphate/carbonate removal step, low ng/L perchlorate concentrations could also be observed in drinking water. The complete on-line method exhibited reproducibility for n = 10 replicate runs of R.S.D. ≤ 3% for peak height/area and R.S.D. = 0.08% for retention time. The optimised method, of 20 min total duration, was applied to the determination of perchlorate by standard addition in 10 rainwater samples and one drinking water sample. Concentrations of perchlorate present ranged from below the detection limit for four rainwater samples, with another three samples showing perchlorate present at between 70 and 100 ng/L, and one sample showing perchlorate present at 2.8 μg/L. Levels of 1.1 μg/L in the drinking water sample were also recorded.     

Simultaneous measurement of trace monoadenosine and diadenosine monophosphate in biomimicking prebiotic synthesis using high-performance liquid chromatography with ultraviolet detection and electrospray ionization mass spectrometry characterization

The method for simultaneous separation and determination of trace monoadenosine and diadenosine monophosphate (i.e. 2′-AMP, 3′-AMP, 5′-AMP and 3′-5′ ApA) in biomimicking prebiotic synthesis was developed using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and electrospray ionization mass spectrometry (ESI-MS) identification. The separation was performed on a Supelco C₁₈ column with a gradient elution (solvent A: 10 mM NH₄Ac aqueous solution; solvent B: MeOH). The flow rate was set at 1.0 ml/min. The quantitative determination was achieved by HPLC with UV detection at 260 nm. The linearity ranged from 0.5 to 100 μg/ml for each nucleotide. The limits of detection (LODs) for the four nucleotides were less than 0.30 μg/ml. The recovery ranged from 95.2 to 100.7%. The intra-day relative standard deviations (RSDs) of the retention times were between 0.7 and 1.1%. Both full-scan ESI-MS and -MS² for the four nucleotides under both positive and negative polarity were carried out and the possible cleavage pathways of them were depicted. The specific ions, [AMP + H]⁺ at m/z 348 and [ApA + H]⁺ at m/z 597, were chosen to characterize the four nucleotides in biomimicking prebiotic synthesis between N-(O,O-diisopropyl) phosphoryl amino acid (Dipp-aa) and adenosine. Using the proposed HPLC/UV/ESI-MS method, the concentration of 2′-AMP, 3′-AMP, 5′-AMP and 3′-5′ ApA in the biomimicking prebiotic synthesis samples were determined.     

A simplified approach to the determination of N-nitroso glyphosate in technical glyphosate using HPLC with post-derivatization and colorimetric detection

A simple and sensitive HPLC post-derivatization method with colorimetric detection has been developed for the determination of N-nitroso glyphosate in samples of technical glyphosate. Separation of the analyte was accomplished using an anionic exchange resin (2.50 mm × 4.00 mm i.d., 15 μm particle size, functional group: quaternary ammonium salt) with Na₂SO₄ 0.0075 M (pH 11.5) (flow rate: 1.0 mL min⁻¹) as mobile phase. After separation, the eluate was derivatized with a colorimetric reagent containing sulfanilamide 0.3% (w/v), [N-(1-naphtil)ethilendiamine] 0.03% (w/v) and HCl 4.5 M in a thermostatized bath at 95 °C. Detection was performed at 546 nm. All stages of the analytical procedure were optimized taking into account the concept of analytical minimalism: less operation times and costs; lower sample, reagents and energy consumption and minimal waste. The limit of detection (k = 3) calculated for 10 blank replicates was 0.04 mg L⁻¹ (0.8 mg kg⁻¹) in the solid sample which is lower than the maximum tolerable accepted by the Food and Agriculture Organization of the United Nations.     

Automated measurement of permeation and dissolution of propranolol HCl tablets using sequential injection analysis

Aim of this study was to automate sampling and quantification of the previously described apparatus for combined determination of dissolution and permeation through Caco-2 monolayer by means of sequential injection analysis (SIA). Native fluorescence of propranolol HCl in Krebs-Ringer buffer (KRB) was used for quantification. Sampling was done at three different locations within the apparatus at a high sampling frequency (approximately 60 h⁻¹). Injection volume delivered to the fluorescence detector was 50 μL for permeation monitoring and 25 μL for dissolution monitoring. Linear regression for 50 μL injection yielded a detection limit calculated as 0.04 μg mL⁻¹ of propranolol HCl in KRB (R² > 0.999). However, linearity for dissolution monitoring was not given for the complete range of concentrations and first order polynomial calibration was established (R² > 0.9999). To conclude, the SIA system was able to monitor simultaneously dissolution and permeation of the immediate release propranolol HCl tablets and the authors succeeded in automating the apparatus for combined measurement of dissolution and permeation. In addition, the obtained data was consistent with data obtained by manual sampling followed by HPLC analysis.     

Development and validation method for determination of fluoxetine and its main metabolite norfluoxetine by nonaqueous capillary electrophoresis in human urine

A simple, rapid and sensitive procedure using nonaqueous capillary electrophoresis (NACE) to measure fluoxetine and its main metabolite norfluoxetine has been developed and validated. Optimum separation of fluoxetine and norfluoxetine, by measuring at 230 nm, was obtained on a 60 cm × 75 μm capillary using a nonaqueous solution system of 7:3 methanol-acetonitrile containing 15 mM ammonium acetate, capillary temperature and voltage 25 °C and 25 kV, respectively and hydrodynamic injection. Paroxetine was used as internal standard. Good results were obtained for different aspects including stability of the solutions, linearity, and precision. Detection limits of 10 μg L⁻¹ were obtained for fluoxetine and its metabolite. This method has been used to determine fluoxetine and it main metabolite at clinically relevant levels in human urine. Before NACE determination, the samples were purified and enriched by means of extraction-preconcentration step with a preconditioned C₁₈ cartridge and eluting the compounds with methanol.