Solid-phase extraction and sample stacking-capillary electrophoresis for the determination of quaternary ammonium herbicides in drinking water

Conditions for the simultaneous determination of paraquat, diquat and difenzoquat by capillary zone electrophoresis were established by combining two preconcentration procedures. Off-line solid-phase extraction was used for the isolation and preconcentration of quats in drinking water. Quats were then analysed by capillary electrophoresis using sample stacking with matrix removal as on-column preconcentration procedure. Two different porous graphitic carbon cartridges were compared. The breakthrough volumes of the three herbicides were calculated and the loading capacity of the sorbents was compared. Recoveries higher than 80% for difenzoquat and around 40% for paraquat and diquat were obtained when a sample volume of 250 ml was percolated. For the stacking-capillary electrophoresis analysis of quats, 50 mM acetic acid-ammonium acetate (pH 4.0), 0.8 mM cetyltrimethylammonium bromide with 5% (v/v) methanol as carrier electrolyte was used. Detection limits, based on a signal-to-noise ratio of 3:1, were lower than 0.3 microg l(-1) for standards in Milli-Q water, and lower than 2.2 microg l(-1) for drinking water samples. Run-to-run and day-to-day precision of the method were established. The two preconcentration procedures used together was successfully applied to the analysis of the three herbicides in spiked drinking water at concentrations below the maximum admissible US Environmental Protection Agency levels.     

Improved capillary electrophoretic separation of nine (fluoro)quinolones with fluorescence detection for biological and environmental samples

A capillary electrophoretic method has been developed for the separation of nine (fluoro)quinolones. Detection is done by fluorescence measurement with broad wavelength band excitation between 240 and 400 nm. Best separation is achieved in a carrier electrolyte containing 50 mM H3PO4 adjusted to pH 7.55-acetonitrile (60:40, v/v). Detection limits are in the low microgl(-1) range. The suitability to real samples has been demonstrated by analyzing blood samples and surface water samples. Sample preconcentration and sample clean-up can easily be done by solid-phase extraction. Different phases based on alkyl- or phenyl-modified silica as well as on polymers have been investigated for this purpose. The method should also be useful for determination of residues of (fluoro)quinolones in food or other matrices.     

Capillary electrophoresis-acid barrage stacking online enrichment method for highly sensitive determination of four isoflavones

A capillary electrophoresis-acid barrage stacking online enrichment method has been established to detect the four isoflavones which are Daidzein, Genistein, Formononetin, and Biochanin A. The proposed method was optimized using a single factor alternative method, and the optimal conditions obtained from the optimization were: the BGE was 25 mM borax and 2 mM β-cyclodextrin, the applied separation voltage was 20 kV, and the detection wavelength was 260 nm. The time ratio of the injection of sample and the injection of acid was 150 s:20 s, and the acid used was 250 mM acetic acid. The sample solvent used was 60% v/v acetonitrile. The established method had the enrichment factor of these four isoflavones at 24.5, 32.0, 29.2, and 33.7, respectively, LOD and LOQ are as low as nanograms per milliliter. Finally, the CE-acid barrage stacking method was successfully applied to the determination of four isoflavones in rat plasma and red clover extract, verifying the applicability and feasibility of the method.     

Determination of pesticides in drinking water by micellar electrokinetic capillary chromatography

A new analytical procedure using a two-step sample preconcentration (solid-phase extraction (SPE) and field-amplified sample stacking) prior to separation by micellar electrokinetic capillary chromatography was developed for the determination of 14 pesticides such as aldicarb, carbofuran, isoproturon, chlorotoluron, metolachlor, mecoprop, dichlorprop, MCPA, 2,4-D, methoxychlor, TDE, DDT, dieldrin, and DDE in drinking water. Good recoveries of pesticides were obtained using SPE with sample pH adjusted to 2-3. Field-amplified sample stacking was found to give enrichment factors up to 30-fold preconcentration of various pesticides under reversed polarity at -2 kV for 50 s. The optimized background electrolyte (BGE) consisted of 50 mM sodium dodecyl sulfate (SDS), 10 mM borate buffer, 15 mM beta-cyclodextrin (beta-CD), and 22% acetonitrile at pH 9.6, running was under 25 kV and detection at 202 nm. Good linearity was obtained for all pesticides with detection limits down to 0.04-0.46 ng/mL and a working range of 0.1-40 ng/mL. The repeatabilities of migration time and peak area were satisfactory with relative standard deviations (RSDs) between 0.66 and 13.6% and 4.1 and 28%, respectively. All pesticides except dieldrin were found to be detected at concentrations at least tenfold lower than the World Health Organization (WHO) guideline values. The analytical procedure developed offers an economic method for fast screening of multiple pesticide residues in drinking water for health protection. It had been applied to determine carbofuran and MCPA in agricultural run-off water samples, giving satisfactory repeatabilities of 10 and 12%, respectively, with n=5 for the determination of pesticides in contaminated water samples.     

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.     

SPE and large-volume sample stacking in MEKC for determination of doxycycline in biological fluids: comparison of direct injection to SPE-MEKC

A novel and simple method has been developed for the determination of doxycycline (DOX) in biological fluids. The method is based on SPE, large-volume sample stacking (LVSS) and MEKC with UV-DAD detection. Six SPE cartridges have been used in investigation for sample clean up and pre-concentration (Supelco LC-8, LC-18, LC-SCX, and LC-WCX, as well as Strata-X and X-C). DOX was determined on a 56 cm (effective length 50 cm) x 50 microm id fused-silica capillary. The BGE was 20 mM borate buffer, pH 9.3, containing 80 mM SDS and 7.5% v/v of methanol (30 sx50 mbar), and the temperature and voltage were 25 degrees C and 30 kV, respectively. The analytical wavelength was set at 210 nm. Under optimized conditions it is possible to determine DOX in human serum, urine, semen, tears and saliva with recovery of 97.5% (RSD 2.5%). The method was shown to be sensitive (LOD is 1 microg/L) and precise (intra-day RSD 0.2 and 2.4%; inter-days 0.4 and 3.5% for migration time and peak area, respectively). Results for developed SPE-LVSS-MEKC were compared with LVSS-MEKC method with direct sample injection. The new LVSS-MEKC method is presented as a useful technique for rapid determination without extraction procedure of DOX in human urine and serum, using 80 mM of SDS, 10% v/v of methanol and 40 mM borate buffer (pH 9.3; 30 s x 50 mbar; 25 degrees C; 30 kV; 350 nm), but not for the other biological fluids, according to lower sensitivity of the method and because of the sample composition.     

Sample stacking microemulsion electrokinetic capillary chromatography induced by reverse migrating pseudostationary phase for the quantification of phenobarbital and its p-hydroxyphenobarbital metabolite in rat urine

For the first time, a capillary electrophoretic (CE) method with sample stacking induced by a reverse migrating pseudostationary phase (SRMP) technique has been developed and validated for sensitive determination of phenobarbital (PB) and its p-hydroxyphenobarbital (PHPB) metabolite in rat urine samples. Separation and determination were optimized on a fused-silica capillary with a total length of 50 cm (effective length 40 cm) and 75 μm ID. The microemulsion background electrolyte consisted of 0.8% (v/v) ethyl acetate, 6.6% (v/v) butan-2-ol, 1.0% (v/v) acetonitrile, 2.0% (w/v) sodium n-dodecyl sulfate (SDS), and 89.6% (v/v) of 7.5 mM ammonium formate at pH 8. When this preconcentration technique was used, the sample stacking and the separation processes took place successively with changing the voltage with an intermediate polarity switching step. For practical application, a solid-phase extraction (SPE), C(18) sorbent with n-hexane/ethyl acetate (1?:?1%, v/v) as the elution solvent was used for sample purification and concentration. The SPE method gave good extraction yields for all the analytes, with absolute recovery values of 96.9% and 99.1% for PB and PHPB, respectively. The regression equations for PB and PHPB showed excellent linearity over a concentration range of 55-1386 ng mL(-1) for PB and PHPB (r = 0.998). The developed microemulsion electrokinetic capillary chromatography (MEEKC) method for separation of the studied compounds with SRMP as the electrophoretic preconcentration technique allowed detection limits in urine samples at 16.8 ng mL(-1) for PB and PHPB which are 15-fold lower than the reported CE method in the literature. The precision results, expressed by the intra-day and inter-day relative standard deviation (RSD) values range from 3.6 to 7.1% (repeatability) and from 3.2 to 7.2% (intermediate precision) for PB and PHPB, respectively, which were in line with Food and Drug Administration (FDA) criteria.     

Sensitive analysis of donepezil in plasma by capillary electrophoresis combining on-column field-amplified sample stacking and its application in Alzheimer's disease

Field-amplified sample stacking (FASS) in capillary electrophoresis (CE) was used to determine the concentration of donepezil, an acetylcholinesterase inhibitor, in human plasma. A sample pretreatment by liquid-liquid extraction with isopropanol/n-hexane (v/v 3:97) and subsequent quantification by FASS-CE was used. Before sample loading, a water plug (0.5 psi, 6 s) was injected to permit FASS. Electrokinetic injection (7 kV, 90 s) was used to introduce sample cations. The separation condition for donepezil was performed in electrolyte solutions containing Tris buffer (60 mM, pH 4.0) with sodium octanesulfonate 40 mM and 0.01% polyvinyl alcohol as a dynamic coating to reduce analytes' interaction with capillary wall. The separation was performed at 28 kV and detected at 200 nm. Using atenolol as an internal standard, the linear ranges of the method for the determination of donepezil in human plasma were over a range of 1-50 ng/mL. The limit of detection was 0.1 ng/mL (S/N=3, sampling 90 s at 7 kV). One female volunteer (54 years old) was orally administered a single dose of 10 mg donepezil (Aricept, Eisai), and blood samples were drawn over a 60 h period for pharmacokinetic study. The method was also applied successfully to monitor donepezil in sixteen Alzheimer's disease patients' plasmas.     

毛细管电泳-场强放大样品堆积法检测染发剂中的7种苯胺类物质

建立了毛细管电泳-场强放大样品堆积测定染发剂中4,4′-二氨基二苯甲烷、苯胺、邻甲氧基苯胺、对氨基苯甲醚、3,4-二甲基苯胺、间氨基苯酚、1-萘胺7种苯胺类物质的分析方法。在优化的缓冲溶液体系(0.15 mol/L NaH2PO4,0.015 mol/L 三乙醇胺, pH 2.3)下7种分析物在6.5 min内实现基线分离。考察了样品中添加的磷酸浓度和乙腈浓度、水柱长度、电动进样时间与电压对场强放大富集效率及重现性的影响。最佳的富集条件为: 水柱注入3.45 kPa(0.5 psi)×6 s,样品中添加40%(v/v)乙腈和0.6×10~3mol/L磷酸,进样电压与进样时间为10 kV×10 s。线性范围为3～1000 μg/L(R2＞0.996),检出限为0.26～2.75 μg/L,将已有方法的检测灵敏度提高了1～3个数量级。在2种市售黑色染发剂中均检测到间氨基苯酚,含量分别为7.32 mg/g和1.34 mg/g。平均加标回收率为74%～108%。该方法灵敏度高、快速、重现性好、成本低,可供多种样品基质中痕量苯胺类污染物及其他阳离子物质的测定借鉴使用。     

Online preconcentration and determination of chondroitin sulfate,dermatan sulfate and hyaluronic acid in biological and cosmetic samples using capillary electrophoresis

Capillary electrophoresis with large‐volume sample stacking using an electroosmotic flow pump was developed for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid. Central composite design was used to simultaneously optimize the parameters for capillary electrophoresis separation. The optimized capillary electrophoresis conditions were 200 mM sodium dihydrogen phosphate, 200 mM butylamine, and 0.5% w/v polyethylene glycol as a background electrolyte, pH 4 and ‐16 kV. Exploiting large‐volume sample stacking using an electroosmotic flow pump, the sensitivity of the proposed capillary electrophoresis system coupled with UV detection was significantly improved with limits of detection of 3, 5, 1 mg/L for chondroitin sulfate, dermatan sulfate, and hyaluronic acid, respectively. The developed method was applied to the determination of chondroitin sulfate and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic products, and supplementary samples with highly acceptable accuracy and precision. Therefore, the proposed capillary electrophoresis approach was found to be simple, rapid, and reliable for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronic acid in cell culture media, cerebrospinal fluid, cosmetic, and supplementary samples without sample pretreatment.     

Field-amplified sample stacking and nonaqueous capillary electrophoresis determination of complex mixtures of polar aromatic sulfonates

Nonaqueous CE and field-amplified sample stacking have been used in the determination of complex mixtures of polar aromatic sulfonates (AS; mainly benzene- and naphthalenesulfonates) of environmental concern. The analytical procedure consists of an on-column aqueous sample enrichment, followed by the nonaqueous electrophoretic determination of stacked aromatic sulfonates. Various organic solvents were used as separation medium, acetonitrile and N-methylformamide gave the best results. Optimum capillary electrophoresis separation is obtained with ammonium acetate (25 mM) dissolved in N-methylformamide-methanol (90:10) as background electrolyte. This combined method was applied to the analysis of surface water samples spiked with selected aromatic sulfonates derivatives.     

Nonaqueous capillary electrophoresis method for the analysis of gleevec and its main metabolite in human urine

The viability of nonaqueous capillary electrophoresis (NACE) was investigated for determination of gleevec and its main metabolite in human urine using a fused-silica capillary. Baseline separation of the studied solutes was obtained using a nonaqueous solution composed of 12 mM ammonium acetate and 87.6 mM acetic acid in methanol-acetonitrile (ACN) (80:20, v:v) providing analysis time shorter than 3 min. Different aspects including stability of the solutions, linearity, accuracy and precision were studied in order to validate the method in the urine matrix. Detection limits of 24 microg L(-1) for gleevec and its metabolite were obtained. A robustness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments. The developed method is simple, rapid and sensitive and has been used to determine gleveec and its metabolite at clinically relevant levels in human urine. Before NACE determination, a solid-phase extraction (SPE) procedure with a C18 cartridge was necessary. Real determination of these analytes in two patient urines were done.     

Determination of chlorophenoxy acid herbicides by capillary electrophoresis with integrated potential gradient detection

This report describes separation and detection of chlorophenoxy acid herbicides spiked in drinking water by the technique combining solid-phase extraction, field-amplified sample stacking, capillary electrophoresis, and potential gradient detection. The herbicide solution (400 mL) was concentrated to 0.1 mL by the solid-phase extraction procedure. The buffer containing 3 mM ammonia and 0.3 mM hydroxypropyl-beta-cyclodextrin was adjusted to pH 9.0 with ammonia. The sample solution was injected into the capillary to 30% of the whole length, and -9 kV and 9 kV were employed for field-amplified sample stacking and separation, respectively. The herbicides were baseline separated and the detection limits with the above combined techniques were in the range of 1-4 x 10(-2) ng/mL.     

Analysis of cytokinin nucleotides in coconut (Cocos nucifera L.) water using capillary zone electrophoresis-tandem mass spectrometry after solid-phase extraction

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is described for the separation and determination of six cytokinin nucleotides in coconut water. The best CZE separation for the six cytokinin nucleotide standards was achieved using a 25 mM ammonium formate/formic acid buffer (pH 3.8) and 2% (v/v) methanol with an applied gradient separation voltage (25 kV for 32 min, and then a linear gradient to 30 kV in 5 min, finally 30 kV to the end of separation) in less than 60 min. MS/MS with multiple reaction monitoring (MRM) detection was carried out to obtain sufficient selectivity and sensitivity for the cytokinin nucleotides. The combined use of on-line sample stacking and CZE-MS/MS achieved limits of detection (LODs) in the range of 0.06-0.19 microM for the six cytokinin nucleotides at a signal-to-noise ratio of 3. Furthermore, a novel dual-step SPE procedure was developed for the pre-concentration and purification of cytokinin nucleotides using Oasis HLB and Oasis MAX cartridges. The recoveries of the cytokinin nucleotides after the dual-step SPE were in the range of 44-71%. The combination of off-line SPE, on-line sample stacking and CZE-MS/MS approach was successfully applied to screen for endogenous cytokinin nucleotides present in coconut water sample. trans-Zeatin riboside-5'-monophosphate (ZMP) was detected and quantified in coconut water by CZE-MS/MS after SPE and on-line sample stacking.     

Nonaqueous capillary electrophoresis for rapid and sensitive determination of fangchinoline and tetrandrine in radix Stephaniae tetrandrae and its medicinal preparations

A simple, rapid, and sensitive non-aqueous capillary electrophoresis procedure with head-column field-amplified sample stacking concentration for the analysis of fangchinoline and tetrandrine is established. Optimum separation and stacking conditions were obtained when the sample was injected at 8 kV for 50 s after preliminary pressure injection of ethanol (16.9 kPa) for 0.6 s and separated with the buffer containing 50 mM ammonium acetate, 0.5% (v/v) acetic acid, and 50% (v/v) acetonitrile in methanol medium at 24 kV applied voltage. The analytes were detected by UV at 214 nm. The two bisbenzylisoquinoline alkaloids can be separated within 6 min and quantified with high sensitivity. The detection limits were 0.30 ng mL(-1) for fangchinoline and 0.34 ng mL(-1) for tetrandrine, which indicated that the sensitivities were at least 1000-fold enhanced over those reported in the literature as obtained by UV detection. The method was applied to the analysis of fangchinoline and tetrandrine in Radix Stephaniae tetrandrae and its medicinal preparations with good results.     

Analysis of naphthalenesulfonate compounds by cyclodextrin-mediated capillary electrophoresis with sample stacking

This study systematically investigates the optimal conditions for analyzing the positional isomers of multi-charged naphthalenesulfonate compounds by cyclodextrin-mediated capillary electrophoresis (CE). Specifically, this work employs large-volume sample injection with the electrode polarity switching technique. The most effective separation and sample stacking conditions were 15 mM borate buffer with a mixture of beta- and gamma-cyclodextrin (concentration ratio 3:7 mM) at pH 9.2, and the sample hydrodynamic injection of up to 60 s at 3 p.s.i. (around 1.8 microl, and 1 p.s.i. = 6.9 kPa). Significantly selective and sensitive improvements were observed and a more than 100-fold enrichment was achieved (based on peak area). The reproducibility of migration time and quantitative results of stacking CE can be improved by using an internal standard. The quantitation limits of these naphthalenesulfonate isomers, based on a signal-to-noise ratio above 10, can be about 4 microg/l with UV detection. This method was successfully applied to determine the trace amount of naphthalenesulfonate isomers in a spiked drinking water sample.     

Different sample stacking strategies to analyse some nonsteroidal anti-inflammatory drugs by micellar electrokinetic capillary chromatography in mineral waters

Three on-column preconcentration techniques were compared to analyse a group of nonsteroidal anti-inflammatory drugs (NSAIDs) using micellar electrokinetic capillary chromatography (MEKC) under pH-suppressed electroosmotic flow (EOF) in water samples. The analysed drugs were ibuprofen, fenoprofen, naproxen, ketoprofen, and diclofenac sodium. The micellar background electrolyte (BGE) solution was formed by 75 mM sodium dodecyl sulfate (SDS), 40% (v/v) acetonitrile, and 25 mM sodium phosphate at pH 2.5. When this BGE solution was used the applied voltage was reversed, -10 kV, and the drugs were separated within 20 min. The on-column preconcentration modes, characterised all of them for the sample matrix removal out of the capillary by itself under a reverse potential at the same time as the EOF was reduced, were stacking with reverse migrating micelles (SRMM), stacking with reverse migrating micelles-anion selective exhaustive injection (SRMM-ASEI), and field-enhanced sample injection with reverse migrating micelles (FESI-RMM). The sensitivity was improved up to 154-, 263-, and 63-fold, respectively when it was calculated through the peaks height. The optimised methods were validated with spiked mineral water by combining off-line solid-phase extraction (SPE) and the proposed on-line sample stacking strategies. The detection limits (LODs) of NSAIDs in mineral water were at ng/L levels.     

Pressure-assisted electrokinetic injection for on-line enrichment in capillary electrophoresis-mass spectrometry: a sensitive method for measurement of ten haloacetic acids in drinking water

Haloacetic acids (HAAs) are by-products of the chlorination of drinking water containing natural organic matter and bromide. A simple and sensitive method has been developed for determination of ten HAAs in drinking water. The pressure-assisted electrokinetic injection (PAEKI), an on-line enrichment technique, was employed to introduce the sample into a capillary electrophoresis (CE)–electrospray ionization–tandem mass spectrometry system (ESI-MS/MS). HAAs were monitored in selected reaction monitoring mode. With 3 min of PAEKI time, the ten major HAAs (HAA10) in drinking water were enriched up to 20,000-fold into the capillary without compromising resolution. A simple solid phase clean-up method has been developed to eliminate the influence of ionic matrices from drinking water on PAEKI. Under conditions optimized for mass spectrometry, PAEKI and capillary electrophoresis, detection limits defined as three times ratio of signal to noise have been achieved in a range of 0.013–0.12 μg L⁻¹ for ten HAAs in water sample. The overall recoveries for all ten HAAs in drinking water samples were between 76 and 125%. Six HAAs including monochloro- (MCAA), dichloro- (DCAA), trichloro- (TCAA), monobromo- (MBAA), bromochloro- (BCAA), and bromodichloroacetic acids (BDCAA) were found in tap water samples collected.     

Online transient micellar phase concentration of anions using CTAB in CE

A transient micellar phase extractor using CTAB was described for the online sample concentration of various anionic analytes (drugs and herbicides) in CE. Stacking and separation was performed at neutral pH in coelectroosmotic flow in a hexadimethrine bromide coated fused‐silica capillary. A micellar plug (e.g. 10 mM CTAB) was injected prior to hydrodynamic injection of the analytes prepared in aqueous organic solvent (e.g. with 30% ACN). In the presence of an electric field, the micelles interacted with the anions inside the capillary. This was followed by selective analyte focusing via the mechanism of micelle to solvent stacking. The micelles acted as transient extractor because the stacking ends when the injected micelles completely migrated through the boundary between the sample and micellar plug. Fundamental studies were performed (effect of surfactant concentration, etc.) and the technique yielded 13‐ to 30‐fold improvements in peak height. A stacking CE method in conjunction with liquid–liquid extraction was also tested for the detection of the herbicides fenoprop and mecoprop in fortified drinking water at analyte concentration levels relevant to Australian Drinking Water Guidelines.     

Optimization of dynamic pH junction for the sensitive determination of amino acids in urine by capillary electrophoresis

A capillary electrophoresis method with UV-absorbance detection was studied and optimized for the determination of underivatized amino acids in urine. To improve concentration sensitivity the utility of in-capillary analyte stacking via dynamic pH junction was investigated with phenylalanine (Phe) and tyrosine (Tyr) as model amino acids. Before sample injection, a plug of ammonium hydroxide solution was injected to enable analyte concentration. Samples were 1:1 (v/v) mixed with background electrolyte (1 M formic acid) prior to injection. The effect of the injected sample volume, and the injected ammonium hydroxide volume and concentration on analyte stacking and separation performance was investigated. The optimal volume of ammonium hydroxide depended on the injected sample volume. Using a dynamic pH junction good resolution (1.4) was obtained for a sample injection volume of 10% of the capillary (196 nl) with Phe and Tyr dissolved in water. Limits of detection (LODs) were 0.036 and 0.049 μM for Phe and Tyr, respectively. For urine samples, the optimized procedure comprised a 1.7-nl injection of 12.5% ammonium hydroxide, followed by a 196-nl injection of urine spiked with Phe and Tyr. Satisfactory resolution was obtained and amino acid peak widths at half height were only 1.6 s indicating efficient stacking. Calibration plots for Phe and Tyr in urine showed good linearity (R(2) > 0.96) in the concentration range 10-175 μM, and LODs for Phe and Tyr were 0.054 and 0.019 μM, respectively. RSDs for peak area and migration time for Phe and Tyr were below 7.5% and 0.75%, respectively.