Separation and determination of l-tyrosine and its metabolites by capillary zone electrophoresis with a wall-jet amperometric detection

A method of capillary electrophoresis with wall-jet amperometric detection (AD) has been developed for separation and determination of l-tyrosine (Tyr) and its metabolites, such as Tyramine (TA), p-hydroxyphenylpyruvic (pHPP), homogentisic acid (HGA) and some dipeptides containing Tyr, such as Tyr-Gly-Gly (YGG), Tyr-Arg (YR) and Tyr-d-Arg (Y-d-R). A carbon disk electrode was used as the working electrode and the optimal detection potential was 1.00 V (versus Ag/AgCl). At 18 kV of applied voltage, the seven compounds were completely separated within 20 min in 110 × 10⁻³ mol/L Na₂HPO₄–NaH₂PO₄ buffer (pH 7.10) containing 3 × 10⁻³ mol/L β-cyclodextrin (β-CD). Good linear relationship was obtained for all analytes and the detection limits of seven analytes were in the range of 0.95–4.25 ng/mL. The proposed method has been applied to examine the metabolic process of l-tyrosine in rabbit's urine.     

Optimization of capillary zone electrophoresis parameters for selenium speciation

The behaviour of four biologically relevant selenium compounds (Se(VI), Se(IV), selenomethionine and selenocystine) in capillary zone electrophoresis (CZE) was investigated. Parameters which affect the separation, detection and sample introduction were investigated to improve the sensitivity of the analysis. Short-term repeatability was evaluated and detection limits were found to be in the g·l^–1 range.     

Capillary Electrophoretic Determination of Gatifloxacin from Pharmaceutical Preparation

Capillary zone electrophoresis has been used for the determination of gatifloxacin from its pharmaceutical preparation (tablets), using fused silica capillary. Separation was performed after hydrodynamic injection; the separation was achieved by applying 21 Kv voltage. Phosphate buffer solution (pH 9.5) was used as separation electrolyte. Detection was at 280 nm using a UV- detector. Under these experimental conditions the analysis takes 8 min. A linearity range for gatifloxacin was between 20.0 g mL^–1 to 60.0 g mL^–1. The method was validated and was found to be specific, precise, accurate, reproducible and robust and can be applied for the routine analysis of gatifloxacin from formulation and bulk drug.     

Kinetics and mechanism of distribution of Am(III) and Eu(III) between thenoyltrifluoroacetone-triphenylarsine oxide mixture in chloroform and aqueous nitrate medium

The kinetics of distribution of Am(III) and Eu(III) between thenoyltrifluoroacetone (HTTA) and triphenylarsine oxide (Ph₃AsO) mixture in chloroform and aqueous nitrate medium has been investigated using a stirred Lewis cell at ionic strength of 0.1M. The effect of the concentration of HTTA, Ph₃AsO, H⁺ and NO ₃ ^– on the rate of distribution of Am(III) and Eu(III) was studied. The results were interpreted by reaction mechanisms where the rate-determining steps are the parallel reactions of Am(OH)²⁺ or Eu(OH)²⁺ with one HTTA molecule and one Ph₃AsO molecule in the aqueous medium. The values at 25 °C of the rate constantk _HLL (HL=HTTA andL=Ph₃AsO) are 1.6±0.3·10⁶M^–2·s^–1 and 2.3±±0.3·10⁸M^–2·s^–1 for Am(III) and Eu(III), respectively.     

Determination of levodopa methyl ester and its metabolites in rat serum by CZE with amperometric detection

A reliable and reproducible method, capillary zone electrophoresis with amperometric detection (CZE–AD), has been developed for separation and quantification of levodopa methyl ester (LDME) and its biotransformation products levodopa (L-DOPA) and dopamine (DA) in rat serum. A carbon-disk electrode was used as working electrode. The optimum conditions for CZE detection were 50 mmol L^–1 phosphate solution at pH 7.0 as running buffer, 17 kV as separation voltage, 1.0 V (vs Ag/AgCl, 3.0 mol L^–1) as detection potential, and sample injection for 8 s at 17 kV. The linear ranges were from 2.4×10^–2 to 2.2 g mL^–1 for LDME, 2.9×10^–1 to 49.5 g mL^–1 for L-DOPA, and 1.4×10^–2 to 1.5 g mL^–1 for DA with correlation coefficients of 0.9997, 0.9994, and 0.9999, respectively. The detection limits for LDME, L-DOPA, and DA were 14.6, 98.0, and 9.7 ng mL^–1, respectively. Recoveries were 80.3% for LDME, 93.5% for L-DOPA, and 86.5% for DA. This method was applied to serum samples after intravenous injection of LDME and L-DOPA to rats.     

Capillary zone electrophoresis for simultaneous determination of seven nonsteroidal anti-inflammatory drugs in pharmaceuticals

A simple capillary zone electrophoresis (CZE) method has been developed for analyzing seven nonsteroidal anti-inflammatory drugs (NSAIDs)—sulindac (SU), ketoprofen (KE), indomethacin (IN), piroxicam (PI), nimesulide (NI), ibuprofen (IB), and naproxen (NA). The separation was run using borate buffer (60 mmol L^–1, pH 8.5) containing 13% (v/v) methanol at 20 kV, and detected at 200 nm. Several conditions were studied, including concentration and pH of borate buffer, methanol percentage, and separation voltage. In method validation, the calibration plots were linear over the range 40.0–500.0 mol L^–1. In intra-day and inter-day analysis, relative standard deviations (RSD) and relative errors (RE) were all less than 5%. The limits of detection were 10 mol L^–1 for SU, IN, PI, and 20 mol L^–1 for KE, NI, IB, NA (S/N = 3, sampling 6 s by pressure). All recoveries were greater than 95%. This method was applied to the quality control of six NSAIDs in pharmaceuticals using NI as internal standard (IS). The assay results were within the labeled amount required by USP 25.     

Capillary electrophoresis of ANTS labelled oligosaccharide ladders and complex carbohydrates

The detection limits of the ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid) label and ANTS maltose as a model carbohydrate conjugate were investigated with on-column UV and laser induced fluorescence detection. Under capillary electrophoresis conditions, the concentration and mass detection limits were found to be 5×10^–7 mol/l or 8 femtomole with UV and 5×10^–8 mol/l or 400 attomole with laser induced fluorescence detection, respectively. Including the derivatization reaction, the best concentration detection limit increases to 1×10^–6 mol/l carbohydrate. A model calculation shows that these detection levels are still insufficient to match those of current protein sequencing protocols.Derivatization conditions for dextran and polygalacturonic acid ladders are described with subsequent fast separation in a capillary electrophoresis system under acidic pH buffer conditions. Up to 30 oligomers could be separated in less than 10 min. The application of ANTS labelled carbohydrate analysis in the food industry is demonstrated with the carbohydrate fraction of sweets and the kinetic monitoring of the hydrolysis of polygalacturonic acid.The described ANTS derivitization protocol works with as little as 5 g carbohydrate as demonstrated with a complex oligosaccharide labelled in a reaction volume as little as 2 l. To demonstrate the applicability of this approach to complex carbohydrate analysis, an oligosaccharide mixture derived from human Immunoglobuline G was labelled and separated within 5 min. Separation efficiency and speed are superior to state-of-the-art chromatographic methods. Both electrophoretic and chromatographic methods are complementary because of their different separation mechanism. The implications of using capillary electrophoresis with laser induced fluorescence and appropriate labelling strategies for structural and compositional analysis of complex carbohydrates are discussed.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday     

On-Line Inhibited Chemiluminescence Detection of 2,3,5,4′-Tetrahydroxystilbene-2-O-β-D-Glucoside and Baicalin Separated by Capillary Electrophoresis

A capillary electrophoresis on-line inhibited luminol-K₃Fe(CN)₆ chemiluminescence method was employed for the determination of 2,3,5,4-tetrahydroxystilbene-2-O--D-glucoside (I) and baicalin (baicalein 7-O-glucuronide, II). In this system, luminol was added to running buffer solution and introduced at the head of the separation capillary, and K₃Fe(CN)₆ was introduced at the end of the capillary. The parameters affecting the separation process and CL detection were studied. Under the optimum conditions, the baseline separation of the two analytes was obtained within 4min. The detection limits (S/N=3) for I and II were 3.0×10^–6M and 1.2×10^–6M, respectively. This proposed method was successfully applied to the determination of II in traditional Chinese medicines.     

Determination and Differentiation of Two Seemingly Identical Medicinal Herbs by Capillary Electrophoresis with Electrochemical Detection

A high-performance capillary electrophoresis with electrochemical detection (CE-ED) method has been employed for the determination of six bioactive ingredients in traditional Chinese herbs, Herba cepbalanoplosis segeti and Herba cirsii japonici. The effects of several factors such as the acidity and concentration of running buffer, the separation voltage, the applied potential and the injection time on CE-ED were investigated. Under the optimum conditions, the six analytes could be well separated within 21 min in a 75 cm length capillary at the separation voltage of 15 kV in a 50 mmol L^–1 borax running buffer (pH 8.4). A 300 m diameter carbon disk electrode was used as the working electrode positioned carefully opposite the outlet of the capillary in a wall-jet configuration at potential of +950 mV (vs. SCE). Good linear relationship was established between peak current and concentration of analytes over two orders of magnitude with detection limits (S/N=3) ranged from 1.5×10^–7 to 6.0×10^–7 g mL^–1 for all six analytes. This proposed method has been successfully applied for the analysis of traditional Chinese herbs after a relatively simple extraction procedure, further on, for the differentiation of these above two seemingly identical herbs based on their electropherograms or characteristic electrochemical profiles.     

Application of co-electroosmotic capillary electrophoresis for the determination of inorganic anions and carboxylic acids in soil and plant extract with direct UV detection

Summary Indirect UV detection in capillary zone electrophoresis (CZE) is frequently used for the determination of inorganic anions and carboxylic acids. However, there are few reports on direct UV detection of these solutes in real samples. This paper describes the use of direct UV detection of inorganic anions and organic acids in environmental samples using co-electroosmotic capillary zone electrophoresis (co-CZE) at 185 nm. The best separation and detection of the solutes was achieved using a fused silica capillary with an electrolyte containing 25 mM phosphate, 0.5 mM tetradecyltrimethylammonium bromide (TTAB) and 15% acetonitrile (v/v) at pH 6.0. Four common inorganic anions (Cl⁻, NO₂ ⁻, NO₃ ⁻ and SO₄ ²⁻) and 11 organic acids (oxalic, formic, fumaric, tartaric, malonic, malic, citric, succinic, maleic, acetic, and lactic acid), were determined simultaneously in 15 min. Linear calibration plots for the test solutes were obtained in the range 0.02–0.5 mM with detection limits ranging from 1–9 μM depending on the analyte. The proposed method was successfully used to determine inorganic anions and carboxylic acids in soil and plant tissue extracts with direct injection of the sample.     

Experimental design-based development and single laboratory validation of a capillary zone electrophoresis method for the determination of the artificial sweetener sucralose in food matrices

A capillary zone electrophoresis (CZE) method, optimised chemometrically, underwent a complete in-house validation protocol for the qualification and quantification of sucralose in various foodstuffs. Separation from matrix components was obtained in a dinitrobenzoic acid (3 mM)/sodium hydroxide (20 mM) background electrolyte with a pH of 12.1, a potential of 0.11 kV cm^–1 and a temperature of 22 °C. Detection was achieved at 238 nm by indirect UV. Screening, optimisation and robustness testing were all carried out with the aid of experimental design. Using standard addition calibration, the CZE method has been applied to still, carbonated and alcoholic beverages, yoghurts and hard-boiled candy. The method allows the detection of sucralose at >30 mg kg^–1, with a linearity range of 50–500 mg kg^–1, making it suitable for implementation of the recently amended Sweeteners for use in foodstuffs Directive (European Parliament and Council (2003) Off J L237:3–12), which set maximum usable doses of sucralose for many foodstuffs, most ranging from 200 mg kg^–1 to 450 mg kg^–1.     

Continuous-flow separation and pre-concentration coupled on-line to solid-surface fluorescence spectroscopy for the simultaneous determination of<Emphasis Type="Italic"> o</Emphasis>-phenylphenol and thiabendazole

A novel and single flow-injection system combined with solid-surface fluorescence detection is proposed in this work for the resolution of a mixture of two widely used pesticides (o-phenylphenol and thiabendazole). The continuous-flow methodology is based on the implementation of on-line pre-concentration and separation of both analytes on the surface of C₁₈ silica gel beads placed just inside the flow cell, implemented with gel-phase fluorimetric multi-wavelength detection (using 305/358 and 250/345 nm as excitation/emission wavelengths for thiabendazole and o-phenylphenol, respectively). The separation of the pesticides was possible owing to the different retention/desorption kinetics of their interactions with the solid support in the zone where the stream impinges on the solid material. No previous separation of the analytes before they reach the flow cell is needed thereby simplifying substantially both the procedure and the manifold. By using a sample volume of 2,600 L, the system was calibrated in the range 0.5–16 and 5–120 ng mL^–1 with detection limits of 0.09 and 0.60 ng mL^–1 for thiabendazole and o-phenylphenol, respectively. The RSD values (n=10) were about 1% for both analytes. The proposed methodology was applied to environmental water samples and also to various commercial pesticide formulations containing both analytes. Recovery percentages were 97–103% and 98–102% for thiabendazole and o-phenylphenol, respectively.     

Determination of dextromethorphan and dextrorphan in urine by capillary zone electrophoresis: Application to the determination of debrisoquin-oxidation metabolic phenotype

Summary A capillary zone electrophoresis method has been developed for the determination of dextromethorphan and its metabolite, dextrorphan, in urine. A linear relationship was observed between the peak area and the concentration of both dextromethorphan and dextrorphan within the range of 490 ng mL^–1 to 500 g mL^–1 with a correlation coefficient of greater than 0.9999. The limit of detection was 80 ng mL^–1 for both compounds. The inter-day coefficients of variation for the concentrations of 2.5 g mL^–1 and 50 g mL^–1 were 6.2% and 4.1% for dextromethorphan, and 5.6% and 2.8% for dextrorphan (n=15). The method could be applied directly to the determination of dextromethorphan and dextrorphan in human urine without any sample pretreatment for the elimination of interfering compounds as is required in published highperformance liquid chromatography and gas chromatography methods. Using dextromethorphan as a probe of the debrisoquin-oxidation metabolic phenotype, the 44 healthy volunteers were phenotyped after oral administration of a 15 mg dose using both this capillary electrophoresis method and a high-performance liquid chromatography assay from the literature. Good agreement was found between the two methods.     

Determination of Levodopa and Benserazide Hydrochloride in Pharmaceutical Formulations by CZE with Amperometric Detection

A novel method, capillary electrophoresis with amperometric detection, has been established for rapid and effective measurement of levodopa (L-dopa), and benserazide (BS) and its impurity (R,S)-2-amino-3-hydroxypropanohydrazide (Ro-04-1419) in co-beneldopa pharmaceutical formulations. Suitable separation and amperometric detection conditions were investigated and optimized. The optimum conditions of CZE detection were 40 mm phosphate solution at pH 5.3 as running buffer, 17 kV separation voltage, carbon-disk working electrode, 0.95 V (relative to Ag/AgCl) as detection potential, and sample injection for 8 s at 17 kV. The linear ranges were from 1.25 to 50 g mL^–1 for L-dopa, 1.2 × 10^–1 to 25.5 g mL^–1 for BS, and 1.0 × 10^–2 to 4.4 × 10^–1 g mL^–1 for Ro-04-1419, with correlation coefficients of 0.9994, 0.9951, and 0.9933, respectively. The detection limits for L-dopa, BS, and Ro-04-1419 were 0.38, 0.02, and 0.004 g mL^–1, respectively. Average recoveries were 100.2% for L-dopa, 102.4% for BS, and 90.8% for Ro-04-1419. This method was successfully applied to co-beneldopa granules and tablets.Revised: 30 November and 22 December 2004     

Determination of rofecoxib,in the presence of its photodegradation product,in pharmaceutical preparations by micellar electrokinetic capillary chromatography

A simple and rapid micellar electrokinetic capillary chromatographic (MEKC) method for analysis of rofecoxib (ROF) and its photodegradation product (PDP) in pharmaceutical preparations has been developed and validated. Analyses were conducted in a fused silica capillary (72 cm effective length, 50 m i.d.) with a background electrolyte consisting of 25 mmol L^–1 borate buffer at pH 7.0 containing 15 mmol L^–1 sodium dodecyl sulfate (SDS) and 10% acetonitrile (ACN). The separation was performed by voltage-controlled system, applying 30 kV at 30 °C, detecting at 225 nm; injection was hydrodynamic at 50 mbar for 2 s. Nifedipine was used as internal standard (IS). Under the optimum conditions ROF, PDP, and IS were well separated with in 10 min. The method was validated with regard to linearity, limit of detection and quantitation, precision, accuracy, specificity, and robustness. The detection limit of the method was low, 0.8 g mL^–1, and the linearity range was wide, 2.5 to 125 g mL^–1. The method was highly efficient—5×10⁵ plates m^–1 for ROF. The method was applied to the tablet form of ROF-containing pharmaceutical preparations. The data were compared with those from the voltammetric method described in literature. No statistically significant difference was found.     

Sorption and separation of some elements of nuclear importance using Chelex-100

Chelex-100, in the anionic form has been studied for its ability to perform selective separation and concentration of some metal ions of nuclear importance from mineral acid solutions. The sorption behavior of Zr(IV)–Nb(V), Mo(VI), Tc(VII), Te(IV) and U(VI) from solutions of hydrochloric and sulphuric acids on Chelex-100 has been studied under static and dynamic conditions. Mo(VI) and Tc(VII) have been concentrated on the resin from hydrochloric or sulphuric acid solutions at low acidities probably, as the anions MoO ₄ ^2– and TcO ₄ ^– , respectively. Te(IV) has been isolated from hydrochloric acid solutions of normalities 6 in the form of the anionic chloro complex TeCl ₆ ^2– . Optimum conditions for elution and separation of Mo(VI), Tc(VII), Te(IV) and U(VI) were recommended.     

Determination of jasmonic acid in bark extracts from <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> by capillary electrophoresis with laser-induced fluorescence detection

A simple and sensitive method is described for determination of jasmonic acid (JA) in plant tissues. The method is based on derivatization of JA with 5-bromomethylfluorescein (5-BMF) and separation and quantification of the resulting 5-BMF–JA derivative by capillary electrophoresis coupled to laser-induced fluorescence detection (CE–LIF). The derivatization conditions were studied in detail. Our results indicated that 5-BMF-labeled JA could be well separated from other plant hormones present in the sample by use of 20 mmol L^–1 borate buffer (pH 8.5). The response to JA was a linear function of concentration in the range 1 to 100 mol L^–1, with a correlation of 0.9986. Our preliminary work showed that the proposed method had fairly good selectivity and sensitivity. Only small amounts of plant sample are needed to complete the analysis. This described method enables the analysis of JA in crude extracts without extra purification and enrichment procedures.     

Changing the Electrophoretic Mobility of Phenols Using Ionenes as Additives in the Buffer Electrolyte

Water-soluble polymers of the ionene class with increased hydrophobicity (2-10-, 6-9-, and 3-X-ionenes) are used as modifiers in capillary electrophoresis in the determination of p-nitrophenol, phenol, and resorcinol. It is shown that 2-10-ionene provides the highest selectivity of phenol separation among all of the selected ionenes. The application of this polymer increases the selectivity of determination compared to commercially available 3-6-ionene (Polybrene) and poly(diallyldimethylammonium chloride). Under optimum conditions (a phosphate buffer solution, pH 11, U = –15 kV, l _cap = 50 cm), the separation efficiency of more than 40000 theoretical plates per capillary has been achieved with an analysis time of about 8 min. Using a stacking procedure, the efficiency of more than 250000 theoretical plates per capillary has been achieved. Changes have been introduced into the design of the capillary cartridge of a Kapel'-103R instrument. As a result, it has been possible to increase the field strength up to 1180 V/cm and, thus, to decrease the analysis time down to 2.5 min. The sensitivity of the instrument is mainly determined by the quality of the equipment used. The limits of detection of phenols are about 40 g/mL for a Kapel'-103R instrument and 0.05–0.1 g/mL for a Hewlett-Packard HP^3D CE.     

Silica Gel Loaded with <Emphasis Type="Italic">o</Emphasis>-Dihydroxybenzene: Design,Metal Sorption Equilibrium Studies and Application to Metal Enrichment Prior to Determination by Flame Atomic Absorption Spectrometry

The aminopropyl silica gel (APSG) prepared by reaction of activated silica gel with 3-aminopropyltriethoxysilane on reaction with 3,4-dihydroxybenzaldehyde has resulted in a new chelating matrix, o-dihydroxybenzene (DHB) anchored silica gel, which is characterized by IR, TGA and elemental analyses. APSG is characterized with ¹³C CPMAS NMR spectroscopy. DHB anchored silica gel sorbs quantitatively (97.4–99.2% recovery) Cu(II), Pb(II), Fe(III), Zn(II), Co(II), Ni(II) and Cd(II) at pH 6.0–7.5, 5.0–7.0, 5.5–7.0, 6.0–8.0, 6.5–8.0, 5.5–7.0 and 6.5–7.5, respectively. The sorption capacity varies from 32 to 348µmolg^–1 and is highest for copper. Desorption was found to be quantitative with 1.0–3.0molL^–1 HCl/HNO₃ (for Pb). The optimum flow rate of the solution for quantitative sorption of metal ions on a column (10cm×10mm) packed with 1g of the modified silica gel is 1.0–4.0mLmin^–1, whereas for desorption it is 2.0 –4.0mLmin^–1. The tolerance limits for NaCl, NaBr, NaI, NaNO₃, Na₂SO₄, Na₃PO₄, humic acid, EDTA, ascorbic acid, citric acid, sodium tartrate, Ca(II) and Mg(II) in the sorption of all the seven metal ions are reported. The preconcentration factors are between 100 and 300 and t_1/2 values 17min. The present matrix coupled with FAAS has been used to enrich and determine the seven metal ions in river and tap water samples (RSD 1.4–7.0%) and in synthetic certified water samples SLRS-4 (NRC, Canada) with an RSD of 2.73–2.83%. The cobalt present in pharmaceutical vitamin tablets and Zn in milk powder was preconcentrated on DHB anchored silica gel and determined by FAAS (RSD of 2.00 to 2.72%).     

Solid phase extraction for sample preparation in trace analysis of ionogenic compounds by capillary isotachophoresis

Various sorbents recommended for solid phase extraction (SPE) in sample preparation procedures were studied for use in combination with capillary isotachophoresis (ITP). They were very efficient in achieving trace concentration levels (low ppb, i.e., low parts per 10⁹) for different types of ITP analytes present in environmental and biological matrices. A macroporous carbon sorbent was convenient for sample preparation in ITP analysis of short chain fatty acids (C₄–C₉) in drinking water. Chelating sorbents based on hydroxyalkyl methacrylate matrix with salicylate, thioglycolate and 8-hydroxyquinolinate functionalities were found to be very suitable for preconcentration of heavy metals with an inherent sample clean-up. An octadecyl-bonded silica sorbent enabled in ITP a photometric detection of -aminobutyrate (labeled with a 2,4,6-trinitrophenyl group) at concentrations considerably lower than required for the determination of this amino acid in cerebrospinal fluid (5·10^–8 mol/l).