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.     

Simultaneous Determination of Glyphosate,Glufosinate, and Aminomethylphosphonic Acid by Capillary Electrophoresis After 9‐Fluorenylmethyl Chloroformate Derivatization

A capillary electrophoresis (CE) with UV absorption detection method is described for the simultaneous determination of glufosinate, glyphosate, and aminomethylphosphoric acid. The 9‐fluorenylmethyl chloroformate (FMOC‐Cl) was used for precolumn derivatization of the non‐absorbing herbicides. The three analytes were separated by CE in 9 min with 25 mM borate buffer at pH 9, followed by detection with a UV detector at 260 nm. We demonstrate how the detection limit can be enhanced by using acetonitrile‐salt mixtures. With acetonitrile‐salt mixtures, the limit of detection (LOD) was in the 10^?7 M range. Linearity of more than two orders of magnitude was generally obtained. Precisions of migration times and peak areas were less than 0.9% and 7.5%, respectively. The applicabilities of the method for the analysis of ground water and lake water were examined.     

Development and validation of analytical methodology using capillary electrophoresis for separation and determination of anions in rainwater

A new capillary electrophoresis (CE) procedure was developed for simultaneous determination of both organic and inorganic anions in rain water using a background electrolyte (BGE) containing 5 mM molybdate, 0.15 mM CTAH, 0.01% PVA and 5 mM Tris buffer to adjust pH at 7.9. Under optimised conditions, good repeatability (RSD for sulphate in migration time=0.36% and peak area=4.2%), low detection limit (2 ppb for chloride) and satisfactory working range (50 ppb-20 ppm for hydrodynamic injection, 10 ppb-3 ppm for electrokinetic injection for chloride) were obtained. The reliability of the CE procedure developed was established by satisfactory recovery tests and good agreement of results obtained by both the CE and ion chromatography (IC) methods. The procedure developed had been successfully applied for field monitoring of rainwater showing good repeatability and capability of detecting trace anions at ppb levels beyond the IC working range. Thus, the new CE procedure developed provides a quick, sensitive, economic and reliable method to meet the need for the simultaneous determination of both organic and inorganic anions in the acid rain monitoring programme.     

Non-aqueous capillary electrophoresis with diode array and electrospray mass spectrometric detection for the analysis of selected steroidal alkaloids in plant extracts

A capillary electrophoresis (CE) assay has been developed for the quantitation and determination of the impurity profile of the potassium channel blockers 3,4-diaminopyridine and 4-aminopyridine. The compounds were separated from related substances using a capillary of 30 cm effective length, a 50 mM phosphate buffer, pH 2.5 and an applied voltage of 25 kV. The assay was validated with respect to specificity, linearity, range, limits of quantitation and detection, precision and robustness. The method allows the detection and quantitation of impurities at the 0.05% level. The feasibility of the assay was demonstrated by analyzing a commercial sample of 3,4-diaminopyridine. All known related substances could be detected in this sample with the present CE method.     

High performance liquid chromatography and capillary electrophoresis determination of sanguinarine in biological matrices

HPLC and CE methods were employed to determine the quaternary benzo[c]phenanthridine alkaloid sanguinarine in biological matrices (rat hepatocytes, human gingival fibroblasts, feed, porcine faeces, body fluids and tissues). HPLC was carried out on a C₁₈ column using gradient elution and ion pairing techniques with 1‐heptanesulfonic acid as ion pairing agent under acidic conditions. The detection limit for fluorimetric detection at λ_ex = 327 nm and λ_em = 577 nm was 3 nM sanguinarine. CE analyses were performed in 50 mM phosphate‐Na buffer pH 2.5, with 150 mM SDS used for pre‐concentration by the sweeping effect. This experimental configuration allows injecting the total capillary length with sanguinarine sample. The detection limit for UV detection at 285 nm was 12 nM. Both methods are suitable for analysing submicromolar quantities of sanguinarine in biological materials. The HPLC method is more sensitive than CE because it uses fluorescence detection.     

Rapid and sensitive determination of phosphoamino acids in phosvitin by N-hydroxysuccinimidyl fluorescein-O-acetate derivatization and capillary zone electrophoresis with laser-induced fluorescence detection

A method was developed for the determination of phosphoamino acids by capillary zone electrophoresis-laser-induced fluorescence detection (argon ion laser, excitation at 488 nm and emission at 520 nm) using derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA). Different variables affecting the derivatization (SIFA concentration, derivatization pH, reaction temperature and reaction time) and the separation (type, pH and concentration of buffer, applied voltage and injection mode) were investigated in detail. The optimized separation conditions were 40 mM boric acid buffer (pH 9.2) for background electrolyte, 25 kV for the separation voltage, 25 degrees C for the capillary temperature and 5 s at 0.5 psi for the sample injection. Under the optimal conditions, the SIFA-labeled phosphoamino acids were fully separated within 7 min. The detection limits ranged from 0.1 to 0.3 nM, which are the lowest values reported for capillary electrophoresis (CE) methods. The proposed methodology allowed the rapid, sensitive and selective determination of phosphoamino acids in hen egg yolk phosvitin by the standard addition method. The recovery of these compounds in real sample was 94.0-103.5%. The developed method surpasses previously published CE methods in terms of detection limit, separation time, stability and simplicity of the electrophoretic procedure.     

Rapid analysis of antimicrobial metabolites monoacetylphloroglucinol and 2,4-diacetylphloroglucinol using capillary zone electrophoresis

A rapid capillary electrophoretic (CE) method was developed for the determination of phloroglucinol compounds, monoacetylphloroglucinol (MAPG) and 2,4-diacetylphloroglucinol (DAPG), in microbial supernatants of Pseudomonas fluorescens F113 over a 24-h growth cycle. Prior to electrophoretic separation, solid-phase extraction of supernatant samples on octadecylsilica for the purpose of sample cleanup is recommended. The optimum electrophoretic conditions were found to be 25 mM sodium tetraborate running buffer at pH 9.3, temperature at 25 degrees C with an applied voltage of 25 kV. The capillary was an Agilent fused-silica capillary of total length 33 cm x 50 microm inner diameter, 375 microm outer diameter, with effective length 24.5 cm. While MAPG and DAPG were monitored at selected wavelengths in the range of 214-320 nm, analysis at 214 nm was used and a CE separation time of less than 2 min was achieved. A partial method validation study was performed in accordance with European Agency for Evaluation of Medicinal Products (EMEA) guidelines. The method displayed linearity over the investigated range of 10-200 microg/mL, with limits of detection of 1.2 microg/mL for MAPG and 1.3 microg/mL for DAPG.     

Analysis of chlortetracycline and related substances by capillary zone electrophoresis: Development and validation

Summary A capillary zone electrophoresis method has been developed and validated for the analysis of chlortetracycline and related substances. The influence of the type of buffer, pH and concentration of the buffer were investigated. In all cases 1 mM EDTA was added to prevent metal ion complexation. Instrumental parameters such as capillary temperature and applied voltage were optimised. The following methods is proposed: capillary: fused silica, 44 cm (36 cm effective length), 50 μm i.d.; buffer: 120 mM sodium tetraborate including 1mM EDTA at pH 8.5; voltage: 10 kV; temperature: 25°C; detection wavelength: 280 nm. The robustness of the method has been examined by means of a full-fraction factorial design. The parameters for validation namely relative standard deviation, linearity, precision, limit of detection and limit of quantitation are also reported.     

Separation and determination of basic dyes formulated in hair care products by capillary electrophoresis

A capillary electrophoretic (CE) method for analyzing five basic dyes (Basic Red 76, Basic Brown 16, Basic Yellow 57, Basic Brown 17 and Basic Blue 99) sold under the trade name Arianor, which are commonly used in hair care products, has been established. A buffer of 100 mM acetic acid-ammonium acetate (50:50) containing 90% (v/v) methanol was employed in a fused-silica capillary of 40.0 cm x 50 microm I.D. with a bubble cell arrangement. Washing the capillary end immediately after injection was effective in preventing peak tailing of the basic dyes, which was due to their adsorption onto the outer wall of the capillary during the injection. Under these optimized conditions, acceptable results for reproducibility, limit of detection and quantitation, and linearity were obtained for the five authentic dyes tested. The recoveries of five authentic basic dyes spiked to three commercial hair care products also provided with acceptable results. This optimized CE method is useful for the analysis of mixed basic dyes in hair care products.     

Therapeutic deferoxamine and deferiprone monitoring in β‐thalassemia patients’ plasma by field‐amplified sample injection and sweeping in capillary electrophoresis

One CE method was established for detecting deferoxamine (DFO) and deferiprone (DFR) in plasma. For β‐thalassemia patients, DFO and DFR are major medicines to treat the iron overload caused by blood transfusion. Field‐amplified sample injection combined with sweeping was used for sensitivity enhancement in CE. This method was performed on an uncoated fused‐silica capillary. After liquid–liquid extraction, the plasma samples were electrokinetically injected into capillary at +10 kV for 180 s. The phosphate buffer (100 mM) containing 50 mM triethanolamine was used as the BGE (pH 6.6). Separation buffer was phosphate buffer (100 mM, pH 3.0) containing 150 mM SDS. This method showed good linearity (r ≥ 0.9960). Precision and accuracy were evaluated by the results of RSD and relative error of intrabatch and interbatch analyses, and all of the absolute values were less than 6.12%. The LODs (S/N = 3) were 200 ng/mL for DFO, and 25 ng/mL for DFR. The LOQ (S/N = 10) of DFO and DFR were 600 and 75 ng/mL, respectively. This method was applied for clinical applications of five β‐thalassemia patients.     

Use of phosphobetaine-type zwitterionic surfactant for the determination of alkali and alkaline earth metal ions and ammonium ion in human saliva by capillary electrophoresis

With conventional capillary electrophoresis (CE), it was difficult to directly analyze samples containing proteins as a result of the irreversible adsorption of proteins onto the inner surface of the capillary column. This difficulty, however, was completely overcome by adding N-dodecylphosphocholine (DPC, a phosphobetaine-type zwitterionic surfactant) to the background electrolyte (BGE). DPC made two essential contributions to the determination of common inorganic cations in the protein-containing samples: protein adsorption onto the capillary walls was completely avoided, and the resolution of the analyte cations was essentially improved. The optimal BGE for analysis of biological samples was found to be 5 mM DPC, 5 mM copper(II) acetate/10 mM ethylenediamine (pH 8). The detection limits (signal-to-noise ratio =3 and UV at 215 nm) of sodium, potassium, calcium, magnesium, and ammonium ions were 25, 31, 24, 45, and 60 micro M, respectively. These five species of the common inorganic cations in human saliva samples were detected successfully within 2 min by the proposed system with direct sample injection.     

Development and validation of an MEKC method for determination of nitrogen-containing drugs in pharmaceutical preparations

A fast and accurate micellar electrokinetic capillary chromatography method was developed for quality control of pharmaceutical preparations containing cold remedies as acetaminophen, salicylamide, caffeine, phenylephrine, pseudoephedrine, norephedrine and chlorpheniramine. The method optimization was realized on a Beckman P/ACE System MDQ instrument. The baseline separation of seven analytes was performed in an uncoated fused silica capillary internal diameter (ID)=50 microm using tris-borate (20 mM, pH=8.5) containing sodium dodecyl sulphate 30 mM BGE. On line-UV detection at 214 nm was performed and the applied voltage was 10 kV. The operating temperature was 25 degrees C. After experimental conditions optimization, the proposed method was validated. The evaluated parameters were: precision of migration time and of corrected peak area ratio, linearity range, limit of detection, limit of quantification, accuracy (recovery), ruggedness and applicability. The method was then successfully applied for the analysis of three pharmaceutical preparations containing some of the analytes listed before.     

Development and validation of a capillary electrophoresis method for the measurement of short-chain organic acids in natural rubber latex

Short-chain organic acid contents in serum of natural latex are interesting to measure and capillary electrophoresis (CE) has proved to be a good tool for their study. In the present work a method has been developed to identify the short-chain organic acids present in sera of natural rubber latex (oxalic, formic, fumaric, aconitic, succinic, malic, glutaric, citric, acetic, glycollic, propionic and quinic acids), the separation was optimised and the quantification method validated. The separation was performed on a CE system with UV detection at 200 nm. The separation was carried out with an uncoated fused-silica capillary (57 cm x 50 microm I.D.) and was operated at -10 kV potential. The separation buffers were prepared with 0.5 M H3PO4, 0.5 mM cetyltrimethylammonium bromide and pH adjusted by adding NaOH to 6.25 except for propionic acid which was better measured at pH 7.00. Validation parameters are adequate and limits of detection range from 0.005 mM to 1.6 mM. Short-chain organic acids were measured with this method in sera of three different types of latex.     

Direct automatic determination of biogenic amines in wine by flow injection-capillary electrophoresis-mass spectrometry

A capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS) method for the separation and determination of nine biogenic amines is proposed. Operational variables, such as the voltage, temperature, sheath liquid composition, flow-rate, and MS parameters, were optimized. Samples are injected in the hydrodynamic mode into a 75 cm x 50 microm ID coated capillary and separated by using 25 mM citric acid at pH 2.0. Heptylamine is used as internal standard. The experimental setup includes a flow manifold coupled to the CE system for automatic insertion of samples into the CE vials. The proposed method allows amines to be determined with limits of detection from 0.018 to 0.09 microg x mL(-1) and relative standard deviation (RSD) values from 2.4% to 5.0% (except 6.8% for histamine). The method was successfully used to determine biogenic amines in red and white wines.     

Metal ion capillary zone electrophoresis with direct UV detection: Separation of metal cyanide complexes

Mixtures of cyanide complexes of iron(III), copper(I), iron(II), nickel(II), chromium(III), mercury(II), palladium(II), silver(I), cadmium(II), zinc(II), cobalt(II), and cobalt(III) have been separated by capillary zone electrophoresis using a fused silica capillary and 20 mM phosphate buffers containing 1–2 mM sodium cyanide. The complexes were detected by direct UV absorpticn at 214 nm; detection limits are in the mid ppb range for all metals except cadmium and zinc. The different detectability of various metal cyanide complexes enables the application of the method to the analysis of complex matrices such as cyanide plating bath solutions.     

CE-electrochemiluminescence with ionic liquid for the facile separation and determination of diester-diterpenoid aconitum alkaloids in traditional Chinese herbal medicine

A CE‐electrochemiluminescence(CE‐ECL) detection system, CE/tris(2,2′‐bipyridyl) ruthenium(II)ECL with ionic liquid, was established for the determination of diester‐diterpenoid aconitum alkaloids (aconitine (AC), mesaconitine (MA) and hypaconitine (HA)) in traditional Chinese herbal medicine. Running buffer containing 25 mM borax‐20 mM 1‐ethyl‐3‐methylimidazolium tetrafluoroborate at pH 9.15 was used, which resulted in significant changes in separation and obvious enhancement in ECL intensity for AC, MA and HA with similar structures. End‐column detection was achieved in 50 mM phosphate buffer with 5 mM (pH 9.15) at applied detection voltage of 1.20 V when the distance between the Pt working electrode and outlet of capillary (50 cm×25 μm id) was set at 150 μm. One single quantitative analysis of three alkaloids was achieved at a separation voltage of 15 kV within 10 min. Moreover, two extraction processes (ethanol extraction and ethyl ether extraction after basification) were investigated. The result showed that ethanol extraction process has higher extraction efficiency than ethyl ether extraction process. Under the optimized conditions, the detection limits of AC, MA and HA were 5.62×10^?8, 2.78×10^?8 and 3.50×10^?9 mol/L (S/N=3), respectively. The method was successfully applied to determine the amounts of AC, MA and HA in the aconitum herbal samples.     

Determination of urea-derived pesticides in fruits and vegetables by solid-phase preconcentration and capillary electrophoresis

A multiresidue analytical method based on solid-phase extraction (SPE) enrichment combined with capillary electrophoresis (CE), using micellar electrokinetic capillary chromatography (MEKC), was developed to determine ten substituted urea pesticides in orange and tomato samples. Several factors such as pH, composition and concentration of the buffer, concentration of surfactant, addition of organic solvent, and working voltage were optimized to obtain the best compound separation in the shortest time. Separation can be achieved in 7 min using a micellar aqueous pH 9 buffer composed of 4 mM borate and 35 mM sodium dodecyl sulfate. After an SPE procedure, which provided a 10-fold enrichment, the limit of detection was about 0.05 mg kg(-1), which is in the order of the maximum residue limits (MRLs) established by the European Union (EU) for most of these compounds. Increasing the enrichment factor by using a larger amount of sample is difficult in oranges due to the matrix interferences, but is possible in tomatoes, which gave cleaner extracts and easily reached a 25-fold enrichment factor. The procedure involving SPE and CE provided acceptable recoveries (ranged 42-118%) and relative standard deviations (RSDs; < 19%) at levels between 0.3 and 5 mg kg(-1).     

A highly sensitive CE-UV method with dynamic coating of silica-fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions

A new highly sensitive capillary electrophoresis (CE) method applying dynamic coating and on-line stacking for the monitoring of nucleotide pyrophosphatases/phosphodiesterases (NPPs) and the screening of inhibitors was developed. NPP1 and NPP3 are membrane glycoproteins that catalyze the hydrolysis nucleotides, e.g. convert adenosine 5'-triphosphate to adenosine 5'-monophosphate (AMP) and pyrophosphate. Enzymatic reactions were performed and directly subjected to CE analysis. Since the enzymatic activity was low, standard methods were insufficient. The detection of nanomolar AMP and other nucleotides could be achieved by field-enhanced sample injection and the addition of polybrene to the running buffer. The polycationic polymer caused a dynamic coating of the silica-fused capillary, resulting in a reversed electroosmotic flow. The nucleotides migrated in the direction of the electroosmotic flow, whereas the positively charged polybrene molecules moved in the opposite direction, resulting in a narrow sample zone over a long injection time. Using this on-line sensitivity enhancement technique, a more than 70-fold enrichment was achieved for AMP (limit of detection, 46 nM) along with a short migration time (5 min) without compromising separation efficiency and peak shape. The optimized CE conditions were as follows: fused-silica capillary (30 cm effective lengthx75 mum), electrokinetic injection for 60 s, 50 mM phosphate buffer pH 6.5, 0.002% polybrene, constant current of -60 muA, UV detection at 210 nm, uridine 5'-monophosphate as the internal standard. The new method was used to study enzyme kinetics and inhibitors. It opens an easy way to determine the activities of slowly metabolizing enzymes such as NPPs, which are of considerable interest as novel drug targets.     

Determination of valproic acid (2-propylpentanoic acid) in human plasma by capillary electrophoresis with indirect UV detection

A rapid capillary zone electrophoresis method with indirect UV detection was developed and validated for the determination of valproic acid (VPA) in human plasma. The analyses were carried out under optimized conditions, using a buffer system composed of 15 mM benzoate and 0.5 mM cetyltrimethylammonium bromide at pH 6.0, and 25% v/v methanol; 2-hydroxybutyric acid was selected as the internal standard (IS). The capillary electrophoresis (CE) separation was carried out at a negative potential of 30 kV and the indirect UV detection was operated at 210 +/- 20 nm for all assays. The influence of buffer pH, ionic strength, concentration of electroosmotic flow (EOF) modifier and organic modifier on indirect signal response and migration behavior of the organic acid was investigated. Isolation of VPA from plasma was accomplished by a carefully implemented procedure using methanol as the precipitant agent. Using a high ratio of methanol to plasma for deproteinization (4:1), good absolute recovery of the analyte and satisfactory selectivity was obtained. The calibration line for VPA was linear over the 1-100 microg/mL concentration range. Sensitivity was high; in fact, the limit of detection (LOD) of VPA was 150 ng/mL and 450 ng/mL the limit of quantitation (LOQ). The results obtained analyzing real plasma samples from schizophrenic patients under polytherapy with VPA as well as antipsychotic drugs were satisfactory in terms of precision, accuracy and sensitivity.     

On-column silver substrate synthesis and surface-enhanced Raman detection in capillary electrophoresis

A new, simple, and efficient approach for on-column surface-enhanced Raman scattering (SERS) detection in capillary electrophoresis (CE) is reported. A ∼50-μm SERS substrate spot was prepared by laser-induced growth of silver particles in the 100-μm inner diameter CE capillary window or in a flow cell consisting of a 250-μm inner diameter fused silica capillary connector. For this purpose, the Raman laser was focused by a 20× objective into the detection window filled with a 0.5 mM silver nitrate and 10 mM citrate buffer solution. During the CE runs, the silver substrate spot was formed in a few seconds after the analyte injection, hence the analytes adsorbed sequentially to the silver surface when the detection window was reached, followed by desorption from the silver surface and continuing the electrophoretic migration to the capillary end. Thus, beyond migration time, valuable molecular specific information was delivered by the SERS spectra. Accurate separations and high-intensity SERS spectra are shown by CE-SERS time-dependent 3D electropherograms for the analytes rhodamine 6G, 4-(2-pyridylazo)resorcinol (PAR), PAR complex with Cu(II) and methylene blue at 0.25–25 ppm concentrations, by using 1.4–3.6 mW HeNe laser power and an acquisition time of 5 s for each spectrum. Before and after each analyte passes the detection window, clean background spectra were recorded and no memory effects perturbed the SERS detection. The silver substrate is characterized by a fast preparation rate, good reproducibility, a preparation success rate of over 95% and no mentionable influence on the electrophoretic migration time, the CE-SERS and CE-UV electropherograms being in good agreement. The successful coupling of CE and on-column SERS detection opens new perspectives for monitoring CE separations.