Simultaneous determination of two active ingredients in Flos daturae by capillary electrophoresis with electrochemiluminescence detection

The coupling of Ru(bpy)₃²⁺ based electrochemiluminescence (ECL) detection with capillary electrophoresis (CE) was developed for the simultaneous determination of the two major active ingredients (atropine and scopolamine) in Flos daturae. Parameters related to the separation and detection were discussed and optimized. It was proved that 20 mM phosphate buffer at pH 8.48 could achieve the most favorable resolution, and the high sensitivity of detection was obtained by maintaining the detection potential at 1.2 V. Under the optimized conditions: ECL detection at 1.2 V, 20 mM phosphate buffer at pH 8.48, 5 mM Ru(bpy)₃²⁺ and 50 mM phosphate buffer at pH 7.48 in the detection reservoir, detection limits of 5 × 10⁻⁸ mol/l for atropine and 1 × 10⁻⁶ mol/l for scopolamine were obtained. Relative standard derivations of the ECL intensity and the migration time were 5.16 and 0.71% for atropine and 5.07 and 1.22% for scopolamine, respectively. Developed method was successfully applied to determine the amounts of both alkaloids in Flos daturae. A baseline separation for atropine and scopolamine was achieved within 11 min.     

Simultaneous determination of atropine,scopolamine, and anisodamine in Flos daturae by capillary electrophoresis using a capillary coated by graphene oxide

A novel CE method was developed for the separation and determination of three main tropane alkaloids in Flos daturae with a capillary coated by graphene oxide (GO). The GO‐coated capillary was characterized by SEM, energy dispersive X‐ray spectroscopy, and Raman spectroscopy, and the results indicated that the inner surface of the capillary was partially coated by GO. A phosphate solution (40 mM, pH7.0) containing 20% v/v methanol and 30% v/v acetonitrile was used as the running buffer for the analysis of the atropine, scopolamine, and anisodamine. The linear ranges of atropine, scopolamine, and anisodamine was 0.5–200 μg/mL with satisfactory correlation coefficients (R²) > 0.9987, and this novel method provided an efficient separation for three tropane alkaloids as well as a good reproducibility and stability. Finally, the method was successfully applied for the determination of these three tropane alkaloids in plant extracts.     

Simultaneous Electrochemiluminescence Detection of Anisodamine,Atropine, and Scopolamine in Flos daturae by Capillary Electrophoresis Using β‐Cyclodextrin as Additive

This work developed a simple and sensitive method for simultaneous determination of three effective ingredients, atropine, scopolamine and anisodamine, in Flos daturae based on capillary electrophoresis coupled with electrochemiluminescence detection. β‐Cyclodextrin was used as an additive to the running buffer for obtaining the absolute separation. The proposed method displayed the linear ranges from 0.2 to 100, 0.2 to 100 and 20 to 200 μM for anisodamine, atropine and scopolamine with correlation coefficients more than 0.99, respectively. This method showed the relative standard deviations less than 4% and 6% for detection of migration time and peak height, respectively, and was suitable for the determination of these tropane alkaloids in plants and valuable in clinical and biochemical laboratories for quality control.     

On-line stacking and sweeping capillary electrophoresis for detecting heroin metabolites in human urine

Heroin metabolites including morphine, codeine, and 6-acetylmorphine were determined by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI–sweep-MEKC). Liquid–liquid extraction was used for urine pretreatment. An uncoated fused silica capillary (Ld = 30 cm, 50 μm ID) was filled with phosphate buffer (50 mM, pH 2.5) containing 30% methanol, then high conductivity buffer (100 mM phosphate, 41.3 kPa for 18 s) was followed. Samples were injected electrokinetically (20 kV, 300 s). The sweeping and separation were performed at −25 kV using phosphate buffer (20 mM, pH 2.5) and 80 mM sodium dodecyl sulfate. The baseline separation was done within 10 min. During method validation, the calibration curves were linear over a range of 50–500 ng/mL (r ≧ 0.994). The RSD and RE values in intra-day and inter-day assays were all below 20%, which showed good precision and accuracy. Their detection limits were 10 ng/mL (S/N = 3). The optimized method was applied to determine real urine samples from addicts. These samples were confirmed by liquid chromatography/mass spectrometry.     

Simultaneous determination of l-ascorbic acid, ascorbic acid-2-phosphate magnesium salt, and ascorbic acid-6-palmitate in commercial cosmetics by micellar electrokinetic capillary electrophoresis

l-Ascorbic acid (LAA) can be used as a whitening agent in cosmetics. Because of its instability, some more stable derivatives have been developed to control melanin production, such as ascorbic acid-2-phosphate magnesium salt (AAPM) and ascorbic acid-6-palmitate (AA6P). To assess the quality of cosmetics, a micellar electrokinetic capillary electrophoresis technique (MEKC) was established for simultaneous analysis of AA and its two derivatives. Separation was performed with 10 mM borate (pH 9.5) containing 50 mM sodium dodecyl sulfate (SDS) at 20 kV. The detection wavelength was 265 nm. Several parameters, including borate concentration, buffer pH, and SDS level, were investigated. On method validation, calibration curves were linear over a concentration range of 150.0-1000.0 μM for LAA and 200.0-1000.0 μM for AAPM and AA6P. For intraday and interday analysis, relative standard deviation and relative errors were all less than 3%. Limits of detection were 70 μM for AAPM and AA6P, and 50 μM for LAA. All recoveries were greater than 95%. This method was applied to quality control of commercial cosmetics.     

Dynamic supported liquid membrane tip extraction of glyphosate and aminomethylphosphonic acid followed by capillary electrophoresis with contactless conductivity detection

A dynamic supported liquid membrane tip extraction (SLMTE) procedure for the effective extraction and preconcentration of glyphosate (GLYP) and its metabolite aminomethylphosphonic acid (AMPA) in water has been investigated. The SLMTE procedure was performed in a semi-automated dynamic mode and demonstrated a greater performance against a static extraction. Several important extraction parameters such as donor phase pH, cationic carrier concentration, type of membrane solvent, type of acceptor stripping phase, agitation and extraction time were comprehensively optimized. A solution of Aliquat-336, a cationic carrier, in dihexyl ether was selected as the supported liquid incorporated into the membrane phase. Quantification of GLYP and AMPA was carried out using capillary electrophoresis with contactless conductivity detection. An electrolyte solution consisting of 12 mM histidine (His), 8 mM 2-(N-morpholino)ethanesulfonic acid (MES), 75 μM cetyltrimethylammonium bromide (CTAB), 3% methanol, pH 6.3, was used as running buffer. Under the optimum extraction conditions, the method showed good linearity in the range of 0.01–200 μg/L (GLYP) and 0.1–400 μg/L (AMPA), acceptable reproducibility (RSD 5–7%, n = 5), low limits of detection of 0.005 μg/L for GLYP and 0.06 μg/L for AMPA, and satisfactory relative recoveries (90–94%). Due to the low cost, the SLMTE device was disposed after each run which additionally eliminated the possibility of carry-over between runs. The validated method was tested for the analysis of both analytes in spiked tap water and river water with good success.     

Determination of sinomenine in Sinomenium acutum by capillary electrophoresis with electrochemiluminescence detection

A Ru(bpy)₃²⁺-based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) has been established for the determination of sinomenine for the first time. Optimum separation was achieved with a fused-silica capillary column (50 cm × 25 μm i.d.) and a background electrolyte of 50 mM sodium phosphate (pH 5.0) at a separation voltage of 15 kV. The content of sinomenine was detected by ECL at the detection voltage of 1.15 V (versus Ag/AgCl) with 5 mM Ru(bpy)₃²⁺ in 75 mM phosphate solution (pH 8.0) when a chemically modified platinum electrode by europium(III)-doped prussian blue analogue (Eu-PB) was used as a working electrode. Under the optimized conditions, the ECL intensity was in proportion to sinomenine concentration in the range from 0.01 to 1.0 μg mL⁻¹ with a detection limit of 2.0 ng mL⁻¹ (3σ). The relative standard derivations of migration time and ECL intensity were 0.93 and 1.11%, respectively. The level of sinomenine in Sinomenium acutum Rehd. et Wils was easily determined with recoveries between 98.6 and 102.7%.     

Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction

A capillary zone electrophoresis (CZE) method for separation of adenosine and N⁶-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20 min with high theoretical plate number (up to 400 000 for isopentenyladenosine triphosphate) was achieved using a 100 mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69–1.27 μmol L⁻¹). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R² > 0.999) was achieved over the concentration range 5–1000 μmol L⁻¹. The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE–ESI-MS using a volatile buffer (30 mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products – isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction.     

Capillary electrophoresis with contactless conductivity detection for uric acid determination in biological fluids

The suitability of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) for the direct determination of uric acid in human plasma and urine was investigated. It was found that a careful optimization of the buffer composition and pH was necessary to achieve selective determination in the complex sample matrices. An electrolyte solution consisting of 10 mM 2-morpholinoethanesulfonic acid (MES), 10 mM histidine and 0.1 mM hexadecyltrimethylammonium bromide (CTAB), pH 6.0, was finally found suitable for use as running buffer for both sample matrices. The limit of detection (3 S/N) was determined as 3.3 μM. The linearity of the response was tested for the range between 10 and 500 μM and a correlation coefficient of 0.9996 was obtained. Intra- and inter-day variabilities were <10%. Quantitative analysis of urine and plasma samples showed a good correlation with the routine enzymatic method currently used at the University Hospital of Basel.     

Determination of scopolamine, atropine and anisodamine in Flos daturae by capillary electrophoresis.

A capillary electrophoresis method was developed for the separation and determination of tropane alkaloids in Flos daturae plants. Separation was performed on a fused silica capillary(42.1 cm x 50 microm i.d.) at an applied voltage of 20 kV. Scopolamine, atropine and anisodamine were well separated in the buffer of 50 mmol/L phosphate buffer (pH 5.0) containing 20% (v/v) tetrahydrofuran (THF). Beer's law was obeyed in the range of concentration of 2.4-21.8 microg/mL for scopolamine, 4.0-36.0 microg/mL for atropine and 2.6-23.7 microg/mL for anisodamine, respectively, and the correlation coefficients were over 0.999 (n = 6). The developed method was applied for the analysis of herb samples.     

Separation of fluoroquinolones in acidic buffer by capillary electrophoresis with contactless conductivity detection

A method to determine five fluoroquinolones (FQs), namely, rufloxacin (RUF), ciprofloxacin (CIP), enrofloxacin (ENO), gatifloxacin (GAT) and moxifloxacin (MOX), in acidic buffer by capillary electrophoresis (CE)-capacitively coupled contactless conductivity detection (C⁴D) technique is presented. Separation was carried out in a fused-silica capillary (42 cm × 50 μm) using a buffer composed of 10 mM tartaric acid, 14 mM sodium acetate and 15% (v/v) methanol at pH 3.8. The RSDs of the migration times and peak areas were 0.65% and 12.3% (intraday), 1.28% and 8.8% (interday), respectively. CE-C⁴D in combination with liquid–liquid extraction (LLE) as clean-up and preconcentration procedure, allows detection of the FQs in fortified chicken muscle samples with detection limits of 6.8–11.7 ng/g. This method shows potential in rapid determination of FQs in samples with complex matrix.     

Simultaneous determination of ascorbic acid,dopamine and uric acid based on tryptophan functionalized graphene

A new type of tryptophan-functionalized graphene nanocomposite (Trp-GR) was synthesized by utilizing a facile ultrasonic method via π–π conjugate action between graphene (GR) and tryptophan (Trp) molecule. The material as prepared had well dispersivity in water and better conductivity than pure GR. The surface morphology of Trp-GR was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopy. The electrochemical behaviors of ascorbic acid (AA), dopamine (DA), and uric acid (UA) were investigated by cyclic voltammetry (CV) on the surface of Trp-GR. The separation of the oxidation peak potentials for AA–DA, DA–UA and UA–AA was about 182 mV, 125 mV and 307 mV, which allowed simultaneously determining AA, DA, and UA. Differential pulse voltammetery (DPV) was used for the determination of AA, DA, and UA in their mixture. Under optimum conditions, the linear response ranges for the determination of AA, DA, and UA were 0.2–12.9 mM, 0.5–110 μM, and 10–1000 μM, with the detection limits (S/N = 3) of 10.09 μM, 0.29 μM and 1.24 μM, respectively. Furthermore, the modified electrode was investigated for real sample analysis.     

A novel tris(2,2′-bipyridine)ruthenium(II)/tripropylamine cathodic electrochemiluminescence in acetonitrile for the indirect determination of hydrogen peroxide

A novel tris(2,2′-bipyridine)ruthenium(II) (Ru(bpy)₃²⁺) cathodic electrochemiluminescence (ECL) was generated at −0.78 V at the Pt electrode in acetonitrile (ACN), which suggested that the cathodic ECL differed from conventional cathodic ECL. It was found that tripropylamine (TPrA) could enhance this cathodic ECL and the linear range (log-log plot) was 0.2 μM-0.2 mM. In addition, hydrogen peroxide (H₂O₂) could inhibit the cathodic ECL and was indirectly detected with the linear range of 27-540 μM. The RSD (n = 12) of the ECL intensity in the presence of 135 μM H₂O₂ was 0.87%. This method was also demonstrated for the fast determination of H₂O₂ in disinfectant sample and satisfactory results were obtained.     

A fast and highly sensitive detection of cholesterol using polymer microfluidic devices and amperometric system

In this work, the rapid detection of cholesterol using poly(dimethylsiloxane) microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, was developed. Direct amperometric detection for poly(dimethylsiloxane) (PDMS) microchip capillary electrophoresis was successfully applied to quantify cholesterol levels. Factors influencing the performance of the method (such as the concentration and pH value of buffer electrolyte, concentration of cholesterol oxidase enzyme (ChOx), effect of solvent on the cholesterol solubility, and interferences) were carefully investigated and optimized. The migration time of hydrogen peroxide, product of the reaction, was less than 100 s when using 40 mM phosphate buffer at pH 7.0 as the running buffer, a concentration of 0.68 U/mL of the ChOx, a separation voltage of +1.6 kV, an injection time of 20 s, and a detection potential of +0.5 V. PDMS microchip capillary electrophoresis showed linearity between 38.7 μg/dL (1 μM) and 270.6 mg/dL (7 mM) for the cholesterol standard; the detection limit was determined as 38.7 ng/dL (1 nM). To demonstrate the potential of this assay, the proposed method was applied to quantify cholesterol in bovine serum. The percentages of recoveries were assessed over the range of 98.9-101.8%. The sample throughput was found to be 60 samples per hour. Therefore, PDMS microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, is very rapid, accurate and sensitive method for the determination of cholesterol levels.     

Sensitive quantitative determination of oxymatrine and matrine in rat plasma by capillary electrophoresis with stacking induced by moving reaction boundary

A quantitative method of capillary electrophoresis with sample stacking induced by moving reaction boundary (MRB) was developed for sensitive determination of oxymatrine (OMT) and matrine (MT) in rat plasma. The experimental conditions were optimized firstly. Below are the optimized experimental conditions: 20 mM sodium formate solution (HCOONa, adjusted to pH 10.70 by ammonia) as sample solution, 3 min 14 mbar sample injection, 40 mM formic buffer (HCOOH-HCOONa, pH 2.60) as stacking buffer, 7 min 14 mbar injection of stacking buffer, 100 mM HCOOH-HCOONa (pH 4.80) as separation buffer, 73 cm capillary (effective length 64 cm), 21 kV voltage, 210 nm wavelength. Under the optimized conditions, higher than 60-fold sensitivity improvement of the stacking was simply achieved as compared with capillary zone electrophoresis, and the detectable limits obtained for OMT and MT were 0.26 and 0.19 μg mL⁻¹, respectively. Then, numerous demonstrations were carefully performed for the methodological validations of OMT and MT in rate plasma, including high specificity of method, good linearity (r = 0.9993 for OMT, r = 0.9991 for MT), fair wide linear concentration range (1.30-65.00 μg mL⁻¹ for OMT, 0.84-42.00 μg mL⁻¹ for MT), low limit of detection (1.03 μg mL⁻¹ for OMT, 0.38 μg mL⁻¹ for MT), less than 5% intra- and inter-day variance value, and higher than 96% recovery of OMT and MT in plasma. The developed method could be used for the trace analyses of OMT and MT in plasma and was finally used for the investigation on pharmacokinetic study of OMT in rat plasma.     

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.     

Development of a microbioreactor with ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) immobilized on a polyacrylamide-coated capillary at the outlet

A simple and fast method of immobilization of cell membrane suspension containing human ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) on a polyacrylamide-coated capillary was developed. The enzyme microbioreactor was prepared by hydrodynamic injection of a small plug of the polycationic electrolyte hexadimethrine bromide (HDB) followed by a suspension of an enzyme-containing membrane preparation. In order to shorten the enzyme assay time and to increase the throughput of the assay, the capillary was coated from the outlet end and all injections were performed from the outlet end of the capillary. For the monitoring of the enzymatic reaction, the substrate ATP dissolved in reaction buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM Hepes, pH 7.4, internal standard: 10 μM UMP) in the absence or presence of inhibitor was injected electrokinetically and incubated in the microbioreactor for 1 min with 1 kV of applied voltage. Then, the electrophoretic separation of the reaction products was initiated by applying a constant current of 60 μA. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by UV absorbance at 260 nm. The new method was compared with an at-capillary-inlet method without immobilization of the enzyme. The results (K_m values, K_i values for inhibitor) obtained with both methods were similar and comparable with literature data. The developed outlet immobilized enzyme microreactor using a coated capillary is very fast, simple and most economic allowing multiple use of the enzyme.     

Simultaneous determination of ascorbic acid,dopamine, uric acid and tryptophan on gold nanoparticles/overoxidized-polyimidazole composite modified glassy carbon electrode

A novel electrode was developed through electrodepositing gold nanoparticles (GNPs) on overoxidized-polyimidazole (PImox) film modified glassy carbon electrode (GCE). The combination of GNPs and the PImox film endowed the GNPs/PImox/GCE with good biological compatibility, high selectivity and sensitivity and excellent electrochemical catalytic activities towards ascorbic acid (AA), dopamine (DA), uric acid (UA) and tryptophan (Trp). In the fourfold co-existence system, the peak separations between AA–DA, DA–UA and UA–Trp were large up to 186, 165 and 285 mV, respectively. The calibration curves for AA, DA and UA were obtained in the range of 210.0–1010.0 μM, 5.0–268.0 μM and 6.0–486.0 μM with detection limits (S/N = 3) of 2.0 μM, 0.08 μM and 0.5 μM, respectively. Two linear calibrations for Trp were obtained over ranges of 3.0–34.0 μM and 84.0–464.0 μM with detection limit (S/N = 3) of 0.7 μM. In addition, the modified electrode was applied to detect AA, DA, UA and Trp in samples using standard addition method with satisfactory results.     

Combination of flow injection with capillary electrophoresis: 8. Miniaturized capillary electrophoresis system with flow injection sample introduction and electrogenerated chemiluminescence detection

The combined flow injection (FI)-capillary electrophoresis (CE) system was further exploited by coupling to an electrogenerated chemiluminescence (ECL) detection system. A low-cost miniaturized CE system was developed on a chip platform to provide easy interface both with FI sample introduction and with ECL detection. A falling-drop interface was employed to perform FI split-flow sample introduction while achieving electrical isolation from the CE high voltage. A plexiglas reservoir at the capillary outlet served as both the reaction and detection cell for the ECL reaction, with Ru(bpy)₃²⁺ reagent continuously flowing through the cell. An optical fiber was positioned within the reservoir close to the capillary outlet for transferring the ECL emission to the PMT. The relative positions of the capillary outlet, working electrode and optical fiber as well as reagent renewal flow-rate were optimized to achieve both good sensitivity and separation efficiency under non-interrupted sampling conditions, involving large numbers of samples. An on-column joint often used in other works for isolating the ECL detection system from the CE separation voltage was not found necessary. The performance of the system was illustrated by the baseline separation of proline, valine and phenylalanine with a high throughput of 50 h⁻¹ and plate height of 14 μm for proline under 147 V cm⁻¹ field strength. Detection limits (3σ) were 1.2, 50 and 25 μM and peak height precisions were 1.4, 5.4 and 4.3% R.S.D. (n=9) for proline, valine and phenylalanine, respectively.     

Enantiomeric analysis of rivastigmine in pharmaceuticals by cyclodextrin-modified capillary zone electrophoresis

Chiral separation method development is usually very time-consuming due to the diversity in chemical structures of pharmaceutical drug substances as well as the suitable separation conditions and the problem to choose the appropriate chiral selector. This paper shows capillary zone electrophoresis (CZE) which was developed for chiral separation of a basic compound - rivastigmine (RIV) using 30 cm × 50 μm i.d. polyacrylamide (PAA)-coated fused-silica capillary (effective length 20 cm), amine-modified phosphate buffer of pH 2.5 and sulfated-β-CD (S-β-CD) as chiral selector. Other selected native or derivatized cyclodextrins (CDs) were also tested: β-CD (5, 30 mM), carboxymethyl-β-CD (5, 30 mM), dimethyl-β-CD (15 mM), hydroxypropyl-β-CD (5, 30 mM), hydroxypropyl-α-CD (5, 30 mM) and hydroxypropyl-γ-CD (5, 30 mM). Complete enantiomeric separation of RIV was achieved at 20 kV, 18 °C and detection at 200 nm within 8 min with R.S.D. for the absolute migration time reproducibility of less than 2.1%. Rectilinear calibration range was 5.0-500.0 μM of each enantiomer (r = 0.9994-0.9995). The CZE method proposed was used for the control of chiral purity of pharmaceutically active S-RIV and for the analysis of Exelon caps preparation.