Nonaqueous capillary electrophoresis conditions for the simultaneous separation of eight alpha-adrenergic blocking agents

The analysis is described for the first time for separating eight alpha-adrenergic blocking agents (oxymetazoline, 5-methylurapidil, prazosin, phentolamine, RS-17053, methoxamine, yohimbine, and BMY7378) by capillary electrophoresis (CE) with UV detection. Optimum separation of the analytes was obtained on a 50 cm × 75 μm i.d. capillary using a buffer containing 20% acetonitrile, 60 mM ammonium acetate, and 1.0% glacial acetic in methanol medium, with applied voltage and capillary temperature of 23 kV and 25 °C, respectively. The relative standard deviations of the migration times and the peak areas of the eight analytes were in the ranges of 0.12–1.29% and 1.02–2.53%, respectively. Detection limits of oxymetazoline, 5-methylurapidil, prazosin, phentolamine, RS-17053, methoxamine, yohimbine, and BMY7378 were 0.5–1.0 μg mL⁻¹. In the tested concentration range, good linear relationships (correlation coefficients >98%) between peak areas and concentrations of the analytes were observed. This method has been successfully applied for determination of prazosin and phentolamine with recoveries of 97.30% and 98.12%, respectively. The proposed method was successfully applied for the rapid CE determination of the frequently applied alpha-adrenergic blocking compounds phentolamine and prazosin in general pharmaceutical preparation.     

Separation and determination of flavonoids using microemulsion EKC with electrochemical detection

A selective and sensitive method of microemulsion EKC (MEEKC) with electrochemical detection (ED) was developed for separation and determination of 14 flavonoids. In order to obtain the better stability for the studied flavonoids, oil (ethyl acetate) with low interfacial surface tension was employed as organic solvent. A running buffer composed of 0.9% (w/v, 30 mM) SDS, 0.9% (w/v, 21 mM) sodium cholate (SC), 0.9% (w/v, 121 mM) butan-1-ol, 0.6% (w/v, 68 mM) ethyl acetate, and 98.2% v/v 10 mM Na(2)B(4)O(7)-20 mM H(3)BO(3) buffer (pH 7.5) was applied for the separation of flavonoids. Under the optimum conditions, the relationship between peak currents and analyte concentrations was linear over about 1.3 and 1.7 orders of magnitude with detection limits (defined as S/N = 3) ranging from 0.02 to 0.5 microg/mL for all analytes. This method was applied for the determination of flavonoids in real samples with simple extraction procedures, and the assay results were satisfactory.     

Separation of fluorescein dyes by capillary electrophoresis using β-cyclodextrin

Summary A capillary electrophoresis method for the separation and determination of five synthetic dyes used in pharmaceutical preparations, cosmetics and as food additives is described. The dyes, fluorescein, dichlorofluorescein, Rose Bengal erythrosine and eosine are well separated in less than 12 min using an electrolyte of 50 mM phosphate buffer (pH 7.5), 10 mM β-cyclodextrin and 5% (v/v) methanol. A linear relationship between concentration and peak area for each dye was obtained in the concentration range 0.3–500 μg mL⁻¹, with a correlation coefficient greater than 0.999. Intra- and inter-day precision of about 0.2–2.6% RSD (n=11) and 4.9–9.7% RSD (n=30), respectively, were obtained. The method has been used for determining the purity of fluorescein and erythrosine in practical samples.     

Determination of guaiphenesin and sodium benzoate in Liqufruta garlic cough medicine by high performance liquid chromatography

A new and simple isocratic high-performance liquid chromatographic method with ultraviolet detection is described for simultaneous determination of active guaiphenesin and preservative sodium benzoate in Liqufruta garlic cough medicine formulation. The chromatographic separation was achieved using a Zorbax CN; 150 mm × 4.6 mm and 5 μm particle size column employing acetonitrile and water (20: 80, v/v) containing 0.1% formic acid (pH 3.5 ± 0.05) as the mobile phase. The method was validated with respect to linearity, range, precision, accuracy, specificity, limit of detection and limit of quantitation. The both analytes were detected by UV-Vis detector at 245 nm. The method was linear over the concentration range of 0.2–0.8 mg/mL and 0.02–0.06 mg/mL for guaiphenesin and sodium benzoate, respectively. The limit of detection was found to be 0.14 μg/mL for GP and 0.06 μg/mL for SB and the quantification limit was 0.54 μg/mL for GP and 0.22 for SB. Accuracy, evaluated as recovery, was in the range of 97.8–100.0%. Intra-day precision and intermediate precision showed relative standard deviation <1% in each case.     

Nonaqueous capillary electrophoresis method for the analysis of tamoxifen, imipramine and their main metabolites in urine

The viability of nonaqueous capillary electrophoresis (NACE) was investigated for the simultaneous determination of tamoxifen, imipramine and their main metabolites (4-hydroxytamoxifen and desipramine, respectively). Baseline separation of the studied solutes was obtained on a 57 cm × 75 μm capillary using a nonaqueous solution composed of 17 mM ammonium acetate and 1.25% acetic acid in 80:20 (v:v) methanol-acetonitrile, temperature and voltage 22 °C and 15 kV, respectively, and hydrodynamic injection. Paroxetine was used as internal standard. Different aspects including linearity, accuracy, ruggedness and precision was studied. Detection limits between 9.0 and 15.0 μg L⁻¹ were obtained for all the studied compounds. The developed method is simple, rapid and sensitive and has been used to determine tamoxifen, imipramine and their metabolites 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 three females urines were done.     

On-line concentration by head-column field amplified sample injection for the analysis of berberine, palmatine and jatrorrhizine in herbal medicine by flow injection-micellar electrokinetic chromatography

A simple, sensitive and continuous on-line stacking technique using head-column (HC)-field amplified sample injection (FASI) and sweeping was developed by combination of flow injection with micellar electrokinetic chromatography. Berberine, palmatine and jatrorrhizine were selected as model mixture to demonstrate this stacking method. Based on the characteristic of a 16-way injection valve (16-V), a sample was injected electrokinetically into a capillary after the introduction of a plug of water. Under optimum conditions, 64–86-fold improvement in the detection sensitivity was obtained for the analytes and the sample throughput can reach up to 24 h⁻¹ using the background electrolyte containing 240 mM ammonium acetate (pH 4.7), 30% (v/v) ethanol, and 2% (v/v) polyoxyethylene sorbitan monolaurate (Tween 20). The repeatabilities (n = 4) reached relative standard deviation values of 1.2, 2.7 and 3.1% for the peak areas and 1.6, 3.3 and 3.8% for peak heights of berberine, palmatine and jatrorrhizine, respectively. The limit of detection for the berberine, palmatine and jatrorrhizine was found to be 27, 26, 22 ng mL⁻¹ (S/N = 3).     

Development of a UPLC-ESI-MS/MS method for the determination of larotaxel in beagle dog plasma: application to the pharmacokinetic study

A UPLC-ESI-MS/MS method has been developed and validated for the determination of larotaxel in beagle dog plasma. After addition of the internal standard, plasma samples were extracted by liquid–liquid extraction with methyl tert-butyl ether and separated on a 50 × 2.1 mm ACQUITY 1.7 μm C₁₈ column (Waters, USA), with acetonitrile and 5 mM ammonium acetate as mobile phase, within a runtime of 3.0 min. The analytes were detected without interference in Multiple Reaction Monitoring mode with positive electrospray ionization. The linear range was 2.5–5,000 ng/mL. The intra-day and inter-day precisions (relative standard deviation, RSD, %) were within 9.3% and 10.2%, respectively, and the accuracy (relative error, RE, %) was less than 11.5%. The validated method was successfully applied to a pharmacokinetic study of larotaxel in beagle dogs after intravenous administration of larotaxel-loaded lipid microsphere with different doses of 0.4, 0.8, and 1.6 mg/kg. The area under the concentration–time curve and the peak concentration of larotaxel seemed to increase with increasing dose proportionally, suggesting linear pharmacokinetics.     

LC-ESI-MS Method for the Determination of Mizolastine in Human Plasma

A sensitive and rapid liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the determination of mizolastine in human plasma using dipyridamole as the internal standard (I.S.). Plasma samples were simply pretreated with methanol for deproteinization. Chromatographic separation was performed on an Agilent Zorbax C₁₈ column with a mobile phase of 10 mM ammonium acetate buffer containing 0.1% formic acid–methanol (20:80, v/v) at a flow rate of 1 mL min⁻¹. The electrospray ionization (ESI) interface was employed in a single quadrupole mass spectrometer. The analytes were protonated in the positive ESI interface and detected in single ion monitoring (SIM) mode. Chromatographic separation was achieved in less than 3.5 min. The linearity was established over the range of 0.5–600 ng mL⁻¹. The lower limited of quantification (LLOQ) of the method was 0.5 ng mL⁻¹. The intra- and inter-run standard deviations were both less than 11.2%. The method was applied to study the pharmacokinetics of the mizolastine sustained-release tablets in healthy volunteers.     

Separation and determination of honokiol and magnolol in herbal medicines by flow injection-capillary electrophoresis

A simple, rapid, and accurate method for the separation and determination of honokiol and magnolol in Magnolia officinalis and related herbal medicines was developed by combination of flow injection (FI) and capillary zone electrophoresis (CZE). The analysis was carried out using an unmodified fused-silica capillary (50-μm I.D.; total length 7.5 cm; effective length 4.5 cm). A series of optimization steps afforded the following conditions: the sample solvent consisted of 150 mM NaOH and a running buffer composed of 10 mM sodium tetraborate/10 mM sodium dihydrogenphosphate (NaH₂PO₄) at pH 12 was applied for the separation of the analytes. The separation could be achieved within 5 min with a sample throughput rate of up to 28 h⁻¹. The repeatability (defined as the relative standard deviation, RSD) for honokiol and magnolol was 2.0% and 1.6% with peak area evaluation, 3.6% and 2.0% with peak height evaluation, and 2.0% and 1.4% with migration time evaluation, respectively. Regression equations revealed linear relationships (r = 0.9991–0.9998) between the peak area of each analyte and the concentration.     

Simultaneous Quantification of Sodium Ferulate, Salicylic Acid, Cinnarizine and Vitamin B1 in Human Plasma by LC Tandem MS Detection

A rapid and simple high performance liquid chromatographic method coupled with tandem mass spectrometry (LC–MS–MS) via electrospray ionization (ESI) has been developed and validated to separate and simultaneously quantify sodium ferulate (SF), salicylic acid (SA), cinnarizine (CIN) and vitamin B1 (VB1) in human plasma. Gemfibrozil (GEM) was used as the internal standard (IS) for SF and SA, whereas lomerizine (LOM) was used as the IS for CIN and VB1. The plasma samples were prepared by one-step protein precipitation followed by an isocratic elution with 10 mM ammonium acetate buffer (pH = 5.0): acetonitrile (35:65, v/v,) on an Agilent Zorbax SB-CN column (150 mm × 2.0 mm ID, 5 μm). The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the selected reaction monitoring mode (SRM) with polarity switch, in the negative-ion mode for SF, SA and GEM, in the positive-ion mode for CIN, VB1 and LOM. The method was validated over the concentration range of 1.5–1,000 ng mL⁻¹ for SF, 20–5,000 ng mL⁻¹ for SA, 2–500 ng mL⁻¹ for CIN, 1–30 ng mL⁻¹ for VB1. The intra- and inter-batch precisions were less than 15% of the relative standard deviation. The recoveries for analytes and IS achieved from spiked plasma samples were consistent and reproducible. The validated LC–MS–MS method has been successfully applied to the pharmacokinetic study of sodium ferulate and aspirin capsule in healthy volunteers.     

Nano polypyrrole-coated magnetic solid phase extraction followed by dispersive liquid phase microextraction for trace determination of megestrol acetate and levonorgestrel

The aim of the present work is combination of the advantages of magnetic solid phase extraction (MSPE) and dispersive liquid phase microextraction (DLLME) followed by filtration-based phase separation. A new pretreatment method was developed for trace determination of megestrol acetate and levonorgestrel by liquid chromatography/ultraviolet detection in biological and wastewater samples. After magnetic solid phase extraction, the eluent of MSPE was used as the disperser solvent for DLLME. Emulsion resulted from DLLME procedure was passed through the in-line filter for phase separation. Finally the retained analytes in the filter was washed with mobile phase of liquid chromatography and transferred to the column for separation. This approach offers the preconcentration factors of 3680 and 3750 for megestrol acetate and levonorgestrel, respectively. This guarantees determination of the organic compounds at trace levels. The important parameters influencing the extraction efficiency were studied and optimized. Under the optimal extraction conditions, a linear range of 0.05–50 ng mL⁻¹ (R² > 0.998) and limit of detection of 0.03 ng mL⁻¹ were obtained for megestrol acetate and levonorgestrel. Under optimal conditions, the method was successfully applied for determination of target analytes in urine and wastewater samples and satisfactory results were obtained (RSDs < 6.8%).     

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.     

On-line concentration by field-enhanced sample injection with reverse migrating micelles in micellar electrokinetic capillary chromatography for the analysis of triterpenoids from traditional chinese medicine

In this work, a simple, reproducible and sensitive micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of five triterpenoids, lupeol (1), 1β-hydroxy-lupeol (2), lup-3β,1α-diol (3), lup-1β,3β,11α-triol (4) and 30-norlupan-3β,11α-diol-20-one (5) in traditional Chinese medicine of Salvia roborowskii Maxim. Field-enhanced sample injection with reverse migrating micelles (FESI-RMM) was used for on-line concentration of triterpenoids. The optimum buffer contained 50 mM H₃PO₄, 160 mM SDS, 20% acetonitrile and 15% 2-propanol and pH of buffer was 2.0. The sample solution was diluted with 10 mM H₃PO₄ (pH 2.5, containing 10 mM SDS) and injected for 15 s with −8 kV after injection of 4 s water plug. The effects of concentrations of sodium dodecyl sulfate (SDS) and organic modifier, the sample matrix, the injection time of water plug, the injection voltage and injection time of sample on the separation and stacking efficiency were investigated. Under the optimum conditions, the analytes were well separated and by optimizing the stacking conditions, about 28–96-fold improvement in the detection sensitivity was obtained for triterpenoids. The contents of five triterpenoids in Salvia roborowskii Maxim were successfully determined with satisfactory repeatability and recovery.     

Analysis of baicalein, baicalin and wogonin in Scutellariae radix and its preparation by microemulsion electrokinetic chromatography with 1-butyl-3-methylimizolium tetrafluoborate ionic liquid as additive

Microemulsion electrokinetic chromatography (MEEKC) using 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) ionic liquid (IL) as additive was developed for the analysis of baicalin, wogonin and baicalein in Scutellariae radix and its preparation. After conducting a series of optimizations, baseline separation was obtained for the analytes within 5min under the optimum conditions (sodium dodecyl sulfate (SDS) 0.88% (m/v) ethyl acetate 0.8% (v/v) butan-1-ol 0.2% (v/v) and the buffer composition were 25% acetonitrile (v/v), 7.5 mM BMIM-BF4 and 10 mM NaH2PO4, pH 8.2, applied voltage 17.5 kV and detection at 254 nm), the method has been successfully applied to the determination and quantification of the analytes in the extracts of S. radix (cooked), S. radix (raw) and Qingfeiyihuowan which was the preparation including S. radix.     

Determination of ibuprofen and tetrazepam in human urine by micellar electrokinetic capillary chromatography

A new micellar electrokinetic capillary chromatography method (MEKC) is proposed for the determination of ibuprofen and tetrazepam in human urine samples over a concentration range of therapeutic interest. A fused silica capillary (60 cm × 75 μm) is used. Ibuprofen and tetrazepam are detected via UV detection at 220 and 228 nm, respectively. Separation is performed at 25 °C and at a separation voltage of 30 kV, with 15 mM borate buffer (pH 10.2) containing 40 mM sodium dodecylsulfate as the electrolyte solution. Under these conditions the analytes were separated in <11 min. Sulfamethazine is used as an internal standard. Prior to determination, the samples are purified and enriched by means of an extraction–preconcentration step with a preconditioned C₁₈ cartridge and by eluting the compounds with methanol. Good linearity, accuracy, precision, robustness and solution stability were achieved for the technique. Detection limits of 200 μg L⁻¹ for ibuprofen and 300 μg L⁻¹ for tetrazepam were obtained. These analytes were then determined in real urine using the technique.     

Separation and sweeping of flavonoids by microemulsion electrokinetic chromatography using mixed anionic and cationic surfactants

In this report, a novel means for the separation and sweeping of flavonoids (quercetin, rutin, calycosin, ononin and calycosin-7-O-β-d-glucoside) by microemulsion electrokinetic chromatography using mixed anionic and cationic surfactants as modified pseudostationary phase was presented. The optimized background electrolyte consisted of 0.5% (w/v) ethyl acetate, 2.0% (w/v) SDS, 9 mM DTAC, 4.0% (w/v) 1-butanol and 10 mM sodium borate or 25 mM phosphoric acid. We systematically investigated the separation and preconcentration conditions, including the concentrations of surfactant, types of sweeping, sample matrix, the effect of high salt or acetonitrile, and sample injection volume. It was found that the use of mixed surfactants significantly enhanced the separation efficiency through the change of the efficient electrophoretic mobility of analytes. Compared with normal sample injection, 185-508-fold sensitivity enhancement in terms of limit of detection was achieved through effective sweeping of large sample volume at 50 mbar pressure (up to 45% capillary length). At last, the proposed method was suitable for the determination of Radix Astragali sample.     

Non-aqueous capillary electrophoresis for separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its medicinal preparations

A non-aqueous capillary electrophoresis method has been developed for the separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its preparation for the first time. Optimum separation of the analytes was obtained on a 47 cm x 75 microm i.d. fused-silica capillary using a non-aqueous buffer system of 60 mM sodium cholate, 20 mM ammonium acetate, 20% acetonitrile and 3% acetic acid at 20 kV and 292 K, respectively. The relative standard deviations (RSDs) of the migration times and the peak heights of the three analytes were in the range of 0.23-0.28 and 2.12-2.60%, respectively. Detection limits of fraxin, esculin and esculetin were 0.1557, 0.4073 and 0.5382 microg/mL, respectively. In the tested concentration range, good linear relationships (correlation coefficients 0.9995 for fraxin, 0.9999 for esculin and 0.9992 for esculetin) between peak heights and concentrations of the analytes were observed. This method has been successfully applied to simultaneous determination of the three bioactive components with the recoveries from 90.2 to 109.2% in the five 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.     

Determination of baicalin, chlorogenic acid and caffeic acid in traditional chinese medicinal preparations by capillary zone electrophoresis

Summary A capillary zone electrophoresis (CZE) method was developed for the simultaneous assay of three bioactive components—baicalin, chlorogenic acid and caffeic acid—in seven traditional Chinese medicinal preparations. The analytes were separated successfully within 3.5 min using 10 mM borate buffer (pH8.6). Regression equations revealed linear relationships (correlation coefficients 0.9942–0.9996) between the peak area and concentration of the three analytes. The relative standard deviations of the migration times and the peak areas of the three constituents were 1.12–2.68% and 1.62–5.73%, respectively. Recovery of the three constituents ranged from 89 to 107%. The extraction efficiencies of different extraction solutions are also discussed. The contents of the three components in seven different Chinese medicinal preparations containing Honeysuckle flower and/orScutellariae radix were determined by the CZE method with satisfactory results.     

Separation of phenolic acids in soil and plant tissue extracts by co-electroosmotic capillary electrophoresis with direct UV detection

Summary A capillary zone electrophoretic method for the analysis of phenolic acids in soil and plant extracts was developed with direct UV detection using a phosphate electrolyte solution. The electrophoretic separation required the phenolic acids to be charged at a pH above their pKa in order to achieve their migration towards the anode. Electroosmotic flow (EOF) was reversed in direction by adding tetradecyltrimethylammonium bromide (TTAB). Factors affecting the separation selectivity, including the buffer pH and EOF modifiers, were investigated systematically. Eight phenolic acids were separated and detected in 10 min using an electrolyte containing 25 mM phosphate, 0.5 mM TTAB and 15% acetonitrile (v/v) at pH of 7.20. Linear plots for the test phenolic acids were obtained in a concentration range of 0.01–1 mM with detection limits in the range of 1.0–7.0 μM. The recoveries ranged from 92.8 to 102.3% in soil and plant tissues samples spiked at 100 μM and the relative standard deviation based on the peak area were ranged 2.0 to 4.5%. The proposed method was used for the determination of phenolic acids in plant tissue and soil extracts with direct injection.