Simultaneous Determination of Four Major Iridoid Glycosides in Scrophularia ningpoensis by CE

A simple, accurate and reproducible capillary electrophoresis method with UV detection has been developed for the simultaneous determination of four iridoid glycosides, 6-O-methyl-catalpol, aucubin, harpagide, and harpagoside, in Scrophularia ningpoensis (Xuan-shen). The running buffer was 100 mM borate (pH 9.3) containing 20% methanol. Applied voltage was 20 kV and temperature was 25 °C. Diphylloside A was used as an internal standard (IS) and detection was at 200 nm. The effects on separation of buffer pH, buffer concentration, and organic modifiers were investigated. The extracts of S. ningpoensis were well separated within 45 min.

     

Simultaneous Determination of the Active Ingredients in Abelmoschus manihot (L.) Medicus by CZE

A capillary zone electrophoretic method has been developed for the quantitative analysis of four active compounds, quercetin-3-O-robinobioside (LXY3), hyperin (LXY4), isoquercetin (LXY5) and myricetin (LXY7) in A. manihot (L.) Medicus with UV detection at 254 nm. The capillary temperature was kept constant at 25 °C. The effects of buffer composition, pH, and concentration and running voltage on migration were studied systematically. Optimum separation was achieved with 20 mmol L^?1 Borax–NaOH buffer (pH 9.50) at 25 °C and 25 kV. Regression equations revealed good linear relationship between the peak area of each compound and its concentration. All the correlation coefficients were higher than 0.9990. The relative standard deviations of migration time and the peak area were <0.53% and 5.34% (inter-day), and <0.56% and 3.81% (intra-day). The contents of the four compounds in A. manihot (L.) Medicus were successfully determined with satisfactory repeatability and recovery.     

Development of a CZE Method for the Determination of Olmesartan Medoxomil in Tablets

A CZE method was developed and validated for the analysis of Olmesartan medoxomil (OLMD) in tablets. The influences of pH, buffer concentration, applied voltage and capillary temperature on the migration time of OLMD were investigated. About 50 mM pH 6.5 phosphate buffer were used as background electrolyte. The optimum instrument parameters were found to be 30 °C temperature with 30 kV applied voltage and diode array detection was carried out at 210 nm. OLMD was hydrodynamically injected (P _inj  = 50 mbar, t _inj  = 3 s) and an internal standard, diflunisal (IS), was used to improve the precision and repeatability. Under these conditions, the migration time of OLMD was 2.32 min and the total analysis time was shorter than 5 min. Linearity range for the developed method was found to be 2.0–50.0 μg mL^?1 and the limit of detection was 0.5 μg mL^?1. The developed method was applied for the analysis of OLMD in pharmaceutical tablet formulations.     

Simultaneous LC–UV Determination of 5-Methyl-1-(3-fluorophenyl)-2-(1H)- pyridone and Its Two Metabolites in Rat Plasma

A simple, rapid, and reproducible isocratic reverse-phase HPLC method was developed to simultaneously determine AKF-PD and its two oxidized metabolites in rat plasma. 5-Carboxyl-1-phenyl-2-(1H)-pyridone and phenacetin were used as internal standards to ensure the precision and accuracy of the method. The analytes were separated on a C₁₈ reversed-phase column with methanol—phosphate buffer (20 mM, pH 2.5) as mobile phase. The limits of detection for AKF-PD and its two oxidized metabolites was 0.1 μg mL^?1. The method is applicable for the pharmacokinetic studies of AKF-PD and its metabolites in rats.     

Analysis of Atorvastatin and Related Substances by MEKC

A new micellar electrokinetic capillary chromatographic (MEKC) method has been developed for simultaneous quantitation of atorvastatin (AT) and its related substances. The separation was carried out in an extended light path capillary at applied voltage of 30 kV using a background electrolyte consisting of 10 mM sodium tetraborate buffer pH 9.5, 50 mM sodium dodecyl sulphate and 20% (v/v) methanol. The addition of methanol to the running buffer resulted in a very effective choice to achieve resolution between the peaks of charged substances adjacent to AT as well as the peaks of neutral drug-related substances. Linear calibration curves were established over the concentration range 100–1,200 μg mL^?1 for AT and 1.0–12.5 μg mL^?1 for related substances. The proposed MEKC procedure has been validated with respect to selectivity, precision, linearity, limits of detection, and quantitation, accuracy and robustness. The method has been successfully applied to the determination of AT and purity evaluation of bulk drug and formulated products.     

Development and Validation of LC-UV for Simultaneous Estimation of Rosiglitazone and Glimepride in Human Plasma

A simple, sensitive and specific liquid chromatographic method with UV detection (228 nm) was developed for the simultaneous estimation of rosiglitazone and glimepride in human plasma. Rosiglitazone and glimepride were extracted from plasma using liquid–liquid extraction. Separation was achieved with an RP C₁₈ Column using a mixture of phosphate buffer (50 mM) with octane sulfonic acid (10 mM), methanol and acetonitrile as a mobile phase (55:10:35, v/v). pH was adjusted to 7.0. Amlodipine was used as an internal standard (IS). LOD of the method was found to be 20 ng mL^?1 for both drugs. Results were linear over the studied range 40.994–2007.556 ng mL^?1 for rosiglitazone (r ² ≥ 0.99) and 41.066–2094.84 ng mL^?1 for glimepride( r ² ≥ 0.99). The method was found to be simple, selective, precise and reproducible for the estimation of both drugs from spiked human plasma.     

An LC Method for the Determination of Bupropion and Its Main Metabolite, Hydroxybupropion in Human Plasma

A new LC method has been developed and validated for the direct determination of bupropion and its main metabolite, hydroxybupropion in human plasma. Plasma samples were analyzed after a simple, one step protein precipitation with trichloroacetic acid using a C₈ column and mobile phase, consisting of methanol/acetonitrile/phosphate buffer (10 mM, pH 3.0) (40:10:50, v/v/v) and 20 mM 1-heptane sulfonic acid sodium salt with carbamazepine as the internal standard. UV detection was performed at 214 and 254 nm. The method was validated over the concentration range of 60–2,400 and 150–4,700 ng mL^?1 for bupropion and hydroxybupropion, respectively. The intra- and inter-day assay variability was less than 15% for the two analytes. Limit of detection values were 24.8 and 63.4 ng mL^?1 for bupropion and hydroxybupropion, respectively. The method developed was applied to quantification of bupropion and hydroxybupropion in human plasma.     

Analysis of carbamazepine and its five metabolites in serum by large-volume sample stacking–sweeping capillary electrophoresis

This study establishes a method, using different buffer conductivities and large-volume sample stacking (LVSS)–sweeping capillary electrophoresis, for analysis of carbamazepine (CBZ) and its five metabolites in serum. The capillary (50/60 cm) was filled with a high concentration of background electrolyte (150 mM phosphate, pH?3.5, containing 15 % methanol), followed by a large volume of samples (10 psi, 20 s) with low-concentration buffers (5 mM phosphate, pH?3.5, with 5 % methanol). When high voltage was applied (?20 kV), the sodium dodecyl sulfate (SDS) started to sweep the analytes to an outlet. Meanwhile, the analytes decelerated at the boundary between low- and high-conductivity buffers. Finally, a narrow sample zone was formed. The procedure of sweeping and separation was simultaneously carried out by a sweeping buffer (150 mM phosphate, pH?3.5) with 15 % methanol and 50 mM SDS added, and the detection was performed by UV at 214 nm. The method was validated for linearity (r?≧?0.997), precision, and accuracy. The calibration curves were established for CBZ and its five metabolites between 0.03–25 and 0.03–3 μg/mL. The limits of detection (S/N?=?3) were 0.01 μg/mL for each analyte. Compared with simple MEKC (0.5 psi, 5 s), this system can improve the sensitivity about 300-fold. Finally, this method was successfully applied to five patients, who had taken 200 mg CBZ daily, and CBZ levels were found to be from 3.72 to 5.82 μg/mL.

CE, with Hydroxypropyl-β-Cyclodextrin as Chiral Selector, for Separation and Determination of the Enantiomers of Amlodipine in the Serum of Hypertension Patients

A capillary electrophoretic method for separation of the enantiomers of amlodipine in the serum of hypertension patients has been established and validated. The two enantiomers were separated in a fused-silica capillary with phosphate running buffer (75 mmol L^?1, pH 2.5) containing 15 mmol L^?1 hydroxypropyl-β-cyclodextrin (HP-β-CD). The effects on the separation of buffer pH and concentration, separation potential, and concentration of HP-β-CD were investigated. The range of quantitation for both enantiomers was 2.0–16.0 μg mL^?1. Intra-day and inter-day relative standard deviation (RSD; n = 5) was <10%. The limits of detection (LOD) and quantification (LOQ) of the amlodipine enantiomers, at 214 nm, were approximately 0.5 and 0.7 μg mL^?1, respectively (S/N = 3 and 10, respectively; 5-s injection). Recovery was always >85%. Results from enantiomer separation and quantification showed that concentrations of the enantiomers of amlodipine in serum from an elderly patient were higher than in serum from a young patient administered the same dose. The method was useful for determining the concentration of the enantiomers of amlodipine in hypertension patient serum and for monitoring the transition behavior of the enantiomers in humans. The method proved suitable for application to the separation of the enantiomers of amlodipine and analysis of clinical samples.     

Simultaneous Determination of the Endogenous Free ��-Lipoic Acid and Dihydrolipoic Acid in Human Plasma and Erythrocytes by RP-HPLC with Electrochemical Detection

A highly sensitive, precise, and accurate reversed-phase high-performance liquid-chromatography/electrochemical detection method for simultaneous determination of the endogenous free ??-lipoic acid and dihydrolipoic acid in biological matrices was developed and validated. The two analytes were extracted from the samples with acetonitrile/10% metaphosphoric acid solution_(aqueous) (50/50 v/v). To determine the total lipoic acid, samples were treated with tris(2-carboxyethyl)phosphine solution in phosphate buffer, pH 2.5 with 85% orthophosphoric acid prior to deproteination. The two analytes were separated on a C₁₈ (150 × 4.6 mm, 5 ??m) analytical column using acetonitrile-50 mM phosphate buffer, pH 2.5 with 85% orthophosphoric acid (35/65 v/v) as the isocratic mobile phase pumped at a flow rate of 2.0 mL min^?1 at the column oven temperature of 35 °C. The column eluents were monitored at a potential of 0.9 V. These analytes were efficiently resolved in <7 min. The present method was sufficiently robust and specific for simultaneous determination of the two analytes and demonstrated acceptable values for linearity (r ² = 0.999 in the range of 0.1?C500 and 0.25?C1,000 ng mL^?1 for ??-lipoic acid and dihydrolipoic acid, respectively), recovery (>97%), precision (RSD% <2), and sensitivity (on column limit of detection, 150 and 375 fg for ??-lipoic acid and dihydrolipoic acid, respectively and limit of quantification: 0.5 and 1.25 pg for ??-lipoic acid and dihydrolipoic acid, respectively), indicating that the proposed method was more sensitive, precise, economical, and versatile, and has higher throughput than the previously reported methods for simultaneous determination of the two analytes.     

Simultaneous LC−UV Analysis of Mirodenafil and Its Two Main Metabolites in Rat Plasma and Urine, and in Tissue Homogenates

A simple, rapid, and reproducible isocratic reversed-phase LC method has been established for simultaneous analysis of mirodenafil and its two main metabolites, SK3541 and SK3544, in rat plasma, urine, and tissue homogenates. Samples were deproteinized with acetonitrile containing sildenafil (internal standard). The compounds were separated on a C₁₈ column with 52:48 (v/v) 0.02 m ammonium acetate buffer (pH 6)—acetonitrile as mobile phase at a flow rate of 1.4 mL min^?1. UV detection was at 254 nm and detection limits of mirodenafil, SK3541, and SK3544 in plasma were 0.03, 0.05, and 0.1 μg mL^?1, respectively. The method is applicable to pharmacokinetic studies of mirodenafil and its metabolites in rats.     

LC with Coulometric Detection for Analysis of 5-Methyltetrahydrofolate in Human Plasma

An isocratic high-performance liquid chromatographic method with coulometric electrochemical detection has been used for analysis of 5-methyltetrahydrofolate (5-MTHF) in human plasma. A 250 mm × 4.6 mm i.d., 5-μm particle, C₁₈ column was used with 12:88 (v/v) acetonitrile ?35 mM sodium phosphate buffer pH 3.8 as mobile phase at a flow rate of 1.0 mL min^?1. The method was validated for 5-MTHF plasma concentrations in the range 2.5–100.0 nM. The method was characterized by a good linearity (regression coefficient r ≥ 0.9989) and limits of detection and quantification of 0.72 and 2.16 nM, respectively. Mean recovery at low and high concentrations ranged from 89.1 to 96.3%, respectively, with a relative standard deviation <4.6%. Between-run imprecision (4.2%) was higher than within-run imprecision (3.4%). The proposed separation and detection procedures were successfully applied to analysis of 5-MTHF in human plasma.     

Direct Determination of Barbiturates in Urine by Capillary Electrophoresis Using a Capillary Coated Dynamically with Polycationic Polymers

A new, simple and rapid capillary electrophoresis method, using hexadimethrine bromide (HDB) as electro-osmotic flow modifier, has been developed for the identification and determination of nine barbiturates, barbital acid, barbital, phenobarbital, pentobarbital, amobarbital, thiobarbituric acid, butobarbital, N-methyl-5-phenyl-ethyl barbital acid and 5-cyclohexenyl-5-ethyl barbital acid in urine with UV detection at 200 nm. The applied voltage was ?25 kV and the capillary temperature was kept constant at 25 °C. The effects of buffer pH, the concentration of HDB and the concentration of α-cyclodextrin were studied systematically. Optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.04% (w/v) HDB and 2.06 mM α-cyclodextrin. Regression equations revealed good linear relationship between the peak area of each compound and its concentration. The correlation coefficients were from 0.9990 to 0.9997. The relative standard deviations of migration times and peak areas were <3.84 and 5.45% (intra-day). The nine barbiturates in urine were successfully determined within 7 min, without a prior preparation step and the method is useful for the investigation of intoxication.     

Cyclodextrin-Modified Gold Nanoparticle Capillary Electrochromatography with Online Sample Stacking for Simultaneous and Sensitive Determination of Aminobenzoic Acid Isomers

A newly-developed method of complete separation and sensitive determination of o-, m-, and p-aminobenzoic acid isomers was achieved by combining open-tubular columns for capillary electrochromatography (OT-CEC) and online sample stacking. In this study, spherical gold nanoparticles were modified by a covalent attachment of mono-6-thio-β-cyclodextrin, and OT-CEC was formed by immobilizing cyclodextrin-modified gold nanoparticles (CD-AuNP) on prederivatized 3-mercaptopropyl-trimethoxysilane fused-silica capillaries. Based on the theory of moving chemical reaction boundary, effects of several important factors such as the pH and concentration of running buffer and the conditions of stacking analytes were optimized. The optimized separations were carried out in 58 mmol/L HAc buffer at pH 3.0 using a capillary coated with CD-AuNP, while the optimized concentration was carried out in 50 mmol/L disodium hydrogen phosphate (pH 9.5). The linear ranges for m-, p-, and o-aminobenzoic acid were from 5.0 × 10^?4–0.1, 5.0 × 10^?4–0.1 and 1.0 × 10^?4–0.1 mmol/L, respectively. And the detection limits (S/N = 3) were as low as 8.22 × 10^?5, 8.21 × 10^?5, and 3.76 × 10^?5 mmol/L for m-, p-, and o-aminobenzoic acid, respectively. The run-to-run, day-to-day, and column-to-column reproducibilities of migration time were satisfactory with relative standard deviation values of less than 4.5 % in all cases. This method was successfully used in determining procaine hydrochloride injection sample with recoveries in the range of 96.1–106.6 % and relative standard deviations less than 5.0 %.     

Chemometrically Assisted Development and Validation of LC for Simultaneous Determination of Carbamazepine and Its Impurities Iminostilbene and Iminodibenzyl in Solid Dosage Form

A chemometrical approach was applied to develop a reversed-phase liquid chromatographic method for simultaneous determination of carbamazepine and its impurities iminostilbene and iminodibenzyl in solid dosage form. According to contemporary literature, no method was developed for simultaneous determination of carbamazepine and these impurities by chemometrical approach. The fractional factorial design was used for selection of variables significantly influencing the chromatographic separation of the investigated substances. The investigated variables were: temperature of the column, the percentage of organic modifier, the acetate buffer concentration and pH of water phase. The first three variables were proved to be significant and were optimized by face centered, central composite design. Investigation was performed using C18 XBridge Shield analytical column (50 mm × 4.6 mm i.d., particle size 3.5 µm). The optimal conditions for the separation were established with the mobile phase composition of methanol–10 mM acetate buffer (pH adjusted to 2.21 with glacial acetic acid) (50:50, v/v) at a flow rate of 1.5 mL min^?1, 25 °C column temperature and detection at 260 nm. Total analysis time was shortened to about 8 min. Finally, the method was successfully validated and subsequently applied to the analysis of commercially available carbamazepine tablets.     

Boronate-Containing Copolymer Grafted on Eupergit C as Matrix for Affinity Chromatography: Isotherms and Kinetics Study

A stability-indicating HPLC method has been developed and subsequently validated for the simultaneous determination of domperidone and pantoprazole in commercial tablets. The proposed HPLC method utilizes Phenomenex^® Gemini C₁₈ column (150 mm × 4.6 mm i.d., 5 μm) and mobile phase consisting of methanol-acetonitrile-20 mM dipotassium hydrogen phosphate and phosphoric acid buffer pH 7.0 (20:33:47, v/v/v) at a flow rate of 1.19 mL min^?1. Quantitation was achieved with UV detection at 285 nm based on peak area with linear calibration curves at concentration ranges 0.5–5.0 μg mL^?1 for domperidone and 1.0–10 μg mL^?1 for pantoprazole (R ² > 0.999 for both drugs). The method was validated in terms of accuracy, precision, linearity, limits of detection, limits of quantitation and robustness. This method has been successively applied to pharmaceutical formulation and no interference from the tablet excipients was found. Domperidone, pantoprazole and their combination drug product were exposed to acid, base and neutral hydrolysis, oxidation, dry heat and photolytic stress conditions and the stressed samples were analyzed by the proposed method. As the proposed method could effectively separate the drug from its degradation products, it can be employed as stability-indicating method for the determination of instability of these drugs in bulk and commercial products.     

In-Capillary Derivatization and Preconcentration for CE of Metal Ions as Their Phenanthroline Complexes

A simple and automated method involving in-capillary derivatization and in-capillary preconcentration was developed for the simultaneous determination of metal ions by capillary zone electrophoresis. Fe(II), Zn(II), Cu(II) and Cd(II) were derivatized using 1,10-phenanthroline as the derivatizing agent. The in-capillary derivatization and in-capillary preconcentration via large volume injection were performed sequentially as follows: 60 mmol L^?1 1,10-phenanthroline was first hydrodynamically injected (0.2 psi) for 2 s; metal ions were introduced by hydrodynamic injection (0.5 psi) for 60 s; 0.2 mol L^?1 acetate pH 5.5 containing 20 % methanol was used as the running buffer. Four metal ions can be determined within 8 min using 16 kV. The resulting preconcentration factors were in the range 12–21. Good linearity was obtained for concentrations of 0.1–8.0 mg L^?1 (r ² > 0.990). The mean recoveries of the metal ions evaluated by fortification of wine samples were in the range 90–102 %. The limits of detection ranged from 0.05 to 0.2 mg L^?1. The proposed method can be applied for directly determining metal ions in wine samples.     

Simultaneous Determination of Ibuprofen and Diphenhydramine Citrate in Tablets by Validated LC

A stability-indicating LC method was developed for the simultaneous determination of ibuprofen and diphenhydramine citrate in pharmaceutical dosage forms. The chromatographic separation was achieved on an Inertsil ODS 3V, 150 × 4.6 mm, 5 μm, column. The mobile phase contained a mixture of 50 mM potassium dihydrogen phosphate buffer:acetonitrile:triethylamine:glacial acetic acid (55:45:0.2:0.2, v/v/v/v). This method allowed the determination of 2.85–9.14 mg mL^?1 of ibuprofen and 0.54–1.73 mg mL^?1 of diphenhydramine citrate, in a diluent consisting of pH 7.2, 50 mM potassium dihydrogen phosphate buffer:acetonitrile (40:60, v/v). The flow rate was 1.2 mL min^?1 and the detection wavelength was 260 nm. The limit of detection for ibuprofen and diphenhydramine citrate was 1.72 and 0.54 μg mL^?1 and the limit of quantification was 5.73 and 1.64 μg mL^?1, respectively. This method was validated for accuracy, precision and linearity. The method was also found to be stability indicating.     

Chiral separation of ketoprofen on an achiral NH<Subscript>2</Subscript> column by HPLC using vancomycin as chiral mobile phase additive

A high-performance liquid chromatographic method for chiral separation of ketoprofen racemate was developed. (R)- and (S)-ketoprofen enantiomers were separated on a LiChrosorb NH₂ column (250 mm × 4.6 mm, i.d 5 µm) at 20 °C, using 2-propanol/potassium dihydrogen phosphate buffer (pH 6.0, 0.05 M) (50:50 v/v). Containing vancomycin as the mobile phase, at a flow rate of 0.8 ml min^?1 and detection wavelength of UV, the detector was set at 310 nm. Under these conditions, ketoprofen enantiomers could be separated with a selectivity factor (α) of 2.172 and a resolution (Rs) of 4.78 using extremely low concentrations of the vancomycin chiral additive.