Determination of Diazepam and associated compounds in pharmaceutical preparations

A method for determining Diazepam and its associated compounds in pharmaceutical products by micellar electrokinetic chromatography (MEKC) is described. The separation was carried out at 30 °C and 25 kV, using a 25 mM borate buffer (pH 9.6) and 35 mM sodium dodecylsulfate (SDS) water solution. Under these conditions the analysis was carried out within 12 min with acceptable limits of detection and quantification. The method has been applied for quantifying Diazepam in different commercial formulations when it is the active drug and when it is employed associated with other drugs (Nortriptyline, Pyridoxine hydrochloride and Sulpiride). Received: 30 October 1998 / Revised: 1 February 1999 / Accepted: 3 February 1999     

Simultaneous determination of nine endogenous steroids in human urine by polymeric-mixed micelle capillary electrophoresis

A new CE system based on the use of polymeric-mixed micelles (cholic acid, SDS and the poloxamine Tetronic(?) 1107) was developed for the simultaneous determination of nine steroids in human urine. This method allows the baseline separation and quantitation of cortisol, androstenedione, estriol, dehydroepiandrosterone sulfate, testosterone, dehydroepiandrosterone, estrone, progesterone and estradiol in less than 25 min showing to be sensitive enough to detect low concentrations of these steroids in urine samples (5-45 ng/mL). The optimized electrophoretic conditions were performed using a 50 cm × 75 μm capillary, 18 kV, 25°C, with 44 mM cholic acid, 10 mM SDS, 0.05% w/v tetronic(?) 1107, 2.5% v/v methanol, 2.5% v/v tetrahydrofuran in 5 mM borate - 5 mM phosphate buffer (pH=8.0) as a background electrolyte and a dual 210/254 UV-detection. The method can simultaneously determine 0.1-120 μg/mL, which corresponds to 5-6000 ng/mL of steroids in 2 mL urine. The recoveries ranged between 82.4 and 101.5%. Due to its simplicity, speed, accuracy and reliability, the proposed method could be a potential alternative to the traditional methodologies used with clinical purposes.     

Microemulsion electrokinetic chromatography for the separation of retinol, cholecalciferol, delta-tocopherol and alpha-tocopherol

A microemulsion electrokinetic chromatographic method was used to separate fat-soluble vitamins. The separation of retinol, cholecalciferol, and delta- and alpha-tocopherol was performed using a microemulsion containing 0.75% (v/v) n-heptane, 30 mM bis(2-ethylhexyl)sodium sulfosuccinate (AOT), 5% (v/v) 1-butanol, 15% (v/v) 1-propanol and 15% (v/v) methanol in 20mM boric acid-sodium borate buffer. The effect of the different microemulsion constituents was studied, including the type and concentration of surfactant, buffer, oil and co-surfactants. The presence of methanol in the microemulsion was found to be necessary to achieve the separation of the tocopherols. Detection was carried out at 200, 265 and 325 nm for the tocopherols, cholecalciferol and retinol, respectively. Calibration curves and precision data were obtained for each analyte. Good linear relationships were found between the analytical signal and the analytes concentration in the 25-500 mg L(-1) for retinol and cholecalciferol, and 25-300 mg L(-1) for tocopherols ranges. The precision of the method afforded relative standard deviations in the 4.0-10% range.     

Development and validation of a capillary electrophoresis method for the determination of escitalopram and sensitive quantification of its enantiomeric impurity in formulations

A rapid capillary zone electrophoresis method has been developed capable of quantifying 0.05% of R-enantiomer and assaying the main component in escitalopram formulations. Many parameters influencing enantioseparation were investigated, which include chiral selectors, buffer composition and pH, applied voltage, capillary length, temperature, and rinsing procedure. Optimal separation conditions were obtained by using a 25 mM phosphate buffer at pH 7.0, containing 1.6% (w/v) sulfated-β-cyclodextrin with short-end injection at 0.5 psi for 5 s. Online UV detection was performed at 205 nm. A voltage of -20 kV was applied and the capillary temperature was kept at 25°C. Separation was achieved in less than 2 min. The method was further validated, including robustness, stability of the solution, selectivity, linearity (escitalopram from 0.25 μg/mL to 600 μg/mL, y = 1528.3 × +1812.9; R2 = 0.9999), LOD and LOQ (0.08 and 0.25 μg/mL, respectively), precision and accuracy. The proposed method was then applied to the quality control of the bulk sample and tablets of escitalopram (10 mg).     

Determination of hydrocortisone,polymyxin B and Zn-bacitracin in pharmaceutical preparations by micellar electrokinetic chromatography

A new, simple and accurate micellar electrokinetic chromatography (MEKC) method is established for quantification of hydrocortisone, polymyxin B and Zn-bacitracin in local pharmaceutical preparations. The separation was carried out at 25 degrees C and 25 kV, using a 15 mmol L(-1) phosphate-15 mmol L(-1) borate buffer (pH 8.2), 60 mmol L(-1) sodium dodecylsulfate (SDS), and 10% methanol-water (v/v) as background electrolyte. Under these conditions the analysis takes about 23 min. The method has been applied for quantifying these compounds in two different commercial pharmaceutical products and the method gave good results when compared with a reference spectrophotometric multivariate calibration method.     

Microemulsion electrokinetic chromatography for separation and analysis of curcuminoids in turmeric samples

Microemulsion EKC (MEEKC) was developed for quantitative analysis of curcuminoids, such as curcumin (C), demethoxycurcumin (D), and bis-demethoxycurcumin (B). MEEKC separation of curcuminoids was optimized, and a change in resolution was explained using a modified equation for resolution in MEEKC without electroosmosis. The suitable MEEKC conditions for separation of curcuminoids were obtained to be the microemulsion buffer containing 50 mM phosphate buffer at pH 2.5, 1.1% v/v n-octane as oil droplets, 180 mM SDS as surfactant, 890 mM 1-butanol as cosurfactant, and 25% v/v 2-propanol as organic cosolvent; applied voltage of -15 kV; and separation temperature 25 degrees C. Achieved baseline resolution of C:D and D:B was obtained with R(s) -2.4 and analysis time within 18 min. In addition, high accuracy and precision of the method were obtained. This MEEKC method was used for quantitative determination of individual curcuminoids in medicinal turmeric capsules and powdered turmeric used as coloring additive in food, with simple sample preparation such as solvent extraction, dilution, and filtration, and without cleaning up by SPE.     

Micellar electrokinetic and high-performance liquid chromatographic determination of potential manufacturing impurities in pholcodine

Quantitative high-performance liquid chromatographic (HPLC) and micellar electrokinetic chromatographic (MEKC) methods have been developed for the determination of four structurally related potential manufacturing impurities, including morphine, of the opiate derivative pholcodine. Pholcodine and the four impurities were separated by MEKC in less than 14 min using a 70 cm x 75 microm I.D. uncoated fused-silica capillary (25 kV at 30 degrees C) and a running buffer consisting of 10% acetonitrile (v/v) in 20 mM borate-phosphate buffer pH 8.0 containing 40 mM sodium dodecyl sulphate (SDS). The MEKC method was compared to a HPLC method using a 5 microm Luna phenyl-hexyl column (150 x 4.6 mm I.D.) eluted with a mobile phase consisting of a mixture of 10% (v/v) acetonitrile, 7% (v/v) tetrahydrofuran in 20 mM phosphate buffer pH 8.0. Both methods were fully validated and a comparison was made regarding selectivity, linearity, precision, robustness and limits of detection and quantitation. The presence of the impurities in different samples of pholcodine drug substance was investigated using both methods.     

Determination of Diazepam and associated compounds in pharmaceutical preparations

A method for determining Diazepam and its associated compounds in pharmaceutical products by micellar electrokinetic chromatography (MEKC) is described. The separation was carried out at 30?°C and 25 kV, using a 25 mM borate buffer (pH 9.6) and 35 mM sodium dodecylsulfate (SDS) water solution. Under these conditions the analysis was carried out within 12 min with acceptable limits of detection and quantification. The method has been applied for quantifying Diazepam in different commercial formulations when it is the active drug and when it is employed associated with other drugs (Nortriptyline, Pyridoxine hydrochloride and Sulpiride).     

SPE and large-volume sample stacking in MEKC for determination of doxycycline in biological fluids: comparison of direct injection to SPE-MEKC

A novel and simple method has been developed for the determination of doxycycline (DOX) in biological fluids. The method is based on SPE, large-volume sample stacking (LVSS) and MEKC with UV-DAD detection. Six SPE cartridges have been used in investigation for sample clean up and pre-concentration (Supelco LC-8, LC-18, LC-SCX, and LC-WCX, as well as Strata-X and X-C). DOX was determined on a 56 cm (effective length 50 cm) x 50 microm id fused-silica capillary. The BGE was 20 mM borate buffer, pH 9.3, containing 80 mM SDS and 7.5% v/v of methanol (30 sx50 mbar), and the temperature and voltage were 25 degrees C and 30 kV, respectively. The analytical wavelength was set at 210 nm. Under optimized conditions it is possible to determine DOX in human serum, urine, semen, tears and saliva with recovery of 97.5% (RSD 2.5%). The method was shown to be sensitive (LOD is 1 microg/L) and precise (intra-day RSD 0.2 and 2.4%; inter-days 0.4 and 3.5% for migration time and peak area, respectively). Results for developed SPE-LVSS-MEKC were compared with LVSS-MEKC method with direct sample injection. The new LVSS-MEKC method is presented as a useful technique for rapid determination without extraction procedure of DOX in human urine and serum, using 80 mM of SDS, 10% v/v of methanol and 40 mM borate buffer (pH 9.3; 30 s x 50 mbar; 25 degrees C; 30 kV; 350 nm), but not for the other biological fluids, according to lower sensitivity of the method and because of the sample composition.     

Micellar electrokinetic capillary chromatography determination of zinc bacitracin and nystatin in animal feed

An MEKC procedure was developed for the separation of zinc bacitracin (Zn-BC) and nystatin (NYS) in mixtures and in animal feedstuff. The running buffer was 15 mM borate/19 mM phosphate, pH 8.2, containing 20 mM SDS and 10% v/v methanol. Samples were run at 25 degrees C, the applied voltage was 25 kV, and an additional pressure of 5 mbar was applied. Both analytes were detected by UV simultaneously at 215 nm, Zn-BC alone at 192 and 254 nm, and NYS alone at 305 nm. The method was shown to be specific, accurate (recoveries were 100.0 +/- 0.6% and 100.1 +/- 0.6% for Zn-BC and NYS, respectively), linear over the tested range (correlation coefficients 0.9991 and 0.9994), and precise (RSD below 1.3% for both analytes). The method was applied to determine Zn-BC and NYS as additives in animal feed.     

Determination of Clobazam, Clorazepate, Flurazepam and Flunitrazepam in pharmaceutical preparations

The separation of four benzodiazepines (Flurazepam, Flunitrazepam, Clobazam and Clorazepate) in pharmaceutical products by micellar electrokinetic chromatography (MEKC) is described. It was carried out at 25 degrees C and 25 kV by using a 57 cm (50 cm to the detector)x75 mum i.d. fused silica capillary and a 15 mM borate buffer (pH 9.2), 35 mM sodium dodecylsulfate (SDS) and 35 mM sodium deoxycholate water solution. Under these conditions, the analysis was performed in 8 min with acceptable limits of quantification (between 3 and 5 mg l(-1)). The method has been applied for quantifying these benzodiazepines in serum and different commercial formulations with recoveries near 100%.     

Determination of ethinylestradiol and gestodene in oral contraceptives by micellar electrokinetic chromatography

Summary A micellar electrokinetic chromatographic method is presented which permits quantification of ethinylestradiol and gestodene in pharmaceutical products. Separation was carried out at 25°C and 25 KV, using a 20 mM borate buffer (pH 9.2), 15 mM sodium dodecylsulfate in 30% acetonitrile-water (v/v). Under these conditions analyses were carried out in 7 min. Four different oral contraceptives were analysed and the results compared favourably with those of a reference liquid chromatographic method.     

Analysis of phospholipids by NACE with on-line ESI-MS

A hyphenated method of nonaqueous capillary electrophoresis coupled to electrospray ionization mass spectrometry (NACE-ESI-MS) is described for the simultaneous analysis of phospholipids. The best results were obtained with a mixed solution of methanol/ACN (40:60 v/v) containing 20 mM ammonium acetate and 0.5% acetic acid, under the applied voltage of 30 kV and capillary temperature of 25 degrees C. ESI-MS measurements were performed in the negative mode with methanol/ACN (40:60 v/v) containing 50 mM ammonium acetate as sheath liquid at a flow rate of 2 microL/min. Different phospholipid classes have been successfully separated within 16 min, and the molecular species of every single class have been identified by using MS(2) or MS(3), which generates characteristic fragments through CID. The developed method has been applied to analyze the phospholipids extracted from rat peritoneal surface and the molecular species of phospholipid classes are presented.     

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.     

Enantiomeric separation of citalopram and its metabolites by capillary electrophoresis

A simple and fast capillary electrophoretic method has been developed for the enantioselective separation of citalopram and its main metabolites, namely N-desmethylcitalopram and N,N-didesmethylcitalopram, using beta-cyclodextrin (beta-CD) sulfate as the chiral selector. For method optimisation several parameters were investigated, such as CD and buffer concentration, buffer pH, and capillary temperature. Baseline enantioseparation of the racemic compounds was achieved in less than 6 min using a fused-silica capillary, filled with a background electrolyte consisting of a 35 mM phosphate buffer at pH 2.5 supplemented with 1% w/v beta-CD sulfate and 0.05% w/v beta-CD at 25 degrees C and applying a voltage of -20 kV. A fast separation method for citalopram was also optimized and applied to the analysis of pharmaceutical formulations. Racemic citalopram was resolved in its enantiomers in less than 1.5 min using short-end injection (8.5 cm, effective length) running the experiments in a background electrolyte composed of a 25 mM citrate buffer at pH 5.5 and 0.04% w/v beta-CD sulfate at a temperature of 10 degrees C.     

Development and validation of a stability-indicating micellar electrokinetic chromatography method for the determination of ezetimibe in pharmaceutical formulations.

A micellar electrokinetic chromatography (MEKC) method was validated for the analysis of ezetimibe. The method was carried out on a fused-silica capillary (50 microm i.d.; effective length, 40 cm). The background electrolyte consisted of a 25 mM borate buffer and 25 mM anionic detergent SDS (pH 9.75)/methanol (90:10, v/v). The capillary temperature was maintained at 35 degrees C, the applied voltage was 30 kV; the injection was performed using a pressure mode at 50 mbar for 5 s, with detection at 232 nm. The method was linear in the range of 2-150 microg/mL (R2=0.9999). The specificity and the stability-indicating capability were proven through degradation studies, which also showed that there was no interference of the excipients. The limits of quantitation and detection were 2 and 0.41 microg/mL, respectively. The method was applied for the analysis of ezetimibe pharmaceutical formulations, and the results were compared to those of the liquid-chromatography method.     

Determination of caffeine and associated compounds in food, beverages, natural products, pharmaceuticals, and cosmetics by micellar electrokinetic capillary chromatography

A method is described for quantitating caffeine, theobromine, theophylline, paracetamol, propyphenazone, acetylsalicylic acid, salicylic acid, and codeine phosphate in corresponding real samples of food, beverages, natural products, pharmaceuticals, and cosmetic preparations by micellar electrokinetic capillary chromatography. The separation is carried out at 25 degrees C and 25 kV, using a 20mM phosphate buffer (pH 9.0), 80mM sodium dodecyl sulfate, and 7.5% (v/v) acetonitrile. UV detection is at 210 nm. The method is shown to be specific, accurate (recoveries over the range 98.9-101.2%), linear over the tested range (correlation coefficients >/= 0.9993), and precise (relative standard deviation below 2.1%). The method is applied for the quantitative analysis of these compounds in different foods, beverages, natural products, pharmaceuticals, and cosmetic products.     

A capillary zone electrophoresis method for determining N-acetylneuraminic acid in glycoproteins and blood sera

A simple capillary zone electrophoresis (CZE) method for the determination of the content of the major sialic acid form N-acetylneuraminic acid (Neu5Ac) in glycoproteins was established. The present method utilizes a simplified hydrolysis-purification procedure consisting of mild acid hydrolysis (25 mM trifluoroacetic acid for 2h at 80 degrees C) to release Neu5Ac and ultrafiltration on Centricon-3 membrane to remove the obtained asialoglycoproteins and other macromolecules present in biologic samples. Derivatization with benzoic anhydride at 80 degrees C for 20 min resulted in complete conversion of Neu5Ac to per-O-benzoylated Neu5Ac. CZE analysis was performed using the operating buffer 25mM phosphate, pH 3.5, containing 50% (v/v) acetonitrile as organic modifier at 30 kV, and detection of the per-O-benzoylated Neu5Ac at 231 nm. The method showed excellent repeatability (RDS<1.98%) and a linearity range from 5 microg/mL to 5mg/mL with a detection limit of 2 microM. Application of the method to microanalysis of human alpha(1)-acid glycoprotein and blood serum samples showed excellent agreement with previously published values, suggesting a high precision for the developed CZE method.     

High-performance liquid chromatographic analysis of dextromethorphan, guaifenesin and benzoate in a cough syrup for stability testing

A method has been developed for the analysis of a cough syrup containing dextromethorphan, guaifenesin, benzoic acid, saccharin and other components. Forced degradation was also studied to demonstrate that the method could be employed during a stability study of the syrup. Final conditions were phosphate buffer (25 mM, pH 2.8) with triethylamine (TEA)-acetonitrile (75:25, v/v). In such conditions, all the actives, excipients and degradation products were baseline resolved in less than 14 min, and different wavelengths were used for the different analytes and related compounds.     

Nonaqueous affinity capillary electrophoresis investigation of small molecule molecular recognition

The conditional binding constants for a bis-guanidinium-like receptor and a series of dicarboxylate ligands have been determined in two buffer/solvent systems, namely 25 mM ammonium acetate/1% acetic acid in acetonitrile/methanol (7:3 v:v) and 30 mM N-methyl morpholine/15 mM methanesulfonic acid in acetonitrile/methanol (9:1 v:v). The latter buffer has not been applied before in capillary electrophoresis. The binding constants in both solvent systems decrease as the dicarboxylate length increases. The binding constants are larger in the less competitive N-methyl morpholine buffer. The dicarboxylates associate only weakly with a dicationic analog of the receptor, p-xylyl trimethylammonium, which is not a hydrogen bond donor.