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Summary
The evaluation of several derivatization procedures for the gas chromatographic analysis of selected anticancer pharmaceuticals (cyclophosphamide, iphosphamide, flutamide, chlorambucil, and melphalan) in the presence of tricyclic antidepressants was carried out. Among the methods, concerning trimethylsilylation, tert-butyldimethylsilylation, and methylation, the most useful was methylation using (trimethylsilyl)diazomethane (TMSD), which is a safe alternative for the common reaction with diazomethane. Most of the anticancer drugs were unstable during derivatization using silylating agents, while antidepressants were stable in all tested conditions. Melphalan was the only compound, for which the results of all tested procedures were not satisfying. The TMSD-based procedure was validated, giving the results possibly suitable for the screening purposes in contaminated environmental samples.
Summary
A stability-indicating gradient reverse-phase liquid chromatographic method was developed for the quantitative determination of process-related impurities and forced degradation products of oxcarbazepine in pharmaceutical formulation. The method was developed by using Inertsil cyano (250 × 4.6 mm) 5 μm column with mobile phase containing a gradient mixture of solvent A (0.01 M sodium dihydrogen phosphate, pH adjusted to 2.7 with orthophosphoric acid and acetonitrile in the ratio of 80:20 v/v) and B (50:40:10 v/v/v mixture of acetonitrile, water, and methanol). The flow rate of mobile phase was 1.0 mL min−1. Column temperature was maintained at 25°C and detection wavelength at 220 nm. Developed reverse-phase high-performance liquid chromatography (RP-HPLC) method can adequately separate and quantitate five impurities of oxcarbazepine, namely imp-A, imp-B, imp-C, imp-D, and imp-E. Oxcarbazepine was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Oxcarbazepine was found to degrade significantly in acid, base, and oxidative stress conditions. The degradation products were well resolved from oxcarbazepine and its impurities. The developed method was validated as per International Conference on Harmonization (ICH) guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, and robustness.
Summary
A simple, rapid, and sensitive high-performance liquid chromatography method was developed and validated for the simultaneous determination and quantification of fusidic acid and steroids (prednisone, betamethasone valerate, hydrocortisone acetate, and dexamethasone sodium) from bulk drugs and human plasma. A RP-HPLC, operated at ambient temperature, was equipped with a UV detector for monitoring the effluents at 235 nm. The mobile phase consisted of methanol, acetonitrile, and 0.05 M phosphoric acid (10:60:30, v/v/v), and separation was achieved on a Medeterrane, C18 (5 μm, 12.5 × 0.46 mm) column at a flow rate of 1.7 mL min−1. Calibration curves were linear over concentration range 0.625–10 μg mL−1 with correlation coefficient (r 2) greater than 0.9999. The coefficient of variation (CV) and relative error (RE) for intra- and interassay were <2% and <1%, respectively. Interference of other already administered common medicaments, such as aspirin, paracetamol, caffeine, nicotine, and other plasma components, were not found.
Summary
Ezetimibe is the first in a new class of antihypercholesterolemic drugs. Since it has not long been available on the market, many of its properties may still be revealed. Analytical methods for its determination are scarce, especially regarding serum samples. A simple, fast, and effective high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the determination of ezetimibe concentration in human serum has therefore been developed. Three mobile phases were analysed, and original modifications to the concentration and flow parameters were made. Of five potential internal standards (IS), only nitrendipine was found to be suitable. The analytical wavelength was chosen based on the absorption spectrum of ezetimibe in the mobile phase. Finally, an extraction analysis was performed using two different solvents, and the extrahent volume was optimised. The final method developed was as follows. Single extraction of 1 mL serum sample, spiked with IS, was performed using 10 mL of methyl-t-butyl ether. Separation was obtained at ambient temperature on a Waters C18 Symmetry Shield (4.6 mm × 250 mm, 5 μm) column. The isocratic mobile phase was composed of acetonitrile and 0.1 M ammonium acetate aqueous solution 55:45 (v/v), set at flow rate of 0.75 mL min−1. Ezetimibe was detected at a wavelength of 232 nm after 5.49 min, and the IS was detected at 8.05 min. The developed method has been validated according to ICH standards. It was found to be specific, precise, accurate and linear over the range 10–800 ng mL−1 with R 2 > 0.998, and detection and quantification limits of 4.60 ng mL−1 and 13.94 ng mL−1, respectively. The method has been applied to clinical serum samples. The developed technique allowed for successful in vivo assessment of ezetimibe concentrations in samples obtained from hypercholesterolemia patients who are chronically receiving the drug.
Summary
A new high-performance liquid chromatography (HPLC) method has been developed and validated for determination of enantiomeric purity of thiazolidine-2-carboxylic acid within a short run time of less than 10 min. The method was based on pre-column derivatization of thiazolidine-2-carboxylic acid with aniline, and complete separation of enantiomers has been achieved on a Chiralcel OD-H analytical column (250 × 4.6 mm) using n-hexane-isopropanol (85:15 v/v) as mobile phase at a flow rate of 1.0 mL min−1 under UV and optical rotation (OR) detection. Detection wavelength was set at 254 nm. Then the effects of mobile phase and temperature on enantioselectivity were further evaluated. The method was validated with respect to precision, accuracy, linearity, limit of detection (LOD), limit of quantification (LOQ), and robustness. The recoveries were between 98.5 and 101.3% with percentage relative standard deviation less than 1.16%. The LOD and LOQ for the aniline derivatives of (+)-thiazolidine-2-carboxylic acid were 4.9 and 16.4 μg mL−1 and for the aniline derivatives (−)-thiazolidine-2-carboxylic acid were 5.1 and 17.2 μg mL−1, respectively.
Summary
A simple, rapid, and sensitive reversed-phase HPLC method was developed and validated for determination of metronidazole and tinidazole in human plasma samples under identical chromatographic conditions. This method involves liquid-liquid extraction using chloroform: isopropylalcohol (95:5). Chromatographic separation was performed using a μ-bondapack C18 (250 mm × 4.6 mm) column. The mobile phase consisted of potassium dihydrogen phosphate solution (0.005 M)/acetonitrile (80/20 v/v). The final pH of the mobile phase was adjusted to 4 ± 0.1 with orthophosphoric acid. The calibration curves were linear over the concentration range 0.1–15 μg/mL for metronidazole and tinidazole with the detection limit of 30 ng/mL. Within- and between-day precision and accuracy did not exceed 9.83% and 10.48%, respectively. Metronidazole and tinidazole were found to be stable in plasma samples with no evidence of degradation during 3 freeze-thaw cycles and 3 months storage in −70 °C. The current validated bio-analytical method was finally applied in bioequivalence studies of two different metronidazole and tinidazole products according to a standard two-way cross-over design with a two-week washout period. No statistically significant difference was observed between the logarithmically transformed AUC0-∞ and C max values. Therefore, generic products were considered bioequivalent with those of standards which could be used interchangeably.
Summary
Dispersive liquid-liquid microextraction in combination with an in situ derivatization is suggested for parabens sampling and preconcentration. The derivatization was carried out with acetic anhydride under alkaline conditions maintained using di-potassium hydrogen phosphate. The effects of an extraction solvent type, extraction and disperser solvents volume, extraction time, and ionic strength of the solution on the extraction efficiency were investigated. Chlorobenzene containing n-hexadecane as internal standard was used as an extracting solvent and acetone was used as a disperser solvent. The calibration graphs were linear up to 10 mg mL−1, correlation coefficients were 0.997–0.999, enrichment factors were from 70 for methylparaben to 210 for butylparaben, and detection limits were 22, 4.2, 3.3, and 2.5 µg L−1 for methylparaben, ethylparaben, propylparaben, and butylparaben, respectively. Repeatabilities of the results were acceptable with relative standard deviations up to 11%. A possibility to apply the proposed method for parabens determination in water samples was demonstrated.
Summary
A simple, rapid, precise, and accurate, stability-indicating reversed phase high performance liquid chromatographic method was developed and validated for simultaneous determination of metformin HCl and repaglinide. The chromatographic separation was achieved on YMC Pack AM ODS (5 μm, 250 mm length × 4.6 mm i.d.) column at a detector wavelength of 210 nm, using an isocratic mobile phase consisting of methanol and 10 mM potassium dihydrogen phosphate buffer (pH 2.5) in a ratio of 70:30 v/v at a flow rate of 1 mL min−1. The retention times for metformin and repaglinide were found to be 2.6 and 11.3 min, respectively. The drugs were exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. Validation of the method was carried out as per International Conference on Harmonization (ICH) guidelines. Linearity was established for metformin and repaglinide in the range of 5–200 μg mL−1 and 1–200 μg mL−1, respectively. The limits of detection were 0.3 μg mL−1 and 0.13 μg mL−1 for metformin and repaglinide, respectively. The method was found to be specific and stability-indicating as no interfering peaks of degradants and excipients were observed. The proposed method is hence suitable for application in quality-control laboratories for quantitative analysis of both the drugs individually and in combination, since it is simple and rapid with good accuracy and precision.
Summary
A simple and sensitive method of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed for the determination of icariin in capsules by precolumn chelation with aluminum. In order to obtain a stable fluorescence signal, the reaction conditions of the fluorescent chelation complex between icariin and aluminum were investigated in detail. Chromatography was carried out on an Agilent Zorbax Extend C18 column (150 mm × 4.6 mm, 5.0 μm) using methanol as mobile phase at a flow rate of 1.0 mL min−1. The excitation and emission wavelengths were set at 430 and 480 nm, respectively. At optimum conditions, the calibration curve was linear in the concentration range from 0.010 to 100.0 μg mL−1 with the limit of detection of 3.5 ng mL−1 (S/N = 3). A comprehensive method was validated for precision and accuracy. The method described here has been successfully applied for the determination of the icariin content in a capsule with satisfactory results.
Summary
An efficient method used to separate five bioactive compounds from Gelidium amansii was optimized by the HCI software. The optimum composition of mobile phase for high-performance liquid chromatographic (HPLC) separation was obtained. The elution profiles were calculated by the polynomial theory based on the retention factor ln k = A + BF + CF 2 (F was the volume percentage of acetonitrile with 1.0% acetic acid); then, the theory was applied to calculate the elution profile in both isocratic and gradient modes by modifying different mobile phase conditions with HCI program. The calculated results of mobile phase condition suggested that acetonitrile-water (containing 1.0% acetic acid) with a linear gradient elution of 0∼30 min from 15:85 to 50:50 (v/v) was the optimized component. In the experimental conditions, the agreement between the experimental elution profiles and the calculated values of eluted concentration was relatively good.
