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.
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 Cmax values. Therefore, generic products were considered bioequivalent with those of standards which could be used interchangeably.
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.
In this research paper we describe validated high-performance liquid chromatographic (HPLC) and high-performance thin-layer chromatographic (HPTLC) methods for simultaneous analysis of tamsulosin hydrochloride and dutasteride in tablet formulations. HPLC was performed on a C18 column with 85:15 (υ/υ) methanol-0.02 m ammonium acetate buffer (pH 9.5, adjusted with triethylamine) as mobile phase. HPTLC was performed on aluminium foil-backed silica gel G60F254 layers with toluene-methanol-triethylamine 9:1.5:1 (υ/υ/υ) as mobile phase. In HPLC, quantification was achieved by photo diode-array (PDA) detection at 274 nm over the concentration range 1–20 μg mL−1 for both; mean recovery was 98.18 ± 0.698 and 99.94 ± 0.611% for TAM and DUTA, respectively. In HPTLC, quantification was achieved by UV detection at 280 nm over the concentration range 200–2000 ng per band for both; mean recovery was 99.66 ± 0.892 and 100.05 ± 1.012% for TAM and DUTA, respectively. These methods are simple, precise, and sensitive, and are suitable for simultaneous analysis of TAM and DUTA in tablet formulations.
A simple isocratic HPLC-UV assay for measurement of total and free melphalan concentrations in human plasma is described. Samples were prepared by methanol precipitation (total melphalan assay) and ultrafiltration (free melphalan assay). On a 25 cm × 4.6 mm C18 column with 0.016 m mixed soldium phosphate citrate buffer (pH 3.75)-acetonitrile 87:13 as mobile phase, at a flow rate of 1 mL min−1, the retention time of melphalan was 11.5 min. Detection was at 254 nm. For total melphalan assay response was a linear function of concentration up to 40 μg mL−1, with excellent interday precision (<6% for 0.5–40 μg mL−1 melphalan), accuracy (<2% deviation from the true concentration), and recovery (91–110%). For free melphalan assay response was a linear function of concentration up to 2.5 μg mL−1, with good precision (<11% for 0.7–2.5 μg mL−1 melphalan) and recovery (89–93%). Detection limits were 0.1 μg mL−1 and 0.05 μg mL−1 for total and free melphalan assays, respectively. The assays were clinically applied in a study of myeloma patients.
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.
A stability-indicating reversed-phase high-performance liquid chromatographic method has been developed for analysis of gemifloxacin in tablet formulations. When the drug was subjected to forced degradation under acidic, basic, thermal, oxidative, and photolytic conditions, the degradation products produced were successfully separated on a 250 mm × 4.6 mm, 5-μm particle, C18 column with ammonium acetate buffer (pH 2.7; 0.05 m)-acetonitrile 70:30 (υ/υ) as mobile phase at a flow rate of 0.7 mL min−1. Diode-array detection was performed at 272 nm. The method was validated in accordance with ICH guidelines. Response was a linear function of concentration over the range 0.256–128 μg mL−1 (correlation coefficient 0.9990). The limits of detection and quantification were 10 and 30 ng mL−1, respectively. Separation of gemifloxacin from its stress-induced degradation products and excipients was adequate; resolution was >1.5 within 11 min. The purity index for the gemifloxacin peak after all types of stress was >0.999, indicating complete separation of the analyte peak from the degradation products. The method can therefore be regarded as stability-indicating. It is rapid, and suitable for purity and assay determination not only for routine quality control but also in stability studies.
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.
This paper reports development and validation of a new microemulsion liquid chromatographic (MELC) method for rapid screening of simvastatin and simvastatin acid in human plasma. Plasma samples were injected directly into the HPLC system after appropriate sample dilution with mobile phase. Separations were performed on a 4.6 mm × 150 mm, 5-μm particle, C18 column, with UV detection at 238 nm. The mobile phase was 0.5% (w/υ) diisopropyl ether, 1.0% (w/υ) sodium dodecylsulphate (SDS), 4.0% (w/v) n-butanol, and 94.5% (w/w) aqueous 25 mM disodium hydrogen phosphate, pH 7.0, at a flow rate of 1 mL min−1. The method was evaluated according to criteria stated in FDA bioanalytical method validation guidance. The unique approach applied in this paper enables direct analysis of simvastatin and simvastatin acid, so the method can be used to obtain reliable results in a rapid and simple way.
An isocratic reversed-phase high-performance liquid chromatographic (RPHPLC) method for analysis of irinotecan HCl has been developed and validated. Separation was achieved on a C18 column with potassium dihydrogen phosphate buffer (pH adjusted to 3.5 with orthophosphoric acid)-acetonitrile-methanol 55:25:20 (v/v) as mobile phase at a flow rate of 1.0 mL min−1. UV detection was performed at 254 nm. The method is simple, sensitive, rapid, and selective, and linear over the range 30–70 μg mL−1 for assay of irinotecan HCl. The precision of the assay method was below 1.0% RSD. Mean recovery was in the range 98.0–102.0%. Recovery of the active pharmaceutical ingredient from dosage forms ranged from 99.0 to 101.0. The method is useful for quality control in bulk manufacture and of the pharmaceutical formulation.