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A simple, rapid and sensitive high performance liquid chromatographic method was developed for the separation and quantification of positional isomers of zafirlukast in bulk drugs and dosage forms using a chiral column. Elution time was 20 min in normal phase mode and ultra violet detection was carried out at 240 nm. Efficient separation was achieved on an immobilized amylose-based Chiralpak-IA column using n-hexane/ethanol/trifluoroacetic acid/diethyl amine (65:35:0.1:0.1, v/v) as the mobile phase. Resolutions between ortho, meta and para isomers of zafirlukast were found to be >3.0. The active pharmaceutical ingredient was extracted from tablets using tetrahydrofuran. The calibration graphs for meta and para isomers of zafirlukast were linear (r 2 > 0.999) when ranging from the limit of quantitation to 0.3%. The method showed excellent recoveries for both zafirlukast isomers identified in bulk and formulated products. The test solution was found to be stable in the mobile phase for 48 h after preparation. The developed LC method was validated with respect to linearity, accuracy, precision and robustness.

A novel liquid chromatographic method for analysis three potential impurities in brimonidine tartrate drug substance has been developed and validated. Efficient chromatographic separation was achieved on a C8 column (250 mm × 4.6 mm, 5-μm particles) with a simple mobile-phase gradient at a flow rate of 1.0 mL min−1. Quantification was achieved by use of ultraviolet detection at 248 nm. Resolution between brimonidine tartrate and its three potential impurities was greater than 3.0. Regression analysis showed the r value (correlation coefficient) was >0.999 for brimonidine and its three impurities. The method was capable of detecting all three impurities of brimonidine tartrate at levels below 0.07 μg in a test concentration of brimonidine tartrate of 1.0 mg mL−1 and for an injection volume of 10 μL. A solution of brimonidine tartrate in acetonitrile–water 2:8 (v/v) was stable for 48 h. The drug was subjected to stress conditions as prescribed by the ICH. Degradation was found to occur slightly under oxidative stress conditions but the drug was stable to aqueous, acidic, and basic hydrolysis, and photolytic and thermal stress. The assay of the stressed samples was calculated relative to a qualified reference standard and the mass balance was found close to 99.8%. The method was validated for linearity, accuracy, precision, and robustness.

A novel liquid chromatographic method has been developed, and validated for the determination of tolterodine tartarate, for its potential three impurities in drug substances and drug products. Efficient chromatographic separation was achieved on a C8 stationary phase (150 × 4.6 mm, 3.5 μm particles) with a simple mobile phase combination delivered in an isocratic mode at a flow rate of 0.8 mL min−1 and quantitation was carried out using ultraviolet detection. Microwave assisted degradation procedure was employed for stress testing studies in addition to the conventional way of a refluxing method. The results of both studies were compared. In the developed LC method, the resolution between tolterodine and its three potential impurities was found to be greater than 2.0. Regression analysis shows an r value (correlation coefficient) greater than 0.999 for tolterodine and for its three impurities. This method was capable to detect all three impurities of tolterodine at a level below 0.0038% with respect to a test concentration of 0.5 mg mL−1 for a 10 μL injection volume. The inter- and intra-day precisions for all three impurities and for tolterodine were found to be within 1.1% RSD at its specification level. The method has shown good, consistent recoveries for tolterodine (98.9–101.6%) and for its three impurities (94.5–103.0%). The test solution was found to be stable in the diluent for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation, as prescribed by ICH. Degradation was found to occur in alkaline stress condition, while the drug was stable to water hydrolysis, acid hydrolysis, oxidative stress, photolytic and thermal stress. The assay of stressed samples was calculated against a qualified reference standard and the mass balance was found close to 99.5%. Microwave degradations were very fast and comparable to the conventional way of the refluxing method. Robustness studies were carried out and suggested that system suitability parameters were unaffected by small changes in critical factors. The validated method was successfully applied for the determination of tolterodine tartarate in drug substances and drug products.

An isocratic reverse phase liquid chromatographic (RP-LC) assay method has been developed for the quantitative determination of nateglinide and its related components namely imp-1 and imp-2 in bulk drug and in pharmaceutical dosage form, used for the treatment of type II diabetes mellitus. The developed method is stability indicating and also can be used for stability testing. The chromatographic separation was achieved on C-8, 150 × 4.6 mm, 3.5 μm stationary phase. The LC method employs solution A as mobile phase. Solution A contains a mixture of phosphate buffer pH 3.0: acetonitrile (50:50 v/v). The flow rate was 1.0 mL min−1 and the detection wavelength was 210 nm. In the developed LC method the resolution between nateglinide and its potential impurities namely imp-1 and imp-2 was found to be greater than 5.0. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid medium, alkaline medium and oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.2%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.

A new simple isocratic chiral RP-LC method has been developed for the separation and quantification of the enantiomer of (R,R)-tadalafil in bulk drugs and dosage forms with an elution time of about 20 min. Chromatographic separation of (R,R)-tadalafil and its enantiomer was achieved on a bonded macro cyclic glycopeptide stationary phase. The method resolves the (R,R)-tadalafil and its enantiomer with a resolution (Rs) greater than 2.4 in the developed chiral RP-LC. The mobile phase used for the separation and quantification of (R,R)-tadalafil and its enantiomer involves a simple mixture of reverse phase solvents and the cost of analysis was drastically decreased. The test concentration is 0.4 mg mL−1 in the mobile phase. This method is capable of detecting the enantiomer of (R,R)-tadalafil up to 0.0048 μg wrt test concentration 400 μg mL−1 for a 20 μL injection volume. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. There was no interference of degradants with (R,R)-tadalafil and its enantiomer in the developed method. The developed chiral RP-LC method was validated with respect to linearity, accuracy, precision and robustness. The percentage recovery for the enantiomer of (R,R)-tadalafil in bulk drug samples and in dosage forms ranged from 97.0 to 102.5%. The test solution was found to be stable in the mobile phase for 48 h after preparation.

A rapid, sensitive, and simple HPLC–MS–MS method, with electro-spray ionization and cetirizine as internal standard (IS), has been developed and validated for simultaneous quantification of fexofenadine and pseudoephedrine in human plasma. The analytes were isolated from plasma by solid-phase extraction (SPE) on Oasis HLB cartridges. The compounds were chromatographed on an RP 18 column with a mixture of ammonium acetate (10 mm, pH 6.4) and methanol as mobile phase. Quantification of the analytes was based on multiple reaction monitoring (MRM) of precursor-to-product ion pairs m/z 502 → 466 for fexofenadine, m/z 166 → 148 for pseudoephedrine, and m/z 389 → 201 for cetirizine. The linear calibration range for both analytes was 2–1,700 ng mL−1 (r = 0.995), based on analysis of 0.1 mL plasma. Extraction recovery was 91.5 and 80.88% for fexofenadine and pseudoephedrine, respectively. The method was suitable for analysis of human plasma samples obtained 72 h after administration of a drug containing both fexofenadine and pseudoephedrine.

Background Caspase-3 inhibition has been demonstrated to be therapeutically effective in moderating excessive programmed cell death. Interest in caspase-3 as a therapeutic target has led many to pursue the development of inhibitors. To date, only a few series of non-peptide inhibitors have been described, and these have limitations on their drug-like properties. Methods Here, we report the screening of 70 novel small molecules against the caspase-3 enzyme which belongs to four different series (indole fluoromethylketone, indole difluoro and tetrafluorophenoxymethylketone, and oxalamide). Selected molecules were subjected for counter-screening, cell-based, ADME/PK assays in order to understand the potency and drug-like properties. Results The screening yielded series of hits with IC50 values ranging from 0.11 to 10 μM with reasonable SAR, irreversible mode of inhibition, and reasonable selectivity against other proteases including caspase-1, cathepsin B and D, and thrombin. On the basis of in vitro profile, the selected molecules were evaluated for their drug-like properties. Among the compounds evaluated, compound 3D exhibited good solubility, low permeability, interaction with efflux pump, and low potential for CYP450 drug-drug interaction. After intravenous administration, compound 3D showed low clearance (588 ml/hr/kg), medium volume of distribution, and good oral bioavailability (90%). Conclusions These results support further advancement of compound 3D in different apoptotic models to develop as a new anti-apoptotic agent in relevant disease conditions.