Lateef Ahmad

A new HPLC method for the simultaneous determination of ascorbic acid and aminothiols in human plasma and erythrocytes using electrochemical detection

A new, simple, economical and validated high-performance liquid chromatography linked with electrochemical detector (HPLC-ECD) method has been developed and optimized for different experimental parameters to analyze the most common monothiols and disulfide (cystine, cysteine, homocysteine, methionine, reduced (GSH) and oxidized glutathione (GSSG)) and ascorbic acid present in human plasma and erythrocytes using dopamine as internal standard (IS). Complete separation of all the targets analytes and IS at 35°C on Discovery HS C18 RP column (250 mm × 4.6mm, 5 μm) was achieved using 0.05% TFA:methanol (97:3, v/v) as a mobile phase pumped at the rate of 0.6 ml min(-1) using electrochemical detector in DC mode at the detector potential of 900 mV. The limits of detection (3 S/N) and limits of quantification (10 S/N) of the studied compounds were evaluated using dilution method. The proposed method was validated according to standard guidelines and optimization of various experimental parameters and chromatographic conditions was carried out. The optimized and validated HPLC-ECD method was successfully applied for the determination of the abovementioned compounds in human plasma and erythrocytes. The method will be quite suitable for the determination of plasma and erythrocyte profile of ascorbic acid and aminothiols in oxidative stress and other basic research studies.

Simultaneous determination of rosuvastatin and atorvastatin in human serum using RP-HPLC/UV detection: Method development, validation and optimization of various experimental parameters

A novel, precise, accurate and rapid isocratic reversed-phase high performance liquid chromatographic/ultraviolet (RP-HPLC/UV) method was developed, optimized and validated for simultaneous determination of rosuvastatin and atorvastatin in human serum using naproxen sodium as an internal standard. Effect of different experimental parameters and various particulate columns on the analysis of these analytes was evaluated. The method showed adequate separation for rosuvastatin and atorvastatin and best resolution was achieved with Brownlee analytical C18 column (150×4.6 mm, 5 μm) using methanol-water (68:32, v/v; pH adjusted to 3.0 with trifluoroacetic acid) as a mobile phase at a flow rate of 1.5 ml/min and wavelength of 241 nm. The calibration curves were linear over the concentration ranges of 2.0-256 ng/ml for rosuvastatin and 3.0-384 ng/ml for atorvastatin. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for rosuvastatin were 0.6 and 2.0 ng/ml while for atorvastatin were 1.0 and 3.0ng/ml, respectively. All the analytes were separated in less than 7.0 min. The proposed method could be applied for routine laboratory analysis of rosuvastatin and atorvastatin in human serum samples, pharmaceutical formulations, drug-drug interaction studies and pharmacokinetics studies.

Simultaneous determination of timolol maleate, rosuvastatin calcium and diclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV.

A novel HPLC-UV method was developed for the simultaneous determination of timolol (TM), rosuvastatin (RST), and diclofenac sodium (DS) in pharmaceuticals, human plasma and aqueous humor using naproxen sodium as internal standard (IS). The target compounds were analyzed on Hypersil BDS C(18) column (250 mm × 4.6 mm, 5 μm), applying 0.2% triethylamine (TEA) and acetonitrile (ACN) (40:60, v/v), in isocratic mode as mobile phase, pH 2.75 adjusted with 85% phosphoric acid at a flow rate of 1 ml/min. The column oven temperature was kept at 45°C and the peak response was monitored at 284 nm after injecting a 50 μl sample into HPLC system. The direct liquid-liquid extraction procedure was applied to human plasma and bovine aqueous humor samples using mobile phase as an extraction solvent after deproteination with methanol. The different HPLC experimental parameters were optimized and the method was validated according to standard guidelines. The recoveries of the suggested method in human plasma were 98.72, 96.04, and 95.14%, for TM, RST, and DS, while in aqueous humor were 94.99, and 98.23%, for TM, and DS, respectively. The LOD values were found to be 0.800, 0.500, and 0.250 ng/ml, for TM, RST, and DS, respectively, while their respective LOQ values were 2.00, 1.50, and 1.00 ng/ml. The co-efficient of variation (CV) were in the range of 0.1492-1.1729% and 1.0516-4.0104%, for intra-day and inter-day studies, respectively. The method was found accurate in human plasma and bovine aqueous humor and will be applied for the quantification of these compounds in plasma, and aqueous humor samples using animal models and in pharmaceuticals.

An optimized and validated RP-HPLC/UV detection method for simultaneous determination of all-trans-Retinol (Vitamin A) and α-Tocopherol (Vitamin E) in human serum : Comparison of different particulate reversed-phase HPLC columns

A novel, simple and fast reversed-phase HPLC/UV method was developed, optimized for various chromatographic conditions, and validated according to international guidelines for simultaneous determination of all-trans-retinol and alpha-tocopherol in human serum using retinyl acetate as internal standard in the concentration of 0.5 microg/ml. A liquid-phase extraction was applied to the 250 microl of serum with n-hexane-dichloromethane mixture (70:30, v/v), in two steps, using ethanol-methanol mixture (95:5, v/v) for protein precipitation and BHT (butylated hydroxy toluene) as stabilizer for sample preparation. Both analytes were analyzed on Kromasil 100 C(18) column (150 mm x 4.6 mm, 5 microm), Brownlee analytical (Perkin Elmer) C(18) column (150 mm x 4.6 mm, 5 microm), and Supelco (Supelcosil) LC-18 column (150 mm x 3 mm, 3 microm), protected by a Perkin Elmer C(18) (30 mm x 4.6 mm, 10 microm; Norwalk, USA) pre-column guard cartridge, at 292 nm wavelength, using methanol-water (99:1, v/v), in isocratic mode as mobile phase applied at flow rate of 1.5 ml/min and 1 ml/min for both 5 microm and 3 microm columns, respectively. Complete separation of all the analytes was achieved in 3 and 6 min on 3 microm and 5 microm columns, respectively by injecting 20 microl of sample into the HPLC system by autosampler, keeping column oven temperature at 25 degrees C. Different particulate reversed-phase chromatographic columns were evaluated in order to select the best column in terms of sensitivity, selectivity, resolution and short run time of both the analytes and it was concluded that 3 microm columns are better to be used in clinical set up as well as in laboratories for the separation of these analytes in a shorter time as compared with 5 microm columns. The method was validated and applied for the analysis of all-trans-retinol and alpha-tocopherol in the serum of human volunteers.

Simultaneous determination of cefdinir and cefixime in human plasma by RP-HPLC/UV detection method: Method development, optimization, validation, and its application to a pharmacokinetic study

A novel isocratic reversed-phase high performance liquid-chromatography/ultraviolet detection method for simultaneous determination of cefdinir and cefixime in human plasma was developed and validated after optimization of various chromatographic conditions and other experimental parameters. Sample preparation based on a simple extraction procedure consisting of deproteination and extraction with 3 parts of 6% trichloroacetic acid aqueous solution followed by volume make up with the aqueous component of the mobile phase obtained best recoveries of the two analytes. Samples were separated on a Supelco Discovery HS C(18) (150 mm × 4.6 mm, 5 μm) analytical column protected by a Perkin Elmer C(18) (30 mm × 4.6 mm, 10 μm) guard cartridge. The mobile phase, methanol/acetonitrile (50/50, v/v):0.05% trifluoroacetic acid (19:81, v/v), operated at 50°C column oven temperature was pumped at a flow rate of 2.0 mL min(-1) and the column eluents were monitored at a wavelength of 285 nm. When Sample was injected into the Perkin Elmer high performance liquid-chromatography system through Rheodyne manual (or auto-sampler) injector equipped with 20 μL loop, separation was achieved within 4 min. The present method demonstrated acceptable values for selectivity, linearity within the expected concentration range (0.004-5.0 μg mL(-1); r(2)>0.999 for both analytes), recovery (>95% for cefdinir and >96% for cefixime), precision (%RSD<2.0 for cefdinir and <2.2 for cefixime), sensitivity (limit of detection: 1 ng mL(-1) and lower limit of quantification: 4 ng mL(-1) for both analytes), stability of solutions, and robustness. The method was efficiently applied to a pharmacokinetic study in healthy volunteers.

Determination of lipoic acid in human plasma by HPLC-ECD using liquid-liquid and solid-phase extraction: method development, validation and optimization of experimental parameters

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001-10μg/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250μl) was carried out with a simple one step liquid-liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40°C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30°C on Discovery HS C18 RP column (250mm×4.6mm, 5μm) was achieved in 6min using 0.05M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5ml/min using electrochemical detector in DC mode at the detector potential of 1.0V. The limit of detection and limit of quantification of lipoic acid were 200pg/ml and 1ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50pg/ml, respectively. The absolute recoveries of lipoic acid with liquid-liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5μg/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases.

Development and evaluation of diclofenac sodium thermorevesible subcutaneous drug delivery system

The objective of current work was to develop and evaluate thermoreversible subcutaneous drug delivery system for diclofenac sodium. The poloxamer 407, methyl cellulose, hydroxypropyl methyl cellulose and polyethylene glycol were used alone and in combination in different ratios to design the delivery system. The physical properties like Tsol-gel, viscosity, clarity of solution and gel were evaluated. The in vitro release of the drug delivery system was evaluated using membrane less method and the drug release kinetics and mechanism was predicted by applying various mathematical models to the in vitro dissolution data. Rabbits were used as in vivo model following subcutaneous injection to predict various pharmacokinetics parameters by applying Pk-Summit software. The in vitro and in vivo data revealed that the system consisting of the poloxamer 407 in concentration of 20% (DP20) was the most capable formulation for extending the drug release and maintaining therapeutic blood level of DS for longer duration (144 h). The data obtained for drug content after autoclaving the solutions indicate that autoclaving results in 6% degradation of DS. The data also suggested that the studied polymers poloxamer, MC and PG are good candidate to extend the drug release possessing a unique thermoreversible property.

OPTIMIZATION AND VALIDATION OF HPLC-UV METHOD FOR SIMULTANEOUS DETERMINATION OF OMEPRAZOLE AND ITS METABOLITES IN HUMAN PLASMA: EFFECTS OF VARIOUS EXPERIMENTAL CONDITIONS AND PARAMETERS

A simple and economical reversed phase HPLC-UV method was developed for omeprazole (OMP) and its two main metabolites, 5-Hydroxy omeprazole (5-OH-OMP) and omeprazole sulfone (OMP-S) in human plasma using pantoprazole as the internal standard. After optimization of various chromatographic conditions and experimental parameters, the method was validated according to standard guidelines. The separation of all the analytes was achieved on a Supelco C18 column using methanol and phosphate buffer (pH 7.2) in the ratio of (42:58, v/v) as the mobile phase in isocratic mode at a flow rate of 0.8 mL/min and wavelength of 302 nm, keeping column oven temperature at 45°C. Extraction of analytes from plasma was achieved through precipitation method followed by injecting a sample volume of 20 µL into the HPLC system. The limits of detection were 3, 1.5, and 1.3 ng/mL and limits of quantification were 10, 5, and 5 ng /mL for 5-0H-OMP, OMP-S, and OMP, respectively. The method was also successfully applied for the determination of S-isomer of omeprazole (esomeprazole) in human plasma.

Enhancement of dissolution rate of class II drugs (Hydrochlorothiazide); a comparative study of the two novel approaches; solid dispersion and liqui-solid techniques

Liqui-solid technique and solid dispersion formation are two novel approaches for enhancement of dissolution rate of BCS class II drugs. Liqui-solid compact converts a liquid drug or drug solution into a free flowing powder with enhanced dissolution rate. In case of solid dispersion drug is molecularly dispersed in a hydrophilic polymer in solid state. In the present study, Liqui-solid and solid dispersion techniques were applied to enhance the dissolution of the Hydrochlorothiazide. Three formulations of Hydrochlorothiazide were prepared by liqui-solid technique using micro crystalline cellulose as carrier material and colloidal silicon dioxide as coating material. Water, poly ethylene glycol-400 and Tween-60 were used as solvent system. Solid dispersions of Hydrochlorothiazide were prepared by solvent fusion method using PEG-4000 as carrier polymer. Tablets were subjected to evaluation of various physical and chemical characteristics. Dissolution profiles of tablets prepared by the novel techniques were compared with marketed conventional tablets. Model independent techniques including similarity factor, dissimilarity factor and dissolution efficiency were applied for comparison of dissolution profiles. The results obtained indicated that liqui-solid compact formulations were more effective in enhancing the dissolution rate compared with solid dispersion technique. The liqui-solid compacts improved the dissolution rate up to 95% while the solid dispersion increased it to 88%.

Development of a method to measure free and bound ropivacaine in human plasma using equilibrium dialysis and Hydrophilic interaction chromatography coupled to high resolution mass spectrometry

The pharmacodynamics of absorption of local anaesthetics used during surgical procedures into tissues is governed by the amount of free drug in plasma. Toxicity may occur during continuous infusions if the levels of free drug become too high which may occur if the binding capacity of the α-1-acid glycoprotein present in plasma is exceeded. In order to monitor this a method was developed for the determination of the amount of free and bound ropivacaine in human plasma during knee and hip surgery. Rapid equilibrium dialysis units were used to separate free and bound drug then protein and buffer salts were removed by solvent precipitation. Analysis was carried out using a ZICHILIC HPLC column interfaced with an LTQ Orbitrap mass spectrometer. The following extracted ion ranges ([M+H](+)) were monitored: m/z 275.21-275.22 for ropivacaine and m/z 235.175-235.185 for lidocaine. The method was calibrated by spiking ropivacaine, and a fixed amount of lidocaine as internal standard, into plasma over the range 0.01-1.5 µg/ml. The equation of the line was y=0.886x±4.2% (n=2), forcing the curve through zero since blank plasma was free of the analyte. The values obtained for the accuracy and precision of the analysis of plasma spiked at 0.03 µg/ml and 1.5 µg/ml were 93.2%±2.8% and 95.4%±1.5% respectively (n=5). Repeat analysis of a patient sample for free and bound drug gave the following values for levels of ropivacaine: bound 1.63 µg/ml±1.48%, unbound 0.0671 µg/ml±1.68% (n=5).