Fazli Nasir

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.

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.

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.

Application of SeDeM Expert system in formulation development of effervescent tablets by direct compression

The SeDeM expert system is a pre formulation tool applied for the prediction of the suitability of a material for direct compression. This innovative tool provides an index of good compressibility of the material indicating its aptitude to be compressed by direct compression. In the study the SeDeM expert system has been applied for the prediction of the behavior of the material to be used in the formulation of effervescent tablets by direct compression. Different formulations were developed on the basis of the results of the SeDeM expert system. Various parameters for the material as per the SeDeM expert system were determined according to their official and reported methods. Powder blend for different formulations was evaluated for their rheological properties while tablets were evaluated for various official and unofficial tests. Suitability of the material for direct compression was successfully predicted using the SeDeM expert system. Domperidone was found unsuitable for direct compression. During formulation all excipients responded as they were predicted as per the SeDeM expert system. Tablets produced using the resultant formulations were having sufficient mechanical strength, free of premature effervescence and were capable to be scaled up for commercial manufacturing.

Development and evaluation of pluronic- and methylcellulose-based thermoreversible drug delivery system for insulin

Abstract The objective of the current work was to develop and evaluate thermoreversible subcutaneous drug delivery system for Insulin. Thermoreversible in-situ gel system was developed and evaluated both in-vitro and in-vivo comprising of pluronic F-127 alone or in combination with methylcellulose in different ratios. The drug release kinetics and mechanism was predicted by applying various mathematical models to the in-vitro dissolution data. Rabbits were used as animal model following subcutaneous injection to predict various pharmacokinetic parameters by applying Pk-Summit® software. The in-vitro and in-vivo data revealed that the formulation IPM 15/3 consisting of the pluronic F-127 (15% w/v) and methylcellulose (3% w/v) was the most robust and capable formulation for extending the drug release and maintaining basal plasma insulin level between 10 and 40 µU/ml for 240 h (10 d).

Method development and validation for simultaneous determination of lumefantrine and its major metabolite, desbutyl lumefantrine in human plasma using RP-HPLC/UV detection

A simple, specific, precise and rapid RP-HPLC-UV method was developed for simultaneous determination of lumefantrine and its metabolite desbutyl lumefantrine in human plasma. Experimental parameters were optimized and the method was validated according to standard guidelines. The method showed adequate separation for lumefantrine and desbutyl lumefantrine and best resolution was achieved with Supelco Discovery HS C18 RP (150mm×4.6mm, 5μm) column using acetonitrile and 0.05% trifluroacetic acid (70:30, v/v) as a mobile phase pumped at a flow rate of 1.0ml/min and wavelength of 335nm. The method was linear over the concentration range of 10-12,000ng/ml. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for lumefantrine were 10.0 and 18.0ng/ml, while for desbutyl lumefantrine were 7.5 and 15.0ng/ml, respectively. The proposed method was efficiently applied for determination of lumefantrine and desbutyl lumefantrine concentrations in plasma samples for pharmacokinetic studies.

Anti-inflammatory study on crude methanol extract and different fractions of Eremostachys laciniata.

Context: Eremostachys laciniata (L.) Bunge (Lamiaceae), which has been reported as a rich source of flavonoids, is one of the rarely explored species of the genus Eremostachys. Objective: In this study, the crude methanol extract and different fractions of E. laciniata were investigated for in vivo anti-inflammatory properties. Material and methods: Shade-dried leaves of E. laciniata were exhaustively extracted by percolation with methanol (80%) to obtain 250 g of crude methanol extract (El), followed by fractionation with different organic solvents to get the n-hexane (Elh), chloroform (Elc), ethyl acetate (Ele), butanol (Elb), and water (Elw) fractions. An in vivo anti-inflammatory study of the crude extract and sub-crude fractions was carried out in rats using the carrageenan model. Results: The Ele fraction was found to be the most potent inhibitor of edema formation by inducing a maximum inhibitory effect of 74.2% at the 300 mg/kg dose, during 3 h post carrageenan injection. The El extract and Elc fraction also showed good anti-inflammatory properties at the same dose. Discussion: The demonstration of excellent anti-inflammatory activity by the plant chiefly concentrating in the Ele fraction and the appearance of peak activity in the latter phase of the experiment suggested the presence of relatively low-polar substances with arachidonic acid metabolite inhibition property. Conclusion: The plant may be an excellent source in the future for activity-guided isolation of important anti-inflammatory substances.