Prakash Katakam

Simultaneous Determination of Ciprofloxacin Hydrochloride and Dexamethasone Sodium Phosphate in Eye Drops by HPLC

A liquid chromatographic method was developed and validated for the simultaneous determination of ciprofloxacin hydrochloride and dexamethasone sodium phosphate in bulk and pharmaceutical formulations. Optimum separation was achieved in less than 5 min using a C18 column (250 mmx4.6 mm i.d, 5μ particle size) by isocratic elution. The mobile phase consisting of a mixture of mixed phosphate buffer (pH 4) and acetonitrile (65:35, v/v) was used. Column effluents were monitored at 254 nm at a flow rate of 1ml/min. Retention times of ciprofloxacin hydrochloride and dexamethasone sodium phosphate were 2.0 and 3.16 min respectively. The linearity of ciprofloxacin hydrochloride and dexamethasone sodium phosphate was in the range of 3-18 μg/ml and 1-6 μg/ml respectively. Developed method was economical in terms of the time taken and amount of solvent consumed for each analysis. The method was validated and successfully applied to the simultaneous determination of ciprofloxacin hydrochloride and dexamethasone sodium phosphate in bulk and pharmaceutical formulations.

Comparative Evaluation of Hypoglycemic Potentials of Eucalyptus Spp. Leaf Extracts and their Encapsulations for Controlled Delivery

Eucalyptus is well represented in different Pharmacopeias for its variant pharmacology and depicts a wide range of photochemicals like triterpenoids, flavonoids, polyphenols, gallotannins and macrocarpals both in its volatile and nonvolatile fractions. Hot aqueous leaf decoctions of Eucalyptus have been recommended as ‘herbal tea’ in different regions of world for its hypoglycemic potentials. However lack of definitive dosage formulations of Eucalyptus bioactive, side effects like nausea, vomiting, gastric irritation, organoleptic unacceptability have limited its application; besides data on toxicology and posology being inconsistent and variant. In the current research work firstly comparative evaluations of hypoglycemic potentials amongst three Eucalyptus spp. E. globulus, E. citriodora, E. camaldulensis have been done by in vitro a-glucosidase assay

Exploration of natural enzyme inhibitors with hypoglycemic potentials amongst Eucalyptus Spp. by in vitro assays.

AIM To investigate the presence and potency of natural enzyme inhibitors with hypoglycemic potentials amongst Eucalyptus Spp. by in vitro assays. METHODS The leaf extracts of the three different Eucalyptus species [E. globulus (EG), E. citriodora (EC), E. camaldulensis (ECA)] were subjected to in vitro assay procedures to explore the prevalence of natural enzyme inhibitors (NEIs) after preliminary qualitative and quantitative phytochemical evaluations, to study their inhibitory actions against the enzymes like α-amylase, α-glucosidase, aldose reductase, angiotensin converting enzyme and dipeptidyl peptidase 4 playing pathogenic roles in type 2 diabetes. The antioxidant potential and total antioxidant capacity of the species were also evaluated. RESULTS Major bioactive compounds like polyphenols (341.75 ± 3.63 to 496.85 ± 3.98) and flavonoids (4.89 ± 0.01 to 7.15 ± 0.02) were found in appreciable quantity in three species. Based on the IC50 values of the extracts under investigation, in all assays the effectivity was in the order of EG > ECA > EC. The results of the ferric reducing antioxidant power assay showed that the reducing ability of the species was also in the order of EG > ECA > EC. A strong correlation (R(2) = 0.81-0.99) was found between the phenolic contents and the inhibitory potentials of the extracts against the targeted enzymes. CONCLUSION These results show immense hypoglycemic potentiality of the Eucalyptus Spp. and a remarkable source of NEIs for a future phytotherapeutic approach in Type 2 diabetes.

Simultaneous determination of ezetimibe and simvastatin in rat plasma by stable-isotope dilution LC-ESI–MS/MS and its application to a pharmacokinetic study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed for simultaneous quantification of ezetimibe and simvastatin in rat plasma. The deuterium isotopes: ezetimibe d4 and simvastatin d6 were used as internal standards for ezetimibe and simvastatin, respectively. MS/MS detection involved a switch of electron spray ionization mode from negative to positive at retention time 3.01 min. Samples were extracted from plasma by liquid–liquid extraction using tertiary butyl methyl ether. Chromatographic separation was achieved with Agilent Eclipse XBD-C18 column using mobile phase that consisted of a mixture of ammonium acetate (pH4.5; 10 mM)–acetonitrile (25:75 v/v). The method was linear and validated over the concentration range of 0.2–40.0 ng/mL for simvastatin and 0.05–15.0 ng/mL for ezetimibe. The transitions selected were m/z 408.3→271.1 and m/z 412.0→275.10 for ezetimibe and ezetimibe d4, and m/z 419.30→285.20 and m/z 425.40→199.20 for simvastatin and simvastatin d6. Intra- and inter-batch precisions for ezetimibe were 1.6–14.8% and 2.1–13.4%; and for simvastatin 0.94–9.56% and 0.79–12%, respectively. The proposed method was sensitive, selective, precise and accurate for the quantification of ezetimibe and simvastatin simultaneously in rat plasma. The method was successfully applied to a pharmacokinetic study by oral co-administration of ezetimibe and simvastatin in SD rats.

Simultaneous Quantification of Mesalamine and Its Metabolite N-Acetyl Mesalamine in Human Plasma by LC-MS/MS and Its Application to a Bioequivalence Study

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used for simultaneous quantification of mesalamine and its metabolite N-acetyl mesalamine in human plasma with N-acetyl mesalamine D3 as an internal standard (IS). Chromatographic separation was performed on a Thermo, HyPURITY C18 (150 x 4.6 mm, 5 m) column with an isocratic mobile phase composed of 10 mM ammonium Original Research Article British Journal of Pharmaceutical Research, 4(13): 1568-1590, 2014 1569 acetate and methanol in the ratio of 85:15 (%v/v), at the flowrate of 0.6 mL/min. The drug, metabolite and internal standard were extracted by liquid-liquid extraction. The method was validated over a linear concentration range of 2-1500 ng/mL for mesalamine and 102000 ng/ml for N-acetyl mesalamine, which demonstrated intra and inter-day precision ranging from 1.60 to 8.63% and 2.14 to 8.67% for mesalamine and 0.99 to 5.67% and 1.72 to 4.89% for N-acetyl mesalamine respectively. Similarly, the intraand inter-day accuracy varied from 102.70 to 105.48% and 100.64 to 103.87% for mesalamine, 99.64 to 106.22% and 100.71 to 104.27% for N-acetyl mesalamine respectively. Both analytes were found to be stable throughout freeze–thawing cycles, bench top and postoperative stability studies. The method was successfully applied to support a bioequivalance study of healthy subjects.

Chemometrics optimized extraction procedures, phytosynergistic blending and in vitro screening of natural enzyme inhibitors amongst leaves of Tulsi, Banyan and Jamun

Background: Tulsi, Banyan, and Jamun are popular Indian medicinal plants with notable hypoglycemic potentials. Now the work reports chemo-profiling of the three species with in-vitro screening approach for natural enzyme inhibitors (NEIs) against enzymes pathogenic for type 2 diabetes. Further along with the chemometrics optimized extraction process technology, phyto-synergistic studies of the composite polyherbal blends have also been reported. Objective: Chemometrically optimized extraction procedures, ratios of polyherbal composites to achieve phyto-synergistic actions, and in-vitro screening of NEIs amongst leaves of Tulsi, Banyan, and Jamun. Materials and Methods: The extraction process parameters of the leaves of three plant species (Ficus benghalensis, Syzigium cumini and Ocimum sanctum) were optimized by rotatable central composite design of chemometrics so as to get maximal yield of bio-actives. Phyto-blends of three species were prepared so as to achieve synergistic antidiabetic and antioxidant potentials and the ratios were optimized by chemometrics. Next, for in vitro screening of natural enzyme inhibitors the individual leaf extracts as well as composite blends were subjected to assay procedures to see their inhibitory potentials against the enzymes pathogenic in type 2 diabetes. The antioxidant potentials were also estimated by DPPH radical scavenging, ABTS, FRAP and Dot Blot assay. Results: Considering response surface methodology studies and from the solutions obtained using desirability function, it was found that hydro-ethanolic or methanolic solvent ratio of 52.46 ± 1.6 and at a temperature of 20.17 ± 0.6 gave an optimum yield of polyphenols with minimal chlorophyll leaching. The species also showed the presence of glycosides, alkaloids, and saponins. Composites in the ratios of 1:1:1 and 1:1:2 gave synergistic effects in terms of polyphenol yield and anti-oxidant potentials. All composites (1:1:1, 1:2:1, 2:1:1, 1:1:2) showed synergistic anti-oxidant actions. Inhibitory activities against the targeted enzymes expressed in terms of IC50 values have shown that hydro-ethanolic extracts in all cases whether individual species or composites in varying ratios gave higher IC50 values thus showing greater effectivity. Conclusion: Current research provides the state-of-the-art of search of NEIs amongst three species by in-vitro assays which can be further utilized for bioactivity-guided isolations of such enzyme inhibitors. Further, it reports the optimized phyto-blend ratios so as to achieve synergistic anti-oxidative actions. SUMMARY The current research work focuses on the optimization of the extraction process parameters and the ratios of phyto-synergistic blends of the leaves of three common medicinal plants viz. banyan, jamun and tulsi by chemometrics. Qualitative and quantitative chemo profiling of the extracts were done by different phytochemical tests and UV spectrophotometric methods. Enzymes like alpha amylase, alpha glucosidase, aldose reductase, dipeptidyl peptidase 4, angiotensin converting enzymes are found to be pathogenic in type 2 diabetes. In vitro screening of natural enzyme inhibitors amongst individual extracts and composite blends were carried out by different assay procedures and the potency expressed in terms of IC50 values. Antioxidant potentials were estimated by DPPH radical scavenging, ABTS, FRAP and Dot Blot assay. Hydroalcoholic solvent (50:50) gave maximal yield of bio-actives with minimal chlorophyll leaching. Hydroethanolic extract of tulsi showed maximal antioxidant effect. Though all composites showed synergism, maximal effects were shown by the composite (1:1:2) in terms of polyphenol yield, antioxidant effect and inhibitory actions against the targeted enzymes. Abbreviations used: DPP4- dipeptidyl peptidase 4; AR- aldose reductase; ACE- angiotensin converting enzyme; PPAR-γ- peroxisome proliferator activated receptor-γ; NEIs- natural enzyme inhibitors; BE- binding energy; GLP-1- Glucagon like peptide -1; ROS- Reactive oxygen species; CAT- catalase; GSH-Px- glutathione per-oxidase; SOD- superoxide dismutase; pNPG- para-nitro phenyl-α-D-gluco-pyranoside solution; DPPH- 1,1-diphenyl-2-picrylhydrazyl; RSM- Response surface methodology; CCD- central composite design; DMSO- dimethyl sulfoxide; HHL- hippuryl-L-histidyl-L-leucine; GPN-Tos- Gly-Pro p-nitroanilide toluenesulfonate salt; ESC- experimental scavenging capacity; TSC- theoretical scavenging capacity; FRAP- Ferric Reducing Assay Procedure; ABTS- 2, 2’- azinobis (3-ethylbenzothiazoline-6 – sulfonic acid.

UV - SPECTROPHOTOMETRIC ESTIMATION OF ACYCLOVIR IN BULK AND PHARMACEUTICAL DOSAGE FORMS

Analytical method development being a vital part of pre formulation - formulation research and development obviates the need to develop reliable, effective, e co friendly and cost effective methodologies for routine analysis of active pharmaceutical ingredients. UV spectroscopy is one of the earliest, yet of wide applications in drug analysis in different stages of formulations and quality control; despite the availabilities of sophisti cated chromatographic techniques and other hyphenated techniques. Current research attempts to develop simple, sensitiv e, accurate, precise and economical UV spectrophotometric methods for the routine analysis of acyclovir in bulk and pharmaceutical dosage forms using two separate alkaline medi a , 0.1N NaOH (method A) and 0.1N KOH (method B) and validate them as per ICH gui delines. In both the methods maximum absorbance was observed at 264 nm. Beer’s law was obeyed in the concentration of 2.5 - 40 µg / mL in method A and 2.5 - 30 µg / mL in method B with correlation co efficient of 0.999. The % recovery carried out by adding know n amount of standard drug to pre - analyzed tablet solutions was 98.75 ± 0.52 % to 99.78 ± 0.69 % (method A) and 98.55 ± 0.31 % to 99.78 ± 0.22 % (method B). Intra and interday precision expressed in % RSD were 0.38 ± 0.01 and 0.27 ± 0.02 - 0.44 ± 0.01 respe ctively and the percent purity was 99.85 ± 0.05 %. The methods were validated statistically as per ICH guidelines and the results obtained were within the acceptance criteria for the param eters relating to linearity, accuracy, precision.

An Experimental Design Approach for Impurity Profiling of Valacyclovir-Related Products by RP-HPLC

Abstract Impurity profiling has become an important phase of pharmaceutical research where both spectroscopic and chromatographic methods find applications. The analytical methodology needs to be very sensitive, specific, and precise which will separate and determine the impurity of interest at the 0.1% level. Current research reports a validated RP-HPLC method to detect and separate valacyclovir-related impurities (Imp-E and Imp-G) using the Box-Behnken design approach of response surface methodology. A gradient mobile phase (buffer: acetonitrile as mobile phase A and acetonitrile: methanol as mobile phase B) was used. Linearity was found in the concentration range of 50–150 μg/mL. The mean recovery of impurities was 99.9% and 103.2%, respectively. The %RSD for the peak areas of Imp-E and Imp-G were 0.9 and 0.1, respectively. No blank interferences at the retention times of the impurities suggest the specificity of the method. The LOD values were 0.0024 μg/mL for Imp-E and 0.04 μg/mL for Imp-G and the LOQ values were obtained as 0.0082 μg/mL and 0.136 μg/mL, respectively, for the impurities. The S/N ratios in both cases were within the specification limits. Proper peak shapes and satisfactory resolution with good retention times suggested the suitability of the method for impurity profiling of valacyclovir-related drug substances.

Bioanalytical method development and validation of milnacipran in rat plasma by LC-MS/MS detection and its application to a pharmacokinetic study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the quantification of milnacipran (MC) in rat plasma by using the liquid–liquid extraction method. Milnacipran-d10 (MCD10) was used as an internal standard (IS). Chromatographic separation was achieved on Zorbax SB-CN (4.6 mm×75 mm, 3.5 µm) column with an isocratic mobile phase composed of 10 mM ammonium acetate (pH 4.0) and methanol in the ratio of 25:75(v/v), at a flow-rate of 0.7 mL/min. MC and MCD10 were detected with proton adducts at m/z 247.2→230.3 and m/z 257.2→240.4 in multiple reaction monitoring (MRM) positive mode respectively. The method was validated over a linear concentration range of 1.00–400.00 ng/mL with a correlation coefficient (r2)≥0.9850. This method demonstrated intra- and inter-day precision within 5.40–10.85% and 4.40–8.29% and accuracy within 97.00–104.20% and 101.64–106.23%. MC was found to be stable throughout three freeze–thaw cycles, bench top and postoperative stability studies. This method was successfully applied to a pharmacokinetic study of rats through i.v. administration.

In Vivo Hypoglycemic Studies of Polyherbal Phytoceuticals, Their Pharmacokinetic Studies and Dose Extrapolation by Allometric Scaling

BACKGROUND This work reports the safety profiling, in vivo hypoglycemic and pharmacokinetic studies of three phytoceuticals viz. conventional and sustained release tablets and microspheres each containing a polyherbal product phytocomposite (PHC) as the active ingredient. PHC is prepared from the leaf extracts of Ficus benghalensis: Syzigium cumini: Ocimum sanctum mixed in the weight ratio of 1:1:2. Further no observed adverse effect level (NOAEL), maximum recommended starting dose (MRSD) in human and prediction of human pharmacokinetic parameters have been accomplished by allometric equations. METHODS Acute and sub chronic studies of the phytoceuticals were done as per OECD and in vivo hypoglycemic studies in STZ induced diabetic rats. Plasma concentrations of the active constituent rutin (pharmacologically active compound of PHC) were determined by HPLC and other pharmacokinetic parameters using PK Solver. Repeated dose toxicity was carried out to determine the NOAEL value, MRSD estimated using allometric formulas of body surface area and clearance (CL) and volume of distribution (Vd) predicted by allometric equations of single species scaling. RESULTS Phytoceuticals showed a wide range of safety profile with a significant lowering of blood gluco-lipid level. The values of the pharmacokinetic parameters for different doses of phytoceuticals showed that the active concentration was maintained in plasma level and each formulation complied with their relevant quality criteria. NOAEL value was 5000 mg/kg/body weight and MRSD was 4864.86 mg. CONCLUSION Phytoceuticals prepared are safe and effectively controlled blood gluco lipid level. Animal to human dose extrapolation and prediction of human pharmacokinetic parameters by allometry was convenient.