Quantification of sibutramine and its two metabolites in human plasma by LC-ESI-MS/MS and its application in a bioequivalence study

Obesity can be considered as a chronic illness of epidemic proportion and its incidents have increased exponentially in recent years.The use of anti-obesity drugs such as sibutramine is somewhat helpful.There is a need to quantify such drugs in biological samples,which is generally quite difficult.In this report,we developed and validated a simple,sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of sibutramine (SB) and its two metabolites N-des methyl sibutramine (DSB) and N-di desmethyl sibutramine (DDSB) in human plasma.Zorbax SBC18 (4.6 mm × 75 mm,3.5 μm,80 (A)) analytical column and 5 mM ammonium formate:acetonitrile (10∶90,v/v) mobile phase were used for chromatographic separation of SB,DSB and DDSB.Multiple reaction monitoring (MRM) in the positive mode was used to detect SB,DSB and DDSB at m/z 280.3/124.9,266.3/125.3 and 252.2/124.9,respectively.Liquid liquid extraction was used for the extraction of analytes and internal standard from human plasma.This method was validated over a linear concentration range of 10.0-10,000.0 pg/mL for SB,DSB and DDSB with correlation coefficients (r) of ≥0.9997.The drug and the two metabolites were stable in plasma samples.The validated method was successfully applied in a bioequivalence and pharmacokinetic study with human volunteers under fasting condition.

Quantification of desloratadine in human plasma by LC-ESI-MS/MS and 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 desloratadine (DL) in human plasma using desloratadine-d5 (DLD5) as an internal standard (IS). Chromatographic separation was performed using an Xbridge C18 column (50 mm 4.6 mm, 5 mm) with an isocratic mobile phase composed of 10 mM ammonium formate: methanol (20:80, v/v), at a flow rate of 0.7 mL/min. DL and DLD5 were detected with proton adducts at m/z 311.2-259.2 and 316.2-264.3 in multiple reaction monitoring (MRM) positive modes, respectively. Liquid–liquid extraction (LLE) method was used to extract the drug and the IS. The method was validated over a linear concentration range of 5.0–5000.0 pg/mL with a correlation coefficient of (r2)Z0.9994. This method demonstrated intra- and inter-day precision within 0.7–2.0% and 0.7–2.7%, and an accuracy within 101.4–102.4%, and 99.5–104.8%. DL was found to be stable throughout the freeze–thaw cycles, bench-top, and postoperative stability studies. This method was successfully applied in the analysis of plasma samples following oral administration of DL (5 mg) in 35 healthy Indian male human volunteers under fasting conditions.

Comparison of Binding Parameter Constants between Sevelamer Carbonate Tablets and Renvela Tablets by a Validated Ion Chromatography Method

Sevelamer Carbonate is a crossolinked polymeric amine, it is the active ingredient in Renvela Tablets. Sevelamer Carbonate is indicated for the control of hyperphosphatamiea in patients with end-stage renal disease. The binding parameter constants of Sevelamer Carbonate were determined using the Langmuir approximation for the dosage form at pH 4.0 and 7.0 by Ion Chromatography. An Ion Chromatogrpahy method has been developed to estimate free phosphate in in-vitro phosphate binding study of Sevelamer Carbonate Tablets. The method is selective and capable of detecting phosphate in the presence of placebo matrix. The method has been validated with a lower limit of quantitation of 0.2 mM for Phosphate. A linear response function was established in the range of concentrations 0.2 - 30.0 mM (r > 0.99) for Phosphate. The intra and inter day precision values for Phosphate met the acceptance as per Food and Drug Administrations guidelines. Phosphate was stable in the set of stability studies viz. bench-top and autosampler. The developed method was applied to in-vitro phosphate binding studies of Sevelamer Carbonate Tablets.

Comparison of Binding Parameter Constants between Sevelamer HCl Tablets and Renagel Tablets by a Validated Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) Method

Sevelamer Hydrochloride is a crossolinked polymeric amine;it is the active ingredient in Renagel Tablets. Sevelamer Hydrochloride is indicated for the control of hyperphosphatamiea in patients with end-stage renal disease. The binding parameter constants of Sevelamer Hydrochloride were determined using the Langmuir approximation for the dosage form at pH 4.0 and 7.0 by Inductively Coupled Plasma-Optical Emission Spectrometry. An ICP-OES method has been developed to estimate free phosphate in In-Vitro phosphate binding study of Sevelamer HCl Tablets. The method is selective and capable of detecting phosphate in the presence of placebo matrix. The method has been validated with a lower limit of quantitation of 0.2 mM for Phosphate. A linear response function was established for the range of concentrations 0.2 - 25.0 mM (r > 0.99) for Phosphate. The intra and inter day precision values for Phosphate met the acceptance as per Food and Drug Administrations guidelines. Phosphate was stable in the set of stability studies viz. bench-top and autosampler. The developed method was applied to in-vitro phosphate binding studies of Sevelamer HCl Tablets.

A Novel Validated Stability Indicative UP-LC Method for Etravirine for the Determination of Process Related and Degradation Impurities

A novel stability indicating reverse phase ultra performance liquid chromatographic (UP-LC) method has been developed for Etravirine along with eight impurities (imp-1, imp-2, imp-3, imp-4, imp-5, imp-6, imp-7 and imp-8) and validated as per ICH recommendations. Stress degradation conditions were established for Etravirine by subjecting it to stress conditions of acid, base, oxidation, humidity, thermal and photolysis. Significant degradation is observed in base stress condition and the major degradant (RRT at about 0.94) is identified by LC-MS and spectral analysis. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.0%. Efficient chromatographic separation was achieved on a Shimpack ODS-II stationary phase with a gradient mobile phase combination. Quantification was carried at 303 nm at a flow rate of 0.6 mL?min–1. The resolution between Etravirine and eight potential impurities is found to be greater than 2.0. Regression analysis shows as r value (correlation coefficient) of greater than 0.999 for Etravirine and eight potential impurities. This method is capable to detect the impurities of Etravirine at a level of 0.003% with respect to test concentration of 1.0 mg·mL–1.

Polarizabilities and Orientational Order Parameter in N-(p-n-Ethoxybenzylidene)- p-n-Alkoxy Anilines, 2O.Om LC Compounds

n.m, n.Om, nO.m and nO.Om compounds play an important role in the fundamental and applied aspects. As a part of our systematic studies of the above homologous series regarding the synthesis, characterization and phase transition studies, here the authors report the polarizabilities and orientational order parameter, S in N-(p-n-ethoxybenzylidene)-p-n-alkoxy anilines, 2O.Om with m = 3 to 10 liquid crystalline compounds except with m = 5 which has been already published. The rest of the compounds exhibit the nematic LC phase except with m = 10 which shows the smectic-C phase along the nematic phase which is originally not detected by Godzwon et al. But the author’s observations with TM and differential scanning calorimeter have shown the existence on SmC phase in addition to nematic phase. Further, SmC is clarified using the histogram technique whose details are given below and are published elsewhere. The orientstonal order parameter, S is estimated using different methods and compared with the value obtained from birefringence, δn = (ne – no) where no field model is used. It is observed in these compounds case as in other LC compounds Vuks isotropic model is favored compared to that of anisotropic model proposed by Neugebauer.

Vibrational spectra of oxygen and nitrogen-bridged iron octaethylporphyrin dimers by an U(2) algebraic

Using U (2) algebraic model Hamiltonian the resonance Raman spectra of Oxygen bridged iron porphyrin dimers (OEPFe)2O and (OEPFe)2N calculated for selected some vibrational modes. Using this model the Hamiltonian so con-structed, we have calculated vibrational energy levels of (OEPFe)2O and (OEPFe)2N accuractly.

Diversity of fungal isolates from fungus-growing termite Macrotermes barneyi and characterization of bioactive compound from Xylaria escharoidea

Owing to their potential applications,as well as their structural diversity,the discovery of novel secondary metabolites from insect-associated fungi has been of interest to researchers in recent years.The aim of this study was therefore to estimate the diversity of fungi associated with fungus-growing termites and bioprospecting these for potential secondary metabolites.In total,18 fungal species were isolated and described from the gut and comb of Macrotermes barneyi based on 18S ribosomal DNA gene sequence analysis.Antimicrobial activity assays were carried out on all the known fungi,and nine isolates were recorded as active against pathogenic fungi.Xylaria escharoidea,the best performing isolate,was grown at laboratory scale and 4,8-dihydroxy-3,4-dihydronaphthalen-l(2H)was isolated and characterized.The minimum inhibitory concentration of this isolated compound against tested pathogenic organisms was found to be 6.25 fig.In addition,molecular docking studies have revealed that 4,8-dihydroxy-3,4-dihydronaphthalen-l(2H)is a prominent antibacterial agent with a marked interaction with key residues on protein A(agrAc)that regulates the accessory gene.The findings of this study support the drug discovery of antimicrobial properties in insect-associated fungi,which may lead to novel secondary metabolites.

Chemical composition, antibacterial and antifungal activities of Saudi Arabian Mentha longifolia L. essential oil

Objective: To investigate the chemical composition, in vitro antibacterial and antifungal activity of Mentha longifolia L. essential oils using aqueous extract. Methods: Mentha longifolia L. essential oils were extracted using hydrodistillation with Clevenger apparatus for 3 h and the yield of the essential oil was calculated. Essential oils were qualitatively and quantitatively analyzed with gas chromatography and gas chromatography-mass spectrometer. For these essential oils, antibacterial and antifungal activity against human pathogens were evaluated. Results: Nineteen chemical constituents representing 99.72% of the essential oil were found, comprising menthone (39.55%), isopulegone (30.49%), eucalyptol (10.38%), and α-terpineol (3.15%);these were major components, and others were minor components. The essential oil showed strong antibacterial activity against Staphylococcus aureus [(35.24 ± 0.13) mm], Enterococcus faecalis [(32.12 ± 0.12) mm] and Bacillus cereus [(30.06 ± 0.04) mm], as well as antifungal activity against Aspergillus flavus [(38.02 ± 0.06) mm], Alternaria alternaria [(35.26 ± 0.12) mm], and Penicillum spp ((34.14 ± 0.02) mm)Conclusions: It seems that the essential oils derived from the Mentha longifolia L. species could be used as a natural source of antimicrobial agents.

Homology modeling and structural validation of human amylin: A type 2 diabetes causing protein

Objective:In the present study, the protein structure model of human amylin was generated to understand the protein's structure, function and mechanism of the action. Methods: The stereo chemical quality of the protein model was checked by usingin silico analysis withSWISSMODEL,SOPMA,PROCHECK, ProSA andQMEAN servers. Results:The 66.7% residues in the core region of Ramachandran plot showing high accuracy of protein model and the QMEAN Z score of –6.57 indicates the overall model quality of amylin protein. Conclusions: These results may be helpful for further investigation on developing target molecules for amylin proteins that play a key role in type 2 diabetes.

In vitro biofilm forming capacity on abiotic contact surfaces by outbreak-associated Vibrio harveyi strains

Objective:To evaluate the in vitro biofilm forming capacity on abiotic food contact surfaces by Vibrio harveyi(V.harveyi)strains.Methods:Thirty six Gram-negative V.harveyi strains were isolated from various street vended seafood outlets in a food processing line and evaluated for their ability to produce mucoid biofilms on food contact surfaces using a microplate assay.Phenotypic characterization of mucoid biofilm producing V.harveyi strains were screened on Congo red agar,thiosulfate-citrate-bile salts-sucrose agar and tryptic soy agar,respectively.Results:Only five V.harveyi strains(14%)were mucoid biofilm producers characterized by formation of black colonies,whereas the remaining 31 strains(86%)were not capable of producing biofilm characterized by formation of red colonies or pinkish-red colonies with darkening at the centre.The morphological,physiological and biochemical characteristics of these isolates were studied using standard protocols.Strain identification was confirmed by polymerase chain reaction targeted to species-specific polymerase chain reaction primers VH-1 and VH-2 corresponding to variable regions of V.harveyi 16S rRNA sequence.All the biofilm-forming strains showed resistance to at least three antimicrobial compounds tested.V.harveyi strains isolated from various seafood were able to form biofilms of different capacity,and the strains VB267,VB238 and VB166 isolated from cat fish,shrimp and eel fish exhibited significantly greater biofilm forming ability compared to other isolates.Conclusions:It can be concluded from the present study that the strain VB166 was able to better attach and form subsequent biofilms on glass and stainless steel compared to high density polyethylene.These properties allow these bacteria to survive,proliferate and persist in street vended seafood outlets.

Isolation and characterization of cobalt-sensitive mutant of Neurospora crassa

Objective:To isolate and demonstrate the mechanism of metal transport in cobalt-sensitive mutant(CSM)of Neurospora crassa(N.crassa).Methods:Isolation of CSM of N.crassa,I_(50)determination,growth measurements,metal ion uptake studies and sexual crosses were performed to determine the mechanism of sensitivity and locus.Results:CSMs of N.crassa were isolated by mutagenesis with diethyl sulfate.More than 500 isolates were screened and out of these isolates,CSM-I was 5-fold and CSM-II was 10-fold sensitive to Co on liquid medium as compared to the wild type.Compositional analysis of cell wall revealed the decrease in total phosphate content.N.crassa CSM bound much less cobalt to cell wall fraction than wild type.The data indicated closer linkage between resistance and mating type locus(mat),which is,located on LG I.Conclusions:A CSM of N.crassa is 5-fold more sensitive than wild type and cross sensitive to nickel and copper and hyper-accumulates 2-4 fold more toxic metal ions over wild type.The mechanism for sensitivity is decreased in cobalt-binding to cell wall fraction and increased intracellular uptake.N.crassa-acon-3 morphologically resembles the CSM,cobalt-sensitive and maps to similar locus.