A UPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF SOFOSBUVIR FROM HUMAN PLASMA

Objective: The present work aimed to develop a simple, rapid, specific and precise ultra-performance liquid chromatography-tandem mass spectrophotometric (LC–MS/MS) validated method for quantification of sofosbuvir and internal standard (ISTD) Sofosbuvir-d3 in human plasma.Methods: Samples prepared by employing liquid-liquid extraction (LLE) using 2.5 ml of ethyl acetate. Chromatographic separation was achieved on Gemini 5µ C18, 50 x 4.6 mm column using a mixture of 0.1% (v/v) formic acid in water to methanol at a ratio of 30:70 v/v as the mobile phase. The flow rate was 0.50 ml/min. The LC eluent was split, and approximately 0.1 ml/min was introduced into Tandem mass spectrometer using turbo Ion Spray interface at 325 °C. Quantitation was performed by transitions of 428.35/279.26 (m/z) for sofosbuvir and 431.38/282.37 (m/z) for sofosbuvir-d3.Results: The concentrations of ten working standards showed linearity between 4.063 to 8000.010ng/ml (r2 ≥ 0.9985). Chromatographic separation was achieved within 2 min. The average extraction recoveries of three quality control concentrations were 75.36% for sofosbuvir and were within the acceptance limits. The coefficient of variation was ≤15% for intra-and inter-batch assays. The %CV of ruggedness ranges 0.35% and 3.09%. The % stability of short term and long term stock solution stability studies was found to be 97.25% and 98.81% respectively.Conclusion: The results obtained for specificity, linearity, accuracy, precision, ruggedness and stability studies were within the acceptance limits. Thus the validated economical method was applied for pharmacokinetic studies of sofosbuvir

A VALIDATED LC-MS/MS METHOD FOR PHARMACOKINETIC STUDY OF CANAGLIFLOZIN IN HEALTHY RABBITS

Objective: A liquid chromatography-tandem mass spectrophotometric (LC-MS/MS) method was developed for quantification of canagliflozin in rabbit plasma employing Liquid-Liquid extraction technique.Methods: Chromatographic separation was achieved on Inertsil ODS 5 µm C18, 50×4.60 mm with 30:70 v/v of 0.01M ammonium acetate: methanol as an isocratic mobile phase with a flow rate of 0.8 ml/min. The developed LC-MS method was applied to assess Cmax, t1/2, AUC0-t, and AUC0-inf of canagliflozin tablet after oral administration in healthy rabbits.Results: The developed method was linear over working range of 5ng/ml to 600ng/ml with a coefficient of correction (r2) = 0.999. The % recovery of the method was found to be 102.05%. The mean intraday and inter-day precision of the method was found to be 0.77 to 3.72%. The Canagliflozin showed Tmax of 1.58±0.2 and mean Cmax, AUC0®t andAUC0®a for Test formulation is 272±13.24, 2571.20±251and 2777.43±276 respectively.Conclusion: The developed method can be applied for routine analysis for quality control and the established LLOQ is sufficiently low to conduct a pharmacokinetic study with any marketing formulation of Canagliflozin in healthy rabbits

Comparative uptake studies on trivalent f-cations from acidic feeds using two extraction chromatography resins containing a diglycolamide in molecular diluent and ionic liquid

Low molecular weight diglycolamide (DGA) extractants were tested for the extraction of europium(III) and americium(III) from nitric acid solutions in n-dodecane, a molecular diluent and 1-butyl-3-methylimidazolium bis(trifluoromethanesulphonyl) imide (C4mim⋅NTf2), a room temperature ionic liquid, as the diluents. N,N,N’,N’-tetra-n-butyl diglycolamide (TBDGA) was selected for extraction chromatography (XC) studies involving Eu(III) and Am(III). While the TBDGA resin containing n-dodecane gave reasonably high Kd values, that containing the ionic liquid showed higher Eu(III) uptake values. Compared to Eu(III), Am(III) was extracted by the resins to a lower extent. The loaded Eu(III) was back extracted from the resin using 0.05 M EDTA solutions in a buffered medium containing 1 M guanidine carbonate. Reusability studies indicated that, while the ionic liquid-based resin can be conveniently recycled five times with very marginal decrease in the percentage extraction values, there was a sharp decrease in the percent extraction after three cycles with the n-dodecane-based resin. The uptake data was fitted into different isotherm models and the results conformed to the Langmuir model. Based on the batch uptake studies, columns were prepared and the breakthrough as well as elution profiles were obtained. The elution profiles were found to be sharp without any significant tailing

Highly selective carrier mediated transport of plutonium(IV) across a supported liquid membrane using two substituted tripodal amides

Plutonium-specific supported liquid membranes were developed and reported for the first time. The studies included in this paper are on the supported liquid membrane transport behavior of Pu(IV), Am(III) and U(VI) investigated using two tripodal amides viz. N,N,N,’N,’N,”N”-hexa-n-octylnitrilotriacetamide (HONTA) and N,N,N,’N,’N,”N”-hexa-n-dodecylnitrilotriacetamide (HDDNTA). The feed phase consisted of 3 M HNO3 while 0.5 M oxalic acid +0.5 M HNO3 was used as the strip phase. The SLM studies indicated 78.2% and 86.2% transport of plutonium(IV) after 4 h using 0.08 M concentrations of HONTA and HDDNTA, respectively, and the transport of Am(III) and U(VI) was <1% under identical feed acid conditions indicating a possible highly selective separation of Pu(IV) using HONTA as the carrier ligand from a mixture of Pu(IV), Am(III) and U(VI). The stability of the membrane was reasonably good up to 9 days. The activation energy of the transport process was determined using temperature variation studies with HONTA and HDDNTA indicating the transport process being kinetically as well as diffusion controlled. This work is highly relevant for the separation of Pu from radioactive feeds containing other actinide elements such as U and Am

Highly efficient and selective extraction of Pu(IV) using two alkyl-substituted amides of nitrilo triacetic acid from nitric acid solutions

Liquid-liquid extraction of the actinide ions UO22+, Pu4+, Am3+ was carried out from nitric acid feed solutions using two tripodal amide extractants, viz., N,N,N’,N’,N”,N”-hexa-n-octylnitrilotriacetamide (HONTA) and N,N,N’,N’,N”,N”-hexa-n-dodecylnitrilotriacetamide (HDDNTA) in 10% isodecanol + 90% n-dodecane diluent. The metal ion extraction trend was: Pu(IV) ≫ U(VI) > Am(III) at 3 M HNO3, HONTA being a superior extractant than HDDNTA. Slope analysis indicated ML2 type species for both Pu(IV) and Am(III) at 3 M HNO3 and pH 2.0, respectively, whereas, only in case of HONTA, a ML type species was obtained with U(VI) at 3 M HNO3. Back extraction data suggested that a mixture of 0.5 M HNO3 + 0.5 M oxalic acid was efficient for quantitative stripping (∼90%) of Pu(IV), whereas 1 M Na2CO3 and 1 M α-HIBA gave satisfactory back extraction for U(VI) and Am(III), respectively. The ligand solutions showed reasonably good radiation stability up to an absorbed total gamma ray dose of 274 kGy. Distribution data were also obtained under U(VI) loading conditions for possible nuclear fuel cycle applications

Highly efficient plutonium scavenging by an extraction chromatography resin containing a tetraaza-12-crown-4 ligand tethered with four diglycolamide pendent arms

An efficient extraction chromatography resin, containing tetraaza-12-crown-4 functionalized with four diglycolamide moieties, was evaluated for the separation of plutonium. This chromatography resin yielded very large distribution coefficients for Pu4+ (>105) in 0.5 – 6 M HNO3 feed solutions. Various physicochemical properties such as sorption kinetics, Pu4+ sorption mechanism, and its sorption capacity were investigated. The sorption kinetics, following a pseudo-second-order model, showed that about 10 minutes of equilibration was sufficient for >99.9% sorption of Pu4+. The sorption of Pu4+ on the resin followed the Langmuir monolayer model, which was confirmed by a theoretical calculation based on the kinetic model. The Pu4+ sorption on the resin was driven by a large exothermic enthalpy change (ΔH = −31.4±2.2 kJ/mol) and a positive entropy change (ΔS = 224±15 J/mol/L). The resin could sorb a maximum of 12.1±0.8 mg of Pu per gram of resin, which is equivalent to 1:2 metal/ligand complex on the resin. The Pu4+ from the resin phase was completely stripped with 0.5 M oxalic acid. A possible application of this resin for the separation / pre-concentration of Pu4+ was successfully demonstrated in the column mode

Carrier mediated transport of actinides using hexa–n-hexylnitrilotriacetamide (HHNTA)

Solutions of N,N,N’,N’,N”,N”-hexa-n-hexylnitrilotriacetamide (HHNTA) in 20 % isodecanol-80 % n-dodecane (v/v) were employed for the supported liquid membrane (SLM) transport of U(VI), Np(IV), and Pu(IV) from nitric acid feed solutions using PTFE (polytetrafluoroethylene) flat sheet membranes. Preliminary transport studies revealed poor transport of U(VI) when compared with that of the tetravalent ions Np(IV) and Pu(IV) which may find application for their separation from acidic radioactive feeds. In view of this, all subsequent studies were carried out using Np(IV) and Pu(IV). The SLM studies performed with 0.08 M HHNTA as the carrier extractant indicated increased transport of the tetravalent actinide ions with increasing nitric acid and carrier extractant concentrations, while using 0.5 M HNO3 + 0.5 M oxalic acid as the strip (or receiver) phase composition. However, while > 80 % transport of Np(IV) and Pu(IV) was obtained for a feed phase of 3 M HNO3, a sharp decrease in the transport rate was seen at a feed acidity of 6 M HNO3 probably due to anionic hexanitrato complex formation of the metal ions and also because of lower availability of the free ligand in the membrane phase at higher nitric acid concentrations. The membrane stability was poor suggesting the need for continuous replenishment for long term use

Muscleblind-Like 1 and Muscleblind-Like 3 Depletion Synergistically Enhances Myotonia by Altering Clc-1 RNA Translation.

Loss of Muscleblind-like 1 (Mbnl1) is known to alter Clc-1 splicing to result in myotonia. Mbnl1(ΔE3/ΔE3)/Mbnl3(ΔE2) mice, depleted of Mbnl1 and Mbnl3, demonstrate a profound enhancement of myotonia and an increase in the number of muscle fibers with very low Clc-1 currents, where gClmax values approach ~&nbsp;1&nbsp;mS/cm(2), with the absence of a further enhancement in Clc-1 splice errors, alterations in polyA site selection or Clc-1 localization. Significantly, Mbnl1(ΔE3/ΔE3)/Mbnl3(ΔE2) muscles demonstrate an aberrant accumulation of Clc-1 RNA on monosomes and on the first polysomes. Mbnl1 and Mbnl3 bind Clc-1 RNA and both proteins bind Hsp70 and eEF1A, with these associations being reduced in the presence of RNA. Thus binding of Mbnl1 and Mbnl3 to Clc-1 mRNA engaged with ribosomes can facilitate an increase in the local concentration of Hsp70 and eEF1A to assist Clc-1 translation. Dual depletion of Mbnl1 and Mbnl3 therefore initiates both Clc-1 splice errors and translation defects to synergistically enhance myotonia. As the HSA(LR) model for myotonic dystrophy (DM1) shows similar Clc-1 defects, this study demonstrates that both splice errors and translation defects are required for DM1 pathology to manifest.Research in contextResearch in context: Myotonic Dystrophy type 1 (DM1) is a dominant disorder resulting from the expression of expanded CUG repeat RNA, which aberrantly sequesters and inactivates the muscleblind-like (MBNL) family of proteins. In mice, inactivation of Mbnl1 is known to alter Clc-1 splicing to result in myotonia. We demonstrate that concurrent depletion of Mbnl1 and Mbnl3 results in a synergistic enhancement of myotonia, with an increase in muscle fibers showing low chloride currents. The observed synergism results from the aberrant accumulation of Clc-1 mRNA on monosomes and the first polysomes. This translation error reflects the ability of Mbnl1 and Mbnl3 to act as adaptors that recruit Hsp70 and eEF1A to the Clc-1 mRNA engaged with ribosomes, to facilitate translation. Thus our study demonstrates that Clc-1 RNA translation defects work coordinately with Clc-1 splice errors to synergistically enhance myotonia in mice lacking Mbnl1 and Mbnl3