Validation of LC/MS electrospray ionisation method for the estimation of ursodiol in human plasma and its application in bioequivalence study

A novel High Performance Liquid Chromatography-electrospray mass spectrometric method has been developed for the estimation of Ursodiol (Ursodeoxycholic acid)--a bile acid, in human plasma using Ornidazole as internal standard. The methodology involved solid phase extraction of the analyte from human plasma matrix. The chromatographic separation was achieved within seven minutes by an isocratic mobile phase containing 1.0 mM ammonium acetate and Acetonitrile (65:35, v/v), flowing through XTerra MS C18, 100 x 2.1, 3.5 microm analytical column, at a flow rate of 0.2 ml/min. Ion signals were measured in negative mode for Ursodiol and internal standard at m/z 391.3 and 278.1, respectively. A detailed validation of the method was performed as per USFDA guidelines and the standard curves were found to be linear in the range 50.0 ng/ml to 3000.0 ng/ml with the mean correlation coefficient more than 0.99. The absolute recovery was more than 54.90% for Ursodiol and 76.51% for internal standard. Ursodiol was stable for sixty-nine days at -70 degrees C and for eight hours at ambient temperature. After extraction from plasma, the reconstituted samples of Ursodiol were stable in autosampler at 10 degrees C for forty-eight hours. Upon subjecting to three freeze thaw cycles, there was no change in the recovery of the analyte. The integrity of the plasma samples remained unaffected even upon four-fold dilution with drug free human plasma. The method was simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. It was successfully applied to the pilot bioequivalence study of Ursodiol in male human subjects.     

Spectrophotometric determination of ruthenium(III) using diphenylcarbazone

Summary The purple violet ruthenium(III)-diphenylcarbazone complex which is formed at p _h 5–7, and has an absorption maximum at 530 nm with molar absorption coefficient 16.2·10⁴l.cm^–1.mole^–1 is suggested for the estimation of 20–125g ruthenium(III) spectrophotometrically in 30–60% ethanol. The complex is stable over p _h range 3.2–8.4. The limits of interference due to foreign ions have been studied.

---

Zusammenfassung Der bei p _h 5 bis7 entstehende Ruthenium(III)-Diphenylcarbazon-Komplex hat ein Absorptionsmaximum bei 530 nm und einen Absorptionskoeffizienten von 16,2·10⁴ l.cm^–1.Mol^–1. Die spektrophotometrische Bestimmung von 20 bis 125g Ruthenium(III) in 30 bis 60%igem Äthanol mit Hilfe dieses zwischen p _h 3,2 und 8,4 beständigen Komplexes wurde vorgeschlagen. Die Störung durch Fremdionen wurde geprüft.

     

Thermal decomposition of cesium hexanitratouranium(IV)

As cesium hexanitratouranium(IV), Cs₂U(NO₃)₆, has the same Cs:U stoichiometry as that of Cs₂UO₄, thermal decomposition of this nitrato complex in air and nitrogen was studied in detail as a possible alternate method of preparing pure Cs₂UO₄. The volatility of cesium nitrate, which is one of the intermediate products, changed this Cs:U ratio during thermal decomposition. Hence, only Cs₂U₂O₇ was obtained on heating the sample to 775 K or higher. A scheme for the thermal decomposition of Cs₂U(NO₃)₆ is given by combining the observed TG, XRD and IR data.     

Purification of recombinant green fluorescent protein by three-phase partitioning

The technique of three-phase partitioning (TPP) was used to purify the green fluorescent protein (GFP) in a single step. TPP uses a combination of ammonium sulphate and tert-butanol to precipitate proteins from their crude extracts. In the first round of TPP with 20% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:1 (v/v), most of the GFP remains in the lower aqueous phase. When subjected to a second round of TPP with 60% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:2 (v/v) gives 78% recovery of GFP with a 20-fold purification. The sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of purified preparation shows single band. The fluorescence excitation and emission spectra agreed with values reported in literature.     

Correlations between topological features and physicochemical properties of molecules

The article reviews in brief, thede novo group additivity approach and, at length, the different topological approaches to obtain predictive and internally consistent correlations between various properties and structural features of molecules. The stress has mainly been on the molecular connectivity method. A new rational scheme for nomenclature of connectivity indices of different orders and types is introduced. The concept of the perturbation connectivity parameter developed by us recently has been applied to obtain correlations for molar refraction, boiling point, molar volume, heat of atomisation, heat of combustion, heat of vaporisation, magnetic susceptibility and critical constants of alkanes, alcohols and alkylbenzenes. A comparative study of various approaches reveals that the present perturbation topological approach has an edge over other similar methods.     

Protein surface recognition by synthetic receptors based on a tetraphenylporphyrin scaffold

[structure: see text] Receptors based on a tetraphenylporphyrin scaffold bearing different charged and hydrophobic groups have been synthesized. The interactions of these with horse heart cytochrome c were studied by fluorescence spectroscopy. Receptor 4 was identified to be the strongest synthetic receptor (K(d) = 20 nM) for cytochrome c. The differences in affinity among the receptors reflected a dependence on the number of anionic and hydrophobic groups.     

A study of nuclear prenylation of 2,4-dihydroxy phenyl benzyl ketone: Syntheses of linear and angular 6″,6″-dimethyl pyrano-isoflavones,and their 2-methyl and 2-phenyl analogues

2,4-Dihydroxyphenyl benzyl ketone (I) condenses with 2-methyl but-3-en-2-ol in the presence of boron trifluoride etherate to afford a mixture of 3-C-, 5-C- and 3,5-di C C-prenyl derivatives separable by chromatography. The above mixture without the 5-C-prenyl but with the 4-O-prenyl derivative results when ketone I is treated with prenyl bromide in the presence of methanolic potash. 3-C-(III a) and 5-C-(IV) prenyl derivatives undergo ready oxidative cyclization with DDQ to yield the corresponding pyrano derivatives VIII and XII respectively which form good starting materials for various isoflavone condensations. Thus both linear and angular 6″,6″-dimethyl pyrano-isoflavones and their 2-methyl and 2-phenyl analogues have been synthesized.     

Role of partially flocculated sols in emulsion stability

Summary The stability of emulsion of Toluene in water stabilized by partially flocculated ferric hydroxide sol has been studied. The size of the emulsion droplets as determined by size frequency analysis is found to be of 1.1 μ radius. The flocculation studies were carried out haemocytometrically at different amount of electrolytes and at different p_H values. The electrokinetic potential ‘ζ’ was determined micro-electrophoretically at different amounts of electrolytes and also at different p_H's. In the case of tri and tetra valent anions the charge reversal was observed. In the light of the D.V.O. theory the interaction energy profiles were drawn and 2–6 kT deep secondary minima obtained showing thereby the occurrence of reversible flocculation. Degree of aggregation has been calculated theoretically at different values of ‘A’ and the value of A was found to be 1.0×10⁻¹² erg to fit the value of degree of aggregation obtained haemocytometrically. The charge densities and binding parameters have also been calculated.     

Structures of E. coli peptide deformylase bound to formate: insight into the preference for Fe2+ over Zn2+ as the active site metal

E. coli peptide deformylase (PDF) catalyzes the deformylation of nascent polypeptides generated during protein synthesis. While PDF was originally thought to be a zinc enzyme, subsequent studies revealed that the active site metal is iron. In an attempt to understand this unusual metal preference, high-resolution structures of Fe-, Co-, and Zn-PDF were determined in complex with its deformylation product, formate. In all three structures, the formate ion binds the metal and forms hydrogen-bonding interactions with the backbone nitrogen of Leu91, the amide side chain of Gln50, and the carboxylate side chain of Glu133. One key difference, however, is how the formate binds the metal. In Fe-PDF and Co-PDF, formate binds in a bidentate fashion, while in Zn-PDF, it binds in a monodentate fashion. Importantly, these structural results provide the first clues into the origins of PDF's metal-dependent activity differences. On the basis of these structures, we propose that the basis for the higher activity of Fe-PDF stems from the better ability of iron to bind and activate the tetrahedral transition state required for cleavage of the N-terminal formyl group.     

Reverse isotope dilution alpha spectrometry using plutionium-239 spike for determining plutonium concentration in high burn-up fuel samples without purification from americium-241 and a bulk of other impurities

A reverse isotope dilution alpha spectrometric /R-IDAS/ method using²³⁹Pu as a spike is described for the determination of plutonium concentration in high burn-up fuel samples wth²³⁸Pu/(²³⁹Pu+²⁴⁰Pu) alpha activity ratio >0.5, without resorting to any purification from²⁴¹Am and a bulk of other impurities. It involves the addition of a pre-clibrated spike solution to a known aliquot of the plutonium sample solution followed by source preparation using TEG as a spreading agent. The results obtained on a number of plutonium samples containing 20–80% of²⁴¹Am /alpha activity wise/ using this method are compared with those achieved by R-IDAS using purification with TTA, with respect to precision and accuracy. Precision and accuracy of 0.5% are demonstrated. This method eliminates the need of any separation and purification of plutonium from²⁴¹Am and a bulk of other impurities like uranium.