Use of biotin-streptavidin system for developing a viable, sensitive and specific antigen heterologous assay for hapten

The present study demonstrates improvement in sensitivity and specificity of hapten assay by using antigen heterology in conjunction with low molecular weight biotin label as compared to high molecular weight horseradish peroxidase (HRP) label. For generation of antiserum, cortisol-3-O-carboxylmethyl-oxime-bovine serum albumin (F-3-CMO-BSA) was used as immunogen whereas, for the preparation of primary label, corticosterone-3-carboxymethyl oxime (B-3-CMO) was coupled with biotinylcaproylhydrazide and HRP by employing N-hydroxysuccinimide mediated carbodiimide reaction. The data of the present study revealed that the antigen heterologous assay which employed high molecular weight HRP label showed 100% cross-reaction with corticosterone. On the contrary, when HRP was replaced with low molecular weight biotin label, less than 0.1% cross-reaction was observed with all analogous C₁₈, C₁₉, C₂₁ and C₂₇ steroids including corticosterone (0.2%). Moreover, the sensitivity of the later assay was 0.09 μg/dL, which is appreciable as compared to previously reported enzyme based assays. The recovery of the exogenously spiked serum pools lies in the range of 90.3-104.2%. The intra-assay and inter-assay coefficient of variation (CVs) ranged from 3.3% to 7.8% and 2.3% to 7.7%, respectively. The serum cortisol values obtained by this method correlated well with those obtained by radioimmunoassay; r = 0.9 (n = 50). The use of much stable biotin label in place of HRP has made the antigen heterologous enzyme linked immunosorbent assay (ELISA) of cortisol assay highly specific and sensitive.     

Enantiomeric LC Separation of Epinephrine on Amylose based sorbent

A simple, rapid, and robust chiral HPLC method has been developed and validated for separation of the enantiomers of epinephrine, l-1-(3,4-dihydroxyphenyl)-2-(methylamino)ethanol, an antihypertensive drug, in the bulk drug. The enantiomers were resolved on an amylose-based stationary phase with n-hexane–2-propanol–methanol–trifluoroacetic acid–diethylamine 90:05:05:0.2:0.2 (v/v) as mobile phase at a flow rate of 1.0 mL min^?1. In the optimized method resolution between the enantiomers was not less than 3.0. The trifluoroacetic acid and diethylamine in the mobile phase were important for enhancing chromatographic efficiency and hence the resolution of the enantiomers. The method was extensively validated and proved to be robust. The calibration plot for the d enantiomer was highly linear over the concentration range 100–2,000 μg mL^?1. The limits of detection and quantification for the d enantiomer were 0.15 and 0.45 μg mL^?1, respectively. Recovery of the d enantiomer from bulk drug samples of epinephrine ranged between 99.5 and 101.5%. Epinephrine sample solution was stable for up to 48 h. The method was suitable for accurate quantitative determination of the d enantiomer in the bulk drug substance     

Development and validation of an RP-HPLC method for the estimation of adenosine and related purines in brain tissues of rats

A new, rapid and sensitive RP-HPLC method with UV spectrophotometric detection was developed and validated for the concomitant estimation of adenosine and related purines in rat brain tissue preparations. The HPLC system consisted of C-18 column with UV-photodiode-array detection ranging from 210 to 400 nm, facilitating the online confirmation of peak purity. The column temperature was maintained at 30 degrees C and the injection volume was 20 muL. Elution with an isocratic mobile phase consisting of water/methanol/acetonitrile (88:5:7 by volume) at a flow rate of 0.8 mL/min yielded sharp, utmost-resolved peaks of adenosine (Ade), inosine (Ino), hypoxanthine (Hypoxan) and adenine (Adn) within 10 min. The method was validated with respect to the linearity, accuracy, precision, sensitivity, selectivity and stability. The method was also employed to estimate the naturally occurring purines in discrete regions of rat brain. A new protocol developed for tissue preparation utilizing H(2)SO(4) and Tris buffer gave well-resolved peaks and high component recoveries (>96%) which eliminated the need of an internal standard. The results show that the method for the determination of Ade, Ino, Hypoxan and Adn by RP-HPLC described here has good linearity, accuracy, precision, sensitivity, selectivity and is simple and rapid to perform.     

Ionic liquid promoted synthesis of bis(indolyl) methanes

1-benzyl-3-methyl imidazolium hydrogen sulphate [bnmim][HSO₄] was found to be an effective catalyst for the condensation reaction of indoles and derivatives with benzaldehydes in microwave irradiation with lower reaction time and higher yields to give bis(indolyl) methanes.      

Efficient synthesis of piperidine derivatives using dendrimer based catalytical pockets

The synthesis of highly functionalized piperidine derivatives using amine functionalized maltitol-cored dendritic polymer (MAL-G0) in acetonitrile as the reaction medium is reported. A variety of piperidine derivatives were synthesized and the reaction gave an excellent yield of 89%–95%. The highly functionalized nature of the catalyst provided large number of active sites which resulted in good yield within a short period of time. Maltitol is a carbohydrate polyol system which was chosen here as the core for the synthesis of the dendritic catalyst; it was an effective approach for the preparation of piperidine like medicinal compounds. Maltitol-cored dendritic polymer was effectively synthesized and characterized using GPC, TG, UV–Visible, IR, and NMR techniques and also all the synthesized piperidine derivatives were characterized using LCMS, IR, and NMR techniques.     

Synthetic and Structural Studies on Novel 4,3′-Bicoumarins

Abstract

A series of directly linked 4-3′ bicoumarins have been synthesized by both Knoevenagel and Perkin reactions. This single-step transformation was accomplished by the reaction of coumarin-4-acetates with substituted salicylaldehydes in presence of piperidine using ethanol as solvent and by the reaction of coumarin-4-acetic acids with substituted salicylaldehydes in the presence of sodium hydride in acetic anhydride. Greater yields have been obtained in the sodium hydride and acetic anhydride condition. An intermediate chalcone with an ortho-hydroxyl group, which is a precursor for lactone formation, has been isolated and its structure has been confirmed by x-ray analysis. The nonplanar S-cis arrangement of two C4-C3’ double bonds has been confirmed by nuclear Overhauser spectroscopy (NOE) and x-ray studies. The UV-fluorescence studies support the formation of a conjugated bicoumarin system.