Tanveer A. Wani

Development of a novel 96-microwell assay with high throughput for determination of olmesartan medoxomil in its tablets

A novel 96-microwell-based spectrophotometric assay has been developed and validated for determination of olmesartan medoxomil (OLM) in tablets. The formation of a colored charge-transfer (CT) complex between OLM as a n-electron donor and 2, 5-dichloro-3, 6-dihydroxy-1, 4-benzoquinone (p-chloranilic acid, pCA) as a π-electron acceptor was investigated, for the first time, and employed as a basis in the development of the proposed assay. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 490 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient was found between the absorbance and the concentration of OLM in the range of 1-200 μg ml-1. The limits of detection and quantitation were 0.3 and 1 μg ml-1, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from hydrochlorothiazide and amlodipine that are co-formulated with OLM in some formulations. The assay was successfully applied to the analysis of OLM in tablets with good accuracy and precision. The assay described herein has great practical value in the routine analysis of OLM in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for OLM, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed.

Application of Box-Behnken design for ultrasonic-assisted extraction of polysaccharides from Paeonia emodi.

The objective of the present work was to study the ultrasonic assisted extraction and optimization of polysaccharides from Paeonia emodi and evaluation of its anti-inflammatory response. Specifically, the optimization of polysaccharides was carried out using Box-Behnken statistical experimental design. Response surface methodology (RSM) of three factors (extraction temperature, extraction time and liquid solid ratio) was employed to optimize the percentage yield of the polysaccharides. The experimental data were fitted to quadratic response surface models using multiple regression analysis with high coefficient of determination value (R) of 0.9906. The highest polysaccharide yield (8.69%) as per the Derringers desirability prediction tool was obtained under the optimal extraction condition (extraction temperature 47.03 °C, extraction time 15.68 min, and liquid solid ratio 1.29 ml/g) with a desirability value of 0.98. These optimized values of tested parameters were validated under similar conditions (n = 6), an average of 8.13 ± 2.08% of polysaccharide yield was obtained in an optimized extraction conditions with 93.55% validity. The anti-inflammatory effect of polysaccharides of P. emodi were studied on carrageenan induced paw edema. In vivo results showed that the P. emodi 200mg/kg of polysaccharide extract exhibited strong potential against inflammatory response induced by 1% suspension of carrageenean in normal saline.

Validation of the LC-MS/MS method for the quantification of mevalonic acid in human plasma and determination of the matrix effect.

A simple, specific, and sufficiently sensitive liquid chromatography-tandem mass spectrometry (negative-ion electrospray ionization) methodology to determine mevalonic acid (MVA) in human plasma is described, and its application to the analysis of rat plasma MVA levels after rosuvastatin administration is demonstrated. The method was validated over the linearity range of 0.5–50.0 ng/ml (r2 > 0.99) using deuterated MVA as an internal standard. The lower limit of quantification was 0.5 ng/ml. The assay procedure involved the isolation of MVA from plasma samples using solid-phase extraction. Chromatographic separation was achieved on a HyPurity Advance column with a mobile phase consisting of ammonium formate buffer (10 mM, pH 8.0) and acetonitrile (70:30, v/v). Excellent precision and accuracy were observed. MVA and deuterated mevalonolactone were stable in water and plasma under different storage and processing conditions. The recovery observed was low, which was attributable to a significant matrix effect. A significant decrease (30–40%; P < 0.05) was observed in rat plasma MVA levels after rosuvastatin administration.

Novel automated flow-based immunosensor for real-time measurement of the breast cancer biomarker CA15-3 in serum

A novel automated immunosensor assay has been developed for real-time measurement of the breast cancer biomarker CA15-3 in serum. The assay employed the kinetic-exclusion analytical technology of the KinExA™ 3200 instrument. Polymethylmethacrylate (PMMA) beads coated with CA15-3 were used as capturing reagent, mouse anti-CA15-3 monoclonal antibody was used as primary antibody, and the fluorescence was monitored and recorded during the flow of the fluorescent-labeled antibody through the beads. The fluorescence signal retained on the beads was plotted versus CA15-3 concentration to generate a calibration curve. The concentrations of CA15-3 in the samples were then obtained by interpolation on the curve. The assay limit of detection was 0.2 IU mL(-1). This highly sensitive automated system allowed rapid and reliable quantification of CA15-3 without any matrix effect; analytical recovery of serum-spiked CA15-3 was 90.7%-108.6%±2.05%-7.45%. The precision of the sensor was satisfactory; relative standard deviation (RSD) was 3.8%-5.1% and 5.2%-7.4% for the intra- and inter-assay precision, respectively. The analytical performance of the proposed sensor was superior to the non-competitive sandwich immunoassays for CA15-3. The automated analysis by the sensor facilitated the processing of a large number of specimens, and the new sensor-based assay is anticipated to have a great value in measurement of CA15-3.

Chiral indolo[3,2-f][3]benzazecine-type dopamine receptor antagonists: synthesis and activity of racemic and enantiopure derivatives.

Racemic and enantiopure 8-substituted derivatives of the lead dopamine receptor antagonist LE 300 (1) were prepared, and their affinities for the dopamine receptors (D(1)-D(5)) were tested. The separate enantiomers showed significantly different affinities; the (8S)-methyl and (8R)-hyroxymethyl derivatives where the substituents point below the reference plane of the indolo[3,2-f][3]benzazecine scaffold were markedly more active than their enantiomeric counterparts. The racemic 8-carboxy derivative was shown to be selective for the D(5)-receptor, even against D(1).

Protective effects of quercetin on cadmium fluoride induced oxidative stress at different intervals of time in mouse liver

Quercetin, a member of the flavonoid family is a major antioxidant acquired in humans by food consumption, while Cadmium fluoride (CdF2) is one of the naturally occurring chemicals having adverse effects. The protective effect of quercetin on time dependent oxidative damage induced in mice liver by CdF2 was studied in the following groups of mice consisting of six mice each: (i) control group; (ii) mice treated with single i.p injection of 2 mg/kg bw CdF2 for 24 h; (iii) mice treated with single i.p injection of 2 mg/kg bw CdF2 for 48 h; (iv) mice treated with single i.p injection of quercetin (100 mg/kg bw); (v) mice treated with i.p injection of 100 mg/kg bw of quercetin followed by i.p injection of CdF2 (2 mg/kg bw) for 24 h; and (vi) mice treated with i.p injection of 100mg/kg bw of quercetin followed by CdF2 (2 mg/kg bw) for 48 h. Administration of quercetin two hours before CdF2 significantly reduced the biochemical alterations in reduced glutathione, ascorbic acid, lipid peroxidation, super oxide dismutase, catalase and total protein (p<0.05). Histopathology also showed the protective effect of quercetin. The livers treated with CdF2 were atrophic, markedly nodular, inflamed and necrotic. However, this effect was reduced to a minimum in the mice pre-treated for two hours with quercetin.

SENSITIVE HPLC METHOD WITH FLUORESCENCE DETECTION AND ON-LINE WAVELENGTH SWITCHING FOR SIMULTANEOUS DETERMINATION OF VALSARTAN AND AMLODIPINE IN HUMAN PLASMA

This study describes the development and validation of a highly sensitive high-performance liquid chromatographic method with fluorescence detection and on-line emission wavelength switching for the simultaneous determination of valsartan (VAL) and amlodipine (AML) in human plasma. Irbesartan (IRB) was used as internal standard (IS). VAL, AML, and IRB were isolated from plasma by nonextractive procedure; simple protein precipitation with methanol. Separations were performed in low pressure gradient mode on Hypersil phenyl 120A analytical column using a mobile phase consisting of phosphate buffer (pH 4.0 ± 0.1):acetonitrile:methanol (60:30:10, v/v/v) at a flow rate of 0.8 mL/min. The detection of VAL and IRB (IS) was carried out at 253 nm (for excitation) and 374 nm (for emission). After elution of VAL and IRB, the detection wavelengths were switched on-line to 393 nm (excitation) and 446 nm (emission) for detection of AML. The linear ranges of the assay were 1–100 and 10–1000 ng/mL for VAL and AML, respectively. The limits of detection (LOD) were 0.3 and 1.6 ng/mL for VAL and AML, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 5.1%. The accuracy of the method was proved; the recovery of VAL and AML from spiked human plasma were 96.6–100.9 and 92.0–104.4% for VAL and AML, respectively. The method had higher throughput as it involved simple sample preparation procedure and short run-time (15 min). The results demonstrated that the proposed method would have great value in pharmacokinetic studies for VAL and AML.

Novel microwell-based spectrophotometric assay for determination of atorvastatin calcium in its pharmaceutical formulations

The formation of a colored charge-transfer (CT) complex between atorvastatin calcium (ATR-Ca) as a n-electron donor and 2, 3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) as a π-electron acceptor was investigated, for the first time. The spectral characteristics of the CT complex have been described, and the reaction mechanism has been proved by computational molecular modeling. The reaction was employed in the development of a novel microwell-based spectrophotometric assay for determination of ATR-Ca in its pharmaceutical formulations. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 460 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient (0.9995) was found between the absorbance and the concentration of ATR-Ca in the range of 10-150 μg/well. The limits of detection and quantitation were 5.3 and 15.8 μg/well, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from the drugs that are co-formulated with ATR-Ca in its combined formulations. The assay was successfully applied to the analysis of ATR-Ca in its pharmaceutical dosage forms with good accuracy and precision. The assay described herein has great practical value in the routine analysis of ATR-Ca in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for ATR-Ca, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed.

Analytical Study for the Charge-Transfer Complexes of Rosuvastatin Calcium with π-Acceptors

Studies were carried out to investigate the charge-transfer (CT) reaction of ROS-Ca, as a n-electron donor with various π-acceptors: tetracyanoethylene, p-chloranilic acid, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, 2,3,5,6-tetrabromo-1,4-benzoquinone, 1,3,5-trinitrobenzene, 2,3,5,6-tetrachloro-1,4-benzoquinone, 7,7,8,8-tetracyano-quinodimethane, and 2,4,7-trinitro-9-fluorenone. Different colored CT complexes were obtained. The reaction mechanism and site of interaction were determined by ultraviolet-visible spectrophotometric techniques and computational molecular modeling. The formation of the colored complexes was utilized in the development of simple, rapid and accurate spectrophotometric methods for the determination of ROS-Ca. Under the optimum reaction conditions, linear relationships with good correlation coefficients (0.9984–0.9995) were found between the absorbances and the concentrations of ROS-Ca in the range of 2–200 μg mL−1. The limits of detection ranged from 0.41 to 12.24 μg mL−1. No interference could be observed from the additives commonly present in the tablets or from the drugs that are co-formulated with ROS-Ca in its combined formulations. The methods were successfully applied to the analysis of tablets with good accuracy and precision; the recovery percentages ranged from 99.54–100.46 ± 1.58–1.82%. The results were compared favorably with the reported method. The proposed methods are practical and valuable for routine application in quality control laboratories for determination of ROS-Ca in its bulk form and tablets.

Morphological changes in vero cells postinfection with dengue virus type-2.

Although no specific antiviral tablets or injections that can kill the dengue virus are currently available, adequate care and treatment could control its morbidity. Interaction of dengue virus to target cells could be an important feature for virus propagation. Ultrastructural analysis of this interaction was studied with vero cells. Vero cells were treated with Dengue virus type‐2 at different time intervals at multiplicity of infection (m.o.i) < 10, m.o.i > 10, and m.o.i = 100. It was found that m.o.i < 10 is best to study morphological changes. At an m.o.i > 10 apoptosis occurs and at m.o.i. = 100, cell necrosis occurs. While studying morphological changes, it was found that at 30 min postinfection cells have morphology very similar to that of the control cells although some have irregular outline and show cytoplasmic projections and intense cytoplasmic vacuolization. After 1–12 hours postinfection (h.p.i), the nuclei ran from normal looking to diffuse. Nuclear membrane begins to disintegrate. Some nucleoli are difficult to be seen. The cytoplasm appears as a mottled, lumps diffuse mass distributed throughout the cytosol, with dense lysosomes and myelin figures, also in the mitochondria. In later hours (24 h.p.i), the intranuclear euchromatin is dispersed and heterochromatin forms peripheral clumps. The cytoplasmic processes are short and few in numbers. A proportion of damaged mitochondria with disrupted cristae appear, suggesting that dengue virus may induce mitochondrial dysfunction and nucleus and mitochondria may be the primary organelles helping in dissemination of virus. Microsc. Res. Tech. 2011.