Novel electrochemical biosensor based on PVP capped CoFe2O4@CdSe core-shell nanoparticles modified electrode for ultra-trace level determination of rifampicin by square wave adsorptive stripping voltammetry.

This work introduces a new electrochemical sensor based on polyvinyl pyrrolidone capped CoFe2O4@CdSe core-shell modified electrode for a rapid detection and highly sensitive determination of rifampicin (RIF) by square wave adsorptive stripping voltammetry. The new PVP capped CoFe2O4@CdSe with core-shell nanostructure was synthesized by a facile synthesis method for the first time. PVP can act as a capping and etching agent for protection of the outer surface nanoparticles and formation of a mesoporous shell, respectively. Another important feature of this work is the choice of the ligand (1,10-phenanthroline) for precursor cadmium complex that works as a chelating agent in order to increase optical and electrical properties and stability of prepared nanomaterial. The nanoparticles have been characterized by field emission scanning electron microscopy (FESEM), transmission electron microscope (TEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), UV-vis, photoluminescence (PL) spectroscopy, FT-IR, and cyclic voltammetry techniques. The PL spectroscopy study of CoFe2O4@CdSe has shown significant PL quenching by the formation of CoFe2O4 core inside CdSe, this shows that CoFe2O4 NPs are efficient electron acceptors with the CdSe. It is clearly observed that the biosensor can significantly enhance electrocatalytic activity towards the oxidation of RIF, under the optimal conditions. The novelty of this work arises from the new synthesis method for the core-shell of CoFe2O4@CdSe. Then, the novel electrochemical biosensor was fabricated for ultra-trace level determination of rifampicin with very low detection limit (4.55×10-17M) and a wide linear range from 1.0×10-16 to 1.0×10-7M. The fabricated biosensor showed high sensitivity and selectivity, good reproducibility and stability. Therefore, it was successfully applied for the determination of ultra-trace RIF amounts in biological and pharmaceutical samples with satisfactory recovery data.

Bone distribution study of anti leprotic drug clofazimine in rat bone marrow cells by a sensitive reverse phase liquid chromatography method.

The aim of the present study was to investigate the distribution of clofazimine (CLF) in rat bone marrow cells by a validated reverse phase high performance liquid chromatography. CLF and chlorzoxazone (I.S) were extracted by liquid-liquid extraction from plasma and rat bone marrow cells. The chromatographic separation was performed in isocratic mode by the mobile phase consisting of 10mM ammonium formate (pH 3.0 with formic acid) and acetonitrile in a ratio of 50:50 (v/v). The method was accurate and precise in the linear range of 15.6-2000.0 ng/mL with a correlation coefficient (r(2)) of 0.996 and 0.995 in rat plasma and bone marrow cells, respectively. After single oral dose of 20mg/kg, the maximum concentration of CLF in plasma and bone marrow cells were obtained at 12h with the concentrations of 593.2 and 915.4 ng/mL, respectively. The AUC0-t and mean elimination half life (t1/2) of CLF in bone marrow cells were 54339.02 ng h/mL and 52.46 h, respectively, which signified the low body clearance and high distribution of CLF in bone marrow cells. The single oral dose pharmacokinetic investigation was confirmed the CLF endure for a long period in rat due to high distribution in various tissues. The developed method was successfully applied to the estimation of the pharmacokinetic parameters of CLF in plasma and bone marrow cells after administration of single oral dose of 20mg/kg to rats.

Individual and simultaneous spectrophotometric determination of dapsone and metoclopramide HCl in pharmaceutical dosage forms and synthetic binary mixtures.

A rapid, sensitive and selective spectrophotometric method has been developed for the quantitative determination of dapsone (DAP) and metoclopramide hydrochloride (MCP) in both pure and dosage forms. Individual and simultaneous methods are based on the diazo coupling reaction of these drugs with benzoylacetone (BAC) in alkaline medium. The resulting azo dyes exhibit maximum absorption at 437 and 411 nm with a molar absorptivity of 4.14x10(4) and 2.97x10(4) l mol-1 cm-1 for DAP and MCP, respectively. Simultaneous determination of DAP and MCP was developed utilizing first-order digital derivative spectrophotometry. All variables have been optimized. No interferences were observed from drug excipients and the validity of the methods was tested against reference methods.

Spectrophotometric determination of dapsone in pharmaceutical products using sodium 1,2-naphthoquinone-4-sulfonic as the chromogenic reagent.

Spectrophotometric determination of dapsone is described. The dapsone reacts with sodium 1,2-naphthoquinone-4-sulfonic in pH 6.98 buffer solution to form a salmon pink compound, and its maximum absorption wavelength is at 525 nm, epsilon525=3.68 x 10(4) l mol(-1) cm(-1). The absorbance of dapsone from 0.40 to 10 microg ml(-1) obeys Beer's law. The linear regression equation of the calibration graph is C=0.2334 A + 0.01288, with a linear regression correlation coefficient of 0.9998, the detection limit is 0.24 microg ml(-1), and recovery is from 99.2 to 102.4%. Effects of pH, surfactant, organic solvents, foreign ions, and standing time on the determination of dapsone have been examined. This method is simple and can be used for the determination of dapsone in injection solution of dapsone. The results obtained by this method agreed with those by the official method (dead-stop titration method [The Chinese Pharmacopoeia, Pharmacopoeia Commission, Ministry of Health, vol. 2, fifth ed., PRC Chemical Industry Press, Beijing, 2000, p.720]).

Liquid chromatography/tandem mass spectrometric bioanalysis using normal-phase columns with aqueous/organic mobile phases - a novel approach of eliminating evaporation and reconstitution steps in 96-well SPE.

Bioanalytical methods using automated 96-well solid-phase extraction (SPE) and liquid chromatography with electrospray tandem mass spectrometry (LC/MS/MS) are widely used in the pharmaceutical industry. SPE methods typically require manual steps of drying of the eluates and reconstituting of the analytes with a suitable injection solvent possessing elution strength weaker than the mobile phase. In this study, we demonstrated a novel approach of eliminating these two steps in 96-well SPE by using normal-phase LC/MS/MS methods with low aqueous/high organic mobile phases, which consisted of 70-95% organic solvent, 5-30% water, and small amount of volatile acid or buffer. While the commonly used SPE elution solvents (i.e. acetonitrile and methanol) have stronger elution strength than a mobile phase on reversed-phase chromatography, they are weaker elution solvents than a mobile phase for normal-phase LC/MS/MS and therefore can be injected directly. Analytical methods for a range of polar pharmaceutical compounds, namely, omeprazole, metoprolol, fexofenadine, pseudoephedrine as well as rifampin and its metabolite 25-desacetyl-rifampin, in biological fluids, were developed and optimized based on the foregoing principles. As a result of the time saving, a batch of 96 samples could be processed in one hour. These bioanalytical LC/MS/MS methods were validated according to "Guidance for Industry - Bioanalytical Method Validation" recommended by the Food and Drug Administration (FDA) of the United States.

Electroanalysis of dapsone, an anti-leprotic drug.

The electrochemical oxidation and adsorption of dapsone, an anti-leprotic drug were studied in aqueous alcohol medium at a stationary glassy carbon electrode. Cyclic voltammetry studies showed one well-defined oxidation peak in the potential range 1.2-1.9 V at pH conditions 1.0, 4.0, 7.0, 9.2 and 13.0. The oxidation was irreversible and exhibited diffusion controlled adsorption. Controlled potential coulometry revealed one electron oxidation of the amino group in the molecule. A systematic study of the experimental parameters that affect the squarewave stripping response was carried out and the optimized experimental conditions were arrived at. A calibration plot was derived for the determination of the compound in solution. This method was used for the determination of dapsone in tablets and urine. The limits of determination was 0.0036 and 3.56 mg/ml and the relative standard deviation (n=10) was 4 ppt (0.4%) at a concentration level 0.100 mg/ml.

A spectrophotometric method for the determination of metoclopramide HCl and dapsone.

A rapid, sensitive and selective spectrophotometric method has been developed for the quantitative determination of metoclopramide hydrochloride (MCP) and dapsone (DAP) in both pure and dosage forms. The method is based on the diazo coupling reaction of the drugs with a new coupling agent, dibenzoyl methane in an alkaline medium. The resulting coloured azo dyes exhibit maximum absorption at 440 nm for MCP and at 470 nm for DAP with a molar absorptivity of 2.85x10(4) and 1.71x10(4) l mol(-1) cm(-1) for MCP and DAP, respectively. All variables have been optimized. No interferences were observed from excipients and the validity of the method was tested against reference method.

Liquid chromatographic determination of thalidomide in tablets, capsules, and raw materials.

A simple, isocratic liquid chromatographic method for assay of thalidomide in tablets, capsules, and raw materials was developed. The method uses a Nova-Pak octadecylsilane bonded-phase column (150 x 3.9 mm, 4 microns particle size), a mobile phase of acetonitrile-water (15 + 85), a flow rate of 1 mL/min, detection at 237 nm, and phenacetin as internal standard. Phosphoric acid was used in preparation of sample solutions to inhibit thalidomide hydrolysis. Assays ranged from 99.3 to 100.4% in raw materials from 4 manufacturers, from 79.7 to 104.8% in tablets from 7 manufacturers, and from 75.3 to 102.6% in capsules from 4 manufacturers. Assay method precisions for triplicate analyses on 5 days were 0.30% for tablets, 0.22% for capsules, and 0.22% for raw materials. Recovery from simulated tablet formulations was 100%. The method has been used to analyze individual tablets and capsules for determination of content uniformity.

Determination of serum and tissue levels of phenazines including clofazimine.

A rapid and sensitive HPLC method is described for the analysis of synthetic phenazines, including clofazimine, from a variety of biological samples. Phenazines were extracted from serum, tissue and fat using a mixture of dichloromethane and sodium hydroxide. The drugs were then quantified on a reversed-phase C18 column using a mobile phase consisting of 594 ml of water, 400 ml of tetrahydrofuran, 6 ml of concentrated acetic acid and 0.471 g of hexanesulfonic acid. In this mobile phase, each phenazine tested had its own retention time. This allowed one phenazine to be used as an internal standard for the analysis of other phenazines. The method was validated for clofazimine [3-(4-chloroanilino)-10-(4-chlorophenyl)-2,10-dihydro-2-(isopro pylimino) phenazine] and B4090 [7-chloro-3-(4-chloranilino)-10-(4-chlorophenyl)-2, 10-dihydro-2-(2,2,6,6-tetramethylpiperid-4-ylimino)phenazine ] (VI) and shown to be accurate and precise across a broad concentration range from 0.01 to 50 micrograms/g (microgram/ml). Extraction was 100% for each agent across this range. This system was used to measure clofazimine and VI levels following their administration to rats. The pharmacokinetic profile of VI was different to that of clofazimine, with high tissue concentrations but lower fat levels.

Intracellular localization of dapsone and rifampicin in skin and nerve of multidrug treated leprosy patients.

Intracellular localization of antileprosy drugs dapsone (DDS) and rifampicin (RFP) was carried out on skin and nerve lesions obtained from multidrug treated, multi (BL-LL)- and pauci (BT-TT) bacillary cases of leprosy using immunocytochemical techniques. Intracellular localization of the above drugs especially in macrophages and Schwann cells was aimed by using rabbit raised anti DDS and RFP polyclonal antibody in an indirect peroxidase assay. Our study records both intra and extracellular staining with anti DDS and RFP antibodies in the skin as well as nerve lesions of MB and PB cases treated with MDT. All the nerves under investigation had moderate to severe pathology and hence free diffusion of the drug could be attributed to the broken barrier. Basal lamina around the Schwann cell did not seem to form a barrier. It was also noted that the drug (metabolite) persisted over a long period of time).

Methods for determining concentrations of antimicrobial agents in human monocytes.

Human monocytes can be derived from the leukocyte-rich by-product of donors' blood available after platelet separation. Large volumes of the monocyte samples obtained from this product provided an opportunity to conduct experiments with relatively high concentrations of the antimicrobial agents sufficient for their detection in bioassays, thus avoiding the necessity of working with the radiolabelled drugs. Washing of the cells after their exposure to the drug may lead to an extraction of the tested agent from the cell, especially if it is a substance of low molecular weight. In our experiments we excluded the washing step, and separated the monocytes from the extracellular medium by velocity gradient centrifugation. In experiments with two rifamycins, the cell pellet as well as the extracellular fluid were subjected to a bioassay using Micrococcus luteus as a target organism. The method showed good reproducibility and consistency in results obtained.

Chromatographic-fluorometric analysis of antileprotic sulfones.

Modifications to the power supply system of a spectrophotofluorometer are described. These modifications stabilize the output of the xenon are lamp and permit the determination of nanogram quantities of antileprotic sulfones. The sulfones are removed from plasma by a single extraction with ethyl acetate, then separated by high-pressure liquid chromatography on silica, and detected in the effluent by their fluorescence. The method is specific, rapid, and reproducible.

Sodium hydnocarpate in leprosy, suggested improvements in administration

1) While the intravenous injection of sodium hydnocarpate may be admitted as the most effective means of administering hydnocarpus preparations, the blocking of veins has hitherto interfered with prolonged administration in this way.2) The sodium salts derived from a special fraction of H. wightiana and also the sodium salts of H. anthelmintica and alpina block the veins less than the salts from the whole H. wightiana oil.3) A new method of mixing the salts prepared from the whole oil of H. wightiana with blood before injection has received vein-blocking to a much more marked extent.4) Alternation of intravenous sodium hydnocarpate with subcutaneous infiltration of hydnocarpus oil with creosote is advised chiefly because of the haemolytic effect of the former preparations.

The irritant constituent of anti-leprotic oils

The constituent fractions of the acids of sapucainha oil have been separated by cold processes as afr as possible and tested for irritant properties. A crystalline acid fraction (chaulmoogric, hydnocarpic and palmitic acids), an oily acid (5 per cent), and a keto-acid (4,5 per cent), gave bland ethyl esters and sodium salts. The only product to exhibit marked irritant properties was a tarry acid fraction (9 per cent), which appeared to consist essentially of a lactonic acid. The ethyl esters of the crystalline acids were not rendered irritant by distillation at 350°/760 mm., but on long exposure in thin layers to ligh and air they change in physical and chemical character and become highly irritant, possibly owing to the production of the lactonic acid above mentioned.