Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase

Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in α-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl α-d-galactopyranoside, for a new α-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK _a of resorufin (~6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease. Zhen-Dan Shi and Omid Motabar contributed equally to this work     

Resonance Rayleigh scattering study on the interaction of gold nanoparticles with berberine hydrochloride and its analytical application

A heavy metals enzymatic-based assay using papain was developed. Papain was assayed using the Casein-coomassie-dye-binding assay. The assay is sensitive to several heavy metals. The IC₅₀ (concentration of toxicant giving 50% inhibition) of Hg²⁺, Ag²⁺, Pb², Zn²⁺ is 0.39, 0.40, 2.16, 2.11 mg l⁻¹, respectively. For Cu²⁺ and Cd²⁺ the LOQ (limits of quantitation) is 0.004 and 0.1 mg l⁻¹, respectively. The IC₅₀ and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox™, 48 h Daphnia magna, and 96 h Rainbow trout. The papain assay is xenobiotics tolerant, has a wide pH for optimum activity, is temperature stable, and has a relatively quick assay time. The papain assay was used to identify polluted water samples from industrial sources in Penang, Malaysia. We found one site where the assay gave a positive toxic response. The toxicity of the site was confirmed using Atomic Emission Spectrometry analysis.     

Conductimetric,Potentiometric and Visual Titrimetric Methods for Analysis of Nadolol

Abstract

Nadolol, a beta-blocking agent, has been assayed in pure form and in pharmaceutical formulation by acidimetric titrations using visual, potentiometric and conductimetric methods for detecting the end point in aqueous as well as in non-aqueous media. The method of assay involves the determination of the amount of nadolol in a given weight of sample. Six samples were run for each method and the percentages of recovery of nadolol from its tablets were calculated. For aqueous titrations, the average percentages of recovery for the visual, potentiometric and conductimetric methods are 100.6 ± 1.0, 99.3 ± 1.9 and 100.1 ± 2.0 respectively. For the non-aqueous titrations, the average percentages of recovery are 98.6 ± 0.8, 99.5 ± 0.5 and 98.4 ± 1.3 for the visual, potentiometric and conductimetric methods respectively.     

A New Method for the HPLC Analysis of Pt(II) in Urine

Abstract

An analytical method has been developed to measure Pt(II) in urine via derivatization and UV or HPLC analysis. A measured quantity of urine is heated briefly with diethyl ammonium diethyl-dithiocarbamate, and the resulting Pt(Et₂NCS₂)₂ is extracted into a measured volume of chloroform. Concentrations of Pt(II) are determined by UV absorption at 346 nm or by reverse phase HPLC analysis. The detection limit for Pt(II) as its dithiocarbamate is ～ 1 ng by HPLC; the concentration limit for HPLC analysis by direct extraction was ～ 25 ng/ml. Chromatographic response was linearly related to Pt(II) concentration over the range 100-4, 000 ng/ml; dilution of more concentrated samples has extended this range to at least 30, 000 ng/ml. This method has been applied to the analysis of Pt(II) in the urine of patients who have received cis-dichlorodiamniineplatinum(II) (CDDP) chemotherapy.     

High-Performance Liquid Chromatographic Assay for Chlorquine and its Two Major Metabolites,Desethylchloroquine and Bidesethylchloroquine in Biological Fluids

Abstract

A high-performance liquid chromatography (HPLC) method for the determination of chloroquine and its two major metabolites in biological fluids is described. Hydroxychloroquine is used as an internal standard (I.S.). Drug, metabolites and I.S. were extracted as bases with diethyl ether by a single step procedure. After drying and evaporation of the organic phase, the residue was dissolved into the mobile phase and injected into the chromatographic system. Separation was performed using a normal phase column (Inertsil sill with mixture of acetonitrile, methanol and ammonia as mobile phase. The detection was carried out by fluorescence measurement : excitation wavelength was set at 325 nm and emission at 380 nm. The limit of detection was near 3.7 ng ml^?1 for chloroquine and metabolites. No chromatographic interference could be detected by endogenous compounds or other antimalarial drugs. Because of the good accuracy of the method, concentrations were determinated with a relative standard deviation lower than 7% at the 25 ng ml^?l level for all substances.

An excellent precision was obtained over the range of concentrations tested, 25–1000ng ml^?l. This method can be applied to therapeutic, pharmacokinetic and epidemial studies.     

A Creatinine Specific Enzyme Electrode

Abstract

An extremely specific creatinine electrode was successfully developed for selective analysis of aqueous creatinine with a linear response over the range from 5 mg/l to 100 mg/l. The endogenous and exogenous species generally present in the serum do not interfere in the assay with the exception of ammonia. With the use of the electrode serum samples containing high creatinine can be assayed directly after pre-treatment. However, the electrode is not sensitive enough to detect the normal level serum creatinine due to the dilution of the serum after treatment.     

An Improved Method to Assay Folates in Milk by a Turbidimetric Microbiological Assay

Abstract

Folates in milk are heat labile and methods used to protect these folates during sample preparation for microbiological estimation of this vitamin result in opaque solutions unsuitable for turbidimetric assays. This has necessitated the use of titrimetric assay for milk folates which are long and cumbersome. By the use of rennin precipitation of casein under conditions which preserve the folate activity, an optically clear solution is obtained which can then be used for turbidimetric assay. This method is described in this paper.     

Simultaneous Quantification of Ceftazidime and Pyridine,its Main Degradation Product,by High-Performance Liquid Chromatography

ABSTRACT

A high performance liquid chromatographic method for the simultaneous assay of ceftazidime and pyridine, the principal degradation product of ceftazidime, is described. The method was fully validated in terms of recovery, linearity, selectivity and precision. An application was done on stability of ceftazidime at 40 mg.mL¹ in infusion solutions 0.9% NaCl and 5% glucose on 24 hours in ambulatory infusion device. Quantification results of pyridine were more linear and accurate than ceftazidime. Pyridine was a good label in ceftazidime stability studies.     

Synthesis,characterization, antiproliferative,and antimicrobial activity of osmium(II) half-sandwich complexes

New osmium(II)-arene complexes [(η⁶-C₆H₆)OsCl(C₅H₄N-2-CH=N-C₆H₅X)](PF₆) (X = p-flouro (1), p-chloro (2), p-methyl (3)) were synthesized by reaction of the corresponding bidentate N,N′-ligands with the osmium-arene precursor [(η⁶-C₆H₆)Os(μ-Cl)Cl]₂ in a 2:1 ratio. These complexes were characterized by UV–Vis, IR, ¹H, ¹³C NMR spectroscopy, and elemental analysis and, for compound 1, a single crystal X-ray structure was also obtained. The complexes were investigated for antiproliferative activity in vitro against MCF-7 (human breast adenocarcinoma), Caco-2 (human epithelial colorectal adenocarcinoma), and HepG2 (human hepatocellular carcinoma) tumor cell lines. To test their selectivity, the non-tumor HEK293 (human embryonic kidney) cell line was used. The compounds were selective toward the tumor cell lines when compared to the known anticancer drug 5-fluorouracil which displayed low selectivity. The compounds were substantially more active against Caco-2 than 5-fluorouracil. They also showed moderate activity against the other two tumor cell lines. In addition, the antimicrobial activity of complex 2 was explored against a panel of antimicrobial-susceptible and -resistant Gram-negative and Gram-positive bacteria. Complex 2 showed anti-mycobacterial activity against Mycobacterium smegmatis and bactericidal activity against drug-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus ATCC 43300.     

Development and validation of UPLC method for simultaneous quantification of carvedilol and ivabradine in the presence of degradation products using DoE concept

A methodical design-of-experiments were performed by applying quality-by-design concepts to establish a design-space for simultaneous and rapid quantification of Carvedilol and Ivabradine by UPLC in the presence of degradation products. Response-surface, central-composite design, and quadratic model were employed for statistical assessment of experimental data using the Design-Expert software. Response variables such as resolution and retention time were analyzed statistically for chromatographic screening. During DoE study, various plots such as perturbation, contour, 3D and design-space plots were considered for method optimization. The method was developed using C8 [100?×?2.1?mm, 1.8?µ] UPLC column, mobile phase comprising 0.5% triethylamine buffer [pH 6.4] and acetonitrile in the ratio of 50:50 v/v, the flow rate of 0.4?mL minute^?1 and UV detection at 285?nm for both Carvedilol and Ivabradine. The method was developed with a short run time of two minutes. The method was found to be linear in the range of 25.0–199.9?µg?mL^?1 and 8.9–21.3?µg?mL^?1 for Carvedilol and Ivabradine, respectively with a correlation coefficient of 0.9998 in each case. The recovery values were found in the range of 99.7–100.8% and 98.9–100.9% for Carvedilol and Ivabradine, respectively. The method was validated according to ICH Q2 (R1) guidelines.