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Continuous-flow fast atom bombardment liquid chromatography-mass spectrometry in bioanalysis study

Le 21 octobre 2016, 11:06 dans Humeurs 0


Medicilon offers comprehensive and FDA/CFDA GLP-compliant bioanalysis services to support preclinical and clinical development for small molecule drugs, biologics, vaccines and biomarkers.

Website:                           Tel: +86 (21) 5859-1500

Development of an on-line coupling of liquid chromatography (LC) with fast atom bombardment (FAB) mass spectrometry (MS) by using continuous-flow fast atom bombardment (CF-FAB) or CF-frit-FAB interfaces, has opened up new opportunities in the analysis of polar, ionic and thermolabile compounds often encountered in bioanalysis study.

The parameters affecting the performance of CF-FAB were studied. Stable CF-FAB ionization conditions are a matter of importance for reproducible performance of CF-FAB. A uniform film of the liquid on the CF-FAB target with equilibrium between the rate of solvent delivery and evaporation was found to be essential for maintenance of stable CF-FAB conditions. Various parameters such as the target material and shape, mobile phase composition, flow-rate and ion source temperature affected the properties of the liquid film on the target. Of those targets tested, the stainless-steel target with a gold-plated drain channel showed the best overall performance.

Intact glucosinolates were separated and identified in standard mixtures and in a plant extract from Brussels sprouts using CF-frit-FAB LC-MS and LC-MS/MS. The mass spectra of glucosinolates in the negative ion FAB LC-MS mode mainly contained molecular weight information. The daughter ion spectra after collision-induced dissociation of the characteristic (M-H) $sp-$ anion gave structural information about the compound, based on the group- and compound-specific fragmentation and group-characteristic neutral losses.

The applicability of the CF-FAB technique in quantitative bioanalysis study was demonstrated. Specific CF-FAB LC-MS methods for quantitative determination of dextromethorphan and erythromycin 2$spprime$-ethylsuccinate in plasma were developed.

The most serious limitation of CF-FAB technique in LC-MS operation is the maximum flow-rate of ca. 15 $mu$l/min, necessitating the splitting of the eluent from conventional LC columns. The solute splitting was avoided completely in target-compound analysis by using the phase-system switching approach prior to CF-FAB LC-MS. Using the phase-system switching approach, a lowering of the flow-rate from 1.0 ml/min in the liquid chromatography system to 15 $mu$l/min going into the mass spectrometer was achieved without splitting. Optimal mobile phases and flow-rates were used for the liquid chromatographic separation as well as for the mass spectrometric detection. A specific method for quantitative determination of erythromycin 2$spprime$-ethylsuccinate in plasma using phase-system switching CF-FAB LC-MS is described.



A Quantitative Method for the Determination of Proteins in Complex Bioanalysis studies

Le 21 octobre 2016, 11:06 dans Humeurs 0

Medicilon offers comprehensive and FDA/CFDA GLP-compliant bioanalysis services to support preclinical and clinical development for small molecule drugs, biologics, vaccines and biomarkers.

Website:                              Tel: +86 (21) 5859-1500

A quantitative method for the determination of proteins in complex bioanalysis studies has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma.

This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography–mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 μl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.


Protein phosphorylation catalyzed by protein kinase is one of the most important post-translational modifications and plays a significant regulatory role in many vital bioanalysis studies. Aberrant protein-phosphorylation states and kinase activity are closely associated with many human diseases. Monitoring the kinase activity and its inhibition is essential for fundamental biochemical research and kinase-targeted drug discovery. Nanomaterial-based kinase assays provide a promising toolkit for exploring protein kinase functions, which have attracted growing interest in academic research, biomedical diagnosis, and pharmaceutical discovery. The recent advances in the development of the protein kinase activity assays based on various nanomaterials, classifies these methods by different analytical techniques, summarizes the general design strategies, and offers perspectives on future developments.

Expression, purification, and assay development for recombinant expressed tryptophan synthase from Escherichia coli

Le 17 octobre 2016, 05:36 dans Humeurs 0


Medicilon offers fully integrated pharmaceutical services for the global scientific community. We focus on providing an exceptional client-centered experience and advancing the drug discovery process.

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The emergence of antibiotic resistant bacteria has been a global concern for numerous decades. Bacteria are able to attain resistance to many current antibiotics due to high mutation rates and the ability to share genomic DNA with other bacterial species. New pathways need to be exploited for drug design studies, not only to limit the spread of resistance but also to generate a greater diversity in antimicrobial mechanisms. Tryptophan is an essential amino acid that is synthesized from enzymes found only in plants and bacteria. The lack of any enzyme homologues for humans makes this pathway an ideal target for inhibition studies. We have previously shown in laboratory studies that bacterial mutants lacking the tryptophan gene are unable to grow in tryptophan deprived media, further demonstrating the potential for drug design studies against it. E. coli Tryptophan synthase (TrpS), the enzyme catalyzing the last two steps in the biosynthetic pathway of tryptophan, was chosen for inhibitor compound screeningstudies. TrpS consists of two distinct subunits (TrpS-α and TrpS-β), each able to catalyze their own reaction. TrpS-α and TrpS-β protein were separately cloned, expressed and purified to >90% purity using recombinant-expression techniques. Assays were developed for the TrpS-α and TrpS-β enzymes to assess their catalytic activity and the TrpS-β assay was converted to a 96-well plate format for initial inhibitor screening. In vitro and in silico screens using a first generation library of compounds generated against IGPS, the enzyme just prior to TrpS in the tryptophan biosynthetic pathway, resulted in several potential inhibitors for future lead compound screening  development. The results of this study demonstrate the potential to develop a new class of inhibitors against E. coli TrpS.

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