Determination of Glimepiride in Human Plasma by LC-MS-MS and Comparison of Sample Preparation Methods for Glimepiride

A sensitive and selective method for quantitation of glimepiride in human plasma was established using liquid chromatography-electrospray ionization tandem mass spectrometry. Three different methods for the sample preparation of glimepiride and an internal standard were investigated (liquid-liquid extraction, solid-phase extraction and protein precipitation). Glipizide was used as an internal standard. Compounds were separated on a C₁₈ column with 80% acetonitrile and 20% deionized water (adjusted to pH 3.5 with acetic acid), as mobile phase at a flow rate of 200 L min^–1. By use of multiple reaction monitoring mode in MS-MS with liquid-liquid extraction and solid-phase extraction, glimepiride and glipizide were detected without severe interference from the human plasma matrix. Glimepiride produced a protonated precursor ion ([M+H]⁺) at m/z 491 and a corresponding product ion at m/z 352, and the internal standard produced a protonated precursor ion ([M+H]⁺) at m/z 446 and a corresponding product ion at m/z 321. The limit of quantitation was 0.1 ng mL^–1, 0.5 ng mL^–1 and 1.0 ng mL^–1 when using liquid-liquid extraction, solid-phase extraction and protein precipitation, respectively. The validation, reproducibility, stability, and recovery of the different sample preparation methods were comparable and all the methods gave reliable results. The method has been successfully applied to pharmacokinetic study of glimepiride in human plasma.     

High-Throughput Analysis of Sofalcone in Human Plasma by Use of Automated 96-Well Protein Precipitation and LC-MS-MS

A sensitive and selective method for the determination of sofalcone in human plasma was established by use of protein precipitation and liquid chromatography-tandem mass spectrometry. Plasma samples were transferred into 96-well plate using an automated sample handling system and spiked with 10 L of 2 g mL^–1 internal standard solution (d₃-sofalcone). 0.5 mL of acetonitrile was added to the 96-well plate and the plasma samples were then vortexed for 30 sec. After centrifugation, the supernatant was transferred into another 96-well plate and completely evaporated at 40 °C under a stream of nitrogen. The dry residue was reconstituted with mobile phase. All sample transfer and protein precipitation was automated through the application of both the PerkinElmer MultiPROBE II HT and TOMTEC Quadra 96 workstation. The limit of quantitation of sofalcone was 2 ng mL^–1 using a sample volume of 0.2 mL for the analysis. The reproducibility of the method was evaluated by analyzing five samples at nine quality control (QC) levels over the nominal concentration range from 2 ng mL^–1 to 1000 ng mL^–1. Validation of the method showed that the assay has good precision and accuracy. Sofalcone and internal standard produced a protonated precursor ion ([M+H]⁺) at m/z 451 and 454, and both gave a corresponding product ion at m/z 315. The high sample throughput of the method has been successfully applied to a pharmacokinetic study of sofalcone in human plasma.     

Supramolecular reactor from self-assembly of rod-coil molecule in aqueous environment

We synthesized an amphiphilic coil-rod-coil triblock molecule consisting of hexa-p-phenylene as a rod block and poly(ethylene oxide) with the number of repeating units of 17 as coil blocks and investigated aggregation behavior in aqueous environment. The rod-coil molecule was observed to aggregate into discrete micelles consisting of hydrophobic disklike rod bundles encapsulated by hydrophilic poly(ethylene oxide) coils. The aromatic bundles of the micelles were demonstrated to be used as an efficient supramolecular reactor for the room temperature Suzuki cross-coupling reaction of a wide range of aryl halides, including even aryl chlorides with phenylboronic acids in aqueous environment. These results demonstrate that self-assembly of amphiphilic rod-coil molecules can provide a useful strategy to construct an efficient supramolecular reactor for aromatic coupling reaction.     

Catalyst- and solvent-free click synthesis of cyclodextrin-based polyrotaxanes exploiting a nitrile N-oxide

A catalyst- and solvent-free synthesis of cyclodextrin-based polyrotaxanes exploiting a stable nitrile N-oxide as an end-capping agent was achieved. The C-C bond-forming end-capping reaction of an allyl-terminated pseudopolyrotaxane with the nitrile N-oxide proceeded smoothly by solid-state grinding in a mortar to afford a polyrotaxane.     

Profiling surface glycans on live cells and tissues using quantum dot-lectin nanoconjugates

The surface of mammalian cells is densely coated with complex glycans, which are directly involved in cell-cell or cell-protein interactions that trigger various biological responses. Here, we present a novel glycomics approach that uses quantum dot (Qdot)-lectin nanoconjugates to interrogate the surface glycans of tissues and patterned cells. Our approach allows highly sensitive in situ monitoring of specific lectin-glycan interactions and quantitative information on surface glycans for each examined cell line and tissue. The results clearly show significant changes in glycosylation for each cell line and tissue sample. We expect that these results will be applicable in cancer diagnostics and promote the development of new analytical tools for glycomics.     

Development of a simultaneous analytical method for selected anorectics,methamphetamine, MDMA,and their metabolites in hair using LC-MS/MS to prove anorectics abuse

Owing to the tight control of methamphetamine, it is presumed that phentermine, an amphetamine-type anorectic, has recently been considered a supplement for methamphetamine abusers in Korea. In addition, the abuse of other anorectics obtained by inappropriate means has become a social issue. Hair is a useful specimen to prove chronic drug use. Therefore, an analytical method for the simultaneous detection of phentermine, phendimetrazine, amfepramone, fenfluramine, mazindol, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA), as well as their metabolites, which covers the major amphetamines and anorectic agents in Korea, in hair was established and validated using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using 1 % HCl in methanol and then filtered and analyzed by LC-MS/MS with electrospray ionization in positive mode. The validation results for selectivity, linearity, matrix effect, recovery, process efficiency, intra- and interassay precision and accuracy, and processed sample stability were satisfactory. The limits of detection ranged from 0.025 to 1 ng/10 mg hair and the limits of quantification were 0.25 ng/10 mg hair for every analyte except mazindol and phentermine, for which they were 10 ng/10 mg hair. The method was successfully applied for the segmental determination of selected anorectics, methamphetamine, MDMA, and their metabolites in hair from 39 drug suspects. Among the anorectics, phentermine and/or phendimetrazine were identified with or without methamphetamine in the hair samples. Closer supervision of the inappropriate use of anorectics is necessary. Also, hair analysis is useful for monitoring the abuse potential of unnoticed drugs.     

Effect of Molecular Structure on Complexation Between Acidic- and Basic-Polypeptides in Solution

Abstract

Interpolymer complex formation between basic polypeptides, poly(L- proline) Form I [PLP(I)], Form II [PLP(II)] and poly-4-hydroxy-L-proline (PHLP), and acidic polypeptides, poly(L-glutamic acid) (PLGA), poly(D- glutamic acid) (PDGA) and poly(L-aspartic acid) (PLAA), has been studied in water-methanol (1:2 v/v) mixed-solvent by viscometry, potentiometry, light scattering and circular dichroism (CD) measurements. It has been found that polymer complexes between basic- and acidic- polypeptides are formed via hydrogen bonding with a stoichiometric ratio of basic/acidic polypeptides =1:2 (in unit mole ratio) and that PLP(II) forms polymer complex more favorably with PLGA than with PLAA, and the complex of PLP(II) with PLGA is also more favorable than the complex formation of PHLP with PLGA. In addition, the complex formation is highly dependent on the conformation, especially the optical structure of the component polymers, i.e., the stereoselective complexation is observed. The PLGA having a right-handed helix at pH 3.2 formed the complex favorably and quickly with left-handed helix PLP(II), whereas PDGA having a left-handed helix at pH 3.2 favorably formed the complex with right-handed helix PLP(I).     

Amide-based nonheme cobalt(III) olefin epoxidation catalyst: partition of multiple active oxidants Co(V)=O, Co(IV)=O, and Co(III)-OO(O)CR

A mononuclear nonheme cobalt(III) complex of a tetradentate ligand containing two deprotonated amide moieties, [Co(bpc)Cl(2)][Et(4)N] (1; H(2)bpc = 4,5-dichloro-1,2-bis(2-pyridine-2-carboxamido)benzene), was prepared and then characterized by elemental analysis, IR, UV/Vis, and EPR spectroscopy, and X-ray crystallography. This nonheme Co(III) complex catalyzes olefin epoxidation upon treatment with meta-chloroperbenzoic acid. It is proposed that complex 1 shows partitioning between the heterolytic and homolytic cleavage of an O-O bond to afford Co(V)=O (3) and Co(IV)=O (4) intermediates, proposed to be responsible for the stereospecific olefin epoxidation and radical-type oxidations, respectively. Moreover, under extreme conditions, in which the concentration of an active substrate is very high, the Co-OOC(O)R (2) species is a possible reactive species for epoxidation. Furthermore, partitioning between heterolysis and homolysis of the O-O bond of the intermediate 2 might be very sensitive to the nature of the solvent, and the O-O bond of the Co-OOC(O)R species might proceed predominantly by heterolytic cleavage, even in the presence of small amounts of protic solvent, to produce a discrete Co(V) ?O intermediate as the dominant reactive species. Evidence for these multiple active oxidants was derived from product analysis, the use of peroxyphenylacetic acid as the peracid, and EPR measurements. The results suggest that a less accessible Co(V)=O moiety can form in a system in which the supporting chelate ligand comprises a mixture of neutral and anionic nitrogen donors.     

Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.