Separation of cationic polymer particles and characterization of avidin-immobilized particles by capillary electrophoresis

Cationic polymer microparticles have received much attention especially in the field of biotechnology, such that their analysis and separation have become important. So far, the separation of cationic polymer particles with different size using CE has not been achieved and the cationic particles migrated as if they are negatively charged, probably due to electrostatic interaction between capillary wall and cationic polymer particles. In this paper, the separation of cationic polymer microparticles by CE was investigated in detail. The separation of cationic particles with different size was achieved in CE by taking into account the interaction between sample particles and the inner surface of capillaries. By employing a poly(vinyl alcohol)-coated capillary, a better size separation of amine-modified latex particles was obtained compared to a Polybrene-coated capillary. It was elucidated that the composition, concentration, and pH of the background solution were also important factors in the separation of colloidal particles to avoid the surface adsorption and the characteristic aggregation of polymer particles. Furthermore, the CE analysis was applied to the characterization of cationic protein-immobilized particles.     

Determination of (R)-xanthoanthrafil, a phosphodiesterase-5 inhibitor, in a dietary supplement promoted for sexual enhancement

We describe here the first case of the finding of xanthoanthrafil, a phosphodiesterase-5 inhibitor, in a dietary supplement. A methanol extract of the supplement product was first analyzed by TLC and HPLC. The results indicated that the extract contained an unknown compound. The molecular weight of the compound was 389 and the accurate mass showed its elemental composition to be C(19)H(23)N(3)O(6). Combined with this data, NMR analysis revealed the planar structure of the unknown compound to be N-(3,4-dimethoxybenzyl)-2-(1-hydroxypropan-2-ylamino)-5-nitrobenzamide. The R-configuration of this compound had been synthesized as a phosphodiesterase-5 inhibitor, formerly reported as FR226807 by Fujisawa Pharmaceutical Co., Ltd. The absolute configuration of the isolated compound was estimated to have R-configuration by its optical rotation. Considering its general properties, this compound is renamed as (R)-xanthoanthrafil with the agreement of Astellas Pharma Inc. which is the successor of Fujisawa Pharmaceutical Co., Ltd. Quantitative analysis revealed that the content of (R)-xanthoanthrafil in the product was about 31 mg/capsule.     

Structures and bonding on a colloidal silver surface of the various length carboxyl terminal fragments of bombesin

Raman (RS) and surface-enhanced Raman scattering spectra (SERS) were measured for various length carboxyl terminal fragments (X-14 of amino acid sequence) of bombesin ( BN): BN13-14, BN12-14, BN11-14, BN10-14, BN9-14, and BN8-14 in silver colloidal solutions. Density functional theory (DFT) calculations of Raman wavenumbers and intensities with extended basis sets (B3LYP/6-31++G**) were performed with the aim of providing the definitive band allocations to the normal coordinates. The proposed band assignment is consistent with the assignment for similar compounds reported in the literature. The nonadsorbed and adsorbed molecular structures were deducted by detailed spectral analysis of the RS and SERS spectra, respectively. This analysis also allowed us to propose the particular surface geometry and orientation of these peptides on silver surface, and their specific interaction with the surface. For example, a SERS spectrum of BN8-14 indicates that the interaction of a thioether atom and Trp8 with the silver surface is favorable and may dictate the orientation and conformation of adsorbed peptide. One of the most prominent and common features in all of the fragments' SERS spectra is a approximately 692 cm (-1) band due to nu(C-S) accompanied by two or three bands of different C-S conformers for all, except BN8-14, which suggests that all of the above-mentioned compounds adsorb on the silver surface through the thioether atom and that the attachment of Trp8 produces limitation in a number of possible C-S conformers adopted on this surface. Our results also show clearly that His12 and CO do not interact with the colloid surface, which supports our earlier results.     

Melt crystallization and crystal transition of poly(butylene adipate) revealed by infrared spectroscopy

The structure evolution of poly(butylene adipate) (PBA) during isothermal melt crystallization and phase transition processes is investigated by Fourier transform infrared spectroscopy (FTIR). Detailed IR spectra analysis and band assignment are performed to disclose the bands sensitive to the alpha-form crystalline order of PBA. It is revealed from the in situ IR study that the functionalities within PBA chains alter simultaneously during the melt crystallization process. From the analysis of the spectral changes, it is found that band shifts take place during the phase transition process of PBA from its metastable beta-form crystal to the stable alpha-form. Notable band shifts in the 1300-1100 cm(-1) region indicate that the twist of polymer chains in the alpha-form is located in the C-O-C and C-O linkages. Moreover, the results elucidated that the different segments of molecular chains tune up their conformations synchronously during the beta to alpha crystal transition process of PBA. It is suggested that the betaalpha phase transition process proceeds randomly throughout the solid at a constant rate.     

Self-modeling curve resolution (SMCR) analysis of near-infrared (NIR) imaging data of pharmaceutical tablets

The idea of quality by design (QbD) has been proposed in pharmaceutical field. QbD is a systematic approach to control the product performance based on the scientific understanding of the product quality and its manufacturing process. In the present study, near-infrared (NIR) imaging is utilized as a tool to achieve this concept. A practical use of a chemometrics technique called self-modeling curve resolution (SMCR) is demonstrated with NIR imaging analysis of pharmaceutical tablets containing two ingredients, a soluble active ingredient, pentoxifylline (PTX), and an insoluble excipient, palmitic acid. Concentration profiles obtained by SMCR reveal that the homogenous distribution of chemical ingredients strongly depends on the grinding time and that its process plays a central role in quantitative control, say sustained-release of PTX. In addition, pure component spectra by SMCR indicate a sequential change of specific NIR peak intensities following the increase of the grinding time. The spectra change shows a molecular structure change related to its crystallinity during grinding process. Accordingly, this study clearly demonstrates that NIR imaging combined with SMCR can be a powerful tool to reveal chemical or physical mechanism induced by the manufacturing process of pharmaceutical products and that it may be a solid solution for QbD of pharmaceutical products.     

Quick analysis of baicalin in Scutellariae Radix by enzyme-linked immunosorbent assay using a monoclonal antibody

To establish an immunoassay for baicalin (BA), a hybridoma cell line (9D6) secreting a monoclonal antibody (MAb) against BA was prepared by cell fusion with splenocytes derived from a mouse immunized with BA-bovine serum albumin (BSA) conjugate and a myeloma cell line, SP2/0-Ag14. MAb 9D6 shows specific reactivity against BA and its aglycone, baicalein, but not against other natural products. We developed an enzyme-linked immunosorbent assay (ELISA) using MAb 9D6 in a competitive manner, ranging from 200 ng/mL to 2 μg/mL. After validating the developed ELISA on the basis of intra- and inter-assays and a recovery experiment, it was found that the ELISA was not only simple, but also sufficiently reliable and accurate for quality control of Scutellariae Radix. It allowed determination of BA in complex and mixed materials, such as Kampo medicines.     

Enantioselective synthesis of silanol

An enantioselective nucleophilic substitution reaction of achiral dialkoxysilane has been developed. The reaction proceeds with efficient stereocontrol on the silicon chirality center to give the enantioenriched silyl ether, which can be converted to the silanol without loss of enantiopurity. We have analyzed the steric course of the reaction by using DFT calculations and propose a transition state model to explain the observed enantioselectivity.     

Mechanism of Mukaiyama-Michael Reaction of Ketene Silyl Acetal: Electron Transfer or Nucleophilic Addition?

Mechanism of Mukaiyama-Michael reaction of ketene silyl acetal has been discussed. The competition reaction employing various types of ketene silyl acetals reveals that those bearing more substituents at the beta-position react preferentially over less substituted ones. However, when ketene silyl acetals involve bulky siloxy and/or alkoxy group(s), less substituted compounds react preferentially. The Lewis acids play an important role in these reactions. Enhanced preference for the more sterically demanding Michael adducts is obtained with Bu(2)Sn(OTf)(2), SnCl(4), and Et(3)SiClO(4) in the former reaction while TiCl(4) gives the highest selectivity for the less sterically demanding products in the latter case. These results are interpreted in terms of alternative reaction mechanisms. The reaction of less bulky ketene silyl acetals are initiated by electron transfer from these compounds to a Lewis acid. On the other hand, bulkier ketene silyl acetals undergo a ubiquitous nucleophilic reaction. Such a mechanistic change is discussed based on a variety of experimental results as well as the semiempirical PM3 MO calculations.     

Determination of total nitrogen content, pH, density, refractive index, and brix in Thai fish sauces and their classification by near-infrared spectroscopy with searching combination moving window partial least squares

Near-infrared (NIR) transflectance spectra in the region of 1100-2500 nm were measured for 100 Thai fish sauces. Quantitative analyses of total nitrogen (TN) content, pH, refractive index, density and brix in the Thai fish sauces and their qualitative analyses were carried out by multivariate analyses with the aid of wavelength interval selection method named searching combination moving window partial least squares (SCMWPLS). The optimized informative region for TN selected by SCMWPLS was the region of 2264-2428 nm. A PLS calibration model, which used this region, yielded the lowest root mean square error of prediction (RMSEP) of 0.100% w/v for the PLS factor of 5. This prediction result is significantly better than those obtained by using the whole spectral region or informative regions selected by moving window partial least squares regression (MWPLSR). As for pH, density, refractive index and brix, the 1698-1722, and 2222-2258 nm regions, the 1358-1438 nm region, the 1774-1846, and 2078-2114 nm regions, and the 1322-1442, and 2000-2076 nm regions were selected by SCMWPLS as the optimized regions. The best prediction results were always obtained by use of the optimized regions selected by SCMWPLS. The lowest RMSEP for pH, density, refractive index and brix were 0.170, 0.007 g cm(-3), 0.0079 and 0.435 degrees Brix, respectively. Qualitative models were developed by using four supervised pattern recognitions, linear discriminant analysis (LDA), factor analysis-linear discriminant analysis (FA-LDA), soft independent modeling of class analog (SIMCA), and K neareat neighbors (KNN) for the optimized combination of informative regions of the NIR spectra of fish sauces to classify fish sauces into three groups based on TN. All the developed models can potentially classify the fish sauces with the correct classification rate of more than 82%, and the KNN classified model has the highest correct classification rate (95%). The present study has demonstrated that NIR spectroscopy combined with SCMWPLS is powerful for both the quantitative and qualitative analyses of Thai fish sauces.