Quantification of the In Vitro and In Vivo Metabolic Fates of 2-, 3- and 4-Bromobenzoic Acids Using High Temperature LC Coupled to ICP-MS and Linear Ion Trap MS

High temperature liquid chromatography (HTLC), with water as the mobile phase, combined with ICP-MS tuned to the detection of Br, for quantification, and a linear ion trap MS, for structural identification, were applied to determine the disposition and metabolic fate of 2-, 3- and 4-bromobenzoic acids (BBAs) following in vitro incubation with rat hepatocytes at 4 mM. The separation of the metabolites was performed using a thermal gradient to increase the eluotropic strength of the aqueous mobile phase through the run to elute less polar components. The use of highly aqueous solvents for separations involving ICP-MS is advantageous because the water does not change the conductivity of the plasma thereby providing a more stable system. The improved system stability resulted in better sensitivity, as shown by the increased signal intensity for HTLC compared to conventional reversed-phase separations. Using HTLC to investigate the in vitro metabolic fate of the BBAs showed the major route of metabolism to be glycine conjugation, irrespective of the structure of the parent, but with different amounts produced depending on the positional isomer. The comparison of HTLC with the conventional methodology showed that chromatography at elevated temperatures had no effect on the observed metabolite profile. HTLC was also applied to urine obtained from an in vivo sample and showed an improved chromatographic peak shape compared to conventional liquid chromatography (LC) whilst providing the same analytical result.     

Photobleaching-based flow measurement in a commercial capillary electrophoresis chip instrument

For microfluidic analytical instruments, a facile, fast, and accurate instrument test is highly demanded. The test includes the quantitative verification of the relationship between pressure drop and flow velocity for the hydrodynamic pump, between the electric voltage and electroosmotic flow (EOF) for the high-voltage supply, and the chip quality. The key point for the test is the measurement of the flow velocity. However, most currently available velocimetries cannot be directly used without any instrumental modification or adding extra instruments. We applied a recently developed Laser Induced Fluorescence Photobleaching Anemometer (LIFPA) for the instrument test through measuring fluid flow velocity in a microfluidic instrument with optical measurement without any modification and extra instrument. We have successfully used the method to test Caliper HTS 250 System from Caliper Life Sciences (Hopkinton, MA) with its own light source and detector. The experimental result demonstrates that this single-point method of measuring flow velocity can be easily used for accurate test of a microfluidic instrument in less than 10 min at extremely low cost without any modification and extra instrument.     

How to realize the linear scale-up process for rapid purification using high-performance counter-current chromatography

This study used the isolation of six constituents from Selaginella tamariscina as an example to demonstrate how to achieve rapid and predictable linear scale-up processes in both normal- and reversed-phase high-performance counter-current chromatography. After systematic optimization of solvent systems, sample concentration, sample loading volume, rotation speed and flow rate on the analytical Mini-DE centrifuge, the optimized parameters obtained were directly transferred to the preparative Midi-DE centrifuge, with nearly the same purities, resolutions and elution times but with 50 times the throughput. Amentoflavone (446.7 mg, 97.8%), robustaflavone (21.6 mg, 89.4%), bilobetin (80.7 mg, 92.7%), hinokiflavone (15.1 mg, 85.5%), isocryptomerin (34.8 mg, 89.6%) and an apigenin-diglucoside (46.3mg, 96.4%) were obtained with amounts and purities shown in parentheses as analysed by HPLC. The process, therefore, offers an efficient and rapid method of obtaining sufficient quantities of target compounds with significantly increased throughput after a linear scale-up.     

Impurities in commercial phytantriol significantly alter its lyotropic liquid-crystalline phase behavior

The lyotropic liquid-crystalline phase behavior of phytantriol is receiving increasing interest in the literature as a result of similarities with glyceryl monooleate, despite its very different molecular structure. Some differences in the phase-transition temperature for the bicontinuous cubic to reverse hexagonal phase have been reported in the literature. In this study, we have investigated the influence that the commercial source and hence the purity has on the lyotropic phase behavior of phytantriol. Suppression of the phase-transition temperatures (by up to 15 degrees C for the bicontinuous cubic to reverse hexagonal phase transition) is apparent with lower-purity phytantriol. In addition, the composition boundaries were also found to depend significantly on the source and purity of phytantriol, with the bicontinuous cubic phase + excess water boundary occurring at a water content above that reported previously (i.e., >5% higher). Both the temperature and compositional changes in phase boundaries have significant implications on the use of these materials and highlight the impact that subtle levels of impurities can play in the phase behavior of these types of materials.     

Cucurbit[7]uril host-guest complexes with small polar organic guests in aqueous solution

The host-guest stability constants for the inclusion of a series of small neutral polar organic guests in cucurbit[7]uril (CB[7]) have been determined in aqueous solution by (1)H NMR titrations. The dependence of the stability constant on the nature of the guests indicates that hydrophobic and dipole-quadrupole interactions are responsible for the binding. The complexation-induced chemical shift changes in the guest proton resonances, coupled with energy-minimization calculations, suggest that the guests are located such that their dipole moment is aligned perpendicular with the quadrupole moment of the CB[7] host. The stability constants for acetone and acetophenone decrease in the presence of Na(+) or K(+) cations as a result of cation capping of the CB[7] portals.     

Surfactant enhanced lipase containing films characterized by confocal laser scanning microscopy

An environmentally benign method for the synthesis of noble metal nanoparticles has been reported using aqueous solution of gum kondagogu (Cochlospermum gossypium). Both the synthesis, as well as stabilization of colloidal Ag, Au and Pt nanoparticles has been accomplished in an aqueous medium containing gum kondagogu. The colloidal suspensions so obtained were found to be highly stable for prolonged period, without undergoing any oxidation. SEM–EDXA, UV–vis spectroscopy, XRD, FTIR and TEM techniques were used to characterize the Ag, Au and Pt nanoparticles. FTIR analysis indicates that –OH groups present in the gum matrix were responsible for the reduction of metal cations into nanoparticles. UV–vis studies showed a distinct surface plasmon resonance at 412 and 525 nm due to the formation of Au and Ag nanoparticles, respectively, within the gum network. XRD studies indicated that the nanoparticles were crystalline in nature with face centered cubic geometry. The noble metal nanoparticles prepared in the present study appears to be homogeneous with the particle size ranging between 2 and 10 nm, as evidenced by TEM analysis. The Ag and Au nanoparticles formed were in the average size range of 5.5 ± 2.5 nm and 7.8 ± 2.3 nm; while Pt nanoparticles were in the size range of 2.4 ± 0.7 nm, which were considerably smaller than Ag and Au nanoparticles. The present approach exemplifies a totally green synthesis using the plant derived natural product (gum kondagogu) for the production of noble metal nanoparticles and the process can also be extended to the synthesis of other metal oxide nanoparticles.     

Polymer brushes on graphene

A critical bottleneck for the widespread use of single layer graphene is the absence of a facile method of chemical modification which does not diminish the outstanding properties of the two-dimensional sp(2) network. Here, we report on the direct chemical modification of graphene by photopolymerization with styrene. We demonstrate that photopolymerization occurs at existing defect sites and that there is no detectable disruption of the basal plane conjugation of graphene. This method thus offers a route to define graphene functionality without degrading its electronic properties. Furthermore, we show that photopolymerization with styrene results in self-organized intercalative growth and delamination of few layer graphene. Under these reaction conditions, we find that a range of other vinyl monomers exhibits no reactivity with graphene. However, we demonstrate an alternative route by which the surface reactivity can be precisely tuned, and these monomers can be locally grafted via electron-beam-induced carbon deposition on the graphene surface.     

Identification and quantification of glutathione adducts of clozapine using ultra-high-performance liquid chromatography with orthogonal acceleration time-of-flight mass spectrometry and inductively coupled plasma mass spectrometry

The application of sulphur-specific detection via ultra-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (UPLC/ICPMS) to detect and quantify the glutathione (GSH)-adducts produced via the in vitro formation of reactive metabolites is demonstrated. The adducts were formed in human liver microsomes supplemented with unlabelled GSH for clozapine. The calculation of adduct concentration was performed via comparison of the peak areas to calibration curves constructed from omeprazole, a sulphur-containing compound over the range of 0.156 to 15.62 μM of sulphur with a detection limit of 1.02 ng of sulphur on-column. Identification of the adducts was performed using conventional UPLC/time-of-flight (TOF)-MS with the calculation of clozapine intrinsic clearance carried out by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). The use of ICPMS in this way appears to offer a novel, rapid and sensitive means of determining the quantity of GSH conjugates with the combined adducts producing 0.9 μM of reactive metabolite out of a total of 3.5 μM of metabolites. The GSH adduct therefore represents 26% of this total produced as a result of the metabolism of drug to reactive species.     

Harmonic vibrational frequencies: scale factors for pure, hybrid, hybrid meta, and double-hybrid functionals in conjunction with correlation consistent basis sets

Scale factors for (a) low (<1000 cm(-1)) and high harmonic vibrational frequencies, (b) thermal contributions to enthalpy and entropy, and (c) zero-point vibrational energies have been determined for five hybrid functionals (B3P86, B3PW91, PBE1PBE, BH&HLYP, MPW1K), five pure functionals (BLYP, BPW91, PBEPBE, HCTH93, and BP86), four hybrid meta functionals (M05, M05-2X, M06, and M06-2X) and one double-hybrid functional (B2GP-PLYP) in combination with the correlation consistent basis sets [cc-pVnZ and aug-cc-pVnZ, n = D(2),T(3),Q(4)]. Calculations for vibrational frequencies were carried out on 41 organic molecules and an additional set of 22 small molecules was used for the zero-point vibrational energy scale factors. Before scaling, approximately 25% of the calculated frequencies were within 3% of experimental frequencies. Upon application of the derived scale factors, nearly 90% of the calculated frequencies deviated less than 3% from the experimental frequencies for all of the functionals when the augmented correlation consistent basis sets were used.