A novel cross-linked chitosan phase was synthesized with a novel cross linking reagent having both aliphatic and aromatic functionalities. Retention behavior for polycyclic aromatic hydrocarbons (PAHs) were compared with that obtained on several commercially available stationary phases including monomeric and polymeric octadecylsilicas (ODSs) in liquid chromatography (LC). The results clearly demonstrated the applicability of the newly-synthesized chitosan phase as the stationary phase in LC, especially for the separation of PAHs having different planarities, and as novel wall-paint or wall-paper materials for reducing volatile organic compounds in indoor air environment.
Keto acids are known to be key intermediates in various metabolic pathways. The development of an accurate method for the determination of keto acids is therefore important for diagnosing metabolic disorders as well as elucidating cellular metabolic processes in the TCA cycle, glycolysis and amino acid biosynthesis. In this study, we have developed a comprehensive and reliable LC-MS/MS method for the analysis of biological samples using a pre-column derivatization process. Ten keto acids, including α- and β-keto acids, were converted to the corresponding O-(2,3,4,5,6-pentafluorobenzyl)oxime derivatives and analyzed by LC-MS/MS. Oxaloacetic acid, which is generally considered to be unstable, was also successfully derivatized under mild reaction conditions. The pretreatment procedure used in this study was simple and did not require any difficult extraction or evaporation processes. The separation and detection of the derivatized keto acids was achieved using an LC-MS/MS system in multiple reaction monitoring mode. This newly developed method was applied to the analysis of keto acids in rat plasma, and showed good reproducibility (1.1–4.7% as CV) and recovery (96–109%) rates. This method also exhibited a low limit of detection in the range of 0.01–0.25 μM and good linearity (r2 > 0.997) over a wide concentration range of up to 300 μM. Based on these performance characteristics, this method could be readily applied to the comprehensive analysis of keto acids in biological samples.
This paper reports an improvement of the detectability in the capillary electrophoretic analysis of glycoproteins by using a sweeping technique based on the complexation between glycoproteins and borate ions: sweeping via borate complexation. In this technique, an anionic glycoprotein solution containing no borate ions is injected as a long plug into a capillary filled with a borate buffer. Due to the complexation between glycoproteins and borate ions, the analytes in a long sample zone are swept by borate ions to a narrow zone. When g1-acid glycoprotein dissolved in a phosphate buffer was injected into poly(vinyl pyrrolidone) coated capillary filled with 50 mM borate buffer (pH 10.0) for 120 s, the 30-40 fold increase in the sensitivity was achieved relative to conventional zone electrophoretic analysis. Furthermore, three glycoproteins could be separated with enhanced sensitivities under the sweeping via borate complexation condition. The enrichment efficiency was dependent on the sugar chain content of the glycoproteins. This indicated that the complexation of glycoproteins with borate ions played a crucial role in the sweeping via borate complexation technique.
A new method was developed for the analysis of age-related steroids, such as dehydroepiandrosterone (DHEA), androstenedione (ASD), and testosterone (TES), utilizing a temperature-responsive polymer of poly(N-isopropylacrylamide)(PNIPAAm) as the stationary phase of LC-MS. A temperature-dependent separation of the steroids was achieved using an aqueous mobile phase. The efficacy of MeOH as an organic modifier in temperature-responsive chromatography (TRC) was also evaluated by LC-MS analysis of steroids. Although the MeOH content in the mobile phase did not significantly influence the resolution, MeOH enhanced the peak intensities of most of the steroids with an atmospheric pressure chemical ionization (APCI) interface. Well-resolved chromatograms of three steroids in plasma and saliva were obtained at 50 °C with a 5% MeOH aqueous solution as the eluent. To increase the detection sensitivity for MS, DHEA was derivatized with isonicotinoyl azide (INA), and subjected to LC-ESI-MS/MS.
Molecular imprinting for (+)-catechin (CA) recognition was successfully demonstrated using naringerin (NG) and 7-hydroxyflavanon (7HF) derivatives as dummy-template molecules. Molecularly imprinted polymer (MIP) films were synthesized using methacryloyl NG (NGMA) by semi-covalent-type imprinting process on gold substrate. The selective binding property toward CA was confirmed by surface plasmon resonance measurements, and we clarified that the imprinting process was effective for the formation of the specific binding cavities from the results of binding experiments for non-imprinted polymers (NIPs), in which the binding amounts towards CA and its analogue were negligible. Moreover, we synthesized successfully the bulk MIPs for CA separation using methacryloyl 7HF (7HFMA) as dummy-template and methacrylamidyl β-cyclodextrin (βCD) as another functional monomers by combination of semi-covalent- and non-covalent-type molecular imprinting processes. For the bulk MIPs, the binding property of obtained βCD-MIP was confirmed by the fluorescent measurements derived from CA remained in the supernatant after interaction with obtained βCD-MIP. From these results, the molecular imprinting with dummy-template is effective technique for preparation of the CA recognition materials (MIPs). The technique is promising for the development of the separation materials for CA as well as the other important compounds.
Binding equilibrium of nine kinds of phenylalkylamines (PAA) as non-charged species to nonionic Brij 58 micelle has been investigated through the measurements of the electrophoretic mobility by capillary zone electrophoresis. Analysis of the binding equilibrium involves: (1) acid dissociation constant of PAA in an aqueous solution, Ka, (2) conditional acid dissociation constant of PAA in Brij 58 micelle solution, Ka', and (3) binding constant of PAA+ as protonated species of PAA to the micelle, KB,HB. Binding constant of non-charged species of PAA to the micelle, KB,B, was then determined through the three equilibrium constants. Both binding constants of KB,HB and KB,B increased with increasing molecular volume of PAA, and linear free energy relationship was verified between the molecular volume and logarithmic value of the binding constants. Binding constants of KB,B are larger than KB,HB for every PAA. This result also suggests the hydrophobic binding that less charged species are more strongly bound to the hydrophobic surfactant micelle.
A method using micellar electrokinetic chromatography (MEKC) was developed for simultaneous determination of flavonoids in citrus fruits, which mainly consist of polymethoxyflavones (PMFs) and flavanone glycosides (FGs). Because there are significant differences in the magnitude of polarity between PMFs and FGs, it was difficult to optimize the conditions of MEKC using sodium dodecyl sulfate (SDS) for proper separation of both PMGs and FGs simultaneously. This was also found to be the case when an organic modifier such as dimethylsulfoxide (DMSO) was added to the running solutions. Simultaneous MEKC separation of PMGs and FGs was achieved by using sodium deoxycholate (SDC) in the presence of DMSO because of the relatively small difference in free energy to transfer from bulk solutions to the SDC micelle between PMFs and FGs. Fourteen flavonoids consisting of 4 flavanones, including three FGs and 10 flavones, including nine PMFs,were separated nearly completely using 10 mM phosphate buffer (pH 7.2), 75 mM SDC, 20% DMSO, and 10% acetonitrile as a running solution for MEKC. Additionally, we proposed a novel index that is an alternative to migration time and corrects variation of migration times in measurements successfully. Some of the flavonoids in fruits such as flat lemon and ponkan orange were identified with the help of this index. Further, fruit rind samples of ponkan oranges were taken monthly from a single tree from July to December and were analyzed for changes in content of four flavonoids.
Spongy monoliths prepared using EVA, poly(ethylene-co-vinyl acetate) were chromatographically analyzed to reveal their bio-compatible properties. EVA formed as monolithic format including the hydrolyzed EVA (EVA-HYD) showed relatively hydrophobic properties as well as different separation selectivity for phenol and benzene diols compared with those on EDMA and GDMA polymer-based columns. Interestingly, planar recognition ability on EVA-HYD changed with higher content of acetonitrile in the mobile phase. IR study suggested that internal hydrogen bonding played as an important role on the chromatographic properties of EVA-HYD monoliths. EVA-HYD monoliths separated polypeptides based on hydrophobic interaction in acidic gradient mode, on the other hand, the recoveries of the polypeptides were almost quantitative in neutral mobile phase. As the results, EVA and EVA-HYD spongy monoliths changed the separation selectivity depending on mobile phase conditions.
Hydrophilic interaction liquid chromatographic (HILIC) separation of biologically important thiols derivatized with 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F) was performed for the first time. After optimization of mobile phase conditions, including acetonitrile content, pH, and buffer concentration, eight ABD-thiols (cysteamine, cysteine, N-acetylcysteine, glutathione, homocysteine, cysteinylglycine, γ-glutamylcysteine, and N-(2-mercaptopropionyl)glycine as an internal standard) were successfully separated in 16 min on ZIC-HILIC column with sulfoalkylbetaine groups. The optimum mobile phase was acetonitrile/50 mM ammonium formate buffer, pH 2.5 (82/18, v/v). The limits of quantitation for ABD-thiols, when signal-to-noise ratio was 10, ranged from 1–120 nM, lower than previous reports that used reversed-phase conditions. The increased sensitivity was partly due to enhancement of fluorescence intensity of ABD-thiols in the acetonitrile-rich mobile phase used in HILIC conditions.