Comparison of the steric selectivity on hydrophilic interaction chromatography columns modified with poly(acrylamide) possessing different morphology.

Poly(acrylamide) (PAAm)-modified hydrophilic interaction chromatography (HILIC) columns were prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) and free radical polymerization (FRP) to generate brush-like and mushroom-like polymer chains on silica particles, respectively. The maltose homologues (MHs) and cyclodextrins (CDs) were chosen as analytes to evaluate steric selectivity by the different polymer morphologies in the ATRP-PAAm and the FRP-PAAm columns. The ATRP-PAAm exhibited superior retention than the FRP-PAAm and three commercial HILIC columns. The house-made PAAm columns provided significant hydrophilicity that enabled to analysis the oligosaccharides even in 60:40 mixture of acetonitrile-aqueous buffer. In the case of three ATRP-PAAm columns characterized by different polymer lengths and the density on the silica particles, those are different thickness of the water-enriched layer, and phase ratio φ, based on hydrophilicity of them columns. The logarithm of the retention factor (ln k) displayed a non-linear dependence on the inverse of the temperature (1/T, T = 278-333 K). Notably, a similar correlation was observed to exist between the logarithm of the phase ratio (ln φ), and 1/T. A van't Hoff plot was used to determine the thermodynamic parameters of the partition process for each MH. The values of the Gibbs free energy (ΔG°) for the analytes partition on the ATRP-PAAm columns were smaller than their counterparts measured for the FRP-PAAm columns; by contrast, the opposite trend was observed for the ΔG° values measured for CDs. The standard entropy ΔS° for MHs and CDs were comparable for the two types PAAm columns, while, the standard enthalpy, ΔH° displays significant difference between the ATRP and the FRP PAAm columns. These findings indicate that the differences between PAAm morphology and polymer densities on the stationary phase surface affect analyte differentiation on the basis of molecular steric factors. The higher selectivity for MHs and CDs displayed by ATRP-PAAm columns with respect to their FRP-PAAm and commercial amide columns will be useful for the fine separation of oligosaccharides.

Functionalization using polymer or silane? A practical test method to characterize hydrophilic interaction chromatography phases in terms of their functionalization method.

Herein, commercially available columns employed in hydrophilic interaction chromatography (HILIC) were characterized by determining their ability to selectively distinguish the minute structural differences between small molecules such as nucleosides and xanthines in complex sample matrices. Principal component analysis (PCA) was applied to the data obtained from structurally similar analytes, and the results showed that HILIC columns could generally be classified into two groups: (i) silane-modified columns that were prepared from either native silica particles or silica particles modified with low-molecular-weight silanes and (ii) polymer-modified columns obtained from silica particles functionalized with organic polymers. These two groups could be further subdivided based on the functionalities attached to the respective stationary phases. These results were confirmed via cluster analysis by preparing a dendrogram using the morphology-based selectivity parameters associated with the respective columns. We were able to determine the selectivity of columns for the OH groups, i.e., α(OH) and the prevailing pH conditions (cation- and anion-exchanging natures) on the surface of the respective stationary phases; α(theobromine/theophylline) was employed to obtain a similar two-dimensional plot. This test scheme, in which five compounds were analyze for each column, was helpful for understanding the impact of factors such as the hydrophilicity, degree of hydration, acidity/basicity, or the weak ion-exchange nature of the respective stationary phases on the separation characteristics of new HILIC stationary phases. The selectivity of columns for the CH2 group was also examined. The cation-exchange nature of the HILIC columns significantly influenced native silica columns and some polymer-modified columns. Herein, 45 commercially available HILIC columns were classified according to this method, and the results proved useful for understanding distinct separation characteristics of each HILIC column, enabling improved column selection.

A selective comprehensive reversed-phase×reversed-phase 2D-liquid chromatography approach with multiple complementary detectors as advanced generic method for the quality control of synthetic and therapeutic peptides.

There is a huge, still increasing market for synthetic and therapeutic peptides. Their quality control is commonly based on a generic reversed-phase liquid chromatography (RPLC) method with C18 stationary phase and acetonitrile gradient with 0.1% trifluoroacetic acid in the mobile phase. It performs exceptionally well for a wide variety of impurities, yet structurally closely related impurities with similar sequences, not resolved in preparative RPLC, may easily coelute in the corresponding QC run as well. To address this problem an advanced generic 2D-LC impurity profiling method was developed in this work. It employs a selective comprehensive (high resolution sampling) RP×RP 2D-LC separation using a 100×2.1 mm ID column with the common acidic generic gradient in the first dimension, while RPLC under basic pH on a short 30×3 mm ID column is used in the second dimension. Recording data with a UV detector at 215 nm after 1D separation provides the common generic 1D chromatogram. However, after the 2D separation a flow splitter enabled recording of the signals of complementary detectors comprising a diode array detector (DAD) in-line with a charged aerosol detector (CAD) and a quadrupole-time-of-flight (QTOF) mass spectrometer (MS) with an electrospray ionization (ESI) source. Generic conditions of this 2D-LC method have been established through optimization of 2D stationary and mobile phase considering different pH values and buffer concentrations. The orthogonal separation principle has been documented by a number of therapeutic peptides including Exenatide, Octreotide, Cyclosporine A and Oxytocin as well as some other proprietary synthetic peptides. The information density can be further enhanced by using the QTOF-MS detector by data independent acquisition with SWATH. Through this sequential window acquisition of all theoretical fragment ion mass spectra it became possible to collect MS/MS data comprehensively in the high-resolution sampling window, thus enabling the extraction of 2D-EICs from fragment ions and the generation of 2D-contour plots of all product ions. Using Oxytocin as an example for an important therapeutic peptide, the ability of this advanced generic sRP-UV×RP-DAD-CAD-ESI-QTOF-MS/MS method with SWATH for peptide quality control is discussed.

Fragment-based Design of Zwitterionic, Strong Cation- and Weak Anion-Exchange Type Mixed-mode Liquid Chromatography Ligands and their Chromatographic Exploration.

The role of individual functional groups has been assessed with regard to surface charge and chromatographic retention. Coatings were prepared from various fragments of the chiral zwitterionic materials Chiralpak ZWIX(+) and ZWIX(-). The different chromatographic ligands allowed fine tuning of the surface charge. Chiralpak ZWIX phases showed strongly negative ζ-potentials over the entire pH-range. Zwitterionic congeners with quinuclidine and sulfonic acid moieties but lacking the quinolone ring in the ligand structure exhibited shifted ζ-potentials of around + 5 to 20 mV depending on the surrounding residues. Capillary electrophoretic mobilitiy measurements with the chromatographic ligands and molecular dynamics simulations were carried out to offer some explanation of these surface charge differences of the distinct zwitterionic stationary phases. The new mixed-mode phases were also chromatographically characterized by simple RP and HILIC tests. The results allowed their positioning within a large variety of different commercially available RP, HILIC and mixed-mode phases, which were evaluated as well, by multivariate data processing using principal component analysis. The new mixed-mode phases overall exhibit reasonable hydrophilicity-lipophilicity balance and enable retention of ionic compounds by additional ionic interactions through weak anion-exchange (WAX-type), strong cation-exchange (SCX-type) or both (RP/ZWIX-type). Hence, the new RP/ZWIX phases can be flexible tools for selectivity tuning in RP and HILIC separations.

Separation of carbohydrate isomers and anomers on poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase by hydrophilic interaction chromatography as well as determination of anomer interconversion energy barriers.

A new commercially available HPLC column, poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase (Daicel DCpak PTZ), was systematically evaluated for its carbohydrate isomer separation capability by hydrophilic interaction liquid chromatography (HILIC) with charged aerosol detection (CAD) or (tandem) mass spectrometry. Reducing sugars tend to split into two anomer peaks which makes carbohydrate isomer separations in non-derivatized form even more complicated. For practical purposes anomer separations are therefore ideally suppressed which can be accomplished by using high temperature or high pH that are both associated with fast interconversion kinetics leading to peak coalescence, or on the other hand by conditions with low chromatographic anomer selectivity. Four major hexoses (glucose, mannose, galactose, fructose), five main pentoses (ribose, ribulose, xylose, xylulose, arabinose) and five most important disaccharides (maltose, cellobiose, lactose, sucrose, trehalose) were analyzed as single carbohydrate standards by isocratic HILIC with 0.1% (v/v) formic acid and 2 mM ammonium acetate at various temperatures to study anomer interconversion equilibria in a pH-dependent manner. Rate constants of forward (α→ß) and backward (ß→α) anomerization and corresponding energy barriers were calculated. The energy barriers of anomerisation were in the range of around 83-91 kJ mol-1 at 298 K and the difference between forward (α→ß) and backward reaction (ß→α) was typically between 1-3 kJ mol-1. The systematic studies finally allowed to pick conditions for the simultaneous analysis of all 14 carbohydrates by HILIC-ESI-MS(/MS) with PTZ in gradient elution mode. A combination of carbohydrate isomer-selective LC (with PTZ), tandem MS (with carbohydrate group-selective MS1 and some species-specific SRM transitions) and a simple deconvolution strategy allowed the determination of all carbohydrates of the complex test mixture except for the disaccharide pair lactose and maltose (which can be determined as sum). Consequently, the proposed method represents a successful step towards a global glycometabolomics profiling method of mono- and disaccharides by HILIC-ESI-MS/MS.

The relationship between polymer structures on silica particles and the separation characteristics of the corresponding columns for hydrophilic interaction chromatography.

Silica particles with various pore sizes were modified with poly(acrylamide) via surface-initiated atom-transfer radical polymerization (SI-ATRP) under different reaction conditions. Twenty different columns were prepared and characterized according to a test method for hydrophilic interaction chromatography (HILIC) columns. Hydrophilic retention by the SI-ATRP columns was much higher than that of poly(acrylamide) columns prepared via free-radical polymerization and many commercially available HILIC columns. The SI-ATRP columns displayed greater selectivity for -OH groups than any of the HILIC columns based on their α(U/2dU) values. SI-ATRP functionalization was used to increase the polymer chain density on the silica particles, which suggested a brush-type morphology, and improved hydrophilic selectivity. This indicated that hydrophilic retention and selectivity could be controlled by adjusting the morphology of the organic stationary phase. This stationary phase design strategy was validated experimentally by the effective separation of highly hydrophilic analytes. The findings of this study will greatly contribute to the creation of better separation media.

Retention characteristics of poly(N-(1H-tetrazole-5-yl)-methacrylamide)-bonded stationary phase in hydrophilic interaction chromatography.

A Poly(N-(1H-tetrazole-5-yl)methacrylamide)-bonded (PTZ) stationary phase has recently gained increased attention in hydrophilic liquid chromatography (HILIC) mode for separation of polar compounds and is now commercially available as DCpak PTZ. It is chromatographically characterized in this work. The property of the new column was proven to be the most hydrophilic and acidic by separation of mixtures of purines and pyrimidines (theophylline, theobromine, uridine and 2'-deoxyuridine) in comparison to other two commercial columns Luna HILIC and LiChrospher Diol column. The retention mechanism of the new column was investigated by design-of-experiment (DoE) approach using factorial design models. Water/acetonitrile ratio in the mobile phase, buffer salt concentration and buffer pH were considered factors employing the purine/pyrimidine mixture and glucose derivatives as test samples. The resultant retention model coefficients and contour plots documented the complementary retention and selectivity profiles of the new PTZ column as compared to Diol and Luna HILIC. Moreover, it became clearly evident that the PTZ column exhibits its best HILIC performance at eluent pH ≥ 5 because the NH group in tetrazolyl moiety is dissociated under these conditions. The applicability and great potential of the new HILIC column was proven by the chromatographic separation of complex mixtures of very hydrophilic glucose and glucose derivatives (sucrose, glucosamine, glucuronic acid, glucose-1-phosphate and trehalose, glucose, maltose, glucosamine-6-phosphate, glucose-6-phosphate and gluconic acid δ-lactone) as well as monosaccharides found in N-glycans. It is concluded that the new DCpak PTZ HILIC column could have good prospects for the separation of polar compounds e.g. in metabolomics and glycomics.

Hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs and therapeutic peptides: A review based on the separation characteristics of the hydrophilic interaction chromatography phases.

Applications of hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs, i.e., glycosylated proteins represented by monoclonal antibodies are discussed in the manner of glycoproteomics. They can be analyzed using hydrophilic interaction chromatography in five different stages as (1) their intact forms, (2) their subunits, (3) N- and O-glycopeptides digested by proteases, (4) N- and O-glycans released from the glycoproteins or glycopeptides, and (5) monosaccharides. Hydrophilic interaction chromatography is a more useful tool in the order of (1) to (5). At the stages (4) and (5), quantitation of glycans and saccharides are also reported. Hydrophilic interaction chromatography is employed not only for analytical uses, but also pretreatment items as solid phase extraction, followed by reversed-phase liquid chromatography separations. Comprehensive search results of these application of hydrophilic interaction chromatography are summarized in tables to show what kind of hydrophilic interaction chromatography columns are suitable for each step of analysis.Relationship of favored and less favored hydrophilic interaction chromatography columns and their separation characteristics such as hydrophilicity, and selectivity for structural difference, is also discussed. Analysis of the therapeutic peptides (not glycosylated) using hydrophilic interaction chromatography is summarized, too.

Method optimization of hydrophilic interaction chromatography separation of nucleotides using design of experiment approaches I: Comparison of several zwitterionic columns.

A systematic method in hydrophilic interaction chromatography (HILIC) was developed for the separation of four monophosphate nucleotides using design of experiment (DOE) approaches. Three HPLC parameters, the buffer concentration (ammonium acetate concentration), gradient time, and temperature, were evaluated within the quality design framework, and the effects on chromatographic parameters were investigated. Four zwitterionic columns (ZIC-HILIC, ZIC-cHILIC, NUCLEODUR HILIC, and PC HILIC) were used to separate four nucleotides, and the HPLC conditions for each column were successfully optimized, although PC HILIC did not give peaks that were suitable for optimization. In addition, it was proved that optimized HPLC conditions differed from column to column even when the same types of zwitterionic sulfobetaine-functionalized columns were applied. This tendency was explained by differences in the separation characteristics of each column, the thickness of the water-enriched layer on the surface of the silica supports, and the pH. DOE for development of the HPLC method provides an effective explanation of the interactions among the variable parameters, especially in HILIC mode. Finally, a robust analytical method could be established by setting the optimum parameters. Among the employed columns, ZIC-cHILIC provided the widest range of suitable analytical conditions. NUCLEODUR HILIC was difficult to build a robust analytical method since the elution order of cytidine monophosphate and guanosine monophosphate was reversed.

Recent Progress in Monolithic Silica Columns for High-Speed and High-Selectivity Separations.

Monolithic silica columns have greater (through-pore size)/(skeleton size) ratios than particulate columns and fixed support structures in a column for chemical modification, resulting in high-efficiency columns and stationary phases. This review looks at how the size range of monolithic silica columns has been expanded, how high-efficiency monolithic silica columns have been realized, and how various methods of silica surface functionalization, leading to selective stationary phases, have been developed on monolithic silica supports, and provides information on the current status of these columns. Also discussed are the practical aspects of monolithic silica columns, including how their versatility can be improved by the preparation of small-sized structural features (sub-micron) and columns (1 mm ID or smaller) and by optimizing reaction conditions for in situ chemical modification with various restrictions, with an emphasis on recent research results for both topics.

Distinction of synthetic dl-α-tocopherol from natural vitamin E (d-α-tocopherol) by reversed-phase liquid chromatography. Enhanced selectivity of a polymeric C18 stationary phase at low temperature and/or at high pressure.

Separation of diastereomers of dl-α-tocopherol was studied by reversed-phase liquid chromatography using three types of stationary phases, polymeric ODS, polymeric C30, and monomeric ODS. Polymeric ODS stationary phase (Inertsil ODS-P, 3mmID, 20cm) was effective for the separation of the isomers created by the presence of three chiral centers on the alkyl chain of synthetic dl-α-tocopherol. Considerable improvement of the separation of isomers was observed on ODS-P phase at high pressure and at low temperature. Complete separation of four pairs of diastereomers was achieved at 12.0°C, 536bar, while three peaks were observed when the separation was carried out either at 12.0°C at low pressure or at 20°C at 488bar. Higher temperature (30.0°C) with the ODS-P phase resulted in only partial separation of the diastereomers even at high pressure. Only slight resolution was observed for the mixture of diastereomers with the C30 stationary phase (Inertsil C30) at 12.0°C and 441bar, although the stationary phase afforded greater resolution for ß- and γ-tocopherol than ODS-P. A monomeric C18 stationary phase did not show any separation at 12.0°C and 463bar. The results suggest that the binding site of the polymeric ODS-P phase is selective for flexible alkyl chains that provided the longest retention for the natural form, (R,R,R) form, and the enantiomer, (S,S,S) form, of dl-α-tocopherol.

Immobilized ß-cyclodextrin-based silica vs polymer monoliths for chiral nano liquid chromatographic separation of racemates.

The enantioselectivity of immobilized ß-cyclodextrin phenyl carbamate-based silica monolithic capillary columns was compared to our previously described polymer counterpart. 2,3,6-Tris(phenylcarbamoyl)-ß-cyclodextrin-6-methacrylate was used as a functional monomer for the preparation of ß-cyclodextrin (ß-CD)-based silica and polymer monoliths. The silica monoliths were prepared via the sol-gel technique in fused silica capillary followed by modification of the bare silica monoliths with an anchor group prior to polymerization with ß-CD methacrylate using either 2,2'-azobis(isobutyronitrile) or benzoylperoxide as radical initiators. On the other hand, the polymer monoliths were prepared via the copolymerization of ß-CD methacrylate and ethylene glycol dimethacrylate in different ratios in situ in fused silica capillary. The prepared silica/polymer monoliths were investigated for the chiral separation of different classes of pharmaceuticals namely; α- and ß-blockers, anti-inflammatory drugs, antifungal drugs, dopamine antagonists, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, diuretics, antihistaminics, anticancer drugs and antiarrhythmic drugs. Baseline separation was achieved for alprenolol, bufuralol, carbuterol, cizolertine, desmethylcizolertine, eticlopride, ifosfamide, 1-indanol, propranolol, tebuconazole, tertatolol and o-methoxymandelic acid under reversed phase conditions using mobile phase composed of methanol and water. The silica-based monoliths showed a comparative enantioselectivity to the polymer monoliths.

Hydrophilic interaction chromatography using a meter-scale monolithic silica capillary column for proteomics LC-MS.

A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 µm for nucleosides and a permeability of 2.1 × 10(-13) m(2), which is comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies.

Effect of pressure on the selectivity of polymeric C18 and C30 stationary phases in reversed-phase liquid chromatography. Increased separation of isomeric fatty acid methyl esters, triacylglycerols, and tocopherols at high pressure.

A high-density, polymeric C18 stationary phase (Inertsil ODS-P) or a polymeric C30 phase (Inertsil C30) provided improved resolution of the isomeric fatty acids (FAs), FA methyl esters (FAMEs), triacylglycerols (TAGs), and tocopherols with an increase in pressure of 20-70MPa in reversed-phase HPLC. With respect to isomeric C18 FAMEs with one cis-double bond, ODS-P phase was effective for recognizing the position of a double bond among petroselinic (methyl 6Z-octadecenoate), oleic (methyl 9Z-octadecenoate), and cis-vaccenic (methyl 11Z-octadecenoate), especially at high pressure, but the differentiation between oleic and cis-vaccenic was not achieved by C30 phase regardless of the pressure. A monomeric C18 phase (InertSustain C18) was not effective for recognizing the position of the double bond in monounsaturated FAME, while the separation of cis- and trans-isomers was achieved by any of the stationary phases. The ODS-P and C30 phases provided increased separation for TAGs and ß- and γ-tocopherols at high pressure. The transfer of FA, FAME, or TAG molecules from the mobile phase to the ODS-P stationary phase was accompanied by large volume reduction (-30∼-90mL/mol) resulting in a large increase in retention (up to 100% for an increase of 50MPa) and improved isomer separation at high pressure. For some isomer pairs, the ODS-P and C30 provided the opposite elution order, and in each case higher pressure improved the separation. The two stationary phases showed selectivity for the isomers having rigid structures, but only the ODS-P was effective for differentiating the position of a double bond in monounsaturated FAMEs. The results indicate that the improved isomer separation was provided by the increased dispersion interactions between the solute and the binding site of the stationary phase at high pressure.

A rare point mutation in the Ras oncogene in hepatocellular carcinoma.

PURPOSE: The Ras gene is one of the oncogenes most frequently detected in human cancers, and codes for three proteins (K-, N-, and H-Ras). The aim of this study was to examine the mutations in codons 12, 13 and 61 of the three Ras genes in cases of human hepatocellular carcinoma (HCC). METHODS: Paired samples of HCC and corresponding non-malignant liver tissue were collected from 61 patients who underwent hepatectomy. A dot-blot analysis was used to analyze the products of the polymerase chain reaction (PCR) amplification of codons 12, 13, and 61 of K-, N- and H-Ras for mutations. RESULTS: Only one mutation (K-Ras codon 13; Gly to Asp) was detected among the 61 patients. Interestingly, this patient had a medical history of surgery for both gastric cancer and right lung cancer. No mutations were found in codons 12 and 61 of K-Ras or codons 12, 13 and 61 of the N-Ras and H-Ras genes in any of the HCCs or corresponding non-malignant tissues. CONCLUSIONS: These findings indicated that the activation of Ras proto-oncogenes by mutations in codons 12, 13, and 61 does not play a major role in hepatocellular carcinogenesis.

Blockage of spontaneous Ca2+ oscillation causes cell death in intraerythrocitic Plasmodium falciparum.

Malaria remains one of the world's most important infectious diseases and is responsible for enormous mortality and morbidity. Resistance to antimalarial drugs is a challenging problem in malaria control. Clinical malaria is associated with the proliferation and development of Plasmodium parasites in human erythrocytes. Especially, the development into the mature forms (trophozoite and schizont) of Plasmodium falciparum (P. falciparum) causes severe malaria symptoms due to a distinctive property, sequestration which is not shared by any other human malaria. Ca(2+) is well known to be a highly versatile intracellular messenger that regulates many different cellular processes. Cytosolic Ca(2+) increases evoked by extracellular stimuli are often observed in the form of oscillating Ca(2+) spikes (Ca(2+) oscillation) in eukaryotic cells. However, in lower eukaryotic and plant cells the physiological roles and the molecular mechanisms of Ca(2+) oscillation are poorly understood. Here, we showed the observation of the inositol 1,4,5-trisphospate (IP(3))-dependent spontaneous Ca(2+) oscillation in P. falciparum without any exogenous extracellular stimulation by using live cell fluorescence Ca(2+) imaging. Intraerythrocytic P. falciparum exhibited stage-specific Ca(2+) oscillations in ring form and trophozoite stages which were blocked by IP(3) receptor inhibitor, 2-aminoethyl diphenylborinate (2-APB). Analyses of parasitaemia and parasite size and electron micrograph of 2-APB-treated P. falciparum revealed that 2-APB severely obstructed the intraerythrocytic maturation, resulting in cell death of the parasites. Furthermore, we confirmed the similar lethal effect of 2-APB on the chloroquine-resistant strain of P. falciparum. To our best knowledge, we for the first time showed the existence of the spontaneous Ca(2+) oscillation in Plasmodium species and clearly demonstrated that IP(3)-dependent spontaneous Ca(2+) oscillation in P. falciparum is critical for the development of the blood stage of the parasites. Our results provide a novel concept that IP(3)/Ca(2+) signaling pathway in the intraerythrocytic malaria parasites is a promising target for antimalarial drug development.

Estimation and optimization of the peak capacity of one-dimensional gradient high performance liquid chromatography using a long monolithic silica capillary column.

An estimation of the performance and optimization of gradient HPLC conditions using various columns for maximum total peak capacity was studied for "one-shot proteomics" which involves one-dimensional gradient HPLC, connected to an electrospray ionization (ESI)-mass spectrometry (MS), using a monolithic silica-C18 capillary column (350 cm long and 100 µm internal diameter) and an over 40 h shallow gradient elution with one injection. Optimization of such special one-dimensional HPLC has been a tedious task if carried out with a trial-and-error approach due to the extremely long analysis time for each run. Here, the optimized separation conditions including the column type, either particle-packed or monolithic, and the column length with a fixed gradient time are proposed by calculating the peak capacity obtainable using a long column and a long gradient time that may promote the "one-shot proteomics" approach. For instance, conventional conditions at less than 20 MPa can be adapted for a 40 h gradient elution for the proteomics experiment, and a ca. 3 m long monolithic silica-C18 capillary column was identified as the optimized medium indicated by our model with peak capacity theory. To verify this model experimentally, the numbers of identified peptides and proteins were investigated with a nano LC/MS/MS system coupled with a 3m monolithic silica-C18 capillary column by using various elution times. The experimental results showed that the numbers of identified peptides and proteins were maximized and reached a plateau with a gradient time of several tens of hours, which indicated that our model to optimize one dimensional HPLC conditions with a long column could be verified and useful.

The long-term outcomes of patients with hepatocellular carcinoma after living donor liver transplantation: a comparison of right and left lobe grafts.

PURPOSE: The feasibility of living donor liver transplantation (LDLT) using left lobe (LL) grafts has been demonstrated. However, the long-term outcome of the hepatocellular carcinoma (HCC) patients with LL grafts has not been elucidated. The aim of this study was to analyze the long-term outcomes after LDLT for HCC according to the graft type. METHODS: A retrospective analysis was performed evaluating the outcomes of LL graft recipients (n = 82) versus recipients of RL grafts (n = 46). The analysis endpoints were the overall and recurrence-free survival after LDLT. The demographics of both recipients and donors, and the tumor characteristics associated with the graft type were also analyzed. RESULTS: The graft volume (436 ± 74 g), as well as the graft volume-standard liver volume rate (38.3 ± 6.2%) of the LL graft group were significantly decreased as compared to those of the RL graft group (569 ± 82 g, 46.3 ± 6.7%; p < 0.01). The 1-, 3-, 5- and 7-year overall survival rates of the LL graft group were 88.2, 80.2, 75.7 and 72.4%, respectively, which were not significantly different compared to those of the RL graft group (95.4, 87.3, 87.3 and 87.3%). The recurrence-free survival rates of the LL graft group (89.1% at 1 year, 78.8% at 3 years, 75.8% at 5 years and 70.3% at 7 years) were similar to those of the RL graft group (88.6, 88.6, 88.6 and 88.6%). The mean peak postoperative total bilirubin levels and duration of hospital stay after surgery for the LL grafting donors were significantly decreased as compared to those of the RL grafting donors (p < 0.01). The rate of severe complications (over Clavien's IIIa) associated with LL graft procurement was 6.2%, which was lower than that in the RL graft group (15.6%). CONCLUSIONS: The long-term outcomes in the HCC patients with LL grafts were similar to those of patients receiving RL grafts, and the outcomes of the donors of LL grafts were more favorable. Therefore, LL grafts should be considered when selecting LDLT for HCC to ensure donor safety.

The impact of pegylated-interferon α-2b on partial and massive hepatectomy model in rats.

BACKGROUND/AIMS: The impact of pegylated-interferon (PEG-IFN) α-2b on liver regeneration has not yet been elucidated. METHODOLOGY: Rats were divided into the following four groups: 70% hepatectomy (Hx); 70% Hx+PEG-IFN; 90% Hx and 90% Hx+PEG-IFN group (n=6 each). Rats were pretreated with subcutaneous of PEGIFN α-2b (1.5 µg/kg) administration 24 hours before Hx. Samples were taken 24, 48 and 72 hours after Hx and the following parameters were investigated: blood analysis (AST, WBC, PLT); liver weight to body weight ratio (Lw/Bw ratio); survival and PCNA labeling index (LI). RESULTS: In the 90% Hx model, there was no significant difference between the Hx+PEG-IFN group and the Hx alone group in blood analysis; AST after postoperative 24 hours (2511 vs. 2466 IU/L), WBC (1200 vs. 1290) and PLT (107 vs. 111 x 104/mm³), in Lw/Bw ratio at postoperative 0, 24, 48, 72 hours, respectively (0.38, 0.60, 1.14, 1.69 vs. 0.37, 0.64, 1.12, 1.63), in postoperative survival (40% vs. 45%), and in PCNA LI at postoperative 0, 24, 48, 72 hours, respectively (10.4%, 16.8%, 14.6%, 12.8% vs. 10.0%, 17.1%, 15.6%, 13.7%). In the 70% Hx model, there was no significant difference between the Hx+PEG-IFN group and the Hx alone group for all parameters. CONCLUSIONS: Our data demonstrated that PEG-IFN α-2b did not affect liver regeneration and the early use of PEG-IFN α-2b would cause no problems after liver transplantation using partial grafts including living donor liver transplantation.

New silica monolith bonded chiral (R)-γ butyrolactone for enantioselective micro high-performance liquid chromatography.

A single low-molecular mass chiral selector namely (R)-acryloyloxy-ß-ß-dimethyl-γ-butyrolactone has been bonded to a modified silica-based monolith to form a new brush-type chiral stationary phase for micro-high performance liquid chromatography (HPLC) separation.